Identification and Functional Characteristics of NR5A1 Gene Variant in Patients with 46,XY Disorders of Sex Development

Background/Objectives: Individuals with 46,XY disorders of sexual development (DSD) present with incomplete genital masculinization, aberrant gonadal development, and occasional retention of Müllerian duct remnants. Genetic factors play a substantial role in DSD pathogenesis, and whole-exome sequencing has expanded the catalog of candidate variants in recent years. However, the underlying molecular pathways remain incompletely characterized, and the functional relevance of most isolated genetic findings has not been systematically determined. This study aimed to identify and functionally characterize novel NR5A1 variants in DSD. Methods: A heterozygous missense variant NR5A1 c.88T>A (p.Cys30Ser) was identified in two patients with DSD, and initial functional characterization of the variant was performed via immunofluorescence analysis, Western Blotting, RNA sequencing, and quantitative real-time PCR analysis. Results: Wildtype NR5A1 protein localized predominantly to the nucleus, whereas the p.Cys30Ser mutant exhibited dual nuclear and cytoplasmic distribution. Compared with the wildtype, the p.Cys30Ser variant altered the expression of 642 genes, with differentially expressed genes primarily enriched in the neuroactive ligand–receptor interaction pathway. The variant impaired the transactivation of canonical NR5A1 downstream targets, resulting in the marked downregulation of 560 genes including key regulators such as KISS1R, CYP11A1, STAR, GABRP, and GRAMD1D. Conclusions: This study is the first to identify and functionally characterize the NR5A1 p.Cys30Ser variant in the context of DSD. Our findings broaden the mutational spectrum of NR5A1-related DSD and provide new insights into the molecular genetic basis of sexual development disorders.