Partial loss of USP9X function leads to a male neurodevelopmental and behavioural disorder converging on TGFβ signalling

Abstract Background The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders (NDDs) primarily in females. USP9X escapes X-inactivation, and in females de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognisable syndrome with intellectual disability (ID), signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male NDDs remains tentative. Methods We collected and interrogated the pathogenicity of 44 male-ascertained USP9X variants associated with NDDs using clinically recommended guidelines. Functional studies in patient derived cell lines and mice were used to determine mechanisms of pathology. Results Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the CNS (white matter disturbances, thin corpus callosum and widened ventricles), global delay with significant alteration of speech, language and behaviour, hypotonia, joint hypermobility, visual system defects and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient derived cell lines we show loss of only specific USP9X substrates which regulate neurodevelopmental signalling pathways and a united defect in TGF signalling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal dependent learning and memory. Conclusion Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioural syndrome in males and identify plausible mechanisms of pathogenesis centred on disrupted TGFβ signalling and hippocampal function.