**Prenatal Diagnosis of Severe Perinatal (Lethal) Hypophosphatasia**

Atsushi Watanabe, Hideo Orimo, Toshiyuki Takeshita and Takashi Shimada *Department of Biochemistry and Molecular Biology, Nippon Medical School Division of Clinical Genetics, Nippon Medical School Hospital Department of Obstetrics and Gynecology, Nippon Medical School Japan* 

### **1. Introduction**

26 Prenatal Diagnosis – Morphology Scan and Invasive Methods

Von Dadelszen,P.;Sermer,M.; Hillier,J.(2005).A randomized controlled trial of biopsy forceps

Wapner, RL.; Johnson, A.; Davis, G.; Urban, A.; Morgan, P.; Jackson, L.(1993). Prenatal

Wilson, RD.(2005) Amended Canadian Guideline for prenatal diagnosis (2005) change to

Welch, RA.; Soha-Salem, EM.;Wiktor, BS.; Van Dyke, DL.; Blessed, WB.(2006). Operator

Wilson, RD. (2007).Letter to Editor: Pregnancy loss rates after midtrimester amniocentesis.

Wurster,KG.;Roemer,VM.;Decker,K.;Hirsch,HA.(1982).Amniotic infection syndrome after amniocentesis.*Geburtshilfe Frauenheilkd*.vol.42,No.9,(September 1982),pp.676-679 Yukobowich, E,.;Anteby ,EY,.;Cohen, SM.; Lavy, Y.;Granat, M.; Yagel, S.(2001). Risk of fetal

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2005—techniques for prenatal diagnosis. SOGC Clinical Practice Guidelines.*Journal of Obstetrics and Gynecology Canada* , vol.27,No. 11, (November 2005). pp.1048–1054 Weiner, S. (1991).Indications, complications ,safety,reliability, and assessment of quality of

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loss in twin pregnancies undergoing second trimester amniocentesis. *Obstet* 

Hypophosphatasia (HPP) is an inherited disorder characterized by defective mineralization of the bone and low activity of alkaline phosphatase (ALP; EC 3.1.3.1) (Mornet, 2008). Screening for mutations in the *ALPL* gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of HPP.

### **2. Hypophosphatasia**

HPP is a clinically heterogeneous disease and classified into at least six forms according to severity and age of onset: perinatal (lethal), perinatal (benign), infantile (MIM [Mendelian Inheritance in Man] # 241500), childhood (MIM# 241510), adult (MIM# 146300), and odontohypophosphatasia (Mornet, 2008) (Table 1). All forms of HPP display reduced activity of unfractionated serum ALP and the presence of either one or two pathologic mutations in the liver/bone/kidney alkaline phosphatase gene (*ALPL,* MIM# 171760), the gene encoding ALP, the tissue-nonspecific isozyme (TNSALP). There is no curative treatment for HPP to date.

### **2.1** *ALPL* **gene**

*ALPL* is the only gene known to be associated with HPP. *ALPL* consists of 12 exons: 11 coding exons and one untranslated exon. More than250 *ALPL* mutations have been described in persons from North America, Japan, and Europe (The Tissue Nonspecific Alkaline Phosphatase Gene Mutations Database). HPP is frequently caused by p.E191K and p.D378V in Caucasian, whereas p.F327L and c.1559delT are more common in Japanese(the first nucleotide (+1) corresponds to the A of the ATG initiation codon using the *ALPL* cDNA number of the standard nomenclature). This variety of mutations in *ALPL* results in highly variable clinical expression and in a great number of compound heterozygous genotypes.

#### **2.2 Perinatal (lethal) form of hypophosphatasia**

The perinatal (lethal) form of HPP (pl-HPP) is the most severe HPP with an autosomal recessive mode of inheritance (Gehring et al., 1999) In the lethal perinatal form, the patients

Prenatal Diagnosis of Severe Perinatal (Lethal) Hypophosphatasia 29

deletions and insertions of nucleotides that change the reading frame will be highly deleterious. Some patients with pl-HPP are homozygous for c.1559delT, with parents who are heterozygous carriers for the mutation but with no evidence of consanguinity (Fig. 2) (Sawai et al. 2003). Patients who are homozygous for the c.1559delT mutation differed in the severity of HPP, including both their symptoms and serum ALP activity. In the c.1559delT mutation, the symptom in HPP of homozygous mutation is responsible for a severe phenotype, but that of compound hetero varies from severe to mild that depends on

