Short-Read NGS of a Long-Range CYP21A2 Amplicon as an Improved Alternative to Sanger Sequencing for Congenital Adrenal Hyperplasia Genetic Testing
Zoia Antysheva, Viktor Bogdanov, Ekaterina Rutkovskaya, Anna Stepanova, Anton Esibov, Julia Krupinova, Victoria Shchekina, Ekaterina Avsievich, Tatyana Frolova, Erzhena Bazarova, Elena Demina, Evgenia Sharibzhanova, Natalia Bodunova, Elena Petryaykina, Ekaterina Petriaikina, Aleksey Ivashechkin, Yulia Katcaran, Anastasia Bukhanova, Vladimir Yudin, Anton Keskinov, Sergey Yudin, Dmitry Svetlichnyy, Mary Woroncow, Veronika Skvortsova, Pavel VolchkovThe standard-of-car and genetic testing for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) is Sanger sequencing of a long-range CYP21A2 amplicon combined with multiplex ligation-dependent probe amplification (MLPA). Many Sanger sequencing protocols capture only selected regions of CYP21A2 and require several sequencing reactions. This retrospective study explores the potential of short-read next-generation sequencing (SRS) to replace Sanger sequencing in CAH genetic testing. A total of 216 participants, including CAH patients, their parents and individuals with no family history of CAH, were included. We assessed the performance of standard SRS analysis and improved upon it with developed computational software AmpliconPipe, which includes copy number calling and caller harmonization for precise variant detection. With these procedures, SRS of the CYP21A2 long-range amplicon achieved 100% sensitivity (CI 95% [94.9, 100]) and 100% specificity (CI 95% [97.4, 100]) for CAH diagnosis, and 100% sensitivity (CI 95% [94.9, 100]) with 98.6% specificity (CI 95% [95.0, 99.6]) for CAH-carrier diagnosis, with better gene coverage outperforming the standard-of-care method, discovering novel variants, and detecting CYP21A2 copy number alterations with high sensitivity. These findings establish SRS as a practical, accurate, and immediately implementable alternative to Sanger sequencing for CAH genetic testing.