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• W2079251282 abstract "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. 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. Intellectual disability (ID) affects ∼2%–3% of the population, and in developed countries the dominant cause is of genetic origin.1Ropers H.H. Genetics of early onset cognitive impairment.Annu. Rev. Genomics Hum. Genet. 2010; 11: 161-187Crossref PubMed Scopus (267) Google Scholar Although ID is one of the most highly heterogeneous human disorders, there is currently an overrepresentation of causative mutations found on the X chromosome.2Gécz J. Shoubridge C. Corbett M. The genetic landscape of intellectual disability arising from chromosome X.Trends Genet. 2009; 25: 308-316Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Recently the reports of a large-scale X-exome resequencing effort coupled with high-resolution copy-number profiling provided plausible explanations for approximately half of a cohort of 208 families with evidence for X-linked ID (XLID).3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar, 4Whibley A.C. Plagnol V. Tarpey P.S. Abidi F. Fullston T. Choma M.K. Boucher C.A. Shepherd L. Willatt L. Parkin G. et al.Fine-scale survey of X chromosome copy number variants and indels underlying intellectual disability.Am. J. Hum. Genet. 2010; 87: 173-188Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Because the cohort was previously excluded from the mutations and large-scale (i.e., 500G banding resolution) cytogenetic alterations known to cause XLID at that time, most variants discovered were located in genes that were not previously associated with XLID.3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar For the vast majority of the variants identified, further genetic and functional evidence was required (e.g., Shoubridge et al.5Shoubridge C. Tarpey P.S. Abidi F. Ramsden S.L. Rujirabanjerd S. Murphy J.A. Boyle J. Shaw M. Gardner A. Proos A. et al.Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability.Nat. Genet. 2010; 42: 486-488Crossref PubMed Scopus (118) Google Scholar) to support their causal involvement in ID of the respective individuals and families studied. Variants in USP9X (MIM 300072) have been identified as being potentially involved in XLID.3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar The X-exome sequencing initially identified a single truncating variant in an X-linked family, and subsequent screening of an additional cohort of male individuals with ID (where X-linkage was not always obvious) identified two additional nonrecurrent missense variants.3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar In two of these three families, the variants segregated as expected for X-linkage; in one case the inheritance could not be established (Figure S1 available online). The study was approved by local ethics committees and institutional review boards of each collaborating insititution, and informed consent for research was obtained from all individuals involved. In family 1, from France, a missense variant (c.6278T>A [RefSeq accession number NM_001039590.2], p.Leu2093His [RefSeq NP_001034679.2]) was found in a singleton affected male, with no other affected individuals known in the family (Figure S1). At 21 months of age, he displayed developmental delay, aggressive behavior, and hypotonia. Other features included relative macrocephaly, facial dysmorphism, broad thumbs and great toes, short stature, constipation, and hyperextensibility of joints and skin (Table 1). In family 2, from the USA, a second missense variant (c.6469C>A [p.Leu2157Ile]) was found in an affected male, his unaffected mother, and his unaffected grandmother (Figures S1 and S2). In addition, the affected individual was subsequently found to have a ∼790 kb deletion at 6q25.3, which includes ARID1B (MIM 614556). Haploinsufficiency of ARID1B is a common de novo cause of ID,6Hoyer J. Ekici A.B. Endele S. Popp B. Zweier C. Wiesener A. Wohlleber E. Dufke A. Rossier E. Petsch C. et al.Haploinsufficiency of ARID1B, a member of the SWI/SNF-a chromatin-remodeling complex, is a frequent cause of intellectual disability.Am. J. Hum. Genet. 