Novel NALCN variant linked to temporal lobe epilepsy.

The sodium leak channel (NALCN) gene encodes a sodium leak channel that plays an important role in the regulation of the resting membrane potential and the control of neuronal excitability. Mutations in the NALCN gene have been reported in patients with infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF) and congenital contractures of the limbs and face with hypotonia and developmental delay (CLIFAHDD syndrome). We describe the case of a father with drug-resistant left temporo-orbitofrontal epilepsy and his son with mildly-symptomatic temporal epilepsy (only recurrent déjà vu auras) whose genetic panels identified a likely pathogenic deletion of exon 27 on the NALCN gene. Our study helps broaden the clinical spectrum of diseases associated with mutations in the NALCN gene.

Usage of Genetic Panels in an Adult Epilepsy Clinic.

BACKGROUND: There is limited data on the utility, yield, and cost efficiency of genetic testing in adults with epilepsy. We aimed to describe the yield and utility of genetic panels in our adult epilepsy clinic. METHODS: We performed a retrospective, cross-sectional study of all patients followed by an epileptologist at a Canadian tertiary care centre's epilepsy clinic between January 2016 and August 2021 for whom a genetic panel was ordered. A panel was generally ordered when the etiology was unknown or in the presence of a malformation of cortical development. We determined the yield of panel positivity and of confirmed genetic diagnoses. We also estimated the proportion of these diagnoses that were clinically actionable. RESULTS: In total, 164 panels were ordered in 164 patients. Most had refractory epilepsy (80%), and few had comorbid intellectual disability (10%) or a positive family history of epilepsy (11%). The yield of panel positivity was 11%. Panel results were uncertain 49% of the time and negative 40% of the time. Genetic diagnoses were confirmed in 7 (4.3%) patients. These genetic conditions involved the following genes: SCARB2, DEPDC5, PCDH19, LGI1, SCN1A, MT-TL1, and CHRNA7. Of the seven genetic diagnoses, 5 (71%) were evaluated to be clinically actionable. CONCLUSION: We report a lower diagnostic yield for genetic panels in adults with epilepsy than what has so far been reported. Although the field of the genetics of epilepsy is a fast-moving one and more data is required, our findings suggest that guidelines for genetic testing in adults are warranted.

Increased frequency of proinflammatory CD4 T cells and pathological levels of serum neurofilament light chain in adult drug-resistant epilepsy.

OBJECTIVE: Adult drug-resistant epilepsy (DRE) is associated with significant morbidity. Infiltration of immune cells is observed in DRE epileptic foci; however, the relation between DRE and the peripheral immune cell compartment remains only partially understood. We aimed to investigate differences in immune cell populations, cytokines, and neurodegenerative biomarkers in the peripheral blood of subjects with epilepsy versus healthy controls, and in DRE compared to well-controlled epilepsy (WCE). METHODS: Peripheral blood mononuclear cells and serum from >120 age- and sex-matched adults suffering from focal onset epilepsy and controls were analyzed by multipanel flow cytometry, multiplex immunoassays, and ultrasensitive single molecule array. RESULTS: Using a data-driven analytical approach, we identified that CD4 T cells in the peripheral blood are present in a higher proportion in DRE patients. Moreover, we observed that the frequency of CD4 T cells expressing proinflammatory cytokines interleukin (IL)-17A, IL-22, tumor necrosis factor, interferon-γ, and granulocyte-macrophage colony-stimulating factor, but not anti-inflammatory cytokines IL-10 and IL-4, is elevated in the peripheral blood of DRE subjects compared to WCE. In parallel, we found that Th17-related circulating proinflammatory cytokines are elevated, but Th2-related cytokine IL-4 is reduced, in the serum of epilepsy and DRE subjects. As Th17 cells can exert neurotoxicity, we measured levels of serum neurofilament light chain (sNfL), a marker of neuronal injury. We found significantly elevated levels of sNfL in DRE compared to controls, especially among older individuals. SIGNIFICANCE: Our data support that DRE is associated with an expansion of the CD4 Tcell subset in the peripheral blood and with a shift toward a proinflammatory Th17/Th1 CD4 Tcell immune profile. Our results further show that pathological levels of sNfL are more frequent in DRE, supporting a potential neurodegenerative component in adult DRE. With this work, we provide evidence for novel potential inflammatory and degenerative biomarkers in DRE.

