Alternative titles; symbols
HGNC Approved Gene Symbol: CLCN4
SNOMEDCT: 1172691004;
Cytogenetic location: Xp22.2 Genomic coordinates (GRCh38) : X:10,156,975-10,237,660 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| Xp22.2 | Raynaud-Claes syndrome | 300114 | X-linked dominant | 3 |
CLCN4 is a voltage-gated chloride channel (van Slegtenhorst et al., 1994).
In the course of constructing a comprehensive transcript map of the Xp22.3 region, van Slegtenhorst et al. (1994) identified an evolutionarily conserved CpG island and cloned the corresponding gene. The predicted 760-amino acid protein contained 12 hydrophobic domains and shared sequence and structural similarities with all the previously isolated members of the family of voltage-gated chloride channels. In contrast with most genes isolated from the Xp22.3 region, CLCN4 did not share homology with the Y chromosome, but was conserved in mouse and hamster. Expression studies demonstrated a 7.5-kb transcript that is particularly abundant in skeletal muscle and also detectable in brain and heart. Thus, this gene encodes a newly identified voltage-gated chloride channel.
Nguyen et al. (2011) compared the CLCN4 exon/intron structure of 8 mammalian species. In all species examined, the ATG translation start site was located at the beginning of exon 3, and the poly(A) tail was within exon 13. The coding sequence of exons 3 to 13 was highly conserved, with an overall 80 to 90% identity between species.
The CLCN4 gene maps to chromosome Xp22.3 (van Slegtenhorst et al., 1994).
Rugarli et al. (1995) found that in the wild Mediterranean mouse Mus spretus, the Cln4 gene maps to the X chromosome as it does in the human; however, in the inbred strain of laboratory mouse C57BL/6J, they found that it maps to chromosome 7. Findings indicated that a recent evolutionary rearrangement occurred in the mouse sex chromosomes very close to the pseudoautosomal region (PAR). The data were considered molecular evidence for a major divergence near the pseudoautosomal region consistent with the hypothesis that hybrid sterility in these species results from abnormal pairing of sex chromosomes during male meiosis. They found that Cln4 is the closest cloned gene to the M. spretus pseudoautosomal region and the most distal locus displaying a conserved position between the human and this mouse locus. The X-inactivation status of the locus in M. spretus was demonstrated by the finding that in F1 females from a cross between M. spretus and an inbred-derived mouse carrying the t(X;16)16H balanced translocation, it was always the normal M. spretus X chromosome that was inactive in adult tissues. This completely skewed X inactivation provided a system for assaying expression of genes from the inactive X chromosome once the parental alleles could be distinguished.
Palmer et al. (1995) likewise found what they referred to as 'contravention of Ohno's law' in the course of mapping a cDNA mouse Cln4 in an interspecific backcross. This was the first example of a gene unique to the X chromosome in 1 eutherian species but autosomal in another. The consequence of this chromosomal rearrangement was that the gene was lost by mendelian segregation in a subset of the male progeny of a (C57BL/6 x Mus spretus) x Mus spretus backcross.
In a 14-month-old boy with Raynaud-Claes syndrome (MRXSRC; 300114) who presented with epileptic encephalopathy, Veeramah et al. (2013) identified a de novo hemizygous missense mutation in the CLCN4 gene (G544R; 302910.0001). The mutation was found by whole-exome sequencing. In vitro functional expression studies in Xenopus oocytes showed that the mutation almost abolished the outwardly rectifying currents, consistent with a loss of function. The patient was 1 of 10 probands with a similar phenotype who underwent whole-exome sequencing.
In affected male members of 5 unrelated families with X-linked intellectual disability, including the family (MRX49) reported by Claes et al. (1997) and the family (MRX15) reported by Raynaud et al. (1996), Hu et al. (2016) identified hemizygous mutations in the CLCN4 gene (302910.0002-302910.0006). The mutations were found by X-chromosome exome sequencing. One of the mutations resulted in a truncated protein, whereas the 4 others were missense mutations. In vitro functional expression studies in Xenopus oocytes showed that all of the missense mutations caused a marked reduction in outwardly-rectifying CLCN4 currents compared to wildtype. Knockdown of the Clcn4 gene in mouse hippocampal neurons resulted in 30% less dendritic branches compared to controls, and primary neurons derived from Clcn4-null mice showed similar, but more subtle, changes. The findings were consistent with a loss of function underlying the cognitive defects in these families.
Palmer et al. (2018) summarized phenotypic and molecular genetic information on 52 individuals from 16 families with a syndromic intellectual disability disorder, including 6 previously reported families, and mutation in the CLCN4 gene. In 5 affected females (see, e.g., 302910.0007) and 2 affected males, the mutations occurred de novo. The mutation spectrum included frameshift, missense, and splice site variants, and one single-exon deletion.
