Search

Cortical spreading depression (CSD), a wave of depolarization followed by depression of cortical activity, is a pathophysiological process implicated in migraine with aura and various other brain pathologies, such as ischemic stroke and traumatic brain injury. To gain insight into the pathophysiology of CSD, we generated a mouse model for a severe monogenic subtype of migraine with aura, familial hemiplegic migraine type 3 (FHM3). FHM3 is caused by mutations in SCN1A, encoding the voltage-gated Na+ channel NaV1.1 predominantly expressed in inhibitory interneurons. Homozygous Scn1aL1649Q knock-in mice died prematurely, whereas heterozygous mice had a normal lifespan. Heterozygous Scn1aL1649Q knock-in mice compared with WT mice displayed a significantly enhanced susceptibility to CSD. We found L1649Q to cause a gain-of-function effect with an impaired Na+-channel inactivation and increased ramp Na+ currents leading to hyperactivity of fast-spiking inhibitory interneurons. Brain slice recordings using K+-sensitive electrodes revealed an increase in extracellular K+ in the early phase of CSD in heterozygous mice, likely representing the mechanistic link between interneuron hyperactivity and CSD initiation. The neuronal phenotype and premature death of homozygous Scn1aL1649Q knock-in mice was partially rescued by GS967, a blocker of persistent Na+ currents. Collectively, our findings identify interneuron hyperactivity as a mechanism to trigger CSD.

Developmental and epileptic encephalopathies (DEE) can be caused by mutations in the KCNA2 gene, coding for the voltage-gated K+ channel K v 1.2. This ion channel belongs to the delayed rectifier class of potassium channels and plays a role during the repolarization phase of an action potential. In this study we reprogrammed fibroblasts from a 30-year-old male patient with DDE carrying a point mutation (c.890G > A, p.Arg297Gln) in KCNA2 to induced pluripotent stem cells. Pluripotency state of the cells was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels., (Copyright © 2019. Published by Elsevier B.V.)

Hereditary neuropathies comprise a wide variety of chronic diseases associated to more than 80 genes identified to date. We herein examined 612 index patients with either a Charcot-Marie-Tooth phenotype, hereditary sensory neuropathy, familial amyloid neuropathy, or small fiber neuropathy using a customized multigene panel based on the next generation sequencing technique. In 121 cases (19.8%), we identified at least one putative pathogenic mutation. Of these, 54.4% showed an autosomal dominant, 33.9% an autosomal recessive, and 11.6% an X-linked inheritance. The most frequently affected genes were PMP22 (16.4%), GJB1 (10.7%), MPZ, and SH3TC2 (both 9.9%), and MFN2 (8.3%). We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4, AARS, BIC2, DHTKD1, FGD4, HK1, INF2, KIF5A, PDK3, REEP1, SBF1, SBF2, SCN9A, and SPTLC2 with a declining frequency. Thirty-four novel variants were considered likely pathogenic not having previously been described in association with any disorder in the literature. In one patient, two homozygous mutations in HK1 were detected in the multigene panel, but not by whole exome sequencing. A novel missense mutation in KIF5A was considered pathogenic because of the highly compatible phenotype. In one patient, the plasma sphingolipid profile could functionally prove the pathogenicity of a mutation in SPTLC2. One pathogenic mutation in MPZ was identified after being previously missed by Sanger sequencing. We conclude that panel based next generation sequencing is a useful, time- and cost-effective approach to assist clinicians in identifying the correct diagnosis and enable causative treatment considerations., (© 2017 International Society for Neurochemistry.)

Objective: To examine the role of mutations in GABRB3 encoding the β 3 subunit of the GABA A receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes., Methods: We performed massive parallel sequencing of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs., Results: We identified 22 patients with heterozygous mutations in GABRB3, including 3 probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies. Electrophysiologic analysis of 7 mutations in Xenopus laevis oocytes, using coexpression of wild-type or mutant β 3 , together with α 5 and γ 2s subunits and an automated 2-microelectrode voltage-clamp system, revealed reduced GABA-induced current amplitudes or GABA sensitivity for 5 of 7 mutations., Conclusions: Our results indicate that GABRB3 mutations are associated with a broad phenotypic spectrum of epilepsies and that reduced receptor function causing GABAergic disinhibition represents the relevant disease mechanism., (© 2017 American Academy of Neurology.)

