Thalassemia Syndromes

#### **Chapter 3**

## Newborn Screening and Thalassaemia Syndrome

*Charity Iheanacho and Christiana Okeke*

#### **Abstract**

Haemoglobin variants or haemoglobin disorders are a group of clinical disorders characterised by impairment of synthesis of normal adult haemoglobin, due to genetically determined abnormality in the formation of the globin moiety of the molecule. These disorders fall into two broad groups, that is qualitative (haemoglobinopathies) and quantitative (thalassaemias). In the anthropoids, the most common congenital single-gene disorder is the alteration of the globin genes which account for about 270 million carriers globally. These globin gene alterations cause low/no globin expression (thalassaemia) or abnormal globin protein production (haemoglobinopathy). The clinical manifestation of haemoglobin disorder is the culminated measure of one's genetic and molecular makeup. Summarily, the study, diagnosis and management of thalassaemia are models of biological principles of human disease. Newborn screening, however, is a system that aims at improving management and/or eradication of genetic disorders from the neonatal stage of life. This chapter will be dealing with the definition and steps involved in newborn screening for thalassaemia.

**Keywords:** thalassaemia, newborn screening, confirmatory diagnosis, haemoglobin disorders, next-generation sequencing (NGS)

#### **1. Introduction**

The haemoglobin (Hb) is a tetramallic, metalloprotein consisting of two alpha α and/or α-like alleles (α or ζ) and two β and/or β-like alleles (ε, γ, δ or β) globin chains with a chemical formula (C2952H4664O832N812S8Fe4) [1, 2]. Each polypeptide globin chain is folded around a haem molecule (**Figure 1**). The major function of Hb is a gaseous transfer between the tissue and the lungs. The globin chains are encrypted by their various genes sited on chromosome 11 and chromosome 16 and they all have more than one allele [4]. These alleles codes for the various globin chains at various stages of human life from the embryonic to adult life in varying concentrations (**Tables 1** and **2**). Many of these alleles undergo point mutations during DNA sequencing resulting in single amino acid substitution in the globin portion, leading to the production of haemoglobin derivatives (variants) [6]. Haemoglobin variants or haemoglobin disorders are a group of clinical disorders characterised by impairment of synthesis of normal adult haemoglobin, due to genetically determined abnormality in the formation of the globin moiety of the molecule. These disorders fall into two broad groups, that is, qualitative (haemoglobinopathies) and quantitative

#### **Figure 1.**

 *Haemoglobin structure. A molecule of haemoglobin is made up of four subunits; two alpha-like subunits and two beta-like subunits. Each subunit contains a haem group with a ferrous core to which an oxygen molecule can reversibly bind. Image adapted from [ 3 ].* 

(thalassaemias) [ 7 ]. In the anthropoids, the most common congenital single-gene disorder is the alteration of the globin genes which account for about 270 million carriers globally and 0.4 million of such births annually [ 8 ]. These globin gene alterations cause low/no globin expression (thalassaemia) or abnormal globin protein production (haemoglobinopathy). The clinical manifestation of haemoglobin disorder is the culminated measure of one's genetic and molecular makeup. Summarily, the study, diagnosis and management of thalassaemia is a model of biological principles of human disease. Newborn screening however is a system that aims at improving management and/or eradication of genetic disorders from the neonatal stage of life.

#### **1.1 The haemoglobin nature and structure**

 The anatomy and genetic structure of the human haemoglobin are demonstrated in the tables and figures below.

*Newborn Screening and Thalassaemia Syndrome DOI: http://dx.doi.org/10.5772/intechopen.109302*


#### **Table 1.**

*Haemoglobins normally present during adult, foetal and embryonic periods of life.*


#### **Table 2.**

*Normal and variant haemoglobin at birth and in older children.*

#### **1.2 What is newborn screening?**

Newborn screening is an entire system of identification, treatment, management and possibly eradication of genetic disorders from the neonatal stage of life. Newborn screening starts from the recruiting stage through the diagnosis of the infant and management. It is generally applied for the early recognition of infants affected by disorders that benefit from early treatment to avoid irreversible health problems [9]. It is supreme for early diagnosis and enrolment of affected children into a comprehensive health care programme. This has created diagnostic and treatment opportunities for several children with genetic or metabolic diseases across the globe with a resultant healthier life. However; in many countries, the screening

programmes have not started or have been limited to a few disease conditions [10]. These delays are tied to financial incapabilities of the citizens and lack of government-established organisation for screening. However, the physician's suspicion and or awareness is heightened by accurate identification of risk factors of haemoglobin disorders and family history [11]. The ultimate benefit of newborn screening programmes is the improved health status in patients diagnosed early and treated optimally. However, issues such as false positives and false negatives results might occur but the use of novel and molecular technologies for confirmation has over that [12]. Each part of the newborn screening system is important and needs evaluation for any weaknesses. Newborn screening for genetic disorders can be undertaken from two dimensions:


The family history method might be cost-effective but will likely miss out on the few misinformed questionnaires, so for a start, a random newborn screening of a population site should be of best interest for subsequent studies, government policies and data storage.

#### *1.2.1 Algorithm for newborn screening*


*Newborn Screening and Thalassaemia Syndrome DOI: http://dx.doi.org/10.5772/intechopen.109302*

8.For the collection of whole blood samples for confirmation, ethylenediaminetetraacetic acid (EDTA) is the typical anti-coagulant used. Heparin may interfere with DNA amplification by polymerase chain reaction (PCR). DBS collected from a finger prick; preferably the last finger or the heel is commonly used. To maintain the integrity of haemoglobin molecules, the medium of transportation and storage of DBS must be dry and cool, possibly by means of dry ice.

#### **1.3 The thalassaemias**

Thalassaemia is a group of heterogenous genetic disorders of haemoglobin synthesis. These disorders arise from a decrease in production rate of one or more globin chain [13]. The thalassaemias are named α, β, δβ- and γδβ-thalassaemias depending on the globin chain that is produced in a reduced amount. In the occasion where one of the globin chain is not synthesised at all, the condition is known as thalassaemia Null (°), that is, α<sup>0</sup> or β<sup>0</sup> thalassaemias. This condition usually occurs amongst the populace with structural Hb abnormalities, therefore the inheritance of one thalassaemia gene from one parent and the second gene with a structural Hb variant from the next parent is a common finding in such places [14]. Other minor haemoglobins in adults include HbF (foetal haemoglobin, α2γ2) and HbA2 (α2δ2) [15].

**The α-Thalassaemias**: These groups of thalassaemias result from the deletion of one or more alpha-globin genes and are subgrouped in order of the number of the α-globin gene deletions. Hence, one gene deletion is α<sup>+</sup> -thalassaemia, α<sup>0</sup> -thalassaemia is two gene deletion from the same chromosome, Hb-H syndrome is a three-gene deletion while hydrops feotalis with the Hb-Barts is a four-gene deletion. The haemoglobin being a tetrametric protein of 4 globin gene α2β2, it has been observed that deletion of only one α gene will not result in a significant haematological abnormality and is therefore referred to as a "silent carrier" state. On the other hand, deletions of two α-genes can occur in two ways i.e. from the same chromosome (in cis) or of the opposite chromosome (in trans). The two α gene deletion is the homozygous state or homozygous α<sup>+</sup> -thalassaemia and has similar clinical presentations as mild hyperchromasia and microcytic anaemia but the cis-genotype is common amongst Asians while the trans-form is common in Black African origins [16].

The Hb-H disease is usually associated with haemolysis due to the excessive accumulation of β-globin subunits that self-bond to form soluble β-chain tetramers which are the Hb-H. Because of the relatively unstable nature of the Hb-H, it does not precipitate as the erythrocytes age leading to the formation of inclusion bodies which distorts the red cells' life span [17].

Hydrop's foetalis with Hb-Barts is usually detected at the third trimester or within the early post-natal period. Haemoglobin Barts is not an effective oxygen transporter because it has a very high affinity for oxygen. It is a tetramer of 4γ globin subunits so the foetus or infant will lack Hb F&A, resulting in hypoxia and extreme organ swelling and subsequent deaths [17].

**The β-Thalassaemias**: The Hb variant resulting from the point-mutation of the β-globin gene is known as the β-thalassaemias. This variant has two main sub-types, that is, the β<sup>0</sup> -thalassaemia in which there is complete absence of normal β-globin subunits. And the second is β<sup>+</sup> -thalassaemia which has remarkably reduced synthesis of normal β-globin. It was noted that some forms of β-thalassaemia might be due to an unequal crossing over of bridges of the δ & β-globin genes leading to a fusion of δβ-globin gene (thalassaemia), εγδβ-thalassaemia and hereditary persistence of foetal haemoglobin (HPFH) syndromes [18]. It has been reported that β-thalassaemia has over 200 molecular different subtypes but in spite of their heterogeneity; they still possess similar clinical manifestations since they all lack HbA with excess accumulation of α-subunits [19].

#### **1.4 Newborn screening methods for thalassaemias**

In the recent past, most newborn screening programmes uses high-performance liquid chromatography (HPLC) as the primary screening method to make a presumptive screening of possible haemoglobinopathy [20]. However, for low-income nations, a simple alkaline or acid globin chain electrophoresis with DL-dithiothreitol (DL-DTT) and urea in Tris EDTA-borate buffer can suffice for the detection of abnormal haemoglobins [21]. Also, manual HbF quantification and inclusion body detection can serve as a good NBS source for low-income states. All suspected abnormal Hbs or neonates can then be subjected to fully automated, high throughput HPLC for identification, and quantitation of Hb F, HbA2 and Hb Bart's, enabling thalassaemic screening and classification in the newborn period [20]. According to literatures, an understanding of the specific HPLC retention times will aid the probable identification of thalassaemic disorders such as a Hb S/Hb A ratio >2.0 is highly suggestive of Hb S/β+ thalassaemia rather than Hb AS trait. Secondary or primary screening with HPLC can thus help to streamline the subsequent tests needed for the identification/confirmation of a thalassaemia syndrome in most cases [22].

**Globin chain electrophoresis**: This is used in the separation of α- and β-globin chains by adding 6 M urea and 2-mercaptoethanol to the buffer. When electrophoresis is applied at alkaline or acid pH, these chains migrate differently revealing the characteristic patterns of migration of abnormal α- and β-chains. This method provides a means of identifying abnormal haemoglobin variants that cannot be identified by routine electrophoretic methods. It is especially helpful when variants other than S and C are present and which have identical migration on both cellulose acetate and citrate agar systems [23].

**Determination of distribution of HbF in red cells**: This is employed to distinguish hereditary persistence of foetal haemoglobin (HPFH) from β thalassaemias. The acid elution test of Betke-Kleihauer is used to evaluate the distribution of HbF, where fresh thin-blood film fixed with ethanol is examined microscopically. The principle is that HbA on fixing readily wash off from red cells by acid solution while HbF resists acid-elution and remains within the cells. Cells containing more HbF appear dark after staining, while those with no HbF appear unstained and empty or ghost-like [24].

**Tests for inclusion bodies**: Inclusion bodies that can be detected in thalassaemias include HbH and α chain inclusion and they can be detected as follows:


the nucleus when peripheral blood or bone marrow sample is incubated with methyl violet, and the prepared films observed under microscope [23].

**High-performance liquid chromatography**: This is used as a screening test for thalassemias and for the detection, identification and quantification of haemoglobin variants. It is also used for the quantitation of HbA2 and HbF. HPLC is well suited for neonatal screening since it can detect small amounts of haemoglobin and needs small amount of blood. Haemoglobins A, F, S, C, E/A2, DPunjab, O-Arab and DPhiladelphia can be separated and identified with HPLC. In this technique, blood sample is introduced into a column packed with silica gel. Different haemoglobins get adsorbed onto the resin. Elution of different haemoglobins is achieved by changing the pH and ionic strength of the buffer. Haemoglobin fractions are detected as they pass through a detector and are recorded by a computer [25].

#### **1.5 Confirmatory diagnostic testing for the thalassaemias**

Demographic information and an EDTA/DBS blood sample from one or both parents are required with that of the newborn to help guide the sequence of confirmatory diagnostic tests for specific thalassaemias. Methods of gene-typing for thalassemia based on PCR techniques are as follows: dot-blot analysis, reverse dot-blot analysis, the amplification refractory mutation system, denaturing gradient gel electrophoresis, mutagenically separated polymerase chain reaction, gap-PCR, restriction endonuclease analysis, real-time polymerase chain reaction, Sanger sequencing, pyrosequencing, multiplex ligation-dependent probe amplification and gene array [26–29] Gap-PCR is used to test for common α-thalassaemia deletions or duplications, as well as all forms of HPFH and Hb Lepore deletions. will identify point mutations in the γ-, α- and β-globin genes are usually captured in direct DNA sequencing but mutations within the alleles as well as large deletions are often missed out. Large β-globin locus deletions account for only a very small number of β-thalassaemia mutations but are the most difficult to detect because gap-PCR relies on knowledge of the deletion breakpoints. Multiplex ligation-dependent amplification (MLPA) becomes a handy method to determine the presence of an unidentified α- or β-globin gene deletion, by assessing DNA ploidy quantity changes [30]. Long-range sequencing using comparative genomic hybridisation (CGH) or microarray-based comparative genomic hybridisation (matrix CGH) method to identify deletion breakpoints and DNA copy numbers with high resolution is employed for beta thalassaemia confirmation [31]. This is a molecular cytogenetic method for analysing "copy number variation" which is related to the number of complete sets of chromosomes in a cell and hence the number of possible alleles for autosomal and pseudo-autosomal genes [32]. These novel methods are summarised in **Table 3**.

