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

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.

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.

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.

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.