Five novel mutations in steroidogenic factor 1 (SF1, NR5A1) in 46,XY patients with severe underandrogenization but without adrenal insufficiency

Abstract

Steroidogenic factor 1 (SF1, NR5A1) is a nuclear receptor that regulates multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Human mutations in SF1 were initially found in two 46,XY female patients with severe gonadal dysgenesis and primary adrenal failure. However, more recent case reports have suggested that heterozygous mutations in SF1 may also be found in patients with 46,XY partial gonadal dysgenesis and underandrogenization but normal adrenal function. We have analyzed the gene encoding SF1 (NR5A1) in a cohort of 27 patients with 46,XY disorders of sex development (DSD) from the German network of DSD. Heterozygous SF1 mutations were found in 5 out of 27 (18.5%) of cases. Four patients with SF1 mutations presented with the similar phenotype of mild gonadal dysgenesis, severe underandrogenization, and absent Müllerian structures. Of these, two patients harbored missense mutations within the DNA-binding region of SF1 (p.C33S, p.R84H), one patient had a nonsense mutation (p.Y138X) and one patient had a frameshift mutation (c.1277dupT) predicted to disrupt RNA stability or protein function. One additional patient ([c.424_427dupCCCA]+[p.G146A]) displayed a more marked phenotype of severe gonadal dysgenesis, normal female external genitalia, and Müllerian structures. Functional studies of the missense mutants (p.C33S, p.R84H) and of one nonsense mutant (p.Y138X) revealed impaired transcriptional activation of SF1-responsive target genes. To date, adrenal insufficiency has not occurred in any of the patients. Thus, SF1 mutations are a relatively frequent cause of 46,XY DSD in humans.

FIGURE 1

Structure of SF1 including the position of SF1 mutations in 46,XYpatients with disorders of sex development. p.G35E and p.R92Q are the first mutations described in SF1 in 46,XY individuals with gonadal dysgenesis and adrenal failure. p.R255L was found in a normal 46,XX female with adrenal failure.The othermutations have been identified in individuals with 46,XY DSD without adrenal insufficiency.The five novel mutations reported here are boxed. DNA RefSeq NM_004959.3.

FIGURE 2

DNA binding, expression, and cellular localization of SF1. A: Electromobility shift assay showing wild–type (WT) and mutant SF1 binding to a labeled probe corresponding to the 3′ SF1 binding site of the Cyp11a promoter. in-vitro translated empty vector (−) (lane 1) and an excess of unlabeled cold probe (with WT SF1 protein) (lane 7) were used as controls. B:Cellular localization of GFP–SF1 fusion proteins (green), generated and expressed in tsa 201 cells using a pAcGFP–C1 vector. Nuclear counterstaining was performed with DAPI (blue), and images merged to conform nuclear localization.WT SF1 showed strong nuclear localization, with relative nucleolar exclusion and very occasional nuclear subfoci. A similar expression and localization pattern to WT was seen for the p.R84H mutant. Clustering in larger subnuclear foci was seen in the cells transfected with the p.C33S mutant.The p.Y138 Xmutant showed di¡use nuclear localization.

FIGURE 3

A: Effect of the SF1 mutants on transcriptional activity of the minimal promoters of Cyp11a in tsa 201 cells as described in themethods section. B: Effect of the SF1 mutants on synergistic activation of the LH beta promoter bySF1 and Egr1.C,D: Effect of the SF1 mutants on transcriptional activity of the Cyp11a and MIS promoter in TM3 (mouse Leydig) and TM4 (mouse Sertoli) cells, respectively. E: Studies of a potential dominant negative e¡ect ofmutant SF1 were performed by transfecting increasing amounts of wild–type (WT) ormutant SF1 (p.C33S, p.R84H, p.Y138X) expression vector (0,1,2,5,10 ng) with1–ng empty vector (−) or1–ngWT (1) and a Cyp11a promoter reporter (100 ng) in tsa 201cells. Additional empty vector was transfected to keep DNA quantities consistent.Data represent the mean 7± standard error of themean (SEM) of three independent experiments, each performed in triplicate.