Uncovering Hidden Genetic Contributors to 46,XY Disorders of Sex Development Through Phenotype-Driven Rare Variant Assessment: A Pilot Study

Background: Despite advances in genetic testing, many 46,XY Disorders of sex development (DSD) cases remain unsolved after whole-exome sequencing (WES). This study intended to explore rare variants in patients with micropenis, cryptorchidism, or hypospadias using bioinformatics analysis to identify potential pathogenic contributors and pathways underlying 46,XY DSD. Methods: A total of 35 patients with specific phenotypes (micropenis/cryptorchidism/hypospadias) and negative whole-exome sequencing results were enrolled. Bioinformatics analysis methods (SKAT-O test and GO enrichment) were applied to identify the putative loss-of-function (pLoF) variation, including nonsense, frameshift, and canonical splice-site variants, and predicted deleterious missense variants (CADD Phred > 20). Literature was reviewed to explore the correlation of detected candidate genes/pathways and 46,XY disorder of sex development. Results: After variant quality filtering, we identified 307,638 pLoF variants and 127,857 predicted deleterious missense variants across all samples. In subgroup A (micropenis, n = 21), we identified 146,268 pLoF variants and 104,746 predicted deleterious missense variants. In subgroup B (cryptorchidism, n = 10), we identified 111,172 pLoF variants and 77,244 predicted deleterious missense variants. In subgroup C (hypospadias, n = 4), we identified 50,198 pLoF variants and 23,111 predicted deleterious missense variants. Using SKAT-O with an initial screening threshold of p < 0.005 (FDR q < 0.05), we obtained 67 candidate genes from the pLoF variant set and 59 candidate genes from the predicted deleterious missense variant set in subgroup A; 81 and 11 candidate genes, respectively, in subgroup B; and 17 and 0 candidate genes, respectively, in subgroup C. Conclusions: Assessment of rare variants helps further explore the genetic contributors to 46,XY disorder of sex development and provide potential candidate genes and associated pathways.