2012; 90: 565-572Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar and because his parents are unaffected, it is most likely that this deletion also occurred de novo (the parents could not be tested). Whether the ARID1B deletion itself is the only cause of ID in this patient or whether the USP9X variant provides an additional, second hit could not be easily determined. Prenatally, the affected male in family 2 displayed a raised maternal serum alpha-fetoprotein, intrauterine growth restriction, and an ectopic left kidney. Postnatally, he had feeding difficulties, hypotonia, tracheomalacia, gastro-esophageal reflux disease, and developmental delay, with speech development most affected. He also had broad thumbs, curving toe nails, and short stature. At 9 years of age, he had ID (he was nonverbal, speech having regressed at age 3), obsessive and autistic behaviors, elevated testosterone and decreased cholesterol levels, and short stature (Table 1). In the third family, from the UK, a single-nucleotide deletion was identified in two half brothers, their unaffected mother and affected uncle, and their unaffected grandmother (Figures S1 and S2). A single-nucleotide deletion in USP9X (c.7574delA [p.Gln2525Argfs∗18]) was the only plausible variant identified in the 700 genes on the X chromosome screened, and a subsequent systematic screening of 550 genes in which mutations are known (or suspected) to cause ID, via a customized exome pull-down platform, did not reveal any other plausible mutation.3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar The deletion introduces a frameshift followed by 17 missense amino acids leading to a premature termination codon (PTC), and a 45 amino acid truncation. Because the PTC falls in the last exon of USP9X, transcripts are not likely to be degraded by the nonsense-mediated mRNA decay pathway. The affected individuals all had ID and hypotonia. In addition, one also displayed obsessive behaviors and another displayed autistic behaviors (Table 1). To summarize, the key clinical features shared by the five affected males from the three families with unique USP9X variants included ID, hypotonia, and short stature (where measured), with additional variable behavioral, gastroenterological, and dysmorphic features (Table 1).Table 1Summary of Key Clinical Features of Affected IndividualsFamily 1Family 2Family 3USP9X mutationc.6278T>A (p.Leu2093His)c.6469C>A (p.Leu2157Ile)c.7574delA (p.Gln2525fs∗18)Number of affected males113Neurological FeaturesID (severity)1/1 (mild)1/1 (mild)3/3 (mild-moderate)Autism0/11/11/3Aggression1/10/10/3Obsessiveness0/11/11/3Hypotonia1/11/13/3Dysmorphic FeaturesCraniofacialrelative macrocephaly and prominent forehead0/10/3Digitalbroad thumbs and great toesbroad thumbs and curling toenails0/3Growthage 21 months:Ht: 78.5 cm (3%)Wt: 11.4 kg (25%)HC: 50.5 cm (95%)age 12 months:Ht: 70.4 cm (3%)Wt: 7.87 Kg (<3%)HC: 44.7 cm (3%–25%)NDage 9 years:Ht: 124 cm (5%)Wt: 24.1 kg (10%)Otherhyperextensible joints and skin, constipationIUGR, ectopic left kidney, tracheomalacia, gastresophageal reflux, upper airway congestion, hypospadia, retractable left testisNDAbbreviations are as follows: Ht, height; Wt, weight, HC, head circumference; IUGR, intrauterine growth restriction; %, percentile; ND, no data. Open table in a new tab Abbreviations are as follows: Ht, height; Wt, weight, HC, head circumference; IUGR, intrauterine growth restriction; %, percentile; ND, no data. USP9X is highly conserved7Khut P.Y. Tucker B. Lardelli M. Wood S.A. Evolutionary and expression analysis of the zebrafish deubiquitylating enzyme, usp9.Zebrafish. 2007; 4: 95-101Crossref PubMed Scopus (10) Google Scholar with a residual variance intolerance score8Petrovski S. Wang Q. Heinzen E.L. Allen A.S. Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar of −1.62, 2.93%, suggesting considerable intolerance to variation. The three USP9X variants are unique, not present in an additional 914 affected males in XLID families, 1,129 control X chromosomes,3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar or dbSNP137, EVS, or the 1000 Genomes Project data sets. Furthermore, the variants were found to change highly conserved amino acid residues residing in the C-terminal region of the protein (Figure S1). In silico prediction programs (PolyPhen2, SIFT, and iPTREE) suggested that the c.6278T>A (p.Leu2093His) variant was deleterious, whereas for the c.6469C>A (p.Leu2157Ile) variant, only iPTREE reported an adverse effect (Figure S1). Additional prediction programs (PANTHER, Mutation Taster) corroborated this last result, suggesting that the p.Leu2157Ile variant was probably deleterious and disease causing. USP9X encodes a very large substrate-specific deubiquitylating enzyme of 2,570 amino acids, with a largely unknown structure outside of its catalytic and ubiquitin-like domains.9Wood S.A. Pascoe W.S. Ru K. Yamada T. Hirchenhain J. Kemler R. Mattick J.S. Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene.Mech. Dev. 1997; 63: 29-38Crossref PubMed Scopus (74) Google Scholar Although the variants did not locate within either of those domains, we tested whether the catalytic activity of the enzyme was affected. Overexpression of both USP9X and variant forms were able to stabilize the level of the known USP9X substrate MCL-110Schwickart M. Huang X. Lill J.R. Liu J. Ferrando R. French D.M. Maecker H. O’Rourke K. Bazan F. Eastham-Anderson J. et al.Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival.Nature. 2010; 463: 103-107Crossref PubMed Scopus (498) Google Scholar (MIM 159552), suggesting that the variants did not affect the ability of USP9X to rescue MCL-1 from proteasomal degradation (Figure S3). We also observed no appreciative difference between the ubiquitylation status of proteins that interacted (i.e., coimmunoprecipitated) with USP9X or variant forms (Figure S3). Together, these data suggest that the variants do not affect the catalytic activity of USP9X. To provide further evidence that the variants are pathogenic, we employed our recently described Usp9x knockout mice11Stegeman S. Jolly L.A. Premarathne S. Gecz J. Richards L.J. Mackay-Sim A. Wood S.A. Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFβ-mediated axonogenesis.PLoS ONE. 2013; 8: e68287Crossref PubMed Scopus (57) Google Scholar to assay the variants’ effect on neurodevelopmental processes. Protocols relating to the use of animals were approved by the Women’s and Children’s Health Network Animal Ethics Committee. During embryogenesis, brain-specific deletion of Usp9x results in early postnatal death, whereas forebrain-specific deletion is compatible with survival to adulthood. In the absence of Usp9x the cortical architecture is disorganized, and neurons display reduced neurite growth in vivo and in vitro.11Stegeman S. Jolly L.A. Premarathne S. Gecz J. Richards L.J. Mackay-Sim A. Wood S.A. Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFβ-mediated axonogenesis.PLoS ONE. 2013; 8: e68287Crossref PubMed Scopus (57) Google Scholar We therefore asked whether the USP9X variants might also effect neurite growth, in particular axonal growth. We introduced the variants into a full-length human USP9X cDNA and placed it under control of an exogenous promoter providing uniform expression levels (Figure S4). We isolated primary hippocampal neurons from both wild-type (Usp9x+/Y) and knockout (Usp9x−/Y) embryonic mice and grew them in vitro. Prior to plating the neurons, they were transfected with expression plasmids encoding EGFP (to track transfected cells), together with either an empty expression plasmid or ones containing wild-type USP9X or USP9X with either of the three DNA variants. After 5 days of growth in vitro, we assayed the length of primary axons and the degree of arborization as reported by the number of axonal termini (Figure 1). The results show that overexpression of USP9X in wild-type neurons has no effect on either length or arborization but that the loss of Usp9x in knockout neurons resulted in a 43% reduction in both axon length and arborization, similar to previous reports.11Stegeman S. Jolly L.A. Premarathne S. Gecz J. Richards L.J. Mackay-Sim A. Wood S.A. Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFβ-mediated axonogenesis.PLoS ONE. 2013; 8: e68287Crossref PubMed Scopus (57) Google Scholar The re-expression of wild-type USP9X successfully rescued these defects, whereas the re-expression of the variant USP9X forms failed to do so (Figure 1). Together these data indicate that the three USP9X variants identified disrupt the ability of USP9X to function in axonal growth. Next we explored whether USP9X variants might alter other neurodevelopmental processes. The C terminus of USP9X (where the variants cluster) binds the regulator of neuronal cell migration Doublecortin (DCX [MIM 300121])12Gleeson J.G. Lin P.T. Flanagan L.A. Walsh C.A. