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

BMC Genetics

卷:19

Salma Sadya¹  Bilquis Banu¹  Golam Sarwardi¹  Waqar Ahmed Khan¹  Manzoor Hussain¹  Hossain Uddin Shekhar²  Emran Kabir Chowdhury²  Abu Ashfaqur Sajib³  Sharif Akhteruzzaman³  Mohammad Abdul Mannan⁴  Syeda Kashfi Qadri⁵  A. K. M. Muraduzzaman⁶  Tahmina Shirin⁶  Golam Sarower Bhuyan⁷  Mohammad Sazzadul Islam⁷  Nusrat Sultana⁸  Firdausi Qadri⁸  Mst. Noorjahan Begum⁸  Kaiissar Mannoor⁸  Aparna Biswas⁸  Syed Saleheen Qadri⁸  Md Tarikul Islam⁸  Suprovath Kumar Sarkar⁸  Pritha Promita Biswas⁸  Md Alauddin⁸  Shezote Talukder⁸ 

[1] Department of Biochemistry and Molecular Biology, Dhaka Shishu Hospital;

[2] Department of Biochemistry and Molecular Biology, University of Dhaka;

[3] Department of Genetic Engineering & Biotechnology, University of Dhaka;

[4] Department of Neonatology, Bangabandhu Sheikh Mujib Medical University;

[5] Department of Paediatric Medicine, KK Women’s and Children’s Hospital;

[6] Department of Virology, Institute of Epidemiology, Disease Control and Research;

[7] Infectious Diseases Laboratory, Institute for Developing Science and Health Initiatives;

[8] Laboratory of Genetics and Genomics, Institute for Developing Science and Health Initiatives;

关键词: Beta-globin gene;    Mutational hot-spot;    Beta-thalassemia;    High resolution melting curve;    Carrier screening;   

DOI  :  10.1186/s12863-017-0594-3

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown