De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function

The UPF3B gene, implicated in intellectual disability, autism, ADHD and childhood onset schizophrenia regulates neural progenitor cell behaviour and neuronal outgrowth

HMG Advance Access published July 2, 2013Loss-of-function mutations in UPF3B result in variable clinical presentations including intellectual disability (ID, syndromic and non-syndromic), autism, childhood onset schizophrenia and attention deficit hyperactivity disorder. UPF3B is a core member of the nonsense-mediatedmRNAdecay (NMD) pathway that functions to rapidly degrade transcripts with premature termination codons (PTCs). Traditionally identified in thousands of human diseases, PTCs were recently also found to be part of 'normal' genetic variation in human populations. Furthermore, many human transcripts have naturally occurring regulatory features compatible with 'endogenous'PTCsstrongly suggesting roles ofNMDbeyondPTCmRNAcontrol. In this study,weinvestigated the role of Upf3b andNMD in neural cells.Weprovide evidence that suggests Upf3b-dependentNMD(Upf3b-NMD) is regulated at multiple levels during development including regulation of expression and sub-cellular localization of Upf3b. Furthermore, complementary expression of Upf3b, Upf3a and Stau1 stratify the developing dorsal telencephalon, suggesting that alternativeNMD,andthe related Staufen1-mediatedmRNAdecay (SMD) pathways are differentially employed. A loss of Upf3b-NMD in neural progenitor cells (NPCs) resulted in the expansion of cell numbers at the expense of their differentiation. In primary hippocampal neurons, loss of Upf3b-NMD resulted in subtle neurite growth effects. Our data suggest that the cellular consequences of loss of Upf3b-NMD can be explained in-part by changes in expression of key NMD-feature containing transcripts, which are commonly deregulated also in patients with UPF3B mutations. Our research identifies novel pathological mechanisms of UPF3B mutations and at least partly explains the clinical phenotype of UPF3B patients.Lachlan A. Jolly, Claire C. Homan, Reuben Jacob, Simon Barry and Jozef Gec

Loss of Usp9x disrupts cell adhesion, and components of the Wnt and Notch signaling pathways in neural progenitors

Development of neural progenitors depends upon the coordination of appropriate intrinsic responses to extrinsic signalling pathways. Here we show the deubiquitylating enzyme, Usp9x regulates components of both intrinsic and extrinsic fate determinants. Nestin-cre mediated ablation of Usp9x from embryonic neural progenitors in vivo resulted in a transient disruption of cell adhesion and apical-basal polarity and, an increased number and ectopic localisation of intermediate neural progenitors. In contrast to other adhesion and polarity proteins, levels of β-catenin protein, especially S33/S37/T41 phospho-β-catenin, were markedly increased in Usp9x embryonic cortices. Loss of Usp9x altered composition of the β-catenin destruction complex possibly impeding degradation of S33/S37/T41 phospho-β-catenin. Pathway analysis of transcriptomic data identified Wnt signalling as significantly affected in Usp9x embryonic brains. Depletion of Usp9x in cultured human neural progenitors resulted in Wnt-reporter activation. Usp9x also regulated components of the Notch signalling pathway. Usp9x co-localized and associated with both Itch and Numb in embryonic neocortices. Loss of Usp9x led to decreased Itch and Numb levels, and a concomitant increase in levels of the Notch intracellular domain as well as, increased expression of the Notch target gene Hes5. Therefore Usp9x modulates and potentially coordinates multiple fate determinants in neural progenitors

Loss of <i>Usp9x</i> reduces neuronal outgrowth.

<p>Embryonic hippocampal neurons were isolated, transfected with a plasmid encoding Enhanced Green Fluorescent Protein, and grown in-vitro for 3, 5 or 7 days. (a) Example immunofluorescent images of wildtype (Nes-<i>Usp9x^+/Y</i>) and null (Nes-<i>Usp9x^−/Y</i>) neurons resolved using GFP expression (Green) and co-stained with the axonal and dendritic specific antibodies, TAU1 (cyan) and MAP2 (red) respectively. (b–c) Morphometric analysis was employed to record mean primary axonal length (b) and number of neurite termini (c). *p<0.05 by student 2-tailed t-test.</p

Loss of <i>Usp9x</i> disrupts TGF-β signalling in hippocampal neurons.