The method employed for the detection of unknown mutations is the restriction fragment length polymorphism (RFLP) analysis. This method is based on the fact that each restriction enzyme targets different nucleotide sequences in a DNA strand hence different enzyme cuts at different sites. The distance between the cleavage sites of a certain restriction endonucleotide differs between individuals. Hence, the length of the DNA fragments produced by a restriction endonuclease will certainly differ from organisms and species [33]. The variations that affect restriction sites and produce different fragmentation sizes after digestion are known as restriction fragment length polymorphisms (RFLPs). This polymorphism serves as 'markers' for genetic disorders, especially thalassemias. If the linkage is not close then the crossing


#### **Table 3.**

*The novel methods of DNA diagnosis for thalassaemia.*

over of chromosomal material between homologous chromosomes during meiosis may 'separate' the polymorphic site from the abnormal gene; this will lead to a false negative result in the foetus [23].

#### **2. Next-generation sequencing (NGS)**

Recently, however, the introduction of technologies such as dosage mutation tests to detect large deletion or duplication mutations and multiple gene panel tests by massively parallel sequencing (next-generation sequencing; NGS) facilitates a more precise molecular diagnosis of thalassaemias and a better understanding of the genomic mechanisms of the disease [34].

In next generation sequencing (NGS), the diagnosis of thalassaemias is based on massively parallel sequencing of clonally amplified DNA molecules, alongside sufficient computational power and appropriate software for efficient data analysis [35]. The procedure can be applied to a whole genome or exome, and to specific targeted regions of the genome. The most critical step in NGS manipulation is the design of the probe-set to be applied for DNA capture which requires a high level of homology between the genes in the alpha and beta clusters. Another critical point for NGS is that it is useful for the detection of single nucleotide substitutions and insertions or small deletions, but it is less accurate for other types of genomic variation.

#### **3. Conclusion**

Newborn screening is a system of identification, treatment, management, and possibly eradication of genetic disorders from the neonatal stage of life. The procedure begins at the recruiting site or stage through the diagnosis of the infant and management. It is generally applied for the early recognition of infants affected by disorders that benefit from early treatment to avoid irreversible health problems. It is supreme for early diagnosis and enrolment of affected children into a comprehensive health care programme thus; thousands of children with genetic and/or metabolic diseases have had an opportunity for a healthy life with early diagnosis and

#### *Newborn Screening and Thalassaemia Syndrome DOI: http://dx.doi.org/10.5772/intechopen.109302*

treatment. Thalassaemia syndrome has a high financial and national health burden, national policies and intervention are needed for its success. Each nation should adopt newborn screening and diagnostic/confirmatory methods for thalassaemia syndromes within their financial or economical capacity, maintaining standards. Communication, documentation and follow-up is the key to the success of newborn screening for thalassaemia syndromes.

### **Author details**

Charity Iheanacho1 \* and Christiana Okeke2

1 Haematology Department, Jos University Teaching Hospital, Jos, Plateau State, Nigeria

2 44 Nigerian Army Reference Hospital, Kaduna, Kaduna State, Nigeria

\*Address all correspondence to: udoanacho@gmail.com

© 2023 The Author(s). Licensee IntechOpen. 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.

### **References**

[1] Pandya PP, Wapner R, Oepkes D, Sebire N. Fetal Medicine E-Book: Basic Science and Clinical Practice. Amsterdam, Netherlands: Elsevier Health Sciences; 2019

[2] Kreitzberg P, Lucke K, Pennington J, Serang O. Selection on \$ X\_1+ X\_1+\ cdots X\_m \$ via Cartesian product tree. arXiv preprint arXiv:2008.07023. 2020

[3] Centers for Disease Control and Prevention. Hemoglobinopathies: Current Practices for Screening, Confirmation and Follow-up. Silver Spring, Maryland, U.S: Association of Public Health Laboratories; 2015

[4] Weatherall DJ. The challenge of haemoglobinopathies in resourcepoor countries. British Journal of Haematology. 2011;**154**(6):736-744

[5] Esan AJ. Hematological differences in newborn and aging: A review study. Hematology Transfusion International Journal. 2016;**3**(3):178-190

[6] Buseri FI, Okonkwo CN. Abnormal hemoglobin genotypes and ABO and rhesus blood groups associated with HIV infection among HIV-exposed infants in north Western Nigeria. Pathology and Laboratory Medicine International. 2014;**6**:15-20

[7] Shiva Raj KC, Basnet S, Gyawali P. Prevalence of hemoglobinopathies and hemoglobin variants. Nepal Medical College Journal. 2017;**19**(3):121-126

[8] Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organization. 2008;**86**(6):480-487

[9] Giordano P. Newborn screening for haemoglobinopathies. In: Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 1: Principles. 2nd ed. Strovolos, Cyprus: Thalassaemia International Federation; 2013

[10] Nnodu OE, Sopekan A, Nnebe-Agumadu U, Ohiaeri C, Adeniran A, Shedul G, et al. Implementing newborn screening for sickle cell disease as part of immunisation programmes in Nigeria: A feasibility study. The Lancet Haematology. 2020;**7**(7):e534-e540

[11] Shorter D, Hong T, Aosborn D. Screening programmes for developmental dysplasia of the hip in newborn infants. Evidence-Based Child Health. 2013;**8**(1):11-54

[12] Wilcken B. Newborn screening: How are we travelling, and where should we be going? Journal of Inherited Metabolic Disease. 2011;**34**(3):569-574

[13] Bain BJ. Haemoglobin and the Genetics of Haemoglobin Synthesis. 2nd ed. New Jersey, USA: Blackwell Publishing Ltd.; 2006. pp. 1-23

[14] Thom CS, Dickson CF, Gell DA, Weiss MJ. Hemoglobin variants: Biochemical properties and clinical correlates. Cold Spring Harbor Perspectives in Medicine. 2013;**3**(3):a011858

[15] Langlois S, Ford JC, Chitayat D, Désilets VA, Farrell SA, Geraghty M, et al. Carrier screening for thalassemia and hemoglobinopathies in Canada. Journal of Obstetrics and Gynaecology Canada. 2008;**30**(10):950-959

[16] Forget BG, Bunn HF. Classification of the disorders of hemoglobin. Cold Spring Harbor Perspectives in Medicine. 2013;**3**(2):a011684

*Newborn Screening and Thalassaemia Syndrome DOI: http://dx.doi.org/10.5772/intechopen.109302*

[17] Higgs DR, Engel JD, Stamatoyannopoulos G. Thalassaemia. Lancet. 2012;**379**:373-383

[18] Thein SL. The molecular basis of β-thalassemia. Cold Spring Harbor Perspectives in Medicine. 2013;**3**(5):a011700

[19] Nienhuis AW, Nathan DG. Pathophysiology and clinical manifestations of the β-thalassemias. Cold Spring Harbor Perspectives in Medicine. 2012;**2**(12):a011726

[20] Hoppe CC. Prenatal and newborn screening for hemoglobinopathies. International Journal of Laboratory Hematology. 2013;**35**(3):297-305

[21] Dacie JV, Lewis SM. Practical Haematology. 10th ed. Edinburgh, London: Churchill Livingstone; 2006. pp. 398-440

[22] Fucharoen S, Winichagoon P. Thalassemia in South East Asia: Problems and strategy for prevention and control. The Southeast Asian Journal of Tropical Medicine and Public Health. 1992;**23**(4):647-655

[23] Adams TL, Latham GJ, Eisses MJ, Bender MA, Haberkern CM. Essentials of hematology. In: A Practice of Anesthesia for Infants and Children. Amsterdam, Netherlands: Elsevier; 2019. pp. 217-239

[24] Amann C, Geipel A, Müller A, Heep A, Ritgen J, Stressig R, et al. Fetal anemia of unknown cause—A diagnostic challenge. Ultraschall in der Medizin - European Journal of Ultrasound. 2011;**32**(S02):E134-E140

[25] Lo L, Singer ST. Thalassemia: Current approach to an old disease. Pediatric Clinics. 2002;**49**(6):1165-1191

[26] Old J, Henderson S. Molecular diagnostics for haemoglobinopathies. Expert Opinion on Medical Diagnostics. 2010;**4**(3):225-240

[27] Harteveld CL, Kleanthous M, Traeger-Synodinos J. Prenatal diagnosis of hemoglobin disorders: Present and future strategies. Clinical Biochemistry. 2009;**42**(18):1767-1779

[28] Fabry M, Old JM. Laboratory methods for diagnosis and evaluation of hemoglobin disorders. In: Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. New York: Cambridge University Press; 2009. pp. 658-686

[29] Old J, Harteveld CL, Traeger-Synodinos J, Petrou M, Angastiniotis M, Galanello R. Molecular diagnosis. In: Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 2: Laboratory Protocols. 2nd ed. Strovolos Cyprus: Thalassaemia International Federation; 2012

[30] Harteveld CL, Voskamp A, Phylipsen M, Akkermans N, den Dunnen JT, White SJ, et al. Nine unknown rearrangements in 16p13. 3 and 11p15. 4 causing α-and β-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. Journal of Medical Genetics. 2005;**42**(12):922-931

[31] Phylipsen M, Chaibunruang A, Vogelaar IP, Balak JR, Schaap RA, Ariyurek Y, et al. Fine-tiling array CGH to improve diagnostics for α-and β-thalassaemia rearrangements. Human Mutation. 2012;**33**(1):272-280

[32] Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science. 1992;**258**(5083):818-821

[33] Cheriyedath S. Restriction Fragment Length Polymorphism (RFLP) Technique. 2018

[34] Shang X, Xu X. Update in the genetics of thalassaemia: What clinicians need to know. Best Practice & Research. Clinical Obstetrics & Gynaecology. 2017;**1**(39):3-15

[35] Lohmann K, Klein C. Next generation sequencing and the future of genetic diagnosis. Neurotherapeutics. 2014;**11**(4):699-707

### **Chapter 4**

## The Key Genetic Determinants Behind the Phenotypic Heterogeneity of HbE/β-thalassemia Patients and the Probable Management Strategy

*Amrita Panja, Brahmarshi Das,Tuphan Kanti Dolai and Sujata Maiti Choudhury*

#### **Abstract**

HbE/β-thalassemia is the most common severe form of thalassemia which is very prominent in South East Asian countries. It is responsible for nearly one-half of all the severe types of β-thalassemia all over the world. It is also known to represent a wide range of phenotypic diversity which varies from asymptomatic to transfusiondependent severe phenotype. The most important predictive factor is mutations within the beta-globin gene (*HBB*). Apart from the primary genetic modifiers, there are certain other determinants regulating the phenotypic heterogeneity including, co-inheritance of alpha thalassemia mutations and other secondary modifiers including *Xmn*1 polymorphism, *HBS1L-MYB*, *GATA-1, BCL11A* polymorphism, and presence of HPFH mutations. Although the degree of severity is also determined by other tertiary genetic modifiers like increase in serum erythropoietin due to anemia, previous infection with malaria, environmental factors, splenectomy, etc. This review aimed to reveal the potential genetic predictors of HbE/β-thalassemia patients and the probable management strategy. This also enhances the generation of "personalized medicine" for better patient care. The instability of clinical phenotype and remarkable variation indicate careful monitoring of treatment for each patient and the therapeutic approaches should be monitored over time.

**Keywords:** HbE/β-thalassemia, genotype, phenotype, genetic modifiers, management strategy

#### **1. Introduction**

Thalassemia is a group of congenital anemias which are characterized by deficient synthesis of one or more globin chain of normal hemoglobin molecules. It is primarily caused due to defective synthesis of globin chain production [1]. It is the most prevalent recessive monogenic disorder and it occurs in about 4.4/10,000 live births all over the world [2]. In European Union, annually 1/10,000 people are symptomatic whereas the global incidence rate is 1/100,000 [3]. It has been found that every year approximately 300,000 and 400,000 babies are born with hemoglobin disorders and most of them are reported from low-income countries [4]. India is now known to possess the largest number of thalassemia major children (150,000) [5]. It has been estimated that approximately 10,000–15,000 new cases are added every year in this country. Moreover, there are 42 million β-thalassemia carriers reported with an average prevalence rate of 3–4% [6].

Hemoglobinopathies are broadly classified into two main groups: thalassemia syndrome and structural hemoglobin variants (abnormal hemoglobins). According to the quantitative reduction in the production of globin chain, thalassemia can be categorised into: 1) thalassemia major (the absence of globin synthesis); 2) thalassemia intermedia (reduced synthesis of globin chain); 3) thalassemia minor (silent type) [7]. The main types of thalassemia include α, β, and δβ thalassemias whereas the clinically important hemoglobin variants include HbS, HbC, HbE, and HbD. So far, >800 different types of mutations and structural variants in the Human beta globin (HBB) gene have been well characterized using the existing genomic protocol. Out of which more than 350 different mutations are known to be associated with β-thalassemia [8, 9]. In the case of α-thalassemia, most of the mutations are deletion type, whereas a wide spectrum of β-thalassemia mutations involved one or a limited number of nucleotides situated within the β-globin gene or its immediate flanking region. Beta-thalassemia has over 200 different point mutations that cause several types of clinical variability due to varying levels of arrangements of compound heterozygous alleles [7, 10]. The structural hemoglobin variants result from the substitution of one or more amino acids in the globin chains of the hemoglobin molecule. The prevalence rate of thalassemia is widely variable depending on the ethnicity of a particular geographical domain. All over the world, HbE/beta-thalassemia represents nearly 50% of all the cases affected from severe beta-thalassemia [11]. It is one of the commonest forms of hemoglobinopathies in many Asian countries including India, Bangladesh, Laos, Indonesia, and Sri Lanka [12]. Moreover, in Southern China, thousands of people are suffering from HbE/beta thalassemia where its gene frequency is about 4% [13]. In certain parts of the world, the number has increased up to 70% like in Thailand and Cambodia [14]. HbE and HbE/β-thalassemia are prominently found in the north-eastern parts of India [15, 16]. The incidence of HbE and beta thalassemia carrier rate is 4.4% and 3.9% correspondingly in this country [17].

