Variant Analysis of BCL11A and AHSP Genes in Beta Thalassemia Patients 1 2 ZIADOON FAWZI MUKHLIF , DR

ISSN 2319-8885 Vol.03,Issue.18 August-2014, Pages:3701-3709 www.semargroup.org, www.ijsetr.com

Variant Analysis of BCL11A and AHSP Genes in Beta Thalassemia Patients 1 2 ZIADOON FAWZI MUKHLIF , DR. B.VIJAYA LAKSHMI 1Research Scholar, Dept of Biotechnology, Institute of Science & Technology, JNTU, Hyderabad, India. 2Dept of Molecular Biology, Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, India.

Abstract: There has been worldwide awareness to control and monitor the genetic disorders by the World Health Organization (WHO). According to WHO estimation, about 3, 32,000 children are born annually inheriting a major hemoglobin disorder throughout the world out of which 56,000 people are (β-thalassemia major who survives on regular transfusions. Among the several other haemoglobinopathies β- thalassemia is the most common hemoglobin disorder found in the Indian subcontinent. Thalassemias are the inherited autosomal recessive blood disorders. In thalassemias (α and β-thalassemia's), the genetic defect results in reduced rate of synthesis or total absence of one of the globin chains that make up hemoglobin. In β- thalassemia the β-globin chain is reduced or totally absent and an excess of α-globin is produced whose precipitation in a cell leads to oxidative stress, red blood cell membrane damage and reduces the lifespan of red blood cells/erythrocytes as a result of hemolysis. Thus, the formation of abnormal hemoglobin molecules in the erythrocytes, results in hemolytic anemia, the characteristic presenting symptom of the disease. An increase in fetal hemoglobin i.e HbF (reactivation) can compensate for lie absence of the β-globin chain thereby reducing the severity of β-thalassemia. Since Thalassemias are difficult to cure, prevention of this disorder is of high priority. Recently, genetic variants that modulate HbF levels but fell outside of the hemoglobin genes have been identified. A common variant (C-T) at position -158 upstream of Gγ-globin gene has been associated with increased HbF levels in normal individuals, in β-thalassemia and in sickle cell anemia BCL11A (B-cell lymphoma/leukemia 11 A) and (AHSP) gene variation has also been shown to moderate the phenotype of homozygous β-thalassemia. Α-Hemoglobin-stabilizing protein (AHSP) is a molecular chaperon binding specifically to free α-glob in. It is considered to be a potential modifier of β- thalassemia. In order to find out if AHSP affects the phenotype of β-thalassemia. The aim of this study is to evaluate the association of BCL11A and AHSP gene polymorphism with severity and other complications in β-thalassemia major patients. Blood samples will be collected from "Thalassemia and Sickle Cell Society". The clinical features of individuals affected with β-thalassemia will be evaluated and the disease will be detected by variant HPLC. DNA will be isolated by phenol-chloroform method. The specific DNA sequences of patients will be amplified by PCR and then RFLP will be done to determine the genotypes. The association of BCL11A and AHSP gene polymorphism with disease severity and other complications in β- thalassemia major patients will be evaluated. In addition to this serum ceruloplasmin and Lipid peroxidation levels will be estimated in β-thalassemia major patients.

Keywords: Α-Hemoglobin-Stabilizing Protein (AHSP), World Health Organization (WHO).

I. INTRODUCTION year with a major and clinical significant emoglobinopathy. A Haemoglobinopathies consist of thalassaemias and Out of these, β-thalassaemia major and clinically significant variant haemoglobins. Carriers of haemoglobinopathies are sickle cell disorders account for almost equal numbers. partially protected against morbidity and mortality of Thalassemia (Thalassa is Greek for the sea, Haema is Greek falciparum malaria and this has resulted in their higher for blood) is the name of a group of genetic blood disorders prevalence in tropical countries. In India, they are responsible characterized by anemia due to enhanced RBC destruction. for the largest number of genetic disorders and hence are of Hemoglobin, the oxygen-carrying component of the RBCs great public health importance. Clinically important consists of two different proteins, an alpha and a beta. If the thalassaemic disorder in India is α-thalassaemia while β- body doesn't produce enough of either of these two proteins, thalassaemia, although more common in tribal population, is the RBCs become defective and cannot carry sufficient free from morbidity. Of the several abnormal haemoglobin oxygen. The resulting anemia is usually severe with several molecules, three which are widely prevalent in India include: health problems like enlarged spleen, bone deformities, HbS, HbE and Hb. The cumulative gene frequency of fatigue and requires regular life-long transfusion, therapy and haemoglobinopathies in India is 4.2%. With a population of medical supervision. Thalassemia is an inherited autosomal over one billion and a birth rate of 28 per thousand, there are recessive blood disease. In thalassemia, the genetic defect over 42 million carriers and over 12,000 infants are born each results in reduced rate of synthesis of one of the globin chains

