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.

A Novel ß-Thalassemia Insertion/Frameshift Mutation Between Codons 77/78 (p.Leu78Profs*13 or HBB: c.235_236insC) Observed in a Family in Bangladesh.

ß-Thalassemia (ß-thal) is one of the most common inherited hemoglobin (Hb) disorders, worldwide. A 28-year-old female and her husband came to Dhaka Shishu (Children) Hospital, Bangladesh for prenatal diagnosis for thalassemia mutations. We identified and characterized a novel ß-thalassemia (ß-thal) mutation due to an insertion of cytosine between codons 77 and 78 (p.Leu78Profs*13) found in mother in a heterozygous state. This mutation caused an insertion in the normal reading frame of the ß-globin coding sequence and the new stop codon being the amino acid 90 (HBB: c.235_236insC) in exon 2 that leads to a ß0-thal phenotype.

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

Screening of mutations that cause ß-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 ß-thalassaemia major male individual was transfusion-dependent and had an atypical ß-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 ß-thalassaemia and Egr1 expression.

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: -92C>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: -92C>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.

Mutation analysis of the HBB gene in selected Bangladeshi beta-thalassemic individuals: presence of rare mutations.

INTRODUCTION AND AIMS: Bangladesh has a large number of thalassemic patients. However, no extensive analysis of the mutations in the HBB gene of thalassemic patients has been previously carried out. We have conducted a systematic research to reveal thalassemia mutations in the Bangladeshi population. In this preliminary analysis of 587 bp of the HBB gene in selected thalassemic individuals, some rare mutations in world perspective have been found to be significantly high in the Bangladeshi population, together with the common mutations for thalassemia. RESULTS: A 587-bp segment of the HBB gene from 32 chromosomes of 16 beta-thalassemic individuals was analyzed for molecular characterization of the disease. Splice junction mutation IVS-I-5 was found to be the most common. The analysis also revealed some rare mutations HBB: c.-80T>C, HBB: c. 92G>C, HBB: c-92C>G, which are not prevalent in geographically adjacent populations. CONCLUSION: This is a first of this kind of study in the Bangladeshi population. Although the small sample size makes it difficult to make any population genetics inference, this study can be regarded as the seminal research for a large-scale study to determine the complete mutation profile underlying thalassemia in the Bangladeshi population. The complete mutation profile will provide invaluable strategies (e.g., prenatal diagnosis and genetic counseling) for better management of thalassemia in the Bangladeshi population.