Objective: To identify the genetic basis of a family segregating episodic ataxia, infantile seizures, and heterogeneous epilepsies and to study the phenotypic spectrum of KCNA2 mutations., Methods: A family with 7 affected individuals over 3 generations underwent detailed phenotyping. Whole genome sequencing was performed on a mildly affected grandmother and her grandson with epileptic encephalopathy (EE). Segregating variants were filtered and prioritized based on functional annotations. The effects of the mutation on channel function were analyzed in vitro by voltage clamp assay and in silico by molecular modeling. KCNA2 was sequenced in 35 probands with heterogeneous phenotypes., Results: The 7 family members had episodic ataxia (5), self-limited infantile seizures (5), evolving to genetic generalized epilepsy (4), focal seizures (2), and EE (1). They had a segregating novel mutation in the shaker type voltage-gated potassium channel KCNA2 (CCDS_827.1: c.765_773del; p.255_257del). A rare missense SCN2A (rs200884216) variant was also found in 2 affected siblings and their unaffected mother. The p.255_257del mutation caused dominant negative loss of channel function. Molecular modeling predicted repositioning of critical arginine residues in the voltage-sensing domain. KCNA2 sequencing revealed 1 de novo mutation (CCDS_827.1: c.890G>A; p.Arg297Gln) in a girl with EE, ataxia, and tremor., Conclusions: A KCNA2 mutation caused dominantly inherited episodic ataxia, mild infantile-onset seizures, and later generalized and focal epilepsies in the setting of normal intellect. This observation expands the KCNA2 phenotypic spectrum from EE often associated with chronic ataxia, reflecting the marked variation in severity observed in many ion channel disorders., (© 2016 American Academy of Neurology.)

Objective: To delineate phenotypic heterogeneity, we describe the clinical features of a cohort of patients with GABRA1 gene mutations., Methods: Patients with GABRA1 mutations were ascertained through an international collaboration. Clinical, EEG, and genetic data were collected. Functional analysis of 4 selected mutations was performed using the Xenopus laevis oocyte expression system., Results: The study included 16 novel probands and 3 additional family members with a disease-causing mutation in the GABRA1 gene. The phenotypic spectrum varied from unspecified epilepsy (1), juvenile myoclonic epilepsy (2), photosensitive idiopathic generalized epilepsy (1), and generalized epilepsy with febrile seizures plus (1) to severe epileptic encephalopathies (11). In the epileptic encephalopathy group, the patients had seizures beginning between the first day of life and 15 months, with a mean of 7 months. Predominant seizure types in all patients were tonic-clonic in 9 participants (56%) and myoclonic seizures in 5 (31%). EEG showed a generalized photoparoxysmal response in 6 patients (37%). Four selected mutations studied functionally revealed a loss of function, without a clear genotype-phenotype correlation., Conclusions: GABRA1 mutations make a significant contribution to the genetic etiology of both benign and severe epilepsy syndromes. Myoclonic and tonic-clonic seizures with pathologic response to photic stimulation are common and shared features in both mild and severe phenotypes., (© 2016 American Academy of Neurology.)

Recently, mutations and deletions in the GRIN2A gene have been identified to predispose to benign and severe idiopathic focal epilepsies (IFE), revealing a higher incidence of GRIN2A alterations among the more severe phenotypes. This study aimed to explore the phenotypic boundaries of GRIN2A mutations by investigating patients with the two most common epilepsy syndromes: (i) idiopathic generalized epilepsy (IGE) and (ii) temporal lobe epilepsy (TLE). Whole exome sequencing data of 238 patients with IGE as well as Sanger sequencing of 84 patients with TLE were evaluated for GRIN2A sequence alterations. Two additional independent cohorts comprising 1469 IGE and 330 TLE patients were screened for structural deletions (>40kb) involving GRIN2A. Apart from a presumably benign, non-segregating variant in a patient with juvenile absence epilepsy, neither mutations nor deletions were detected in either cohort. These findings suggest that mutations in GRIN2A preferentially are involved in genetic variance of pediatric IFE and do not contribute significantly to either adult focal epilepsies as TLE or generalized epilepsies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To test whether mutations in γ-aminobutyric acid type A receptor (GABAA -R) subunit genes contribute to the etiology of rolandic epilepsy (RE) or its atypical variants (ARE)., Methods: We performed exome sequencing to compare the frequency of variants in 18 GABAA -R genes in 204 European patients with RE/ARE versus 728 platform-matched controls. Identified GABRG2 variants were functionally assessed for protein stability, trafficking, postsynaptic clustering, and receptor function., Results: Of 18 screened GABAA -R genes, we detected an enrichment of rare variants in the GABRG2 gene in RE/ARE patients (5 of 204, 2.45%) in comparison to controls (1 of 723, 0.14%; odds ratio = 18.07, 95% confidence interval = 2.01-855.07, p = 0.0024, pcorr  = 0.043). We identified a GABRG2 splice variant (c.549-3T>G) in 2 unrelated patients as well as 3 nonsynonymous variations in this gene (p.G257R, p.R323Q, p.I389V). Functional assessment showed reduced surface expression of p.G257R and decreased GABA-evoked currents for p.R323Q. The p.G257R mutation displayed diminished levels of palmitoylation, a post-translational modification crucial for trafficking of proteins to the cell membrane. Enzymatically raised palmitoylation levels restored the surface expression of the p.G257R variant γ2 subunit., Interpretation: The statistical association and the functional evidence suggest that mutations of the GABRG2 gene may increase the risk of RE/ARE. Restoring the impaired membrane trafficking of some GABRG2 mutations by enhancing palmitoylation might be an interesting therapeutic approach to reverse the pathogenic effect of such mutants., (© 2015 American Neurological Association.)

Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as particular electroencephalographic changes, with or without seizures. Photosensitivity is prominent in a very rare epileptic encephalopathy due to de novo CHD2 mutations, but is also seen in epileptic encephalopathies due to other gene mutations. We determined whether CHD2 variation underlies photosensitivity in common epilepsies, specific photosensitive epilepsies and individuals with photosensitivity without seizures. We studied 580 individuals with epilepsy and either photosensitive seizures or abnormal photoparoxysmal response on electroencephalography, or both, and 55 individuals with photoparoxysmal response but no seizures. We compared CHD2 sequence data to publicly available data from 34 427 individuals, not enriched for epilepsy. We investigated the role of unique variants seen only once in the entire data set. We sought CHD2 variants in 238 exomes from familial genetic generalized epilepsies, and in other public exome data sets. We identified 11 unique variants in the 580 individuals with photosensitive epilepsies and 128 unique variants in the 34 427 controls: unique CHD2 variation is over-represented in cases overall (P = 2.17 × 10(-5)). Among epilepsy syndromes, there was over-representation of unique CHD2 variants (3/36 cases) in the archetypal photosensitive epilepsy syndrome, eyelid myoclonia with absences (P = 3.50 × 10(-4)). CHD2 variation was not over-represented in photoparoxysmal response without seizures. Zebrafish larvae with chd2 knockdown were tested for photosensitivity. Chd2 knockdown markedly enhanced mild innate zebrafish larval photosensitivity. CHD2 mutation is the first identified cause of the archetypal generalized photosensitive epilepsy syndrome, eyelid myoclonia with absences. Unique CHD2 variants are also associated with photosensitivity in common epilepsies. CHD2 does not encode an ion channel, opening new avenues for research into human cortical excitability., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.

Objective: We report a consanguineous family with 2 affected individuals whose clinical symptoms closely resembled MERRF (myoclonus epilepsy with ragged red fibers) syndrome including severe myoclonic epilepsy, progressive spastic tetraparesis, progressive impairment of vision and hearing, as well as progressive cognitive decline., Methods: After excluding the presence of pathogenic mitochondrial DNA mutations, whole-exome sequencing of blood DNA from the index patient was performed. Detected homozygous mutations and their cosegregation were confirmed by Sanger sequencing. CARS2 (cysteinyl-tRNA synthetase 2, mitochondrial) messenger RNA analysis was performed by reverse transcription PCR and sequencing., Results: We identified a homozygous c.655G>A mutation in the CARS2 gene cosegregating in the family. The mutation is localized at the last nucleotide of exon 6 and thus is predicted to cause aberrant splicing. Analysis of the CARS2 messenger RNA showed that the presence of the mutation resulted in removal of exon 6. This leads to an in-frame deletion of 28 amino acids in a conserved sequence motif of the protein involved in stabilization of the acceptor end hairpin of tRNA(Cys)., Conclusion: CARS2 is a novel disease gene associated with a severe progressive myoclonic epilepsy most resembling MERRF syndrome., (© 2014 American Academy of Neurology.)

Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes.

