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## **Meet the editor**

Dr. Subrata Dey is the Director of School of Biotechnology and Biological Sciences and Professor of Biotechnology and Human Molecular Genetics at the West Bengal University of Technology. His laboratory has long been involved in research on Molecular Genetics of Down syndrome. His current research interests include the identification of genes involved in the development of

Congenital heart disease and Alzheimer's disease in both Down syndrome and healthy individuals. His group also studies the mechanism of radiation induced genomic instability and radioprotection. Dr. Dey has been teaching courses in Genetics, Molecular Biology, Evolution and Developmental Biology for more than thirty years. He is also the founding Director of Centre for Genetic Studies. Dr. Dey is the author of many scientific papers and has edited two books on Down syndrome.

Contents

**Preface VII**

**Section 1 Prenatal Diagnosis and Genetic Counseling 1**

Eny Maria Goloni Bertollo

Chapter 3 **Prenatal Screening and Diagnosis 35**

Manuel Rosety-Rodriguez

Chapter 6 **Heart Diseases in Down Syndrome 95** A. K. M. Mamunur Rashid

Kazuko Kudo

**Section 2 Diseases in Children with Down Syndrome 63**

**Patients with Down Syndrome 65**

Jaana Marttala

Chapter 1 **Down Syndrome: Clinical and Genetic Aspects, Genetic**

Chapter 2 **Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome 21** Ksenija Gersak, Darija M. Strah and Maja Pohar-Perme

Chapter 4 **Control of Dental Biofilm and Oral Health Maintenance in**

Ana Paula Teitelbaum and Gislaine Denise Czlusniak

Chapter 5 **How to Design an Exercise Program TO Reduce Inflammation in Obese People With Down Syndrome 83**

Chapter 7 **Myeloid Leukemia Associated with Down Syndrome 107**

Francisco J. Ordonez, Gabriel Fornieles, Alejandra Camacho, Miguel A. Rosety, Antonio J Diaz, Ignacio Rosety, Natalia Garcia and

**Counseling and Prenatal Screening and Diagnosis 3**

Érika Cristina Pavarino, Joice Matos Biselli, Walter Pinto Junior and

## Contents

## **Preface XI**



#### X Contents


Preface

and environmental factors.

This book provides recent developments and advances in research on Down syndrome. It covers a wide range of topics, including investigations on prenatal diagnosis and screening, genetic counseling, neoplastic disease, congenital heart disease, dentistry and oral health, obesity, molecular genetics and neurological disorders in Down syn‐ drome. It is also a resource for scientists and research workers who wish to learn more about Down syndrome. To date, well over one hundred chromosome syndromes have been reported. Whilst on an individual basis many of these are rare, together they make a major contribution to human morbidity and mortality. Chromosome aneuploidies are now known to account for a large proportion of spontaneous pregnancy loss and child‐ hood disability, and can also contribute to the genesis of a significant proportion of ma‐ lignancy. Trisomy 21 in humans, commonly referred as Down syndrome, is the most common genetic cause of mental retardation and most frequent autosomal trisomies in liveborns. In approximately ninety five percent of cases , the extra chromosome occurs as a result of meiotic nondisjunction or abnormal segregation of chromosome. The cause of nondisjunction of chromosome 21 is largely unknown. Although several hy‐ potheses have been suggested, it is still unclear as to whether particular gene loci on chromosome 21 are sufficient to cause Down syndrome and its associated features. The risk factors associated with the birth of Down syndrome are enigmatic. The overall ma‐ ternal risk factors for Down syndrome birth are multifactorial and include both genetic

This book is organized into four sections. All sections include chapters on recent advan‐ ces in research on Down syndrome. The editor endeavored to keep the big picture and overarching philosophy of the review articles in focus while editing the text and illus‐

The first section deals with our present knowledge on common diseases in Down syn‐ drome. The second one discusses the present status of investigations on molecular ge‐ netics of Down syndrome. The third section covers the recent investigations on neurological disorders in Down syndrome, and the concluding section focuses on pre‐

This book provides a concise yet comprehensive source of current information on Down syndrome. Research workers, scientists, medical graduates and pediatricians

The editor wants to acknowledge the superb assistance of staff members and manage‐ ment of InTech Publisher. In particular, Mr. Dejan Grgur for co-ordination and editorial assistance. We are grateful to all contributing authors and scientists who made this

will find the book Down syndrome an excellent source for reference and review.

trations for consistent use of scientific terminology and level of exposition.

natal diagnosis, screening and genetic counseling in Down syndrome.


## Preface

**Section 3 Genetics of Down Syndrome 115**

**VI** Contents

**Region Genes 117**

**Phenotype 173**

**Syndrome 209**

**Syndrome Brain 237** Jie Lu and Volney Sheen

Chapter 8 **Molecular Pathways of Down Syndrome Critical**

Chapter 9 **Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology 149**

Chapter 10 **RCAN1 and Its Potential Contribution to the Down Syndrome**

Chapter 11 **Laterality Explored: Atypical Hemispheric Dominance in Down**

George Grouios, Antonia Ypsilanti and Irene Koidou

Melanie A. Pritchard and Katherine R. Martin

Ferdinando Di Cunto and Gaia Berto

Sujay Ghosh and Subrata Kumar Dey

**Section 4 Neural Development in Down Syndrome 207**

Chapter 12 **Genetic and Epigenetic Mechanisms in Down**

This book provides recent developments and advances in research on Down syndrome. It covers a wide range of topics, including investigations on prenatal diagnosis and screening, genetic counseling, neoplastic disease, congenital heart disease, dentistry and oral health, obesity, molecular genetics and neurological disorders in Down syn‐ drome. It is also a resource for scientists and research workers who wish to learn more about Down syndrome. To date, well over one hundred chromosome syndromes have been reported. Whilst on an individual basis many of these are rare, together they make a major contribution to human morbidity and mortality. Chromosome aneuploidies are now known to account for a large proportion of spontaneous pregnancy loss and child‐ hood disability, and can also contribute to the genesis of a significant proportion of ma‐ lignancy. Trisomy 21 in humans, commonly referred as Down syndrome, is the most common genetic cause of mental retardation and most frequent autosomal trisomies in liveborns. In approximately ninety five percent of cases , the extra chromosome occurs as a result of meiotic nondisjunction or abnormal segregation of chromosome. The cause of nondisjunction of chromosome 21 is largely unknown. Although several hy‐ potheses have been suggested, it is still unclear as to whether particular gene loci on chromosome 21 are sufficient to cause Down syndrome and its associated features. The risk factors associated with the birth of Down syndrome are enigmatic. The overall ma‐ ternal risk factors for Down syndrome birth are multifactorial and include both genetic and environmental factors.

This book is organized into four sections. All sections include chapters on recent advan‐ ces in research on Down syndrome. The editor endeavored to keep the big picture and overarching philosophy of the review articles in focus while editing the text and illus‐ trations for consistent use of scientific terminology and level of exposition.

The first section deals with our present knowledge on common diseases in Down syn‐ drome. The second one discusses the present status of investigations on molecular ge‐ netics of Down syndrome. The third section covers the recent investigations on neurological disorders in Down syndrome, and the concluding section focuses on pre‐ natal diagnosis, screening and genetic counseling in Down syndrome.

This book provides a concise yet comprehensive source of current information on Down syndrome. Research workers, scientists, medical graduates and pediatricians will find the book Down syndrome an excellent source for reference and review.

The editor wants to acknowledge the superb assistance of staff members and manage‐ ment of InTech Publisher. In particular, Mr. Dejan Grgur for co-ordination and editorial assistance. We are grateful to all contributing authors and scientists who made this

book possible by providing valuable research and review articles. Finally, I would like to dedicate this book to children with Down syndrome who need our love and care to lead a healthy life.

> **Dr. Subrata Dey** Director, School of Biotechnology & Biological Sciences, West Bengal University of Technology, India

**Section 1**

**Prenatal Diagnosis and Genetic Counseling**

**Prenatal Diagnosis and Genetic Counseling**

book possible by providing valuable research and review articles. Finally, I would like to dedicate this book to children with Down syndrome who need our love and care to

**Dr. Subrata Dey**

School of Biotechnology & Biological Sciences,

West Bengal University of Technology,

Director,

India

lead a healthy life.

VIII Preface

**Chapter 1**

**Down Syndrome: Clinical and Genetic Aspects, Genetic**

Down syndrome (DS) or trisomy 21 is the most common genetic disorder with a prevalence of 1 in 660 live births [1]. In 1959, Lejeune and colleagues discovered the genetic basis of DS and named as trisomy of chromosome 21, which is the smallest human autosomal chromo‐ some [2]. Trisomy 21 can occur as three types of chromosomal abnormalities: free trisomy 21, translocation or mosaicism. Free trisomy 21 is characterized by the presence of three complete copies of chromosome 21, occurring in about 90-95% of DS cases [3-5]. More than 90% of the cases of chromosomal nondisjunction are of maternal origin, mainly during meiosis I, about 5% involve an additional paternal extra chromosome and a small propor‐ tion (2%) is consequence of post-zygotic mitotic non-disjunction [6]. Translocations are at‐ tributed to 1-7% of the cases, with Robertsonian translocation involving chromosomes 14 and 21 being the most common type. Mosaicism, characterized by some cells containing 46 chromosomes and others with 47 chromosomes (with an extra chromosome 1), is reported in

DS phenotype is complex and varies among individuals, who may present a combination of dysmorphic features and developmental delay [7]. The intellectual disability is a characteristic observed in all cases and the most frequent clinical features include muscular hypotonia (99%), diastasis of the muscle rectus of abdomen (90%), upslanted palpebral fissures (90%), microce‐ phaly (85%), flat occipital (80%), joint hyperextension (80%), broad hands with short fingers (70%), short stature (60%), clinodactyly of fifth finger (50%), epicanthal fold (40%), low-set ears (50%), single palmar crease (40%), atlantoaxial instability (15%) and label-femoral instability (10%) [8]. On average, 50-70% of children with DS have congenital heart defects, such as ventric‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Pavarino et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Counseling and Prenatal Screening and Diagnosis**

Érika Cristina Pavarino, Joice Matos Biselli,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52950

**1.1. Clinical and genetic aspects**

**1. Introduction**

1-7% of DS cases [3-5].

Walter Pinto Junior and Eny Maria Goloni Bertollo

## **Down Syndrome: Clinical and Genetic Aspects, Genetic Counseling and Prenatal Screening and Diagnosis**

Érika Cristina Pavarino, Joice Matos Biselli, Walter Pinto Junior and Eny Maria Goloni Bertollo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52950

## **1. Introduction**

#### **1.1. Clinical and genetic aspects**

Down syndrome (DS) or trisomy 21 is the most common genetic disorder with a prevalence of 1 in 660 live births [1]. In 1959, Lejeune and colleagues discovered the genetic basis of DS and named as trisomy of chromosome 21, which is the smallest human autosomal chromo‐ some [2]. Trisomy 21 can occur as three types of chromosomal abnormalities: free trisomy 21, translocation or mosaicism. Free trisomy 21 is characterized by the presence of three complete copies of chromosome 21, occurring in about 90-95% of DS cases [3-5]. More than 90% of the cases of chromosomal nondisjunction are of maternal origin, mainly during meiosis I, about 5% involve an additional paternal extra chromosome and a small propor‐ tion (2%) is consequence of post-zygotic mitotic non-disjunction [6]. Translocations are at‐ tributed to 1-7% of the cases, with Robertsonian translocation involving chromosomes 14 and 21 being the most common type. Mosaicism, characterized by some cells containing 46 chromosomes and others with 47 chromosomes (with an extra chromosome 1), is reported in 1-7% of DS cases [3-5].

DS phenotype is complex and varies among individuals, who may present a combination of dysmorphic features and developmental delay [7]. The intellectual disability is a characteristic observed in all cases and the most frequent clinical features include muscular hypotonia (99%), diastasis of the muscle rectus of abdomen (90%), upslanted palpebral fissures (90%), microce‐ phaly (85%), flat occipital (80%), joint hyperextension (80%), broad hands with short fingers (70%), short stature (60%), clinodactyly of fifth finger (50%), epicanthal fold (40%), low-set ears (50%), single palmar crease (40%), atlantoaxial instability (15%) and label-femoral instability (10%) [8]. On average, 50-70% of children with DS have congenital heart defects, such as ventric‐

© 2013 Pavarino et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ular septal defect, atrial septal defect, tetralogy of Fallot, patent ductus arteriosus and atrioven‐ tricular septal defect [3,4,9]. There are also ocular problems, such as refractive errors, nystagmus, abnormalities of the retina, among others [10]. About 80% of cases present hearing loss, which can be conductive, sensorineural, or mixed [11]. Thyroid dysfunction, particularly hypothyroidism [9], periodontal diseases [10], upper airway obstruction [12] and hypogonad‐ ism [14] are more frequent in individuals with DS than in the general population. Other impor‐ tant clinical aspects of DS include immunodeficiency [15], increased risk for hematological disorders and leukemia [16] and early onset of Alzheimer's disease [17].

is the age-related risk times 3.5. For those with maternal age ≥35 at previous trisomy 21, the revised risk is the age-related risk times 1.7 [26]. So, these risk times implies that other fac‐ tors might influence the risk for DS in young mothers [27]. On the other hand, translocation may be recurrent. If neither parent carries a balanced translocation, the DS recurrence risk is low, probably similar to that of free trisomy 21. However, if one of the parents is the carrier of a balanced translocation, the risk of recurrence is dependent on the type of translocation and the sex of the carrier parent. In the case of Robertsonian translocations involving chro‐ mosome 13, 14, 15 or 22 and the chromosome 21, the recurrence risk at time of amniocentesis is of up to 17% when the mother is the carrier and of up to 1.4% when the carrier of this balanced translocation is the father. On the other hand, if one of the parents is the carrier of a balanced translocation involving two chromosomes 21, the recurrence risk of DS is 100% [26]. Thus, once diagnosed as a case of DS due to a translocation, a karyotype analysis of

Down Syndrome: Clinical and Genetic Aspects, Genetic Counseling and Prenatal Screening and Diagnosis

http://dx.doi.org/10.5772/52950

5

For an individual with DS, the theoretical chance to have a child with DS is 50%, and 66% when both partners have DS. However, empiric risks are difficult to estimate, once the re‐ production rates are low. Empiric data indicate a 30–50% chance of a woman with DS have a child with DS [26]. However, considering that the rate of fetal death between 11 weeks and term is about 43% for trisomy 21 [28], the chance of birth of a child with DS decreases. For individuals with mosaicism, the maximum theoretical recurrence risk is as high as 50%, but is dependent upon the proportion of trisomic gonadal cells and whether the other partner

Genetic counseling is also important to guide the parents about caring for the child with DS. Be‐ cause individuals with DS often experience delays in reaching various developmental mile‐ stones, early intervention with speech therapy, occupational therapy, and physical therapy is recommended as it maximizes long-term outcomes [29]. As healthcare has improved for indi‐ viduals with DS, the average life expectancy has increased by more than 30 years, from an aver‐ age of 25 years of age in 1983 to almost 60 years of age in 2000 [30]. A study performed between 1985–2004 in England showed that the one-year survival of live births with DS increased, espe‐ cially in babies with cardiovascular malformations, reaching almost 100% [31], and a more re‐

Genetic counselors should balance the negative aspects of DS, such as birth defects, medical complications, and developmental delay, with positive aspects like available treatments, thera‐ pies, and the ability for people with DS and their families to enjoy a high quality of life [33].

There are several methods that allow the early detection of DS in prenatal phase. At this point, it is not possible avoid congenital malformations or genetic diseases, but the objective is its early detection, looking for emotional and psychological preparation for parents and family and adequate medical support and monitoring for the child's birth. Furthermore, ear‐ ly detection allows treatment of malformations of the complications that may occur, pre‐

cent study showed that the 25-year survival of DS individuals is about 87.5% [32].

venting or attenuating their evolution through surgical correction in utero.

**3. Prenatal screening and diagnosis**

both parents is recommended.

has DS as well [26].

The development of secondary sexual characteristics in DS is similar to other adolescents. The fetal oogenesis of women with the syndrome appears to be normal and, therefore, they are capable of reproduction [18]. On the other hand, men have diminished reproductive ca‐ pacity, showing testicular histology compatible with oligospermia and, frequently, hypogo‐ nadism [19]. However, there have been reports of men with Down syndrome who have fathered pregnancies [20].

## **2. Genetic counseling**

Genetic counseling can be defined as a communication process that takes care of the human problems associated with the occurrence or recurrence of a genetic disease in a family with the purpose of providing individuals and families comprehensive understanding of all the implications related to genetic disease under discussion, the options that the current medi‐ cine offers for therapy or for reducing the risk of occurrence or recurrence of the disease and psychotherapeutic support [21,22].

For DS, a well-established risk factor is advanced maternal age at conception [23,24]. The es‐ timated risk for fetal trisomy 21 for a woman aged 20 years at 12 weeks of gestation is about 1 in 1000, and the risk of such woman delivering an affected baby at term is 1 in 1500. The risk for this aneuploidy for a woman aged 35 years at 12 weeks of gestation is about 1 in 250 and the risk of delivering an affected baby at term is 1 in 350 [25].

Although there is considerable variation in the physical features of individuals with DS, most individuals present with a range of characteristics that enable clinical diagnosis of the syn‐ drome [3,4,7]. However, cytogenetic investigation of individuals who present with clinical characteristics of DS is fundamental to establish a precise diagnosis, which may have implica‐ tions in the genetic counseling process, once it is very important in determining the recurrence risk of the syndrome. In addition, the karyotype analysis of affected individuals identifies cases that may have been inherited making necessary the investigation of the parents' karyotypes. In this case, the cytogenetic investigation of the genitors is essential to establish the risk of recur‐ rence of the syndrome in future generations. Thus, all individuals with a diagnosis suggestive of DS should be referred to a genetic counseling service.

Accurate estimation of recurrence risks depends upon the verification of the individual's karyotype. Cases of free trisomy 21 and mosaicism generally do not recur in siblings of indi‐ viduals with DS. For women with maternal age <35 at previous trisomy 21, the revised risk is the age-related risk times 3.5. For those with maternal age ≥35 at previous trisomy 21, the revised risk is the age-related risk times 1.7 [26]. So, these risk times implies that other fac‐ tors might influence the risk for DS in young mothers [27]. On the other hand, translocation may be recurrent. If neither parent carries a balanced translocation, the DS recurrence risk is low, probably similar to that of free trisomy 21. However, if one of the parents is the carrier of a balanced translocation, the risk of recurrence is dependent on the type of translocation and the sex of the carrier parent. In the case of Robertsonian translocations involving chro‐ mosome 13, 14, 15 or 22 and the chromosome 21, the recurrence risk at time of amniocentesis is of up to 17% when the mother is the carrier and of up to 1.4% when the carrier of this balanced translocation is the father. On the other hand, if one of the parents is the carrier of a balanced translocation involving two chromosomes 21, the recurrence risk of DS is 100% [26]. Thus, once diagnosed as a case of DS due to a translocation, a karyotype analysis of both parents is recommended.

For an individual with DS, the theoretical chance to have a child with DS is 50%, and 66% when both partners have DS. However, empiric risks are difficult to estimate, once the re‐ production rates are low. Empiric data indicate a 30–50% chance of a woman with DS have a child with DS [26]. However, considering that the rate of fetal death between 11 weeks and term is about 43% for trisomy 21 [28], the chance of birth of a child with DS decreases. For individuals with mosaicism, the maximum theoretical recurrence risk is as high as 50%, but is dependent upon the proportion of trisomic gonadal cells and whether the other partner has DS as well [26].

Genetic counseling is also important to guide the parents about caring for the child with DS. Be‐ cause individuals with DS often experience delays in reaching various developmental mile‐ stones, early intervention with speech therapy, occupational therapy, and physical therapy is recommended as it maximizes long-term outcomes [29]. As healthcare has improved for indi‐ viduals with DS, the average life expectancy has increased by more than 30 years, from an aver‐ age of 25 years of age in 1983 to almost 60 years of age in 2000 [30]. A study performed between 1985–2004 in England showed that the one-year survival of live births with DS increased, espe‐ cially in babies with cardiovascular malformations, reaching almost 100% [31], and a more re‐ cent study showed that the 25-year survival of DS individuals is about 87.5% [32].

Genetic counselors should balance the negative aspects of DS, such as birth defects, medical complications, and developmental delay, with positive aspects like available treatments, thera‐ pies, and the ability for people with DS and their families to enjoy a high quality of life [33].

## **3. Prenatal screening and diagnosis**

ular septal defect, atrial septal defect, tetralogy of Fallot, patent ductus arteriosus and atrioven‐ tricular septal defect [3,4,9]. There are also ocular problems, such as refractive errors, nystagmus, abnormalities of the retina, among others [10]. About 80% of cases present hearing loss, which can be conductive, sensorineural, or mixed [11]. Thyroid dysfunction, particularly hypothyroidism [9], periodontal diseases [10], upper airway obstruction [12] and hypogonad‐ ism [14] are more frequent in individuals with DS than in the general population. Other impor‐ tant clinical aspects of DS include immunodeficiency [15], increased risk for hematological

The development of secondary sexual characteristics in DS is similar to other adolescents. The fetal oogenesis of women with the syndrome appears to be normal and, therefore, they are capable of reproduction [18]. On the other hand, men have diminished reproductive ca‐ pacity, showing testicular histology compatible with oligospermia and, frequently, hypogo‐ nadism [19]. However, there have been reports of men with Down syndrome who have

Genetic counseling can be defined as a communication process that takes care of the human problems associated with the occurrence or recurrence of a genetic disease in a family with the purpose of providing individuals and families comprehensive understanding of all the implications related to genetic disease under discussion, the options that the current medi‐ cine offers for therapy or for reducing the risk of occurrence or recurrence of the disease and

For DS, a well-established risk factor is advanced maternal age at conception [23,24]. The es‐ timated risk for fetal trisomy 21 for a woman aged 20 years at 12 weeks of gestation is about 1 in 1000, and the risk of such woman delivering an affected baby at term is 1 in 1500. The risk for this aneuploidy for a woman aged 35 years at 12 weeks of gestation is about 1 in 250

Although there is considerable variation in the physical features of individuals with DS, most individuals present with a range of characteristics that enable clinical diagnosis of the syn‐ drome [3,4,7]. However, cytogenetic investigation of individuals who present with clinical characteristics of DS is fundamental to establish a precise diagnosis, which may have implica‐ tions in the genetic counseling process, once it is very important in determining the recurrence risk of the syndrome. In addition, the karyotype analysis of affected individuals identifies cases that may have been inherited making necessary the investigation of the parents' karyotypes. In this case, the cytogenetic investigation of the genitors is essential to establish the risk of recur‐ rence of the syndrome in future generations. Thus, all individuals with a diagnosis suggestive

Accurate estimation of recurrence risks depends upon the verification of the individual's karyotype. Cases of free trisomy 21 and mosaicism generally do not recur in siblings of indi‐ viduals with DS. For women with maternal age <35 at previous trisomy 21, the revised risk

and the risk of delivering an affected baby at term is 1 in 350 [25].

of DS should be referred to a genetic counseling service.

disorders and leukemia [16] and early onset of Alzheimer's disease [17].

fathered pregnancies [20].

4 Down Syndrome

**2. Genetic counseling**

psychotherapeutic support [21,22].

There are several methods that allow the early detection of DS in prenatal phase. At this point, it is not possible avoid congenital malformations or genetic diseases, but the objective is its early detection, looking for emotional and psychological preparation for parents and family and adequate medical support and monitoring for the child's birth. Furthermore, ear‐ ly detection allows treatment of malformations of the complications that may occur, pre‐ venting or attenuating their evolution through surgical correction in utero.

There are some methods used to screen fetus with DS that allow the prenatal diagnosis of the syndrome. Among the screening methods are the nuchal translucency test, the measure‐ ment of maternal serum concentrations of various fetoplacental products and fetal ultra‐ sound. The nuchal translucency (NT) test is the measurement of the fluid filled fold at the back of the fetal neck in the first trimester of pregnancy, performed through transabdominal or transvaginal sonography. The test is performed between the 11th and 13th weeks of ges‐ tation and the minimum fetal crown–rump length (CRL) should be 45 mm and the maxi‐ mum 84 mm. Fetal NT increases with CRL and therefore it is essential to take gestation into account when determining whether a given NT thickness is increased [25]. The excess skin in the fetus may be the consequence of excessive accumulation of subcutaneous fluid behind the fetal neck which could be visualized by ultrasonography as increased NT in the third month of intrauterine life [34]. Nowadays, it is well established that the measurement of fe‐ tal NT thickness provides effective and early screening for trisomy 21 and other major aneu‐ ploidies, such as Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13) [34-36] besides for screening of congenital heart disease [37]. In case of abnormality in NT measure‐ ment, additional tests are needed to elucidate the cause of increased nuchal fold.

Importantly, any suspect result of the markers mentioned implies the genetic analysis of the fetus, the only way to accurate diagnosis. The methods for obtaining fetal cells for analysis vary with gestational age. Among the invasive methods for obtaining fetal cells, chorionic villus sampling (CVS) allows diagnosis in the first trimester of pregnancy (between the 10th and 13th weeks of gestation) [50]. The procedure involves aspiration of trophoblastic tissue under continuous ultrasound monitoring, performed via trans-cervical or trans-abdominal. Studies have showed that the risk miscarriage associated to this procedure is about 0.6-1.1% [51,52] and the procedure is not recommended for pregnant women that present bleeding

Down Syndrome: Clinical and Genetic Aspects, Genetic Counseling and Prenatal Screening and Diagnosis

http://dx.doi.org/10.5772/52950

7

The amniocentesis is the method indicated for obtaining fetal cells after 15 weeks of gesta‐ tion [53]. This requires taking a small sample of amniotic fluid transabdominally under ul‐ trasound guidance. The procedure-related fetal loss rate is about 0.4-0.8 % [51,52]. After 20th week of gestation, the option is percutaneous umbilical blood sampling or cordocentesis, which involves direct sampling of fetal blood from the umbilical cord. The procedure-relat‐ ed loss rate is about 1.0-1.5% and cordocentesis with placenta penetration had a significantly

Considering the risks which accompany invasive methods for obtaining fetal cells [51,52,56], the use of noninvasive methods could be a good option. Several methods to develop a non‐ invasive prenatal test for trisomy 21 and other aneuploidies have been investigated, includ‐ ing the use of cell-free fetal nucleic acids [57-60] and nucleated red blood fetal cells present in maternal peripheral blood [61,62]. Although studies have showed that noninvasive meth‐ ods for obtaining fetal cells allow noninvasive prenatal diagnosis for a variety of genetic conditions and may in future form part of national antenatal screening programs for DS and other common genetic disorders, a major obstacle in the widespread application of noninva‐ sive methods for obtaining fetal cells in clinical diagnostics is still that fetal cells / DNA con‐ stitutes a small percentage of total cell / DNA in maternal blood and the inconsistencies in

After obtaining fetal cells, conventional karyotype analysis has been used for the past few decades as the gold standard for the prenatal diagnosis of numerical and major structural chromosomal abnormalities. Nevertheless, it is labor intensive and requires skilled chromo‐ somal analysis with an average reporting time of 14 days. However, the availability of mo‐ lecular techniques such as fluorescence in situ hybridization (FISH) has allowed the prenatal diagnosis of most frequent trisomies (21, 13, 18) and aneuploidy of sex chromosomes quick‐ ly and accurately, obtaining result from one to two days [64,65]. In addition, the technique of polymerase chain reaction quantitative fluorescent (QF-PCR), besides other molecular techniques such as the multiplex ligation-dependent probe amplification (MLPA) test and DNA sequencing, can also be used for a rapid diagnosis of aneuploidies [66-68]. It has been showed that QF-PCR technique presents 95.4% sensitivity, 100% specificity, 99.5% efficiency and is less laborious than the FISH technique, less time consuming, and some results were obtained in eight hours. The sensitivity, specificity, and efficiency of the assay for detecting DS using this technique are about 95.4%, 100%, and 99.5%, respectively [69]. Molecular tech‐ niques also enable the diagnosis of pre-implantation embryos in assisted reproduction [70].

due to an increase in the procedure-related fetal loss rate [51].

enrichment strategies of these fetal samples [62,63].

higher rate of fetal loss [54-56].

Pregnancies with fetal aneuploidies are associated with altered maternal serum concentrations of various fetoplacental products, including alpha-fetoprotein (AFP), free chorionic gonadotro‐ pin (β-hCG), unconjugated estriol (uE3), inhibin A (INH-A) and pregnancy associated plasma protein-A (PAPP-A) [38-42]. The measurement of concentrations of maternal serum AFP, βhCG and uE3, the triple test, is one of a range of screening tests that are used to identify pregnant women whose fetus is likely to be affected by trisomy 21 and who should then be offered a diag‐ nostic test. AFP is produced in the yolk sac and fetal liver, while uE3 and hCG are produced by the placenta. Elevated β-hCG concentration and low levels of AFP and uE3 suggests the pres‐ ence of a fetus with DS [38-40]. The test is performed in second trimester of pregnancy and the values should be adjusted to gestational age. The expected detection rate and false-positive rate are about 73 - 78% and 7.5 - 9%, respectively [43].

The incorporation of INH-A into maternal serum DS screening in the second trimester, along with AFP, hCG and uE3, is named quadruple test. INH-A is a glycoprotein mainly se‐ creted from the corpus luteum and the placenta [44] and its concentration is raised in the serum of pregnant women carrying a fetus with DS [42]. The quadruple test presents expect‐ ed detection rate and false-positive rate about 79 - 82% and 6.5 - 7.8%, respectively [43]. The measurement of PAPP-A is also used as a screening gestations of fetus with DS in the first trimester, once the maternal serum concentration of this protein are reduced in these women [41]. The measurement of PAPP-A at 10–14 weeks of pregnancy is used to screen for fetal DS during the first trimester of pregnancy [45,47].

The fetal ultrasound is also considered a method of screening for DS, once any change in the development of organs or structures is easily visualized. The objective is the detection of major and soft markers of aneuploidy, including alterations in central nervous system, face, neck, heart, gastrointestinal tract, genitourinary tract among others [47]. Besides increased nuchal translucency in the first trimester, alterations commonly detected in DS in the second trimester of gestation include lack of visualization of the nasal bone [48], reduced femur and humerus, mild pyelectasis, hyperechoic bowel and echogenic intracardiac focus [47,49].

Importantly, any suspect result of the markers mentioned implies the genetic analysis of the fetus, the only way to accurate diagnosis. The methods for obtaining fetal cells for analysis vary with gestational age. Among the invasive methods for obtaining fetal cells, chorionic villus sampling (CVS) allows diagnosis in the first trimester of pregnancy (between the 10th and 13th weeks of gestation) [50]. The procedure involves aspiration of trophoblastic tissue under continuous ultrasound monitoring, performed via trans-cervical or trans-abdominal. Studies have showed that the risk miscarriage associated to this procedure is about 0.6-1.1% [51,52] and the procedure is not recommended for pregnant women that present bleeding due to an increase in the procedure-related fetal loss rate [51].

There are some methods used to screen fetus with DS that allow the prenatal diagnosis of the syndrome. Among the screening methods are the nuchal translucency test, the measure‐ ment of maternal serum concentrations of various fetoplacental products and fetal ultra‐ sound. The nuchal translucency (NT) test is the measurement of the fluid filled fold at the back of the fetal neck in the first trimester of pregnancy, performed through transabdominal or transvaginal sonography. The test is performed between the 11th and 13th weeks of ges‐ tation and the minimum fetal crown–rump length (CRL) should be 45 mm and the maxi‐ mum 84 mm. Fetal NT increases with CRL and therefore it is essential to take gestation into account when determining whether a given NT thickness is increased [25]. The excess skin in the fetus may be the consequence of excessive accumulation of subcutaneous fluid behind the fetal neck which could be visualized by ultrasonography as increased NT in the third month of intrauterine life [34]. Nowadays, it is well established that the measurement of fe‐ tal NT thickness provides effective and early screening for trisomy 21 and other major aneu‐ ploidies, such as Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13) [34-36] besides for screening of congenital heart disease [37]. In case of abnormality in NT measure‐

ment, additional tests are needed to elucidate the cause of increased nuchal fold.

are about 73 - 78% and 7.5 - 9%, respectively [43].

6 Down Syndrome

during the first trimester of pregnancy [45,47].

Pregnancies with fetal aneuploidies are associated with altered maternal serum concentrations of various fetoplacental products, including alpha-fetoprotein (AFP), free chorionic gonadotro‐ pin (β-hCG), unconjugated estriol (uE3), inhibin A (INH-A) and pregnancy associated plasma protein-A (PAPP-A) [38-42]. The measurement of concentrations of maternal serum AFP, βhCG and uE3, the triple test, is one of a range of screening tests that are used to identify pregnant women whose fetus is likely to be affected by trisomy 21 and who should then be offered a diag‐ nostic test. AFP is produced in the yolk sac and fetal liver, while uE3 and hCG are produced by the placenta. Elevated β-hCG concentration and low levels of AFP and uE3 suggests the pres‐ ence of a fetus with DS [38-40]. The test is performed in second trimester of pregnancy and the values should be adjusted to gestational age. The expected detection rate and false-positive rate

The incorporation of INH-A into maternal serum DS screening in the second trimester, along with AFP, hCG and uE3, is named quadruple test. INH-A is a glycoprotein mainly se‐ creted from the corpus luteum and the placenta [44] and its concentration is raised in the serum of pregnant women carrying a fetus with DS [42]. The quadruple test presents expect‐ ed detection rate and false-positive rate about 79 - 82% and 6.5 - 7.8%, respectively [43]. The measurement of PAPP-A is also used as a screening gestations of fetus with DS in the first trimester, once the maternal serum concentration of this protein are reduced in these women [41]. The measurement of PAPP-A at 10–14 weeks of pregnancy is used to screen for fetal DS

The fetal ultrasound is also considered a method of screening for DS, once any change in the development of organs or structures is easily visualized. The objective is the detection of major and soft markers of aneuploidy, including alterations in central nervous system, face, neck, heart, gastrointestinal tract, genitourinary tract among others [47]. Besides increased nuchal translucency in the first trimester, alterations commonly detected in DS in the second trimester of gestation include lack of visualization of the nasal bone [48], reduced femur and humerus, mild pyelectasis, hyperechoic bowel and echogenic intracardiac focus [47,49].

The amniocentesis is the method indicated for obtaining fetal cells after 15 weeks of gesta‐ tion [53]. This requires taking a small sample of amniotic fluid transabdominally under ul‐ trasound guidance. The procedure-related fetal loss rate is about 0.4-0.8 % [51,52]. After 20th week of gestation, the option is percutaneous umbilical blood sampling or cordocentesis, which involves direct sampling of fetal blood from the umbilical cord. The procedure-relat‐ ed loss rate is about 1.0-1.5% and cordocentesis with placenta penetration had a significantly higher rate of fetal loss [54-56].

Considering the risks which accompany invasive methods for obtaining fetal cells [51,52,56], the use of noninvasive methods could be a good option. Several methods to develop a non‐ invasive prenatal test for trisomy 21 and other aneuploidies have been investigated, includ‐ ing the use of cell-free fetal nucleic acids [57-60] and nucleated red blood fetal cells present in maternal peripheral blood [61,62]. Although studies have showed that noninvasive meth‐ ods for obtaining fetal cells allow noninvasive prenatal diagnosis for a variety of genetic conditions and may in future form part of national antenatal screening programs for DS and other common genetic disorders, a major obstacle in the widespread application of noninva‐ sive methods for obtaining fetal cells in clinical diagnostics is still that fetal cells / DNA con‐ stitutes a small percentage of total cell / DNA in maternal blood and the inconsistencies in enrichment strategies of these fetal samples [62,63].

After obtaining fetal cells, conventional karyotype analysis has been used for the past few decades as the gold standard for the prenatal diagnosis of numerical and major structural chromosomal abnormalities. Nevertheless, it is labor intensive and requires skilled chromo‐ somal analysis with an average reporting time of 14 days. However, the availability of mo‐ lecular techniques such as fluorescence in situ hybridization (FISH) has allowed the prenatal diagnosis of most frequent trisomies (21, 13, 18) and aneuploidy of sex chromosomes quick‐ ly and accurately, obtaining result from one to two days [64,65]. In addition, the technique of polymerase chain reaction quantitative fluorescent (QF-PCR), besides other molecular techniques such as the multiplex ligation-dependent probe amplification (MLPA) test and DNA sequencing, can also be used for a rapid diagnosis of aneuploidies [66-68]. It has been showed that QF-PCR technique presents 95.4% sensitivity, 100% specificity, 99.5% efficiency and is less laborious than the FISH technique, less time consuming, and some results were obtained in eight hours. The sensitivity, specificity, and efficiency of the assay for detecting DS using this technique are about 95.4%, 100%, and 99.5%, respectively [69]. Molecular tech‐ niques also enable the diagnosis of pre-implantation embryos in assisted reproduction [70].

It is important to note that the examinations of prenatal diagnosis should not be offered without the guidance of a geneticist to explain the risks to the parents and especially the im‐ plications of possible results. Early diagnosis helps couples to program for the treatment of the consequences of the syndrome diagnosed, preventing further damage and making possi‐ ble the early stimulation of the patients, aiming their better integration into society.

## **4. Gene expression and DS phenotype**

In a recent review of chromosome 21 content, 552 genes were identified in the long arm of the chromosome (21q) [71], including 161 protein-coding genes cataloged in the Reference Sequence database of the National Center for Biotechnology Information (NCBI). The re‐ maining 391 gene models are referred to as novel genes or non-cataloged genes, which could be protein-coding genes or functional RNA genes. Considering that the genetic basis of DS is the presence of three copies of chromosome 21, the first and most commonly accept‐ ed hypothesis for DS phenotype is that the genes in triplicate are overexpressed and, thus, the dosage imbalance of genes on chromosome 21 is responsible for the molecular dysfunc‐ tions in DS [72]. Among the genes present in chromosome 21, may be highlighted some de‐ scribed in the literature with overexpression associated with phenotypes of DS, most influencing the structure or function of the central nervous system (Table 1). Location of these genes on chromosome 21 is presented in Figure 1.

**Figure 1.** Location of genes overexpressed in DS influencing the structure or function of the central nervous system.

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9

However, although elevated levels of gene expression on chromosome 21 in trisomy 21 tissues have been reported in several studies, there are evidences that increased copy number does not always correspond with increased gene expression level or even less with increased gene func‐ tion [86,87]. In addition, studies have showed up- or downregulation of genes located on diso‐ mic chromosomes, indicating that the phenotype is due to an unstable environment resulting from the dosage imbalance of the hundreds of genes on chromosome 21 which determines a

Besides altered pattern of gene expression, regulatory mechanisms are also altered in triso‐ my 21. Individuals with DS present altered pattern of DNA methylation in genes present in two or three copies with functional consequences in gene expression [91,92]. More recent studies have shown that trisomy 21 results in altered expression of microRNAs, small mole‐ cules of noncoding RNA involved in post-transcriptional gene regulation, which could re‐ sult in abnormal expression of specific proteins and contribute to the DS phenotype [93-97]

The complete sequencing of chromosome 21 provided basis for the identification of candi‐ date genes for DS phenotype manifestations. Currently, there are several genes located on chromosome 21 associated to DS phenotype and the involvement of other genes still will be elucidated with advances of genomics and proteomics. The knowing of these gene functions and their contribution for DS phenotype are fundamental for the understanding of the syn‐ drome and for providing basis for the planning of therapeutic strategies that could contrib‐

Figure adapted from the NCBI Map Viewer database (http://www.ncbi.nlm.nih.gov/mapview/).

non-specific disturbance of genomic regulation and expression [88-90].

ute to improve the quality of life of DS individuals.


\* http://www.ncbi.nlm.nih.gov/gene

**Table 1.** Chromosome 21 gene-located with overexpression in DS influencing the structure or function of the central nervous system.

Down Syndrome: Clinical and Genetic Aspects, Genetic Counseling and Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52950 9

It is important to note that the examinations of prenatal diagnosis should not be offered without the guidance of a geneticist to explain the risks to the parents and especially the im‐ plications of possible results. Early diagnosis helps couples to program for the treatment of the consequences of the syndrome diagnosed, preventing further damage and making possi‐

In a recent review of chromosome 21 content, 552 genes were identified in the long arm of the chromosome (21q) [71], including 161 protein-coding genes cataloged in the Reference Sequence database of the National Center for Biotechnology Information (NCBI). The re‐ maining 391 gene models are referred to as novel genes or non-cataloged genes, which could be protein-coding genes or functional RNA genes. Considering that the genetic basis of DS is the presence of three copies of chromosome 21, the first and most commonly accept‐ ed hypothesis for DS phenotype is that the genes in triplicate are overexpressed and, thus, the dosage imbalance of genes on chromosome 21 is responsible for the molecular dysfunc‐ tions in DS [72]. Among the genes present in chromosome 21, may be highlighted some de‐ scribed in the literature with overexpression associated with phenotypes of DS, most influencing the structure or function of the central nervous system (Table 1). Location of

**Gene symbol\* Gene location\* Candidate gene for Reference** APP 21q21.3 Neurodegeneration [73,74] BACH1 21q22.11 Alzheimer's disease-like neuropathological changes [75] DOPEY2 21q22.2 Functional brain alterations and mental retardation [76] DSCAM 21q22.2 Mental retardation and the precocious dementia [77] DYRK1A 21q22.13 Leukemogenesis [78]

ERG 21q22.3 Alzheimer's disease-like neuropathological changes [75] OLIG2 21q22.11 Developmental brain defects [81] SIM2 21q22.13 Impairment of learning and memory [82]

SOD1 21q22.11 Neurodegeneration [84] PCP4 21q22.2 Abnormal neuronal development [85]

**Table 1.** Chromosome 21 gene-located with overexpression in DS influencing the structure or function of the central

Impaired brain development [79] Early onset of neurofibrillary degeneration [80]

Pathogenesis of mental retardation [83]

ble the early stimulation of the patients, aiming their better integration into society.

**4. Gene expression and DS phenotype**

8 Down Syndrome

these genes on chromosome 21 is presented in Figure 1.

\* http://www.ncbi.nlm.nih.gov/gene

nervous system.

**Figure 1.** Location of genes overexpressed in DS influencing the structure or function of the central nervous system. Figure adapted from the NCBI Map Viewer database (http://www.ncbi.nlm.nih.gov/mapview/).

However, although elevated levels of gene expression on chromosome 21 in trisomy 21 tissues have been reported in several studies, there are evidences that increased copy number does not always correspond with increased gene expression level or even less with increased gene func‐ tion [86,87]. In addition, studies have showed up- or downregulation of genes located on diso‐ mic chromosomes, indicating that the phenotype is due to an unstable environment resulting from the dosage imbalance of the hundreds of genes on chromosome 21 which determines a non-specific disturbance of genomic regulation and expression [88-90].

Besides altered pattern of gene expression, regulatory mechanisms are also altered in triso‐ my 21. Individuals with DS present altered pattern of DNA methylation in genes present in two or three copies with functional consequences in gene expression [91,92]. More recent studies have shown that trisomy 21 results in altered expression of microRNAs, small mole‐ cules of noncoding RNA involved in post-transcriptional gene regulation, which could re‐ sult in abnormal expression of specific proteins and contribute to the DS phenotype [93-97]

The complete sequencing of chromosome 21 provided basis for the identification of candi‐ date genes for DS phenotype manifestations. Currently, there are several genes located on chromosome 21 associated to DS phenotype and the involvement of other genes still will be elucidated with advances of genomics and proteomics. The knowing of these gene functions and their contribution for DS phenotype are fundamental for the understanding of the syn‐ drome and for providing basis for the planning of therapeutic strategies that could contrib‐ ute to improve the quality of life of DS individuals.

## **5. Conclusion**

Although individuals with trisomy 21 present several characteristics that make possible the clinical diagnosis of DS, the confirmation of the diagnosis by cytogenetic analysis is essential to establish the recurrence risks of the syndrome. We highlight the importance of the prena‐ tal diagnosis of DS to provide the needed healthcare for the child, to prepare the family emotional and psychologically and to plan early intervention therapies. The successful con‐ trol of pharmacological and clinical problems of patients with DS is the biggest medical challenge and depends on the understanding of unbalanced metabolism induced by high expression of the genes located on chromosome 21.

[3] Ahmed I, Ghafoor T, Samore NA, Chattha MN. Down syndrome: clinical and cytoge‐

Down Syndrome: Clinical and Genetic Aspects, Genetic Counseling and Prenatal Screening and Diagnosis

http://dx.doi.org/10.5772/52950

11

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## **Acknowledgments**

The authors acknowledge support from FAPESP (São Paulo Research Foundation), CNPq (National Council for Scientific and Technological Development) and CAPES (Coordination for the Improvement of Higher Education Personnel).

## **Author details**

Érika Cristina Pavarino1,2\*, Joice Matos Biselli1 , Walter Pinto Junior3 and Eny Maria Goloni Bertollo1,2

\*Address all correspondence to: erika@famerp.br

1 Department of Molecular Biology, Sao Jose do Rio Preto Medical School (FAMERP), Ge‐ netics and Molecular Biology Research Unit (UPGEM), Sao Jose do Rio Preto, Brazil

2 Ding-Down multidisciplinary group, Sao Jose do Rio Preto Medical School (FAMERP), Sao Jose do Rio Preto, Brazil

3 Medical and Forensic Genetics Ltd, Campinas, Brazil

## **References**


[3] Ahmed I, Ghafoor T, Samore NA, Chattha MN. Down syndrome: clinical and cytoge‐ netic analysis. J Coll Physicians Surg Pak. 2005 Jul;15(7):426-9.

**5. Conclusion**

10 Down Syndrome

**Acknowledgments**

**Author details**

Eny Maria Goloni Bertollo1,2

Jose do Rio Preto, Brazil

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**Chapter 2**

**Increased Fetal Nuchal Translucency Thickness and**

Ksenija Gersak, Darija M. Strah and

**Figure 1.** Normal nuchal translucency thickness (NT)

Additional information is available at the end of the chapter

Maja Pohar-Perme

**1. Introduction**

http://dx.doi.org/10.5772/53112

**Normal Karyotype: Prenatal and Postnatal Outcome**

Nuchal translucency (NT) is the assessment of the amount of fluid behind the neck of the fetus, also known as the nuchal fold. An anechoic space is visible and measurable sonographically in all fetuses between the 11th and the 14th week of the pregnancy (Figure 1). Underlying patho‐ physiological mechanisms for nuchal fluid collection under the skin include cardiac dysfunc‐ tion, venous congestion in the head and neck, altered composition of the extracellular matrix, failure of lymphatic drainage, fetal anemia or hypoproteinemia and congenital infection [1].

> © 2013 Gersak et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

The abnormal accumulation of nuchal fluid decreases after the 14th week.


## **Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome**

Ksenija Gersak, Darija M. Strah and Maja Pohar-Perme

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53112

## **1. Introduction**

polymorphisms related to phenotypes. Am J Hum Genet. 2007 Aug;81(2):405-13. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950808/?tool=pubmed (accessed 6

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June 2012).

20 Down Syndrome

2012).

June 2012).

June 2012).

tool=pubmed (accessed 26 June 2012).

Nuchal translucency (NT) is the assessment of the amount of fluid behind the neck of the fetus, also known as the nuchal fold. An anechoic space is visible and measurable sonographically in all fetuses between the 11th and the 14th week of the pregnancy (Figure 1). Underlying patho‐ physiological mechanisms for nuchal fluid collection under the skin include cardiac dysfunc‐ tion, venous congestion in the head and neck, altered composition of the extracellular matrix, failure of lymphatic drainage, fetal anemia or hypoproteinemia and congenital infection [1]. The abnormal accumulation of nuchal fluid decreases after the 14th week.

**Figure 1.** Normal nuchal translucency thickness (NT)

© 2013 Gersak et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1.1. Increased NT in chromosomally abnormal fetuses**

The association between the increased NT and the chromosomal abnormalities has been well documented (Figure 2). It helps us identify the high-risk fetuses for trisomy 21 and oth‐ er chromosomal abnormalities [2,3].

positive. At the invasive testing, chromosomal abnormalities were identified in 8.6% of high risk cases (34 out of 394), which represented one case of fetal chromosomal abnormality, de‐ tected per 12 invasive diagnostic procedures. Consequently we believe that the effective screening for trisomy 21 can be achieved in the first trimester of pregnancy by the combina‐ tion of maternal age, sonographic measurement of the fetal NT thickness and assessment of

Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome

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23

the fetal nasal bone, with detection rate of 85% at a false positive rate of less than 3%.

**Karyotype n** Trisomy 21 20 Trisomy 18 10 Trisomy 13 2 45,X (Turner syndrom) 3 47,XXY 2 Mosaic structure 3 Unbalanced structural rearrangements 5 Total 45

**Table 1.** Chromosomal abnormalities in fetuses and newborns in our sample of 13,049 women with singleton

The NT can be increased also in chromosomally normal fetuses. When the karyotype is nor‐ mal, the fetus is still at a significant risk of adverse pregnancy outcome e.g. fetal loss, struc‐ tural abnormalities, particularly cardiac defects, various genetic syndromes and delayed neurodevelopment [9,13]. The prevalence of fetal abnormalities and adverse pregnancy out‐

The impact of the increased nuchal fluid collection, seen during the ultrasound examination, raises the parents' great anxiety about future fetal development [13]. The risks of adverse pregnancy outcomes have to be discussed with the parents and an objective counseling has to be offered to them together with detailed ultrasound examinations later in the pregnancy. But even in the absence of clear fetal abnormalities, some couples request pregnancy termi‐

Therefore, the aim of this study was to evaluate the pregnancy outcomes of fetuses with in‐

The retrospective study included unselected population of pregnant women of Caucasian ethnic origin appointed for the first trimester ultrasound screening examination at a single

creased NT thickness and normal karyotype in an unselected pregnant population.

**1.2. Increased NT in chromosomally normal fetuses**

comes increases with the thickness of NT.

nation in such circumstances [14].

**2. Subjects and methods**

**2.1. Study design**

pregnancies [12].

**Figure 2.** Increased nuchal translucency thickness (NT)

The findings of numerous studies suggest that an effective first trimester screening for triso‐ my 21 can be obtained by the combination of maternal age and measurement of fetal NT [4-11]. At a risk cut-off of 1 in100, the detection rate of trisomy 21 is about 75%, at a false positive rate of about 2%. The detection rate can be improved to 85% by the additional as‐ sessment of the fetal nasal bone and even more by the Doppler assessment of blood flow across the tricuspid valve or blood flow in the ductus venosus, which has increased the de‐ tection rate to about 95% at a false positive rate of 2.5% [11].

Our retrospective study of the first trimester screening for trisomy 21 in 5-year period from 2005 to 2010 by employing the combination of maternal age, sonographic measurement of the fetal NT thickness and assessment of the fetal nasal bone, included 13,049 pregnant women [12]. The sample represented an unselected population of women with singleton pregnancies. The cut-off risk for trisomy 21 was set at 1 in 300. The distribution of maternal age of the examined women was compared to the age distribution in the pregnant popula‐ tion in Slovenia for the same time interval (2005-2010). The balance between the false posi‐ tive rate and the detection rate was studied and the trends were inspected graphically. The cut-off risk that would yield 5% false positives was calculated for trisomy 21. The average gestation was 12 4/7 weeks (range from 11 1/7 weeks to 14 0/7 weeks). The average fetal CRL was 63.2 mm (from 45 mm to 83 mm). The average NT thickness was 1.7 mm (range from 0.9 mm to 13.4 mm). The NT was above the 95th centile of the normal range for the CRL in 75% (15 out of 20) of trisomy 21 pregnancies and in 64% (16 out of 25) pregnancies with oth‐ er chromosomal abnormalities. At the time of the testing the estimated risk for trisomy 21 was 1 in 300 or higher in 3% of all the pregnancies (394 out of 13,049), considering the calcu‐ lation based on FMF program. Three hundred and sixty cases (2.8%) turned out to be false positive. At the invasive testing, chromosomal abnormalities were identified in 8.6% of high risk cases (34 out of 394), which represented one case of fetal chromosomal abnormality, de‐ tected per 12 invasive diagnostic procedures. Consequently we believe that the effective screening for trisomy 21 can be achieved in the first trimester of pregnancy by the combina‐ tion of maternal age, sonographic measurement of the fetal NT thickness and assessment of the fetal nasal bone, with detection rate of 85% at a false positive rate of less than 3%.


**Table 1.** Chromosomal abnormalities in fetuses and newborns in our sample of 13,049 women with singleton pregnancies [12].

#### **1.2. Increased NT in chromosomally normal fetuses**

The NT can be increased also in chromosomally normal fetuses. When the karyotype is nor‐ mal, the fetus is still at a significant risk of adverse pregnancy outcome e.g. fetal loss, struc‐ tural abnormalities, particularly cardiac defects, various genetic syndromes and delayed neurodevelopment [9,13]. The prevalence of fetal abnormalities and adverse pregnancy out‐ comes increases with the thickness of NT.

The impact of the increased nuchal fluid collection, seen during the ultrasound examination, raises the parents' great anxiety about future fetal development [13]. The risks of adverse pregnancy outcomes have to be discussed with the parents and an objective counseling has to be offered to them together with detailed ultrasound examinations later in the pregnancy. But even in the absence of clear fetal abnormalities, some couples request pregnancy termi‐ nation in such circumstances [14].

Therefore, the aim of this study was to evaluate the pregnancy outcomes of fetuses with in‐ creased NT thickness and normal karyotype in an unselected pregnant population.

## **2. Subjects and methods**

#### **2.1. Study design**

**1.1. Increased NT in chromosomally abnormal fetuses**

er chromosomal abnormalities [2,3].

22 Down Syndrome

**Figure 2.** Increased nuchal translucency thickness (NT)

tection rate to about 95% at a false positive rate of 2.5% [11].

The association between the increased NT and the chromosomal abnormalities has been well documented (Figure 2). It helps us identify the high-risk fetuses for trisomy 21 and oth‐

The findings of numerous studies suggest that an effective first trimester screening for triso‐ my 21 can be obtained by the combination of maternal age and measurement of fetal NT [4-11]. At a risk cut-off of 1 in100, the detection rate of trisomy 21 is about 75%, at a false positive rate of about 2%. The detection rate can be improved to 85% by the additional as‐ sessment of the fetal nasal bone and even more by the Doppler assessment of blood flow across the tricuspid valve or blood flow in the ductus venosus, which has increased the de‐

Our retrospective study of the first trimester screening for trisomy 21 in 5-year period from 2005 to 2010 by employing the combination of maternal age, sonographic measurement of the fetal NT thickness and assessment of the fetal nasal bone, included 13,049 pregnant women [12]. The sample represented an unselected population of women with singleton pregnancies. The cut-off risk for trisomy 21 was set at 1 in 300. The distribution of maternal age of the examined women was compared to the age distribution in the pregnant popula‐ tion in Slovenia for the same time interval (2005-2010). The balance between the false posi‐ tive rate and the detection rate was studied and the trends were inspected graphically. The cut-off risk that would yield 5% false positives was calculated for trisomy 21. The average gestation was 12 4/7 weeks (range from 11 1/7 weeks to 14 0/7 weeks). The average fetal CRL was 63.2 mm (from 45 mm to 83 mm). The average NT thickness was 1.7 mm (range from 0.9 mm to 13.4 mm). The NT was above the 95th centile of the normal range for the CRL in 75% (15 out of 20) of trisomy 21 pregnancies and in 64% (16 out of 25) pregnancies with oth‐ er chromosomal abnormalities. At the time of the testing the estimated risk for trisomy 21 was 1 in 300 or higher in 3% of all the pregnancies (394 out of 13,049), considering the calcu‐ lation based on FMF program. Three hundred and sixty cases (2.8%) turned out to be false

The retrospective study included unselected population of pregnant women of Caucasian ethnic origin appointed for the first trimester ultrasound screening examination at a single outpatient clinic between January 4, 2005 and April 30, 2010. Included in the study popula‐ tion were only singleton pregnancies with live fetus from the 11th to the 14th week of gesta‐ tion with the CRL of 45-83 mm.

**2.4. Statistical analysis**

2.14.

**3. Results**

April 30, 2010.

**3.2. Fetal loss**

Outcome

**3.1. Study population**

Descriptive statistics were used to describe our sample. Means, standard deviations and ranges are reported for continuous variables, numbers and proportions are reported for cat‐ egorical variables. Statistical analysis was performed using R statistical package, version

Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome

http://dx.doi.org/10.5772/53112

25

The sample represented 11,980 unselected pregnant women appointed for the first trimester ultrasound screening examination at a single outpatient clinic between January 4, 2005 and

Five hundred and fifty-eight fetuses had an increased fetal NT and normal karyotype (558/11,980; 4.7%). In 46 cases (46/558; 8.2%) the outcome of the pregnancy was unknown;

The mean maternal age was 30.2 years (range from 17 to 46 years, SD=4.8). There were 421 out of 512 pregnancies (82.2%) conceived naturally and 91 (17.8%; 91/512) after in vitro fertil‐

The fetal loss was registered in 36 pregnancies (36/512; 7%). Twelve women (2.3%; 12/512) had miscarriage, 19 pregnancies (3.7%; 19/512) were terminated at parental request or due to the finding of structural abnormalities, and 1% of pregnancies (5/512) ended with intrauter‐ ine death. The outcomes with respect to the NT thickness are presented in Table 2. Table 3 provides details on all the types of fetal loss. The most common causes of termination were

therefore 512 singleton pregnancies were included in the further analysis.

hydrops fetalis, increased NT or cystic hygroma (Figure 3).

**Table 2.** Outcome of pregnancies with respect to the NT thickness.

ization. The mean NT ≥95th percentile was of 2.5 mm (range from 1.3 to 13.4 mm).

**NT (mm) ≤ 3.4 3.5-4.4 4.5-5.4 5.5-6.4 ≥ 6.5 Total**

Delivery 436 34 5 1 0 476 (93%) Miscarriage 11 0 1 0 0 12 (2.3%) Intrauterine death 1 3 0 0 1 5 (1%) Termination 8 2 3 2 4 19 (3.7%) Total 456 39 9 3 5 512

Before the screening they had all received counseling by their level one gynecologists and an information leaflet about the ultrasound examination and the aim of the screening. In the majority of cases the examination of early fetal morphology and other measurements was performed transabdominally within 20 minutes. In less than 1% of the cases a transvaginal ultrasound examination had to be carried out.

For the examinations we used 2-5 MHz and 3.7-9.3 MHz transducers GE Healthcare Volu‐ son 730 Pro, Milwaukee, USA, 4–6 MHz, 4–7 MHz, 5–9 MHz and 7–9 MHz transducers Acu‐ son S2000, Siemens Medical Solution, Mountain View CA, USA.

The measurement of fetal NT followed the criteria recommended by the Fetal Medicine Foundation (FMF). The increased NT thickness was defined as a measurement above the 95th percentile for the normal range. Risks were calculated according to the FMF program, following its guidelines [15,16].

The women with an increased risk for chromosomal anomalies (≥ 1:300) calculated on the basis of maternal age, NT and fetal crown-rump length (CRL) were offered invasive testing for fetal karyotyping. The karyotyping was performed by using chorionic villus sampling or amniocentesis in three cytogenetic laboratories.

The fetuses with increased fetal NT and normal karyotype were followed by detailed struc‐ tural ultrasound evaluation between the 20th and the 24th week of gestation. Fetal echocar‐ diography was performed in cases in which NT exceeded 3.5 mm.

After an informed consent had been signed, pregnancy outcomes were obtained from the participating women by written questionnaires. In cases of non-responders or uncertainty, telephone contact with the parents was established. The length of follow-up ranged from 18 months to 5 years.

#### **2.2. Exclusion criteria**

The exclusion criteria were the loss to follow-up, the chromosomal abnormalities or no in‐ formation on karyotype in a fetal loss.

#### **2.3. Classification of adverse outcome**

Adverse pregnancy outcome was defined as fetal loss (miscarriage, intrauterine death, ter‐ mination of pregnancy), and as liveborn infant with structural abnormality, genetic disor‐ ders and/or neurodevelopmental delay diagnosed before or after delivery. Stillbirth <22 weeks of pregnancy was defined as miscarriage, and stillbirth ≥22 weeks of pregnancy or birth of a child of at least 500 g weight without vital signs as intrauterine fetal death.

#### **2.4. Statistical analysis**

Descriptive statistics were used to describe our sample. Means, standard deviations and ranges are reported for continuous variables, numbers and proportions are reported for cat‐ egorical variables. Statistical analysis was performed using R statistical package, version 2.14.

## **3. Results**

outpatient clinic between January 4, 2005 and April 30, 2010. Included in the study popula‐ tion were only singleton pregnancies with live fetus from the 11th to the 14th week of gesta‐

Before the screening they had all received counseling by their level one gynecologists and an information leaflet about the ultrasound examination and the aim of the screening. In the majority of cases the examination of early fetal morphology and other measurements was performed transabdominally within 20 minutes. In less than 1% of the cases a transvaginal

For the examinations we used 2-5 MHz and 3.7-9.3 MHz transducers GE Healthcare Volu‐ son 730 Pro, Milwaukee, USA, 4–6 MHz, 4–7 MHz, 5–9 MHz and 7–9 MHz transducers Acu‐

The measurement of fetal NT followed the criteria recommended by the Fetal Medicine Foundation (FMF). The increased NT thickness was defined as a measurement above the 95th percentile for the normal range. Risks were calculated according to the FMF program,

The women with an increased risk for chromosomal anomalies (≥ 1:300) calculated on the basis of maternal age, NT and fetal crown-rump length (CRL) were offered invasive testing for fetal karyotyping. The karyotyping was performed by using chorionic villus sampling or

The fetuses with increased fetal NT and normal karyotype were followed by detailed struc‐ tural ultrasound evaluation between the 20th and the 24th week of gestation. Fetal echocar‐

After an informed consent had been signed, pregnancy outcomes were obtained from the participating women by written questionnaires. In cases of non-responders or uncertainty, telephone contact with the parents was established. The length of follow-up ranged from 18

The exclusion criteria were the loss to follow-up, the chromosomal abnormalities or no in‐

Adverse pregnancy outcome was defined as fetal loss (miscarriage, intrauterine death, ter‐ mination of pregnancy), and as liveborn infant with structural abnormality, genetic disor‐ ders and/or neurodevelopmental delay diagnosed before or after delivery. Stillbirth <22 weeks of pregnancy was defined as miscarriage, and stillbirth ≥22 weeks of pregnancy or

birth of a child of at least 500 g weight without vital signs as intrauterine fetal death.

tion with the CRL of 45-83 mm.

24 Down Syndrome

following its guidelines [15,16].

months to 5 years.

**2.2. Exclusion criteria**

formation on karyotype in a fetal loss.

**2.3. Classification of adverse outcome**

ultrasound examination had to be carried out.

amniocentesis in three cytogenetic laboratories.

son S2000, Siemens Medical Solution, Mountain View CA, USA.

diography was performed in cases in which NT exceeded 3.5 mm.

#### **3.1. Study population**

The sample represented 11,980 unselected pregnant women appointed for the first trimester ultrasound screening examination at a single outpatient clinic between January 4, 2005 and April 30, 2010.

Five hundred and fifty-eight fetuses had an increased fetal NT and normal karyotype (558/11,980; 4.7%). In 46 cases (46/558; 8.2%) the outcome of the pregnancy was unknown; therefore 512 singleton pregnancies were included in the further analysis.

The mean maternal age was 30.2 years (range from 17 to 46 years, SD=4.8). There were 421 out of 512 pregnancies (82.2%) conceived naturally and 91 (17.8%; 91/512) after in vitro fertil‐ ization. The mean NT ≥95th percentile was of 2.5 mm (range from 1.3 to 13.4 mm).

#### **3.2. Fetal loss**

The fetal loss was registered in 36 pregnancies (36/512; 7%). Twelve women (2.3%; 12/512) had miscarriage, 19 pregnancies (3.7%; 19/512) were terminated at parental request or due to the finding of structural abnormalities, and 1% of pregnancies (5/512) ended with intrauter‐ ine death. The outcomes with respect to the NT thickness are presented in Table 2. Table 3 provides details on all the types of fetal loss. The most common causes of termination were hydrops fetalis, increased NT or cystic hygroma (Figure 3).


**Table 2.** Outcome of pregnancies with respect to the NT thickness.


**NT (mm)≤ 3.4 3.5-4.4 4.5-5.4 5.5-6.4 ≥ 6.5 Total**

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27

Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome

Healthy 393 29 5 1 0 431 Heart defect 6 2 0 0 0 8 Structural abnormalities 29 1 0 0 0 30 Genetic syndromes 5 0 0 0 0 5 Neurodevelopmental delay 4 1 0 0 0 5 **Total 437 33 5 1 0 476**

**Disorder n NT (mm)**

VSD 3 2.6/3.0/3.2

Hydronephrosis (isolated) 4 1.8/2.5/2.8/3.1

Cleft lip and/or cleft palate 4 1.5/2.1/3.1/3.1

Hydronephrosis and ureteral stenosis 2 2.8/3.1 Vesicouretheral reflux 2 2.6/2.8

Craniosynostosis 1 2.3 Hypoplasia of the corpus callosum 1 2.5 Hydrocephalus 1 3.2 Micrognathia 1 2.6 Hemangioma 2 1.8/1.9 Cystic adenomatoid malformation 1 2.4 Diaphragmatic hernia 1 3.2 Atresia of the duodenum 1 3.0 Unilateral renal agenesis 1 2.7

VSD, ASD and aortic coarctation 1 3.9 VSD, ASD and tricuspid valve anomaly 1 4.0 Hypoplastic left ventricle 1 3.4 Isolated valve anomaly 2 3.4/2.6

**Table 4.** Clinical findings in liveborn infants with respect to the NT thickness.

**Heart defects:** 8

**Other abnormalities:** 30

**Clinical findings**

**Table 3.** Fetal loss with respect to the NT thickness.

#### **3.3. Liveborn infants with abnormalities**

Four hundred and seventy-six pregnancies ended with delivery of a viable infant (93%). Among them we found 48 newborns (9.5%; 48/476) with either single or multiple abnormali‐ ties. The clinical findings in 476 liveborn infants with respect to the NT thickness are pre‐ sented in Table 4. There were 8 cases (1.7%, 8/476) born with heart defects, other structural abnormalities were found in 30 newborns (6.3%; 30/476). During the first year of life some genetic syndromes or neurodevelopmental delay were recorded in 10 cases (2.1%; 10/476). All abnormalities were found in the group of newborns with mildly enlarged NT, between 95th percentiles to 4.4 mm. Among healthy babies, there was no NT thicker than 6.4 mm. Table 5 describes the disorders of 48 babies in more detail.

**Figure 3.** Hydrops fetalis


**Table 4.** Clinical findings in liveborn infants with respect to the NT thickness.

**Fetal loss n NT (mm)**

Spontaneous abortion after amniocentesis 2 2.5/3.0 Intrauterine death (unspecified) 4 3.0/3.5/3.7/4.2

Intrauterine death/tetralogy of Fallot 1 7.8 Termination: 19

**Table 3.** Fetal loss with respect to the NT thickness.

26 Down Syndrome

**3.3. Liveborn infants with abnormalities**

**Figure 3.** Hydrops fetalis

Table 5 describes the disorders of 48 babies in more detail.

Miscarriage (unspecified) 10 2.1/2.2/2.3/2.5/2.6/

Hydrops fetalis 5 2.7/3.7/6.4/7.4/12.0 Increased NT 4 2.9/3.0/5.0/5.2 Cystic hygroma 3 12.2/13.4 Hydrocephalus 2 2.5/4.9 VSD/valve anomaly 1 3.7 Dandy-Walker malformation 1 5.6 Diaphragmatic hernia 1 3.2 Renal dysplasia 1 2.2 Neurofibromatosis type I (inherit) 1 2.1

Four hundred and seventy-six pregnancies ended with delivery of a viable infant (93%). Among them we found 48 newborns (9.5%; 48/476) with either single or multiple abnormali‐ ties. The clinical findings in 476 liveborn infants with respect to the NT thickness are pre‐ sented in Table 4. There were 8 cases (1.7%, 8/476) born with heart defects, other structural abnormalities were found in 30 newborns (6.3%; 30/476). During the first year of life some genetic syndromes or neurodevelopmental delay were recorded in 10 cases (2.1%; 10/476). All abnormalities were found in the group of newborns with mildly enlarged NT, between 95th percentiles to 4.4 mm. Among healthy babies, there was no NT thicker than 6.4 mm.

2.9/2.9/3.0/3.0/4.6



**4. Discussion**

**4.1. Fetal loss**

We evaluated the pregnancy outcome of a subgroup of 512 fetuses with increased NT thick‐ ness and normal karyotype referring to 11,980 unselected pregnancies. According to the da‐ ta of fetal loss, structural abnormalities, genetic disorders and neurodevelopmental delay, one out of 6 fetuses had an increased risk ≥1:300 of trisomy 21 calculated on the basis of ma‐ ternal age, NT and fetal crown-rump length. The study confirms that 16.4% fetuses (84/512)

Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome

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29

The number of studies, which examined unselected pregnancy population with clear de‐ scription of all adverse pregnancy outcomes, is limited [9]. Bilardo CM et al [14] noted that one out of five fetuses with increased NT had an adverse pregnancy outcome. Their study provides an overview of the selected pregnancy of 675 fetuses referred from other centers

In some studies it is not clear whether the fetuses with an unknown karyotype were includ‐ ed [17,18]. This is particularly important in the cases of fetal loss. We included only fetuses with known normal karyotypes. Karyotyping was provided in all cases of miscarriages, in‐ trauterine deaths and terminations of pregnancies using amniocentesis or tissue samples ob‐

The increased NT thickness augments the risk of fetal loss. The allover fetal loss in our sub‐ group of fetuses was 7% (Table 2). We share the opinion that fetal loss in studies without a control group is very difficult to interpret [9,17-20]. The reported rates of spontaneous loss

Fifteen women terminated their pregnancies because of the fetal abnormalities (Table 3). But in 4 cases the pregnancy was terminated at the request of the parents because of an in‐ creased risk of trisomy 21, despite of the fact that no fetal malformation had been detected at

Our study shows similar weakness compared to the related studies, namely not all fetuses lost having undergone autopsy for ascertainment of fetal abnormalities, especially in the

The prevalence of structural abnormalities in our subgroup of newborns with increased NT was 8%. The percentage is higher than expected in general population (2-3%). A similar

Heart defects were confirmed in 8 out of 38 infants with structural abnormalities. The me‐ dian NT thickness was significantly higher in fetuses with major heart defects compared to those with normal hearts [21-24]. In 8 infants with heart defects we found NT measurement between 3.4 and 4.4 mm. Although the measurement of NT thickness alone appears to be a

finding can be encountered in the studies without a control group (9.5-30.3%) [9].

are 0.5-3.8% and the reported rates of termination of pregnancy are 2.3-16.9%.

the ultrasound examination. Westin M et al [9] describe similar experiences.

were at increased risk of adverse pregnancy outcome.

tained during surgical evacuation of the products of conception.

because of an increased NT measurement.

group of miscarriages [9,17-20].

**4.2. Liveborn infants with abnormalities**

**Table 5.** Disorders described in forty-eight euploid infants.

#### **3.4. Gender and preterm labor**

The overall male: female ratio was 1.37:1. In the group of fetuses with NT thickness between 95th percentile to ≤ 3.4 mm the ratio was 1.27:1 and in the group with NT ≥ 3.5 mm 2.64:1.

The gender distribution of liveborn infants with respect to the abnormalities is presented in Table 6. Male gender was predominant among healthy infants and in the group with genetic syndromes neurodevelopmental delay.

Preterm delivery was registered in 41 cases (41/476; 8.6%). Thirty-one healthy babies (31/431; 9.5%) and 10 infants with abnormalities (10/48; 20.8%) were born preterm.


## **4. Discussion**

**Disorder n NT (mm)** Cryptorchidism 2 3.2/4.4 Hypospadias 1 3.2 Polydactyly 1 2.3

Hip dysplasia 3 1.8/1.8/2.2

Neurodevelopmental delay 3 2.5/2.9/3.5

The overall male: female ratio was 1.37:1. In the group of fetuses with NT thickness between 95th percentile to ≤ 3.4 mm the ratio was 1.27:1 and in the group with NT ≥ 3.5 mm 2.64:1.

The gender distribution of liveborn infants with respect to the abnormalities is presented in Table 6. Male gender was predominant among healthy infants and in the group with genetic

Preterm delivery was registered in 41 cases (41/476; 8.6%). Thirty-one healthy babies (31/431;

8 2

**Infants Males Females** Healthy 253 175 Heart defect 1 7 Structural abnormalities 15 15

Total 276 198

9.5%) and 10 infants with abnormalities (10/48; 20.8%) were born preterm.

2 2.6/3.0

Talipes 1 2.9

Adrenogenital syndrome 1 3.0 Lipid metabolism disorder 1 1.9 Coeliac disease 1 1.6 Polycystic kidney disease 2 2.4/2.4

**Genetic syndromes and neurodevelopmental delay**: 10

Unspecific genetic syndrome and neurodevelopmental delay

28 Down Syndrome

**3.4. Gender and preterm labor**

syndromes neurodevelopmental delay.

Genetic syndromes/ neurodevelopmental delay

**Table 6.** Gender distribution of liveborn infants with respect to the abnormalities.

**Table 5.** Disorders described in forty-eight euploid infants.

We evaluated the pregnancy outcome of a subgroup of 512 fetuses with increased NT thick‐ ness and normal karyotype referring to 11,980 unselected pregnancies. According to the da‐ ta of fetal loss, structural abnormalities, genetic disorders and neurodevelopmental delay, one out of 6 fetuses had an increased risk ≥1:300 of trisomy 21 calculated on the basis of ma‐ ternal age, NT and fetal crown-rump length. The study confirms that 16.4% fetuses (84/512) were at increased risk of adverse pregnancy outcome.

The number of studies, which examined unselected pregnancy population with clear de‐ scription of all adverse pregnancy outcomes, is limited [9]. Bilardo CM et al [14] noted that one out of five fetuses with increased NT had an adverse pregnancy outcome. Their study provides an overview of the selected pregnancy of 675 fetuses referred from other centers because of an increased NT measurement.

#### **4.1. Fetal loss**

In some studies it is not clear whether the fetuses with an unknown karyotype were includ‐ ed [17,18]. This is particularly important in the cases of fetal loss. We included only fetuses with known normal karyotypes. Karyotyping was provided in all cases of miscarriages, in‐ trauterine deaths and terminations of pregnancies using amniocentesis or tissue samples ob‐ tained during surgical evacuation of the products of conception.

The increased NT thickness augments the risk of fetal loss. The allover fetal loss in our sub‐ group of fetuses was 7% (Table 2). We share the opinion that fetal loss in studies without a control group is very difficult to interpret [9,17-20]. The reported rates of spontaneous loss are 0.5-3.8% and the reported rates of termination of pregnancy are 2.3-16.9%.

Fifteen women terminated their pregnancies because of the fetal abnormalities (Table 3). But in 4 cases the pregnancy was terminated at the request of the parents because of an in‐ creased risk of trisomy 21, despite of the fact that no fetal malformation had been detected at the ultrasound examination. Westin M et al [9] describe similar experiences.

Our study shows similar weakness compared to the related studies, namely not all fetuses lost having undergone autopsy for ascertainment of fetal abnormalities, especially in the group of miscarriages [9,17-20].

#### **4.2. Liveborn infants with abnormalities**

The prevalence of structural abnormalities in our subgroup of newborns with increased NT was 8%. The percentage is higher than expected in general population (2-3%). A similar finding can be encountered in the studies without a control group (9.5-30.3%) [9].

Heart defects were confirmed in 8 out of 38 infants with structural abnormalities. The me‐ dian NT thickness was significantly higher in fetuses with major heart defects compared to those with normal hearts [21-24]. In 8 infants with heart defects we found NT measurement between 3.4 and 4.4 mm. Although the measurement of NT thickness alone appears to be a moderately effective screening pool for the detection of heart abnormalities, its role in detec‐ tion of specific congenital heart defects seems more promising [24]. When an increased NT is found, the fetus has to be screened for additional sonographic markers such as tricuspid regurgitation and abnormal ductus venosus Doppler flow profile. We share the opinion that in fetuses with an NT measurement ≥99th percentile, and/or in which tricuspid regurgita‐ tion and/or abnormal ductus venosus Doppler flow pattern is found, an earlier fetal echocar‐ diograpy is indicated [23,24].

**Author details**

Ksenija Gersak1

Ljubljana, Slovenia

867-9.

Gynecol 1998;12:163–9.

1998;105:58–62.

2002;20:219–25.

necol Scand 2004;83:1130–4.

Obstet Gynecol 2006;27:632–9.

**References**

, Darija M. Strah2

2 Diagnostic Centre Strah, Domzale, Slovenia

and Maja Pohar-Perme3

Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome

http://dx.doi.org/10.5772/53112

31

1 Department of Obstetrics and Gynecology, University Medical Center Ljubljana, Slovenia

3 Institute for Biostatistics and Medical Informatics, Faculty of Medicine, University of

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[2] Nicolaides KH, Azar G, Byrne D, et al. Fetal nuchal translucency: ultrasound screen‐ ing for chromosomal defects in the first trimester of pregnancy. BMJ 1992;304(6831):

[3] Nicolaides KH, Brizot ML, Snijders RJM. Fetal nuchal translucency: ultrasound screening for fetal trisomy in the first trimester of pregnancy. BJOG 1994;101:782-6.

[4] Pajkrt E, van Lith JMM, Mol BWJ, et al. Screening for Down's syndrome by fetal nu‐ chal translucency measurement in a general obstetric population. Ultrasound Obstet

[5] Economides DL, Whitlow BJ, Kadir R, et al. First trimester sonografic detection of chromosomal abnormalities in an unselected population. Br J Obstet Gynaecol

[6] Bindra R, Heath V, Liao A, et al. One stop assessment of risk for trisomy 21 at 11–14 weeks a prospective study of 15030 pregnancies. Ultrasound Obstet Gynecol

[7] Liu SS, Lee FK, Lee JL, et al. Pregnancy outcomes in unselected singleton pregnant women with an increased risk of first-trimester Down's syndrome. Acta Obstet Gy‐

[8] Rozenberg P, Bussières L, Chevret S, et al. Screening for Down syndrome using firsttrimester combined screening followed by second-trimester ultrasound examination

[9] Westin M, Saltvedt S, Bergman G, et al. Is measurement of nuchal translucency thick‐ ness a useful screening tool for heart defects? A study of 16,383 fetuses. Ultrasound

in an unselected population. Am J Obstet Gynecol 2006;195:1379–87.

The second most common isolated structural abnormality was hydronephrosis followed by cleft lip and/or cleft palate (Table 5).

In 5 cases genetic syndromes were found. There were two cases of inherited polycystic kid‐ ney disease, and three "de novo" genetic syndromes. In comparison with other studies we detected no infants with neuromuscular disorders [13,14].

As Bilardo CM et al [14] pointed out, the most unpredictable aspect of increased NT is neu‐ rodevelopmental delay, which could be manifested unexpectedly, in the postnatal period. The reported incidence of neurodevelopmental delay in fetuses with or without recogniza‐ ble genetic syndrome varies from 0 to 13% [14,25,26]. In our study 10.4% of newborns (5 out of 48) were diagnosed during the follow-up period of at least 18-months.

#### **4.3. Gender and preterm labor**

In our population of fetuses with increased NT thickness, male gender was predominant, es‐ pecially in the group with NT ≥ 3.5 mm. The impact of male: female ratio on the degree of nuchal fluid accumulation has been reported with controversial results. Yaron et al [27] and Prefumo et al [28] did not find NT to be significantly related to gender, but Lam et al [29] and Timmerman et al [30] reported significantly larger NT in male fetuses. Also Spencer et al [31] found NT to be 3-4% smaller in both chromosomally normal and Down syndrome female fetuses.

## **5. Conclusion**

Many couples enter any of the screening programs without an intricate understanding of the potential fetal and newborn complications. While it is reasonable for the future parents to consider normal karyotype as a "good" result, the healthcare professionals should coun‐ sel them that enlarged NT thickness is a strong marker for adverse pregnancy outcome, as‐ sociated with miscarriage, intrauterine death, heart defects, numerous other structural abnormalities and genetic syndromes. Although the measurement of the nuchal translucen‐ cy thickness was introduced over 15 years ago, we share the opinion that a general consen‐ sus on how to counsel parents of an euploid fetus with enlarged NT has not yet been achieved [13]. The larger studies with uniform protocols and long-term follow-up are need‐ ed to recommend the guidelines for objective parental counseling.

## **Author details**

moderately effective screening pool for the detection of heart abnormalities, its role in detec‐ tion of specific congenital heart defects seems more promising [24]. When an increased NT is found, the fetus has to be screened for additional sonographic markers such as tricuspid regurgitation and abnormal ductus venosus Doppler flow profile. We share the opinion that in fetuses with an NT measurement ≥99th percentile, and/or in which tricuspid regurgita‐ tion and/or abnormal ductus venosus Doppler flow pattern is found, an earlier fetal echocar‐

The second most common isolated structural abnormality was hydronephrosis followed by

In 5 cases genetic syndromes were found. There were two cases of inherited polycystic kid‐ ney disease, and three "de novo" genetic syndromes. In comparison with other studies we

As Bilardo CM et al [14] pointed out, the most unpredictable aspect of increased NT is neu‐ rodevelopmental delay, which could be manifested unexpectedly, in the postnatal period. The reported incidence of neurodevelopmental delay in fetuses with or without recogniza‐ ble genetic syndrome varies from 0 to 13% [14,25,26]. In our study 10.4% of newborns (5 out

In our population of fetuses with increased NT thickness, male gender was predominant, es‐ pecially in the group with NT ≥ 3.5 mm. The impact of male: female ratio on the degree of nuchal fluid accumulation has been reported with controversial results. Yaron et al [27] and Prefumo et al [28] did not find NT to be significantly related to gender, but Lam et al [29] and Timmerman et al [30] reported significantly larger NT in male fetuses. Also Spencer et al [31] found NT to be 3-4% smaller in both chromosomally normal and Down syndrome

Many couples enter any of the screening programs without an intricate understanding of the potential fetal and newborn complications. While it is reasonable for the future parents to consider normal karyotype as a "good" result, the healthcare professionals should coun‐ sel them that enlarged NT thickness is a strong marker for adverse pregnancy outcome, as‐ sociated with miscarriage, intrauterine death, heart defects, numerous other structural abnormalities and genetic syndromes. Although the measurement of the nuchal translucen‐ cy thickness was introduced over 15 years ago, we share the opinion that a general consen‐ sus on how to counsel parents of an euploid fetus with enlarged NT has not yet been achieved [13]. The larger studies with uniform protocols and long-term follow-up are need‐

diograpy is indicated [23,24].

30 Down Syndrome

**4.3. Gender and preterm labor**

female fetuses.

**5. Conclusion**

cleft lip and/or cleft palate (Table 5).

detected no infants with neuromuscular disorders [13,14].

of 48) were diagnosed during the follow-up period of at least 18-months.

ed to recommend the guidelines for objective parental counseling.

Ksenija Gersak1 , Darija M. Strah2 and Maja Pohar-Perme3

1 Department of Obstetrics and Gynecology, University Medical Center Ljubljana, Slovenia

2 Diagnostic Centre Strah, Domzale, Slovenia

3 Institute for Biostatistics and Medical Informatics, Faculty of Medicine, University of Ljubljana, Slovenia

## **References**


[10] Czuba B, Borowski D, Cnota W, et al. Ultrasonographic assessment of fetal nuchal translucency (NT) at 11th and 14th week of gestation-Polish multicentre study. Neu‐ ro Endocrinol Lett 2007;28:175–81.

[24] Clur SA, Ottenkamp J, Bilardo CM. The nuchal translucency and the fetal heart: a lit‐

Increased Fetal Nuchal Translucency Thickness and Normal Karyotype: Prenatal and Postnatal Outcome

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[25] Maymon R, Herman A. The clinical evaluation and pregnancy outcome of euploid

[26] Souka AP, Von Kaisenberg CS, Hyett JA, et al. Increased nuchal translucency with

[27] Yaron Y, Wolman I, Kupferminc MJ, et al. Effect of fetal gender on first trimester markers and on Down syndrome screening. Prenat Diagn 2001;21:1027-30.

[28] Prefumo F, Venturini PL, De Biasio P. Effect of fetal gender on first-trimester ductus‐

[29] Lam YH, Tang MH, Lee CP, et al. The effect of fetal gender on nuchal translucency at

[30] Timmerman E, Pajkrt E, Bilardo CM. Male gender as a favorable prognostic factor in pregnancies with enlarged nuchal translucency. Ultrasound Obstet Gynecol

[31] Spencer K, Ong CY, Liao AW, et al. The influence of fetal sex in screening for trisomy 21 by fetal nuchal translucency, maternal serum free beta-hCG and PAPP-A at 10-14

fetuses with increased nuchal translucency. Clin Genet 2004;66:426-36.

normal karyotype. Am J Obstet Gynecol 2005;192:1005-21.

venosus blood flow. Ultrasound Obstet Gynecol 2003;22:268-70.

10-14 weeks of gestation. Prenat Diagn 2001;21:627-9.

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[10] Czuba B, Borowski D, Cnota W, et al. Ultrasonographic assessment of fetal nuchal translucency (NT) at 11th and 14th week of gestation-Polish multicentre study. Neu‐

[11] Kagan KO, Staboulidou I, Cruz J, et al. Two-stage first-trimester screening for triso‐ my 21 by ultrasound assessment and biochemical testing. Ultrasound Obstet Gynecol

[12] Gersak K, Pohar-Perme M, Strah DM. First trimester screening for trisomy 21 by ma‐ ternal age, nuchal translucency and fetal nasal bone in unselected pregnancies. In: Day S, ed. Genetics and etiology of Down syndrome. Rijeka: Intech; 2011:301-312. [13] Bilardo CM, Timmerman E, Pajkrt E, et al. Increased nuchal translucency in euploid fetuses-what should we be telling the parents? Prenat Diagn 2010;30:93-102.

[14] Bilardo CM, Müller MA, Pajkrt, E, et al. Increased nuchal translucency thickness and normal karyotype: time for parental reassurance. Ultrasound Obstet Gynecol

[15] Snijders RJM, Noble P, Sebire N, et al. UK multicentre project on assessment of risk for trisomy 21 by maternal age and fetal nuchal translucency thickness at 10 – 14

[16] The Fetal Medicine Centre. Ultrasound scan procedures. http://www.fetalmedi‐

[17] Maymon R, Jauniaux E, Cohen O, et al. Pregnancy outcome and infant follow-up of fetuses with abnormally increased first trimester nuchal translucency. Hum Reprod

[18] Cheng CC, Bahado-Singh RO, Chen SC, et al. Pregnancy outcomes with increased nuchal translucency after routine Down syndrome screening. Int J Gynaecol Obstet

[19] Souka AP, Krampl E, Bakalis S, et al. Outcome of pregnancy in chromosomally nor‐ mal fetuses with increased nuchal translucency in the first trimester. Ultrasound Ob‐

[20] Senat MV, De Keersmaecker B, Audibert F, et al. Pregnancy outcome in fetuses with increased nuchal translucency and normal karyotype. Prenat Diagn 2002;22:345-9. [21] Ghi T, Huggon IC, Zosmer N, et al. Incidence of major structural cardiac defects as‐ sociated with increased nuchal translucency but normal karyotype. Ultrasound Ob‐

[22] Müller MA, Bleker P, Bonsel GJ, et al. Nuchal translucency screening and anxiety lev‐ els in pregnancy and puerperium. Ultrasound Obstet Gynecol 2007;27:357-61. [23] Vogel M, Sharland GK, McElhinney DB, et al. Prevalence of increased nuchal translu‐ cency in fetuses with congenital cardiac disease and a normal karyotype. Cardiol

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2000;15:2023-7.

2004;84:5-9.


**Chapter 3**

**Prenatal Screening and Diagnosis**

Additional information is available at the end of the chapter

In recent years the prevalence of Down syndrome has been increasing. The increase in the prevalence might be partly explained by better compilation of statistics on Down syndrome today. Also, the mean maternal age at first delivery as well as the proportion of older moth‐ ers is increasing in all western countries and the risk of Down syndrome increases with ad‐ vancing maternal age [1]. The proportion of mothers aged 35 years or older in France, Finland, Germany, Greece and United Kingdom were 15.8 %, 19.0 %, 17.0 %, 14.2 % and 17.2 % in 2001, respectively, in 2008 the proportions were 18.9 %, 18.2 %, 21.8 %, 20.9 % and 20.1 %, respectively (Eurostat). Screening for Down syndrome was first performed in 1970's us‐ ing advanced maternal age or previous history of chromosomal abnormality. The preva‐ lence of Down syndrome at term rises from 1/1527 at the maternal age of 20 years to 1/895 at age 30 and to 1/97 at age 40 [11]. Also the gestational age affects the prevalence of Down syndrome. The estimated rate of fetal loss in Down syndrome pregnancies is 43 % between gestational week 10 and term, 23 % between gestational week 15 and term and 12 % of births are stillbirths or result in a neonatal death [12]. Therefore, the risk of Down syndrome decreases as the pregnancy progresses. Table 1 presents the prevalence of Down syndrome

pregnancies in different maternal age groups according to the gestational age.

Maternal age of 35 years or more used as a screening method can detect approximately 43-61 % of Down syndrome cases [13, 14]. However, the false positive rate (FPR) is high since the proportion of women aged 35 years or older is approximately 20 % in western countries. Chorionic villus sampling (CVS) and amniocentesis (AC) carry a 0.5-1.0 % risk of fetal loss [15]. Maternal age of 35 is an arbitrary threshold and there are better screening methods available today. The invasive test should not be offered only because of increased

> © 2013 Marttala; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Jaana Marttala

**1. Introduction**

**1.1. Maternal age**

maternal age.

http://dx.doi.org/10.5772/52861

## **Chapter 3**

## **Prenatal Screening and Diagnosis**

## Jaana Marttala

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52861

## **1. Introduction**

#### **1.1. Maternal age**

In recent years the prevalence of Down syndrome has been increasing. The increase in the prevalence might be partly explained by better compilation of statistics on Down syndrome today. Also, the mean maternal age at first delivery as well as the proportion of older moth‐ ers is increasing in all western countries and the risk of Down syndrome increases with ad‐ vancing maternal age [1]. The proportion of mothers aged 35 years or older in France, Finland, Germany, Greece and United Kingdom were 15.8 %, 19.0 %, 17.0 %, 14.2 % and 17.2 % in 2001, respectively, in 2008 the proportions were 18.9 %, 18.2 %, 21.8 %, 20.9 % and 20.1 %, respectively (Eurostat). Screening for Down syndrome was first performed in 1970's us‐ ing advanced maternal age or previous history of chromosomal abnormality. The preva‐ lence of Down syndrome at term rises from 1/1527 at the maternal age of 20 years to 1/895 at age 30 and to 1/97 at age 40 [11]. Also the gestational age affects the prevalence of Down syndrome. The estimated rate of fetal loss in Down syndrome pregnancies is 43 % between gestational week 10 and term, 23 % between gestational week 15 and term and 12 % of births are stillbirths or result in a neonatal death [12]. Therefore, the risk of Down syndrome decreases as the pregnancy progresses. Table 1 presents the prevalence of Down syndrome pregnancies in different maternal age groups according to the gestational age.

Maternal age of 35 years or more used as a screening method can detect approximately 43-61 % of Down syndrome cases [13, 14]. However, the false positive rate (FPR) is high since the proportion of women aged 35 years or older is approximately 20 % in western countries. Chorionic villus sampling (CVS) and amniocentesis (AC) carry a 0.5-1.0 % risk of fetal loss [15]. Maternal age of 35 is an arbitrary threshold and there are better screening methods available today. The invasive test should not be offered only because of increased maternal age.

© 2013 Marttala; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The median MoMs and standard deviations in the populations influence the degree of the overlap. A patient-specific risk for each screened woman is calculated by multiplying the a

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861 37

Maternal serum biochemistry reflects the degree of maturity of the placenta rather than direct‐ ly measuring the presence or absence of Down syndrome. These markers have also limita‐ tions, such as the relatively narrow gestational window in which they can be used. In pregnancies that are affected by fetal chromosomal abnormalities the placental function is im‐ paired and the levels of fβ-hCG and PAPP-A differ from normal pregnancies. The results of the maternal serum biochemistry are reported as multiples of the median (MoM) specific to the gestational week. MoM values are calculated by dividing a woman's marker level by the median level of that marker for the entire population at that gestational age in each laborato‐ ry. The use of MoM values therefore also allows the interpretation between the results from different laboratories in different countries. The expected levels of maternal serum markers are not only affected by maternal age and gestational age but also other factors like maternal weight, ethnic origin, the presence of insulin dependent diabetes mellitus, multiple pregnan‐ cy, smoking and vaginal bleeding. Screening program takes into account certain variables.

Human chorionic gonadotropin (hCG) was first purified from the pregnant women's urine. hCG is produced by the trophoblastic cells of the placenta from the 10th to 12th day after con‐ ception and it reaches its peak value in maternal circulation at 8 to 10 weeks of gestation. Then, a rapid decrease is seen and a plateau is reached at 20th week of gestation [22]. hCG was first used as a second trimester screening marker for Down syndrome. Later, it was shown that the free beta subunit of hCG (fβ-hCG) is an effective screening marker for Down syndrome in the first trimester of the pregnancy. In Down syndrome pregnancies maternal serum fβ-hCG lev‐ els are higher than in normal pregnancies during the first trimester of the pregnancy. The re‐ ported DRs for fβ-hCG alone are around 19-42 % for a 5 % FPR [9, 16]. The DR of fβ-hCG for

The association between abnormal levels of maternal serum pregnancy associated plasma protein-A (PAPP-A) and fetal aneuploidy was made in late 1980's and early 1990's [22, 23]. PAPP-A levels normally rise during pregnancy all the way to the delivery. PAPP-A is a metal‐ loproteinase that cleaves insulin-growth factor binding protein-4 (IGFBP-4) which binds IGFs with high affinity thus preventing their interaction with the IGF-receptors that mediate cell growth and survival signals [24, 25]. IGFs are important in implantation, placental physiolo‐ gy and fetal growth [25]. Therefore, PAPP-A is believed to function as a growth factor of both fetus and placenta during the pregnancy. PAPP-A levels are lower in Down syndrome preg‐ nancies during the first trimester of the pregnancy but the deviation from normal decreases with gestational age [20]. The DR for PAPP-A alone is approximately 52 % for a 5 % FPR [16]. Fetal NT in the first trimester of the pregnancy was described as the fluid-filled space under the skin behind the fetal neck in 1992 [7, 8]. NT is measured during first trimester ultra‐ sound scan at gestational weeks 10-13. Ultrasound scan also offers accurate dating of the

Down syndrome is better at gestational week 13 than at gestational week 10 [9].

priori risk based on maternal age with the LR [18-21].

**2.1. Screening markers**

**Table 1.** The prevalence of Down syndrome according to maternal age and gestational age. (Modified from Snijders *et al.* 1999).

#### **1.2. Second trimester screening**

Abnormal levels of specific maternal serum markers were associated with Down syndrome in 1980's. Second trimester screening with maternal age and maternal serum markers was developed consisting of either double, triple or quadruple serum screening. Optimal win‐ dow for second trimester serum screening is between 15 and 22 weeks of gestation. Double test includes maternal age, maternal serum free beta human chorionic gonadotropin (fβhCG) and alfafetoprotein (AFP). The additional serum markers are unconjugated oestriol (uE3) in triple screening and uE3 and inhibin-A in quadruple screening. The estimated FPRs for an 85 % detection rate (DR) for double, triple and quadruple screening are 13.1 %, 9.3-14 % and 6.2-7.3 %, respectively [9, 16]. For a 5 % FPR the DRs for double, triple and quadruple screening are approximately 59 %, 63 % and 72 %, respectively.

#### **2. Screening for Down syndrome today**

Screening for Down syndrome has moved from second trimester to first trimester during the last two decades. The most popular screening method today is combined first trimester screening where maternal serum biomarkers fβ-hCG and pregnancy associated plasma pro‐ tein-A (PAPP-A) are used in combination with fetal nuchal translucency (NT) measurement, ultrasound dated gestational age and maternal age to calculate a woman's risk for Down syndrome using a computer based program. The serum markers and NT do not correlate with each other in chromosomally normal or abnormal fetuses [17]. Each screened woman has a priori risk which is based on her age and the gestational age. The risk calculation soft‐ ware program uses the Gaussian distributions of NT and serum values of normal and affect‐ ed cases to calculate the LRs. These are described by their means of log10 MoMs, standard deviations and correlation coefficients between the markers. The screening test performs well if the Gaussian distributions of the markers in the normal and affected populations are separated. Alternatively, the screening test is impractical if the distributions overlap widely. The median MoMs and standard deviations in the populations influence the degree of the overlap. A patient-specific risk for each screened woman is calculated by multiplying the a priori risk based on maternal age with the LR [18-21].

Maternal serum biochemistry reflects the degree of maturity of the placenta rather than direct‐ ly measuring the presence or absence of Down syndrome. These markers have also limita‐ tions, such as the relatively narrow gestational window in which they can be used. In pregnancies that are affected by fetal chromosomal abnormalities the placental function is im‐ paired and the levels of fβ-hCG and PAPP-A differ from normal pregnancies. The results of the maternal serum biochemistry are reported as multiples of the median (MoM) specific to the gestational week. MoM values are calculated by dividing a woman's marker level by the median level of that marker for the entire population at that gestational age in each laborato‐ ry. The use of MoM values therefore also allows the interpretation between the results from different laboratories in different countries. The expected levels of maternal serum markers are not only affected by maternal age and gestational age but also other factors like maternal weight, ethnic origin, the presence of insulin dependent diabetes mellitus, multiple pregnan‐ cy, smoking and vaginal bleeding. Screening program takes into account certain variables.

#### **2.1. Screening markers**

**Maternal age**

36 Down Syndrome

*et al.* 1999).

**1.2. Second trimester screening**

**(years) Gestational age (weeks)**

screening are approximately 59 %, 63 % and 72 %, respectively.

**2. Screening for Down syndrome today**

 12 14 16 20 40 1/983 1/1068 1/1140 1/1200 1/1295 1/1527 1/870 1/946 1/1009 1/1062 1/1147 1/1352 1/576 1/626 1/668 1/703 1/759 1/895 1/229 1/249 1/266 1/280 1/302 1/356 1/62 1/68 1/72 1/76 1/82 1/97 1/15 1/16 1/17 1/18 1/19 1/23

**Table 1.** The prevalence of Down syndrome according to maternal age and gestational age. (Modified from Snijders

Abnormal levels of specific maternal serum markers were associated with Down syndrome in 1980's. Second trimester screening with maternal age and maternal serum markers was developed consisting of either double, triple or quadruple serum screening. Optimal win‐ dow for second trimester serum screening is between 15 and 22 weeks of gestation. Double test includes maternal age, maternal serum free beta human chorionic gonadotropin (fβhCG) and alfafetoprotein (AFP). The additional serum markers are unconjugated oestriol (uE3) in triple screening and uE3 and inhibin-A in quadruple screening. The estimated FPRs for an 85 % detection rate (DR) for double, triple and quadruple screening are 13.1 %, 9.3-14 % and 6.2-7.3 %, respectively [9, 16]. For a 5 % FPR the DRs for double, triple and quadruple

Screening for Down syndrome has moved from second trimester to first trimester during the last two decades. The most popular screening method today is combined first trimester screening where maternal serum biomarkers fβ-hCG and pregnancy associated plasma pro‐ tein-A (PAPP-A) are used in combination with fetal nuchal translucency (NT) measurement, ultrasound dated gestational age and maternal age to calculate a woman's risk for Down syndrome using a computer based program. The serum markers and NT do not correlate with each other in chromosomally normal or abnormal fetuses [17]. Each screened woman has a priori risk which is based on her age and the gestational age. The risk calculation soft‐ ware program uses the Gaussian distributions of NT and serum values of normal and affect‐ ed cases to calculate the LRs. These are described by their means of log10 MoMs, standard deviations and correlation coefficients between the markers. The screening test performs well if the Gaussian distributions of the markers in the normal and affected populations are separated. Alternatively, the screening test is impractical if the distributions overlap widely.