Fig. 2. Direct sequencing results around 1559delT in *ALPL* from a pl-HPP patient both parents, and healthy control. The sequence of the parents could not be determined in progress at cDNA number 1559 of the *ALPL* and these results indicate both parents were

c.1559delT, a deletion of T at 1559, which caused a frameshift downstream from leucine (Leu) at codon 503, resulted in the elimination of the termination codon at 508 and the addition of 80 amino acid residues at the C-terminus. The mutant protein caused by 1559delT formed an aggregate, was polyubiquitinated, and was then degraded in the proteome (Komaru et al., 2005), thus allowing us to directly correlate the phenotype

heterogenous carriers for 1559delT. The sequence of the pl-HPP patient could be determined in progress containing the deletion of T at nucleotide 1559, which was different from that of a healthy control and indicates that the fetus is homozygous for a

mutation position in other allele.

1559delT of the *ALPL.*

(perinatal type) and the genotype (1559delT).

show markedly impaired mineralization *in utero* (Fig.1). Pregnancies may end in stillbirth. Some infants survive a few days with pulmonary complication due to hypoplastic lungs and rachitic deformities of the chest. Hypercalcemia is common and may be associated with apnea or seizures.


Table 1. Clinical Features of Hypophosphatasia by Type. AD; autosomal dominant, AR; autosomal recessive

Fig. 1. Ultrasonography examination at 19 weeks' gestation of pl-HPP fetus The upper limb(A) femur(B) at 19 weeks' gestation was shortened with no evidence of fractures. The cranium(C) at 19 weeks' gestation was thin with marked hypomineralization.

pl-HPP is more common in Japan than in other countries (Satoh et al. 2009). Parents of pl-HPP are heterozygous carriers of *ALPL* mutations. They show no clinical symptoms, but have reduced serum ALP activity and increased urinary phosphoethanolamine (PEA).

#### **2.3 c.1559delT in** *ALPL***, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japan**

c.1559delT in *ALPL* is a common mutation resulting in pl-HPP in Japan and has only been found in Japanese to date (Orimo et al., 2002; Michigami et al., 2005). Symptoms caused by

show markedly impaired mineralization *in utero* (Fig.1). Pregnancies may end in stillbirth. Some infants survive a few days with pulmonary complication due to hypoplastic lungs and rachitic deformities of the chest. Hypercalcemia is common and may be associated with

Type Inheritance MIM Symptoms

Childhood AR or AD 241510 Short stature, skeletal deformity,

Adult AR or AD 146300 Stress fractures: metatarsal, tibia;

Odontohypophosphatasia AR or AD Exfoliation (incisors), dental caries

Table 1. Clinical Features of Hypophosphatasia by Type. AD; autosomal dominant, AR;

Fig. 1. Ultrasonography examination at 19 weeks' gestation of pl-HPP fetus

The upper limb(A) femur(B) at 19 weeks' gestation was shortened with no evidence of fractures. The cranium(C) at 19 weeks' gestation was thin with marked hypomineralization.

pl-HPP is more common in Japan than in other countries (Satoh et al. 2009). Parents of pl-HPP are heterozygous carriers of *ALPL* mutations. They show no clinical symptoms, but have reduced serum ALP activity and increased urinary phosphoethanolamine (PEA).

**2.3 c.1559delT in** *ALPL***, a common mutation resulting in the perinatal (lethal) form of** 

c.1559delT in *ALPL* is a common mutation resulting in pl-HPP in Japan and has only been found in Japanese to date (Orimo et al., 2002; Michigami et al., 2005). Symptoms caused by

osteochondral spurs

Hypomineralization, rachitic ribs, hypercalciuria, Premature loss,

bone pain/fractures, Premature

postnatal course

deciduous teeth

loss, deciduous teeth

chondrocalcinosis

, Alveolar bone loss

Perinatal (lethal) AR Hypomineralization,

Infantile AR 241500 Craniosynostosis,

Perinatal (benign) AR or AD Long-bone bowing, benign

apnea or seizures.

autosomal recessive

**hypophosphatasia in Japan** 

deletions and insertions of nucleotides that change the reading frame will be highly deleterious. Some patients with pl-HPP are homozygous for c.1559delT, with parents who are heterozygous carriers for the mutation but with no evidence of consanguinity (Fig. 2) (Sawai et al. 2003). Patients who are homozygous for the c.1559delT mutation differed in the severity of HPP, including both their symptoms and serum ALP activity. In the c.1559delT mutation, the symptom in HPP of homozygous mutation is responsible for a severe phenotype, but that of compound hetero varies from severe to mild that depends on mutation position in other allele.