Copyright @ 2014 SEMAR GROUPS TECHNICAL SOCIETY. All rights reserved. ZIADOON FAWZI MUKHLIF, DR.B.VIJAYA LAKSHMI that make up hemoglobin. Deficient synthesis of hemoglobin chain production. A decrease in the rate of production of a occurs in thalassemia, a group of hereditary hemolytic certain globin chain or chains (α, β, γ, δ) impedes Hb anemias. The RBCs are small, pale & short lived. (Tortora et synthesis and creates an imbalance with the other, normally al., 2005). Thalassemia is a quantitative problem of too few produced globin chains. Because 2 types of chains (α and globins synthesized, whereas sickle-cell anemia (a non-α) pair with each other at a ratio close to 1:1 to form haemoglobinopathy) is a qualitative problem of synthesis of normal Hbs, an excess of the normally produced type is an incorrectly functioning globin. Thalassemias usually result present and accumulates in the cell as an unstable product, in underproduction of normal globin proteins, often through leading to the destruction of the cell. This imbalance is the mutations in regulatory genes. Haemoglobinopathies imply hallmark of all forms of thalassemia. The reduction varies structural abnormalities in the globin proteins themselves. from a slight decrease to a complete absence of production. The two conditions may overlap, however, since some The consequences of impaired production of globin chains conditions which cause abnormalities in globin proteins ultimately result in the deposition of less Hb into each RBC, (haemoglobinopathy) also affect their production leading to hypochromasia. (thalassemia). Thus, some thalassemias are haemoglobinopathies, but most are not. Either or both of these conditions The Hb deficiency causes RBCs to be smaller, leading to may cause anemia. (Park et al., 2005). the classic hypochromic and microcytic picture of thalassemia. However, this does not occur in the silent carrier II. CAUSES [ETIOLOGY] state, since both Hb level and RBC indices remain normal. Normal hemoglobin, also called hemoglobin A, has four Large deletions that may involve the entire β gene, or even protein chains, two alpha globin and two beta globin. The extend to delete the neighboring δ gene, have been previously two major types of thalassemia, alpha and beta, are named reported. Four new such mutations were recently identified, 3 after defects in these protein chains. Four genes are needed to of these mutations, the deletion has extended to involve the δ make enough alpha globin protein chains. Alpha thalassemia gene, resulting in failure to produce any Hb A2. In such trait occurs when one or two of the four genes are missing. If cases, the β/δ thalassemia is to be differentiated from the more than two genes are missing, the result is moderate to phenotypically similar condition known as hereditary severe anemia. The most severe form of alpha thalassemia is persistence of fetal hemoglobin (HPFH). The importance of known as alpha thalassemia major or hydrops fetalis. Babies differentiating the conditions is reflected in prenatal and with this disorder usually die before or shortly after birth. newborn screening for hemoglobinopathy. The significant Two genes (one from each parent) are needed to make excess of free α chain caused by the deficiency of β chains enough beta globin protein chains. Beta thalassemia occurs causes destruction of the RBC precursors in the bone marrow when one or both genes are altered. The severity of beta (i.e., ineffective erythropoiesis) (Harsh et al., 2006). thalassemia depends on how badly one or both genes are affected. If both genes are affected, the result is moderate to III.TYPES OF THALASSEMIAS severe anemia. Thalassemia is a common inherited disease in A. Alpha Thalassemias the world. India accounts for 10% of the total world People whose hemoglobin does not produce enough alpha thalassemia population and approximately1 in 30 in the protein have alpha thalassemia. Four genes (two from each general population is carrier of the mutated gene. Every year parent) are needed to make enough alpha globin protein about 15,000 infants are born with haemoglobinopathies in chains. If one or more of the genes is missing, one will have India. alpha thalassemia trait or disease. This means that one don't make enough alpha globin protein. If one has only 1 missing Nearly 28 mutations are reported in beta Thalassemia gene, you're a silent carrier and won't have any signs of Indian population of which eight accounts for 95% of the illness. If one have 2 missing genes, one have alpha cases. Alpha Thalassemia is generally caused by deletions on thalassemia trait (also called alpha thalassemia minor). One alpha globin gene. Mutations are specific to population and may have mild anemia. There are four subtypes of alpha state specific mutations are reported. The thalassemias are thalassemia. Each type represents the loss of or damage to classified according to which chain of the hemoglobin one, two, three, or four genes. molecule is affected. In α thalassemias, production of α globin chain is affected, while in β thalassemia production of One Gene: If one alpha-globin gene is missing or damaged, the β globin chain is affected. Thalassemia produces a one will have no symptoms and will not need treatment. But deficiency of α or β globin, unlike sickle-cell disease which he/she is a silent carrier. This means one doesn’t have the produces a specific mutant form of β globin. β globin chains disease but one can pass the defective gene onto your child. are encoded by a single gene on chromosome 11; α globin Smaller-than-normal blood cells may be the only sign of the chains are encoded by two closely linked genes on condition. chromosome 16. Thus in a normal person with two copies of each chromosome, there are two loci encoding the β chain, Two genes: If two alpha-globin genes are missing or and four loci encoding the α-chain. Deletion of one of α loci damaged, one will have very mild anemia that will not need has a high prevalence in more likely to develop α treatment. This is known as alpha thalassemia minor or alpha thalassemias. The thalassemias are inherited disorders of Hb thalassemia trait. synthesis that result from an alteration in the rate of globin International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709 Variant Analysis of BCL11A and AHSP Genes in Beta Thalassemia Patients Three genes: If three alpha-globin genes are missing, one will have mild to moderately severe anemia. This is sometimes called hemoglobin H disease, because it produces heavy hemoglobin. The body removes this heavy hemoglobin faster than it does normal hemoglobin. The more severe forms may need treatment with blood transfusions.