HbE/β-thalassemia is formed due to differential interaction between β-thalassemia mutant allele and HbE allele. In many cases, there is interface between α-thalassemia allele with HbE which results in a complex series of phenotypes, although the clinical severity is comparatively milder [18]. It is not always possible to predict the appropriate phenotype from the genotype and it needs adequate genetic counseling. The objective of the present study is to describe the wide range of clinical spectrum of HbE/β-thalassemia and the probable primary and secondary genetic modifiers which influence the variable phenotype. At the same time, the probable management strategy and future therapeutic approach of thalassemia have been focussed.

*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

#### **2. Pathophysiology**

HbE is a hemoglobin variant which appears mildly unstable and shows increased sensitivity to oxidants. The blood oxygen dissociation curves of patients with homozygous HbE appear to be normal or very slightly right-shifted. HbE is synthesized at a mildly reduced rate and globin chain imbalance. It is formed due to substitution mutation at codon 26 of the β-globin gene (GAG>AAG) which leads to the substitute of lysine for glutamic acid. This mutation activates the cryptic splice site toward the 30 end of exon 1, which causes abnormal messenger RNA processing [19]. Therefore, the normally spliced β<sup>E</sup> messenger RNA is declined as the normal donor site must complete with the newly formed spliced site (**Figure 1**).

In the case of HbE patients, hemoglobin constitutes about 25–30% of the total hemoglobin and the level widely varies between 3 and 11 g/dl. The HbE patients with β + thalassemia mutations are characterized by low levels of HbA% and elevated HbE%. The blood smear reveals hypochromic, microcytic red cells with considerable morphologically altered blood cells with increased numbers of target cells. HbE (EE) homozygous red blood cells are not very flexible while moving through the blood vessels. These blood cells have a smaller outside surface area to carry oxygen. EE red blood cells have a reduced capacity to hold oxygen. The lifespan of these RBCs is also shorter than that of the normal. They appear mildly anemic and their hematological findings are very similar to that of heterozygous β-thalassemia [20].

#### **3. The interactions of hemoglobin E with different forms of thalassemia**

The interaction of HbE with other types of hemoglobinopathies can be complex and puzzling. Due to a lack of proper diagnosis and genetic counseling, the chance of different types of hemoglobinopathies can be enhanced [21]. HbE alone cannot lead to any significant clinical complications, although the co-association with α and β-thalassemia leads to a diverse range of clinical syndromes of varying severity.

**Figure 1.**

*Simplified schematic representation of abnormal splicing of β<sup>E</sup> -globin mRNA. The black box denotes 16 nucleotides at the 3*<sup>0</sup> *-end of exon-1 deleted by abnormal splicing mechanism.*


#### **Table 1.**

*Common HbE syndrome and their respective genotype.*

In Thailand and different South East Asian countries, the association between HbE and α-thalassemia (α/αα) causes a various range of phenotypic diversity. Clinical parameters exhibit that level of HbE is almost similar in the case of HbE heterozygous and compound heterozygous for <sup>α</sup><sup>+</sup> thalassemia (<sup>α</sup>/αα), whereas the coassociation of α<sup>0</sup> -thalassemia (α/) have mild thalassemia like syndrome with HbE ranges between 19 and 21%. In certain extreme cases where HbE is associated with HbH disease (/α) which is characterized by 13–15% of HbE and it is called HbAE Bart's Disease [22]. On other hand, the compound heterozygote condition for HbE and β-thalassemia leads to the formation of HbE/β-thalassemia which exhibits a remarkably heterogenous range of phenotypic variability. The phenotypic variability may be influenced by the inheritance of α<sup>0</sup> and α<sup>+</sup> mutant alleles [23]. Heterogenous types of HbE syndromes are also observed due to interaction with other hemoglobinopathies. The symptomatic and asymptomatic forms of HbE syndrome are summarized in **Table 1**. The blood smears of different types of HbE-thalassemia and its coassociation with other types of hemoglobinopathies have been depicted in **Figure 2**.

#### **4. Phenotypic heterogeneity of HbE/β-thalassemia**

The clinical heterogeneity of HbE/β-thalassemia is not well understood. The condition may present as mild, asymptomatic anemia or life-threatening disorder that may lead to lethality from anemia in the first years of life. The phenotype of HbE/β-thalassemia seems to be unstable. Scanty reports have been found on the clinical heterogeneity of these patients. At one end of the spectrum, there are patients whose clinical severity is alike to that of β-thalassemia major; whereas at another end there are patients who can lead a normal life without the need for regular blood transfusions.

At the time of birth, infants with severe HbE/β-thalassemia patients are asymptomatic as the HbF level becomes high. As the production of HbF becomes low with increasing age and is replaced by HbE, gradually anemia and splenomegaly develop during the first decade of life [18]. Moreover, deficient blood transfusion can lead to *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

**Figure 2.**

*The peripheral blood film in the (A) homozygous state for hemoglobin E shows large numbers of target cells, (B) HbH disease indicating tear drop cells and anisopoikilocytes, (C) HbE/β-thalassemia contains numerous fragmented RBC, hypochromic RBC, and target cells, (D) HbSE disease having sickle-shaped RBC in blood smear.*

anemia, jaundice, hepatomegaly, and growth retardation. Sometimes chronic leg ulcer is also associated with delayed sexual development. Decreased oxygen delivery leads to ineffective erythropoiesis which is like β-thalassemia major. Patients with milder forms of HbE/β-thalassemia tend to grow normally and are generally active. Usually, there is a delayed pubertal growth pattern and under-developed secondary sexual characteristics. Although it is still not distinct whether they further develop complications in the near future. Gradual iron absorption may develop endocrine complications like diabetes. The clinical symptoms of HbE/β-thalassemia has been depicted in **Figure 3**.

**Figure 3.** *Clinical heterogeneity of HbE/β-thalassemia patients.*

#### **4.1 Asymptomatic forms**

*HbE trait:* Individuals with the HbE trait are clinically normal with minimal changes in blood count and erythrocyte indices. Haemoglobin electrophoresis reveals the presence of HbE is approximately 28.5 1.5%. Likewise, the hemolysate in compound heterozygotes for HbE and α + thalassemia contains 25–30%. Due to the coinheritance of α<sup>0</sup> -thalassemia, HbE levels are reduced up to 19–21% and in the case of HbAE Bart's disease syndrome, there is a markedly reduction in HbE (13–15%). On the other hand, the interaction of β-thalassemia can cause the elevation of HbE (>39%). Iron deficiency also causes lower amounts of HbE and MCV, MCH in the case of HbE trait [24].

*Homozygous HbE:* The clinical symptoms of HbE homozygous appear as normal individuals except few clinical conditions like jaundice and hepatosplenomegaly; although the reticulocyte count and hemoglobin level appear as normal (>10 g/dl). The hematological profile reveals nearly 85–95% of HbE and about 20–80% target cells with reduced osmotic fragility. In these patients, HbE/A2 level is high (10–90%) with lower HbA and HbF levels [25]. The interaction of HbE with different types of hemoglobinopathies has been depicted in **Figure 4** where chromatograms of high performance liquid chromatography have been described.

#### **4.2 Symptomatic forms**

The co-inheritance of the HbE and β<sup>0</sup> -thalassemia trait can lead to transfusiondependent form of thalassemia major. Likewise, the co-association between HbE homozygote and Hb CS causes mild anemic condition. In contrast, the association between HbE and β<sup>0</sup> mutant allele can cause moderate to severe thalassemia. The coinheritance of HbE homozygotes with HbH disease (α<sup>o</sup> -Thal/<sup>α</sup> <sup>+</sup> thal and <sup>β</sup><sup>E</sup> /β<sup>E</sup> )

#### **Figure 4.**

*Chromatograms of HPLC showing interactions of HbE with different hemoglobinopathies (A) HbE homozygous, (B) HbE/β-thalassemia, (C) Hb Lapore, (D) HbAE Bart, (E) HbE-CS, (F) HbSE.*

*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*


**Table 2.**

*Different types of HbE thalassemia and their clinical symptoms.*

and HbH-CS (α<sup>o</sup> -Thal/Hb CS and β<sup>E</sup> /β<sup>E</sup> ) lead to form moderate to severe anemia. Severe anemia may also form due to the presence of HbE/β-thalassemia along with HbH disease (α<sup>o</sup> -Thal/α<sup>+</sup> -thal & β<sup>o</sup> /β<sup>E</sup> ) or HbH-CS disease (α<sup>o</sup> -Thal/Hb CS and β<sup>o</sup> /β<sup>E</sup> ) [26].

The different forms of symptomatic and asymptomatic forms of HbE disease have been enlisted in **Table 2**.

#### **5. Genotype-phenotype interaction**

The exact reason behind the phenotypic heterogeneity of HbE/β-thalassaemia is not properly understood. For a proper understanding of the clinical severity there is a need for a standardized, robust classification of disease severity; although a lack of

suitable clinical severity scoring may impair deciphering of the proper clinical spectrum of HbE/β-thalassemia. According to the phenotypic heterogeneity, patients have been classified into "severe," "moderate," and "mild." There are considerable number of patients who are transfusion-independent while others are regular transfusiondependent [27–29]. According to the severity of the disease, patients are classified into five groups. Group 1 included those patients who need minimal transfusion requirements as well as normal growth and sexual maturity. Group 2 comprised patients with similar types of quality of life to that of Group 1 except for transfusion history as these patients usually experience longer history of transfusion. Group 3 includes patients who have undergone splenectomy and have an advantageous response to splenectomy. Group 4 comprises patients who are transfusion-dependent and their secondary sexual characteristics and growth rate are not satisfactory. Likewise, Group 5 includes patients who are unable to maintain their regular lifestyle without transfusion [29].

#### **6. Genetic modifiers of HbE/β-thalassemia**

The wide range of clinical phenotypes of HbE/β-thalassemia is believed to be regulated through several genetic as well as environmental factors. There is an emerging understanding of the interaction between genetic and environmental factors which triggers the clinical progression and severity.

According to some previous findings, alterations in the *HBB* gene play a crucial role in modulating phenotypic features of HbE/β-thalassemia. Although *HBB* mutations are not only responsible for modulating phenotypic alterations by changing the patterns of gene expression. Currently, genome-wide association study (GWAS) has shown the linked genetic loci to predict phenotype diversity. The genetic modifiers can be classified into the following three groups: the first one is the primary genetic factors including the β-globin gene mutations which are responsible for the manifestation of β-thalassemia; the second one includes loci involved in globin chain synthesis; and the third one is the tertiary factors which are not involved directly in globin chain synthesis but might modulate the disease severity [30].

#### **6.1 Primary modifier**

The primary modifier is one of the most important factors responsible for regulating the phenotype variability in β-thalassemia disorder. Such type of defects occurs in the β-globin gene itself [31]. The mutant allele can reduce the synthesis of the β-globin chain or lead to the complete absence of the β<sup>0</sup> -globin chain. The patients who inherit a mild β-thalassemia allele with HbE might exhibit minor disease, whereas patients who co-inherit severe β + or β0-thalassemia alleles might exhibit the severe form of the disease [32]. In addition, the severity of β-mutation is an important parameter for determining the clinical diversity of HbE/β-thalassemia. Most of the mutations in the *HBB* gene are point mutation, deletion, or insertion type and situated in the promoter, exon, intron, or at the junction between the intron-exon boundary, and polyadenylation site. Defects in single base substitution in the coding sequence of β-polypeptides will lead to premature stop codon whereas small insertion or deletion may lead to alteration in the reading form of mRNA. Likewise, β + allele defects are generally caused due to single base substitution which causes alteration of mRNA reading frame. The list of mutations responsible for mild and silent types of thalassemia is enlisted in **Table 3**.


*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

#### **Table 3.**

*List of* HBB *gene mutations responsible for mild β + and silent type thalassemia.*

In the Indian population, five mutations are the most frequently found β-globin gene including IVS 1-5 (G ! C), IVS 1-1 (G ! T), Codon 41/42 (� TCTT), Codon 8/9 (+G), and 619 bp deletion which account for over 90% of all the mutations associated with β-thalassemia [33, 34], whereas in Malaysian population, IVS1-5(G > C), IVS1-1 (G > T) mutations and Chinese population, CD41/42 (-TCTT), CD71/72(+A), CD 17 (A-T) and � 28 (A-G) mutations are most commonly found. Among the non-sense mutations CD43(G > T), CD35(C > A), CD15(A > G) mutations lead in formation of nonfunctional mRNA, whereas CD8/9(+G), CD15(�T) are frame shift mutations. Certain mutations cause alterations in RNA processing including splice junction changes [CD27/28 + C, CD14/15 + G, CD 95 + A, CD41(-C), CD26 (G > T), IVS1-1 (G > T)]. There are certain deletion mutations including 619 bp del, 3.5 kb del, 45 kb del, and 105 bp del. [35]. Mutation at nucleotide �28 in the ATA box of the β globin gene, was reported earlier among the HbE/β-thalassemia patients [36]. The interaction of two β + globin alleles which is IVS1-5(G > C) and � 28(A > G) resulting in the mild phenotype The beta-thalassemia alleles in "*trans*"-condition to HbE do not seem to have a significant role in regulating phenotypic variation of HbE/β-thalassemia and there must be some other modifying factors. The position of different *HBB* mutations is schematically represented in **Figure 5.** The co-inheritance of β<sup>0</sup> -allele with HbE gives rise to widely variable clinical phenotype. Therefore, β-globin gene mutations alone cannot determine the clinical severity.