Human chorionic gonadotropin (hCG) was first purified from the pregnant women's urine. hCG is produced by the trophoblastic cells of the placenta from the 10th to 12th day after con‐ ception and it reaches its peak value in maternal circulation at 8 to 10 weeks of gestation. Then, a rapid decrease is seen and a plateau is reached at 20th week of gestation [22]. hCG was first used as a second trimester screening marker for Down syndrome. Later, it was shown that the free beta subunit of hCG (fβ-hCG) is an effective screening marker for Down syndrome in the first trimester of the pregnancy. In Down syndrome pregnancies maternal serum fβ-hCG lev‐ els are higher than in normal pregnancies during the first trimester of the pregnancy. The re‐ ported DRs for fβ-hCG alone are around 19-42 % for a 5 % FPR [9, 16]. The DR of fβ-hCG for Down syndrome is better at gestational week 13 than at gestational week 10 [9].

The association between abnormal levels of maternal serum pregnancy associated plasma protein-A (PAPP-A) and fetal aneuploidy was made in late 1980's and early 1990's [22, 23]. PAPP-A levels normally rise during pregnancy all the way to the delivery. PAPP-A is a metal‐ loproteinase that cleaves insulin-growth factor binding protein-4 (IGFBP-4) which binds IGFs with high affinity thus preventing their interaction with the IGF-receptors that mediate cell growth and survival signals [24, 25]. IGFs are important in implantation, placental physiolo‐ gy and fetal growth [25]. Therefore, PAPP-A is believed to function as a growth factor of both fetus and placenta during the pregnancy. PAPP-A levels are lower in Down syndrome preg‐ nancies during the first trimester of the pregnancy but the deviation from normal decreases with gestational age [20]. The DR for PAPP-A alone is approximately 52 % for a 5 % FPR [16].

Fetal NT in the first trimester of the pregnancy was described as the fluid-filled space under the skin behind the fetal neck in 1992 [7, 8]. NT is measured during first trimester ultra‐ sound scan at gestational weeks 10-13. Ultrasound scan also offers accurate dating of the pregnancy, ascertainment of viable fetus or missed abortion, detection of multiple pregnan‐ cies, accurate dating of the pregnancy, identification of chorionicity and detection of some major fetal anomalies. NT measurement is not altered in multiple pregnancies or by assisted reproduction techniques. Large studies in low risk populations have shown the association between increased NT and chromosomal defects. The combination of maternal age and NT was reported to have a DR of 63.0-90.0 % for a FPR of 5.0-13.0 % [16, 26]. Therefore, NT measurement is the best single marker in screening for Down syndrome [16, 27].

**Study Gestation Sample size Trisomy 21**

12 13

12 13

Engels *et al.* 2011 [16] 9-14 26274

Peuhkurinen *et al.* 2012

[55]

Malone *et al.* 2005 [9] 11

Borrell *et al.* 2009 [46] 11

**(N)**

**Incidence of trisomy 21**

Bindra *et al.* 2002 [40] 11 – 14 15030 82 1:175 34.0 47.1 1:300 90.2 5.0 Crossley *et al.* 2002 [41] 10 – 14 17229 45 1:383 29.9 15.4 1:250 82 5 Wald *et al.* 2003 [16] 10 – 13 39983 85 1:470 - - 1:310 83 5 Wapner *et al.* 2003 [42] 10 – 14 8514 61 1:135 34.5 50.0 1:270 85.2 9.4

Rozenberg *et al.* 2006 [43] 11 – 13 14934 51 1:293 30.9 - 1:250 79.6 2.7 Kagan *et al.* 2008 [44] 11 – 13 56771 395 1:143 35.4 - 1:200 89 4.6 Okun *et al.* 2008 [45] 11 – 13 14487 62 1:234 34.0 - 1:200 83.9 4.0

Kagan *et al.* 2009 [47] 11-13 19736 122 1:162 34.5 - 1:150 91.0 3.1 Leung *et al.* 2009 [48] 11 – 13 10363 38 1:272 32.0 27.4 1:300 91.2 5.4 Schaelike *et al.* 2009 [49] 11-13 10668 59 1:181 - 31.0 1:300 88.1 4.9

Salomon *et al.* 2010 [51] 11-13 21492 80 1:269 30.7 - 1:250 80.0 8.8 Wright *et al.* 2010 [52] 7-14 223361 886 1:252 31.9 - 1:100 90.0 3.0

Marttala *et al.* 2011 [53] 9 – 13 76949 188 1:409 29.3 19.3 1:250 81.9 4.3 Yeo *et al.* 2012 [54] 10-13 12585 31 1:406 - - 1:300 87.1 5.1

1:217 <36 1:346 ≥36 1:120

<35 1:876 ≥35 1:113

Wortelboer *et al.* 2009 [50] 10 – 14 20293 87 1:233 34.3 "/>36 yr

121 <36 52 ≥36 69

<35 73 ≥35 115

**Table 2.** Performance of first trimester combined screening of Down syndrome in different studies.

<36 17970 ≥36 8304

> <35 50941 ≥35 13004

9-13 63945

**Median age**

38167 117 1:326 30.1 21.6 1:300 87

7250 66 1:110 32.0 - 1:250 86

**Women at age of ≥ 35 (%)**

38.7

31.6

34.1 ≥36 yr

<35 27.9 ≥35 37.8 **Cut-off level DR**

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861

**%**

85 82

84 83

1:250 75.9 3.3

1:200 95.2 <36 94.5 ≥36 95.8

16.9 1:250 <35

74.0 ≥35 87.0 **FPR %**

39

5.0

4.9 5.4 6.1

6.6 <36 4.1 ≥36 13.0

<35 2.8 ≥35 11.9

The incidence of chromosomal defects is related to the thickness rather than the appearance of NT [28]. In initial studies, single millimeter cut-offs like 2.5 mm or 3.0 mm were used to define screen positivity but as it was learned that NT increases with CRL it was realized that it is important to take gestational age into account [29]. Later certain percentile cut-offs, like the fetal NT measurement equal to or above the 95th or 99th centile for CRL, were used. To‐ day, most current screening programs advocate the use of gestational age based cut-offs for risk assessment of MoMs. However, some recent studies like a study of 36120 singleton pregnancies with complete first trimester NT and serum marker data have concluded that immediate invasive testing should be offered to all patients with NT measurement of 3 mm or greater since the addition of the first trimester serum markers do not seem to significantly reduce the final risk of fetal aneuploidy [30].

Also the risk of other adverse pregnancy outcomes increases with enlarging NT measure‐ ment. Between NT values of the 95th and 99th percentiles, the prevalence of major anomalies is 2.5 %. With NT measurement of 6.5 mm or larger, the risk is approximately 45 % [31]. The causes behind increased NT measurement are heterogenic which is in relation to the variety of adverse pregnancy outcomes that increased fetal NT has been associated with [32]. Con‐ genital heart defect is the most common adverse pregnancy outcome that has been associat‐ ed with increased NT [31]. The prevalence of congenital heart defects in children with Down syndrome is approximately 43 % [33]. Other suggested mechanisms include impaired or de‐ layed development of lymphatic drainage [34], mediastinal compression and impedence to venous return caused by for example diaphragmatic hernia or skeletal dysplasias [35, 36], over-expression of certain collagen genes in trisomic fetuses [37], exomphalos, body stalk anomaly, fetal akinesia deformation sequence and genetic syndromes [38, 39].

#### **2.2. Performance of the combined first trimester screening**

Screening works better among a population where the incidence of the screened condition is high. Therefore, since the risk of Down syndrome increases with advancing maternal age, screening works better among the older women. Overall, more than half of the Down syn‐ drome cases occur among the women aged 35 years or older [13, 14]. Reported screening perfomances are better in studies that have been conducted in high risk populations where the median maternal age is high and thereby the incidence of Down syndrome is also high. When the screened population reflects well the general low risk population and united screening strategy and high quality ultrasound machines are used, reliable screening results are drawn. Table 2 summarizes the performance of combined first trimester screening for trisomy 21 in large studies reported in the literature.


pregnancy, ascertainment of viable fetus or missed abortion, detection of multiple pregnan‐ cies, accurate dating of the pregnancy, identification of chorionicity and detection of some major fetal anomalies. NT measurement is not altered in multiple pregnancies or by assisted reproduction techniques. Large studies in low risk populations have shown the association between increased NT and chromosomal defects. The combination of maternal age and NT was reported to have a DR of 63.0-90.0 % for a FPR of 5.0-13.0 % [16, 26]. Therefore, NT

The incidence of chromosomal defects is related to the thickness rather than the appearance of NT [28]. In initial studies, single millimeter cut-offs like 2.5 mm or 3.0 mm were used to define screen positivity but as it was learned that NT increases with CRL it was realized that it is important to take gestational age into account [29]. Later certain percentile cut-offs, like the fetal NT measurement equal to or above the 95th or 99th centile for CRL, were used. To‐ day, most current screening programs advocate the use of gestational age based cut-offs for risk assessment of MoMs. However, some recent studies like a study of 36120 singleton pregnancies with complete first trimester NT and serum marker data have concluded that immediate invasive testing should be offered to all patients with NT measurement of 3 mm or greater since the addition of the first trimester serum markers do not seem to significantly

Also the risk of other adverse pregnancy outcomes increases with enlarging NT measure‐ ment. Between NT values of the 95th and 99th percentiles, the prevalence of major anomalies is 2.5 %. With NT measurement of 6.5 mm or larger, the risk is approximately 45 % [31]. The causes behind increased NT measurement are heterogenic which is in relation to the variety of adverse pregnancy outcomes that increased fetal NT has been associated with [32]. Con‐ genital heart defect is the most common adverse pregnancy outcome that has been associat‐ ed with increased NT [31]. The prevalence of congenital heart defects in children with Down syndrome is approximately 43 % [33]. Other suggested mechanisms include impaired or de‐ layed development of lymphatic drainage [34], mediastinal compression and impedence to venous return caused by for example diaphragmatic hernia or skeletal dysplasias [35, 36], over-expression of certain collagen genes in trisomic fetuses [37], exomphalos, body stalk

Screening works better among a population where the incidence of the screened condition is high. Therefore, since the risk of Down syndrome increases with advancing maternal age, screening works better among the older women. Overall, more than half of the Down syn‐ drome cases occur among the women aged 35 years or older [13, 14]. Reported screening perfomances are better in studies that have been conducted in high risk populations where the median maternal age is high and thereby the incidence of Down syndrome is also high. When the screened population reflects well the general low risk population and united screening strategy and high quality ultrasound machines are used, reliable screening results are drawn. Table 2 summarizes the performance of combined first trimester screening for

anomaly, fetal akinesia deformation sequence and genetic syndromes [38, 39].

**2.2. Performance of the combined first trimester screening**

trisomy 21 in large studies reported in the literature.

measurement is the best single marker in screening for Down syndrome [16, 27].

reduce the final risk of fetal aneuploidy [30].

38 Down Syndrome

**Table 2.** Performance of first trimester combined screening of Down syndrome in different studies.

Improving the screening means increase in the DR and decrease in the FPR and thus de‐ crease in the number of invasive procedures needed to detect one case of Down syndrome and number of procedure related miscarriages. However, with current screening strategies, increase in DR means an increase also in the FPR. A decrease in invasive procedures is an important goal and therefore special attention should be given to decreasing the FPR.

significantly increased and NT measurements significantly reduced in women carrying fe‐ male fetuses compared to women carrying male fetuses [51]. In future, NIPD may replace contemporary prenatal diagnosis in those women who are at risk of fetal chromosomal ab‐ normality after Down syndrome screening. However, at the moment, research should also focus on improving the sensitivity and specificity of the combined screening. This might

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861 41

After a positive screening result, a diagnostic test is offered. Also women who are in in‐ creased risk for Down syndrome due to increased maternal age or have a family history of Down syndrome are offered invasive testing. CVS can be performed at 11-14 weeks of gesta‐ tion and AC from 15 weeks of gestation. CVS and AC carry an approximately 0.5-1 % risk of

Ductus venosus (DV) shunts approximately half of the well-oxygenated blood from the um‐ bilical vein directly into the inferior vena cava thus bypassing the liver. The blood flow in the DV is normally forward and triphasic. The waveform of the blood flow has a peak dur‐ ing ventricular systole (S-wave) and diastole (D-wave), during the atrial contraction in late diastole there is a nadir (A-wave). Abnormal flow in the DV in the first trimester of the preg‐ nancy has been associated with chromosomal abnormalities. The abnormal DV flow has been reported to detect approximately 65-75 % of the Down syndrome cases for a FPR of 5.0-21 % [56, 57]. Addition of DV assessment to combined screening can improve the DR

Fetal tachycardia has been associated with Down syndrome. However, the results have been controversial and even when the association has been made the authors have not always sug‐ gested the use of fetal heart rate (FHR) in the screening program. Addition of FHR to com‐ bined screening improves the DR only marginally, from 89 % to 90 % for a FPR of 3.0 % [58]. Frontomaxillary facial (FMF) angle decreases normally with CRL from 85˚ at 45 mm to 75˚ at 84 mm [59]. The FMF angle measurements are above the 95th centile in approximately 69 % of Down syndrome fetuses and 5 % of euploid fetuses. Addition of FMF angle to combined

Nasal bone (NB) has been found to be absent or hypoplastic in fetuses with Down syn‐ drome. NB is classified as being absent in cases where NB appears as a thin line, or less echogenic than the overlying skin suggesting that the NB is not yet ossified. The DR for NB alone is approximately 73 % for a FPR of 0.5 % [83]. Addition of NB to combined screening

Tricuspid regurgitation (TR) is defined by the Fetal Medicine Foundation as when the veloc‐ ity of the flow exceeds 60 cm/s and occurs during at least half of the systole. In some studies,

from 89 % to 96 % with an increase in FPR from 2.3 % to 2.5 % [58].

screening can improve the DR from 90 % to 95 % for a FPR of 5.0 % [60].

can improve the DR from 89 % to 91 % for a FPR of 2.5 % [58].

happen by adding new biochemical and sonographic markers into screening.

**2.3. Invasive testing**

miscarriage [15].

**2.4. Other investigated screening markers**

*2.4.1. Additional ultrasound markers*

As screening performance depends on maternal age the screening program takes into ac‐ count maternal age [55]. DR and FPR increase with advancing maternal age. Worst screen‐ ing performance is among the women aged 25-29 years [14, 43]. The overall screening performance may be an underestimation or overestimation on individual level depending on the screened woman's age. More focus on individual risk in counseling is needed. Among younger women, the possibility of a false negative screening result is higher and among older women the possibility of false positive screening result is higher. Possibly, low‐ ering the screening cut-off level among women aged 35 or more could improve the balance between DR and FPR [14]. For example, in USA, improved prenatal screening tests and in‐ creased availability of screening for also the older women has declined the uptake of inva‐ sive testing over the past decade. Also the risk of procedure related miscarriage affects women's decision. The possibility for earlier screening during the first trimester has de‐ creased the number of invasive tests more than the second trimester screening. Also a screening strategy that excludes maternal age, called advanced first trimester screening, might be an option among older women.

Most common factor for a false negative screening result is NT. Therefore, appropriate train‐ ing and constant audit as well as possibly the certification of the competence should be re‐ quired from the examiners performing ultrasound scans and NT measurements. Even more competence will be required if additional ultrasound markers like nasal bone will be includ‐ ed into the screening program. The quality of ultrasound machines is also important.

It is possible to provide pretest counseling, biochemical testing of the mother, and NT meas‐ urement at the same visit and post-test counseling on a combined risk estimate within a onehour visit to a one-stop clinic [40]. However, screening performance differs according to the gestational age. The difference between fβ-hCG MoM values increases between unaffected and affected pregnancies as the pregnancy progresses. On the contrary, the difference in PAPP-A values decreases and PAPP-A is more effective screening marker than fβ-hCG. The maximum separation in PAPP-A levels is seen at 9-10 gestational weeks. Therefore, screen‐ ing works better when PAPP-A is measured during 9-10 weeks of gestation rather than dur‐ ing gestational weeks 7-8 or 11-14. First trimester ultrasound scan is more accurate during the late first trimester. Therefore it would be rational to draw blood samples for the meas‐ urements of PAPP-A and fβ-hCG at gestational weeks 9-10 and have another visit at 12th gestational week for the ultrasound scan. Another option could be to measure PAPP-A at gestational weeks 9-10 and NT and fβ-hCG at 12th gestational week. This could improve DRs from 90 % to 92 % for a FPR of 3 % and from 93 % to 95 % for a FPR of 5 % [20, 47, 48, 50].

Also, fetal gender has been shown to affect the levels of maternal serum PAPP-A and fβhCG in Down syndrome pregnancies. The levels of fβ-hCG and PAPP-A were shown to be significantly increased and NT measurements significantly reduced in women carrying fe‐ male fetuses compared to women carrying male fetuses [51]. In future, NIPD may replace contemporary prenatal diagnosis in those women who are at risk of fetal chromosomal ab‐ normality after Down syndrome screening. However, at the moment, research should also focus on improving the sensitivity and specificity of the combined screening. This might happen by adding new biochemical and sonographic markers into screening.

#### **2.3. Invasive testing**

Improving the screening means increase in the DR and decrease in the FPR and thus de‐ crease in the number of invasive procedures needed to detect one case of Down syndrome and number of procedure related miscarriages. However, with current screening strategies, increase in DR means an increase also in the FPR. A decrease in invasive procedures is an

As screening performance depends on maternal age the screening program takes into ac‐ count maternal age [55]. DR and FPR increase with advancing maternal age. Worst screen‐ ing performance is among the women aged 25-29 years [14, 43]. The overall screening performance may be an underestimation or overestimation on individual level depending on the screened woman's age. More focus on individual risk in counseling is needed. Among younger women, the possibility of a false negative screening result is higher and among older women the possibility of false positive screening result is higher. Possibly, low‐ ering the screening cut-off level among women aged 35 or more could improve the balance between DR and FPR [14]. For example, in USA, improved prenatal screening tests and in‐ creased availability of screening for also the older women has declined the uptake of inva‐ sive testing over the past decade. Also the risk of procedure related miscarriage affects women's decision. The possibility for earlier screening during the first trimester has de‐ creased the number of invasive tests more than the second trimester screening. Also a screening strategy that excludes maternal age, called advanced first trimester screening,

Most common factor for a false negative screening result is NT. Therefore, appropriate train‐ ing and constant audit as well as possibly the certification of the competence should be re‐ quired from the examiners performing ultrasound scans and NT measurements. Even more competence will be required if additional ultrasound markers like nasal bone will be includ‐

It is possible to provide pretest counseling, biochemical testing of the mother, and NT meas‐ urement at the same visit and post-test counseling on a combined risk estimate within a onehour visit to a one-stop clinic [40]. However, screening performance differs according to the gestational age. The difference between fβ-hCG MoM values increases between unaffected and affected pregnancies as the pregnancy progresses. On the contrary, the difference in PAPP-A values decreases and PAPP-A is more effective screening marker than fβ-hCG. The maximum separation in PAPP-A levels is seen at 9-10 gestational weeks. Therefore, screen‐ ing works better when PAPP-A is measured during 9-10 weeks of gestation rather than dur‐ ing gestational weeks 7-8 or 11-14. First trimester ultrasound scan is more accurate during the late first trimester. Therefore it would be rational to draw blood samples for the meas‐ urements of PAPP-A and fβ-hCG at gestational weeks 9-10 and have another visit at 12th gestational week for the ultrasound scan. Another option could be to measure PAPP-A at gestational weeks 9-10 and NT and fβ-hCG at 12th gestational week. This could improve DRs from 90 % to 92 % for a FPR of 3 % and from 93 % to 95 % for a FPR of 5 % [20, 47, 48,

Also, fetal gender has been shown to affect the levels of maternal serum PAPP-A and fβhCG in Down syndrome pregnancies. The levels of fβ-hCG and PAPP-A were shown to be

ed into the screening program. The quality of ultrasound machines is also important.

important goal and therefore special attention should be given to decreasing the FPR.

might be an option among older women.

50].

40 Down Syndrome

After a positive screening result, a diagnostic test is offered. Also women who are in in‐ creased risk for Down syndrome due to increased maternal age or have a family history of Down syndrome are offered invasive testing. CVS can be performed at 11-14 weeks of gesta‐ tion and AC from 15 weeks of gestation. CVS and AC carry an approximately 0.5-1 % risk of miscarriage [15].

#### **2.4. Other investigated screening markers**

#### *2.4.1. Additional ultrasound markers*

Ductus venosus (DV) shunts approximately half of the well-oxygenated blood from the um‐ bilical vein directly into the inferior vena cava thus bypassing the liver. The blood flow in the DV is normally forward and triphasic. The waveform of the blood flow has a peak dur‐ ing ventricular systole (S-wave) and diastole (D-wave), during the atrial contraction in late diastole there is a nadir (A-wave). Abnormal flow in the DV in the first trimester of the preg‐ nancy has been associated with chromosomal abnormalities. The abnormal DV flow has been reported to detect approximately 65-75 % of the Down syndrome cases for a FPR of 5.0-21 % [56, 57]. Addition of DV assessment to combined screening can improve the DR from 89 % to 96 % with an increase in FPR from 2.3 % to 2.5 % [58].

Fetal tachycardia has been associated with Down syndrome. However, the results have been controversial and even when the association has been made the authors have not always sug‐ gested the use of fetal heart rate (FHR) in the screening program. Addition of FHR to com‐ bined screening improves the DR only marginally, from 89 % to 90 % for a FPR of 3.0 % [58].

Frontomaxillary facial (FMF) angle decreases normally with CRL from 85˚ at 45 mm to 75˚ at 84 mm [59]. The FMF angle measurements are above the 95th centile in approximately 69 % of Down syndrome fetuses and 5 % of euploid fetuses. Addition of FMF angle to combined screening can improve the DR from 90 % to 95 % for a FPR of 5.0 % [60].

Nasal bone (NB) has been found to be absent or hypoplastic in fetuses with Down syn‐ drome. NB is classified as being absent in cases where NB appears as a thin line, or less echogenic than the overlying skin suggesting that the NB is not yet ossified. The DR for NB alone is approximately 73 % for a FPR of 0.5 % [83]. Addition of NB to combined screening can improve the DR from 89 % to 91 % for a FPR of 2.5 % [58].

Tricuspid regurgitation (TR) is defined by the Fetal Medicine Foundation as when the veloc‐ ity of the flow exceeds 60 cm/s and occurs during at least half of the systole. In some studies, however, TR has been defined as when the flow exceeds 80 cm/s [62]. The DR for TR alone is approximately 59.4 % for a FPR of 8.8 % [63]. Addition of TR to combined screening can im‐ prove the DR from 75 % to 87 % for a FPR of 1.0 % [62]. Table 3 presents the reported DRs and FPRs for additional ultrasound markers alone and in combination with first trimester combined screening.

club foot. The genetic sonogram has been reported to have a DR of 66.6 – 83.0 % for a 6.7 – 19.3 % FPR depending on the population. The screening performance is naturally lower in a low risk population [67, 68]. Combining the genetic sonogram into combined first trimester

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861 43

If major defects are detected during the scan, fetal karyotyping is offered to determine the underlying cause and the risk of recurrence. Even if the condition, like diaphragmatic her‐ nia, is treatable by a surgery, there might be a chromosomal abnormality behind it. Unlike major defects, minor defects are common and rarely associated with any other handicap than chromosomal abnormality. Therefore, detection of a minor defect should lead to a thor‐ ough search for other defects. The risk of a fetal anomaly should be individually evaluated since it increases with the number of minor defects detected. Second trimester ultrasound scan will likely have an important role also in the future in the detection of fetal Down syn‐

New biochemical screening markers are under investigation. A disintegrin and metallopro‐ tease 12 (ADAM12) is a glycoprotein that is synthesized by placenta. Lowered levels of ADAM12 in maternal serum have been associated with Down syndrome and other chromo‐ somal abnormalities such as trisomies 18 and 13 during the early first trimester of the preg‐ nancy but its deviation from normality decreases as the pregnancy progresses [69-71]. ADAM12 is not a good screening marker for Down syndrome during gestational weeks 11-13 since its levels are not significantly different from normal. Although in other chromo‐ somal abnormalities the levels differ significantly from normal, there is a significant associa‐ tion between ADAM12 and fβ-hCG and PAPP-A [95, 96]. Modeled DRs for ADAM12 in combination with first trimester combined screening markers are 97 % and 89 % for FPRs of 5 % and 1 % at gestational week 12 [70]. However, it seems that no additional benefit could

be obtained be the inclusion of ADAM12 into the first trimester combined screening.

Inhibin A has been long used as a part of second trimester quadruple screening. High levels of inhibin A in Down syndrome pregnancies have also been found during the first trimester of the pregnancy. Using inhibin A with combined screening markers during the gestational weeks 9-11 can achieve an approximately 82.6 % DR for a 1.0 % FPR which is close to the

Placental protein 13 (PP13) levels are not altered significantly in Down syndrome pregnan‐ cies but its levels are significantly decreased in trisomy 18 and 13, Turner syndrome and triploidy pregnancies [72, 73]. Placental growth factor (PlGF) levels in maternal serum have been reported to be decreased, increased or the same in Down syndrome pregnancies com‐ pared to unaffected pregnancies during the first and second trimester of the pregnancy. Ac‐ cording to the literature, maternal serum PlGF is potentially useful in first trimester

Using second trimester serum markers AFP, inhibin A and uE3 during the first trimester has also been studied. For the combination of PAPP-A, fβ-hCG, AFP and NT the estimated DR

screening can improve the DR from 81 % to 90 % for a 5 % FPR [69].

drome and other chromosomal abnormalities.

*2.4.3. Other biochemical screening markers*

performance of the integrated test [5].

screening for fetal chromosomal abnormalities.


**Table 3.** Screening performance of the additional ultrasound markers used alone and in combination with combined first trimester screening markers.

There is no significant association between DV flow, FMF angle, NB or TR and the com‐ bined screening markers PAPP-A, fβ-hCG and NT [59, 64]. New sonographic markers may also be used in combination. Inclusion of the new sonographic markers in to screening re‐ quires appropriate training of the examiners and the imagining protocols need to be stand‐ ardized.

#### *2.4.2. Genetic sonogram*

Genetic sonogram is an ultrasound examination performed during the second trimester of the pregnancy. During the genetic sonogram fetuses are evaluated for structural malforma‐ tions and also searched for the sonographic markers of Down syndrome. Main markers in‐ clude nuchal fold, short femur and humerus, pyelectasis, echogenic intracardiac focus, hyperechoic bowel and any major anomaly. Major abnormalities can be recognized approxi‐ mately in 25 % of the Down syndrome pregnancies [65]. If there are one or more sonograph‐ ic markers present, the baseline risk of Down syndrome increases. Similarly, the absence of markers conveys a reduction in the risk based on for example combined first trimester screening, previous chromosomal abnormality or advanced maternal age [66].

Besides major markers there are also minor, "soft", markers that can be evaluated during the scan. These include nuchal skinfold of 6 mm or more, choroid plexus cyst, enlarged cisterna magna over 10 mm, ventriculomegaly 10 mm or more, echogenic intracardiac focus, pericar‐ dial effusion, hydrops, two-vessel umbilical cord, polydactyly, clinodactyly, sandal gap, and club foot. The genetic sonogram has been reported to have a DR of 66.6 – 83.0 % for a 6.7 – 19.3 % FPR depending on the population. The screening performance is naturally lower in a low risk population [67, 68]. Combining the genetic sonogram into combined first trimester screening can improve the DR from 81 % to 90 % for a 5 % FPR [69].

If major defects are detected during the scan, fetal karyotyping is offered to determine the underlying cause and the risk of recurrence. Even if the condition, like diaphragmatic her‐ nia, is treatable by a surgery, there might be a chromosomal abnormality behind it. Unlike major defects, minor defects are common and rarely associated with any other handicap than chromosomal abnormality. Therefore, detection of a minor defect should lead to a thor‐ ough search for other defects. The risk of a fetal anomaly should be individually evaluated since it increases with the number of minor defects detected. Second trimester ultrasound scan will likely have an important role also in the future in the detection of fetal Down syn‐ drome and other chromosomal abnormalities.

#### *2.4.3. Other biochemical screening markers*

however, TR has been defined as when the flow exceeds 80 cm/s [62]. The DR for TR alone is approximately 59.4 % for a FPR of 8.8 % [63]. Addition of TR to combined screening can im‐ prove the DR from 75 % to 87 % for a FPR of 1.0 % [62]. Table 3 presents the reported DRs and FPRs for additional ultrasound markers alone and in combination with first trimester

**Ultrasound marker Ultrasound marker + maternal age Combined screening + ultrasound**

**(%)**

**Detection rate (%) False positive rate**

**Ductus venosus flow** 65-75 5-21 96 2.5 **Fetal heart rate** - - 90 3 **Frontomaxillary facial angle** 69 5 95 5

**Table 3.** Screening performance of the additional ultrasound markers used alone and in combination with combined

There is no significant association between DV flow, FMF angle, NB or TR and the com‐ bined screening markers PAPP-A, fβ-hCG and NT [59, 64]. New sonographic markers may also be used in combination. Inclusion of the new sonographic markers in to screening re‐ quires appropriate training of the examiners and the imagining protocols need to be stand‐

Genetic sonogram is an ultrasound examination performed during the second trimester of the pregnancy. During the genetic sonogram fetuses are evaluated for structural malforma‐ tions and also searched for the sonographic markers of Down syndrome. Main markers in‐ clude nuchal fold, short femur and humerus, pyelectasis, echogenic intracardiac focus, hyperechoic bowel and any major anomaly. Major abnormalities can be recognized approxi‐ mately in 25 % of the Down syndrome pregnancies [65]. If there are one or more sonograph‐ ic markers present, the baseline risk of Down syndrome increases. Similarly, the absence of markers conveys a reduction in the risk based on for example combined first trimester

Besides major markers there are also minor, "soft", markers that can be evaluated during the scan. These include nuchal skinfold of 6 mm or more, choroid plexus cyst, enlarged cisterna magna over 10 mm, ventriculomegaly 10 mm or more, echogenic intracardiac focus, pericar‐ dial effusion, hydrops, two-vessel umbilical cord, polydactyly, clinodactyly, sandal gap, and

screening, previous chromosomal abnormality or advanced maternal age [66].

**Nasal bone** 59.8-73 0.5-2.6 91

**Tricuspid regurgitation** 59.4 8.8 87

**marker**

**Detection rate (%) False positive rate**

97

96

**(%)**

2.5 5

1 2.6

combined screening.

42 Down Syndrome

first trimester screening markers.

*2.4.2. Genetic sonogram*

ardized.

New biochemical screening markers are under investigation. A disintegrin and metallopro‐ tease 12 (ADAM12) is a glycoprotein that is synthesized by placenta. Lowered levels of ADAM12 in maternal serum have been associated with Down syndrome and other chromo‐ somal abnormalities such as trisomies 18 and 13 during the early first trimester of the preg‐ nancy but its deviation from normality decreases as the pregnancy progresses [69-71]. ADAM12 is not a good screening marker for Down syndrome during gestational weeks 11-13 since its levels are not significantly different from normal. Although in other chromo‐ somal abnormalities the levels differ significantly from normal, there is a significant associa‐ tion between ADAM12 and fβ-hCG and PAPP-A [95, 96]. Modeled DRs for ADAM12 in combination with first trimester combined screening markers are 97 % and 89 % for FPRs of 5 % and 1 % at gestational week 12 [70]. However, it seems that no additional benefit could be obtained be the inclusion of ADAM12 into the first trimester combined screening.

Inhibin A has been long used as a part of second trimester quadruple screening. High levels of inhibin A in Down syndrome pregnancies have also been found during the first trimester of the pregnancy. Using inhibin A with combined screening markers during the gestational weeks 9-11 can achieve an approximately 82.6 % DR for a 1.0 % FPR which is close to the performance of the integrated test [5].

Placental protein 13 (PP13) levels are not altered significantly in Down syndrome pregnan‐ cies but its levels are significantly decreased in trisomy 18 and 13, Turner syndrome and triploidy pregnancies [72, 73]. Placental growth factor (PlGF) levels in maternal serum have been reported to be decreased, increased or the same in Down syndrome pregnancies com‐ pared to unaffected pregnancies during the first and second trimester of the pregnancy. Ac‐ cording to the literature, maternal serum PlGF is potentially useful in first trimester screening for fetal chromosomal abnormalities.

Using second trimester serum markers AFP, inhibin A and uE3 during the first trimester has also been studied. For the combination of PAPP-A, fβ-hCG, AFP and NT the estimated DR is 87.2 %, when AFP is replaced with uE3 the estimated DR is 87.9 % and for all the markers, 88.3 % for a 5 % FPR [20]. Inhibin A with combination of first trimester combined screening markers has been shown to achieve DRs of 81.4 % and 82.6 % at gestational weeks 7-8 and 9-11, respectively, for FPRs of 0.9 % and 1 % [5]. The studies on inhibin A have been contro‐ versial and some have found that inhibin A does not increase the screening performance in the first trimester [74].

**3. Screening for Down syndrome in the future**

One of the hottest topics in prenatal medicine today is the noninvasive prenatal diagnosis (NIPD). Since 1997 many approaches have been made in the field of NIPD and today it is possible to determine fetal sex, fetal Rhesus D status and diagnose genetic disorders or carri‐ er status for paternally inherited mutations [81]. Women in high risk of X-linked disorders like hemophilia can be offered noninvasive fetal sex determination. Y chromosome derived sequences can be found in maternal blood as early as eight weeks of gestation [82]. The de‐ tection of Y chromosome material indicates further investigations but if no evidence of de‐ tectable Y chromosome is found, unnecessary invasive testing with the risk of pregnancy loss, can be avoided. The costs of NIPD of fetal gender and invasive testing are similar [83, 84]. Y chromosome sequences can be detected with approximately 95.4 % sensitivity and 98.6 % specificity. Best test performance reported is for the real-time quantitative polymer‐ ase chain reaction (RTQ-PCR) after 20 weeks of gestation. Tests performed before seventh

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861 45

gestational week or using urine sample have been reported to be unreliable [85].

Detection of fetal rhesus D status can reduce the use of D immunoglobulin to prevent im‐ mune hemolytic disease of the newborn. The reported sensitivities and specificities for fetal Rhesus D sequence are greater than 95 % [86]. Reported false negative results are mainly due to a lack of fetal DNA in maternal blood sample due to too early gestation or insensitive methods. The presence of pseudogenes, mainly in African women, can lead to false positive results. However, current genotyping protocols in molecular diagnostic laboratories ac‐ knowledge the possibility of the pseudogene and do not amplify this region of the genome [87]. The first study evaluating the national clinical application of NIPD of fetal Rhesus D status conducted in Denmark, reported a sensitivity of 99.9 % and specificity of 96.5 % [88].

Fetal hemoglobin in maternal circulation was detected in 1956 indicating transplacental transmission of fetal erythrocytes [89]. Fetal cells were found in maternal blood during preg‐ nancy in 1958 [90]. Nucleated red blood cells have a relatively short lifespan in maternal blood but other cells can reside in maternal blood for decades after delivery and therefore cause false positive or negative test results in subsequent pregnancies [87, 91]. Other prob‐ lems besides the possibility of the presence of previous pregnancy include the rare number of fetal cells in maternal plasma, one cell per ml, and low efficiency of enrichment methods.

CffDNA, originating from the apoptotic trophoblasts derived from the embryo, was first de‐ tected in maternal circulation in 1997 [92, 93]. It has been shown that cffDNA is present in maternal circulation even before placental circulation has been established. It is present also in anembryonic gestations. Detected cffDNA sequences in maternal blood have been shown to reflect the placental genotype in cases of confined placental mosaicism [87]. Compared to intact fetal cells cffDNA has many advantages; it is almost a thousand times more present in maternal circulation than fetal cells, its mean half-life in maternal blood is approximately 16-30 minutes making it a marker of the current pregnancy [94, 95]. Even though the concen‐ tration of cffDNA in maternal blood is higher than that of the intact fetal cells, it is still low

**3.1. Non-Invasive Prenatal Diagnosis (NIPD)**

Besides the biomarkers mentioned above, also other maternal serum proteins have been shown to be more abundant in control versus Down syndrome pregnancies in both first and second trimester of the pregnancy [75]. Large scale prospective studies in low risk popula‐ tions evaluating the new maternal serum biomarkers need to be conducted before these markers could be implemented into the routine first trimester screening.

#### **2.5. Integrated screening and contingent screening**

In 1999 first trimester and second trimester screening were combined to create an integrated screening method which has been shown to achieve DRs around 85 %, 90 % and 94 % for FPRs of 1 %, 2 % and 5 %, respectively [76]. After first trimester combined screening is per‐ formed, no risk assessment is provided, instead, women return between gestational weeks 15 and 20 for measurements of serum quadruple markers. These screening methods are then combined with maternal age and an individual risk for Down syndrome is calculated. The advantage of integrated screening is its high sensitivity and specificity. However, first tri‐ mester screening results are withheld and the screening results are not available until the second trimester of the pregnancy. In the FaSTER trial, with a 5 % FPR, modeled DRs for integrated screening method were 96 %, 95 % and 94 % when the PAPP-A was measured during the gestational weeks 11, 12 or 13, respectively [9]. In the SURUSS study, integrated screening achieved a 93 % DR for a 5 % FPR. At an 85 % DR the FPR was 1.2 % [16].

Contingent screening policy was developed to reduce the number of NT measurements needed. This can be beneficial in the areas where there are no qualified personnel or highquality ultrasound machines available or where distances are long. Firstly, first trimester se‐ rum sample is analyzed for the levels of PAPP-A and fβ-hCG. Secondly, women are divided into three groups, women in low, intermediate and high risk for chromosomal abnormalities according to the serum markers. Women in low risk are offered no further screening. Wom‐ en in high risk are offered immediate invasive testing. NT screening is offered for those in intermediate risk and new risk calculation using first trimester serum markers and NT measurement is performed and invasive testing is offered for those in high risk. This meth‐ od has been estimated to achieve DRs of 67.6 % and 88.6 % for FPRs of 2.3 % and 6.4 %, re‐ spectively [77, 78]. Contingent screening might put women in unequal positions as first trimester combined screening is known to achieve higher screening performances. More‐ over, major structural abnormalities can be detected during the first trimester ultrasound scan [79, 80]. Also other variations of contingent screening including for example new sono‐ graphic markers have been developed.

## **3. Screening for Down syndrome in the future**

## **3.1. Non-Invasive Prenatal Diagnosis (NIPD)**

is 87.2 %, when AFP is replaced with uE3 the estimated DR is 87.9 % and for all the markers, 88.3 % for a 5 % FPR [20]. Inhibin A with combination of first trimester combined screening markers has been shown to achieve DRs of 81.4 % and 82.6 % at gestational weeks 7-8 and 9-11, respectively, for FPRs of 0.9 % and 1 % [5]. The studies on inhibin A have been contro‐ versial and some have found that inhibin A does not increase the screening performance in

Besides the biomarkers mentioned above, also other maternal serum proteins have been shown to be more abundant in control versus Down syndrome pregnancies in both first and second trimester of the pregnancy [75]. Large scale prospective studies in low risk popula‐ tions evaluating the new maternal serum biomarkers need to be conducted before these

In 1999 first trimester and second trimester screening were combined to create an integrated screening method which has been shown to achieve DRs around 85 %, 90 % and 94 % for FPRs of 1 %, 2 % and 5 %, respectively [76]. After first trimester combined screening is per‐ formed, no risk assessment is provided, instead, women return between gestational weeks 15 and 20 for measurements of serum quadruple markers. These screening methods are then combined with maternal age and an individual risk for Down syndrome is calculated. The advantage of integrated screening is its high sensitivity and specificity. However, first tri‐ mester screening results are withheld and the screening results are not available until the second trimester of the pregnancy. In the FaSTER trial, with a 5 % FPR, modeled DRs for integrated screening method were 96 %, 95 % and 94 % when the PAPP-A was measured during the gestational weeks 11, 12 or 13, respectively [9]. In the SURUSS study, integrated

screening achieved a 93 % DR for a 5 % FPR. At an 85 % DR the FPR was 1.2 % [16].

Contingent screening policy was developed to reduce the number of NT measurements needed. This can be beneficial in the areas where there are no qualified personnel or highquality ultrasound machines available or where distances are long. Firstly, first trimester se‐ rum sample is analyzed for the levels of PAPP-A and fβ-hCG. Secondly, women are divided into three groups, women in low, intermediate and high risk for chromosomal abnormalities according to the serum markers. Women in low risk are offered no further screening. Wom‐ en in high risk are offered immediate invasive testing. NT screening is offered for those in intermediate risk and new risk calculation using first trimester serum markers and NT measurement is performed and invasive testing is offered for those in high risk. This meth‐ od has been estimated to achieve DRs of 67.6 % and 88.6 % for FPRs of 2.3 % and 6.4 %, re‐ spectively [77, 78]. Contingent screening might put women in unequal positions as first trimester combined screening is known to achieve higher screening performances. More‐ over, major structural abnormalities can be detected during the first trimester ultrasound scan [79, 80]. Also other variations of contingent screening including for example new sono‐

markers could be implemented into the routine first trimester screening.

**2.5. Integrated screening and contingent screening**

graphic markers have been developed.

the first trimester [74].

44 Down Syndrome

One of the hottest topics in prenatal medicine today is the noninvasive prenatal diagnosis (NIPD). Since 1997 many approaches have been made in the field of NIPD and today it is possible to determine fetal sex, fetal Rhesus D status and diagnose genetic disorders or carri‐ er status for paternally inherited mutations [81]. Women in high risk of X-linked disorders like hemophilia can be offered noninvasive fetal sex determination. Y chromosome derived sequences can be found in maternal blood as early as eight weeks of gestation [82]. The de‐ tection of Y chromosome material indicates further investigations but if no evidence of de‐ tectable Y chromosome is found, unnecessary invasive testing with the risk of pregnancy loss, can be avoided. The costs of NIPD of fetal gender and invasive testing are similar [83, 84]. Y chromosome sequences can be detected with approximately 95.4 % sensitivity and 98.6 % specificity. Best test performance reported is for the real-time quantitative polymer‐ ase chain reaction (RTQ-PCR) after 20 weeks of gestation. Tests performed before seventh gestational week or using urine sample have been reported to be unreliable [85].

Detection of fetal rhesus D status can reduce the use of D immunoglobulin to prevent im‐ mune hemolytic disease of the newborn. The reported sensitivities and specificities for fetal Rhesus D sequence are greater than 95 % [86]. Reported false negative results are mainly due to a lack of fetal DNA in maternal blood sample due to too early gestation or insensitive methods. The presence of pseudogenes, mainly in African women, can lead to false positive results. However, current genotyping protocols in molecular diagnostic laboratories ac‐ knowledge the possibility of the pseudogene and do not amplify this region of the genome [87]. The first study evaluating the national clinical application of NIPD of fetal Rhesus D status conducted in Denmark, reported a sensitivity of 99.9 % and specificity of 96.5 % [88].

Fetal hemoglobin in maternal circulation was detected in 1956 indicating transplacental transmission of fetal erythrocytes [89]. Fetal cells were found in maternal blood during preg‐ nancy in 1958 [90]. Nucleated red blood cells have a relatively short lifespan in maternal blood but other cells can reside in maternal blood for decades after delivery and therefore cause false positive or negative test results in subsequent pregnancies [87, 91]. Other prob‐ lems besides the possibility of the presence of previous pregnancy include the rare number of fetal cells in maternal plasma, one cell per ml, and low efficiency of enrichment methods.

CffDNA, originating from the apoptotic trophoblasts derived from the embryo, was first de‐ tected in maternal circulation in 1997 [92, 93]. It has been shown that cffDNA is present in maternal circulation even before placental circulation has been established. It is present also in anembryonic gestations. Detected cffDNA sequences in maternal blood have been shown to reflect the placental genotype in cases of confined placental mosaicism [87]. Compared to intact fetal cells cffDNA has many advantages; it is almost a thousand times more present in maternal circulation than fetal cells, its mean half-life in maternal blood is approximately 16-30 minutes making it a marker of the current pregnancy [94, 95]. Even though the concen‐ tration of cffDNA in maternal blood is higher than that of the intact fetal cells, it is still low and it only comprises 3-6 % of the total cell-free DNA in maternal blood since the majority of cell-free DNA is of maternal origin. Also, half of the fetal genome is inherited from the mother and there are individual differences in the concentration of the total cffDNA [94, 96].

Another method used is the measurement of the total concentration of PLAC4 mRNA in maternal plasma is increased in Down syndrome pregnancies because of the extra gene copy in the placenta [112]. The mRNA quantification method can be used for pregnancies with homozygous fetuses. However, it is not yet known if there are other factors such as in‐ creased apoptosis in aneuploid placentas that might contribute to the increase of circulating PLAC4 mRNA in maternal plasma. The diagnostic accuracies of RNA-SNP approach, using blood samples from women carrying heterozygous fetuses for the PLAC4 mRNA, on the MS and digital-PCR platforms have identical sensitivities and specificities of 90-100 % and

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861 47

Also gene sequences present in neonatal and maternal whole blood have been studied [81, 87]. In amniotic fluid, abundant amounts of both cffRNA and cffDNA can be found and the present cell-free nucleic acids (cffNA) are nearly exclusively of fetal origin. Also, the cffNA appears to originate from fetal tissues that are either in direct contact with the amniotic fluid or drain into the amniotic fluid and there seems to be no NA derived from the placenta. In‐ tial studies on the molecular pathophysiology in the living fetus suggest that the majority of dysregulated gene espression in aneuploid fetuses occurs in genes present in other chromo‐ somes than the one involved in the chromosomal abnormality. Another finding is the oxida‐ tive stress in fetuses affected by Down syndrome which may result in the mental retardation and Alzheimer's disease [87]. After birth, analysis of cffNA from neonatal saliva can be used to monitor neonatal health and development. This offers comprehensive, real-time informa‐ tion regarding many organs and tissues which could allow the monitoring of premature ne‐

The reported data indicates that highly accurate NIPD of chromosomal abnormalities by maternal blood sample is achievable during the first trimester of the pregnancy. However, the gestational window of NIPD is still to be researched. Although studies have reported high sensitivities and specificities, approximately 1 % FPRs have been reported. Therefore, at the moment, invasive testing is still required after positive test result and the method might be more incisively regarded as an "advanced screening test" rather than a diagnostic test and pregnancy termination should not be offered only based on a positive NIPD test. However, it has been estimated that 98 % of the invasive procedures could be avoided if AC or CVS were based on the MPS test results [101]. Most studies to date have been small and conducted in high risk women. Large-scale objective clinical trials are needed to evaluate the sensitivity and specificity of NIPD in low risk general populations. The future costs of NIPD can be only estimated and are dependent on the relative costs of NIPD, Down syndrome

NIPD of fetal Rhesus D genotype has been widely validated in Europe but it is slower been undertaken in United States of America. It is anticipated that besides fetal sex determination and Rhesus D detection, over the next few years also the NIPD of fetal aneuploidy will be possible and NIPD will be refined to include also other trisomies than trisomy 21. However, it may take longer to develop proper techniques to detect other pathogenic rearrangements. Ultrasound scan during the early pregnancy will be necessary even if NIPD would become a routine screening method. Increased levels of cffNA in maternal blood have been associated,

89.7-96.5 %, respectively [100, 112].

onates in terms of health, disease and development [113].

screening and number of invasive tests that are performed.

The newest strategy for noninvasive prenatal gene profiling is the maternal blood analysis of fetal mRNA. Discovery of fetal placenta-specific expressed mRNAs in the maternal serum and plasma was made in 2000 [97]. Fetal mRNA molecules have been shown to be easily de‐ tectable since they are very stable in maternal blood probably due to the association with particulate matters [99]. Numerous pregnancy-specific, fetal-specific mRNA transcripts that are independent from fetal gender and fetal genetic polymorphisms have been identified in maternal circulation [99, 100]. Studied noninvasive prenatal screening mRNA markers in‐ clude for example placenta-specific 4 (PLAC4) which is cleared rapidly after delivery and has been reported to have a 90 % DR for a 3.5 % FPR for Down syndrome [100].

## *3.1.1. Current state of art in NIPD*

Various methods for NIPD using cffNA in maternal circulation have been introduced. Mas‐ sively parallel sequencing (MPS) of fetal DNA has high sensitivity and specificity for the de‐ tection of trisomy 21. The reported sensitivities range between 79.1 % and 100 % and specificities between 97.9 % and 100 %, respectively [101-104]. Similar sensitivities and spe‐ cificities for trisomies 21 and 18 have been reported for targeted MPS method and for triso‐ my 21 with differential methylation and real-time multiplex ligation-dependent probe amplification (RT-MLPA). One study achieved a 100 % sensitivity and specificity for trisomy 21 by a targeted approach that was based on calculation of haplotype ratios from tandem single nucleotide polymorphisms (SNP) sequences on chromosome 21 combined with a quantitative DNA measurement technology [105].

The use of MPS as the screening strategy has been reported to achieve sensitivities of 91.9-100 % and 100 % with specificities of 98.9-100 % and 98.4-100 % for trisomy 18 and tris‐ omy 13, respectively [106-108]. MPS combined with improved z-score test methodology, was reported to achieve 100 % DR with a 0 % FPR for Down syndrome, trisomy 18, trisomy 13, Turner syndrome and Klinefelter syndrome [109]. High troughput DNA sequencing has many advantages as the entire process can be automated and multiple samples be analyzed simultaneously so that thousands of sequencing reactions can occur in parallel as the test DNA is bound to a solid support such as an array.

One method called the RNA-SNP approach measures the ratio of alleles for a SNP in placen‐ ta-derived mRNA molecules in maternal plasma [100]. PLAC4 mRNA has been used for this method [110]. The RNA-SNP method detects the deviated RNA-SNP allelic ratio on PLAC4 mRNA which is caused by the imbalance in chromosome 21 dosage. The RNA-SNP strategy is only suitable to women with a fetus heterozygous for the studied SNP in the PLAC4 gene. Method can be based on a mass spectrometry (MS) method or digital-PCR which enhances the precision [100, 111]. Digital-PCR method is more costly but it can be used in analysis of plasma samples with low concentration of PLAC4 mRNA such in early pregnancy samples.

Another method used is the measurement of the total concentration of PLAC4 mRNA in maternal plasma is increased in Down syndrome pregnancies because of the extra gene copy in the placenta [112]. The mRNA quantification method can be used for pregnancies with homozygous fetuses. However, it is not yet known if there are other factors such as in‐ creased apoptosis in aneuploid placentas that might contribute to the increase of circulating PLAC4 mRNA in maternal plasma. The diagnostic accuracies of RNA-SNP approach, using blood samples from women carrying heterozygous fetuses for the PLAC4 mRNA, on the MS and digital-PCR platforms have identical sensitivities and specificities of 90-100 % and 89.7-96.5 %, respectively [100, 112].

and it only comprises 3-6 % of the total cell-free DNA in maternal blood since the majority of cell-free DNA is of maternal origin. Also, half of the fetal genome is inherited from the mother and there are individual differences in the concentration of the total cffDNA [94, 96].

The newest strategy for noninvasive prenatal gene profiling is the maternal blood analysis of fetal mRNA. Discovery of fetal placenta-specific expressed mRNAs in the maternal serum and plasma was made in 2000 [97]. Fetal mRNA molecules have been shown to be easily de‐ tectable since they are very stable in maternal blood probably due to the association with particulate matters [99]. Numerous pregnancy-specific, fetal-specific mRNA transcripts that are independent from fetal gender and fetal genetic polymorphisms have been identified in maternal circulation [99, 100]. Studied noninvasive prenatal screening mRNA markers in‐ clude for example placenta-specific 4 (PLAC4) which is cleared rapidly after delivery and

Various methods for NIPD using cffNA in maternal circulation have been introduced. Mas‐ sively parallel sequencing (MPS) of fetal DNA has high sensitivity and specificity for the de‐ tection of trisomy 21. The reported sensitivities range between 79.1 % and 100 % and specificities between 97.9 % and 100 %, respectively [101-104]. Similar sensitivities and spe‐ cificities for trisomies 21 and 18 have been reported for targeted MPS method and for triso‐ my 21 with differential methylation and real-time multiplex ligation-dependent probe amplification (RT-MLPA). One study achieved a 100 % sensitivity and specificity for trisomy 21 by a targeted approach that was based on calculation of haplotype ratios from tandem single nucleotide polymorphisms (SNP) sequences on chromosome 21 combined with a

The use of MPS as the screening strategy has been reported to achieve sensitivities of 91.9-100 % and 100 % with specificities of 98.9-100 % and 98.4-100 % for trisomy 18 and tris‐ omy 13, respectively [106-108]. MPS combined with improved z-score test methodology, was reported to achieve 100 % DR with a 0 % FPR for Down syndrome, trisomy 18, trisomy 13, Turner syndrome and Klinefelter syndrome [109]. High troughput DNA sequencing has many advantages as the entire process can be automated and multiple samples be analyzed simultaneously so that thousands of sequencing reactions can occur in parallel as the test

One method called the RNA-SNP approach measures the ratio of alleles for a SNP in placen‐ ta-derived mRNA molecules in maternal plasma [100]. PLAC4 mRNA has been used for this method [110]. The RNA-SNP method detects the deviated RNA-SNP allelic ratio on PLAC4 mRNA which is caused by the imbalance in chromosome 21 dosage. The RNA-SNP strategy is only suitable to women with a fetus heterozygous for the studied SNP in the PLAC4 gene. Method can be based on a mass spectrometry (MS) method or digital-PCR which enhances the precision [100, 111]. Digital-PCR method is more costly but it can be used in analysis of plasma samples with low concentration of PLAC4 mRNA such in early pregnancy samples.

has been reported to have a 90 % DR for a 3.5 % FPR for Down syndrome [100].

*3.1.1. Current state of art in NIPD*

46 Down Syndrome

quantitative DNA measurement technology [105].

DNA is bound to a solid support such as an array.

Also gene sequences present in neonatal and maternal whole blood have been studied [81, 87]. In amniotic fluid, abundant amounts of both cffRNA and cffDNA can be found and the present cell-free nucleic acids (cffNA) are nearly exclusively of fetal origin. Also, the cffNA appears to originate from fetal tissues that are either in direct contact with the amniotic fluid or drain into the amniotic fluid and there seems to be no NA derived from the placenta. In‐ tial studies on the molecular pathophysiology in the living fetus suggest that the majority of dysregulated gene espression in aneuploid fetuses occurs in genes present in other chromo‐ somes than the one involved in the chromosomal abnormality. Another finding is the oxida‐ tive stress in fetuses affected by Down syndrome which may result in the mental retardation and Alzheimer's disease [87]. After birth, analysis of cffNA from neonatal saliva can be used to monitor neonatal health and development. This offers comprehensive, real-time informa‐ tion regarding many organs and tissues which could allow the monitoring of premature ne‐ onates in terms of health, disease and development [113].

The reported data indicates that highly accurate NIPD of chromosomal abnormalities by maternal blood sample is achievable during the first trimester of the pregnancy. However, the gestational window of NIPD is still to be researched. Although studies have reported high sensitivities and specificities, approximately 1 % FPRs have been reported. Therefore, at the moment, invasive testing is still required after positive test result and the method might be more incisively regarded as an "advanced screening test" rather than a diagnostic test and pregnancy termination should not be offered only based on a positive NIPD test. However, it has been estimated that 98 % of the invasive procedures could be avoided if AC or CVS were based on the MPS test results [101]. Most studies to date have been small and conducted in high risk women. Large-scale objective clinical trials are needed to evaluate the sensitivity and specificity of NIPD in low risk general populations. The future costs of NIPD can be only estimated and are dependent on the relative costs of NIPD, Down syndrome screening and number of invasive tests that are performed.

NIPD of fetal Rhesus D genotype has been widely validated in Europe but it is slower been undertaken in United States of America. It is anticipated that besides fetal sex determination and Rhesus D detection, over the next few years also the NIPD of fetal aneuploidy will be possible and NIPD will be refined to include also other trisomies than trisomy 21. However, it may take longer to develop proper techniques to detect other pathogenic rearrangements. Ultrasound scan during the early pregnancy will be necessary even if NIPD would become a routine screening method. Increased levels of cffNA in maternal blood have been associated, besides chromosomal abnormalities, with various pathological conditions like pre-eclamp‐ sia, hemolytic anemia, elevated liver enzymes, low platelets syndrome and placental abnor‐ malities like placenta accrete [87, 114].

FPR. When specific algorithm for trisomy 18 is used, the DR for trisomy 18 is reported to be 74.0 - 88 % with a slight increase of 0.1 % in the FPR. Using the specific algorithm for triso‐ my 13 improves the DR for trisomy 13 to approximately 54.5 - 73 % for an additional 0.1 %

Prenatal Screening and Diagnosis http://dx.doi.org/10.5772/52861 49

Adverse pregnancy outcomes like pregnancy loss, hypertension, preeclampsia, eclampsia, preterm delivery, small for gestational age newborns and fetal death cannot yet be predicted in the early pregnancy. Closer surveillance and possible new treatments could be studied on women in high risk to avoid the adverse pregnancy outcomes in the future. As well as in‐ creased NT measurements, also abnormal levels of maternal serum biochemical markers

Participating in the screening for chromosomal abnormalities and the diagnostic testing is voluntary. Women have an opportunity to retrieve screening at any point. It is essential that women make an informed decision when they decide to participate in the screening. When a positive screening result is received, detailed and objective counseling should be offered about the condition at issue and about the procedure and its risks. Health professionals' per‐ sonal opinions should not affect the woman's decision. However, it is known that the many issues like the age, level of medical knowledge, opinion about the screening test, specialty and attitudes towards the patients affect the counseling. Due to the complexity of the screen‐ ing, women need to assimilate a lot of information which might not always be successful. If the possibility of a chromosomal abnormality is introduced for the first time when the screening is offered, worry can be caused. The possibility to terminate the pregnancy after a chromosomal abnormality is detected raises many ethical issues about the right of the disa‐ bled to be born regardless of their disability. Screening is also thought to be insulting to‐ wards people with a chromosomal or a structural anomaly. Screening does not produce diagnoses, only risks for chromosomal abnormalities. The limitations of the screening should be told for the women participating in the screening. One redeeming feature of the screening is that it provides a great deal of knowledge about chromosomal and structural

increase in the FPR [116, 117].

have been associated with pregnancy complications.

abnormalities equally for every screened woman.

**6. Screening in multiple pregnancies and in ART pregnancies**

Screening in multiple pregnancies is more difficult than in singleton pregnancies. Firstly, maternal serum biochemistry is less effective in multiple pregnancies since placental analy‐ tes from normal fetus/fetuses can mask abnormal levels in the affected fetus. Moreover, ab‐ normal levels of maternal serum biochemical markers cannot distinguish which fetus is the affected one [118]. Secondly, second trimester ultrasound examination is more challenging because of the limitations due to the positions of the fetuses and interposition of fetal parts.

**5. Ethical aspects of the first trimester screening**

#### *3.1.2. Ethics in NIPD*

NIPD has many benefits as definitive diagnoses can be made earlier in the pregnancy when termination of an affected pregnancy is safer, parental anxiety is reduced and costs are de‐ creased. As testing becomes safer the uptake will probably increase and thus additional health and economic benefits can be reached. However, NIPD also raises many ethical is‐ sues. Counseling needs to be informative so that women could make the decision fully aware of the consequences of possible findings. At the moment, counseling is offered for ev‐ ery woman but only those who have received a positive screening result are offered more detailed information about Down syndrome as they are offered invasive testing. The nature of NIPD, however, is closer to invasive diagnosis than screening. Therefore, all women should be comprehensively counseled before the testing. This probably requires much more genetic counselors than are currently available.

In recent years, private sector has been funding research around NIPD. This might lead to expensive testing. Until now, Down syndrome screening has had a minimal effect on birth incidence of genetic disorders. As testing becomes safer and more accurate than before more affected pregnancies may be found and possibly terminated. This might affect the public at‐ titudes towards affected individuals and their families. Women might feel more pressured by the society to test and terminate affected pregnancies. Also commercial and insurance sectors might perceive economic benefits in decreasing the prevalence of disorders. As the technology develops, also less severe disorders, late-onset disorders, nonmedical traits and predispositions can be detected prenatally. Codes of practice should be developed as well as regulatory recommendations made [158]. In United States of America, several professional organizations have stated that noninvasive fetal gender determination should only be of‐ fered for medical indications. However, via the internet the test is available directly to the consumers and the technology might also be used for fetal sex selection.

Women seem to feel positive about the new improvements in the screening field. However, they find it hard to fully realize the new choices and consequences that will follow with NIPD [115]. Among the healthcare providers there seems to be a lack of knowledge or con‐ viction about using NIPD. Healthcare providers hold genetic counseling and professional society approval important and they are more willing to offer cffDNA testing for chromoso‐ mal abnormalities and single-gene disorders than determination of sex and behavioral or late-onset conditions. Standards of care and professional guidelines are necessary.

## **4. Other implications for combined Down syndrome screening method**

Using the algorithm for Down syndrome, combined screening detects approximately 55.6 % of trisomy 18 cases, 36.4 % of trisomy 13 cases and 60 % of other aneuploidies for a 4.3 % FPR. When specific algorithm for trisomy 18 is used, the DR for trisomy 18 is reported to be 74.0 - 88 % with a slight increase of 0.1 % in the FPR. Using the specific algorithm for triso‐ my 13 improves the DR for trisomy 13 to approximately 54.5 - 73 % for an additional 0.1 % increase in the FPR [116, 117].

Adverse pregnancy outcomes like pregnancy loss, hypertension, preeclampsia, eclampsia, preterm delivery, small for gestational age newborns and fetal death cannot yet be predicted in the early pregnancy. Closer surveillance and possible new treatments could be studied on women in high risk to avoid the adverse pregnancy outcomes in the future. As well as in‐ creased NT measurements, also abnormal levels of maternal serum biochemical markers have been associated with pregnancy complications.

## **5. Ethical aspects of the first trimester screening**

besides chromosomal abnormalities, with various pathological conditions like pre-eclamp‐ sia, hemolytic anemia, elevated liver enzymes, low platelets syndrome and placental abnor‐

NIPD has many benefits as definitive diagnoses can be made earlier in the pregnancy when termination of an affected pregnancy is safer, parental anxiety is reduced and costs are de‐ creased. As testing becomes safer the uptake will probably increase and thus additional health and economic benefits can be reached. However, NIPD also raises many ethical is‐ sues. Counseling needs to be informative so that women could make the decision fully aware of the consequences of possible findings. At the moment, counseling is offered for ev‐ ery woman but only those who have received a positive screening result are offered more detailed information about Down syndrome as they are offered invasive testing. The nature of NIPD, however, is closer to invasive diagnosis than screening. Therefore, all women should be comprehensively counseled before the testing. This probably requires much more

In recent years, private sector has been funding research around NIPD. This might lead to expensive testing. Until now, Down syndrome screening has had a minimal effect on birth incidence of genetic disorders. As testing becomes safer and more accurate than before more affected pregnancies may be found and possibly terminated. This might affect the public at‐ titudes towards affected individuals and their families. Women might feel more pressured by the society to test and terminate affected pregnancies. Also commercial and insurance sectors might perceive economic benefits in decreasing the prevalence of disorders. As the technology develops, also less severe disorders, late-onset disorders, nonmedical traits and predispositions can be detected prenatally. Codes of practice should be developed as well as regulatory recommendations made [158]. In United States of America, several professional organizations have stated that noninvasive fetal gender determination should only be of‐ fered for medical indications. However, via the internet the test is available directly to the

Women seem to feel positive about the new improvements in the screening field. However, they find it hard to fully realize the new choices and consequences that will follow with NIPD [115]. Among the healthcare providers there seems to be a lack of knowledge or con‐ viction about using NIPD. Healthcare providers hold genetic counseling and professional society approval important and they are more willing to offer cffDNA testing for chromoso‐ mal abnormalities and single-gene disorders than determination of sex and behavioral or

late-onset conditions. Standards of care and professional guidelines are necessary.

**4. Other implications for combined Down syndrome screening method**

Using the algorithm for Down syndrome, combined screening detects approximately 55.6 % of trisomy 18 cases, 36.4 % of trisomy 13 cases and 60 % of other aneuploidies for a 4.3 %

consumers and the technology might also be used for fetal sex selection.

malities like placenta accrete [87, 114].

genetic counselors than are currently available.

*3.1.2. Ethics in NIPD*

48 Down Syndrome

Participating in the screening for chromosomal abnormalities and the diagnostic testing is voluntary. Women have an opportunity to retrieve screening at any point. It is essential that women make an informed decision when they decide to participate in the screening. When a positive screening result is received, detailed and objective counseling should be offered about the condition at issue and about the procedure and its risks. Health professionals' per‐ sonal opinions should not affect the woman's decision. However, it is known that the many issues like the age, level of medical knowledge, opinion about the screening test, specialty and attitudes towards the patients affect the counseling. Due to the complexity of the screen‐ ing, women need to assimilate a lot of information which might not always be successful. If the possibility of a chromosomal abnormality is introduced for the first time when the screening is offered, worry can be caused. The possibility to terminate the pregnancy after a chromosomal abnormality is detected raises many ethical issues about the right of the disa‐ bled to be born regardless of their disability. Screening is also thought to be insulting to‐ wards people with a chromosomal or a structural anomaly. Screening does not produce diagnoses, only risks for chromosomal abnormalities. The limitations of the screening should be told for the women participating in the screening. One redeeming feature of the screening is that it provides a great deal of knowledge about chromosomal and structural abnormalities equally for every screened woman.

## **6. Screening in multiple pregnancies and in ART pregnancies**

Screening in multiple pregnancies is more difficult than in singleton pregnancies. Firstly, maternal serum biochemistry is less effective in multiple pregnancies since placental analy‐ tes from normal fetus/fetuses can mask abnormal levels in the affected fetus. Moreover, ab‐ normal levels of maternal serum biochemical markers cannot distinguish which fetus is the affected one [118]. Secondly, second trimester ultrasound examination is more challenging because of the limitations due to the positions of the fetuses and interposition of fetal parts. Nuchal translucency measurement together with maternal age, however, has been shown to be an effective screening method in multiple pregnancies. The DR is comparable to that in singleton pregnancies for a slightly higher FPR. Also, determination of fetus-specific risk is possible with this technique. The limitation of ultrasound in twins is that they can be influ‐ enced by hemodynamic imbalance between the twins' circulation. Other possible screening markers in multiple pregnancies are DV flow and NB [119-121].

**Author details**

Department of Obstetrics and Gynecology, Oulu, Finland

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[7] Nicolaides KH, Azar G, Byrne D, Mansur C, Marks K. Fetal nuchal translucency: Ul‐ trasound screening for chromosomal defects in first trimester of pregnancy. BMJ.

[8] Ville Y, Lalondrelle C, Doumerc S, Daffos F, Frydman R, Oury JF, et al. First-trimes‐ ter diagnosis of nuchal anomalies: Significance and fetal outcome. Ultrasound Obstet

[9] Malone FD, Canick JA, Ball RH, Nyberg DA, Comstock CH, Bukowski R, et al. Firstand Second-Trimester Evaluation of Risk (FASTER) Research Consortium. First-tri‐ mester or second-trimester screening, or both, for down's syndrome. N Engl J Med.

[10] de Graaf IM, Tijmstra T, Bleker OP, van Lith JM. Womens' preference in down syn‐

down's syndrome early in the first trimester. Clin Genet. 2005; 68: 35-9.

Jaana Marttala

**References**

Screening in pregnancies conceived using assisted reproductive technologies (ART) has been studied by different research groups and contradictory results have been reported. In some studies fβ-hCG and NT have been enlarged in ART pregnancies and PAPP-A levels decreased, while others have reported no significant differences in these markers. It seems that decreased PAPP-A levels in ART pregnancies is the most discriminating factor leading to increased FPR in these pregnancies. However, some have reported no significant differ‐ ence in FPR in ART pregnancies compared with spontaneous pregnancies [191].

## **7. Cost-effectiveness of the screening and international differences in screening strategies**

The demands for the prenatal screening performance are high. Also, the cost-effectiveness of the screening should be good. There are some estimations about the screening costs in differ‐ ent countries but overall, the cost and patient acceptability of the alternative policies of screen‐ ing tests depend on the existing infrastructure of antenatal care, which varies between different countries and centers. Screening and diagnostic tests for chromosomal abnormali‐ ties have been developed and been available for several decades and the research for new strategies is ongoing. National committees review available evidence and national screening statistics and each country adopts testing modalities in its own way. In dissimilar healthcare systems guidelines for best practice evolve different ways. There are differences in what tests are offered, insurance coverage, counseling and the national legal situation for terminating an affected pregnancy. Global knowledge about testing practices gets more and more important for the counselors as people immigrate between the countries and into different cultures. In Europe, almost 90 % of couples who receive a prenatal diagnosis of Down syndrome decide to terminate the pregnancy. However, the legal situation concerning pregnancy termination dif‐ fers between countries [123]. Most couples that feel that they would continue the pregnancy even though the fetus would be diagnosed with a chromosomal abnormality do not partici‐ pate in the screening program [124]. There are significant differences in screening modalities between for example United Kingdom and the United States of America despite many simi‐ larities between the countries [125]. The introduction of prenatal screening has, however, led to a reduction in live-births of Down syndrome cases internationally.

## **Author details**

Jaana Marttala

Nuchal translucency measurement together with maternal age, however, has been shown to be an effective screening method in multiple pregnancies. The DR is comparable to that in singleton pregnancies for a slightly higher FPR. Also, determination of fetus-specific risk is possible with this technique. The limitation of ultrasound in twins is that they can be influ‐ enced by hemodynamic imbalance between the twins' circulation. Other possible screening

Screening in pregnancies conceived using assisted reproductive technologies (ART) has been studied by different research groups and contradictory results have been reported. In some studies fβ-hCG and NT have been enlarged in ART pregnancies and PAPP-A levels decreased, while others have reported no significant differences in these markers. It seems that decreased PAPP-A levels in ART pregnancies is the most discriminating factor leading to increased FPR in these pregnancies. However, some have reported no significant differ‐

ence in FPR in ART pregnancies compared with spontaneous pregnancies [191].

to a reduction in live-births of Down syndrome cases internationally.

**7. Cost-effectiveness of the screening and international differences in**

The demands for the prenatal screening performance are high. Also, the cost-effectiveness of the screening should be good. There are some estimations about the screening costs in differ‐ ent countries but overall, the cost and patient acceptability of the alternative policies of screen‐ ing tests depend on the existing infrastructure of antenatal care, which varies between different countries and centers. Screening and diagnostic tests for chromosomal abnormali‐ ties have been developed and been available for several decades and the research for new strategies is ongoing. National committees review available evidence and national screening statistics and each country adopts testing modalities in its own way. In dissimilar healthcare systems guidelines for best practice evolve different ways. There are differences in what tests are offered, insurance coverage, counseling and the national legal situation for terminating an affected pregnancy. Global knowledge about testing practices gets more and more important for the counselors as people immigrate between the countries and into different cultures. In Europe, almost 90 % of couples who receive a prenatal diagnosis of Down syndrome decide to terminate the pregnancy. However, the legal situation concerning pregnancy termination dif‐ fers between countries [123]. Most couples that feel that they would continue the pregnancy even though the fetus would be diagnosed with a chromosomal abnormality do not partici‐ pate in the screening program [124]. There are significant differences in screening modalities between for example United Kingdom and the United States of America despite many simi‐ larities between the countries [125]. The introduction of prenatal screening has, however, led

markers in multiple pregnancies are DV flow and NB [119-121].

**screening strategies**

50 Down Syndrome

Department of Obstetrics and Gynecology, Oulu, Finland

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[88] Clausen FB, Christiansen M, Steffensen R, Jorgensen S, Nielsen C, Jakobsen MA, et al. Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis.

[89] Bromberg YM, Salzberger M, Abrahamov A. Transplacental transmission of fetal er‐ ythrocytes with demonstration of fetal hemoglobin in maternal circulation. Obstet

[90] Weiner W, Child RM, Garvie JM, Peek WH. Foetal cells in the maternal circulation

[91] Lurie S, Mamet Y. Red blood cell survival and kinetics during pregnancy. Eur J Ob‐

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**Section 2**

**Diseases in Children with Down Syndrome**

**Diseases in Children with Down Syndrome**

**Chapter 4**

**Control of Dental Biofilm**

http://dx.doi.org/10.5772/53348

**1. Introduction**

cases [2], [3].

capacity of saliva [4].

**and Oral Health Maintenance**

**in Patients with Down Syndrome**

Additional information is available at the end of the chapter

Ana Paula Teitelbaum and Gislaine Denise Czlusniak

The greatest prophylaxis challenge in dentistry is the control of dental biofilm and conse‐ quently, avoiding dental caries and gingival inflammation [1]. This control is generally car‐ ried out through mechanical and ⁄ or chemical methods. Although the mechanical methods (toothbrush and dental floss) are considered efficient, they are not sufficiently so in certain

Individuals with Down syndrome (DS) present various oral diseases, such as the presence of pseudoprognatismo, hard palate and lower ogival shape; pseudomacroglossia due to hypo‐ tonia tongue; high prevalence and susceptibility to periodontal problems due to error in the autoregulatory mechanism immune, and poor occlusal relationship, with a predominance of anterior crossbite and / or later. The position of the tongue protruded, produces abnormal strength in the lower anterior teeth, which normally are in a position to cross-bite. These fac‐ tors favor the onset of severe periodontitis, leading to early loss of teeth. However, there is a lower incidence of dental caries, which has been attributed mainly to the increase in buffer

Some dental anomalies can be observed, as the presence of hypodontia or oligodontia, tooth conoids, microteeth, hypocalcification enamel, fusion and twinning can also be an increase in the size of the clinical crown of molars and the inclination of the occlusal surface to the lingual, making access to restorative procedures. Furthermore, the rash and exfoliation of the primary teeth and eruption of the permanent are delayed, and there is a high prevalence

and reproduction in any medium, provided the original work is properly cited.

© 2013 Teitelbaum and Czlusniak; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

of bruxism [4], and these alterations interfere with the quality of toothbrushing.

## **Control of Dental Biofilm and Oral Health Maintenance in Patients with Down Syndrome**

Ana Paula Teitelbaum and Gislaine Denise Czlusniak

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53348

## **1. Introduction**

The greatest prophylaxis challenge in dentistry is the control of dental biofilm and conse‐ quently, avoiding dental caries and gingival inflammation [1]. This control is generally car‐ ried out through mechanical and ⁄ or chemical methods. Although the mechanical methods (toothbrush and dental floss) are considered efficient, they are not sufficiently so in certain cases [2], [3].

Individuals with Down syndrome (DS) present various oral diseases, such as the presence of pseudoprognatismo, hard palate and lower ogival shape; pseudomacroglossia due to hypo‐ tonia tongue; high prevalence and susceptibility to periodontal problems due to error in the autoregulatory mechanism immune, and poor occlusal relationship, with a predominance of anterior crossbite and / or later. The position of the tongue protruded, produces abnormal strength in the lower anterior teeth, which normally are in a position to cross-bite. These fac‐ tors favor the onset of severe periodontitis, leading to early loss of teeth. However, there is a lower incidence of dental caries, which has been attributed mainly to the increase in buffer capacity of saliva [4].

Some dental anomalies can be observed, as the presence of hypodontia or oligodontia, tooth conoids, microteeth, hypocalcification enamel, fusion and twinning can also be an increase in the size of the clinical crown of molars and the inclination of the occlusal surface to the lingual, making access to restorative procedures. Furthermore, the rash and exfoliation of the primary teeth and eruption of the permanent are delayed, and there is a high prevalence of bruxism [4], and these alterations interfere with the quality of toothbrushing.

© 2013 Teitelbaum and Czlusniak; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

decisively etiologic agents in the origin and development of caries and periodontal also (Kö‐ nig et al. [9] 2002). In 1965, Löe et al. [10] demonstrated the direct relationship between the biofilm and the development of gingivitis in humans, concluding that the removal of biofilm employing brushing and flossing, could result in reversal in health (Löe et al. [10] 1965, Theilade et al. [11] 1966). For this reason, control of the biofilm has an important role in the

Control of Dental Biofilm and Oral Health Maintenance in Patients with Down Syndrome

http://dx.doi.org/10.5772/53348

67

The mechanical control is to remove biofilm employing proper technique of brushing, com‐ bined with a dentifrice and auxiliary materials such as wire or dental tape (Owens et al. [12]

The ability to remove dental biofilm by the use of different types of brushes is basically the same. There is no ideal brush, and your choice should be guided by the needs of each indi‐ vidual patient and clinical observations of the professional. However, there are characteris‐ tics that facilitate the oral hygiene procedures, as the presence of small head multitufuladas,

The toothbrushing is an effective procedure for the maintenance of proper oral hygiene. However, to get a good cleaning of the oral cavity, in addition to toothbrushes, other factors must be considered such as time, frequency, brushing technique, manual skills and motiva‐

Several authors report that, although brushing is the most widespread and universally suit‐ able for the mechanical removal of the plate are not known techniques ideal nor brushes which, by itself, may promote a perfect cleaning. All this technical device should be associat‐

prevention, treatment and maintenance of periodontal health.

soft bristle, rounded second study by Panzeri et al. [13] (1993).

**2. Mechanical control of dental biofilm**

tion of patients (Halla [14] 1982).

**Figure 2.** Motivation of patients and toothbrushing

ed with constant motivation [12],[15]-[16].

1997).

**Figure 1.** Pseudomacroglossia due to hypotonia tongue

There is agreement among many authors on the existence of factors predisposing to perio‐ dontal disease in patients with Down syndrome. Although poor oral hygiene, poor nutrition and local irritants may exacerbate this problem, they can not be regarded as its main cause. The greater predisposition to periodontal disease has been attributed to characteristics of pa‐ tients with chromosomal abnormalities of trisomy [5]. It is therefore essential to establish strategies to p revent periodontal disease in these individuals.

Second Cornejo et al. [6] (1996), which conducted a study in 86 individuals with DS living in Argentina, aged between 3 and 19 years, the presence of the changes described above puts them at a disadvantage in relation to oral health, compared with noncarriers.

Besides the inherent disadvantage to the individual, access to dental care is also difficult for these people. Allison et al. [7] (2000), in a study conducted in France, compared the levels of care received dental services and oral hygiene habits among children with DS and their sib‐ lings. According to parents and / or guardians, the group with DS had difficulty finding ac‐ cess to dental services and oral care compared to their phenotypically normal siblings. In Brazil, studying the prevalence of dental caries in primary and permanent teeth of children with DS in Sao Jose dos Campos (SP), Moraes et al. [8] (2002) found that the values of ceo and CPO-D were similar to those identified by the Municipal Health Department, in a sur‐ vey of dental caries in children from public schools. However, the authors found a frequen‐ cy of 9.25% and 4.76% decayed teeth restored among the children examined, against the values of 3.98% and 5.88%, respectively, obtained by the Municipal Health.

All these mentioned aspects can be inferred that it would be essential to adopt appropriate measures aimed at controlling biofilm among the DS patients, to prevent the installation of dental caries and gingival inflammation, because the microorganisms in the biofilm and act decisively etiologic agents in the origin and development of caries and periodontal also (Kö‐ nig et al. [9] 2002). In 1965, Löe et al. [10] demonstrated the direct relationship between the biofilm and the development of gingivitis in humans, concluding that the removal of biofilm employing brushing and flossing, could result in reversal in health (Löe et al. [10] 1965, Theilade et al. [11] 1966). For this reason, control of the biofilm has an important role in the prevention, treatment and maintenance of periodontal health.

## **2. Mechanical control of dental biofilm**

**Figure 1.** Pseudomacroglossia due to hypotonia tongue

66 Down Syndrome

tients with chromosomal abnormalities of trisomy [5].

strategies to p revent periodontal disease in these individuals.

There is agreement among many authors on the existence of factors predisposing to perio‐ dontal disease in patients with Down syndrome. Although poor oral hygiene, poor nutrition and local irritants may exacerbate this problem, they can not be regarded as its main cause. The greater predisposition to periodontal disease has been attributed to characteristics of pa‐

Second Cornejo et al. [6] (1996), which conducted a study in 86 individuals with DS living in Argentina, aged between 3 and 19 years, the presence of the changes described above puts

Besides the inherent disadvantage to the individual, access to dental care is also difficult for these people. Allison et al. [7] (2000), in a study conducted in France, compared the levels of care received dental services and oral hygiene habits among children with DS and their sib‐ lings. According to parents and / or guardians, the group with DS had difficulty finding ac‐ cess to dental services and oral care compared to their phenotypically normal siblings. In Brazil, studying the prevalence of dental caries in primary and permanent teeth of children with DS in Sao Jose dos Campos (SP), Moraes et al. [8] (2002) found that the values of ceo and CPO-D were similar to those identified by the Municipal Health Department, in a sur‐ vey of dental caries in children from public schools. However, the authors found a frequen‐ cy of 9.25% and 4.76% decayed teeth restored among the children examined, against the

All these mentioned aspects can be inferred that it would be essential to adopt appropriate measures aimed at controlling biofilm among the DS patients, to prevent the installation of dental caries and gingival inflammation, because the microorganisms in the biofilm and act

them at a disadvantage in relation to oral health, compared with noncarriers.

values of 3.98% and 5.88%, respectively, obtained by the Municipal Health.

It is therefore essential to establish

The mechanical control is to remove biofilm employing proper technique of brushing, com‐ bined with a dentifrice and auxiliary materials such as wire or dental tape (Owens et al. [12] 1997).

The ability to remove dental biofilm by the use of different types of brushes is basically the same. There is no ideal brush, and your choice should be guided by the needs of each indi‐ vidual patient and clinical observations of the professional. However, there are characteris‐ tics that facilitate the oral hygiene procedures, as the presence of small head multitufuladas, soft bristle, rounded second study by Panzeri et al. [13] (1993).

The toothbrushing is an effective procedure for the maintenance of proper oral hygiene. However, to get a good cleaning of the oral cavity, in addition to toothbrushes, other factors must be considered such as time, frequency, brushing technique, manual skills and motiva‐ tion of patients (Halla [14] 1982).

**Figure 2.** Motivation of patients and toothbrushing

Several authors report that, although brushing is the most widespread and universally suit‐ able for the mechanical removal of the plate are not known techniques ideal nor brushes which, by itself, may promote a perfect cleaning. All this technical device should be associat‐ ed with constant motivation [12],[15]-[16].

**Figure 5.** Constant guidance and motivation

tions (Medeiros [28]1991).

tiano, Bignelli [30]1995).

Second Nielsen [26] (1990), the type and degree of disability are also important factors, since

Control of Dental Biofilm and Oral Health Maintenance in Patients with Down Syndrome

http://dx.doi.org/10.5772/53348

69

The obstacles inherent to children with Down syndrome and the difficulties faced by pa‐ rents and ⁄ or people in charge for toothbrushing, lead the professional in dentistry to look for a substance capable of aiding and stimulating these patients in the mechanical control of the dental biofilm. Studies suggest the use of disclosing agents, such as erythrosine, to re‐ move dental biofilm more easily. For this reason, the presence of a disclosing agent in the

Are disclosing the chemicals used for staining bacteria, which show the colonies, invisible or barely visible, that adhere to tooth surfaces, making them visible, thus supporting the main‐ tenance of oral hygiene while facilitating their removal (Bellini et al. [27]1974). Among the forms of application of disclosing the most commonly used in dentistry are tablets or solu‐

The proven merit of disclosing meant that its use became a source of motivation (Toassi, Pet‐ ry [29]2002), are indicated as excellent aids in determining the state of oral hygiene. Shown to be valuable as a teaching tool in education, not only by convincing the population for the presence of dental biofilm, as well as raising awareness about the need for its removal (Cris‐

Second Bouquet [31] (1971) and Gillings [32] (1977), the disclosing must provide ease of ap‐ plication and handling, good flavor, not blushing residually plastic restorations or tooth cracks, do not stain the mucosal lip, cheek and gum, to be of contrasting color facilitate the

There are a variety of disclosing the market, among them are methylene blue, eosin, erythro‐ sin, fluorescein sodium, neutral red and proflavine monosulfate. According to the work of

differentiation from the marginal gingiva and does not cause tissue irritation.

the greater the degree of mental deficiency the worse the level of hygiene.

formulation of the dentifrice could aid in the removal of the dental biofilm [27].

**3. Dentifrices with disclosing agent dental biofilm**

**Figure 3.** Toothbrushing technique and manual skills

The control of dental biofilm is a preventive action that involves a number of aspects, such as health education, which is achieved through constant guidance and motivation for people on oral hygiene (Bijella [17]1999).

The manual dexterity and, many times, the motivation, are indispensable factors for efficient oral hygiene through mechanical means in patients with Down syndrome [18]-[22]. Thus, the key to success in promoting and maintaining a satisfactory oral health in these patients is the application of a rigorous program of oral hygiene constant [23].

Mental disability is another aspect to be considered as difficult to awareness of the impor‐ tance of oral health, difficulty in learning the techniques of brushing and lack of concentra‐ tion at the time of toothbrushing [24],[25]. This difficulty leads these patients to have high levels of plaque-dependent oral diseases, especially periodontal changes 6 . It is therefore es‐ sential to establish strategies to prevent periodontal disease in these individuals.

**Figure 5.** Constant guidance and motivation

**Figure 3.** Toothbrushing technique and manual skills

**Figure 4.** Lecture to motivate the control of biofilm

on oral hygiene (Bijella [17]1999).

68 Down Syndrome

The control of dental biofilm is a preventive action that involves a number of aspects, such as health education, which is achieved through constant guidance and motivation for people

The manual dexterity and, many times, the motivation, are indispensable factors for efficient oral hygiene through mechanical means in patients with Down syndrome [18]-[22]. Thus, the key to success in promoting and maintaining a satisfactory oral health in these patients

Mental disability is another aspect to be considered as difficult to awareness of the impor‐ tance of oral health, difficulty in learning the techniques of brushing and lack of concentra‐ tion at the time of toothbrushing [24],[25]. This difficulty leads these patients to have high

. It is therefore es‐

levels of plaque-dependent oral diseases, especially periodontal changes 6

sential to establish strategies to prevent periodontal disease in these individuals.

is the application of a rigorous program of oral hygiene constant [23].

Second Nielsen [26] (1990), the type and degree of disability are also important factors, since the greater the degree of mental deficiency the worse the level of hygiene.

## **3. Dentifrices with disclosing agent dental biofilm**

The obstacles inherent to children with Down syndrome and the difficulties faced by pa‐ rents and ⁄ or people in charge for toothbrushing, lead the professional in dentistry to look for a substance capable of aiding and stimulating these patients in the mechanical control of the dental biofilm. Studies suggest the use of disclosing agents, such as erythrosine, to re‐ move dental biofilm more easily. For this reason, the presence of a disclosing agent in the formulation of the dentifrice could aid in the removal of the dental biofilm [27].

Are disclosing the chemicals used for staining bacteria, which show the colonies, invisible or barely visible, that adhere to tooth surfaces, making them visible, thus supporting the main‐ tenance of oral hygiene while facilitating their removal (Bellini et al. [27]1974). Among the forms of application of disclosing the most commonly used in dentistry are tablets or solu‐ tions (Medeiros [28]1991).

The proven merit of disclosing meant that its use became a source of motivation (Toassi, Pet‐ ry [29]2002), are indicated as excellent aids in determining the state of oral hygiene. Shown to be valuable as a teaching tool in education, not only by convincing the population for the presence of dental biofilm, as well as raising awareness about the need for its removal (Cris‐ tiano, Bignelli [30]1995).

Second Bouquet [31] (1971) and Gillings [32] (1977), the disclosing must provide ease of ap‐ plication and handling, good flavor, not blushing residually plastic restorations or tooth cracks, do not stain the mucosal lip, cheek and gum, to be of contrasting color facilitate the differentiation from the marginal gingiva and does not cause tissue irritation.

There are a variety of disclosing the market, among them are methylene blue, eosin, erythro‐ sin, fluorescein sodium, neutral red and proflavine monosulfate. According to the work of Silva et al. [33] (2002), among all the solutions mentioned, eosin, erythrosine and neutral red showed the greatest ability to blush, ease of removal and absence of antimicrobial activity, are essential requirements in studies evaluating methods of hygiene and guidance patient.

cording to the manual skills of each participant. The time was recorded in seconds since the beginning of each experiment (opening the packages), to its end. To evaluate the remaining plate, disclosure was made with basic fuchsin after each experiment, and recorded the num‐ ber of stained surfaces, indicating the remaining plaque. The authors found that the average time of tooth brushing with toothpaste containing erythrosine has become more than double when compared to an ordinary toothpaste. Regarding the plaque index, the authors ob‐ served that the impregnation of the dye this is most efficient method III (Dentplaque ®), be‐ cause the dye is rubbed on the plaque while toothbrushing, when compared to other (MI toothbrushing with dentifrice common; M II - use of disclosing tablets plaque and

Control of Dental Biofilm and Oral Health Maintenance in Patients with Down Syndrome

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71

However, Rodrigues et al. [39] (1994) found different result. Undertook a study on the effec‐ tiveness of the dentifrices containing erythrosine Dentplaque ® in the stimulation process to dental hygiene for 45 male children, aged 6-12 years living in an orphanage in the city of Rio de Janeiro. These children were divided randomly into three groups of 15 patients main‐ tained the same dietary habits. Initially, all received instructions on oral hygiene and tooth‐ brushing technique, through lectures and posters illustrative devices. Were given tuition every 30 days during the 90 days of the survey. The brushing technique recommended in this study was to headphones, and recommended its implementation soon after meals. The control group made use of the brush with your regular dentifrice, the second group made use of a disclosing in tablet form before each brushing, and use your usual toothpaste, and the third group used a dentifrice containing erythrosine for toothbrushing routine. These children were supervised daily by an official of the orphanage properly oriented. In the ini‐ tial evaluation, all were subjected to three more evaluations, with a 30-day interval between them being given the simplified oral hygiene index of Greene & Vermillion. The authors concluded that there were no statistical differences in relation to reducing the level of dental plaque in the three groups, but it was observed that the dentifrice was the easiest way of disclosure, and inserts a method of assimilation more difficult for children aged 6-12 years.

The same result of the work of Rodrigues et al. [39] (1994) found in Silva et al. [97] (2004), with 62 students at a public school in the city of Piracicaba, aged between 12 and 14 years. Participants were divided into groups: dentifrice with erythrosine Dentplaque ® (Group I) and the use of disclosing tablets (Group II). The plaque reduction was observed in all groups did not show statistically significant differences between them. However, the au‐ thors noted that factors that had limited the completion of this study, as the amount of sam‐ ple, the low amount of plaque revealed by the index and the small amount of plaque shown by the students may have influenced the results, covering the response of the methods. In addition, the fact that some individuals participated in this study only the initial assessment, refusing to participate in the final evaluation, the amount of the sample was reduced to 18

In this context, the use of a dentifrice with erythrosine, as an agent for plaque removal should encourage the completion of a thorough toothbrushing, presumably more closely in‐

toothbrushing with dentifrice common).

participants.

dividual (Silva et al. [40] 2003).

Erythrosine a dye consisting of the disodium salt of 3 ', 6' - dihydroxy - 2 ', 4', 5 ', 7' - tetraio‐ dospiro [isobenzenofurano -1 (3H), 9 - [9H] xateno] - 3 - one and may contain up to 4.0% Fluoresceins a lesser degree of iodination, and chloride and / or sodium sulfate and water of crystallization. Must contain at least 85% calculated as total dye C20H6I4O5Na2. Presented as physical characteristics: fine powder, red or brown odorless, soluble in water and hidroscó‐ pio giving red solution should not exhibit fluorescent room light, also soluble in ethanol, glycerin and propylene glycol. Practically insoluble in ether, mineral oil and fats (Standing Committee Review of the Brazilian Pharmacopoeia [34] 1996).

**Figure 6.** Structural formula erythrosine

The use of a dentifrice containing the color erythrosine as agent for removal of dental bio‐ film during toothbrushing is an excellent resource to stimulate the patient in your dental hy‐ giene (Quintanilla, Bastos [35]1988), because the presence of this dye to facilitate parents and individuals / or guardians to view the plaque, especially in places where there is greater dif‐ ficulty of removal during brushing (Duarte et al. [36]1990).

The use of toothpaste containing erythrosine, Dentplaque ®, was approved by the ADA, and is used as part of a program to promote oral health, being distributed by the Ministry of Health in 1999, the Health Secretariat of São Paul, including the Regional Health of Piracica‐ ba, Piracicaba encompassing than 25 cities in the region (Silva et al. [37] 2004).

According to research Quintanilla et al. [38] (1989) where they studied the clinical behavior of the dentifrice added erythrosine Dentplaque ® 0.5% by comparing the new proposal to existing, as the common dentifrice and dental plaque disclosing in tablet form coadjuntor dentifrice common comparing the percentage of plaque remaining and the time taken to perform each of the three proposals in nine females with mean age of 21.33 years, and all with private have never experienced the use of a plaque disclosing. Individuals selected for the sample received no instruction on brushing technique, since the aim was to assess whether humans would be able to remove plaque evident on the surfaces of the teeth, ac‐ cording to the manual skills of each participant. The time was recorded in seconds since the beginning of each experiment (opening the packages), to its end. To evaluate the remaining plate, disclosure was made with basic fuchsin after each experiment, and recorded the num‐ ber of stained surfaces, indicating the remaining plaque. The authors found that the average time of tooth brushing with toothpaste containing erythrosine has become more than double when compared to an ordinary toothpaste. Regarding the plaque index, the authors ob‐ served that the impregnation of the dye this is most efficient method III (Dentplaque ®), be‐ cause the dye is rubbed on the plaque while toothbrushing, when compared to other (MI toothbrushing with dentifrice common; M II - use of disclosing tablets plaque and toothbrushing with dentifrice common).

Silva et al. [33] (2002), among all the solutions mentioned, eosin, erythrosine and neutral red showed the greatest ability to blush, ease of removal and absence of antimicrobial activity, are essential requirements in studies evaluating methods of hygiene and guidance patient.

Erythrosine a dye consisting of the disodium salt of 3 ', 6' - dihydroxy - 2 ', 4', 5 ', 7' - tetraio‐ dospiro [isobenzenofurano -1 (3H), 9 - [9H] xateno] - 3 - one and may contain up to 4.0% Fluoresceins a lesser degree of iodination, and chloride and / or sodium sulfate and water of crystallization. Must contain at least 85% calculated as total dye C20H6I4O5Na2. Presented as physical characteristics: fine powder, red or brown odorless, soluble in water and hidroscó‐ pio giving red solution should not exhibit fluorescent room light, also soluble in ethanol, glycerin and propylene glycol. Practically insoluble in ether, mineral oil and fats (Standing

The use of a dentifrice containing the color erythrosine as agent for removal of dental bio‐ film during toothbrushing is an excellent resource to stimulate the patient in your dental hy‐ giene (Quintanilla, Bastos [35]1988), because the presence of this dye to facilitate parents and individuals / or guardians to view the plaque, especially in places where there is greater dif‐

The use of toothpaste containing erythrosine, Dentplaque ®, was approved by the ADA, and is used as part of a program to promote oral health, being distributed by the Ministry of Health in 1999, the Health Secretariat of São Paul, including the Regional Health of Piracica‐

According to research Quintanilla et al. [38] (1989) where they studied the clinical behavior of the dentifrice added erythrosine Dentplaque ® 0.5% by comparing the new proposal to existing, as the common dentifrice and dental plaque disclosing in tablet form coadjuntor dentifrice common comparing the percentage of plaque remaining and the time taken to perform each of the three proposals in nine females with mean age of 21.33 years, and all with private have never experienced the use of a plaque disclosing. Individuals selected for the sample received no instruction on brushing technique, since the aim was to assess whether humans would be able to remove plaque evident on the surfaces of the teeth, ac‐

ba, Piracicaba encompassing than 25 cities in the region (Silva et al. [37] 2004).

Committee Review of the Brazilian Pharmacopoeia [34] 1996).

ficulty of removal during brushing (Duarte et al. [36]1990).

**Figure 6.** Structural formula erythrosine

70 Down Syndrome

However, Rodrigues et al. [39] (1994) found different result. Undertook a study on the effec‐ tiveness of the dentifrices containing erythrosine Dentplaque ® in the stimulation process to dental hygiene for 45 male children, aged 6-12 years living in an orphanage in the city of Rio de Janeiro. These children were divided randomly into three groups of 15 patients main‐ tained the same dietary habits. Initially, all received instructions on oral hygiene and tooth‐ brushing technique, through lectures and posters illustrative devices. Were given tuition every 30 days during the 90 days of the survey. The brushing technique recommended in this study was to headphones, and recommended its implementation soon after meals. The control group made use of the brush with your regular dentifrice, the second group made use of a disclosing in tablet form before each brushing, and use your usual toothpaste, and the third group used a dentifrice containing erythrosine for toothbrushing routine. These children were supervised daily by an official of the orphanage properly oriented. In the ini‐ tial evaluation, all were subjected to three more evaluations, with a 30-day interval between them being given the simplified oral hygiene index of Greene & Vermillion. The authors concluded that there were no statistical differences in relation to reducing the level of dental plaque in the three groups, but it was observed that the dentifrice was the easiest way of disclosure, and inserts a method of assimilation more difficult for children aged 6-12 years.

The same result of the work of Rodrigues et al. [39] (1994) found in Silva et al. [97] (2004), with 62 students at a public school in the city of Piracicaba, aged between 12 and 14 years. Participants were divided into groups: dentifrice with erythrosine Dentplaque ® (Group I) and the use of disclosing tablets (Group II). The plaque reduction was observed in all groups did not show statistically significant differences between them. However, the au‐ thors noted that factors that had limited the completion of this study, as the amount of sam‐ ple, the low amount of plaque revealed by the index and the small amount of plaque shown by the students may have influenced the results, covering the response of the methods. In addition, the fact that some individuals participated in this study only the initial assessment, refusing to participate in the final evaluation, the amount of the sample was reduced to 18 participants.

In this context, the use of a dentifrice with erythrosine, as an agent for plaque removal should encourage the completion of a thorough toothbrushing, presumably more closely in‐ dividual (Silva et al. [40] 2003).

## **4. Chemical control of dental biofilm**

Studies have shown that mechanical control produces significant reductions in gingivitis in people with special needs. However, many patients with Down syndrome, besides being unable to cooperate, do not have sufficient manual dexterity to do toothbrushing or to use dental floss. Consequently, the use of chemical and ⁄ or antimicrobial agents as aids in pla‐ que control can be indicated for these individuals. Considering the fact that toothbrushing with dentifrice is the most common tool for good oral hygiene, adding chlorhexidine to den‐ tifrices could be seen as a practical means of improving the quality of oral hygiene [2, 41-47].

The chemicals and / or antimicrobial agents are often used in dental plaque reduction and can be used in conjunction with the mechanical control in preserving health and treatment of gingivitis, in some patients (Mandel [48] 1994), especially those that have little manual dexterity to the realization of toothbrushing (Fischman [49] 1979).

