Hypospadias is a common congenital malformation of the male external genitalia, with severe cases presenting considerable surgical and long-term challenges. Despite the clinical importance of severe hypospadias demonstrated by prolonged hospital stays, repeated surgeries, and substantial costs, the genetic etiology of severe hypospadias remains incompletely understood, particularly in diverse populations. To determine the molecular basis, we performed whole-exome sequencing (WES) on 30 Chinese patients from southeastern China with confirmed 46,XY karyotypes. Our analysis identified clinically relevant genetic variants, including single-nucleotide variants (SNVs) and copy number variations (CNVs), with subsequent phenotypic correlation. Clinically relevant genetic variants were identified in 33.3% (10/30) of cases, including novel SNVs in gonadal regulators (nuclear receptor subfamily 5 group A member 1 [NR5A1] c.1344dupC/c.244+1G>T and SRY-box 3 [SOX3] c.1273G>C), morphogenetic modulators (GLI family zinc finger 3 [GLI3] c.4731delA and aristaless-related homeobox [mARX] c.644C>G), and syndromic genes (patched domain containing 1 [PTCHD1] c.667G>A and euchromatic histone lysine methyltransferase 1 [EHMT1] c.3081C>T). Additionally, recurrent CNVs at 22q12.3 and a novel CNV exon 18 deletion in myelin regulatory factor (MYRF) and 18q11.2 were identified. Mutation carriers showed a significantly higher frequency of cryptorchidism (40.0% vs 5.0%, P < 0.01) and a higher prevalence of ≥3 associated malformations (80.0% vs 35.0%, P < 0.05) than non-carriers, highlighting genotype–phenotype correlations. The 33.3% diagnostic yield tripled conventional estimates, demonstrating WES efficacy in identifying SNVs and CNVs in severe phenotypes. These findings reveal the genetic heterogeneity of severe hypospadias and support WES utility in uncovering novel variants and structural genomic alterations.

Keywords: copy number variations; genotype–phenotype correlations; hypospadias; whole-exome sequencing