Combination of two rare mutations causes β-thalassaemia in a Bangladeshi patient

Screening of mutations that cause β-thalassaemia in the Bangladeshi population led to the identification of a patient with a combination of two rare mutations, Hb Monroe and HBB: −92 C > G. The β-thalassaemia major male individual was transfusion-dependent and had an atypical β-globin gene cluster haplotype. Of the two mutations, Hb Monroe has been characterized in detail. Clinical effects of the other mutation, HBB: −92 C > G, are unknown so far. Bioinformatics analyses were carried out to predict the possible effect of this mutation. These analyses revealed the presence of a putative binding site for Egr1, a transcription factor, within the HBB: −92 region. Our literature survey suggests a close relationship between different phenotypic manifestations of β-thalassaemia and Egr1 expression

Spectrum of types of thalassemias and hemoglobinopathies: study in a tertiary level children hospital in Bangladesh

Thalassemias and hemoglobinopathies are the most common hemolytic congenital disorders in Bangladesh as in many parts of the world. This study was done to find the common types of thalassemias and abnormal hemoglobin variants seen in Bangladeshi populations. A total of 4813 samples were analyzed for hemoglobin disorders out of which 2308 (49.95%) showed abnormalities. The samples were analyzed by Bio Rad D 10 Analyzer in 3914 (81.32%) cases, BIORAD VARIANTβ thalassemia short program using the principle of high performance liquid chromatography in 474 (9.85%) cases and by CAPILLARYS 2 FLEX PIERCING utilizing capillary electrophoresis in 425 (8.83%) cases. The samples were analyzed in the Department of Biochemistry and Molecular Biology of Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. The common hemoglobin disorders seen were β trait 863 (17.94%), Hb E trait 601 (12.50%), Hb E β thalassemia 524 (10.87%), β thalassemia major 192(4.00 %), Hb E disease 99 (2.05%). Other Hb abnormalities detected were Hb D trait 17 (0.35%), Sickle cell trait 4 (0.08%), hereditary persistence of fetal hemoglobin (HPFH) 2 (0.04%), and Hb Lepore, δ β thalassemia, sickle cell β thalassemia, Sickle cell disease, compound heterozygote for HbE+D and Hb Q band one case each (0.02%).   与世界许多地方一样，地中海和血红蛋白病是孟加拉国最常见的溶血性先天性疾病。本研究的目的是找到孟加拉国人群的常见地中海贫血类型和异常血红蛋白变体。总共对4813例样本进行了血红蛋白疾病分析，其中2308例（49.95%）显示异常。3914例（81.32%）样本使用Bio Rad D 10分析仪分析，由BIORAD VARIANTβ地中海贫血症短期计划使用高效液相色谱法分析了474例（9.85%），由CAPILLARYS 2 FLEX PIERCING使用毛细管电泳分析了425例（8.83%）。样本在孟加拉达卡的达卡生化与分子生物学教研室进行分析。观察到的常见血红蛋白疾病是863例（17.94%）β特征、601例（12.50%）Hb E特征、524例（10.87%）Hb E β地中海贫血、192例（4.00 %）β重型地中海贫血、99例（2.05%）Hb E疾病。检测到的其他Hb异常为17例（0.35%）Hb D特征、4例（0.08%）镰状细胞性状、2例（0.04%）遗传性持续性胎儿血红蛋白综合征（HPFH）以及Hb Lepore δ β地中海贫血、镰状细胞β地中海贫血、镰状细胞病、HbE+D和Hb Q带的复合杂合体各一例（0.02%）

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

Abstract 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

Additional file 2: Figure S1. of 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

Normalized melt curve patterns of homozygous and heterozygous c.79G > A and c.92 + 5G > C mutations. (TIFF 232 kb

Additional file 5: Figure S4. of 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

Normalized melt curve patterns of heterozygous c.92 + 130G > C, c.126delC and c.135delC mutations. (TIFF 189 kb

Additional file 6: Figure S5. of 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

Temperature shifted difference curves of unknown carrier parents subjected to HRM analysis by 1st set of primers. (TIFF 530 kb

Additional file 1: Table S1. of 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

Mutational status of 40 parents of beta-thalassemia patients recruited for initial evaluation of HRM study. (DOCX 11 kb

Additional file 3: Figure S2. of 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

Normalized melt curve patterns of homozygous and heterozygous c.126_129delCTTT mutations. (TIFF 173 kb