#### **Figure 5.**

*The position of different* HBB *gene mutations within the promoter, 5*<sup>0</sup> *UTR, Exon 1, Intron 1, Exon 2, Intron 2, and Exon 3 region which act as the primary genetic modifiers in HbE/β-thalassemia.*

#### **6.2 Other modifying factors**

#### *6.2.1 α-thalassemia*

HbE/β-thalassemia patients who co-inherit determinants for α-thalassemia may have some unmatched α-globin chains leading to more balanced globin chain synthesis and resulting in a milder phenotype. According to some previous studies, HbE/β-thalassemia patients with α<sup>+</sup> -thalassemia allele demonstrate higher state of hemoglobin in comparison to those who do not have α-thalassemia [37, 38]. One of the studies done on Thai HbE patients revealed that the mean age of clinical presentation was more than those patients who did not possess α-thalassemia mutations [39]. Another study done on Indian HbE/β-thalassemia patients presented that coinheritance of the triplicated α-globin gene led to the formation of mild phenotype and their transfusion requirement was also minimal [40]. Therefore, the coinheritance of the alpha thalassemia gene appears as a major genetic factor regulating the clinical phenotype.

#### *6.2.2 Determining factors for regulating increased HbF level*

*Xmn1 polymorphism:* The presence of G to T substitution at �158 position 5<sup>0</sup> to Gγ gene (*Xmn*1 polymorphism) is known to be associated with increased HbF production. Earlier investigations showed that patients with the *Xmn*1 (+/+) genotype were identified only in mildly affected patients; in contrast, patients having the *Xmn*1 (�/�) genotype exhibited severe phenotypes including early age of onset and more transfusion dependency [41, 42]. Overall homozygosity for *Xmn*1 appeared as a crucial genetic determinants for HbE/β-thalassemia; although some conflicting data were presented.

*Additional genetic factors:* Recently, genome-wide association studies elucidate the role of several additional genetic factors in regulating clinical variability. Numerous single nucleotide polymorphisms (SNPs) in the *BCL11A* gene on chromosome 2p16.1 are known to increase the F-cell number [43]. An extensive genetic association study revealed the presence of quantitative trait loci (QTL) on chromosomes 6q23, 8q, and Xp22 may facilitate the amount of HbF production [44]. Apart from these loci, there are certain other protein molecules including erythropoietin, β-protein 1, *EKLF, GATA-1,* and *NF-E2* which have significant role in regulating the HbF level. The strongest correlation was observed in SNPs in the β-globin gene cluster (chr.11p15),

*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

rs2071348 of the *HBBP*1 and intergenic region between the *HBS1L* and *MYB* genes (chr.6q23) which had important significant role in modulating the clinical heterogeneity.

*α-hemoglobin stabilizing protein (AHSP) gene:* The alpha hemoglobin stabilizing protein (AHSP) is an erythroid-specific protein which has a potential role as a molecular chaperone for binding with the free α-chains of hemoglobin molecule. AHSP participated in the hemoglobin synthesis and reduced the cytotoxic effect of excessive α-globin chain accumulation [45]. Since these proteins are essential for conformational change in many essential proteins required for erythropoiesis, it might have a certain important role in Hb E/β-thalassaemia.

#### *6.2.3 Bilirubin metabolism*

The phenotype of HbE disease is also altered by the presence of chronic hyperbilirubinemia, and gallstone formation. The increased level of bilirubin is associated with the polymorphism of the promoter of the UDP-glucuronosyltransferase-1 (UGT1) gene [46]. The UGT1 gene polymorphism is also important in the genesis of gall stone. Investigators found significantly higher bilirubin levels in HbE/β-thalassemia patients [47].

#### *6.2.4 Coinheritance of other hematologic disorders*

Coinheritance of other hematologic aberrations may play important role in phenotypic alterations within the patients with HbE/β-thalassemia. The deficiency of pyrimidine 5 nucleotidase 1 (P5N-I) is found to be resulting in hemolytic anemia while co-associated with homozygous HbE [48]. Therefore, the inhibition of P5N-I activity may lead to severe hemolysis related to HbE.

#### *6.2.5 Variation in iron overload*

Poor growth and delayed sexual maturation are the common complications found in a majority of HbE/β-thalassemia and it may be resulted in chronic anemia, iron overload, or a combination of these. Many patients with a transfusion history have substantial iron overload and end-organ damage. Some of the previous studies reported that mutations within the *SLC40A1*, hepcidin, and hemojuvelin might play a crucial role in iron overload [49–51]. Although the complete profile of iron overload among the HbE/β-thalassemia patients is not completely understood.

#### **7. Environmental influences on the phenotype of HbE/beta-thalassemia**

There are scanty reports available on the environmental influence of HbE/betathalassemia. In tropical regions and developing countries, there is a higher incidence of malaria. It is one of the major health issues in many Asian countries and mostly the transmission occurs through *P. falciparum* and *P. vivax*. A population-based study among the HbE/β-thalassemia revealed the level of malarial antibody is significantly high, especially among splenectomized patients rather than those with the intact spleen. Overall, the quantity of malarial antibodies was quite higher in HbE/β-thalassemia patients rather than control population [52]. Studies reported

that the growth of malarial parasites was inhibited in HbE cells [53]. Children with HbE/β-thalassemia are more prone to *P. vivax* due to the production of an increased amount of young red cell population. *P. vivax* has high potentiality for invasion within the young red blood cell [54]. Although, the biological pathways linking HbE thalassemia and malarial infection are not completely still understood and yet not investigated so much. Further research is required to elucidate whether there is a positive selection for HbE due to protection against malarial infection. The finding is very important for the treatment purpose of malaria, especially in developing countries like India and other South-East Asian countries where HbE/β-thalassemia is very common. Patients with HbE/β-thalassemia should be provided with prophylaxis against common forms of malaria.

#### **7.1 Treatment strategy**

The time of diagnosis, physical examination, and clinical history may give proper information for providing potentially effective treatment strategies. The proper history of appetite, weight gain, energy level, irritability, different major and minor infections, and daily functioning details may help in defining the proper clinical status of a child with thalassemia. Moreover, the genotype of the patients, the presence of αthalassemia, and polymorphism associated with increased HbF production should also be investigated intermittently. Among the pathological features, Hb concentration, and platelet count should be determined in each visit. Serum EPO level, ferritin, or transferrin saturation should also be considered in the regular interval [55]. In the case of HbE/β-thalassemia patients, there is a significant decrease in the oxygen affinity of hemoglobin in comparison to other types of β-thalassemia diseases which indicates that HbE thalassemia might adapt better to anemia; therefore, proper treatment guidelines should be followed which depends on the clinical severity score of the patient.

#### **7.2 Transfusions**

Patients with severe forms of HbE need lifelong RBC transfusions, iron chelation therapy, and management of complications. In contrast, patients with milder forms of disease severity only require occasional blood transfusions. The pre-transfusion hemoglobin concentration of 9–10 g/dl is recommended as this is required for preventing ineffective erythropoiesis. The hemoglobin threshold for determining the increased frequency of disease complications was 7–8 g/dl [56]. The optimal quantity of hemoglobin should be maintained with accessibility to iron chelation therapy. Some patients may need regular transfusion as it is essential to identify phenotypic heterogeneity between "mild" and "severe" within a narrow range of stable stage hemoglobin values [27, 28]. Generally, transfusion-dependent patients should be monitored carefully especially their quality of life, spleen size, signs, and symptoms of anemia. Sometimes, many HbE/β-thalassemia children suffer from different complications due to unnecessary administration of regular transfusion therapy for several years.

#### **7.3 Splenectomy**

One of the treatment strategies for transfusion-dependent thalassemia patients is splenectomy which is believed to ameliorate red blood cell transfusion. Although not

#### *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

all patients respond equally to splenectomy. Moreover, splenectomy might facilitate many unfavorable consequences like postoperative infections and thromboembolic events [57]. In recent years, the use of splenectomy is characteristically reduced due to an adequate amount of blood transfusion. When the annual blood transfusion volume exceeds 225–250 ml/kg, splenectomy is prescribed among patients with increased demand for transfusion, hypersplenism, or splenomegaly due to severe hemolysis [58]. Splenectomy might have some benefits in HbE/β-thalassemia patients. Although it is not clear that morbidity and mortality due to infection after splenectomy are higher in all groups of patients.

#### **7.4 Iron chelation therapy**

Regular transfusion-dependent patients require iron chelation therapy. Even in the absence of transfusions, chelation therapy may be required in some patients with HbE thalassemia because of excess intestinal iron absorption. In the case of regular blood transfusion, each RBC contains 200 mg of iron which leads to nearly 0.3–0.6 mg/kg per day iron accumulation. Iron chelators are classified into three classes: Deferasirox (DFX), Deferiprone (DFP), and Deferoxamine (DFO) [59]. DFX (Exjade) is recommended as an iron chelator after 2005 for transfusion overloaded [60, 61]. It is found to be effective both in the case of offspring and grownups. Generally, 20–30 mg/ kg daily dose is recommended; although in certain cases, the dose is recommended to enhance up to 40 mg/kg daily. DFP is generally absorbed by the gastrointestinal tract and the half-life period of DFP is 1.5–4 hours in plasma. The dose is recommended as 75 mg/kg daily. This dose might be elevated to 100 mg/kg daily [62]. It has efficiency for improving cardiac function by removing iron from cardiac muscle [63]. Deferoxamine (DFO) enters the parenchymal cells of the liver where it chelates the iron as the iron chelator DFO in plasma and bile. The dose and duration of administration differ from patient to patient and depend on how much amount of iron is accumulated after transfusion [64]. Chelation therapy is generally initiated after 20–25 RBC units are transfused between 2 and 4 years of age [65].

Patients should be prescribed iron chelation treatment according to proper guidelines. Quantitative assessment of iron is now recommended among patients before starting chelation therapy [37]. In patients who fail to respond sufficiently to a single iron-chelating drug, the dose can be augmented for betterment [66, 67]. The low molecular weight orally absorbed DFP and DFX quickly access intracellular iron in cytosol and organelles whereas larger DFO molecule contacts with these intracellular iron pools relatively slowly; although it interacts more effectively with lysosomal ferritin iron [68].

#### **7.5 Bone marrow transplantation (BMT)**

The bone marrow transplantation is regarded as the main conclusive treatment approach for thalassemia patients [69]. The most effective transplantation was completed in the 1980s. The thalassemia-free survival rate is 70% in very young individuals after satisfactory BMT whereas the rejection rate is 23%, and the mortality rate is 7% [70]. In this therapy, hematopoietic stem cells (HSC) from the bone marrow of healthy individuals are collected and transmitted to thalassemia patients [71]. Although BMT has a few disadvantages like human leukocyte antigen-matched compatible donor is required for the successful attempt. Graft versus host disease (GVHD) is the most clinically important problem associated with bone marrow transplantation which may lead to lethality [72]. In low socioeconomic developing countries, treatment through BMT is still not accessible for all patients and the accessible treatment includes chelation therapy and transfusion of packed red cells.

#### **7.6 Gene therapy**

Gene transfer therapy helps in introducing genetic materials into the cells. If the altered gene leads to forming the necessary protein being defective, gene transfer therapy can bring about a normal copy of the gene to regain the proper function of the targeted protein. In gene therapy initially, a hematopoietic stem and progenitor cells (HSPCs) of patients are harvested from the peripheral blood, bone marrow, and umbilical cord blood. Through a lentiviral vector, normal β or γ-gene is transferred into the genome of host cells. Hemoglobin genome is transferred into pluripotent hematopoietic cells and is also performed carefully in humans [73]. The cells that contain expected genes are again implanted into patients where they multiply and proliferate in the bone marrow.

Induced pluripotent stem cells (iPSCs) are also used in future gene therapies. Recently, iPSCs are used as in vitro models to reveal the pathophysiological mechanisms of human diseases. In this technique, somatic cells are first isolated from the patients and then remodeled into a pluripotent form [74]. Induced pluripotent stem cells (iPSCs) are susceptible to acchieve alterations in the gene. Then after, these cells are distinguished into hematopoietic stem and progenitor cells and then transferred back into the individuals.

Gene editing is another approach to future gene therapy. According to this method, human DNA can be cut at specific nucleases, like CRISPR/Cas9 and zincfinger nucleases [75]. They can either enhance the production of HbF, by reorienting the mutations seen in the hereditary persistence of fetal hemoglobin or act specifically on the erythroid enhancer region that regulates the switch from the γ-globin gene to βglobin gene. Recently, gene editing was done by treating with CRISPR/Cas9 geneediting method in a thalassemia patient with β0/IVS-1-110 genotype. By editing *BCL11A* gene (chromosome 2) the HbF level was elevated and the patient was transfusion-independent at 12-months follow-up [76].

#### **8. Induction of fetal hemoglobin production**

Various drugs are used to induce the production of HbF including Hydroxyurea (HU). It is used for the treatment of sickle cell anemia as well as thalassemia. HU enhances the production of gamma-globin gene and improves the hematological profile of thalassemia patients. It increases the expression of fetal hemoglobin by regulating the expression of GATA-2 (fetal hemoglobin regulating gene) which is related to the cell cycle and apoptosis. It may also facilitate the propagation of progenitor cells and enhance the quantity of erythropoietin [77]. At the same time, 5-azacytidine and butyrate analogs have also been used most frequently to elevate the HbF level [78, 79].

Several HbF inducers inhibit the histone deacetylase (HDAC) activity [80] and can stimulate the HbF level without disturbing the growth and proliferation of other cells. The combined use of HbF inducers may improve the result. Presently, different oligonucleotide (ODN)-based approaches might help design specific treatment strategies for different types of β-thalassemia [81].

#### **9. Molecular therapy for HbE/β-thalassemia**

The severity of β-thalassemia, as well as HbE/β-thalassemia, can be reduced by regulating the amount of free α-globin chain synthesis by the coinheritance of αthalassemia which may reduce the disease severity. The upregulation of AHSP protein or synthesis of such type of similar agent can limit the formation of α inclusion bodies and ineffective erythropoiesis [45].