Mutations in SCN1A and other ion channel genes can cause different epileptic phenotypes, but the precise mechanisms underlying the development of hyperexcitable networks are largely unknown. Here, we present a multisystem analysis of an SCN1A mouse model carrying the NaV1.1-R1648H mutation, which causes febrile seizures and epilepsy in humans. We found a ubiquitous hypoexcitability of interneurons in thalamus, cortex, and hippocampus, without detectable changes in excitatory neurons. Interestingly, somatic Na(+) channels in interneurons and persistent Na(+) currents were not significantly changed. Instead, the key mechanism of interneuron dysfunction was a deficit of action potential initiation at the axon initial segment that was identified by analyzing action potential firing. This deficit increased with the duration of firing periods, suggesting that increased slow inactivation, as recorded for recombinant mutated channels, could play an important role. The deficit in interneuron firing caused reduced action potential-driven inhibition of excitatory neurons as revealed by less frequent spontaneous but not miniature IPSCs. Multiple approaches indicated increased spontaneous thalamocortical and hippocampal network activity in mutant mice, as follows: (1) more synchronous and higher-frequency firing was recorded in primary neuronal cultures plated on multielectrode arrays; (2) thalamocortical slices examined by field potential recordings revealed spontaneous activities and pathological high-frequency oscillations; and (3) multineuron Ca(2+) imaging in hippocampal slices showed increased spontaneous neuronal activity. Thus, an interneuron-specific generalized defect in action potential initiation causes multisystem disinhibition and network hyperexcitability, which can well explain the occurrence of seizures in the studied mouse model and in patients carrying this mutation., (Copyright © 2014 the authors 0270-6474/14/3414874-16$15.00/0.)

Mutations in SLC2A1, encoding the glucose transporter type 1 (Glut1), cause a wide range of neurological disorders: (1) classical Glut1 deficiency syndrome (Glut1-DS) with an early onset epileptic encephalopathy including a severe epilepsy, psychomotor delay, ataxia and microcephaly, (2) paroxysmal exercise-induced dyskinesia (PED) and (3) various forms of idiopathic/genetic generalized epilepsies such as different forms of absence epilepsies. Up to now, focal epilepsy was not associated with SLC2A1 mutations. Here, we describe four cases in which focal seizures present the main or at least initial category of seizures. Two patients suffered from a classical Glut1-DS, whereas two individuals presented with focal epilepsy related to PED. We identified three novel SLC2A1 mutations in these unrelated individuals. Our study underscores that focal epilepsy can be caused by SLC2A1 mutations or that focal seizures may present the main type of seizures. Patients with focal epilepsy and PED should undergo genetic testing and can benefit from a ketogenic diet. But also individuals with pharmaco-resistant focal epilepsy and cognitive impairment might be candidates for genetic testing in SLC2A1.

Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human β1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-clamp analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, dampened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease., (© The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Recent studies reported DEPDC5 loss-of-function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n=11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes., (© 2014 American Neurological Association.)

Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10(-9), odds ratio (A) = 1.42, 95% confidence interval: 1.26-1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures.

Partial deletions of the gene encoding the neuronal splicing regulator RBFOX1 have been reported in a range of neurodevelopmental diseases, including idiopathic generalized epilepsy. The RBFOX1 protein and its homologues (RBFOX2 and RBFOX3) regulate alternative splicing of many neuronal transcripts involved in the homeostatic control of neuronal excitability. In this study, we explored if structural microdeletions and exonic sequence variations in RBFOX1, RBFOX2, RBFOX3 confer susceptibility to rolandic epilepsy (RE), a common idiopathic focal childhood epilepsy. By high-density SNP array screening of 289 unrelated RE patients, we identified two hemizygous deletions, a 365 kb deletion affecting two untranslated 5'-terminal exons of RBFOX1 and a 43 kb deletion spanning exon 3 of RBFOX3. Exome sequencing of 242 RE patients revealed two novel probably deleterious variants in RBFOX1, a frameshift mutation (p.A233Vfs*74) and a hexanucleotide deletion (p.A299_A300del), and a novel nonsense mutation in RBFOX3 (p.Y287*). Although the three variants were inherited from unaffected parents, they were present in all family members exhibiting the RE trait clinically or electroencephalographically with only one exception. In contrast, no deleterious mutations of RBFOX1 and RBFOX3 were found in the exomes of 6503 non-RE subjects deposited in the Exome Variant Server database. The observed RBFOX3 exon 3 deletion and nonsense mutation suggest that RBFOX3 represents a novel risk factor for RE, indicating that exon deletions and truncating mutations of RBFOX1 and RBFOX3 contribute to the genetic variance of partial and generalized idiopathic epilepsy syndromes.