**Figure 7.** Structural formula chlorhexidine

component (Bonacorsi et al. [57] 2000).

ble (Bonacorsi et al. [57] 2000).

biofilm:

is safe and effective (Quagliato [58] 1991).

avoiding the formation of the acquired pellicle.

Chlorhexidine is a cationic agent, a bis-guanidine non-toxic molecule is a symmetrical, with two rings and two 4-chlorophenyl groups ethane pentânicos connected by a central hexam‐ ethylene chain. Is prepared in the form of various salts, and gluconate, the digluconate or chlorhexidine acetate in its composition (Vinholis et al. [56] 1996). The chlorhexidine digluc‐ onate salt is one of the most widely used in the preparation of therapeutic formulations, be‐ cause of its greater solubility in water and physiological pH, dissociates releasing the active

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The main site of action of chlorhexidine, both in prokaryotes and in eukaryotic cells is the cytoplasmic membrane. The mechanism of action of chlorhexidine begins with a call in the bacterial cell wall, when the adsorption of positive charges in the molecule of the substance to the surface of the negative charges increases the permeability of the bacterial cell walls of microorganism and allows the agent to penetrate the cytoplasmic occurring disruption of cell membrane leakage of intracellular components and low molecular weight, as potassium ions. At this stage the bacteriostatic effect is considered and reversible. While in high con‐ centrations, lead to enzyme inhibition (ATPase), extravasation of macromolecules (nucleoti‐ des) and clotting components of the cytoplasm, due to the interaction of chlorhexidine with cytoplasmic proteins and nucleic acid, thus reaching the stage of bactericidal and irreversi‐

The chlorhexidine is usually effective against Gram positive and Gram-negative bacteria, fungi, yeasts and Candida albicans. It has broad spectrum antibacterial, high substantivity,

Second Vinholis et al. [56] (1996), there are three mechanisms for chlorhexidine inhibition of

Chlorhexidine is connected by means of electrostatic forces to the groups of acidic proteins such as phosphates, sulphates and carboxyl ions found in saliva and mouth tissues, there

The attributes required for a chemical agent can play its effectiveness in controlling supra‐ gingival biofilm was postulated by Loesche [50] (1976). According to the author, the chemi‐ cal agent to be effective against microorganisms responsible for inflammation and must have substantivity, ie, the intraoral retention capacity, to achieve a contact time sufficient to act on the microorganisms existing, and to maintain inhibition dental biofilm formation by a longer period. Furthermore, the product must be stable at room temperature for a consider‐ able time and safe for human use.

Other features should also be observed for a chemical agent to be considered effective, such as lack of toxicity, not to be allergenic, have clinical evidence of significant reductions of pla‐ que and gingivitis, be selective and have specificity to act on pathogenic microbiota, provide a pleasant taste have to be affordable and easy to use (Van Der Ouderra [51] 1991).

Chemical control of biofilm can be made to prophylactic or therapeutic. In the first case, the goal would be that there were an imbalance in the microbiota, when mechanical methods are ineffective. In the therapeutic sense with respect to individuals who already have changes in order to achieve the predominant bacteria-related diseases, aiming at restoring the microbiota and its harmony with the host (Marsh [52] 1992).

In 1954, Davies et al. [53] synthesized in the laboratory substance large bacterial action against Gram + and Gram -, and fungi. From this time, the chlorhexidine is now used as a general disinfectant for the treatment of various infections.

It was marketed in the 60s, by Imperial Chemical Industries (England), and one of the first applications of chlorhexidine in dentistry to control biofilm was performed by Schiott and Löe [54] (1970). The authors recommend the use of 10 mL of chlorhexidine digluconate 0.2% twice a day for one minute in order to prevent the accumulation of plaque and gingivitis subsequent. Since then, this compound has been considered the most effective agent in the chemical control dental biofilm (Souza, Abreu [55] 2003).

Control of Dental Biofilm and Oral Health Maintenance in Patients with Down Syndrome http://dx.doi.org/10.5772/53348 73

**Figure 7.** Structural formula chlorhexidine

**4. Chemical control of dental biofilm**

72 Down Syndrome

able time and safe for human use.

Studies have shown that mechanical control produces significant reductions in gingivitis in people with special needs. However, many patients with Down syndrome, besides being unable to cooperate, do not have sufficient manual dexterity to do toothbrushing or to use dental floss. Consequently, the use of chemical and ⁄ or antimicrobial agents as aids in pla‐ que control can be indicated for these individuals. Considering the fact that toothbrushing with dentifrice is the most common tool for good oral hygiene, adding chlorhexidine to den‐ tifrices could be seen as a practical means of improving the quality of oral hygiene [2, 41-47].

The chemicals and / or antimicrobial agents are often used in dental plaque reduction and can be used in conjunction with the mechanical control in preserving health and treatment of gingivitis, in some patients (Mandel [48] 1994), especially those that have little manual

The attributes required for a chemical agent can play its effectiveness in controlling supra‐ gingival biofilm was postulated by Loesche [50] (1976). According to the author, the chemi‐ cal agent to be effective against microorganisms responsible for inflammation and must have substantivity, ie, the intraoral retention capacity, to achieve a contact time sufficient to act on the microorganisms existing, and to maintain inhibition dental biofilm formation by a longer period. Furthermore, the product must be stable at room temperature for a consider‐

Other features should also be observed for a chemical agent to be considered effective, such as lack of toxicity, not to be allergenic, have clinical evidence of significant reductions of pla‐ que and gingivitis, be selective and have specificity to act on pathogenic microbiota, provide

Chemical control of biofilm can be made to prophylactic or therapeutic. In the first case, the goal would be that there were an imbalance in the microbiota, when mechanical methods are ineffective. In the therapeutic sense with respect to individuals who already have changes in order to achieve the predominant bacteria-related diseases, aiming at restoring

In 1954, Davies et al. [53] synthesized in the laboratory substance large bacterial action against Gram + and Gram -, and fungi. From this time, the chlorhexidine is now used as a

It was marketed in the 60s, by Imperial Chemical Industries (England), and one of the first applications of chlorhexidine in dentistry to control biofilm was performed by Schiott and Löe [54] (1970). The authors recommend the use of 10 mL of chlorhexidine digluconate 0.2% twice a day for one minute in order to prevent the accumulation of plaque and gingivitis subsequent. Since then, this compound has been considered the most effective agent in the

a pleasant taste have to be affordable and easy to use (Van Der Ouderra [51] 1991).

dexterity to the realization of toothbrushing (Fischman [49] 1979).

the microbiota and its harmony with the host (Marsh [52] 1992).

general disinfectant for the treatment of various infections.

chemical control dental biofilm (Souza, Abreu [55] 2003).

Chlorhexidine is a cationic agent, a bis-guanidine non-toxic molecule is a symmetrical, with two rings and two 4-chlorophenyl groups ethane pentânicos connected by a central hexam‐ ethylene chain. Is prepared in the form of various salts, and gluconate, the digluconate or chlorhexidine acetate in its composition (Vinholis et al. [56] 1996). The chlorhexidine digluc‐ onate salt is one of the most widely used in the preparation of therapeutic formulations, be‐ cause of its greater solubility in water and physiological pH, dissociates releasing the active component (Bonacorsi et al. [57] 2000).

The main site of action of chlorhexidine, both in prokaryotes and in eukaryotic cells is the cytoplasmic membrane. The mechanism of action of chlorhexidine begins with a call in the bacterial cell wall, when the adsorption of positive charges in the molecule of the substance to the surface of the negative charges increases the permeability of the bacterial cell walls of microorganism and allows the agent to penetrate the cytoplasmic occurring disruption of cell membrane leakage of intracellular components and low molecular weight, as potassium ions. At this stage the bacteriostatic effect is considered and reversible. While in high con‐ centrations, lead to enzyme inhibition (ATPase), extravasation of macromolecules (nucleoti‐ des) and clotting components of the cytoplasm, due to the interaction of chlorhexidine with cytoplasmic proteins and nucleic acid, thus reaching the stage of bactericidal and irreversi‐ ble (Bonacorsi et al. [57] 2000).

The chlorhexidine is usually effective against Gram positive and Gram-negative bacteria, fungi, yeasts and Candida albicans. It has broad spectrum antibacterial, high substantivity, is safe and effective (Quagliato [58] 1991).

Second Vinholis et al. [56] (1996), there are three mechanisms for chlorhexidine inhibition of biofilm:

Chlorhexidine is connected by means of electrostatic forces to the groups of acidic proteins such as phosphates, sulphates and carboxyl ions found in saliva and mouth tissues, there avoiding the formation of the acquired pellicle.

The ability of bacteria to bind to the tooth can be reduced by the absorption of chlorhexidine to the capsule of extracellular polysaccharides.

significant differences for the group that used chlorhexidine. However, clinically, the acute signs of inflammation are gone. The author stated that the conventional techniques of oral hygiene can be difficult to implement for this group of patients and chlorhexidine, in its var‐ ious forms of application, an agent is extremely useful for maintaining oral health of pa‐

Control of Dental Biofilm and Oral Health Maintenance in Patients with Down Syndrome

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75

Russell and Bay [44] (1981) observed that the use of toothpaste the basis of 1% chlorhexidine in daily brushing of children with epilepsy and mental retardation, reflected in a significant

Dolles and Gjermo [41] (1980) evaluated the effect of three dentifrices in reducing dental ca‐ ries and gingivitis (DI - dentifrice containing chlorhexidine (2%), IBD - with fluoride tooth‐ paste (0.1% NaF) and DIII - chlorhexidine dentifrice with the two % and fluorine (0.1% NaF) for two years. Ninety-one students from 13 to 15 years of age participated in the research. the group using the dentifrice with fluoride and chlorhexidine showed a lower rate of dental caries, although the gingival conditions improved in the three groups, showing no statistical

In a study of experimental gingivitis, Jenkins et al. 42 (1993), found that a dentifrice formula‐ tion of 1% chlorhexidine and 1000 ppm F (NaF) produced statistically significant reductions in plaque and gingivitis, compared with the placebo dentifrice. Subsequently, Yates et al. 47 1993, proposed to assess the clinical effects of chlorhexidine dentifrice 1%, with or without the 1000ppmF (NaF) previously tested by Jenkins et al. 42 1993. This study aimed to evalu‐ ate the control of plaque and gingivitis using: a) dentifrice containing 1% chlorhexidine called single asset, b) 1% of dentifrice containing fluoride clorexidina/1000ppm called active double c) negative control for six months. The sample consisted of two hundred ninety-sev‐ en individuals aged between 18 and 61 years. The periodontal parameters used were the plaque index, gingival bleeding and staining that were recorded at the beginning, six, 0,24 weeks, along with the index calculation was also recorded in the sixth, twelfth and twentyfourth week. After prophylaxis performed at baseline, the subjects used the assigned denti‐ frice twice a day for one minute, without any other additional information on oral hygiene were given, just the direction we should use enough toothpaste to cover the head of the toothbrush. It was not permitted to use any other adjunctive oral hygiene product. At the end of the study all subjects were examined by a hygienist and extrinsic staining, supragin‐ gival plaque and calculus were removed. The results showed reduction of plaque index and bleeding in all groups, but a significant improvement occurred in the chlorhexidine group. In contrast to these results, staining and calculus indices were more significant in the test groups compared with the control group. The authors concluded that the side effects of chlorhexidine are acceptable, the dentifrice containing chlorhexidine can be recommended for the same clinical applications than the other products based on chlorhexidine. The com‐ patibility of fluoride with chlorhexidine in one of the products could be effective in prevent‐ ing tooth decay, and fluoride dentifrice containing chlorhexidine and could provide benefits

to gingival health than preventive and therapeutic applications in clinical dentistry.

The action of a dentifrice containing 1% chlorhexidine in reducing dental plaque and gingi‐ val bleeding in 156 children over a period of twelve weeks, residents in Ga-Rankuwa (Preto‐

improvement in plaque and gingival index in this group of patients.

tients with special needs.

differences.

Chlorhexidine can compete with Ca + + ions. The mechanism is probably due to a direct competition between ions and / or availability of the drug and the carboxylic groups in the oral tissues. Can also inhibit the formation of bridges between the Ca + bacteria and surfa‐ ces, and the bacteria together. Due to its cationic properties, chlorhexidine can bind to the hydroxyapatite of enamel, and the acquired pellicle salivary proteins (Gjermo 59 1989).

## **5. Dentifrices with chlorhexidine**

The chemical agent chlorhexidine as deputy in the control of dental biofilm is useful in sit‐ uations where oral hygiene is inefficient, is compromised or is impossible to be realized. This antimicrobial agent is particularly suited to individuals who, because of physical or mental limitations, they are incapable, in whole or in part, the appropriate mechanical re‐ moval of plaque, were considered patients with special needs (Al-Tannir, Goodman [60] 1994).

That the dentifrices are used in conjunction with toothbrushing, causes the addition of chlo‐ rhexidine greater deserves attention, since it does not represent changes to the patient, as is routine in the same. Importantly, most studies of dentifrices containing chlorhexidine has been made with experimental formulations (Sathler, Fischer [61] 1996).

Experimental studies have shown that dentifrices with 0.5% chlorhexidine were less effec‐ tive than rinsing mouthwash with 0.2% chlorhexidine (Addy et al. [62] 1989, Jenkins et al. [63] 1990). In a study by Gjermo and Rolla [64] (1970), the use of dentifrices with 0.6% chlo‐ rhexidine and 0.8% applied in trays on the teeth to avoid interference from the mechanical action of toothbrushing, showed a reduction in the rate of plate, and these results were con‐ sistent with those obtained with mouthwash.

Second Jenkins et al. [42] (1993), introduction of 1% chlorhexidine dentifrices promoted to an improvement in gingival index and plaque index, similar to those experienced in rinsing with 0.2% chlorhexidine. The authors also state that the association of fluoride with chlo‐ rhexidine dentifrices does not inhibit chlorhexidine.

The use of chlorhexidine dentifrice is a controversial subject. Some research on the shortterm clinical effect of reducing plaque and gingival show the effectiveness of this substance (Torres 65 2000). This was proved in the study of Storhaug 46 (1977), which evaluated the use of toothpaste containing 0.8% chlorhexidine in 27 patients with special needs, from 4 to 12 years in a clinic held by the government of Norway. These patients were selected to test the effects of toothbrushing performed with the plaque index, gingival index, according to the criteria proposed by Löe and Silness. Patients were then divided into two groups: 17 children were using toothpaste containing chlorhexidine (GI) and 10 children used a placebo dentifrice (GII). After 6 weeks of study, there was significant reduction in plaque index of the group that used chlorhexidine compared with the control group and gingival index, no significant differences for the group that used chlorhexidine. However, clinically, the acute signs of inflammation are gone. The author stated that the conventional techniques of oral hygiene can be difficult to implement for this group of patients and chlorhexidine, in its var‐ ious forms of application, an agent is extremely useful for maintaining oral health of pa‐ tients with special needs.

The ability of bacteria to bind to the tooth can be reduced by the absorption of chlorhexidine

Chlorhexidine can compete with Ca + + ions. The mechanism is probably due to a direct competition between ions and / or availability of the drug and the carboxylic groups in the oral tissues. Can also inhibit the formation of bridges between the Ca + bacteria and surfa‐ ces, and the bacteria together. Due to its cationic properties, chlorhexidine can bind to the hydroxyapatite of enamel, and the acquired pellicle salivary proteins (Gjermo 59 1989).

The chemical agent chlorhexidine as deputy in the control of dental biofilm is useful in sit‐ uations where oral hygiene is inefficient, is compromised or is impossible to be realized. This antimicrobial agent is particularly suited to individuals who, because of physical or mental limitations, they are incapable, in whole or in part, the appropriate mechanical re‐ moval of plaque, were considered patients with special needs (Al-Tannir, Goodman [60]

That the dentifrices are used in conjunction with toothbrushing, causes the addition of chlo‐ rhexidine greater deserves attention, since it does not represent changes to the patient, as is routine in the same. Importantly, most studies of dentifrices containing chlorhexidine has

Experimental studies have shown that dentifrices with 0.5% chlorhexidine were less effec‐ tive than rinsing mouthwash with 0.2% chlorhexidine (Addy et al. [62] 1989, Jenkins et al. [63] 1990). In a study by Gjermo and Rolla [64] (1970), the use of dentifrices with 0.6% chlo‐ rhexidine and 0.8% applied in trays on the teeth to avoid interference from the mechanical action of toothbrushing, showed a reduction in the rate of plate, and these results were con‐

Second Jenkins et al. [42] (1993), introduction of 1% chlorhexidine dentifrices promoted to an improvement in gingival index and plaque index, similar to those experienced in rinsing with 0.2% chlorhexidine. The authors also state that the association of fluoride with chlo‐

The use of chlorhexidine dentifrice is a controversial subject. Some research on the shortterm clinical effect of reducing plaque and gingival show the effectiveness of this substance (Torres 65 2000). This was proved in the study of Storhaug 46 (1977), which evaluated the use of toothpaste containing 0.8% chlorhexidine in 27 patients with special needs, from 4 to 12 years in a clinic held by the government of Norway. These patients were selected to test the effects of toothbrushing performed with the plaque index, gingival index, according to the criteria proposed by Löe and Silness. Patients were then divided into two groups: 17 children were using toothpaste containing chlorhexidine (GI) and 10 children used a placebo dentifrice (GII). After 6 weeks of study, there was significant reduction in plaque index of the group that used chlorhexidine compared with the control group and gingival index, no

been made with experimental formulations (Sathler, Fischer [61] 1996).

to the capsule of extracellular polysaccharides.

**5. Dentifrices with chlorhexidine**

sistent with those obtained with mouthwash.

rhexidine dentifrices does not inhibit chlorhexidine.

1994).

74 Down Syndrome

Russell and Bay [44] (1981) observed that the use of toothpaste the basis of 1% chlorhexidine in daily brushing of children with epilepsy and mental retardation, reflected in a significant improvement in plaque and gingival index in this group of patients.

Dolles and Gjermo [41] (1980) evaluated the effect of three dentifrices in reducing dental ca‐ ries and gingivitis (DI - dentifrice containing chlorhexidine (2%), IBD - with fluoride tooth‐ paste (0.1% NaF) and DIII - chlorhexidine dentifrice with the two % and fluorine (0.1% NaF) for two years. Ninety-one students from 13 to 15 years of age participated in the research. the group using the dentifrice with fluoride and chlorhexidine showed a lower rate of dental caries, although the gingival conditions improved in the three groups, showing no statistical differences.

In a study of experimental gingivitis, Jenkins et al. 42 (1993), found that a dentifrice formula‐ tion of 1% chlorhexidine and 1000 ppm F (NaF) produced statistically significant reductions in plaque and gingivitis, compared with the placebo dentifrice. Subsequently, Yates et al. 47 1993, proposed to assess the clinical effects of chlorhexidine dentifrice 1%, with or without the 1000ppmF (NaF) previously tested by Jenkins et al. 42 1993. This study aimed to evalu‐ ate the control of plaque and gingivitis using: a) dentifrice containing 1% chlorhexidine called single asset, b) 1% of dentifrice containing fluoride clorexidina/1000ppm called active double c) negative control for six months. The sample consisted of two hundred ninety-sev‐ en individuals aged between 18 and 61 years. The periodontal parameters used were the plaque index, gingival bleeding and staining that were recorded at the beginning, six, 0,24 weeks, along with the index calculation was also recorded in the sixth, twelfth and twentyfourth week. After prophylaxis performed at baseline, the subjects used the assigned denti‐ frice twice a day for one minute, without any other additional information on oral hygiene were given, just the direction we should use enough toothpaste to cover the head of the toothbrush. It was not permitted to use any other adjunctive oral hygiene product. At the end of the study all subjects were examined by a hygienist and extrinsic staining, supragin‐ gival plaque and calculus were removed. The results showed reduction of plaque index and bleeding in all groups, but a significant improvement occurred in the chlorhexidine group. In contrast to these results, staining and calculus indices were more significant in the test groups compared with the control group. The authors concluded that the side effects of chlorhexidine are acceptable, the dentifrice containing chlorhexidine can be recommended for the same clinical applications than the other products based on chlorhexidine. The com‐ patibility of fluoride with chlorhexidine in one of the products could be effective in prevent‐ ing tooth decay, and fluoride dentifrice containing chlorhexidine and could provide benefits to gingival health than preventive and therapeutic applications in clinical dentistry.

The action of a dentifrice containing 1% chlorhexidine in reducing dental plaque and gingi‐ val bleeding in 156 children over a period of twelve weeks, residents in Ga-Rankuwa (Preto‐ ria, South Africa), aged between 12 and 14 years were evaluated by Gugushe et al. [2] (1994). The children were divided into three groups, which used conventional dentifrice (group A - 51 subjects), placebo dentifrice (group B - 49 individuals) and chlorhexidine dentifrice (group C - 56 individuals). Before starting the experiment, they were instructed on oral hy‐ giene, had their records of plaque index, gingival taken and received professional dental prophylaxis. The record of the indices was repeated in the sixth and twelfth weeks. All pa‐ tients were instructed to make tooth brushing morning and night. In the presence of plaque, it was observed that the rate decreased in all groups, with reductions substantially equal groups A and B and further reduction to the group C In relation to the gingival index, a re‐ duction very similar in all groups (approximately 4%) without significant differences. How‐ ever, the dentifrice with 1% chlorhexidine was more effective in controlling dental plaque as compared with the conventional dentifrice and placebo.

sults. The dentifrice containing an association of chlorhexidine and erythrosine gave the best results. Thus, with the methodology employed, it was possible to conclude that the combi‐ nation of drugs (chlorhexidine, fluorine and erythrosine) within one dentifrice can be useful

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1 Department of Dentistry, Ponta Grossa Dental School, Center for Higher Education of

2 Department of Dentistry, School of Dentistry, Ponta Grossa State University, Ponta Grossa,

[1] Sekino S, Ramberg P, Uzel NG, Socransky S, Lindhe J. Effect of various chlorhexidine regimens on salivary bacteria and de novo plaque formation. J Clin Periodontol

[2] Gugushe TS, de Wet FA, Rojas-Silva O. Efficacy of an experimental dentifrice formu‐ lation on primary school children in Ga-Rankuwa, Pretoria. J Dent Assoc S Afr

[3] Owens J, Addy M, Faulkner J, Lockwood C, Adair R. A short-term clinical study de‐ sign to investigate the chemical plaque inhibitory properties of mouthrinses when used as adjuncts to toothpastes: applied to chlorhexidine. J Clin Periodontol

[4] Sabbagh-Haddad A, Ciamponi AL, Guaré RO. Pacientes especiais. In Guedes-Pinto

[5] Reuland-Bosma W, Dijk J. Periodontal disease in Down's syndrome: a review. J Clin

[6] Cornejo LS, Zak GA, Dorronsoro de Cattoni ST, Calamari SE, Azcurra AI, Battellino LJ. S. Bucodental health condition in patients with Down syndrome of Cordoba City,

[7] Alisson PJ, Hennequin M, Faulks D. Dental care access among individuals with

[8] Moraes MEL, Bastos MS, Moraes LC,Rocha JC. Prevalência de cárie pelo índice CPO-D em portadores de síndrome de Down. Pós-Grad Rev Odontol. 2002; 5(2): 64-73.

AC. Odontopediatria. São Paulo: Santos; 2003. p. 893-931.

Argentina. Acta Odontol Latinoam. 1996; 9(2):65-79.

Down syndrome in France. Spec Care Dent. 2000; 20(1):28-34.

in controlling dental biofilm and in the reduction of gingival bleeding [68,69].

and Gislaine Denise Czlusniak1

Campos Gerais (CESCAGE), Ponta Grossa, Paraná, Brazil

**Author details**

Paraná, Brazil

**References**

2003;30:919–25.

1994;49:209–12.

1997;24:732–7.

Periodontol. 1986; 13(1);64-73.

Ana Paula Teitelbaum1

In a clinical study by Sanz et al. [45] (1994), the experimental dentifrice containing chlorhexi‐ dine 0.4% and 0.345 mg of zinc, contributed significantly to the improvement of oral hy‐ giene, both in relation to the plaque and gingivitis and bleeding, resulting in fewer spots than those found in the group who used mouthwash with chlorhexidine 0.12%. The investi‐ gators concluded that the tested dentifrice can be viewed as a promising alternative for the use of substances effective in reducing plaque and gingivitis, and offer minimal side effects.

In respect the effect on the microflora of the mouth, the dentifrices to 1% chlorhexidine and tested for a period of 6 months, promoted reduction of aerobic microorganisms and aneróbi‐ cos (Maynard et al. [66] 1993).

Considering the fact that toothbrushing with dentifrice is the most common habits of oral hygiene (Owens et al. [12] 1997), this practice can be seen as a plausible way for the intro‐ duction of chemicals to improve the oral health (Yates et al. [47] 1993).

According to Newman [67] (1986), the introduction of antimicrobial agents in dentifrices aims to improve the effectiveness of toothbrushing, promoting a positive effect in reducing biofilm.

Thus, Teltelbaum et al. [68,69] (2009, 2010) conducted a study with patients with SD, where he developed a dentifrice containing these two substances, chlorhexidine and erythrosine and evaluated the mechanical and chemical control of dental biofilm. The mechanical and chemical control of dental biofilm in patients with Down syndrome, of using different ex‐ perimental dentifrices in forty institutionalized children between ages 7 and 13 years in the mixed dentition in an experimental cross-over, blind clinical trial where we used the follow‐ ing protocols: fluoridated dentifrice (protocol G1); fluoridated dentifrice + chlorhexidine (protocol G2); fluoridated dentifrice + chlorhexidine + plaquedisclosing agent (protocol G3); and fluoridated dentifrice + plaque-disclosing agent (protocol G4). Each experimental stage lasted 10 days with a 15-day washout. The evaluated parameters were plaque index and gingival bleeding and initial clinical conditions between each stage were similar. The denti‐ frices containing plaque-disclosing agent, irrespective of their association with chlorhexi‐ dine, produced a greater reduction in the final plaque index. As for gingival bleeding, the dentifrice containing erythrosine and the one containing chlorhexidine produced similar re‐ sults. The dentifrice containing an association of chlorhexidine and erythrosine gave the best results. Thus, with the methodology employed, it was possible to conclude that the combi‐ nation of drugs (chlorhexidine, fluorine and erythrosine) within one dentifrice can be useful in controlling dental biofilm and in the reduction of gingival bleeding [68,69].

## **Author details**

ria, South Africa), aged between 12 and 14 years were evaluated by Gugushe et al. [2] (1994). The children were divided into three groups, which used conventional dentifrice (group A - 51 subjects), placebo dentifrice (group B - 49 individuals) and chlorhexidine dentifrice (group C - 56 individuals). Before starting the experiment, they were instructed on oral hy‐ giene, had their records of plaque index, gingival taken and received professional dental prophylaxis. The record of the indices was repeated in the sixth and twelfth weeks. All pa‐ tients were instructed to make tooth brushing morning and night. In the presence of plaque, it was observed that the rate decreased in all groups, with reductions substantially equal groups A and B and further reduction to the group C In relation to the gingival index, a re‐ duction very similar in all groups (approximately 4%) without significant differences. How‐ ever, the dentifrice with 1% chlorhexidine was more effective in controlling dental plaque as

In a clinical study by Sanz et al. [45] (1994), the experimental dentifrice containing chlorhexi‐ dine 0.4% and 0.345 mg of zinc, contributed significantly to the improvement of oral hy‐ giene, both in relation to the plaque and gingivitis and bleeding, resulting in fewer spots than those found in the group who used mouthwash with chlorhexidine 0.12%. The investi‐ gators concluded that the tested dentifrice can be viewed as a promising alternative for the use of substances effective in reducing plaque and gingivitis, and offer minimal side effects.

In respect the effect on the microflora of the mouth, the dentifrices to 1% chlorhexidine and tested for a period of 6 months, promoted reduction of aerobic microorganisms and aneróbi‐

Considering the fact that toothbrushing with dentifrice is the most common habits of oral hygiene (Owens et al. [12] 1997), this practice can be seen as a plausible way for the intro‐

According to Newman [67] (1986), the introduction of antimicrobial agents in dentifrices aims to improve the effectiveness of toothbrushing, promoting a positive effect in reducing

Thus, Teltelbaum et al. [68,69] (2009, 2010) conducted a study with patients with SD, where he developed a dentifrice containing these two substances, chlorhexidine and erythrosine and evaluated the mechanical and chemical control of dental biofilm. The mechanical and chemical control of dental biofilm in patients with Down syndrome, of using different ex‐ perimental dentifrices in forty institutionalized children between ages 7 and 13 years in the mixed dentition in an experimental cross-over, blind clinical trial where we used the follow‐ ing protocols: fluoridated dentifrice (protocol G1); fluoridated dentifrice + chlorhexidine (protocol G2); fluoridated dentifrice + chlorhexidine + plaquedisclosing agent (protocol G3); and fluoridated dentifrice + plaque-disclosing agent (protocol G4). Each experimental stage lasted 10 days with a 15-day washout. The evaluated parameters were plaque index and gingival bleeding and initial clinical conditions between each stage were similar. The denti‐ frices containing plaque-disclosing agent, irrespective of their association with chlorhexi‐ dine, produced a greater reduction in the final plaque index. As for gingival bleeding, the dentifrice containing erythrosine and the one containing chlorhexidine produced similar re‐

duction of chemicals to improve the oral health (Yates et al. [47] 1993).

compared with the conventional dentifrice and placebo.

cos (Maynard et al. [66] 1993).

biofilm.

76 Down Syndrome

Ana Paula Teitelbaum1 and Gislaine Denise Czlusniak1

1 Department of Dentistry, Ponta Grossa Dental School, Center for Higher Education of Campos Gerais (CESCAGE), Ponta Grossa, Paraná, Brazil

2 Department of Dentistry, School of Dentistry, Ponta Grossa State University, Ponta Grossa, Paraná, Brazil

## **References**


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80 Down Syndrome


**Chapter 5**

**How to Design an Exercise Program TO Reduce**

Francisco J. Ordonez, Gabriel Fornieles, Alejandra Camacho, Miguel A. Rosety,

Manuel Rosety-Rodriguez

http://dx.doi.org/10.5772/ 52984

**1. Introduction**

Antonio J Diaz, Ignacio Rosety, Natalia Garcia and

Additional information is available at the end of the chapter

*Obesity in people with Down syndrome: a big problem.*

higher risk of metabolic syndrome in this group [4].

also for its negative impact on their health status and quality of life.

**Inflammation in Obese People With Down Syndrome**

Over the last decade, a significant increase in the life expectancy of people with Down syn‐ drome (DS) has been observed. The higher life expectancy has caused a higher incidence of morbidity as they age [1]. Many of these disorders have been associated to obesity that is a major health problem in people with intellectual disabilities. Not only for its prevalence but

In a more detailed way, it is widely accepted that obesity is a serious problem that is over‐ whelmingly prevalent in the general population. However, the magnitude of this problem is even worse in people with intellectual disability in general and Down syndrome in particu‐ lar. A cross-sectional study with adult clients (n=470) of three Dutch intellectual disability care providing organizations and found that healthy behavior was low, with 98.9% of the partici‐ pants having an unhealthy diet and 68.3% a lack of exercise [2]. In a more detailed way, women

Obesity and overweight are independent risk factors for chronic disease and have been shown to make a significant contribution to the reduced life expectancy of adults with intel‐ lectual disability. Further, the increased visceral fat in females with DS might indicate a

and reproduction in any medium, provided the original work is properly cited.

© 2013 Ordonez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and people with Down syndrome were significantly more at risk of being obese [3].

## **How to Design an Exercise Program TO Reduce Inflammation in Obese People With Down Syndrome**

Francisco J. Ordonez, Gabriel Fornieles, Alejandra Camacho, Miguel A. Rosety, Antonio J Diaz, Ignacio Rosety, Natalia Garcia and Manuel Rosety-Rodriguez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/ 52984

**1. Introduction**

## *Obesity in people with Down syndrome: a big problem.*

Over the last decade, a significant increase in the life expectancy of people with Down syn‐ drome (DS) has been observed. The higher life expectancy has caused a higher incidence of morbidity as they age [1]. Many of these disorders have been associated to obesity that is a major health problem in people with intellectual disabilities. Not only for its prevalence but also for its negative impact on their health status and quality of life.

In a more detailed way, it is widely accepted that obesity is a serious problem that is over‐ whelmingly prevalent in the general population. However, the magnitude of this problem is even worse in people with intellectual disability in general and Down syndrome in particu‐ lar. A cross-sectional study with adult clients (n=470) of three Dutch intellectual disability care providing organizations and found that healthy behavior was low, with 98.9% of the partici‐ pants having an unhealthy diet and 68.3% a lack of exercise [2]. In a more detailed way, women and people with Down syndrome were significantly more at risk of being obese [3].

Obesity and overweight are independent risk factors for chronic disease and have been shown to make a significant contribution to the reduced life expectancy of adults with intel‐ lectual disability. Further, the increased visceral fat in females with DS might indicate a higher risk of metabolic syndrome in this group [4].

© 2013 Ordonez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Accordingly, recent studies have concluded that more attention needs to be paid to the ris‐ ing fat mass percentages seen in individuals with Down syndrome in order to minimize negative, long-term health consequences [5,6].

[18]. These data are of particular interest since increased low-grade inflammation is associat‐ ed with increased arterial stiffness, a recognized marker for increased cardiovascular risk in

How to Design an Exercise Program TO Reduce Inflammation in Obese People With Down Syndrome

http://dx.doi.org/10.5772/ 52984

85

Importantly, some frequently diagnosed comorbidities could affect systemic inflammation in people with intellectual disability. In fact, obstructive sleep apnea, is a syndrome that has itself been linked to increased low-grade inflammation both in general population [20] and

Adipokines and acute phase proteins are important mediators of adverse effects (insulin re‐ sistance) so that the normalization of their levels has been reported as a therapeutic target in

Contradictory data have been reported about the effect of statins on adiponectin plasma lev‐ els. In this respect, atorvastatin (10-80 mg/day) increased adiponectin plasma levels in sub‐ jects at high cardiovascular risk. Further, adiponectin concentrations were positively correlated with high-density lipoprotein-cholesterol both before and after atorvastatin treat‐ ment [23]. Similar results were found using simvastatin (40mg/day) suggesting a novel anti-

Fortunately several studies have reported both endurance and resistance training programs at low/moderate intensity may reduce proinflammatory adipokines both at early life stages and elderly in obese people without intellectual disability [25]. However, to the best of our knowledge, there is a lack of information in people, especially women, with intellectual dis‐ abilities. Accordingly additional studies based on specific training programs that are adapta‐

In addition, it would be of interest to reduce the length of training programs previously published. In fact, it is expected shorter training programs may facilitate their follow-up, re‐

The benefits of physical activity are universal for general population, including those with

In fact, the participation of people with disabilities in sports and recreational activities pro‐ motes social inclusion, minimizes deconditioning, optimizes physical functioning, and en‐ hances overall welfare [14,28]. Further sports participation enhances the psychological wellbeing of people with disabilities through the provision of opportunities to form friendships, express creativity, increase self-esteem, develop a self-identity, and foster meaning and pur‐

Physical consequences of inactivity for persons with disabilities include among others: re‐ duced cardiovascular fitness, osteoporosis and impaired circulation. In addition, the psycho‐ social implications of inactivity include decreased self-esteem, decreased social acceptance,

and ultimately, greater dependence on others for daily living [14].

ble to the needs of individuals with intellectual disability are strongly required [26].

people with Prader-Willi syndrome [19].

people with Prader-Willi syndrome [19].

subjects at high cardiovascular risks [21,22].

inflammatory effect of this drug [24].

ducing drop-out rates.

disabilities [14, 27].

pose in life [29].

*Regular exercise in Down Syndrome*

In reviewing the current evidence, the effectiveness of interventions was judged on both the extent of and the maintenance of weight reduction.

It is widely accepted promoting appropriate levels of physical activity remains an important component for both weight loss and management and should have its place as a lifestyle and behavioral change in people with Down syndrome [7,8,9].

However, the interventions that have been conducted has achieved a degree of success in promoting weight reduction in the short term. There is less evidence about whether inter‐ vention programs can maintain weight loss effectively in the long term. In fact, current guidelines highlight the interventions that lead to modest, maintainable weight lose for peo‐ ple with intellectual disability will have significant benefits on both health and welfare.

The latter authors also concluded that much of the research on obesity in adults with Down syndrome has design weaknesses, including small sample sizes and a lack of con‐ trolled studies [10].

#### *Association between obesity and low-grade systemic inflammation*

Accumulating evidence derived from both clinical and experimental studies highlight obesi‐ ty may be viewed as a chronic low-grade inflammatory disease as well as a metabolic dis‐ ease [11,12]. Therefore, it is widely accepted adipose tissue is not merely a fat storage depot. In contrast, endocrine and paracrine aspects of adipose tissue have become an active re‐ search area in the last years.

Recent studies have reported that parenchymal and stromal cells (fibroblats, endothelial cells and immune cells) in adipose tissue change dramatically in number and cell type during the course of obesity, which is referred to as "adipose tissue remodeling." In this regard, recent evidence suggests that the intimate crosstalk between mature adypocytes and stromal cells in adipose tissue plays a critical role in the dysregulation of adipocytokine production [13].

These findings were of particular interest since adults with intellectual disabilities experi‐ ence high rates of obesity. Although Down syndrome has been traditionally considered an atheroma free model [14] recent studies have also reported individuals with intellectual dis‐ ability suffer from low-graded systemic inflammation that has been proposed as a patho‐ genic mechanism of several disorders [15]. Previous studies showing increased levels of soluble intercellular adhesion molecule (sICAM-3) and soluble vascular cell adhesion mole‐ cule (sVCAM-1) in plasma, also suggested the presence of a moderate dysfunction of endo‐ thelial cells in subjects with Down syndrome [16].

Similarly, plasmatic concentrations of IL-6, IL-18 and CRP (C-reactive protein) levels were highly correlated with measures of total and visceral adiposity in obese adults with Prader-Willi Syndrome (PWS) [17]. The reported excessive visceral adiposity in subjects with PWS may be associated with decreased production and lower circulating levels of adiponectin [18]. These data are of particular interest since increased low-grade inflammation is associat‐ ed with increased arterial stiffness, a recognized marker for increased cardiovascular risk in people with Prader-Willi syndrome [19].

Importantly, some frequently diagnosed comorbidities could affect systemic inflammation in people with intellectual disability. In fact, obstructive sleep apnea, is a syndrome that has itself been linked to increased low-grade inflammation both in general population [20] and people with Prader-Willi syndrome [19].

Adipokines and acute phase proteins are important mediators of adverse effects (insulin re‐ sistance) so that the normalization of their levels has been reported as a therapeutic target in subjects at high cardiovascular risks [21,22].

Contradictory data have been reported about the effect of statins on adiponectin plasma lev‐ els. In this respect, atorvastatin (10-80 mg/day) increased adiponectin plasma levels in sub‐ jects at high cardiovascular risk. Further, adiponectin concentrations were positively correlated with high-density lipoprotein-cholesterol both before and after atorvastatin treat‐ ment [23]. Similar results were found using simvastatin (40mg/day) suggesting a novel antiinflammatory effect of this drug [24].

Fortunately several studies have reported both endurance and resistance training programs at low/moderate intensity may reduce proinflammatory adipokines both at early life stages and elderly in obese people without intellectual disability [25]. However, to the best of our knowledge, there is a lack of information in people, especially women, with intellectual dis‐ abilities. Accordingly additional studies based on specific training programs that are adapta‐ ble to the needs of individuals with intellectual disability are strongly required [26].

In addition, it would be of interest to reduce the length of training programs previously published. In fact, it is expected shorter training programs may facilitate their follow-up, re‐ ducing drop-out rates.

#### *Regular exercise in Down Syndrome*

Accordingly, recent studies have concluded that more attention needs to be paid to the ris‐ ing fat mass percentages seen in individuals with Down syndrome in order to minimize

In reviewing the current evidence, the effectiveness of interventions was judged on both the

It is widely accepted promoting appropriate levels of physical activity remains an important component for both weight loss and management and should have its place as a lifestyle

However, the interventions that have been conducted has achieved a degree of success in promoting weight reduction in the short term. There is less evidence about whether inter‐ vention programs can maintain weight loss effectively in the long term. In fact, current guidelines highlight the interventions that lead to modest, maintainable weight lose for peo‐ ple with intellectual disability will have significant benefits on both health and welfare.

The latter authors also concluded that much of the research on obesity in adults with Down syndrome has design weaknesses, including small sample sizes and a lack of con‐

Accumulating evidence derived from both clinical and experimental studies highlight obesi‐ ty may be viewed as a chronic low-grade inflammatory disease as well as a metabolic dis‐ ease [11,12]. Therefore, it is widely accepted adipose tissue is not merely a fat storage depot. In contrast, endocrine and paracrine aspects of adipose tissue have become an active re‐

Recent studies have reported that parenchymal and stromal cells (fibroblats, endothelial cells and immune cells) in adipose tissue change dramatically in number and cell type during the course of obesity, which is referred to as "adipose tissue remodeling." In this regard, recent evidence suggests that the intimate crosstalk between mature adypocytes and stromal cells in adipose tissue plays a critical role in the dysregulation of adipocytokine production [13].

These findings were of particular interest since adults with intellectual disabilities experi‐ ence high rates of obesity. Although Down syndrome has been traditionally considered an atheroma free model [14] recent studies have also reported individuals with intellectual dis‐ ability suffer from low-graded systemic inflammation that has been proposed as a patho‐ genic mechanism of several disorders [15]. Previous studies showing increased levels of soluble intercellular adhesion molecule (sICAM-3) and soluble vascular cell adhesion mole‐ cule (sVCAM-1) in plasma, also suggested the presence of a moderate dysfunction of endo‐

Similarly, plasmatic concentrations of IL-6, IL-18 and CRP (C-reactive protein) levels were highly correlated with measures of total and visceral adiposity in obese adults with Prader-Willi Syndrome (PWS) [17]. The reported excessive visceral adiposity in subjects with PWS may be associated with decreased production and lower circulating levels of adiponectin

negative, long-term health consequences [5,6].

trolled studies [10].

84 Down Syndrome

search area in the last years.

extent of and the maintenance of weight reduction.

and behavioral change in people with Down syndrome [7,8,9].

*Association between obesity and low-grade systemic inflammation*

thelial cells in subjects with Down syndrome [16].

The benefits of physical activity are universal for general population, including those with disabilities [14, 27].

In fact, the participation of people with disabilities in sports and recreational activities pro‐ motes social inclusion, minimizes deconditioning, optimizes physical functioning, and en‐ hances overall welfare [14,28]. Further sports participation enhances the psychological wellbeing of people with disabilities through the provision of opportunities to form friendships, express creativity, increase self-esteem, develop a self-identity, and foster meaning and pur‐ pose in life [29].

Physical consequences of inactivity for persons with disabilities include among others: re‐ duced cardiovascular fitness, osteoporosis and impaired circulation. In addition, the psycho‐ social implications of inactivity include decreased self-esteem, decreased social acceptance, and ultimately, greater dependence on others for daily living [14].

Despite the benefits associated to regular exercise, subjects with disabilities are still, to a large extent, more restricted in their participation than their peers without disabilities. They may experience negative societal stereotypes and low performance expectations, rendering them with limited opportunities for participation in physical activities [30].

**2. Body**

abilities.

*Application area*

help them to access activities [34,38].

*Research course & method used*

*Problem statement*

Accumulating evidence derived from both clinical and experimental studies highlight the association of visceral obesity with a proinflammatory status in general population [11,12]. Recent studies have also reported individuals with intellectual disability suffer from lowgrade systemic inflammation that has been proposed as a pathogenic mechanism of several disorders [15]. The adipokines are important mediators of these adverse effects so that the

How to Design an Exercise Program TO Reduce Inflammation in Obese People With Down Syndrome

http://dx.doi.org/10.5772/ 52984

87

Fortunately several studies have reported both endurance and resistance training programs at low/moderate intensity may reduce proinflammatory adipokines both at early life stages and elderly in obese people without intellectual disability [25]. However, to the best of our knowledge, there is a lack of information in people, especially women, with intellectual dis‐

Accordingly, this study was designed to assess the influence of a 10-week aerobic training

Healthcare costs are continuously increasing because of the increasing life expectancy among people with disabilities [1]. This is a strong argument for strengthening the role of

However, researchers suggest that people with an intellectual disability undertake less physical activity than the general population and many rely, to some extent, on others to

Currently, a wide variety of sporting activities are accessible to people with disabilities, and guidelines are available to assist caregivers, volunteers, educators and healthcare-providers in recommending activities appropriate for those people with specific conditions. These training programs should be not only effective but safe since previous studies have reported

A 10-week aerobic training program was designed by a multidisciplinary team to re‐ duce plasmatic adipokines in obese women with Down syndrome. In order to achieve this goal, twenty obese adult women with Down syndrome volunteered for the present interventional study. They had an intelligence quotient (IQ) range of 50–69, determined by Stanford-Binet Scale, being diagnosed as having mild intellectual disability. Eleven of them were randomly assigned to perform a 10-week aerobic training program, 3 sessions/ week, consisting of warming-up followed by a main part in a treadmill (30-40 min [increas‐ ing 2 minutes and half each two weeks]) at a work intensity of 55-65% of peak heart rate (increasing a 2.5% each two weeks) and a cooling-down period. Control group included

program on plasmatic levels of adipokines in obese women with Down syndrome.

preventive strategies, such as exercise, with the aim to reduce future costs.

their sport-related injury risk may be complicated by preexisting disability.

normalization of their levels has been reported as a therapeutic target [21].

In this regard, people with Down syndrome are especially at risk because of physical and health impairments, as well as perceived and real barriers to participation in exercise [31].

In a more detailed way, it is accepted that persons with Down syndrome exhibit low peak aerobic capacities and maximal heart rates when compared with healthy non-disabled peers. These findings may be explained by a lower walking economy that is mainly related to their inability to adapt efficiently to positive variations in walking speed [32]. Furthermore, they present a different catecholamine response to exercise [33]. Accordingly, intervention pro‐ grams based on regular exercise should be designed by taking into account their chrono‐ tropic incompetence. On the contrary, sessions theoretically designed at moderate intensity for the general population become exhausting for participants with Down syndrome, lead‐ ing to undesired results and increased withdrawal rates.

However, it is important to note that environmental and family factors seem to be more sig‐ nificant determinants of participation than characteristics of the subjects themselves. In fact, families who engage in physical activities themselves tend to promote similar participation for their relatives with disabilities. Conversely, inactive role models, competing demands and time pressures, unsafe environments, lack of adequate facilities, insufficient funds, and inadequate access to quality daily physical education seem to be more prevalent among populations with special needs. The establishment of short-term goals, emphasizing variety and enjoyment, and positive reinforcement through documented progress toward goals can help spark and sustain the motivation for participation [14, 27,34].

In summary, misconceptions and attitudinal barriers at the level of the individual, the fami‐ ly, and the community need to be addressed to integrate people with disabilities into recrea‐ tional and sports activities [14].

Another point of interest is that physical activity comes with an inherent risk for injury. For people with intellectual disability, previous studies have reported their injury risk may be complicated by preexisting disability [26]. Accordingly it is important for caregivers, educa‐ tors and others to identify strategies to minimize risks of illness and injury related to partici‐ pation through activity adaptations and safety precautions.

Fortunately, little or no sport-related injuries are reported in the literature during interven‐ tion programs based on regular exercise [32,35,36]. It may be explained, at least in part, due to the preparticipation physical examination (PPPE) and the design of specific training pro‐ grams that are adaptable to the needs of individuals with intellectual disability. This is of particular interest since injuries and discomfort may lead to participants to interrupt their training program, increasing withdrawal rates and sedentary lifestyle [37].

## **2. Body**

Despite the benefits associated to regular exercise, subjects with disabilities are still, to a large extent, more restricted in their participation than their peers without disabilities. They may experience negative societal stereotypes and low performance expectations, rendering

In this regard, people with Down syndrome are especially at risk because of physical and health impairments, as well as perceived and real barriers to participation in exercise [31].

In a more detailed way, it is accepted that persons with Down syndrome exhibit low peak aerobic capacities and maximal heart rates when compared with healthy non-disabled peers. These findings may be explained by a lower walking economy that is mainly related to their inability to adapt efficiently to positive variations in walking speed [32]. Furthermore, they present a different catecholamine response to exercise [33]. Accordingly, intervention pro‐ grams based on regular exercise should be designed by taking into account their chrono‐ tropic incompetence. On the contrary, sessions theoretically designed at moderate intensity for the general population become exhausting for participants with Down syndrome, lead‐

However, it is important to note that environmental and family factors seem to be more sig‐ nificant determinants of participation than characteristics of the subjects themselves. In fact, families who engage in physical activities themselves tend to promote similar participation for their relatives with disabilities. Conversely, inactive role models, competing demands and time pressures, unsafe environments, lack of adequate facilities, insufficient funds, and inadequate access to quality daily physical education seem to be more prevalent among populations with special needs. The establishment of short-term goals, emphasizing variety and enjoyment, and positive reinforcement through documented progress toward goals can

In summary, misconceptions and attitudinal barriers at the level of the individual, the fami‐ ly, and the community need to be addressed to integrate people with disabilities into recrea‐

Another point of interest is that physical activity comes with an inherent risk for injury. For people with intellectual disability, previous studies have reported their injury risk may be complicated by preexisting disability [26]. Accordingly it is important for caregivers, educa‐ tors and others to identify strategies to minimize risks of illness and injury related to partici‐

Fortunately, little or no sport-related injuries are reported in the literature during interven‐ tion programs based on regular exercise [32,35,36]. It may be explained, at least in part, due to the preparticipation physical examination (PPPE) and the design of specific training pro‐ grams that are adaptable to the needs of individuals with intellectual disability. This is of particular interest since injuries and discomfort may lead to participants to interrupt their

them with limited opportunities for participation in physical activities [30].

ing to undesired results and increased withdrawal rates.

help spark and sustain the motivation for participation [14, 27,34].

pation through activity adaptations and safety precautions.

training program, increasing withdrawal rates and sedentary lifestyle [37].

tional and sports activities [14].

86 Down Syndrome

#### *Problem statement*

Accumulating evidence derived from both clinical and experimental studies highlight the association of visceral obesity with a proinflammatory status in general population [11,12]. Recent studies have also reported individuals with intellectual disability suffer from lowgrade systemic inflammation that has been proposed as a pathogenic mechanism of several disorders [15]. The adipokines are important mediators of these adverse effects so that the normalization of their levels has been reported as a therapeutic target [21].

Fortunately several studies have reported both endurance and resistance training programs at low/moderate intensity may reduce proinflammatory adipokines both at early life stages and elderly in obese people without intellectual disability [25]. However, to the best of our knowledge, there is a lack of information in people, especially women, with intellectual dis‐ abilities.

Accordingly, this study was designed to assess the influence of a 10-week aerobic training program on plasmatic levels of adipokines in obese women with Down syndrome.

#### *Application area*

Healthcare costs are continuously increasing because of the increasing life expectancy among people with disabilities [1]. This is a strong argument for strengthening the role of preventive strategies, such as exercise, with the aim to reduce future costs.

However, researchers suggest that people with an intellectual disability undertake less physical activity than the general population and many rely, to some extent, on others to help them to access activities [34,38].

Currently, a wide variety of sporting activities are accessible to people with disabilities, and guidelines are available to assist caregivers, volunteers, educators and healthcare-providers in recommending activities appropriate for those people with specific conditions. These training programs should be not only effective but safe since previous studies have reported their sport-related injury risk may be complicated by preexisting disability.

#### *Research course & method used*

A 10-week aerobic training program was designed by a multidisciplinary team to re‐ duce plasmatic adipokines in obese women with Down syndrome. In order to achieve this goal, twenty obese adult women with Down syndrome volunteered for the present interventional study. They had an intelligence quotient (IQ) range of 50–69, determined by Stanford-Binet Scale, being diagnosed as having mild intellectual disability. Eleven of them were randomly assigned to perform a 10-week aerobic training program, 3 sessions/ week, consisting of warming-up followed by a main part in a treadmill (30-40 min [increas‐ ing 2 minutes and half each two weeks]) at a work intensity of 55-65% of peak heart rate (increasing a 2.5% each two weeks) and a cooling-down period. Control group included 9 age, sex and BMI matched women with Down syndrome. Fat mass percentage and fat distribution were measured.

provided the evidence that abdominal fat was significantly correlated to plasmatic levels of

How to Design an Exercise Program TO Reduce Inflammation in Obese People With Down Syndrome

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89

To the best of our knowledge this is the first study conducted exclusively in premenopausal women with intellectual disability, in attempt to keep our sample homogeneous. To date, many studies focused on the influence of regular exercise in people with intellectual disabil‐ ity have recruited mixed (males and females) groups in order to increase their sample size to strengthen research designs and increase generalization of study findings [43,44,45]. A few

However, less attention has been paid to women in spite of the higher prevalence of obe‐ sity in the latter [4]. This finding may contribute to explain women with DS are ob‐ served to have a shorter life expectancy than men with DS [47]. A major strength of the present study was that we discarded gender mismatching, which itself influences total

Further, it should be emphasized that our sample size was similar to the largest ones report‐ ed in previous exercise intervention research on persons with trisomy 21[35,36,43,44]. This is of particular interest since studying subjects with intellectual disabilities is associated with

The present protocol lasted just 10 weeks, so that it may be considered more feasible and practical for participants and guidance. In order to promote sustainability of these healthy programs based on exercise, it is essential targeting not only participants but also their pa‐ rents, caregivers, educators, etc. However the latter have received little attention so that fu‐

As was hypothesized, peak VO2max was also significantly increased after being exercised for 10 weeks. These results are lower than that of male adults with Down syndrome.[46] In this respect, it is widely known that persons with Down syndrome exhibit low peak aerobic capacities and maximal heart rates when compared with healthy non-disabled peers. This finding may be explained by a lower walking economy that is mainly related to their inabili‐ ty to adapt efficiently to positive variations in walking speed.[32] Furthermore, they present a different catecholamine response to exercise.[33] Accordingly, intervention programs based on regular exercise should be designed by taking into account their chronotropic in‐ competence. On the contrary, sessions theoretically designed at moderate intensity for the general population become exhausting for participants with Down syndrome, leading to un‐

Finally, despite the high prevalence of obesity in people with Down syndrome, it should be pointed out it may be even more prevalent in several genetic syndromes such as Prader-Willy syndrome, Bardet-Biedl syndrome, Cohen syndrome etc. Accordingly further studies

ture studies designed as cluster-randomized interventions are highly required [38].

many challenges that restrict the number of participants investigated.

studies have been conducted in males [35,36,46].

desired results and increased withdrawal rates.

on these populations are also required [10].

adiposity and fat distribution.

CRP.

Plasmatic levels of TNF-α, IL-6 and leptin were assessed by commercial ELISA kits (Immu‐ notech, MA, USA). High-sensitive C-Reactive Protein (hs-CRP) in plasma was assessed by nephelometric methods on a BN-II analyzer (Dade-Behring Diagnostics, Marburg, Germa‐ ny). Fat mass percentage was assessed by bioelectrical impedance analysis BIA (Tanita TBF521). To determine waist to hip ratio, waist and hip circumferences were measured with an anthropometric tape (Holtain Ltd). Furthermore, each participant underwent a maximal continuous treadmill graded exercised test. All outcomes at individual level were assessed firstly at baseline and secondly 72-h after the end of the intervention. Written informed con‐ sent was obtained from all their parents or legal representatives. Further this protocol was approved by an Institutional Ethics Committee.

The results were expressed as a mean (SD). The statistical analysis of the data was per‐ formed using Student's *t*-test for paired data. Pearson´s correlation coefficient (r) was used to identify potential associations among tested parameters. The significance of the changes observed was ascertained to be p<0.05.

#### *Results*

When compared to baseline results, plasmatic levels of TNF-α (11.7±2.6 vs. 10.2±2.3 pg/ml; p=0.022), IL-6 (8.0±1.7 vs. 6.6±1.4 pg/ml; p=0.014) and leptin (54.2±6.7 vs. 45.7±6.1 ng/ml; p=0.026) were significantly reduced in interventional group. Similarly, C-reactive protein level was significantly decreased after being exercised (0.62±0.11 vs. 0.53± 0.09mg/ dl;p=0.009). Regarding anthropometric measurements, both fat mass percentage (38.9±4.6 vs. 35.0±4.2%; p=0.041) and WHR (1.12±0.006 vs. 1.00±0.005 cm; p=0.038) were also reduced. We also found significant associations between WHR and IL-6 (r=0.51; p<0.001). VO2max was al‐ so increased in exercised at the end of the experience (20.2±5.8 vs. 23.7±6.3 ml/kg/min; p=0.0007) suggesting an improvement of their physical fitness.

In contrast, control group showed no changes in any of the tested parameters.

#### *Further research and Discussion*

The main finding of this study was that aerobic training reduced significantly plasmatic lev‐ els of adipokines (TNF-α, IL-6 and leptin) as well as C-reactive protein (CRP) in adult wom‐ en with Down syndrome. Similar results regarding anti-inflammatory effect of a 16-week aerobic training program have been reported in young women without intellectual disabili‐ ty [39]. Furthermore, a 6-month aerobic training program (four times/week, 45-60 min/ session) reduced plasmatic levels of TNF in adults with type 2 diabetes [40].

Another challenge of this study was to identify significant associations between plasmatic adipokines and indices of obesity in order to provide an easier, quicker, cheaper and noinvasive assessment of the outcomes. The strongest correlation was found between IL-6 and waist-to-hip ratio (WHR). Our findings not only confirmed adipokines correlated with indi‐ rect body fat mass measures in obese women without intellectual disability [41,42]. It also provided the evidence that abdominal fat was significantly correlated to plasmatic levels of CRP.

9 age, sex and BMI matched women with Down syndrome. Fat mass percentage and fat

Plasmatic levels of TNF-α, IL-6 and leptin were assessed by commercial ELISA kits (Immu‐ notech, MA, USA). High-sensitive C-Reactive Protein (hs-CRP) in plasma was assessed by nephelometric methods on a BN-II analyzer (Dade-Behring Diagnostics, Marburg, Germa‐ ny). Fat mass percentage was assessed by bioelectrical impedance analysis BIA (Tanita TBF521). To determine waist to hip ratio, waist and hip circumferences were measured with an anthropometric tape (Holtain Ltd). Furthermore, each participant underwent a maximal continuous treadmill graded exercised test. All outcomes at individual level were assessed firstly at baseline and secondly 72-h after the end of the intervention. Written informed con‐ sent was obtained from all their parents or legal representatives. Further this protocol was

The results were expressed as a mean (SD). The statistical analysis of the data was per‐ formed using Student's *t*-test for paired data. Pearson´s correlation coefficient (r) was used to identify potential associations among tested parameters. The significance of the changes

When compared to baseline results, plasmatic levels of TNF-α (11.7±2.6 vs. 10.2±2.3 pg/ml; p=0.022), IL-6 (8.0±1.7 vs. 6.6±1.4 pg/ml; p=0.014) and leptin (54.2±6.7 vs. 45.7±6.1 ng/ml; p=0.026) were significantly reduced in interventional group. Similarly, C-reactive protein level was significantly decreased after being exercised (0.62±0.11 vs. 0.53± 0.09mg/ dl;p=0.009). Regarding anthropometric measurements, both fat mass percentage (38.9±4.6 vs. 35.0±4.2%; p=0.041) and WHR (1.12±0.006 vs. 1.00±0.005 cm; p=0.038) were also reduced. We also found significant associations between WHR and IL-6 (r=0.51; p<0.001). VO2max was al‐ so increased in exercised at the end of the experience (20.2±5.8 vs. 23.7±6.3 ml/kg/min;

The main finding of this study was that aerobic training reduced significantly plasmatic lev‐ els of adipokines (TNF-α, IL-6 and leptin) as well as C-reactive protein (CRP) in adult wom‐ en with Down syndrome. Similar results regarding anti-inflammatory effect of a 16-week aerobic training program have been reported in young women without intellectual disabili‐ ty [39]. Furthermore, a 6-month aerobic training program (four times/week, 45-60 min/

Another challenge of this study was to identify significant associations between plasmatic adipokines and indices of obesity in order to provide an easier, quicker, cheaper and noinvasive assessment of the outcomes. The strongest correlation was found between IL-6 and waist-to-hip ratio (WHR). Our findings not only confirmed adipokines correlated with indi‐ rect body fat mass measures in obese women without intellectual disability [41,42]. It also

distribution were measured.

88 Down Syndrome

approved by an Institutional Ethics Committee.

p=0.0007) suggesting an improvement of their physical fitness.

In contrast, control group showed no changes in any of the tested parameters.

session) reduced plasmatic levels of TNF in adults with type 2 diabetes [40].

observed was ascertained to be p<0.05.

*Further research and Discussion*

*Results*

To the best of our knowledge this is the first study conducted exclusively in premenopausal women with intellectual disability, in attempt to keep our sample homogeneous. To date, many studies focused on the influence of regular exercise in people with intellectual disabil‐ ity have recruited mixed (males and females) groups in order to increase their sample size to strengthen research designs and increase generalization of study findings [43,44,45]. A few studies have been conducted in males [35,36,46].

However, less attention has been paid to women in spite of the higher prevalence of obe‐ sity in the latter [4]. This finding may contribute to explain women with DS are ob‐ served to have a shorter life expectancy than men with DS [47]. A major strength of the present study was that we discarded gender mismatching, which itself influences total adiposity and fat distribution.

Further, it should be emphasized that our sample size was similar to the largest ones report‐ ed in previous exercise intervention research on persons with trisomy 21[35,36,43,44]. This is of particular interest since studying subjects with intellectual disabilities is associated with many challenges that restrict the number of participants investigated.

The present protocol lasted just 10 weeks, so that it may be considered more feasible and practical for participants and guidance. In order to promote sustainability of these healthy programs based on exercise, it is essential targeting not only participants but also their pa‐ rents, caregivers, educators, etc. However the latter have received little attention so that fu‐ ture studies designed as cluster-randomized interventions are highly required [38].

As was hypothesized, peak VO2max was also significantly increased after being exercised for 10 weeks. These results are lower than that of male adults with Down syndrome.[46] In this respect, it is widely known that persons with Down syndrome exhibit low peak aerobic capacities and maximal heart rates when compared with healthy non-disabled peers. This finding may be explained by a lower walking economy that is mainly related to their inabili‐ ty to adapt efficiently to positive variations in walking speed.[32] Furthermore, they present a different catecholamine response to exercise.[33] Accordingly, intervention programs based on regular exercise should be designed by taking into account their chronotropic in‐ competence. On the contrary, sessions theoretically designed at moderate intensity for the general population become exhausting for participants with Down syndrome, leading to un‐ desired results and increased withdrawal rates.

Finally, despite the high prevalence of obesity in people with Down syndrome, it should be pointed out it may be even more prevalent in several genetic syndromes such as Prader-Willy syndrome, Bardet-Biedl syndrome, Cohen syndrome etc. Accordingly further studies on these populations are also required [10].

## **3. Conclusion**

In summary, it was concluded a 10-week aerobic training program reduced plasmatic levels of adipokines and acute phase proteins in adult obese women with Down syndrome. There‐ fore, additional long-term, well-conducted studies are required to determine whether cor‐ rection of this low-grade proinflammatory status improves clinical outcomes of people with trisomy 21.

[5] Bhaumik, S., Watson, J. M., Thorp, C. F., Tyrer, F., & Mc Grother, C. W. (2008). Body mass index in adults with intellectual disability: distribution, associations and service implications: a population-based prevalence study. *Journal of Intellectual Disability Re‐*

How to Design an Exercise Program TO Reduce Inflammation in Obese People With Down Syndrome

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91

[6] Sohler, N., Lubetkin, E., Levy, J., Soghomonian, C., & Rimmerman, A. (2009). Factors associated with obesity and coronary heart disease in people with intellectual disabil‐

[7] Elmahgoub, S. M., Lambers, S., Stegen, S., Van Laethem, C., Cambier, D., & Calders, P. (2009). The influence of combined exercise training on indices of obesity, physical fitness and lipid profile in overweight and obese adolescents with mental retarda‐

[8] Melville, CA, Boyle, S., Miller, S., Macmillan, S., Penpraze, V., Pert, C., Spanos, D., Matthews, L., Robinson, N., Murray, H., & Hankey, C. R. (2011). An open study of the effectiveness of a multi-component weight-loss intervention for adults with intel‐

[9] Ordonez, F. J., Rosety, M., & Rosety-Rodriguez, M. (2006). Influence of 12-week exer‐ cise training on fat mass percentage in adolescents with Down syndrome. *Medicine*

[10] Hamilton, S., Hankey, C. R., Miller, S., Boyle, S., & Melville, CA. (2007). A review of weight loss interventions for adults with intellectual disabilities. *Obesity Review*, 8,

[11] Inadera, H. (2008). The usefulness of circulating adipokine levels for the assessment of obesity-related health problems. *International Journal of Medical Sciences*, 5, 248-262.

[12] Popko, K., Gorska, E., Stelmaszczyk-Emmel, A., Plywaczewski, R., Stoklosa, A., Gor‐ ecka, D., Pyrzak, B., & Demkow, U. (2010). Proinflammatory cytokines Il-6 and TNFα and the development of inflammation in obese subjects. *European Journal of Medical*

[13] Suganami, T., Tanaka, M., & Ogawa, Y. (2012). Adipose tissue inflammation and ec‐ topic lipid accumulation. Endocrinology Journal Aug 9. [Epub ahead of print] [14] Murdoch, J. C., Rodger, J. C., Rao, S. S., Fletcher, C. D., & Dunnigan, M. G. (1977). Down's syndrome: an atheroma-free model? *British Medical Journal*, 2, 226-8.

[15] De Winter, C. F., Magilsen, K. W., van Alfen, J. C., Willemsen, S. P., & Evenhuis, H. M. (2011). Metabolic syndrome in 25% of older people with intellectual disability.

[16] Licastro, F., Chiappelli, M., Porcellini, E., Trabucchi, M., Marocchi, A., & Corsi, MM. (2006). Altered vessel signalling molecules in subjects with Downs syndrome. *Inter‐*

[17] Caixàs, A., Giménez-Palop, O., Broch, M., Vilardell, C., Megía, A., Simón, I., Gimé‐ nez-Pérez, G., Mauricio, D., Vendrell, J., Richart, C., & González-Clemente, J. M.

*national Journal of Immunopathology and Pharmacology*, 19, 181-5.

tion. *Journal of Strength and Conditioning Research*, 168, 1327-33.

lectual disabilities and obesity. *British Journal of Nutrition*, 105, 1553-62.

*search*, 52, 287-98.

ities. *Social Work in Health Care*, 48, 76-89.

*and Science Monitor*, 12, CR416-9.

339-45.

*Research*, 15, 120-122.

*Family Practice*, 28, 141-4.

Authors gratefully acknowledge financial support (Exp Nº211/10) by Women´s Institute (Ministry of Health and Consumer Affairs, Spanish Government)

## **Author details**

Francisco J. Ordonez1 , Gabriel Fornieles2 , Alejandra Camacho3 , Miguel A. Rosety1 , Antonio J Diaz2 , Ignacio Rosety1 , Natalia Garcia4 and Manuel Rosety-Rodriguez2\*

\*Address all correspondence to: manuel.rosetyrodriguez@uca.es

1 Human Anatomy Department. School of Sports Medicine, Spain

2 Medicine Department. School of Sports Medicine, Spain

3 Juan Ramon Jimenez Hospital, Spain

4 Pathology Department. School of Medicine, Spain

## **References**


[5] Bhaumik, S., Watson, J. M., Thorp, C. F., Tyrer, F., & Mc Grother, C. W. (2008). Body mass index in adults with intellectual disability: distribution, associations and service implications: a population-based prevalence study. *Journal of Intellectual Disability Re‐ search*, 52, 287-98.

**3. Conclusion**

90 Down Syndrome

trisomy 21.

**Author details**

Antonio J Diaz2

**References**

Francisco J. Ordonez1

In summary, it was concluded a 10-week aerobic training program reduced plasmatic levels of adipokines and acute phase proteins in adult obese women with Down syndrome. There‐ fore, additional long-term, well-conducted studies are required to determine whether cor‐ rection of this low-grade proinflammatory status improves clinical outcomes of people with

Authors gratefully acknowledge financial support (Exp Nº211/10) by Women´s Institute

, Alejandra Camacho3

[1] Tenenbaum, A., Chavkin, M., Wexler, I. D., Korem, M., & Merrick, J. (2012). Morbidi‐ ty and hospitalizations of adults with Down syndrome. *Research in Developmental Dis‐*

[2] de Winter, C. F., Magilsen, K. W., van Alfen, J. C., Penning, C., & Evenhuis, H. M. (2009). Prevalence of cardiovascular risk factors in older people with intellectual dis‐ ability. *American Journal on Intellectual and Developmental Disabilities*, 114, 427-36.

[3] De Winter, C. F., Bastiaanse, L. P., Hilgenkamp, T. I., Evenhuis, H. M., & Echteld, M. A. (2012). Overweight and obesity in older people with intellectual disability. *Re‐*

[4] González-Agüero, A., Ara, I., Moreno, L. A., Vicente-Rodríguez, G., & Casajús, J. A. (2011). Fat and lean masses in youths with Down syndrome: gender differences. *Re‐*

, Miguel A. Rosety1

and Manuel Rosety-Rodriguez2\*

,

(Ministry of Health and Consumer Affairs, Spanish Government)

, Gabriel Fornieles2

\*Address all correspondence to: manuel.rosetyrodriguez@uca.es

1 Human Anatomy Department. School of Sports Medicine, Spain

2 Medicine Department. School of Sports Medicine, Spain

*search in Developmental Disabilities*, 33, 398-405.

*search in Developmental Disabilities*, 32, 1685-93.

4 Pathology Department. School of Medicine, Spain

, Natalia Garcia4

, Ignacio Rosety1

3 Juan Ramon Jimenez Hospital, Spain

*abilities*, 33, 435-41.


(2008). Adult subjects with Prader-Willi syndrome show more low-grade systemic inflammation than matched obese subjects. *Journal of Endocrinological Investigation*, 31, 169-75.

[28] Grandisson, M., Tétreault, S., & Freeman, A. R. (2012). Enabling integration in sports for adolescents with intellectual disabilities. *Journal of Applied Research in Intellectual*

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[29] Weiss, J., Diamond, T., Demark, J., & Lovald, B. (2003). Involvement in Special Olym‐ pics and its relations to self-concept and actual competency in participants with de‐

[30] King, G., Law, M., King, S., Rosenbaum, P., Kertoy, M. K., & Young, N. L. (2003). A conceptual model of the factors affecting the recreation and leisure participation of children with disabilities. *Physical and Occupational Therapy in Pediatrics*, 23, 63-90. [31] Terblanche, E., & Boer, P. H. (2012). The functional fitness capacity of adults with Down syndrome in South Africa. Journal of Intellectual Disability Research Jul 10.

[32] Mendonca, G. V., Pereira, F. D., Morato, P. P., & Fernhall, B. (2010). Walking econo‐ my of adults with Down syndrome. *International Journal of Sports Medicine*, 31, 10-15.

[33] Fernhall, B., Baynard, T., Collier, S. R., et al. (2009). Catecholamine response to maxi‐ mal exercise in persons with Down syndrome. *American Journal of Cardiology*, 103,

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92 Down Syndrome

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[40] Kadoglou, N. P., Iliadis, F., Angelopoulou, N., Perrea, D., Ampatzidis, G., Liapis, C. D., & Alevizos, M. (2007). The anti-inflammatory effects of exercise training in pa‐ tients with type 2 diabetes mellitus. *European Journal of Cardiovascular Prevention and Rehabilitation*, 14, 837-43.

**Chapter 6**

**Heart Diseases in Down Syndrome**

Additional information is available at the end of the chapter

Down syndrome (trisomy 21) is the common disorder among chromosomal anomalies. Trisomy 21 remains the commonest with its incidence 1:650 – 1: 1000 live births (Hassold TA and Sherman S 2000). The clinical manifestations of Down syndrome (DS) are numer‐ ous and can present in any body system. The most significant include intellectual impair‐ ment, short stature, heart disease, digestive disorders and orthopedic abnormalities

Cardiac malformations present at birth are an important component of pediatric cardiovas‐ cular disease and contribute a major percentage of clinically significant birth defects with an estimated prevalence of 4 to 5 per 1000 live births. It is estimated that 4 to 10 live born in‐ fants per 1000 have cardiac malformation, 40% of which are diagnosed in the first year of life.(Hoffman J I, 1990 ; Moller J H et al, 1993). Congenital heart defect are the most common of all birth defects, which is found to affect nearly 1% of newborns, and their frequency in spontaneously aborted pregnancies is estimated to be tenfold higher (Behrman RE et al., 2000). In the year 2000, prevalence of CHD in the pediatric population was estimated at ap‐ proximately 623000 (320000 with single lesion, 165000 with moderately complex disease, and 138000 with highly complex CHD). (Hoffman J I et al, 2004) Among the CHD the inci‐ dence of ventricular septal defect (VSD) has been demonstrated to be high as 5% in 2 inde‐ pendent cohorts of 5000 serial newborns, 5000 serial premature infants. (Roguin N et al.,

The causes for CHD can be categorized in to three major groups such as chromosomal, sin‐

Its association of congenital heart disease is well known. Among all cases of congenital heart diseases, 4%-10% are associated with Down syndrome, and 40%-60% of Down syndrome patients present congenital heart disease. Cardiac malformation in DS is the principal cause

and reproduction in any medium, provided the original work is properly cited.

© 2013 Rashid; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

gle gene disorder (10-15%) and multiple factors (85-90%). (Payne M et al., 1995)

A. K. M. Mamunur Rashid

http://dx.doi.org/10.5772/46009

**1. Introduction**

(Ramakrishnan V, 2011).

1995; Du Z D et al., 1996)


## **Chapter 6**

## **Heart Diseases in Down Syndrome**

A. K. M. Mamunur Rashid

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46009

## **1. Introduction**

[40] Kadoglou, N. P., Iliadis, F., Angelopoulou, N., Perrea, D., Ampatzidis, G., Liapis, C. D., & Alevizos, M. (2007). The anti-inflammatory effects of exercise training in pa‐ tients with type 2 diabetes mellitus. *European Journal of Cardiovascular Prevention and*

[41] Ackermann, D., Jones, J., Barona, J., Calle, M. C., Kim, J. E., La Pia, B., Volek, J. S., Mc Intosh, M., Kalynych, C., Najm, W., Lerman, R. H., & Fernandez, M. L. (2011). Waist circumference is positively correlated with markers of inflammation and negatively with adiponectin in women with metabolic syndrome. *Nutrition Research*, 31, 197-204.

[42] Bahceci, M., Gokalp, D., Bahceci, S., Tuzcu, A., Atmaca, S., & Arikan, S. (2007). The correlation between adiposity and adiponectin, tumor necrosis factor alpha, interleu‐ kin-6 and high sensitivity C-reactive protein levels. Is adipocyte size associated with

[43] González-Agüero, A., Vicente-Rodríguez, G., Gómez-Cabello, A., Ara, I., Moreno, L. A., & Casajús, J. A. (2012 A). A 21-week bone deposition promoting exercise pro‐ gramme increases bone mass in young people with Down syndrome. *Dev Med Child*

[44] Cowley, P. M., Ploutz-Snyder, L. L., Baynard, T., Heffernan, K. S., Jae, S. Y., Hsu, S., Lee, M., Pitetti, K. H., Reiman, M. P., & Fernhall, B. (2011). The effect of progressive resistance training on leg strength, aerobic capacity and functional tasks of daily liv‐ ing in persons with Down syndrome. *Disability and Rehabilitation*, 33, 2229-2236.

[45] Mendonca, G. V., Pereira, F. D., & Fernhall, B. (2011). Effects of combined aerobic and resistance exercise training in adults with and without Down syndrome. *Archives*

[46] Mendonca, G. V., & Pereira, F. D. (2009). Influence of long-term exercise training on submaximal and peak aerobic capacity and locomotor economy in adult males with

[47] Tyrer, F., Smith, L. K., & Mc Grother, C. W. (2007). Mortality in adults with moderate to profound intellectual disability: a population-based study. *Journal of Intellectual*

*of Physical Medicine and Rehabilitation*, 92, 37-45.

Down's syndrome. *Med Sci Monit*, 15, 33-39.

*Disability Research*, 51, 520-7.

inflammation in adults? *Journal of Endocrinological Investigation*, 30, 210-214.

*Rehabilitation*, 14, 837-43.

94 Down Syndrome

*Neurol*, 54, 552-6.

Down syndrome (trisomy 21) is the common disorder among chromosomal anomalies. Trisomy 21 remains the commonest with its incidence 1:650 – 1: 1000 live births (Hassold TA and Sherman S 2000). The clinical manifestations of Down syndrome (DS) are numer‐ ous and can present in any body system. The most significant include intellectual impair‐ ment, short stature, heart disease, digestive disorders and orthopedic abnormalities (Ramakrishnan V, 2011).

Cardiac malformations present at birth are an important component of pediatric cardiovas‐ cular disease and contribute a major percentage of clinically significant birth defects with an estimated prevalence of 4 to 5 per 1000 live births. It is estimated that 4 to 10 live born in‐ fants per 1000 have cardiac malformation, 40% of which are diagnosed in the first year of life.(Hoffman J I, 1990 ; Moller J H et al, 1993). Congenital heart defect are the most common of all birth defects, which is found to affect nearly 1% of newborns, and their frequency in spontaneously aborted pregnancies is estimated to be tenfold higher (Behrman RE et al., 2000). In the year 2000, prevalence of CHD in the pediatric population was estimated at ap‐ proximately 623000 (320000 with single lesion, 165000 with moderately complex disease, and 138000 with highly complex CHD). (Hoffman J I et al, 2004) Among the CHD the inci‐ dence of ventricular septal defect (VSD) has been demonstrated to be high as 5% in 2 inde‐ pendent cohorts of 5000 serial newborns, 5000 serial premature infants. (Roguin N et al., 1995; Du Z D et al., 1996)

The causes for CHD can be categorized in to three major groups such as chromosomal, sin‐ gle gene disorder (10-15%) and multiple factors (85-90%). (Payne M et al., 1995)

Its association of congenital heart disease is well known. Among all cases of congenital heart diseases, 4%-10% are associated with Down syndrome, and 40%-60% of Down syndrome patients present congenital heart disease. Cardiac malformation in DS is the principal cause

of mortality in the first two years of life. (Rodriguez LH, 1984; Stoll C, et al., 1998) This con‐ genital heart disease contributes significantly to the morbidity and mortality of children with Down syndrome, who may develop congestive heart failure, pulmonary vascular dis‐ ease, pneumonia, or failure to thrive. In the first few days life symptoms or signs may be absent or minimal despite the presence of significant congenital heart disease. The character‐ istic heart defects seen in Down syndrome derives from the abnormal development of endo‐ cardial cushions and results in a spectrum of defects involving the atrioventricular septum and valves. Accounting for approximately 63% of all DS-CHD, their lesion varies in severity from persistent of the common atrioventricular canal and membranous ventricular septal defects to ostiumprimum patency with valvular anomalies. (Cooney T P et al., 1982; Ander‐ son R H, 1991) The specificity of atriventricularseptal defects for trisomy 21 is emphasized by the observation that individuals with Down syndrome account for 70% of all atriventri‐ cularseptal defects. (Ferencz C et al.,1997) This is followed by patent ductus arteriosus and atrial septal defects. Other forms of complex heart disease can occur including overriding aorta and Tetralogy of fallot. (Berr C and Borghi E, 1990) The hypothesis suggests the exis‐ tence of a gene or gene clusters on chromosome 21 which is involved in cell adhesion and likely plays an important role in valvuloseptal morphogenesis, but when over expressed, re‐ sult in the defects of Down syndrome – congenital heart disease. (Barlow G M et al., 2001)

MX1/2.(Hubert RS et al., 1997) Recent study speculate that the over expression of Down syndrome cell adhesion molecule may have the potential to perturb epithelial-mesenchy‐ mal transformation and/or the migration and proliferation of mesenchymal cells, and possibly thus contribute to the increased intercellular adhesion seen in DS cushion fibro‐ blasts and abnormal cushion development seen in DS-CHD. The DSCAM gene consti‐ tutes a large part of the DS-CHD region, spanning more than 840Kb of the region between D21S3 and (PFKL) as determined from BAC contigs (Yamakawa K et al., 1998) and genomic sequence analysis.( Hattori M et al., 2000) The study for DS-CHD suggests that the candidate region for DS-CHD may be narrowed to D21S3 (Defined by VSD), through PFKL (defined by TOF), comprising 5.5 Mb. This represents significant reduction of the previously described candidate region, which spanned 10.5 Mb from D21S55 to the telomere. (Korenberg JR et al., 1992; Korenberg JR et al., 1994) This study supports the hypothesis that trisomy for a gene in the DS-CHD candidate region is essential for the production of DS-CHD including TOF and VSD, trisomy for additional genes located in the telomere and other regions likely contributes the phenotypic variability of DS-CHD.

Heart Diseases in Down Syndrome http://dx.doi.org/10.5772/46009 97

**3. Type of heart defects in children with Down syndrome**

defects, and three combinations of heart defects.)(Tubman TRJ et al., 1991)

other heart defects 20(12.5%). (Ramakrishnan, V. 2011)

**4.1. Atroventricular Septal Defects (AVSDs)**

**•** Atrioventricular septal defects (AVSDs)- These are the most common in children with

In a study by TRJ Tubman & et al. among 34 babies of Down syndrome had congenital heart disease detected by echocardiography (13 had atrioventricularseptal defects, seven secen‐ dum atrial septal defects, six solitary patent ductusarteriosus, five isolated ventricular septal

Another study showed the association between CHD and DS in atrioventricularseptal defect 56 (35%), ventricular septal defect 48 (30%),ASD 14 (8.7%), TOF 8(5%), PDA 18 (11.2%) and

These heart defects are marked by a hole in the wall between the top chambers (atria) and bottom chambers (ventricles) and one common valve between the two atria. In some cases,

(Barlow GM et al., 2001)

Down syndrome.

**•** Atrial Septal Defects (ASDs)

**•** Tetralogy of Fallot (TOF)

**4. Presentations**

**•** Patent Ductus arteriosus (PDA)

## **2. Etiology and genetics**

Down syndrome which is normally caused by trisomy 21 is a major cause of congenital heart disease and provides an important model with which to link individual to the pathways controlling heart development. The characteristic heart defect seen in Down syndrome derives from the abnormal development of the endocardial cushions and re‐ sults in a spectrum of defects involving atrioventricular septum and valves. Accounting for approximately 63% of all DS-CHD,( Van PR et al., 1996) these lesions vary in severity from persistence of the common atrioventricular canal and membranous ventricular sep‐ tal defects to Ostium primum patency with valvular anomalies. (Cooney TP et al., 1982; Anderson RH, 1991) Independent and intersecting approaches to identifying the gene(s) for DS-CHD have included mapping genes known to be involved in cardiac develop‐ ment (none of which localized to chromosome 21) and studying rare individuals with CHD and partial duplications of chromosome 21. There are number of genetic tests that can assist the clinician in diagnosing genetic alterations in the child with CHD. These in‐ clude cytogenetic technique, fluorescence in situ hybridization (FISH), and DNA muta‐ tion analysis.(Pierpont ME et al., 2007) The studies initially suggested that subsets of the DS phenotype were associated with three copies of chromosome band 21q22.2-22.3(Rah‐ mani Z et al., 1989; McCormick MK et al., 1989; Korenberg JR et al., 1990) and later, that DS-CHD was caused by the over expression of genes in the region including D21S55 through the telomere.(Korenberg JR etal., 1992; Delabar JM et al.,1993; Korenberg JR et al., 1994) Another work focused on the identification of a transcriptional map of DS-CHD region using a 3.5 Mb contiguous clone array covering the interval from D21S55 through MX1/2.(Hubert RS et al., 1997) Recent study speculate that the over expression of Down syndrome cell adhesion molecule may have the potential to perturb epithelial-mesenchy‐ mal transformation and/or the migration and proliferation of mesenchymal cells, and possibly thus contribute to the increased intercellular adhesion seen in DS cushion fibro‐ blasts and abnormal cushion development seen in DS-CHD. The DSCAM gene consti‐ tutes a large part of the DS-CHD region, spanning more than 840Kb of the region between D21S3 and (PFKL) as determined from BAC contigs (Yamakawa K et al., 1998) and genomic sequence analysis.( Hattori M et al., 2000) The study for DS-CHD suggests that the candidate region for DS-CHD may be narrowed to D21S3 (Defined by VSD), through PFKL (defined by TOF), comprising 5.5 Mb. This represents significant reduction of the previously described candidate region, which spanned 10.5 Mb from D21S55 to the telomere. (Korenberg JR et al., 1992; Korenberg JR et al., 1994) This study supports the hypothesis that trisomy for a gene in the DS-CHD candidate region is essential for the production of DS-CHD including TOF and VSD, trisomy for additional genes located in the telomere and other regions likely contributes the phenotypic variability of DS-CHD. (Barlow GM et al., 2001)

## **3. Type of heart defects in children with Down syndrome**


of mortality in the first two years of life. (Rodriguez LH, 1984; Stoll C, et al., 1998) This con‐ genital heart disease contributes significantly to the morbidity and mortality of children with Down syndrome, who may develop congestive heart failure, pulmonary vascular dis‐ ease, pneumonia, or failure to thrive. In the first few days life symptoms or signs may be absent or minimal despite the presence of significant congenital heart disease. The character‐ istic heart defects seen in Down syndrome derives from the abnormal development of endo‐ cardial cushions and results in a spectrum of defects involving the atrioventricular septum and valves. Accounting for approximately 63% of all DS-CHD, their lesion varies in severity from persistent of the common atrioventricular canal and membranous ventricular septal defects to ostiumprimum patency with valvular anomalies. (Cooney T P et al., 1982; Ander‐ son R H, 1991) The specificity of atriventricularseptal defects for trisomy 21 is emphasized by the observation that individuals with Down syndrome account for 70% of all atriventri‐ cularseptal defects. (Ferencz C et al.,1997) This is followed by patent ductus arteriosus and atrial septal defects. Other forms of complex heart disease can occur including overriding aorta and Tetralogy of fallot. (Berr C and Borghi E, 1990) The hypothesis suggests the exis‐ tence of a gene or gene clusters on chromosome 21 which is involved in cell adhesion and likely plays an important role in valvuloseptal morphogenesis, but when over expressed, re‐ sult in the defects of Down syndrome – congenital heart disease. (Barlow G M et al., 2001)

Down syndrome which is normally caused by trisomy 21 is a major cause of congenital heart disease and provides an important model with which to link individual to the pathways controlling heart development. The characteristic heart defect seen in Down syndrome derives from the abnormal development of the endocardial cushions and re‐ sults in a spectrum of defects involving atrioventricular septum and valves. Accounting for approximately 63% of all DS-CHD,( Van PR et al., 1996) these lesions vary in severity from persistence of the common atrioventricular canal and membranous ventricular sep‐ tal defects to Ostium primum patency with valvular anomalies. (Cooney TP et al., 1982; Anderson RH, 1991) Independent and intersecting approaches to identifying the gene(s) for DS-CHD have included mapping genes known to be involved in cardiac develop‐ ment (none of which localized to chromosome 21) and studying rare individuals with CHD and partial duplications of chromosome 21. There are number of genetic tests that can assist the clinician in diagnosing genetic alterations in the child with CHD. These in‐ clude cytogenetic technique, fluorescence in situ hybridization (FISH), and DNA muta‐ tion analysis.(Pierpont ME et al., 2007) The studies initially suggested that subsets of the DS phenotype were associated with three copies of chromosome band 21q22.2-22.3(Rah‐ mani Z et al., 1989; McCormick MK et al., 1989; Korenberg JR et al., 1990) and later, that DS-CHD was caused by the over expression of genes in the region including D21S55 through the telomere.(Korenberg JR etal., 1992; Delabar JM et al.,1993; Korenberg JR et al., 1994) Another work focused on the identification of a transcriptional map of DS-CHD region using a 3.5 Mb contiguous clone array covering the interval from D21S55 through

**2. Etiology and genetics**

96 Down Syndrome


In a study by TRJ Tubman & et al. among 34 babies of Down syndrome had congenital heart disease detected by echocardiography (13 had atrioventricularseptal defects, seven secen‐ dum atrial septal defects, six solitary patent ductusarteriosus, five isolated ventricular septal defects, and three combinations of heart defects.)(Tubman TRJ et al., 1991)

Another study showed the association between CHD and DS in atrioventricularseptal defect 56 (35%), ventricular septal defect 48 (30%),ASD 14 (8.7%), TOF 8(5%), PDA 18 (11.2%) and other heart defects 20(12.5%). (Ramakrishnan, V. 2011)

## **4. Presentations**

#### **4.1. Atroventricular Septal Defects (AVSDs)**

These heart defects are marked by a hole in the wall between the top chambers (atria) and bottom chambers (ventricles) and one common valve between the two atria. In some cases, there might not be a hole between the bottom chambers. Or the valves may be joined togeth‐ er, but either or both might leak.

**4.4. Patent Ductus Anteriosus (PDA)**

This anomaly includes four different heart problems:

**•** Thickening of the right bottom chamber (ventricle)

patient of TOF with DS at birth. (AKMM Rashid et al., 2009)

to bypass the baby's lungs.

poor weight gain.

of fallot.

all three

**5. Case**

**4.5. Tetralogy of fallot**

This defect is the continuance of a direct connection between the aorta and the lung (pulmo‐ nary) artery, which normally closes shortly after birth. A baby in the womb is supplied oxy‐ gen by the placenta via the umbilical cord. The baby's lungs are not expanded and require only a small amount blood for them to grow. The ductus is a blood vessel that allows blood

Heart Diseases in Down Syndrome http://dx.doi.org/10.5772/46009 99

If the ductus has partially closed and only a narrow connection remains, the baby won't show symptoms. If the connection is larger, the baby may be breathless and tired and show

A small percentage of babies with Down syndrome have this complex heart condition which combines the most common defect associated with Down syndrome, AVSD, with Tetralogy

**•** Narrowed pulmonary artery (from heart to lungs) or the area under or above the valve, or

The combination of these defects early in life almost seems to balance out such that the child may be rather blue, but not too breathless. There can, of course, be too much blueness or too

In Tetralogy of fallot (TOF), often caynosis is not present at birth but increasing hypertrophy of the right ventricular infundibulum and cyanosis occur usually in the later part of infancy. But cyanosis is present since birth if Tetralogy of Fallot is accompanied with Down Syn‐ drome. This may be due to increased hypertrophy of the right ventricular infundibulum in

A case of eleven months boy was admitted in a hospital with the complaints of bluish dis‐ coloration of lip and finger since birth and low grade fever, cough for seven days. Bluish discoloration aggravates during crying. He was born to an elderly mother and was com‐ pletely immunized. There was no such illness in the family. On examination the child was cyanosed, heart rate 130/m, weight 7.5 kg. He had got mongoloid face with flat occiput, de‐ pressed nasal bridge, upward slanting of eyes, medial epicanthic fold. There was gap be‐

**•** A hole between the top chambers and a hole between the bottom chambers

**•** Combined mitral and tricuspid valves (common atrioventricular valve)

much breathlessness, depending on the severity of the different conditions.

Because of the high pressure in the left ventricle which is needed to pump the blood around the body, blood is forced through the holes in the central heart wall (septum) when the ven‐ tricles contracts. This increases the pressure in the right ventricles. This increased pressure (pulmonary hypertension) results in excess blood flow to the lung.

Some of the early symptoms seen are difficulty in eating, weight gain, fast irregular breath‐ ing and a degree of cyanosis (blueness) particularly noticeable around the mouth, fingers and toes. Clinical examination may show an enlarged heart and liver, and a diagnosis of heart failure may be given. This term, not all children will exhibit symptoms early in life, and those that do will not always show all of these features.

#### **4.2. Ventricular Septal Defects (VSDs)**

In this defect there is a hole between the bottom clambers (pumping chambers or ventricles). Because of the higher pressure in the left side of the heart this allows oxygenated blood to flow through the hole from the left to the right side of the heart and back to the lungs in addition to the normal flow. The amount of blood flow from the left to right ventricle de‐ pends on the size of the hole and on the pressure between the ventricles. In other words, the higher the rate of flow means more strain on the heart. The abnormal blood flow is responsi‐ ble for the murmur that may be heard.

Generally patients with a small VSD will not exhibit symptoms (they are asymptomatic) and the problem may only be found when a murmur is detected upon routine examination. Pa‐ tients with a moderate VSD may breathe quickly, exhibit poor weight gain and be slower at eating. These children are also much more prone to chest infection. This tends to be more pronounced when the hole is large.

#### **4.3. Atrial Septal Defects (ASDs)**

In this defect there is a hole between the top chambers (receiving chambers or atria). Because of the higher pressure in the left side of the heart, oxygenated blood flows through the hole from the left to the right side, and back to the lungs, in addition to the normal flow.

There are three types of atrial septal defects; the most common is when there is a hole in the middle of the central heart wall. Holes in the lower part of the septum, called primum defect (partial atrioventricularseptal defect), are often associated with a problem of the mitral valve that often results in a leak. Less common are sinus venosus defects or holes in the top of the septum. These are associated with an abnormality of the right upper lung vein.

Generally patients with an ASD defect will exhibit no symptoms and the problem is only found when a routine clinical examination detects a heart murmur. Occasionally children with this problem will exhibit poor weight gain and a failure to thrive, and if there is mitral valve leakage there may be early symptoms of breathlessness.

## **4.4. Patent Ductus Anteriosus (PDA)**

This defect is the continuance of a direct connection between the aorta and the lung (pulmo‐ nary) artery, which normally closes shortly after birth. A baby in the womb is supplied oxy‐ gen by the placenta via the umbilical cord. The baby's lungs are not expanded and require only a small amount blood for them to grow. The ductus is a blood vessel that allows blood to bypass the baby's lungs.

If the ductus has partially closed and only a narrow connection remains, the baby won't show symptoms. If the connection is larger, the baby may be breathless and tired and show poor weight gain.

#### **4.5. Tetralogy of fallot**

there might not be a hole between the bottom chambers. Or the valves may be joined togeth‐

Because of the high pressure in the left ventricle which is needed to pump the blood around the body, blood is forced through the holes in the central heart wall (septum) when the ven‐ tricles contracts. This increases the pressure in the right ventricles. This increased pressure

Some of the early symptoms seen are difficulty in eating, weight gain, fast irregular breath‐ ing and a degree of cyanosis (blueness) particularly noticeable around the mouth, fingers and toes. Clinical examination may show an enlarged heart and liver, and a diagnosis of heart failure may be given. This term, not all children will exhibit symptoms early in life,

In this defect there is a hole between the bottom clambers (pumping chambers or ventricles). Because of the higher pressure in the left side of the heart this allows oxygenated blood to flow through the hole from the left to the right side of the heart and back to the lungs in addition to the normal flow. The amount of blood flow from the left to right ventricle de‐ pends on the size of the hole and on the pressure between the ventricles. In other words, the higher the rate of flow means more strain on the heart. The abnormal blood flow is responsi‐

Generally patients with a small VSD will not exhibit symptoms (they are asymptomatic) and the problem may only be found when a murmur is detected upon routine examination. Pa‐ tients with a moderate VSD may breathe quickly, exhibit poor weight gain and be slower at eating. These children are also much more prone to chest infection. This tends to be more

In this defect there is a hole between the top chambers (receiving chambers or atria). Because of the higher pressure in the left side of the heart, oxygenated blood flows through the hole

There are three types of atrial septal defects; the most common is when there is a hole in the middle of the central heart wall. Holes in the lower part of the septum, called primum defect (partial atrioventricularseptal defect), are often associated with a problem of the mitral valve that often results in a leak. Less common are sinus venosus defects or holes in the top of the

Generally patients with an ASD defect will exhibit no symptoms and the problem is only found when a routine clinical examination detects a heart murmur. Occasionally children with this problem will exhibit poor weight gain and a failure to thrive, and if there is mitral

from the left to the right side, and back to the lungs, in addition to the normal flow.

septum. These are associated with an abnormality of the right upper lung vein.

valve leakage there may be early symptoms of breathlessness.

(pulmonary hypertension) results in excess blood flow to the lung.

and those that do will not always show all of these features.

er, but either or both might leak.

98 Down Syndrome

**4.2. Ventricular Septal Defects (VSDs)**

ble for the murmur that may be heard.

pronounced when the hole is large.

**4.3. Atrial Septal Defects (ASDs)**

A small percentage of babies with Down syndrome have this complex heart condition which combines the most common defect associated with Down syndrome, AVSD, with Tetralogy of fallot.

This anomaly includes four different heart problems:


The combination of these defects early in life almost seems to balance out such that the child may be rather blue, but not too breathless. There can, of course, be too much blueness or too much breathlessness, depending on the severity of the different conditions.

In Tetralogy of fallot (TOF), often caynosis is not present at birth but increasing hypertrophy of the right ventricular infundibulum and cyanosis occur usually in the later part of infancy. But cyanosis is present since birth if Tetralogy of Fallot is accompanied with Down Syn‐ drome. This may be due to increased hypertrophy of the right ventricular infundibulum in patient of TOF with DS at birth. (AKMM Rashid et al., 2009)

## **5. Case**

A case of eleven months boy was admitted in a hospital with the complaints of bluish dis‐ coloration of lip and finger since birth and low grade fever, cough for seven days. Bluish discoloration aggravates during crying. He was born to an elderly mother and was com‐ pletely immunized. There was no such illness in the family. On examination the child was cyanosed, heart rate 130/m, weight 7.5 kg. He had got mongoloid face with flat occiput, de‐ pressed nasal bridge, upward slanting of eyes, medial epicanthic fold. There was gap be‐ tween the first and second toes with clinodactyly. On examination of the precordium there was left parasternal heave, pansystolic murmur was present in the lower sternal border. There was motor developmental delay. The boy was clinically diagnosed with congenital cy‐ anotic heart disease with Down syndrome. On investigation his hemoglobin was 78%, Total leucocyte count 14700/cum, Neutrophil 82%, X – Ray chest had the feature of boot shaped cardiac shadow. ECG showed right ventricular hypertrophy. Karyotyping showed trisomy 21. Tetralogy of fallot was detected by Echocardiogram. Finally the child was diagnosed as Down Syndrome with Tetralogy of Fallot. (AKMM Rashid et al., 2009)

**Figure 3.** X-ray chest showing : boot shaped heart.

**7. Diagnosis**

**6. Other heart related problems in Down syndrome**

complete AV canals or large ventricular septal defects.

the absence of physical findings.(Shashi V et al., 2002)

In addition to the heart defects associated with Down syndrome, high blood pressure in the lungs (pulmonary hypertension) is more common in people with Down syndrome. This high blood pressure may be a result of malformation of the lung tissue, but the exact cause is not known. High blood pressure may limit the amount of blood flow to the lungs and therefore decrease the likelihood of symptoms of congestive heart failure seen in babies with

Heart Diseases in Down Syndrome http://dx.doi.org/10.5772/46009 101

All babies that have been diagnosed with Down syndrome should have a cardiology evalua‐ tion because of the high incidence of associated congenital heart defects. A good history and physical examination should be performed in all Down syndrome children to rule out any obvious heart defect. Early diagnosis of congenital heart disease particularly of large left to right shunts, could enable a paediatrician to follow the baby carefully, to start medical treat‐ ment with diuretics and digoxin at an earlier stage and possibly to plan for earlier surgical intervention should this be indicated. Babies should be seen as early in life as possible, pref‐

Electocardiogram can be very helpful in making the diagnosis of AV canal defect, even in

Echocardiography has to be performed routinely early in life in Down syndrome can detect congenital heart disease that might otherwise be missed. Early detection may help prevent

erably in the first six months of life before pulmonary vascular disease can develop.

