The Complete Spectrum of Beta (β) Thalassemia Mutations in Bangladeshi Population

Research Article

Austin Biomark Diagn. 2016; 3(1): 1024.

The Complete Spectrum of Beta (β) Thalassemia Mutations in Bangladeshi Population

Sultana GNN1*, Begum R1, Akhter H2, Shamim Z3, Rahim MA4 and Chubey G5

1Centre for Advanced Research in Sciences, University of Dhaka, Bangladesh

2Bangabandhu Sheikh Mujib Medical University (BSMMU), Bangladesh

3Popular Diagnostic Center, Dhanmondi, Dhaka, Bangladesh

4Thalassemia Foundation Hospital, Chamelibag, Bangladesh

5Estonia Biocenter, Tartu, Estonia

*Corresponding author: Sultana GNN, Centre for Advanced Research in Sciences, University of Dhaka, Bangladesh,

Received: March 18, 2016; Accepted: April 06, 2016; Published: April 12, 2016

Abstract

Background: In Bangladesh, 3% people are carriers of β-thalassemia and 4% people are E- β-thalassemia. However to date, very little data about HBB gene mutations in the population of Bangladesh are available. This study was done to determine the complete spectrum of β-thalassemia mutations in the population of Bangladesh. We analyzed the entire HBB gene including regulatory regions in 19 unrelated β-thalassemia major and 51 E- β-thalassemia patients and 33 healthy controls using DNA direct sequencing method.

Result: Nine β-thalassemia polymorphisms were identified in Bangladeshi thalassemia patients. The most common polymorphisms were IVSI -5 G > C (81.4%), CD 26/ HbE: G>A (72.85%), CD 2: T>C (57.1%) and IVSII-16: G>C (57.1%). The less common polymorphisms were -90 C>T (1.43%), CD1 T>A (2.86%), CD2 C>A (5.71%), CD30 G>C (1.43%), and IVS-II-81 C>T (1.43%). These polymorphisms were distributed as 6 genotyping patterns in β-thalassemia major and 8 in E- β-thalassemia patients. IVS1-5: G>C alone or combined with other β thalassemia polymorphisms are responsible for pathogenic condition in β-thalassemia major and along with CD26/E: G>A and other β thalassemia mutations in most E- β-thalassemia. CD2: T>C alone cannot produce pathogenic condition but when it combined with CD1: T>A and CD2: C>A it can generated thalassemia. This CD2: T>C and another common polymorphism IVSII-16: G>C along with IVSII-74: T>G were also identified in healthy controls. Altogether 17 haplotypes among the Bangladeshi populations was observed and two shared haplotypes among patients and controls. The haplotype diversity was substantially higher among patients and lower among controls.

Conclusion: The genotypic profile of β—thalassemia in Bangladesh patients shows great variability. IVS1-5: G>C most common polymorphism for β- thalassemia and a combination of IVS1-5: G>C and CD26/E: most common polymorphism for E β-thalassemia disease in Bangladeshi population.

Keywords: β-thalassemia; Mutations; Bangladesh; Heterozygous; Homozygous

Introduction

β-thalassemia is a genetic disorder that is prevalent in certain parts of the world including Bangladesh [1,2]. The incidence for this disease is high in tropical and subtropical areas including Southeast Asia [3]. In Bangladesh the carrier rate of β-thalassemia is 3.0% and Hb-E/β- thalassemia is 4.0% and affected birth per thousand of β-thalassemia and Hb-E/ β-thalassemia is 0.106 & 3.000 respectively [4]. About 10% of the world’s thalassemia major children are born in India [5]. Gene flow from Indian sub-continent to Bangladesh may be one of the reasons of prevalence of this disease in Bangladesh [5-6]. Other reasons of prevalence of thalassemia disease are intermarriage between different ethnic groups, lack of awareness for blood test before marriage [7]. Besides accurate data on carrier rates in Bangladesh is lacking, insufficient technologies for prenatal diagnosis also increases the prevalence of beta thalassemia in Bangladesh.

The function of hemoglobin in human and animal is very important which carries oxygen from lungs to other parts of the body. It is metalloprotein having quaternary structure which contains iron and performs the important function of transporting oxygen via RBCs in blood in mammals as well as other animals [8]. It also effect modulation and gas transport duties, although it differ from species to species and most probably is altogether different in vertebrates. As long as more oxygen is bound to hemoglobin more oxygen is reached to every part of the body. The external chemical factor which helps in regulation of oxyhemoglobin affinity induces, pH, 2, 3-diphospahoglycerate and carbon dioxide.

β-thalassemia can be divided into three main types depends on the clinical phenotypes: thalassemia major, thalassemia trait and thalassemia intermediate. β-thalassemia major is a severe form that requires transfusions from infancy for survival. This clinical phenotypic variability β-thalassemia occurs due to the mutation in three exons and two intervening sequences, 5´ UTR (untranslated region) and 3´ UTR of β-globin (HBB) gene [9-10]. Mutation in exons and intervening sequences that may produces non-functional beta globin protein or β0 allele and mutation in HBB promoter, 5´ UTR and 3´ UTR that may produces reduced quantity of beta globin protein or β+ allele [10,11]. Approximately 600 mutations have been identified in the β-globin (HBB) gene, of which more than 200 are associated with β-thalassemia phenotype [11,12]. The distribution and frequency of different mutations regarding thalassemia vary from population to population [13-15]. Haemoglobin E (HbE) allele, point mutation (G > A) in codon 26 of β - globin gene, can induce alternative splicing and thus result in decreased β -globin E chains [16]. Hb E/β-thalassemia results from co-inheritance of a β-thalassemia allele from one parent and Haemoglobin E from the other [17]. HbE/ β thalassemia causes a surprisingly variable anaemia, ranging from nearly asymptomatic states to severe anaemia and transfusion-dependency [18].