Global characterization of copy number variants in epilepsy patients from whole genome sequencing.

Epilepsy will affect nearly 3% of people at some point during their lifetime. Previous copy number variants (CNVs) studies of epilepsy have used array-based technology and were restricted to the detection of large or exonic events. In contrast, whole-genome sequencing (WGS) has the potential to more comprehensively profile CNVs but existing analytic methods suffer from limited accuracy. We show that this is in part due to the non-uniformity of read coverage, even after intra-sample normalization. To improve on this, we developed PopSV, an algorithm that uses multiple samples to control for technical variation and enables the robust detection of CNVs. Using WGS and PopSV, we performed a comprehensive characterization of CNVs in 198 individuals affected with epilepsy and 301 controls. For both large and small variants, we found an enrichment of rare exonic events in epilepsy patients, especially in genes with predicted loss-of-function intolerance. Notably, this genome-wide survey also revealed an enrichment of rare non-coding CNVs near previously known epilepsy genes. This enrichment was strongest for non-coding CNVs located within 100 Kbp of an epilepsy gene and in regions associated with changes in the gene expression, such as expression QTLs or DNase I hypersensitive sites. Finally, we report on 21 potentially damaging events that could be associated with known or new candidate epilepsy genes. Our results suggest that comprehensive sequence-based profiling of CNVs could help explain a larger fraction of epilepsy cases.

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Testing association of rare genetic variants with resistance to three common antiseizure medications.

OBJECTIVE: Drug resistance is a major concern in the treatment of individuals with epilepsy. No genetic markers for resistance to individual antiseizure medication (ASM) have yet been identified. We aimed to identify the role of rare genetic variants in drug resistance for three common ASMs: levetiracetam (LEV), lamotrigine (LTG), and valproic acid (VPA). METHODS: A cohort of 1622 individuals of European descent with epilepsy was deeply phenotyped and underwent whole exome sequencing (WES), comprising 575 taking LEV, 826 LTG, and 782 VPA. We performed gene- and gene set-based collapsing analyses comparing responders and nonresponders to the three drugs to determine the burden of different categories of rare genetic variants. RESULTS: We observed a marginally significant enrichment of rare missense, truncating, and splice region variants in individuals who were resistant to VPA compared to VPA responders for genes involved in VPA pharmacokinetics. We also found a borderline significant enrichment of truncating and splice region variants in the synaptic vesicle glycoprotein (SV2) gene family in nonresponders compared to responders to LEV. We did not see any significant enrichment using a gene-based approach. SIGNIFICANCE: In our pharmacogenetic study, we identified a slightly increased burden of damaging variants in gene groups related to drug kinetics or targeting in individuals presenting with drug resistance to VPA or LEV. Such variants could thus determine a genetic contribution to drug resistance.

Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy.

PURPOSE: Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy. METHOD: We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene. RESULTS: We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4ß2 (wild-type), α4ß2-Thr26Met (homozygote), and α4ß2/ß2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation. CONCLUSIONS: This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

Human copy number variants are enriched in regions of low mappability.

Copy number variants (CNVs) are known to affect a large portion of the human genome and have been implicated in many diseases. Although whole-genome sequencing (WGS) can help identify CNVs, most analytical methods suffer from limited sensitivity and specificity, especially in regions of low mappability. To address this, we use PopSV, a CNV caller that relies on multiple samples to control for technical variation. We demonstrate that our calls are stable across different types of repeat-rich regions and validate the accuracy of our predictions using orthogonal approaches. Applying PopSV to 640 human genomes, we find that low-mappability regions are approximately 5 times more likely to harbor germline CNVs, in stark contrast to the nearly uniform distribution observed for somatic CNVs in 95 cancer genomes. In addition to known enrichments in segmental duplication and near centromeres and telomeres, we also report that CNVs are enriched in specific types of satellite and in some of the most recent families of transposable elements. Finally, using this comprehensive approach, we identify 3455 regions with recurrent CNVs that were missing from existing catalogs. In particular, we identify 347 genes with a novel exonic CNV in low-mappability regions, including 29 genes previously associated with disease.