Idiopathic focal epilepsy (IFE) with rolandic spikes is the most common childhood epilepsy, comprising a phenotypic spectrum from rolandic epilepsy (also benign epilepsy with centrotemporal spikes, BECTS) to atypical benign partial epilepsy (ABPE), Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and waves during slow-wave sleep (CSWS). The genetic basis is largely unknown. We detected new heterozygous mutations in GRIN2A in 27 of 359 affected individuals from 2 independent cohorts with IFE (7.5%; P = 4.83 × 10(-18), Fisher's exact test). Mutations occurred significantly more frequently in the more severe phenotypes, with mutation detection rates ranging from 12/245 (4.9%) in individuals with BECTS to 9/51 (17.6%) in individuals with CSWS (P = 0.009, Cochran-Armitage test for trend). In addition, exon-disrupting microdeletions were found in 3 of 286 individuals (1.0%; P = 0.004, Fisher's exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE.

Purpose: The group of the rare progressive myoclonic epilepsies (PME) include a wide spectrum of mitochondrial and metabolic diseases. In juvenile and adult ages, MERRF (myoclonic epilepsy with ragged red fibres) is the most common form. The underlying genetic defect in most patients with the syndrome of MERRF is a mutation in the tRNALys gene, but mutations were also detected in the tRNAPhe gene., Method: Here, we describe a 40 year old patient with prominent myoclonic seizures since 39 years of age without a mutation in the known genes who underwent intensive clinical, genetic and functional workup., Results: The patient had a slight mental retardation and a severe progressive hearing loss based on a defect of the inner ear on both sides. Ictal electroencephalography (EEG) showed bilateral occipital and generalized spikes and polyspikes induced and aggravated by photostimulation. A cranial magnetic resonance imaging (cMRI) detected a global cortical atrophy of the brain and mild periventricular white matter lesions. The electromyography (EMG) was normal but the muscle biopsy showed abundant ragged red fibres. Sequencing of the mitochondrial DNA from the skeletal muscle biopsy revealed a novel heteroplasmic mutation (m.4279A>G) in the tRNAIle gene which was functionally relevant as tested in single skeletal muscle fibre investigations., Conclusion: Mutations in tRNAIle were described in patients with chronic progressive external ophthalmoplegia (CPEO), prominent deafness or cardiomyopathy but, up to now, not in patients with myoclonic epilepsy. The degree of heteroplasmy of this novel mitochondrial DNA mutation was 70% in skeletal muscle but only 15% in blood, pointing to the diagnostic importance of a skeletal muscle biopsy also in patients with myoclonic epilepsy., (Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Recent studies reported mutations in the gene encoding the proline-rich transmembrane protein 2 (PRRT2) to be causative for paroxysmal kinesigenic dyskinesia (PKD), PKD combined with infantile seizures (ICCA), and benign familial infantile seizures (BFIS). PRRT2 is a presynaptic protein which seems to play an important role in exocytosis and neurotransmitter release. PKD is the most common form of paroxysmal movement disorder characterized by recurrent brief involuntary hyperkinesias triggered by sudden movements. Here, we sequenced PRRT2 in 14 sporadic and 8 familial PKD and ICCA cases of Caucasian origin and identified three novel mutations (c.919C>T/p.Gln307, c.388delG/p.Ala130Profs 46, c.884G>A/p.Arg295Gln) predicting two truncated proteins and one probably damaging point mutation. A review of all published cases is also included. PRRT2 mutations occur more frequently in familial forms of PRRT2-related syndromes (80-100 %) than in sporadic cases (33-46 %) suggesting further heterogeneity in the latter. PRRT2 mutations were rarely described in other forms of paroxysmal dyskinesias deviating from classical PKD, as we report here in one ICCA family without kinesigenic triggers. Mutations are exclusively found in two exons of the PRRT2 gene at a high rate across all syndromes and with one major mutation (c.649dupC) in a mutational hotspot of nine cytosines, which is responsible for 57 % of all cases in all phenotypes. We therefore propose that genetic analysis rapidly performed in early stages of the disease is highly cost-effective and can help to avoid further unnecessary diagnostic and therapeutic interventions.

