A recurrent p.Arg92Trp variant in steroidogenic factor-1 (NR5A1) can act as a molecular switch in human sex development

Abstract

Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.

Figure 1.

Identification and modelling of the NR5A1 p.Arg92Trp variant. (A) Shows the pedigrees of the four families with the affected members shaded. Squares denote individuals brought up as boys and circles denote individuals brought up as girls, with respective karyotypes shown below. (B) Shows testicular histology from individual 4.2 with 46,XX testicular DSD. (C) Shows sequence chromatograms of the wild-type and p.Arg92Trp NR5A1. (D) Shows the sequence alignment of the A-box motif in selected vertebrae, together with a schematic representation of the NR5A1 protein. (E) Shows the model of wild-type NR5A1 and p.Arg92Trp DNA-binding domain in complex with DNA. The RGGR motif of the A-box interacts with the minor groove of DNA. The basic arginine residue (Arg, R) in wild-type NR5A1 (left panel) is replaced by a neutral tryptophan residue (Trp, W) with a bulky indole side chain (right panel).

Figure 2.

Expression, functional analyses and model of NR5A1 p.Arg92Trp acting as a cell fate switch. (A) Shows expression of NR5A1 in somatic cells of the developing ovary with germ cells shown by POU5F1 (OCT4) (9 wpc) (upper panel). Negative controls without primary antibody are shown below. Nuclei are stained blue with DAPI. Scale bar denotes 50 µm. (B) Demonstrates that the mutant NR5A1 p.Arg92Trp lacks the ability to bind to a known consensus target sequence. (C) Shows a reduced ability of the NR5A1 mutant protein to activate reporter activity using the Tesco enhancer element as a target in HEK 293-T cells. (D) HEK 293-T cells were transfected with multimerized TCF/LEF1 binding sites containing the luciferase reporter construct (TOPFlash). Co-transfection with the vectors pCSβ-catenin with pCMX-NR5A1 WT, pCMX-NR5A1p.Arg92Gln or pCMX-NR5A1p.Arg92Trp showed that in contrast to both the NR5A1 WT and NR5A1 p.Arg92Gln proteins the NR5A1 p.Arg92Trp showed a reduced ability to synergise with β-catenin to up-regulate reporter gene activity. Similar results were obtained using KGN cells (data not shown). All results are expressed as mean +/- S.D. of at least three experiments. (E) shows a potential model based on the in vitro data. Left: in a 46,XY individual NR5A1 synergises with SRY to upregulate SOX9 expression leading to testis formation. Left centre: in 46,XX individuals, NR5A1 syngerises with β-catenin to upregulate the expression of anti-testis genes (e.g. DAX-1/NR0B1). Right centre: In the 46,XY DSD case (4.1) the p.Arg92Trp mutant shows a reduced ability to up-regulate SOX9 gene expression leading to a lack of testis formation. Right: In a 46,XX child with TDSD/OTDSD, the same mutant shows reduced ability to synergise with β-catenin to up-regulate the expression of anti-testis genes. As a consequence of this lack of repression, the expression of pro-testis genes (e.g. SOX9) leads to testis formation.