Nationwide carrier detection and molecular characterization of ß-thalassemia and hemoglobin E variants in Bangladeshi population.

BACKGROUND: ß-thalassemia is one of the most common inherited blood disorders in the world and a major deterrent to the public health of Bangladesh. The management of thalassemia patients requires lifelong frequent blood transfusion and the available treatment options are unsatisfactory. A national policy on thalassemia prevention is mandatory in Bangladesh. However, precise and up-to-date information on the frequency of ß-thalassemia carriers are missing due to lack of accurate diagnostic approaches, limited access to information and absence of national screening program. This study aims to determine the nationwide carrier frequency of hemoglobin E (HbE) and ß- thalassemia and mutation spectrum among the carriers using molecular, hematological and biochemical methods. METHODS: The study enrolled a total of 1877 individuals (60.1% male and 39.9% female) aged between 18 and 35 years. Total sample size and its division-wise breakdown were calculated in proportion to national and division-wise population. Venous blood was collected and subjected to CBC analysis and Hb-electrophoresis for each participant. Serum ferritin was measured to detect coexistence of iron deficiency anemia with thalassemia carrier. DNA-based High Resolution Melting (HRM) curve analysis was performed for confirmation of carrier status by mutation detection. RESULTS: Of 11.89% (95% CI, 10.43-13.35) carriers of ß-globin gene mutations, 8.68% (95% CI, 7.41-9.95) had HbE trait (ETT) and 2.24% (95% CI, 1.57-2.91) had beta-thalassemia trait (BTT). 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 ß-globin gene. CONCLUSIONS: Since carrier frequency for both HbE and ß-thalassemia is alarmingly high in Bangladesh, a nationwide awareness and prevention program should be made mandatory to halt the current deteriorating situations. Mutation-based confirmation is highly recommended for the inconclusive cases with conventional carrier screening methods to avoid any faulty detection of thalassemia carriers.

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

BACKGROUND: Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. RESULTS: Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. CONCLUSIONS: Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.

Glycated haemoglobin--a marker and predictor of cardiovascular disease.

Glycated haemoglobin is formed by simple chemical reaction between haemoglobin and blood glucose. It represents a reliable and moving average of blood glucose over preceding three months. In 2009 the International Expert Committee recommended the use of HbA1c to diagnose diabetes with a cut-off of 6.5%. Studies have shown that HbA1c even in the range of 5.5% to 6.5% poses considerably high risk of morbidity and mortality due to cardiovascular disease. HbA1c as a test is important because about 220 million people have diabetes and with increasing life expectancy and emergence of type 2 diabetes in children makes it even more important. Each 1% increase in HbA1c poses 15-18% relative risk of cardiovascular disease in T1DM and T2DM respectively. CVD is a major cause of death and disability among diabetes patients and glycated haemoglobin itself is proportionately linked with excess CV morbidity and mortality. UKPDS-35 demonstrated 14% reduction in the incidence of acute myocardial infarction with only 1% reduction in HbA1c in T2DM patients. The DCCT reported significant reduction in retinopathy and nephropathy in T1DM, the follow up trial EDIC demonstrated 42% reduction in CVD with intensive reduction of HbAlc. This review is written to remind ourselves of the importance of this simple test which can predict early CV mortality in patients without prior CVD and poor prognosis in established cardiovascular disease. In a country like ours, Pakistan; where diabetes is prevalent with poor health awareness and limited resources, a test like HbA1c which costs Rs, 500.00 twice a year should be considered a cost effective way to avoid the long term diabetes complications, which once start unfolding put enormous burden on already stretched healthcare resources which could easily be avoided by intensive control of diabetes.