NR5A1 is a novel disease gene for 46,XX testicular and ovotesticular disorders of sex development

Abstract

Purpose: We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD).

Methods: Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of mutation were investigated using luciferase assays, localization studies, and RNA-seq.

Results: We identified a novel heterozygous NR5A1 mutation, c.274C>T p.(Arg92Trp), in three unrelated patients. The Arg92 residue is highly conserved and located in the Ftz-F1 region, probably involved in DNA-binding specificity and stability. There were no consistent changes in transcriptional activation or subcellular localization. Transcriptomics in patient-derived lymphocytes showed upregulation of MAMLD1, a direct NR5A1 target previously associated with 46,XY DSD. In gonads of affected individuals, ovarian FOXL2 and testicular SRY-independent SOX9 expression observed.

Conclusions: We propose NR5A1, previously associated with 46,XY DSD and 46,XX primary ovarian insufficiency, as a novel gene for 46,XX (ovo)testicular DSD. We hypothesize that p.(Arg92Trp) results in decreased inhibition of the male developmental pathway through downregulation of female antitestis genes, thereby tipping the balance toward testicular differentiation in 46,XX individuals. In conclusion, our study supports a role for NR5A1 in testis differentiation in the XX gonad.Genet Med 19 4, 367-376.

Figure 1

Pedigrees and haplotypes of mutation-positive families and schematic representation of the NR5A1 protein, mutation location, and conservation. (a) Pedigrees for families 1–3. Affected individuals heterozygous for NR5A1 mutation c.274C>T p.(Arg92Trp) are indicated by filled symbols. Asymptomatic individuals with NR5A1 mutation p.(Arg92Trp) are represented by a black spot in the symbol. In total, 29 microsatellite markers were used to assess the haplotypes; only the ones building up the common haplotype and flanking markers are shown here. The common haplotype (in red box) shared by the three mutation carriers has a maximum size of 1.5 Mb. (b) The different domains found in NR5A1, a DNA-binding domain at the C-terminus, followed by the Ftz-F1 region, which is important for DNA-binding stability, a hinge region, and a ligand-binding domain. An electropherogram showing the c.274C>T substitution is depicted below. The protein alignment of p.(Arg92Trp) and its surrounding amino acids is shown on top. The affected amino acid and the surrounding region are conserved up to zebrafish.

Figure 2

Structural analysis of NR5A1 (PDB code 2FF0). Position of Arg92 (R92) in the wild-type NR5A1 DNA-binding domain (DBD). (a) R92 is part of the C-terminal A-box and its side chain fits in the minor groove, making a hydrogen bond with a deoxycytosine base. Models of the p.(Arg92Trp) (Trp=W) (b) and p.(Arg92Gln) (Gln=Q) (c) mutants show how this mutated side chain binds in the DNA minor groove without hydrogen bond formation to the DNA. The affected amino acid R92 is located in a region C-terminal to the classical DBD with its two zinc (Zn) fingers. This additional domain is found in family members of NR5A1, including NGFI-B, and was called the A-box or Ftz-F1 box. This A-box domain has been implicated in interactions with the minor groove of the target DNA. Prior to structure determination, binding studies with NR5A1 and NGFI-B mutants showed that this interaction is important for their binding specificity toward different DNA targets. For NR5A1, this specificity is determined by amino acids 91–93, which are predicted to interact with three specific basepairs 5ʹ of the classical half-site bound by the Zn fingers. The results of this study are in line with those for the later NMR structure of the NR5A1 DBD bound to a DNA fragment of the inhibin-alpha subunit promoter. In this structure, the side chain of R92 inserts deeply into the minor groove, and its guanidine head group binds to two of the three basepairs at the 5ʹ of classical half-sites. Mutation R92A abolished DNA-binding and signaling via the inhibin-alpha subunit promoter in this study by removing the interaction with the basepairs of the minor groove. The p.(Arg92Trp) mutation is not allowed in the structure without gross structural rearrangements; the W side chain cannot insert into the minor groove and cannot be accommodated in the current DNA-bound structure without inducing steric clashes.

Figure 3

Immunohistochemistry on gonadal biopsies. (a) Case 1. Testis. Supportive cells have differentiated as Sertoli cells as indicated by SOX9 positivity. SOX9 staining, 200×. (b) Case 1. Scarce germ cells (stained in brown) populate the testis tubules. DDX4 staining, 200×. (c) Case 2. Ovotestis. Sertoli cells in the testicular component (right) express SOX9; no SOX9 expression is seen in the ovarian part (left). SOX9 staining, 200×. (d) Same gonad. Ovarian granulosa cells but not testicular Sertoli cells display FOXL2 positivity. FOXL2 staining, 200×. (e,f) Germ cells (ova, black arrows) are only present in the ovarian and not in the testicular part. DDX4 staining, 200×. (g) Case 3. In this patient, bilateral infantile testes with a Sertoli cell–only pattern were observed. All Sertoli cells are SOX9-positive. SOX9 staining, 200×. (h) Same gonad. FOXL2 staining is negative. FOXL2, 200×. (i) Case 3. DDX4 staining indicates absence of germ cells, thus confirming the Sertoli cell–only pattern. DDX4 staining, 200×.

Figure 4

Schematic overview of the sex development gene regulation network. Blue: testis-promoting activity of NR5A1 (ref. 1). Orange: counteractive connections to suppress the opposite pathway. Green: ovary-promoting activity of NR5A1 as hypothesized here and supported by other works.^, (a) General scheme: NR5A1 is known to initiate the male developmental pathway through upregulation of SOX9 (synergistically with SRY). SOX9 then maintains its own expression via Fgf9 signaling. In the female embryo, in the absence of SRY, NR5A1 induces WNT4 and RSPO1 expression as shown by Combes, leading to upregulation of FOXL2 and other ovary-specific genes. High FOXL2 expression results in stable repression of SOX9 and, possibly, NR5A1, and hence the male pathway. (b) Possible mechanism by which c.274C<T leads to ovotesticular disorders of sex development. We hypothesize that this novel mutation affects the activation of female-specific genes such as WNT4/β-catenin (as indicated by the dotted lines), leading to decreased FOXL2 expression. In this way, FOXL2 can no longer prosecute its pro-ovarian functions and, at the same time, male-promoting genes escape firm suppression, ultimately resulting in NR5A1-mediated and/or independent SOX9 upregulation and enhancement of testicular differentiation.