Four genes: If all four alpha-globin genes are missing (alpha thalassemia major), the fetus will be stillborn or the child will die shortly after birth. The hemoglobin produced by this condition is sometimes called hemoglobin Barts.

B. Beta Thalassemias People whose hemoglobin does not produce enough beta protein have beta thalassemia. Two genes (one from each Fig.1. Pathophysiology of β-thalassemia. parent) are needed to make enough beta globin protein chains. If one or both of these genes are altered, one will Homozygous β-thalassaemia is a severe disease. Children have beta thalassemia. This means that one don't make with β-thalassaemia major appear healthy at birth because Hb enough beta globin protein. If one has one altered gene, F is still functioning. Pathology occurs when the α-globin he/she is a carrier. This condition is called beta thalassemia gene is switched off at 8 to 12 months after birth but there is trait or beta thalassemia minor. It causes mild anemia. There no β-globin expression. Defects in the globin genes result in are two subtypes of beta thalassemia. Each type represents impaired globin chain synthesis leading to the reduced the loss of or damage to one & two. haemoglobin content of red cells and consequent red cell pathology. Ineffective erythropoiesis and shortened red cell One gene: If one of your beta hemoglobin genes is defective, survival will lead to chronic anaemia in thalassaemia. one has mild signs and symptoms. This condition is called Without treatment, the spleen, liver, and heart become greatly beta thalassemia minor or as a beta-thalassemia trait. enlarged. During the first decade of their life they become pale, listless and fussy, and have a poor appetite. They grow Two genes: If both of your beta hemoglobin genes are slowly and often develop jaundice. Bones turn thin and defective, your signs and symptoms will be moderate to brittle, osteoporosis and osteopenia, and face bones become severe. This condition is called beta-thalassemia major or distorted. In this region many children with major Cooley's anemia. Babies born with two defective beta thalassaemic diseases are still treated by a minimal hemoglobin genes usually are healthy at birth, but develop transfusion programme and only a few of them receive signs and symptoms within the first two years of life. regular iron chelation. Most of these thalassaemia major patients die in the paediatric age group. Heart failure due to C. Pathophysiology of β-thalassemia anaemia and infection are the leading causes of death among The basic molecular defect in β-thalassemia results in untreated children. either absence (βo) or reduced (β+) beta chain production, however, β chain synthesis proceeds at a normal rate. The D. Clinical heterogeneity of α-thalassaemia first consequence of reduced β-chain production is reduced In general, the clinical severity of α-thalassaemia phenotype production of the adult hemoglobin (HbA: α2 β2). A second relates to the number of affected α-globin genes. Individuals consequence is imbalanced globin chain synthesis, in which who have a deletion or inactivation of one α-globin gene α chain synthesis proceeds at a normal rate and hence there is usually do not present significant haematologic changes. an excess of α chain in the erythrocytes. The excess α chains When two α-globin genes are deleted either on the same are unstable and precipitate in the bone marrow red cell chromosome (α-thalassaemia 1 heterozygote) or one on each precursors, giving rise to a large intracellular inclusions that chromosome (α-thalassaemia 2 homozygote), hypochromic interfere with the red cell maturation, function and survival and microcytic red blood cells are observed without anaemia. (Fig.1) (Weather all et al., 2000). The interference