**Figure 1.** Patient with Down syndrome.

**Figure 2.** Echocardiogram showing Tetralogy of Fallot.

**Figure 3.** X-ray chest showing : boot shaped heart.

## **6. Other heart related problems in Down syndrome**

In addition to the heart defects associated with Down syndrome, high blood pressure in the lungs (pulmonary hypertension) is more common in people with Down syndrome. This high blood pressure may be a result of malformation of the lung tissue, but the exact cause is not known. High blood pressure may limit the amount of blood flow to the lungs and therefore decrease the likelihood of symptoms of congestive heart failure seen in babies with complete AV canals or large ventricular septal defects.

## **7. Diagnosis**

tween the first and second toes with clinodactyly. On examination of the precordium there was left parasternal heave, pansystolic murmur was present in the lower sternal border. There was motor developmental delay. The boy was clinically diagnosed with congenital cy‐ anotic heart disease with Down syndrome. On investigation his hemoglobin was 78%, Total leucocyte count 14700/cum, Neutrophil 82%, X – Ray chest had the feature of boot shaped cardiac shadow. ECG showed right ventricular hypertrophy. Karyotyping showed trisomy 21. Tetralogy of fallot was detected by Echocardiogram. Finally the child was diagnosed as

Down Syndrome with Tetralogy of Fallot. (AKMM Rashid et al., 2009)

**Figure 1.** Patient with Down syndrome.

100 Down Syndrome

**Figure 2.** Echocardiogram showing Tetralogy of Fallot.

All babies that have been diagnosed with Down syndrome should have a cardiology evalua‐ tion because of the high incidence of associated congenital heart defects. A good history and physical examination should be performed in all Down syndrome children to rule out any obvious heart defect. Early diagnosis of congenital heart disease particularly of large left to right shunts, could enable a paediatrician to follow the baby carefully, to start medical treat‐ ment with diuretics and digoxin at an earlier stage and possibly to plan for earlier surgical intervention should this be indicated. Babies should be seen as early in life as possible, pref‐ erably in the first six months of life before pulmonary vascular disease can develop.

Electocardiogram can be very helpful in making the diagnosis of AV canal defect, even in the absence of physical findings.(Shashi V et al., 2002)

Echocardiography has to be performed routinely early in life in Down syndrome can detect congenital heart disease that might otherwise be missed. Early detection may help prevent complications such as pulmonary vascular disease that may adversely affect the outcome of cardiac surgery.

**9. Long-term outlook**

**Abbreviation**

ASD – Atrial Septal Defect

BAC- Beta-site APP –Cleaving

CHD- Congenital Heart Disease

DS- Down syndrome

MX- Myxovirus resistance

TOF- Tetralogy of fallot

BT- Blalock Taussig

**Author details**

A. K. M. Mamunur Rashid

PDA- Patent Ductus Arteriosus

VSD-Ventricular Septal defect

PFKL- Phosphofructo-kinase liver types

AVSD- Atrioventricular Septal Defect

DSCAM- Down syndrome cell adhesion molecule

Dept. of Pediatrics, Khulna Medical College, Khulna, Bangladesh

Over all, survival beyond one year of age is 85 percent in all children with Down syndrome. Over 50 percent of individuals with Down syndrome live to be greater than 50 years of age.

Heart Diseases in Down Syndrome http://dx.doi.org/10.5772/46009 103

Congenital heart disease is the most common causes of death in early childhood. However, as of the late 1980s, 70 percent of children with Down syndrome and congenital heart dis‐ ease lived beyond their first birth day with improved medical and surgical care, these num‐

bers continue to improve. (Cincinnati Children's hospital medical Center, 2009)

Occasionally a repeat electrocardiogram, chest x-ray, or echocardiogram is performed to fur‐ ther evaluate clinical changes. These tests are likely to be repeated before surgical repair is recommended.

Rarely, a cardiac catheterization is required for complete evaluation prior to corrective sur‐ gery especially in patients where elevated pressures in the lungs are a concern.

## **8. Treatment**

Children with Down syndrome and symptoms of congestive heart failure can be initially managed medically with the use of diuretics, blood pressure medications to allow the heart to eject more blood out to the body rather than out to the lungs; and/or digoxin, a medica‐ tion and to improve the pumping ability of the heart.

If the baby is having difficulty with feeding and weight gain, nasogastric tube feeding with calorie formula or fortified breast milk can be used to help with growth.

These are all temporary solutions to allow the baby to grow while deciding if and when surgery is indicated. If the baby has no signs of heart failure or is controlled well with medications, the decisions for surgical closure can be delayed. The decision must be indi‐ vidualized to each child's physical state as well as the family's concerns. The majority of cases of AVSD usually require surgical intervention; this generally takes place within the first six months of life.

Many VSD, will close spontaneously or get much smaller, so, it is normal practice to leave a child with a small or moderate VSD and monitor their progress before deciding to operate. Surgery may be needed if there is failure to thrive despite medication, or concern about pul‐ monary hypertension. If a large VSD is present, surgery is almost always recommended.

Small holes in ASD which allows little blood flow from left to right generally causes no problems. If they are located in the middle portion of the central heart wall, they may even close on their own. However, moderate and large holes do not close, and the extra work over the years places a strain of the right side of the heart causing an enlargement of both pumping chambers. Therefore, Surgery is recommended in the first few years of life or larg‐ er holes, before excessive strain has been placed on the heart.

If the ductus open for more than three months, it is unlikely to close on its own and surgical closure is imperative.

The types of surgery in TOF depend on the severity of the AVSD or the Fallots. Usually the children are quite blue and require a BT shunt to increase the amount of blue going to the lungs. Then another operation is performed later- usually at 1-2 years of age- so, that the holes can be closed, the valves repaired and the way out to the lung artery widened. (Cin‐ cinnati Children's hospital medical Center, 2006)

## **9. Long-term outlook**

complications such as pulmonary vascular disease that may adversely affect the outcome of

Occasionally a repeat electrocardiogram, chest x-ray, or echocardiogram is performed to fur‐ ther evaluate clinical changes. These tests are likely to be repeated before surgical repair is

Rarely, a cardiac catheterization is required for complete evaluation prior to corrective sur‐

Children with Down syndrome and symptoms of congestive heart failure can be initially managed medically with the use of diuretics, blood pressure medications to allow the heart to eject more blood out to the body rather than out to the lungs; and/or digoxin, a medica‐

If the baby is having difficulty with feeding and weight gain, nasogastric tube feeding with

These are all temporary solutions to allow the baby to grow while deciding if and when surgery is indicated. If the baby has no signs of heart failure or is controlled well with medications, the decisions for surgical closure can be delayed. The decision must be indi‐ vidualized to each child's physical state as well as the family's concerns. The majority of cases of AVSD usually require surgical intervention; this generally takes place within the

Many VSD, will close spontaneously or get much smaller, so, it is normal practice to leave a child with a small or moderate VSD and monitor their progress before deciding to operate. Surgery may be needed if there is failure to thrive despite medication, or concern about pul‐ monary hypertension. If a large VSD is present, surgery is almost always recommended.

Small holes in ASD which allows little blood flow from left to right generally causes no problems. If they are located in the middle portion of the central heart wall, they may even close on their own. However, moderate and large holes do not close, and the extra work over the years places a strain of the right side of the heart causing an enlargement of both pumping chambers. Therefore, Surgery is recommended in the first few years of life or larg‐

If the ductus open for more than three months, it is unlikely to close on its own and surgical

The types of surgery in TOF depend on the severity of the AVSD or the Fallots. Usually the children are quite blue and require a BT shunt to increase the amount of blue going to the lungs. Then another operation is performed later- usually at 1-2 years of age- so, that the holes can be closed, the valves repaired and the way out to the lung artery widened. (Cin‐

gery especially in patients where elevated pressures in the lungs are a concern.

calorie formula or fortified breast milk can be used to help with growth.

tion and to improve the pumping ability of the heart.

er holes, before excessive strain has been placed on the heart.

cinnati Children's hospital medical Center, 2006)

cardiac surgery.

102 Down Syndrome

recommended.

**8. Treatment**

first six months of life.

closure is imperative.

Over all, survival beyond one year of age is 85 percent in all children with Down syndrome. Over 50 percent of individuals with Down syndrome live to be greater than 50 years of age.

Congenital heart disease is the most common causes of death in early childhood. However, as of the late 1980s, 70 percent of children with Down syndrome and congenital heart dis‐ ease lived beyond their first birth day with improved medical and surgical care, these num‐ bers continue to improve. (Cincinnati Children's hospital medical Center, 2009)

## **Abbreviation**

ASD – Atrial Septal Defect AVSD- Atrioventricular Septal Defect BAC- Beta-site APP –Cleaving CHD- Congenital Heart Disease DSCAM- Down syndrome cell adhesion molecule DS- Down syndrome MX- Myxovirus resistance PDA- Patent Ductus Arteriosus PFKL- Phosphofructo-kinase liver types TOF- Tetralogy of fallot VSD-Ventricular Septal defect BT- Blalock Taussig

## **Author details**

A. K. M. Mamunur Rashid

Dept. of Pediatrics, Khulna Medical College, Khulna, Bangladesh

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[3] Barlow G M., Xiao-Ning Chen, Zheng Y. Shi, Gary E Lyons, David M. Kurnit, Livija Celle, Nancy B. Spinner, Elaine Zackai, Mark J. Pettenati, Alexander J. Van Riper, Mi‐ chael J. Vekemans, Corey H. Mjaatvedt, Julie R Korenberg. Genetics in Medicine

[4] Behrman RE, Kliegman RM and Jenson HB. From congenital heart disease. Philadel‐ phia: Harcourt Asia Pvt. Ltd. Nelson Textbook of Pediatrics. 2000; 16 1362-63.

[5] Berr C and Borghi E. Risk of Down syndrome in relatives of trisomy 21 children. A

[6] Cooney TP, Thurlbeck WM. Pulmonary hypoplasia in Down's syndrome. N Engl J

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[14] Hattori M, Fujiyama A, Taylor TD, Watanabe H, Yada T, Park HS, Toyoda A, Ishii K, Totoki Y, Choi DK, Soeda E, Ohki M, Takagi T, Sakaki T, Taudien S, Blechschmidt K,


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**Chapter 7**

**Myeloid Leukemia Associated with Down Syndrome**

Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute leukemia. [1-4] The relative risk of developing acute megakaryoblastic leukemia (AMKL) is estimated to be 500 times higher in children with DS than in those without DS. Interestingly, five to 10 % of neonates with DS develop transient abnormal myelopoiesis (TAM). In most cases, it resolves spontaneously within 3 months. However, approximately 15% of the severe cases are fatal and 20% of patients develop AMKL until 3 year-old (Fig.1). AMKL in DS has a number of distinct features and it is now considered a specific subtype of acute myeloid leukemia (AML) in the 4th edition of the World Health Organization (WHO) classification

The majority of cases of AML with DS (70-100%) are megakaryoblastic [5] and occur within the first 4 years of life. [6] The characteristic antecedent preleukaemic TAM is observed in 20– 30% of cases. Overt leukemia in DS children is preceded in 20- 60% of cases by an indolent myelodysplasia, characterised by thrombocytopenia and bone marrow fibrosis, which may last several months before overt AML. [1, 7] The median age at presentation of AML is 1.8 years. [7] The bone marrow aspirate shows dysplasia, increased blasts, abnormal megakaryo‐ cytes and variable myelofibrosis.[5, 7-8] Immunophenotypically, ML-DS blasts typically express megakaryocytic (CD42b and CD41) and erythroid markers (CD36 and Glycophorin A) as well as the T cell marker, CD7. [9]Neither the favorable cytogenetic changes, such as t(8;21), t(15;17), t(9;11) and inv(16), nor the AMKL-associated translocations, t(1;22) and t(1;3), occur in ML-DS.[1] Additional copies of chromosome 8 and/or 21 (in addition to the +21c,

> © 2013 Kudo; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

10-15%), monosomy 7 and –5/5q- (together in 10–20%) are observed. [10]

Kazuko Kudo

**1. Introduction**

http://dx.doi.org/10.5772/52784

Additional information is available at the end of the chapter

called Myeloid Leukemia of Down syndrome (ML-DS).

**2. Acute Myeloid Leukemia (AML)**


## **Myeloid Leukemia Associated with Down Syndrome**

Kazuko Kudo

D21S55 region on chromosome 21 in the pathogenesis of Down syndrome. Proc Natl

[26] Ramakrishnan V. Research Article: Genetic aspects of congenital heart disease in

[27] Roguin N, Du ZD, Barak M, Nasser N, hershkowitz S, Milgram E. High prevalence of muscular ventricular septal defect in neonates. J Am Coll Cardiol. 1995; 26:

[28] Rodriguez LH, and Reyes JN. Cardiopatias en el syndrome de Down. Bol Med Hosp

[29] Shashi V, Berry MN, Covitz W. A combination of physical examination and ECG de‐ tects the majority of hemodynamically significant heart defects in neonates with

[30] Stoll C, Alembik Y, Dott B, Roth MP. 1998. Study of Down syndrome in 238,942 con‐

[31] T R J Tubman, M D Shields, B G Craig, H C Mulholland, N c Nevin. Congenital Heart Disease in Down syndrome; two year prospective early screening study. B M J,

[32] Van Praagh R, Papagiannis J, Bar-EI YI, Schwint OA. The heart in Down syndrome: Pathologic anatomy. In: Marino B, Pueschel SM, editors. Heart disease in persons with Down syndrome. Baltimore, MD: Paul H Brookers Publishing Co. 1996: 69-110.

[33] Yamakawa K, Huo YK, Haendelt MA, Hubert R, Chen X-N, Lyones GE, Korenberg JR. DSCAM: a novel member of the immunoglobulin super family maps in a Down syndrome region and is involved in the development of the nervous system. Hum

Acad Sci U S A 1989; 86: 5958-62.

Infant Mex. 1984; 41: 622-25.

secutive births. Ann Genet. 41: 44-51.

Volume 302, 15 June, 1991, 1425-27.

Mol Genet 1998; 7: 227-37.

1545-48.

106 Down Syndrome

Down syndrome. Inter J Cur Res 2011; 3(6): 165-70.

Down syndrome. Am J Med Genet 2002 Mar 15;108(3):205-8.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52784

## **1. Introduction**

Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute leukemia. [1-4] The relative risk of developing acute megakaryoblastic leukemia (AMKL) is estimated to be 500 times higher in children with DS than in those without DS. Interestingly, five to 10 % of neonates with DS develop transient abnormal myelopoiesis (TAM). In most cases, it resolves spontaneously within 3 months. However, approximately 15% of the severe cases are fatal and 20% of patients develop AMKL until 3 year-old (Fig.1). AMKL in DS has a number of distinct features and it is now considered a specific subtype of acute myeloid leukemia (AML) in the 4th edition of the World Health Organization (WHO) classification called Myeloid Leukemia of Down syndrome (ML-DS).

## **2. Acute Myeloid Leukemia (AML)**

The majority of cases of AML with DS (70-100%) are megakaryoblastic [5] and occur within the first 4 years of life. [6] The characteristic antecedent preleukaemic TAM is observed in 20– 30% of cases. Overt leukemia in DS children is preceded in 20- 60% of cases by an indolent myelodysplasia, characterised by thrombocytopenia and bone marrow fibrosis, which may last several months before overt AML. [1, 7] The median age at presentation of AML is 1.8 years. [7] The bone marrow aspirate shows dysplasia, increased blasts, abnormal megakaryo‐ cytes and variable myelofibrosis.[5, 7-8] Immunophenotypically, ML-DS blasts typically express megakaryocytic (CD42b and CD41) and erythroid markers (CD36 and Glycophorin A) as well as the T cell marker, CD7. [9]Neither the favorable cytogenetic changes, such as t(8;21), t(15;17), t(9;11) and inv(16), nor the AMKL-associated translocations, t(1;22) and t(1;3), occur in ML-DS.[1] Additional copies of chromosome 8 and/or 21 (in addition to the +21c, 10-15%), monosomy 7 and –5/5q- (together in 10–20%) are observed. [10]

© 2013 Kudo; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

23.3 g/m2 in the AMLBFM93 study, respectively. Outcome improved significantly for patients treated in the AMLBFM98 study, with a 3-year EFS of 91% plus or minus 4% versus 70% plus

> **death in CCR (%)**

POG, Pediatric Oncology Group; CCG, Children's Cancer Group; COG, Children's Oncology Group; NOPHO, Nordic Society for Paediatric Haematology and Oncology; BFM, Berlin-Frankfurt-Munster; MRC, Medical Research Council; JCCLSG; Japan Children's Cancer and Leukemia Study Group DS, Down syndrome;

taining high-dose and/or continuous infusion of intermediate-dose cytarabine.

A treatment regimen specifically designed for AML-DS has been used in Japan since the mid- 1980s.[15, 16] AML 99 DS protocol consisted of pirarubicin (25 mg/m2/d, on days 1 and 2), which was estimated to be equivalent as 25mg/m2/d of daunomycin (DNR), cy‐ tarabine (100 mg/m2/d on day 1 through 7), and etoposide (150 mg/m2/d on day 3 through 5). Pirarubicin is much less cardiotoxic and more myelosuppressive than daunor‐ ubicin.A total of 70 of the 72 patients (97.2%) achieved a CR. The 4-year EFS was 83.3% plus or minus 9.1% and the 4-year OS was 83.7% plus or minus 9.5%. The regimen-relat‐ ed toxicities were relatively tolerable. Only one patient died as a result of pneumonia in the second course of intensification. The 3-year EFS in the five patients with monosomy 7 was significantly worse than in the 65 patients without monosomy 7 (40,0% plus or mi‐ nus 26.3% v 86.2% plus or minus 8.8%). Future treatment protocols could include adher‐ ence to a very low-intensity chemotherapy for the majority of ML-DS patients, identification of the subgroup with a poor prognosis using minimal residual disease (MRD), and stratification of these patients to receive a more intensive chemotherapy con‐

POG942112 57 77 (5y) 7 14 20.7 135 80 1,000 CCG28915 161 77 (6y) 14 4 15.8 320 0 1,600 COG-A297113 132 79 (5y) 11 3 24.8 80 0 0 NOPHO-AML9314 41 85 (8y) 7 5 49.6 150 30 1,600 AML-BFM987 67 89 (3y) 6 5 23-29 Ida; 26-36 0-14 950 MRC-AML10/128 46 74 (5y) 3 15 7.8 300 50 1,500 AT-DS(Japan)15 33 80 (8y) 6 9 4.2 100-400 0 2,700 AML99 DS16 72 83 (4y) 12.5 1.4 3.5 THP; 250 0 2,250 JCCLSG 9805DS17 24 83 (5y) 0 13 12.6 THP; 135 10 200

**Cytarabine (g/m2)**

**Drugs administered**

Myeloid Leukemia Associated with Down Syndrome

**Mitoxantron e (mg/m2)**

http://dx.doi.org/10.5772/52784

**Etoposide (mg/m2)**

109

**Daunorubici n (mg/m2)**

or minus 7% in the AMLBFM93 study.

**No of**

**patients EFS (%) Relapse**

**(%)**

**Protocol**

Ida, Idarubicin; THP, pirarubicin

**Table 1.** Comparison of the results in DS-AML patients

**Figure 1.** Multi-step model of myeloid leukemogenesis in DS. Trisomy 21 enhances the proliferation of fetal liver meg‐ akaryo-erythroid progenitors via PDGF and/or TGF beta. The acquisition of GATA1 mutation further enhances the clo‐ nal proliferation of immature magakaryoblasts diagnosed at birth as TAM. GATA1 mutations are necessary but insufficient for the development of AMKL. Additional genetic events such as trisomy 8, or JAK2/3 mutations have been proposed in progression from TAM to AMKL.

#### **2.1. Treatment for AML-DS**

Conventional treatment of AML-DS has been associated with excessive treatment-related mortality (TRM), cardiac toxicity due to anthracyclines and serious infections. Zwaan et al demonstrated a 12-fold increase in sensitivity to cytarabine in DS-AML cells compared with non-DS AML cells, as well as increased sensitivity to anthracyclines (two- to seven-fold) and etoposide (20-fold).[11] Several collaborative study groups have adapted their standard AML protocol for AML-DS by reducing the dose of drugs (Table 1).[5, 8, 12-17] In the Children's Oncology Group (COG) trial A2971 (n=132), [13] etoposide, dexamethasone, and the mainte‐ nance course were eliminated from the previous CCG2891 protocol. COG A2971 achieved a 5-year EFS rate of 79% plus or minus 7% (versus 77% plus or minus 7% in the CCG2891 trial) while maintaining a low induction failure rate of 6.4%, attaining a 0% CNS relapse rate, and sustaining an acceptably low 5-year postremission. In the AML-BFM98 study (n=66), [7] AML-DS patients were treated with reduced doses of anthracyclines and cytarabine compared with the previous AMLBFM93 protocol (n = 44). The cumulative doses of anthracyclines and cytarabine were 220 to 240mg/m2 and 23 to 29g/m2 in the BFM98 study, and 440mg/m2 and 23.3 g/m2 in the AMLBFM93 study, respectively. Outcome improved significantly for patients treated in the AMLBFM98 study, with a 3-year EFS of 91% plus or minus 4% versus 70% plus or minus 7% in the AMLBFM93 study.


POG, Pediatric Oncology Group; CCG, Children's Cancer Group; COG, Children's Oncology Group; NOPHO,

Nordic Society for Paediatric Haematology and Oncology; BFM, Berlin-Frankfurt-Munster; MRC, Medical

Research Council; JCCLSG; Japan Children's Cancer and Leukemia Study Group DS, Down syndrome;

Ida, Idarubicin; THP, pirarubicin

**2.1. Treatment for AML-DS**

108 Down Syndrome

been proposed in progression from TAM to AMKL.

Conventional treatment of AML-DS has been associated with excessive treatment-related mortality (TRM), cardiac toxicity due to anthracyclines and serious infections. Zwaan et al demonstrated a 12-fold increase in sensitivity to cytarabine in DS-AML cells compared with non-DS AML cells, as well as increased sensitivity to anthracyclines (two- to seven-fold) and etoposide (20-fold).[11] Several collaborative study groups have adapted their standard AML protocol for AML-DS by reducing the dose of drugs (Table 1).[5, 8, 12-17] In the Children's Oncology Group (COG) trial A2971 (n=132), [13] etoposide, dexamethasone, and the mainte‐ nance course were eliminated from the previous CCG2891 protocol. COG A2971 achieved a 5-year EFS rate of 79% plus or minus 7% (versus 77% plus or minus 7% in the CCG2891 trial) while maintaining a low induction failure rate of 6.4%, attaining a 0% CNS relapse rate, and sustaining an acceptably low 5-year postremission. In the AML-BFM98 study (n=66), [7] AML-DS patients were treated with reduced doses of anthracyclines and cytarabine compared with the previous AMLBFM93 protocol (n = 44). The cumulative doses of anthracyclines and cytarabine were 220 to 240mg/m2 and 23 to 29g/m2 in the BFM98 study, and 440mg/m2 and

**Figure 1.** Multi-step model of myeloid leukemogenesis in DS. Trisomy 21 enhances the proliferation of fetal liver meg‐ akaryo-erythroid progenitors via PDGF and/or TGF beta. The acquisition of GATA1 mutation further enhances the clo‐ nal proliferation of immature magakaryoblasts diagnosed at birth as TAM. GATA1 mutations are necessary but insufficient for the development of AMKL. Additional genetic events such as trisomy 8, or JAK2/3 mutations have

**Table 1.** Comparison of the results in DS-AML patients

A treatment regimen specifically designed for AML-DS has been used in Japan since the mid- 1980s.[15, 16] AML 99 DS protocol consisted of pirarubicin (25 mg/m2/d, on days 1 and 2), which was estimated to be equivalent as 25mg/m2/d of daunomycin (DNR), cy‐ tarabine (100 mg/m2/d on day 1 through 7), and etoposide (150 mg/m2/d on day 3 through 5). Pirarubicin is much less cardiotoxic and more myelosuppressive than daunor‐ ubicin.A total of 70 of the 72 patients (97.2%) achieved a CR. The 4-year EFS was 83.3% plus or minus 9.1% and the 4-year OS was 83.7% plus or minus 9.5%. The regimen-relat‐ ed toxicities were relatively tolerable. Only one patient died as a result of pneumonia in the second course of intensification. The 3-year EFS in the five patients with monosomy 7 was significantly worse than in the 65 patients without monosomy 7 (40,0% plus or mi‐ nus 26.3% v 86.2% plus or minus 8.8%). Future treatment protocols could include adher‐ ence to a very low-intensity chemotherapy for the majority of ML-DS patients, identification of the subgroup with a poor prognosis using minimal residual disease (MRD), and stratification of these patients to receive a more intensive chemotherapy con‐ taining high-dose and/or continuous infusion of intermediate-dose cytarabine.

## **3. Transient Abnormal Myelopoiesis (TAM)**

Transient abnormal myelopoiesis (TAM), also known as transient leukemia (TL) or transient myeloproliferative disorder (TMD) occurs in approximately 10% of infants with DS.[1, 4] TAM was considered to be ''self-limiting''; the prognosis of TAM was favorable, except for the risk of the subsequent development of acute leukemia. Most of newborns are asymptomatic and only present with circulating blast cells, with or without leucocytosis. Other clinical features include hepatomegaly, splenomegaly, serous effusions and, in up to 10% of patients, liver fibrosis due to blast cell infiltration that can rarely cause fulminant liver failure. Leucocytosis and thrombocytopenia are common. About a quarter of patients have abnormal liver transa‐ minases and abnormal laboratory coagulation tests. The blast cells in TAM usually have the 'blebby' appearance characteristic of megakaryoblasts and typically express CD41, CD42b. Most neonates with TAM do not need chemotherapy as the clinical and laboratory abnormal‐ ities spontaneously resolve within 3–6 months after birth. However, symptomatic babies with TAM, especially those with high blast counts or liver dysfunction, may benefit from low-dose cytarabine.

complications may be caused by blast cell infiltration into visceral organs. In the Pediatric Oncology Group (POG) study 9481, 10 mg/m2 per dose or 1.2–1.5 mg/kg per dose was given subcutaneously or intravenously by slow injection twice a day for 7 days (Table 2). [18] In the AML-BFM study, 0.5–1.5 mg/kg was administered for 3–12 days. [19] As TAM blasts are highly sensitive to cytarabine, there is generally a rapid response, characterized by the disappearance

> **Leukemia (%)**

POG948118 <sup>48</sup> <sup>17</sup> <sup>19</sup> 78 (3y) <sup>2</sup> 10mg/m2 x 2 x 1-2

AML-BFM19 <sup>146</sup> <sup>15</sup> 23.4\* 85 (5y) <sup>28</sup> 0.5-1.5 mg/kg x 3-12

COGA297120 <sup>135</sup> <sup>21</sup> <sup>16</sup> 77 (3y) <sup>29</sup> 3.33mg/kg/24 hrs x 5

Tokai (Japan)21 <sup>70</sup> <sup>23</sup> 22\* 74.3(1y) <sup>3</sup> 0.7 mg/kg x 5days,

Although TAM resolves in the majority of DS infants, 20– 30% subsequently develop ML-DS, usually within in the first 4 years of life. [18-22] In the COG study 2971, twenty-one patients among total 135 TAM patients (16%) developed ML-DS, including 3 received cytarabine.[20] The development of AMKL after remission of TAM has been interested as a model of myeloid leukaemogenesis, presumably from a subclone of persisting TMD cells that acquire a selective advantage. This hypothesis can be verified by monitoring minimal residual disease, either by

Division of Hematology and OncologyShizuoka Children's Hospital, Urushiyama, Aoi-ku,

**OS (%)** **No of treated**

Myeloid Leukemia Associated with Down Syndrome

http://dx.doi.org/10.5772/52784

111

**patients Cytarabine**

days

days

days

10mg/m2 x 2/day

of peripheral blasts by day 7 of treatment.

**No of patients**

POG, Pediatric Oncology Group; BFM, Berlin-Frankfurt-Munster;

COG, Children's Oncology Group; \*. Alive > 6 mo

**Early death (%)**

Kikuchi (Japan)22 73 22 23 71.2(3y) 9

immunophenotype or quantitative GATA1[23] polymerase chain reaction.

**Table 2.** The outcomes of transient abnormal myelopoiesis with Down syndrome.

Address all correspondence to: kazukok@sch.pref.shizuoka.jp

**Study group**

**Author details**

Kazuko Kudo

Shizuoka, Japan

In 2006, Children's Oncology Group (COG) reported a prospective study of the natural history of 48 children with DS and TAM. [18] Early death occurred in 17% of infants and was signifi‐ cantly correlated with higher WBC count at diagnosis, increased bilirubin and liver enzymes, and failure to normalize the blood count. Recurrence of leukemia occurred in 19% of infants at a mean of 20 months. In the AML-BFM study, 22 children among total 146 children (15%) died within the first 6 months. The 5-year OS and EFS were 85% plus or minus 3% and 63% plus or minus 4%, respectively. [19]A total of 28 children received a short course of cytarabine treatment. Interestingly, EFS and OS did not differ significantly in the treated versus the untreated group. Among the 124 children who survived the first 6 months of life, 29 (23.4%) subsequently developed ML-DS. The 5-year EFS after diagnosis of ML-DS for all 29 patients was 91% plus or minus 5%, which is significantly higher than the 5-year EFS of those of ML-DS patients without documented TAM (70% plus or minus 4%). According to the retrospective study from Japan, estimated gestational age (EGA), higher WBC counts and higher direct bilirubin levels were significant predictive factors for poor prognosis. [20, 21] Muramatsu et al devised a simple risk stratification system based on the EGA and the peak WBC count. The high-risk group (HR) was defined as preterm infants with WBC >100 x 109 /l, the intermediaterisk group (IR) was defined as preterm infants with WBC <100 x 109 /l and term infants with WBC >100 x 109 /l, and the low-risk group (LR) was defined as term infants with WBC< 100 x 109 /l. In the LR group, only three of 39 patients (7.7 %) died early. Based on their data, patients in the LR group should receive no interventions. However, since the probability of early death in patients in the HR group exceeded 50%, active intervention including low dose cytarabine should be tried in the context of a clinical trial for these patients.

#### **3.1. Treatment for TAM**

In patients with a severe form of TAM, the main causes of death in early life are progressive hepatic fibrosis, cardiopulmonary failure, and disseminated intravascular coagulation. These complications may be caused by blast cell infiltration into visceral organs. In the Pediatric Oncology Group (POG) study 9481, 10 mg/m2 per dose or 1.2–1.5 mg/kg per dose was given subcutaneously or intravenously by slow injection twice a day for 7 days (Table 2). [18] In the AML-BFM study, 0.5–1.5 mg/kg was administered for 3–12 days. [19] As TAM blasts are highly sensitive to cytarabine, there is generally a rapid response, characterized by the disappearance of peripheral blasts by day 7 of treatment.


POG, Pediatric Oncology Group; BFM, Berlin-Frankfurt-Munster;

COG, Children's Oncology Group; \*. Alive > 6 mo

**Table 2.** The outcomes of transient abnormal myelopoiesis with Down syndrome.

Although TAM resolves in the majority of DS infants, 20– 30% subsequently develop ML-DS, usually within in the first 4 years of life. [18-22] In the COG study 2971, twenty-one patients among total 135 TAM patients (16%) developed ML-DS, including 3 received cytarabine.[20] The development of AMKL after remission of TAM has been interested as a model of myeloid leukaemogenesis, presumably from a subclone of persisting TMD cells that acquire a selective advantage. This hypothesis can be verified by monitoring minimal residual disease, either by immunophenotype or quantitative GATA1[23] polymerase chain reaction.

## **Author details**

Kazuko Kudo

**3. Transient Abnormal Myelopoiesis (TAM)**

cytarabine.

110 Down Syndrome

WBC >100 x 109

**3.1. Treatment for TAM**

109

Transient abnormal myelopoiesis (TAM), also known as transient leukemia (TL) or transient myeloproliferative disorder (TMD) occurs in approximately 10% of infants with DS.[1, 4] TAM was considered to be ''self-limiting''; the prognosis of TAM was favorable, except for the risk of the subsequent development of acute leukemia. Most of newborns are asymptomatic and only present with circulating blast cells, with or without leucocytosis. Other clinical features include hepatomegaly, splenomegaly, serous effusions and, in up to 10% of patients, liver fibrosis due to blast cell infiltration that can rarely cause fulminant liver failure. Leucocytosis and thrombocytopenia are common. About a quarter of patients have abnormal liver transa‐ minases and abnormal laboratory coagulation tests. The blast cells in TAM usually have the 'blebby' appearance characteristic of megakaryoblasts and typically express CD41, CD42b. Most neonates with TAM do not need chemotherapy as the clinical and laboratory abnormal‐ ities spontaneously resolve within 3–6 months after birth. However, symptomatic babies with TAM, especially those with high blast counts or liver dysfunction, may benefit from low-dose

In 2006, Children's Oncology Group (COG) reported a prospective study of the natural history of 48 children with DS and TAM. [18] Early death occurred in 17% of infants and was signifi‐ cantly correlated with higher WBC count at diagnosis, increased bilirubin and liver enzymes, and failure to normalize the blood count. Recurrence of leukemia occurred in 19% of infants at a mean of 20 months. In the AML-BFM study, 22 children among total 146 children (15%) died within the first 6 months. The 5-year OS and EFS were 85% plus or minus 3% and 63% plus or minus 4%, respectively. [19]A total of 28 children received a short course of cytarabine treatment. Interestingly, EFS and OS did not differ significantly in the treated versus the untreated group. Among the 124 children who survived the first 6 months of life, 29 (23.4%) subsequently developed ML-DS. The 5-year EFS after diagnosis of ML-DS for all 29 patients was 91% plus or minus 5%, which is significantly higher than the 5-year EFS of those of ML-DS patients without documented TAM (70% plus or minus 4%). According to the retrospective study from Japan, estimated gestational age (EGA), higher WBC counts and higher direct bilirubin levels were significant predictive factors for poor prognosis. [20, 21] Muramatsu et al devised a simple risk stratification system based on the EGA and the peak WBC count. The

/l, and the low-risk group (LR) was defined as term infants with WBC< 100 x

/l. In the LR group, only three of 39 patients (7.7 %) died early. Based on their data, patients in the LR group should receive no interventions. However, since the probability of early death in patients in the HR group exceeded 50%, active intervention including low dose cytarabine

In patients with a severe form of TAM, the main causes of death in early life are progressive hepatic fibrosis, cardiopulmonary failure, and disseminated intravascular coagulation. These

/l, the intermediate-

/l and term infants with

high-risk group (HR) was defined as preterm infants with WBC >100 x 109

risk group (IR) was defined as preterm infants with WBC <100 x 109

should be tried in the context of a clinical trial for these patients.

Address all correspondence to: kazukok@sch.pref.shizuoka.jp

Division of Hematology and OncologyShizuoka Children's Hospital, Urushiyama, Aoi-ku, Shizuoka, Japan

## **References**

[1] Lange B: The management of neoplastic disorders of haematopoiesis in children with Down's syndrome. Br J Haematol 110(3): 512-24, 2000

[13] Sorrell AD, Alonzo TA, Hilden JM, et al. Favorable survival maintained in children who have myeloid leukemia associated with Down syndrome using reduced-dose chemotherapy on Children's Oncology Group trial A2971: A report from the Children's

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[14] Abildgaard L, Ellebaek E, Gustafsson G, et al: Optimal treatment intensity in children with Down syndrome and myeloid leukaemia: data from 56 children treated on NOPHO-AML protocols and a review of the literature. Ann Hematol 85(5): 275-80, 2006

[15] Kojima S, Sako M, Kato K, et al: An effective chemotherapy regimen for acute myeloid leukemia and myelodysplastic syndrome with Down's syndrome. Leukemia 14: 786-91,

[16] Kudo K, Kojima S, Tabuchi K, et al: Prospective study of a pirarubicin, intermediatedose cytarabine, and etoposide regimen in children with Down syndrome and acute myeloid leukemia: the Japanese Childhood AML Cooperative Study Group. J Clin

[17] Taga T, Shimomura Y, Horikoshi Y, et al: Continuous and high-dose cytarabine combined chemotherapy in children with Down syndrome and acute myeloid leuke‐ mia: Report from the Japanese Children's Cancer and Leukemia Study Group (JCCLSG)

[18] Massey GV, Zipursky A, Chang MN,et al: A prospective study of the natural history of transient leukemia (TL) in neonates with Down syndrome (DS): Children's Oncology

[19] Klusmann JH, Creutzig U, Zimmermann M, et al: Treatment and prognostic impact of transient leukemia in neonates with Down syndrome Blood 111(6): 2991-8, 2008

[20] Gamis AS, Alonzo TA, Gerbing RB, et al. Natural history of transient myeloproliferative disorder clinically diagnosed in Down syndrome neonates: a report from the Children's Oncology Group Study A2971. Blood. 2011 Dec 22; 118(26):6752-9; quiz 6996. Epub 2011

[21] Muramatsu H, Kato K, Watanabe N, et al: Risk factors for early death in neonates with Down syndrome and transient leukaemia. Br J Haematol 142(4): 610-5, 2008

[22] Kikuchi A: Transient abnormal myelopoiesis in Down's syndrome. JPH23: 58-61, 2009

[23] Wechsler J, Greene M, McDevitt MA et al: Acquired mutations in GATA 1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet 32: 148-52, 2002

AML 9805 Down Study. Pediatr Blood Cancer 2011; 57(1): 36-40.

Group (COG) study POG-9481. Blood 107(12): 4606-13, 2006

Oncology Group.Cancer 2012 .Mar 5 [Epub ahead of print]

2000

Aug 17.

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[13] Sorrell AD, Alonzo TA, Hilden JM, et al. Favorable survival maintained in children who have myeloid leukemia associated with Down syndrome using reduced-dose chemotherapy on Children's Oncology Group trial A2971: A report from the Children's Oncology Group.Cancer 2012 .Mar 5 [Epub ahead of print]

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Review.

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1355-60, 2005

[1] Lange B: The management of neoplastic disorders of haematopoiesis in children with

[2] Zwaan CM, Reinhardt D, Hitzler J, et al: Acute leukemias in children with Down

[3] Izraeli S, Rainis L, Hertzberg L, et al. Trisomy of chromosome 21 in leukemogenesis.

[4] Roy A, Roberts I, Norton A, et al. Acute megakaryoblastic leukaemia (AMKL) and transient myeloproliferative disorder (TMD) in Down syndrome: a multi-step model of myeloid leukaemogenesis. Br J Haematol. 2009 Oct;147(1):3-12. Epub 2009 Jul 6.

[5] Gamis AS, Woods WG, Alonzo TA, et al. Increased age at diagnosis has a significantly negative effect on outcome in children with Down syndrome and acute myeloid leukemia: a report from the Children's Cancer Group Study 2891. J Clin Oncol. 2003

[6] Hasle H, Abrahamsson J, Arola M,et al. Myeloid leukemia in children 4 years or older with Down syndrome often lacks GATA1 mutation and cytogenetics and risk of relapse

[7] Creutzig U, Reinhardt D, Diekamp S, et al: AML patients with Down syndrome have a high cure rate with AML-BFM therapy with reduced dose intensity. Leukemia 19(8):

[8] Rao A, Hills RK, Stiller C, et al. Treatment for myeloid leukaemia of Down syndrome: population-based experience in the UK and results from the Medical Research Council

[9] Yumura-Yagi K, Hara J, Kurahashi H, et al. Mixed phenotype of blasts in acute megakaryocytic leukaemia and transient abnormal myelopoiesis in Down's syndrome.

[10] Forestier E, Izraeli S, Beverloo B, et al. Cytogenetic features of acute lymphoblastic and myeloid leukemias in pediatric patients with Down syndrome: an iBFM-SG study.

[11] Zwaan CM, Kaspers GJ, Pieters R, et al. Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells

in children with and without Down syndrome. Blood. 2002 Jan 1; 99(1):245-51.

Group Study POG 9421. J Clin Oncol. 2008 Jan 20; 26(3):414-20.

[12] O'Brien MM, Taub JW, Chang MN, et al. Cardiomyopathy in children with Down syndrome treated for acute myeloid leukemia: a report from the Children's Oncology

are more akin to sporadic AML. Leukemia. 2008 Jul; 22(7): 1428-30.

AML 10 and AML 12 trials. Br J Haematol. 2006 Mar; 132(5): 576-83.

Down's syndrome. Br J Haematol 110(3): 512-24, 2000

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syndrome. Hematol Oncol Clin North Am 24(1): 19-34, 2010


**Section 3**

**Genetics of Down Syndrome**

**Genetics of Down Syndrome**

**Chapter 8**

**Molecular Pathways of**

http://dx.doi.org/10.5772/53000

**1. Introduction**

Ferdinando Di Cunto and Gaia Berto

**1.1. Identification and annotation of the DSCR**

Down syndrome (DS) is a very complex disorder that requires, even more than other human genetics diseases, a "system level" understanding [1,2], both under the clinical and under the molecular genetics perspectives. Under the clinical point of view, all individuals affected by Down syndrome are characterized by learning disabilities, distinctive facial features, and low muscle tone (hypotonia) in early infancy. However, in most cases the clinical picture is complicated by additional problems, such as heart defects, leukemia, and early-onset Alz‐ heimer's disease [3,4]. The degree to which an individual is affected by these characteristics varies from mild to severe. After the pioneering description by J.L. Down in 1866, almost one century was needed to decipher the etiology of the syndrome. The work of Lejeune proved that DS was caused by an extra copy of chromosome 21 (HSA21) [5], thus providing the first evidence for a genetic basis of intellectual disability. The main implication of this seminal discovery is that the complex phenotype seen in DS patients [6] must be caused by overdosage of HSA21 genes. However, it also raised the outstanding questions of whether one or few HSA21 genes may play a dominant role in the syndrome and whether specific HSA21 genes could contribute to specific phenotypic tracts. Answering these questions is still of paramount importance, because the identification of one or few 'dominant' molecular players could pave the road for the development of targeted therapeutic approaches. The development of molecular karyotyping has provided strong support to the view that a restricted region of HSA21, commonly referred to as Down Syndrome Crtitical Region (DSCR) might be respon‐ sible for the different phenotypes that characterize DS. In 1976 Poissonnier and coworkers, by using chromosome staining methods, found that one DS patient not possessing an extra HSA21 had only a partial trisomy, involving 21q22.1 and 21q22.2 bands [7]. Afterwards, it turned out

> © 2013 Cunto and Berto; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

**Down Syndrome Critical Region Genes**

## **Chapter 8**

## **Molecular Pathways of Down Syndrome Critical Region Genes**

Ferdinando Di Cunto and Gaia Berto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53000

## **1. Introduction**

#### **1.1. Identification and annotation of the DSCR**

Down syndrome (DS) is a very complex disorder that requires, even more than other human genetics diseases, a "system level" understanding [1,2], both under the clinical and under the molecular genetics perspectives. Under the clinical point of view, all individuals affected by Down syndrome are characterized by learning disabilities, distinctive facial features, and low muscle tone (hypotonia) in early infancy. However, in most cases the clinical picture is complicated by additional problems, such as heart defects, leukemia, and early-onset Alz‐ heimer's disease [3,4]. The degree to which an individual is affected by these characteristics varies from mild to severe. After the pioneering description by J.L. Down in 1866, almost one century was needed to decipher the etiology of the syndrome. The work of Lejeune proved that DS was caused by an extra copy of chromosome 21 (HSA21) [5], thus providing the first evidence for a genetic basis of intellectual disability. The main implication of this seminal discovery is that the complex phenotype seen in DS patients [6] must be caused by overdosage of HSA21 genes. However, it also raised the outstanding questions of whether one or few HSA21 genes may play a dominant role in the syndrome and whether specific HSA21 genes could contribute to specific phenotypic tracts. Answering these questions is still of paramount importance, because the identification of one or few 'dominant' molecular players could pave the road for the development of targeted therapeutic approaches. The development of molecular karyotyping has provided strong support to the view that a restricted region of HSA21, commonly referred to as Down Syndrome Crtitical Region (DSCR) might be respon‐ sible for the different phenotypes that characterize DS. In 1976 Poissonnier and coworkers, by using chromosome staining methods, found that one DS patient not possessing an extra HSA21 had only a partial trisomy, involving 21q22.1 and 21q22.2 bands [7]. Afterwards, it turned out

© 2013 Cunto and Berto; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

that partial trisomies are responsible for approximately 1% of DS cases [8,9]. These patients show variable phenotypes, depending on the extension of the triplicated region. Therefore, partial trisomies of genes carried by chromosome 21 have been extremely valuable in inves‐ tigating the involvement in DS. The analysis of 10 partial trisomy patients, [10] suggested that two regions of chromosome 21 were linked to most of the Jackson signs [3], including cognitive disorders. These regions, referred to has DCR-1 and DCR-2, respectively, encompassed the 21q22.2 band and were located around the D21S55 Site Targeted Sequence (STS) and between D21S55 and the MX1 gene, respectively. Korenberg and coworkers studied a different population and observed that the proximal and distal regions of the 21q arm were also associated with the full DS phenotype [11]. Although these studies confirmed the strong association of DS phenotypes with the DCR-1 region, they also suggested that DS is a contig‐ uous gene syndrome, arguing against a single DS chromosomal region responsible for most of the DS phenotypic features [11]. More recently, an additional causal link of the region located between D21S17 and ETS2 to clinical features of DS was confirmed through lattice analysis [12]. Although the notion of a DSCR has gained wide acceptance in DS research, it must be underscored that some of the data that support it remain controversial and that its existence has recently come under considerable question. Indeed, a detailed study of segmental trisomy 21 in DS subjects, performed by using array comparative genome hybridization (GCH), excludes the implication of a single but rather suggest that multiple regions of HSA21 contribute to many of the phenotypes of DS, including intellectual disability DSCR [13]. Despite these apparent inconsistencies, we think that, in practical terms, the crucial point is not to prove whether one or more "critical region" exist, but rather to understand which dosage-sensitive genes contribute to specific DS phenotypes. Indeed, it is quite clear that the classical "reductionist" approach of identifying one or few master genes, which has been very successful in the case of Mendelian disorders, is not appropriate to unravel the extremely more complicated case of DS. In this case, the overall phenotype is certainly produced by the combined action of several genes, causing complex rearrangements of different molecular networks [14]. The relevance of the mentioned studies has been to restrict the list of HSA21 genes that may contribute more significantly to the clinical manifestations.

coding sequences, whose number is approximately of 40, on the basis of a comprehensive definition of the DSCR and of the present annotation of the human genome (Table 1). However, systematic studies performed in the last few years revealed that many genomic sequences that have been initially considered as "junk DNA", are endowed with extremely relevant functional potential [16]. Indeed, genome-wide interrogations have revealed that a large majority of the human genome is transcribed and that a significant proportion of transcripts appears to be non-protein coding (ncRNA). Although it is well recognized that some ncRNAs play essential enzymatic activities in translation, splicing and ribosome biogenesis, the functions of most ncRNAs are still unknown. It is now believed that they could participate in complex regulatory circuits responsible for the fine-tuning of gene expression at both the transcriptional and posttranscriptional levels [16]. The best known ncRNAs are miRNAs, ~22 nucleotide-long mole‐ cules that mediate post-transcriptional gene silencing by binding complementary sequences located in the 3' UTR of the mRNAs. Long intergenic ncRNAs (lincRNA) represent a less characterized but more abundant and heterogeneous class, and comprise transcripts longer than 200 nt involved in many biological processes, including transcriptional control, epigenetic modification and post-transcriptional control on mRNAs [16]. A recent discovery demonstrat‐ ed that both mRNAs and ncRNAs can deploy their functions by contributing to an extensive RNA-RNA interaction network, based on the competition of these molecules for the binding of shared miRNAs (the ceRNA hypothesis) [17-20]. Importantly, transcribed pseudogenes could also be involved in these complex regulatory interactions [21]. In light of this growing complexity, we think that the presence of many 'non conventional' sequences within the DSCR should be taken into consideration when exploring the molecular consequences of an increased

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dosage of this region. We provide an updated list of them in Table 2.

1 CBR3 874 Enzyme [165]

1 MORC3 23515 RNA-binding [167]

1 CLDN14 23562 Tight junctions component [169]

 RCAN1 1827 CaN inhibitor See main text Yes CLIC6 54102 Channel See main text Yes RUNX1 861 Transcription factor See main text Yes SETD4 54093 Unknown No information Yes CBR1 873 Enzyme [165] Yes

1 DOPEY2 9980 Unknown [166] Yes

1 CHAF1B 8208 Chromatin assembly [168] Yes

1 SIM2 6493 Transcription factor See main text Yes 1 HLCS 3141 Enzyme [170] Yes

**Main molecular function Essential references Expression in**

**adult brain**

**Entrez Gene ID**

**DCR Gene Name**

For these motivations, in Tables 1 and 2 we adopt an inclusive definition of the DSCR, which extends from the RCAN1 gene to the MX1 gene. This definition takes into account not only the putative borders that have been identified in the mentioned studies, but also the fact that the RCAN1 gene as been commonly considered as part of the DSCR, even though a precise mapping on the current release of the human genome sequence (HG19) would locate it outside the centromeric border defined by [12]. Obviously, the usefulness of this information will strongly depend on the degree of functional characterization of the genes comprised in the interval. Under this respect, as it is generally true for the human genome, it must be recognized that our knowledge is still quite limited.

HSA21 was one of the first human chromosomes to be fully sequenced [15]. Nevertheless, the list of the possible functional sequences located in the DSCR has progressively changed, not only for the uncertainty of defining precise borders, but especially for the changes in the current view of what a human gene is. Obviously, the initial emphasis has been to identify the proteincoding sequences, whose number is approximately of 40, on the basis of a comprehensive definition of the DSCR and of the present annotation of the human genome (Table 1). However, systematic studies performed in the last few years revealed that many genomic sequences that have been initially considered as "junk DNA", are endowed with extremely relevant functional potential [16]. Indeed, genome-wide interrogations have revealed that a large majority of the human genome is transcribed and that a significant proportion of transcripts appears to be non-protein coding (ncRNA). Although it is well recognized that some ncRNAs play essential enzymatic activities in translation, splicing and ribosome biogenesis, the functions of most ncRNAs are still unknown. It is now believed that they could participate in complex regulatory circuits responsible for the fine-tuning of gene expression at both the transcriptional and posttranscriptional levels [16]. The best known ncRNAs are miRNAs, ~22 nucleotide-long mole‐ cules that mediate post-transcriptional gene silencing by binding complementary sequences located in the 3' UTR of the mRNAs. Long intergenic ncRNAs (lincRNA) represent a less characterized but more abundant and heterogeneous class, and comprise transcripts longer than 200 nt involved in many biological processes, including transcriptional control, epigenetic modification and post-transcriptional control on mRNAs [16]. A recent discovery demonstrat‐ ed that both mRNAs and ncRNAs can deploy their functions by contributing to an extensive RNA-RNA interaction network, based on the competition of these molecules for the binding of shared miRNAs (the ceRNA hypothesis) [17-20]. Importantly, transcribed pseudogenes could also be involved in these complex regulatory interactions [21]. In light of this growing complexity, we think that the presence of many 'non conventional' sequences within the DSCR should be taken into consideration when exploring the molecular consequences of an increased dosage of this region. We provide an updated list of them in Table 2.

that partial trisomies are responsible for approximately 1% of DS cases [8,9]. These patients show variable phenotypes, depending on the extension of the triplicated region. Therefore, partial trisomies of genes carried by chromosome 21 have been extremely valuable in inves‐ tigating the involvement in DS. The analysis of 10 partial trisomy patients, [10] suggested that two regions of chromosome 21 were linked to most of the Jackson signs [3], including cognitive disorders. These regions, referred to has DCR-1 and DCR-2, respectively, encompassed the 21q22.2 band and were located around the D21S55 Site Targeted Sequence (STS) and between D21S55 and the MX1 gene, respectively. Korenberg and coworkers studied a different population and observed that the proximal and distal regions of the 21q arm were also associated with the full DS phenotype [11]. Although these studies confirmed the strong association of DS phenotypes with the DCR-1 region, they also suggested that DS is a contig‐ uous gene syndrome, arguing against a single DS chromosomal region responsible for most of the DS phenotypic features [11]. More recently, an additional causal link of the region located between D21S17 and ETS2 to clinical features of DS was confirmed through lattice analysis [12]. Although the notion of a DSCR has gained wide acceptance in DS research, it must be underscored that some of the data that support it remain controversial and that its existence has recently come under considerable question. Indeed, a detailed study of segmental trisomy 21 in DS subjects, performed by using array comparative genome hybridization (GCH), excludes the implication of a single but rather suggest that multiple regions of HSA21 contribute to many of the phenotypes of DS, including intellectual disability DSCR [13]. Despite these apparent inconsistencies, we think that, in practical terms, the crucial point is not to prove whether one or more "critical region" exist, but rather to understand which dosage-sensitive genes contribute to specific DS phenotypes. Indeed, it is quite clear that the classical "reductionist" approach of identifying one or few master genes, which has been very successful in the case of Mendelian disorders, is not appropriate to unravel the extremely more complicated case of DS. In this case, the overall phenotype is certainly produced by the combined action of several genes, causing complex rearrangements of different molecular networks [14]. The relevance of the mentioned studies has been to restrict the list of HSA21

genes that may contribute more significantly to the clinical manifestations.

that our knowledge is still quite limited.

118 Down Syndrome

For these motivations, in Tables 1 and 2 we adopt an inclusive definition of the DSCR, which extends from the RCAN1 gene to the MX1 gene. This definition takes into account not only the putative borders that have been identified in the mentioned studies, but also the fact that the RCAN1 gene as been commonly considered as part of the DSCR, even though a precise mapping on the current release of the human genome sequence (HG19) would locate it outside the centromeric border defined by [12]. Obviously, the usefulness of this information will strongly depend on the degree of functional characterization of the genes comprised in the interval. Under this respect, as it is generally true for the human genome, it must be recognized

HSA21 was one of the first human chromosomes to be fully sequenced [15]. Nevertheless, the list of the possible functional sequences located in the DSCR has progressively changed, not only for the uncertainty of defining precise borders, but especially for the changes in the current view of what a human gene is. Obviously, the initial emphasis has been to identify the protein-



**DCR Gene Name Ensembl ID**

**Entrez Gene ID**

 LINC00160 ENSG00000230978 54064 36096105 - 36109478 lincRNA AP000330.8 ENSG00000234380 100506385 36118054 - 36157183 Antisense

EZH2P1 ENSG00000231300 266693 36972030 - 36972320 Pseudogene

 MIR802 ENSG00000211590 768219 37093013 - 37093106 miRNA RPS20P1 ENSG00000229761 54025 37097045 - 37097398 Pseudogene PPP1R2P2 ENSG00000234008 54036 37259493 - 37260105 Pseudogene

RIMKLBP1 ENSG00000189089 54031 37422512 - 37423675 Pseudogene

 U6 ENSG00000200213 1497008 37438843 - 37438950 snRNA AP000688.14 ENSG00000230212 100133286 37441940 - 37498938 sense intronic

 MEMO1P1 ENSG00000226054 728556 37502669 - 37504208 Pseudogene CBR3-AS1 ENSG00000236830 100506428 37504065 - 37528605 lincRNA RPS9P1 ENSG00000214889 8410 37504748 - 37505330 Pseudogene RPL3P1 ENSG00000228149 8488 37541268 - 37542478 Pseudogene Metazoa\_SRP ENSG00000265882 37585858 - 37586136 miscellaneous RNA snoU13 ENSG00000238851 37630724 - 37630829 snoRNA SRSF9P1 ENSG00000214867 54021 37667471 - 37668000 Pseudogene

ATP5J2LP ENSG00000224421 54100 37761176 - 37761410 Pseudogene

AP000695.4 ENSG00000233818 37818029 - 37904706 Antisense

RN5S491 ENSG00000199806 100873733 38224211 - 38224328 rRNA

DSCR9 ENSG00000230366 257203 38580804 - 38594037 lincRNA

 Y\_RNA ENSG00000207416 38359039 - 38359151 miscellaneous RNA MRPL20P1 ENSG00000215734 359737 38366943 - 38367375 Pseudogene U6 ENSG00000212136 1497008 38417830 - 38417936 snRNA

 AF015262.2 ENSG00000234703 36508935 - 36511519 lincRNA + RPL34P3 ENSG00000223671 54026 36844395 - 36844730 Pseudogene +

AF015720.3 ENSG00000230794 37085437 - 37105240 processed transcript +

 AP000688.8 ENSG00000231106 37377636 - 37379899 lincRNA + RPL23AP3 ENSG00000214914 8489 37388377 - 37388844 Pseudogene ++

AP000688.11 ENSG00000236677 37432730 - 37436706 Antisense +

 AP000688.15 ENSG00000236119 37455157 - 37462712 lincRNA + AP000688.29 ENSG00000233393 37477179 - 37481988 lincRNA +

AP000692.9 ENSG00000228107 37732928 - 37734338 processed transcript +

AP000695.6 ENSG00000230479 37802658 - 37853368 Antisense +

 PSMD4P1 ENSG00000223741 54035 37858281 - 37859709 Pseudogene + AP000696.2 ENSG00000231324 38004979 - 38009331 lincRNA ++ AP000697.6 ENSG00000224269 38071073 - 38073864 Antisense + HLCS-IT1 ENSG00000237646 100874294 38176285 - 38178585 sense intronic ++

AP000704.5 ENSG00000224790 38338812 - 38344128 lincRNA ++

TTC3-AS1 ENSG00000228677 100874006 38559967 - 38566227 Antisense ++

**HSA21 coordinates Gene Biotype**

Molecular Pathways of Down Syndrome Critical Region Genes

**Evidence of expression (EST)**

http://dx.doi.org/10.5772/53000

**Table 1.** Summary of the protein-coding genes contained by the DSCR. The first column indicates whether the genes belong to the DCR-1, to the DCR-2 or to the overlap region. The evidence for expression in adult brain is derived from the EVOC data [186] contained in the Ensembl genome browser. Genes are given in their physical order, starting from the more centromeric sequence.

#### Molecular Pathways of Down Syndrome Critical Region Genes http://dx.doi.org/10.5772/53000


**DCR Gene Name**

Down Syndrome

**Entrez Gene ID** **Main molecular function Essential references Expression in**

 DSCR6 53820 Unknown [171] Yes PIGP 51227 Enzyme [172] Yes TTC3 7267 E3 ligase See main text Yes DSCR3 10311 Unknown [173] Yes DYRK1A 1859 Protein kinase See main text Yes

1-2 ERG 2078 Transcription factor See main text Yes 1-2 ETS2 2114 Transcription factor See main text Yes PSMG1 8624 Chaperone [177] Yes BRWD1 54014 Transcription factor See main text Yes HMGN1 3150 Transcription factor See main text Yes WRB 7485 Protein trafficking [178] Yes

SH3BGR 6450 Unknown No information Yes

C21orf88 114041 Unknown No information Yes

PCP4 5121 Unknown [182] Yes

BACE2 25825 Protease See main text Yes

MX1 4599 Unknown [185] Yes

**Table 1.** Summary of the protein-coding genes contained by the DSCR. The first column indicates whether the genes belong to the DCR-1, to the DCR-2 or to the overlap region. The evidence for expression in adult brain is derived from the EVOC data [186] contained in the Ensembl genome browser. Genes are given in their physical order, starting from

1-2 KCNJ6 3763 Channel See main text 1-2 DSCR4 10281 Unknown [174] 1-2 DSCR8 84677 Unknown [175] 1-2 KCNJ15 3772 Channel [176]

LCA5L 150082 Ciliary protein [179]

B3GALT5 10317 Enzyme [180]

IGSF5 150084 Adhesion molecule [181]

DSCAM 1826 Adhesion molecule [183]

 FAM3B 54097 Cytokine [184] MX2 4600 Unknown [185]

the more centromeric sequence.

**adult brain**


**DCR Gene Name Ensembl ID**

phenotype.

**Entrez Gene ID**

2 YRDCP3 ENSG00000230859 100861429 42235920 - 42236399 Pseudogene 2 LINC00323 ENSG00000226496 284835 42513427 - 42520060 Antisense 2 MIR3197 ENSG00000263681 100423023 42539484 - 42539556 miRNA

**2. Functional analysis of the DSCR through mouse models**

potentiation (LTP) in the hippocampus and fascia dentata (FD) [34-36].

2 AF064863.1 ENSG00000221396 41949429 - 41949538 miRNA + 2 DSCAM-IT1 ENSG00000233756 100874326 41987304 - 42002693 sense intronic ++

2 AL773572.7 ENSG00000225745 42548249 - 42558715 processed transcript ++ 2 BACE2-IT1 ENSG00000224388 282569 42552024 - 42552553 Antisense + 2 AP001610.5 ENSG00000228318 42813321 - 42814669 Antisense +

**Table 2.** Summary of the non-protein-coding elements contained by the DSCR. The first column indicates whether the genes belong to the DCR-1, to the DCR-2 or to the overlap region. Elements are given in their physical order, starting from the more centromeric sequence. Genomic coordinates refer to the HG19 version of the human genome sequence. The evidence for expression is derived from the ESTs linked to the Ensembl genome browser. + = at least one EST sequence supporting the Ensemble prediction. ++ prediction supported by several EST sequences.

Animal models are essential to understand the molecular pathogenesis of DS. Moreover, although none of them can faithfully mimic the human situation, they are crucial for the preclinical development of new therapeutic strategies. The availability of sophisticated tools for mouse genetics and the conserved synteny between mouse chromosome 16 (MMU16) and HSA21 have provided the basis for the development of many mouse models of DS, allowing to test the critical region concept and to perform a genetic dissection of the complex DS

The first mouse models have been obtained by studying the effects of partial trisomies of MMU16 derived from Robertsonian translocations. These mice live until adulthood and show many clinical phenotypes similar to DS patients, in particular the neuropathological and neurobiological alterations, including learning and behavioral abnormalities [22-25]. The most studied mouse model for DS is theTs65Dn mouse, which possesses an extra copy of the distal 13 Mbp part of MMU16, including ~ 104 mouse genes orthologous to those on HSA21 [23]. These mice show a number of developmental and functional parallels with DS, including craniofacial abnormalities and behavioural changes [26-32]. Moreover, they show alterations in the structure of dendritic spines in cortex and hippocampus [33] and reduced long-term

Ts1Cje mice, which are trisomic for a shorter but fully overlapping segment of MMU16 (~81 genes), show similar changes, usually to a lesser degree [24,25,37,38]. Comparison of the behavioral performances of the Ts1Cje and Ts65Dn showed that the learning deficits of Ts1Cje mice are similar to those of Ts65Dn. The data obtained from these models strongly supported the concept of DSCR, because they indicated that conserved genes are capable to influence

**HSA21 coordinates Gene Biotype**

Molecular Pathways of Down Syndrome Critical Region Genes

**Evidence of expression (EST)**

123

http://dx.doi.org/10.5772/53000


**Table 2.** Summary of the non-protein-coding elements contained by the DSCR. The first column indicates whether the genes belong to the DCR-1, to the DCR-2 or to the overlap region. Elements are given in their physical order, starting from the more centromeric sequence. Genomic coordinates refer to the HG19 version of the human genome sequence. The evidence for expression is derived from the ESTs linked to the Ensembl genome browser. + = at least one EST sequence supporting the Ensemble prediction. ++ prediction supported by several EST sequences.

## **2. Functional analysis of the DSCR through mouse models**

**DCR Gene Name Ensembl ID**

122 Down Syndrome

**Entrez Gene ID**

1 Metazoa\_SRP ENSG00000263969 38587906 - 38588202 miscellaneous RNA

1-2 snoU13 ENSG00000238581 39559551 - 39559656 snoRNA 1-2 DSCR10 ENSG00000233316 259234 39578250 - 39580738 lincRNA

1-2 SPATA20P1 ENSG00000231123 100874060 39610149 - 39610586 Pseudogene

1-2 SNRPGP13 ENSG00000231480 100874428 39874369 - 39874545 Pseudogene 1-2 LINC00114 ENSG00000223806 400866 40110825 - 40140898 lincRNA 2 AP001042.1 ENSG00000229986 40218171 - 40220568 lincRNA 2 AF064858.6 ENSG00000205622 400867 40249215 - 40328392 lincRNA

2 SNORA62 ENSG00000252384 40266709 - 40266791 snoRNA 2 RPSAP64 ENSG00000227721 40266841 - 40267176 Pseudogene

2 PCBP2P1 ENSG00000235701 54040 40543056 - 40544032 Pseudogene 2 TIMM9P2 ENSG00000232608 100862727 40588550 - 40589432 Pseudogene

2 METTL21AP1 ENSG00000229623 100421629 40607312 - 40607946 Pseudogene

2 Y\_RNA ENSG00000252915 40716463 - 40716554 miscellaneous RNA 2 snoU13 ENSG00000238556 40717300 - 40717383 snoRNA 2 RNF6P1 ENSG00000227406 100420924 40745689 - 40748992 Pseudogene

2 AF121897.4 ENSG00000235012 40897510 - 40901782 Pseudogene

2 MIR4760 ENSG00000263973 100616148 41584279 - 41584358 miRNA 2 DSCAM-AS1 ENSG00000235123 100506492 41755010 - 41757285 Antisense 2 SNORA51 ENSG00000207147 41885071 - 41885206 snoRNA

1 AP001432.14 ENSG00000242553 38593720 - 38610045 lincRNA + 1-2 KCNJ6-IT1 ENSG00000233213 100874329 39089405 - 39091872 sense intronic + 1-2 AP001427.1 ENSG00000264691 39334968 - 39335068 miRNA + 1-2 DSCR4-IT1 ENSG00000223608 100874327 39378846 - 39382920 sense intronic +

1-2 AP001434.2 ENSG00000226012 39609139 - 39610123 lincRNA +

1-2 AP001422.3 ENSG00000231231 39695557 - 39705343 lincRNA ++

2 AP001043.1 ENSG00000229925 40260696 - 40275829 processed transcript +

 AP001044.2 ENSG00000234035 40285093 - 40287072 lincRNA + AF064858.7 ENSG00000232837 40346355 - 40349700 lincRNA + AF064858.8 ENSG00000235888 40360633 - 40378079 lincRNA + AF064858.11 ENSG00000237721 40378574 - 40383255 lincRNA + AF064858.10 ENSG00000237609 40400461 - 40401053 lincRNA + RPL23AP12 ENSG00000228861 391282 40499494 - 40499966 Pseudogene +

2 BRWD1-IT1 ENSG00000237373 40589019 - 40591731 processed transcript +

2 BRWD1-AS1 ENSG00000238141 100874093 40687633 - 40695144 Antisense +

2 MYL6P2 ENSG00000235808 100431168 40860253 - 40860686 Pseudogene ++ 2 RPS26P4 ENSG00000228349 692146 40863470 - 40863824 Pseudogene +

2 AF064860.5 ENSG00000225330 41002198 - 41098012 processed transcript + 2 AF064860.7 ENSG00000231713 41099682 - 41102607 lincRNA +

**HSA21 coordinates Gene Biotype**

**Evidence of expression (EST)**

> Animal models are essential to understand the molecular pathogenesis of DS. Moreover, although none of them can faithfully mimic the human situation, they are crucial for the preclinical development of new therapeutic strategies. The availability of sophisticated tools for mouse genetics and the conserved synteny between mouse chromosome 16 (MMU16) and HSA21 have provided the basis for the development of many mouse models of DS, allowing to test the critical region concept and to perform a genetic dissection of the complex DS phenotype.

> The first mouse models have been obtained by studying the effects of partial trisomies of MMU16 derived from Robertsonian translocations. These mice live until adulthood and show many clinical phenotypes similar to DS patients, in particular the neuropathological and neurobiological alterations, including learning and behavioral abnormalities [22-25]. The most studied mouse model for DS is theTs65Dn mouse, which possesses an extra copy of the distal 13 Mbp part of MMU16, including ~ 104 mouse genes orthologous to those on HSA21 [23]. These mice show a number of developmental and functional parallels with DS, including craniofacial abnormalities and behavioural changes [26-32]. Moreover, they show alterations in the structure of dendritic spines in cortex and hippocampus [33] and reduced long-term potentiation (LTP) in the hippocampus and fascia dentata (FD) [34-36].

> Ts1Cje mice, which are trisomic for a shorter but fully overlapping segment of MMU16 (~81 genes), show similar changes, usually to a lesser degree [24,25,37,38]. Comparison of the behavioral performances of the Ts1Cje and Ts65Dn showed that the learning deficits of Ts1Cje mice are similar to those of Ts65Dn. The data obtained from these models strongly supported the concept of DSCR, because they indicated that conserved genes are capable to influence

cognition through their dosage lie in a region spanning from Sod1 to Mx1, which contains the mouse counterpart of the human DCR-1.

proaches, in which a single human or mouse gene is inserted in the mouse genome in the form

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On the other hand, the combination of gene targeting technologies with the "classical" DS model discussed above allows a subtractive strategy, providing the most stringent test to address the relevance of single genes for the overall phenotype. Indeed, once a null allele for a DSCR gene is available, a compound mutant can be generated, carrying the specific mutation in a trisomic background. The subtractive approach allowed to detect a significant rescue of the phenotype in the case of some DS-related genes, belonging to the DSCR as in the case of

**3. Functional role of DSCR genes in DS intellectual disability: Towards the**

In the following section we will summarize the most relevant functional information available on DSCR genes, trying to especially underscore their implication in molecular networks relevant to intellectual disability. As it is obvious from the previous sections, this discussion will involve not only genes that strictly belong to the DSCR, but also their interactions with other HSC21 genes, whose functional involvement is supported by abundant literature. In particular, we will try to discuss as much as possible the single DSCR genes on the basis of their common features. The essential information about genes not included in this section is reported in Tables 1 and 2. While deploying this summary, we will also provide a perspective of how this information can be useful for progressing towards the development of new

In order to evaluate the possible degree of functional involvement for specific genes, it is very important to briefly analyze the principal biological processes that have been to cogni‐ tive impairment in the DS. To this regard, studies performed both in humans and in ani‐ mal models have shown that trisomy 21 leads to an unbalance of key cellular events, such as neuronal cell proliferation and differentiation, which can be detected during develop‐ ment and post-natal life using morphological methods [52,53]. Importantly, these defects may coexist with or may be causally related to functional deficits, that can be revealed us‐ ing sophisticated physiological methods [52,53]. Reduced neurons number is found in cor‐ tex, hippocampus and cerebellum of DS brain and are accompanied by impaired neuronal function. Brain hypocellularity is acquired during early developmental stages and is paral‐ leled by impaired cognitive development leading to intellectual disabilities. Further deteri‐ oration of cognitive abilities occurs in adolescence and adulthood, possibly due to degenerative mechanisms [28]. Although the syndrome invariably results in AD-like neuro‐ pathology, the actual onset of dementia is quite variable. The availability of genetic models of trisomy 21 has been instrumental in gaining insights into the pathogenic mechanisms leading to DS cognitive disability. Morphological abnormalities of neuronal dendritic com‐

DSCR1[48], Olig1 and Olig2 [49], or even external to it, as in the case of APP [50,51].

therapeutic strategies that may take into account the complex nature of DS.

of a cDNA driven by a non-physiological promoter [47].

**identification of drugable pathways**

**3.1. Pathogenesis of intellectual disability in DS**

Probably, the most elegant studies that have addressed the role of the mouse genome region syntenic to the human DSCR are those undertaken by Roger H. Reeves and coworkers. Using chromosome engineering, this group has generated a mouse line referred to as Ts1Rhr, trisomic for a segment closely corresponding to the DCR-1 region, as defined by [10] and [11] and including 33 genes [39]. Moreover, they obtained the corresponding deletion, resulting in the monosomic line Ms1Rhr. Interestingly, the first results produced by the analysis of these models did not confirm strongly the DSCR hypothesis. Indeed, the craniofacial dysmorphol‐ ogies of Ts1Rhr are less marked and distinct from those detected in Ts65Dn and Ts1Cje mice [39]. Furthermore, no differences were initially detected between Ts1Rhr and normal controls in the Morris water maze, in the induction of LTP in the hippocampal CA1 Region and in the hippocampal and in cerebellum volume [39-41]. These results seemed to suggest that tripli‐ cation of the Ts1Rhr segment is not sufficient to produce these correlates of DS phenotypes. However, the intercross of the monosomic line Ms1Rhr with the Ds65Dn line, which restored in a disomic condition for DCR-1 genes, generated mice showing normal performances in the Morris water maze, indicating that trisomy of DCR-1 is necessary for these cognitive pheno‐ types [41]. Importantly, a more recent report established that, if the Ts1Rhr mutation is analyzed on the same genetic background of the Ts65Dn and Ts1Cje mice and with more stringent tests, important cognitive and synaptic neurobiological phenotypes can be detected [42]. In particular, 20 of 48 phenotypes, many of which are shared with Ts65Dn mice, distin‐ guished Ts1Rhr animals from their 2N controls. In addition to the genetic background difference, it must be noticed that the task used in this work was less stressful and more sensitive than the water maze, which may further account for the initial discrepancy [42]. These phenotypes were correlated with changes in synaptic density and in dendritic spine morphol‐ ogy, further indicating that DCR-1 genes strongly contribute to these abnormalities [42]. In conclusion, taken together, these results provide strong support to the view that increased dosage of DCR1 genes is necessary and sufficient to confer to mice some of the neurobiological phenotypes characteristic of DS.

The use of mouse genetic tools has allowed the production of even more restricted models, addressing the role of specific subregions of the human or mouse DSCR, or even the role of single DSCR genes. For instance, the isolation from the DSCR of huge genomic clones main‐ tained as Yeast Artificial Chromosomes (YAC) or as Bacterial Artificial Chromosomes (BAC) and their microinjection in mouse oocytes has allowed the generation of transgenic lines covering the entire length of the human DSCR [43-45]. The characterization of these mice has shown that the approach can be very useful to study the function of specific genes. However, it became also clear that this strategy is of limited usefulness to establish genes contribution to the phenotype. For instance, BAC transgenesis allowed the production of a mouse line carrying a single extra copy of the DYRK1A gene [46]. Interestingly, these mice showed impaired cognitive behaviours, but they were characterized by increased hippocampal LTP, while all the models discussed above show depressed hippocampal LTP [46]. The same conclusion applies even better to the models obtained through classical transgenesis ap‐ proaches, in which a single human or mouse gene is inserted in the mouse genome in the form of a cDNA driven by a non-physiological promoter [47].

On the other hand, the combination of gene targeting technologies with the "classical" DS model discussed above allows a subtractive strategy, providing the most stringent test to address the relevance of single genes for the overall phenotype. Indeed, once a null allele for a DSCR gene is available, a compound mutant can be generated, carrying the specific mutation in a trisomic background. The subtractive approach allowed to detect a significant rescue of the phenotype in the case of some DS-related genes, belonging to the DSCR as in the case of DSCR1[48], Olig1 and Olig2 [49], or even external to it, as in the case of APP [50,51].

## **3. Functional role of DSCR genes in DS intellectual disability: Towards the identification of drugable pathways**

In the following section we will summarize the most relevant functional information available on DSCR genes, trying to especially underscore their implication in molecular networks relevant to intellectual disability. As it is obvious from the previous sections, this discussion will involve not only genes that strictly belong to the DSCR, but also their interactions with other HSC21 genes, whose functional involvement is supported by abundant literature. In particular, we will try to discuss as much as possible the single DSCR genes on the basis of their common features. The essential information about genes not included in this section is reported in Tables 1 and 2. While deploying this summary, we will also provide a perspective of how this information can be useful for progressing towards the development of new therapeutic strategies that may take into account the complex nature of DS.

#### **3.1. Pathogenesis of intellectual disability in DS**

cognition through their dosage lie in a region spanning from Sod1 to Mx1, which contains the

Probably, the most elegant studies that have addressed the role of the mouse genome region syntenic to the human DSCR are those undertaken by Roger H. Reeves and coworkers. Using chromosome engineering, this group has generated a mouse line referred to as Ts1Rhr, trisomic for a segment closely corresponding to the DCR-1 region, as defined by [10] and [11] and including 33 genes [39]. Moreover, they obtained the corresponding deletion, resulting in the monosomic line Ms1Rhr. Interestingly, the first results produced by the analysis of these models did not confirm strongly the DSCR hypothesis. Indeed, the craniofacial dysmorphol‐ ogies of Ts1Rhr are less marked and distinct from those detected in Ts65Dn and Ts1Cje mice [39]. Furthermore, no differences were initially detected between Ts1Rhr and normal controls in the Morris water maze, in the induction of LTP in the hippocampal CA1 Region and in the hippocampal and in cerebellum volume [39-41]. These results seemed to suggest that tripli‐ cation of the Ts1Rhr segment is not sufficient to produce these correlates of DS phenotypes. However, the intercross of the monosomic line Ms1Rhr with the Ds65Dn line, which restored in a disomic condition for DCR-1 genes, generated mice showing normal performances in the Morris water maze, indicating that trisomy of DCR-1 is necessary for these cognitive pheno‐ types [41]. Importantly, a more recent report established that, if the Ts1Rhr mutation is analyzed on the same genetic background of the Ts65Dn and Ts1Cje mice and with more stringent tests, important cognitive and synaptic neurobiological phenotypes can be detected [42]. In particular, 20 of 48 phenotypes, many of which are shared with Ts65Dn mice, distin‐ guished Ts1Rhr animals from their 2N controls. In addition to the genetic background difference, it must be noticed that the task used in this work was less stressful and more sensitive than the water maze, which may further account for the initial discrepancy [42]. These phenotypes were correlated with changes in synaptic density and in dendritic spine morphol‐ ogy, further indicating that DCR-1 genes strongly contribute to these abnormalities [42]. In conclusion, taken together, these results provide strong support to the view that increased dosage of DCR1 genes is necessary and sufficient to confer to mice some of the neurobiological

The use of mouse genetic tools has allowed the production of even more restricted models, addressing the role of specific subregions of the human or mouse DSCR, or even the role of single DSCR genes. For instance, the isolation from the DSCR of huge genomic clones main‐ tained as Yeast Artificial Chromosomes (YAC) or as Bacterial Artificial Chromosomes (BAC) and their microinjection in mouse oocytes has allowed the generation of transgenic lines covering the entire length of the human DSCR [43-45]. The characterization of these mice has shown that the approach can be very useful to study the function of specific genes. However, it became also clear that this strategy is of limited usefulness to establish genes contribution to the phenotype. For instance, BAC transgenesis allowed the production of a mouse line carrying a single extra copy of the DYRK1A gene [46]. Interestingly, these mice showed impaired cognitive behaviours, but they were characterized by increased hippocampal LTP, while all the models discussed above show depressed hippocampal LTP [46]. The same conclusion applies even better to the models obtained through classical transgenesis ap‐

mouse counterpart of the human DCR-1.

124 Down Syndrome

phenotypes characteristic of DS.

In order to evaluate the possible degree of functional involvement for specific genes, it is very important to briefly analyze the principal biological processes that have been to cogni‐ tive impairment in the DS. To this regard, studies performed both in humans and in ani‐ mal models have shown that trisomy 21 leads to an unbalance of key cellular events, such as neuronal cell proliferation and differentiation, which can be detected during develop‐ ment and post-natal life using morphological methods [52,53]. Importantly, these defects may coexist with or may be causally related to functional deficits, that can be revealed us‐ ing sophisticated physiological methods [52,53]. Reduced neurons number is found in cor‐ tex, hippocampus and cerebellum of DS brain and are accompanied by impaired neuronal function. Brain hypocellularity is acquired during early developmental stages and is paral‐ leled by impaired cognitive development leading to intellectual disabilities. Further deteri‐ oration of cognitive abilities occurs in adolescence and adulthood, possibly due to degenerative mechanisms [28]. Although the syndrome invariably results in AD-like neuro‐ pathology, the actual onset of dementia is quite variable. The availability of genetic models of trisomy 21 has been instrumental in gaining insights into the pathogenic mechanisms leading to DS cognitive disability. Morphological abnormalities of neuronal dendritic com‐ partment are paralleled by functional electrophysiological deficits and impairment of learn‐ ing and memory, pointing to the existence of defective neural network connectivity and faulty neuronal communication as primary determinants of DS cognitive disabilities [34-38,42,54]. Such pathological scenario arises from a combination of neurodevelopmental abnormalities and neurodegenerative processes. Addressing which processes are irreversi‐ ble and which ones can be prevented or reverted by manipulating genes and pathways is of paramount importance for the development of new therapeutic strategies. Although the crossover between neurogenesis dysfunction and neurodegeneration is still poorly under‐ stood, it is likely that common pathways differentially affect various cellular functions dur‐ ing development and aging. Thus, the developmental aspects are fundamental in defining the most important functional consequences of the genetic imbalance in DS at the cognitive level. However, the IQ of DS patients decreases in the first decade of life, indicating that the maturation of central nervous system is compromised [8]. Indeed, on one side, different observations suggest that neurogenesis impairment starting from the earliest stages of de‐ velopment may underlie the widespread brain atrophy of DS, the delayed and disorgan‐ ized lamination in the DS fetal cortex [55] and hippocampal hypoplasia [56]. On the other, postmortem studies show that DS patients start their lives with an apparently normal neu‐ ronal architecture that progressively degenerates. During the peak period of dendritic growth and differentiation (2.5 months old infants), no significant differences were detect‐ ed in dendritic differentiation between euploid and DS cases in pyramidal neurons of pre‐ frontal cortex [57]. Similarly, DS infants younger than 6 months showed greater dendritic branching and length than normal infants [58] [59] in contrast to the reduced number of dendrites and degenerative changes in DS children older than two years [60].

characterized example is RCAN1, which was initially named DSCR1 [70]. The gene name was then changed after realizing that the encoded protein inhibits calcineurin-dependent tran‐ scriptional responses by binding to the catalytic domain of calcineurin A and interfering with the phosphorylation of the NFAT transcription factor [71,72]. RCAN1 is overexpressed in DS brain [14,73] and seems to play a key role in the regulation of mitochondrial function and oxidative stress. Indeed, the *Drosophila* homolog of RCAN1 especially affects the activity of the mitochondrial ADP/ATP translocator [74]. Moreover, it has been shown that, when RCAN1 is overexpressed in PC12 cells, it induces the expression of superoxide dismutase type 1 (SOD1) [75], which is encoded by another HSA21 gene [15] and is upregulated in DS brain [76]. Importantly, RCAN1 acts as a stress response element: its acute overexpression protects cells from oxidative stress [77]. Indeed, RCAN1 overexpression may have beneficial effects by counteracting the oxidative damage associated with DS. Elevated levels of DNA damage, lipid peroxidation [78] and pro-oxidant state develop early in life in DS subjects [79]. Nevertheless, it is very likely that the benefits arising from these actions on oxidative stress may be overcome by the long-term detrimental effects on synaptic functions and neuronal survival due to the

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127

chronic RCAN1 overexpression, which will be discussed in sections 3.4 and 3.5.

Modifications of the cellular cytoskeleton in response to extracellular stimuli, such as growth factor engagement and cell-cell contacts are essential for neuronal proliferation, for the formation of axons and Dendrites, for the differentiation and for the establishment, maintenance and remodeling of neuronal connections. Many of the well-characterized DSCR genes, such as DSCAM, CLDN14, PIGP, LCA5L, IGSF5 and FAM3B are implicated in these processes. However, the best characterized proteins belonging to this category are

DYRK1A, dual-specificity tyrosine-phosphorilation-regulated kinase1A, encodes a protein kinase capable to phosphorylate serine, threonine and tyrosine residues, highly conserved at the aminoacidic level across vertebrates and invertebrates [80]. The orthologus *Drosophila* gene is involved in neuroblast proliferation and it is named *minibrain* (MNB), because null mutations affect post-embrionic neurogenesis, resulting in reduced brain size [81]. The highly conserved structure of this kinase and its mapping to the DSCR prompted extensive studies on its vertebrate homologues [82]. These studies have revealed that the dosage of DYRK1A is extremely important to normal brain development. Indeed, mice homozygous for a null mutation of DYRK1A die early in development and even heterozygous mice display reduced viability and a smaller brain, characterized by reduction of neuronal counts in specific regions [83]. Accordingly, truncation of the human MNB⁄DYRK1A gene has been reported to cause microcephaly [84,27]. Furthermore transgenic mice overexpressing DYRK1A show severe impairment in spatial learning and memory in the Morris water maze tests, indicating hippocampal and prefrontal cortical function alteration [45,85]. Moreover, these transgenic mice show abnormal LTP and LTD, indicating synaptic plasticity alterations [46]. These defects

**3.3. Signaling proteins encoded by the DSCR**

DYRK1A and TTC3.

*3.3.1. DYRK1A*

#### **3.2. Transcription factors and co-factors encoded by the DSCR**

The DSCR contains 6 genes encoding for transcription factors (Table 1), which are likely to play crucial roles in determining DS phenotypes, considering their potential to affect many cellular networks. Two of them, ERG and ETS2 belong to the erythroblast transformationspecific (ETS) family. Members of this family are key regulators of embryonic development, cell proliferation, differentiation, angiogenesis, inflammation, and apoptosis [61]. ERG is required for vascular cell remodeling and hematopoesis [62,63], while ETS2 has been linked to thymocytes development and apoptosis [64]. Together with RUNX1 [65], these proteins are very likely to contribute to the hematological abnormalities that characterize DS, but not to contribute significantly to ID. In contrast, BRWD1 and HGMN1 are two proteins highly expressed in brain that is involved in chromatin-remodeling [66,67]. Importantly, HGMN1 has been found to regulate the expression of the ID gene MeCP2 [67]. Under the same perspective, another interesting candidate is the bHLH factor SIM2 that together with its paralog SIM1 is the homolog of *Drosophila* single-minded (sim) gene. The *Drosophila* sim gene encodes a transcription factor that is a master regulator of fruit fly neurogenesis [68], raising the possi‐ bility that SIM2 could perform a similar function in mammals. However, a role of SIM2 in mammalian neurogenesis has not been so far confirmed, while this gene has been shown to repress myogenesis in mouse [69]. Besides to directly regulating transcription, DSCR genes could strongly modulate the activity of transcription factors encoded by other loci. The best characterized example is RCAN1, which was initially named DSCR1 [70]. The gene name was then changed after realizing that the encoded protein inhibits calcineurin-dependent tran‐ scriptional responses by binding to the catalytic domain of calcineurin A and interfering with the phosphorylation of the NFAT transcription factor [71,72]. RCAN1 is overexpressed in DS brain [14,73] and seems to play a key role in the regulation of mitochondrial function and oxidative stress. Indeed, the *Drosophila* homolog of RCAN1 especially affects the activity of the mitochondrial ADP/ATP translocator [74]. Moreover, it has been shown that, when RCAN1 is overexpressed in PC12 cells, it induces the expression of superoxide dismutase type 1 (SOD1) [75], which is encoded by another HSA21 gene [15] and is upregulated in DS brain [76]. Importantly, RCAN1 acts as a stress response element: its acute overexpression protects cells from oxidative stress [77]. Indeed, RCAN1 overexpression may have beneficial effects by counteracting the oxidative damage associated with DS. Elevated levels of DNA damage, lipid peroxidation [78] and pro-oxidant state develop early in life in DS subjects [79]. Nevertheless, it is very likely that the benefits arising from these actions on oxidative stress may be overcome by the long-term detrimental effects on synaptic functions and neuronal survival due to the chronic RCAN1 overexpression, which will be discussed in sections 3.4 and 3.5.

#### **3.3. Signaling proteins encoded by the DSCR**

Modifications of the cellular cytoskeleton in response to extracellular stimuli, such as growth factor engagement and cell-cell contacts are essential for neuronal proliferation, for the formation of axons and Dendrites, for the differentiation and for the establishment, maintenance and remodeling of neuronal connections. Many of the well-characterized DSCR genes, such as DSCAM, CLDN14, PIGP, LCA5L, IGSF5 and FAM3B are implicated in these processes. However, the best characterized proteins belonging to this category are DYRK1A and TTC3.

### *3.3.1. DYRK1A*

partment are paralleled by functional electrophysiological deficits and impairment of learn‐ ing and memory, pointing to the existence of defective neural network connectivity and faulty neuronal communication as primary determinants of DS cognitive disabilities [34-38,42,54]. Such pathological scenario arises from a combination of neurodevelopmental abnormalities and neurodegenerative processes. Addressing which processes are irreversi‐ ble and which ones can be prevented or reverted by manipulating genes and pathways is of paramount importance for the development of new therapeutic strategies. Although the crossover between neurogenesis dysfunction and neurodegeneration is still poorly under‐ stood, it is likely that common pathways differentially affect various cellular functions dur‐ ing development and aging. Thus, the developmental aspects are fundamental in defining the most important functional consequences of the genetic imbalance in DS at the cognitive level. However, the IQ of DS patients decreases in the first decade of life, indicating that the maturation of central nervous system is compromised [8]. Indeed, on one side, different observations suggest that neurogenesis impairment starting from the earliest stages of de‐ velopment may underlie the widespread brain atrophy of DS, the delayed and disorgan‐ ized lamination in the DS fetal cortex [55] and hippocampal hypoplasia [56]. On the other, postmortem studies show that DS patients start their lives with an apparently normal neu‐ ronal architecture that progressively degenerates. During the peak period of dendritic growth and differentiation (2.5 months old infants), no significant differences were detect‐ ed in dendritic differentiation between euploid and DS cases in pyramidal neurons of pre‐ frontal cortex [57]. Similarly, DS infants younger than 6 months showed greater dendritic branching and length than normal infants [58] [59] in contrast to the reduced number of

126 Down Syndrome

dendrites and degenerative changes in DS children older than two years [60].

The DSCR contains 6 genes encoding for transcription factors (Table 1), which are likely to play crucial roles in determining DS phenotypes, considering their potential to affect many cellular networks. Two of them, ERG and ETS2 belong to the erythroblast transformationspecific (ETS) family. Members of this family are key regulators of embryonic development, cell proliferation, differentiation, angiogenesis, inflammation, and apoptosis [61]. ERG is required for vascular cell remodeling and hematopoesis [62,63], while ETS2 has been linked to thymocytes development and apoptosis [64]. Together with RUNX1 [65], these proteins are very likely to contribute to the hematological abnormalities that characterize DS, but not to contribute significantly to ID. In contrast, BRWD1 and HGMN1 are two proteins highly expressed in brain that is involved in chromatin-remodeling [66,67]. Importantly, HGMN1 has been found to regulate the expression of the ID gene MeCP2 [67]. Under the same perspective, another interesting candidate is the bHLH factor SIM2 that together with its paralog SIM1 is the homolog of *Drosophila* single-minded (sim) gene. The *Drosophila* sim gene encodes a transcription factor that is a master regulator of fruit fly neurogenesis [68], raising the possi‐ bility that SIM2 could perform a similar function in mammals. However, a role of SIM2 in mammalian neurogenesis has not been so far confirmed, while this gene has been shown to repress myogenesis in mouse [69]. Besides to directly regulating transcription, DSCR genes could strongly modulate the activity of transcription factors encoded by other loci. The best

**3.2. Transcription factors and co-factors encoded by the DSCR**

DYRK1A, dual-specificity tyrosine-phosphorilation-regulated kinase1A, encodes a protein kinase capable to phosphorylate serine, threonine and tyrosine residues, highly conserved at the aminoacidic level across vertebrates and invertebrates [80]. The orthologus *Drosophila* gene is involved in neuroblast proliferation and it is named *minibrain* (MNB), because null mutations affect post-embrionic neurogenesis, resulting in reduced brain size [81]. The highly conserved structure of this kinase and its mapping to the DSCR prompted extensive studies on its vertebrate homologues [82]. These studies have revealed that the dosage of DYRK1A is extremely important to normal brain development. Indeed, mice homozygous for a null mutation of DYRK1A die early in development and even heterozygous mice display reduced viability and a smaller brain, characterized by reduction of neuronal counts in specific regions [83]. Accordingly, truncation of the human MNB⁄DYRK1A gene has been reported to cause microcephaly [84,27]. Furthermore transgenic mice overexpressing DYRK1A show severe impairment in spatial learning and memory in the Morris water maze tests, indicating hippocampal and prefrontal cortical function alteration [45,85]. Moreover, these transgenic mice show abnormal LTP and LTD, indicating synaptic plasticity alterations [46]. These defects are similar to those found in murine models of DS with trisomy of chromosome 16, suggesting a causative role of DYRK1A in cognitive disorders present in DS patients. DYRK1A is ex‐ pressed in the cortex, in the hippocampus and in the cerebellum [86,18] and is overexpressed in the mouse trisomic model Ts65Dn [87], in DS fetal brain and other trisomic tissues [88]. These data obtained from different experimental systems have revealed various possible functions of DYRK1A in central nervous system (CNS) development, including its influence on proliferation, neurogenesis, neuronal differentiation, cell death and synaptic plasticity [46, 89-92]. These multiple biological functions of DYRK1A are due to its interactions with numerous cytoskeletal, synaptic and nuclear proteins, including transcription and splicing factors [93]. Together with other studies [85,94-96], these data strongly support the involve‐ ment of Dyrk1A in several neuropathological phenotypes and in the cognitive deficits that characterize Down syndrome. More recently, the observation that DYRK1A is overexpressed in the adult DS brain [97] implicated this protein also in the DS neurodegenerative phenotype. In particular, DYRK1A overexpression appears to be the cause of gene dosage-dependent modifications of several mechanisms that may contribute to the early onset of neurofibrillary degeneration. In fact, it has been demonstrated that Dyrk1A phosphorylates tau at several sites *in vitro* [98] and such sites are phosphorylated in DS brain [99]. Dyrk1A-induced tau phos‐ phorylation inhibits the biological activity of tau, primes it for further phosphorylation by glycogen synthetase-3β (GSK- 3β) and promotes its self-aggregation into neurofibrillary tangles (NFTs) [99]. Interestingly, besides to phosphorylating protein, DYRK1A also colocal‐ izes with NFTs [100]. In addition, neuropathological and molecular studies indicate that overexpressed nuclear DYRK1A contributes to the modification of the alternative splicing of Tau leading to neurofibrillary degeneration [101,102]. Neurofibrillary degeneration is the leading cause of neuronal death and dementia in Alzheimer's disease (AD) and in DS⁄AD. The multi-pathway involvement of DYRK1A in neurofibrillary degeneration indicates that therapeutic inhibition of the activity of overexpressed DYRK1A may delay the age of onset and inhibit the progression of neurodegeneration in DS. To this regard, the studies recently performed by the group of Delabar [103] represent, arguably, the best example of how the functional knowledge about DSCR genes can be translated into new potential therapeutic strategy. Indeed, this research group has found that Epigallocatechin gallate (EGCG) - a member of a natural polyphenols family, found in great amount in green tea leaves - is a specific and safe DYRK1A inhibitor and that its administration can revert the brain defects induced by overexpression of DYRK1A [103]. Together with a previous report showing that EGCG administration may beneficially affect the LTP abnormalities detected in Ts65Dn mice [104], this study paved the way for the promotion of clinical trials, which are already in Phase 2 (see for instance http://clinicaltrials.gov/ct2/show/NCT01394796).

observed in DS patients [55,107]. In particular, TTC3 is expressed at highest levels in the postmitotic areas of central nervous system (CNS), suggesting a role in neuronal cell differentiation [108,109]. Moreover, it has been reported that the expression of TTC3 is increased in tissues and in cells derived from DS experimental models [110] and from DS individuals [111,112]. In 2007, on the basis of both overexpression and knockdown experiments performed in PC12 neuroblastoma cells, we demonstrated that the TTC3 protein may play a pivotal role in regulating the differentiation program of neuronal cells, starting from the earliest stages [113]. More specifically, increased TTC3 function strongly prevents the neurite sprouting normally elicited by NGF-treatment, while TTC3 knockdown increases neurite length [113]. Important‐ ly, TTC3 may affect not only the generation of neuronal processes, but also their maintenance (Berto et al., unpublished)., and its effects on neuronal differentiation are mediated by the activation of a specific pathway comprising the master cytoskeletal regulator RhoA and its effettor proteins, namely Citron-isoforms [113] Rho kinases (ROCKs) and LIM-kinase (Berto et al., in preparation), which have been implicated in all the different aspects of the neuronal differentiation program [114] and in different aspect of cognitive disorders [115]. Importantly, specific inhibitors of ROCKs, such as Fasudil, have been already approved by FDA, and therefore represent ideal candidates for testing in the experimental models [116]. In addition, a recent report by the group of Dr. M. Noguchi has shown that TTC3 can down-modulate the activity of the Akt kinases (AKTs), by promoting their ubiquitination and degradation [111]. This observation is particularly important, not only because AKTs have been shown to regulate neuronal survival [117], axonogenesis [118], dendritogenesis and synaptogenesis [119], but especially because these proteins are effectors of the PI3K pathway, which is the subject of extensive pharmacological investigation, in light of its centrality in cancer and inflammation

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**3.4. Gene networks affecting the excitatory-inhibitory balance in DS**

The majority of forebrain is comprised of excitatory glutamatergic projection neurons and approximately 10% inhibitory γ-amminobutyric acid (GABA) interneurons. The normal functioning of the neural networks underlying cognitive functions depend on a finely-tuned balance of excitatory and inhibitory activities [122]. Accordingly, different reports have supported the possibility that cognitive impairment in DS models can be related to specific alterations of the excitatory/inhibitory balance, which may result from the direct action of DSCR genes or from more indirect mechanisms. For instance, it has been hypothesized that the increased dosage of HSA21 gene could favor the excitatory inputs in the hippocampus by increasing the activity of N-methyl-D-aspartate (NMDA) receptor (NMDAR), with potential effects on synaptic plasticity and neuron survival [123]. This theory was based on the obser‐ vation that that several HSA21 genes, such as APP, SOD1, RCAN1 and DYRK1A, directly interact or indirectly affect the activity of the NMDARs. The best characterized pathway is that involving RCAN1, which regulates NMDARs by directly binding and inhibiting the calci‐ neurin protein phosphatase (CaN) [71,77,124]. NMDARs are CaN targets [125] [126] and CaN inhibition leads to increased NMDARs [127] activity, by decreasing channel open probability and mean time [127]. On this basis Costa and co-workers hypothesized that the noncompetitive NMDA antagonist memantine, which acts as open channel blocker and is currently approved

research [120,121].

#### *3.3.2. TTC3*

Since its discovery in 1996, the TTC3 gene has been considered an important candidate for the CNS-related phenotypes that characterize DS, because of its mapping within the DSCR [105,106]. This hypothesis was further supported by the analysis of TTC3 expression during normal development. Indeed, during mouse and human brain embryogenesis, TTC3 expres‐ sion shows regional and cellular specificities well correlated with the anatomical defects observed in DS patients [55,107]. In particular, TTC3 is expressed at highest levels in the postmitotic areas of central nervous system (CNS), suggesting a role in neuronal cell differentiation [108,109]. Moreover, it has been reported that the expression of TTC3 is increased in tissues and in cells derived from DS experimental models [110] and from DS individuals [111,112]. In 2007, on the basis of both overexpression and knockdown experiments performed in PC12 neuroblastoma cells, we demonstrated that the TTC3 protein may play a pivotal role in regulating the differentiation program of neuronal cells, starting from the earliest stages [113]. More specifically, increased TTC3 function strongly prevents the neurite sprouting normally elicited by NGF-treatment, while TTC3 knockdown increases neurite length [113]. Important‐ ly, TTC3 may affect not only the generation of neuronal processes, but also their maintenance (Berto et al., unpublished)., and its effects on neuronal differentiation are mediated by the activation of a specific pathway comprising the master cytoskeletal regulator RhoA and its effettor proteins, namely Citron-isoforms [113] Rho kinases (ROCKs) and LIM-kinase (Berto et al., in preparation), which have been implicated in all the different aspects of the neuronal differentiation program [114] and in different aspect of cognitive disorders [115]. Importantly, specific inhibitors of ROCKs, such as Fasudil, have been already approved by FDA, and therefore represent ideal candidates for testing in the experimental models [116]. In addition, a recent report by the group of Dr. M. Noguchi has shown that TTC3 can down-modulate the activity of the Akt kinases (AKTs), by promoting their ubiquitination and degradation [111]. This observation is particularly important, not only because AKTs have been shown to regulate neuronal survival [117], axonogenesis [118], dendritogenesis and synaptogenesis [119], but especially because these proteins are effectors of the PI3K pathway, which is the subject of extensive pharmacological investigation, in light of its centrality in cancer and inflammation research [120,121].