Moreover, there are some additional prognostic indicators including *Xmn*1 polymorphism, co-existence of Alpha globin gene mutations, and age of onset [82]. Ethnicity and environment are also significant parameters for the analysis of genotype and phenotype correlation. Genetic modifiers that enhance the secondary complications resulting from severe anemia or excessive iron overload due to frequent transfusion are also considered for determining the disease's severity and progression [83]. Therapeutic antisense m RNA is used for correcting aberrent RNA splicing. The use of morpholino oligonucleotides has the ability for high level correction of transcribed mutant β-globin m RNA. These oligonucleotides have been shown to correct the aberrant splice site in a HeLa cell line bearing β<sup>E</sup> /IVS1–6 mutations. The repaired β<sup>E</sup> mRNA was stable and translated into mature β<sup>E</sup> globin polypeptide [84]. Another approach for molecular therapy of HbE/β -thalassemia in the Ubiquitin-dependent αchain proteolysis. The excess α-globin chains in β -thalassemia and HbE/β -thalassemia erythrocytes are degraded by ATP and ubiquitin-dependent mechanisms. The cytosolic α-chain precipitation and subsequent cellular damage and haemolysis may be reduced due to efficient proteolysis. Previous experiments demonstrated that radiolabelled α-chains in hemolysates obtained from β-thalassaemia patients were degraded in increased level due to hydrolysis through Ubiquitin aldehyde [85]. *Role of antioxidants in cellular damage* several studies have demonstrated that reactive oxygen species (ROS) play a crucial role in the pathophysiology of thalassemia. Thalassemia patients have very high level of malonyldialdehyde, a biproduct of lipid peroxidation. Transfusion-dependent HbE/β-thalassemia patients have very high level of serum iron and correlates positively with levels of malonyldialdehyde [86]. Treatment with Vitamin C and E is well known to improve the oxidative profile of thalassemia patients [87].

The phenotype heterogeneity of HbE/β-thalassemia causes difficulty in the proper management and classification of the disease. Although some of the genetic factors have been recognized as possible modifiers, the wide range of phenotypic alterations cannot be well understood and it requires thorough investigation from early childhood before substantial medical intervention [88, 89]. With the advancement of molecular technologies, the association studies between genetic polymorphism and thalassemia may help explore potential clinical applications by providing possible risk markers and therapeutic targets. The development of personalized medicine is the main objective and genetic counseling should be included in providing patient care programs in the proper management of HbE/β thalassemia patients in the future.

#### **Acknowledgements**

We thank our colleagues for their assistance and constant support provided by them.

### **Author contributions**

Conceptualization, Supervision and Editing: TKD and SMC. Conceptualization, Original Draft writing formatting and Editing: AP. Writing and formatting: BD.

### **Conflict of interest**

The authors declare that they have no competing interests.

**Appendix A: Mutations in the human beta globin gene cluster-responsible for β0 or β+ type of thalassemias**


*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*



#### *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*


#### *Thalassemia Syndromes – New Insights and Transfusion Modalities*


#### *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*


#### *Thalassemia Syndromes – New Insights and Transfusion Modalities*


#### *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*


#### *Thalassemia Syndromes – New Insights and Transfusion Modalities*


#### *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*


#### *Thalassemia Syndromes – New Insights and Transfusion Modalities*


*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

#### **Author details**

Amrita Panja<sup>1</sup> , Brahmarshi Das<sup>2</sup> , Tuphan Kanti Dolai<sup>3</sup> \* and Sujata Maiti Choudhury<sup>1</sup> \*

1 Biochemistry, Molecular Endocrinology, and Reproductive Physiology Laboratory, Department of Human Physiology, Vidyasagar University, Paschim Medinipore, West Bengal, India

2 Department of Biochemistry, Midnapore Medical College, Paschim Medinipore, West Bengal, India

3 Department of Haematology, Nilratan Sircar Medical College and Hospital, Kolkata, West Bengal, India

\*Address all correspondence to: tkdolai@hotmail.com, sujata\_vu@mail.vidyasagar.ac.in and sujata.vu2009@gmail.com

© 2023 The Author(s). Licensee IntechOpen. 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 Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

#### **References**

[1] Origa R. β-Thalassemia. Genetics in Medicine. 2017;**19**(6):609-619. DOI: 10.1038/gim.2016.173

[2] Muncie HL Jr, Campbell J. Alpha and beta thalassemia. American Family Physician. 2009;**80**(4):339-344 PMID: 19678601

[3] Malagù M, Marchini F, Fiorio A, Sirugo P, Clò S, Mari E, et al. Atrial fibrillation in β-thalassemia: Overview of mechanism, significance and clinical management. Biology (Basel). 2022;**11** (1):148. DOI: 10.3390/biology11010148

[4] Williams TN, Weatherall DJ. World distribution, population genetics, and health burden of the hemoglobinopathies. Cold Spring Harbor Perspectives in Medicine. 2012;**2**(9):a011692. DOI: 10.1101/cshperspect.a011692

[5] Colah R, Italia K, Gorakshakar A. Burden of thalassemia in India: The road map for control. Pediatric Hematology Oncology Journal. 2017;**2**(4):79-84. DOI: doi.org/10.1016/j.phoj.2017.10.002

[6] MOHFW (2016) National Health Mission - Guidelines on hemoglobinopathies in India: Prevention and control of hemoglobinopathies in India [Internet]. Available from: https://nh m.gov.in/images/pdf/programmes/RBSK/ Resource\_Documents/Guidelines\_on\_He moglobinopathies\_in India.pdf

[7] Lee JS, Cho SI, Park SS, Seong MW. Molecular basis and diagnosis of thalassemia. Blood Research. 2021;**56** (S1):S39-S43. DOI: 10.5045/ br.2021.2020332

[8] Kountouris P, Lederer CW, Fanis P, Feleki X, Old J, Kleanthous M. IthaGenes: An interactive database for haemoglobin variations and

epidemiology. PLoS One. 2014;**9**(7): e103020. DOI: 10.1371/journal. pone.0103020

[9] Thein SL. Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells, Molecules & Diseases. 2018;**70**:54-65. DOI: 10.1016/j. bcmd.2017.06.001

[10] Giardine B, Borg J, Viennas E, Pavlidis C, Moradkhani K, Joly P, et al. Updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Research. 2014;**42**(Database issue): D1063-D1069. DOI: 10.1093/nar/gkt911

[11] Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organization. 2008; **86**(6):480-487. DOI: 10.2471/blt.06. 036673

[12] Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford, UK: Blackwell Science Ltd; 2001. DOI: 10.1002/9780470696705

[13] Angastiniotis M, Modell B. Global epidemiology of hemoglobin disorders. Annals of the New York Academy of Sciences. 1998;**850**:251-269. DOI: 10.1111/j.1749-6632.1998.tb10482.x

[14] Apidechkul T, Yeemard F, Chomchoei C, Upala P, Tamornpark R. Epidemiology of thalassemia among the hill tribe population in Thailand. PLoS One. 2021;**16**(2):e0246736. DOI: 10.1371/ journal.pone.0246736

[15] Kishore B, Khare P, Gupta RJ, Bisht S, Majumdar K. Hemoglobin E disease in North Indian population: A report of 11 cases. Hematology. 2007;**12**(4):343-347. DOI: 10.1080/10245330701255247

[16] Saha, S., Ghosh, S., Basu, K. Bhattacharyya, M. Prevalence of βhaemoglobinopathies in Eastern India and development of a novel formula for carrier detection. Journal of Hematopathology 2020; 13: 159–164. https://doi.org/10.1007/s12308-020- 00407-7

[17] Nigam N, Kushwaha R, Yadav G, Singh PK, Gupta N, Singh B, et al. A demographic prevalence of β thalassemia carrier and other hemoglobinopathies in adolescent of Tharu population. Journal of Family Medicine and Primary Care. 2020;**9**(8):4305-4310. DOI: 10.4103/ jfmpc.jfmpc\_879\_20

[18] Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring Harbor Perspectives in Medicine. 2012;**2** (8):a011734. DOI: 10.1101/cshperspect. a011734

[19] Roca X, Sachidanandam R, Krainer AR. Intrinsic differences between authentic and cryptic 5<sup>0</sup> splice sites. Nucleic Acids Research. 2003;**31**(21): 6321-6333. DOI: 10.1093/nar/gkg830

[20] Brancaleoni V, Di Pierro E, Motta I, Cappellini MD. Laboratory diagnosis of thalassemia. International Journal of Laboratory Hematology. 2016;**38**(Suppl 1):32-40. DOI: 10.1111/ijlh.12527

[21] Fucharoen S, Sanchaisuriya K, Fucharoen G, Panyasai S, Devenish R, Luy L. Interaction of hemoglobin E and several forms of alpha-thalassemia in Cambodian families. Haematologica. 2003;**88**(10):1092-1098 PMID: 14555303

[22] Traivaree C, Boonyawat B, Monsereenusorn C, Rujkijyanont P, Photia A. Clinical and molecular genetic features of Hb H and AE Bart's diseases in central Thai children. The Application of Clinical Genetics. 2018;**11**:23-30. DOI: 10.2147/TACG.S161152

[23] Hariharan P, Gorivale M, Sawant P, Mehta P, Nadkarni A. Significance of genetic modifiers of hemoglobinopathies leading towards precision medicine. Scientific Reports. 2021;**11**(1):20906. DOI: 10.1038/s41598-021-00169-x

[24] Angastiniotis M, Eleftheriou A, Galanello R, Harteveld CL, Petrou M, Traeger-Synodinos J, Giordano P, Jauniaux E, Modell B, Serour G. Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 1: Principles [Internet]. Old J, editor. 2nd ed. Nicosia (Cyprus): Thalassaemia International Federation; 2013. PMID: 24672827.

[25] Tyagi S, Pati HP, Choudhry VP, Saxena R. Clinico-haematological profile of HbE syndrome in adults and children. Hematology. 2004;**9**(1):57-60. DOI: 10.1080/10245330310001638983

[26] Old J, Harteveld CL, Traeger-Synodinos J, Petrou M, Angastiniotis M, Galanello R. Prevention of Thalassaemias and Other Haemoglobin Disorder: Volume 2: Laboratory Protocols [Internet]. 2nd ed. Nicosia (Cyprus): Thalassaemia International Federation; 2012. PMID: 24672828

[27] Premawardhena A, Fisher CA, Olivieri NF, de Silva S, Arambepola M, Perera W, et al. Haemoglobin E beta thalassaemia in Sri Lanka. Lancet. 2005; **366**(9495):1467-1470. DOI: 10.1016/ S0140-6736(05)67396-5

[28] Olivieri NF, Muraca GM, O'Donnell A, Premawardhena A, Fisher C, Weatherall DJ. Studies in haemoglobin E beta-thalassaemia. British Journal of Haematology. 2008;**141**(3):388-397. DOI: 10.1111/j.1365-2141.2008.07126.x

[29] Olivieri NF, Pakbaz Z, Vichinsky E. HbE/β-thalassemia: Basis of marked clinical diversity. Hematology/Oncology Clinics of North America. 2010;**24**(6):

*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

1055-1070. DOI: 10.1016/j.hoc.2010. 08.008

[30] Weatherall DJ. Phenotype-genotype relationships in monogenic disease: Lessons from the thalassaemias. Nature Reviews. Genetics. 2001;**2**(4):245-255. DOI: 10.1038/35066048

[31] George E. HbE β thalassemia in Malaysia: Revisited. Journal of Hematology and Thromboembolic Diseases. 2013;**1**(1):1-3. DOI: 10.4172/ 2329-8790.1000101

[32] Winichagoon P, Thonglairoam V, Fucharoen S, Wilairat P, Fukumaki Y, Wasi P. Severity differences in betathalassaemia/haemoglobin E syndromes: Implication of genetic factors. British Journal of Haematology. 1993;**83**(4):633- 639. DOI: 10.1111/j.1365-2141.1993. tb04702.x

[33] Panigrahi I, Marwaha RK. Mutational spectrum of thalassemias in India. Indian Journal of Human Genetics. 2007;**13**(1):36-37. DOI: 10.4103/0971- 6866.32034

[34] Shrivastava M, Bathri R, Chatterjee N. Mutational analysis of thalassemia in transfusion-dependent beta-thalassemia patients from central India. Asian Journal of Transfusion Science. 2019;**13**(2):105- 109. DOI: 10.4103/ajts.AJTS\_115\_18

[35] Sherva R, Sripichai O, Abel K, Ma Q, Whitacre J, Angkachatchai V, et al. Genetic modifiers of Hb E/betathalassemia identified by a two-stage genome-wide association study. BMC Medical Genetics. 2010;**11**:51. DOI: 10.1186/1471-2350-11-51

[36] Winichagoon P, Fucharoen S, Chen P, Wasi P. Genetic factors affecting clinical severity in beta-thalassemia syndromes. Journal of Pediatric Hematology/Oncology. 2000;**22**(6):573580. DOI: 10.1097/00043426- 200011000-00026

[37] Olivieri NF, Pakbaz Z, Vichinsky E. Hb E/beta-thalassaemia: A common & clinically diverse disorder. The Indian Journal of Medical Research. 2011;**134** (4):522-531 PMID: 22089616

[38] Panigrahi I, Agarwal S, Gupta T, Singhal P, Pradhan M. Hemoglobin Ebeta thalassemia: Factors affecting phenotype. Indian Pediatrics. 2005;**42** (4):357-362 PMID: 15876597

[39] Sripichai O, Munkongdee T, Kumkhaek C, Svasti S, Winichagoon P, Fucharoen S. Coinheritance of the different copy numbers of alpha-globin gene modifies severity of betathalassemia/Hb E disease. Annals of Hematology. 2008;**87**(5):375-379. DOI: 10.1007/s00277-007-0407-2

[40] Sharma V, Saxena R. Effect of alphagene numbers on phenotype of HbE/beta thalassemia patients. Annals of Hematology. 2009;**88**(10):1035-1036. DOI: 10.1007/s00277-009-0723-9