of these Inactivation of 3 α-globin genes due to deletions with or intracellular inclusions with red-cell maturation subsequently without non-deletional α-thalassaemia leads to the gives rise to intramedullarly destruction of red-cell thalassaemia inter media, called Hb H disease. Whereas the precursors, i.e. ineffective erythropoiesis. However, those red deletion of all four α-globin genes results in Hb Bart’s cells that become mature and enter the circulation contain α hydrops fetalis, the most severe and lethal thalassaemia chain inclusion, which interfere with their passage through disease. the microcirculation, particularly in the spleen, and hence extramedullarly destruction of red cells becomes the norm. Homozygous α-thalassaemia 1 is also known as Hb Bart’s Thus, the anemia of β-thalassemias results from both hydrops fetalis due to the presence of tetrameric unbound γ- ineffective erythropoiesis and a shortened red cell survival like foetal globin chains (γ4, Hb Bart’s). Although different (Weatherall et al., 1996). forms of α-thalassaemia alleles have a worldwide distribution, the occurrence of Hb Bart’s hydrops fetalis is International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709 ZIADOON FAWZI MUKHLIF, DR.B.VIJAYA LAKSHMI almost confined to this region where the highest incidence of excellent candidate region for mutations associated with α-thalassaemia 1 exists. Because of the absence of α-globin decreased AHSP gene expression. chain synthesis, the foetus does not have either Hb F or Hb A. Hemoglobin electrophoresis shows large amounts of Hb IV. RESULTS Bart's and about 10-15 per cent Hb Portland (δ2γ2). The A. Sample Collection foetus dies in utero or soon after birth because Hb Bart’s does 50 Blood samples were collected from patients who not release O2 to the tissues. The affected foetus is hydropic visited the Institute of Genetics and Hospital for Genetic with abnormal development of vital organs such as brain and Diseases Hyderabad, for the diagnosis of β-Thalassemia lung, and is thus incompatible with life. Maternal during the course of project. Blood samples were also complications such as toxaemia of pregnancy have been collected from the “Thalassemia and Sickle cell Society”, observed in almost all pregnancies (Vaeusorn, personal Hyderabad. Osmotic fragility test was carried for all the 50 communication). Ultrasonography provides unambiguous samples. Then they were subjected to HPLC for confirmation detection of Hb Bart’s hydrops fetalis at 18-20 wk of of the diagnosis. Out of 50 patient samples 25 samples were pregnancy. DNA diagnosis from chorionic villi or amniotic diagnosed as β-Thalassemia major. DNA isolation, PCR and fluid fibroblasts can detect Hb Bart’s hydrops fetalis as early RFLP were carried out to detect BCL11A and AHSP gene as 10-16 wk gestation. Therapeutic abortion is suggested for polymorphism. the foetus diagnosed as having this disease. TABLE I: Osmotic Fragility Results More recently, major advances have been made in the discovery of critical modifier genes, such as BCL11A (B cell lymphoma 11A), a master regulator of HbF (fetal hemoglobin) and hemoglobin switching (CAO ET AL., 2011) BCL11A gene encodes a C2H2 type zinc-finger protein, which is similar to the mouse Bcl11a/Evi9 protein. BCL11A on chromosome 2p16 was shown to be a major quantitative trait locus for HbF level and F-cell number in several populations with or without β-hemoglobinopathy (Chen et al., 2009). Moreover, BCL11A plays a central role in the evolutionarily different globin gene switches of mammals. As an aspect crucial for gamma-globin gene silencing, BCL11A can supposed to be consider as a beneficial target to increase HbF in a directed manner in beta-thalassemia patients (Sankaran et al ., 2010).

Hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds and stabilizes α hemoglobin during normal erythropoiesis and in pathological states of α hemoglobin excess. AHSP has been proposed as a candidate gene in some Heinz body hemolytic anemias and as a modifier gene in the β thalassemia syndromes. To gain additional insight into the molecular mechanisms controlling the erythroid-specific expression of the AHSP gene and provide the necessary tools for further genetic studies of these disorders, we have initiated identification and characterization of the regulatory elements controlling the human AHSP gene. We mapped the 5-end of the AHSP erythroid cDNA and cloned the 5- flanking genomic DNA containing the putative AHSP gene promoter. In vitro studies using transfection of promoter/ reporter plasmids in human tissue culture cell lines, DNase I foot printing analyses and gel mobility shift assays, identified an AHSP gene erythroid promoter with functionally important binding sites for GATA-1- and Oct-1-related proteins. In transgenic mice, a reporter gene directed by a minimal human AHSP promoter was expressed in bone marrow, spleen, and reticulocytes, but not in nonerythroid tissues. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of the promoter region and occupancy by GATA-1. The AHSP promoter is an

Number of positive cases : 25 Number of negative cases : 20 Number of cases to be confirmed : 5

Out of 50 samples subjected to osmotic fragility test, 25 turned out to be positive. 20 were negative and 5sample needed other tests to confirm the disease as shown in Figs.2 to 4. International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709 ZIADOON FAWZI MUKHLIF, DR.B.VIJAYA LAKSHMI

Fig.2. HPLC Chromatogram (Normal).

Fig.3. HPLC Chromatogram (β-thalassemia carrier).

Table III: BCL11A Genotypic and Allelic Frequencies Fig.4.HPLC Chromatogram (β-thalassemia major).

Among the controls considered for the present study 36% were to heterozygotes with CT genotype, 64% homozygous normal with CC genotype, none of controls turned to be homozygous mutant (TT genotypes)were not seen in our

 TT-Homozygous population. 36 % of thalassemia patients were heterozygotes, 48% were homozygotes for CC and 16% were homozygous  TC-Heterozygous mutant. Further the T allele frequency was found to be high  CC-Homozygous mutant in thalassemia patients, when compared to controls. International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709 Variant Analysis of BCL11A and AHSP Genes in Beta Thalassemia Patients TABLE V: Association of BCL11A and AHSP Genotypes with HBF Levels

Fig.5. Amplification of BCL11A gene.

Lanes 1, 2, 3, 5, 6, 7 and 8 represents the amplified product of BCL11A gene and lane 4 represents 100bp ladder as shown in Fig.5.

Table IV: AHSP Genotypic and Allelic Frequencies

 TT-Homozygous  TC-Heterozygous TABLE VI: Results of Serum Ceruloplasmin in Patients  CC-Homozygous mutant

Among the controls in the present study 60.0% were heterozygotes with GA genotype, 40.0% homozygous normal with GG genotype, none of controls turned to be homozygous mutant (CC genotypes)were not seen in our population. 56.0% of thalassemia patients were heterozygotes, 28.0% were homozygotes for GG and 16.0% were homozygous mutant. The frequency of A allele was found to be high in thalassemia patients, when compared to controls.

B. PCR Amplification of AHSP Gene

Fig.6. Amplification of AHSP gene.

Lanes 1, 2, 3, 5, 6, 7 and 8 represents the amplified product of AHSP gene and lane 4 represents 100bp ladder as shown in Fig.6. International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709 ZIADOON FAWZI MUKHLIF, DR.B.VIJAYA LAKSHMI TABLE VII: Results of Serum Ceruloplasmin in Controls TABLE IX: MDA Levels in Patients Samples

TABLE X: MDA Levels in Control Samples

TABLE VIII: Serum Ceruloplasmin levels in patients and controls

In the above TABLE 6 and 7 The Mean level of serum Ceruloplasmin level in patient samples was found to be41.3764 mg/100ml serum where as the mean level in case of control was 31.29615 mg/100ml serum. Iron levels were elevated in patients in comparison with controls. The mean lipid peroxidation level in patient samples was found to be 5.8357mol/ml whereas the mean level in case of controls was 2.945 n mol/ml. Lipid peroxidation levels were elevated in patients in comparison with controls.

