Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling

Abstract

Background: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative.

Methods: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. 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 central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; 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 that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. 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.

Conclusions: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function.

Keywords: Brain malformation; Deubiquitylating enzyme; Hippocampus; Neurodevelopmental disorder; TGFβ; USP9X.

Figure 1.. Likely pathogenic USP9X variants cause a characteristic NDD in males.

A. Constellation and penetrance of defining clinical features. n = the number of subjects whose information contributed to the data. B. Magnetic Resonance Imaging of the brains of individuals with likely pathogenic USP9X variants. Examples highlight evidence of white matter loss and ventricular widening in all, and in particular peri-ventricular leukomalacia (p.Ile79Val), loss of myelination / gliosis of posterior peri-ventricular white matter (p.Asn971Ser), cerebellar vermis hypoplasia (p.Arg2085His) and hypoplastic corpus callosum (p.Ser2233Pro). C-D. Photographs of individuals with USP9X variants. Note short, tapered fingers.

Figure 2.. USP9X VUS share in silico signatures with likely pathogenic variants.

A. Protein location of USP9X variants and common variants extracted from gnomAD data base. B. Bulk comparison of common, benign, likely pathogenic and variants of unknown significance by a suite of in silico prediction tools. *significantly different from common variants p<0.05 by Student’s t-test. C. Comparison of CADD and MUT_PRED2 scores reveal clustering of variants of unknown significance with likely pathogenic variants in upper-right quadrant consistent with pathogenicity (CADD >25, MUT_PRED >0.7). Scores are significantly correlated (Pearson’s correlation given). Colour scheme as in A and B. Inset identifies each variant in the ‘pathogenic quadrant’. Graphs show percent of each type of variant, and the overall composition of variant types within the pathogenic quadrant.

Figure 3.. USP9X variants impact substrates that regulate neurodevelopmental signalling pathways.

A. qRT-PCR of USP9X mRNA expression in male control and patient derived fibroblasts. B. Quantitation of n=3 western blot experiments analysing USP9X protein expression (See Figure S8). *p<0.05 Student’s t-test. C. Representative immunofluorescence images from control and USP9X variant fibroblast cell lines. D. Western-blot of representative USP9X immunoprecipitation (IP) experiment from control and USP9X variant fibroblast lysates. Immunoprecipitated proteins from n=3 independent experiments (See Figure S10) were analysed by tandem mass tag mass spectroscopy for quantitation. E. Relative protein quantities of significantly enriched USP9X interactors (enriched in USP9X IPs compared to IgG IPs in control cells) in variant USP9X IP experiments. *p<0.05 paired Student’s t-test. F. Representative western blot analysis of USP9X substrates implicated in neurodevelopmental signalling pathways in control and variant USP9X fibroblast cell lines. G. Quantitation of western-blots in C and replicates experiments (Figure S8; n=3 experiments). Values represent relative abundance compared to controls (n=3 cell lines); values underlined are significantly reduced (p<0.05 Student’s t-test).

Figure 4.. TGFβ signalling is disrupted in USP9X variant fibroblast cell lines.

Cells were serum starved (0.2% serum) for 16 hours prior to addition of TGFβ and assayed 24 hours later. A. In the absence of added TGFβ, cells display similar basal levels of signalling as assessed by TGFβ luciferase reporter assay. B. Relative increase of TGFβ signalling following addition of ligand as assessed by TGFβ luciferase reporter assays. Experiment done in quadruplicate. C. Representative immunofluorescent images of SMAD4 localisation before (time = 0 hr) and after (time = 24 hours) addition of TGFβ. Arrow heads indicate nuclear localisation. D. Quantitation of SMAD4 nuclear translocation following addition of TGFβ. n=5 replicates. E. Representative images of scratch migration assay. F. Quantitation of the relative stimulation of migration of cells into the scratch area following addition of TGFβ. n=3 technical x 3 biological replicates. * statistical difference between +/− TGFβ. # statistical difference between controls and USP9X variant cell lines. n.s.: non-significant difference between controls and USP9X variant cell lines. #* p<0.05 Student’s t-test.

Figure 5.. Behavioral deficits in Usp9x knockout mice.

A. Adult Usp9x forebrain-specific knockout mice (Usp9x^−/Y; Emx1-Cre) travel further than wildtype littermate controls (Usp9x^LoxP/Y) in an open field test. B. Knockout mice also exhibited significant differences in various parameters of the modified SHIRPA neurological screening protocol (also see Supplementary Table 4); * p < 0.05; 2-tailed unpaired t test. C. Schematic of the active place avoidance (APA) arena. D-I. Knockout mice exhibited significantly reduced performance on different aspects of the APA task. Statistics relate to comparisons between wild-type and knockout animals on individual days of the five day test. * p < 0.05, ** p < 0.01, *** p < 0.001; two way ANOVA (also see Figure S16). J-L. Coronal sections of adult wild-type (left 2 panels) and mutant (right two panels) at the level of the hippocampus. OCT6 (red) was used a marker for CA1 hippocampal neurons, and DAPI (blue) was used to label nuclei. Whereas the density of OCT6-expressing neurons was not different between control and mutant animals (K), the total number of OCT6-expressing neurons per CA1 region was reduced within the hippocampus of mutant animals. * p < 0.05; t test. Scale bars in J; 250 μm in low magnification images; 30 μm in high magnification images.