<p>(a–b) TGF-β luciferase reporter assays conducted in either wildtype (Nes-<i>Usp9x^+/Y</i>) or null (Nes-<i>Usp9x^−/Y</i>) embryonic hippocampal neuronal cultures. Hippocampal neurons were isolated and transfected with both renilla control and pGL3-TGF-β luciferase reporter plasmids. (a) Cells were grown for 3 days before analysis using dual-luciferase reporter assays and data normalised relative to wildtype readings. (b) Luciferase reporter activity in response to increasing concentrations of TGF-β. Data normalised to controls in the absence of TGF-β. All luciferase data from 6 biological replicates (i.e. cultures isolated from 6 <i>Usp9x^+/y</i> and 6 <i>Usp9x^−/Y</i> embryos). (c) Response of established TGFβ target genes in presence or absence of <i>Usp9x</i>, analysed by RT-qPCR. Isolated hippocampal neurons grown for 2 days prior to the addition of 1 ng/ml TGF-β. (d–e). Morphological analysis of hippocampal neurons exposed to 1 ng/ml TGF-β in the presence or absence of Usp9x. (d) Comparison of mean primary axonal length. (e). Comparison of number of neurite termini.</p

<i>Usp9x</i> loss affects neuronal and astrocytic projections.

<p>NF160 antibodies decorate axonal projection from the neocortex (Nc) to the hippocampus (Hp) in E18.5 Nes-<i>Usp9x^+/Y</i> mice (A). These projections were absent in Nes-<i>Usp9x^−/Y</i> mice (B). GFAP staining is reduced in both the hippocampus and neocortex of E18.5 <i>Usp9x ^−/Y</i> embryos (D) compared with littermate controls (C). In the hippocampus GFAP-labeled projections extended toward the CA3 region in control embryos (arrowhead in E) but not in the absence of <i>Usp9x</i> (F). Scale bar = 20 µm (A,B), 160×µm (C,D), 80×µm (E,F).</p

Loss of <i>Usp9x</i> disrupts the architecture of the embryonic neocortex.

<p>The Nestin-cre mediated deletion of Usp9x (B,D) results in loss of demarcation between the cells of the ventricular and sub-ventricular zones (VZ/SVZ), the more disperse cellular density of the intermediate zone (IZ) and the neurons of the cortical plate (CP) seen in control littermates (A,C). C and D are higher magnification images of A and B, respectively. Nestin (E,F) and BLBP (G,H) staining in E18.5 embryos indicated that neural progenitors were more loosely organized in the VZ/SVZ. Neurons of the cortical plate were disorganized in the absence of <i>Usp9x</i> (J) compared with littermates (I). Nissl stain of <i>Usp9x^+/Y</i> (A,C,G) and <i>Usp9x^−/Y</i> (B,D,H) in E16.5 embryos (A–D, G–H). V = ventricle. Scale bar = 100 µm (A), 50 µm (C), 40 µm (E), 100 µm (G), 40 µm (I).</p

Impaired neural differentiation of MPS IIIA patient induced pluripotent stem cell-derived neural progenitor cells

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.Rebecca J. Lehmann, Lachlan A. Jolly, Brett V. Johnson, Megan S. Lord, Ha Na Kim, Jennifer T. Saville, Maria Fuller, Sharon Byers, Ainslie L.K. Derrick-Robert

Mutations in USP9X Are Associated with X-Linked Intellectual Disability and Disrupt Neuronal Cell Migration and Growth

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual's phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved

<i>Usp9x</i> is required for hippocampal development.

<p>The hippocampi of adult Emx1-<i>Usp9x^−/Y</i> mice (B) were reduced in area compared with <i>Usp9x^+/Y</i> littermates (A). Nissl stain of 7–8week old mice. (C) Quantification of hippocampal area of <i>Usp9x^+/Y</i> adult males (Control, n = 4) compared with <i>Usp9x^−/Y</i> (cKO, n = 4) (** p<0.01). (D–F) Higher magnification identifying disruption and reduction of the CA3 region in <i>Usp9x ^−/Y</i> males. (D - <i>Usp9x^+/Y</i>; E,F independent <i>Usp9x^−/Y</i> males) Scale bar = 100 µm (A,B), 150 µm (D–F).</p