However, to date, the genetic basis of β- thalassemia in Bangladeshi patients is still poorly understood. Here, we attempt to identify the complete spectrum of β-thalassemia mutations in Bangladeshi population. To acquire the data we sequenced entire 1.6 kb HBB gene, 163 bp upstream and 153bp downstream regulatory region in 70 patients with β-thalassemia or Hb-E/ β-thalassemia phenotypes and 33 healthy controls. During the analysis, nine point mutations were identified, of them IVSI -5G > C in β-thalassemia major and CD26 (G-A) + IVSI -5G > C in Hb-E/β-thalassemia were frequent. Therefore our findings could provide genetic insight of beta thalassemia disease occurrence in Bangladeshi population and are useful in genetic counseling, diagnostic application and treatment.

Materials and Methods

Subjects

As Bengali people are less diverse, mutations of β-thalassemia are not regional specific [19]. Therefore, a total of 70 patients with β-thalassemia were included in this study came from different states of Bangladesh who visited Bangladesh Thalassemia Hospital for treatment after diagnosis. Samples were randomly selected from thousand and fourty five cases of our previous study [20]. Out of them 19 patients were β-thalassemia major and 51 were HbE-β- thalassemia. 33 healthy control samples were also chosen to evaluate the mutations profile of β-thalassemia in Bangladesh. Blood samples were collected from the patients under an agreement with the Head of the Bangladesh Thalassemia Hospital with Centre for Advanced Research in Sciences (CARS) and Department of Biochemistry and Molecular Biology, University of Dhaka. The protocol for this study was approved by ethics committee of Bangladesh Medical Research Council (BMRC). Informed consent was obtained from each individual or parents of individuals younger than 18 years old. Approximate 3-5 ml of blood sample was collected in EDTA coated vacutainer from both patients and controls and finally stored at -20o C until analysis.

Hematological analysis and Clinical data collection

Hematological analyzer (Sysmex XE-2100) was used to determine the complete blood counts and red blood cells. The data of Mean Corpuscular Volume (MCV) and Mean Corpuscular Haemoglobin (MCH) were evaluated. The Hemoglobin Electrophoresis was carried out by using Sebia’s Capillary Electrophoresis System (CAPILLARYS™ 2) and the levels of HbA, A2 and F were analyzed according to Sultana et al. [20]. Clinical data e.g. the information about blood transfusions was obtained by retrospective clinical data.

Beta (β)-Thalassemia mutations analysis

Genomic DNA was extracted from whole blood by a standard procedure of phenol/ chloroform/ isoamyl alcohol extraction [21]. The quality and quantity of extracted DNA was measured by Nano Drop spectrophotometer 2000 and visualized by 0.8% agarose gel electrophoresis in 1×TAE buffer.

A total of 1922 bp genomic sequence containing entire HBB gene, 163 bp upstream and 153bp downstream regulatory region was amplified and sequenced in 70 patients and 33 controls. This region was amplified using three pairs of primers and sequenced by these six end primers that were designed using Primer 3 software (http://frodo. wi.mit.edu/primer3/). The sequence of the primers will be available on request from the authors. Amplifications were performed in a 20 ml volume containing one unit of AmpliTaq Gold (Promega, USA), 1× Polymerase Chain Reaction (PCR) buffer, 1.87 mM MgCl2, 200 μM deoxynucleotides triphosphates, 5 pmol each of forward and reverse primer and 40-50ng of genomic DNA. The PCR program consisted an initial denaturation at 95° C for 10 min, followed by 35 cycles of 1 min at 950 C, 1 min at 600 C, 1 min at 720 C and with a final elongation at 720 C for 10 min. PCR products were visually verified on 1% agarose gels and directly sequenced using a Big Dye Terminator cycle sequencing kit V3.1 (Applied Biosystems, Foster City, CA) and by ABI PRISM® 3130 Genetic analyzer (Center for Advanced Research in Sciences or CARS, Dhaka University, Bangladesh). Each sequence was reconfirmed by second run of sequencing.

The chromatograms generated from the genetic analyzer along with the base sequences were analysed by Bio-edit Sequence Alignment editor (V 7.0) [22] and Mac-based software (Auto Assembler V 3.0). Patient’s sequences were compared with National Centre for Biotechnology Information (NCBI) RefSeq entry of HBB (NG_000007.3) using NCBI BLAST (bl2seq) tools. HbVar database was used for the identification of the presence of reported mutations in other populations [23].

Statistical analysis

Data were expressed as mean (±SD) and number (percentage) as appropriate. The mean (±SD) of each haematological and clinical parameter were calculated using Microsoft Excel 2010. The statistical analysis was of these data was calculated by using GraphPad statistical software (http://www.graphpad.com/quickcalcs/index.cfm ).

Statistical significance was determined by one way Analysis Of Variance (ANOVA). P value < 0.05 was considered to be statistically significant. We have used phased the data by using DNASP software [24]. The haplotypes were used to construct the Network (Fluxus Engineering). Arlequin 3.5 [25] was used to measure diversity indices.

Results

Basic clinical characteristics of β thalassemia patients of Bangladesh

The basic clinical characteristics of β thalassemia patients of Bangladesh revealed that there were no significant differences between patients with β thalassemia major and E-β thalassemia regarding WBC, RBC, Hb level, MCV, MCH and Body Mass Index (BMI) (Table 1). There were also no significance difference was found in time of blood transfusion taken from and frequency of blood transfusion (Table 1).