Functional characterization of fiveNR5A1gene mutations found in patients with 46,XY disorders of sex development

Abstract

Steroidogenic factor-1 (SF1), encoded by the NR5A1 gene, is a key regulator of steroidogenesis and reproductive development. NR5A1 mutations described in 46,XY patients with disorders of sex development (DSD) can be associated with a range of conditions of phenotypes; however, the genotype-phenotype correlation remains elusive in many cases. In the present study, we describe the impact of five NR5A1 variants (three novel: p.Arg39Cys, p.Ser32Asn, and p.Lys396Argfs*34; and two previously described: p.Cys65Tyr and p.Cys247*) on protein function, identified in seven patients with 46,XY DSD. In vitro functional analyses demonstrate that NR5A1 mutations impair protein functions and result in the DSD phenotype observed in our patients. Missense mutations in the DNA binding domain and the frameshift mutation p.Lys396Argfs*34 lead to both, markedly affected transactivation assays, and loss of DNA binding, whereas the mutation p.Cys247* retained partial transactivation capacity and the ability to bind a consensus SF1 responsive element. SF1 acts in a dose-dependent manner and regulates a cascade of genes involved in the sex determination and steroidogenesis, but in most cases reported so far, still lead to a sufficient adrenal steroidogenesis and function, just like in our cases, in which heterozygous mutations are associated to 46,XY DSD with intact adrenal steroid biosynthesis.

NovelNR5A1mutations found in Chinese patients with 46,XYdisorders of sex development

“…8 When Ser32 mutates to asparagine, the SF-1 mutant decreases both its binding and transactivational function to target genes. 28 Our study shows that S32N also decreases the transactivation activity of SF-1 to 39%. Asn44 is located between the first and F I G U R E 2 SF-1 protein structure and the location of the heterozygous variants.…”

“…Ser32 is in the first zinc fingers module within the P‐box, which determines DNA sequence recognition . When Ser32 mutates to asparagine, the SF‐1 mutant decreases both its binding and transactivational function to target genes . Our study shows that S32N also decreases the transactivation activity of SF‐1 to 39%.…”

“…The seven patients with 46, XY DSD were shown to have heterozygotic rare NR5A1 variants by targeted next‐generation sequencing, validated by Sanger sequencing, with a detection rate of approximately 12% (7/60) (Figure S1). Four novel variants, including one missense (c.272G>A), two deletion (c.132_134delCAA, c.1083delG) and one insertion (c.763_764insTGCAGCTG) variant, and three recurrent mutations (c.95G>A, c.250C>T, c.691_699dupCTGCAGCTG) were identified. All mutations were distributed in SF‐1 domains, including the DNA‐binding domain, A‐box, the hinge region and the ligand‐binding domain (Figure ).…”

Novel<b><i> NR5A1</i></b> Pathogenic Variants Cause Phenotypic Heterogeneity in 46,XY Disorders of Sex Development

“…Sequencing of the NR5A1 coding region (exon 2-7) revealed a total of 3 pathogenic variants of which 2 were novel: c.64G>A (p.Gly22Ser) and c.428G>A (p.Se-r143Asn).One pathogenic variant, c.95G>A (p.Se-r32Asn), was recently reported in patients with 46,XY DSD [Fabbri-Scallet et al, 2018;Yu et al, 2018]. The 2 pathogenic variants p.Ser32Asn and p.Gly22Ser (encoded by exon 2) identified in family 1 and family 2, respectively, were located in the first zinc finger (DNA recognition helix in the C-terminus) of the DBD.…”

Mutant NR5A1/SF-1 in patients with disorders of sex development shows defective activation of theSOX9TESCO enhancer

“…The precise mechanism of SF-1 action that fails in DSDs is not fully understood, nor has an explanation for the wide-ranging phenotypes associated with SF-1 mutations been determined. Attempts to explain this phenotypic variability have focused on analyzing defective SF-1 function at target promoters of either steroidogenic genes, such as CYP11A1, CYP17A1, and CYP19A1, or sex differentiation genes such as AMH and INSL3 (see Supporting Information for full gene names; Allali et al, 2011;Camats et al, 2012;Fabbri-Scallet et al, 2018;Ito, Achermann, & Jameson, 2000;Kohler et al, 2008;Lin et al, 2007;Philibert et al, 2007;WuQiang et al, 2003). However, no correlation between SF-1 function and patient phenotype has been identified.…”