In his classic monograph entitled 'Sex Chromosomes and Sex-Linked Genes,' Ohno (1967) promulgated his famous law based on numerous examples of conservation of X-linked genes, cementing the concept of the X chromosome in placental mammals as an immutable element (Ellis, 1995). Motivating most of Ohno's thesis was the compelling but complex evolutionary argument that heteromorphic sex chromosomes in mammals evolved originally from a pair of homomorphic autosomes. Mutations occurred in 2 (or more) genes on 1 of these chromosomes (the proto-Y chromosome) but caused the genes to become factors in the sexual differentiation of the male. These genes had to segregate together; thus, recombination was suppressed between the incipient sex chromosomes. As a consequence of recombination suppression, the proto-Y chromosome suffered gradual genetic deterioration that eventually left it a 'dummy chromosome.' At the same time, a dosage compensation mechanism evolved that doubled the rate of product output of X-linked genes (because monosomy is deleterious) and led to the inactivation of the extra X chromosome in females. The X-chromosome inactivation mechanism became an evolutionary barrier to the shuffling of genes between the X and autosomes because, as had previously been shown, such rearrangements disrupt the compensation mechanism. Laxity in Ohno's law was previously discovered in relation to the pseudoautosomal region in which DNA segments of the X and Y pair and show crossing-over, generally presumed to be necessary for the proper segregation of the X and Y chromosomes. Genes in the PAR escape X inactivation which removes them from the evolutionary force that would retain them on the X chromosome. The genes that are known to be in the PAR include ASMT (300015) and ASMTY (402500); the first such gene to be discovered, MIC2 (313470) and MIC2Y (450000); the XG blood group gene (300879 and XGPY); IL3RA (308385) and IL3RAY (430000); AMELX (300391) and AMELY (410000); CSF2RA (306250) and CSF2RY (425000); and ANT3 (300151) and ANT3Y (403000). Consistent with this laxity, syntenic conservation of genes in the PAR is not a hard and fast rule. For example, CSF2RA and IL3RA are pseudoautosomal in the human and autosomal in the mouse; see Disteche et al. (1992) and Milatovich et al. (1993), respectively. A further wrinkle on the evolution of this region of the X chromosome is demonstrated by the steroid sulfatase gene (STS; 300747), which escapes X inactivation in both humans and the mouse but is X-linked in humans while pseudoautosomal in the mouse. The PAR has been implicated in the mouse and perhaps in other species in providing an explanation for Haldane's rule (Haldane, 1922) that in interspecific hybrids, the heterogametic sex of the F1 offspring is absent, rare, or sterile. In crosses between M. spretus and M. musculus, the male F1 offspring are sterile. Their testes are small, probably due to breakdown of spermatogenesis after meiosis 1. Almost all X-Y bivalents dissociate by diakinesis, probably because of the failure of the sex chromosomes from the 2 different species to pair and recombine properly. Lack of homology between the PARs may be the underlying cause.
Schnur and Wick (1995) detected a TaqI RFLP within the CLCN4 gene which lies between the loci for ocular albinism (OA1; 300500) and microphthalmia with linear skin defects (MLS; 309801). No recombination was observed between the RFLP and the OA1 mutation in 3 informative families, indicating that the marker would be useful for genetic counseling in OA1.
In a 14-month-old boy with Raynaud-Claes syndrome (MRXSRC; 300114) who presented with epileptic encephalopathy, Veeramah et al. (2013) identified a de novo hemizygous c.1630G-A transition in the CLCN4 gene, resulting in a gly544-to-arg (G544R) substitution at a highly conserved residue within an intramembrane 4-residue loop that connects intramembrane helices P and Q. The mutation, which was found by whole-exome sequencing, was not present in the 1000 Genomes Project or Exome Sequencing Project databases. The patient developed refractory complex partial seizures with secondary generalization at age 4 months. He had microcephaly, delayed psychomotor development, hypotonia, and dystonia. Transfection of the mutation into HeLa cells showed that the mutant protein had normal localization to structures resembling ER membranes. However, in vitro functional expression studies in Xenopus oocytes showed that the mutation almost abolished the outwardly rectifying currents, consistent with a loss of function. The patient was 1 of 10 probands with a similar phenotype who underwent whole-exome sequencing.
