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

BackgroundBangladesh 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.ResultsOur 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.ConclusionsTargeting 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.Electronic supplementary materialThe online version of this article (10.1186/s12863-017-0594-3) contains supplementary material, which is available to authorized users.

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

“…From the borderline suspected Group 2, a participant with 3.5% HbA2 generated a HRM curve pattern different from the wild type cluster (without mutation in HBB gene) and also did not match with any of the HRM curves previously established for all the reported mutations in Bangladesh [2]. Sanger sequencing identified and confirmed the suspected mutation as c.151A > G (ACT>GCT; Thr > Ala) in the HBB gene and upon BLAST with databases it was found to be a novel mutation, thereby confirming the carrier status of this participant ( Fig.…”

“…Since screening by hematological indices may result in misdetection of carriers due to factors like co-inheritance of αthalassemia, mutations in βand δ-globin genes, mutations in KLF1 gene and iron deficiency anemia [15,27,28], this study applied a comparatively cheaper and easy-to-perform DNA-based HRM curve analysis method to confirm and characterize mutations in the β-globin gene. This method supplements the conventional hematological and electrophoretic parameter based approaches for identification of the thalassemia carriers and patients [2]. For example, although the parameters MCV ≥ 80 fL and MCH ≥ 27 pg are usually considered as a negative indicator for HbE trait, our study using electrophoresis had identified two HbE carriers with MCV ≥ 80 fL and MCH ≥ 27 pg and a case having MCV > 80 fL and MCH < 27 pg as HbE carrier, indicating the shortcomings of hematological indices in screening of HbE carriers.…”

“…Therefore, although the combination of MCV, MCH and Hb electrophoresis resulted in high sensitivity and specificity, the DNA-based approaches like HRM curve analysis and Sanger sequencing had been proved to be very useful to avoid false positive and false negative results by detecting mutations in the β-globin gene, and thereby confirming the true thalassemia carrier status of the participants. In a previous study, we de [2]. Notably, this real time PCR-based high throughput HRM curve analysis is easy to perform and time-saving as there is no need of post PCR amplification processes like PCR product purification, gel electrophoresis etc.…”

“…Real-time PCR and HRM curve analysis using Precision Melt Analysis™ Software (BioRad) were performed on Bio-Rad CFX96 Real-Time System. This HRM method was previously developed for mutation screening in the β-globin gene of Bangladeshi and regional population of thalassemic endemic countries [2,17,18]. This high throughput approach enables to screen mutation(s) in unknown specimens in the presence of reference samples without nucleotide sequencing as well as to screen a large number of samples in a quick and cost-effective manner.…”

“…β-thalassemia, characterized by reduced or absent βglobin chain synthesis, is one of the most common inherited blood disorders in the world and hence a major deterrent to public health. Although widespread, the major at-risk populations are mainly from Mediterranean, Middle East and Southeast Asian countries including Bangladesh [1,2]. WHO reported that approximately 1.5% of global population are carriers of β-thalassemia and 68, 000 children are born each year with various thalassemia syndromes [3].…”