Prevalence and Mutation Spectrum of Thalassemia Carriers and Study of Genetic Modifiers Affecting the Disease Severity of HbE/β-Thalassemia in Bangladeshi Population

Abstract

Background: As being a country of the global thalassemia belt, β-thalassemia, a hereditary hemolytic disorder caused by reduced or absent synthesis of the β-globin chain of the hemoglobin tetramer (α2β2) is the most common congenital single gene disorder in Bangladesh. However, it is a matter of deep concern that thalassemia is not recognized as priorities in public health sectors of the country and there is no exact data representing the current prevalence of thalassemia in different regions Bangladesh. Also, molecular study for mutation profiling of the carriers is lacking. The only available remedy for thalassemia is an overly expensive allogeneic bone marrow transplant, otherwise lifelong regular blood transfusion that gradually damages the vital organs of the body due to excess iron accumulation. However, due to having autosomal recessive transmission this deadly incurable disease is completely preventable by carrier detection followed by genetic counselling and discouraging marriage between the carriers. For development of a proper prevention strategy, it is important to know the carrier frequency of thalassemia with complete mutational profiling involving participants from all over the country. To get an accurate prevalence data, a large-scale population survey, which should not be hospital-based, is mandatory using proper screening methods combining hematological and molecular approaches. Another challenge is the clinical manifestations of thalassemia, particularly the Hb E/β-thalassemia thst is highly variable in terms of severity which might be influenced a number of genetic factors like types of β-globin mutations, polymorphisms in modifier genes of γ-globin expression, coinheritance of α-thalassemia etc. In Bangladesh, so far there is no any precise study on the genetic modifiers of the clinical variability of HbE/β-thalassemia patients. The proposed work will evaluate the genetic factors modulating the phenotypic heterogeneity of HbE/β-thalassemia in Bangladeshi patients. Hypothesis: 1. Actual prevalence of thalassemia carriers is high in Bangladesh and molecular approaches will complement the conventional methods for carrier detection of thalassemia to provide an accurate data on frequency of the carriers in Bangladesh. 2. Specific mutations in the genetic modifiers affect the disease severity in HbE/β-thalassemia patients of Bangladesh. Aim: One of the objectives of our study was to determine the nationwide (1) carrier frequency of hemoglobin (Hb) E (ETT) and β-thalassemia (BTT) and (2) mutation spectrum among the carriers using molecular, hematological and biochemical methods. Another objective was to study the effect of genetic modifiers influencing disease severity in HbE/β-thalassemia patients of Bangladesh that includes types of β-globin mutation and coexistance of α-thalassemia along with fetal hemoglobin inducer 8 single nucleotide polymorphisms in 4 major modifier genes. Methods: For prevalence determination, the study enrolled a total of 1877 individuals (60.1% male and 39.9% female) aged between 18-35 years. The total sample size and its division-wise breakdown were statistically calculated in proportion to national and division-wise population size. About 5.0 mL blood was collected and hematological indices were measured using Complete Blood Count (CBC) analysis and Hb-electrophoresis. Serum ferritin was measured to detect coexistence of IDA with thalassemia carrier. DNA-based High Resolution Melting curve analysis was performed for confirmation of carrier status and detection of mutations. To determine the effect of genetic modifier of the HbE/β-thalassemia, a cross-sectional was performed on a total of 130 of HbE/β-thalassemia patients and 50 unrelated healthy individuals over a period of 11 months from September 2018 to August 2019. These patients, with the age range of 6 to 65 years (80 males, 50 females), were enrolled from the Bangladesh Thalassemia Samity Hospital located in Dhaka where they came for follow-up examination and blood transfusion. The patients were categorized into three severity groups – mild, moderate, and severe following previously