NR5A1 gene variants repress the ovarian-specific WNT signaling pathway in 46,XX disorders of sex development patients

Abstract

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.

Keywords: 46,XX ovotesticular DSD; 46,XX testicular DSD; NR5A1; SF1; disorders of sex development.

Figure 1

Variants in NR5A1 identified in a cohort of individuals with 46,XX DSD. (a) Schematic representation of the predicted protein structure of NR5A1 showing the approximate location of the two variants identified in a cohort of individuals with 46,XX DSD. The protein domains are as follows: DNA binding domain (DBD) containing two zinc finger motifs (Zn) and the Fushi‐tarazu factor 1 box (Ftz‐F1), the hinge region, and ligand binding domain (LBD). P‐Box, T‐box, A‐box, as well as two activational domains—AF1 and AF2. (b) Evolutionary conservation of the NR5A1 protein sequence around the two missense variants identified in our cohort

Figure 2

Protein conformation and cellular expression. (a) To investigate the potential effect of the variants on protein conformation, we performed an in silico prediction with the wild‐type NR5A1 and both variants using I‐Tasser and PyMol modeling. (a: i and ii) Wild‐type arginine (Arg, R) at position 92 falls within the DNA binding domain of the protein. The mutant tryptophan (Trp, W) is larger than the arginine and has less hydrogen bonding potential. (a: iii and iv) The residue at position 260 falls within alpha helix 3 of the ligand binding domain. The wild‐type alanine (Ala, A) is smaller than the mutant valine (val, V), this is located on the protein surface. (b) Protein expression of both variant and wild‐type NR5A1 was assessed in COS‐7 cells with an NR5A1 antibody (green). Cells were transfected with an equal amount of NR5A1 expression vector (wild‐type or variant). Nuclear counterstaining was performed with DAPI (blue). Wild‐type NR5A1 showed strong nuclear staining with nucleolar exclusions (b: i and ii). The variant NR5A1 protein expression and localization was unaffected (b: iii–vi)

Figure 3

NR5A1 mutants show altered function in luciferase assays using sex differentiation‐specific reporters. (a) Both NR5A1 variants as well as a positive control variant (loss of function from 46,XY DSD) show decreased transactivation of SOX9 mTESCO when co‐transfected into COS‐7 cells with SOX9. This is also observed when the female pathway SOX9 repressor, FOXL2, is also transfected. (b) Co‐transfection of HEK 293‐T cells with NR5A1, SOX9, and increasing concentrations of NR0B1 showed that mutant NR5A1 does not affect NR0B1‐mediated repression of SOX9. SOX9 activity was measured using the TESCO reporter. (c) Co‐transfection of COS‐7 cells with wild‐type or mutant NR5A1 shows no change in activity of the NR0B1 promoter for both NR5A1 mutants (d) Co‐transfection of COS‐7 cells with wild‐type or mutant NR5A1 and β‐catenin results in repression of the NR0B1 promoter for both NR5A1 mutants. (e) TOPFlash activation is reduced when HEK 293‐T cells are transfected with β‐catenin and mutant NR5A1 compared to wild‐type NR5A1. Data represent the mean with the standard error of three independent experiments performed in triplicate. An unpaired t‐test was applied to obtain P‐values, ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns = P > 0.05