International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709 Variant Analysis of BCL11A and AHSP Genes in Beta Thalassemia Patients TABLE XI: Mean Lipid Peroxidation Levels in Patients VI. REFERENCES and Controls [1]http://genetech.co.in/downloads/patthalassemia. pdf. [2] Tortora J. Gerard, Derrickson Bryan. Principles of Anatomy & hysiology. 11th Edition. John Wiley & Sons Inc Publication, United States. 2005, 689. [3] Park K. Park’s Textbook of Preventive & Social Medicine. 18th Edition. M/S Banarsidas Bhanot Publishers.

V. CONCLUSION 2005; 626. [4]http://rarediseases.aboutcom/cs/thalassemia/a/062102.htm. β-Thalassemia is an autosomal recessive genetic disorder th with many mutations β-globin gene. Homozygous β- [5] Harsh Mohan. Textbook of Pathology. 5 Edition. Jaypee thalassemia is accounting for a majority of β thalassemic Brothers Publishers, New Delhi. 2006; 397-402. syndromes in India. Despite remarkable success in the [6] Weatherall D. (2000): Single gene disorders or complex treatment of β thalassemia in the past decades it is still traits: lessons from the thalassaemias and other monogenic the leading cause of death and premature disability in diseases. British Medical Journal 321: 1117- 1121. developed and developing countries. Possible factors that [7] Weatherall D. and Clegg J. (1996): Thalassemia. a global influence the severity of anemia in thalassemia may be public health problem. Nature Medicine 2: 847-849. inherited or not inherited. The inherited factors include type [8] Cao A, Moi P, Galanello R. 2011. Recent advances in of β-thalassemia mutation and factors that stimulate HbF beta-thalassemias. Pediatr. Rep., 3(2): e17. production. Up to present time it is still speculative whether [9] Chen Z, Luo HY, Steinberg MH, Chui DH. 2009. genotypes could be predictive of phenotype. Despite BCL11A represses HBG transcription in K562 cells. Blood seemingly genotypes, the patients, especially those with Cells Mol. Dis., 42(2): 144-149. β thalassemia, have a remarkable variability in anemia, [10] Sankaran VG, Xu J, Orkin SH. 2010. Transcriptional growth development, hepatosplenomegaly and transfusion silencing of fetal hemoglobin by BCL11A. Ann. N. Y. Acad. requirements. The genetic factors may differ in each race/ Sci., 1202: 64-68. ethnic group and variations in genes may alter these [11] Perutz MF. Science is not a quiet life: unraveling the parameters. Thus, it is very interesting to investigate the atomic mechanism of haemoglobin. London Imperial College associations between these genes and β thalassemia in each Press; 1997. ethnic group for therapy and prevention. The BCL11A gene [12] Lecomte JT, Videtich DA, Lesk AM. Structural has been shown to play a key role in silencing of the γ-glibin divergence and distant relationships in proteins: evolution of gene during various stages of development and in the globins. Curr Opin Struct Biol. 2005; 15:290-301. reactivation of HbF in adult erythroblasts. AHSP is good candidate for ameliorating the clinical severity in β- thalassemia patients, since it selectively binds to newly synthesized α-globin chain, stabilizes and folds before transferring them to β-globin chain to form functional haemoglobin molecule. Depending on the amount of excess α-globin present, there is a significant negative correlation of AHSP expression in consistentcy with HbF levels. AHSP is also highly expressed in Hb-synthesizing eythroid precursors. AHSP knockout mice show increased α and β-globin precipitation leading to impaired erythropoiesis and therefore, more severe β-thalassemia picture in these mice. In the present study, we found that the patients who had BCL11A (rs11886868) gene polymorphism had high percentage of HbF. The results suggested that this polymorphism may have a synergistic effect on severity and percentage of HbF among β-thalassemia. The frequency of C allele of AHSP gene was found to be high in thalassemia patients, when compared to controls. The serum ceruloplasmin levels were elevated in patients when compared to controls. Mean serum ceruloplasmin levels in patients were found to be 41.3764 mg/100ml whereas in controls it was about 31.29mg/dl. The MDA levels were elevated in patents when compared to controls. Mean MDA levels in patents were found to be 5.8357mol/ml whereas in controls it was about was 2.945nmol/ml showing a significant association with the disease.

International Journal of Scientific Engineering and Technology Research Volume.03, IssueNo.18, August-2014, Pages: 3701-3709