ESPE Abstracts

Background: Steroidogenic factor-1 (SF-1), denominated as nuclear receptor subfamily five group A member 1 (NR5A1), is an orphan receptor that regulates several steps of adrenal and gonadal development. Mutations in its gene are responsible for different phenotypes of disorders of sex development (DSD).

Objective and hypotheses: To study the functional impact of two novel NR5A1 mutations, the p.C247* and p.K396Rfs*34, both identified within the ligand-binding domain (LBD).

Method: In order to evaluate the impact of those mutations at the protein function, normal and mutated SF-1 were expressed in HeLa cells and the expression efficiency was monitored using Western blot. Their transactivation abilities were tested in vitro using AMH and STAR promoter containing luciferase reporter genes and electrophoretic mobility shift assays (EMSA).

Results: Luciferase reporter gene expression was reduced for both p.C247* and p.K396Rfs*34 when tested with either promoters. Whereas the transactivation activity for p.K396Rfs*34 was completely null, p.C247* retained a very low activity. Western blot showed that normal and mutant proteins were expressed in similar amounts. EMSA was also performed to analyze if those mutations would disturb SF-1 DNA binding ability. Results showed that the mutation p.K396Rfs*34 abolished the ability to bind DNA, whereas the formation of a protein-DNA complex was still observed for p.C247*.

Conclusion: It is already known that, mutations at SF-1 LBD, may result in variable effects depending on their location and alterations in the ligand specificity/recognition. This was also observed here, once both mutations localized in the LBD had completely different effects on DNA binding. However, both patients present partial gonadal dysgenesis, suggesting that the genotype-phenotype correlation, especially for mutations within the LBD, remains elusive. SF-1 function/regulation is very complex and must be increasingly studied, mainly because the number of different phenotypes correlated with mutations on this gene has been constantly increased.