Utility of magnetic source imaging in nonlesional focal epilepsy: a prospective study.

OBJECTIVE: For patients with nonlesional refractory focal epilepsy (NLRFE), localization of the epileptogenic zone may be more arduous than for other types of epilepsy and frequently requires information from multiple noninvasive presurgical modalities and intracranial EEG (icEEG). In this prospective, blinded study, the authors assessed the clinical added value of magnetic source imaging (MSI) in the presurgical evaluation of patients with NLRFE. METHODS: This study prospectively included 57 consecutive patients with NLRFE who were considered for epilepsy surgery. All patients underwent noninvasive presurgical evaluation and then MSI. To determine the surgical plan, discussion of the results of the presurgical evaluation was first undertaken while discussion participants were blinded to the MSI results. MSI results were then presented. MSI influence on the initial management plan was assessed. RESULTS: MSI results influenced patient management in 32 patients. MSI results led to the following changes in surgical strategy in 14 patients (25%): allowing direct surgery in 6 patients through facilitating the detection of subtle cortical dysplasia in 4 patients and providing additional concordant diagnostic information to other presurgical workup in another 2 patients; rejection of surgery in 3 patients originally deemed surgical candidates; change of plan from direct surgery to icEEG in 2 patients; and allowing icEEG in 3 patients deemed not surgical candidates. MSI results led to changed electrode locations and contact numbers in another 18 patients. Epilepsy surgery was performed in 26 patients influenced by MSI results and good surgical outcome was achieved in 21 patients. CONCLUSIONS: This prospective, blinded study showed that information provided by MSI allows more informed icEEG planning and surgical outcome in a significant percentage of patients with NLRFE and should be included in the presurgical workup in those patients.

De novo mutations in epileptic encephalopathies.

Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown. Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies: infantile spasms (n = 149) and Lennox-Gastaut syndrome (n = 115). We sequenced the exomes of 264 probands, and their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 × 10(-3)). Among these are GABRB3, with de novo mutations in four patients, and ALG13, with the same de novo mutation in two patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P = 4.1 × 10(-10) and P = 7.8 × 10(-12), respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR and NEDD4L. Finally, we show that the de novo mutations observed are enriched in specific gene sets including genes regulated by the fragile X protein (P < 10(-8)), as has been reported previously for autism spectrum disorders.

Effects of SYN1Q555X mutation on cortical gray matter microstructure.

A new Q555X mutation on the SYN1 gene was recently found in several members of a family segregating dyslexia, epilepsy, and autism spectrum disorder. To describe the effects of this mutation on cortical gray matter microstructure, we performed a surface-based group study using novel diffusion and quantitative multiparametric imaging on 13 SYN1Q555X mutation carriers and 13 age- and sex-matched controls. Specifically, diffusion kurtosis imaging (DKI) and neurite orientation and dispersion and density imaging (NODDI) were used to analyze multi-shell diffusion data and obtain parametric maps sensitive to tissue structure, while quantitative metrics sensitive to tissue composition (T1, T2* and relative proton density [PD]) were obtained from a multi-echo variable flip angle FLASH acquisition. Results showed significant microstructural alterations in several regions usually involved in oral and written language as well as dyslexia. The most significant changes in these regions were lowered mean diffusivity and increased fractional anisotropy. This study is, to our knowledge, the first to successfully use diffusion imaging and multiparametric mapping to detect cortical anomalies in a group of subjects with a well-defined genotype linked to language impairments, epilepsy and autism spectrum disorder (ASD).

γ-Aminobutyric acid receptor alpha 1 subunit loss of function causes genetic generalized epilepsy by impairing inhibitory network neurodevelopment.

OBJECTIVE: In humans, mutations of the γ-aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy-causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. METHODS: We generated a gabra1-/- zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1-/- larval brains. RESULTS: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic-clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. SIGNIFICANCE: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1-deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

The clinical impact of integration of magnetoencephalography in the presurgical workup for refractory nonlesional epilepsy.

