Clinical application of long-read sequencing in newborn genetic screening for congenital adrenal hyperplasia.

BACKGROUND: Screening for congenital adrenal hyperplasia (CAH) relying solely on 17α-hydroxyprogesterone (17α-OHP) presents limited diagnostic performance, highlighting an urgent need to develop more robust screening strategies for neonates. METHODS: We conducted retrospective and prospective cohort studies to explore the clinical applicability of long-read sequencing (LRS) in CAH genetic testing within primary and secondary newborn screening (NBS) systems, respectively. The retrospective cohort comprised 100,145 neonates who underwent routine 17α-OHP primary CAH screening at the Fujian Provincial Newborn Screening Center from January 1 to December 31, 2019. Among these infants, 52 full-term screen-positive neonates received further LRS-based CAH genotyping. The prospective cohort enrolled 2,100 newborns recruited from Fujian Maternity and Child Health Hospital between May 1 and May 31, 2023, who underwent simultaneous 17α-OHP measurement and LRS-mediated CAH genetic analysis. RESULTS: In the retrospective cohort, the positive rate of 17α-OHP screening was 0.19% (190/100,145, 95% CI: 0.17%-0.21%), and five infants were definitively diagnosed with CAH, corresponding to a disease prevalence of 1:20,029. LRS genotyping successfully identified five neonates harboring pathogenic CYP21A2 mutations consistent with confirmed genetic diagnosis. In this prospective cohort study, two newborns (1/1050) with normal 17α-OHP concentrations were found to carry biallelic pathogenic variants in the CYP21A2 gene. In addition, 88 neonates (4.2%) with normal 17α-OHP levels were found to carry heterozygous CAH-related variants. Among these heterozygotes, 85 individuals harbored CYP21A2 variants, representing 32 distinct genotypes. The calculated carrier frequencies were 1 in 78 for classic CAH and 1 in 40 for non-classic CAH. CONCLUSION: LRS-integrated genetic newborn screening exhibits favorable efficacy for CAH identification. This combined screening modality holds great promise for wide implementation in routine neonatal screening practice.