Several studies have reported the role of CLCN4 in tumor progression. However, its mechanism remains to be thoroughly studied. The objective of this study was to explore the potential pathogenic role of CLCN4 in endometrial carcinoma (UCEC) with a better understanding of the pathological mechanisms involved. The potential roles of CLCN4 in different tumors were explored based on The Cancer Genome Atlas (TCGA), the expression difference, mutation, survival, pathological stage, Immunity subtypes, Immune infiltration, tumor microenvironment (TME), tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR) related to CLCN4 were analyzed. Then, the expression, prognosis, mutation, and functional enrichment of CLCN4 in UCEC were analyzed. Immunohistochemical experiment was used to verify the expression of CLCN4 in endometrial cancer tissues and normal tissues. In vitro, we knocked down of CLCN4 in HEC-1-A cells and performed CCK8, WB, RT-PCR, wound-healing, transwell assays to further validation of the molecular function. Results revealed that high expression of CLCN4 was observed in 20 cancer types of TCGA. CLCN4 expression correlates with poor survival in MESO, BLCA, THCA, especially UCEC tumors. CLCN4 expression was significantly associated with CD4+ T-cell infiltration, especially CD4+ Th1-cell. Immunohistochemical experiment reveals that CLCN4 is high expressed in endometrial tumors, in vitro experiment reveals that knockdown of CLCN4 inhibits the cells proliferation, migration and invasion. Our study is the first to offer a comprehensive understanding of the oncogenic roles of CLCN4 on different tumors. CLCN4 may become a potential biomarker in UCEC.

Introduction: Epilepsy is a group of neurological disorders characterized by recurring seizures and fits. The Epilepsy genes can be classified into four distinct groups, based on involvement of these genes in different pathways leading to Epilepsy as a phenotype. Genetically the disease has been associated with various pathways, leading to pure epilepsy-related disorders caused by CNTN2 variations, or involving physical or systemic issues along with epilepsy caused by CARS2 and ARSA, or developed by genes that are putatively involved in epilepsy lead by CLCN4 variations. Methods: In this study, five families of Pakistani origin (EP-01, EP-02, EP-04, EP-09, and EP-11) were included for molecular diagnosis. Results: Clinical presentations of these patients included neurological symptoms such as delayed development, seizures, regression, myoclonic epilepsy, progressive spastic tetraparesis, vision and hearing impairment, speech problems, muscle fibrillation, tremors, and cognitive decline. Whole exome sequencing in index patients and Sanger sequencing in all available individuals in each family identified four novel homozygous variants in genes CARS2: c.655G>A p.Ala219Thr (EP-01), ARSA: c.338T>C: p.Leu113Pro (EP-02), c.938G>T p.Arg313Leu (EP-11), CNTN2: c.1699G>T p.Glu567Ter (EP-04), and one novel hemizygous variant in gene CLCN4: c.2167C>T p.Arg723Trp (EP-09). Conclusion: To the best of our knowledge these variants were novel and had not been reported in familial epilepsy. These variants were absent in 200 ethnically matched healthy control chromosomes. Three dimensional protein analyses revealed drastic changes in the normal functions of the variant proteins. Furthermore, these variants were designated as \"pathogenic\" as per guidelines of American College of Medical Genetics 2015. Due to overlapping phenotypes, among the patients, clinical subtyping was not possible. However, whole exome sequencing successfully pinpointed the molecular diagnosis which could be helpful for better management of these patients. Therefore, we recommend that exome sequencing be performed as a first-line molecular diagnostic test in familial cases.