OBJECTIVE: For patients with nonlesional refractory focal epilepsy (NLRFE), localization of the epileptogenic zone is more arduous, and intracranial electroencephalography (EEG) (icEEG) is frequently required. Planning for icEEG is dependent on combined data from multiple noninvasive modalities. We report the negative impact of lack of integration of magnetoencephalography (MEG) in the presurgical workup in NLRFE. METHODS: Observational MEG case series involving 31 consecutive patients with NLRFE in an academic epilepsy center. For various reasons, MEG data were not analyzed in a timely manner to be included in the decision-making process. The presumed impact of MEG was assessed retrospectively. RESULTS: Magnetoencephalography would have changed the initial management in 21/31 (68%) had MEG results been available by reducing the number of intracranial electrodes, modifying their position, allowing for direct surgery, canceling the intracranial study, or providing enough evidence to justify one. Good surgical outcome was achieved in 11 out of 17 patients who proceeded to epilepsy surgery. Nine out of eleven had MEG clusters corresponding to the resection area, and MEG findings would have allowed for direct surgery (avoiding icEEG) in 2/11. Six patients had poor outcome including three patients where MEG would have significantly changed the outcome by modifying the resection margin. Magnetoencephalography provided superior information in 3 patients where inadequate coverage precluded accurate mapping of the epileptogenic zone. CONCLUSION: In this single center retrospective study, MEG would have changed patient management, icEEG planning, and surgical outcome in a significant percentage of patients with NLRFE and should be considered in the presurgical workup in those patients.

Exome sequencing reveals a novel PLP1 mutation in a Moroccan family with connatal Pelizaeus-Merzbacher disease: a case report.

BACKGROUND: Epilepsy regroups a common and diverse set of chronic neurological disorders that are characterized by spontaneous, unprovoked, and recurrent epileptic seizures. Epilepsies have a highly heterogeneous background with a strong genetic contribution and various mode of inheritance. X-linked epilepsy usually manifests as part of a syndrome or epileptic encephalopathy. The variability of clinical manifestations of X-linked epilepsy may be attributed to several factors including the causal genetic mutation, making diagnosis, genetic counseling and treatment decisions difficult. We report the description of a Moroccan family referred to our genetic department with X-linked epileptic seizures as the only initial diagnosis. CASE PRESENTATION: Knowing the new contribution of Next-Generation Sequencing (NGS) for clinical investigation, and given the heterogeneity of this group of disorders we performed a Whole-Exome Sequencing (WES) analysis and co-segregation study in several members of this large family. We detected a novel pathogenic PLP1 missense mutation c.251C > A (p.Ala84Asp) allowing us to make a diagnosis of Pelizaeus-Merzbacher Disease for this family. CONCLUSION: This report extends the spectrum of PLP1 mutations and highlights the diagnostic utility of NGS to investigate this group of heterogeneous disorders.

Correction to: Exome sequencing reveals a novel PLP1 mutation in a Moroccan family with connatal Pelizaeus-Merzbacher disease: a case report.

After publication of the original article [1] it was brought to our attention that author Bouchra Ouled Amar Bencheikh was incorrectly included as Bouchra Oulad Amar Bencheikh.

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth.

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD.

The genetic landscape of infantile spasms.

Infantile spasms (IS) is an early-onset epileptic encephalopathy of unknown etiology in ∼40% of patients. We hypothesized that unexplained IS cases represent a large collection of rare single-gene disorders. We investigated 44 children with unexplained IS using comparative genomic hybridisation arrays (aCGH) (n = 44) followed by targeted sequencing of 35 known epilepsy genes (n = 8) or whole-exome sequencing (WES) of familial trios (n = 18) to search for rare inherited or de novo mutations. aCGH analysis revealed de novo variants in 7% of patients (n = 3/44), including a distal 16p11.2 duplication, a 15q11.1q13.1 tetrasomy and a 2q21.3-q22.2 deletion. Furthermore, it identified a pathogenic maternally inherited Xp11.2 duplication. Targeted sequencing was informative for ARX (n = 1/14) and STXBP1 (n = 1/8). In contrast, sequencing of a panel of 35 known epileptic encephalopathy genes (n = 8) did not identify further mutations. Finally, WES (n = 18) was very informative, with an excess of de novo mutations identified in genes predicted to be involved in neurodevelopmental processes and/or known to be intolerant to functional variations. Several pathogenic mutations were identified, including de novo mutations in STXBP1, CASK and ALG13, as well as recessive mutations in PNPO and ADSL, together explaining 28% of cases (5/18). In addition, WES identified 1-3 de novo variants in 64% of remaining probands, pointing to several interesting candidate genes. Our results indicate that IS are genetically heterogeneous with a major contribution of de novo mutations and that WES is significantly superior to targeted re-sequencing in identifying detrimental genetic variants involved in IS.