In a 10-year-old Dutch boy (Family O) with a severe intellectual disability and seizure disorder, Palmer et al. (2018) identified a G-to-C transversion (chrX:10181774G-C) in the CLCN4 gene, resulting in a gly544-to-arg substitution in the transmembrane domain. The boy was nonverbal, showed apathy and social disinhibition, and had absence and tonic clonic seizures diagnosed at age 3 that responded to lamotrigine. He had a round face, downslanting palpebral fissures, and an open mouth. His growth parameters were low, below the 2nd centile for head circumference and below the 1st centile for weight and height (-3.4 SD). He was not hypotonic in infancy. He showed progressive spasticity, an unsteady gait, tics, and stereotypies. A brain MRI at age 7 years was normal.
In 4 male patients from a family (MRX49) with X-linked intellectual disability (MRXSRC; 300114) originally reported by Claes et al. (1997), Hu et al. (2016) identified a hemizygous 13-bp deletion (chrX.101,531,111-101,531,123del13, GRCh37) in the CLCN4 gene, resulting in a frameshift and premature termination (Asp15SerfsTer18). The mutation, which was found by X-chromosome exome sequencing, was filtered against the dbSNP (build 135), Exome Variant Server, and 1000 Genomes Project databases, as well as against 200 Danish exomes.
In 2 male patients from a family (MRX15) with X-linked intellectual disability (MRXSRC; 300114) originally reported by Raynaud et al. (1996), Hu et al. (2016) identified a hemizygous G-to-A transition (chrX.10,188,916G-A, GRCh37) in the CLCN4 gene, resulting in a gly731-to-arg (G731R) substitution in the cytosolic cystathionine-beta-synthase domain. The mutation, which was found by X-chromosome exome sequencing, was filtered against the dbSNP (build 135), Exome Variant Server, and 1000 Genomes Project databases, as well as against 200 Danish exomes. In vitro functional expression studies in Xenopus oocytes showed that the mutation caused a marked reduction in outwardly-rectifying CLCN4 currents compared to wildtype.
In a male patient (family N70) with X-linked intellectual disability (MRXSRC; 300114), Hu et al. (2016) identified a hemizygous G-to-A transition (chrX.10,155,642G-A, GRCh37) in the CLCN4 gene, resulting in a gly78-to-ser (G78S) substitution in the transmembrane domain. The patient was deceased and had a deceased similarly affected brother, but the brother was not tested for the mutation. The mutation, which was found by X-chromosome exome sequencing, was filtered against the dbSNP (build 135), Exome Variant Server, and 1000 Genomes Project databases, as well as against 200 Danish exomes. In vitro functional expression studies in Xenopus oocytes showed that the mutation caused a marked reduction in outwardly-rectifying CLCN4 currents compared to wildtype.
In 2 affected males from a family (family AU27) with X-linked intellectual disability (MRXSRC; 300114), Hu et al. (2016) identified a hemizygous C-to-G transversion (chrX.10,174,503C-G, GRCh37) in the CLCN4 gene, resulting in a leu221-to-val (L221V) substitution in the transmembrane domain. The mutation, which was found by X-chromosome exome sequencing, was filtered against the dbSNP (build 135), Exome Variant Server, and 1000 Genomes Project databases, as well as against 200 Danish exomes. In vitro functional expression studies in Xenopus oocytes showed that the mutation caused a marked reduction in outwardly-rectifying CLCN4 currents compared to wildtype. There were 2 additional male patients in the family with a similar disorder, but they were not tested for the mutation. One unaffected female was confirmed to be a carrier.
In 2 affected males from a family with X-linked intellectual disability (MRXSRC; 300114), Hu et al. (2016) identified a hemizygous G-to-A transition (chrX.10,181,750G-A, GRCh37) in the CLCN4 gene, resulting in a val536-to-met (V536M) substitution in the transmembrane domain. The mutation, which was found by X-chromosome exome sequencing, was filtered against the dbSNP (build 135), Exome Variant Server, and 1000 Genomes Project databases, as well as against 200 Danish exomes. In vitro functional expression studies in Xenopus oocytes showed that the mutation caused a marked reduction in outwardly-rectifying CLCN4 currents compared to wildtype. There were 5 additional male patients and 1 female in the family with a similar disorder, but they were not tested for the mutation. One unaffected female was confirmed to be a carrier. (In the article by Hu et al. (2016), this family was designated family AU4 in figure 1 and supplement table 7, but as family AU9 in table 2.)
In a 15-year-old Scottish girl (Family M) with severe intellectual disability and a history of brief absence seizures (MRXSRC; 300114), Palmer et al. (2018) identified a de novo C-to-T transition (chrX:10188877C-T) in the CLCN4 gene, resulting in an arg718-to-trp (R718W) substitution within the second cytosolic cystathionine-beta-synthase domain. The patient was nonverbal, drooled, and exhibited self-abusive behavior. No X-inactivation studies were performed. Expression of the variant in Xenopus oocytes showed reduced steady-state current compared to wildtype.
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