Palmer EE , Stuhlmann T , Weinert S , Haan E , Van Esch H , Holvoet M , Boyle J , Leffler M , Raynaud M , Moraine C , van Bokhoven H , Kleefstra T , Kahrizi K , Najmabadi H , Ropers HH , Delgado MR , Sirsi D , Golla S , Sommer A , Pietryga MP , Chung WK , Wynn J , Rohena L , Bernardo E , Hamlin D , Faux BM , Grange DK , Manwaring L , Tolmie J , Joss S , DDD Study , Cobben JM , Duijkers FAM , Goehringer JM , Challman TD , Hennig F , Fischer U , Grimme A , Suckow V , Musante L , Nicholl J , Shaw M , Lodh SP , Niu Z , Rosenfeld JA , Stankiewicz P , Jentsch TJ , Gecz J , Field M , Kalscheuer VM .

Wellcome Trust , R01 HD057036 NICHD NIH HHS , UL1 RR024156 NCRR NIH HHS , WT098051 Wellcome Trust , Department of Health

             inactivation of paternal X chromosome

                syndromic X-linked intellectual disability

	Figure 1. Pedigrees of affected families A–G, J and P. Filled square=affected male; Filled circle=affected female; *Familial CLCN4 variant present; wt, familial CLCN4 variant absent; NT, not tested. Note unaffected male IV:5 in family B has Klinefelter syndrome (47,XXY). Probands from families H, I, K, L, M, N and O (pedigrees not shown) have de novo variants.
	Figure 2. Neuroimaging of affected individuals with CLCN4-related condition at different ages. F1–4: CT images of individual F:IV:3, aged 66. Non-contrast multi-slice helical acquisitions, thick-slice reconstructions only. F1: sagittal midline image depicts callosal thinning, atrophic midbrain and cerebellar atrophy with prominent quadri-cerebellar cistern. F2: axial image of posterior fossa depicting cerebellar vermian atrophy with increased caliber of the adjacent CSF spaces. F3: axial image depicting prominent Sylvian fissures and increased caliber of lateral and third ventricles with surrounding atrophic change and evidence of a right frontal infarct. F4: coronal image showing disproportionate cerebellar atrophy and prominence of the adjacent CSF spaces. J1–6: MRI images of individual J:III:2 aged 4 and 8. J1: Axial FLAIR image from individual J:III:2, aged 4, depicting enlarged Sylvian fissures secondary to surrounding volume loss, particularly affecting the frontal lobes. J2: coronal T2 weighted image from individual J:III:2 aged 4 depicting generalized white matter volume loss. J3 Axial FLAIR image from individual J:III:2 aged 8 demonstrating periventricular leukomalacia. J4: coronal FLAIR image from individual J:III:2 aged 8. Images depict that despite interval progression of myelination between the ages of 4 and 8, there is still significantly delayed myelination involving the anterior frontal, posterior parietal and temporal lobes. The corpus callosum is normally formed but attenuated. There is pronounced global volume loss, preferentially affecting the white matter. Prominent perivascular spaces are seen bilaterally. L1–3: MRI images from family L proband at four months of age. Diffuse cortical hypoplasia with resultant prominence of the extra-axial CSF spaces and thinning of the corpus callosum without other morphologic abnormalities. Myelination is otherwise grossly normal. N1–2 MRI images from proband from family N aged 10. N1: sagittal T1 weighted midline image showing dilated third ventricle. N2: Coronal T2 weighted image showing dilated third ventricle. I1–2: images from proband from family I aged 13 months. I1: sagittal T1 weighted midline image showing dilated lateral ventricles. I:2: axial T2 weighted image showing volume loss, periventricular leukomalacia and dilated ventricles. CSF, cerebrospinal fluid; MRI, magnetic resonance imaging.
	Figure 3. Facial features of affected individuals with CLCN4-related disorder in age order. In older males note the elongated face, straight nose and pointed prominent chin that becomes relatively ‘squared off’ with age that is particularly prominent in affected adult males from families A, B, C and F (F:IV:3), and mildly present in the 9-year-old F:V:1 and 12-year-old affected male from family P. A relatively flat midface is noticeable in several individuals (particularly A:III:6 and affected members of family C). Individuals F:IV:3 and F:IV:5 have relative ‘coarsening’ of facial features with age; however, they were both treated with phenytoin, which can coarsen features and F:IV:5 had multiple facial injuries secondary to epileptic drop attacks. Although the facial phenotype is less distinctive in younger children and females (family F:V:1 and F:V2 as infants, and affected individuals from families O, I, L, H, N and M) some similarities include young children having relatively rounded faces and affected females from families H, L and F (F:IV:5) having down-sloping palpebral fissures and depressed nasal bridges. Clinical pictures from family C were first published in Raynaud et al.11 and reproduced from John Wiley & Sons with permission.
	Figure 4. Genotypic information, three-dimensional modeling and functional studies of CLCN4 variants. (a) Position of CLCN4 variants in families A–P (information adapted from Protein Data Bank P51793). Missense variants in families D (p.Gly78Ser), G (p.Val212Gly), L (p.Ala555Val) and O (p.Gly544Arg) are within helical transmembrane domains of the protein. Missense variants in families F (p.Val536Met), I (p.Ser534Leu) and N (p.Val275Met) are within helical-intramembrane domains. Missense variants in families E (p.Leu221Val) and H (p.Leu221Pro) lie within a loop between transmembrane domains. Missense variants in families C (p.Gly731Arg) and M (p.Arg718Trp) lie within the second cytosolic cystathionine-β-synthase (CBS) domain and therefore may affect gating. (b) Analogous positions of amino acids mutated in ClC-4 highlighted in the crystal structure of CmClC. Amino acids are displayed as spheres in colors. CLC transporters are (homo)dimers of identical subunits with two ion translocation pathways. Each subunit is composed of a transmembrane domain (TMD) containing several intramembrane helices and cytosolic CBS domains. Asterisks indicate previously published analogous positions of amino acids.5, 6 (c) Current voltage relationships of the electrogenic 2Cl−/H+ exchanger ClC-4 and the CIC-4 variants expressed in Xenopus oocytes, shown as mean values of normalized steady-state currents from several oocytes (numbers indicated in figure, in parentheses: number of independent cRNA batches injected). The current of wild-type (WT) CIC-4 is strongly outwardly rectifying, that is, increases steeply at inside-positive voltages. The human missense mutations p.Val212Gly, p.Leu221Pro, p.Val275Met, p.Ser543Leu, p.Ala555Val and p.Arg718Trp reduced, or even abolished, these ClC-4 currents. Only variant p.Asp15Asn exhibited normal wild-type like currents. Error bars, s.e.m. Noninjected (n.i.) oocytes served as control.

