Di Mao, Lele Li, Lijun Fan, Yueni Zhang, Chunxiu Gong
No clear genotype-phenotype correlation was identified. Functional assays alone could not explain the wide phenotypic variability observed in individuals carrying novel NR5A1 variants; however, variants located within the A-box or AF-2 domains appeared to display greater functional tolerance.
INTRODUCTION: NR5A1 variants are a common cause of 46,XY disorders of sex development (DSD) and are associated with a wide spectrum of clinical phenotypes. However, genotype-phenotype correlations and the underlying pathogenic mechanisms remain incompletely understood. In this study, we characterized 27 novel NR5A1 variants identified in Chinese patients with 46,XY DSD and investigate their pathogenic mechanisms.
METHODS: In silico tools were used to predict the functional impact of novel NR5A1 variants. Functional analyses were performed in HEK293 cells. Protein level of coding missense variants was assessed by Western blotting, subcellular localization was examined by immunofluorescence, and transcriptional activity was evaluated using dual-luciferase reporter assays with pGL4.11-CYP11A1 and pGL4.11-CYP17A1 promoters.
RESULTS: 27 patients carrying novel NR5A1 variants exhibited a broad phenotypic spectrum, with external masculinization scores ranging from 0 to 9. Among the 27 variants, missense variants were the most frequent (9/27, 33.3%). Functional validation was performed for the 9 missense variants. Western blot analysis showed markedly reduced protein level for variants located in the DBD, A-box, and LBD, whereas variants within the AF-2 domain showed increased protein level. Subcellular localization analysis demonstrated preserved nuclear localization for all variants. Transcriptional assays revealed that variants in the DBD and LBD generally exhibited reduced transactivation activity, while variants in other domains showed greater variability in transcriptional activity.
CONCLUSION: No clear genotype-phenotype correlation was identified. Functional assays alone could not explain the wide phenotypic variability observed in individuals carrying novel NR5A1 variants; however, variants located within the A-box or AF-2 domains appeared to display greater functional tolerance.