Benign familial neonatal seizures (BFNS) are a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels K(V) 7.2 and K(V) 7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of K(V) 7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-of-function with a dominant-negative effect of the mutant on WT K(V) 7.2 and K(V) 7.3 channels. Since single-channel recordings of K(V) 7.3-K(V) 7.2 and K(V) 7.3-N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells-either at lower temperatures of <30°C or in presence of the agonist retigabine (RTG)-the N258S-derived currents increased fivefold in contrast to the WT. The obtained results represent a first example of temperature and pharmacological rescue of a K(V) 7 mutation and suggest a folding and trafficking deficiency as the cause of reduced current amplitudes with a dominant-negative effect of N258S mutant proteins., (© 2011 Wiley-Liss, Inc.)

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the beta1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type beta1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the beta1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with beta1(C121W) subunits. We therefore conclude that Na+ channel beta1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.

Many idiopathic epilepsy syndromes have a characteristic age dependence, the underlying molecular mechanisms of which are largely unknown. Here we propose a mechanism that can explain that epileptic spells in benign familial neonatal-infantile seizures occur almost exclusively during the first days to months of life. Benign familial neonatal-infantile seizures are caused by mutations in the gene SCN2A encoding the voltage-gated Na(+) channel Na(V)1.2. We identified two novel SCN2A mutations causing benign familial neonatal-infantile seizures and analysed the functional consequences of these mutations in a neonatal and an adult splice variant of the human Na(+) channel Na(V)1.2 expressed heterologously in tsA201 cells together with beta1 and beta2 subunits. We found significant gating changes leading to a gain-of-function, such as an increased persistent Na(+) current, accelerated recovery from fast inactivation or altered voltage-dependence of steady-state activation. Those were restricted to the neonatal splice variant for one mutation, but more pronounced for the adult form for the other, suggesting that a differential developmental splicing does not provide a general explanation for seizure remission. We therefore analysed the developmental expression of Na(V)1.2 and of another voltage-gated Na(+) channel, Na(V)1.6, using immunohistochemistry and real-time reverse transcription-polymerase chain reaction in mouse brain slices. We found that Na(V)1.2 channels are expressed early in development at axon initial segments of principal neurons in the hippocampus and cortex, but their expression is diminished and they are gradually replaced as the dominant channel type by Na(V)1.6 during maturation. This finding provides a plausible explanation for the transient expression of seizures that occur due to a gain-of-function of mutant Na(V)1.2 channels.

Idiopathic epilepsies are considered to be genetically determined. The inheritance can be monogenic and the detected mutation considered sufficient to cause the phenotype. In contrast, when the inheritance is complex, the epileptic phenotype is determined by several minor genetic defects that are much more difficult to discover. In recent years, an increasing number of mutations, mainly associated with rare monogenic idiopathic epilepsy syndromes, have been identified in genes encoding subunits of voltage- or ligand-gated ion channels. A few mutations have also been found in the frequent classical forms of idiopathic generalized epilepsies which are thought to follow a complex genetic trait, for example, in absence or juvenile myoclonic epilepsies. Functional studies characterizing the molecular defects of the mutant channels point to an important role of GABAergic synaptic inhibition in the pathophysiology of idiopathic epilepsies. As a result of genetic and functional investigations, not only will the pathophysiology of epilepsy be better understood, but newly discovered genes and pathophysiological pathways may also determine novel targets for pharmacotherapy, as has been shown for the anticonvulsant drug retigabine, which enhances the activity of neuronal KCNQ potassium channels.

Paroxysmal exercise-induced dyskinesia (PED) can occur in isolation or in association with epilepsy, but the genetic causes and pathophysiological mechanisms are still poorly understood. We performed a clinical evaluation and genetic analysis in a five-generation family with co-occurrence of PED and epilepsy (n = 39), suggesting that this combination represents a clinical entity. Based on a whole genome linkage analysis we screened SLC2A1, encoding the glucose transporter of the blood-brain-barrier, GLUT1 and identified heterozygous missense and frameshift mutations segregating in this and three other nuclear families with a similar phenotype. PED was characterized by choreoathetosis, dystonia or both, affecting mainly the legs. Predominant epileptic seizure types were primary generalized. A median CSF/blood glucose ratio of 0.52 (normal >0.60) in the patients and a reduced glucose uptake by mutated transporters compared with the wild-type as determined in Xenopus oocytes confirmed a pathogenic role of these mutations. Functional imaging studies implicated alterations in glucose metabolism in the corticostriate pathways in the pathophysiology of PED and in the frontal lobe cortex in the pathophysiology of epileptic seizures. Three patients were successfully treated with a ketogenic diet. In conclusion, co-occurring PED and epilepsy can be due to autosomal dominant heterozygous SLC2A1 mutations, expanding the phenotypic spectrum associated with GLUT1 deficiency and providing a potential new treatment option for this clinical syndrome.

