HBB gene mutation spectrum in an Indian cohort of 1530 cases using an in-house targeted next-generation sequencing assay
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ORIGINAL ARTICLE
HBB gene mutation spectrum in an Indian cohort of 1530 cases using an in-house targeted next-generation sequencing assay Ketki Kelkar 1,2,3 & Vijay Ramanan 2,4 & Siddharth Anand 1 & Purvi Majethia 1 & Shatakshi Ranade 1 & Kunal Patil 1 & Priyanka Gangodkar 1 & Ashwini Bapat 1 & Asawari Pilankar 1 & Vidula Sengaokar 1 & Kavita Khatod 1 & Meenal Agarwal 1,5 & Nikhil Phadke 1,5,6 Received: 20 February 2020 / Accepted: 3 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Beta thalassemia major is a common genetic disorder characterized by the reduced production or absence of beta globin, a product of the haemoglobin subunit beta (HBB) gene. Every year, approximately 10,000–12,000 children with thalassemia major are born in India. Molecular methodologies like ARMS (amplification-refractory mutation system)-PCR (polymerase chain reaction) and capillary sequencing are used to detect HBB gene mutations. It is, however, challenging to achieve comprehensive coverage of the HBB gene by these methods. Next-generation sequencing (NGS) can be used to circumvent these problems. Commercial NGS panels are prohibitively expensive and hence are not routinely implemented in most laboratories. We have developed a cost-effective, highly sensitive and specific, indigenous targeted NGS assay for detecting mutations in the HBB gene. Using this custom NGS assay, we processed 1530 samples (3017 alleles), in which we detected a spectrum of 48 pathogenic/ likely pathogenic variants (mutations); IVS-I-5 (c.92+5G>C) was the most common mutation detected (allele frequency (AF) 44.55%), followed by the 619-bp deletion (AF 10.74%), c.92+1G>T (AF 6.99%), c.27_28insG (AF 6.23%), c.47G>A (AF 5.77%) and c.126_129delCTTT (AF 4.71%). Additionally, we discovered a novel mutation (c.7delC) that was submitted to the HbVar database (HbVar ID 3193) as a variant of unknown significance (VUS), probably pathogenic. The targeted NGS assay developed during this study was validated using orthogonal methods and showed excellent correlation with currently available molecular methods. Additionally, this targeted NGS assay was used to analyse the mutation spectrum of the largest beta thalassemia cohort from India. Keywords Beta thalassemia . Mutation spectrum . Novel mutations . Next generation sequencing . ARMS PCR . Capillary sequencing
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12308-020-00414-8) contains supplementary material, which is available to authorized users. * Nikhil Phadke [email protected] 1
GenePath Diagnostics India Private Limited (GenePath Dx), Pune, India
2
MVR Welfare Foundation, Pune, India
3
Anjali Diagnostic Pathology Laboratory, Pune, India
4
Yashoda Hematology Clinic, Pune, India
5
I-SHARE Foundation, Pune, India
6
Genepath Diagnostics Inc, Ann Arbor, Michigan, USA
Beta thalassemia major is one of the most common genetic disorders in India with reported carrier frequencies between 3 and 18% [1]. The carr
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