[41] Bashir S, Mahmood S, Mohsin S, Tabassum I, Ghafoor M, Sajjad O. Modulatory effect of single nucleotide polymorphism in Xmn1, BCL11A and HBS1L-MYB loci on foetal haemoglobin levels in β-thalassemia major and Intermedia patients. The Journal of the Pakistan Medical Association. 2021;**71** (5):1394-1398. DOI: 10.47391/JPMA.1351

[42] Jaing TH, Chang TY, Chen SH, Lin CW, Wen YC, Chiu CC. Molecular genetics of β-thalassemia: A narrative review. Medicine (Baltimore). 2021;**100** (45):e27522. DOI: 10.1097/ MD.0000000000027522

[43] Menzel S, Garner C, Gut I, Matsuda F, Yamaguchi M, Heath S, et al. A QTL

influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15. Nature Genetics. 2007; **39**(10):1197-1199. DOI: 10.1038/ng2108

[44] Thein SL, Menzel S, Peng X, Best S, Jiang J, Close J, et al. Intergenic variants of HBS1L-MYB are responsible for a major quantitative trait locus on chromosome 6q23 influencing fetal hemoglobin levels in adults. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104** (27):11346-11351. DOI: 10.1073/ pnas.0611393104

[45] Favero ME, Costa FF. Alphahemoglobin-stabilizing protein: An erythroid molecular chaperone. Biochemistry Research International. 2011;**2011**:373859. DOI: 10.1155/2011/ 373859

[46] Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, et al. The genetic basis of the reduced expression of bilirubin UDPglucuronosyltransferase 1 in Gilbert's syndrome. The New England Journal of Medicine. 1995;**333**(18):1171-1175. DOI: 10.1056/NEJM199511023331802

[47] Premawardhena A, Fisher CA, Fathiu F, de Silva S, Perera W, Peto TE, et al. Genetic determinants of jaundice and gallstones in haemoglobin E beta thalassaemia. Lancet. 2001;**357**(9272): 1945-1946. DOI: 10.1016/s0140-6736 (00)05082-0

[48] Rees DC, Duley J, Simmonds HA, Wonke B, Thein SL, Clegg JB, et al. Interaction of hemoglobin E and pyrimidine 5<sup>0</sup> nucleotidase deficiency. Blood. 1996;**88**(7):2761-2767 PMID: 8839873

[49] Merryweather-Clarke AT, Pointon JJ, Jouanolle AM, Rochette J, Robson KJ. Geography of HFE C282Y and H63D mutations. Genetic Testing. 2000;**4**(2): 183-198. DOI: 10.1089/109065700 50114902

[50] Pointon JJ, Viprakasit V, Miles KL, Livesey KJ, Steiner M, O'Riordan S, et al. Hemochromatosis gene (HFE) mutations in South East Asia: A potential for iron overload. Blood Cells, Molecules & Diseases. 2003;**30**(3):302-306. DOI: 10.1016/s1079-9796(03)00041-x

[51] Jones E, Pasricha SR, Allen A, Evans P, Fisher CA, Wray K, et al. Hepcidin is suppressed by erythropoiesis in hemoglobin E β-thalassemia and β-thalassemia trait. Blood. 2015;**125**(5): 873-880. DOI: 10.1182/blood-2014-10- 606491

[52] Ha J, Martinson R, Iwamoto SK, Nishi A. Hemoglobin E, malaria and natural selection. Evolution, Medicine, and Public Health. 2019;**2019**(1):232- 241. DOI: 10.1093/emph/eoz034

[53] Yuthavong Y, Butthep P, Bunyaratvej A, Fucharoen S. Inhibitory effect of beta zero-thalassaemia/ haemoglobin E erythrocytes on *Plasmodium falciparum* growth in vitro. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1987;**81** (6):903-906. DOI: 10.1016/0035-9203 (87)90344-0

[54] O'Donnell A, Premawardhena A, Arambepola M, Samaranayake R, Allen SJ, Peto TE, et al. Interaction of malaria with a common form of severe thalassemia in an Asian population. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**(44):18716-18721. DOI: 10.1073/pnas.0910142106

[55] Mettananda S, Pathiraja H, Peiris R, Bandara D, de Silva U, Mettananda C, et al. Health related quality of life among

*The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

children with transfusion dependent β-thalassaemia major and haemoglobin E β-thalassaemia in Sri Lanka: A case control study. Health and Quality of Life Outcomes. 2019;**17**(1):137. DOI: 10.1186/ s12955-019-1207-9

[56] Lal A, Wong T, Keel S, Pagano M, Chung J, Kamdar A, et al. The transfusion management of beta thalassemia in the United States. Transfusion. 2021;**61**(10):3027-3039. DOI: 10.1111/trf.16640

[57] Sari TT, Gatot D, Akib AA, Bardosono S, Hadinegoro SR, Harahap AR, et al. Immune response of thalassemia major patients in Indonesia with and without splenectomy. Acta Medica Indonesiana. 2014;**46**(3):217-225 PMID: 25348184

[58] Rachmilewitz EA, Giardina PJ. How I treat thalassemia. Blood. 2011;**118**(13): 3479-3488. DOI: 10.1182/blood-2010- 08-300335

[59] Entezari S, Haghi SM, Norouzkhani N, Sahebnazar B, Vosoughian F, Akbarzadeh D, et al. Iron chelators in treatment of iron overload. Journal of Toxicology. 2022;**2022**:4911205. DOI: 10.1155/2022/4911205

[60] Jaiswal S, Hishikar R, Khandwal O, Agarwal M, Joshi U, Halwai A, et al. Efficacy of deferasirox as an oral iron chelator in paediatric thalassaemia patients. Journal of Clinical and Diagnostic Research. 2017;**11**(2):FC01- FC03. DOI: 10.7860/JCDR/2017/ 22650.9395

[61] Olivieri NF, Sabouhanian A, Gallie BL. Single-center retrospective study of the effectiveness and toxicity of the oral iron chelating drugs deferiprone and deferasirox. PLoS One. 2019;**14**(2): e0211942. DOI: 10.1371/journal. pone.0211942

[62] Ali S, Mumtaz S, Shakir HA, Khan M, Tahir HM, Mumtaz S, et al. Current status of beta-thalassemia and its treatment strategies. Molecular Genetics & Genomic Medicine. 2021;**9**(12):e1788. DOI: 10.1002/mgg3.1788

[63] Hider RC, Hoffbrand AV. The role of deferiprone in iron chelation. The New England Journal of Medicine. 2018;**379** (22):2140-2150. DOI: 10.1056/ NEJMra1800219

[64] Bayanzay K, Alzoebie L. Reducing the iron burden and improving survival in transfusion-dependent thalassemia patients: Current perspectives. Journal of Blood Medicine. 2016;**7**:159-169. DOI: 10.2147/JBM.S61540

[65] Taher AT, Cappellini MD. How I manage medical complications of βthalassemia in adults. Blood. 2018;**132** (17):1781-1791. DOI: 10.1182/blood-2018-06-818187

[66] Davis BA, Porter JB. Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in high-risk betathalassemia. Blood. 2000;**95**(4):1229- 1236 PMID: 10666195

[67] Tanner MA, Galanello R, Dessi C, Smith GC, Westwood MA, Agus A, et al. A randomized, placebo-controlled, double-blind trial of the effect of combined therapy with deferoxamine and deferiprone on myocardial iron in thalassemia major using cardiovascular magnetic resonance. Circulation. 2007; **115**(14):1876-1884. DOI: 10.1161/ CIRCULATIONAHA.106.648790

[68] De Domenico I, Ward DM, Kaplan J. Specific iron chelators determine the route of ferritin degradation. Blood. 2009;**114**(20):4546-4551. DOI: 10.1182/ blood-2009-05-224188

[69] Majolino I, Othman D, Rovelli A, Hassan D, Rasool L, Vacca M, et al. The start-up of the first hematopoietic stem cell transplantation center in the Iraqi Kurdistan: A Capacity-Building Cooperative Project by the Hiwa Cancer Hospital, Sulaymaniyah, and the Italian Agency for Development Cooperation: An Innovative Approach. Mediterranean Journal of Hematology and Infectious Diseases. 2017;**9**(1):e2017031. DOI: 10.4084/MJHID.2017.031

[70] Jeengar R K, Upadhyaya A, Agarwal N, Mehta A. Red cell alloimmunization in repeatedly transfused children with beta thalassemia major. International Journal of Contemporary Pediatrics, 2017;4(3): 775–779. https://doi.org/ 10.18203/2349-3291.ijcp20171486.

[71] Bernardo ME, Piras E, Vacca A, Giorgiani G, Zecca M, Bertaina A, et al. Allogeneic hematopoietic stem cell transplantation in thalassemia major: Results of a reduced-toxicity conditioning regimen based on the use of treosulfan. Blood. 2012;**120**(2): 473-476. DOI: 10.1182/blood-2012-04- 423822

[72] Cario H. Hemoglobinopathies: Genetically diverse, clinically complex, and globally relevant. European Medical Oncology2018;11(3): 235–240. https:// doi.org/10.1007/s12254-018-0402-4.

[73] Morgan RA, Gray D, Lomova A, Kohn DB. Hematopoietic stem cell gene therapy: Progress and lessons learned. Cell Stem Cell. 2017;**21**(5):574-590. DOI: 10.1016/j.stem.2017.10.010

[74] Yang J, Li S, He XB, Cheng C, Le W. Induced pluripotent stem cells in Alzheimer's disease: Applications for disease modeling and cell-replacement therapy. Molecular Neurodegeneration. 2016;**11**(1):39. DOI: 10.1186/s13024-016- 0106-3

[75] Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: A review of the challenges and approaches. Drug Delivery. 2018;**25**(1):1234-1257. DOI: 10.1080/10717544.2018.1474964

[76] Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: Mechanisms, advances and prospects. Signal Transduction and Targeted Therapy. 2020;**5**(1):1. DOI: 10.1038/s41392-019- 0089-y

[77] Zivot A, Lipton JM, Narla A, Blanc L. Erythropoiesis: Insights into pathophysiology and treatments in 2017. Molecular Medicine. 2018;**24**(1):11. DOI: 10.1186/s10020-018-0011-z

[78] Alebouyeh M, Moussavi F, Haddad-Deylami H, Vossough P. Hydroxyurea in the treatment of major beta-thalassemia and importance of genetic screening. Annals of Hematology. 2004;**83**(7):430- 433. DOI: 10.1007/s00277-003-0836-5

[79] Dixit A, Chatterjee TC, Mishra P, Choudhry DR, Mahapatra M, Tyagi S, et al. Hydroxyurea in thalassemia intermedia – a promising therapy. Annals of Hematology. 2005;**84**(7):441- 446. DOI: 10.1007/s00277-005-1026-4

[80] Cao H. Pharmacological induction of fetal hemoglobin synthesis using histone deacetylase inhibitors. Hematology. 2004;**9**(3):223-233. DOI: 10.1080/ 10245330410001701512

[81] Gambari R, Fibach E. Medicinal chemistry of fetal hemoglobin inducers for treatment of beta-thalassemia. Current Medicinal Chemistry. 2007;**14** (2):199-212. DOI: 10.2174/ 092986707779313318

[82] Suwanmanee T, Sierakowska H, Fucharoen S, Kole R. Repair of a splicing *The Key Genetic Determinants Behind the Phenotypic Heterogeneity… DOI: http://dx.doi.org/10.5772/intechopen.109999*

defect in erythroid cells from patients with beta-thalassemia/HbE disorder. Molecular Theory. 2002;**6**:718-726. DOI: 10.1006/mthe.2002.0805

[83] Shaeffer JR, Cohen RE. Ubiquitin aldehyde increases adenosine triphosphate-dependent proteolysis of hemoglobin alpha-subunits in betathalassemic hemolysates. Blood. 1997;**90** (3):1300-1308

[84] Hashemieh M, Saadatmandi ZAS, Azarkeivan A, Najmabadi H. The Effect of Xmn -1 Polymorphism and Coinheritance of Alpha Mutations on Age at First Blood Transfusion in Iranian Patients with Homozygote IVSI-5 Mutation. Int J Hematol Oncol Stem Cell Res. 2022;**16**(1):47-54. DOI: 10.18502/ ijhoscr.v16i1.8441

[85] Cao A, Galanello R. Betathalassemia. Genetics in Medicine. 2010; **12**(2):61-76. DOI: 10.1097/ GIM.0b013e3181cd68ed

[86] de Dreuzy E, Bhukhai K, Leboulch P, Payen E. Current and future alternative therapies for beta-thalassemia major. Biomedical Journal. 2016;**39**(1):24-38. DOI: 10.1016/j.bj.2015.10.001

[87] Ben Salah N, Bou-Fakhredin R, Mellouli F, Taher AT. Revisiting beta thalassemia intermedia: Past, present, and prospects. Hematology. 2017;**22**(10): 607-616. DOI: 10.1080/ 10245332.2017.1333246

[88] Cighetti G, Duca L, Bortone L, Sala S, Nava I, Fiorelli G, et al. Oxidative status and malondialdehyde in betathalassaemia patients. European Journal of Clinical Investigation. 2002;**32**(1):55- 60. DOI: 10.1046/j.1365-2362.2002. 0320s1055.x

[89] Dissayabutra T, Tosukhowong P, Seksan P. The benefits of vitamin C and vitamin E in children with betathalassemia with high oxidative stress. Journal of the Medical Association of Thailand. 2005;**88**(4):S317-S321

#### **Chapter 5**

## Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship

*Manit Nuinoon*

#### **Abstract**

Thalassemia and hemoglobinopathies are characterized by globin gene mutations affecting the production of quantitative and structural defects of the globin chain. α-Thalassemia, β-thalassemia, hemoglobin E (Hb E), and hemoglobin Constant Spring (Hb CS) are very common in Southeast Asian countries. Complex interactions of thalassemia and Hb variants are also common and affect the thalassemia diagnosis with several techniques including Hb typing and DNA analysis. A family study (family pedigree) is required in the proband with a complex interaction of several globin gene defects with rare types. Homozygous β-thalassemia, Hb E/β-thalassemia, and Hb Bart's hydrops fetalis are severe thalassemia and these diseases have been concerned and included in the prevention and control program in several countries. Understanding the genotype-phenotype could help with the proper laboratory tests, genetic counseling, and effective treatment for the patients.

