Novel CYP17A1 variants and functional validation in a large Chinese cohort of complete 17α-hydroxylase deficiency

Background Complete 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is a rare autosomal recessive form of congenital adrenal hyperplasia caused by CYP17A1 variants. Large-scale studies integrating clinical, genetic, and functional data remain limited. Methods We recruited 113 genetically confirmed 17-OHD patients from 107 unrelated families. Comprehensive clinical manifestations, hormonal, and imaging examination data were collected. CYP17A1 variants were identified by Sanger sequencing, whole-exome sequencing or long-read sequencing. In vitro functional studies including enzyme activity assays and minigene splicing assays were performed to verify the pathogenicity of the variants. Results All patients presented as phenotypic females, with a median diagnostic age of 17 years. Hypertension (93.58%) and hypokalemia (74.31%) were the dominant clinical features, and 91.26% exhibited Tanner stage I breast development. We identified 50 pathogenic CYP17A1 variants, including 6 novel ones (c.286C>G, c.436+1G>T, c.1241C>T, c.1300C>T, c.1348C>T, c.1433G>T). The mutation spectrum was dominated by a founder hotspot, c.985_987delinsAA, accounting for 59.29% of alleles, with mutations clustering in exons 6 and 8. In vitro enzyme activity assays confirmed near-complete loss of both 17α-hydroxylase and 17,20-lyase activities in all variants except p.Val236Gly, which retained minimal residual activity. Notably, 5 variants (including 2 missense changes, c.1084C>T and c.1085G>A) were shown to disrupt normal pre-mRNA splicing in minigene assays, revealing a dual molecular mechanism of pathogenicity involving both protein dysfunction and aberrant RNA processing. Conclusion This study defines the most comprehensive CYP17A1 variant spectrum for complete 17-OHD in China, identifies 6 novel pathogenic variants, and uncovers splicing disruption as an under-recognized mechanism in missense mutations. These findings expand the molecular and clinical understanding of 17-OHD, highlight the founder effect of c.985_987delinsAA in the Chinese population, and provide critical insights for genetic diagnosis and counseling.