Endocrine Abstracts

Background: Variants in NR5A1/SF-1 are associated with a wide spectrum of conditions, ranging from primary ovarian insufficiency to 46,XY Partial Gonadal Dysgenesis (PGD) with variable degrees of virilization of the external genitalia. This study aimed to determine the in vitro consequences of two missense NR5A1 variants identified in three 46,XY partial gonadal dysgenesis individuals.

Methods: The c.76G>C, p.Gly26Arg variant was found in two siblings. The proband presented female external genitalia with discrete clitoromegaly and palpable testis in the inguinal region, whereas her brother presented micropenis, penoscrotal hypospadias and testis located in the scrotal region. The c.232A>T, p.Met78Leu variant was identified in a 46,XY PGD patient with micropenis, penoscrotal hypospadias and testis located in the scrotal region. Whole-exome sequencing (WES) was performed in the proband’s sibling to identify other genetic variants that could explain the phenotype disparity. Site-directed mutagenesis was performed to insert the variants of interest within NR5A1 cDNA contained on the expression vector pMyc-SF1. Transactivation capacity of WT and mutant NR5A1 were compared using human AMH or STAR promoter containing reporter genes in transiently transfected HEK-293T cells.

Results: WES performed in the proband’s sibling did not identify additional variants in known DSD-associated genes. Luciferase Assay results indicated a reduction of approximately 50% of SF-1 transcriptional activity on AMH promoter region for the p.Met78Leu variant (P < 0.05) and no statistical significance on the STAR promoter, while the p.Gly26Arg variant indicated a near null transcriptional activity (P < 0.05) in both promoters.

Conclusions: Both variants were found to significantly reduce NR5A1 transcriptional activity. As p.Gly26Arg is located in the DNA-binding domain (DBD), which is essential for protein DNA-binding capability, a more severe outcome was expected. Additionally, our negative WES result demonstrates a classical example of phenotypic heterogeneity due to a same NR5A1 variant, extensively documented in the literature. Further in vitro studies, such as Western blot analysis, are currently being performed to determine the consequences of both variants on protein expression. Therefore, these findings support the pathogenic role of these variants in gonadal development, while reinforcing the importance of functional assays for the interpretation of NR5A1 variants in patients with 46,XY PGD.