**Keywords:** thalassemia, Hb variants, southeast Asian countries, thalassemia interaction, genotype-phenotype, DNA analysis

#### **1. Introduction**

Southeast Asia (SEA) is composed of 11 countries such as Burma (Myanmar), Laos, Thailand, Cambodia, Vietnam, Malaysia, Singapore, Brunei, Indonesia, the Philippines, and Timor-Leste (**Figure 1**). As of 2021, around 676 million people live in the region [1]. The ethnic origins of people living in SEA countries are very heterogeneous according to religion, culture, and history. This chapter focused on the genotype-phenotype relationship between thalassemia and hemoglobinopathies in the Southeast Asian population. Both common and rare types of thalassemia and Hb variant are demonstrated in homozygous, double heterozygous, and compound heterozygous states for clinical and red blood cell phenotypes.

**Figure 1.** *The map of southeast Asian countries [2].*

#### **2. Globin gene cluster, functional globin genes, and normal adult hemoglobin**

In humans, two globin gene clusters are responsible for hemoglobin synthesis in all developmental stages, including embryonic, fetal, and adult stages (**Figure 2**). The α-like gene cluster contains three functional genes, including the ζ2, α2, and α1 globin genes in chromosome 16 (16p13.3), and encoded to form the ζ- and α-globin chains which consist of 141 amino acids. In addition, the β -like gene cluster contains 5 functional genes including the ε, Gγ, Aγ, δ, and β-globin genes, in chromosome 11 (11p15.5), and encoded to form the ε, γ, δ, and β-globin chains which consist of 146 amino acids. During normal humans, each globin gene from 2 globin gene clusters is activated and expressed according to the specific developmental stage such as the embryonic stage (Hb Portland, Hb Gower I, and Hb Gower II), fetal stage (Hb F or fetal hemoglobin), and adult stage (Hb A and Hb A2) [3, 4].

Hemoglobin (Hb), an iron-containing protein in erythrocytes (red blood cells), is responsible for transporting oxygen (O2) from the lungs to tissues and to transporting carbon dioxide (CO2) from tissues. In adult life, Hb A (α2β2), or adult hemoglobin is the major component of normal adult hemoglobin (more than 95% of the total hemoglobin). Hb A2 (α2δ2) is the second component about less than 3.5% in normal adults. Hb F (α2γ2) or fetal hemoglobin with 1–2% is found in normal individuals [5].

**Figure 2.** *Schematic representation of the globin gene cluster.*

*Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship DOI: http://dx.doi.org/10.5772/intechopen.110001*

#### **Figure 3.**

*General structure functional α -globin genes (A)and the β-globin gene (B).*

According to Hb A (α2β2) is the major component of total hemoglobin and contributes to gas transport in the human body. In the adult stage, α and β-globin genes are the two most important for globin chain synthesis and build up to form the tetramerization of 2 α-globin chains and 2 β-globin chains and each globin chain bound heme group (an iron atom bound within a protoporphyrin IX ring) [6]. Therefore, α and β-globin gene mutations were the most considered condition in the adult for thalassemia or Hb variants. An approximate 50 bp of the 5′ untranslated region (5'UTR) and codons for amino acid sequences 1–31 in the *HBA1*(or *HBA2*) and 1–30 in the *HBB* genes are represented as the first exon. The second exon encodes amino acids 32–99 and 31–104, respectively. The third exon encodes amino acids 101–141 for the α-globin gene and 105–146 for the β-globin gene, together with about 100 bp of 3′UTR (**Figure 3**) [3].

#### **3. Genotype-phenotype relationship**

In the human globin gene clusters, the α-globin gene cluster is located at the short arm of chromosome 16 (two copies of the α-globin gene per chromatid, *HBA2*, and *HBA1* genes) whereas the β-globin gene cluster is located at the short arm of chromosome 11 (one copy of each β-globin gene per chromatid, *HBB* gene). Both chromosomes 11 and 16 are autosomal chromosomes (*2n*, diploid cell). Therefore, a total of four genes per diploid cell of the α-globin genotype (αα/αα) and a total of two genes per diploid cell for the β -globin genotype (βA/βA). The mutations of the human globin gene can inherit from the parent ranging from 1 allele to 4 alleles of α- and β-globin genes and resulting in various forms of the carrier or thalassemia disease. αand β-Globin genotyping can be characterized by several PCR-based methods [7]. In the context of globin gene defects, phenotype refers to the observable hematological (red blood cell morphology, osmotic fragility test, abnormal Hb screening, and Hb analysis) or clinical characteristics of the carriers or patients. Recently, the clinical classification of thalassemia is divided into two phenotypes according to the patient's clinical severity and transfusion requirements such as non-transfusion-dependent thalassemia (NTDT) and transfusion-dependent thalassemia (TDT) [8]. Therefore,

genotype-phenotype correlation is a relationship between specific globin mutations and hematological profiles or clinical symptoms. The red blood cell phenotypes and other related screening methods are the primary results for predicting a possible type of thalassemia carrier or disease [9, 10]. However, globin genotyping is required for a definitive and precise diagnosis of thalassemia for proper management and treatment [11].

#### **4. Thalassemia and hemoglobinopathy**

Thalassemia, a quantitative defect of globin chain synthesis, is caused by globin gene mutation and characterized by the absence (designed with a "0" superscript) or reduced (designed with a "+" superscript) synthesis of one or more of the normal globin chains. The α- and β-thalassemia are major types during the adult stage. In contrast, hemoglobinopathy is characterized by a qualitative or structural defect of globin chain synthesis. Thalassemic hemoglobinopathy is the combination of quantitative and qualitative features of globin chain synthesis such as Hb Constant Spring (Hb CS, α+ -thalassemia-like effect) and hemoglobin E (Hb E, β<sup>+</sup> -thalassemia) [12]. Hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia are characterized by elevated fetal hemoglobin (Hb F) levels in adult life. There are no morphological changes to the red blood cells and red cell indices in HPFH whereas more abnormal red blood cells are observed in δβ-thalassemia [13]. In Southeast Asia α-thalassemia, β-thalassemia, Hb E, and Hb CS are prevalent and the gene frequencies vary in different countries. In Thailand, the carrier frequencies of 10–30% for α-thalassemia, 3–9% for β-thalassemia, and 10–53% for Hb E [14, 15]. The combinations of different globin gene mutations lead to over 60 different thalassemia syndromes and the most complex thalassemia genotypes were found among Southeast Asians [15]. According to common globin gene mutations found in the Southeast Asian population, the four major thalassemia diseases are Hb Bart's hydrops fetalis (−−/−−), homozygous β-thalassemia (β\*/β\*), Hb E/β-thalassemia (β<sup>E</sup> /β\*), and Hb H diseases (deletional Hb H disease, −−/−α; non-deletional Hb H disease, −−/α<sup>T</sup> α) [ 15–17]. Only the first three thalassemia diseases were concerned with prevention and control programs for severe thalassemia in Thailand and other Southeast Asian countries [18–21]. Clinical manifestations of thalassemia range from asymptomatic with mild microcytic hypochromic red blood cells to the totally lethal Hb Bart's hydrops fetalis [16, 22]. Moreover, the interaction of the thalassemias and hemoglobin variants from multiple globin gene mutations may not be uncommon in Southeast Asians. The hematological and complex hemoglobin profile has been reported in several publications and DNA analysis is required to characterize disease-causing mutation [7, 21]. Therefore, understanding the genotype-phenotype relationship is very useful for precise diagnosis with proper laboratory tests and economic benefits [23]. In Southeast Asia α-Thalassemia is associated with variable numbers of α-globin gene deletions by combining 2 alleles such as −α3.7, −α4.2, −(α) −20, −−SEA, −−THAI, −−FIL, and −−CR with other alleles such as normal (αα) or α-globin chain variants (α<sup>T</sup> α or αα<sup>T</sup> ) [15, 24–27]. The clinical phenotype of α-thalassemia relates to the number of affected α-globin genes ranging from no clinical symptom (hypochromic and microcytic red cells without anemia) to lethal thalassemia disease [16, 28]. β-Thalassemias are very heterogenous and various β-globin gene mutations have been characterized. β-Thalassemia mutations could be classified as β++, β<sup>+</sup> , or β<sup>0</sup> thalassemia phenotypes according to different molecular mechanisms [11, 29–31]. In addition, several Hb chain variants of α-globin genes

*Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship DOI: http://dx.doi.org/10.5772/intechopen.110001*

(*HBA1* and *HBA2*), β-globin gene (*HBB*), and δ-globin gene (*HBD*) have been found among the Southeast Asian population which are summarized in **Table 1**.

#### **5. Interaction of common thalassemia and hemoglobin variants**

α- and β-globin genes can be inherited independently by the next generation. There are 4 possible genotypes of the α-globin gene and 4 possible genotypes of the β -globin gene. Therefore, the maximum genotypes of α- and β-globin genes are 16 possible genotypes. This model is useful for the prediction of severe thalassemia for the child in preconception counseling or prenatal diagnosis (PND) process (**Figure 4**).

In Southeast Asian countries, the complex interaction of thalassemia and the Hb variant is common. The dihybrid cross with the mutations in both α- and β-globin genes from the father (CS EA Bart's disease) and mother (double heterozygosity for β<sup>0</sup> -thalassemia and α<sup>0</sup> -thalassemia) is used to give an example for the reader. All globin genotypes obtained from the parent are essential information for evaluating the risk ratio of being severe thalassemia. The list of possible α-globin genotypes are 4 distinct genotypes as follows; αCSα/αα (Hb Constant Spring heterozygote), −−SEA/αα (α<sup>0</sup> -thalassemia heterozygote), −−SEA/αCSα (Hb H-Constant Spring), and −−SEA/−−SEA (Hb Bart's hydrops fetalis or homozygous α<sup>0</sup> -thalassemia). In addition, the list of possible β-globin genotypes are 4 distinct genotypes as follows; βA/β<sup>A</sup> (normal genotype), βA/β<sup>0</sup> (β<sup>0</sup> -thalassemia heterozygote), β<sup>E</sup> /βA (Hb E heterozygote), and β<sup>E</sup> /β0 (Hb E/β<sup>0</sup> -thalassemia). According to 4 possible α- and 4 possible β-globin genotypes, 16 distinct combinations are obtained. In this case, Hb Bart's hydrops fetalis and Hb E/β<sup>0</sup> -thalassemia are concerned and 7 combined genotypes (1, 2, 3, 4, 7, 11, and 15) are risk genotypes and this couple is a true risk couple with 7/16 (43.75%) for being severe thalassemia in the child (**Figure 5**).

Because of the high frequency of thalassemias and Hb variants, the interactions of thalassemias and Hb variants especially in two major globin chains (α- and β-globin) were observed in the Southeast Asian population. Hb E and Hb CS are the two most common Hb variants represented for β- and α-globin genes. Commonly, interactions of Hb E with other thalassemias or Hb variants resulting in Hb E-related syndromes such as Hb E/β-thalassemia with or without α-thalassemia interaction, AE Bart's disease, EF Bart's disease, etc. (**Table 2**). In an area where Hb E, β-thalassemia, and α-thalassemia are prevalent, the interaction of Hb E with several types of thalassemia is frequently observed. Among Hb E heterozygotes, a proportion of Hb A2/E lower than 25% has been used for suspecting α-thalassemia interaction and confirmed by DNA analysis [9]. Various forms of α-thalassemia are common and interaction of thalassemia with heterozygous Hb E can result in a reduced Hb A2/E level and hematological changes [35]. In contrast, the interaction of homozygous Hb E with α-thalassemia could not be differentially diagnosed by red cell indices and Hb-HPLC analysis [36]. Hb analysis by capillary electrophoresis can separate Hb A2 from Hb E and Hb A2 could be reported in the presence of Hb E [37, 38]. Interestingly, an increased Hb A2 level is a useful biomarker for differentiation of Hb E homozygote with or without α<sup>0</sup> -thalassemia [39]. The combination of heterozygous Hb E and Hb H disease or Hb H-Constant Spring disease has a marked decrease of Hb E (13–15%) with thalassemia intermedia, which is called AE Bart's disease [22]. Co-inheritance of Hb H disease with homozygous Hb E resulted in EF Bart's disease with mild anemia and increased Hb F levels and Hb


*Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship DOI: http://dx.doi.org/10.5772/intechopen.110001*


#### **Table 1.**

*Hb variants in southeast Asian countries [32–34].*

#### *Thalassemia Syndromes – New Insights and Transfusion Modalities*


#### **Figure 4.**

*The model of the dihybrid cross of α- and β-globin genotypes.*

#### **Figure 5.**

*The model of dihybrid cross of CS EA Bart's disease and double heterozygosity for β<sup>0</sup> -thalassemia and α0 -thalassemia.*

Bart's [22]. The compound heterozygous state for β-thalassemia and Hb E namely Hb E-β-thalassemia is variable disease severity ranging from transfusion-dependent thalassemia to thalassemia intermedia. An ameliorating effect of α-thalassemia interactions and high Hb F determinants has been well studied [40–42]. Moreover, the interaction of thalassemia and Hb variants has been reported in several publications in the Thai population such as compound heterozygosity for Hb Korle-Bu and Hb E with α<sup>+</sup> -thalassemia, complex interactions between Hb Lepore-Hollandia and Hb E with α<sup>+</sup> -thalassemia and interaction between Hb E and Hb Yala resulting in Hb E/β<sup>0</sup> -thalassemia, double heterozygosity of Hb Hope and α<sup>0</sup> -thalassemia and compound heterozygotes for Hb Hope and β<sup>0</sup> -thalassemia [43–46]. Hereditary persistence (HPFH) and δβ-thalassemia are characterized by elevated fetal hemoglobin levels in adult life. There are several mutations reported in the Thai population such as <sup>G</sup>γ<sup>A</sup>γ (δβ) 0 -thalassemia, deletional HPFH-6, and deletion-inversion <sup>G</sup>γ( <sup>A</sup>γδβ) 0 thalassemia [47–49].

