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. 1. Research Scientist, GenePath Diagnostics India Private Limited, Pune, Maharashtra, India. 2. Consultant Pediatric Endocrinologist, Hirabai Cowasji Jehangir Medical Research Institute, Jehangir hospital, Pune, Maharashtra, India. 3. Pediatric Endocrinologist, Indira Gandhi Institute of Child Health, Bengaluru, Karnataka, India. 4. Endocrinology and Diabetes Unit, Department of Pediatrics, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research (PGIMER), Sector 12, Chandigarh, India. 5. Consultant Pediatric Endocrinologist, Sir Gangaram Hospital, Delhi, India. 6. Pediatric Endocrinology & Diabetes Unit, PGIMER, Chandigarh, India. 7. Consultant Pediatric Endocrinologist and Diabetologist, GKNM hospital, Coimbatore, Tamil Nadu, India. 8. Consultant Pediatric Endocrinologist, Harmony Health Hub, Nashik, Maharashtra, India. 9. Professor and Pediatric Endocrinologist, Bharati Vidyapeeth University Medical College, Pune, Maharashtra, India. 10. Chief Pediatric Endocrinologist, Rainbow Children's Hospital, Bengaluru, Karnataka, India. 11. Division of Genetics, Department of Pediatrics, AIIMS, New Delhi, India. 12. Addl. Prof. & HOD of Endocrinology, JIPMER, Puducherry, India. 13. Consultant, Medical Geneticist & dysmorphologist, Mediscan, Chennai, Tamil Nadu, India. 14. Senior Research Scientist, GenePath Diagnostics india Private Limited, Pune, Maharashtra, India. 15. Fellow in Pediatric Endocrinology, Hirabai Cowasji Jehangir Medical Research Institute, Jehangir hospital, Pune, Maharashtra, India. 16. Research Scientist, Hirabai Cowasji Jehangir Medical Research Institute, Jehangir hospital, Pune, Maharashtra, India. 17. Chief Technology Officer, GenePath Diagnostics Inc., Pune, India. 18. Head of Lab operations, GenePath Diagnostics India Private Limited, Pune, Maharashtra, India. 19. Chief Clinical Officer, GenePath Diagnostics India Private Limited, Pune, Maharashtra, India. 20. Chief Scientific Officer, GenePath Diagnostics India Private Limited, Pune, Maharashtra, India. 21. Consultant Pediatrician and Deputy Director, Hirabai Cowasji Jehangir medical research Institute, Jehangir Hospital, Pune, Maharashtra, India. anuradhavkhadilkar@gmail.com.
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.
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.
Entities:
Keywords:
Congenital Adrenal Hyperplasia; India; Next Generation Sequencing; children
Authors: William McLaren; Laurent Gil; Sarah E Hunt; Harpreet Singh Riat; Graham R S Ritchie; Anja Thormann; Paul Flicek; Fiona Cunningham Journal: Genome Biol Date: 2016-06-06 Impact factor: 13.583