BACKGROUND: Raynaud-Claes syndrome is a very rare X-linked condition, characterized by intellectual disability, impaired language development, brain abnormalities, facial dysmorphisms and drug-resistant epilepsy. It is caused by loss-of-function variants in the CLCN4 gene, which encodes the 2Cl-/H + exchanger ClC-4, prominently expressed in the hippocampus and cerebellum. Different genotypic variants have been described, each exhibiting specific phenotypic characteristics. The loss-of-function variant p.Gly544Arg in the CLCN4 gene has been described in only two male probands, but there are no reports on phenotypic characterization in females.
METHODS: We present a 30-year-old Italian woman with early-onset drug-resistant epilepsy, developmental and epileptic encephalopathy, developmental delay, absence of verbal language development, behavioral impairment with autistic features, and clusters of seizures during catamenial periods. The interictal EEG showed slight inconstant slowing of the background rhythm, with abnormal frontal predominant mu like rhythm and generalized spike and polyspike wave discharges, which increased in frequency during drowsiness. A brain MRI showed slight cranio-encephalic asymmetry and a smaller size of the left hippocampus. The whole exome sequencing (WES) revealed a de novo heterozygous c.1630G > A variant in the CLCN4 gene, resulting in the amino acid substitution p.Gly544Arg (rs587777161), consistent with Raynaud-Claes syndrome.
CONCLUSIONS: Our patient is the first case of a de novo p.Gly544Arg variant of the CLCN4 gene in a female proband, confirming that female patients with Raynaud-Claes syndrome can be as severely affected as the male counterparts. Our case expands the phenotypic characterization of different genotypic CLCN4 variants, which can become crucial in the future for early diagnosis if targeted therapy becomes available.

Thauvin-Robinet-Faivre syndrome (TROFAS; OMIM #617107) is a rare autosomal recessive overgrowth syndrome characterized by generalized overgrowth, dysmorphic facial features, and delayed psychomotor development caused by biallelic pathogenic variants in the FGF-1 intracellular binding protein (FIBP) gene. To date, only four patients from two families have been reported. In this report, we present a 4-year-old male patient with generalized overgrowth and delayed developmental milestones consistent with this syndrome. In addition, he has unique features that were not reported in previous patients, including drooling, recurrent pulmonary infections, chronic pulmonary disease, hyperextensible elbow joints, hypoplastic nipples, unilateral cryptorchidism, and frequent spontaneous erections. We identified a homozygous, likely pathogenic variant, c.415_416insCAGTTTG (p.Asp139AlafsTer3), which causes a frameshift in the FIBP. Additionally, we identified a homozygous missense variant in the Toll-like receptor 5(TLR5) gene and a hemizygous missense variant in the chloride voltage-gated channel 4 (CLCN4) gene, with uncertain significance in either case. In this article, we set out the new observations and also discuss the frequency of the characteristic findings of the syndrome in the patients so far reported.

Early/late endosomes, recycling endosomes, and lysosomes together form the endo-lysosomal recycling pathway. This system plays a crucial role in cell differentiation and survival, and dysregulation of the endo-lysosomal system appears to be important in the pathogenesis of neurodevelopmental and neurodegenerative diseases. Each endo-lysosomal compartment fulfils a specific function, which is supported by ion transporters and channels that modify ion concentrations and electrical gradients across endo-lysosomal membranes. CLC-type Cl-/H+ exchangers are a group of endo-lysosomal transporters that are assumed to regulate luminal acidification and chloride concentration in multiple endosomal compartments. Heterodimers of ClC-3 and ClC-4 localize to various internal membranes, from the endoplasmic reticulum and Golgi to recycling endosomes and late endosomes/lysosomes. The importance of ClC-4-mediated ion transport is illustrated by the association of naturally occurring CLCN4 mutations with epileptic encephalopathy, intellectual disability, and behavioral disorders in human patients. However, how these mutations affect the expression, subcellular localization, and function of ClC-4 is insufficiently understood. We here studied 12 CLCN4 variants that were identified in patients with X-linked intellectual disability and epilepsy and were already characterized to some extent in earlier work. We analyzed the consequences of these mutations on ClC-4 ion transport, subcellular trafficking, and heterodimerization with ClC-3 using heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings. The mutations led to a variety of changes in ClC-4 function, ranging from gain/loss of function and impaired heterodimerization with ClC-3 to subtle impairments in transport functions. Our results suggest that even slight functional changes to the endosomal Cl-/H+ exchangers can cause serious neurological symptoms.

To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches.

OBJECTIVE: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses.
METHODS: We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing.
RESULTS: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures.
CONCLUSIONS: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.