Newborn genetic screening of congenital adrenal hyperplasia using long-read sequencing

<h4>Objective</h4>To explore the use of genomic screening for congenital adrenal hyperplasia (CAH) based on long-read sequencing (LRS), aiming to provide an effective method for LRS-based screening (LRSBCS).<h4>Methods</h4>All newborns underwent traditional CAH screening via the collection of dried blood spots. We conducted a retrospective clinical study of 73 cases, including 12 confirmed cases of CAH, 18 cases with false-positive biochemical screening results, and 43 healthy newborns as control. Full-length CAH-related genes, including CYP21A2, CYP11B1, CYP17A1, HSD3B2, and STAR were amplified and sequenced on a Sequel II platform (Pacific Biosciences).<h4>Results</h4>Among the 235,999 newborns, 12 were confirmed to have CAH, based on biochemical and/or genetic testing. The positive-predictive values of the initial and positive recall results were 0.60% (12/1958) and 3.68% (12/326), respectively. The 12 children with CAH were accurately diagnosed using LRSBCS. For LRS, the Bayesian-estimated sensitivity is 96.2% (95% CrI: 80.3%-99.9%) and the specificity is 99.2% (95% CrI: 98.0%-99.9%). Eleven pathogenic variants of CYP21A2 were detected, including eight SNVs/indels and three deletions. The most frequent variants were c.293-13 C > G (7/11) and c.518T > A (7/11). Furthermore, LRSBCS can directly report the characteristics of gene variants (cis or trans mutations) and effectively distinguish between functional genes and pseudogenes.<h4>Conclusions</h4>LRSBCS represents a novel molecular screening approach tailored specifically for CAH, demonstrating preliminary feasibility in clinical settings.<h4>Clinical trial number</h4>Not applicable.