IMPE Abstracts

Background: Steroidogenic factor 1 (SF-1/NR5A1) is a nuclear receptor that regulates sex development, steroidogenesis and reproduction. Genetic variants in SF-1/NR5A1 are common among differences of sex development (DSD) and associate with a wide range of phenotypes, but their pathogenic mechanisms remain unclear. We aimed at characterizing novel missense, likely disease-causing SF-1/NR5A1 variants in a large cohort of DSD individuals using cell-based methods.

Methods: Within the framework of the SF1next study, we identified novel SF-1/NR5A1 variants using the databases ClinVar and HGMD. We used different in-silico tools to predict the impact of SF-1/NR5A1 variants on protein function and classified them according to American College of Medical Genetics and Genomics (ACMG) guidelines for pathogenicity. We then searched the literature for the best functional studies to investigate the pathogenic effect of the novel variants in a classic cell culture model. We generated SF-1/NR5A1 expression vectors containing the novel missense variants by site-directed mutagenesis using specific primers and performed functional assays with the promoter luciferase reporter vector -152CYP11A1_pGL3 in non-steroidogenic HEK293T cells. The study is ongoing, we are performing localization studies for nuclear shift of wild-type (WT) vs mutant SF-1/NR5A1 proteins by Western blot (WB) of nuclear and cytoplasmic lysates with the corresponding antibodies.

Results: In total, the SF1next cohort (https://www.dropbox.com/s/mpxzw9rhueewf8s/SF1next%20study%20group_IMPE.pdf?dl=0) included 115 individuals with SF-1/NR5A1 variants. We identified 84 different SF-1/NR5A1 variants, of which 28 were novel. Literature review showed that SF-1/NR5A1 variants located in the DNA binding domain (DBD) had the most significant pathogenic effect, based on gene reporter transactivation assays in different cell lines, as well as WB analyses showing impaired nuclear localization. Variants located elsewhere showed diverse results. From our novel 28 SF-1/NR5A1 variants, we selected 15 missense variants, of which 9 were located in the DBD and another 6 were in the ligand binding domain (LBD) or at the C-terminus. Our cell-based studies showed that among the 9 DBD variants, only 2 had impaired activity on the CYP11A1 promoter, while the rest had similar activity compared to the WT. As expected, variants located in the LBD and C-terminus showed diverse results.

Conclusions: Novel SF-1/NR5A1variants are still being found. Our results highlight the limitations of the classic cell-based studies for assessing the pathogenicity of the SF-1/NR5A1 variants, and additional different methods and models are needed to characterize the disease mechanism in these patients; for instance, ex vivo models and methods with reprogrammed donor-specific human induced stem cells.