Fig. 2. Direct sequencing results around 1559delT in *ALPL* from a pl-HPP patient both parents, and healthy control. The sequence of the parents could not be determined in progress at cDNA number 1559 of the *ALPL* and these results indicate both parents were heterogenous carriers for 1559delT. The sequence of the pl-HPP patient could be determined in progress containing the deletion of T at nucleotide 1559, which was different from that of a healthy control and indicates that the fetus is homozygous for a 1559delT of the *ALPL.*

c.1559delT, a deletion of T at 1559, which caused a frameshift downstream from leucine (Leu) at codon 503, resulted in the elimination of the termination codon at 508 and the addition of 80 amino acid residues at the C-terminus. The mutant protein caused by 1559delT formed an aggregate, was polyubiquitinated, and was then degraded in the proteome (Komaru et al., 2005), thus allowing us to directly correlate the phenotype (perinatal type) and the genotype (1559delT).

Prenatal Diagnosis of Severe Perinatal (Lethal) Hypophosphatasia 31

will be indispensable before starting the treatment, and perhaps the characterization of the

Fig. 3. Pedigree examples of two situations in genetic counseling for pl-HPP: A) family with an affected first child (index case) or B) fortuitous prenatal skeletal dysplasia in a family

A prenatal diagnosis should be provided in a supportive, noncoercive atmosphere that allows the couple to make informed choices regarding what is are best for them in view of their values and parenting goals. Genetic counseling is particularly important before prenatal diagnosis to enable parents to make an informed choice. Counseling before testing makes counseling after testing (for those with an affected fetus) less difficult because prospective parents are better prepared. Careful counseling regarding if and how to inform the parents about the child can help to overcome this potential problem. A prenatal genetic

To diagnose pl-HPP in prenatal stage, collaborations between obstetricians and clinical geneticists are important and could provide support for parents of prenatal patients

This work was supported in part by grants from the Ministry of Health and Welfare of Japan.

Gehring B., Mornet E., Plath H., Hansmann M., Bartmann P. & Brenner R. E. (1999).

Komaru K., Ishida Y., Amaya Y., Goseki-Sone M., Orimo H. & Oda K. (2005). Novel

Retarded secretion and proteasomal degradation. *FEBS J.* 272, 1704-1717.

Perinatal hypophosphatasia: diagnosis and detection of heterozygote carriers

aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension.

diagnosis may also help the professional team to prepare for a difficult delivery.

mutations will orient and personalize the treatment in future.

without history of HP (no index case)

suspected of having skeletal dysplasia.

within the family. *Clin. Genet.* 56, 313–317.

**3. Conclusion** 

**5. References** 

**4. Acknowledgment** 

The c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1,562-1/284) in Japanese (Watanabe et al. 2011). This indicates that approximately 1 in 900,000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. The majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine PEA. The only way to reliably detect the pl-HPP carriers is to perform the *ALPL* mutation analysis.

### **2.4 Prenatal diagnosis for perinatal (lethal) form of hypophosphatasia**

pl-HPP has been diagnosed in *utero* by ultrasonography performed with careful attention to marked hypomineralization of the limbs and the skull (Fig.1) (Tongsong & Pongsatha, 2000). The differential diagnosis of HPP depends on the age at which the diagnosis is considered. Ultrasonography examination in prenatal stage may lead to a consideration of osteogenesis imperfecta type II, campomelic dysplasia, and chondrodysplasias with defects in bone mineralization, as well as pl-HPP. Experienced sonographers usually have little difficulty in distinguishing among these disorders. However, pl-HPP is occasionally not diagnosed with sonographic examination in the first trimester because incomplete ossification is a normal finding at this stage of development (Zankl, 2008).

Prenatal assessment for pregnancies at increased risk of severe HPP by mutation analysis is possible if two HPP causing *ALPL* mutations of an affected family member are identified (Watanabe et al., 2007). Most of pl-HPPs are related to c.1559delT in Japan (Watanabe et al., 2011), but to usually compound heterozygotes, carrying two distinct mutations in US and France (Simon-Bouy et al., 2008). In Japan,screeing of c.1559delT is important to diagnose pl-HPPs. In out of Japan, mutations occur throughout the entire gene without hot spots. To detect different mutations, all exon screening of *ALPL* is needed. In prenatal genetic diagnosis, fetal genomic DNA was extracted from chorionic villus at approximately ten to 12 weeks' gestation or cultured cells of amniotic fluid at approximately 15 to 18 weeks' gestation.

