Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congen
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ORIGINAL ARTICLE
Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India Priyanka Gangodkar1 Vaman Khadilkar2 P. Raghupathy3 Rakesh Kumar4 Archana Arya Dayal5 Devi Dayal6 Ahila Ayyavoo7 Tushar Godbole8 Rahul Jahagirdar9 Kavitha Bhat10 Neerja Gupta11 Sadishkumar Kamalanathan12 Sujatha Jagadeesh13 Shatakshi Ranade14 Nikhil Lohiya15 Rashmi Lote Oke16 Karthik Ganesan17 Kavita Khatod18 Meenal Agarwal19 Nikhil Phadke20 Anuradha Khadilkar21 ●
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Received: 2 May 2020 / Accepted: 7 September 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Purpose Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. Methods Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband’s and parent’s(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). Results Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. Conclusions We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH. Keywords Congenital Adrenal Hyperplasia children India Next Generation Sequencing ●
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Introduction Congenital adrenal hyperplasia (CAH) is a disorder of the adrenal cortex that impairs steroidogenic activity, which is essential for cortisol biosynthesis [1]. While the incidence of CAH in literature is quoted to be ~1:5000–1:15,000 infants [2–4], reports suggest that the incidence is higher in India (~1:3000 live births) [4], possibly because
* Anuradha Khadilkar [email protected] Extended author information available on the last page of the article
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