#### **3.4. Gene networks affecting the excitatory-inhibitory balance in DS**

are similar to those found in murine models of DS with trisomy of chromosome 16, suggesting a causative role of DYRK1A in cognitive disorders present in DS patients. DYRK1A is ex‐ pressed in the cortex, in the hippocampus and in the cerebellum [86,18] and is overexpressed in the mouse trisomic model Ts65Dn [87], in DS fetal brain and other trisomic tissues [88]. These data obtained from different experimental systems have revealed various possible functions of DYRK1A in central nervous system (CNS) development, including its influence on proliferation, neurogenesis, neuronal differentiation, cell death and synaptic plasticity [46, 89-92]. These multiple biological functions of DYRK1A are due to its interactions with numerous cytoskeletal, synaptic and nuclear proteins, including transcription and splicing factors [93]. Together with other studies [85,94-96], these data strongly support the involve‐ ment of Dyrk1A in several neuropathological phenotypes and in the cognitive deficits that characterize Down syndrome. More recently, the observation that DYRK1A is overexpressed in the adult DS brain [97] implicated this protein also in the DS neurodegenerative phenotype. In particular, DYRK1A overexpression appears to be the cause of gene dosage-dependent modifications of several mechanisms that may contribute to the early onset of neurofibrillary degeneration. In fact, it has been demonstrated that Dyrk1A phosphorylates tau at several sites *in vitro* [98] and such sites are phosphorylated in DS brain [99]. Dyrk1A-induced tau phos‐ phorylation inhibits the biological activity of tau, primes it for further phosphorylation by glycogen synthetase-3β (GSK- 3β) and promotes its self-aggregation into neurofibrillary tangles (NFTs) [99]. Interestingly, besides to phosphorylating protein, DYRK1A also colocal‐ izes with NFTs [100]. In addition, neuropathological and molecular studies indicate that overexpressed nuclear DYRK1A contributes to the modification of the alternative splicing of Tau leading to neurofibrillary degeneration [101,102]. Neurofibrillary degeneration is the leading cause of neuronal death and dementia in Alzheimer's disease (AD) and in DS⁄AD. The multi-pathway involvement of DYRK1A in neurofibrillary degeneration indicates that therapeutic inhibition of the activity of overexpressed DYRK1A may delay the age of onset and inhibit the progression of neurodegeneration in DS. To this regard, the studies recently performed by the group of Delabar [103] represent, arguably, the best example of how the functional knowledge about DSCR genes can be translated into new potential therapeutic strategy. Indeed, this research group has found that Epigallocatechin gallate (EGCG) - a member of a natural polyphenols family, found in great amount in green tea leaves - is a specific and safe DYRK1A inhibitor and that its administration can revert the brain defects induced by overexpression of DYRK1A [103]. Together with a previous report showing that EGCG administration may beneficially affect the LTP abnormalities detected in Ts65Dn mice [104], this study paved the way for the promotion of clinical trials, which are already in Phase 2 (see

for instance http://clinicaltrials.gov/ct2/show/NCT01394796).

Since its discovery in 1996, the TTC3 gene has been considered an important candidate for the CNS-related phenotypes that characterize DS, because of its mapping within the DSCR [105,106]. This hypothesis was further supported by the analysis of TTC3 expression during normal development. Indeed, during mouse and human brain embryogenesis, TTC3 expres‐ sion shows regional and cellular specificities well correlated with the anatomical defects

*3.3.2. TTC3*

128 Down Syndrome

The majority of forebrain is comprised of excitatory glutamatergic projection neurons and approximately 10% inhibitory γ-amminobutyric acid (GABA) interneurons. The normal functioning of the neural networks underlying cognitive functions depend on a finely-tuned balance of excitatory and inhibitory activities [122]. Accordingly, different reports have supported the possibility that cognitive impairment in DS models can be related to specific alterations of the excitatory/inhibitory balance, which may result from the direct action of DSCR genes or from more indirect mechanisms. For instance, it has been hypothesized that the increased dosage of HSA21 gene could favor the excitatory inputs in the hippocampus by increasing the activity of N-methyl-D-aspartate (NMDA) receptor (NMDAR), with potential effects on synaptic plasticity and neuron survival [123]. This theory was based on the obser‐ vation that that several HSA21 genes, such as APP, SOD1, RCAN1 and DYRK1A, directly interact or indirectly affect the activity of the NMDARs. The best characterized pathway is that involving RCAN1, which regulates NMDARs by directly binding and inhibiting the calci‐ neurin protein phosphatase (CaN) [71,77,124]. NMDARs are CaN targets [125] [126] and CaN inhibition leads to increased NMDARs [127] activity, by decreasing channel open probability and mean time [127]. On this basis Costa and co-workers hypothesized that the noncompetitive NMDA antagonist memantine, which acts as open channel blocker and is currently approved for AD therapy, could mimic the actions of CaN and restore normal NMDARs function, possibly improving learning and memory [123]. Indeed, memantine ameliorates contextual fear conditioning learning in 4–6- and 10–14-month old Ts65Dn mice when administered at 5 mg/kg by acute intraperitoneal injection before context exposure. Despite these studies, a recently published clinical trial reported that memantine is not an effective pharmacological treatment for cognitive decline or dementia in DS patients who are above 40 years old [128]. This suggests that therapies that are effective in DS models and in AD patients may not necessarily confer benefits in DS.

earlier in developing DS brains and decreases to below normal levels by birth [139]. Moreover reduced 5-HT levels are present in adults with DS [140]. Since 5-HT depletion causes a permanent reduction in neuron number in the adult brain [138], it is conceivable that altera‐ tions in the serotonergic systems during early life stages may contribute to the reduced neurogenesis of the DS brain. Activity of the serotonin receptor 1A (5HTR1A) is required for adult neurogenesis in the hippocampus [141] and is mediated by the potassium channel KCNJ6. Overexpression of KCNJ6, as in the Ts65Dn, may over-inhibit presynaptic 5HTR1A, causing reduced levels of serotonin. Fluoxetine, an antidepressant that inhibits serotonin (5- HT) reuptake, inhibits KCNJ6 and increases presynaptic levels of serotonin. Consistent with this, it has been already demonstrated that fluoxetine is able to rescue neurogenesis in the adult Ts65Dn [135]. Recently, treatment during the early postnatal period restored neurogenesis and the total number of neurons in the dentate gyrus. This effect was accompanied by the full recovery of a cognitive task [142]. The releance of these data is even greater if considering that fluoxetin is an antidepressant widely used by adults and prescribed in children and adoles‐ cents [143] and that it does not seem to have negative effects on post-natal development [144].

Molecular Pathways of Down Syndrome Critical Region Genes

http://dx.doi.org/10.5772/53000

131

Most DS patients experience a decline in cognition during adulthood, followed by the development of classical Alzheimer's disease (AD) neuropathology, characterized by the accumulation of amyloid plaques containing high levels of the A-beta fragments of the APP protein, by neurofibrillary tangles containing high levels of hyperphosphorylated Tau protein and by massive neurodegeneration [145]. Increased dosage of the APP gene, which is located outside the DSCR, is very likely the most important factor that underlies this phenomenon [146]. Indeed, increased dosage of APP is sufficient to strongly increase the risk of AD, since APP gene duplication has been detected as the mutation responsible for some early-onset familial cases of AD [147]. The link between AD and the APP gene has been further strength‐ ened by the finding that an extra copy of APP seems to be necessary for the development of AD in DS. Indeed, it has been reported the case of an old patient affected by DS but not showing any signs of dementia [148]. At autopsy, plaques and tangles were absent in the brain of this individual. The patient had a segmental trisomy HSA21, not including the APP gene [148]. These data strongly support that the early onset of AD pathology in DS is in part due to overexpression of the APP gene. The data obtained from experimental models further support the crucial role of APP in DS [51]. Indeed, it has been shown that APP overexpression in Ts65Dn impairs the retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain, causing the degeneration of BFCN [51], which significantly degenerates in Ts65Dn. Importantly, APP is one of the few genes for which a successful subtractive genetic approach has been reported, since restoring APP gene dosage to two copies in the Ts65Dn model corrected the water maze phenotype and prevented BFCN degeneration [50,51]. Finally, APP-mediated pathological mechanism may also contribute to the developmental abnormal‐ ities detected in mouse models, since it has been suggested that APP overexpression can result in increased Notch signaling pathway, which is crucial for neuronal and glial differentiation [149]. However, it is conceivable that also some of the DSCR genes may cooperate with APP in accelerating the AD-related neuropathological phenotypes observed in DS patients. In

**3.5. The DSCR and Alzheimer-related molecular networks**

More consistent reports have shown that the LTP phenotypes and the reduced performance in cognitive tests observed in mouse models could be the result of excessive GABA-ergic responses, producing a net decrease of synaptic output [36,37,129]. This phenomenon could be a direct effect of the overexpression of at least three proteins encoded by the DSCR, namely the chloride channel CLIC6 and the rectifying potassium channels KCNJ6 and KCNJ15. Accordingly, primary hippocampal neurons derived from Ts65Dn mice display a significant increase in GABA-mediated GIRK currents, consistent with the increased expression of KCNJ6/GIRK2 [130]. However, some of the data are also consistent with an increased presynaptic availability of GABA [129], produced by undefined and probably indirect mecha‐ nisms. On this basis, several pharmacological interventions have been proposed to restore the excitatory-inhibitory imbalance by decreasing the excessive inhibition of GABAergic neuro‐ transmission prevalent in DS mouse models [131]. In particular, Ts65Dn mice have been treated with non-competitive GABAA antagonists, pentylenetetrazol (PTZ) and picrotoxin (PTX), which inhibit GABAA receptors. Chronic treatment with PTZ reversed the deficits seen in the novel object recognition task (NORT) and spontaneous alternation tasks in Ts65Dn mice [129,132]. Surprisingly, the improvement in cognition and LTP was sustained for up to 2 months after initial treatment, suggesting a long-lasting effect on neuronal circuit modification. Chronic treatment with PTZ for 8 weeks in Ts65Dn mice did not modify sensorimotor abilities and locomotor activity in home cages. However it did rescue learning and memory perform‐ ance in the Morris water maze (MWM) task [133]. Recently, chronic treatment in Ts65Dn mice with an inverse agonist selective for the α5 subunit of the GABAA benzodiazepine receptor (α5IA) improved cognitive deficits in the MWM and normalized Sod1 overexpression with an enhancement in learning-evoked immediate early genes expression levels [134]. Encouraged by this body of evidence, Roche, a healthcare company, recently announced the commence‐ ment of a trial to examine the cognitive impact of reducing GABA-ergic neurotransmission in the hippocampus using a drug selective for the α5 subunit of GABAA receptors (http:// www.roche-trials.com).

Finally, the imbalance in excitatory/inhibitory ratio could be the result of abnormal neurogen‐ esis. Indeed, reduced cell numbers in the DS hippocampus could be caused by impaired adult neurogenesis, which has been observed in Ts65Dn [135] [136] and Ts1Cje mice [137]. Therefore, approaches targeting neurogenesis seem very promising for DS therapy. Interestingly, a fascinating connection has been documented between the DSCR gene KCNJ6 and adult neurogenesis, mediated by serotonin signaling. DS has long been associated with defects in the serotonergic system [138]. In particular, the serotonin 5-HT1A receptor expression peaks earlier in developing DS brains and decreases to below normal levels by birth [139]. Moreover reduced 5-HT levels are present in adults with DS [140]. Since 5-HT depletion causes a permanent reduction in neuron number in the adult brain [138], it is conceivable that altera‐ tions in the serotonergic systems during early life stages may contribute to the reduced neurogenesis of the DS brain. Activity of the serotonin receptor 1A (5HTR1A) is required for adult neurogenesis in the hippocampus [141] and is mediated by the potassium channel KCNJ6. Overexpression of KCNJ6, as in the Ts65Dn, may over-inhibit presynaptic 5HTR1A, causing reduced levels of serotonin. Fluoxetine, an antidepressant that inhibits serotonin (5- HT) reuptake, inhibits KCNJ6 and increases presynaptic levels of serotonin. Consistent with this, it has been already demonstrated that fluoxetine is able to rescue neurogenesis in the adult Ts65Dn [135]. Recently, treatment during the early postnatal period restored neurogenesis and the total number of neurons in the dentate gyrus. This effect was accompanied by the full recovery of a cognitive task [142]. The releance of these data is even greater if considering that fluoxetin is an antidepressant widely used by adults and prescribed in children and adoles‐ cents [143] and that it does not seem to have negative effects on post-natal development [144].

#### **3.5. The DSCR and Alzheimer-related molecular networks**

for AD therapy, could mimic the actions of CaN and restore normal NMDARs function, possibly improving learning and memory [123]. Indeed, memantine ameliorates contextual fear conditioning learning in 4–6- and 10–14-month old Ts65Dn mice when administered at 5 mg/kg by acute intraperitoneal injection before context exposure. Despite these studies, a recently published clinical trial reported that memantine is not an effective pharmacological treatment for cognitive decline or dementia in DS patients who are above 40 years old [128]. This suggests that therapies that are effective in DS models and in AD patients may not

More consistent reports have shown that the LTP phenotypes and the reduced performance in cognitive tests observed in mouse models could be the result of excessive GABA-ergic responses, producing a net decrease of synaptic output [36,37,129]. This phenomenon could be a direct effect of the overexpression of at least three proteins encoded by the DSCR, namely the chloride channel CLIC6 and the rectifying potassium channels KCNJ6 and KCNJ15. Accordingly, primary hippocampal neurons derived from Ts65Dn mice display a significant increase in GABA-mediated GIRK currents, consistent with the increased expression of KCNJ6/GIRK2 [130]. However, some of the data are also consistent with an increased presynaptic availability of GABA [129], produced by undefined and probably indirect mecha‐ nisms. On this basis, several pharmacological interventions have been proposed to restore the excitatory-inhibitory imbalance by decreasing the excessive inhibition of GABAergic neuro‐ transmission prevalent in DS mouse models [131]. In particular, Ts65Dn mice have been treated with non-competitive GABAA antagonists, pentylenetetrazol (PTZ) and picrotoxin (PTX), which inhibit GABAA receptors. Chronic treatment with PTZ reversed the deficits seen in the novel object recognition task (NORT) and spontaneous alternation tasks in Ts65Dn mice [129,132]. Surprisingly, the improvement in cognition and LTP was sustained for up to 2 months after initial treatment, suggesting a long-lasting effect on neuronal circuit modification. Chronic treatment with PTZ for 8 weeks in Ts65Dn mice did not modify sensorimotor abilities and locomotor activity in home cages. However it did rescue learning and memory perform‐ ance in the Morris water maze (MWM) task [133]. Recently, chronic treatment in Ts65Dn mice with an inverse agonist selective for the α5 subunit of the GABAA benzodiazepine receptor (α5IA) improved cognitive deficits in the MWM and normalized Sod1 overexpression with an enhancement in learning-evoked immediate early genes expression levels [134]. Encouraged by this body of evidence, Roche, a healthcare company, recently announced the commence‐ ment of a trial to examine the cognitive impact of reducing GABA-ergic neurotransmission in the hippocampus using a drug selective for the α5 subunit of GABAA receptors (http://

Finally, the imbalance in excitatory/inhibitory ratio could be the result of abnormal neurogen‐ esis. Indeed, reduced cell numbers in the DS hippocampus could be caused by impaired adult neurogenesis, which has been observed in Ts65Dn [135] [136] and Ts1Cje mice [137]. Therefore, approaches targeting neurogenesis seem very promising for DS therapy. Interestingly, a fascinating connection has been documented between the DSCR gene KCNJ6 and adult neurogenesis, mediated by serotonin signaling. DS has long been associated with defects in the serotonergic system [138]. In particular, the serotonin 5-HT1A receptor expression peaks

necessarily confer benefits in DS.

130 Down Syndrome

www.roche-trials.com).

Most DS patients experience a decline in cognition during adulthood, followed by the development of classical Alzheimer's disease (AD) neuropathology, characterized by the accumulation of amyloid plaques containing high levels of the A-beta fragments of the APP protein, by neurofibrillary tangles containing high levels of hyperphosphorylated Tau protein and by massive neurodegeneration [145]. Increased dosage of the APP gene, which is located outside the DSCR, is very likely the most important factor that underlies this phenomenon [146]. Indeed, increased dosage of APP is sufficient to strongly increase the risk of AD, since APP gene duplication has been detected as the mutation responsible for some early-onset familial cases of AD [147]. The link between AD and the APP gene has been further strength‐ ened by the finding that an extra copy of APP seems to be necessary for the development of AD in DS. Indeed, it has been reported the case of an old patient affected by DS but not showing any signs of dementia [148]. At autopsy, plaques and tangles were absent in the brain of this individual. The patient had a segmental trisomy HSA21, not including the APP gene [148]. These data strongly support that the early onset of AD pathology in DS is in part due to overexpression of the APP gene. The data obtained from experimental models further support the crucial role of APP in DS [51]. Indeed, it has been shown that APP overexpression in Ts65Dn impairs the retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain, causing the degeneration of BFCN [51], which significantly degenerates in Ts65Dn. Importantly, APP is one of the few genes for which a successful subtractive genetic approach has been reported, since restoring APP gene dosage to two copies in the Ts65Dn model corrected the water maze phenotype and prevented BFCN degeneration [50,51]. Finally, APP-mediated pathological mechanism may also contribute to the developmental abnormal‐ ities detected in mouse models, since it has been suggested that APP overexpression can result in increased Notch signaling pathway, which is crucial for neuronal and glial differentiation [149]. However, it is conceivable that also some of the DSCR genes may cooperate with APP in accelerating the AD-related neuropathological phenotypes observed in DS patients. In particular BACE2 could promote the beta-cleavage of APP, further increasing the amount of generated A-beta peptides [150-152]. DYRK1A can also play an important role, because it can stimulate the phosphorylation of APP and Tau, resulting in increased cleavage and aggrega‐ tion, respectively [98,153]. Finally, Tau hyperphosphorylation can be stimulated by increased expression of RCAN1, since phosphorylated Tau is one of the substrates of calcineurin [154]. Moreover, it has been shown that this activity of RCAN1 can be modulated by DYRK1A [155] Therefore it is very likely that the development of new approaches aimed at targeting these proteins could turn out to be beneficial both for AD and for DS management.

**4. Concluding remarks**

**Acknowledgements**

edged.

**Author details**

**References**

Ferdinando Di Cunto\*

disorders such as Alzheimer's disease and Rett syndrome.

and Gaia Berto

of human disease. Circ Res 111: 359-374.

Clin Genet 9: 483-487.

Med 12: 473-479.

University of Torino, Molecular Biotechnology Centre, Torino, Italy

\*Address all correspondence to: ferdinando.dicunto@unito.it; gaia.berto@unito.it

[1] Piro RM (2012) Network medicine: linking disorders. Hum Genet.

oid children. C R Hebd Seances Acad Sci 248: 1721-1722.

[2] Chan SY, Loscalzo J (2012) The emerging paradigm of network medicine in the study

[3] Jackson JF, North ER, 3rd, Thomas JG (1976) Clinical diagnosis of Down's syndrome.

[4] Antonarakis SE, Epstein CJ (2006) The challenge of Down syndrome. Trends Mol

[5] Lejeune J, Gautier M, Turpin R (1959) Study of somatic chromosomes from 9 mongol‐

Functional information on HSA21 genes is still quite partial and mostly limited to a subset of protein-coding genes. However, the recent success in DS models of therapeutic strategies targeted either on specific DSCR genes, or even on much broader mechanisms, justifies to our opinion an optimistic view of the future. In particular, we think that it will be reasonable to expect that a high level of understanding of the complex networks implicating DSCR genes through systems biology approaches will provide very useful insight, which could be trans‐ lated into new therapies that could turn out to be useful not only for DS, but also for other

Molecular Pathways of Down Syndrome Critical Region Genes

http://dx.doi.org/10.5772/53000

133

We are grateful to Dr. Christian Damasco for his help in the production of Tables 1 and 2. The financial contribution of the Jerome Lejeune Foundation FDC and GB is gratefully acknowl‐

#### **3.6. DSCR-dependent RNA-networks**

As it is generally the case for the human genome, besides to protein coding genes, the DSCR contains many sequences that have been so far almost completely neglected, because they are not predicted to encode for proteins [16]. However, as we show in Table 2, on the basis of the current knowledge, many of these loci display features indicating that they could be func‐ tionally relevant and could contribute to the pathogenesis of DS phenotypes. Indeed, besides to the two copies of snRNAs and five copies of snoRNAs associated to splicing factors, the DSCR contains many regions that are transcribed to produce processed transcripts, devoid of coding potential. Some of these sequences, such as antisense transcripts, processed pseudo‐ genes and sequences located in proximity of promoters, are closely associated to functioning genes, and could be involved in their regulation, as it has been shown in many other cases [156-158]. In many other cases, the genes appear to produce llincRNAs, that could act in cis to modify chromatin structure, or in trans to modify gene expression at the transcriptional and post transcriptional level, as it has been shown in the cases of HOTAIR [159] and of LincRNAp21 [160,161]. Although the function of these molecules is at the moment completely unknown, their study could be extremely interesting. Indeed many of these sequences have been implicated in the epigenetic and in the post-transcriptional control of gene expression. Moreover, since these sequences diverge much more rapidly than the sequences of proteincoding genes, it is very likely that they could be strongly implicated in the control of humanspecific features and phenotypes. Therefore, it seems reasonable to anticipate that the functional study of lincRNA-encoding genes in DS models and the study of their variation in humans will be a fertile ground for future research. Finally, the DSCR contains at least three genes encoding miRNA precursors (probably five, if considering also those that have only been predicted). Interestingly, mir-802, which is encoded by the DSCR, and mir-155, which is located on HSA21 in a more centromeric position, have been shown to repress the expression of MeCP2 [162], whose inactivation is the cause of Rett syndrome. Since MeCP2 is also repressed by HMGN1, this study further underscore the potential relevance of MeCP2 repression in DS and provides a very interesting example of how the intertwining of tran‐ scription and post-transcriptional regulatory networks dependent on DSCR genes can produce intellectual disability. Considering the reported reversibility of MeCP2 downregulation phenotypes [163] and the great efforts that are being dedicated to identify drugable pathways downstream of MeCP2 [164], it is conceivable that the functional exploration of these networks in DS could be also relevant for the development of future therapies.

## **4. Concluding remarks**

particular BACE2 could promote the beta-cleavage of APP, further increasing the amount of generated A-beta peptides [150-152]. DYRK1A can also play an important role, because it can stimulate the phosphorylation of APP and Tau, resulting in increased cleavage and aggrega‐ tion, respectively [98,153]. Finally, Tau hyperphosphorylation can be stimulated by increased expression of RCAN1, since phosphorylated Tau is one of the substrates of calcineurin [154]. Moreover, it has been shown that this activity of RCAN1 can be modulated by DYRK1A [155] Therefore it is very likely that the development of new approaches aimed at targeting these

As it is generally the case for the human genome, besides to protein coding genes, the DSCR contains many sequences that have been so far almost completely neglected, because they are not predicted to encode for proteins [16]. However, as we show in Table 2, on the basis of the current knowledge, many of these loci display features indicating that they could be func‐ tionally relevant and could contribute to the pathogenesis of DS phenotypes. Indeed, besides to the two copies of snRNAs and five copies of snoRNAs associated to splicing factors, the DSCR contains many regions that are transcribed to produce processed transcripts, devoid of coding potential. Some of these sequences, such as antisense transcripts, processed pseudo‐ genes and sequences located in proximity of promoters, are closely associated to functioning genes, and could be involved in their regulation, as it has been shown in many other cases [156-158]. In many other cases, the genes appear to produce llincRNAs, that could act in cis to modify chromatin structure, or in trans to modify gene expression at the transcriptional and post transcriptional level, as it has been shown in the cases of HOTAIR [159] and of LincRNAp21 [160,161]. Although the function of these molecules is at the moment completely unknown, their study could be extremely interesting. Indeed many of these sequences have been implicated in the epigenetic and in the post-transcriptional control of gene expression. Moreover, since these sequences diverge much more rapidly than the sequences of proteincoding genes, it is very likely that they could be strongly implicated in the control of humanspecific features and phenotypes. Therefore, it seems reasonable to anticipate that the functional study of lincRNA-encoding genes in DS models and the study of their variation in humans will be a fertile ground for future research. Finally, the DSCR contains at least three genes encoding miRNA precursors (probably five, if considering also those that have only been predicted). Interestingly, mir-802, which is encoded by the DSCR, and mir-155, which is located on HSA21 in a more centromeric position, have been shown to repress the expression of MeCP2 [162], whose inactivation is the cause of Rett syndrome. Since MeCP2 is also repressed by HMGN1, this study further underscore the potential relevance of MeCP2 repression in DS and provides a very interesting example of how the intertwining of tran‐ scription and post-transcriptional regulatory networks dependent on DSCR genes can produce intellectual disability. Considering the reported reversibility of MeCP2 downregulation phenotypes [163] and the great efforts that are being dedicated to identify drugable pathways downstream of MeCP2 [164], it is conceivable that the functional exploration of these networks

proteins could turn out to be beneficial both for AD and for DS management.

in DS could be also relevant for the development of future therapies.

**3.6. DSCR-dependent RNA-networks**

132 Down Syndrome

Functional information on HSA21 genes is still quite partial and mostly limited to a subset of protein-coding genes. However, the recent success in DS models of therapeutic strategies targeted either on specific DSCR genes, or even on much broader mechanisms, justifies to our opinion an optimistic view of the future. In particular, we think that it will be reasonable to expect that a high level of understanding of the complex networks implicating DSCR genes through systems biology approaches will provide very useful insight, which could be trans‐ lated into new therapies that could turn out to be useful not only for DS, but also for other disorders such as Alzheimer's disease and Rett syndrome.

## **Acknowledgements**

We are grateful to Dr. Christian Damasco for his help in the production of Tables 1 and 2. The financial contribution of the Jerome Lejeune Foundation FDC and GB is gratefully acknowl‐ edged.

## **Author details**

Ferdinando Di Cunto\* and Gaia Berto

\*Address all correspondence to: ferdinando.dicunto@unito.it; gaia.berto@unito.it

University of Torino, Molecular Biotechnology Centre, Torino, Italy

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Molecular Pathways of Down Syndrome Critical Region Genes

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**Chapter 9**

**Risk Factors for Down Syndrome Birth: Understanding**

Aneuploidy can be defined as presence of erroneous number of chromosome in organisms and in human aneuploidy is the major cause of birth wastage. Among all known recogniza‐ ble human aneuploidies, trisomy 21 shows the highest frequency of occurrence, estimating approximately 1 in 700 live-births (Kanamori *et al*., 2000). The trisomy 21 condition origi‐ nates due to non-separation or nondisjunction (NDJ) of chromosome 21(Ch21) during game‐ togenesis and as a result disomic gametes with two copies of a particular chromosome are formed and upon fertilization by haploid gamete from opposite sex lead to the formation and implantation of trisomic fetus. The trisomy 21 condition is popularly known as Down syndrome (DS) after the name of John Langdon Down who described the syndrome for the first time in 1866 (Down, 1866). Beside chromosomal NDJ, a small proportion of DS occurs due to post zygotic mitotic error or translocation of chromosome 21 to other autosomes.

Within the category of free trisomy 21 due to NDJ, overwhelming majority of errors occurs in maternal oogenesis particularly at meiosis I (MI) stage (Table 1). A little fraction of NDJ errors arise at paternal spermatogenesis. This preferential occurrence of maternal meiotic er‐ ror is probably due to the mechanism of oocyte maturation in the ovary. Meiosis is initiated in the human foetal ovary at 11–12 weeks of gestation (Gondos *et al*., 1986), but becomes ar‐ rested after completion of homologous chromosome pairing and recombination. This meiot‐ ic-halt lasts for several years until the elevated level of LH and FSH resume the process at the onset of puberty. Then the oocyte completes meiosis I (MI) and enters meiosis II (MII) and again undergoes a phase of pause. It completes the meiosis II after the sperm enter its cytoplasm following fertilization. Thus, the oocyte, whose ovulation marks the menarche, remains in pause for shortest period and that ovulates just preceding menopause experien‐ ces longest period of arrest. This long tenure of oocyte development makes it vulnerable to

and reproduction in any medium, provided the original work is properly cited.

© 2013 Ghosh and Dey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**the Causes from Genetics and Epidemiology**

Sujay Ghosh and Subrata Kumar Dey

http://dx.doi.org/10.5772/52903

**1. Introduction**

Additional information is available at the end of the chapter

## **Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology**

Sujay Ghosh and Subrata Kumar Dey

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52903

## **1. Introduction**

Aneuploidy can be defined as presence of erroneous number of chromosome in organisms and in human aneuploidy is the major cause of birth wastage. Among all known recogniza‐ ble human aneuploidies, trisomy 21 shows the highest frequency of occurrence, estimating approximately 1 in 700 live-births (Kanamori *et al*., 2000). The trisomy 21 condition origi‐ nates due to non-separation or nondisjunction (NDJ) of chromosome 21(Ch21) during game‐ togenesis and as a result disomic gametes with two copies of a particular chromosome are formed and upon fertilization by haploid gamete from opposite sex lead to the formation and implantation of trisomic fetus. The trisomy 21 condition is popularly known as Down syndrome (DS) after the name of John Langdon Down who described the syndrome for the first time in 1866 (Down, 1866). Beside chromosomal NDJ, a small proportion of DS occurs due to post zygotic mitotic error or translocation of chromosome 21 to other autosomes.

Within the category of free trisomy 21 due to NDJ, overwhelming majority of errors occurs in maternal oogenesis particularly at meiosis I (MI) stage (Table 1). A little fraction of NDJ errors arise at paternal spermatogenesis. This preferential occurrence of maternal meiotic er‐ ror is probably due to the mechanism of oocyte maturation in the ovary. Meiosis is initiated in the human foetal ovary at 11–12 weeks of gestation (Gondos *et al*., 1986), but becomes ar‐ rested after completion of homologous chromosome pairing and recombination. This meiot‐ ic-halt lasts for several years until the elevated level of LH and FSH resume the process at the onset of puberty. Then the oocyte completes meiosis I (MI) and enters meiosis II (MII) and again undergoes a phase of pause. It completes the meiosis II after the sperm enter its cytoplasm following fertilization. Thus, the oocyte, whose ovulation marks the menarche, remains in pause for shortest period and that ovulates just preceding menopause experien‐ ces longest period of arrest. This long tenure of oocyte development makes it vulnerable to

© 2013 Ghosh and Dey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


acquire environmental hazards within its microenvironment which inevitably increases the risk of chromosomal NDJ.

eggs more vulnerable to the aging effect than sperms. This long period of oocyte maturation results in the aging associated deteriorative changes to accumulate over time either in the oocyte or its milieu. Examples of such factors would be a diminishing amount of a meiotic proteins, like those maintaining sister chromatid adhesion (Hodges *et al*., 2005; Hunt & Has‐ sold, 2008) or meiotic checkpoints components (Garcia-Cruz *et al*., 2010) or weakening of centromere cohesion due to age-related reduction in centromere associated proteins MCAK (Eichenlaub-Ritter *et al*. 2010). This list of age related risks may also include the accumula‐ tion of environmentally induced damage to the meiotic machinery over time or genetic changes such as mitochondrial deletions (Van Blerkom, 2011). Among all these variables, the spindle assembly check point (SAC) components and sister chromatid cohesion (SCC) were investigated thoroughly (Chiang *et al*.; 2010), as they are prospective genetic candidates that may explain the aging effect on aneuploid oocyte formation. The SAC is a molecular ma‐ chine that ensures proper chromosome separation in both mitosis and meiosis. In meiosis SAC prevents anaphase until all chromosomes properly attach to the spindle. The SAC in‐ cludes *MAD2L1, BUB1B*, and *TTK* (Hached *et al*., 2011; Niault *et al*., 2007) which show de‐ cline in concentration with age in mouse leading to misaligned chromosomes (Pan *et al*., 2008) and errors in SAC function contribute in age-related aneuploidy. Disrupted spindles, misaligned chromosomes and decreased expression of SAC components *Mad2L1* and *Bub1* have evident in aged human oocytes (Mc Guinness *et al*., 2009; Steuerwald *et al*., 2001) and these findings are consistent with aging hypothesis. On the other hand, the SSC mediates physical pairing of duplicated chromosomes which is essential for appropriate distribution of chromosomes. The cohesion along chromosome arms keeps the bivalents intact in MI and centromere cohesion holds sister chromatids together in MII. A defect in cohesion distal to crossover sites may result in a shift in chiasmata placement (alternatively known as 'chiasma slippage') or even premature bivalent separation in MI, whereas reduced centromere cohe‐ sion may result in premature separation of sister chromatids in MII (Steuerwald *et al*., 2001). The loss of cohesion with maternal age for distally placed chiasma (Subramanian and Bickel, 2008) is consistent with the idea that cohesion defects may contribute to age related aneu‐ ploidy (Chiang *et al*., 2012). Another component that supposed to decline with age and con‐ tributes significantly to aging effect on DS birth is the meiosis surveillance system of ovary that ensures achiasmate chromosome segregation (Oliver *et al*., 2008). Chiasma formation and subsequent recombination are prerequisite of faithful separation of homologues at mei‐ otic anaphase. Absences of chiasma, faulty configurations of chiasma and reduction in chias‐ ma frequency have been attributed to NDJ of Ch21 and subsequent DS birth (Lamb *et al*., 2005; Ghosh *et al*., 2010). A high proportion of achiasmate Ch21 tetrad was reported among the mothers of DS having age >35 year (Oliver *et al*., 2008). As the decision regarding chias‐ ma formation is taken in foetal ovary, high frequency of achisamate nondisjoined Ch21 in older oocyte can only be explained by down regulation of surveillance system. Human pro‐ teins involved in segregation of nonexchange chromosome show down regulation with in‐

Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology

http://dx.doi.org/10.5772/52903

151

creasing ovarian age (Steuerwald *et al*., 2001; Baker *et al*., 2004).

**Table 1.** Distribution of mean parental age for Down syndrome birth and nondisjunctional errors of chromosome 21 stratified by parent and meiotic stage of origin

In search of etiology of Ch21 NDJ, researchers have unambiguously identified two risk fac‐ tors namely advancing maternal age and altered pattern of meiotic recombination. Beside these two risk factors, other environmental and behavioural factors have also been identi‐ fied as risk of Ch21 NDJ and they exhibit several degrees of interactions with advancing ma‐ ternal age and recombination pattern of Ch21. These make the etiology of DS birth a puzzle in the field of medical genetics.

## **2. Genetic risk factors**

#### **2.1. Advanced maternal age and related hypotheses**

The age of the mother at the time of the conception of a fetus with DS is, by far, the most significant risk factor for meiotic NDJ of Ch21. As a woman ages, her risk for having a fetus with trisomy 21 significantly increases. This association was noted initially by Penrose in 1933 (Penrose, 1933). For all the populations studied so far, estimated mean maternal age of conception of DS baby is higher than that of controls i.e., having euploid baby and women with MII NDJ is older than women affected with MI NDJ.

Several hypotheses have been put forward to explain the link between advancing maternal age and higher incidence of aneuploid oocyte formation but no one has proved to be com‐ pletely satisfactory. The most popular hypothesis (Gondos *et al*., 1986) holds that the pro‐ tracted tenure of oogenesis interrupted with meiotic halts (Figure 1), probably makes the acquire environmental hazards within its microenvironment which inevitably increases the

Maternal Meiosis I 79.03% 29.07±6.11 34.98±3.88

Paternal Meiosis I 39.23% 24.07±6.22 33.02±5.9

**Table 1.** Distribution of mean parental age for Down syndrome birth and nondisjunctional errors of chromosome 21

In search of etiology of Ch21 NDJ, researchers have unambiguously identified two risk fac‐ tors namely advancing maternal age and altered pattern of meiotic recombination. Beside these two risk factors, other environmental and behavioural factors have also been identi‐ fied as risk of Ch21 NDJ and they exhibit several degrees of interactions with advancing ma‐ ternal age and recombination pattern of Ch21. These make the etiology of DS birth a puzzle

The age of the mother at the time of the conception of a fetus with DS is, by far, the most significant risk factor for meiotic NDJ of Ch21. As a woman ages, her risk for having a fetus with trisomy 21 significantly increases. This association was noted initially by Penrose in 1933 (Penrose, 1933). For all the populations studied so far, estimated mean maternal age of conception of DS baby is higher than that of controls i.e., having euploid baby and women

Several hypotheses have been put forward to explain the link between advancing maternal age and higher incidence of aneuploid oocyte formation but no one has proved to be com‐ pletely satisfactory. The most popular hypothesis (Gondos *et al*., 1986) holds that the pro‐ tracted tenure of oogenesis interrupted with meiotic halts (Figure 1), probably makes the

**Frequency Maternal**

Meiosis II 29.97% 32.54±2.45 35.02±4.66

Meiosis II 59.26% 28.03±4.6 34.09±3.9

**Age at Conception (Years±SD)**

2.2% 29.66±7.3 32.08±5.32

**Paternal Age at Conception (Years±SD**

**Meiotic Origin of Nondisjunction**

risk of chromosomal NDJ.

**Parental Origin**

150 Down Syndrome

Post Zygotic Mitotic Error

stratified by parent and meiotic stage of origin

in the field of medical genetics.

**2. Genetic risk factors**

**2.1. Advanced maternal age and related hypotheses**

with MII NDJ is older than women affected with MI NDJ.

eggs more vulnerable to the aging effect than sperms. This long period of oocyte maturation results in the aging associated deteriorative changes to accumulate over time either in the oocyte or its milieu. Examples of such factors would be a diminishing amount of a meiotic proteins, like those maintaining sister chromatid adhesion (Hodges *et al*., 2005; Hunt & Has‐ sold, 2008) or meiotic checkpoints components (Garcia-Cruz *et al*., 2010) or weakening of centromere cohesion due to age-related reduction in centromere associated proteins MCAK (Eichenlaub-Ritter *et al*. 2010). This list of age related risks may also include the accumula‐ tion of environmentally induced damage to the meiotic machinery over time or genetic changes such as mitochondrial deletions (Van Blerkom, 2011). Among all these variables, the spindle assembly check point (SAC) components and sister chromatid cohesion (SCC) were investigated thoroughly (Chiang *et al*.; 2010), as they are prospective genetic candidates that may explain the aging effect on aneuploid oocyte formation. The SAC is a molecular ma‐ chine that ensures proper chromosome separation in both mitosis and meiosis. In meiosis SAC prevents anaphase until all chromosomes properly attach to the spindle. The SAC in‐ cludes *MAD2L1, BUB1B*, and *TTK* (Hached *et al*., 2011; Niault *et al*., 2007) which show de‐ cline in concentration with age in mouse leading to misaligned chromosomes (Pan *et al*., 2008) and errors in SAC function contribute in age-related aneuploidy. Disrupted spindles, misaligned chromosomes and decreased expression of SAC components *Mad2L1* and *Bub1* have evident in aged human oocytes (Mc Guinness *et al*., 2009; Steuerwald *et al*., 2001) and these findings are consistent with aging hypothesis. On the other hand, the SSC mediates physical pairing of duplicated chromosomes which is essential for appropriate distribution of chromosomes. The cohesion along chromosome arms keeps the bivalents intact in MI and centromere cohesion holds sister chromatids together in MII. A defect in cohesion distal to crossover sites may result in a shift in chiasmata placement (alternatively known as 'chiasma slippage') or even premature bivalent separation in MI, whereas reduced centromere cohe‐ sion may result in premature separation of sister chromatids in MII (Steuerwald *et al*., 2001). The loss of cohesion with maternal age for distally placed chiasma (Subramanian and Bickel, 2008) is consistent with the idea that cohesion defects may contribute to age related aneu‐ ploidy (Chiang *et al*., 2012). Another component that supposed to decline with age and con‐ tributes significantly to aging effect on DS birth is the meiosis surveillance system of ovary that ensures achiasmate chromosome segregation (Oliver *et al*., 2008). Chiasma formation and subsequent recombination are prerequisite of faithful separation of homologues at mei‐ otic anaphase. Absences of chiasma, faulty configurations of chiasma and reduction in chias‐ ma frequency have been attributed to NDJ of Ch21 and subsequent DS birth (Lamb *et al*., 2005; Ghosh *et al*., 2010). A high proportion of achiasmate Ch21 tetrad was reported among the mothers of DS having age >35 year (Oliver *et al*., 2008). As the decision regarding chias‐ ma formation is taken in foetal ovary, high frequency of achisamate nondisjoined Ch21 in older oocyte can only be explained by down regulation of surveillance system. Human pro‐ teins involved in segregation of nonexchange chromosome show down regulation with in‐ creasing ovarian age (Steuerwald *et al*., 2001; Baker *et al*., 2004).

prediction comes from the recent data from prenatal diagnosis after a previous trisomic con‐ ception which shows that the risk of a subsequent trisomy birth is about 1.7 times the mater‐ nal age-related risk (Warburton *et al*., 2005). Mathematical model proposed by Kline and Levin (1992) estimated that women with trisomy pregnancy experience 0.9 years early men‐ opause which suggests that such women suffer from advanced ovarian aging than the wom‐ en with chromosomally normal pregnancies. Population sample survey for calculating the median age of menopause among the women with trisomic pregnancy loss also suggested an early cessation of menstrual cycle among them than the mothers with chromosomally normal foetus (Kline *et al*., 2000). Elevated level of FSH is reported among the women with DS pregnancy (Nasseri *et al*., 1991; van Montfrans *et al*., 2002) which suggests precocious ag‐ ing among them. Very recently, Kline *et al*. (2011) conducted the survey on the hormonal level of women with trisomic pregnancy and supported the '*reduced oocyte pool hypothesis'*, suggesting that some women have smaller follicle content than the others of same chrono‐ logical age. The former group are susceptible for rapid ovarian aging and associated triso‐ mic conceptions. All these findings suggest intuitive existence of some predisposing factors

Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology

http://dx.doi.org/10.5772/52903

153

among some women for their earlier aging that relates their trisomic conception too.

chromosome segregating apparatus at molecular level.

**2.2. Altered pattern of recombination and its interaction with maternal age**

Aside from maternal age, there is only one other factor that has been shown to associate in‐ creased susceptibility of maternal NDJ, namely altered recombination patterns. Warren *et al*. (1987) provided the first evidence to suggest that a proportion of maternal NDJ errors were associated with reduced recombination along Ch 21. Further examination has shown that, in addition to the absence of an exchange along the nondisjoined Ch 21, the placement of an exchange is an important susceptibility factor for NDJ. Examination of recombination along the maternal nondisjoined Ch 21 has suggested three susceptible exchange patterns: 1) no exchange leads to an increased risk of MI errors, 2) a single telomeric exchange leads to an increased risk of MI errors, and 3) a pericentromeric exchange leads to an increased risk of so-called MII errors. These patterns are similar to those observed in model organisms where

The third hypothesis is concerned with 'genetic age' of women and stated that it is the ge‐ netic aging that underlies the all kind of degenerative changes in ovary and oocyte. The hy‐ pothesis was proposed by Ghosh *et al*., (2010). The authors estimated the telomere length of peripheral lymphocyte of women with DS child and compared with age matched controls. They found that beyond of age 29 years the DS bearing mothers exhibit rapid telomere shortening and hence rapid genetic aging than the controls. The authors inferred that DS bearing younger mothers do not experience any accelerated genetic aging; it is only the chronological older age when DS bearing mothers suffer from rapid genetic and molecular aging than the age matched mothers of euploid child. The authors proposed 'Genetic aging hypothesis' which stated that some women are predisposed to rapid genetic and molecular aging and its effect is exacerbated at advance age when age-related deteriorative changes al‐ so affect the chromosome separation system leading to NDJ. The notion has suggested some intuitive link between telomere maintenance system (i.e., system of molecular aging) and

**Figure 1.** Time line for oocyte development in human and probable time of occurrence of risk factors for chromosome 21 nondisjunction.

A second hypothesis relates the "biological aging" or "ovarian aging" with the increasing rate of meiotic errors (Warburton, 1989; 2005). The central theme of this hypothesis is the prediction that biological aging is different among women of the same chronological age and that the frequency of trisomic conceptions depends upon the biological age of the wom‐ an rather than the chronological age (Warburton, 2005). The biological age of women can usually be assessed by counting the falling number of antral follicles with chronological age together with decrease in total oocyte pool size (Scheffer *et al*. 1999; Kline *et al*. 2004). These altogether alter the optimum hormonal balance in ovary, which is marked by falling concen‐ tration of serum inhibin A and B, decline in estrogens surge and elevated level of FSH (War‐ burton, 2005). This change in hormone balance is related to increased rate of aneuploidy at advanced maternal age. Support to this prediction is available from the experiment on mouse model (Robert *et al*. 2005). Alternative to this prediction was provided in the 'limited oocyte pool hypothesis' (Warburton, 2005), which stated that with biological age there is a decrease in the number of antral follicles, leaving only the premature or post mature oocyte to ovulate. The "biological aging" hypothesis predicts that women with a trisomic concep‐ tion should on the average have an older "ovarian age" than other women of the same chro‐ nological age with a normal conception (Warburton, 2005) and women having trisomic pregnancy have average earlier (~1 year) age of menopause (Kline *et al.*, 2000). If these were the facts, one would expect that after a trisomic conception, the risk of a subsequent trisomy for any chromosome should be higher than the maternal age-related risk. Support to this prediction comes from the recent data from prenatal diagnosis after a previous trisomic con‐ ception which shows that the risk of a subsequent trisomy birth is about 1.7 times the mater‐ nal age-related risk (Warburton *et al*., 2005). Mathematical model proposed by Kline and Levin (1992) estimated that women with trisomy pregnancy experience 0.9 years early men‐ opause which suggests that such women suffer from advanced ovarian aging than the wom‐ en with chromosomally normal pregnancies. Population sample survey for calculating the median age of menopause among the women with trisomic pregnancy loss also suggested an early cessation of menstrual cycle among them than the mothers with chromosomally normal foetus (Kline *et al*., 2000). Elevated level of FSH is reported among the women with DS pregnancy (Nasseri *et al*., 1991; van Montfrans *et al*., 2002) which suggests precocious ag‐ ing among them. Very recently, Kline *et al*. (2011) conducted the survey on the hormonal level of women with trisomic pregnancy and supported the '*reduced oocyte pool hypothesis'*, suggesting that some women have smaller follicle content than the others of same chrono‐ logical age. The former group are susceptible for rapid ovarian aging and associated triso‐ mic conceptions. All these findings suggest intuitive existence of some predisposing factors among some women for their earlier aging that relates their trisomic conception too.

The third hypothesis is concerned with 'genetic age' of women and stated that it is the ge‐ netic aging that underlies the all kind of degenerative changes in ovary and oocyte. The hy‐ pothesis was proposed by Ghosh *et al*., (2010). The authors estimated the telomere length of peripheral lymphocyte of women with DS child and compared with age matched controls. They found that beyond of age 29 years the DS bearing mothers exhibit rapid telomere shortening and hence rapid genetic aging than the controls. The authors inferred that DS bearing younger mothers do not experience any accelerated genetic aging; it is only the chronological older age when DS bearing mothers suffer from rapid genetic and molecular aging than the age matched mothers of euploid child. The authors proposed 'Genetic aging hypothesis' which stated that some women are predisposed to rapid genetic and molecular aging and its effect is exacerbated at advance age when age-related deteriorative changes al‐ so affect the chromosome separation system leading to NDJ. The notion has suggested some intuitive link between telomere maintenance system (i.e., system of molecular aging) and chromosome segregating apparatus at molecular level.

#### **2.2. Altered pattern of recombination and its interaction with maternal age**

**Figure 1.** Time line for oocyte development in human and probable time of occurrence of risk factors for chromosome

A second hypothesis relates the "biological aging" or "ovarian aging" with the increasing rate of meiotic errors (Warburton, 1989; 2005). The central theme of this hypothesis is the prediction that biological aging is different among women of the same chronological age and that the frequency of trisomic conceptions depends upon the biological age of the wom‐ an rather than the chronological age (Warburton, 2005). The biological age of women can usually be assessed by counting the falling number of antral follicles with chronological age together with decrease in total oocyte pool size (Scheffer *et al*. 1999; Kline *et al*. 2004). These altogether alter the optimum hormonal balance in ovary, which is marked by falling concen‐ tration of serum inhibin A and B, decline in estrogens surge and elevated level of FSH (War‐ burton, 2005). This change in hormone balance is related to increased rate of aneuploidy at advanced maternal age. Support to this prediction is available from the experiment on mouse model (Robert *et al*. 2005). Alternative to this prediction was provided in the 'limited oocyte pool hypothesis' (Warburton, 2005), which stated that with biological age there is a decrease in the number of antral follicles, leaving only the premature or post mature oocyte to ovulate. The "biological aging" hypothesis predicts that women with a trisomic concep‐ tion should on the average have an older "ovarian age" than other women of the same chro‐ nological age with a normal conception (Warburton, 2005) and women having trisomic pregnancy have average earlier (~1 year) age of menopause (Kline *et al.*, 2000). If these were the facts, one would expect that after a trisomic conception, the risk of a subsequent trisomy for any chromosome should be higher than the maternal age-related risk. Support to this

21 nondisjunction.

152 Down Syndrome

Aside from maternal age, there is only one other factor that has been shown to associate in‐ creased susceptibility of maternal NDJ, namely altered recombination patterns. Warren *et al*. (1987) provided the first evidence to suggest that a proportion of maternal NDJ errors were associated with reduced recombination along Ch 21. Further examination has shown that, in addition to the absence of an exchange along the nondisjoined Ch 21, the placement of an exchange is an important susceptibility factor for NDJ. Examination of recombination along the maternal nondisjoined Ch 21 has suggested three susceptible exchange patterns: 1) no exchange leads to an increased risk of MI errors, 2) a single telomeric exchange leads to an increased risk of MI errors, and 3) a pericentromeric exchange leads to an increased risk of so-called MII errors. These patterns are similar to those observed in model organisms where absence or reduced recombination, along with sub-optimally placed recombinant events, in‐ creases the likelihood of NDJ (Rasooly *et al*., 1991; Moore *et al*., 1994; Sears *et al*.1995; Zetka and Rose, 1995; Koehler *et al*., 1996; Ross *et al*., 1996; Krawchuk and Wahls, 1999). Exchanges too close to the centromere or single exchange too close to the telomere seem to confer chro‐ mosomal instability.

Subsequently, researchers have identified a potential interaction between maternal age and pattern of recombination. The study on US population (Sherman *et al.,* 1994) provided the first evidence in this regard and proved an age related reduction in recombination frequen‐ cy among the MI cases, with older women (35 yrs. and more) having less recombination along 21q than younger women (< 35 yrs.), as suggested by estimated length (cM) of agespecific linkage map of Ch21. In exploring the interaction between maternal age and recom‐ bination and to gain further insight into the potential mechanisms of abnormal chromosome segregation, comparison had been made for frequency and location of meiotic exchanges along 21q (Lamb et al. 2005) among women of various ages who had an infant with DS due to a maternal MI error. While there was no significant association between maternal age and overall frequency of exchange, the placement of meiotic exchange differed significantly by age of conception. In particular, single telomeric recombination event was present in highest proportion among the youngest age group (80%), while the proportion in the oldest group of women and in control group were almost equal (14% and 10% respectively). Moreover, studies (Lamb *et al*., 1996, 2005) suggested that in maternal MI error cases, majority of single exchanges were located in the telomeric end of Ch21, whereas the single exchange within the peri-centromeric region was associated with maternal MII errors. In the independent age-stratified analysis on the US population by Oliver *et al*., (2008) and on the Indian popu‐ lation by Ghosh et al., (2009) a universal pattern of interactions among maternal age groups, chiasma placement and amount of meiotic recombination has been discovered. In these studies a major fraction of MI errors was recorded due to absence of any detectable ex‐ change between non-sister chromatids of nondisjoined homologues. A trend of decreasing frequency of achiasmate meiosis (meiosis without recombination) with increasing maternal age is also observed in both the studies (Oliver *et al*., 2008; Ghosh *et al*., 2009), which sug‐ gests achiasmate meiosis without any recombination is maternal age-independent risk. Ac‐ cording to the model of maternal risk factors for DS birth proposed by Oliver *et al*., (2008) and supported by (Ghosh *et al*. 2009, Ghosh *et al*.,. 2010) that any risk factor which is mater‐ nal age independent should present in highest frequency in the younger mother, the age group in which other risk factors are usually absent. In contrast, any risk factors whose fre‐ quency increases with increasing maternal age is regarded as maternal age dependent risk factor as its effect gets exacerbated in interaction with increasing maternal age. The chiasma stabilizes the tetrad and counter balances the pull from opposite poles which ensure the faithful segregation of homologues. In absence of chiasma, the chromosomes move random‐ ly at MI, resulting in formation of disomic gametes. As the chiasma formation takes place in foetal ovary, the achisamate chromosome containing disomic oocyte may ovulate at any time in reproductive life and hence it is maternal age independent risk factor of Ch21 NDJ.

**Figure 2.** Model for mechanism of nondisjunction of chromosome 21: a) Normal segregation of chromosomes; b) First meiotic nondisjunction; c) Second meiotic nondisjunction. The first meiotic nondisjunction involves telomeric chiasma with premature sister chromatid separation followed by mono-orientation of homologous chromosome at MI. The second meiotic nondisjunction involves peri-centromeric chiasma formation with chromosome entanglement. Noted

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155

that the error actually arises at MI but its effect appeared at MII.

absence or reduced recombination, along with sub-optimally placed recombinant events, in‐ creases the likelihood of NDJ (Rasooly *et al*., 1991; Moore *et al*., 1994; Sears *et al*.1995; Zetka and Rose, 1995; Koehler *et al*., 1996; Ross *et al*., 1996; Krawchuk and Wahls, 1999). Exchanges too close to the centromere or single exchange too close to the telomere seem to confer chro‐

Subsequently, researchers have identified a potential interaction between maternal age and pattern of recombination. The study on US population (Sherman *et al.,* 1994) provided the first evidence in this regard and proved an age related reduction in recombination frequen‐ cy among the MI cases, with older women (35 yrs. and more) having less recombination along 21q than younger women (< 35 yrs.), as suggested by estimated length (cM) of agespecific linkage map of Ch21. In exploring the interaction between maternal age and recom‐ bination and to gain further insight into the potential mechanisms of abnormal chromosome segregation, comparison had been made for frequency and location of meiotic exchanges along 21q (Lamb et al. 2005) among women of various ages who had an infant with DS due to a maternal MI error. While there was no significant association between maternal age and overall frequency of exchange, the placement of meiotic exchange differed significantly by age of conception. In particular, single telomeric recombination event was present in highest proportion among the youngest age group (80%), while the proportion in the oldest group of women and in control group were almost equal (14% and 10% respectively). Moreover, studies (Lamb *et al*., 1996, 2005) suggested that in maternal MI error cases, majority of single exchanges were located in the telomeric end of Ch21, whereas the single exchange within the peri-centromeric region was associated with maternal MII errors. In the independent age-stratified analysis on the US population by Oliver *et al*., (2008) and on the Indian popu‐ lation by Ghosh et al., (2009) a universal pattern of interactions among maternal age groups, chiasma placement and amount of meiotic recombination has been discovered. In these studies a major fraction of MI errors was recorded due to absence of any detectable ex‐ change between non-sister chromatids of nondisjoined homologues. A trend of decreasing frequency of achiasmate meiosis (meiosis without recombination) with increasing maternal age is also observed in both the studies (Oliver *et al*., 2008; Ghosh *et al*., 2009), which sug‐ gests achiasmate meiosis without any recombination is maternal age-independent risk. Ac‐ cording to the model of maternal risk factors for DS birth proposed by Oliver *et al*., (2008) and supported by (Ghosh *et al*. 2009, Ghosh *et al*.,. 2010) that any risk factor which is mater‐ nal age independent should present in highest frequency in the younger mother, the age group in which other risk factors are usually absent. In contrast, any risk factors whose fre‐ quency increases with increasing maternal age is regarded as maternal age dependent risk factor as its effect gets exacerbated in interaction with increasing maternal age. The chiasma stabilizes the tetrad and counter balances the pull from opposite poles which ensure the faithful segregation of homologues. In absence of chiasma, the chromosomes move random‐ ly at MI, resulting in formation of disomic gametes. As the chiasma formation takes place in foetal ovary, the achisamate chromosome containing disomic oocyte may ovulate at any time in reproductive life and hence it is maternal age independent risk factor of Ch21 NDJ.

mosomal instability.

154 Down Syndrome

**Figure 2.** Model for mechanism of nondisjunction of chromosome 21: a) Normal segregation of chromosomes; b) First meiotic nondisjunction; c) Second meiotic nondisjunction. The first meiotic nondisjunction involves telomeric chiasma with premature sister chromatid separation followed by mono-orientation of homologous chromosome at MI. The second meiotic nondisjunction involves peri-centromeric chiasma formation with chromosome entanglement. Noted that the error actually arises at MI but its effect appeared at MII.

In both the studies on US and Indian populations (Oliver *et al.*, 2008; Ghosh *et al*., 2009), the single telomeric chiasma and subsequent recombination were found in highest frequency among the women of younger age group i.e., age group below 29 years, who had a NDJ er‐ ror at meiosis I stage of oogenesis and there was a gradual decrease in telomeric chiasma frequency with advancing maternal age. This observation suggests that the single telomeric chiasma formation is the risk of NDJ of Ch 21 even in younger women who otherwise do not suffer from deterioration related to the aging. Thus within the total risk probability of Ch21 NDJ, the single telomeric chiasma formation represent the highest proportion among the younger women of MI NDJ category. Two important inferences have been drawn from this finding. The first one is that the single telomeric chiasma formation is maternal age in‐ dependent risk of Ch21 NDJ. The second is that the single telomeric chiasma probably indu‐ ces some structural instability of Ch21 that segregates randomly at meiosis I which takes place in fetal ovary.

bility to move randomly into the same product of meiosis at MII, resulting in an apparent MII NDJ. Similar observation is reported from the study in Yeast in which centromere-proxi‐ mal crossover promotes local loss of sister-chromatid cohesion (Rockmill *et al*., 2006). Stud‐ ies of NDJ in both humans (Angell, 1995) and *Drosophila* (Miyazaki & Orr-Weaver, 1992)

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The effect of pericentromeric exchange on meiotic chromosome separation gets exacerbated with maternal age related insults in ovarian environment, as suggested by greater propor‐ tion of DS births among older women who have experienced the particular pattern of chias‐ ma formation. This relationship can be interpreted in two different ways: 1) pericentromeric exchange set up a sub-optimal configuration that initiates or exacerbates the susceptibility to maternal age-related risk factors, perhaps leading to an increase in premature sister chroma‐ tid segregation or 2) a pericentromeric exchange protect the bivalent against age related risk factor, allowing proper segregation of homologues, but not the sister chromatids at MII (Oliver *et al.*, 2008). The former explanation is likely to the '*two hit model*' proposed previous‐ ly by Lamb *et al.,* (1996). Alternatively, a pericentromeric exchange may protect the bivalent from maternal age related risk factors. The effect of degradation of centromere or sister chro‐ matid cohesion complexes or of spindle proteins with age of oocyte may lead to premature sister chromatid separation. Perhaps the pericentromeric exchanges help to stabilize the compromised tetrad through MI. This would lead to an enrichment of MII errors among the

As far as effect of multiple chiasmata formation on the nondisjoined Ch 21 is concerned, two important reports have been published very recently. In their study Ghosh *et al.* (2010) found that two or more chiasmata formation is prevalent particularly in older age group (≥ 34 years). This infers that the older oocyte suffers from nondisjunctional errors even when Ch21 experiences formation of two or more chiasmata which are believed to be protective of NDJ; this is due to aging effects that imparts various degenerative changes in ovary. Analyz‐ ing the effect of multiple chiasmata of the 21q, Oliver *et al*. (2011) found a decrease in the interval between two simultaneous chisamata on the chromosome that disjoined at MI and this closeness is due to shifting of distal chiasma towards centromere. The author argued that as the proximal chiasma remains at its usual position, similar to that on the normally disjoined chromosome, it is the distal chiasma whose dislocation towards the proximal chiasma nullifies the 'good-effect' of the latter that is needed for faithful segregation of the chromosome. The Ch21 experiences such distal chiasma dislocation in association with cor‐ rectly placed proximal chiasma disjoines erroneously at MI. Moreover, the authors found more intimate positioning of proximal chiasma with the centromere of the chromosomes with two exchanges and this tendency increases with advancing age. This pattern is very similar to the single chiasma shifting related to MII errors reported in earlier studies (Oliver et al., 2008; Ghosh et al., 2009). Moreover, the authors further extend their realization that the centromeric chiasma may not be protective of NDJ, the notion previously assumed both

have provided preliminary supports for this model.

older oocytes which is a maternal age dependent risk for NDJ of Ch21.

by Oliver *et al.* (2008) and Ghosh *et al*. (2009).

Understanding the exact mechanism how does single telomeric chiasma cause chromosomal mis-segregation has been obtained from the observations in model organisms like *Drosophila* (Koehler *et al.,* 1996), *Saccharomyces* (Ross *et al*. 1996) and *Caenorhabditis elegans* (Zetka and Rose, 1995). As the telomeric chiasma located far from the kinetochore, the point of spindleattachment links the homologues less efficiently and orients each kinetochore to the same spindle pole and prevents bi-orientation of homologues (Nicklas, 1974; Hawley *et al*., 1994; Koehler *et al.,* 1996). Most likely, this susceptibility is related to the minimal amount of sister chromatid cohesion complex (Figure 2b) remaining distal to the exchange event (Orr-Wea‐ ver, 1996). Alternatively, the integrity of chiasma may be compromised when a minimum amount of cohesin remains to hold homologue together. Thus bivalent may act as pair of functional univalent during MI, as has been evident in human oocyte (Angell, 1994; 1995).

Another chiasma configuration that poses susceptibility for NDJ of Ch21 is the pericentro‐ meric exchange. In both the studies on US and Indian DS populations (Oliver *et al.,* 2008; Ghosh *et al.,* 2009), highest frequency of pericentromeric exchange was scored in older wom‐ en having age >34 years. A trend of gradual increase in centromeric chiasma frequency with increasing age was recorded in both the studies with gradual shifting of chiasma from mid‐ dle of the chromosome in younger age group to more proximal to centromere in older age group. In explaining the effect on chromosome segregation that single centromeric chiasma imparts two hypotheses have been put forward by the authors. The chiasma that is posi‐ tioned very close to centromere may cause 'chromosomal entanglement' at MI, with the bi‐ valent being unable to separate, passing intact to MII metaphase plate (Lamb *et al*., 1996). Upon MII division, the bivalent divides reductionally, resulting in disomic gamete with identical centromeres (Figure 2c). In this manner, proximal pericentromeric exchange, which occurs at MI, is resolved and visualized as MII error. According to an alternate model, stud‐ ied in *Drosophila* (Koehler *et al*., 1996), proximal chiasma leads to a premature sister chroma‐ tid separation just prior to anaphase I. Resolution of chiasma requires the release of sister chromatid cohesion distal to the site of exchange (Hawley *et al.,* 1994). Attempt to resolve chiasma that is very close to centromere could result in premature separation of chromatids (Figure 2c). If the sister chromatids migrate to a common pole at MI, they have 50% proba‐ bility to move randomly into the same product of meiosis at MII, resulting in an apparent MII NDJ. Similar observation is reported from the study in Yeast in which centromere-proxi‐ mal crossover promotes local loss of sister-chromatid cohesion (Rockmill *et al*., 2006). Stud‐ ies of NDJ in both humans (Angell, 1995) and *Drosophila* (Miyazaki & Orr-Weaver, 1992) have provided preliminary supports for this model.

In both the studies on US and Indian populations (Oliver *et al.*, 2008; Ghosh *et al*., 2009), the single telomeric chiasma and subsequent recombination were found in highest frequency among the women of younger age group i.e., age group below 29 years, who had a NDJ er‐ ror at meiosis I stage of oogenesis and there was a gradual decrease in telomeric chiasma frequency with advancing maternal age. This observation suggests that the single telomeric chiasma formation is the risk of NDJ of Ch 21 even in younger women who otherwise do not suffer from deterioration related to the aging. Thus within the total risk probability of Ch21 NDJ, the single telomeric chiasma formation represent the highest proportion among the younger women of MI NDJ category. Two important inferences have been drawn from this finding. The first one is that the single telomeric chiasma formation is maternal age in‐ dependent risk of Ch21 NDJ. The second is that the single telomeric chiasma probably indu‐ ces some structural instability of Ch21 that segregates randomly at meiosis I which takes

Understanding the exact mechanism how does single telomeric chiasma cause chromosomal mis-segregation has been obtained from the observations in model organisms like *Drosophila* (Koehler *et al.,* 1996), *Saccharomyces* (Ross *et al*. 1996) and *Caenorhabditis elegans* (Zetka and Rose, 1995). As the telomeric chiasma located far from the kinetochore, the point of spindleattachment links the homologues less efficiently and orients each kinetochore to the same spindle pole and prevents bi-orientation of homologues (Nicklas, 1974; Hawley *et al*., 1994; Koehler *et al.,* 1996). Most likely, this susceptibility is related to the minimal amount of sister chromatid cohesion complex (Figure 2b) remaining distal to the exchange event (Orr-Wea‐ ver, 1996). Alternatively, the integrity of chiasma may be compromised when a minimum amount of cohesin remains to hold homologue together. Thus bivalent may act as pair of functional univalent during MI, as has been evident in human oocyte (Angell, 1994; 1995).

Another chiasma configuration that poses susceptibility for NDJ of Ch21 is the pericentro‐ meric exchange. In both the studies on US and Indian DS populations (Oliver *et al.,* 2008; Ghosh *et al.,* 2009), highest frequency of pericentromeric exchange was scored in older wom‐ en having age >34 years. A trend of gradual increase in centromeric chiasma frequency with increasing age was recorded in both the studies with gradual shifting of chiasma from mid‐ dle of the chromosome in younger age group to more proximal to centromere in older age group. In explaining the effect on chromosome segregation that single centromeric chiasma imparts two hypotheses have been put forward by the authors. The chiasma that is posi‐ tioned very close to centromere may cause 'chromosomal entanglement' at MI, with the bi‐ valent being unable to separate, passing intact to MII metaphase plate (Lamb *et al*., 1996). Upon MII division, the bivalent divides reductionally, resulting in disomic gamete with identical centromeres (Figure 2c). In this manner, proximal pericentromeric exchange, which occurs at MI, is resolved and visualized as MII error. According to an alternate model, stud‐ ied in *Drosophila* (Koehler *et al*., 1996), proximal chiasma leads to a premature sister chroma‐ tid separation just prior to anaphase I. Resolution of chiasma requires the release of sister chromatid cohesion distal to the site of exchange (Hawley *et al.,* 1994). Attempt to resolve chiasma that is very close to centromere could result in premature separation of chromatids (Figure 2c). If the sister chromatids migrate to a common pole at MI, they have 50% proba‐

place in fetal ovary.

156 Down Syndrome

The effect of pericentromeric exchange on meiotic chromosome separation gets exacerbated with maternal age related insults in ovarian environment, as suggested by greater propor‐ tion of DS births among older women who have experienced the particular pattern of chias‐ ma formation. This relationship can be interpreted in two different ways: 1) pericentromeric exchange set up a sub-optimal configuration that initiates or exacerbates the susceptibility to maternal age-related risk factors, perhaps leading to an increase in premature sister chroma‐ tid segregation or 2) a pericentromeric exchange protect the bivalent against age related risk factor, allowing proper segregation of homologues, but not the sister chromatids at MII (Oliver *et al.*, 2008). The former explanation is likely to the '*two hit model*' proposed previous‐ ly by Lamb *et al.,* (1996). Alternatively, a pericentromeric exchange may protect the bivalent from maternal age related risk factors. The effect of degradation of centromere or sister chro‐ matid cohesion complexes or of spindle proteins with age of oocyte may lead to premature sister chromatid separation. Perhaps the pericentromeric exchanges help to stabilize the compromised tetrad through MI. This would lead to an enrichment of MII errors among the older oocytes which is a maternal age dependent risk for NDJ of Ch21.

As far as effect of multiple chiasmata formation on the nondisjoined Ch 21 is concerned, two important reports have been published very recently. In their study Ghosh *et al.* (2010) found that two or more chiasmata formation is prevalent particularly in older age group (≥ 34 years). This infers that the older oocyte suffers from nondisjunctional errors even when Ch21 experiences formation of two or more chiasmata which are believed to be protective of NDJ; this is due to aging effects that imparts various degenerative changes in ovary. Analyz‐ ing the effect of multiple chiasmata of the 21q, Oliver *et al*. (2011) found a decrease in the interval between two simultaneous chisamata on the chromosome that disjoined at MI and this closeness is due to shifting of distal chiasma towards centromere. The author argued that as the proximal chiasma remains at its usual position, similar to that on the normally disjoined chromosome, it is the distal chiasma whose dislocation towards the proximal chiasma nullifies the 'good-effect' of the latter that is needed for faithful segregation of the chromosome. The Ch21 experiences such distal chiasma dislocation in association with cor‐ rectly placed proximal chiasma disjoines erroneously at MI. Moreover, the authors found more intimate positioning of proximal chiasma with the centromere of the chromosomes with two exchanges and this tendency increases with advancing age. This pattern is very similar to the single chiasma shifting related to MII errors reported in earlier studies (Oliver et al., 2008; Ghosh et al., 2009). Moreover, the authors further extend their realization that the centromeric chiasma may not be protective of NDJ, the notion previously assumed both by Oliver *et al.* (2008) and Ghosh *et al*. (2009).

## **2.3. Genetic polymorphism and increasing susceptibility of Down syndrome birth**

Maternal genetic factors such as polymorphism of certain gene probably make them suscep‐ tible for NDJ error. Experimental organisms have been used to identify genes that are im‐ portant in the proper segregation of chromosomes. The potential candidates are those genes involved in the meiotic process such as homologue pairing, assembly of the synaptonemal complex, chiasmata formation and chiasma positioning, sister chromatid cohesion, spindle formation. Genetic variations of these genes are predisposing factors for chromosome NDJ.

polymorphisms (Coppedè *et al.,* 2010). Cyril *et al*., (2009) conducted such association study on Indian women and confirmed the association of *MTHFR* 677C→T polymorphism with

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**Figure 3.** Role of *MTHFR* gene in folate metabolism pathway and effect of its polymorphism on chromosome 21 seg‐ regation. a) The left panel shows wild *MTHFR* genes and its involvement in chromosome segregation system; b) The mutation in MTHFR gene disrupts the folate metabolism pathway leading to missegregation of chromosome.

The other way to find out the genes involved in human NDJ is to analyze the association of consanguinity and trisomy 21(Sherman *et al*., 2005). If such an association really does exist, it would provide evidence for a genetic effect for NDJ. The study of Alfi *et al*., (1980) provided one of the earlier reports suggesting an association between increased consanguinity among parents of individuals with DS in a study population in Kuwait. Authors postulated the ex‐ istence of a gene that increases the risk for mitotic NDJ. Alternatively, they suggested that increased rates of consanguinity among parents would be correlated with those in grand‐ parents and therefore, an autosomal recessive gene may be postulated to be involved in mei‐ otic NDJ in the homozygous parents. But the reports from subsequent studies in other populations are contradictory and did not find any evidence for an association between con‐ sanguinity and human NDJ (Devoto *et al*., 1985; Hamamy *et al*., 1990; Roberts *et al*., 1991;

Lastly, differences in the prevalence of DS among different racial groups may provide indi‐ rect evidence for genetic factors involved in human NDJ. However, such studies are difficult to conduct and to interpret. Differences (or similarities) may reflect the maternal age distri‐ bution of the population, accuracy of diagnosis, cultural preference and/or access to selec‐

Basaran *et al.*, 1992; Zlotogora, 1997; Sayee & Thomas, 1998; Rittler *et al*., 2001).

DS birth risk.

The gene that has been identified first in this category is *MTHFR* (methylene tetrahydrofo‐ late reductase), which is not directly related to the meiotic process. The case-control study by James *et al*., (1999) provided primary evidence that the 677C→T polymorphism in the *MTHFR* gene increases the risk of having a child with DS (Odds Ratio = 2.6) in North Ameri‐ can population. This polymorphism is associated with elevated plasma homocysteine and/or low folate status (Sherman *et al*., 2005). Folate is essential for the production of S-ade‐ nosylmethionine, which is the primary methyl donor (Figure 3a) for epigenetic DNA meth‐ ylation essential for gene expression regulation and maintenance of chromosomal integrity at centromere (James *et al*., 1999; Dworkin *et al*., 2009; Sciandrello *et al*., 2004). Folate deficien‐ cy reduces S-adenosylmethionine synthesis, leading to DNA hypomethylation (Pogribny *et al*., 1997; Beetstra *et al*., 2005; Wang *et al*., 2004). The pericentromeric hypomethylation could impair the heterochromatin formation and kinetochore establishment (Figure 3b )resulting in chromosomal NDJ (James *et al*., 1999). This happens because the stable centromeric chro‐ matin depends on the epigenetic inheritance of specific centromeric methylation patterns and it binds with specific methyl-sensitive proteins in order to maintain the higher-order DNA architecture necessary for kinetochore assembly (Migliore *et al.,* 2009).

This initial report had inspired several follow-up studies on the *MTHFR* 677C→T polymor‐ phism, as well as several other allelic variants in the folate pathway genes to identify genetic risk factors for having a child with DS. But the results are inconsistent (James *et al*. 2004a, 2004b), especially those that have evaluated genotype alone without biomarkers of metabol‐ ic phenotype. Those who have examined blood homocysteine levels, a broad-spectrum indi‐ cator of nutritional and/or genetic impairment in folate/B12 metabolism have documented a significantly higher level among the mothers of children with DS compared with control mothers from the same country. One possible explanation for the inconsistent results among the numerous studies may reflect the complex interaction between effects of genetic variants and nutritional intake (James *et al.,* 2004b). Nevertheless, support to the notion regarding the association between MTHFR 677C-T polymorphism and risk of DS birth was provided by other studies in different populations. Wang *et al*., (2004) reported significant increase in the risk of DS conception among Chinese women bearing two polymorphisms namely, poly‐ morphisms of *MTHFR* 677C→T and the polymorphism *MTRR* (Methionine synthase reduc‐ tase) 66A→G. The estimated risks were more than three folds and five folds for *MTHFR* (Odd Ratio=3.7; 95% CI, 1.78~8.47) and *MTRR* (Odd Ratio= 5.2; 95% CI, 1.90~14.22) respec‐ tively. The combined presence of both polymorphisms was associated with a greater risk of DS than the presence of either alone, with an odds ratio of 6.0 (95% CI, 2.058~17.496). The study on Italian population also agreed the link between DS birth and *MTHFR* and *MTRR* polymorphisms (Coppedè *et al.,* 2010). Cyril *et al*., (2009) conducted such association study on Indian women and confirmed the association of *MTHFR* 677C→T polymorphism with DS birth risk.

**2.3. Genetic polymorphism and increasing susceptibility of Down syndrome birth**

158 Down Syndrome

Maternal genetic factors such as polymorphism of certain gene probably make them suscep‐ tible for NDJ error. Experimental organisms have been used to identify genes that are im‐ portant in the proper segregation of chromosomes. The potential candidates are those genes involved in the meiotic process such as homologue pairing, assembly of the synaptonemal complex, chiasmata formation and chiasma positioning, sister chromatid cohesion, spindle formation. Genetic variations of these genes are predisposing factors for chromosome NDJ.

The gene that has been identified first in this category is *MTHFR* (methylene tetrahydrofo‐ late reductase), which is not directly related to the meiotic process. The case-control study by James *et al*., (1999) provided primary evidence that the 677C→T polymorphism in the *MTHFR* gene increases the risk of having a child with DS (Odds Ratio = 2.6) in North Ameri‐ can population. This polymorphism is associated with elevated plasma homocysteine and/or low folate status (Sherman *et al*., 2005). Folate is essential for the production of S-ade‐ nosylmethionine, which is the primary methyl donor (Figure 3a) for epigenetic DNA meth‐ ylation essential for gene expression regulation and maintenance of chromosomal integrity at centromere (James *et al*., 1999; Dworkin *et al*., 2009; Sciandrello *et al*., 2004). Folate deficien‐ cy reduces S-adenosylmethionine synthesis, leading to DNA hypomethylation (Pogribny *et al*., 1997; Beetstra *et al*., 2005; Wang *et al*., 2004). The pericentromeric hypomethylation could impair the heterochromatin formation and kinetochore establishment (Figure 3b )resulting in chromosomal NDJ (James *et al*., 1999). This happens because the stable centromeric chro‐ matin depends on the epigenetic inheritance of specific centromeric methylation patterns and it binds with specific methyl-sensitive proteins in order to maintain the higher-order

DNA architecture necessary for kinetochore assembly (Migliore *et al.,* 2009).

This initial report had inspired several follow-up studies on the *MTHFR* 677C→T polymor‐ phism, as well as several other allelic variants in the folate pathway genes to identify genetic risk factors for having a child with DS. But the results are inconsistent (James *et al*. 2004a, 2004b), especially those that have evaluated genotype alone without biomarkers of metabol‐ ic phenotype. Those who have examined blood homocysteine levels, a broad-spectrum indi‐ cator of nutritional and/or genetic impairment in folate/B12 metabolism have documented a significantly higher level among the mothers of children with DS compared with control mothers from the same country. One possible explanation for the inconsistent results among the numerous studies may reflect the complex interaction between effects of genetic variants and nutritional intake (James *et al.,* 2004b). Nevertheless, support to the notion regarding the association between MTHFR 677C-T polymorphism and risk of DS birth was provided by other studies in different populations. Wang *et al*., (2004) reported significant increase in the risk of DS conception among Chinese women bearing two polymorphisms namely, poly‐ morphisms of *MTHFR* 677C→T and the polymorphism *MTRR* (Methionine synthase reduc‐ tase) 66A→G. The estimated risks were more than three folds and five folds for *MTHFR* (Odd Ratio=3.7; 95% CI, 1.78~8.47) and *MTRR* (Odd Ratio= 5.2; 95% CI, 1.90~14.22) respec‐ tively. The combined presence of both polymorphisms was associated with a greater risk of DS than the presence of either alone, with an odds ratio of 6.0 (95% CI, 2.058~17.496). The study on Italian population also agreed the link between DS birth and *MTHFR* and *MTRR*

**Figure 3.** Role of *MTHFR* gene in folate metabolism pathway and effect of its polymorphism on chromosome 21 seg‐ regation. a) The left panel shows wild *MTHFR* genes and its involvement in chromosome segregation system; b) The mutation in MTHFR gene disrupts the folate metabolism pathway leading to missegregation of chromosome.

The other way to find out the genes involved in human NDJ is to analyze the association of consanguinity and trisomy 21(Sherman *et al*., 2005). If such an association really does exist, it would provide evidence for a genetic effect for NDJ. The study of Alfi *et al*., (1980) provided one of the earlier reports suggesting an association between increased consanguinity among parents of individuals with DS in a study population in Kuwait. Authors postulated the ex‐ istence of a gene that increases the risk for mitotic NDJ. Alternatively, they suggested that increased rates of consanguinity among parents would be correlated with those in grand‐ parents and therefore, an autosomal recessive gene may be postulated to be involved in mei‐ otic NDJ in the homozygous parents. But the reports from subsequent studies in other populations are contradictory and did not find any evidence for an association between con‐ sanguinity and human NDJ (Devoto *et al*., 1985; Hamamy *et al*., 1990; Roberts *et al*., 1991; Basaran *et al.*, 1992; Zlotogora, 1997; Sayee & Thomas, 1998; Rittler *et al*., 2001).

Lastly, differences in the prevalence of DS among different racial groups may provide indi‐ rect evidence for genetic factors involved in human NDJ. However, such studies are difficult to conduct and to interpret. Differences (or similarities) may reflect the maternal age distri‐ bution of the population, accuracy of diagnosis, cultural preference and/or access to selec‐ tive prenatal termination of pregnancies with trisomic fetuses, and as yet unidentified environmental factors (Sherman *et al*., 2005). Only one such study by Allen *et al*., (2009) re‐ ported demographic differences in mean maternal age of DS conception recorded in two dif‐ ferent sample sets from USA. This study included DS samples from Atlanta Down syndrome project and National Down syndrome project and found that mothers enrolled in National Down syndrome project were on an average older than those of Atlanta. Moreover, the authors have also reported some ethnic differences in maternal age distribution. The At‐ lanta Down syndrome project had a higher proportion of cases and controls that were black and a significantly smaller proportion of Hispanics than did the National Down syndrome project. Comparison of mean maternal ages indicated variation by ethnic groups. In both the Atlanta Down syndrome project and National Down syndrome project, white mothers tend‐ ed to be older than their black or Hispanic counterparts. Specifically, for both cases and con‐ trols, white mothers were found to be significantly older than black mothers (P< 0.01) and Hispanic mothers (P< 0.01); blacks and Hispanics were not significantly different from each other (P>0.05). To confirm such effect of demographic and ethnic differences on the etiology of DS birth, further large scale population based studies are needed to be conducted.

show various degrees of associations with DS birth. The list includes maternal cigarette smoking, use of oral contraceptive, peri-conceptional alcohol consumption by mother, expo‐ sure to radiation and low socio-economic status. Number of studies reported a negative as‐ sociation between maternal smoking around the time of conception and the risk for DS birth (Kline *et al*., 1983, 1993; Hook & Cross, 1985, 1988; Shiono *et al*., 1986; Chen *et al*., 1999). One explanation for the negative association was that trisomic conceptuses were selectively lost prenatally among women who smoke (Hook and Cross, 1985; Kline *et al*., 1993). But evi‐ dence against this speculation is also available (Cuckle *et al*., 1990; Kallen, 1997; Torf & Christianson, 2000). Study conducted by Yang *et al*., (1999) suggested that maternal-smoking was significantly associated with MII error and probably due to compromise in blood and oxygen supply surrounding the developing follicles. Besides smoking, the other maternal risk factor for which epidemiological studies have been conducted most is oral contracep‐ tive. The use of oral contraceptive by women at the time of conception is subject of specula‐ tion as risk for DS births (Yang *et al*., 1999). The study by Martinez-Frias *et al*., (2001) showed that the risk for DS in infants born to mothers with less than 35 years of age (as a group) who became pregnant while taking oral-contraceptive is near the risk for mothers of DS with more than 35 years of age. In their epidemiological study, Yang *et al*., (1999) found that women having simultaneous habits of smoking and using oral contraceptive have seven folds increased risk of having DS pregnancy and they argued that this is due to anoxic con‐ dition in ovarian microenvironment related to toxicant induced reduction in blood flow sur‐ rounding ovary. This speculation is similar to that proposed by Gaulden (1992) to explain the cause of maternal-age related NDJ. She suggested that the follicular microcirculation may be compromised in an aging ovary because of abnormal hormone signaling. Although sufficient evidence is lacking (Henderson *et al*., 2007), alcohol consumption by women in‐

Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology

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161

creases the chance of having DS pregnancy as suggested by Kaufman (1983).

Very recently, population based epidemiological study by Ghosh *et al*., (2011) analyzed the effect of chewing tobacco and contraceptive pill use on the Ch21 NDJ in interaction with known risk variables like maternal age, meiotic stage of NDJ and pattern of recombination i.e., amount of exchange and positioning of chiasma on the recombining homologues. Vari‐ ous logistic regression models have been designed to examine every possible interaction among all above mentioned risk factors. Smokeless chewing tobacco was associated with significant risk for MII NDJ and achiasmate (nonexchange) MI error among the younger mothers. For both of these groups, the highest frequency of tobacco user was recorded in young age group (≤28 yrs) with successive gradual decrease in middle (29-34 years) and old (≥35 years) age group. According to risk prediction model (mentioned above) of DS birth, the chewing tobacco may impart some maternal age-independent risk of DS birth. In ex‐ plaining the possible adverse influence of chewing tobacco on subcellular components of oo‐ cyte, the authors speculated that, regardless of oocyte age and the amount and location of recombination, tobacco probably affects some molecular system common both to meiosis I and meiosis II stages, for example the spindle apparatus. Conversely, the prevalence of oral contraceptive pill exhibited a trend of increasing frequency of occurrence with advancing

#### **2.4. Paternal risk factor for chromosome 21 nondisjunction**

The paternal error constitutes nearly 5 to 10% of total occurrence of live born DS cases, de‐ pending upon the populations studied. Unlike maternal cases the studies on the etiology of paternal NDJ are limited by insufficient sample size. The first significant report was provid‐ ed by Savage *et al*., (1998) who found reduction in recombination in MI nondisjoined cases, but not in MII errors. Moreover, the authors inferred that altered chiasma positioning may not associate with NDJ in spermatogenesis, as the authors recorded very concordant pattern of chiasma distribution among DS cases and control. In their extension study with more pa‐ ternally derived samples, Oliver *et al*., (2009) determined that majority of Ch21 NDJ errors in spermatogenesis occurs at MII (32%MI:68%MII), and the authors did not found significant reduction in recombination either in MI or in MII errors. Moreover, their sample did not ex‐ hibit any advanced age effect for either of meiotic outcome groups. The authors argued that the time scale of spermatogenesis is much shorter starting at puberty runs continuously without meiotic halt and this explains why advancing paternal age does not exacerbate and associate Ch21 NDJ in spermatogenesis. This study is significant in the realization that etiol‐ ogy of Ch21 NDJ differs in two sexes and case of paternal errors remains an enigma. In gen‐ eral the frequency of recombination for normally segregating chromosome is less in male than in female. But further reduction in recombination frequency may not cause NDJ in male. Moreover, epidemiological study on the risk factors for paternal NDJ of Ch21 is yet to be conducted.

## **3. Habitual risk factor for chromosome 21 nondisjunction**

Beside maternal age and altered pattern of recombination, set of prospective environmental or habitual risk factors have been identified in several epidemiological studies. These factors show various degrees of associations with DS birth. The list includes maternal cigarette smoking, use of oral contraceptive, peri-conceptional alcohol consumption by mother, expo‐ sure to radiation and low socio-economic status. Number of studies reported a negative as‐ sociation between maternal smoking around the time of conception and the risk for DS birth (Kline *et al*., 1983, 1993; Hook & Cross, 1985, 1988; Shiono *et al*., 1986; Chen *et al*., 1999). One explanation for the negative association was that trisomic conceptuses were selectively lost prenatally among women who smoke (Hook and Cross, 1985; Kline *et al*., 1993). But evi‐ dence against this speculation is also available (Cuckle *et al*., 1990; Kallen, 1997; Torf & Christianson, 2000). Study conducted by Yang *et al*., (1999) suggested that maternal-smoking was significantly associated with MII error and probably due to compromise in blood and oxygen supply surrounding the developing follicles. Besides smoking, the other maternal risk factor for which epidemiological studies have been conducted most is oral contracep‐ tive. The use of oral contraceptive by women at the time of conception is subject of specula‐ tion as risk for DS births (Yang *et al*., 1999). The study by Martinez-Frias *et al*., (2001) showed that the risk for DS in infants born to mothers with less than 35 years of age (as a group) who became pregnant while taking oral-contraceptive is near the risk for mothers of DS with more than 35 years of age. In their epidemiological study, Yang *et al*., (1999) found that women having simultaneous habits of smoking and using oral contraceptive have seven folds increased risk of having DS pregnancy and they argued that this is due to anoxic con‐ dition in ovarian microenvironment related to toxicant induced reduction in blood flow sur‐ rounding ovary. This speculation is similar to that proposed by Gaulden (1992) to explain the cause of maternal-age related NDJ. She suggested that the follicular microcirculation may be compromised in an aging ovary because of abnormal hormone signaling. Although sufficient evidence is lacking (Henderson *et al*., 2007), alcohol consumption by women in‐ creases the chance of having DS pregnancy as suggested by Kaufman (1983).

tive prenatal termination of pregnancies with trisomic fetuses, and as yet unidentified environmental factors (Sherman *et al*., 2005). Only one such study by Allen *et al*., (2009) re‐ ported demographic differences in mean maternal age of DS conception recorded in two dif‐ ferent sample sets from USA. This study included DS samples from Atlanta Down syndrome project and National Down syndrome project and found that mothers enrolled in National Down syndrome project were on an average older than those of Atlanta. Moreover, the authors have also reported some ethnic differences in maternal age distribution. The At‐ lanta Down syndrome project had a higher proportion of cases and controls that were black and a significantly smaller proportion of Hispanics than did the National Down syndrome project. Comparison of mean maternal ages indicated variation by ethnic groups. In both the Atlanta Down syndrome project and National Down syndrome project, white mothers tend‐ ed to be older than their black or Hispanic counterparts. Specifically, for both cases and con‐ trols, white mothers were found to be significantly older than black mothers (P< 0.01) and Hispanic mothers (P< 0.01); blacks and Hispanics were not significantly different from each other (P>0.05). To confirm such effect of demographic and ethnic differences on the etiology

of DS birth, further large scale population based studies are needed to be conducted.

The paternal error constitutes nearly 5 to 10% of total occurrence of live born DS cases, de‐ pending upon the populations studied. Unlike maternal cases the studies on the etiology of paternal NDJ are limited by insufficient sample size. The first significant report was provid‐ ed by Savage *et al*., (1998) who found reduction in recombination in MI nondisjoined cases, but not in MII errors. Moreover, the authors inferred that altered chiasma positioning may not associate with NDJ in spermatogenesis, as the authors recorded very concordant pattern of chiasma distribution among DS cases and control. In their extension study with more pa‐ ternally derived samples, Oliver *et al*., (2009) determined that majority of Ch21 NDJ errors in spermatogenesis occurs at MII (32%MI:68%MII), and the authors did not found significant reduction in recombination either in MI or in MII errors. Moreover, their sample did not ex‐ hibit any advanced age effect for either of meiotic outcome groups. The authors argued that the time scale of spermatogenesis is much shorter starting at puberty runs continuously without meiotic halt and this explains why advancing paternal age does not exacerbate and associate Ch21 NDJ in spermatogenesis. This study is significant in the realization that etiol‐ ogy of Ch21 NDJ differs in two sexes and case of paternal errors remains an enigma. In gen‐ eral the frequency of recombination for normally segregating chromosome is less in male than in female. But further reduction in recombination frequency may not cause NDJ in male. Moreover, epidemiological study on the risk factors for paternal NDJ of Ch21 is yet to

**2.4. Paternal risk factor for chromosome 21 nondisjunction**

**3. Habitual risk factor for chromosome 21 nondisjunction**

Beside maternal age and altered pattern of recombination, set of prospective environmental or habitual risk factors have been identified in several epidemiological studies. These factors

be conducted.

160 Down Syndrome

Very recently, population based epidemiological study by Ghosh *et al*., (2011) analyzed the effect of chewing tobacco and contraceptive pill use on the Ch21 NDJ in interaction with known risk variables like maternal age, meiotic stage of NDJ and pattern of recombination i.e., amount of exchange and positioning of chiasma on the recombining homologues. Vari‐ ous logistic regression models have been designed to examine every possible interaction among all above mentioned risk factors. Smokeless chewing tobacco was associated with significant risk for MII NDJ and achiasmate (nonexchange) MI error among the younger mothers. For both of these groups, the highest frequency of tobacco user was recorded in young age group (≤28 yrs) with successive gradual decrease in middle (29-34 years) and old (≥35 years) age group. According to risk prediction model (mentioned above) of DS birth, the chewing tobacco may impart some maternal age-independent risk of DS birth. In ex‐ plaining the possible adverse influence of chewing tobacco on subcellular components of oo‐ cyte, the authors speculated that, regardless of oocyte age and the amount and location of recombination, tobacco probably affects some molecular system common both to meiosis I and meiosis II stages, for example the spindle apparatus. Conversely, the prevalence of oral contraceptive pill exhibited a trend of increasing frequency of occurrence with advancing maternal age, suggesting maternal age dependent risk of contraceptive pill in both the mei‐ otic I and meiotic II error groups. Moreover, both risk factors, when present together, exhib‐ ited a strong age-dependent effect.

deleterious effect of ionizing radiation on the chromosome segregation system in oocyte of the women who are exposed to the radiation. After conducting month wise birth prevalence study on DS birth in West Germany from January 1980 to December 1989, Sperling *et al*., (1994) suggested that low dose of ionizing radiation might cause birth of cluster of triso‐ my21 children in that area. Further they hypothesized that the effect of radiation got worse owing to error susceptible process of oogenesis and rapid accumulation of radioactive io‐ dine (I131) in body, as the people of that area suffered from iodine deficiency. Although the notion is intuitive, it is very compelling and needs further scientific investigation. Similarly, the effect of irradiation to which the women remained exposed for medical purpose has also been evaluated as DS birth risk in few studies (Uchida *et al*., 1979; Strigini *et al*., 1990; Pad‐ manabhan *et al*., 2004), which suggest radiation may affect the younger women more severe‐

Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology

http://dx.doi.org/10.5772/52903

163

Attempt to resolve the etiology of DS birth is a continuous process and we hope this will bring new insight in the understanding the hidden truth in near future. But the problem lies in its multi factorial nature (Table 2) which inevitably suggests necessity of multi-faceted re‐ search efforts from the several directions. For example, it is needed to analyze the polymor‐ phisms of certain genes that regulate meiotic recombination or genes that control maternal molecular aging or those who are involved in faithful chromosome segregation system in meiosis. In searching the cause of recombination anomaly, *PRDM9* would be the good target of investigation, as it is a documented regulator of mammalian recombination (Borel *et al*., 2012). Telomere maintenance system and their genetic components such as *TERT* and *TERC* may be the other targets of research and exploration of these genes would help us to realize the cause of molecular aging and related genetic susceptibility of NDJ. The component of sister chromatid cohesion complex and their role in chromosome segregation have been evi‐ dent in mammals and non-mammalian model organisms. Their functional impairment is known to associate with increased rate of chromosomal missegregation and aneuploidy. But their role and allelic variations have not been explored in the context of Ch21 NDJ and sub‐ sequent DS birth. Apart from genetic components, several environmental influences are known to associate with DS birth as risk factors. But proper molecular study on how their adverse effect interacts and imperils faithful chromosome separation apparatus is tantaliz‐ ingly low. At this level it is almost certain that environmental hazards or aneugen in various forms are associated with accidental increase in DS birth rate at different parts of world. But scientific evidence in favor of their interaction with genetic component is lacking and needs in depth study. If these could be resolved properly in future great advances will be made in the field of medical science and potential couple would enjoy their parenthood with physi‐

ly and may increase the chance of having DS conception.

**5. Future research**

cally and mentally healthy babies.

## **4. Epidemiology of environmental pollutants associated with Down syndrome birth**

The epidemiological evidences in favour of the association between DS birth and environ‐ mental pollution are also surprisingly high, although controversial. Several pollution events are known to be followed by higher incidence of DS birth in an affected geographical locali‐ ty. Early reports in the 1950s from USA suggested that fluoridation of water supplies might result in an increase in the frequency of DS birth (Dolk & Vrijheid, 2003). Subsequent com‐ parison of overall DS birth rates in fluoridated and non-fluoridated areas in Massachusetts found no evidence for a difference (Needleman *et al*., 1974). In this study prevalence rates of DS at birth were compared for Massachusetts residents ingesting fluoridated and non-fluo‐ ridated water. The observations included nearly all children born alive with DS in Massa‐ chusetts during the 17-year period 1950–1966. A rate of 1.5 cases per 1000 births was found both for fluoride-related births and appropriate comparison groups. Analysis of data from 51 American cities also found no difference in maternal age-specific DS rates between fluori‐ dated and non-fluoridated areas (Erickson, 1980).

Similarly, water contamination with pesticide trichlorfon has been reported to cause an out‐ break of DS birth incidence. It was reported in the village of Hungary in 1990s (Czeizel *et al*., 1993) to increase in teratogenic births, including that of DS. In Woburn, Massachusetts, toxic chemicals (industrial solvents, mainly trichloroethylene) from a waste disposal site were de‐ tected in municipal drinking water wells (Dolk & Vrijheid, 2003) and people of this area re‐ ported increased incidence of several congenital anomalies. Lagakos *et al*., (1986) followed up this finding by compiling an exposure score for residential zones in Woburn, using infor‐ mation on what fraction of the water supply in each zone had come from the contaminated wells annually since the start of the wells. The authors found a positive correlation between contaminated water use and higher birthrate of DS in this locality.

The increase in DS birth incidence due to accidental exposure to radioactive materials or ra‐ diation remains as a subject of research interest for long time. The disaster at nuclear power plant of Chernobyl, located in former Soviet Union, now at Ukraine, is the worst nuclear ac‐ cident of the century. The immediate fallout of the incidence was the exposure of a large number of people to the various degree of ionizing radiation, which created a new situation for epidemiological investigation. The accidental event prompted numerous studies on the genetic effects of low dose ionizing radiation in man and almost all studies reported a signif‐ icant increase in Down syndrome birth along with other birth defects in the parts of Germa‐ ny, Scandinavia and the Lothian region of central Scotland, nine months after the disaster (Burkart *et al*., 1997; Sperling *et al*., 1994; Verger, 1997). This incidence was suggestive for the deleterious effect of ionizing radiation on the chromosome segregation system in oocyte of the women who are exposed to the radiation. After conducting month wise birth prevalence study on DS birth in West Germany from January 1980 to December 1989, Sperling *et al*., (1994) suggested that low dose of ionizing radiation might cause birth of cluster of triso‐ my21 children in that area. Further they hypothesized that the effect of radiation got worse owing to error susceptible process of oogenesis and rapid accumulation of radioactive io‐ dine (I131) in body, as the people of that area suffered from iodine deficiency. Although the notion is intuitive, it is very compelling and needs further scientific investigation. Similarly, the effect of irradiation to which the women remained exposed for medical purpose has also been evaluated as DS birth risk in few studies (Uchida *et al*., 1979; Strigini *et al*., 1990; Pad‐ manabhan *et al*., 2004), which suggest radiation may affect the younger women more severe‐ ly and may increase the chance of having DS conception.

