Pathophysiological mechanism
The NR5A1 mutations identified in DSD-related conditions thus far are all inactivating mutations. The mutational spectrum including sequence variants as well as heterozygous deletions confirms that NR5A1 haploinsufficiency is the main underlying pathogenic mechanism.
NR5A1 encodes a member of the nuclear receptor (NR) transcription factor (TF) family and harbours all the important protein domains present in NRs: a DNA-binding domain, a hinge-region, a ligand-binding domain and two activation functions (AF-1 and AF-2), involved in transcriptional activation and co-factor binding. Additionally, the NR5A1 protein contains the Ftz-F1 domain, which is highly important for DNA-binding specificity. Usually, these TFs exert their function by translocating to the nucleus upon ligand binding. However, NR5A1 is known to be an orphan receptor for which no ligand exists or has yet been identified. Furthermore, NR5A1 is known to bind its targets as a monomer in contrast to other NRs that can only bind after dimerization.
NR5A1 is mainly expressed in the adrenal glands and gonads. It is known to activate expression of nearly all genes encoding the steroidogenic enzymes (the cytochrome P450 steroid hydroxylases (CYPs) and 3β-steroid dehydrogenase (3β-HSD)), as well as genes involved in early sex determination and differentiation (e.g. SOX9, NR0B1, AMH, INSL3). Furthermore, NR5A1 directly affects the hypothalamic-pituitary-gonadal axis by stimulating the Gonadotropin Releasing Hormone Receptor (GnRH) gene and the genes encoding the β chain of LH and FSH.
Heterozygous NR5A1 variants clearly demonstrate that proper dosage is essential for adrenal and gonadal development and functioning. In vitro assays have shown that the identified mutations result in a protein with lowered DNA-binding capacity or with decreased transcriptional activity. It is hypothesized that the resulting phenotype will depend on the affected target genes and residuals transcriptional activity. For instance, when a mutation perturbs the DNA binding of NR5A1 to the promotor region of genes involved in early gonadal determination a more severe phenotype will develop compared to mutations that only affect binding and activation of genes involved in steroidogenesis. Functional validation of some of the identified variants generated more thorough insights in the exact pathogenetic mechanisms and the perturbed pathways. Luciferase assays have already shown that variants resulting in 46,XY DSD fail to upregulate genes of the male specific pathways e.g. SOX9, AMH and CYP11B1.
When the first 46,XX DSD causing NR5A1 variant, giving rise to testicular and ovotesticular DSD was found, it was hypothesized that this variant would have a gain-of-function effect and result in increased activation of the male specific genes. However, this could not be confirmed by luciferase assays. Furthermore, the R92Q variant, previously identified in a 46,XY DSD case, also shows a decreased activation of these male genes and is now also shown to be involved in 46,XX DSD. The recently identified A260V variant also results in increased SOX9 activation.
Additional testing revealed that these variants, besides disrupting the testicular pathways, also show a disruption of the ovarian pathways. During ovarian development, NR5A1 and β-catenin form a complex that upregulates the NR0B1 gene. NR0B1 in its turn is involved in SOX9 repression in female embryo’s. It was hypothesized that the mutation resulting in 46,XX DSD would perturb the action of the NR5A1/β-catenin complex and thus undo the NR0B1-mediated repression of SOX9. Luciferase assays in which the complex and a NR0B1 promoter construct were co-transfected confirmed this hypothesis.
Genotype-phenotype correlations
So far, no strict genotype-phenotype correlations could be made, however some mutations are found in specific conditions and some general trends can be discerned.
- 46,XY DSD and primary adrenal insufficiency (PAI)
To date only two NR5A1 variants have been found in patients with 46,XY DSD and PAI, p.(Gly35Glu) or G35E and p.(Arg92Gln) or R92Q. The G35E variant is a heterozygous de novo variant and affects an amino acid in the DNA-binding domain of the protein, while the R92Q variant was found in a biallelic state and changes the hinge region, more specifically the FtzF1 domain. Both domains are essential for correct binding of the NR5A1 transcription factor to target gene promoter regions and for transcriptional activation.
- Isolated 46,XY DSD
Mutations associated with 46,XY DSD without PAI are spread over the entire coding region of NR5A1 and include frameshift and nonsense variants as well as missense variants that perturb DNA binding and transcriptional activation. These heterozygous mutations are usually de novo, however approximately one third of mutations are maternally inherited. Women carrying these variants are at risk for developing POI. Furthermore, few cases of paternal transmission have been described, these men usually have a history of hypospadias but appear to have preserved fertility. In vitro functional validation for some of these variants shows reduced transcriptional activity. In addition, heterozygous NR5A1 encompassing deletions have been found in 46,XY DSD cases. In general, it is estimated the approximately 15% of 46,XY DSD cases can be explained by underlying NR5A1 mutations.
- 46,XX DSD
Currently, only three different NR5A1 mutations have been associated with 46,XX DSD and remarkably two of them affect the same codon. The first mutation c.274C>T (p.(Arg92Trp), R92W) was reported at approximately the same time by three independent research groups. The second mutation was previously found in patients with 46,XY DSD and PAI, namely the R92Q variant. Recently, a third variant was added to the list: c.779C>T (p.(Ala260Val), A260V.
Diagnostic testing
- Genetic testing
NR5A1 targeted testing (sequencing of the coding region, copy number analysis) or DSD panel testing (e.g. exome-based panel testing). Copy number assessment: array-based methods or shallow whole genome sequencing.
- Biochemical analyses
Measurements of androgen precursors, (dihydro)testosterone, gonadotropin levels, AMH, Inhibin B.