Adkins, Potential role of cardiac chloride channels and transporters as novel therapeutic targets. 2015, Pubmed

Adkins, Potential role of cardiac chloride channels and transporters as novel therapeutic targets. 2015, Pubmed
Blanz, Leukoencephalopathy upon disruption of the chloride channel ClC-2. 2007, Pubmed
Brunklaus, Genotype phenotype associations across the voltage-gated sodium channel family. 2014, Pubmed
Claes, Regional localization of two genes for nonspecific X-linked mental retardation to Xp22.3-p22.2 (MRX49) and Xp11.3-p11.21 (MRX50). 1997, Pubmed
Depienne, Brain white matter oedema due to ClC-2 chloride channel deficiency: an observational analytical study. 2013, Pubmed
Dierssen, Dendritic pathology in mental retardation: from molecular genetics to neurobiology. 2006, Pubmed
Feng, Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle. 2010, Pubmed
Fong, Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata. 1998, Pubmed , Xenbase
Grantham, Amino acid difference formula to help explain protein evolution. 1974, Pubmed
Hu, X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes. 2016, Pubmed
Hu, Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing. 2009, Pubmed
Jentsch, Discovery of CLC transport proteins: cloning, structure, function and pathophysiology. 2015, Pubmed
Kasper, Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. 2005, Pubmed
Kircher, A general framework for estimating the relative pathogenicity of human genetic variants. 2014, Pubmed
Lau, Glutamate receptors, neurotoxicity and neurodegeneration. 2010, Pubmed
Leisle, ClC-7 is a slowly voltage-gated 2Cl(-)/1H(+)-exchanger and requires Ostm1 for transport activity. 2011, Pubmed
Mohammad-Panah, The chloride channel ClC-4 contributes to endosomal acidification and trafficking. 2003, Pubmed
Nguyen, Clcn4-2 genomic structure differs between the X locus in Mus spretus and the autosomal locus in Mus musculus: AT motif enrichment on the X. 2011, Pubmed
Okkenhaug, The human ClC-4 protein, a member of the CLC chloride channel/transporter family, is localized to the endoplasmic reticulum by its N-terminus. 2006, Pubmed
Picollo, Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. 2005, Pubmed
Poët, Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. 2006, Pubmed
Raynaud, X-linked mental retardation with neonatal hypotonia in a French family (MRX15): gene assignment to Xp11.22-Xp21.1. 1996, Pubmed
Rickheit, Role of ClC-5 in renal endocytosis is unique among ClC exchangers and does not require PY-motif-dependent ubiquitylation. 2010, Pubmed
Scheel, Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. 2005, Pubmed , Xenbase
Snijders Blok, Mutations in DDX3X Are a Common Cause of Unexplained Intellectual Disability with Gender-Specific Effects on Wnt Signaling. 2015, Pubmed
Stobrawa, Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. 2001, Pubmed
Suzuki, Protein-protein interactions in the mammalian brain. 2006, Pubmed
Tanaka, Mutations in SPATA5 Are Associated with Microcephaly, Intellectual Disability, Seizures, and Hearing Loss. 2015, Pubmed
Tarpey, A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. 2009, Pubmed
Tufan, Ultrastructure protection and attenuation of lipid peroxidation after blockade of presynaptic release of glutamate by lamotrigine in experimental spinal cord injury. 2008, Pubmed
Veeramah, Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. 2013, Pubmed , Xenbase
Verkman, Chloride channels as drug targets. 2009, Pubmed
Wright, Genetic diagnosis of developmental disorders in the DDD study: a scalable analysis of genome-wide research data. 2015, Pubmed
Wu, Cellular resolution maps of X chromosome inactivation: implications for neural development, function, and disease. 2014, Pubmed
Yang, Clinical whole-exome sequencing for the diagnosis of mendelian disorders. 2013, Pubmed
Yu, Increased burden of de novo predicted deleterious variants in complex congenital diaphragmatic hernia. 2015, Pubmed
Zweier, Females with de novo aberrations in PHF6: clinical overlap of Borjeson-Forssman-Lehmann with Coffin-Siris syndrome. 2014, Pubmed
van Slegtenhorst, A gene from the Xp22.3 region shares homology with voltage-gated chloride channels. 1994, Pubmed