The genetic landscape of hypospadias: clinical insights into inherited and de novo risk factors.
Hypospadias is one of the most common birth defects in China and a key feature of differences in sex development (DSD), yet its genetic etiology remains largely unresolved. Current diagnostic approaches using DSD-targeted gene panels have a low rate of definitive diagnoses (5.5%), highlighting the need for more comprehensive genetic investigation. In this study, we performed next-generation sequencing (NGS) on the largest trio-based cohort of hypospadias to date, comprising 106 pediatric cases and their parents (92 trios). We achieved a definitive genetic diagnosis in 6.6% of patients, identifying pathogenic variants in canonical DSD genes such as AR, NR5A1 and WT1. By incorporating a broader spectrum of potentially clinically significant variants, we increased the overall genetic identification rate to 33.0% (35/106). Strikingly, trio analysis uncovered a significant burden of de novo loss-of-function (LoF) variants (2.2-fold enrichment, P=0.001), primarily driven by variants in genes associated with ciliopathies (10.35-fold, P=0.016), a previously underappreciated gene class in hypospadias. Furthermore, we identified and functionally validated two high-confidence risk genes, PRKCZ and HRNR, based on recurrent de novo variants. Functional assays confirmed that these variants disrupt key biological mechanisms, including cell proliferation, migration, and androgen signaling. Our large-scale trio approach substantially expands the genetic landscape of hypospadias, demonstrates the critical value of trio-based sequencing for improving diagnostic yield, and decisively implicates ciliary genes in its pathogenesis.