Innovative approach for reliable screening of beta-globin gene mutations in Bangladesh

Innovative approach for reliable screening of all common as well as most of the rare beta-globin gene mutations in Bangladesh

The alarmingly risen carrier density of beta-thalassemia and hemoglobin E trait in Bangladesh warrants the development of a homegrown DNA based method for detection of carrier that in conjunction with hematological and electrophoretic indices can succeed in dealing with the hurdles of carrier screening.


Researchers at ideSHi aimed exactly at that by establishing a high resolution melt (HRM) curve-based swift and dependable method that specifically targets mutational hotspots of South Asian and Southeast Asian countries for detection. Till date at least 893 beta-globin gene mutations have been reported. Despite, the reporting of large numbers of beta-globin gene mutations from around the world, only a small number of mutations can be found in β-thalassemic individuals of each ethnic population. These comprise of both frequently occurring as well as some rare mutations. For example, over 90% of mutations in beta-thalassemic patients in India are accounted for by five common mutations, these are c.92 + 5G > C, c.92 + 1G > T, c.126_129delCTTT, c.27_28insG and the 619 bp deletion. Not much different from India, only five mutations, including c.-78A > G, c.52A > T, c.126_129delCTTT, c.216_217insA and c.316-197C > T in HBB gene are responsible for 90% of beta-thalassemia cases in China

The mutation spectrum in Bangladesh entails most frequently, c.79G > A, c.92 + 5G > C and c.126_129delCTTT; and less frequently c.92G > C, c.27_28insG, c.47G > A, c.92G > A, c.46delT, c.92 + 130G > C, and c.51delC which together account for more than 95% of beta thalassemia patients in Bangladesh

All the aforementioned mutations together constitute the mutational hot-spot (c.1 – c.92 of exon 1, c.92 + 1 – c.92 + 130 intron-1 and c.93 – c.217 of exon-2) in the HBB gene of Bangladeshi thalassemia patients. The same hot spot harbors majority of HBB gene mutations in many other countries of South Asia and Southeast Asia and therefore, the hot-spot could be a useful target to screen the beta-globin gene mutations and can also serve as a supplement to hematological and electrophoretic methods to overcome certain limitations of these approaches. For example, sole dependence on hematological and electrophoretic parameters for screening of beta-thalassemia carrier may miss a significant number of carriers due to adequacy of mild variant c.79G > A and complex inheritance of other factors such as presence of α-globin gene mutations, mutation in Krüppel-like Factor 1 (KLF1) gene, promoter mutations of gamma-globin gene, iron deficiency anemia, hereditary persistence of fetal Hb etc.

High-resolution DNA melting is a very simple, economic, and high-throughput solution for genotyping of known variants and scanning for unknown variants within polymerase chain reaction products.

When DNA is melted or denatured, double-stranded DNA (dsDNA) is separated into random coils of single stranded DNA. Using dyes that fluoresce in the presence of dsDNA this melting of DNA can be tracked. When a polymerase chain reaction (PCR) product is melted in the presence of a dsDNA dye, the fluorescence is monitored continuously and plotted against the temperature. As the temperature increases, there is a characteristic drop in fluorescence at high temperature that coincides with the denaturation of the PCR product. The melting profile of a PCR product depends on its guanine-cytosine (GC) content, length, and sequence. The HRM analysis enables rapid detection and categorization of mutations, as well as identification of new genetic variants without sequencing. Successfull utilization of this technique has been in the detection of mutations in various types of genetic diseases including autosomal recessive, autosomal dominant and X-linked recessive disorders as well as diseases involving somatic mutations

“Our data have already demonstrated that both common as well as rare mutations could be screened using HRM. Next, as a means of cost-saving approach, we wanted to observe whether the compound heterozygous mutations could be separated from their respective wild type alleles for direct genotyping of beta-thalassemia patients. The HRM curve analysis with reference samples of known mutations has already demonstrated that HRM could be a very good approach for distinguishing different types of mutations from the wild type alleles. Combinations of mutations could also be distinguished from one another by HRM. The findings prompted us to investigate whether the HRM approach could be applied for genotyping of beta-thalassemia samples for the confirmation of the disease status. In order to test this hypothesis, we performed HRM analysis on 41 unknown samples without any prior knowledge of their mutational status. The reference samples with known mutations were also run side-by-side to calibrate the data. All of these forty one samples differed in HRM curve patterns from the control wild type alleles. Each one of them had exhibited melting patterns similar to that of the corresponding reference samples with known homozygous, heterozygous or compound heterozygous mutations”, says Tarikul Islam, the principal investigator who led the study.

Tarikul added, “In our laboratory settings, it cost $145.00 ($3.50 per sample) for HRM-based screening of forty one samples by targeting the HBB gene hot-spot using two sets of primers that flanked all the major mutations and even some rare mutations. On the other hand, the cost of ARMS PCR for forty one samples was $125.00 ($3.00 per sample) when only one mutation was targeted, indicating that the cost would be much higher for screening of multiple mutations by ARMS PCR than that of the HRM. Finally, detection of mutations by Sanger Sequencing required approximately $565.00 for forty one samples ($13.75 per sample) which was four times higher than that of HRM-based screening. Thus, on the one hand, the HRM-based screening of mutations is a rapid, reliable and high throughput approach, and, on the other hand, it is less expensive than other genotyping approaches.”

The findings of this study was published in BMC Genetics

Paper(s) cited:

Islam MT, Sarkar SK, Sultana N, Begum MN, Bhuyan GS, Shithi ST et al. 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 BangladeshBMC Genetics. 2018;19(1).