Mutations in MAP3K1 cause 46,XY disorders of sex development and implicate a common signal transduction pathway in human testis determination

Abstract

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.

Figure 1

Pedigrees from Two Families of Interest Exhibiting Sex-Limited Autosomal-Dominant Mendelian Inheritance of 46,XY DSD Shading indicates that the individual has 46,XY DSD or 46,XY complete gonadal dysgenesis. Circles indicate female sex of rearing, and squares indicate male sex of rearing. Asterisks (^∗) indicate individuals who were tested and found to have the c.IVS2-8T>A mutation. Plus signs (+) indicate individuals who were tested and found to have the p.Gly616Arg mutation. A strikethrough indicates a deceased individual.

Figure 2

Whole-Mount In Situ Hybridization of Wild-Type Embryonic Gonads Reveals Strong Expression of Map3k1 at the Sex-Determining Stage of Mouse Gonad Development (A and B) Whole-mount staining of 13.5 dpc ovary (A) and testis (B). Expression is detected in the mesonephric tubules, Wolffian duct, and Müllerian duct of both sexes and in the testis cords in males. (C) Negative control with sense RNA probe used. (D) Longitudinal section through 13.5 dpc testis after Map3k1 whole-mount in situ hybridization (WMISH). The staining is detected throughout the testis cords, and the smooth edge of this profile (arrowheads in inset showing high magnification image of cord in hatched box) indicates expression in Sertoli cells that abut the basement membrane. (E and F) Whole-mount expression analysis of 11.5 dpc female (E) and male (F) gonads. The staining is prominent throughout the gonads of both sexes and is detected in the mesonephric tubules and associated Wolffian duct. (G) Negative control with sense RNA probe used. (H) Section of gonad shown in (B) (at level indicated by dashed line). Staining is detected throughout the gonad (g), in contrast to negligible staining in the adjacent mesonephric mesenchyme (m), but the signal is highest in the coelomic epithelium and subepithelial mesenchyme. The dashed line in (G) indicates the boundary between gonad and mesonephros. Abbreviations are as follows: WD, Wolffian duct; MD, Müllerian duct; MT, mesonephric tubules; CE, coelomic epithelium; t, testis; m, mesonephros.

Figure 3

Mutations in MAP3K1 in Patients with 46,XY DSD (A) Schematic of MAP3K1 structure and mutation locations. Sequencing chromatograms of four mutations identified an amino acid conservation taken from the UCSC Genome Browser. Alignment 1: invariant conservation of amino acids in ten species in the region of exon 2 where sporadic mutation 1 and sporadic mutation 2 lie. Alignment 2: the mutation in family 2 lies in a less highly conserved region. (B) SYBR-green quantitative PCR dissociation curves. Control samples exhibited one form of cDNA with a single dissociation curve peak, whereas samples from family 1 exhibited heteroduplex and homoduplex cDNAs with two dissociation curve peaks indicating the presence of the mutant and wild-type forms. (C) Chromatogram of MAP3K1 cDNA demonstrating insertion of six nucleotides in-frame into the mutant mRNA (NM_005921.1:c.633_634insAATCAG) and two amino acid residues (p.Val211_Val212insIleGln) into the mutant protein.

Figure 4

Effects of MAP3K1 Mutations on Phosphorylation of Downstream Targets and Binding of RHOA (A) Dysregulation of MAPK signaling activity by MAP3K1 mutations. Immunoblot analysis of cell lysates from human lymphoblastoid cell lines cultured in suspension in RPMI medium without serum supplemented with 1% penicillin and streptomycin for 24 hr, then refed serum for 1 hr. The blots were dual-hybridized overnight at 4°C with primary antibodies against phosphorylated and total p38 and ERK1/2 (both at 1:2000) and detected with the use of infrared dye-conjugated secondary antibodies (1:5000). The cell lines are designated as “WT” for wild-type or by the specific mutation and affected individual in family 1. Infrared absorbance detects phosphorylated signal at 680 nm (red) and total target activities at 800 nm (green). The histone H3 loading controls are also shown. (B) Phosphorylated (phospho) p38 and ERK intensities normalized to histone H3 loading control intensity. The normalized intensities are indicated on the y axis, and the cell lines are indicated on the x axis. A box and whisker plot of p.Leu189Arg and p.Leu189Pro was represented by five measurements, whereas the p.Val211_Val212insIleGln and WT plots were represented by 15 and 25 measurements, respectively. The bottom and top whisker hash lines indicate the minimum and maximum values, the box length represents the interquartile range, and the dark horizontal line indicates the median of the data. The p values were determined by comparing the mutants to WT controls for each set of mutations with the use of the Student's t-test. Comparison of all mutant lines to all WT lines is denoted. (C) Increased binding of RHOA to mutant MAP3K1. Proteins were immunoprecipitated with MAP3K1 antibody from lysates collected from mutant (p.Val211_Val212insIleGln) and wild-type cell lines and were probed on immunoblots with the RHOA . Upper panel: Mutant (p.Val211_Val212insIleGln) and wild-type cells were seeded in triplicates of 3 million cells. Triplicates were pooled and immunoprecipitated for each cell line. RHOA binding to MAP3K1 complexes in the wild-type (lane 1) was enhanced by the presence of the p.Val211_Val212insIleGln mutation in MAP3K1 (lane 2). Lower panel: Equal loading of MAP3K1 complexes in each lane. (D) Increased binding of mutant MAP3K1 to RHOA detected by immunoprecipitation. Mutant (p.Val211_Val212insIleGln, p.Leu189Pro, and p.Leu189Arg) and two wild-type cell line lysates were pooled from three independent experiments, totaling ∼9 million cells per experiment, and pulled down (immunoprecipitated) with the use of anti-RHOA, then probed for MAP3K1 on immunoblots and normalized to the histone bands. Upper panel: MAP3K1 binding was enhanced by the presence of the p.Val211_Val212insIleGln, p.Leu189Arg, and p.Leu189Pro mutations in MAP3K1. Lower panel: Histone loading control of RHOA complexes in each lane. (E) MAP3K1 intensities normalized to loading control intensity. The normalized intensities are indicated on the y axis, and the five cell lines are indicated on the x axis. A box and whisker plot of p.Leu189Arg and p.Leu189Pro was represented by five measurements, whereas the p.Val211_Val212insIleGln and WT plots were represented by 15 and 25 measurements, respectively. The p values were determined by comparing the mutants to WT controls for each mutation set with the use of the Student's t-test. Comparison of all mutant lines to all WT lines is denoted.