A prenatal genetic diagnosis for HPP gives a couple important information about the fetus. Prenatal genetic diagnosis for HPP in combination with ultrasonography is thus considered useful for confirming a diagnosis of HPP, which presents with a wide variety of phenotypes.

### **2.5 Genetic counseling for perinatal (lethal) form of hypophosphatasia**

Genetic counseling for pl-HPP has two situations, family with an affected first child (index case) or fortuitous prenatal skeletal dysplasia in a family without history of HP (no index case) (Fig. 3) (Simon-Bouy et al., 2008). First, a couple with an index case with recessive form of pl-HPP will have in subsequent pregnancies affected children similar to the index case with a 25% chance of recurrence. However, the severity of symptoms in HPP may differ from one child to another even in the same mutation (Nakamura-Utsunomiya, 2010). Second, in pl-HPP, pregnancies with clinical symptoms could be detected by ultrasound with no familial history of pl-HPP (no index case). The screening for c.1559delT in *ALPL* may be useful for diagnosis of pl-HPP in Japanese. Detection of 1559delT mutation confirms the diagnosis of severe HPP, and an attempt to predict the severity of the disease. Postnatal molecular genetic analysis using the cord tissue can provide a diagnosis of pl-HPP allows time for parental counseling and delivery planning. In addition, Enzyme replacement therapy (Millán JL et al., 2008) and gene therapy (Yamamoto et al., 2011) will be certainly the most promising challenge. Confirmation of the diagnosis of HPP by *ALPL* genetic testing will be indispensable before starting the treatment, and perhaps the characterization of the mutations will orient and personalize the treatment in future.

Fig. 3. Pedigree examples of two situations in genetic counseling for pl-HPP: A) family with an affected first child (index case) or B) fortuitous prenatal skeletal dysplasia in a family without history of HP (no index case)

A prenatal diagnosis should be provided in a supportive, noncoercive atmosphere that allows the couple to make informed choices regarding what is are best for them in view of their values and parenting goals. Genetic counseling is particularly important before prenatal diagnosis to enable parents to make an informed choice. Counseling before testing makes counseling after testing (for those with an affected fetus) less difficult because prospective parents are better prepared. Careful counseling regarding if and how to inform the parents about the child can help to overcome this potential problem. A prenatal genetic diagnosis may also help the professional team to prepare for a difficult delivery.

### **3. Conclusion**

30 Prenatal Diagnosis – Morphology Scan and Invasive Methods

The c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1,562-1/284) in Japanese (Watanabe et al. 2011). This indicates that approximately 1 in 900,000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. The majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine PEA. The only way

pl-HPP has been diagnosed in *utero* by ultrasonography performed with careful attention to marked hypomineralization of the limbs and the skull (Fig.1) (Tongsong & Pongsatha, 2000). The differential diagnosis of HPP depends on the age at which the diagnosis is considered. Ultrasonography examination in prenatal stage may lead to a consideration of osteogenesis imperfecta type II, campomelic dysplasia, and chondrodysplasias with defects in bone mineralization, as well as pl-HPP. Experienced sonographers usually have little difficulty in distinguishing among these disorders. However, pl-HPP is occasionally not diagnosed with sonographic examination in the first trimester because incomplete ossification is a normal

Prenatal assessment for pregnancies at increased risk of severe HPP by mutation analysis is possible if two HPP causing *ALPL* mutations of an affected family member are identified (Watanabe et al., 2007). Most of pl-HPPs are related to c.1559delT in Japan (Watanabe et al., 2011), but to usually compound heterozygotes, carrying two distinct mutations in US and France (Simon-Bouy et al., 2008). In Japan,screeing of c.1559delT is important to diagnose pl-HPPs. In out of Japan, mutations occur throughout the entire gene without hot spots. To detect different mutations, all exon screening of *ALPL* is needed. In prenatal genetic diagnosis, fetal genomic DNA was extracted from chorionic villus at approximately ten to 12 weeks' gestation

A prenatal genetic diagnosis for HPP gives a couple important information about the fetus. Prenatal genetic diagnosis for HPP in combination with ultrasonography is thus considered useful for confirming a diagnosis of HPP, which presents with a wide variety of phenotypes.

