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Greenwood, United States

Robinson A.,University College London | Escuin S.,University College London | Vekemans K.D.,University College London | Vekemans K.D.,University of Paris Descartes | And 5 more authors.
Human Mutation | Year: 2012

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein- protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.

Kerzendorfer C.,University of Sussex | Whibley A.,University of Cambridge | Carpenter G.,University of Sussex | Outwin E.,University of Sussex | And 6 more authors.
Human Molecular Genetics | Year: 2010

CUL4A and B encode subunits of E3-ubiquitin ligases implicated in diverse processes including nucleotide excision repair, regulating gene expression and controlling DNA replication fork licensing. But, the functional distinction between CUL4A and CUL4B, if any, is unclear. Recently, mutations in CUL4B were identified in humans associated with mental retardation, relative macrocephaly, tremor and a peripheral neuropathy. Cells from these patients offer a unique system to help define at the molecular level the consequences of defective CUL4B specifically. We show that these patient-derived cells exhibit sensitivity to camptothecin (CPT), impaired CPT-induced topoisomerase I (Topo I) degradation and ubiquitination, thereby suggesting Topo I to be a novel Cul4-dependent substrate. Consistent with this, we also find that these cells exhibit increased levels of CPT-induced DNA breaks. Furthermore, over-expression of known CUL4-dependent sub- strates including Cdt1 and p21 appear to be a feature of these patient-derived cells. Collectively, our findings highlight the interplay between CUL4A and CUL4B and provide insight into the pathogenesis of CUL4B- deficiency in humans. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

Schwartz C.E.,Jc Self Research Institute
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Polyamines, small positively charged molecules, are vital for cell proliferation and differentiation. They are found ubiquitously in eukaryotic cells. Additionally, they interact with a wide range of other molecules and some membrane associated receptors. Polyamines, spermidine and spermine, are synthesized by two aminopropyltransferases, spermidine synthase and spermine synthase. Recently, mutations in the latter enzyme have been shown to be responsible for an X-linked intellectual disability condition known as Snyder-Robinson syndrome. Spermine synthase deficiency is thus far the only known polyamine deficiency syndrome in humans.

Brookes E.,Harvard University | Laurent B.,Harvard University | Ounap K.,University of Tartu | Carroll R.,University of Adelaide | And 4 more authors.
Human Molecular Genetics | Year: 2015

Mutations in KDM5C are an important cause of X-linked intellectual disability in males. KDM5C encodes a histone demethylase, suggesting that alterations in chromatin landscape may contribute to disease.We used primary patient cells and biochemical approaches to investigate the effects of patient mutations on KDM5C expression, stability and catalytic activity.We report and characterize a novel nonsense mutation, c.3223delG (p.V1075Yfs*2), which leads to loss of KDM5C protein.We also characterize twoKDM5Cmissense mutations, c.1439C>T (p.P480L) and c.1204G>T (p.D402Y) that are compatible with protein production, but compromise stability and enzymatic activity. Finally,we demonstrate that a c.2T>C mutation in the translation initiation codon of KDM5C results in translation re-start and production of a N-terminally truncated protein (p.M1_E165del) that is unstable and lacks detectable demethylase activity. Patient fibroblasts do not show global changes in histone methylation but we identify several up-regulated genes, suggesting local changes in chromatin conformation and gene expression. This thorough examination of KDM5C patient mutations demonstrates the utility of examining the molecular consequences of patient mutations on several levels, ranging from enzyme production to catalytic activity, when assessing the functional outcomes of intellectual disability mutations. © The Author 2015. Published by Oxford University Press. All rights reserved.

Homan C.C.,University of Adelaide | Kumar R.,Womens and Childrens Health Research Institute | Kumar R.,University of Adelaide | Nguyen L.S.,University of Adelaide | And 10 more authors.
American Journal of Human Genetics | Year: 2014

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. © 2014 The American Society of Human Genetics.

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