#### **6. Conclusions**

The understanding of the genotype-phenotype relationship is essential for proper laboratory testing, genetic counseling, and treatments. The concept of thalassemia interaction could be applied in a country with high frequency and heterogeneity of thalassemia and hemoglobinopathies. DNA analysis is very important for definitive diagnosis, as well as the family study, and could be helped in complex thalassemia with a rare hemoglobin variant. Characterization of globin *Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship DOI: http://dx.doi.org/10.5772/intechopen.110001*


*CS, Constant Spring; PS, Pakse; HPFH, hereditary persistence of fetal hemoglobin; NTDT, non-transfusion-dependent thalassemia; TDT, transfusion-dependent thalassemia; Thal, Thalassemia; TI, thalassemia intermedia; TM, thalassemia major.\*Hb type is based on HPLC technique.*

#### **Table 2.**

*Phenotypes of thalassemias, Hb variants and interaction of thalassemia and Hb variants in the southeast Asian population.*

gene mutations in the population is important and a globin gene mutation database in each country is required for improving prevention and control program for severe thalassemia.

### **Conflict of interest**

The author declares no conflict of interest.

### **Author details**

Manit Nuinoon1,2

1 Hematology and Transfusion Science Research Center, Walailak University, Nakhon Si Thammarat, Thailand

2 School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, Thailand

\*Address all correspondence to: manit.nu@wu.ac.th

© 2023 The Author(s). Licensee IntechOpen. 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.

*Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship DOI: http://dx.doi.org/10.5772/intechopen.110001*

#### **References**

[1] Southeast Asian. Available from: https://en.wikipedia.org/wiki/ Southeast\_Asia

[2] Map chart. Available from: https:// www.mapchart.net/index.html

[3] Weatherall DJ. Globin Genes, Human. In: Maloy S, Hughes K, editors. Brenner's Encyclopedia of Genetics. 2nd ed. San Diego: Academic Press; 2013. pp. 337-339

[4] Higgs D, Thein S, Wood W, Human haemoglobin. The Thalassaemia Syndromes. Oxford: Blackwell Science Ltd; 2001. pp. 65-120

[5] Otto CN. 7 - Hemoglobin metabolism. In: Keohane EM, Otto CN, Walenga JM, editors. Rodak's Hematology. 6th ed. St. Louis (MO): Elsevier; 2020. pp. 91-103

[6] Steinberg MH, Benz EJ, Adewoye AH, Ebert BL. Chapter 33 - Pathobiology of the Human erythrocyte and its Hemoglobins. In: Hoffman R, Benz EJ, Silberstein LE, Heslop HE, Weitz JI, Anastasi J, et al., editors. Hematology. 7th ed. Philadelphia (PA): Elsevier; 2018. pp. 447-457

[7] Munkongdee T, Chen P, Winichagoon P, Fucharoen S, Paiboonsukwong K. Update in laboratory diagnosis of thalassemia. Frontiers in Molecular Biosciences. 2020;**7**:74

[8] Viprakasit V, Ekwattanakit S. Clinical classification, screening and diagnosis for thalassemia. Hematology/ Oncology Clinics of North America. 2018;**32**(2):193-211

[9] Fucharoen G, Sanchaisuriya K, Sae-ung N, Dangwibul S, Fucharoen S. A simplified screening strategy for thalassaemia and

haemoglobin E in rural communities in south-East Asia. Bulletin of the World Health Organization. 2004;**82**(5):364-372

[10] Savongsy O, Fucharoen S, Fucharoen G, Sanchaisuriya K, Sae-Ung N. Thalassemia and hemoglobinopathies in pregnant Lao women: Carrier screening, prevalence and molecular basis. Annals of Hematology. 2008;**87**(8):647-654

[11] Danjou F, Anni F, Galanello R. β-thalassemia: From genotype to phenotype. Haematologica. 2011;**96**(11):1573-1575

[12] Steinberg MH, Adams JG. Thalassemic hemoglobinopathies. The American Journal of Pathology. 1983;**113**(3):396-409

[13] Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Annals of the New York Academy of Sciences. 1998;**850**:38-44

[14] Fucharoen S, Winichagoon P, Siritanaratkul N, Chowthaworn J, Pootrakul P. α- and β-thalassemia in Thailand. Annals of the New York Academy of Sciences. 1998;**850**:412-414

[15] Fucharoen S, Winichagoon P. Haemoglobinopathies in Southeast Asia. The Indian Journal of Medical Research. 2011;**134**(4):498-506

[16] Vichinsky EP. Clinical manifestations of α-thalassemia. Cold Spring Harbor Perspectives in Medicine. 2013;**3**(5):a011742

[17] Farashi S, Najmabadi H. Diagnostic pitfalls of less well recognized HbH disease. Blood Cells, Molecules, and Diseases. 2015;**55**(4):387-395

[18] Yamsri S, Sanchaisuriya K, Fucharoen G, Sae-Ung N, Ratanasiri T, Fucharoen S. Prevention of severe thalassemia in Northeast Thailand: 16 years of experience at a single university center. Prenatal Diagnosis. 2010;**30**(6):540-546

[19] Fucharoen S, Winichagoon P. Prevention and control of thalassemia in Asia. Asian Biomedicine. 2007;**1**:1-6

[20] Fucharoen S, Winichagoon P. Thalassemia in SouthEast Asia: Problems and strategy for prevention and control. The Southeast Asian Journal of Tropical Medicine and Public Health. 1992;**23**(4):647-655

[21] Nopparatana C, Nopparatana C, Saechan V, Karnchanaopas S, Srewaradachpisal K. Prenatal diagnosis of α- and β-thalassemias in southern Thailand. International Journal of Hematology. 2020;**111**(2):284-292

[22] Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring Harbor Perspectives in Medicine. 2012;**2**(8):a011734

[23] Cao A, Galanello R, Rosatelli MC. Genotype-phenotype correlations in β-thalassemias. Blood Reviews. 1994;**8**(1):1-12

[24] Tritipsombut J, Sanchaisuriya K, Phollarp P, Bouakhasith D, Sanchaisuriya P, Fucharoen G, et al. Micromapping of thalassemia and hemoglobinopathies in diferent regions of Northeast Thailand and Vientiane, Laos People's Democratic Republic. Hemoglobin. 2012;**36**(1):47-56

[25] Charoenkwan P, Taweephon R, Sae-Tung R, Thanarattanakorn P, Sanguansermsri T. Molecular and clinical features of Hb H disease in northern Thailand. Hemoglobin. 2005;**29**(2):133-140

[26] Tangvarasittichai O, Jeenapongsa R, Sitthiworanan C, Sanguansermsri T. Laboratory investigations of Hb constant spring. Clinical and Laboratory Haematology. 2005;**27**(1):47-49

[27] Nittayaboon K, Nopparatana C. Molecular characterization of Hb H disease in southern Thailand. International Journal of Hematology. 2018;**108**(4):384-389

[28] Piel FB, Weatherall DJ. The α-Thalassemias. New England Journal of Medicine. 2014;**371**(20):1908-1916

[29] Yamsri S, Singha K, Prajantasen T, Taweenan W, Fucharoen G, Sanchaisuriya K, et al. A large cohort of β+ -thalassemia in Thailand: Molecular, hematological and diagnostic considerations. Blood Cells, Molecules & Diseases. 2015;**54**(2):164-169

[30] Abdullah UYH, Ibrahim HM, Mahmud NB, Salleh MZ, Teh LK, Noorizhab M, et al. Genotype-phenotype correlation of β-thalassemia in Malaysian population: Toward effective genetic Counseling. Hemoglobin. 2020;**44**(3):184-189

[31] Yamsri S, Sanchaisuriya K, Fucharoen G, Sae-Ung N, Fucharoen S. Genotype and phenotype characterizations in a large cohort of β-thalassemia heterozygote with different forms of α-thalassemia in Northeast Thailand. Blood Cells, Molecules & Diseases. 2011;**47**(2):120-124

[32] Giardine B, Borg J, Viennas E, Pavlidis C, Moradkhani K, Joly P, et al. Updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Research. 2014;**42**(Database issue):D1063-D1069

[33] Saechan V, Nopparatana C, Nopparatana C, Fucharoen S. Molecular basis and hematological features of

*Interaction of Thalassemia and Hb Variants in Southeast Asia: Genotype-Phenotype Relationship DOI: http://dx.doi.org/10.5772/intechopen.110001*

hemoglobin variants in southern Thailand. International Journal of Hematology. 2010;**92**(3):445-450

[34] Srivorakun H, Singha K, Fucharoen G, Sanchaisuriya K, Fucharoen S. A large cohort of hemoglobin variants in Thailand: Molecular epidemiological study and diagnostic consideration. PLoS One. 2014;**9**(9):e108365

[35] Sanchaisuriya K, Fucharoen G, Sae-ung N, Jetsrisuparb A, Fucharoen S. Molecular and hematologic features of hemoglobin E heterozygotes with different forms of α-thalassemia in Thailand. Annals of Hematology. 2003;**82**(10):612-616

[36] Fucharoen G, Trithipsombat J, Sirithawee S, Yamsri S, Changtrakul Y, Sanchaisuriya K, et al. Molecular and hematological profiles of hemoglobin EE disease with different forms of α-thalassemia. Annals of Hematology. 2006;**85**(7):450-454

[37] Sangkitporn S, Sangkitporn SK, Tanjatham S, Suwannakan B, Rithapirom S, Yodtup C, et al. Multicenter validation of fully automated capillary electrophoresis method for diagnosis of thalassemias and hemoglobinopathies in Thailand. The Southeast Asian Journal of Tropical Medicine and Public Health. 2011;**42**(5):1224-1232

[38] Hafiza A, Malisa MY, Khirotdin RD, Azlin I, Azma Z, Thong MC, et al. HbA2 levels in normal, β-thalassaemia and haemoglobin E carriers by capillary electrophoresis. The Malaysian Journal of Pathology. 2012;**34**(2):161-164

[39] Singha K, Srivorakun H, Fucharoen G, Fucharoen S. Co-inheritance of α<sup>0</sup> -thalassemia elevates Hb A2 level in homozygous Hb E: Diagnostic implications. International

Journal of Laboratory Hematology. 2017;**39**(5):508-512

[40] Sripichai O, Munkongdee T, Kumkhaek C, Svasti S, Winichagoon P, Fucharoen S. Coinheritance of the different copy numbers of α-globin gene modifies severity of β-thalassemia/ Hb E disease. Annals of Hematology. 2008;**87**(5):375-379

[41] Winichagoon P, Fucharoen S, Chen P, Wasi P. Genetic factors affecting clinical severity in β-thalassemia syndromes. Journal of Pediatric Hematology/Oncology. 2000;**22**(6):573-580

[42] Winichagoon P, Thonglairoam V, Fucharoen S, Wilairat P, Fukumaki Y, Wasi P. Severity differences in β-thalassaemia/haemoglobin E syndromes: Implication of genetic factors. British Journal of Haematology. 1993;**83**(4):633-639

[43] Pornprasert S, Panyasai S, Kongthai K. Comparison of capillary electrophoregram among heterozygous Hb Hope, Hb Hope/α-thalassemia-1 SEA type deletion and Hb Hope/ β0 -thalassemia. Clinical Chemistry and Laboratory Medicine. 2012;**50**(9):1625-1629

[44] Ekwattanakit S, Riolueang S, Viprakasit V. Interaction between Hb E and Hb Yala (HBB:c.129delT); a novel frameshift β globin gene mutation, resulting in Hemoglobin E/ β0 -thalassemia. Hematology. 2018;**23**(2):117-121

[45] Viprakasit V, Pung-Amritt P, Suwanthon L, Clark K, Tanphaichtr VS. Complex interactions of δβ hybrid haemoglobin (Hb Lepore-Hollandia) Hb E (β(26G-->A)) and α<sup>+</sup> thalassaemia in a Thai family. European Journal of Haematology. 2002;**68**(2):107-111

[46] Changtrakun Y, Fucharoen S, Ayukarn K, Siriratmanawong N, Fucharoen G, Sanchaisuriya K. Compound heterozygosity for Hb Korle-Bu (beta(73); Asp-Asn) and Hb E (beta(26); Glu-Lys) with a 3.7-kb deletional α-thalassemia in Thai patients. Annals of Hematology. 2002;**81**(7):389-393

[47] Svasti S, Paksua S, Nuchprayoon I, Winichagoon P, Fucharoen S. Characterization of a novel deletion causing (δβ)0-thalassemia in a Thai family. American Journal of Hematology. 2007;**82**(2):155-161

[48] Panyasai S, Fucharoen S, Surapot S, Fucharoen G, Sanchaisuriya K. Molecular basis and hematologic characterization of δβ-thalassemia and hereditary persistence of fetal hemoglobin in Thailand. Haematologica. 2004;**89**(7):777-781

[49] Fucharoen S, Pengjam Y, Surapot S, Fucharoen G, Sanchaisuriya K. Molecular and hematological characterization of HPFH-6/Indian deletion-inversion Gγ(Aγδβ) 0 -thalassemia and Gγ(Aγδβ) 0 thalassemia/HbE in Thai patients. American Journal of Hematology. 2002;**71**(2):109-113

## Section 3
