X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4−/− mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

Summary Purpose The management of epilepsy in children is particularly challenging when seizures are resistant to anti-epileptic 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 etiological diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurological disorders, especially those associated with abnormal brain development. Here we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiological diagnoses. Methods We performed WES (mean coverage ~40X) 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. Key Findings In nine of ten 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%. Interestingly, these seven probands all presented with seizures within the first six months of life, and six of these have intractable seizures. Significance The finding that seven of ten 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.

Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive–compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4’s function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.

Glucose homeostasis depends critically on insulin that is secreted by pancreatic β-cells. Serum glucose, which is directly sensed by β-cells, stimulates depolarization- and Ca2+-dependent exocytosis of insulin granules. Here we show that pancreatic islets prominently express LRRC8A and LRRC8D, subunits of volume-regulated VRAC anion channels. Hypotonicity- or glucose-induced β-cell swelling elicits canonical LRRC8A-dependent VRAC currents that depolarize β-cells to an extent that causes electrical excitation. Glucose-induced excitation and Ca2+ responses are delayed in onset, but not abolished, in β-cells lacking the essential VRAC subunit LRRC8A. Whereas Lrrc8a disruption does not affect tolbutamide- or high-K+-induced insulin secretion from pancreatic islets, it reduces first-phase glucose-induced insulin secretion. Mice lacking VRAC in β-cells have normal resting serum glucose levels but impaired glucose tolerance. We propose that opening of LRRC8/VRAC channels increases glucose sensitivity and insulin secretion of β-cells synergistically with KATP closure. Neurotransmitter-permeable LRRC8D-containing VRACs might have additional roles in autocrine/paracrine signaling within islets.