Assessment of Long-Read Sequencing-Based Congenital Adrenal Hyperplasia Genotyping Assay for Newborns in Fujian, China

Long-read sequencing (LRS) provides comprehensive genetic information, but research of LRS applied to congenital adrenal hyperplasia (CAH) newborn screening is limited. This study aimed to evaluate the clinical utility of LRS in genetic diagnosis and second-tier newborn screening. Neonates born between January 2017 and December 2022 in Fujian, China, were recruited for biochemical and LRS-based genetic screening assay. The LRS covers the entire gene regions and exon-intron boundary regions for <i>CYP21A2</i>, <i>CYP11B1</i>, <i>CYP17A1</i>, <i>HSD3B2</i>, and <i>StAR</i>. In this retrospective study, 1,774,555 newborns received 17α-OHP screening, yielding a screening positive rate of 0.20%. Of these high-risk neonates, 3411 were successfully recalled for re-evaluation. Finally, 66 neonates were diagnosed with CAH, with a positive predictive value of 28.82%. Based on this data, the overall prevalence of CAH in Fujian was estimated to be 1:26,883. LRS was performed on 57 neonates with 21-hydroxylase deficiency (21-OHD) and 109 variant alleles were identified. The c.293-13C>G variant (31.19%) was the most prevalent in Fujian. Additionally, 647 neonates with suspected positive results were genotyped, and 41 were identified as carriers, with carrier frequencies of 1:18 for <i>CYP21A2</i>, 1:162 for <i>HSD3B2</i>, and 1:324 for <i>CYP17A1</i> in Xiamen. Therefore, LRS can provide comprehensive genotypes in approximately 1.5 days at a cost of less than $20 USD per sample, and effectively improve screening efficiency, reduce anxiety of parents during newborn screening (NBS), and shorten the time to referral of CAH patients (approximately 10 days). Such a combined screening strategy is worthy to be recommended for NBS programs in the future.