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.Neuron. 1999; 23: 257-271Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar, 13Friocourt G. Kappeler C. Saillour Y. Fauchereau F. Rodriguez M.S. Bahi N. Vinet M.C. Chafey P. Poirier K. Taya S. et al.Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in neuronal processes.Mol. Cell. Neurosci. 2005; 28: 153-164Crossref PubMed Scopus (36) Google Scholar and the related Doublecortin-like Kinase (DCLK1 [MIM 604742]).13Friocourt G. Kappeler C. Saillour Y. Fauchereau F. Rodriguez M.S. Bahi N. Vinet M.C. Chafey P. Poirier K. Taya S. et al.Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in neuronal processes.Mol. Cell. Neurosci. 2005; 28: 153-164Crossref PubMed Scopus (36) Google Scholar Furthermore, mutations in DCX cause XLID.14des Portes V. Francis F. Pinard J.M. Desguerre I. Moutard M.L. Snoeck I. Meiners L.C. Capron F. Cusmai R. Ricci S. et al.doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH).Hum. Mol. Genet. 1998; 7: 1063-1070Crossref PubMed Scopus (230) Google Scholar Therefore, we asked whether the loss of Usp9x might also affect neuronal migration. We isolated neural progenitor cells (NPCs) from both wild-type and knockout embryonic brains and grew them in vitro as nonadherent neurosphere cultures. Next we employed an in vitro neuronal migration assay, wherein neurospheres are adhered to a poly-L-lysine substrate and the migration of neurons outward from the sphere boundary is recorded.15Angata K. Fukuda M. Roles of polysialic acid in migration and differentiation of neural stem cells.Methods Enzymol. 2010; 479: 25-36Crossref PubMed Scopus (19) Google Scholar, 16Ocbina P.J. Dizon M.L. Shin L. Szele F.G. Doublecortin is necessary for the migration of adult subventricular zone cells from neurospheres.Mol. Cell. Neurosci. 2006; 33: 126-135Crossref PubMed Scopus (38) Google Scholar, 17Durbec P. Franceschini I. Lazarini F. Dubois-Dalcq M. In vitro migration assays of neural stem cells.Methods Mol. Biol. 2008; 438: 213-225Crossref PubMed Scopus (12) Google Scholar We found a highly significant decrease in the migration of neurons from the neurospheres in the absence of Usp9x (Figure 2). Next, we tested whether re-expression of the wild-type USP9X and variant USP9X forms could rescue this defect. We transfected wild-type and knockout neurospheres by the same regime as described above for the axonal growth assay. We found that overexpression of wild-type USP9X had no effect, whereas loss of Usp9x resulted in a 42% reduction in neuronal migration, similar to our previous finding (Figure 2). This migration defect could be partially rescued when wild-type USP9X or the c.6278T>A (p.Leu2093His) variant were re-expressed in the knockout cells, but re-expression of c.6469C>A (p.Leu2157Ile) or c.7574delA (p.Gln2525Argfs∗18) USP9X variants failed to do so (Figure 2). These data reveal that USP9X is required for normal neuronal cell migration and that two of the three variant USP9X forms probably disrupt this process during brain development of the affected individuals. Reduced axonal growth and neuronal migration are also features of neurons lacking DCX.18Deuel T.A. Liu J.S. Corbo J.C. Yoo S.Y. Rorke-Adams L.B. Walsh C.A. Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth.Neuron. 2006; 49: 41-53Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 19Fu X. Brown K.J. Yap C.C. Winckler B. Jaiswal J.K. Liu J.S. Doublecortin (Dcx) family proteins regulate filamentous actin structure in developing neurons.J. Neurosci. 2013; 33: 709-721Crossref PubMed Scopus (46) Google Scholar, 20Jean D.C. Baas P.W. Black M.M. A novel role for doublecortin and doublecortin-like kinase in regulating growth cone microtubules.Hum. Mol. Genet. 2012; 21: 5511-5527Crossref PubMed Scopus (38) Google Scholar, 21Koizumi H. Higginbotham H. Poon T. Tanaka T. Brinkman B.C. Gleeson J.G. Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain.Nat. Neurosci. 2006; 9: 779-786Crossref PubMed Scopus (193) Google Scholar, 22Koizumi H. Tanaka T. Gleeson J.G. Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration.Neuron. 