FUNCTIONAL CHARACTERIZATION OF TWO MUTATIONS (R313H and S303R) IN THE NR5A1 (SF1) GENE IN TWO PATIENTS WITH 46,XY DISORDERS OF SEX DEVELOPMENT (DSD)

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 586-595-Reproductive Axis Determination, Development & Transgender Medicine
Basic/Clinical
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-586
Natalia Perez Garrido*, Nora Isabel Saraco, Roxana Marino, Pablo Ramirez, Marta Ciaccio, Mariana Costanzo, Gabriela Guercio, Diana Monica Warman, Valeria De Dona, Marco A Rivarola and Alicia Belgorosky
Hospital de Pediatria Garrahan, Buenos Aires, Argentina
The steroidogenic factor 1 (SF1/NR5A1) plays a key role in the regulation of adrenal and reproductive development. In humans, NR5A1 mutations are associated with a wide phenotypic spectrum. Two patients with 46, XY familial DSD were evaluated. Both had severe hypospadias at birth, elevated serum FSH, low AMH, normal steroidogenic response to hCG stimulation and evidence of Müllerian structures. The NR5A1 gene molecular study revealed the mutation c.938G> A (R313H) in the first family and a novel mutation, c.909G> A (S303R) in the second family. Both mutations are located in the highly conserved helix 5 of the ligand binding domain of the protein (LBD). In silico analysis (SIFT; Polyphen) indicated that both amino acid changes are predicted to affect protein function. The aim of our study was to functionally characterize the two mutations (R313H and S303R) in the NR5A1 gene. Expression vector containing human wild type (WT) SF1 cDNA was constructed in the pcDNA3 vector (p-SF1wt). Expression vectors containing each mutant (p-R313H and p-S303R) were generated by site-directed mutagenesis using p-SF1wt as a template. Each expression vector (p-SF1wt, p-R313H or p-S303R) was co-transfected into SMAT1 cell line with reporter plasmid containing the hAMH promoter and into Y1 cell line with reporter plasmid containing the h3βHSDII promoter. Luciferase activity was evaluated by Dual Luciferase Reporter Assay System (Promega) using Renilla luciferase as transfection control. Results are presented as fold activity of the empty vector activity. Each assay was repeated three times each one in triplicate. In SMAT1 cells, hAMH promoter luciferase activity increased significantly in the presence of p-SF1wt (3.00 ± 0.09, mean ± SEM, p <0.05 ANOVA) while mutants R313H and S303R significantly decreased luciferase activity compared to p-SF1wt (1.32 ± 0.05 and 1.09 ± 0.01 respectively, mean±SEM, p <0.05 ANOVA). Similar results were obtained in Y1 cells (h3βHSDII promoter) with p-SF1wt (3.16 ± 0.02, mean ± SEM, p <0.05 ANOVA) and mutants p-R313H and p-S303R (2.35 ± 0.08 and 2.44 ± 0.04 respectively, mean ± SEM, p <0.05 vs SF1wt, ANOVA). The effect of both mutations in reducing SF1 activity was more evident on hAMH promoter in SMAT1 cells than on h3βHSDII promoter in Y1 cells. These results confirm that both mutations cause a decrease in the action of SF1 and could explain the clinical phenotypes found in these DSD patients.

Nothing to Disclose: NP, NIS, RM, PR, MC, MC, GG, DMW, VD, MAR, AB

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Supported by FONCYT, PICT-2008-0347,Argentina
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