Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India |
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| Affiliation: | 1. Department of Endocrinology, Diabetes and Metabolism, Christian Medical College, Ida Scudder Road, Vellore, Tamil Nadu, India;2. Department of Paediatric Endocrinology, Christian Medical College, Ida Scudder Road, Vellore, Tamil Nadu, India;3. Department of Biochemistry, Christian Medical College, Ida Scudder Road, Vellore, Tamil Nadu, India;4. Department of Clinical Genetics, Aster MIMS, Calicut, Kerala, India;1. Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran;2. Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran;3. Medical Imaging Research Center, Department of Radiology, Shiraz University of Medical Sciences, Shiraz, Iran;1. i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;2. IBMC – Institute for Molecular and Cell Biology, Universidade do Porto, Portugal;3. Centre for Predictive and Preventive Genetics (CGPP), Universidade do Porto, Portugal;4. Faculty of Psychology and Education Sciences, University of Porto, Porto, Portugal;5. CHUC - Medical Genetics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Portugal;6. CHULN-HSM – Serviço de Genética Médica, Centro Hospitalar Universitário de Lisboa Norte – Hospital de Santa Maria, Portugal;7. EPER – Hospital de Santo Espírito da Ilha Terceira, Portugal;8. HB - Unidade de Genética Médica, Hospital de Braga, Portugal;9. ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal;10. ICVS/3B''s, PT Government Associate Laboratory, Braga/Guimarães, Portugal;11. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal;12. Centro de Psicologia, Universidade do Porto, Porto, Portugal;1. Laboratory of Molecular and Cellular Screening Processes (LPCMC), Center of Biotechnology of Sfax, University of Sfax, Tunisia;2. Medical Genetics Department, University Hedi Chaker Hospital of Sfax, Tunisia;3. Child Neurology Department, University Hedi Chaker Hospital of Sfax, Tunisia;4. Research Laboratory “Neuropédiatrie" LR19ES15, Sfax University, Tunisia;5. Radiology Department, Hedi Chaker University Hospital, University of Sfax, Sfax, Tunisia;6. Department of Ophthalmology, Habib Bourguiba Hospital, Sfax, Tunisia;7. Department of Otorhinolaryngology, University Habib Bourguiba Hospital of Sfax, Tunisia;8. Laboratory of Human Molecular Genetics, LR33ES99, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia;9. Department of Applied Biology, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates;10. Human Genetics and Stem Cell Laboratory, Research Institute of Sciences and Engineering, University of Sharjah, Sharjah, United Arab Emirates;11. Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates;1. Department of Medical Sciences, University of Turin, Turin, Italy;2. Immunogenetics and Transplant Biology Service, Città della Salute e della Scienza University Hospital, Turin, Italy;3. Pediatric Nephrology Dialysis and Transplantation Unit, Città della Salute e della Scienza University Hospital, Turin, Italy;4. Department of Pediatrics, AOU Città della Salute e della Scienza di Torino, University of Turin, Turin, Italy;1. Department of Neurology, Children''s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, 400014, China;2. China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing, 400014, China;3. Chongqing Key Laboratory of Pediatrics, Chongqing, 400014, China;1. Greenwood Genetic Center, Greenwood, SC, USA;2. Department of Pediatrics, Section of Genetics and Metabolism, Children''s Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, USA;3. Divsion of Genetics, Department of Pediatrics, Medical University of South Carolina, Charleston, SC, USA;4. Dell Children''s Medical Group, Austin, TX, USA |
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| Abstract: | Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting. |
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| Keywords: | Congenital Adrenal Hyperplasia (CAH) Targeted Next Generation Sequencing (NGS) Comprehensive genetic screening strategy India |
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