2006; 49: 55-66Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar Given this overlap, the shared aspects of respective knockout mice phenotypes,18Deuel T.A. Liu J.S. Corbo J.C. Yoo S.Y. Rorke-Adams L.B. Walsh C.A. Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth.Neuron. 2006; 49: 41-53Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 23Jolly L.A. Taylor V. Wood S.A. USP9X enhances the polarity and self-renewal of embryonic stem cell-derived neural progenitors.Mol. Biol. Cell. 2009; 20: 2015-2029Crossref PubMed Scopus (45) Google Scholar, 24Tanaka T. Koizumi H. Gleeson J.G. The doublecortin and doublecortin-like kinase 1 genes cooperate in murine hippocampal development.Cereb. Cortex. 2006; 16: i69-i73Crossref PubMed Scopus (35) Google Scholar and the overlap of the variants with the known DCX-interacting domain of USP9X, we asked whether the variant USP9X proteins disrupted interactions with DCX. We first overexpressed USP9X and variant forms together with DCX in HEK293T cells and conducted coimmunoprecipitation experiments with USP9X as bait. DCX coimmunoprecipitated with USP9X and all three variant forms in this assay, suggesting that the overexpressed proteins could interact in these cells (Figure S5). DCX is, however, endogenously expressed only in the highly polarized newly born neurons, and DCX and USP9X are known to colocalize in the growth cones of extending axons in such cells.13Friocourt G. Kappeler C. Saillour Y. Fauchereau F. Rodriguez M.S. Bahi N. Vinet M.C. Chafey P. Poirier K. Taya S. et al.Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in neuronal processes.Mol. Cell. Neurosci. 2005; 28: 153-164Crossref PubMed Scopus (36) Google Scholar Therefore, we asked whether in these cells we could see evidence of altered interactions. Given the fact that DCX is not a USP9X substrate (rather, it is an interacting protein), we reasoned that this might present as an alteration in subcellular localization and/or changes in colocalization. In cultured Usp9x knockout neurons, the localization of Dcx was unaffected (Figure S6). Therefore, we asked whether the localization of USP9X was altered by the variants. We re-expressed either USP9X or the variant forms in knockout neurons as described above. We observed that Usp9x and Dcx colocalized in the cell soma and in the axonal growth cones of wild-type neurons as previously described13Friocourt G. Kappeler C. Saillour Y. Fauchereau F. Rodriguez M.S. Bahi N. Vinet M.C. Chafey P. Poirier K. Taya S. et al.Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in neuronal processes.Mol. Cell. Neurosci. 2005; 28: 153-164Crossref PubMed Scopus (36) Google Scholar (Figure 3). Likewise, when we re-expressed USP9X in knockout neurons, its localization overlapped substantially with Dcx in axonal growth cones (Figure 3). When we re-expressed the variant forms, however, we observed a clear reduction in the localization of USP9X in the axonal growth cones, whereas expression in the cell soma was unaffected (Figure 3). This in turn led to a drastic reduction in the level of colocalization of USP9X and Dcx in the axonal growth cones of neurons. These data suggest that variant USP9X proteins are unable to be targeted or maintained in the growth cones of axons, which probably hinders its ability to interact with DCX in these structures. With the exception of the c.6278T>A (p.Leu2093His) in the migration assay, the variant forms of USP9X behaved similar to a complete loss of function in our neuronal growth and migration assays. To gain further insight into the molecular pathways behind the neuronal cell defects, we employed a global, unbiased approach. Because Usp9x is a substrate-specific deubiquitylating enzyme, and thus regulates the proteome, we sought to identify proteins that were differentially expressed in Usp9x knockout neurons. We isolated and grew primary cortical neuronal cultures from four wild-type and four knockout E18.5 embryos. Lysates were isolated at day 5 of culture and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE; Figure S7). We identified 50 protein spots that were differentially represented in the gels (p < 0.05; DeCyder Software Module, GE Health). We manually inspected all protein spots and removed those with un-spot-like appearance, noise spikes, or poor resolution. The remaining spots were excised from the gel and proteins