reported Mahidol disease severity scoring system. The ethical clearance certificate and consent form regarding this research was approved by Bangladesh Medical Research Council (BMRC) of National Ethics Review Committee (NERC), Dhaka, Bangladesh. Upon obtaining written informed consent form along with a structured questionnaire of information about the age of onset, age of first transfusion, transfusion interval, and splenectomy status of the patients venous blood samples (~ 5 ml) were collected from the patients and subjected to CBC and Hb electrophoresis. After genomic DNA extraction, PCR-RFLP method was used to identify rs7482144 (-158 Xmn1-Gγ) in γ-globin promoter. As PCR-RFLP is a lengthy time consuming method requiring a number of steps, we established Real-time PCR followed by HRM curve analysis based SNP detection method for rest of the 7 SNPs namely, rs4895441, rs28384513, rs28384512, rs11886868, rs4671393, rs766432 and rs2071348. Conventional Gap-PCR was used for detection of -α3.7 deletion and αααanti 3.7 triplication while --SEA deletion was detected using Real-time Gap-PCR followed by HRM curve analysis. Result: Total carrier frequency of HbE plus β-thalassemia was 10.92%, where ETT had the highest frequency of 8.68% followed by BTT 2.24%. Among eight divisions, Rangpur had the highest carrier frequency of 27.1% (ETT-25%, BTT-2.1%), whereas Khulna had the lowest frequency of 4.2% (ETT-4.2% only). Moreover, α- thalassemia, HbD trait, HbE disease, hereditary persistence of HbF were detected in 0.11, 0.16, 0.43 and 0.16% participants, respectively. HRM could identify two individuals with reported pathogenic mutations in both alleles who were erroneously interpreted as carriers by hematological indices. Finally, a total of nine different mutations including a novel mutation (c.151A > G) were detected in the β-globin gene. Low levels of HbF and HbE were found to be significant determinants of high disease severity in patients with HbE/β-thalassemia as both were inversely related to the disease severity score as well as showing the higher was the level of HbF, the higher was the blood transfusion interval and age of 1st blood transfusion. 11 different causative mutations were found in the the β- globin allele trans to HbE allele in the patients with HbE/β-thalassemia in Bangladesh. c.79 G>A (E allele)+IVS1_5 G>C combination was found as most predominant mutation pair across all the 3 severity groups of patients covering 73.8% of HbE/β-thalassemia in the country. On the other hand, heterozygous silent carrier and homozygous trait of α3.7 deletion were found only in 1 moderate and 2 sever cases in the present study while α-globin triplication (αααanti 3.7/αααanti 3.7) was found in 4 moderate and 8 sever patients However, the α--SEA thalassemia allele was not detected in our HbE/β-thalassemia patients. Among the 8 SNPs, the highest MAF (minor allele frequency) was found for HBS1L-MYB rs28384513 with the frequency of 0.46 followed by -158 Xmn1-Gγ in HBG2 (MAF: 0.45), HBS1L-MYB rs28384512 (MAF: 0.45) and rs2071348 in HBBP1 gene (MAF: 0.28). The lowest MAF of 0.1 was found for the rs11886868 in BCL11A gene. Upon association study, only 4 SNPs, namely, -158 Xmn1-Gγ, rs4895441, rs28384513 and rs2071348 showed statistically significant association with elevated level of HbF in the study population and 3 SNPs namely -158 Xmn1, rs4895441 and rs2071348 showed significant association with the clinical scores of the patients with HbE/β-thalassemia. The strongest association in terms of both increasing HbF level and decreasing severity, was observed with SNPs in HBG2 (-158Xmn1-Gγ) and HBBP1 (rs2071348) gene in the β globin gene cluster followed by HBS1L_MYB rs4895441 and rs28384513. However, none of the 3 SNPs in BCL11A exhibited significant association with either HbF level or the severity score Conclusion: Carrier frequencies of both HbE and β-thalassemia are alarmingly high in Bangladesh. A nationwide awareness and prevention program is in mandate to halt further aggravation of the current burden of thalassemia patients in Bangladesh. Detection of the HbF inducer SNPs for our population will help in planning appropriate management and treatment strategy revealing new therapeutic targets for increasing HbF levels in HbE/β-thalassemia patients in our country.