Abstract
Background: 46,XY disorders/differences of sex development (46,XY DSD) are congenital conditions that result from abnormal gonadal development (gonadal dysgenesis) or abnormalities in androgen synthesis or action. During early embryonic development, several genes are involved in regulating the initiation and maintenance of testicular or ovarian-specific pathways. Recent reports have shown that MAP3K1 genes mediate the development of the 46,XY DSD, which present as complete or partial gonadal dysgenesis. Previous functional studies have demonstrated that some MAP3K1 variants result in the gain of protein function. However, data on possible mechanisms of MAP3K1 genes in modulating protein functions remain scant. Methods: This study identified a Han Chinese family with the 46,XY DSD. To assess the history and clinical manifestations for the 46,XY DSD patients, the physical, operational, ultra-sonographical, pathological, and other examinations were performed for family members. Variant analysis was conducted using both trio whole-exome sequencing (trio WES) and Sanger sequencing. On the other hand, we generated transiently transfected testicular teratoma cells (NT2/D1) and ovary-derived granular cells (KGN), with mutant or wild-type MAP3K1 gene. We then performed functional assays such as determination of steady-state levels of gender related factors, protein interaction and luciferase assay system. Results: Two affected siblings were diagnosed with 46,XY DSD. Our analysis showed a missense c.556A > G/p.R186G variant in the MAP3K1 gene. Functional assays demonstrated that the MAP3K1R186G variant was associated with significantly decreased affinity to ubiquitin (Ub; 43–49%) and increased affinity to RhoA, which was 3.19 ± 0.18 fold, compared to MAP3K1. The MAP3K1R186G led to hyperphosphorylation of p38 and GSK3β, and promoted hyperactivation of the Wnt4/β-catenin signaling. In addition, there was increased recruitment of β-catenin into the nucleus, which enhanced the expression of pro-ovarian transcription factor FOXL2 gene, thus contributing to the 46,XY DSD. Conclusion: Our study identified a missense MAP3K1 variant associated with 46,XY DSD. We demonstrated that MAP3K1R186G variant enhances binding to the RhoA and improves its own stability, resulting in the activation of the Wnt4/β-catenin/FOXL2 pathway. Taken together, these findings provide novel insights into the molecular mechanisms of 46,XY DSD and promotes better clinical evaluation.
Highlights
Disorders/differences of sex development (DSD) are a set of congenital conditions, characterized by atypical development in chromosomal, gonadal, or other anatomical sex characteristics (Lee et al, 2006; Ono and Harley, 2013)
Stunted uterine-like tissue was observed during the operation, but no obvious abnormalities were found in the bilateral fallopian tubes
The MAP3K1 missense variant was predicted to be deleterious by all five predictors (PredictSNP, polyphen1, polyphen2, SIFT, and SNAP), with a high confidence score of 0.65, 0.59, 0.60, 0.79, and 0.81, respectively (Supplementary Figure S3). These findings suggested that the c.556A > G (p.R186G) variant in MAP3K1 gene might be pathogenic
Summary
Disorders/differences of sex development (DSD) are a set of congenital conditions, characterized by atypical development in chromosomal, gonadal, or other anatomical sex characteristics (Lee et al, 2006; Ono and Harley, 2013). The overall incidence of genitally ambiguous patients at birth remains undefined. Wholeexome sequencing has become the mainstay clinical test for analysis of known or novel genes associated with DSD, only 13–20% of DSD patients are estimated to have an accurate clinical genetic diagnosis (Lee et al, 2006; Eggers et al, 2016). Out of the total tests, only 35–43% resulted in identification of a specific pathogenic gene (Baxter et al, 2015; Eggers et al, 2016), suggesting that the causes of 46,XY DSD remain undefined. Accurate genetic diagnosis of the 46,XY DSD patients remains crucial for early treatment and prediction of associated risks of malignant tumors
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