identified by liquid chromatography-electrospray ionization tandem mass spectroscopy (LC-ESI-MS/MS). From this analysis, we identified 28 proteins that were differentially expressed. We did not identify any known substrates of USP9X, but we found evidence for all identified proteins being regulated by ubiquitylation (Table S1). Furthermore, 27/28 proteins were downregulated in the absence of Usp9x, consistent with its role in antagonizing the ubiquitin-proteasome pathway (Figure 4). Therefore, the list may contain potential substrates of Usp9x. It is, however, likely that at least some of these deregulated proteins are indirectly affected by the absence of Usp9x, and instead report on the molecular pathways deregulated in Usp9x knockout neurons. The deregulated gene list was submitted to the DAVID annotation as well,25Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. DAVID: Database for Annotation, Visualization, and Integrated Discovery.Genome Biol. 2003; 4: 3Crossref PubMed Google Scholar and significant (p < 0.05) gene ontology and pathway terms were identified. The highest ranking gene ontology terms and PANTHER pathways reveal that loss of Usp9x affected proteins involved in regulation and structure of the cytoskeleton (Figure 4 and Table S2). Together, the data suggest that the neuronal migration and axonal growth defects observed in the absence of Usp9x, and also probably in neurons of the affected individuals, are based on a disruption of the neuronal cytoskeleton. The recent advances in sequencing and genome annotation are revolutionizing the discovery of genetic causes of disease. These approaches have found many variants that appear likely, based on initial genetic evidence, to be pathogenic. However, their acceptance as disease-causing mutations should be treated with caution, and supported by additional evidence, including variant frequency, segregation, genotype-phenotype correlations, and in silico and wet laboratory functional investigation. Here we focused on validating three unique variants discovered in USP9X that associated with ID in three unrelated families.3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (469) Google Scholar For family 3, the clear X-linked mode of inheritance of a truncation type USP9X variant, coupled with the lack of any other plausible genetic alterations, provided persuasive genetic support of pathogenicity. Further evidence of the involvement of this USP9X variant in ID was derived from the variants discovered in families 1 and 2, but" @default.
• W2079251282 created "2016-06-24" @default.
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• W2079251282 date "2014-03-01" @default.
• W2079251282 modified "2023-10-16" @default.
• W2079251282 title "Mutations in USP9X Are Associated with X-Linked Intellectual Disability and Disrupt Neuronal Cell Migration and Growth" @default.
• W2079251282 cites W1964426200 @default.
• W2079251282 cites W1985966348 @default.
• W2079251282 cites W1991249033 @default.
• W2079251282 cites W1999823034 @default.
• W2079251282 cites W2009304445 @default.
• W2079251282 cites W2012271876 @default.
• W2079251282 cites W2013745892 @default.
• W2079251282 cites W2013883770 @default.
• W2079251282 cites W2026566803 @default.
• W2079251282 cites W2027922031 @default.
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• W2079251282 cites W2063377436 @default.
• W2079251282 cites W2063511140 @default.
• W2079251282 cites W2071476740 @default.
• W2079251282 cites W2073374817 @default.
• W2079251282 cites W2074244952 @default.
• W2079251282 cites W2099133509 @default.
• W2079251282 cites W2104617652 @default.
• W2079251282 cites W2110272715 @default.
• W2079251282 cites W2121121587 @default.
• W2079251282 cites W2122683221 @default.
• W2079251282 cites W2133082302 @default.
• W2079251282 cites W2135805609 @default.
• W2079251282 cites W2151114041 @default.
• W2079251282 cites W2152231606 @default.
• W2079251282 cites W2153170275 @default.
• W2079251282 cites W2155139530 @default.
• W2079251282 cites W2160734046 @default.
• W2079251282 cites W2160930779 @default.
• W2079251282 cites W2163529521 @default.
• W2079251282 cites W2163876042 @default.
• W2079251282 cites W53174471 @default.
• W2079251282 cites W67982678 @default.
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