Voltage-gated K+channels of the K(V)7 (KCNQ) family have been identified in the last 10-15 years by discovering the causative genes for three autosomal dominant diseases: cardiac arrhythmia (long QT syndrome) with or without congenital deafness (KCNQ1), a neonatal epilepsy (KCNQ2 and KCNQ3) and progressive deafness alone (KCNQ4). A fifth member of this gene family (KCNQ5) is not affected in a disease so far. Four genes (KCNQ2-5) are expressed in the nervous system. This review is focused on recent findings on the neuronal K(V)7 channelopathies, in particular on benign familial neonatal seizures (BFNS) and peripheral nerve hyperexcitability (PNH, neuromyotonia, myokymia) caused by KCNQ2 mutations. The phenotypic spectrum associated with KCNQ2 mutations is probably broader than initially thought, as patients with severe epilepsies and developmental delay, or with Rolando epilepsy have been described. With regard to the underlying molecular pathophysiology, it has been shown that mutations with very subtle changes restricted to subthreshold voltages can cause BFNS thereby proving in a human disease model that this is the relevant voltage range for these channels to modulate neuronal firing. The two mutations associated with PNH induce much more severe channel dysfunction with a dominant negative effect on wild type (WT) channels. Finally, K(V)7 channels present interesting targets for new therapeutic approaches to diseases caused by neuronal hyperexcitability, such as epilepsy, neuropathic pain, and migraine. The molecular mechanism of K(V)7 activation by retigabine, which is in phase III clinical testing to treat pharmacoresistant focal epilepsies, has been recently elucidated as a stabilization of the open conformation by binding to the pore region.

The voltage-gated potassium channels KV7.2 and KV7.3 (genes KCNQ2 and KCNQ3) constitute a major component of the M-current controlling the firing rate in many neurons. Mutations within these two channel subunits cause benign familial neonatal convulsions (BFNC). Here we identified a novel BFNC-causing mutation (E119G) in the S1-S2 region of KV7.2. Electrophysiological investigations in Xenopus oocytes using two-microelectrode voltage clamping revealed that the steady-state activation curves for E119G alone and its coexpressions with KV7.2 and/or KV7.3 wild-type (WT) channels were significantly shifted in the depolarizing direction compared to KV7.2 or KV7.2/KV7.3. These shifts reduced the relative current amplitudes for mutant channels particularly in the subthreshold range of an action potential (about 45% reduction at --50 mV for E119G compared to KV7.2, and 33% for E119G/KV7.3 compared to KV7.2/KV7.3 channels). Activation kinetics were significantly slowed for mutant channels. Our results indicate that small changes in channel gating at subthreshold voltages are sufficient to cause neonatal seizures and demonstrate the importance of the M-current for this voltage range. This was confirmed by a computer model predicting an increased burst duration for the mutation. On a molecular level, these results reveal a critical role in voltage sensing of the negatively charged E119 in S1-S2 of KV7.2, a region that-- according to molecular modelling - might interact with a positive charge in the S4 segment.

Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy, the idiopathic generalized epilepsies. Although the mode of inheritance is usually complex, mutations in single genes have been shown to cause the disease in some families with autosomal dominant inheritance. The first mutation in a multigeneration JME family has been recently found in the alpha1-subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing alpha1-, beta2- and gamma2-subunits in human embryonic kidney (HEK293) cells. By using an ultrafast application system, mutant receptors have shown reduced macroscopic current amplitudes at saturating GABA concentrations and a highly reduced affinity to GABA compared to the wild-type (WT). Dose-response curves for current amplitudes, activation kinetics, and GABA-dependent desensitization parameters showed a parallel shift towards 30- to 40-fold higher GABA concentrations. Both deactivation and resensitization kinetics were considerably accelerated in mutant channels. In addition, mutant receptors labelled with enhanced green fluorescent protein (EGFP) were not integrated in the cell membrane, in contrast to WT receptors. Therefore, the A322D mutation leads to a severe loss-of-function of the human GABAA receptor by several mechanisms, including reduced surface expression, reduced GABA-sensitivity, and accelerated deactivation. These molecular defects could decrease and shorten the resulting inhibitory postsynaptic currents (IPSCs) in vivo, which can induce a hyperexcitability of the postsynaptic membrane and explain the occurrence of epileptic seizures.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.

