Pathogenic mutations disrupt stress granules assembly in patients with DDX3X neurodevelopmental disorder

Abstract DDX3X neurodevelopmental disorder (DDX3X-NDD) represents a recently identified genetic syndrome characterized by intellectual disability (ID) and developmental delays, primarily caused by pathogenic variants in the DDX3X gene. The physiological ramifications of these mutations remain largely unexplored. In this study, we reported 21 DDX3X variants from 22 Chinese patients with DDX3X-NDD by whole exome sequencing. We selected five variants for further functional analyses, including two previously reported by our group. Three frameshift variants (c.280_281dup p.R95Efs*127, c.669_670del p.A224Pfs*70, and c.1579del p.H527Ifs*9) resulted in either the loss of DDX3X protein or the production of truncated proteins. Additionally, two missense variants (c.1051C > G p.R351G and c.1501G > A p.A501T) significantly reduced DDX3X protein expression. Notably, variants DDX3X-R95Efs*127 and DDX3X-A224Pfs*70 triggered marked apoptosis induction and failed to form stress granules in HEK293T cells compared to wild-type DDX3X. This defect may stem from their inability to interact with the stress particle marker PABPC1, as evidenced by co-immunoprecipitation assays. Moreover, DDX3X-H527Ifs*9 and DDX3X-R351G variants were found to disrupt the cell cycle, extending the S phase relative to the wild type. Collectively, our findings provide mechanistic insights into the pathogenic consequences of DDX3X-NDD associated mutations, suggesting that the loss-of-function variants of DDX3X lack a context-dependent survival advantage, potentially contributing to the pathology of this syndrome.