Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India
- 18 September 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Endocrine
- Vol. 71 (1), 189-198
- https://doi.org/10.1007/s12020-020-02494-z
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.Funding Information
- Department of Biotechnology India (BT/PR17363/MED/97/305)
This publication has 37 references indexed in Scilit:
- Molecular genetic analysis of CYP21A2 gene in patients with congenital adrenal hyperplasiaIndian Journal of Endocrinology and Metabolism, 2012
- Clinical Phenotype and Mutation Spectrum of the CYP21A2 Gene in Patients with Steroid 21-Hydroxylase DeficiencyExperimental and Clinical Endocrinology & Diabetes, 2011
- Comprehensive Genetic Analysis of 182 Unrelated Families with Congenital Adrenal Hyperplasia due to 21-Hydroxylase DeficiencyJournal of Clinical Endocrinology & Metabolism, 2011
- Sequencing technologies — the next generationNature Reviews Genetics, 2009
- Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiencyMolecular Genetics and Metabolism, 2007
- Emerging technologies in DNA sequencingGenome Research, 2005
- Newborn screening in IndiaIndian Journal of Pediatrics, 2004
- Congenital Adrenal HyperplasiaThe New England Journal of Medicine, 2003
- The P450 Superfamily: Update on New Sequences, Gene Mapping, and Recommended NomenclatureDNA and Cell Biology, 1991
- Newborn screening, prenatal diagnosis, and prenatal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiencyTrends in Endocrinology & Metabolism, 1990