Familial focal epilepsy with focal cortical dysplasia due to DEPDC5 mutations.

OBJECTIVE: The DEPDC5 (DEP domain-containing protein 5) gene, encoding a repressor of the mTORC1 signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies. We aimed to further extend the role of DEPDC5 to focal cortical dysplasias (FCDs). METHODS: Seven patients from 4 families with DEPDC5 mutations and focal epilepsy associated with FCD were recruited and investigated at the clinical, neuroimaging, and histopathological levels. The DEPDC5 gene was sequenced from genomic blood and brain DNA. RESULTS: All patients had drug-resistant focal epilepsy, 5 of them underwent surgery, and 1 had a brain biopsy. Electroclinical phenotypes were compatible with FCD II, although magnetic resonance imaging (MRI) was typical in only 4 cases. Histopathology confirmed FCD IIa in 2 patients (including 1 MRI-negative case) and showed FCD I in 2 other patients, and remained inconclusive in the last 2 patients. Three patients were seizure-free postsurgically, and 1 had a worthwhile improvement. Sequencing of blood DNA revealed truncating DEPDC5 mutations in all 4 families; 1 mutation was found to be mosaic in an asymptomatic father. A brain somatic DEPDC5 mutation was identified in 1 patient in addition to the germline mutation. INTERPRETATION: Germline, germline mosaic, and brain somatic DEPDC5 mutations may cause epilepsy associated with FCD, reinforcing the link between mTORC1 pathway and FCDs. Similarly to other mTORopathies, a "2-hit" mutational model could be responsible for cortical lesions. Our study also indicates that epilepsy surgery is a valuable alternative in the treatment of drug-resistant DEPDC5-positive focal epilepsies, even if the MRI is unremarkable.

Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy.

The KCC2 cotransporter establishes the low neuronal Cl(-) levels required for GABAA and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl(-)-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EG ly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.

Epileptogenic Q555X SYN1 mutant triggers imbalances in release dynamics and short-term plasticity.

Synapsin I (SynI) is a synaptic vesicle (SV) phosphoprotein playing multiple roles in synaptic transmission and plasticity by differentially affecting crucial steps of SV trafficking in excitatory and inhibitory synapses. SynI knockout (KO) mice are epileptic, and nonsense and missense mutations in the human SYN1 gene have a causal role in idiopathic epilepsy and autism. To get insights into the mechanisms of epileptogenesis linked to SYN1 mutations, we analyzed the effects of the recently identified Q555X mutation on neurotransmitter release dynamics and short-term plasticity (STP) in excitatory and inhibitory synapses. We used patch-clamp electrophysiology coupled to electron microscopy and multi-electrode arrays to dissect synaptic transmission of primary SynI KO hippocampal neurons in which the human wild-type and mutant SynI were expressed by lentiviral transduction. A parallel decrease in the SV readily releasable pool in inhibitory synapses and in the release probability in excitatory synapses caused a marked reduction in the evoked synchronous release. This effect was accompanied by an increase in asynchronous release that was much more intense in excitatory synapses and associated with an increased total charge transfer. Q555X-hSynI induced larger facilitation and post-tetanic potentiation in excitatory synapses and stronger depression after long trains in inhibitory synapses. These changes were associated with higher network excitability and firing/bursting activity. Our data indicate that imbalances in STP and release dynamics of inhibitory and excitatory synapses trigger network hyperexcitability potentially leading to epilepsy/autism manifestations.