Genetic counseling for pl-HPP has two situations, family with an affected first child (index case) or fortuitous prenatal skeletal dysplasia in a family without history of HP (no index case) (Fig. 3) (Simon-Bouy et al., 2008). First, a couple with an index case with recessive form of pl-HPP will have in subsequent pregnancies affected children similar to the index case with a 25% chance of recurrence. However, the severity of symptoms in HPP may differ from one child to another even in the same mutation (Nakamura-Utsunomiya, 2010). Second, in pl-HPP, pregnancies with clinical symptoms could be detected by ultrasound with no familial history of pl-HPP (no index case). The screening for c.1559delT in *ALPL* may be useful for diagnosis of pl-HPP in Japanese. Detection of 1559delT mutation confirms the diagnosis of severe HPP, and an attempt to predict the severity of the disease. Postnatal molecular genetic analysis using the cord tissue can provide a diagnosis of pl-HPP allows time for parental counseling and delivery planning. In addition, Enzyme replacement therapy (Millán JL et al., 2008) and gene therapy (Yamamoto et al., 2011) will be certainly the most promising challenge. Confirmation of the diagnosis of HPP by *ALPL* genetic testing

to reliably detect the pl-HPP carriers is to perform the *ALPL* mutation analysis.

**2.4 Prenatal diagnosis for perinatal (lethal) form of hypophosphatasia** 

or cultured cells of amniotic fluid at approximately 15 to 18 weeks' gestation.

**2.5 Genetic counseling for perinatal (lethal) form of hypophosphatasia** 

finding at this stage of development (Zankl, 2008).

To diagnose pl-HPP in prenatal stage, collaborations between obstetricians and clinical geneticists are important and could provide support for parents of prenatal patients suspected of having skeletal dysplasia.

### **4. Acknowledgment**

This work was supported in part by grants from the Ministry of Health and Welfare of Japan.

### **5. References**


**3** 

**Skeletal Dysplasias of the Human Fetus:** 

Congenital skeletal disorders comprise a heterogenous group of abnormalities of the bones related to their shape, growth and integrity. They are present at birth or become manifest during gestation causing abnormal development of the fetal skeleton that can be prenatally detected by ultrasonography. They make part of a large group of genetic skeletal disorders, formerly called constitutional disorders of bone. They all refer to abnormal skeletal development on the basis of a defective genetic background. Excluding chromosomal abnormalities affecting the skeleton, the large and heterogeneous family of genetic skeletal disorders comprise (1) disorders with significant skeletal involvement corresponding to the definition of skeletal dysplasias (alternatively called osteochondrodysplasias), (2) metabolic and molecular bone disorders, (3) dysostoses, (4) skeletal malformation and/or reduction syndromes and (5) multiple congenital malformation syndromes with a prominent skeletal involvement. . The genetic skeletal disorders, although individually rare, are not uncommon as a whole group. The latest 2010 Revision of the Nosology and Classification of Genetic Skeletal Disorders (Warman et al., 2011) includes 456 entities. Some 50 of them are perinatally lethal and can be diagnosed at birth (Nikkels, 2009), while some others, non lethal and compatible with short or long term survival, may present with abnormal phenotypic findings at birth or with abnormal ultrasonographic findings in utero and raise a prenatal diagnostic dilemma, as pertains to the possible lethality or morbidity of the affected fetus. With the advent of prenatal ultrasonographic examination, many of the affected fetuses are aborted at an early gestational age. A correct diagnosis and typing of the skeletal disorder is essential for the prognosis and genetic counselling of the family, as well as for the possibility of prenatal diagnosis in subsequent pregnancies. The molecular defects underlying the genetic skeletal disorders are increasingly being identified and have shed some light on the pathogeneses of these conditions. One important example is that of the fibroblast growth factor receptor (FGFR3) defects underlying skeletal dysplasias such as Thanatophoric dysplasia, Achondroplasia etc. Nevertheless, in only a restricted subgroup of fetal skeletal dysplasias is the molecular genetic analysis part of a routine prenatal control able to provide an accurate diagnosis. In most instances, the responsibility of the final diagnosis of a fetal skeletal dysplasia lies on the post-mortem examination and in many

institutions it is largely or uniquely the task of the pathologist.

The objective of this chapter is to provide an overview on the role of the pathologist in the handling of the congenital skeletal disorders and enable the postmortem diagnostic

**1. Introduction** 

**Postmortem Diagnosis** 

Anastasia Konstantinidou

*University of Athens* 

*Greece* 