## **5. Future research**

maternal age, suggesting maternal age dependent risk of contraceptive pill in both the mei‐ otic I and meiotic II error groups. Moreover, both risk factors, when present together, exhib‐

The epidemiological evidences in favour of the association between DS birth and environ‐ mental pollution are also surprisingly high, although controversial. Several pollution events are known to be followed by higher incidence of DS birth in an affected geographical locali‐ ty. Early reports in the 1950s from USA suggested that fluoridation of water supplies might result in an increase in the frequency of DS birth (Dolk & Vrijheid, 2003). Subsequent com‐ parison of overall DS birth rates in fluoridated and non-fluoridated areas in Massachusetts found no evidence for a difference (Needleman *et al*., 1974). In this study prevalence rates of DS at birth were compared for Massachusetts residents ingesting fluoridated and non-fluo‐ ridated water. The observations included nearly all children born alive with DS in Massa‐ chusetts during the 17-year period 1950–1966. A rate of 1.5 cases per 1000 births was found both for fluoride-related births and appropriate comparison groups. Analysis of data from 51 American cities also found no difference in maternal age-specific DS rates between fluori‐

Similarly, water contamination with pesticide trichlorfon has been reported to cause an out‐ break of DS birth incidence. It was reported in the village of Hungary in 1990s (Czeizel *et al*., 1993) to increase in teratogenic births, including that of DS. In Woburn, Massachusetts, toxic chemicals (industrial solvents, mainly trichloroethylene) from a waste disposal site were de‐ tected in municipal drinking water wells (Dolk & Vrijheid, 2003) and people of this area re‐ ported increased incidence of several congenital anomalies. Lagakos *et al*., (1986) followed up this finding by compiling an exposure score for residential zones in Woburn, using infor‐ mation on what fraction of the water supply in each zone had come from the contaminated wells annually since the start of the wells. The authors found a positive correlation between

The increase in DS birth incidence due to accidental exposure to radioactive materials or ra‐ diation remains as a subject of research interest for long time. The disaster at nuclear power plant of Chernobyl, located in former Soviet Union, now at Ukraine, is the worst nuclear ac‐ cident of the century. The immediate fallout of the incidence was the exposure of a large number of people to the various degree of ionizing radiation, which created a new situation for epidemiological investigation. The accidental event prompted numerous studies on the genetic effects of low dose ionizing radiation in man and almost all studies reported a signif‐ icant increase in Down syndrome birth along with other birth defects in the parts of Germa‐ ny, Scandinavia and the Lothian region of central Scotland, nine months after the disaster (Burkart *et al*., 1997; Sperling *et al*., 1994; Verger, 1997). This incidence was suggestive for the

**4. Epidemiology of environmental pollutants associated with Down**

ited a strong age-dependent effect.

dated and non-fluoridated areas (Erickson, 1980).

contaminated water use and higher birthrate of DS in this locality.

**syndrome birth**

162 Down Syndrome

Attempt to resolve the etiology of DS birth is a continuous process and we hope this will bring new insight in the understanding the hidden truth in near future. But the problem lies in its multi factorial nature (Table 2) which inevitably suggests necessity of multi-faceted re‐ search efforts from the several directions. For example, it is needed to analyze the polymor‐ phisms of certain genes that regulate meiotic recombination or genes that control maternal molecular aging or those who are involved in faithful chromosome segregation system in meiosis. In searching the cause of recombination anomaly, *PRDM9* would be the good target of investigation, as it is a documented regulator of mammalian recombination (Borel *et al*., 2012). Telomere maintenance system and their genetic components such as *TERT* and *TERC* may be the other targets of research and exploration of these genes would help us to realize the cause of molecular aging and related genetic susceptibility of NDJ. The component of sister chromatid cohesion complex and their role in chromosome segregation have been evi‐ dent in mammals and non-mammalian model organisms. Their functional impairment is known to associate with increased rate of chromosomal missegregation and aneuploidy. But their role and allelic variations have not been explored in the context of Ch21 NDJ and sub‐ sequent DS birth. Apart from genetic components, several environmental influences are known to associate with DS birth as risk factors. But proper molecular study on how their adverse effect interacts and imperils faithful chromosome separation apparatus is tantaliz‐ ingly low. At this level it is almost certain that environmental hazards or aneugen in various forms are associated with accidental increase in DS birth rate at different parts of world. But scientific evidence in favor of their interaction with genetic component is lacking and needs in depth study. If these could be resolved properly in future great advances will be made in the field of medical science and potential couple would enjoy their parenthood with physi‐ cally and mentally healthy babies.


**Acknowledgements**

drome research.

**Author details**

gal, India

**References**

Sujay Ghosh1,2 and Subrata Kumar Dey1

\*Address all correspondence to: g.sujoy.g@gmail.com

ed to University of Calcutta), Pathankhali, West Bengal, India

tion in man. Am J Hum Genet , 32, 477-483.

Reprod 9:1199-2000). 9, 1199-2000.

in mice. Nat Genet , 36, 744-749.

Clin Biol Res , 393, 13-26.

Genet , 42, 13-15.

We are extremely grateful to Prof. Eleanor Feingold, Pittsburgh University, USA and Prof. Stephanie Sherman, Emory University, Atlanta, USA for their cooperation in Down syn‐

Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology

http://dx.doi.org/10.5772/52903

165

1 Centre for Genetic Studies, Department of Biotechnology, School of Biotechnology and Bi‐ ological Sciences, West Bengal University of Technology, Salt Lake City, Kolkata, West Ben‐

2 Genetics Research Unit, Department of Zoology, Sundarban Hazi Desarat College (Affiliat‐

[1] Alfi, O. S., Chang, R., & Azen, S. P. (1980). Evidence for genetic control of nondisjunc‐

[2] Allen, E. G., Freeman, S. B., Druschel, C., Hobbs, C. A., O'Leary, L. A., Romitti, P. A., Royle, M. H., Torfs, C. P., & Sherman, S. L. (2009). Maternal age and risk for trisomy 21 assessed by the origin of chromosome nondisjunction: a report from the Atlanta

[3] (Angell, R. (1994). Higher rates of aneuploidy in oocytes from older women. Hum

[4] Angell, R. (1995). Mechanism of chromosome nondisjunction in human oocytes. Prog

[5] Baker, D. J., Jeganathan, K. B., Cameron, J. D., Thompson, M., Juneja, S., Kopecka, A., Kumar, R., Jenkins, R. B., de Groen, P. C., Roche, P., & van Deursen, J. M. (2004). BubR1 insufficiency causes early onset of aging associated phenotypes and infertility

[6] Basaran, N., Cenani, A., Sayli, B. S., Ozkinay, C., Artan, S., Seven, H., Basaran, A., & Dincer, S. (1992). Consanguineous marriages among parents of Down patients. Clin

and National Down Syndrome Projects. Hum Genet , 125, 41-52.

**Table 2.** Summary of maternal risk factors for Ch21 nondisjunction and their probable mode of action

## **Acknowledgements**

Risk Factors **Relation with maternal**

Telomeric single chiasma Maternal age

Reduced meiotic recombination

164 Down Syndrome

Pericentromeric single chiasma

Shifting of distal chiasma towards proximal one when two simultaneous recombination occur

Shifting of proximal chiasma towards centromere when two simultaneous recombination occur

Genetic polymorphisms: MTHFR 677C→T, MTRR 66A→G

> Maternal cigarette smoking

> Maternal chewing tobacco use

Maternal oral contraceptive use

Combined exposure to tobacco and oral contraceptive

Maternal low socioeconomic exposure **age**

Maternal age independent

independent

Maternal age dependent

Maternal age independent

Maternal age dependent

Possibly maternal age independent

> Maternal age independent

> Maternal age independent

> Maternal age dependent

> Maternal age independent

**Interaction with other risk factors**

Not clear, possibly affected by genetic polymorphisms influence chiasma formation

The risk exacerbates with increasing maternal age

The risk exacerbates with increasing maternal age

Possibly affects system that ensure non recombinant chromosome segregation and some components common to both MI and MII phases

hormone level

The risk exacerbates with increasing maternal age

Debatable Supposed to affect ovarian

**Table 2.** Summary of maternal risk factors for Ch21 nondisjunction and their probable mode of action

**Meiotic stage of errors**

Not evident MI Oliver et al. (2008),

Not evident MI Oliver et al. (2011)

Not evident Not analyzed James et al. (2004),

Not evident Not analyzed Kline et al. (1983),

Not evident MII Christianson et al.

**Reference**

Oliver et al. (2008), Ghosh et al. (2009), Ghosh et al. (2011).

Ghosh et al. (2009).

Ghosh et al. (2009).

Wang et al. (2004).

Hook & Cross (1985); Yang et al. (1999).

Both MI and MII Ghosh et al. (2011)

MII Martı´nez-Frı´as et al

Both MI and MII Yang et al. (1999).

(2001), Ghosh et al. (2011)

Ghosh et al. (2011)

(2004)

MI Lamb et al. (2005),

MII Oliver et al. (2008),

MII Oliver et al. (2011)

We are extremely grateful to Prof. Eleanor Feingold, Pittsburgh University, USA and Prof. Stephanie Sherman, Emory University, Atlanta, USA for their cooperation in Down syn‐ drome research.

## **Author details**

Sujay Ghosh1,2 and Subrata Kumar Dey1

\*Address all correspondence to: g.sujoy.g@gmail.com

1 Centre for Genetic Studies, Department of Biotechnology, School of Biotechnology and Bi‐ ological Sciences, West Bengal University of Technology, Salt Lake City, Kolkata, West Ben‐ gal, India

2 Genetics Research Unit, Department of Zoology, Sundarban Hazi Desarat College (Affiliat‐ ed to University of Calcutta), Pathankhali, West Bengal, India

## **References**


[7] Beetstra, S., Thomas, P., Salisbury, C., Turner, J., & Fenech, M. (2005). Folic acid defi‐ ciency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei. *Mutation Research*, 578, 317-326.

overview and update on MCAK in mammalian oocytes. Biochem Soc Trans , 38,

Risk Factors for Down Syndrome Birth: Understanding the Causes from Genetics and Epidemiology

http://dx.doi.org/10.5772/52903

167

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**Chapter 10**

**RCAN1 and Its Potential Contribution**

**to the Down Syndrome Phenotype**

Melanie A. Pritchard and Katherine R. Martin

Additional information is available at the end of the chapter

Down Syndrome (DS) is caused by trisomy of *Hsa21* in humans [1]. It is the most common autosomal aneuploidy, occurring in about 1 in 700 live births [2]. The clinical features of DS are variable and affect many different aspects of development. In any given individual, there may be over 80 different clinical traits [3]. Major clinical features associated with DS include the distinctive craniofacial appearance, reduced size and altered morphology of the brain, cognitive impairments, hearing loss and defects of the gastrointestinal, immune and endocrine systems [3]. Whilst this constellation of anomalies has been described we are still far from understanding their cause. How does an extra set of normal *Hsa21* genes result in whole body system disturbances and what are the molecular genetics bases for these disturbances?

A large number of genes are simultaneously expressed at abnormal levels in DS, therefore, it is a challenge to determine which genes contribute to specific abnormalities, and then identify the key molecular pathways involved. We are advocates of the approach articulated by Nadel [4] that a careful and detailed analysis of the clinical defects in humans be followed by the crea‐ tion of mouse models that over-express only some of the genes triplicated on *Hsa21*, so that the genes responsible for specific featuresoftheDSphenotype canbe identified.Wegeneratedmice in which the *RCAN1* gene is over-expressed (RCAN1-TG) to study the consequences of excess RCAN1 and thus investigate its potential contribution to the DS phenotype. Our research adds to the growing body of work assigning specific functions to particular *Hsa21* genes. Other examples under study with a particular focus on brain function include, *DYRK1A* [5], *SOD1* [6], *APP* [7] [8] [9], *SNYJ1* [10] and *ITSN1* [11]. Once we understand the abnormalities caused by subtle over-expression of single genes, we can embark on a programme to generate mice expressing combinations of genes to examine potential additive effects. This sort of approach is consistent with the idea that the DS phenotype results from disturbances in biological path‐

> © 2013 Pritchard and Martin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5772/52977

**1. Introduction**


## **Chapter 10**

## **RCAN1 and Its Potential Contribution to the Down Syndrome Phenotype**

Melanie A. Pritchard and Katherine R. Martin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52977

## **1. Introduction**

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172 Down Syndrome

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Down Syndrome (DS) is caused by trisomy of *Hsa21* in humans [1]. It is the most common autosomal aneuploidy, occurring in about 1 in 700 live births [2]. The clinical features of DS are variable and affect many different aspects of development. In any given individual, there may be over 80 different clinical traits [3]. Major clinical features associated with DS include the distinctive craniofacial appearance, reduced size and altered morphology of the brain, cognitive impairments, hearing loss and defects of the gastrointestinal, immune and endocrine systems [3]. Whilst this constellation of anomalies has been described we are still far from understanding their cause. How does an extra set of normal *Hsa21* genes result in whole body system disturbances and what are the molecular genetics bases for these disturbances?

A large number of genes are simultaneously expressed at abnormal levels in DS, therefore, it is a challenge to determine which genes contribute to specific abnormalities, and then identify the key molecular pathways involved. We are advocates of the approach articulated by Nadel [4] that a careful and detailed analysis of the clinical defects in humans be followed by the crea‐ tion of mouse models that over-express only some of the genes triplicated on *Hsa21*, so that the genes responsible for specific featuresoftheDSphenotype canbe identified.Wegeneratedmice in which the *RCAN1* gene is over-expressed (RCAN1-TG) to study the consequences of excess RCAN1 and thus investigate its potential contribution to the DS phenotype. Our research adds to the growing body of work assigning specific functions to particular *Hsa21* genes. Other examples under study with a particular focus on brain function include, *DYRK1A* [5], *SOD1* [6], *APP* [7] [8] [9], *SNYJ1* [10] and *ITSN1* [11]. Once we understand the abnormalities caused by subtle over-expression of single genes, we can embark on a programme to generate mice expressing combinations of genes to examine potential additive effects. This sort of approach is consistent with the idea that the DS phenotype results from disturbances in biological path‐

© 2013 Pritchard and Martin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ways due to an accumulation of subtle changes brought about by the effects of the overexpressionofmanysinglegenes.Indeed, suchanapproachisbearingfruit already-*RCAN1* and *DYRK1A* have been shown to act cooperatively to destabilise a calcineurin regulatory circuit when the genes are over-expressed in a combinatorial fashion [12].

and RCAN1-4. RCAN1-1 protein consists of 252 amino acids, while RCAN1-4 is a shorter, 197 amino acid protein [22, 23]. Using Northern blot analysis, *RCAN1-1* and *RCAN1-4* were found to be similarly distributed throughout the body [22]. *RCAN1-1* was highly expressed in the foetal brain and in the adult brain, heart and skeletal muscle. Lower levels were detected in the foetal lung, liver and kidney and in the adult pancreas, lung, liver and placenta. High levels of *RCAN1-4* were detected in the foetal kidney and in adult heart, skeletal muscle and placental tissues, with lower levels in the foetal brain, lung and liver and adult lung, liver, kidney and pancreas. While both isoforms exhibited a similar expression pattern, only very low levels of *RCAN1-4* were found in the adult brain and *RCAN1-1* expression could not be detected in the adult kidney [13, 22]. Northern blot and RT-PCR failed to detect exon 3 in any of the foetal or

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adult tissue studied, while isoform 2 was found only in the foetal brain and liver [22].

expression [24].

RCAN1-1 and RCAN1-4, the most predominantly expressed isoforms, are under the control of different promoters and are therefore likely to have different regulatory mechanisms and possibly even different functions. For example, *RCAN1-4* expression is regulated by calcium signalling. Experiments in PC-12 cells (a neuronal like cell line) found that when intracellular calcium levels increased through membrane depolarisation, *RCAN1-4* gene expression was rapidly induced [24] and this was mediated by the calcineurin/Nuclear factor activated T cells (NFAT) signalling pathway [24]. Studies on the *RCAN1-4* promoter identified the presence of putative NFAT binding sites. No study published to date has demonstrated Ca2+/calcineurinmediated expression of *RCAN1-1*. Interestingly, RCAN1 is able to function in an autoinhibitory manner as over-expression of any RCAN1 isoform resulted in an inhibition of *RCAN1-4* gene

The subcellular location of RCAN1 protein was initially determined using tranfection of a RCAN1-GFP protein construct in C2C12 cells, a mouse myoblast cell line. RCAN1 protein was located in both the nuclear and cytosolic compartments and in the absence of treatments to activate the calcineurin signalling pathway, resided predominantly in the nucleus [25]. Various physiological and biochemical stresses have been demonstrated to influence the location of RCAN1 within a cell. For example, under normal circumstances RCAN1 was located within the nuclear compartment in various cell lines, including HT-1080 fibrosarcoma and I251 astroglioma cells. However, when these cells were subjected to oxidative stress, RCAN1 protein was redistributed to the cytoplasm [26]. The same observation was made following activation of the calcineurin signalling pathway, which resulted in the translocation of RCAN1

Initial studies found that both RCAN1 isoforms encode a proline rich protein consisting of a putative acidic domain, a serine proline motif, a putative DNA binding domain and a proline rich region typical of a SH3 domain ligand [22, 28]. These structural motifs are typically seen in proteins involved in transcriptional regulation and signal transduction. A more recent study on RCAN1 proteins in dozens of species revealed 4 highly conserved regions separated by

from the nucleus into the cytosolic compartment [27].

**4. Functional domains of the RCAN1 protein**

The focus of this chapter will be to provide insight into *RCAN1* and its functions, and examine the evidence to suggest that this gene plays a role in the neurological, immune and vascular systems. We will firstly give an overview of the gene family to which *RCAN1* belongs; followed by a description of the functional domains of the protein product, including post translational modification domains; its tissue expression pattern; cellular pathways involving RCAN1; and finally, how its over-expression may contribute to the neurological, immune and cancer phenotypes associated with DS.

## **2. The RCAN gene family**

*DSCR1*,renamed*RCAN1,*wasfirstdescribedbyourgroupin1995afterasearchforgeneslocated on*Hsa21*withthepotentialtobeinvolvedinDS[13].*RCAN1*isamemberofafamilyofcalcineurin binding proteins and is conserved across species, from lower unicellular eukaryotes such as yeast to complex organisms including humans [14]. The high level of interspecies homology of this protein has been taken to indicate a conserved role during evolution [15] [16]. A number of differentgenesbelongingtothis familyhavenowbeenidentifiedinhumans,including,*RCAN1, RCAN1L2, RCAN2* and *RCAN3* [15, 17]. The family was identified based on the presence of a short "signature"polypeptideFLISPPxSPP(partofthe socalledSPmotif)[18]butthere is ahigh degreeof similarityacross the entireproteininallRCANfamilymembers.Allmembersperform similar functions. For example, RCAN2 interacts with calcineurin with similar efficiency to RCAN1[19]andthehumangenecanfunctionallyreplacetheyeastgene[18].Interestingly,while RCANfamilymembersareallexpressedinsimilartissues,eachfamilymemberdisplaysadistinct expression profile. For instance, while all family members were expressed in the brain, each displayed different levels of expression, depending on the region and developmental stage examined [20]. Within these regions there were also differences in the cellular and subcellular location of the family members. RCAN1 was highly expressed in neurones and in the neutro‐ pil,whileRCAN1L2wasexpressedinscatteredneuronesandwastheonlyRCANfamilymember detected in glial cells [20, 21]. The differential expression pattern of the RCAN family mem‐ bers in the brain indicates that they are all likely to be important in brain development and function, yet each member may be functionally distinct [20].

## **3. General tissue and cellular expression of RCAN1**

The *RCAN1* gene spans about 100 kb of genomic DNA and consists of seven exons and six introns. Of the seven exons, the first four are alternative first exons (*RCAN1-1* to *RCAN1-4* containing exons 1 to 4, respectively). *RCAN1* encodes two major protein isoforms, RCAN1-1 and RCAN1-4. RCAN1-1 protein consists of 252 amino acids, while RCAN1-4 is a shorter, 197 amino acid protein [22, 23]. Using Northern blot analysis, *RCAN1-1* and *RCAN1-4* were found to be similarly distributed throughout the body [22]. *RCAN1-1* was highly expressed in the foetal brain and in the adult brain, heart and skeletal muscle. Lower levels were detected in the foetal lung, liver and kidney and in the adult pancreas, lung, liver and placenta. High levels of *RCAN1-4* were detected in the foetal kidney and in adult heart, skeletal muscle and placental tissues, with lower levels in the foetal brain, lung and liver and adult lung, liver, kidney and pancreas. While both isoforms exhibited a similar expression pattern, only very low levels of *RCAN1-4* were found in the adult brain and *RCAN1-1* expression could not be detected in the adult kidney [13, 22]. Northern blot and RT-PCR failed to detect exon 3 in any of the foetal or adult tissue studied, while isoform 2 was found only in the foetal brain and liver [22].

ways due to an accumulation of subtle changes brought about by the effects of the overexpressionofmanysinglegenes.Indeed, suchanapproachisbearingfruit already-*RCAN1* and *DYRK1A* have been shown to act cooperatively to destabilise a calcineurin regulatory circuit

The focus of this chapter will be to provide insight into *RCAN1* and its functions, and examine the evidence to suggest that this gene plays a role in the neurological, immune and vascular systems. We will firstly give an overview of the gene family to which *RCAN1* belongs; followed by a description of the functional domains of the protein product, including post translational modification domains; its tissue expression pattern; cellular pathways involving RCAN1; and finally, how its over-expression may contribute to the neurological, immune and cancer

*DSCR1*,renamed*RCAN1,*wasfirstdescribedbyourgroupin1995afterasearchforgeneslocated on*Hsa21*withthepotentialtobeinvolvedinDS[13].*RCAN1*isamemberofafamilyofcalcineurin binding proteins and is conserved across species, from lower unicellular eukaryotes such as yeast to complex organisms including humans [14]. The high level of interspecies homology of this protein has been taken to indicate a conserved role during evolution [15] [16]. A number of differentgenesbelongingtothis familyhavenowbeenidentifiedinhumans,including,*RCAN1, RCAN1L2, RCAN2* and *RCAN3* [15, 17]. The family was identified based on the presence of a short "signature"polypeptideFLISPPxSPP(partofthe socalledSPmotif)[18]butthere is ahigh degreeof similarityacross the entireproteininallRCANfamilymembers.Allmembersperform similar functions. For example, RCAN2 interacts with calcineurin with similar efficiency to RCAN1[19]andthehumangenecanfunctionallyreplacetheyeastgene[18].Interestingly,while RCANfamilymembersareallexpressedinsimilartissues,eachfamilymemberdisplaysadistinct expression profile. For instance, while all family members were expressed in the brain, each displayed different levels of expression, depending on the region and developmental stage examined [20]. Within these regions there were also differences in the cellular and subcellular location of the family members. RCAN1 was highly expressed in neurones and in the neutro‐ pil,whileRCAN1L2wasexpressedinscatteredneuronesandwastheonlyRCANfamilymember detected in glial cells [20, 21]. The differential expression pattern of the RCAN family mem‐ bers in the brain indicates that they are all likely to be important in brain development and

The *RCAN1* gene spans about 100 kb of genomic DNA and consists of seven exons and six introns. Of the seven exons, the first four are alternative first exons (*RCAN1-1* to *RCAN1-4* containing exons 1 to 4, respectively). *RCAN1* encodes two major protein isoforms, RCAN1-1

when the genes are over-expressed in a combinatorial fashion [12].

function, yet each member may be functionally distinct [20].

**3. General tissue and cellular expression of RCAN1**

phenotypes associated with DS.

174 Down Syndrome

**2. The RCAN gene family**

RCAN1-1 and RCAN1-4, the most predominantly expressed isoforms, are under the control of different promoters and are therefore likely to have different regulatory mechanisms and possibly even different functions. For example, *RCAN1-4* expression is regulated by calcium signalling. Experiments in PC-12 cells (a neuronal like cell line) found that when intracellular calcium levels increased through membrane depolarisation, *RCAN1-4* gene expression was rapidly induced [24] and this was mediated by the calcineurin/Nuclear factor activated T cells (NFAT) signalling pathway [24]. Studies on the *RCAN1-4* promoter identified the presence of putative NFAT binding sites. No study published to date has demonstrated Ca2+/calcineurinmediated expression of *RCAN1-1*. Interestingly, RCAN1 is able to function in an autoinhibitory manner as over-expression of any RCAN1 isoform resulted in an inhibition of *RCAN1-4* gene expression [24].

The subcellular location of RCAN1 protein was initially determined using tranfection of a RCAN1-GFP protein construct in C2C12 cells, a mouse myoblast cell line. RCAN1 protein was located in both the nuclear and cytosolic compartments and in the absence of treatments to activate the calcineurin signalling pathway, resided predominantly in the nucleus [25]. Various physiological and biochemical stresses have been demonstrated to influence the location of RCAN1 within a cell. For example, under normal circumstances RCAN1 was located within the nuclear compartment in various cell lines, including HT-1080 fibrosarcoma and I251 astroglioma cells. However, when these cells were subjected to oxidative stress, RCAN1 protein was redistributed to the cytoplasm [26]. The same observation was made following activation of the calcineurin signalling pathway, which resulted in the translocation of RCAN1 from the nucleus into the cytosolic compartment [27].

## **4. Functional domains of the RCAN1 protein**

Initial studies found that both RCAN1 isoforms encode a proline rich protein consisting of a putative acidic domain, a serine proline motif, a putative DNA binding domain and a proline rich region typical of a SH3 domain ligand [22, 28]. These structural motifs are typically seen in proteins involved in transcriptional regulation and signal transduction. A more recent study on RCAN1 proteins in dozens of species revealed 4 highly conserved regions separated by other regions that are less well conserved. These four regions consist of: a region at the amino terminus capable of forming an RNA recognition motif; the gene family signature domain consisting of the highly conserved SP motif; a PxIxIT-like domain (x represents any amino acid) and a C-terminal TxxP motif [29] (see Figure 1). The functions of these highly conserved regions in RCAN1 proteins are yet to be fully explored.

Other studies have shown that RCAN1 is cleaved by calpain and this cleavage appears to increase the stability of the protein by decreasing its proteasome-dependent degradation [38]. Further, the cleavage of RCAN1 by calpain also affects its interactions with other proteins. For example, cleavage of RCAN1-4 by calpain abolished its ability to bind to Raf-1 [38]. Yet another pathway involved in the post translational regulation of RCAN1 is the ubiquitin-proteasome system (UPS). The UPS is important in the regulation of protein turnover in response to changing cellular conditions and facilitates the degradation of defective proteins [39]. Ubiq‐ uitin is a polypeptide able to bind to lysine residues on proteins targeted for degradation. This binding occurs through sequential steps mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3) [40]. Following this sequence of events, the 26s proteasome is able to recognise and degrade the poly-ubiquinated protein. The first evidence to suggest that RCAN1 was degraded by the ubiquitin pathway came from yeast two hybrid and co-immunoprecipitation experiments which found that RCAN1-4 interacted with ubiquitin [41]. More recent studies demonstrated that RCAN1 interacts with other members of the UPS, including, Skp1, Cullin/Cdc53, F-box protein Cdc4 (SCFCdc4) [42] and SCFβ TrCP1/2 [40]. The interaction between RCAN1 and the UPS is not only important in regulating turnover of the RCAN1 protein but may also influence its function. For example, increased degradation of RCAN1 by SCFCdc4 diminished its ability to inhibit

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Interest in *RCAN1* surged after the discovery that it encoded a protein capable of inhibiting the protein serine/threonine phosphatase calcineurin (PP2B/PPP3C) [19] [27] [31] [43] [44]. RCAN1 has since been implicated in a variety of cellular processes, including oxidative stress [45] [46] [47] [48], angiogenesis [49], mitochondrial function [50] and immune responses and inflammation [44] [51]. Participation of RCAN1 in these processes has been mostly attributed to its interaction with the calcineurin pathway. Nonetheless, calcineurin-independent activi‐ ties have been demonstrated [51] [52] [53] [54] [55]. Recently, RCAN1 mRNA and protein was found to increase in the peri-infarct region following middle cerebral artery occlusion (MCAO)

The calcineurin pathway plays an integral role in the development and homeostatic regulation of a number of different cell types, including immune cells and neurones. The pathway is activated by increases in intracellular calcium (Ca2+) due to oxidative stresses, chemicalmediated calcium increases and in response to biomechanical strain [58]. An increase in intracellular Ca2+ leads to the activation of calmodulin, which forms a complex with calcineurin to activate its phosphatase function. Activated calcineurin then dephosphorylates cytosolic NFAT leading to its translocation to the nucleus where it complexes with GATA-4 [59] allowing

DNA binding and facilitation of the transcription of numerous gene targets [60].

calcineurin signalling [42].

**5.1. The calcineurin pathway**

**5. RCAN1 function—Signal transduction pathways**

in mice [56] and its up regulation was found to be protective [57].

**Figure 1. Schematic representation of the major RCAN1 protein isoforms.** Protein motifs are shown: the RRM (RNA recognition motif); the SP (serine / proline) motif incorporating the LxxP, family signature and ExxP domains; the PxIxIT-like domain; and the TxxP motif. Serines 108 and 112 in RCAN1-4 are also indicated.

The most highly conserved region in the RCAN1 protein is the SP motif. This motif is similar to that present in NFAT proteins [30]. *In vitro,* the SP motif is able to bind to and inhibit calcineurin activity, however studies in cell lines have suggested that it is not necessary or sufficient to achieve this. By generating various deletion-constructs of the RCAN1 coding sequence it was found that RCAN1 was able to inhibit calcineurin in C2C12 myoblasts even when the SP domain was absent [31]. This study determined that two additional domains, one at the N-terminus, the other in the distal C-terminal region, were required to inhibit calcineurin activity [31]. Use of a truncated version of the RCAN1 protein also demonstrated that the last 33 amino acids were essential for nuclear localisation. In the absence of this 33 amino acid domain (which contains the SP motif and a region identified as a SH2 domain) RCAN1 protein accumulated in the cytoplasm [25].

Site-directed mutagenesis studies have shown that phosphorylation of the RCAN1 protein regulates its function, subcellular location and stability. Indeed, RCAN1 can be phosphory‐ lated by various kinases at a number of different sites to change its activity towards calcineurin. For example, the serine residue within the SP domain at position 112 (Ser112) (Ser167 in RCAN1-1) is variously phosphorylated by BMK1 [32], NIK [33] and DYRK1 [34] and acts as a priming site for subsequent phosphorylation at Ser108 (Ser163 in RCAN1-1) by GSK-3 [35] [31] [34]. Phosphorylation by TAK1 at Ser94 and Ser136 [36] and by DYRK1A at Thr193 [34] also change the activity of RCAN1 towards calcineurin (see later). NIK-mediated phosphorylation [33] or phosphorylation by PKA [37] augmented the half-life of RCAN1 protein. And, phosphoryla‐ tion of a threonine residue (Thr166 in RCAN1-4) in the SH2 domain controlled its subcellular localisation since exchanging the threonine for an alanine resulted in an accumulation of RCAN1 protein within the cytoplasm [25]. Thus, nuclear localisation of RCAN1 is controlled, at least in part, by phosphorylation.

Other studies have shown that RCAN1 is cleaved by calpain and this cleavage appears to increase the stability of the protein by decreasing its proteasome-dependent degradation [38]. Further, the cleavage of RCAN1 by calpain also affects its interactions with other proteins. For example, cleavage of RCAN1-4 by calpain abolished its ability to bind to Raf-1 [38]. Yet another pathway involved in the post translational regulation of RCAN1 is the ubiquitin-proteasome system (UPS). The UPS is important in the regulation of protein turnover in response to changing cellular conditions and facilitates the degradation of defective proteins [39]. Ubiq‐ uitin is a polypeptide able to bind to lysine residues on proteins targeted for degradation. This binding occurs through sequential steps mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3) [40]. Following this sequence of events, the 26s proteasome is able to recognise and degrade the poly-ubiquinated protein. The first evidence to suggest that RCAN1 was degraded by the ubiquitin pathway came from yeast two hybrid and co-immunoprecipitation experiments which found that RCAN1-4 interacted with ubiquitin [41]. More recent studies demonstrated that RCAN1 interacts with other members of the UPS, including, Skp1, Cullin/Cdc53, F-box protein Cdc4 (SCFCdc4) [42] and SCFβ TrCP1/2 [40]. The interaction between RCAN1 and the UPS is not only important in regulating turnover of the RCAN1 protein but may also influence its function. For example, increased degradation of RCAN1 by SCFCdc4 diminished its ability to inhibit calcineurin signalling [42].

## **5. RCAN1 function—Signal transduction pathways**

Interest in *RCAN1* surged after the discovery that it encoded a protein capable of inhibiting the protein serine/threonine phosphatase calcineurin (PP2B/PPP3C) [19] [27] [31] [43] [44]. RCAN1 has since been implicated in a variety of cellular processes, including oxidative stress [45] [46] [47] [48], angiogenesis [49], mitochondrial function [50] and immune responses and inflammation [44] [51]. Participation of RCAN1 in these processes has been mostly attributed to its interaction with the calcineurin pathway. Nonetheless, calcineurin-independent activi‐ ties have been demonstrated [51] [52] [53] [54] [55]. Recently, RCAN1 mRNA and protein was found to increase in the peri-infarct region following middle cerebral artery occlusion (MCAO) in mice [56] and its up regulation was found to be protective [57].

#### **5.1. The calcineurin pathway**

other regions that are less well conserved. These four regions consist of: a region at the amino terminus capable of forming an RNA recognition motif; the gene family signature domain consisting of the highly conserved SP motif; a PxIxIT-like domain (x represents any amino acid) and a C-terminal TxxP motif [29] (see Figure 1). The functions of these highly conserved

**Figure 1. Schematic representation of the major RCAN1 protein isoforms.** Protein motifs are shown: the RRM (RNA recognition motif); the SP (serine / proline) motif incorporating the LxxP, family signature and ExxP domains; the

The most highly conserved region in the RCAN1 protein is the SP motif. This motif is similar to that present in NFAT proteins [30]. *In vitro,* the SP motif is able to bind to and inhibit calcineurin activity, however studies in cell lines have suggested that it is not necessary or sufficient to achieve this. By generating various deletion-constructs of the RCAN1 coding sequence it was found that RCAN1 was able to inhibit calcineurin in C2C12 myoblasts even when the SP domain was absent [31]. This study determined that two additional domains, one at the N-terminus, the other in the distal C-terminal region, were required to inhibit calcineurin activity [31]. Use of a truncated version of the RCAN1 protein also demonstrated that the last 33 amino acids were essential for nuclear localisation. In the absence of this 33 amino acid domain (which contains the SP motif and a region identified as a SH2 domain) RCAN1 protein

Site-directed mutagenesis studies have shown that phosphorylation of the RCAN1 protein regulates its function, subcellular location and stability. Indeed, RCAN1 can be phosphory‐ lated by various kinases at a number of different sites to change its activity towards calcineurin. For example, the serine residue within the SP domain at position 112 (Ser112) (Ser167 in RCAN1-1) is variously phosphorylated by BMK1 [32], NIK [33] and DYRK1 [34] and acts as a priming site for subsequent phosphorylation at Ser108 (Ser163 in RCAN1-1) by GSK-3 [35] [31] [34]. Phosphorylation by TAK1 at Ser94 and Ser136 [36] and by DYRK1A at Thr193 [34] also change the activity of RCAN1 towards calcineurin (see later). NIK-mediated phosphorylation [33] or phosphorylation by PKA [37] augmented the half-life of RCAN1 protein. And, phosphoryla‐ tion of a threonine residue (Thr166 in RCAN1-4) in the SH2 domain controlled its subcellular localisation since exchanging the threonine for an alanine resulted in an accumulation of RCAN1 protein within the cytoplasm [25]. Thus, nuclear localisation of RCAN1 is controlled,

PxIxIT-like domain; and the TxxP motif. Serines 108 and 112 in RCAN1-4 are also indicated.

accumulated in the cytoplasm [25].

at least in part, by phosphorylation.

regions in RCAN1 proteins are yet to be fully explored.

176 Down Syndrome

The calcineurin pathway plays an integral role in the development and homeostatic regulation of a number of different cell types, including immune cells and neurones. The pathway is activated by increases in intracellular calcium (Ca2+) due to oxidative stresses, chemicalmediated calcium increases and in response to biomechanical strain [58]. An increase in intracellular Ca2+ leads to the activation of calmodulin, which forms a complex with calcineurin to activate its phosphatase function. Activated calcineurin then dephosphorylates cytosolic NFAT leading to its translocation to the nucleus where it complexes with GATA-4 [59] allowing DNA binding and facilitation of the transcription of numerous gene targets [60].

RCAN1 interacts directly with calcineurin [19] [27]. Calcineurin is a heterodimer, consist‐ ing of a catalytic A subunit and a calcium binding regulatory B subunit [61]. RCAN1 is able to bind to the A subunit in a linker region between the calcineurin A catalytic domain and the calcineurin B binding region [19]. Deletion of the carboxyl-terminal half of the catalytic domain of calcineurin A abolished binding with RCAN1, indicating that this region was critical for the interaction [27]. Studies with RCAN1 have shown that exon 7 is able to bind to and regulate the activity of calcineurin and this binding occurs with a very high affinity [62]. While binding of RCAN1 to calcineurin did not interfere with the interaction between calcineurin and calmodulin, it is believed to interfere with the ability of calcineurin to bind NFAT by competing with the NFAT binding site [31]. Indeed, when *RCAN1* was overexpressed, it inhibited the activity of an exogenously added constitutively active calcineur‐ in and transcription of a number of calcineurin-dependent genes including *IL-2* and *MEF2* was prevented [27]. *RCAN1* over-expression was found to inhibit NFAT translocation to the nucleus, thus inhibiting calcineurin-dependent gene transcription [19] but was unable to inhibit a constitutively active form of NFAT demonstrating that the inhibition of calcineur‐ in signalling was through calcineurin, rather than interference with downstream compo‐ nents of the pathway [27].

aforementioned studies – RCAN1 was an inhibitor at low levels but a facilitator when levels were high [66]. Another study indicated that 4 highly conserved domains in the RCAN1 protein were important in determining its activity towards calcineurin. Specifically, that preferential binding of RCAN1 to calcineurin prevented NFAT binding resulting in inhibition of calcineurin signal transduction due to competition between RCAN1 and NFAT for calci‐ neurin docking sites [29]. This preferential binding occurred in the presence of high levels of Rcan1 and required the LxxP domain within the SP motif and the PxIxIT domain [29]. Conversely, when Rcan1 was expressed at lower levels, the protein was able to stimulate calcineurin signalling. This stimulatory effect required the LxxP and ExxP domains within the

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Other studies have suggested that it is the phosphorylation status of RCAN1 that determines its action as either an inhibitor or facilitator of calcineurin activity. A study in yeast found that for Rcan1 to facilitate calcineurin signalling it required phosphorylation of both serine residues located within the SP motif by a priming kinase (in this case MAPK) and Mck1, a member of the glycogen synthase kinase 3 (GSK-3) protein family. When the serines were mutated to alanines or in the absence of Mck1, Rcan1 was no longer able to stimulate calcineurin signalling resulting in inhibition [35]. Phosphorylation by TAK1, DYRK1A and NIK all switch RCAN1 from an inhibitor to a calcineurin facilitator [33] [32] [34]. At odds with most studies, phos‐ phorylation of the serine residues within the SP motif of RCAN1 was reported to enhance its

In summary, although the mechanisms responsible for the dual role of RCAN1 in the calci‐ neurin signalling pathway is still under investigation, the results so far indicate that the primary function of RCAN1 is to facilitate calcineurin activity and this occurs when RCAN1 is expressed at lower or physiological levels. On the other hand, when RCAN1 is highly expressed, it has a secondary role of inhibiting calcineurin signalling by interfering with the

Numerous studies outlined above have shown that GSK-3 phosphorylates RCAN1 to regulate its function. Interestingly, GSK-3 activity can also be regulated by RCAN1. PC-12 cells overexpressing RCAN1 displayed an increase in the absolute levels of GSK-3β protein, which in turn increased its kinase activity towards Tau [67]. Tau protein is a known target of GSK-3 which in its hyperphosphorylated form has been implicated in the aetiology of Alzheimer's disease [67]. Exactly how RCAN1 regulates the abundance of GSK-3 remains undetermined, but it seems that RCAN1 is acting at a post-transcriptional level as the amount of *GSK-3β*

The MAPK/ERK signalling pathway mediates signal transduction from cell surface receptors to downstream transcription factors. This pathway plays a role in a number of cellular processes including proliferation, growth, motility, survival and apoptosis [68]. As indicated

SP motif as mutations within both of these domains prevented stimulation.

ability to inhibit calcineurin [23].

**5.2. GSK–3 signalling**

interaction between calcineurin and NFAT.

**5.3. The MAPK/ERK signalling pathway**

mRNA did not change upon increasing RCAN1 expression [67].

Interestingly, activation of calcineurin signalling induces *RCAN1-4* expression [18, 19]. This occurs through a 900 base pair sequence located between exons 3 and 4 in an intragenic promoter region for *RCAN1-4*, which contains a dense cluster of consensus binding sites for the NFAT transcription factor [61]. The existence of such a site suggested that RCAN1 participates in a negative feedback loop, presumed to exist to prevent the adverse effects of unrestrained calcineurin activity following prolonged Ca2+ stimulation [27]. Indeed, following induction of the calcineurin pathway, levels of *RCAN1-4* mRNA increased within 1.5 hours and peaked 6 hours after treatment with a calcium stressor [45].

As more and more studies have emerged on RCAN1 and the propagation of calcium signals in the cell, it has become clear that the role of RCAN1 is not always to inhibit the calcineurin pathway. While the earliest studies found RCAN1 to negatively regulate the pathway, in other circumstances it seems to facilitate calcineurin activity. Indeed, contrary to expectations it was found that the absence of *Rcan1* diminished calcineurin signalling in yeast [18]. Similar results were found when *Rcan1* expression was disrupted in mice. *Rcan1*-null mice exhibited an unexpected decrease in calcineurin activity in the heart under normal physiological conditions and after stress [63] and a reduction of calcineurin activity was concomitant with reduced nuclear distribution of NFAT and a loss of NFAT-dependent gene transcription [64].

These apparently paradoxical actions of RCAN1 may be explained, at least in part, by its cellular concentration, its nuclear or cytosolic localisation and/or its phosphorylation status [64] [35] [32] [65] [25]. For example, the abundance of RCAN1 in the cell may determine its ability to either enhance or inhibit calcineurin signalling. Low or intermediate levels of RCAN1 were shown to facilitate calcineurin signalling while very high levels of over-expression were inhibitory, suggesting that RCAN1 oscillates between stimulatory and inhibitory forms depending on its concentration [35] [138]. In contrast, in another study, the functional role of RCAN1 was found to change in a dose-dependent fashion, but in the opposite direction to the aforementioned studies – RCAN1 was an inhibitor at low levels but a facilitator when levels were high [66]. Another study indicated that 4 highly conserved domains in the RCAN1 protein were important in determining its activity towards calcineurin. Specifically, that preferential binding of RCAN1 to calcineurin prevented NFAT binding resulting in inhibition of calcineurin signal transduction due to competition between RCAN1 and NFAT for calci‐ neurin docking sites [29]. This preferential binding occurred in the presence of high levels of Rcan1 and required the LxxP domain within the SP motif and the PxIxIT domain [29]. Conversely, when Rcan1 was expressed at lower levels, the protein was able to stimulate calcineurin signalling. This stimulatory effect required the LxxP and ExxP domains within the SP motif as mutations within both of these domains prevented stimulation.

Other studies have suggested that it is the phosphorylation status of RCAN1 that determines its action as either an inhibitor or facilitator of calcineurin activity. A study in yeast found that for Rcan1 to facilitate calcineurin signalling it required phosphorylation of both serine residues located within the SP motif by a priming kinase (in this case MAPK) and Mck1, a member of the glycogen synthase kinase 3 (GSK-3) protein family. When the serines were mutated to alanines or in the absence of Mck1, Rcan1 was no longer able to stimulate calcineurin signalling resulting in inhibition [35]. Phosphorylation by TAK1, DYRK1A and NIK all switch RCAN1 from an inhibitor to a calcineurin facilitator [33] [32] [34]. At odds with most studies, phos‐ phorylation of the serine residues within the SP motif of RCAN1 was reported to enhance its ability to inhibit calcineurin [23].

In summary, although the mechanisms responsible for the dual role of RCAN1 in the calci‐ neurin signalling pathway is still under investigation, the results so far indicate that the primary function of RCAN1 is to facilitate calcineurin activity and this occurs when RCAN1 is expressed at lower or physiological levels. On the other hand, when RCAN1 is highly expressed, it has a secondary role of inhibiting calcineurin signalling by interfering with the interaction between calcineurin and NFAT.

#### **5.2. GSK–3 signalling**

RCAN1 interacts directly with calcineurin [19] [27]. Calcineurin is a heterodimer, consist‐ ing of a catalytic A subunit and a calcium binding regulatory B subunit [61]. RCAN1 is able to bind to the A subunit in a linker region between the calcineurin A catalytic domain and the calcineurin B binding region [19]. Deletion of the carboxyl-terminal half of the catalytic domain of calcineurin A abolished binding with RCAN1, indicating that this region was critical for the interaction [27]. Studies with RCAN1 have shown that exon 7 is able to bind to and regulate the activity of calcineurin and this binding occurs with a very high affinity [62]. While binding of RCAN1 to calcineurin did not interfere with the interaction between calcineurin and calmodulin, it is believed to interfere with the ability of calcineurin to bind NFAT by competing with the NFAT binding site [31]. Indeed, when *RCAN1* was overexpressed, it inhibited the activity of an exogenously added constitutively active calcineur‐ in and transcription of a number of calcineurin-dependent genes including *IL-2* and *MEF2* was prevented [27]. *RCAN1* over-expression was found to inhibit NFAT translocation to the nucleus, thus inhibiting calcineurin-dependent gene transcription [19] but was unable to inhibit a constitutively active form of NFAT demonstrating that the inhibition of calcineur‐ in signalling was through calcineurin, rather than interference with downstream compo‐

Interestingly, activation of calcineurin signalling induces *RCAN1-4* expression [18, 19]. This occurs through a 900 base pair sequence located between exons 3 and 4 in an intragenic promoter region for *RCAN1-4*, which contains a dense cluster of consensus binding sites for the NFAT transcription factor [61]. The existence of such a site suggested that RCAN1 participates in a negative feedback loop, presumed to exist to prevent the adverse effects of unrestrained calcineurin activity following prolonged Ca2+ stimulation [27]. Indeed, following induction of the calcineurin pathway, levels of *RCAN1-4* mRNA increased within 1.5 hours

As more and more studies have emerged on RCAN1 and the propagation of calcium signals in the cell, it has become clear that the role of RCAN1 is not always to inhibit the calcineurin pathway. While the earliest studies found RCAN1 to negatively regulate the pathway, in other circumstances it seems to facilitate calcineurin activity. Indeed, contrary to expectations it was found that the absence of *Rcan1* diminished calcineurin signalling in yeast [18]. Similar results were found when *Rcan1* expression was disrupted in mice. *Rcan1*-null mice exhibited an unexpected decrease in calcineurin activity in the heart under normal physiological conditions and after stress [63] and a reduction of calcineurin activity was concomitant with reduced

nuclear distribution of NFAT and a loss of NFAT-dependent gene transcription [64].

These apparently paradoxical actions of RCAN1 may be explained, at least in part, by its cellular concentration, its nuclear or cytosolic localisation and/or its phosphorylation status [64] [35] [32] [65] [25]. For example, the abundance of RCAN1 in the cell may determine its ability to either enhance or inhibit calcineurin signalling. Low or intermediate levels of RCAN1 were shown to facilitate calcineurin signalling while very high levels of over-expression were inhibitory, suggesting that RCAN1 oscillates between stimulatory and inhibitory forms depending on its concentration [35] [138]. In contrast, in another study, the functional role of RCAN1 was found to change in a dose-dependent fashion, but in the opposite direction to the

and peaked 6 hours after treatment with a calcium stressor [45].

nents of the pathway [27].

178 Down Syndrome

Numerous studies outlined above have shown that GSK-3 phosphorylates RCAN1 to regulate its function. Interestingly, GSK-3 activity can also be regulated by RCAN1. PC-12 cells overexpressing RCAN1 displayed an increase in the absolute levels of GSK-3β protein, which in turn increased its kinase activity towards Tau [67]. Tau protein is a known target of GSK-3 which in its hyperphosphorylated form has been implicated in the aetiology of Alzheimer's disease [67]. Exactly how RCAN1 regulates the abundance of GSK-3 remains undetermined, but it seems that RCAN1 is acting at a post-transcriptional level as the amount of *GSK-3β* mRNA did not change upon increasing RCAN1 expression [67].

#### **5.3. The MAPK/ERK signalling pathway**

The MAPK/ERK signalling pathway mediates signal transduction from cell surface receptors to downstream transcription factors. This pathway plays a role in a number of cellular processes including proliferation, growth, motility, survival and apoptosis [68]. As indicated above, MAPK was able to phosphorylate RCAN1 at S112 within the SP motif to prime its subsequent phosphorylation by GSK-3. Moreover, the same study demonstrated that phos‐ phorylation of RCAN1 by MAPK allowed RCAN1 to become a substrate for calcineurin [31], thus introducing a further level of control to keep the pathway operating at an optimal level.

cardiovascular disease [71]. Angiogenesis is orchestrated by a balance between pro-angiogenic factors and angiogenic inhibitors [72]. A critical mediator of angiogenesis is Vascular endo‐ thelial growth factor (VEGF) which acts to stimulate angiogenesis and vascular permeability [73-75]. VEGF stimulation of cells causes the rapid activation and translocation of NFAT into the nucleus which in turn results in the up regulation of numerous genes associated with angiogenesis [76]. A number of studies have implicated RCAN1 in angiogenesis. Early studies found that *RCAN1* mRNA increased by 6-fold when endothelial cell lines were treated with VEGF [77, 78] and RCAN1 protein increased in human aortic endothelial cells (HUVECs) similarly treated [49, 79]. RCAN1 gene expression was also up regulated by other mediators

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181

Both major RCAN1 isoforms are involved in angiogenesis and appear to be regulated by different mechanisms. When human endothelial cells were treated with VEGF, there was an induction of *RCAN1-4* mRNA after 30 min, with the highest levels observed after 1 hour. Expression returned to basal levels by 24 hours after treatment [79, 81]. Others reported that up regulation of *RCAN1-4* during angiogenesis was mediated by calcium and calcineurin signalling, because treatment with cyclosporine A (CsA), a calcineurin inhibitor, or intracel‐ lular calcium chelators prevented its up regulation [80, 82]. Further evidence to suggest that *RCAN1-4* was regulated by calcineurin signalling came from studies demonstrating that *RCAN1-4* expression following VEGF and thrombin treatment was dependent upon the cooperative binding of transcription factors NFAT and GATA to the *RCAN1-4* promoter [80]. RCAN1-1 expression also appears to be modulated during angiogenesis. While initial studies found that *RCAN1-1* was not induced following VEGF treatment [79, 81], more recent reports have indicated that RCAN1-1 is up regulated in cultured endothelial cells treated with VEGF and during angiogenesis *in vivo* [49, 83]. However, unlike expression of *RCAN1-4* during angiogenesis, *RCAN1-1* expression does not appear to be regulated by the calcineurin signalling pathway as its expression was unaffected by treatment with either CsA or

A number of reports have suggested that RCAN1-1 and RCAN1-4 may play opposing roles in angiogenesis, where RCAN1-1 appears to be pro-angiogenic and is capable of inducing the formation of new blood vessels, while RCAN1-4 inhibits angiogenesis and vessel formation. For example, siRNA-mediated silencing of *RCAN1-1* in HUVECs inhibited VEGF-induced endothelial cell proliferation and angiogenic responses [49]. Further, when *RCAN1-1* was overexpressed in these cells it induced angiogenesis even in the absence of VEGF. This effect was also observed *in vivo* when human skin melanoma (SK-MEL-2) cells, which over-express VEGF-A, were transfected with *RCAN1-1*, implanted into a matrigel and transplanted into mice. In this situation, exogenous expression of *RCAN1-1* in SK-MEL-2 cells induced angio‐ genesis and vessel formation [49]. In contrast, RCAN1-4 appears to be anti-angiogenic as overexpression of *RCAN1-4* in SK-MEL-2 cells inhibited angiogenesis and siRNA-mediated silencing of *RCAN1-4* enhanced VEGF-induced proliferation [49]. Another study [80] found that forced up regulation of *RCAN1-4* in primary endothelial cells resulted in a reduction in the expression of many pro-angiogenic genes, including cell cycle inhibitors and growth factors and cytokines involved in the formation of new blood vessels, and moreover, the

of angiogenesis including thrombin [80].

intracellular calcium chelators [80, 82].

### **5.4. The NFκβ inflammatory pathway**

RCAN1 is also able to regulate the Nuclear factor κβ (NFκB) signalling pathway. NFκB is a transcription factor that regulates target genes involved in many physiological processes, including immunity, inflammation, cancer, synaptic plasticity and memory. Under normal circumstances, NFκB exists as a dimer and is sequestered in the cytoplasm through its interaction with an inhibitory molecule known as Inhibitor of κB (IκB). Upon stimulation of the NFκB signalling pathway, IκB is degraded by the ubiquitin/proteasome pathway releasing its inhibitory action on NFκB [69]. Degradation of IκB allows NFκB to translocate to the nucleus where it acts to induce the expression of various target genes including the inflammatory genes cyclooxygenase-2 (*Cox-2*) and interleukin 1 (*IL-1*) [69]. RCAN1 is able to negatively regulate the NFκB signalling pathway by attenuating NFκB activation. When RCAN1 was overexpressed in a glioblastoma cell line, it resulted in a decrease in the expression of a number of NFκB target genes including COX-2, IL-8, monocyte chemoattractant protein 1 (MCP-1), ICAM1 and VCAM1 [51]. This study demonstrated that RCAN1 inhibited NFκB signalling through a mechanism that reduced the basal turnover rate of IκBα thereby enhancing its stability [51]. By increasing the level of steady state IκBα, RCAN1 was able to exert antiinflammatory effects by preventing NFκB activation following stimulation with inflammatory mediators such as TNFα and IL-1β.

Studies have also linked RCAN1 to NFκB signalling via other members of the pathway. For example, RCAN1 is able to negatively regulate the mRNA expression of NFκB inducing kinase (*NIK*) in PC-12 cells [70]. NIK is a member of the MAP kinase family which acts to phosphorylate and activate IκB kinase α (IKKα). Once active, IKKα phosphorylates IκBα, which in turn causes it to dissociate from NFκB, allowing the transcription factor to migrate into the nucleus and activate target genes. If RCAN1 negatively regulates the expression of *NIK*, IκB would remain bound to NFκB and inhibit NFκB signalling [33]. Interestingly, while RCAN1 regulates *NIK* expression, NIK also acts on RCAN1. As mentioned above, NIK phosphorylates the C-terminal region of RCAN1, the end result of which is to reduce RCAN1 proteasomal-dependent degradation and increase the stability of RCAN1 protein [33]. The functional consequences of this increased stability of RCAN1 on NFκB signalling have yet to be determined; however consistent with the study described above [51] it seems likely that elevated levels of RCAN1 would increase the stability of Iκβ which would in turn inhibit the NFκB signalling pathway.

#### **5.5. Angiogenesis**

Angiogenesis is a physiological process involving the growth of new blood vessels essential for embryonic development as well as growth and development throughout life. This process has also been associated with disease states including inflammation, tumourigenesis and cardiovascular disease [71]. Angiogenesis is orchestrated by a balance between pro-angiogenic factors and angiogenic inhibitors [72]. A critical mediator of angiogenesis is Vascular endo‐ thelial growth factor (VEGF) which acts to stimulate angiogenesis and vascular permeability [73-75]. VEGF stimulation of cells causes the rapid activation and translocation of NFAT into the nucleus which in turn results in the up regulation of numerous genes associated with angiogenesis [76]. A number of studies have implicated RCAN1 in angiogenesis. Early studies found that *RCAN1* mRNA increased by 6-fold when endothelial cell lines were treated with VEGF [77, 78] and RCAN1 protein increased in human aortic endothelial cells (HUVECs) similarly treated [49, 79]. RCAN1 gene expression was also up regulated by other mediators of angiogenesis including thrombin [80].

above, MAPK was able to phosphorylate RCAN1 at S112 within the SP motif to prime its subsequent phosphorylation by GSK-3. Moreover, the same study demonstrated that phos‐ phorylation of RCAN1 by MAPK allowed RCAN1 to become a substrate for calcineurin [31], thus introducing a further level of control to keep the pathway operating at an optimal level.

RCAN1 is also able to regulate the Nuclear factor κβ (NFκB) signalling pathway. NFκB is a transcription factor that regulates target genes involved in many physiological processes, including immunity, inflammation, cancer, synaptic plasticity and memory. Under normal circumstances, NFκB exists as a dimer and is sequestered in the cytoplasm through its interaction with an inhibitory molecule known as Inhibitor of κB (IκB). Upon stimulation of the NFκB signalling pathway, IκB is degraded by the ubiquitin/proteasome pathway releasing its inhibitory action on NFκB [69]. Degradation of IκB allows NFκB to translocate to the nucleus where it acts to induce the expression of various target genes including the inflammatory genes cyclooxygenase-2 (*Cox-2*) and interleukin 1 (*IL-1*) [69]. RCAN1 is able to negatively regulate the NFκB signalling pathway by attenuating NFκB activation. When RCAN1 was overexpressed in a glioblastoma cell line, it resulted in a decrease in the expression of a number of NFκB target genes including COX-2, IL-8, monocyte chemoattractant protein 1 (MCP-1), ICAM1 and VCAM1 [51]. This study demonstrated that RCAN1 inhibited NFκB signalling through a mechanism that reduced the basal turnover rate of IκBα thereby enhancing its stability [51]. By increasing the level of steady state IκBα, RCAN1 was able to exert antiinflammatory effects by preventing NFκB activation following stimulation with inflammatory

Studies have also linked RCAN1 to NFκB signalling via other members of the pathway. For example, RCAN1 is able to negatively regulate the mRNA expression of NFκB inducing kinase (*NIK*) in PC-12 cells [70]. NIK is a member of the MAP kinase family which acts to phosphorylate and activate IκB kinase α (IKKα). Once active, IKKα phosphorylates IκBα, which in turn causes it to dissociate from NFκB, allowing the transcription factor to migrate into the nucleus and activate target genes. If RCAN1 negatively regulates the expression of *NIK*, IκB would remain bound to NFκB and inhibit NFκB signalling [33]. Interestingly, while RCAN1 regulates *NIK* expression, NIK also acts on RCAN1. As mentioned above, NIK phosphorylates the C-terminal region of RCAN1, the end result of which is to reduce RCAN1 proteasomal-dependent degradation and increase the stability of RCAN1 protein [33]. The functional consequences of this increased stability of RCAN1 on NFκB signalling have yet to be determined; however consistent with the study described above [51] it seems likely that elevated levels of RCAN1 would increase the stability of Iκβ which would in turn inhibit

Angiogenesis is a physiological process involving the growth of new blood vessels essential for embryonic development as well as growth and development throughout life. This process has also been associated with disease states including inflammation, tumourigenesis and

**5.4. The NFκβ inflammatory pathway**

180 Down Syndrome

mediators such as TNFα and IL-1β.

the NFκB signalling pathway.

**5.5. Angiogenesis**

Both major RCAN1 isoforms are involved in angiogenesis and appear to be regulated by different mechanisms. When human endothelial cells were treated with VEGF, there was an induction of *RCAN1-4* mRNA after 30 min, with the highest levels observed after 1 hour. Expression returned to basal levels by 24 hours after treatment [79, 81]. Others reported that up regulation of *RCAN1-4* during angiogenesis was mediated by calcium and calcineurin signalling, because treatment with cyclosporine A (CsA), a calcineurin inhibitor, or intracel‐ lular calcium chelators prevented its up regulation [80, 82]. Further evidence to suggest that *RCAN1-4* was regulated by calcineurin signalling came from studies demonstrating that *RCAN1-4* expression following VEGF and thrombin treatment was dependent upon the cooperative binding of transcription factors NFAT and GATA to the *RCAN1-4* promoter [80]. RCAN1-1 expression also appears to be modulated during angiogenesis. While initial studies found that *RCAN1-1* was not induced following VEGF treatment [79, 81], more recent reports have indicated that RCAN1-1 is up regulated in cultured endothelial cells treated with VEGF and during angiogenesis *in vivo* [49, 83]. However, unlike expression of *RCAN1-4* during angiogenesis, *RCAN1-1* expression does not appear to be regulated by the calcineurin signalling pathway as its expression was unaffected by treatment with either CsA or intracellular calcium chelators [80, 82].

A number of reports have suggested that RCAN1-1 and RCAN1-4 may play opposing roles in angiogenesis, where RCAN1-1 appears to be pro-angiogenic and is capable of inducing the formation of new blood vessels, while RCAN1-4 inhibits angiogenesis and vessel formation. For example, siRNA-mediated silencing of *RCAN1-1* in HUVECs inhibited VEGF-induced endothelial cell proliferation and angiogenic responses [49]. Further, when *RCAN1-1* was overexpressed in these cells it induced angiogenesis even in the absence of VEGF. This effect was also observed *in vivo* when human skin melanoma (SK-MEL-2) cells, which over-express VEGF-A, were transfected with *RCAN1-1*, implanted into a matrigel and transplanted into mice. In this situation, exogenous expression of *RCAN1-1* in SK-MEL-2 cells induced angio‐ genesis and vessel formation [49]. In contrast, RCAN1-4 appears to be anti-angiogenic as overexpression of *RCAN1-4* in SK-MEL-2 cells inhibited angiogenesis and siRNA-mediated silencing of *RCAN1-4* enhanced VEGF-induced proliferation [49]. Another study [80] found that forced up regulation of *RCAN1-4* in primary endothelial cells resulted in a reduction in the expression of many pro-angiogenic genes, including cell cycle inhibitors and growth factors and cytokines involved in the formation of new blood vessels, and moreover, the formation of tube structures (as a model for blood vessel development) formed from primary human endothelial cells *in vitro* was inhibited. Consistent with this, B16 melanoma cells engineered to over-express *RCAN1-4* and implanted subcutaneously into C57BL6 mice displayed a reduction in tumour growth due to a decrease in blood vessel density [80]. Interestingly, RCAN1-4 is thought to exert its anti-angiogenic effects by providing a negative feedback loop to inactivate calcineurin, preventing nuclear translocation and transcriptional activity of NFAT after VEGF stimulation. In support of this, ablation of *RCAN1-4* expression in endothelial cells increased NFAT activity and was associated with increased transcription of NFAT-regulated genes, such as *E-selectin* and *VCAM1* [78]. Intriguingly, RCAN1-1 was found to activate NFAT activity and enhance is pro-angiogenic functions [49]. Thus, RCAN1-4 inhibits the calcineurin/NFAT pathway while RCAN1-1 activates it.

and memory and many of the behavioural and cognitive defects seen in DS are hippocampaldependent [85]. The difference in hippocampal volume is most likely due to various structural abnormalities, including a decrease in the mean area of the dentate gyrus (DG) and inadequate migration of cells into the pyramidal cell layer [96]. Notably, in adults there is an additional age-related decrease in the volume of the hippocampus, most likely due to some degree of

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Smaller brains in DS individuals probably result from a reduction in the total number of neurones, with certain regions preferentially affected. DS brains exhibit a decrease in neuronal density by adulthood of between 10-50% [91]. The cortex of DS adults exhibits decreases in neuronal number and density in addition to abnormal distribution of neurones [97]. This same pattern of neuronal loss was also observed in the hippocampus and visual cortex. Interestingly, DS foetuses exhibited the same pattern of neuronal development as normal foetuses, with similar neuronal morphology, dendritic spine number and density [98]. However shortly after birth defects were evident and became more pronounced with age [99]. This indicates that something happens after birth which results in alterations in neuronal number and morphol‐ ogy. Using Golgi staining which allows for the visualisation of neurones including their cell bodies, axons, dendrites and spines, the brains of DS infants exhibited shorter basilar dendrites with a significant decrease in the absolute number of spines [100], which was postulated to correlate with a 20-35% decrease in surface area per synaptic contact [91]. Why and how this decline in neuronal development occurs is currently undetermined. These same defects were observed in adults with DS, who exhibited decreased dendritic branching, dendrite length and spine density [101]. Biochemical examination of adult DS brains also revealed a significant reduction in the concentrations of various neurotransmitter markers including, noradrenaline, serotonin or 5-hydroxytraptamine (5-HT) and choline acetyltransferase (ChAT) [102, 103],

On top of the neurodevelopmental problems associated with DS, all individuals with the disorder develop the neuropathological and neurochemical changes associated with AD by the third decade of life [89]. This includes the accumulation of amyloid β (Aβ), formation of hyperphosphorylated Tau-containing neurofibrillary tangles (NFT) and senile plaques. The progression of AD-neuropathology is analogous in both DS and AD, despite occurring decades

RCAN1 has been implicatedindevelopment andfunction ofthe brain.*Rcan1* is expressedin the developing mouse neural tube from embryonic day (E) E9.5 onwards and at E11.5-E12.5 was detected in the telencephalic vesicles, the caudal hypothalamus, the pretectum and the basal plate of the hindbrain and spinal cord. In later stages of embryonic development, *Rcan1* was highlyexpressedintheneuralproliferativeanddifferentiationzoneswithinthebrainwithlower expression observed in other regions, including the telecephalon, hypothalamus, pretectum, cortical plate, striatum, amygdala, midbrain, hindbrain and spinal cord. In the post natal brain *Rcan1*geneexpressionwaswidelydistributedthroughout,withthehighestlevelsintheolfactory bulb, the cerebral cortex, hippocampus and dentate gyrus, striatum and septum, amygdala,

neurodegeneration [95].

earlier in DS [104].

**6.2. RCAN1 in the brain**

again signifying neuro-functional deficits in the brain.

## **6. The consequences of RCAN1 over-expression in the DS brain**

#### **6.1. Down syndrome and the neural system**

DS is the leading genetic cause of intellectual impairment in the general population and is thought to contribute to around 30% of all cases of moderate to severe mental retardation [84]. Mental retardation in DS is characterised by behavioural and cognitive impairments which include low IQ, language deficits and defects in both short and long term memory. Later these deficits are compounded by the early onset of dementia [85].

People with DS exhibit a reduced performance on a number of different tests designed to demonstrate short term or working memory, including visual perception, visual imagery and spatial imagery tasks [86]. Long term memory is also affected by DS with both implicit (defined as improvement in perceptual, cognitive or motor tasks without any conscious reference to previous experience) and explicit (intentional recall or recognition of experiences or informa‐ tion) memory impaired [87]. In addition to the cognitive defects observed throughout life, neuropsychological tests showed that there is a cognitive decline in DS individuals with age and these cognitive changes equate to those observed following the onset of dementia [88]. DS participants with early stage dementia displayed severely diminished long term memory as well as a decreased ability to retrieve stored information compared with the non-demented DS controls [88]. The decline in these forms of cognition, particularly the ability to form new long term memories, is analogues to the cognitive deterioration seen in early to moderate Alzheimer's disease (AD) [89]. Interestingly, the cognitive defects that characterise DS are associated with hippocampal-based learning and memory while prefrontal-mediated execu‐ tive function and cognition remain relatively unaffected [85].

The cognitive impairments in DS are accompanied by many neuro-morphological changes. Individuals with DS have a significant reduction in brain weight and volume [90], despite brain weight falling within the normal range at birth [91]. DS brains have a shorter anterior-posterior diameter, a reduction in the size of the frontal lobes, a flatter occipital lobe and a smaller brain stem and cerebellum [91]. The anterior and posterior corpus callosum regions and hippocam‐ pus are also smaller [92-95]. The hippocampus is a key brain structure involved in learning and memory and many of the behavioural and cognitive defects seen in DS are hippocampaldependent [85]. The difference in hippocampal volume is most likely due to various structural abnormalities, including a decrease in the mean area of the dentate gyrus (DG) and inadequate migration of cells into the pyramidal cell layer [96]. Notably, in adults there is an additional age-related decrease in the volume of the hippocampus, most likely due to some degree of neurodegeneration [95].

Smaller brains in DS individuals probably result from a reduction in the total number of neurones, with certain regions preferentially affected. DS brains exhibit a decrease in neuronal density by adulthood of between 10-50% [91]. The cortex of DS adults exhibits decreases in neuronal number and density in addition to abnormal distribution of neurones [97]. This same pattern of neuronal loss was also observed in the hippocampus and visual cortex. Interestingly, DS foetuses exhibited the same pattern of neuronal development as normal foetuses, with similar neuronal morphology, dendritic spine number and density [98]. However shortly after birth defects were evident and became more pronounced with age [99]. This indicates that something happens after birth which results in alterations in neuronal number and morphol‐ ogy. Using Golgi staining which allows for the visualisation of neurones including their cell bodies, axons, dendrites and spines, the brains of DS infants exhibited shorter basilar dendrites with a significant decrease in the absolute number of spines [100], which was postulated to correlate with a 20-35% decrease in surface area per synaptic contact [91]. Why and how this decline in neuronal development occurs is currently undetermined. These same defects were observed in adults with DS, who exhibited decreased dendritic branching, dendrite length and spine density [101]. Biochemical examination of adult DS brains also revealed a significant reduction in the concentrations of various neurotransmitter markers including, noradrenaline, serotonin or 5-hydroxytraptamine (5-HT) and choline acetyltransferase (ChAT) [102, 103], again signifying neuro-functional deficits in the brain.

On top of the neurodevelopmental problems associated with DS, all individuals with the disorder develop the neuropathological and neurochemical changes associated with AD by the third decade of life [89]. This includes the accumulation of amyloid β (Aβ), formation of hyperphosphorylated Tau-containing neurofibrillary tangles (NFT) and senile plaques. The progression of AD-neuropathology is analogous in both DS and AD, despite occurring decades earlier in DS [104].

#### **6.2. RCAN1 in the brain**

formation of tube structures (as a model for blood vessel development) formed from primary human endothelial cells *in vitro* was inhibited. Consistent with this, B16 melanoma cells engineered to over-express *RCAN1-4* and implanted subcutaneously into C57BL6 mice displayed a reduction in tumour growth due to a decrease in blood vessel density [80]. Interestingly, RCAN1-4 is thought to exert its anti-angiogenic effects by providing a negative feedback loop to inactivate calcineurin, preventing nuclear translocation and transcriptional activity of NFAT after VEGF stimulation. In support of this, ablation of *RCAN1-4* expression in endothelial cells increased NFAT activity and was associated with increased transcription of NFAT-regulated genes, such as *E-selectin* and *VCAM1* [78]. Intriguingly, RCAN1-1 was found to activate NFAT activity and enhance is pro-angiogenic functions [49]. Thus, RCAN1-4

inhibits the calcineurin/NFAT pathway while RCAN1-1 activates it.

deficits are compounded by the early onset of dementia [85].

tive function and cognition remain relatively unaffected [85].

**6.1. Down syndrome and the neural system**

182 Down Syndrome

**6. The consequences of RCAN1 over-expression in the DS brain**

DS is the leading genetic cause of intellectual impairment in the general population and is thought to contribute to around 30% of all cases of moderate to severe mental retardation [84]. Mental retardation in DS is characterised by behavioural and cognitive impairments which include low IQ, language deficits and defects in both short and long term memory. Later these

People with DS exhibit a reduced performance on a number of different tests designed to demonstrate short term or working memory, including visual perception, visual imagery and spatial imagery tasks [86]. Long term memory is also affected by DS with both implicit (defined as improvement in perceptual, cognitive or motor tasks without any conscious reference to previous experience) and explicit (intentional recall or recognition of experiences or informa‐ tion) memory impaired [87]. In addition to the cognitive defects observed throughout life, neuropsychological tests showed that there is a cognitive decline in DS individuals with age and these cognitive changes equate to those observed following the onset of dementia [88]. DS participants with early stage dementia displayed severely diminished long term memory as well as a decreased ability to retrieve stored information compared with the non-demented DS controls [88]. The decline in these forms of cognition, particularly the ability to form new long term memories, is analogues to the cognitive deterioration seen in early to moderate Alzheimer's disease (AD) [89]. Interestingly, the cognitive defects that characterise DS are associated with hippocampal-based learning and memory while prefrontal-mediated execu‐

The cognitive impairments in DS are accompanied by many neuro-morphological changes. Individuals with DS have a significant reduction in brain weight and volume [90], despite brain weight falling within the normal range at birth [91]. DS brains have a shorter anterior-posterior diameter, a reduction in the size of the frontal lobes, a flatter occipital lobe and a smaller brain stem and cerebellum [91]. The anterior and posterior corpus callosum regions and hippocam‐ pus are also smaller [92-95]. The hippocampus is a key brain structure involved in learning

RCAN1 has been implicatedindevelopment andfunction ofthe brain.*Rcan1* is expressedin the developing mouse neural tube from embryonic day (E) E9.5 onwards and at E11.5-E12.5 was detected in the telencephalic vesicles, the caudal hypothalamus, the pretectum and the basal plate of the hindbrain and spinal cord. In later stages of embryonic development, *Rcan1* was highlyexpressedintheneuralproliferativeanddifferentiationzoneswithinthebrainwithlower expression observed in other regions, including the telecephalon, hypothalamus, pretectum, cortical plate, striatum, amygdala, midbrain, hindbrain and spinal cord. In the post natal brain *Rcan1*geneexpressionwaswidelydistributedthroughout,withthehighestlevelsintheolfactory bulb, the cerebral cortex, hippocampus and dentate gyrus, striatum and septum, amygdala, hypothalamus and the habenula. Within the hippocampus and dentate gyrus, highest levels of expression were observed in the pyramidal and granular cell layers [105].

together results from this study suggested that the absence of *Rcan1* selectively affects some,

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These behavioural deficits in RCAN1-KO mice were accompanied by abnormal synaptic transmissions and impaired long term potentiation (LTP). LTP is a form of synaptic plasticity hypothesised to be a biological substrate for some forms of memory [109]. Two forms of LTP can be examined: early-component LTP (E-LTP), a weak and short-lived enhancement of synaptic transmission; and late-component LTP (L-LTP) which is a robust enhancement of synaptic transmission lasting many hours [110, 111]. Paired-pulse facilitation (PPF) is also a component of LTP and is a measure of pre-synaptic short-term plasticity and neurotransmitter release [112]. Absence of RCAN1 did not affect the basal level of synaptic transmission but did result in a reduction in PPF compared with the WT controls, suggesting that pre-synaptic short term plasticity was affected by the lack of *Rcan1*. While there was no difference in the E-LTP, L-LTP was adversely affected by the ablation of *Rcan1*, with RCAN1-KO mice exhibiting a reduction in initial amplitude of L-LTP as well as a reduction in duration of the potentiation [54]. This is significant because the amplitude and duration are the biological correlates of

The strongest evidence to suggest a role for RCAN1 in the neurological defects observed in DS comes from a recent study by our group examining *RCAN1* transgenic (RCAN1-TG) mice. Using mice engineered to over-express *RCAN1-1* at a level analogous to that observed in DS, we found up regulation of RCAN1 contributed to some of the neurological defects character‐ istic of DS. For example, RCAN1 over-expression resulted in multiple defects in the formation, structure and function of the hippocampus [55]. Specifically, there was a significant reduction in the overall size of the hippocampus and analysis of the various structures within the hippocampal formation revealed a decrease in the absolute volume and cellularity of the dentate gyrus [55], mirroring the structural hippocampal defects and marked neuronal loss observed in DS. Our study suggested that the decrease in neuronal cellularity within the hippocampus of RCAN1-TG mice was the result of defective neurogenesis because fewer terminally differentiated neurones within the dentate gyrus formed and progenitor cells isolated and cultured from the sub ventricular zone had diminished ability to differentiate into neurones. This also reflects changes observed in DS [113]. RCAN1 transgenic mice also exhibited neuro-physiological impairments. In particular, over-expression of RCAN1 resulted in a defect in the maintenance phase of LTP which may be explained in part, by the reduction in post-synaptic spine density observed in the brains of these mice. Failure to maintain LTP in hippocampal slices was accompanied by deficits in hippocampal-dependent spatial learning and in short and long term memory. At a molecular level, in response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2, signifying that the processes essential for the maintenance of LTP and formation of

RCAN1 has also been shown by our group to be involved in neurotransmission. Using chromaffin cells cultured from the adrenal gland as a model for the neuronal system, cells from both RCAN1-TG and RCAN1-KO mice displayed a reduction in neurotransmitter release. Our study demonstrated that the normal function of RCAN1 was to regulate the number of synaptic

synaptic strength required to reinforce the laying down of memory.

memory [55] are defective in mice with an excess of RCAN1.

but not all, types of memory.

Western blot analysis using an antibody designed to detect both RCAN1-1 and RCAN1-4 proteins found that the two isoforms were differentially expressed in the adult mouse brain. RCAN1-1 was abundant throughout the brain, with the highest levels of expression detected in the cortex and hippocampus [20, 54, 106]. RCAN1-4 was generally found at lower levels in the hippocampus, striatum, cortex and prefrontal cortex [54]. Similar results have been observed in the adult human brain where RCAN1-1 was most highly expressed in the cerebral cortex, hippocampus, substantia nigra, thalamus and medulla oblongata [21]. It is worth noting that while one study indicated that both isoforms of RCAN1 were located exclusively within neurones and not in astrocytes or microglial cells [107], another study found a wider distri‐ bution pattern [106], with RCAN1-1 and RCAN1-4 detected in multiple cell types including astrocytes and microglia. The highest levels of expression were observed in neurones [106]. Moreover, RCAN1-1 was also detected in primary glial-like cell cultures containing microglial cells and expression of RCAN1-4 was strongly induced following calcium stress [106].

Experimental evidence suggests that RCAN1 has a role in brain function. For example, studies on the RCAN1 orthologue in *Drosophila* known as *nebula*, demonstrated that a loss-of-function mutation of *nebula* displayed a decrease in learning and memory acquisition and performed significantly worse on learning and memory tests after a single trial compared with WT controls. Testing after 1 hour found no difference in the short term memory performance, however tests of long term memory (after 24 hours) found that *nebula*-deficient flies displayed virtually no long term memory [108]. This defect was apparent despite the normal presence of mushroom bodies (the learning and memory centres in *Drosophila*). The decrease in learning and memory observed was attributed to abnormal calcineurin signalling, as *nebula* loss-offunction mutants exhibited a 40% increase in calcineurin activity [108]. Interestingly, overexpression of *nebula* resulted in a similar phenotype. When *Drosophila* over-expressing *nebula* were generated and tested, they displayed virtually no ability to learn. This study also found that transient over-expression of *nebula* was sufficient to cause learning and memory deficits, indicating that a biochemical defect was responsible for learning and memory rather than a pre-existing developmental abnormality [108], a finding that may have implications for DS treatment options.

Similar behavioural abnormalities were observed in RCAN1-KO mice. While the absence of *Rcan1* did not result in any gross anatomical changes within the brain, RCAN1-KO mice exhibited various behavioural and synaptic deficiencies. For example, RCAN1-KO mice were shown to have impaired learning and memory in the Morris Water Maze (MWM), a wellestablished paradigm of hippocampal-dependent learning and memory. During the acquisi‐ tion phase of the trial, RCAN1-KO mice displayed a decreased ability to learn the location of the platform compared with WT controls. This indicated that RCAN1-KO mice had a spatial learning impairment. These mice also displayed a poor spatial memory because when the escape platform was removed, RCAN1-KO mice did not demonstrate a specific preference for the target quadrant. On the other hand, a passive avoidance test using electric shock found that long- and short-term contextual fear memory was normal in these mice [54]. Taken together results from this study suggested that the absence of *Rcan1* selectively affects some, but not all, types of memory.

hypothalamus and the habenula. Within the hippocampus and dentate gyrus, highest levels of

Western blot analysis using an antibody designed to detect both RCAN1-1 and RCAN1-4 proteins found that the two isoforms were differentially expressed in the adult mouse brain. RCAN1-1 was abundant throughout the brain, with the highest levels of expression detected in the cortex and hippocampus [20, 54, 106]. RCAN1-4 was generally found at lower levels in the hippocampus, striatum, cortex and prefrontal cortex [54]. Similar results have been observed in the adult human brain where RCAN1-1 was most highly expressed in the cerebral cortex, hippocampus, substantia nigra, thalamus and medulla oblongata [21]. It is worth noting that while one study indicated that both isoforms of RCAN1 were located exclusively within neurones and not in astrocytes or microglial cells [107], another study found a wider distri‐ bution pattern [106], with RCAN1-1 and RCAN1-4 detected in multiple cell types including astrocytes and microglia. The highest levels of expression were observed in neurones [106]. Moreover, RCAN1-1 was also detected in primary glial-like cell cultures containing microglial cells and expression of RCAN1-4 was strongly induced following calcium stress [106].

Experimental evidence suggests that RCAN1 has a role in brain function. For example, studies on the RCAN1 orthologue in *Drosophila* known as *nebula*, demonstrated that a loss-of-function mutation of *nebula* displayed a decrease in learning and memory acquisition and performed significantly worse on learning and memory tests after a single trial compared with WT controls. Testing after 1 hour found no difference in the short term memory performance, however tests of long term memory (after 24 hours) found that *nebula*-deficient flies displayed virtually no long term memory [108]. This defect was apparent despite the normal presence of mushroom bodies (the learning and memory centres in *Drosophila*). The decrease in learning and memory observed was attributed to abnormal calcineurin signalling, as *nebula* loss-offunction mutants exhibited a 40% increase in calcineurin activity [108]. Interestingly, overexpression of *nebula* resulted in a similar phenotype. When *Drosophila* over-expressing *nebula* were generated and tested, they displayed virtually no ability to learn. This study also found that transient over-expression of *nebula* was sufficient to cause learning and memory deficits, indicating that a biochemical defect was responsible for learning and memory rather than a pre-existing developmental abnormality [108], a finding that may have implications for DS

Similar behavioural abnormalities were observed in RCAN1-KO mice. While the absence of *Rcan1* did not result in any gross anatomical changes within the brain, RCAN1-KO mice exhibited various behavioural and synaptic deficiencies. For example, RCAN1-KO mice were shown to have impaired learning and memory in the Morris Water Maze (MWM), a wellestablished paradigm of hippocampal-dependent learning and memory. During the acquisi‐ tion phase of the trial, RCAN1-KO mice displayed a decreased ability to learn the location of the platform compared with WT controls. This indicated that RCAN1-KO mice had a spatial learning impairment. These mice also displayed a poor spatial memory because when the escape platform was removed, RCAN1-KO mice did not demonstrate a specific preference for the target quadrant. On the other hand, a passive avoidance test using electric shock found that long- and short-term contextual fear memory was normal in these mice [54]. Taken

expression were observed in the pyramidal and granular cell layers [105].

treatment options.

184 Down Syndrome

These behavioural deficits in RCAN1-KO mice were accompanied by abnormal synaptic transmissions and impaired long term potentiation (LTP). LTP is a form of synaptic plasticity hypothesised to be a biological substrate for some forms of memory [109]. Two forms of LTP can be examined: early-component LTP (E-LTP), a weak and short-lived enhancement of synaptic transmission; and late-component LTP (L-LTP) which is a robust enhancement of synaptic transmission lasting many hours [110, 111]. Paired-pulse facilitation (PPF) is also a component of LTP and is a measure of pre-synaptic short-term plasticity and neurotransmitter release [112]. Absence of RCAN1 did not affect the basal level of synaptic transmission but did result in a reduction in PPF compared with the WT controls, suggesting that pre-synaptic short term plasticity was affected by the lack of *Rcan1*. While there was no difference in the E-LTP, L-LTP was adversely affected by the ablation of *Rcan1*, with RCAN1-KO mice exhibiting a reduction in initial amplitude of L-LTP as well as a reduction in duration of the potentiation [54]. This is significant because the amplitude and duration are the biological correlates of synaptic strength required to reinforce the laying down of memory.

The strongest evidence to suggest a role for RCAN1 in the neurological defects observed in DS comes from a recent study by our group examining *RCAN1* transgenic (RCAN1-TG) mice. Using mice engineered to over-express *RCAN1-1* at a level analogous to that observed in DS, we found up regulation of RCAN1 contributed to some of the neurological defects character‐ istic of DS. For example, RCAN1 over-expression resulted in multiple defects in the formation, structure and function of the hippocampus [55]. Specifically, there was a significant reduction in the overall size of the hippocampus and analysis of the various structures within the hippocampal formation revealed a decrease in the absolute volume and cellularity of the dentate gyrus [55], mirroring the structural hippocampal defects and marked neuronal loss observed in DS. Our study suggested that the decrease in neuronal cellularity within the hippocampus of RCAN1-TG mice was the result of defective neurogenesis because fewer terminally differentiated neurones within the dentate gyrus formed and progenitor cells isolated and cultured from the sub ventricular zone had diminished ability to differentiate into neurones. This also reflects changes observed in DS [113]. RCAN1 transgenic mice also exhibited neuro-physiological impairments. In particular, over-expression of RCAN1 resulted in a defect in the maintenance phase of LTP which may be explained in part, by the reduction in post-synaptic spine density observed in the brains of these mice. Failure to maintain LTP in hippocampal slices was accompanied by deficits in hippocampal-dependent spatial learning and in short and long term memory. At a molecular level, in response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2, signifying that the processes essential for the maintenance of LTP and formation of memory [55] are defective in mice with an excess of RCAN1.

RCAN1 has also been shown by our group to be involved in neurotransmission. Using chromaffin cells cultured from the adrenal gland as a model for the neuronal system, cells from both RCAN1-TG and RCAN1-KO mice displayed a reduction in neurotransmitter release. Our study demonstrated that the normal function of RCAN1 was to regulate the number of synaptic vesicles fusing with the plasma membrane and undergoing exocytosis, and the speed at which the vesicle pore opens and closes [53]. Although our study showed that the final outcome was the same whether RCAN1 was in excess or deficit, increased expression of *RCAN1* had the opposite effect to *Rcan1* ablation on vesicle fusion kinetics - ablation slowed fusion pore kinetics while over-expression accelerated fusion pore kinetics.

hyperphosphorylated tau protein and cytoskeletal changes in the brain similar to those observed in AD accumulate when the phosphatase activity of calcineurin is reduced [120]. Thus, if RCAN1 is behaving as a calcineurin inhibitor it is possible that increased levels of

RCAN1 and Its Potential Contribution to the Down Syndrome Phenotype

http://dx.doi.org/10.5772/52977

187

RCAN1, via its role as an inhibitor of calcineurin, has also been implicated in the pathogenesis of Huntington's disease (HD). In a mouse model of HD, phosphorylation of huntingtin at serine residue 421 was protective and treatment of HD neuronal cells with calcineurin inhibitors prevented their death by maintaining their phosphorylation status at Ser421 [122]. RCAN1-1L protein was significantly down regulated in human HD post mortem brains and exogenous expression of RCAN1-1L in a cell culture model of HD protected the cells against toxicity caused by mutant huntingtin [123]. This protection was attributed to the ability of excess RCAN1 to inhibit calcineurin phosphatase activity, indicating that in this circumstance

Another connection between RCAN1 and neurodegeneration may be through the formation of aggregates. When proteins accumulate within a cell a mitrotubule-based apparatus known as an aggresome acts to sequester proteins within the cytoplasm. The formation of aggresomes within cells is most likely a defence mechanism against the presence of misfolded or abnormal proteins. However if these misfolded proteins are not cleared appropriately it can lead to abnormal protein accumulation and eventual neurotoxicity [124]. The formation of aggre‐ somes is believed to contribute to many neurodegenerative disorders including AD, Hun‐ tington's disease and cerebral ataxia [125]. When RCAN1 was over-expressed in various neuronal cell lines and in primary neurones, formation of aggregates occurred [124] and the aggregates were associated with microtubules, indicating that they had formed inclusion bodies within the cells. When RCAN1 was aggregated within neurones, neuronal abnormal‐ ities characterised by a decreased number and density of synapses were observed, which in turn altered synaptic function [124]. This constitutes another example of the damaging effects

Finally, two polymorphisms located in the *RCAN1-4* promoter region have been associated with AD in the Chinese Han population [126]. One of these, rs71324311, in the heterozygousdeletion genotype confers protection while the other, rs10550296, also in the heterozygousdeletion configuration, is a risk factor. The functional consequences of these sequence variants

**7. The consequences of RCAN1 over-expression in the DS immune system**

DS is associated with a multitude of immune system defects. People with DS are more susceptible to infections, particularly respiratory tract infections with pneumonia one of the major causes of early death [127]. The incidence of viral hepatitis and haematopoietic malig‐ nancies is also increased in people with DS as is their tendency to develop certain types of

RCAN1, as occurs in DS and AD, promote the development of AD [21] [121].

RCAN1 over-expression is advantageous.

of excess RCAN1.

are yet to be determined.

**7.1. The Down syndrome immune system**

### **6.3. RCAN1 in neurodegeneration**

Although it has not been proven, there is circumstantial evidence to suggest that RCAN1 plays a role in neurodegenerative conditions (other than DS). For example, Northern blot analysis of human brain samples found that *RCAN1* expression was increased about 2-fold in brains of AD patients [21, 107]. This increased gene expression was confined to the regions of the brain affected by AD, such as the hippocampus and cerebral cortex. This study also found that regions of the brain containing NFT had up to 3 times more *RCAN1* mRNA compared with the same regions of the brain without tangles [21]. Immunohistochemistry on human brain tissue using a RCAN1-specific antibody, found that RCAN1 protein levels increased in abundance with normal ageing in pyramidal neurones with further increases observed in brains affected by moderate to severe AD [65, 107]. In addition to increased protein levels, there was an alteration in the subcellular location of RCAN1 in AD-affected neurones, with a significant increase in the amount of RCAN1 within the nucleus compared with non-diseased tissue [65]. Interestingly, there was an up regulation of RCAN1-1 mRNA and protein in the hippocampus of AD patients, with no changes observed in the abundance of RCAN1-4 [65, 107], suggesting divergent functions of the major isoforms.

While these observations are intriguing, the question remains, what effect does increased RCAN1 expression have on the ageing brain and does it play a role in AD-like neuropathology? While this question remains unanswered, there are a number of possible reasons as to why increased RCAN1 expression might lead to neurodegeneration. One proposed explanation invokes a possible relationship between elevated RCAN1 expression, AD-like neurodegener‐ ation and Tau protein. Tau is involved in the stabilisation of the microtubule networks within neurones and its hyperphosphorylation has been linked to the pathogenesis of AD. Tau can be phosphorylated by a number of different kinases, including GSK-3β and Ca2+/calmodulindependent protein kinases (CaMK). Hyperphosphorylation of Tau is detrimental and can lead to AD neuropathology, including formation of NFT [114-116]. During normal cellular proc‐ esses, there is a proteasome-dependent degradation of Tau protein but when Tau becomes hyperphosphorylated, it is resistant to this degradation and accumulates within the cell [117]. Some studies have found that increased levels of RCAN1 result in a concomitant increase in the phosphorylation of Tau and thus may contribute to its neuronal accumulation [67, 117] and we showed an accumulation of hyperphosphorylated Tau in the brains of aged RCAN1- TG mice [118]. This observed enhancement in Tau phosphorylation may be due to the effect of RCAN1 on GSK-3 activity, since increased RCAN1 expression in PC-12 cells resulted in an increase in the absolute level of GSK-3β, which in turn enhanced its ability to phosphorylate Tau [67]. There have also been suggestions that excess RCAN1 can exacerbate AD-like neuropathology by inhibiting calcineurin. Calcineurin activity is decreased in AD [119] and hyperphosphorylated tau protein and cytoskeletal changes in the brain similar to those observed in AD accumulate when the phosphatase activity of calcineurin is reduced [120]. Thus, if RCAN1 is behaving as a calcineurin inhibitor it is possible that increased levels of RCAN1, as occurs in DS and AD, promote the development of AD [21] [121].

RCAN1, via its role as an inhibitor of calcineurin, has also been implicated in the pathogenesis of Huntington's disease (HD). In a mouse model of HD, phosphorylation of huntingtin at serine residue 421 was protective and treatment of HD neuronal cells with calcineurin inhibitors prevented their death by maintaining their phosphorylation status at Ser421 [122]. RCAN1-1L protein was significantly down regulated in human HD post mortem brains and exogenous expression of RCAN1-1L in a cell culture model of HD protected the cells against toxicity caused by mutant huntingtin [123]. This protection was attributed to the ability of excess RCAN1 to inhibit calcineurin phosphatase activity, indicating that in this circumstance RCAN1 over-expression is advantageous.

Another connection between RCAN1 and neurodegeneration may be through the formation of aggregates. When proteins accumulate within a cell a mitrotubule-based apparatus known as an aggresome acts to sequester proteins within the cytoplasm. The formation of aggresomes within cells is most likely a defence mechanism against the presence of misfolded or abnormal proteins. However if these misfolded proteins are not cleared appropriately it can lead to abnormal protein accumulation and eventual neurotoxicity [124]. The formation of aggre‐ somes is believed to contribute to many neurodegenerative disorders including AD, Hun‐ tington's disease and cerebral ataxia [125]. When RCAN1 was over-expressed in various neuronal cell lines and in primary neurones, formation of aggregates occurred [124] and the aggregates were associated with microtubules, indicating that they had formed inclusion bodies within the cells. When RCAN1 was aggregated within neurones, neuronal abnormal‐ ities characterised by a decreased number and density of synapses were observed, which in turn altered synaptic function [124]. This constitutes another example of the damaging effects of excess RCAN1.

Finally, two polymorphisms located in the *RCAN1-4* promoter region have been associated with AD in the Chinese Han population [126]. One of these, rs71324311, in the heterozygousdeletion genotype confers protection while the other, rs10550296, also in the heterozygousdeletion configuration, is a risk factor. The functional consequences of these sequence variants are yet to be determined.

## **7. The consequences of RCAN1 over-expression in the DS immune system**

#### **7.1. The Down syndrome immune system**

vesicles fusing with the plasma membrane and undergoing exocytosis, and the speed at which the vesicle pore opens and closes [53]. Although our study showed that the final outcome was the same whether RCAN1 was in excess or deficit, increased expression of *RCAN1* had the opposite effect to *Rcan1* ablation on vesicle fusion kinetics - ablation slowed fusion pore kinetics

Although it has not been proven, there is circumstantial evidence to suggest that RCAN1 plays a role in neurodegenerative conditions (other than DS). For example, Northern blot analysis of human brain samples found that *RCAN1* expression was increased about 2-fold in brains of AD patients [21, 107]. This increased gene expression was confined to the regions of the brain affected by AD, such as the hippocampus and cerebral cortex. This study also found that regions of the brain containing NFT had up to 3 times more *RCAN1* mRNA compared with the same regions of the brain without tangles [21]. Immunohistochemistry on human brain tissue using a RCAN1-specific antibody, found that RCAN1 protein levels increased in abundance with normal ageing in pyramidal neurones with further increases observed in brains affected by moderate to severe AD [65, 107]. In addition to increased protein levels, there was an alteration in the subcellular location of RCAN1 in AD-affected neurones, with a significant increase in the amount of RCAN1 within the nucleus compared with non-diseased tissue [65]. Interestingly, there was an up regulation of RCAN1-1 mRNA and protein in the hippocampus of AD patients, with no changes observed in the abundance of RCAN1-4 [65,

While these observations are intriguing, the question remains, what effect does increased RCAN1 expression have on the ageing brain and does it play a role in AD-like neuropathology? While this question remains unanswered, there are a number of possible reasons as to why increased RCAN1 expression might lead to neurodegeneration. One proposed explanation invokes a possible relationship between elevated RCAN1 expression, AD-like neurodegener‐ ation and Tau protein. Tau is involved in the stabilisation of the microtubule networks within neurones and its hyperphosphorylation has been linked to the pathogenesis of AD. Tau can be phosphorylated by a number of different kinases, including GSK-3β and Ca2+/calmodulindependent protein kinases (CaMK). Hyperphosphorylation of Tau is detrimental and can lead to AD neuropathology, including formation of NFT [114-116]. During normal cellular proc‐ esses, there is a proteasome-dependent degradation of Tau protein but when Tau becomes hyperphosphorylated, it is resistant to this degradation and accumulates within the cell [117]. Some studies have found that increased levels of RCAN1 result in a concomitant increase in the phosphorylation of Tau and thus may contribute to its neuronal accumulation [67, 117] and we showed an accumulation of hyperphosphorylated Tau in the brains of aged RCAN1- TG mice [118]. This observed enhancement in Tau phosphorylation may be due to the effect of RCAN1 on GSK-3 activity, since increased RCAN1 expression in PC-12 cells resulted in an increase in the absolute level of GSK-3β, which in turn enhanced its ability to phosphorylate Tau [67]. There have also been suggestions that excess RCAN1 can exacerbate AD-like neuropathology by inhibiting calcineurin. Calcineurin activity is decreased in AD [119] and

while over-expression accelerated fusion pore kinetics.

107], suggesting divergent functions of the major isoforms.

**6.3. RCAN1 in neurodegeneration**

186 Down Syndrome

DS is associated with a multitude of immune system defects. People with DS are more susceptible to infections, particularly respiratory tract infections with pneumonia one of the major causes of early death [127]. The incidence of viral hepatitis and haematopoietic malig‐ nancies is also increased in people with DS as is their tendency to develop certain types of autoimmune disorders such as autoimmune thyroid disease (AITD) (Hashimoto type), coeliac disease and diabetes [127] [128]. Thus, DS appears to include a combination of immunodefi‐ ciency and immune dysfunction. Although the precise cause of this immune dysfunction is unclear, the DS immune system is characterised by a number of abnormalities thought to originate from defective innate and adaptive immunity.

altered apoptosis of lymphocytes may also contribute to the decrease in overall numbers of T cells in the periphery, as well as to the alterations observed in the abundance of the various T

T lymphocytes isolated from DS people are also functionally compromised. Under conditions designed to simulate an infection using anti-CD3 antibodies or the non-specific mitogen, phytohemagglutinin to activate T cells, DS lymphocytes were diminished in their proliferative capacity [136, 137]. Not only did the DS-derived T cells have a proliferative defect, they showed increased expression of apoptotic markers including APO-I/Fas (CD95) antigen, a T cell death marker, and increased apoptosis was demonstrated in cultured T cells using Annexin V [138].

 or cytotoxic T lymphocytes (CTLs) isolated from DS individuals were also compromised in their ability to kill target cells [139], indicating a functional defect in this cell type also. DSderived T cells also produce abnormal levels of cytokines, the small proteins produced by immune cells that are involved in signalling and controlling immune responses. IL-2 is central to the proliferation and differentiation of T cells and is produced by T lymphocytes once activated. Inhibition or reduction in IL-2 results in suppression of the immune system. One study on adults with DS found that the levels of IL-2 secreted from cultured stimulated T cells were significantly reduced compared with T cells cultured from normal individuals [140]. Other studies have suggested that IL-2 is produced at comparable levels in both DS and normal individuals, but in DS the response to IL-2 may be defective [141]. Levels of IFN-γ and TNF α are also altered in DS and although the number of DS studies is small, the consensus is that

In addition to T cell lymphopenia, DS individuals have marked B lymphopenia [143-145]. As well as a reduction in the number and proportions of B lymphocytes, there is a skewing of the B cell subpopulations, suggesting that maturation of B cells is defective in DS [146] akin to the situation with T cells, although the exact nature of this defect has not been explored. Immu‐ noglobulin levels in DS are also abnormal, with DS B lymphocytes producing lower levels of IgM, IgG2 and IgG4 and higher levels of IgG1 [146, 147]. IgG3 and IgA levels were unchanged. Also suggesting a B cell functional deficit is the finding that antibody responses to a variety of antigens are low in DS, including the responses to pneumococcal and bacteriophage ØX174

There is evidence to indicate that RCAN1 has a role in innate immunity and inflammation. For example, when human mononuclear cells were activated with *Candida albicans,* a pathogen capable of eliciting an innate immune response, RCAN1 gene expression was rapidly induced [151]. RCAN1 expression was also induced in response to various pro-inflammatory cytokines involved in the innate immune system such as TNFα [78]. Other studies have found that RCAN1 regulates inflammatory mediators and cytokines that have previously been identified as components of the innate immune system. For example, forced over-expression of RCAN1 in endothelial cells using adenoviral vectors resulted in a decrease in the expression of inflammatory markers such as E-selectin, VCAM1, TNF and COX-2 mRNA [78]. This sug‐

antigens and to vaccine antigens such as tetanus, influenza A and polio [148-150].

