Mutation analysis of NR5A1 encoding steroidogenic factor 1 in 77 patients with 46, XY disorders of sex development (DSD) including hypospadias

Abstract

Background: Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias.

Methodology/principal findings: We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein.

Conclusions/significance: Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations.

Figure 1. Mutations in NR5A1 associated with 46,XY DSD.

(A). Distribution of NR5A1 mutations in relation to the functional domains of the protein. The DNA-binding domain (DBD) containing two zinc-finger motifs is indicated. The FtzF1 box stabilizes protein binding to DNA. The hinge region is important for stabilizing the ligand-binding domain and interacts with other proteins that control NR5A1 transcriptional activity. The AF2 domain recruits cofactors necessary for NR5A1 transactivating activity. (B) The sequence alignment of the distal portion of the hinge and the ligand-binding domain (LBD) of human NR5A1 protein as compared with those of other mammals. 1 to 10 of the predicted alpha helixes in the ligand-binding domain of NR5A1 are indicated as solid boxes, and corresponding amino acids in bold text. The position of the p.Trp279Arg and p.Arg313Cys are highlighted. The mutations fall either in the highly conserved Helix 3 (p.Trp279Arg) or in Helix 5 (p.Arg313Cys) of the ligand-binding domain. The mutation c.390delG was described previously . (C) Evolutionary conservation of DNA binding domain of NR5A1. Sequence alignment of the evolutionary conserved DNA-binding domain of NR5A1 showing the position of the two zinc finger domains and the p.Arg39Pro mutation.

Figure 2. Plasma AMH and inhibin B concentrations in each patient group.

In each AMH graph the broken lines correspond to the upper and lower limits of the normal range . For inhibin B, the solid line corresponds to the median and the broken lines to the 5^th and 95^th percentiles , . The red asterisk indicates individuals carrying an NR5A1 heterozygote mutation and the numbers indicate the patients described in table 1.

Figure 3. Assays of NR5A1 transactivation activity.

The transcriptional activity of wild-type (WT) NR5A1 and mutant p.Gly35Glu (control) , p.Arg39Pro and p.Arg313Cys were studied using the human AMH promoter in HEK293-T cells. The human AMH reporter construct was transfected into HEK293-T cells with either the wild type or mutant NR5A1 expression vectors. Mutants p.Arg39Pro and p.Arg313Cys exhibited a dramatic reduction in transactivation activity (blue bars). The human AMH reporter construct was transfected into HEK293-T cells with either the wild type or mutant NR5A1 expression vector in the absence (blue) or presence (red) of the GATA4 expression vector. Data represent the mean±SEM of three independent experiments, each performed in triplicate. Results are expressed as fold activity of the empty vector activity.

Figure 4. Far Western blot analysis of the interaction between in-vitro translated wild type GATA4 and wild-type NR5A1, NR5A1p.Arg39Pro and p.Arg313Cys.

Blots containing wild-type NR5A1, NR5A1p.Arg39Pro and NR5A1p.Arg313Cys proteins were incubated with GATA4 protein and probed by anti-GATA4 antibody. Both the wild-type and mutant NR5A1 proteins can bind to GATA4 protein.