B cells expressed significantly higher

http://dx.doi.org/10.5772/52977

189

RCAN1 and Its Potential Contribution to the Down Syndrome Phenotype

T cells and CD19+

levels of early apoptotic markers compared with control cells [135].

cell subsets. For example, DS CD3+

IFN- γ and TNF α levels are increased [142] [134].

**7.4. RCAN1 in innate immunity**

CD8+

#### **7.2. Impairments in innate immunity**

Innate immunity is the body's first line of defence against invasion. This arm of the immune system either prevents the entry of pathogens into the body, or upon entry, eliminates them before they can cause any damage or disease. If a pathogen is able to gain entry into the body, innate immunity includes various non-specific mechanisms which can eliminate and destroy foreign invaders. These mechanisms include phagocytosis and inflammation. DS is associated with defects in the innate immune system. For example, natural killer (NK) cells, components of the innate immune system involved in the recognition and elimination of bacteria, viruses and tumour cells, are defective in DS individuals [129]. Also, neutrophils from DS people exhibited a decreased ability to phagocytose [130] and the ability of DS-derived neutrophils and monocytes to migrate towards a site of injury or infection in response to chemokine release was reduced [131].

#### **7.3. Impairments in adaptive immunity**

T cell development and maturation occurs within the thymus. Bone marrow (BM) derived precursor cells migrate into the thymus where they receive developmental cues from the thymic microenvironment. Here they progress through a number of different stages of development broadly defined by the expression of CD4 and CD8 on the cell surface. Once cells become fully mature, expressing only CD4 or CD8 on the surface, they are able to migrate to the periphery and populate the immune system. The DS immune system is characterised by a number of abnormalities thought to originate from defective T cell development in the thymus. Typically, the DS thymus is small and morphologically abnormal. It exhibits cortical atrophy, loss of cortico-medullary demarcation and lymphopenia due to a defect in the development of thymocytes [114]. The number of cells expressing high levels of the TCR α-β-CD3 complex is reduced [132] as are the numbers of helper (CD4+ ) T (Th) cells resulting in the inversion of the normal CD4+ /CD8+ ratio in favour of the CD8+ population. Th cells can be further subcategorised into either Th1 or Th2 cells where Th1 cells participate in the elimination of intra-vesicular pathogens, including bacteria and parasites via the activation of macro‐ phages, while Th2 cells clear extracellular pathogens and toxins by assisting antibody pro‐ duction in B cells. There is an imbalance in the T helper responses of DS individuals, although there is some disagreement as to whether it is an alteration in the Th1 or Th2 phenotype. Some studies have suggested that Th2 responses are augmented in DS based on the observation that there is an increased number of circulating CD3+ /CD30 Th2 lymphocytes [133]. Others report an increase in the Th1 population in DS and this has been attributed to increased IFNγ production [134] because IFNγ polarises Th0 cells towards the Th1 phenotype. While there is no doubt that a defect in T cell development and maturation within the DS thymus exists, altered apoptosis of lymphocytes may also contribute to the decrease in overall numbers of T cells in the periphery, as well as to the alterations observed in the abundance of the various T cell subsets. For example, DS CD3+ T cells and CD19+ B cells expressed significantly higher levels of early apoptotic markers compared with control cells [135].

T lymphocytes isolated from DS people are also functionally compromised. Under conditions designed to simulate an infection using anti-CD3 antibodies or the non-specific mitogen, phytohemagglutinin to activate T cells, DS lymphocytes were diminished in their proliferative capacity [136, 137]. Not only did the DS-derived T cells have a proliferative defect, they showed increased expression of apoptotic markers including APO-I/Fas (CD95) antigen, a T cell death marker, and increased apoptosis was demonstrated in cultured T cells using Annexin V [138]. CD8+ or cytotoxic T lymphocytes (CTLs) isolated from DS individuals were also compromised in their ability to kill target cells [139], indicating a functional defect in this cell type also. DSderived T cells also produce abnormal levels of cytokines, the small proteins produced by immune cells that are involved in signalling and controlling immune responses. IL-2 is central to the proliferation and differentiation of T cells and is produced by T lymphocytes once activated. Inhibition or reduction in IL-2 results in suppression of the immune system. One study on adults with DS found that the levels of IL-2 secreted from cultured stimulated T cells were significantly reduced compared with T cells cultured from normal individuals [140]. Other studies have suggested that IL-2 is produced at comparable levels in both DS and normal individuals, but in DS the response to IL-2 may be defective [141]. Levels of IFN-γ and TNF α are also altered in DS and although the number of DS studies is small, the consensus is that IFN- γ and TNF α levels are increased [142] [134].

In addition to T cell lymphopenia, DS individuals have marked B lymphopenia [143-145]. As well as a reduction in the number and proportions of B lymphocytes, there is a skewing of the B cell subpopulations, suggesting that maturation of B cells is defective in DS [146] akin to the situation with T cells, although the exact nature of this defect has not been explored. Immu‐ noglobulin levels in DS are also abnormal, with DS B lymphocytes producing lower levels of IgM, IgG2 and IgG4 and higher levels of IgG1 [146, 147]. IgG3 and IgA levels were unchanged. Also suggesting a B cell functional deficit is the finding that antibody responses to a variety of antigens are low in DS, including the responses to pneumococcal and bacteriophage ØX174 antigens and to vaccine antigens such as tetanus, influenza A and polio [148-150].

#### **7.4. RCAN1 in innate immunity**

autoimmune disorders such as autoimmune thyroid disease (AITD) (Hashimoto type), coeliac disease and diabetes [127] [128]. Thus, DS appears to include a combination of immunodefi‐ ciency and immune dysfunction. Although the precise cause of this immune dysfunction is unclear, the DS immune system is characterised by a number of abnormalities thought to

Innate immunity is the body's first line of defence against invasion. This arm of the immune system either prevents the entry of pathogens into the body, or upon entry, eliminates them before they can cause any damage or disease. If a pathogen is able to gain entry into the body, innate immunity includes various non-specific mechanisms which can eliminate and destroy foreign invaders. These mechanisms include phagocytosis and inflammation. DS is associated with defects in the innate immune system. For example, natural killer (NK) cells, components of the innate immune system involved in the recognition and elimination of bacteria, viruses and tumour cells, are defective in DS individuals [129]. Also, neutrophils from DS people exhibited a decreased ability to phagocytose [130] and the ability of DS-derived neutrophils and monocytes to migrate towards a site of injury or infection in response to chemokine release

T cell development and maturation occurs within the thymus. Bone marrow (BM) derived precursor cells migrate into the thymus where they receive developmental cues from the thymic microenvironment. Here they progress through a number of different stages of development broadly defined by the expression of CD4 and CD8 on the cell surface. Once cells become fully mature, expressing only CD4 or CD8 on the surface, they are able to migrate to the periphery and populate the immune system. The DS immune system is characterised by a number of abnormalities thought to originate from defective T cell development in the thymus. Typically, the DS thymus is small and morphologically abnormal. It exhibits cortical atrophy, loss of cortico-medullary demarcation and lymphopenia due to a defect in the development of thymocytes [114]. The number of cells expressing high levels of the TCR α-β-

ratio in favour of the CD8+

further subcategorised into either Th1 or Th2 cells where Th1 cells participate in the elimination of intra-vesicular pathogens, including bacteria and parasites via the activation of macro‐ phages, while Th2 cells clear extracellular pathogens and toxins by assisting antibody pro‐ duction in B cells. There is an imbalance in the T helper responses of DS individuals, although there is some disagreement as to whether it is an alteration in the Th1 or Th2 phenotype. Some studies have suggested that Th2 responses are augmented in DS based on the observation that

an increase in the Th1 population in DS and this has been attributed to increased IFNγ production [134] because IFNγ polarises Th0 cells towards the Th1 phenotype. While there is no doubt that a defect in T cell development and maturation within the DS thymus exists,

) T (Th) cells resulting in the

population. Th cells can be

/CD30 Th2 lymphocytes [133]. Others report

originate from defective innate and adaptive immunity.

**7.2. Impairments in innate immunity**

**7.3. Impairments in adaptive immunity**

inversion of the normal CD4+

CD3 complex is reduced [132] as are the numbers of helper (CD4+

/CD8+

there is an increased number of circulating CD3+

was reduced [131].

188 Down Syndrome

There is evidence to indicate that RCAN1 has a role in innate immunity and inflammation. For example, when human mononuclear cells were activated with *Candida albicans,* a pathogen capable of eliciting an innate immune response, RCAN1 gene expression was rapidly induced [151]. RCAN1 expression was also induced in response to various pro-inflammatory cytokines involved in the innate immune system such as TNFα [78]. Other studies have found that RCAN1 regulates inflammatory mediators and cytokines that have previously been identified as components of the innate immune system. For example, forced over-expression of RCAN1 in endothelial cells using adenoviral vectors resulted in a decrease in the expression of inflammatory markers such as E-selectin, VCAM1, TNF and COX-2 mRNA [78]. This sug‐ gested that increased expression of RCAN1 may dampen inflammation and inhibit induction of the innate immune system. Conversely, knockdown of RCAN1 using siRNA resulted in an increase in the expression of inflammatory mediators [78].

**7.5. RCAN1 in adaptive immunity**

, CD8+

, CD3+

bers of CD4+

preparation).

The first evidence to suggest that RCAN1 functions in adaptive immunity came from experi‐ ments investigating T cell responses in human Jurkat cells, an immortalised T lymphocyte cell line. When these cells were stimulated with the T cell mitogens, CD3 and CD28, expression of *RCAN1-4* mRNA was induced [26]. This result was confirmed by stimulating primary T cells cultured from humans [156]. A more definitive role for RCAN1 in the adaptive immune system came fromexaminingRCAN1-KOmice [44].While thesemicedisplayednormalTcelldevelop‐ ment and maturation with comparable numbers of mature thymocytes and equivalent num‐

the T cells were isolated from the spleen and cultured *ex vivo*, the RCAN1-KO cells were functionally defective. Specifically, these T cells exhibited a 50% reduction in proliferation in response to mitogenic stimulation as well as a decrease in the production of IFNγ. This loss of IFNγ indicated that the Th1 population was especially affected by the lack of *Rcan1* expres‐ sion. Indeed, these mice exhibited defective Th1 responses due to the premature death of this population of cells as a result of an up regulation of FasL and a loss of viability. Antibody class switching was also altered in RCAN1-KO mice, with a decrease in IgG2 production. Notably, theTcelldefectinRCAN1-KOmicecouldberescuedbytreatmentwiththecalcineurininhibitor, CsA, suggesting that the defect was calcineurin/NFAT-dependent and presumably due to hyperactivation of the calcineurin signal transduction pathway [44]. However, despite restora‐ tion of T cell function in RCAN1-KO mice following CsA treatment, genetic loss of calcineurin Aβ superimposed on the *Rcan1* deficiency by crossing RCAN1-KO mice with CnAβ knockout mice, could not rescue the T cell defects [64]. In fact, loss of calcineurin Aβ in addition to the loss of *Rcan1* resulted in an increase in the severity of the T cell defect. This observation suggests that in these mice RCAN1 is acting to facilitate calcineurin activity rather than inhibit it as the use of CsAtreatmenthadsuggested.OurgroupalsohasevidenceofRCAN1'sinvolvementinadaptive immunity; our RCAN1-TG mice have T and B cell defects (unpublished data and manuscript in

Inadditiontoits functioninTcells,RCAN1 is involvedinthenormalfunctionofmast cells.Mast cells are specialised immune cells that contain granules rich in histamine and heparin and are known to play a role in wound healing, defence against pathogens and the pathology of IgEdependent allergic disease and anaphylaxis [157]. Mast cells are activated through the high affinity IgE receptor (FcεRI) on their cell surface and this activation is controlled by a number of activating and inhibitory molecules. The down regulation of mast cell activity by inhibitory signals is essential in preventing allergic disease and anaphylaxis [157]. RCAN1 is believed to be one of these inhibitory signals. Evidence to suggest this comes from experiments conduct‐ ed on RCAN1-KO mice, which displayed an exaggerated mast cell response. While RCAN1- KO mice displayed normal mast cell maturation, many of the signalling pathways following mast cell activationwereperturbed.For example,mast cells isolatedfromRCAN1-KOmice and stimulated with FcεRI had an increase in the activation of both the NFAT and NFκB signalling pathways. As expected, there was also an increase in the expression of many pro-inflammato‐ ry genes regulated by these two pathways including *IL-6*, *IL-13* and *TNFα* [158]. Further, when mice lacking *Rcan1* were sensitised with an intravenous injection of anti-IgE antibody and then later treated with an agent designed to elicit an anaphylactic reaction, *Rcan1* deficiency led to

T cells in the periphery, these cells exhibited functional deficits. When

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191

Importantly, RCAN1 also mediates inflammatory responses *in vivo*. When mice were administered with lipopolysaccharide (LPS), a component of gram negative bacteria cell wall used experimentally to activate innate immune responses, *Rcan1* gene expression was induced [152]. Interestingly, RCAN1-KO mice had lower survival following LPS-induced endotoxae‐ mia compared with their WT littermates [152]. Knockout mice had an accentuated re‐ sponse to LPS treatment, including lower heart rate, blood pressure and body temperature. An increase in the concentration of circulating IL-6 protein, a pro-inflammatory cytokine believed to be detrimental during infection was also found, along with a significant increase in the mRNA expression of inflammatory mediators such as *E-selectin*, *ICAM1* and *VCAM1* in organs including the heart and lung. There was a concomitant increase in the number of infiltrating leukocytes within these organs [152]. On the other hand, over-expression of *RCAN1-4* achieved by the intravenous injection of mice with a *RCAN1-4*-containing adeno‐ virus, conferred a survival advantage upon LPS administration. A decrease in the levels of circulating IL-6 and an attenuation of the physiological responses to systemic LPS treat‐ ment were evident [152]. Induction of inflammatory mediators was also reduced and there was a marked reduction in leukocyte infiltrate in the heart, liver and lungs [152]. Another study found that following infection with the bacteria *Fransicella tularensis*, induction of proinflammatory cytokines including MCP1, IL6, IFNγ, and TNFα was significantly higher in *Rcan1*-deficient spleen and lung [153]. All this suggests that over-expression of RCAN1 is protective.

Other studies on the role of RCAN1 in innate immunity have focussed on identifying the mechanisms by which RCAN1 regulates inflammation. One plausible means is by modula‐ tion of the NFκB signal transduction pathway. As described earlier, RCAN1 is able to inhibit NFκB signalling by increasing the stability of IκB protein [51]. Given that NFκB is a transcription factor that controls the expression of pro-inflammatory genes and the subse‐ quent activation of innate immune cells, negative regulation of this pathway by RCAN1 would result in inhibition of inflammation. Such a proposition is consistent with published *in vitro* and *in vivo* data. However, another study investigating the potential involvement of RCAN1 in the Toll-like receptor (TLR) pathway arrived at the opposite conclusion [154]. The TLR pathway is activated as a first line defence mechanism during microbial infection and culminates in the induction of interleukins and other pro-inflammatory mediators [155]. When RCAN1-4 (DSCR1-1S) was exogenously expressed in HEK293 cells, the end result was activation of NFκB-mediated inflammatory responses [154], not suppression. Here, RCAN1 was found to regulate the TLR pathway through a direct interaction with the adaptor protein known as Toll-interacting protein (Tollip). The normal cellular role of Tollip is to suppress TLR signalling by sequestering IL-1 receptor associated kinase 1 (IRAK-1). Exogenously added RCAN1 bound Tollip, causing the release of IRAK-1 from the complex thereby removing the block on IRAK-1 activity [154]. The end result was an enhancement of the inflammatory response and thus represents yet another example of the sometimes contradic‐ tory actions of RCAN1.

#### **7.5. RCAN1 in adaptive immunity**

gested that increased expression of RCAN1 may dampen inflammation and inhibit induction of the innate immune system. Conversely, knockdown of RCAN1 using siRNA resulted in an

Importantly, RCAN1 also mediates inflammatory responses *in vivo*. When mice were administered with lipopolysaccharide (LPS), a component of gram negative bacteria cell wall used experimentally to activate innate immune responses, *Rcan1* gene expression was induced [152]. Interestingly, RCAN1-KO mice had lower survival following LPS-induced endotoxae‐ mia compared with their WT littermates [152]. Knockout mice had an accentuated re‐ sponse to LPS treatment, including lower heart rate, blood pressure and body temperature. An increase in the concentration of circulating IL-6 protein, a pro-inflammatory cytokine believed to be detrimental during infection was also found, along with a significant increase in the mRNA expression of inflammatory mediators such as *E-selectin*, *ICAM1* and *VCAM1* in organs including the heart and lung. There was a concomitant increase in the number of infiltrating leukocytes within these organs [152]. On the other hand, over-expression of *RCAN1-4* achieved by the intravenous injection of mice with a *RCAN1-4*-containing adeno‐ virus, conferred a survival advantage upon LPS administration. A decrease in the levels of circulating IL-6 and an attenuation of the physiological responses to systemic LPS treat‐ ment were evident [152]. Induction of inflammatory mediators was also reduced and there was a marked reduction in leukocyte infiltrate in the heart, liver and lungs [152]. Another study found that following infection with the bacteria *Fransicella tularensis*, induction of proinflammatory cytokines including MCP1, IL6, IFNγ, and TNFα was significantly higher in *Rcan1*-deficient spleen and lung [153]. All this suggests that over-expression of RCAN1 is

Other studies on the role of RCAN1 in innate immunity have focussed on identifying the mechanisms by which RCAN1 regulates inflammation. One plausible means is by modula‐ tion of the NFκB signal transduction pathway. As described earlier, RCAN1 is able to inhibit NFκB signalling by increasing the stability of IκB protein [51]. Given that NFκB is a transcription factor that controls the expression of pro-inflammatory genes and the subse‐ quent activation of innate immune cells, negative regulation of this pathway by RCAN1 would result in inhibition of inflammation. Such a proposition is consistent with published *in vitro* and *in vivo* data. However, another study investigating the potential involvement of RCAN1 in the Toll-like receptor (TLR) pathway arrived at the opposite conclusion [154]. The TLR pathway is activated as a first line defence mechanism during microbial infection and culminates in the induction of interleukins and other pro-inflammatory mediators [155]. When RCAN1-4 (DSCR1-1S) was exogenously expressed in HEK293 cells, the end result was activation of NFκB-mediated inflammatory responses [154], not suppression. Here, RCAN1 was found to regulate the TLR pathway through a direct interaction with the adaptor protein known as Toll-interacting protein (Tollip). The normal cellular role of Tollip is to suppress TLR signalling by sequestering IL-1 receptor associated kinase 1 (IRAK-1). Exogenously added RCAN1 bound Tollip, causing the release of IRAK-1 from the complex thereby removing the block on IRAK-1 activity [154]. The end result was an enhancement of the inflammatory response and thus represents yet another example of the sometimes contradic‐

increase in the expression of inflammatory mediators [78].

protective.

190 Down Syndrome

tory actions of RCAN1.

The first evidence to suggest that RCAN1 functions in adaptive immunity came from experi‐ ments investigating T cell responses in human Jurkat cells, an immortalised T lymphocyte cell line. When these cells were stimulated with the T cell mitogens, CD3 and CD28, expression of *RCAN1-4* mRNA was induced [26]. This result was confirmed by stimulating primary T cells cultured from humans [156]. A more definitive role for RCAN1 in the adaptive immune system came fromexaminingRCAN1-KOmice [44].While thesemicedisplayednormalTcelldevelop‐ ment and maturation with comparable numbers of mature thymocytes and equivalent num‐ bers of CD4+ , CD8+ , CD3+ T cells in the periphery, these cells exhibited functional deficits. When the T cells were isolated from the spleen and cultured *ex vivo*, the RCAN1-KO cells were functionally defective. Specifically, these T cells exhibited a 50% reduction in proliferation in response to mitogenic stimulation as well as a decrease in the production of IFNγ. This loss of IFNγ indicated that the Th1 population was especially affected by the lack of *Rcan1* expres‐ sion. Indeed, these mice exhibited defective Th1 responses due to the premature death of this population of cells as a result of an up regulation of FasL and a loss of viability. Antibody class switching was also altered in RCAN1-KO mice, with a decrease in IgG2 production. Notably, theTcelldefectinRCAN1-KOmicecouldberescuedbytreatmentwiththecalcineurininhibitor, CsA, suggesting that the defect was calcineurin/NFAT-dependent and presumably due to hyperactivation of the calcineurin signal transduction pathway [44]. However, despite restora‐ tion of T cell function in RCAN1-KO mice following CsA treatment, genetic loss of calcineurin Aβ superimposed on the *Rcan1* deficiency by crossing RCAN1-KO mice with CnAβ knockout mice, could not rescue the T cell defects [64]. In fact, loss of calcineurin Aβ in addition to the loss of *Rcan1* resulted in an increase in the severity of the T cell defect. This observation suggests that in these mice RCAN1 is acting to facilitate calcineurin activity rather than inhibit it as the use of CsAtreatmenthadsuggested.OurgroupalsohasevidenceofRCAN1'sinvolvementinadaptive immunity; our RCAN1-TG mice have T and B cell defects (unpublished data and manuscript in preparation).

Inadditiontoits functioninTcells,RCAN1 is involvedinthenormalfunctionofmast cells.Mast cells are specialised immune cells that contain granules rich in histamine and heparin and are known to play a role in wound healing, defence against pathogens and the pathology of IgEdependent allergic disease and anaphylaxis [157]. Mast cells are activated through the high affinity IgE receptor (FcεRI) on their cell surface and this activation is controlled by a number of activating and inhibitory molecules. The down regulation of mast cell activity by inhibitory signals is essential in preventing allergic disease and anaphylaxis [157]. RCAN1 is believed to be one of these inhibitory signals. Evidence to suggest this comes from experiments conduct‐ ed on RCAN1-KO mice, which displayed an exaggerated mast cell response. While RCAN1- KO mice displayed normal mast cell maturation, many of the signalling pathways following mast cell activationwereperturbed.For example,mast cells isolatedfromRCAN1-KOmice and stimulated with FcεRI had an increase in the activation of both the NFAT and NFκB signalling pathways. As expected, there was also an increase in the expression of many pro-inflammato‐ ry genes regulated by these two pathways including *IL-6*, *IL-13* and *TNFα* [158]. Further, when mice lacking *Rcan1* were sensitised with an intravenous injection of anti-IgE antibody and then later treated with an agent designed to elicit an anaphylactic reaction, *Rcan1* deficiency led to enhanced mast cell activation, degranulation and passive cutaneous anaphylaxis [158]. These results indicate that RCAN1 may be an inhibitor molecule that negatively controls mast cell function.

some protection. Firstly, a number of cancers display abnormal expression of RCAN1 and this expression varies depending on the stage of the cancer. For example, studies have shown that RCAN1 is up regulated in most primary papillary thyroid tumours but this expression is lost in the metastatic tissue of thyroid tumours [169]. This is interesting given that RCAN1 has been identified as a target gene for metastatin, a protein that functions to suppress metastatic tumour growth. It is possible that loss of metastatin in tumour cells leads to a loss of RCAN1 expression which may in turn contribute to tumour metastasis [169]. RCAN1 has also been linked to other cancers including colorectal cancer. Peroxisome proliferator-activated receptor γ (PPARγ) is a member the nuclear hormone receptor family of transcription factors and has been identified as a tumour suppressor gene in colon cancer. This gene is important in a number of cellular processes including inflammation, proliferation, apoptosis as well as adipocyte and intestinal epithelial cell differentiation and has been shown to suppress experimental colon carcinogen‐ esis in mice (reviewed in [170]). Loss of *RCAN1-4* in *MOSER colon carcinoma cells* resulted in an inhibition of PPARγ-mediated tumour suppression and increased tissue invasion [171]. While not conclusive, these results indicate that RCAN1 may be required for PPARγ suppres‐ sion of colorectal cancers [171]. Again this is consistent with the idea that RCAN1 can act as a

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The strongest genetic evidence to suggest a role for *RCAN1* in tumourigenesis comes from experiments conducted on RCAN1-KO and RCAN1-TG mice. When RCAN1-KO mice were injected subcutaneously with renal carcinoma or colon carcinoma tumour cells, there was a significant suppression of tumour growth [172]. Tumour growth was suppressed due to an inability to form and maintain tumour vasculature within the solid tumours. Further investigation showed that RCAN1-KO mice had hyperactive VEGF-calcineurin-NFAT signalling, which resulted in a suppression of endothelial cell proliferation and an increase in apoptosis [172]. Tumour growth in the RCAN1-KO mice could be restored following treatment with CsA, suggesting that suppression of tumour cell growth in RCAN1-KO mice was dependent on hyperactive calcineurin signalling. Perhaps counterintuitively, but similar to the situation with the RCAN1-KO, mice over-expressing *RCAN1-4* were also resistant to tumour growth when injected subcutaneously with Lewis lung or B16F10 tumour cells [173]. Tumours isolated from these mice also displayed a decrease in the density of microvessels and the vessels lacked a functional lumen. Moreover, it appeared that *RCAN1-4* mediated tumour growth through the calcineurin pathway as *RCAN1-4* transgenic tumour cells had a decrease in both calcineurin and NFAT activity [173]. The exact mechanisms by which RCAN1 suppresses solid tumour growth remain unknown, but both studies strongly suggest that regulation of angiogenesis by RCAN1 underpins the inhibition of tumour growth by reducing the formation of blood vessels throughout the tumour. It is interesting to note that RCAN1- KO and RCAN1-TG mice displayed a similar phenotype, with both exhibiting a decrease in tumour formation due to an inhibition of angiogenesis preventing the formation of microves‐ sels required to support tumour growth. Perhaps more intriguing is that opposite effects on the calcineurin pathway produced the same end result. Also intriguing is that microvessel formation was also decreased in teratomas generated from human DS-derived pluripotent stem cells transplanted into WT mice, indicating that decreased angiogenesis may be

tumour suppressor.

responsible for tumour suppression in DS [173].

Eosinophils, another immune cell type, are predominant effector cells in allergic asthma and their presence in the lungs of asthma sufferers is regarded as a defining feature of this inflammatory disease. Absence of *Rcan1* was shown to prevent experimentally-induced allergic asthma in a mouse model due to an almost complete absence of eosinophils infiltrating the lungs [159]. Although the exact mechanism for this protection is not fully understood, it seems that a lack of *Rcan1* blocks the development and migration of eosinophil progenitors from the bone marrow and selectively lowers their production of the inflammatory mediator IL-4. This study implies that over-expression of RCAN1 would exacerbate the allergic response and in this regard it is interesting to note that a recent study reported an increased incidence of allergic asthma in people with DS [160]. Therefore, it would be very informative to test allergic asthma responses in RCAN1-TG mice.

## **8. The consequences of RCAN1 over–expression on the incidence of solid tumours in DS**

#### **8.1. Down syndrome and cancer**

Individuals with DS are more likely to develop certain malignancies, especially of the immune system. There is a well-established link between leukaemia and DS, with an increased inci‐ dence in DS compared with the general population. Large population based studies conduct‐ ed in different countries around the world have consistently found that the rates of leukaemia were between 10- to 19-fold higher in people with DS in comparison with the average popula‐ tion and there was an increased incidence of both lymphoid and myeloid leukaemias [140, 161-163]. While the incidence of both acute myeloid leukaemia (AML) and acute lymphatic leukaemia (ALL) was significantly higher in DS subjects than expected in the general popula‐ tion, there were significantly more cases of AML compared to ALL in DS [163]. This increased riskismostevidentatayoungerage,howeverremainedthroughoutlife.Thereisalsoasignificant increase inthe incidenceofneoplasticdisorders suchasmegakaryoblastic leukaemia,where the incidence is increased about 500-fold in DS [164, 165]. In males, there is also a link between DS and testicular cancer, possibly due to higher levels of follicular stimulating hormone, hypogo‐ nadismor cryptorchidism[166,167].Notably,thosewithDSare less likelytodevelopother solid tumours such as neuroblastomas and breast and lung cancers [162, 163, 168]. Indeed, DS individuals had a 50% reduction in the incidence of solid tumours compared to the number of cases expected in the general population and this was observed over all age groups examined [162]. Thus it seems likely that a number of tumour suppressor genes reside on *Hsa21*.

#### **8.2. RCAN1 and tumourigenesis**

While the identities of the *Hsa21* genes responsible for the reduction in solid tumour formation in DS remain unknown, there is evidence to suggest that up regulation of RCAN1 may afford some protection. Firstly, a number of cancers display abnormal expression of RCAN1 and this expression varies depending on the stage of the cancer. For example, studies have shown that RCAN1 is up regulated in most primary papillary thyroid tumours but this expression is lost in the metastatic tissue of thyroid tumours [169]. This is interesting given that RCAN1 has been identified as a target gene for metastatin, a protein that functions to suppress metastatic tumour growth. It is possible that loss of metastatin in tumour cells leads to a loss of RCAN1 expression which may in turn contribute to tumour metastasis [169]. RCAN1 has also been linked to other cancers including colorectal cancer. Peroxisome proliferator-activated receptor γ (PPARγ) is a member the nuclear hormone receptor family of transcription factors and has been identified as a tumour suppressor gene in colon cancer. This gene is important in a number of cellular processes including inflammation, proliferation, apoptosis as well as adipocyte and intestinal epithelial cell differentiation and has been shown to suppress experimental colon carcinogen‐ esis in mice (reviewed in [170]). Loss of *RCAN1-4* in *MOSER colon carcinoma cells* resulted in an inhibition of PPARγ-mediated tumour suppression and increased tissue invasion [171]. While not conclusive, these results indicate that RCAN1 may be required for PPARγ suppres‐ sion of colorectal cancers [171]. Again this is consistent with the idea that RCAN1 can act as a tumour suppressor.

enhanced mast cell activation, degranulation and passive cutaneous anaphylaxis [158]. These results indicate that RCAN1 may be an inhibitor molecule that negatively controls mast cell

Eosinophils, another immune cell type, are predominant effector cells in allergic asthma and their presence in the lungs of asthma sufferers is regarded as a defining feature of this inflammatory disease. Absence of *Rcan1* was shown to prevent experimentally-induced allergic asthma in a mouse model due to an almost complete absence of eosinophils infiltrating the lungs [159]. Although the exact mechanism for this protection is not fully understood, it seems that a lack of *Rcan1* blocks the development and migration of eosinophil progenitors from the bone marrow and selectively lowers their production of the inflammatory mediator IL-4. This study implies that over-expression of RCAN1 would exacerbate the allergic response and in this regard it is interesting to note that a recent study reported an increased incidence of allergic asthma in people with DS [160]. Therefore, it would be very informative to test

**8. The consequences of RCAN1 over–expression on the incidence of solid**

Individuals with DS are more likely to develop certain malignancies, especially of the immune system. There is a well-established link between leukaemia and DS, with an increased inci‐ dence in DS compared with the general population. Large population based studies conduct‐ ed in different countries around the world have consistently found that the rates of leukaemia were between 10- to 19-fold higher in people with DS in comparison with the average popula‐ tion and there was an increased incidence of both lymphoid and myeloid leukaemias [140, 161-163]. While the incidence of both acute myeloid leukaemia (AML) and acute lymphatic leukaemia (ALL) was significantly higher in DS subjects than expected in the general popula‐ tion, there were significantly more cases of AML compared to ALL in DS [163]. This increased riskismostevidentatayoungerage,howeverremainedthroughoutlife.Thereisalsoasignificant increase inthe incidenceofneoplasticdisorders suchasmegakaryoblastic leukaemia,where the incidence is increased about 500-fold in DS [164, 165]. In males, there is also a link between DS and testicular cancer, possibly due to higher levels of follicular stimulating hormone, hypogo‐ nadismor cryptorchidism[166,167].Notably,thosewithDSare less likelytodevelopother solid tumours such as neuroblastomas and breast and lung cancers [162, 163, 168]. Indeed, DS individuals had a 50% reduction in the incidence of solid tumours compared to the number of cases expected in the general population and this was observed over all age groups examined

[162]. Thus it seems likely that a number of tumour suppressor genes reside on *Hsa21*.

While the identities of the *Hsa21* genes responsible for the reduction in solid tumour formation in DS remain unknown, there is evidence to suggest that up regulation of RCAN1 may afford

function.

192 Down Syndrome

**tumours in DS**

**8.1. Down syndrome and cancer**

**8.2. RCAN1 and tumourigenesis**

allergic asthma responses in RCAN1-TG mice.

The strongest genetic evidence to suggest a role for *RCAN1* in tumourigenesis comes from experiments conducted on RCAN1-KO and RCAN1-TG mice. When RCAN1-KO mice were injected subcutaneously with renal carcinoma or colon carcinoma tumour cells, there was a significant suppression of tumour growth [172]. Tumour growth was suppressed due to an inability to form and maintain tumour vasculature within the solid tumours. Further investigation showed that RCAN1-KO mice had hyperactive VEGF-calcineurin-NFAT signalling, which resulted in a suppression of endothelial cell proliferation and an increase in apoptosis [172]. Tumour growth in the RCAN1-KO mice could be restored following treatment with CsA, suggesting that suppression of tumour cell growth in RCAN1-KO mice was dependent on hyperactive calcineurin signalling. Perhaps counterintuitively, but similar to the situation with the RCAN1-KO, mice over-expressing *RCAN1-4* were also resistant to tumour growth when injected subcutaneously with Lewis lung or B16F10 tumour cells [173]. Tumours isolated from these mice also displayed a decrease in the density of microvessels and the vessels lacked a functional lumen. Moreover, it appeared that *RCAN1-4* mediated tumour growth through the calcineurin pathway as *RCAN1-4* transgenic tumour cells had a decrease in both calcineurin and NFAT activity [173]. The exact mechanisms by which RCAN1 suppresses solid tumour growth remain unknown, but both studies strongly suggest that regulation of angiogenesis by RCAN1 underpins the inhibition of tumour growth by reducing the formation of blood vessels throughout the tumour. It is interesting to note that RCAN1- KO and RCAN1-TG mice displayed a similar phenotype, with both exhibiting a decrease in tumour formation due to an inhibition of angiogenesis preventing the formation of microves‐ sels required to support tumour growth. Perhaps more intriguing is that opposite effects on the calcineurin pathway produced the same end result. Also intriguing is that microvessel formation was also decreased in teratomas generated from human DS-derived pluripotent stem cells transplanted into WT mice, indicating that decreased angiogenesis may be responsible for tumour suppression in DS [173].

Finally, the significance of RCAN1 in tumour suppression in DS was elegantly demonstrated using yet another DS genetic model. TS65Dn mice that harbour a third copy of many *Hsa21* orthologous genes, including *Rcan1*, were bred with RCAN1-KO mice, thereby returning the gene dosage of *Rcan1* to normal. When tumour cells were injected into these mice, there was a significant increase in the formation of microvessels within solid tumours compared with their TS65Dn littermates expressing 3 copies of *Rcan1* [173]. This is more evidence to support the idea that elevated levels of *RCAN1* are responsible, at least in part, for the decrease in the incidence of solid tumour formation in DS.

**9. Conclusion**

**Author details**

tralia

**References**

its activity to ameliorate/treat pathology.

Melanie A. Pritchard and Katherine R. Martin

diatr, (1959). , 962-963.

Rev, (2007). , 221-227.

Behav, (2003). , 156-166.

*mice.* Eur J Neurosci, (1998). , 538-544.

(1997). , 28-36.

In this review we have attempted to summarise what is currently known about the function of the *RCAN1* gene and its pleiotropic actions in three areas of relevance to DS (see Figure 2). No matter which system you look at, the reports on RCAN1 function are often contradictory – we still have much to learn. Researchers with a passionate interest in DS and its molecular genetic aetiology have suggested that specific down regulation of a few of the genes produced in excess in DS tissues may provide an avenue for therapies. We and others have suggested that inhibition of RCAN1 signalling may have pharmacological potential for reducing neuronal loss and treating cognitive decline in DS and AD, but we still have much to learn about the molecular function and physiological role of RCAN1 and how we can manipulate

RCAN1 and Its Potential Contribution to the Down Syndrome Phenotype

http://dx.doi.org/10.5772/52977

195

Department Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Aus‐

[1] Lejeune, J, Turpin, R, & Gautier, M. *Chromosomic diagnosis of mongolism].* Arch Fr Pe‐

[2] Sherman, S. L, et al. *Epidemiology of Down syndrome.* Ment Retard Dev Disabil Res

[3] Reeves, R, Baxter, L, & Richtsmeier, J. *Too much of a good thing: mechanisms of gene ac‐*

[4] Nadel, L. *Down's syndrome: a genetic disorder in biobehavioral perspective.* Genes Brain

[5] Smith, D. J, et al. *Functional screening of 2 Mb of human chromosome 21q22.2 in transgen‐ ic mice implicates minibrain in learning defects associated with Down syndrome.* Nat Genet,

[6] Gahtan, E, et al. *Reversible impairment of long-term potentiation in transgenic Cu/Zn-SOD*

*tion in Down syndrome.* TRENDS in Genetics, (2001). , 83-88.

**Figure 2. Summary of the positive and negative effects of excess RCAN1.** Effects on the brain, immune system and solid tumour formation in Down syndrome are shown. The putative contributions of an over abundance of RCAN1 have either been demonstrated in mouse models or in cell lines or implied from *Rcan1*-KO studies where, in the absence of data to the contrary, the assumption is that over-expression will produce the opposite effect to the deficiency. Detrimental effects are shown in blue and protective effects in yellow.

## **9. Conclusion**

Finally, the significance of RCAN1 in tumour suppression in DS was elegantly demonstrated using yet another DS genetic model. TS65Dn mice that harbour a third copy of many *Hsa21* orthologous genes, including *Rcan1*, were bred with RCAN1-KO mice, thereby returning the gene dosage of *Rcan1* to normal. When tumour cells were injected into these mice, there was a significant increase in the formation of microvessels within solid tumours compared with their TS65Dn littermates expressing 3 copies of *Rcan1* [173]. This is more evidence to support the idea that elevated levels of *RCAN1* are responsible, at least in part, for the decrease in the

**Figure 2. Summary of the positive and negative effects of excess RCAN1.** Effects on the brain, immune system and solid tumour formation in Down syndrome are shown. The putative contributions of an over abundance of RCAN1 have either been demonstrated in mouse models or in cell lines or implied from *Rcan1*-KO studies where, in the absence of data to the contrary, the assumption is that over-expression will produce the opposite effect to the

deficiency. Detrimental effects are shown in blue and protective effects in yellow.

incidence of solid tumour formation in DS.

194 Down Syndrome

In this review we have attempted to summarise what is currently known about the function of the *RCAN1* gene and its pleiotropic actions in three areas of relevance to DS (see Figure 2). No matter which system you look at, the reports on RCAN1 function are often contradictory – we still have much to learn. Researchers with a passionate interest in DS and its molecular genetic aetiology have suggested that specific down regulation of a few of the genes produced in excess in DS tissues may provide an avenue for therapies. We and others have suggested that inhibition of RCAN1 signalling may have pharmacological potential for reducing neuronal loss and treating cognitive decline in DS and AD, but we still have much to learn about the molecular function and physiological role of RCAN1 and how we can manipulate its activity to ameliorate/treat pathology.

## **Author details**

Melanie A. Pritchard and Katherine R. Martin

Department Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Aus‐ tralia

## **References**


[7] Cataldo, A. M, et al. *App gene dosage modulates endosomal abnormalities of Alzheimer's disease in a segmental trisomy 16 mouse model of down syndrome.* J Neurosci, (2003). , 6788-6792.

[21] Ermak, G, Morgan, T. E, & Davies, K. J. *Chronic overexpression of the calcineurin inhibi‐ tory gene DSCR1 (Adapt78) is associated with Alzheimer's disease.* J Biol Chem, (2001). ,

RCAN1 and Its Potential Contribution to the Down Syndrome Phenotype

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197

[22] Fuentes, J. J, Pritchard, M. A, & Estivill, X. *Genomic organization, alternative splicing, and expression patterns of the DSCR1 (Down syndrome candidate region 1) gene.* Genom‐

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[133] Bertotto, A, et al. *CD3+/CD30+ circulating T lymphocytes are markedly increased in older subjects with Down's syndrome (Trisomy 21).* Pathobiology, (1999). , 108-110.

[134] Franciotta, D, et al. *Interferon-gamma- and interleukin-4-producing T cells in Down's syn‐*

[135] Elsayed, S. M, & Elsayed, G. M. *Phenotype of apoptotic lymphocytes in children with*

[136] Bertotto, A, et al. *T cell response to anti-CD3 antibody in Down's syndrome.* Arch Dis

[137] Burgio, G. R, et al. *Derangements of immunoglobulin levels, phytohemagglutinin respon‐ siveness and T and B cell markers in Down's syndrome at different ages.* Eur J Immunol,

[138] Corsi, M. M, et al. *Proapoptotic activated T-cells in the blood of children with Down's syn‐ drome: relationship with dietary antigens and intestinal alterations.* Int J Tissue React,

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[140] Park, E, et al. *Partial impairment of immune functions in peripheral blood leukocytes from*

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[145] Spina, C. A, et al. *Altered cellular immune functions in patients with Down's syndrome.*

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453-467.

204 Down Syndrome

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**Section 4**

**Neural Development in Down Syndrome**


**Neural Development in Down Syndrome**

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[164] Zipursky, A. *Transient leukaemia--a benign form of leukaemia in newborn infants with tris‐*

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[170] Zou, B, Qiao, L, & Wong, B. C. *Current Understanding of the Role of PPARgamma in*

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1019-1025.

206 Down Syndrome

**Chapter 11**

**Laterality Explored:**

http://dx.doi.org/10.5772/53829

**1. Introduction**

spheric dominance in DS.

**Atypical Hemispheric Dominance in Down Syndrome**

Down syndrome (DS) is a genetic disorder caused by an extra copy of chromosome 21 (triso‐ my 21), with an incidence in 1 in 700 live births. The third chromosome causes a series of physical, biological and behavioural characteristics that are syndrome-specific including in‐ tellectual disability, heart defects, problems in the endocrine and immune system and other medical conditions (Epstein et al., 1991). Moreover, there is established evidence for the lan‐ guage difficulties in people with DS particularly in expressive vocabulary and grammar. Re‐ search on language has documented a specific pattern of cerebral lateralization that commonly characterizes these individuals, that is unique to the syndrome compared to typi‐ cally developing individuals and individuals with intellectual disability (ID) non-DS. This realization has triggered the interest of neuropsychologists to investigate atypical hemi‐

Atypical hemispheric Dominance, or otherwise termed "anomalous dominance" or "anoma‐ lous cerebral organization", refers to the atypical lateralization of language areas within the brain (Geschwind & Galaburda, 1985). Usually, most right-handed individuals (97%) exhibit left-hemisphere lateralization for language. The remaining 3% of right-handed individuals exhibit bilateral or right hemisphere lateralization for language (Bishop, 1990). In left-hand‐ ed individuals this distribution is very different. About 60% of left-handed individuals ex‐ hibit left-hemisphere lateralization for language, 30% bilateral lateralization and 10% righthemisphere lateralization for language (Bishop, 1990). Geschwind and Behan (1982) termed anomalous dominance that in which the pattern of language laterality differed from the "… standard dominance pattern" (pp. 70). Bryden, McManus and Bulman-Fleming (1994) criti‐ cized this definition, highlighting that if one accepts this description "… we run the risk of defining the majority of the population as being anomalous" (pp. 111). According to Gesch‐

> © 2013 Grouios et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

George Grouios, Antonia Ypsilanti and Irene Koidou

Additional information is available at the end of the chapter

## **Laterality Explored: Atypical Hemispheric Dominance in Down Syndrome**

George Grouios, Antonia Ypsilanti and Irene Koidou

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53829

## **1. Introduction**

Down syndrome (DS) is a genetic disorder caused by an extra copy of chromosome 21 (triso‐ my 21), with an incidence in 1 in 700 live births. The third chromosome causes a series of physical, biological and behavioural characteristics that are syndrome-specific including in‐ tellectual disability, heart defects, problems in the endocrine and immune system and other medical conditions (Epstein et al., 1991). Moreover, there is established evidence for the lan‐ guage difficulties in people with DS particularly in expressive vocabulary and grammar. Re‐ search on language has documented a specific pattern of cerebral lateralization that commonly characterizes these individuals, that is unique to the syndrome compared to typi‐ cally developing individuals and individuals with intellectual disability (ID) non-DS. This realization has triggered the interest of neuropsychologists to investigate atypical hemi‐ spheric dominance in DS.

Atypical hemispheric Dominance, or otherwise termed "anomalous dominance" or "anoma‐ lous cerebral organization", refers to the atypical lateralization of language areas within the brain (Geschwind & Galaburda, 1985). Usually, most right-handed individuals (97%) exhibit left-hemisphere lateralization for language. The remaining 3% of right-handed individuals exhibit bilateral or right hemisphere lateralization for language (Bishop, 1990). In left-hand‐ ed individuals this distribution is very different. About 60% of left-handed individuals ex‐ hibit left-hemisphere lateralization for language, 30% bilateral lateralization and 10% righthemisphere lateralization for language (Bishop, 1990). Geschwind and Behan (1982) termed anomalous dominance that in which the pattern of language laterality differed from the "… standard dominance pattern" (pp. 70). Bryden, McManus and Bulman-Fleming (1994) criti‐ cized this definition, highlighting that if one accepts this description "… we run the risk of defining the majority of the population as being anomalous" (pp. 111). According to Gesch‐

© 2013 Grouios et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

wind and Galaburda (1985a; 1985b), atypical dominance may involve the inverse or weak dominance of three features; hand dominance, language dominance and visuospatial domi‐ nance. Previc (1994) distinguished the term atypical laterality into anatomical atypical asym‐ metry, which involves the decreased volume of the left hemisphere compared to the right hemisphere, particularly in the temporal region, and is observed in approximately 30-35% of the normal population, and functional atypical asymmetry, which relates to the bilateral or right hemisphere language dominance.

A scientific procedure is strictly defined as non-invasive when no break in the skin is created and there is no contact with the mucosa, or skin break, or internal body cavity, beyond a natural or artificial body orifice. Νon-invasive techniques for the assessment of cerebral lateralization can be further subdivided into neuroimaging techniques and be‐

Laterality Explored: Atypical Hemispheric Dominance in Down Syndrome

http://dx.doi.org/10.5772/53829

211

Neuroimaging techniques include both anatomical techniques, which create "constructed" images of brain structure, and functional techniques, which generate a series of dynamic brain images reflecting ongoing brain activity (Ganou, Kollias, Koidou, & Grouios, 2012). The anatomical techniques, which are the classical methods to image the brain, comprise computed tomography and structural magnetic resonance imaging. The functional techni‐ ques contain both direct (electroencephalography and magnetoencephalography) and indi‐ rect (positron-emissiontomography, single photon emission computed tomography and functional magnetic resonance imaging) measures of neural activity, which basically meas‐ ure haemodynamic responses or differences in metabolic concentrations to cognitive stimu‐

lation (for more information see Cohen & Sweet, 2011; Hüsing, Jäncke, & Tag, 2006).

Neuroimaging have offered a broad range of investigative tools to basic (e.g., Aziz-Zadeh, Koski, Zaidel, Mazziotta, & Iacoboni, 2006; Jansen, Menke, Sommer, Forster, Bruchmann et al., 2006; Tomasi & Volkow, 2012) and clinical (e.g., Desmond, Sum, Wagner, Demb, Shear et al., 1995; khondi-Asi, Jafari-Khouzani, Elisevich, & Soltanian-Zadeh, 2011; Oertel, Knöchel, Rotarska-Jagiela, Schönmeyer, Lindner et al., 2010) laterality research that fulfill the popular fantasy of being able to ''read the mind,'' albeit in the form of ''seeing the brain'' both struc‐

Over the past 20 years, evidence for atypical cerebral lateralization in individuals with DS has been adduced using various neuroimaging techniques (Azari, Horwitz, Pettigrew, Gra‐ dy, Haxby, et al., 1994; Menghini, Costanzo, & Vicari, 2011; Pinter, Eliez, Schmitt, Capone, & Reiss, 2001). However, despite the large and growing literature describing patterns of brain structure and function in the healthy and diseased human brain, scientific research on Down syndrome has not been well integrated into the mainstream of human neuroimaging re‐ search. Nevertheless, a few investigators have demonstrated success in applying digital

For example, Uecker, Mangan, Obrzut and Nadel (1993) argued that diffuse language later‐ alization in individuals with DS is likely to be a contributor to their poor visuospatial per‐ formance. Frangou, Aylward, Warren, Sharma, Barta et al. (1997) investigated whether the anatomic substrate for language are abnormal in DS. They examined volumetric Magnetic Resonance Imaging (MRI) measures of the superior temporal gyrus and the planum tempo‐ rale for community-dwelling individuals with DS and matched healthy comparison sub‐ jects. It was found that brain abnormalities in DS were not uniform. Specifically, the planum temporale volume of the individuals with DS was smaller than that of the healthy subjects. The volume of the superior temporal gyrus in the DS individuals was proportionally similar to that of the comparison group. For the subjects with DS, neither superior temporal gyrus nor planum temporale volume was significantly correlated with performance on language tests. Losin, Rivera, O'Hare, Sowell, and Pinter (2009) compared functional Magnetic Reso‐

havioural techniques.

turally and functionally (Kerr & Denk, 2008).

imaging technology in individuals with DS.

During the past decades atypical laterality has been studied in a number of pathological conditions, including individuals with intellectual disability (ID) (e.g., Grouios, Sakadami, Poderi, & Alevriadou, 1999), DS (e.g., Heath & Elliott, 1999), autism (Cornish & McManus, 1996), Turner syndrome (Ganou & Grouios, 2008), Klinefelter syndrome (Ganou, Grouios, koidou, & Alevriadou, 2010), Williams syndrome (Järvinen-Pasley, Pollak, Yam, Hill, Gri‐ chanik et al., 2010), fragile-X syndrome (Cornish, Pigram, & Shaw, 1997), developmental stuttering (Foundas, Corey, Angeles, Bollich, Crabtree-Hartman et al., 2003), developmental dyslexia (Illingworth & Bishop, 2009), disabled reading (Dalby & Gibson, 1981), attentiondeficit/hyperactivity disorder (Hale, Zaidel, McGough, Phillips, & McCracken, 2006), de‐ pression (Pinea, Kentgena, Bruderb, Leiteb, Bearmana et al., 2000), schizophrenia (Giotakos, 1999) and epilepsy (Slezicki, Cho, Brock, Pfeiffer, McVearry et al., 2009). The aim of the present review is to present and discuss research on atypical cerebral laterality in DS.

## **2. Laterality measures**

There are several techniques with which one can assess the laterality of cognitive functions. A broad division of these techniques is that between invasive and non-invasive laterality measures.

An invasive technique is one, which penetrates or breaks the skin or enters a body cavity. The only available invasive technique for the assessment of lateralization of cognitive func‐ tions is the intracarotid amobarbital procedure (IAP) or Wada test. The IAP is a procedure first described by Wada (1949) and Wada and Rasmussen (1960) for anaesthetizing cerebral hemispheres for the purpose of lateralizing language and memory functions. The procedure consists of unilateral injection of sodium amobarbital into the internal carotid, which tempo‐ rarily anaesthetizes the hemisphere ipsilateral to the injection site. While one hemisphere is anaesthetized, language and memory functions of the hemisphere contralateral to the injec‐ tion site can be tested. After the effect of the anaesthesia has dissipated, the process is re‐ peated with the other hemisphere. Determining the lateralization of language and memory functions is of both theoretical and practical interest, establishing cerebral language laterali‐ zation, predicting patients who are at risk for developing a post-surgical amnestic syndrome and identifying lateralized dysfunction to help confirm seizure onset laterality (Loring & Meador, 2000). Scientific investigation of cerebral lateralization in individuals with ID using the IAP is generally hampered for obvious moral and ethical reasons.

A scientific procedure is strictly defined as non-invasive when no break in the skin is created and there is no contact with the mucosa, or skin break, or internal body cavity, beyond a natural or artificial body orifice. Νon-invasive techniques for the assessment of cerebral lateralization can be further subdivided into neuroimaging techniques and be‐ havioural techniques.

wind and Galaburda (1985a; 1985b), atypical dominance may involve the inverse or weak dominance of three features; hand dominance, language dominance and visuospatial domi‐ nance. Previc (1994) distinguished the term atypical laterality into anatomical atypical asym‐ metry, which involves the decreased volume of the left hemisphere compared to the right hemisphere, particularly in the temporal region, and is observed in approximately 30-35% of the normal population, and functional atypical asymmetry, which relates to the bilateral or

During the past decades atypical laterality has been studied in a number of pathological conditions, including individuals with intellectual disability (ID) (e.g., Grouios, Sakadami, Poderi, & Alevriadou, 1999), DS (e.g., Heath & Elliott, 1999), autism (Cornish & McManus, 1996), Turner syndrome (Ganou & Grouios, 2008), Klinefelter syndrome (Ganou, Grouios, koidou, & Alevriadou, 2010), Williams syndrome (Järvinen-Pasley, Pollak, Yam, Hill, Gri‐ chanik et al., 2010), fragile-X syndrome (Cornish, Pigram, & Shaw, 1997), developmental stuttering (Foundas, Corey, Angeles, Bollich, Crabtree-Hartman et al., 2003), developmental dyslexia (Illingworth & Bishop, 2009), disabled reading (Dalby & Gibson, 1981), attentiondeficit/hyperactivity disorder (Hale, Zaidel, McGough, Phillips, & McCracken, 2006), de‐ pression (Pinea, Kentgena, Bruderb, Leiteb, Bearmana et al., 2000), schizophrenia (Giotakos, 1999) and epilepsy (Slezicki, Cho, Brock, Pfeiffer, McVearry et al., 2009). The aim of the

present review is to present and discuss research on atypical cerebral laterality in DS.

There are several techniques with which one can assess the laterality of cognitive functions. A broad division of these techniques is that between invasive and non-invasive laterality

An invasive technique is one, which penetrates or breaks the skin or enters a body cavity. The only available invasive technique for the assessment of lateralization of cognitive func‐ tions is the intracarotid amobarbital procedure (IAP) or Wada test. The IAP is a procedure first described by Wada (1949) and Wada and Rasmussen (1960) for anaesthetizing cerebral hemispheres for the purpose of lateralizing language and memory functions. The procedure consists of unilateral injection of sodium amobarbital into the internal carotid, which tempo‐ rarily anaesthetizes the hemisphere ipsilateral to the injection site. While one hemisphere is anaesthetized, language and memory functions of the hemisphere contralateral to the injec‐ tion site can be tested. After the effect of the anaesthesia has dissipated, the process is re‐ peated with the other hemisphere. Determining the lateralization of language and memory functions is of both theoretical and practical interest, establishing cerebral language laterali‐ zation, predicting patients who are at risk for developing a post-surgical amnestic syndrome and identifying lateralized dysfunction to help confirm seizure onset laterality (Loring & Meador, 2000). Scientific investigation of cerebral lateralization in individuals with ID using

the IAP is generally hampered for obvious moral and ethical reasons.

right hemisphere language dominance.

**2. Laterality measures**

measures.

210 Down Syndrome

Neuroimaging techniques include both anatomical techniques, which create "constructed" images of brain structure, and functional techniques, which generate a series of dynamic brain images reflecting ongoing brain activity (Ganou, Kollias, Koidou, & Grouios, 2012). The anatomical techniques, which are the classical methods to image the brain, comprise computed tomography and structural magnetic resonance imaging. The functional techni‐ ques contain both direct (electroencephalography and magnetoencephalography) and indi‐ rect (positron-emissiontomography, single photon emission computed tomography and functional magnetic resonance imaging) measures of neural activity, which basically meas‐ ure haemodynamic responses or differences in metabolic concentrations to cognitive stimu‐ lation (for more information see Cohen & Sweet, 2011; Hüsing, Jäncke, & Tag, 2006).

Neuroimaging have offered a broad range of investigative tools to basic (e.g., Aziz-Zadeh, Koski, Zaidel, Mazziotta, & Iacoboni, 2006; Jansen, Menke, Sommer, Forster, Bruchmann et al., 2006; Tomasi & Volkow, 2012) and clinical (e.g., Desmond, Sum, Wagner, Demb, Shear et al., 1995; khondi-Asi, Jafari-Khouzani, Elisevich, & Soltanian-Zadeh, 2011; Oertel, Knöchel, Rotarska-Jagiela, Schönmeyer, Lindner et al., 2010) laterality research that fulfill the popular fantasy of being able to ''read the mind,'' albeit in the form of ''seeing the brain'' both struc‐ turally and functionally (Kerr & Denk, 2008).

Over the past 20 years, evidence for atypical cerebral lateralization in individuals with DS has been adduced using various neuroimaging techniques (Azari, Horwitz, Pettigrew, Gra‐ dy, Haxby, et al., 1994; Menghini, Costanzo, & Vicari, 2011; Pinter, Eliez, Schmitt, Capone, & Reiss, 2001). However, despite the large and growing literature describing patterns of brain structure and function in the healthy and diseased human brain, scientific research on Down syndrome has not been well integrated into the mainstream of human neuroimaging re‐ search. Nevertheless, a few investigators have demonstrated success in applying digital imaging technology in individuals with DS.

For example, Uecker, Mangan, Obrzut and Nadel (1993) argued that diffuse language later‐ alization in individuals with DS is likely to be a contributor to their poor visuospatial per‐ formance. Frangou, Aylward, Warren, Sharma, Barta et al. (1997) investigated whether the anatomic substrate for language are abnormal in DS. They examined volumetric Magnetic Resonance Imaging (MRI) measures of the superior temporal gyrus and the planum tempo‐ rale for community-dwelling individuals with DS and matched healthy comparison sub‐ jects. It was found that brain abnormalities in DS were not uniform. Specifically, the planum temporale volume of the individuals with DS was smaller than that of the healthy subjects. The volume of the superior temporal gyrus in the DS individuals was proportionally similar to that of the comparison group. For the subjects with DS, neither superior temporal gyrus nor planum temporale volume was significantly correlated with performance on language tests. Losin, Rivera, O'Hare, Sowell, and Pinter (2009) compared functional Magnetic Reso‐ nance Imaging (fMRI) activation patterns during passive story listening in young adults with DS and approximately age-matched, typically developing controls. They found that in‐ dividuals with DS exhibited differences in blood oxygen level dependant activation patterns compared to a typically developing group during the fMRI story-listening task. In particu‐ lar, their results indicated that the DS group showed almost no difference in activation pat‐ terns between the language (forward speech) and non-language (backward speech) conditions. Menghini, Costanzo and Vicari (2011) investigated regional grey matter density in adolescents with DS compared to age-matched controls and correlated MRI data with neuropsychological measures in the DS group. Their findings revealed that a number of brain regions subserved the neuropsychological abilities of participants with DS. Although adolescents with DS showed typical organization of brain structures related to some cogni‐ tive abilities, in particular spatial memory and visuoperception, they presented abnormal brain organization related to other cognitive domains, such as linguistic and verbal memory. Jacola, Byars, Chalfonte-Evans, Schmithorst, Hickey et al. (2011) used fMRI to investigate neural activation during a semantic-classification/object-recognition task in individuals with DS and typically developing control participants. A comparison between groups suggested atypical patterns of brain activation for the individuals with DS.

ly in recognition of abstract rather than concrete nouns (Ellis & Shepard, 1974, Hines, 1978) and also of words that only elicit a visual imagination with difficulty (Day, 1979). Righthanders usually show a right visual field advantage for verbal stimuli, as determined by the speed and correctness of the responses (Belin, Jullien, Perrier, & Larmande, 1990). A limited body of literature, using dichoptic presentation techniques, has documented the existence of perceptual asymmetries in individuals with DS (e.g., Chua, Weeks, & Elliott, 1996; Weeks,

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213

The dichaptic stimulation technique requires the subject to feel two different objects with meaningless shapes presented one to each hidden hand at the same time (Witelson, 1974). Upon dichaptic examination, the subject is asked to identify the two shapes from among a collection of six visually displayed shapes (Springer & Deutsch, 1981). Thus, hemispheric differences in haptic perception might be uncovered because of the complexity of the task, by making verbal mediation impossible, or by interfering with the interhemispheric transfer of information through the activation of homologous cortical areas. It has been shown that when meaningless stimuli are used, perceptual asymmetries are usually found in favor of the left hand for right-handed individuals (Benton, Harvey, & Varney, 1973; Dodds, 1978; Verjat, 1988), which reflects a better treatment of spatial information by the right hemi‐ sphere. Experimental data, using dichaptic stimulation techniques, have supported the exis‐ tence of perceptual asymmetries in individuals with DS (e.g., Chua, Weeks, & Elliott, 1996;

Elliott, Pollock, Chua, & Weeks, 1995; Weeks, Chua, Elliot, Lyons, & Pollock, 1995).

Laterality researchers have increasingly come to recognize the importance of sensory asym‐ metries in determining observed patterns of cerebral dominance (Dittmar, 2002). Lateral asymmetries in the use of sensory organs, based on their preferential use or/and functional primacy in a specific situation, are among the most obvious functional lateral preferences (Hellige, 1993), and they figure prominently in explanations of our evolutionary past (Cor‐ ballis, 1989), of ontogenetic development (Best, 1988; Levy, 1981), and of various abnormali‐ ties (Geschwind & Galaburda, 1985). The rationale for using the sensory asymmetries paradigm in the n the context of brain laterality is based on the presumption that difference in sensory performance between sensory stimuli presented to a sensory organ contralateral or ipsilateral to the dominant hemisphere would reflect a hemispheric bias in their attribu‐ tion strategy (Porac, Coren, Steiger, & Duncan, 1980). Sensory asymmetries are most promi‐ nent with respect to the auditory (e.g., Reiss & Reiss, 1998), visual (e.g., Porac & Coren, 1976), tactile (e.g., Harada, Saito, Kashikura, Sato, Yonekura et al., 2004) and chemical senses [taste (e.g., Faurion, Cerf, Van De Moortele, Lobel, MacLeod et al., 1999) and smell (e.g., Royet & Plailly, 2004)]. As far as we know, no study to date has examined sensory asymme‐

Motor indices of laterality, namely hand and foot preference and performance, have been used extensively to explore fundamental properties of the human brain, such as lateraliza‐ tion of brain functions, both in typically developing individuals (e.g., De Agostini & Dellato‐ las, 2001; Reiss, Tymnik, Kogler, Kogler, & Reiss 1999) and individuals with DS (e.g., Porac, Coren & Duncan, 1980; Grouios, Sakadami, Poderi & Aleuriadou, 1999). The most common‐ ly used index of laterality is handedness. The main consideration in the assessment of hand‐

Chua, Elliot, Lyons, & Pollock, 1995).

tries in DS individuals.

Behavioural techniques that have frequently been used to assess cerebral lateralization in‐ clude those that involve measurement of perceptual asymmetries, those that engage evalua‐ tion of sensory asymmetries and those that implicate determination of motor (or manual) asymmetries.

Studies of perceptual asymmetries have been utilized to explore lateral dominance of brain function and comprise dichotic, dichoptic and dichaptic stimulation. The rationale underly‐ ing the dichotic listening technique is that contralateral projections from each ear override ipsilateral projections when both ears are simultaneously presented with an auditory stimu‐ lus (e.g. a speech sound, digit or a musical tone) and the subject has to report what he/she has heard (Kimura 1967). Individuals with left hemisphere dominance for speech generally show a right-ear advantage for verbal stimuli. The stimuli, most commonly consonant vow‐ el syllables or monosyllabic words, are presented to the participant via ear-phones. Righthanders commonly exhibit a right ear advantage for verbal stimuli (e.g., Elliot & Weeks, 1993; Hugdahl, 2005), although individual differences seem to affect performance (e.g., gen‐ der, age) (Cowell & Hugdahl 2000). Empirical research, using dichotic listening techniques, has stressed asymmetry at the perceptual level in individuals with DS (e.g., Bowler, Cufflin, & Kiernan, 1985; Bunn, Welsh, Simon, Howarth, & Elliott, 2003; Hartley, 1981).

In the dichoptic presentation technique (or divided visual field technique), the subject is asked to report verbal stimuli (letters, words) that are rapidly flashed tachistoscopically into one visual half-field, thereby, limiting visual input to the contralateral hemisphere (Banich, 2003). The very short tachistoscopic presentation time prevents possible eye movements and, thus, bilateral cortical projection of the stimuli. Speech stimuli presented in the right visual field and, thus, transmitted primarily to the left hemisphere are recognized and named more rapidly and certainly than stimuli presented in the left visual field (McKeever & Huling, 1970; Hines, 1972). The dominance of the left hemisphere is shown more distinct‐ ly in recognition of abstract rather than concrete nouns (Ellis & Shepard, 1974, Hines, 1978) and also of words that only elicit a visual imagination with difficulty (Day, 1979). Righthanders usually show a right visual field advantage for verbal stimuli, as determined by the speed and correctness of the responses (Belin, Jullien, Perrier, & Larmande, 1990). A limited body of literature, using dichoptic presentation techniques, has documented the existence of perceptual asymmetries in individuals with DS (e.g., Chua, Weeks, & Elliott, 1996; Weeks, Chua, Elliot, Lyons, & Pollock, 1995).

nance Imaging (fMRI) activation patterns during passive story listening in young adults with DS and approximately age-matched, typically developing controls. They found that in‐ dividuals with DS exhibited differences in blood oxygen level dependant activation patterns compared to a typically developing group during the fMRI story-listening task. In particu‐ lar, their results indicated that the DS group showed almost no difference in activation pat‐ terns between the language (forward speech) and non-language (backward speech) conditions. Menghini, Costanzo and Vicari (2011) investigated regional grey matter density in adolescents with DS compared to age-matched controls and correlated MRI data with neuropsychological measures in the DS group. Their findings revealed that a number of brain regions subserved the neuropsychological abilities of participants with DS. Although adolescents with DS showed typical organization of brain structures related to some cogni‐ tive abilities, in particular spatial memory and visuoperception, they presented abnormal brain organization related to other cognitive domains, such as linguistic and verbal memory. Jacola, Byars, Chalfonte-Evans, Schmithorst, Hickey et al. (2011) used fMRI to investigate neural activation during a semantic-classification/object-recognition task in individuals with DS and typically developing control participants. A comparison between groups suggested

Behavioural techniques that have frequently been used to assess cerebral lateralization in‐ clude those that involve measurement of perceptual asymmetries, those that engage evalua‐ tion of sensory asymmetries and those that implicate determination of motor (or manual)

Studies of perceptual asymmetries have been utilized to explore lateral dominance of brain function and comprise dichotic, dichoptic and dichaptic stimulation. The rationale underly‐ ing the dichotic listening technique is that contralateral projections from each ear override ipsilateral projections when both ears are simultaneously presented with an auditory stimu‐ lus (e.g. a speech sound, digit or a musical tone) and the subject has to report what he/she has heard (Kimura 1967). Individuals with left hemisphere dominance for speech generally show a right-ear advantage for verbal stimuli. The stimuli, most commonly consonant vow‐ el syllables or monosyllabic words, are presented to the participant via ear-phones. Righthanders commonly exhibit a right ear advantage for verbal stimuli (e.g., Elliot & Weeks, 1993; Hugdahl, 2005), although individual differences seem to affect performance (e.g., gen‐ der, age) (Cowell & Hugdahl 2000). Empirical research, using dichotic listening techniques, has stressed asymmetry at the perceptual level in individuals with DS (e.g., Bowler, Cufflin,

& Kiernan, 1985; Bunn, Welsh, Simon, Howarth, & Elliott, 2003; Hartley, 1981).

In the dichoptic presentation technique (or divided visual field technique), the subject is asked to report verbal stimuli (letters, words) that are rapidly flashed tachistoscopically into one visual half-field, thereby, limiting visual input to the contralateral hemisphere (Banich, 2003). The very short tachistoscopic presentation time prevents possible eye movements and, thus, bilateral cortical projection of the stimuli. Speech stimuli presented in the right visual field and, thus, transmitted primarily to the left hemisphere are recognized and named more rapidly and certainly than stimuli presented in the left visual field (McKeever & Huling, 1970; Hines, 1972). The dominance of the left hemisphere is shown more distinct‐

atypical patterns of brain activation for the individuals with DS.

asymmetries.

212 Down Syndrome

The dichaptic stimulation technique requires the subject to feel two different objects with meaningless shapes presented one to each hidden hand at the same time (Witelson, 1974). Upon dichaptic examination, the subject is asked to identify the two shapes from among a collection of six visually displayed shapes (Springer & Deutsch, 1981). Thus, hemispheric differences in haptic perception might be uncovered because of the complexity of the task, by making verbal mediation impossible, or by interfering with the interhemispheric transfer of information through the activation of homologous cortical areas. It has been shown that when meaningless stimuli are used, perceptual asymmetries are usually found in favor of the left hand for right-handed individuals (Benton, Harvey, & Varney, 1973; Dodds, 1978; Verjat, 1988), which reflects a better treatment of spatial information by the right hemi‐ sphere. Experimental data, using dichaptic stimulation techniques, have supported the exis‐ tence of perceptual asymmetries in individuals with DS (e.g., Chua, Weeks, & Elliott, 1996; Elliott, Pollock, Chua, & Weeks, 1995; Weeks, Chua, Elliot, Lyons, & Pollock, 1995).

Laterality researchers have increasingly come to recognize the importance of sensory asym‐ metries in determining observed patterns of cerebral dominance (Dittmar, 2002). Lateral asymmetries in the use of sensory organs, based on their preferential use or/and functional primacy in a specific situation, are among the most obvious functional lateral preferences (Hellige, 1993), and they figure prominently in explanations of our evolutionary past (Cor‐ ballis, 1989), of ontogenetic development (Best, 1988; Levy, 1981), and of various abnormali‐ ties (Geschwind & Galaburda, 1985). The rationale for using the sensory asymmetries paradigm in the n the context of brain laterality is based on the presumption that difference in sensory performance between sensory stimuli presented to a sensory organ contralateral or ipsilateral to the dominant hemisphere would reflect a hemispheric bias in their attribu‐ tion strategy (Porac, Coren, Steiger, & Duncan, 1980). Sensory asymmetries are most promi‐ nent with respect to the auditory (e.g., Reiss & Reiss, 1998), visual (e.g., Porac & Coren, 1976), tactile (e.g., Harada, Saito, Kashikura, Sato, Yonekura et al., 2004) and chemical senses [taste (e.g., Faurion, Cerf, Van De Moortele, Lobel, MacLeod et al., 1999) and smell (e.g., Royet & Plailly, 2004)]. As far as we know, no study to date has examined sensory asymme‐ tries in DS individuals.

Motor indices of laterality, namely hand and foot preference and performance, have been used extensively to explore fundamental properties of the human brain, such as lateraliza‐ tion of brain functions, both in typically developing individuals (e.g., De Agostini & Dellato‐ las, 2001; Reiss, Tymnik, Kogler, Kogler, & Reiss 1999) and individuals with DS (e.g., Porac, Coren & Duncan, 1980; Grouios, Sakadami, Poderi & Aleuriadou, 1999). The most common‐ ly used index of laterality is handedness. The main consideration in the assessment of hand‐ edness is the use of different handedness measures, which produce different types of handedness. For example, hand preference can be assessed using questionnaires (e.g., Briggs & Nebes, 1972; Oldfield, 1971) on a five-scale continuum ranging from strong left-handers to strong right-handers. Alternatively, researchers have used preference measures to distin‐ guish between left and right-handers (2 categories), excluding intermittent hand preferences (e.g., Coren & Porac, 1980), or right and non-right handers (2 categories) (e.g., Ypsilanti, 2009) or right-handers, left handers and ambiguous (or mixed) handers (3 categories) (e.g., Cornish & McManus, 1996).

questionnaires (even if those are read to them). It has become very common during the pasts decades to use behavioural measures of hand preference (e.g., Bryden, Pryde, & Roy, 2000; Bishop, Ross, Daniel, & Bright, 1996) or observation of hand preference on a number of tasks (Porac & Coren, 1981). These tasks are comprised of 10-12 preference measures (to assess de‐ gree of hand preference), which are examined twice (to assess hand consistency) and hand‐ edness is usually evaluated on a three point scale of preference; left, right, mixed. However, studies have used the demonstration of hand preference based on the items of an inventory and a five-point scale has been used classifying individuals as strongly left, weak left, ambi‐ texter, weak right, strongly right (Van Strein, Lagers, van Haselen, van Hagen, de Coo, Frens, & van der Geest, 2005). An alternative example of such a task is the WatHand Box Test (Bryden, Pryde, & Roy, 2000), which assesses direction and consistency of hand prefer‐ ence using a variety of unimanual tasks (e.g., lifting a cupboard door, using a toy hammer, placing rings on hooks and tossing a ball). In addition, Bishop's card reaching task (Bishop, Ross, Daniel, & Bright, 1996) that provides a measure of the degree and the direction of hand preference has commonly been used in individuals with neurodevelopmental disor‐

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215

ders (see Desplanches, Deruelle, Stefanini, Ayoun, Volterra, Vicari et al, 2006).

McManus, 1996; Cornish, Pigram, & Paw, 1997).

ative foot performance in individuals with DS.

**listening studies)**

Performance measures of handedness are used less often to assess the relative proficiency of on hand over the other in individuals with neurodevelopmental disorders. Tasks that have commonly been utilized to assess hand skill are finger tapping (Elliott, Edwards, Weeks, Lindley, & Carnahan, 1987; Elliott, Weeks, & Jones 1986) and the pegboard (e.g., Cornish &

Other laterality indexes, such as ear, eye and foot, are also assessed both as preference and as performance. For example, foot preference can be assessed using a questionnaire or using a demonstration of foot preference across a number of tasks (e.g., Porac & Coren, 1981). Moreover, foot performance can also be examined by assessing the relative proficiency of one foot over the other. Up until now, no study that we know has specifically addressed rel‐

**3. Atypical laterality in individuals with Down syndrome (Dichotic**

In dichotic listening studies the participants selectively attend one of the two messages pre‐ sented simultaneously in both ears indicating a left or right ear advantage for linguistic ma‐ terial. Most evidence agrees that right-handed individuals with DS exhibit a unique pattern of ear dominance that is syndrome-specific and cannot be attributed to the mental retarda‐ tion per se (Heath & Elliot, 1999). Support for this dissociation in ear preference comes from various studies assessing individual with DS, individuals with mental retardation (non-DS) and typically developing participants (e.g., Hartley, 1981; Pipe, 1983; Elliot & Weeks, 1993; Heath & Elliot, 1999; Giencke & Lewandowski, 1989). There is increased evidence for left ear/right hemisphere dominance for language in right-handed individuals with DS, which is indicative of a reversed cerebral specialization for speech perception (see Elliot, Weeks &

In attempting to clarify both the conceptual and theoretical issues surrounding handedness assessment methodology, it is important to discriminate between "direction of hand prefer‐ ence", "degree of hand preference" and "consistency of hand preference" (Cornish & McManus, 1996). Direction of hand preference refers to the degree of dexterity or sinistrality that an individual exhibits (Bishop, 1990). Degree of hand preference is determined by whether an individual consistently exhibits a specific hand preference *across* several tasks or behaviours (Cornish & McManus, 1996). Consistency of hand preference is ascertained by whether an individual exhibits a specific hand preference for the same task on several occa‐ sions (Cornish & McManus, 1996). Consistency of hand preference was previously described by Palmer (1964), which he termed "variable hand preference" and postulated to be in‐ creased in left-handers. Moreover, the degree of hand preference was also previously descri‐ bed by Palmer (1964) which he termed "ambidexterity or mixed motor preference" referring to the degree of hand differentiation across different tasks.

Classification of handedness is further complicated by the fact that a researcher may assess hand preference (be that the direction, degree, or consistency) by a self-reported question‐ naire (e.g., Briggs & Nebes, 1972) or a behavioural measure of hand preference (e.g., Bryden, Pryde, & Roy, 2000) or observation of hand preference (Porac & Coren, 1981) and/or hand performance or hand skill, which evaluates the proficiency of one hand over the other in performing a specific task (e.g., pegboard). The advantage of accessing hand preference is that one can evaluate several tasks (e.g., writing, throwing, cutting and dealing cards), rath‐ er than assessing hand performance on one task. However, assessing hand performance as‐ sists in the more qualitative understanding of handedness by allowing individuals to document their relative proficiency of one hand over the other. Most researchers (e.g., Porac & Coren, 1981; Bishop, 1990) agree that the assessments of hand preference and hand skill are two qualitatively different measures (i.e., they measure different things) of handedness. The mechanisms that mediate preference and performance are different representing two di‐ mensions of laterality. In essence hand preference is mediated more by cognitive mecha‐ nisms that support the choice of hand-use, while hand skill may be less mediated by cognitive mechanisms and more supported by motoric systems. Annett, Hudson and Turner (1974) have supported the use of performance measures, suggesting that the relative profi‐ ciency of one hand over the other would most likely lead to increased preference of the more skilled hand.

The assessment of preference in populations with DS using questionnaires has been scarce since most clinical groups document ID, which may interfere with the process of answering questionnaires (even if those are read to them). It has become very common during the pasts decades to use behavioural measures of hand preference (e.g., Bryden, Pryde, & Roy, 2000; Bishop, Ross, Daniel, & Bright, 1996) or observation of hand preference on a number of tasks (Porac & Coren, 1981). These tasks are comprised of 10-12 preference measures (to assess de‐ gree of hand preference), which are examined twice (to assess hand consistency) and hand‐ edness is usually evaluated on a three point scale of preference; left, right, mixed. However, studies have used the demonstration of hand preference based on the items of an inventory and a five-point scale has been used classifying individuals as strongly left, weak left, ambi‐ texter, weak right, strongly right (Van Strein, Lagers, van Haselen, van Hagen, de Coo, Frens, & van der Geest, 2005). An alternative example of such a task is the WatHand Box Test (Bryden, Pryde, & Roy, 2000), which assesses direction and consistency of hand prefer‐ ence using a variety of unimanual tasks (e.g., lifting a cupboard door, using a toy hammer, placing rings on hooks and tossing a ball). In addition, Bishop's card reaching task (Bishop, Ross, Daniel, & Bright, 1996) that provides a measure of the degree and the direction of hand preference has commonly been used in individuals with neurodevelopmental disor‐ ders (see Desplanches, Deruelle, Stefanini, Ayoun, Volterra, Vicari et al, 2006).

edness is the use of different handedness measures, which produce different types of handedness. For example, hand preference can be assessed using questionnaires (e.g., Briggs & Nebes, 1972; Oldfield, 1971) on a five-scale continuum ranging from strong left-handers to strong right-handers. Alternatively, researchers have used preference measures to distin‐ guish between left and right-handers (2 categories), excluding intermittent hand preferences (e.g., Coren & Porac, 1980), or right and non-right handers (2 categories) (e.g., Ypsilanti, 2009) or right-handers, left handers and ambiguous (or mixed) handers (3 categories) (e.g.,

In attempting to clarify both the conceptual and theoretical issues surrounding handedness assessment methodology, it is important to discriminate between "direction of hand prefer‐ ence", "degree of hand preference" and "consistency of hand preference" (Cornish & McManus, 1996). Direction of hand preference refers to the degree of dexterity or sinistrality that an individual exhibits (Bishop, 1990). Degree of hand preference is determined by whether an individual consistently exhibits a specific hand preference *across* several tasks or behaviours (Cornish & McManus, 1996). Consistency of hand preference is ascertained by whether an individual exhibits a specific hand preference for the same task on several occa‐ sions (Cornish & McManus, 1996). Consistency of hand preference was previously described by Palmer (1964), which he termed "variable hand preference" and postulated to be in‐ creased in left-handers. Moreover, the degree of hand preference was also previously descri‐ bed by Palmer (1964) which he termed "ambidexterity or mixed motor preference" referring

Classification of handedness is further complicated by the fact that a researcher may assess hand preference (be that the direction, degree, or consistency) by a self-reported question‐ naire (e.g., Briggs & Nebes, 1972) or a behavioural measure of hand preference (e.g., Bryden, Pryde, & Roy, 2000) or observation of hand preference (Porac & Coren, 1981) and/or hand performance or hand skill, which evaluates the proficiency of one hand over the other in performing a specific task (e.g., pegboard). The advantage of accessing hand preference is that one can evaluate several tasks (e.g., writing, throwing, cutting and dealing cards), rath‐ er than assessing hand performance on one task. However, assessing hand performance as‐ sists in the more qualitative understanding of handedness by allowing individuals to document their relative proficiency of one hand over the other. Most researchers (e.g., Porac & Coren, 1981; Bishop, 1990) agree that the assessments of hand preference and hand skill are two qualitatively different measures (i.e., they measure different things) of handedness. The mechanisms that mediate preference and performance are different representing two di‐ mensions of laterality. In essence hand preference is mediated more by cognitive mecha‐ nisms that support the choice of hand-use, while hand skill may be less mediated by cognitive mechanisms and more supported by motoric systems. Annett, Hudson and Turner (1974) have supported the use of performance measures, suggesting that the relative profi‐ ciency of one hand over the other would most likely lead to increased preference of the

The assessment of preference in populations with DS using questionnaires has been scarce since most clinical groups document ID, which may interfere with the process of answering

Cornish & McManus, 1996).

214 Down Syndrome

more skilled hand.

to the degree of hand differentiation across different tasks.

Performance measures of handedness are used less often to assess the relative proficiency of on hand over the other in individuals with neurodevelopmental disorders. Tasks that have commonly been utilized to assess hand skill are finger tapping (Elliott, Edwards, Weeks, Lindley, & Carnahan, 1987; Elliott, Weeks, & Jones 1986) and the pegboard (e.g., Cornish & McManus, 1996; Cornish, Pigram, & Paw, 1997).

Other laterality indexes, such as ear, eye and foot, are also assessed both as preference and as performance. For example, foot preference can be assessed using a questionnaire or using a demonstration of foot preference across a number of tasks (e.g., Porac & Coren, 1981). Moreover, foot performance can also be examined by assessing the relative proficiency of one foot over the other. Up until now, no study that we know has specifically addressed rel‐ ative foot performance in individuals with DS.

## **3. Atypical laterality in individuals with Down syndrome (Dichotic listening studies)**

In dichotic listening studies the participants selectively attend one of the two messages pre‐ sented simultaneously in both ears indicating a left or right ear advantage for linguistic ma‐ terial. Most evidence agrees that right-handed individuals with DS exhibit a unique pattern of ear dominance that is syndrome-specific and cannot be attributed to the mental retarda‐ tion per se (Heath & Elliot, 1999). Support for this dissociation in ear preference comes from various studies assessing individual with DS, individuals with mental retardation (non-DS) and typically developing participants (e.g., Hartley, 1981; Pipe, 1983; Elliot & Weeks, 1993; Heath & Elliot, 1999; Giencke & Lewandowski, 1989). There is increased evidence for left ear/right hemisphere dominance for language in right-handed individuals with DS, which is indicative of a reversed cerebral specialization for speech perception (see Elliot, Weeks & Chua, 1994 for a meta-analysis). This reversed pattern has been linked to the poor linguistic abilities of these individuals although dissociation between laterality for speech perception and speech production that involves oral motor systems has also been suggested (Elliot, Weeks, & Elliot, 1987; Giencke & Lewandowski, 1989; Heath & Elliot, 1999). During the past decade, studies explored the issue of the dissociation of lateralized systems for speech per‐ ception and speech production in individuals with DS using a verbal-motor task that tapped interhemispheric integration (Welsh, Elliot, & Simon, 2003). Their results supported their model of functional dissociation between perception and oral-motor production for speech stimuli that are typically supported by the same cerebral hemisphere in typically developing individuals. Moreover, this atypical pattern of cerebral specialization is specific to DS and is not observed in other populations with mental retardation (non-DS) of unknown etiology.

studies provide insight into the functioning of the brain and its lateralization. They also provide evidence for the representation of cognitive systems within the brain. For exam‐ ple, it may be suggested that the brains of individuals with DS may represent processing centers bilaterally causing a delay in the production of relevant cognitive and motor ma‐ terial. In addition, by combining neuroimaging with behavioral laterality techniques one can infer that certain brain areas are predominately involved in specific processes, while other areas are unable to execute their intended function. For instance, perhaps the weak collaboration of the two hemispheres is due to the thinner corpus callosum in individu‐ als with DS (Wang, Doherti, Hesselink, & Bellugi, 1992) that may cause the isolation of the functions of the hemispheres enhancing weak intra-hemispheric integration at least

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217

for verbal-motor stimuli (Welsh, Elliot, & Simon, 2003).

standing of atypical laterality in individuals with DS.

**4. Atypical laterality and Down syndrome (handedness studies)**

exhibited an almost twofold increased prevalence of left-handedness (31%).

in the non-clinical groups (age matched controls=11% left-handers).

Ear preference using dichotic listening tasks indicates a syndrome- specific pattern of cere‐ bral laterality in individuals with DS. This pattern can perhaps be documented using other laterality indexes, such as hand, foot and eye preference. To date there has not been a study assessing individuals with DS on various laterality indexes using preference and perform‐ ance measures and controlling for the effect of age, gender and degree of mental retardation. Such studies are currently been undertaken in our laboratory to assist further in the under‐

However, handedness studies in individuals with DS have been reported since the 70's. Pickersgill and Pank (1970) assessed the prevalence of left handedness in individuals with DS, individuals with mental retardation non-DS and typically developing individuals. They found a higher prevalence of left-handedness in individuals with mental retardation non-DS compared to individuals with DS and typically developing adults. More specifically, the prevalence of left-handedness in typically developing individuals in their sample was 15.6% and that of individuals with DS 18.7%, while individuals with mental retardation non-DS

In a later study, Batheja and Mc Manus (1985) explored the prevalence of left-handedness in individuals with DS, individuals with mental retardation (non-DS) and typically developing Individuals, matched for age, and found no difference between the two clinical groups (DS=27% left-handers, non-DS= 29% left-handers), although there was a marked difference

In a similar study, Pipe (1987) assessed hand preference in individuals with DS, individuals with mental retardation non-DS and age-matched controls including her families to deter‐ mine whether familial sinistrality is documented in these populations. Their results indicat‐ ed that the two clinical groups, regardless of their etiology (DS or non-DS) exhibited 35-36% of non-right handedness (i.e., left and mixed handedness) and increased familial sinistrality compared to the non-clinical population. The authors explained that the increased preva‐ lence of mixed handedness and familial sinistrality in individuals with mental retardation

Unlike typically developing individuals, DS people exhibit right hemisphere lateraliza‐ tion for receptive language and a left hemisphere lateralization for the production of simple and complex movement. This separation of speech perception and motor move‐ ment in addition to the morphological callosal deficiencies (causing poor intrahemispher‐ ic communication) may be responsible for the verbal difficulties of DS individuals (Heath, Grierson, Binsted, & Elliott, 2007).

Pipe (1983) used dichotic listening tasks to assess language laterality in young children with DS, individuals with mental retardation (non-DS) and typically developing individuals. Their results indicated an atypical left-ear right-hemisphere advantage for speech stimuli in individuals with DS a pattern that was only observed in this clinical group. Non-DS individ‐ uals with mental retardation exhibited a right-ear left-hemisphere advantage for speech stimuli a pattern that was similar to typically developing individuals. In accordance with With Elliott, Edwards, Weeks, Lindley and Carnahan's (1987) study, Pipe (1983) observed the unique pattern of ear preference in individuals with DS, which seems to be expressed over and above the degree of mental retardation and may be described as syndrome-specif‐ ic. It should be noted here that most researchers (e.g., Pipe, 1983; Elliott, Edwards, Weeks, Lindley, & Carnahan, 1987 Heath & Elliot, 1999) have linked this unique pattern of cerebral laterality for language in individuals with DS with the weak linguistic abilities that they ex‐ hibit. However, further research assessing different clinical syndromes that also exhibit lin‐ guistic deficits (e.g., Williams syndrome) using dichotic listening tasks is needed to support this hypothesis.

On the other hand, Paquette, Bourassa and Peretz (1996) documented a left ear advantage in individuals with ID of unknown etiology. Their results indicated a left ear/ right hemisphere advantage for speech stimuli in both impaired groups and the opposite pattern in typically developing individuals. This pattern of ear preference supports the notion of atypical cere‐ bral laterality in individuals with mental retardation as a consequence of the early brain damage that affects intellectual functioning and cerebral specialization.

The importance of studies using non-invasive techniques, such as dichotic listening and handedness, to assess cerebral laterality in individuals with mental retardation is of vast importance. Firstly, non-invasive measures are easy and safe to administer to such popu‐ lations and produce significant information to researchers in this field. Secondly, such studies provide insight into the functioning of the brain and its lateralization. They also provide evidence for the representation of cognitive systems within the brain. For exam‐ ple, it may be suggested that the brains of individuals with DS may represent processing centers bilaterally causing a delay in the production of relevant cognitive and motor ma‐ terial. In addition, by combining neuroimaging with behavioral laterality techniques one can infer that certain brain areas are predominately involved in specific processes, while other areas are unable to execute their intended function. For instance, perhaps the weak collaboration of the two hemispheres is due to the thinner corpus callosum in individu‐ als with DS (Wang, Doherti, Hesselink, & Bellugi, 1992) that may cause the isolation of the functions of the hemispheres enhancing weak intra-hemispheric integration at least for verbal-motor stimuli (Welsh, Elliot, & Simon, 2003).

## **4. Atypical laterality and Down syndrome (handedness studies)**

Chua, 1994 for a meta-analysis). This reversed pattern has been linked to the poor linguistic abilities of these individuals although dissociation between laterality for speech perception and speech production that involves oral motor systems has also been suggested (Elliot, Weeks, & Elliot, 1987; Giencke & Lewandowski, 1989; Heath & Elliot, 1999). During the past decade, studies explored the issue of the dissociation of lateralized systems for speech per‐ ception and speech production in individuals with DS using a verbal-motor task that tapped interhemispheric integration (Welsh, Elliot, & Simon, 2003). Their results supported their model of functional dissociation between perception and oral-motor production for speech stimuli that are typically supported by the same cerebral hemisphere in typically developing individuals. Moreover, this atypical pattern of cerebral specialization is specific to DS and is not observed in other populations with mental retardation (non-DS) of unknown etiology. Unlike typically developing individuals, DS people exhibit right hemisphere lateraliza‐ tion for receptive language and a left hemisphere lateralization for the production of simple and complex movement. This separation of speech perception and motor move‐ ment in addition to the morphological callosal deficiencies (causing poor intrahemispher‐ ic communication) may be responsible for the verbal difficulties of DS individuals

Pipe (1983) used dichotic listening tasks to assess language laterality in young children with DS, individuals with mental retardation (non-DS) and typically developing individuals. Their results indicated an atypical left-ear right-hemisphere advantage for speech stimuli in individuals with DS a pattern that was only observed in this clinical group. Non-DS individ‐ uals with mental retardation exhibited a right-ear left-hemisphere advantage for speech stimuli a pattern that was similar to typically developing individuals. In accordance with With Elliott, Edwards, Weeks, Lindley and Carnahan's (1987) study, Pipe (1983) observed the unique pattern of ear preference in individuals with DS, which seems to be expressed over and above the degree of mental retardation and may be described as syndrome-specif‐ ic. It should be noted here that most researchers (e.g., Pipe, 1983; Elliott, Edwards, Weeks, Lindley, & Carnahan, 1987 Heath & Elliot, 1999) have linked this unique pattern of cerebral laterality for language in individuals with DS with the weak linguistic abilities that they ex‐ hibit. However, further research assessing different clinical syndromes that also exhibit lin‐ guistic deficits (e.g., Williams syndrome) using dichotic listening tasks is needed to support

On the other hand, Paquette, Bourassa and Peretz (1996) documented a left ear advantage in individuals with ID of unknown etiology. Their results indicated a left ear/ right hemisphere advantage for speech stimuli in both impaired groups and the opposite pattern in typically developing individuals. This pattern of ear preference supports the notion of atypical cere‐ bral laterality in individuals with mental retardation as a consequence of the early brain

The importance of studies using non-invasive techniques, such as dichotic listening and handedness, to assess cerebral laterality in individuals with mental retardation is of vast importance. Firstly, non-invasive measures are easy and safe to administer to such popu‐ lations and produce significant information to researchers in this field. Secondly, such

damage that affects intellectual functioning and cerebral specialization.

(Heath, Grierson, Binsted, & Elliott, 2007).

this hypothesis.

216 Down Syndrome

Ear preference using dichotic listening tasks indicates a syndrome- specific pattern of cere‐ bral laterality in individuals with DS. This pattern can perhaps be documented using other laterality indexes, such as hand, foot and eye preference. To date there has not been a study assessing individuals with DS on various laterality indexes using preference and perform‐ ance measures and controlling for the effect of age, gender and degree of mental retardation. Such studies are currently been undertaken in our laboratory to assist further in the under‐ standing of atypical laterality in individuals with DS.

However, handedness studies in individuals with DS have been reported since the 70's. Pickersgill and Pank (1970) assessed the prevalence of left handedness in individuals with DS, individuals with mental retardation non-DS and typically developing individuals. They found a higher prevalence of left-handedness in individuals with mental retardation non-DS compared to individuals with DS and typically developing adults. More specifically, the prevalence of left-handedness in typically developing individuals in their sample was 15.6% and that of individuals with DS 18.7%, while individuals with mental retardation non-DS exhibited an almost twofold increased prevalence of left-handedness (31%).

In a later study, Batheja and Mc Manus (1985) explored the prevalence of left-handedness in individuals with DS, individuals with mental retardation (non-DS) and typically developing Individuals, matched for age, and found no difference between the two clinical groups (DS=27% left-handers, non-DS= 29% left-handers), although there was a marked difference in the non-clinical groups (age matched controls=11% left-handers).

In a similar study, Pipe (1987) assessed hand preference in individuals with DS, individuals with mental retardation non-DS and age-matched controls including her families to deter‐ mine whether familial sinistrality is documented in these populations. Their results indicat‐ ed that the two clinical groups, regardless of their etiology (DS or non-DS) exhibited 35-36% of non-right handedness (i.e., left and mixed handedness) and increased familial sinistrality compared to the non-clinical population. The authors explained that the increased preva‐ lence of mixed handedness and familial sinistrality in individuals with mental retardation couldn't support Satz's (1973) model of pathological left-handedness. If non-right handed‐ ness is caused by early brain insult, as the model suggests, then there should not be an in‐ creased prevalence of familial sinistrality in these populations. Rather as Batheja and McManus (1985) suggested non-right handedness may be the result of any biological dis‐ turbance causing variability in cerebral asymmetry. Alternatively, specific hormones such as testosterone, delays the development of left-hemisphere functions resulting in increased prevalence of non-right handedness in clinical populations.

**Study no. Reference Participants N1 Preference/**

3 Lenneberg, Nickols and Rosenberger (1964)

9 Porac, Coren, & Duncan (1980)

14 Bradshaw, Hick &

16 Batheja & McManus (1985)

17 Elliott, Weeks & Jones (1986).

Kinsbourne (1984)

1 Gordon (1921) ID Preference Hand LH 18%

4 Clausen (1966) ID 276 Preference Hand LH 17%

5 Rengstroff (1967) ID 395 Preference Hand, eye 81.8% RH 18.2 LH -

6 Peckersgill & Pank (1970) ID, DS 32 Performance Hand LH 18% in Ds

7 Hicks & Barton (1975) ID 550 Preference Hand LH 20.7% \*

8 Silva & Satz (1979) ID 1409 Performance Hand LH 15.5

11 Hartley (1981) ID, DS Performance Ear LEA in DS

13 Pipe (1983) ID, DS Performance Ear LEA in DS

10 Burns & Zeaman (1980) ID 20 Preference Hand, eye,

12 McManus (1983) ID 68 Preference

15 Elliot, D (1985) ID DS 38 Preference/

ID 128 Preference Hand, eye,

(mother's report)

ID 232 Performance Hand More LH

performance

ID, DS 130 Performance Hand LH 27%

DS Performance Hand DS same asymmetry

2 Merphy (1962) ID, DS 64 Preference Hand LH 31% of ID

**performance**

Laterality Explored: Atypical Hemispheric Dominance in Down Syndrome

DS 61 Preference Hand M 42.6%

**Indices Results**

LH 7%

http://dx.doi.org/10.5772/53829

219

LH 13% of DS

48.5% RE 51.4 LE%

LH 31% in ID

(13%)

(28%)

M 12.7%

LH 15.9 M 44.2%

groups.

LH 29% LH 11%

on finger-tapping

Hand LH 13.2

Hand is more lateralized than foot, ear, eye in both

ear, foot

ear, foot

Hand

Mild & Moderate:

Severe & Profound:

Lewin, Kohen and Mathew (1993) investigated handedness in individuals with DS, epilepsy and autism. Their results indicated a significantly increased prevalence of non-right handers in all three populations with no differences between the three groups and no differences as‐ sociated with the level of mental retardation as reported elsewhere (e.g., Hicks & Barton, 1975). It was proposed that the theory of left-handedness (Satz, 1972) may explain the in‐ creased incidence of non-right handers in individuals with epilepsy in which focal brain damage may be assumed, however, it may not hold true for individuals with DS or autism. The theory of increased randomness (Palmer, 1964) may explain this pattern in individuals with learning disabilities, since the arrested development of the nervous system may lead to the undifferentiation of the two hemispheres documented by the increased prevalence of non-right handers in these populations. Table 1 below presents research using laterality in‐ dexes in DS and ID.

Findings from our laboratory confirm the existence of an atypical pattern of handedness preference in individuals with DS (n=50) and ID (n=50), compared to typically developing (TD) individuals (n=100) (Ypsilanti, 2009) (Figure 1). Specifically, our results demonstrate no significant differences between DS and ID individuals with similar level of intellectual func‐ tioning (mean IQ=43). However, they indicate statistically significant differences between both clinical groups and TD individuals (χ<sup>2</sup> = 46.86, d.f.=2, p<0.01).

In reviewing studies of atypical laterality in individuals with DS, compared to individuals with ID (non-DS) and typically developing individuals, two conclusions can be drawn. First‐ ly, in the existing literature there seems to be inconsistent findings even when similar meth‐ odologies are employed. For example, Pickersgill and Pank (1970) found no significant differences in laterality in individuals with DS and typically developing individuals, while other studies have found such differences consistently (e.g., Batheja & McManus, 1985; Pipe, 1987). The reason for this discrepancy may be linked to various laterality measures that have been used to assess hand preference in individuals with neurodevelopmental disorders as well as the different age groups that have been selected in each case. Moreover, differences in the degree of mental retardation may have interfered with the results of different studies. Secondly, few studies have taken into account the fact that individuals with DS do not ex‐ hibit focal brain lesion during fetal development, which has converted them from natural right-handers to pathological left-handers as in the cases of individuals with focal brain in‐ jury in the left hemisphere (Satz, 1972). As Batheja and McManus (1985) proposed it is more likely that the difference in the prevalence of hand preference may be due to "… any form of biological noise" (pp. 66) (Batheja & McManus, 1985) that disrupts the development of typi‐ cal asymmetry in these individuals at its genesis.


couldn't support Satz's (1973) model of pathological left-handedness. If non-right handed‐ ness is caused by early brain insult, as the model suggests, then there should not be an in‐ creased prevalence of familial sinistrality in these populations. Rather as Batheja and McManus (1985) suggested non-right handedness may be the result of any biological dis‐ turbance causing variability in cerebral asymmetry. Alternatively, specific hormones such as testosterone, delays the development of left-hemisphere functions resulting in increased

Lewin, Kohen and Mathew (1993) investigated handedness in individuals with DS, epilepsy and autism. Their results indicated a significantly increased prevalence of non-right handers in all three populations with no differences between the three groups and no differences as‐ sociated with the level of mental retardation as reported elsewhere (e.g., Hicks & Barton, 1975). It was proposed that the theory of left-handedness (Satz, 1972) may explain the in‐ creased incidence of non-right handers in individuals with epilepsy in which focal brain damage may be assumed, however, it may not hold true for individuals with DS or autism. The theory of increased randomness (Palmer, 1964) may explain this pattern in individuals with learning disabilities, since the arrested development of the nervous system may lead to the undifferentiation of the two hemispheres documented by the increased prevalence of non-right handers in these populations. Table 1 below presents research using laterality in‐

Findings from our laboratory confirm the existence of an atypical pattern of handedness preference in individuals with DS (n=50) and ID (n=50), compared to typically developing (TD) individuals (n=100) (Ypsilanti, 2009) (Figure 1). Specifically, our results demonstrate no significant differences between DS and ID individuals with similar level of intellectual func‐ tioning (mean IQ=43). However, they indicate statistically significant differences between

In reviewing studies of atypical laterality in individuals with DS, compared to individuals with ID (non-DS) and typically developing individuals, two conclusions can be drawn. First‐ ly, in the existing literature there seems to be inconsistent findings even when similar meth‐ odologies are employed. For example, Pickersgill and Pank (1970) found no significant differences in laterality in individuals with DS and typically developing individuals, while other studies have found such differences consistently (e.g., Batheja & McManus, 1985; Pipe, 1987). The reason for this discrepancy may be linked to various laterality measures that have been used to assess hand preference in individuals with neurodevelopmental disorders as well as the different age groups that have been selected in each case. Moreover, differences in the degree of mental retardation may have interfered with the results of different studies. Secondly, few studies have taken into account the fact that individuals with DS do not ex‐ hibit focal brain lesion during fetal development, which has converted them from natural right-handers to pathological left-handers as in the cases of individuals with focal brain in‐ jury in the left hemisphere (Satz, 1972). As Batheja and McManus (1985) proposed it is more likely that the difference in the prevalence of hand preference may be due to "… any form of biological noise" (pp. 66) (Batheja & McManus, 1985) that disrupts the development of typi‐

= 46.86, d.f.=2, p<0.01).

prevalence of non-right handedness in clinical populations.

dexes in DS and ID.

218 Down Syndrome

both clinical groups and TD individuals (χ<sup>2</sup>

cal asymmetry in these individuals at its genesis.


**Figure 1.** Frequencies of right and non-right handed individuals with DS, ID and TD.

Several accounts have been put forward to explain the increased incidence of atypical laterality in individuals with ID. It has been suggested that theories on atypical laterality fall into two categories; namely, pathological and natural (Satz, 1973). However, for the purposes of clarity this discrimination will not be adapted in the present paper. Rather a detailed analysis of all the theories will be presented including those that are scarcely

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221

One of the most prominent theories has been put forward by Geschwind and Galaburda (1987), who implicated the levels of testosterone in the development of atypical laterality. According to the theory, several genetic factors, such as chromosomes and antigens, as well as environmental factors that affect fetal development, like the endocrine environment and the cyclic variation, alter the levels of testosterone to the fetus. This effect is directly linked to both the delayed growth of the left hemisphere and the increased growth of the right hemisphere particularly in the posterior regions. The decreased growth of the left hemi‐ sphere has been linked to mental retardation and poor verbal ability, which are some of the characteristics of individuals with neurodevelopmental disorders. In essence, the model pre‐ dicts that the increased levels of testosterone will have an impact on the development of the left hemisphere, causing reduced language and visual-spatial dominance. Therefore, indi‐ viduals with this condition will exhibit increased left and mixed handedness compared to the normal population. In support of their theory, Geschiwind and Galaburda (1985a, 1985b,

**5. Theoretical explanations of atypical laterality**

discussed in the literature.

1Sorted by year of study

**Table 1.** Laterality indices in ID and DS.

**Figure 1.** Frequencies of right and non-right handed individuals with DS, ID and TD.

## **5. Theoretical explanations of atypical laterality**

**Study no. Reference Participants N1 Preference/**

21 Elliot et al. (1987) DS 12 Preference/

18 Searleman, Cunningham & Goodwin (1987)

220 Down Syndrome

27 Vlachos & Karapetsas, (1999)

30 Leconte and Fagard (2006)

32 Mulvey, Ringenbach & Jung, 2011

**Table 1.** Laterality indices in ID and DS.

1Sorted by year of study

33 Carlier et al., 2011 DS, WS, DiGeorge

**performance**

performance

performance

DS 41 Preference Hand LH & LRH in DS

ID 30 Preference Hand Eye

perfromance

Preference Hand Eye,

ID 90 Preference/

19 Soper et al., (1987) ID 73 Preference Hand LH 9.6%, M 45.2%,

20 Pipe (1987) ID, DS 318 Preference Hand M 35%\*

22 Lucas et al., (1989) ID 238 Preference Hand LH 17.4% mild

24 Paquatte et al (1996) ID 16 Performance Ear LEA in ID

28 Heath & Elliot (1999) DS 10 Performance Ear

29 Carlier et al., (2006) DS, WS 79 Preference

31 Desplanches et al, 2006 Preference

syndrome

23 Morris & Romski, (1993) ID 50 Preference Hand LH 19%, LRH 32%,

25 Mandal et al (1998) ID 50 Preference Hand Mixed handedness. 26 Grouios et al. (1999) ID 73 Preference Hand LH 17.8%, LRH

DS 25 Preference &

**Indices Results**

Hand LH 17.8

Hand

Foot

Ear & Foot

M 5.6

RH 45.2%

LRH 36%\* LRH 18%

LH 28.0% severe

38.4%, RH 43.8% LH 9.6%, LRH 4.1%,

Crossed Eye-hand

asymmetry in bimanual coordination

Increased mixed handedness and footedness in all groups, related to degree of ID.

Hand Reduced hand

RH 86.3%

RH 49%

Several accounts have been put forward to explain the increased incidence of atypical laterality in individuals with ID. It has been suggested that theories on atypical laterality fall into two categories; namely, pathological and natural (Satz, 1973). However, for the purposes of clarity this discrimination will not be adapted in the present paper. Rather a detailed analysis of all the theories will be presented including those that are scarcely discussed in the literature.

One of the most prominent theories has been put forward by Geschwind and Galaburda (1987), who implicated the levels of testosterone in the development of atypical laterality. According to the theory, several genetic factors, such as chromosomes and antigens, as well as environmental factors that affect fetal development, like the endocrine environment and the cyclic variation, alter the levels of testosterone to the fetus. This effect is directly linked to both the delayed growth of the left hemisphere and the increased growth of the right hemisphere particularly in the posterior regions. The decreased growth of the left hemi‐ sphere has been linked to mental retardation and poor verbal ability, which are some of the characteristics of individuals with neurodevelopmental disorders. In essence, the model pre‐ dicts that the increased levels of testosterone will have an impact on the development of the left hemisphere, causing reduced language and visual-spatial dominance. Therefore, indi‐ viduals with this condition will exhibit increased left and mixed handedness compared to the normal population. In support of their theory, Geschiwind and Galaburda (1985a, 1985b, 1985c) presented a series of studies associating atypical laterality (or "anomalous domi‐ nance") with developmental learning disorders, autism and immune disorders.

these disorders, in addition to other neurodevelopmental disorders, that exhibit abnormali‐ ties in the brain stem, the basal ganglia, the cerebellum and the temporal lobes, since these systems are directly affected or affect the vestibular system. Also, increased percentages of poor motoric dominance (i.e., non-right handedness) are likely to exist in pre-term infants, since they have not been exposed long enough to the right face position allowing for right handedness to be established. Previc's (1991) theory initiated a new era in the research of human laterality. The presence of prenatal factors that affect and essentially define motoric dominance in humans in combination with genetic, environmental and cultural theories could provide an important framework for the development of a stronger and more inclu‐

Laterality Explored: Atypical Hemispheric Dominance in Down Syndrome

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223

An alternative model attempting to explain the increased incidence of atypical laterality in individuals with neurodevelopmental disorders is the theory of pathological left-handed‐ ness (Satz, 1973) According to this account, there is a subgroup of left-handed individuals which are described as pathological left handers. This subgroup was genetically natural right-handers, but suffered early brain insult to the left hemisphere causing a mild dysfunc‐ tion of the contralateral hand for motor movements. The result of this dysfunction was a switch of hand dominance to the other hand (i.e., left hand) to perform complex motor tasks. Therefore, although these individuals were genetically programmed to become right-hand‐ ers having left hemisphere dominance for language an early brain insult (before the age of six) caused a switch hand preference making them pathological left-handers. This subgroup is differentiated for natural left-handers who have no history of brain insult early in devel‐ opment and are naturally born with left hand dominance. In addition, the model describes a subgroup of pathological right-handers who were natural left-handers but an early brain in‐ jury in the right hemisphere caused them to switch hand preference to the opposite hand, thus becoming pathological right-handers. The account of pathological left-handedness can predict the increased incidence of left-handers in populations with ID and epilepsy, since both groups seem to have brain abnormalities exhibited early in development. Therefore, within a population of individuals with mental retardation, there will be an 8% of natural left-handers, as in the typical population, and approximately another 8-9% who are patho‐ logical left-handers. This model would explain the almost twofold percentage of manifest

Several studies have provided evidence for the model of pathological left-handedness, since the initial account was put forward (Satz, 1973). However, the theory has been test‐ ed in cross cultural studies (Satz, Baymure, & Van der Vlugt, 1979), in studies using EEG recordings (Silva & Satz, 1979), in studies with individuals with left or right con‐ genital hemiplegia (Carlsson, Hugdahl, Uvenbrant, Wiklund, & Von Wentd, 1992), in re‐ lation to familial sinistrality (Orsini, Satz, Soper, & Light, 1985; Pipe 1987) and degree of ID (Bradshaw-McAnulty, Hicks, & Kinsbourne, 1984) and has been termed the pathologi‐ cal left handedness syndrome (Satz, Orsini, Saslow, & Henry, 1985). Since the original study (Satz, 1973) Soper and Satz (1984) incorporated one more type of pathological handedness in their model, termed ambiguous handedness, to explain the increased inci‐ dence of mixed handedness in individuals with early brain insult. The new explanatory

sive theory that encompasses strengths of all other theories.

left-handers in individual with mental retardation.

Although Geschwind's and Galaburda's (1987) theory has been considered one of the most prominent theories in the field of cognitive neuropsychology, it has been strongly criticized for its complexity and its arbitrary predictions (e.g., McManus & Bryden, 1991; McManus, Bryden, & Bulman-Fleming, 1994; Annett, 1994; Previc, 1994). Bryden McManus and Bul‐ man-Fleming (1994) suggest that the relationship between language dominance and hand‐ edness, as discussed by Geschwind and Galaburda (1987), is weak and the conclusions drawn based on this assumption are poorly supported by empirical findings. Moreover, the predictions made by Geshwind and Galaburda (1987) are farfetched and the experimental data cannot support the numerous associations that are predicted by theory. On the other hand, the theory, although long and complex, contributed greatly to the understanding of the biological factors (i.e., hormones) that may be linked to atypical laterality and triggered a large number of studies in atypical laterality and neurodevelopmental disorders.

Genetic theories have also been put forward to explain atypical laterality (Annett & Alexander, 1996; Bryden & McManus, 1985). The main focus of these genetic theories was to explain the origin of left and right-handedness in normal populations (Annett 1972, 1985). More specifically, Annett's (1972) theory, referred to as "right-shift theory", explained the exhibition of right and left handedness as the outcome of left hemisphere speech induced by a single gene. In the case of atypical handedness Annett (1994) suggested that atypical de‐ velopmental effects could trigger randomness in the absence of the right-shift gene and in‐ hibit the "natural" cerebral asymmetry that is observed in typical development. Moreover, individuals lacking the gene for right hemisphere speech (rs+ gene) are at risk for various difficulties that affect language expression and phonology such as dyslexia. In other words, Annett (1985) proposed that atypical laterality may be a "... natural variation in cerebral asymmetry" (pp. 241) triggered by the absence of the right-shift gene (Annett, 1994).

Previc (1991) postulated that cerebral asymmetry derives from the asymmetric development of the vestibular system (left ear dominance in approximately 70% of the population), which is established during prenatal life and is directly linked to the postural position of the fetus and the pattern of maternal movements during the final trimester of the pregnancy. More‐ over, the anatomy of the female uterus induces fetuses in the final trimester of pregnancy to be positioned "… with their head to the left side of the mother's midline and their right ear facing outward" (pp. 301) (Previc, 1991). This postural asymmetry of the fetus and the moth‐ er favours a sinistral vestibular dominance at birth, which is documented by the dextral lie preference of newborns and is correlated with the development of right hand preference lat‐ er in life. The asymmetrical development of the two vestibular organs, the ear and the laby‐ rinth, may be responsible for the asymmetry of the left and right hemisphere and the difference in ear preference documented in the literature using dichotic listening tasks (e.g., Heath & Elliott, 1999). Previc (1991) proposed a link between poor motoric lateralization (i.e., mixed or left handedness), the vestibular system and neurodevelopmental disorders that are associated with vestibular dysfunction; namely autism, dyslexia and deafness. In es‐ sence, Previc's (1991) theory predicted increased percentages of non-right handedness in these disorders, in addition to other neurodevelopmental disorders, that exhibit abnormali‐ ties in the brain stem, the basal ganglia, the cerebellum and the temporal lobes, since these systems are directly affected or affect the vestibular system. Also, increased percentages of poor motoric dominance (i.e., non-right handedness) are likely to exist in pre-term infants, since they have not been exposed long enough to the right face position allowing for right handedness to be established. Previc's (1991) theory initiated a new era in the research of human laterality. The presence of prenatal factors that affect and essentially define motoric dominance in humans in combination with genetic, environmental and cultural theories could provide an important framework for the development of a stronger and more inclu‐ sive theory that encompasses strengths of all other theories.

1985c) presented a series of studies associating atypical laterality (or "anomalous domi‐

Although Geschwind's and Galaburda's (1987) theory has been considered one of the most prominent theories in the field of cognitive neuropsychology, it has been strongly criticized for its complexity and its arbitrary predictions (e.g., McManus & Bryden, 1991; McManus, Bryden, & Bulman-Fleming, 1994; Annett, 1994; Previc, 1994). Bryden McManus and Bul‐ man-Fleming (1994) suggest that the relationship between language dominance and hand‐ edness, as discussed by Geschwind and Galaburda (1987), is weak and the conclusions drawn based on this assumption are poorly supported by empirical findings. Moreover, the predictions made by Geshwind and Galaburda (1987) are farfetched and the experimental data cannot support the numerous associations that are predicted by theory. On the other hand, the theory, although long and complex, contributed greatly to the understanding of the biological factors (i.e., hormones) that may be linked to atypical laterality and triggered a

nance") with developmental learning disorders, autism and immune disorders.

222 Down Syndrome

large number of studies in atypical laterality and neurodevelopmental disorders.

asymmetry" (pp. 241) triggered by the absence of the right-shift gene (Annett, 1994).

Previc (1991) postulated that cerebral asymmetry derives from the asymmetric development of the vestibular system (left ear dominance in approximately 70% of the population), which is established during prenatal life and is directly linked to the postural position of the fetus and the pattern of maternal movements during the final trimester of the pregnancy. More‐ over, the anatomy of the female uterus induces fetuses in the final trimester of pregnancy to be positioned "… with their head to the left side of the mother's midline and their right ear facing outward" (pp. 301) (Previc, 1991). This postural asymmetry of the fetus and the moth‐ er favours a sinistral vestibular dominance at birth, which is documented by the dextral lie preference of newborns and is correlated with the development of right hand preference lat‐ er in life. The asymmetrical development of the two vestibular organs, the ear and the laby‐ rinth, may be responsible for the asymmetry of the left and right hemisphere and the difference in ear preference documented in the literature using dichotic listening tasks (e.g., Heath & Elliott, 1999). Previc (1991) proposed a link between poor motoric lateralization (i.e., mixed or left handedness), the vestibular system and neurodevelopmental disorders that are associated with vestibular dysfunction; namely autism, dyslexia and deafness. In es‐ sence, Previc's (1991) theory predicted increased percentages of non-right handedness in

Genetic theories have also been put forward to explain atypical laterality (Annett & Alexander, 1996; Bryden & McManus, 1985). The main focus of these genetic theories was to explain the origin of left and right-handedness in normal populations (Annett 1972, 1985). More specifically, Annett's (1972) theory, referred to as "right-shift theory", explained the exhibition of right and left handedness as the outcome of left hemisphere speech induced by a single gene. In the case of atypical handedness Annett (1994) suggested that atypical de‐ velopmental effects could trigger randomness in the absence of the right-shift gene and in‐ hibit the "natural" cerebral asymmetry that is observed in typical development. Moreover, individuals lacking the gene for right hemisphere speech (rs+ gene) are at risk for various difficulties that affect language expression and phonology such as dyslexia. In other words, Annett (1985) proposed that atypical laterality may be a "... natural variation in cerebral An alternative model attempting to explain the increased incidence of atypical laterality in individuals with neurodevelopmental disorders is the theory of pathological left-handed‐ ness (Satz, 1973) According to this account, there is a subgroup of left-handed individuals which are described as pathological left handers. This subgroup was genetically natural right-handers, but suffered early brain insult to the left hemisphere causing a mild dysfunc‐ tion of the contralateral hand for motor movements. The result of this dysfunction was a switch of hand dominance to the other hand (i.e., left hand) to perform complex motor tasks. Therefore, although these individuals were genetically programmed to become right-hand‐ ers having left hemisphere dominance for language an early brain insult (before the age of six) caused a switch hand preference making them pathological left-handers. This subgroup is differentiated for natural left-handers who have no history of brain insult early in devel‐ opment and are naturally born with left hand dominance. In addition, the model describes a subgroup of pathological right-handers who were natural left-handers but an early brain in‐ jury in the right hemisphere caused them to switch hand preference to the opposite hand, thus becoming pathological right-handers. The account of pathological left-handedness can predict the increased incidence of left-handers in populations with ID and epilepsy, since both groups seem to have brain abnormalities exhibited early in development. Therefore, within a population of individuals with mental retardation, there will be an 8% of natural left-handers, as in the typical population, and approximately another 8-9% who are patho‐ logical left-handers. This model would explain the almost twofold percentage of manifest left-handers in individual with mental retardation.

Several studies have provided evidence for the model of pathological left-handedness, since the initial account was put forward (Satz, 1973). However, the theory has been test‐ ed in cross cultural studies (Satz, Baymure, & Van der Vlugt, 1979), in studies using EEG recordings (Silva & Satz, 1979), in studies with individuals with left or right con‐ genital hemiplegia (Carlsson, Hugdahl, Uvenbrant, Wiklund, & Von Wentd, 1992), in re‐ lation to familial sinistrality (Orsini, Satz, Soper, & Light, 1985; Pipe 1987) and degree of ID (Bradshaw-McAnulty, Hicks, & Kinsbourne, 1984) and has been termed the pathologi‐ cal left handedness syndrome (Satz, Orsini, Saslow, & Henry, 1985). Since the original study (Satz, 1973) Soper and Satz (1984) incorporated one more type of pathological handedness in their model, termed ambiguous handedness, to explain the increased inci‐ dence of mixed handedness in individuals with early brain insult. The new explanatory model predicted increased incidence of ambiguous handedness in the more severe groups with neurodevelopmental disorders, such as infantile autism and severe ID (So‐ per & Satz, 1984), which has also been reported elsewhere (e.g., Tsai, 1982).

another measure of non-verbal intelligence (e.g., Raven, 1985), would be more appropriate for matching control groups. Further, research in the area of motor development and the as‐

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225

A link has also been postulated between literacy and handedness, suggesting that cerebral organization may change as a result of schooling and literacy, although the evidence for this link is contradictory (Tzavaras, Kaprinis, & Gatzouas, 1981). In controversy with genetic the‐ ories, this approach suggests that literacy reinforces the left hemisphere dominance for lan‐ guage. According to the theory, there should be an increased number of individuals with atypical laterality among illiterate populations exhibiting right or bilateral dominance for language. Tzavaras, Kaprinis and Gatzouas (1981) examined this possibility using the di‐ chotic listening technique as a measure of language dominance in an illiterate population and found an increased left-right ear difference in the illiterate population compared to the literate individuals. The authors suggested that this difference might be due to the poor stra‐ tegic techniques used by illiterate subjects, which do not enhance bi-hemispheric participa‐ tion for speech as in the educated brain. However, it has been found that aphasia is less severe and more provisional in illiterate patients suggesting a right hemisphere involvement of language in these individuals (Lecours, Mehler, Parente, Behrami, Tolipan, Cary, et al, 1988; Cameron, Currier, & Haerer, 1971). Castro-Caldas, Reis and Geurreiro (1997) in a re‐ view on literacy and laterality concluded that the empirical findings of studies from aphasic patients and dichotic listening tasks are inconclusive about the link between atypical lateral‐ ity and literacy and further research is need to clarify this postulation. To the authors' knowledge, no studies have been reported linking the observed atypical laterality of indi‐ viduals with neurodevelopmental disorders with literacy and schooling. However, a num‐ ber of researchers propose that lateral preferences may be affected by the type of task used and may be related to the level of experience and practice that a group of individuals have (e.g., Bishop, 1983). If one accepts this notion, it is probable that individuals with mental re‐ tardation are less skilled than typically developing individuals with objects like pencils, scis‐ sors and playing cards, which are commonly used to assess hand preference in these populations. In this case, inconsistent hand preference when manipulating such objects may be affected by the immature behaviour exhibited by these individuals due to decreased ex‐ perience. More specifically, the effect of limited schooling and skilfulness in individuals with mental retardation may have an indirect impact on lateral preferences particularly

sessment of handedness in relation to motor age are needed to clarify the issue.

when the preference measures presented are school-related utilities.

counterparts.

Another line of research suggests that individuals with DS exhibit atypical neural activation in left/right hemisphere regions compared to typically developing individuals (Jacola, Byars, Chalfonte-Evans, Schmithorst, Hickey, Patterson, et al., 2011). In an fMRI study with 13 DS individuals, there was a positive association between visual-spatial ability and occipito-pari‐ etal and dorso-frontal activation exclusively in individuals with DS compared to control

Although the above-mentioned theories contribute to the understanding of the increased in‐ cidence of non-right handers in individuals with neurodevelopmental disorders, the evi‐ dence for this link is far from conclusive. Satz's (1973) theory of pathological left handedness could account for the increased incidence of left handers in individual with focal brain in‐ jury, but in clinical populations with defuse brain damage (e.g., DS, Williams syndrome) and lack of hand preference (i.e., increased mixed handedness) the theory seems inadequate. Particularly in individuals with ID, it has long been recognized that ambiguous handedness rather than left-handedness is most commonly observed (e.g., Porac, Coren, Steiger, & Dun‐ can, 1980). This lack of handedness would be documented by random hand preference in preference measures.

Palmer (1964) termed this observation "increased randomness" referring to the increased ambiguous hand preference in individuals with mental retardation. In particular, he postu‐ lated that handedness is a developmental process and could be utilized as an index of typi‐ cal motor development. This developmental process progresses from a bilateral undifferentiated state early in infancy to a unilateral state that is viewed as a "… differentia‐ tion from a whole" (pp. 258) (Palmer, 1964), since it initiates from the trunks before the shoulders and then the hands. Therefore, Palmer (1964) proposed a maturational process that is linked to typical cerebral laterality and one-sidedness. If this maturational process is arrested or lagged it could cause increased randomness, which would be documented by lack of hand preference (i.e., ambiguous handedness). One of the main conclusions that could be drawn from Palmer's (1964) theory is that mixed and left-handedness has long been considered differentiated states and should be studied separately. Particularly in popu‐ lations with neurodevelopmental disorders, "lack of hand preference" (i.e., mixed handed‐ ness) may be a more significant indicator of atypical cerebral laterality than left-handedness.

Along this vein, Bishop (1983, 1990) postulated that non right-handedness is an indicator of an immature development of the motor system, caused by diffuse brain abnormalities in in‐ dividuals with mental retardation. In contrast to Satz's (1973) theory and other genetic theo‐ ries, Bishop (1990) suggests that differentiated hand preference indicates mature motor development. According to Bishop (1990), studies assessing hand preference in individuals with mental retardation should utilize a control group matched for motor development rather than chronological or mental age. The question remains whether there is correspond‐ ence between motor and mental age and whether measuring motor age when assessing handedness can further contribute to the existing literature. To our knowledge, there are no published data on of handedness in neurodevelopmental disorders that utilises a control group matched for motor age. On the other hand, mental age as assessed using the WISC III (Wechsler, 1992) may also be problematic because the verbal subtests of the WISC III (Wechsler, 1992) may undermine the motor development of an individual with mental retar‐ dation. The link between mental retardation and motor retardation has not been widely in‐ vestigated. Perhaps using the performance subscales of the WISC III (Wechsler, 1992), or another measure of non-verbal intelligence (e.g., Raven, 1985), would be more appropriate for matching control groups. Further, research in the area of motor development and the as‐ sessment of handedness in relation to motor age are needed to clarify the issue.

model predicted increased incidence of ambiguous handedness in the more severe groups with neurodevelopmental disorders, such as infantile autism and severe ID (So‐

Although the above-mentioned theories contribute to the understanding of the increased in‐ cidence of non-right handers in individuals with neurodevelopmental disorders, the evi‐ dence for this link is far from conclusive. Satz's (1973) theory of pathological left handedness could account for the increased incidence of left handers in individual with focal brain in‐ jury, but in clinical populations with defuse brain damage (e.g., DS, Williams syndrome) and lack of hand preference (i.e., increased mixed handedness) the theory seems inadequate. Particularly in individuals with ID, it has long been recognized that ambiguous handedness rather than left-handedness is most commonly observed (e.g., Porac, Coren, Steiger, & Dun‐ can, 1980). This lack of handedness would be documented by random hand preference in

Palmer (1964) termed this observation "increased randomness" referring to the increased ambiguous hand preference in individuals with mental retardation. In particular, he postu‐ lated that handedness is a developmental process and could be utilized as an index of typi‐ cal motor development. This developmental process progresses from a bilateral undifferentiated state early in infancy to a unilateral state that is viewed as a "… differentia‐ tion from a whole" (pp. 258) (Palmer, 1964), since it initiates from the trunks before the shoulders and then the hands. Therefore, Palmer (1964) proposed a maturational process that is linked to typical cerebral laterality and one-sidedness. If this maturational process is arrested or lagged it could cause increased randomness, which would be documented by lack of hand preference (i.e., ambiguous handedness). One of the main conclusions that could be drawn from Palmer's (1964) theory is that mixed and left-handedness has long been considered differentiated states and should be studied separately. Particularly in popu‐ lations with neurodevelopmental disorders, "lack of hand preference" (i.e., mixed handed‐ ness) may be a more significant indicator of atypical cerebral laterality than left-handedness.

Along this vein, Bishop (1983, 1990) postulated that non right-handedness is an indicator of an immature development of the motor system, caused by diffuse brain abnormalities in in‐ dividuals with mental retardation. In contrast to Satz's (1973) theory and other genetic theo‐ ries, Bishop (1990) suggests that differentiated hand preference indicates mature motor development. According to Bishop (1990), studies assessing hand preference in individuals with mental retardation should utilize a control group matched for motor development rather than chronological or mental age. The question remains whether there is correspond‐ ence between motor and mental age and whether measuring motor age when assessing handedness can further contribute to the existing literature. To our knowledge, there are no published data on of handedness in neurodevelopmental disorders that utilises a control group matched for motor age. On the other hand, mental age as assessed using the WISC III (Wechsler, 1992) may also be problematic because the verbal subtests of the WISC III (Wechsler, 1992) may undermine the motor development of an individual with mental retar‐ dation. The link between mental retardation and motor retardation has not been widely in‐ vestigated. Perhaps using the performance subscales of the WISC III (Wechsler, 1992), or

per & Satz, 1984), which has also been reported elsewhere (e.g., Tsai, 1982).

preference measures.

224 Down Syndrome

A link has also been postulated between literacy and handedness, suggesting that cerebral organization may change as a result of schooling and literacy, although the evidence for this link is contradictory (Tzavaras, Kaprinis, & Gatzouas, 1981). In controversy with genetic the‐ ories, this approach suggests that literacy reinforces the left hemisphere dominance for lan‐ guage. According to the theory, there should be an increased number of individuals with atypical laterality among illiterate populations exhibiting right or bilateral dominance for language. Tzavaras, Kaprinis and Gatzouas (1981) examined this possibility using the di‐ chotic listening technique as a measure of language dominance in an illiterate population and found an increased left-right ear difference in the illiterate population compared to the literate individuals. The authors suggested that this difference might be due to the poor stra‐ tegic techniques used by illiterate subjects, which do not enhance bi-hemispheric participa‐ tion for speech as in the educated brain. However, it has been found that aphasia is less severe and more provisional in illiterate patients suggesting a right hemisphere involvement of language in these individuals (Lecours, Mehler, Parente, Behrami, Tolipan, Cary, et al, 1988; Cameron, Currier, & Haerer, 1971). Castro-Caldas, Reis and Geurreiro (1997) in a re‐ view on literacy and laterality concluded that the empirical findings of studies from aphasic patients and dichotic listening tasks are inconclusive about the link between atypical lateral‐ ity and literacy and further research is need to clarify this postulation. To the authors' knowledge, no studies have been reported linking the observed atypical laterality of indi‐ viduals with neurodevelopmental disorders with literacy and schooling. However, a num‐ ber of researchers propose that lateral preferences may be affected by the type of task used and may be related to the level of experience and practice that a group of individuals have (e.g., Bishop, 1983). If one accepts this notion, it is probable that individuals with mental re‐ tardation are less skilled than typically developing individuals with objects like pencils, scis‐ sors and playing cards, which are commonly used to assess hand preference in these populations. In this case, inconsistent hand preference when manipulating such objects may be affected by the immature behaviour exhibited by these individuals due to decreased ex‐ perience. More specifically, the effect of limited schooling and skilfulness in individuals with mental retardation may have an indirect impact on lateral preferences particularly when the preference measures presented are school-related utilities.

Another line of research suggests that individuals with DS exhibit atypical neural activation in left/right hemisphere regions compared to typically developing individuals (Jacola, Byars, Chalfonte-Evans, Schmithorst, Hickey, Patterson, et al., 2011). In an fMRI study with 13 DS individuals, there was a positive association between visual-spatial ability and occipito-pari‐ etal and dorso-frontal activation exclusively in individuals with DS compared to control counterparts.

## **6. Epilogue**

Research in laterality in individuals with DS has been fruitful. Findings from dichotic listen‐ ing studies suggest that individuals with DS exhibit a unique pattern of lateralization of lan‐ guage, which is syndrome specific. Specifically, it has been repeatedly supported that there is a left-ear, right-hemisphere advantage for speech stimuli, unlike that observed in typical populations or individuals with ID of other aetiologies. Moreover, handedness studies dem‐ onstrate that lateralization of language may be pathological with increased incidence of lefthandedness, left- footedness, left-eyedness and cross eye-hand preferences. Several theories have been put forward to explain this atypicality, including, hormonal, structural and neu‐ ral anomalies related to the syndrome. This atypical pattern of functional lateralization, most likely contributes to the linguistic difficulties observed in individuals with DS, which are rather permanent. At the same time, limited educational and motor training leaves little space for improvement in linguistic and motor efficiency in individuals with DS. Other de‐ velopmental milestones that are fundamentally delayed in individuals with DS obstruct the developmental transition from an undifferentiated state to a lateralized state.

volving language areas in young adults with Down syndrome. *Brain and Language*,

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[8] Aziz-Zadeh, L., Koski, L., Zaidel, E., Mazziotta, J., & Iacoboni, M. (2006). Lateraliza‐ tion of the human mirror neuron system. *The Journal of Neuroscience*, 26, 2964-2970.

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## **Author details**

George Grouios, Antonia Ypsilanti and Irene Koidou

Laboratory of Motor Control and Learning, Department of Physical Education and Sport Sciences, Aristotle University of Thessaloniki, Greece

## **References**


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**6. Epilogue**

226 Down Syndrome

**Author details**

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**Chapter 12**

**Genetic and Epigenetic Mechanisms**

Additional information is available at the end of the chapter

caused by epigenetic factors including DNA methylation.

Down syndrome (DS) is the most common congenital disorder in children, affecting one in 800 live births. While the large number of contiguous genes from a trisomy of chromosome 21 (HSA21) is expected to broadly affect various organ systems during development, significant advances in medicine have been made in this disorder such that those with DS live fairly long life spans. Individuals with DS, however, uniformly demonstrate some degree of mental retardation. Arguably, cognitive disabilities are the more devastating aspect of DS disorder. Part of the cognitive dysfunction lies not only in the progressive neuronal degeneration/cell death and impaired neurogenesis seen in this developmental and degenerative disorder, but also in the reduction in dendrite formation and spine density, resulting in a disruption of synaptic function. These neurological endophenotypes seen in DS may not be merely due to genomic imbalance from triplication of HSA21 genes, but also to additive influences on associated genes within a given network or pathway and modification of gene expressions

Epigenetic factors regulate gene expression largely through DNA modification. Histones are alkaline proteins that package and order DNA into structural nucleosomes. Acetylation and deacetylation, as well as methylation, of histones can modify the density of chromatin and thereby regulate gene transcription through chromatin remodeling. In a parallel manner, biochemical modification of DNA can occur through DNA methylation. This process involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring. DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression by physically impeding the binding of transcrip‐ tional proteins to the gene itself, or by recruiting protein complexes including methyl-CpGbinding domain proteins (MBDs), histone deacetylases (HDACs) and other chromatin

and reproduction in any medium, provided the original work is properly cited.

© 2013 Lu and Sheen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**in Down Syndrome Brain**

Jie Lu and Volney Sheen

http://dx.doi.org/10.5772/52807

**1. Introduction**


## **Genetic and Epigenetic Mechanisms in Down Syndrome Brain**

Jie Lu and Volney Sheen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52807

## **1. Introduction**

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236 Down Syndrome

Down syndrome (DS) is the most common congenital disorder in children, affecting one in 800 live births. While the large number of contiguous genes from a trisomy of chromosome 21 (HSA21) is expected to broadly affect various organ systems during development, significant advances in medicine have been made in this disorder such that those with DS live fairly long life spans. Individuals with DS, however, uniformly demonstrate some degree of mental retardation. Arguably, cognitive disabilities are the more devastating aspect of DS disorder. Part of the cognitive dysfunction lies not only in the progressive neuronal degeneration/cell death and impaired neurogenesis seen in this developmental and degenerative disorder, but also in the reduction in dendrite formation and spine density, resulting in a disruption of synaptic function. These neurological endophenotypes seen in DS may not be merely due to genomic imbalance from triplication of HSA21 genes, but also to additive influences on associated genes within a given network or pathway and modification of gene expressions caused by epigenetic factors including DNA methylation.

Epigenetic factors regulate gene expression largely through DNA modification. Histones are alkaline proteins that package and order DNA into structural nucleosomes. Acetylation and deacetylation, as well as methylation, of histones can modify the density of chromatin and thereby regulate gene transcription through chromatin remodeling. In a parallel manner, biochemical modification of DNA can occur through DNA methylation. This process involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring. DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression by physically impeding the binding of transcrip‐ tional proteins to the gene itself, or by recruiting protein complexes including methyl-CpGbinding domain proteins (MBDs), histone deacetylases (HDACs) and other chromatin

© 2013 Lu and Sheen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

remodeling proteins. Furthermore, environmental factors such as chemical toxins or oxidative stress can accumulate over time and effect gene transcription. Collectively, these processes modify DNA transcription and may affect many neurodevelopmental processes.

**2.1. Genetic mechanisms underlying oxidative stress in DS**

**2.2. Genetics mechanisms underlying neurogenesis in DS**

expression of a single gene.

Increased levels of oxidative stress and reactive oxygen species (ROS) have commonly been associated with the DS brain. Free radicals are thought to disrupt the mitochondrial respiratory system, induce apoptosis of neurons and stimulate gliosis, which can further promote neuronal damage. This cyclical pathway may contribute to neuronal losses during neurogenesis as well as neuronal degeneration in adulthood. Several HSA21 genes have been implicated in generation of ROS including *DYRK1A*, *DSCR1*, *SOD1*, *ETS2, S100B, APP* and *BACH1* [15, 17]. Additionally, more recent studies would suggest a synergistic role for various HSA21 genes in induction of this pathological process. For example, over-expression of HSA21 genes *APP* and *S100B* synergistically increase hydrogen peroxide levels and decrease membrane potential in the mitochondria of human DS neuroprogenitor cells. The combination of a loss of mito‐ chondrial integrity and an increase of oxidative stress promotes apoptosis (changes in caspase and respiratory chain protein expression) and gliosis (increase of GFAP). S100B induction can occur through RAGE (Receptor for Advanced Glycation Endproducts) with consequent activation of JNK/p38 and JAK/STAT signaling. These stress response pathways are known to serve as downstream effectors potentially relevant to reactive gliosis, induction of S100B and glial associated aquaporin 4 [18, 19]. Increased levels of S100B and APP further enhance this cyclical cascade by promoting RAGE activation and inflammation with reactive gliosis. Lastly, multiple HSA21 genes have demonstrated enhanced APP-dependent toxic effects on the mitochondria whereas network prediction analyses have shown that four HSA21 proteins are components of the JAK/STAT pathway. These studies imply that an additional 19 HSA21 (among 2004 in total) proteins interact with components in this pathway [20]. These findings reiterate the large cascade of molecules that can be perturbed in a pathway following over-

Genetic and Epigenetic Mechanisms in Down Syndrome Brain

http://dx.doi.org/10.5772/52807

239

Although oxidative stress in DS patients is considered to be a primary contributor of neuro‐ degeneration such as Alzheimer's Disease (AD) in adult patients, evidences from both human and animal models suggest that these same processes could also affect neurodevelopment and cognitive function at a much earlier age [19, 21-23]. Oxidative stress could therefore not only alter neuronal numbers through degeneration and changes in synaptic plasticity through impaired mitochondrial function, but also affect the generation of neurons during develop‐ ment. In this respect, ongoing effects from over-expression of HSA21 genes likely promote the cognitive dysfunction in DS throughout the lifetime of an individual with this disorder.

The observation of reduced cortical volume and decreased neuronal numbers in DS patients and animal models could in part be attributed to a reduction in the generation of neurons [24-27]. Over-expression of several HSA21 genes has been implicated in neurogenesis by either altering the rate or proliferation or by changing cell fate specification. By over-expressing HSA21-associated OLIG2, we observed a phenotypic shift in the neural progenitor pool toward glial progenitor phenotypes, accompanied by a corresponding decrease in the number of neuronal progenitors. This change can partly be explained by OLIG2-dependent inhibition of the expression and activity of KCNA3 outward rectifying potassium channels whose activa‐

Recent advances in high throughput screening of both mRNA expression and DNA methyl‐ ation have provided a means to examine changes in gene activation and expression, and to understand the integral relationship between gene clusters in effecting particular pathways. The following review begins by exploring the potential contribution of both genetic and epigenetic factors in regulation of various DS endophenotypes. More specifically, our prior work has examined changes in DS neural progenitor mRNA expression and has led us to identify several important pathways affected in this disorder, such as oxidative stress, mitochondrial dysfunction and gliogenesis. Ongoing studies suggest that changes in DNA methylation in DS may have an effect on oxidative phosphorylation, ubiquitin proteolysis and insulin signaling. The confirmation of mRNA and DNA methylation changes and the clarifi‐ cation of these possible causal pathways may have implications for impaired synaptic function and neurogenesis, which contribute to the cognitive impairment seen in DS. These ongoing studies may further provide informative targets for early pharmaceutical interference to ameliorate the symptoms of mental retardation (MR) in DS.

## **2. Genetic mechanisms underlying the DS phenotype**

The triplication of genes on HSA21 causes a wide spectrum of neurological phenotypes in DS, including mental retardation. DS individual displays not only delayed linguistic skills and a relatively low IQ (Intelligent Quotient) but also behavioral issues such as attention-deficit disorder (sometimes with hyperactivity) and autism [1-5]. The cognitive impairments extend further after development, as individuals with DS are more prone to develop Alzheimer's type dementia [6]. In addition, individuals with DS are susceptible to epilepsy in the form of infantile spasms and tonic clonic seizures with myoclonus at early ages [7-9]. These patholog‐ ical abnormalities in humans are, in part, replicated in DS animal models which show defects in learning, social interactions, memory, and seizures [10-14].

Several genes on HSA21 are implicated in the abnormal neurodevelopment in DS [15]. They can affect cellular function at every stage of neural development, such as proliferation and differentiation of neuroprogenitor cells, neuronal survival and death, synapse formation, maturation and plasticity, as well as myelination. Disruption of each of these pathways can conceptually contribute to the MR seen in DS. Moreover, HSA21 genes have global effects on other genes; a meta-analysis of heterogeneous DS data identified 324 genes with consistent dosage effects, 77 on HSA21 and 247 on non-HSA21 [16]. Therefore, the over-expression of a not so small group of genes on HSA21 may initiate cascades of other signaling pathways on other chromosomes thorough an interactive network. The combinatorial effects from activa‐ tion of these processes may further contribute to the impairments seen during neurodevelop‐ ment in DS.

#### **2.1. Genetic mechanisms underlying oxidative stress in DS**

remodeling proteins. Furthermore, environmental factors such as chemical toxins or oxidative stress can accumulate over time and effect gene transcription. Collectively, these processes

Recent advances in high throughput screening of both mRNA expression and DNA methyl‐ ation have provided a means to examine changes in gene activation and expression, and to understand the integral relationship between gene clusters in effecting particular pathways. The following review begins by exploring the potential contribution of both genetic and epigenetic factors in regulation of various DS endophenotypes. More specifically, our prior work has examined changes in DS neural progenitor mRNA expression and has led us to identify several important pathways affected in this disorder, such as oxidative stress, mitochondrial dysfunction and gliogenesis. Ongoing studies suggest that changes in DNA methylation in DS may have an effect on oxidative phosphorylation, ubiquitin proteolysis and insulin signaling. The confirmation of mRNA and DNA methylation changes and the clarifi‐ cation of these possible causal pathways may have implications for impaired synaptic function and neurogenesis, which contribute to the cognitive impairment seen in DS. These ongoing studies may further provide informative targets for early pharmaceutical interference to

The triplication of genes on HSA21 causes a wide spectrum of neurological phenotypes in DS, including mental retardation. DS individual displays not only delayed linguistic skills and a relatively low IQ (Intelligent Quotient) but also behavioral issues such as attention-deficit disorder (sometimes with hyperactivity) and autism [1-5]. The cognitive impairments extend further after development, as individuals with DS are more prone to develop Alzheimer's type dementia [6]. In addition, individuals with DS are susceptible to epilepsy in the form of infantile spasms and tonic clonic seizures with myoclonus at early ages [7-9]. These patholog‐ ical abnormalities in humans are, in part, replicated in DS animal models which show defects

Several genes on HSA21 are implicated in the abnormal neurodevelopment in DS [15]. They can affect cellular function at every stage of neural development, such as proliferation and differentiation of neuroprogenitor cells, neuronal survival and death, synapse formation, maturation and plasticity, as well as myelination. Disruption of each of these pathways can conceptually contribute to the MR seen in DS. Moreover, HSA21 genes have global effects on other genes; a meta-analysis of heterogeneous DS data identified 324 genes with consistent dosage effects, 77 on HSA21 and 247 on non-HSA21 [16]. Therefore, the over-expression of a not so small group of genes on HSA21 may initiate cascades of other signaling pathways on other chromosomes thorough an interactive network. The combinatorial effects from activa‐ tion of these processes may further contribute to the impairments seen during neurodevelop‐

modify DNA transcription and may affect many neurodevelopmental processes.

ameliorate the symptoms of mental retardation (MR) in DS.

in learning, social interactions, memory, and seizures [10-14].

ment in DS.

238 Down Syndrome

**2. Genetic mechanisms underlying the DS phenotype**

Increased levels of oxidative stress and reactive oxygen species (ROS) have commonly been associated with the DS brain. Free radicals are thought to disrupt the mitochondrial respiratory system, induce apoptosis of neurons and stimulate gliosis, which can further promote neuronal damage. This cyclical pathway may contribute to neuronal losses during neurogenesis as well as neuronal degeneration in adulthood. Several HSA21 genes have been implicated in generation of ROS including *DYRK1A*, *DSCR1*, *SOD1*, *ETS2, S100B, APP* and *BACH1* [15, 17]. Additionally, more recent studies would suggest a synergistic role for various HSA21 genes in induction of this pathological process. For example, over-expression of HSA21 genes *APP* and *S100B* synergistically increase hydrogen peroxide levels and decrease membrane potential in the mitochondria of human DS neuroprogenitor cells. The combination of a loss of mito‐ chondrial integrity and an increase of oxidative stress promotes apoptosis (changes in caspase and respiratory chain protein expression) and gliosis (increase of GFAP). S100B induction can occur through RAGE (Receptor for Advanced Glycation Endproducts) with consequent activation of JNK/p38 and JAK/STAT signaling. These stress response pathways are known to serve as downstream effectors potentially relevant to reactive gliosis, induction of S100B and glial associated aquaporin 4 [18, 19]. Increased levels of S100B and APP further enhance this cyclical cascade by promoting RAGE activation and inflammation with reactive gliosis. Lastly, multiple HSA21 genes have demonstrated enhanced APP-dependent toxic effects on the mitochondria whereas network prediction analyses have shown that four HSA21 proteins are components of the JAK/STAT pathway. These studies imply that an additional 19 HSA21 (among 2004 in total) proteins interact with components in this pathway [20]. These findings reiterate the large cascade of molecules that can be perturbed in a pathway following overexpression of a single gene.

Although oxidative stress in DS patients is considered to be a primary contributor of neuro‐ degeneration such as Alzheimer's Disease (AD) in adult patients, evidences from both human and animal models suggest that these same processes could also affect neurodevelopment and cognitive function at a much earlier age [19, 21-23]. Oxidative stress could therefore not only alter neuronal numbers through degeneration and changes in synaptic plasticity through impaired mitochondrial function, but also affect the generation of neurons during develop‐ ment. In this respect, ongoing effects from over-expression of HSA21 genes likely promote the cognitive dysfunction in DS throughout the lifetime of an individual with this disorder.

#### **2.2. Genetics mechanisms underlying neurogenesis in DS**

The observation of reduced cortical volume and decreased neuronal numbers in DS patients and animal models could in part be attributed to a reduction in the generation of neurons [24-27]. Over-expression of several HSA21 genes has been implicated in neurogenesis by either altering the rate or proliferation or by changing cell fate specification. By over-expressing HSA21-associated OLIG2, we observed a phenotypic shift in the neural progenitor pool toward glial progenitor phenotypes, accompanied by a corresponding decrease in the number of neuronal progenitors. This change can partly be explained by OLIG2-dependent inhibition of the expression and activity of KCNA3 outward rectifying potassium channels whose activa‐ tion stimulates proliferation of neural progenitors [28]. With respect to proliferation, APP overexpression can antagonistically compete with APPBP1, a protein required for the cell cycle progression from G1 to S phase [29]. Similarly, increased S100B levels stimulate p53 nuclear accumulation and inhibit proliferation [30]. DYRK1A has alternatively been shown to phos‐ phorylate p53, impair G1/S phase transition and inhibit proliferation [31]. Finally, many HSA21 genes regulate neurogenesis through their effects on NGF, hedgehog, WNT, Notch and insulin signaling pathways [20]. Changes in expression of various HSA21 genes can also regulate subpopulations of progenitors. For example, microarray profiling of DS human neuroproge‐ nitors implicated a defect in interneuron neurogenesis through increased expression of glial progenitor genes such as *OLIG1*, *OLIG2*, *OMG* and *COUP*-*TF1/NR2F1* and downregulation of the interneuron related genes *DLX1*, *DLX2* and *DLX5* [32].

DNMT3L. DNMT1 is responsible for DNA methylation maintenance while DNMT3A and DNMT3B are involved in *de novo* DNA methylation. DNMT2 is involved in RNA methylation. DNMT3L (DNA methyltransferase 3-like) does not have enzymatic activity but can stimulate DNMT3A and DNMT3B activation [55-57]. The addition of a methyl group to cytosine may physically impede the binding of transcriptional factors to the gene itself, or by recruiting protein complexes including methyl-CpG-binding protein 2 (MECP2), methyl-CpG-binding domain proteins (MBDs), HDACs and other chromatin remodeling proteins [58]. Alternative‐ ly, other enzymes involved in DNA demethylation can reverse this process. These molecules include cytidine deamination (AID, APOBEC) for deamination of cytosine and 5-methylcyto‐ sine and hydroxylation (TETs) for converting 5-methylcytosine to 5-hydroxymethylcytosine [59]. DNA modification, especially in the promoter region, by these various regulators may alter gene expression, and thereby affect many physiological processes [60]. In this context, proteins that affect the methylation machinery in DS are likely to alter gene expression and

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Epigenetic modification is thought to be an important contributor to development and numerous diseases. Several disorders associated with cognitive impairment such as X-linked alpha-thalassemia mental retardation (ATRX) syndrome, Rett syndrome, and Rubinstein– Taybi Syndrome involve some level of disruption in gene regulation through epigenetic effects [61]. The pathology is medicated by different mechanisms including histone modification, chromosome remodeling, small RNAs (siRNA, miRNA and other non-coding RNA) regula‐ tion and DNA methylation. More directly, *DNMT3B* mutations are associated with Immuno‐ deficiency, Centromere instability and Facial anomalies syndrome (ICF) with MR, suggesting that epigenetic alterations in the expression of genes regulating neurogenesis, axon branching, and neuronal migration such as IGF1 and ROBO1, contribute to cognitive impairment [62]. Certain features in DS may, in a similar fashion, be caused by epigenetic changes. For instance, HSA21 genes *DYRK1A*, *BRWD1* and *RUNX1* are associated with SWI/SNF complex, a chromatin remodeling complex that regulates the expression of subsets of genes such as *HDMTs*, *HMTs* and *HDACs*- histone modification proteins involved in controlling the expression of various interacting genes [63-65]. HSA21 genes *CHAF1B* and *HMGN1* express chromatin constitutive proteins involved in nucleosome assembly, which controls gene expression through DNA methylation and histone methylation or acetylation [66, 67]. Overexpression of HSA21 derived miRNA miR-155, miR802 in DS brain could also inhibit MECP2 expression, thereby mimicking *MECP2* loss of function in Rett syndrome with mental retar‐ dation. MECP2 transcriptionally activates and silences *CREB1* and *MEF2C*, genes that are critical in neurodevelopment [68-70]. DNA methylation is another extensively studied epigenetic regulator, being shown as impaired in many diseases. Although its importance has been recognized in cancers, its involvement in neurological disorders such as DS has not been

Several observations suggest that DNA methylation may play an important role in the DS endophenotype. Oxidative stress from over-expression of various HSA21 genes [15] could modulate DNA methylation directly through DNA damage or modification at the CpG sites, thereby preventing normal binding of DNMTs to DNA [71, 72]. DNMT3L is localized on

contribute to the DS phenotype.

well studied yet.

#### **2.3. Genetics mechanisms underlying synaptic formation, maturation and plasticity in DS**

A reduction in brain volume in DS has been attributed to impaired dendritic and synaptic maturation. Dendritic branching and spine number are dramatically reduced in pyramidal neurons in the hippocampus, visual cortex and motor cortex after 4 months postnatal age in individuals with DS [33-35]. The decreased number of spines is usually accompanied by aberrant spine morphology including enlarged or irregular spine heads, and sparse, small, short stalks intermingled with unusually long spines [34, 36]. In addition, DS brains also show changes in expression levels of various synaptic proteins such as decreased SEPT6, SYN1, SNAP-25, SYP and increased SYNJ1 levels [37-41]. Similar morphological changes have been observed in DS animal models and correlate on a molecular level with synaptic protein level changes and functionally with synaptic plasticity defects, observed through LTP, LTD and imbalance of excitatory-inhibitory neurotransmission [42-50]. Many genes on HSA21 (*TINM1*, *SYNJ1*, *ITSN1*; *KCNJ6*, *KCNJ15*, *KCNE1*, *KCNE2*; *NRIP1*, *ETS2*, *PCP4*, *DSCR1*, *DYRK1A*, *S100B*, *APP*, *OLIG1*, *OLIG2*) have been implicated in the synaptic pathology in DS, and the resulting phenotype likely involves a complex interrelationship between these various genes and their direct or indirect effect on various synaptic proteins [15, 48]. For instance, Dyrk1A overexpression could impair synaptic vesicle endocytosis, reduce dendrite branching and spine density of neurons; these phenotypes might be attributed to Dyrk1A induced hyperphos‐ phorylation of Tau and APP, or other synaptic proteins such as SYNJ1, resulting in impaired hippocampal-dependent learning [51-53]. Moreover, the multiple genetic interactions can additively promote the pathological DS synaptic endophenotype, as more severe defects were observed in Ts65dn mice than in Ts1Cje mice, the former of which contain a larger number of HSA21 associated genes [54].

## **3. Epigenetic mechanisms underlying the DS phenotype**

DNA methylation refers to a process of DNA modification that involves the enzymatic transfer of a methyl group from a methyl donor S-adenosylmethionine to carbon 5 of cytosine at 5'- CpG-3' sites. The enzymes carrying out this reaction are called DNA methyltransferases (DNMTs). There are five members in this family: DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. DNMT1 is responsible for DNA methylation maintenance while DNMT3A and DNMT3B are involved in *de novo* DNA methylation. DNMT2 is involved in RNA methylation. DNMT3L (DNA methyltransferase 3-like) does not have enzymatic activity but can stimulate DNMT3A and DNMT3B activation [55-57]. The addition of a methyl group to cytosine may physically impede the binding of transcriptional factors to the gene itself, or by recruiting protein complexes including methyl-CpG-binding protein 2 (MECP2), methyl-CpG-binding domain proteins (MBDs), HDACs and other chromatin remodeling proteins [58]. Alternative‐ ly, other enzymes involved in DNA demethylation can reverse this process. These molecules include cytidine deamination (AID, APOBEC) for deamination of cytosine and 5-methylcyto‐ sine and hydroxylation (TETs) for converting 5-methylcytosine to 5-hydroxymethylcytosine [59]. DNA modification, especially in the promoter region, by these various regulators may alter gene expression, and thereby affect many physiological processes [60]. In this context, proteins that affect the methylation machinery in DS are likely to alter gene expression and contribute to the DS phenotype.

tion stimulates proliferation of neural progenitors [28]. With respect to proliferation, APP overexpression can antagonistically compete with APPBP1, a protein required for the cell cycle progression from G1 to S phase [29]. Similarly, increased S100B levels stimulate p53 nuclear accumulation and inhibit proliferation [30]. DYRK1A has alternatively been shown to phos‐ phorylate p53, impair G1/S phase transition and inhibit proliferation [31]. Finally, many HSA21 genes regulate neurogenesis through their effects on NGF, hedgehog, WNT, Notch and insulin signaling pathways [20]. Changes in expression of various HSA21 genes can also regulate subpopulations of progenitors. For example, microarray profiling of DS human neuroproge‐ nitors implicated a defect in interneuron neurogenesis through increased expression of glial progenitor genes such as *OLIG1*, *OLIG2*, *OMG* and *COUP*-*TF1/NR2F1* and downregulation of

**2.3. Genetics mechanisms underlying synaptic formation, maturation and plasticity in DS** A reduction in brain volume in DS has been attributed to impaired dendritic and synaptic maturation. Dendritic branching and spine number are dramatically reduced in pyramidal neurons in the hippocampus, visual cortex and motor cortex after 4 months postnatal age in individuals with DS [33-35]. The decreased number of spines is usually accompanied by aberrant spine morphology including enlarged or irregular spine heads, and sparse, small, short stalks intermingled with unusually long spines [34, 36]. In addition, DS brains also show changes in expression levels of various synaptic proteins such as decreased SEPT6, SYN1, SNAP-25, SYP and increased SYNJ1 levels [37-41]. Similar morphological changes have been observed in DS animal models and correlate on a molecular level with synaptic protein level changes and functionally with synaptic plasticity defects, observed through LTP, LTD and imbalance of excitatory-inhibitory neurotransmission [42-50]. Many genes on HSA21 (*TINM1*, *SYNJ1*, *ITSN1*; *KCNJ6*, *KCNJ15*, *KCNE1*, *KCNE2*; *NRIP1*, *ETS2*, *PCP4*, *DSCR1*, *DYRK1A*, *S100B*, *APP*, *OLIG1*, *OLIG2*) have been implicated in the synaptic pathology in DS, and the resulting phenotype likely involves a complex interrelationship between these various genes and their direct or indirect effect on various synaptic proteins [15, 48]. For instance, Dyrk1A overexpression could impair synaptic vesicle endocytosis, reduce dendrite branching and spine density of neurons; these phenotypes might be attributed to Dyrk1A induced hyperphos‐ phorylation of Tau and APP, or other synaptic proteins such as SYNJ1, resulting in impaired hippocampal-dependent learning [51-53]. Moreover, the multiple genetic interactions can additively promote the pathological DS synaptic endophenotype, as more severe defects were observed in Ts65dn mice than in Ts1Cje mice, the former of which contain a larger number of

the interneuron related genes *DLX1*, *DLX2* and *DLX5* [32].

240 Down Syndrome

HSA21 associated genes [54].

**3. Epigenetic mechanisms underlying the DS phenotype**

DNA methylation refers to a process of DNA modification that involves the enzymatic transfer of a methyl group from a methyl donor S-adenosylmethionine to carbon 5 of cytosine at 5'- CpG-3' sites. The enzymes carrying out this reaction are called DNA methyltransferases (DNMTs). There are five members in this family: DNMT1, DNMT2, DNMT3A, DNMT3B and

Epigenetic modification is thought to be an important contributor to development and numerous diseases. Several disorders associated with cognitive impairment such as X-linked alpha-thalassemia mental retardation (ATRX) syndrome, Rett syndrome, and Rubinstein– Taybi Syndrome involve some level of disruption in gene regulation through epigenetic effects [61]. The pathology is medicated by different mechanisms including histone modification, chromosome remodeling, small RNAs (siRNA, miRNA and other non-coding RNA) regula‐ tion and DNA methylation. More directly, *DNMT3B* mutations are associated with Immuno‐ deficiency, Centromere instability and Facial anomalies syndrome (ICF) with MR, suggesting that epigenetic alterations in the expression of genes regulating neurogenesis, axon branching, and neuronal migration such as IGF1 and ROBO1, contribute to cognitive impairment [62]. Certain features in DS may, in a similar fashion, be caused by epigenetic changes. For instance, HSA21 genes *DYRK1A*, *BRWD1* and *RUNX1* are associated with SWI/SNF complex, a chromatin remodeling complex that regulates the expression of subsets of genes such as *HDMTs*, *HMTs* and *HDACs*- histone modification proteins involved in controlling the expression of various interacting genes [63-65]. HSA21 genes *CHAF1B* and *HMGN1* express chromatin constitutive proteins involved in nucleosome assembly, which controls gene expression through DNA methylation and histone methylation or acetylation [66, 67]. Overexpression of HSA21 derived miRNA miR-155, miR802 in DS brain could also inhibit MECP2 expression, thereby mimicking *MECP2* loss of function in Rett syndrome with mental retar‐ dation. MECP2 transcriptionally activates and silences *CREB1* and *MEF2C*, genes that are critical in neurodevelopment [68-70]. DNA methylation is another extensively studied epigenetic regulator, being shown as impaired in many diseases. Although its importance has been recognized in cancers, its involvement in neurological disorders such as DS has not been well studied yet.

Several observations suggest that DNA methylation may play an important role in the DS endophenotype. Oxidative stress from over-expression of various HSA21 genes [15] could modulate DNA methylation directly through DNA damage or modification at the CpG sites, thereby preventing normal binding of DNMTs to DNA [71, 72]. DNMT3L is localized on HSA21, and its triplication in DS suggests aberrant levels of expression. DNMT3L can form a heterotetramer with DNMT3A, and increased DNMT3L levels could potentially promote release of DNMT3A as well as increase its methylation activity [56]. DNMT3L can also stimulate DNMT3B activity directly [57, 73]. In addition, Dnmt3a modulates neurogenesis and synaptic plasticity in developing mouse neuroprogenitors and mature neurons by regulating related genes expression, such as *Bdnf, Reln, Dlx2, Gbx2, Sp8* and *Stat1* [74-77]. It remains to be seen whether other HSA21 genes in addition to DNMT3L can change the expression or activity of various epigenetic modifiers including the DNMTs, MBDs, HDACs or TETs. Overall, epigenetic modification provides an added layer of complexity to the interactive network established from over-expression of genes on HSA21. These modifiers also server as attractive candidates for targeting in DS given the broad effects they potentially have on a particular phenotype.

neurodegeneration, and they do so by regulating several genes on HSA21 involved in oxidative stress. Moreover, HSA21 genes associated with oxidative stress can influence the methylation

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DNA methylation regulates neurogenesis. Dnmts are broadly expressed in the brain and are dynamically regulated [90, 91]. For example, Dnmt1 is expressed in both dividing neuropro‐ genitors and postmitotic neurons [91, 92]. Dnmt3b is mainly expressed in neuroprogenitor cells during neurogenesis, whereas Dnmt3a is predominantly expressed in maturing brain (including neural precursors, neurons, astrocytes and oligodendrocytes). Dnmt3a expression peaks at three weeks after birth and then declines in adulthood [93, 94]. Dnmt3l directly regulates Dnmt3a and Dnmt3b but is weakly expressed in the brain and does not appear to disrupt normal cortical development. As for function, Hutnick et al used Emx1-cre to condi‐ tionally knockdown Dnmt1 exclusively in telencephalic precursors of mice, which induced hypomethylation in excitatory neurons and astrocytes of cortex and hippocampus. The methylation change increased neuronal apoptosis coupled with upregulation of apoptosisrelated genes such as *Gadd45a*, *Casp4* and *Ngfr*. Loss of Dnmt1 also impaired neurogenesis, maturation, learning and memory and was associated with downregulation of layer specific gene such as *Lhx2*, neuronal channel genes such as *Kcnh5*, *Kcnj9* and *Scnn1a* [95]. Interestingly, Gadd45b could contribute to DNA demethylation of pro-neuronal genes such as *BDNF* and *FGF* [96]. Studies using postnatal neural stem cells (NSC) in Dnmt3a knockout mice suggest that Dnmt3a promotes non-promoter DNA methylation of neurogenesis genes such as *Dlx2*, *Gbx2* and *Sp8* by functionally antagonizing Polycomb repression, resulting in increased expression of these genes [77]. Finally, the expression pattern of Dnmt3b suggests that it may

DNA methylation may directly effect neural progenitor development in DS. In normal development, Dnmt3l does not appear to have a significant phenotype in the developing mouse cerebral cortex, likely due to its relatively low expression levels in the brain [97, 98], (personal communications, Dr. Yi E. Sun, UCLA). DNMT3L, however, is located on chromo‐ some 21 and its triplication results in aberrantly high levels of expression in DS neuroproge‐ nitors (personal observations). Given that DNMT3L directly regulates both DNMT3A/B and both these proteins have been implicated in neural progenitor development, a pathological

role for methylation genes such as DNMT3L in contributing to neurogenesis is likely.

**3.3. Epigenetic mechanisms underlying synaptic formation, maturation and plasticity in DS**

Several HSA21 genes can indirectly regulate epigenetic factors involved in synaptic function. For example, SWI/SNF (SWItch/Sucrose NonFermentable) is a nucleosome-remodeling complex that can destabilize histone-DNA interactions in an ATP-dependent manner. HSA21 localized *DYRK1A* binds the SWI/SNF complex and subsequently induces a coordinated deregulation of multiple genes that are responsible for dendritic growth [65]. Likewise, APP has been shown to alter CpG methylation in three target genes CTIF (CBP80/CBP20-dependent translation initiation factor), NXT2 (nuclear exporting factor 2), and hypermethylated DDR2

**3.2. Epigenetic mechanisms underlying neurogenesis in DS**

be important for the early phase of neurogenesis (Feng et al., 2005).

status of other genes.

Next, we will discuss how DNA methylation could be involved in some important neurode‐ velopmental phenotypes in DS.

#### **3.1. Epigenetic mechanisms underlying oxidative stress in DS**

While excessive oxidative stress leading to mitochondrial dysfunction is a main feature of DS neurodevelopment, its effects on DNA methylation are not known. Currently no direct evidence demonstrates a role for oxidative stress in regulating DNA methylation changes in DS brain. However, DNA methylation studies from cancer seem to provide some clues. For instance, hydroxyl radicals generated from hydrogen peroxide can cause DNA damage including base modifications, deletions, and breakages, which could consequently interfere with normal function of DNMTs, leading to global hypomethylation in cancer cells [78]. 8- OHdG in CpG dinucleotides or the presence of O6-methylguanine could inhibit adjacent cytosine methylation [79-82] by inhibiting DNMTs or MBDs binding [83]. By extension, some of these same pathological mechanisms in cancer cells will likely be relevant in DS.

Methylation changes in the subset of DS genes involved in oxidative stress can contribute to similar phenotypes seen in DS development and disease. For instance, Dnmt1 conditional knockout in neural progenitor cells induced precocious astrogliogenesis through demethyla‐ tion of *S100b*, *Gfap* and *Stat1* promoters and activation of the JAK-STAT pathway. Silencing of these genes occurs through Mecp2 mediated inactivation of chromatin remodeling [84], with demethylation resulting in an increase in S100B, GFAP and STAT1 expression. Enhanced expression of these genes further promotes oxidative stress, cell death and gliosis. HSA21 localized APP could also be regulated by promoter dependent DNA methylation. The methylation pattern in the APP promoter is different in different tissues and even in different brain areas [85]. Hypomethylation of APP is found in the cerebral cortex of aging people and AD patients [86, 87]; the methylation frequency of CpG sites on APP promoter in younger people (26%) is higher than that in older people (8%), suggesting an age related methylation difference [86]. Altered methylation patterns have also been implicated in deregulation of APP processing enzymes PS1 and BACE in AD [88]. Finally, APP can also regulate the expression of other genes such as *CTIF, NTX2* and *DDR2* through DNA methylation [89]. Overall, these studies suggest that DNMTs appear to play some role in regulation of neurogenesis and neurodegeneration, and they do so by regulating several genes on HSA21 involved in oxidative stress. Moreover, HSA21 genes associated with oxidative stress can influence the methylation status of other genes.

### **3.2. Epigenetic mechanisms underlying neurogenesis in DS**

HSA21, and its triplication in DS suggests aberrant levels of expression. DNMT3L can form a heterotetramer with DNMT3A, and increased DNMT3L levels could potentially promote release of DNMT3A as well as increase its methylation activity [56]. DNMT3L can also stimulate DNMT3B activity directly [57, 73]. In addition, Dnmt3a modulates neurogenesis and synaptic plasticity in developing mouse neuroprogenitors and mature neurons by regulating related genes expression, such as *Bdnf, Reln, Dlx2, Gbx2, Sp8* and *Stat1* [74-77]. It remains to be seen whether other HSA21 genes in addition to DNMT3L can change the expression or activity of various epigenetic modifiers including the DNMTs, MBDs, HDACs or TETs. Overall, epigenetic modification provides an added layer of complexity to the interactive network established from over-expression of genes on HSA21. These modifiers also server as attractive candidates for targeting in DS given the broad effects they potentially have on a particular

Next, we will discuss how DNA methylation could be involved in some important neurode‐

While excessive oxidative stress leading to mitochondrial dysfunction is a main feature of DS neurodevelopment, its effects on DNA methylation are not known. Currently no direct evidence demonstrates a role for oxidative stress in regulating DNA methylation changes in DS brain. However, DNA methylation studies from cancer seem to provide some clues. For instance, hydroxyl radicals generated from hydrogen peroxide can cause DNA damage including base modifications, deletions, and breakages, which could consequently interfere with normal function of DNMTs, leading to global hypomethylation in cancer cells [78]. 8- OHdG in CpG dinucleotides or the presence of O6-methylguanine could inhibit adjacent cytosine methylation [79-82] by inhibiting DNMTs or MBDs binding [83]. By extension, some

of these same pathological mechanisms in cancer cells will likely be relevant in DS.

Methylation changes in the subset of DS genes involved in oxidative stress can contribute to similar phenotypes seen in DS development and disease. For instance, Dnmt1 conditional knockout in neural progenitor cells induced precocious astrogliogenesis through demethyla‐ tion of *S100b*, *Gfap* and *Stat1* promoters and activation of the JAK-STAT pathway. Silencing of these genes occurs through Mecp2 mediated inactivation of chromatin remodeling [84], with demethylation resulting in an increase in S100B, GFAP and STAT1 expression. Enhanced expression of these genes further promotes oxidative stress, cell death and gliosis. HSA21 localized APP could also be regulated by promoter dependent DNA methylation. The methylation pattern in the APP promoter is different in different tissues and even in different brain areas [85]. Hypomethylation of APP is found in the cerebral cortex of aging people and AD patients [86, 87]; the methylation frequency of CpG sites on APP promoter in younger people (26%) is higher than that in older people (8%), suggesting an age related methylation difference [86]. Altered methylation patterns have also been implicated in deregulation of APP processing enzymes PS1 and BACE in AD [88]. Finally, APP can also regulate the expression of other genes such as *CTIF, NTX2* and *DDR2* through DNA methylation [89]. Overall, these studies suggest that DNMTs appear to play some role in regulation of neurogenesis and

**3.1. Epigenetic mechanisms underlying oxidative stress in DS**

phenotype.

242 Down Syndrome

velopmental phenotypes in DS.

DNA methylation regulates neurogenesis. Dnmts are broadly expressed in the brain and are dynamically regulated [90, 91]. For example, Dnmt1 is expressed in both dividing neuropro‐ genitors and postmitotic neurons [91, 92]. Dnmt3b is mainly expressed in neuroprogenitor cells during neurogenesis, whereas Dnmt3a is predominantly expressed in maturing brain (including neural precursors, neurons, astrocytes and oligodendrocytes). Dnmt3a expression peaks at three weeks after birth and then declines in adulthood [93, 94]. Dnmt3l directly regulates Dnmt3a and Dnmt3b but is weakly expressed in the brain and does not appear to disrupt normal cortical development. As for function, Hutnick et al used Emx1-cre to condi‐ tionally knockdown Dnmt1 exclusively in telencephalic precursors of mice, which induced hypomethylation in excitatory neurons and astrocytes of cortex and hippocampus. The methylation change increased neuronal apoptosis coupled with upregulation of apoptosisrelated genes such as *Gadd45a*, *Casp4* and *Ngfr*. Loss of Dnmt1 also impaired neurogenesis, maturation, learning and memory and was associated with downregulation of layer specific gene such as *Lhx2*, neuronal channel genes such as *Kcnh5*, *Kcnj9* and *Scnn1a* [95]. Interestingly, Gadd45b could contribute to DNA demethylation of pro-neuronal genes such as *BDNF* and *FGF* [96]. Studies using postnatal neural stem cells (NSC) in Dnmt3a knockout mice suggest that Dnmt3a promotes non-promoter DNA methylation of neurogenesis genes such as *Dlx2*, *Gbx2* and *Sp8* by functionally antagonizing Polycomb repression, resulting in increased expression of these genes [77]. Finally, the expression pattern of Dnmt3b suggests that it may be important for the early phase of neurogenesis (Feng et al., 2005).

DNA methylation may directly effect neural progenitor development in DS. In normal development, Dnmt3l does not appear to have a significant phenotype in the developing mouse cerebral cortex, likely due to its relatively low expression levels in the brain [97, 98], (personal communications, Dr. Yi E. Sun, UCLA). DNMT3L, however, is located on chromo‐ some 21 and its triplication results in aberrantly high levels of expression in DS neuroproge‐ nitors (personal observations). Given that DNMT3L directly regulates both DNMT3A/B and both these proteins have been implicated in neural progenitor development, a pathological role for methylation genes such as DNMT3L in contributing to neurogenesis is likely.

#### **3.3. Epigenetic mechanisms underlying synaptic formation, maturation and plasticity in DS**

Several HSA21 genes can indirectly regulate epigenetic factors involved in synaptic function. For example, SWI/SNF (SWItch/Sucrose NonFermentable) is a nucleosome-remodeling complex that can destabilize histone-DNA interactions in an ATP-dependent manner. HSA21 localized *DYRK1A* binds the SWI/SNF complex and subsequently induces a coordinated deregulation of multiple genes that are responsible for dendritic growth [65]. Likewise, APP has been shown to alter CpG methylation in three target genes CTIF (CBP80/CBP20-dependent translation initiation factor), NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 [89]. DDR2 is a tyrosine kinase that functions as a cell surface receptor for fibrillar collagen and regulates cell differentiation, remodeling of the extracellular matrix, cell migration, cell proliferation, and cell cycle progression. More evidences from DNA methylation changing synaptic function come from Dnmt transgenic mice. Dnmt1 and Dnmt3a knockout mice show reduced LTP, deficits in learning and memory and deregulated genes expression associated with synaptic plasticity [74]. Dnmt3a overexpression increases spine density in nucleus accumbens [75]. DNMT3B is the gene mutated in ICF syndrome. Its mutation in lymphoblas‐ toid cell line from patients led to altered genes expression of several systems including regulators of neurogenesis and synaptic function, such as ROBO1, JPH4, FRY, MAP4K4, PCDHGC3, IGF1, SNCA, GABRA4 and BCHE [62]. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, whose mutation leads to reduced neurogenesis, decreased LTP and impaired spatial learning [99]. The involvement of Dnmts and Hdacs in synaptic function is further supported by pharmacological manipulations [100-102]. For instance, Dnmt inhibitors zebularine and 5-aza-2-deoxycytidine can alter DNA methylation at promoters for Reln and Bdnf, and block the induction of LTP in synapses of mouse hippocampus [103].

**4.1. Oxidative phosphorylation**

**4.2. Insulin signaling**

Oxidative phosphorylation involves cellular metabolism through oxidation to produce ATP. The broad methylation and gene expression changes in this pathway suggest its role as a primary consequence of DS genes' overdose effects. Plasma membrane NADPH oxidase is considered a major producer of ROS in neurons or astrocytes in brain and is activated by S100B through a RAGE-dependent pathway [108-111]. Over-expression of HSA21 genes such as *S100B* and *APP* likely promote this pathway and cause cell death in DS neurons [19]. Small amounts of superoxide anion and peroxide are also produced by the electron transport chain in mitochondria [112-114]. The global deregulation of enzymes in this mitochondrial pathway could thus disrupt the balance between oxidant generation and ATP production, result in enhanced ROS generation and lead to diminished ATP levels [115, 116]. Several DS genes have been implicated in this process. For instance, three HSA21 genes, *ATP5J*, *ATP5O* and *NDUFV3* are components of ATP synthase and NADH dehydrogenase, though their expression and regulation in DS brain are not known yet. In addition, other HSA21 genes may indirectly affect this pathway. Alternatively, HSA21 gene *S100B* may target mitochondrial proteins such as p53 and ATPase ATAD3A, thereby assisting the cytoplasmic processing of proteins for proper folding and subcellular localization [117-121]. Another HSA21 gene APP and its product beta amyloid can interact with import receptors to gain entry into mitochondrial compartment, where they accumulate and affect the normal function of this pathway [122, 123]. Finally, gene expression in mitochondrial oxidative phosphorylation may be modulated by DNA methyl‐ ation. For instance, prenatal protein diet excess or restriction leads to hypomethylation of CpG sites in the cytochrome C *CYCS* gene promoter, including those representing putative transcription factor-binding sites. Elevation of this protein can alter electron transport chain function in mitochondria and initiate apoptosis [124]. Our preliminary studies suggest there is a broad change of DNA methylation and genes expression in this oxidative phosphorylation pathway. Given the importance of ATP/ROS metabolism in mitochondrial function, further

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studies will be needed to understand the epigenetic contribution to this pathway.

The insulin/insulin growth factor (IGF)-I pathway is a conserved pathway required for neurogenesis and neuroprotection. It acts through IR/IGF-IR, IRS, and RAS/MAPK or PI3K/AKT in regulating neurogenic cell fate [125]. Decreased levels of IGF-I have been found to associate with growth retardation in DS patients, which could be rescued by GH therapy [126, 127]. In addition, the insulin receptor knockout mouse suggests that neurons without insulin receptor exhibit significant reduction of Akt and Gsk3beta and increased tau hyper‐ phosphorylation, characteristics of neurotoxicity in DS and AD [128]. Inhibition of the brain insulin signaling pathways have been report in AD brain, with decreased expression of IR, IRS1, IRS2, PI3K and AKT [129, 130]. This deficiency may, in part, involve DNA methylation changes, given reports of co-localization of Hdac2 with insulin signaling components (Ir, Irs) in postsynaptic glutamatergic neurons of the mouse hippocampus [131]. DNA methylation changes in human DS progenitors (personal observations Lu and Sheen) also suggest that the insulin-associated pathways may contribute to the DS endophenotype during development.

## **4. Global effects of DNA methylation in causing DS phenotypes**

Several reports have shown global DNA methylation changes in DS [104, 105]. For example, individual proteins on HSA21 such as beta amyloid (the protein encoded by HSA21 localized APP) can induce global hypomethylation [106, 107]. Comparison of normal and DS methyla‐ tion in DS leukocytes and T lymphocytes using microarray-based profiling (MSNP (single nucleotide polymorphism (SNP) chip-based method for profiling DNA methylation) identi‐ fied a small subset of genes with altered methylation, specific to the DS cell population [104]. Among the genes identified, five candidates (*TMEM131*, *CD3Z*, *NOD2* and *NPDC1*) showed correlation with RNA expression, and the methylation changes could be recapitulated by exposing normal lymphocytes to the demethylation drug 5-aza-cytidine. These genes have known or predicted roles in lymphocyte development. In order to gain some insights into the DNA methylation deregulation in DS brain, we have performed some preliminary studies by comparing the methylation profiles of control (CON) and DS frontal cortex from 18 gestational weeks' fetal brain using Illumina 450 Infinium Beadchip assay. Approximately 4% of the CpG sites showed significant changes at the methylation level. When compared to CON baseline methylated and unmethylated states, more CON unmethylated CpG sites became methylated in DS than CON methylated states that became unmethylated. Moreover, there was overall greater global hyper versus hypomethylation in DS compared to CON across all chromosomes, except on HSA21. Chromosome 21 actually demonstrated a greater degree of hypo versus hypermethylation in DS (unpublished data). Hypomethylation generally results in increased gene transcription, whereas hypermethylation leads to the converse. Cross comparison of DNA methylation states with the differential mRNA expression genes from previous micro‐ array studies, suggested epigenetic effects on several specific pathways (oxidative phosphor‐ ylation, insulin signaling and ubiquitination).

#### **4.1. Oxidative phosphorylation**

[89]. DDR2 is a tyrosine kinase that functions as a cell surface receptor for fibrillar collagen and regulates cell differentiation, remodeling of the extracellular matrix, cell migration, cell proliferation, and cell cycle progression. More evidences from DNA methylation changing synaptic function come from Dnmt transgenic mice. Dnmt1 and Dnmt3a knockout mice show reduced LTP, deficits in learning and memory and deregulated genes expression associated with synaptic plasticity [74]. Dnmt3a overexpression increases spine density in nucleus accumbens [75]. DNMT3B is the gene mutated in ICF syndrome. Its mutation in lymphoblas‐ toid cell line from patients led to altered genes expression of several systems including regulators of neurogenesis and synaptic function, such as ROBO1, JPH4, FRY, MAP4K4, PCDHGC3, IGF1, SNCA, GABRA4 and BCHE [62]. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, whose mutation leads to reduced neurogenesis, decreased LTP and impaired spatial learning [99]. The involvement of Dnmts and Hdacs in synaptic function is further supported by pharmacological manipulations [100-102]. For instance, Dnmt inhibitors zebularine and 5-aza-2-deoxycytidine can alter DNA methylation at promoters for Reln and Bdnf, and block the induction of LTP in synapses of

**4. Global effects of DNA methylation in causing DS phenotypes**

Several reports have shown global DNA methylation changes in DS [104, 105]. For example, individual proteins on HSA21 such as beta amyloid (the protein encoded by HSA21 localized APP) can induce global hypomethylation [106, 107]. Comparison of normal and DS methyla‐ tion in DS leukocytes and T lymphocytes using microarray-based profiling (MSNP (single nucleotide polymorphism (SNP) chip-based method for profiling DNA methylation) identi‐ fied a small subset of genes with altered methylation, specific to the DS cell population [104]. Among the genes identified, five candidates (*TMEM131*, *CD3Z*, *NOD2* and *NPDC1*) showed correlation with RNA expression, and the methylation changes could be recapitulated by exposing normal lymphocytes to the demethylation drug 5-aza-cytidine. These genes have known or predicted roles in lymphocyte development. In order to gain some insights into the DNA methylation deregulation in DS brain, we have performed some preliminary studies by comparing the methylation profiles of control (CON) and DS frontal cortex from 18 gestational weeks' fetal brain using Illumina 450 Infinium Beadchip assay. Approximately 4% of the CpG sites showed significant changes at the methylation level. When compared to CON baseline methylated and unmethylated states, more CON unmethylated CpG sites became methylated in DS than CON methylated states that became unmethylated. Moreover, there was overall greater global hyper versus hypomethylation in DS compared to CON across all chromosomes, except on HSA21. Chromosome 21 actually demonstrated a greater degree of hypo versus hypermethylation in DS (unpublished data). Hypomethylation generally results in increased gene transcription, whereas hypermethylation leads to the converse. Cross comparison of DNA methylation states with the differential mRNA expression genes from previous micro‐ array studies, suggested epigenetic effects on several specific pathways (oxidative phosphor‐

mouse hippocampus [103].

244 Down Syndrome

ylation, insulin signaling and ubiquitination).

Oxidative phosphorylation involves cellular metabolism through oxidation to produce ATP. The broad methylation and gene expression changes in this pathway suggest its role as a primary consequence of DS genes' overdose effects. Plasma membrane NADPH oxidase is considered a major producer of ROS in neurons or astrocytes in brain and is activated by S100B through a RAGE-dependent pathway [108-111]. Over-expression of HSA21 genes such as *S100B* and *APP* likely promote this pathway and cause cell death in DS neurons [19]. Small amounts of superoxide anion and peroxide are also produced by the electron transport chain in mitochondria [112-114]. The global deregulation of enzymes in this mitochondrial pathway could thus disrupt the balance between oxidant generation and ATP production, result in enhanced ROS generation and lead to diminished ATP levels [115, 116]. Several DS genes have been implicated in this process. For instance, three HSA21 genes, *ATP5J*, *ATP5O* and *NDUFV3* are components of ATP synthase and NADH dehydrogenase, though their expression and regulation in DS brain are not known yet. In addition, other HSA21 genes may indirectly affect this pathway. Alternatively, HSA21 gene *S100B* may target mitochondrial proteins such as p53 and ATPase ATAD3A, thereby assisting the cytoplasmic processing of proteins for proper folding and subcellular localization [117-121]. Another HSA21 gene APP and its product beta amyloid can interact with import receptors to gain entry into mitochondrial compartment, where they accumulate and affect the normal function of this pathway [122, 123]. Finally, gene expression in mitochondrial oxidative phosphorylation may be modulated by DNA methyl‐ ation. For instance, prenatal protein diet excess or restriction leads to hypomethylation of CpG sites in the cytochrome C *CYCS* gene promoter, including those representing putative transcription factor-binding sites. Elevation of this protein can alter electron transport chain function in mitochondria and initiate apoptosis [124]. Our preliminary studies suggest there is a broad change of DNA methylation and genes expression in this oxidative phosphorylation pathway. Given the importance of ATP/ROS metabolism in mitochondrial function, further studies will be needed to understand the epigenetic contribution to this pathway.

#### **4.2. Insulin signaling**

The insulin/insulin growth factor (IGF)-I pathway is a conserved pathway required for neurogenesis and neuroprotection. It acts through IR/IGF-IR, IRS, and RAS/MAPK or PI3K/AKT in regulating neurogenic cell fate [125]. Decreased levels of IGF-I have been found to associate with growth retardation in DS patients, which could be rescued by GH therapy [126, 127]. In addition, the insulin receptor knockout mouse suggests that neurons without insulin receptor exhibit significant reduction of Akt and Gsk3beta and increased tau hyper‐ phosphorylation, characteristics of neurotoxicity in DS and AD [128]. Inhibition of the brain insulin signaling pathways have been report in AD brain, with decreased expression of IR, IRS1, IRS2, PI3K and AKT [129, 130]. This deficiency may, in part, involve DNA methylation changes, given reports of co-localization of Hdac2 with insulin signaling components (Ir, Irs) in postsynaptic glutamatergic neurons of the mouse hippocampus [131]. DNA methylation changes in human DS progenitors (personal observations Lu and Sheen) also suggest that the insulin-associated pathways may contribute to the DS endophenotype during development.

#### **4.3. Ubiquitin proteolysis**

The ubiquitin proteasome/lysome system (UPLS) is responsible for the removal of excessive proteins from multiple cellular compartments (especially mitochondria and synapses) in order to maintain normal cellular function [132, 133]. Progression in DS cognitive impairment is associated with accumulation of NF plaques and tangles, which have been shown to contain ubiquitin [134]. Dystrophic neurites in DS also contain ubiquitin and the UPLS-associated molecules PSMA5 and USP5 are upregulated in DS fetal brain [135]. Beta amyloid could regulate synaptic protein degradation and function through ubiquitin pathway [136, 137]. Moreover, several E3 ubiquitin ligases have been shown to promote APP degradation [138, 139]. Additionally, HSA21 located genes *AIRE* and *UBE2G2* are directly involved in the ubiquitin pathway and could contribute to the phenotype. Taken in this context, disruption of mitochondrial function (i.e. through S100B, APP, OLIG2 or disruption of the oxidative phosphorylation pathway) might consequently impair ubiquitin-dependent lysosomal and proteosomal clearance, because it is an ATP-dependent process. Finally, our preliminary studies suggest that DNA methylation may also directly impair ubiquitin function. Loss of ubiquitin function would have direct effects on synaptic function and structure (through beta amyloid or synaptic proteins) but would also possibly enhance oxidative stress and mito‐ chondrial dysfunction. It is interesting to note that the high throughput DNA methylation screen in DS invoked changes in methylation involving three networks (oxidative phosphor‐ ylation, insulin signaling, and ubiquitin function), which are highly dependent on one another. features in DS and neuronal survival [19, 145]. These observations would suggest combinato‐ rial and interactive effects between these genes in contributing to the MR seen in DS. It remains to be seen whether DNMT3L effects on DNMT3A/B are responsible for the part of the preliminary methylation defects seen in the several pathways discussed above. It is also not known how the trisomy of HSA21 genes will effect methylation, but it is highly likely that DNMT3L alters at least a subset of genes. In this respect, it will be important to identify the causative methylation defects due to this single gene, as it will have implications for other DS

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The epigenetic screens in DS predict involvement of several mutually interactive pathways in contributing to the neurological endophenotype in this disorder: oxidative phosphorylation, insulin signaling, and ubiquitination. Approaches for therapeutic intervention possibly involve either altering the methylation patterns or directly targeting specific pathways.

If global hypermethylation in DS neuroprogenitors is confirmed, then inhibition of DNMT or DNA deamination could be used to rescue or treat the pathological phenotypes. There are two clinical licensed DNMT inhibitors currently used in myelodysplastic syndrome, where they relieve the repression of tumor suppressor genes: 5-aza-cytidine (Vidaza®) and 5-aza-2' deoxycytidine (Dacogen®) [59]. In addition, because of the occurrence of hypomethylation, especially on HSA21, it would be desirable to develop a more specific methylation inhibitor/ activator or deamination activator/inhibitor in order to target specific promoters of genes in

Dysfunction of the UPLS system causes protein accumulation or over-degradation in cellular organelles. Thus developing activator or inhibitor of proteasomes would have therapeutic meaning. Most currently available activators/inhibitors of the ubiquitin-proteasome pathway directly target the subunits of proteasome, the core of the proteolysis machinery, instead of targeting upstream ubiquitination and recognition of ubiquitinated protein substrates by more specific E3 ubiquitin ligases. Proteasome inhibitors such as Bortezomib, (Velcade®) are in clinical treatment for multiple myeloma [146, 147]. Proteasome activators including 11s

Preservation of oxidative phosphorylation pathway and mitochondrial function can be achieved through a new investigational drug EPI-743, currently in phase 2B/3 pivotal clinical trials in Inherited Mitochondrial Respiratory Chain Disease [148]. EPI-743 is an orally absorb‐ able small molecule that readily crosses into the central nervous system. It works by targeting an enzyme NADPH quinone oxidoreductase 1 (NQO1). Its mode of action is to synchronize energy generation in mitochondria with the need to counter cellular redox stress [149].

**6. Possible targets for pharmaceutical interference**

activator, Blm10/PA200, and 19s activator are still under research.

phenotypes.

important pathways.

## **5. Possible functions of** *DNMT3L* **in DS**

Given that DNMT3A and DNMT3B are involved in neurogenesis and synaptic plasticity, HSA21 localized *DNMT3L* regulates activities of *DNMT3A/3B*, suggesting that over-expres‐ sion of this gene will have pathological implications in methylation patterns involved in neural development. Moreover, DNMT3L represses transcription by recruiting HDACs, which may also affect the neurodevelopment [140, 141]. *Dnmt3l* null mice do not demonstrate a neuro‐ logical phenotype due to low levels of expression but rather exhibits defects in reproductive organs where it is highly expressed and leads to imprinting and differentiation defect in early stages of embryonic development [97, 98]. *DNMT3L (R271Q)* variant is associated with significant DNA hypomethylation at the subtelomeric region in healthy human, though it does not seem to cause any diseases [142]. On the other hand, over-expression of DNMT3L in Hela cells mimics the characteristics of iPS cells and carcinogenesis by upregulating SOX2, HOX genes and DNMTs including DNMT1 and DNMT3B expression, suggesting that DNMT3L over-expression may change the DNA methylation profile in later stages of embryo develop‐ ment through activating DNMT3A/DNMT3B when neurogenesis and synapse formation happen [143]. Interestingly, a recently developed DS model Dp(10)1Yey/+ mice harboring a duplication spanning the entire HSA21 syntenic region on mouse chromosome 10 (Mmu10), which contains Dnmt3l and S100b, did not show alterations in cognitive behaviors or hippo‐ campal LTP [144]. However, other mouse transgenic studies with over-expression of select HSA21 genes (i.e. APP and S100b) have shown combinatorial effects in contributing to AD features in DS and neuronal survival [19, 145]. These observations would suggest combinato‐ rial and interactive effects between these genes in contributing to the MR seen in DS. It remains to be seen whether DNMT3L effects on DNMT3A/B are responsible for the part of the preliminary methylation defects seen in the several pathways discussed above. It is also not known how the trisomy of HSA21 genes will effect methylation, but it is highly likely that DNMT3L alters at least a subset of genes. In this respect, it will be important to identify the causative methylation defects due to this single gene, as it will have implications for other DS phenotypes.

## **6. Possible targets for pharmaceutical interference**

**4.3. Ubiquitin proteolysis**

246 Down Syndrome

**5. Possible functions of** *DNMT3L* **in DS**

The ubiquitin proteasome/lysome system (UPLS) is responsible for the removal of excessive proteins from multiple cellular compartments (especially mitochondria and synapses) in order to maintain normal cellular function [132, 133]. Progression in DS cognitive impairment is associated with accumulation of NF plaques and tangles, which have been shown to contain ubiquitin [134]. Dystrophic neurites in DS also contain ubiquitin and the UPLS-associated molecules PSMA5 and USP5 are upregulated in DS fetal brain [135]. Beta amyloid could regulate synaptic protein degradation and function through ubiquitin pathway [136, 137]. Moreover, several E3 ubiquitin ligases have been shown to promote APP degradation [138, 139]. Additionally, HSA21 located genes *AIRE* and *UBE2G2* are directly involved in the ubiquitin pathway and could contribute to the phenotype. Taken in this context, disruption of mitochondrial function (i.e. through S100B, APP, OLIG2 or disruption of the oxidative phosphorylation pathway) might consequently impair ubiquitin-dependent lysosomal and proteosomal clearance, because it is an ATP-dependent process. Finally, our preliminary studies suggest that DNA methylation may also directly impair ubiquitin function. Loss of ubiquitin function would have direct effects on synaptic function and structure (through beta amyloid or synaptic proteins) but would also possibly enhance oxidative stress and mito‐ chondrial dysfunction. It is interesting to note that the high throughput DNA methylation screen in DS invoked changes in methylation involving three networks (oxidative phosphor‐ ylation, insulin signaling, and ubiquitin function), which are highly dependent on one another.

Given that DNMT3A and DNMT3B are involved in neurogenesis and synaptic plasticity, HSA21 localized *DNMT3L* regulates activities of *DNMT3A/3B*, suggesting that over-expres‐ sion of this gene will have pathological implications in methylation patterns involved in neural development. Moreover, DNMT3L represses transcription by recruiting HDACs, which may also affect the neurodevelopment [140, 141]. *Dnmt3l* null mice do not demonstrate a neuro‐ logical phenotype due to low levels of expression but rather exhibits defects in reproductive organs where it is highly expressed and leads to imprinting and differentiation defect in early stages of embryonic development [97, 98]. *DNMT3L (R271Q)* variant is associated with significant DNA hypomethylation at the subtelomeric region in healthy human, though it does not seem to cause any diseases [142]. On the other hand, over-expression of DNMT3L in Hela cells mimics the characteristics of iPS cells and carcinogenesis by upregulating SOX2, HOX genes and DNMTs including DNMT1 and DNMT3B expression, suggesting that DNMT3L over-expression may change the DNA methylation profile in later stages of embryo develop‐ ment through activating DNMT3A/DNMT3B when neurogenesis and synapse formation happen [143]. Interestingly, a recently developed DS model Dp(10)1Yey/+ mice harboring a duplication spanning the entire HSA21 syntenic region on mouse chromosome 10 (Mmu10), which contains Dnmt3l and S100b, did not show alterations in cognitive behaviors or hippo‐ campal LTP [144]. However, other mouse transgenic studies with over-expression of select HSA21 genes (i.e. APP and S100b) have shown combinatorial effects in contributing to AD The epigenetic screens in DS predict involvement of several mutually interactive pathways in contributing to the neurological endophenotype in this disorder: oxidative phosphorylation, insulin signaling, and ubiquitination. Approaches for therapeutic intervention possibly involve either altering the methylation patterns or directly targeting specific pathways.

If global hypermethylation in DS neuroprogenitors is confirmed, then inhibition of DNMT or DNA deamination could be used to rescue or treat the pathological phenotypes. There are two clinical licensed DNMT inhibitors currently used in myelodysplastic syndrome, where they relieve the repression of tumor suppressor genes: 5-aza-cytidine (Vidaza®) and 5-aza-2' deoxycytidine (Dacogen®) [59]. In addition, because of the occurrence of hypomethylation, especially on HSA21, it would be desirable to develop a more specific methylation inhibitor/ activator or deamination activator/inhibitor in order to target specific promoters of genes in important pathways.

Dysfunction of the UPLS system causes protein accumulation or over-degradation in cellular organelles. Thus developing activator or inhibitor of proteasomes would have therapeutic meaning. Most currently available activators/inhibitors of the ubiquitin-proteasome pathway directly target the subunits of proteasome, the core of the proteolysis machinery, instead of targeting upstream ubiquitination and recognition of ubiquitinated protein substrates by more specific E3 ubiquitin ligases. Proteasome inhibitors such as Bortezomib, (Velcade®) are in clinical treatment for multiple myeloma [146, 147]. Proteasome activators including 11s activator, Blm10/PA200, and 19s activator are still under research.

Preservation of oxidative phosphorylation pathway and mitochondrial function can be achieved through a new investigational drug EPI-743, currently in phase 2B/3 pivotal clinical trials in Inherited Mitochondrial Respiratory Chain Disease [148]. EPI-743 is an orally absorb‐ able small molecule that readily crosses into the central nervous system. It works by targeting an enzyme NADPH quinone oxidoreductase 1 (NQO1). Its mode of action is to synchronize energy generation in mitochondria with the need to counter cellular redox stress [149].

## **7. Conclusion**

DS is a contiguous gene syndrome which gives rise to MR, dementia, and seizures. These clinical outcomes are mirrored by endophenotypes including increased oxidative stress, decreased neurogenesis and synaptic dysfunction. While these characteristics have largely been attributed to HSA21 gene dosage effects, recent progresses in epigenetic studies have raised the high likelihood that DNA methylation have significant effects on DS neurodevel‐ opment. Methylome screening suggests disruption of pathways involving oxidative phos‐ phorylation, ubiquitination and insulin signaling in DS. Candidate gene analyses suggest that DNMT3L is over-expressed in DS given its location on chromosome 21. Alternatively, other studies have implicated several HSA21 genes in altering methylation sites on genes involved in these same pathways. The pathways invoked through epigenetic regulation contribute directly to known pathological mechanisms identified on prior gene expression profiling such as oxidative stress, gliosis, and mitochondrial dysfunction. In this respect, the DS brain endophenotypes likely arise from the integration of various genetic and epigenetic factors on chromosome 21.

[3] Gath A, Gumley D. Behaviour problems in retarded children with special reference to Down's syndrome. The British journal of psychiatry : the journal of mental science. 1986

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249

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## **Acknowledgements**

This work was supported by grants to V.L.S from NINDS 1R01NS063997 and NICHD 1R21HD054347.

## **Author details**

Jie Lu and Volney Sheen\*

\*Address all correspondence to: vsheen@bidmc.harvard.edu

Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA, USA

## **References**


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**7. Conclusion**

248 Down Syndrome

chromosome 21.

1R21HD054347.

**Author details**

**References**

Jie Lu and Volney Sheen\*

\*Address all correspondence to: vsheen@bidmc.harvard.edu

**Acknowledgements**

DS is a contiguous gene syndrome which gives rise to MR, dementia, and seizures. These clinical outcomes are mirrored by endophenotypes including increased oxidative stress, decreased neurogenesis and synaptic dysfunction. While these characteristics have largely been attributed to HSA21 gene dosage effects, recent progresses in epigenetic studies have raised the high likelihood that DNA methylation have significant effects on DS neurodevel‐ opment. Methylome screening suggests disruption of pathways involving oxidative phos‐ phorylation, ubiquitination and insulin signaling in DS. Candidate gene analyses suggest that DNMT3L is over-expressed in DS given its location on chromosome 21. Alternatively, other studies have implicated several HSA21 genes in altering methylation sites on genes involved in these same pathways. The pathways invoked through epigenetic regulation contribute directly to known pathological mechanisms identified on prior gene expression profiling such as oxidative stress, gliosis, and mitochondrial dysfunction. In this respect, the DS brain endophenotypes likely arise from the integration of various genetic and epigenetic factors on

This work was supported by grants to V.L.S from NINDS 1R01NS063997 and NICHD

Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA, USA

(Clinical Trial Controlled Clinical Trial). 1999 Apr;29(2):149-56.

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## *Edited by Subrata Kumar Dey*

Down syndrome, the most cutting-edge book in the field congenital disorders. This book features up-to-date, well referenced research and review articles on Down syndrome. Research workers, scientists, medical graduates and pediatricians will find it to be an excellent source for references and review. It is hoped that such individuals will view this book as a resource that can be consulted during all stages of their research and clinical investigations. Key features of this book are: Common diseases in Down syndrome; Molecular Genetics; Neurological Disorders; Prenatal Diagnosis and Genetic Counselling. Whilst aimed primarily at research workers on Down syndrome, we hope that the appeal of this book will extend beyond the narrow confines of academic interest and be of interest to a wider audience, especially parents, relatives and health-care providers who work with infants and children with Down syndrome.

Down Syndrome

Down Syndrome

*Edited by Subrata Kumar Dey*

Photo by Cappan / iStock