Hypokalaemic periodic paralysis (hypoPP) is a dominantly inherited muscle disorder characterized by episodes of flaccid weakness. Previous genetic studies revealed mutations in the voltage-gated calcium channel alpha1-subunit (CACNA1S gene) in families with hypoPP (type I). Electrophysiological studies on these mutants in different expression systems could not explain the pathophysiology of the disease. In addition, several mutations (Arg669His, Arg672His, Arg672Gly and Arg672Ser) in the voltage sensor of the skeletal muscle sodium channel alpha-subunit (SCN4A gene) have been found in families with hypoPP (type II). For Arg672Gly/His a fast inactivation defect was described, and for Arg669His an impairment of slow inactivation was reported. Except for the substitution for serine, we have now expressed all mutants in a human cell-line and studied them electrophysiologically. Patch-clamp recordings show an enhanced fast inactivation for all three mutations, whereas two of them reveal enhanced slow inactivation. This may reduce the number of functional sodium channels at resting membrane potential and contribute to the long-lasting periods of paralysis experienced by hypoPP patients. The gating of both histidine mutants (Arg669His, Arg672His) can be modulated by changes of extra- or intracellular pH. The inactivation defects of Arg669His and Arg672His can be alleviated by low pH to a significant degree, suggesting that the decrease of pH in muscle cells (e.g. during muscle work) might lead to an auto-compensation of functional defects. This may explain a delay or prevention of paralytic attacks in patients by slight physical activity. Moreover, the histidine residues may be the target for a potential therapeutic action by acetazolamide.

OBJECTIVE: The aim of this study was to expand the spectrum of epilepsy syndromes related to STX1B, encoding the presynaptic protein syntaxin-1B, and establish genotype-phenotype correlations by identifying further disease-related variants. METHODS: We used next-generation sequencing in the framework of research projects and diagnostic testing. Clinical data and EEGs were reviewed, including already published cases. To estimate the pathogenicity of the variants, we used established and newly developed in silico prediction tools. RESULTS: We describe 17 new variants in STX1B, which are distributed across the whole gene. We discerned 4 different phenotypic groups across the newly identified and previously published patients (49 patients in 23 families): (1) 6 sporadic patients or families (31 affected individuals) with febrile and afebrile seizures with a benign course, generally good drug response, normal development, and without permanent neurologic deficits; (2) 2 patients with genetic generalized epilepsy without febrile seizures and cognitive deficits; (3) 13 patients or families with intractable seizures, developmental regression after seizure onset and additional neuropsychiatric symptoms; (4) 2 patients with focal epilepsy. More often, we found loss-of-function mutations in benign syndromes, whereas missense variants in the SNARE motif of syntaxin-1B were associated with more severe phenotypes. CONCLUSION: These data expand the genetic and phenotypic spectrum of STX1B-related epilepsies to a diverse range of epilepsies that span the International League Against Epilepsy classification. Variants in STX1B are protean and contribute to many different epilepsy phenotypes, similar to SCN1A, the most important gene associated with fever-associated epilepsies.

Emerging evidence indicates that epileptic encephalopathies are genetically highly heterogeneous, underscoring the need for large cohorts of well-characterized individuals to further define the genetic landscape. Through a collaboration between two consortia (EuroEPINOMICS and Epi4K/EPGP), we analyzed exome-sequencing data of 356 trios with the "classical" epileptic encephalopathies, infantile spasms and Lennox Gastaut syndrome, including 264 trios previously analyzed by the Epi4K/EPGP consortium. In this expanded cohort, we find 429 de novo mutations, including de novo mutations in DNM1 in five individuals and de novo mutations in GABBR2, FASN, and RYR3 in two individuals each. Unlike previous studies, this cohort is sufficiently large to show a significant excess of de novo mutations in epileptic encephalopathy probands compared to the general population using a likelihood analysis (p = 8.2 × 10(-4)), supporting a prominent role for de novo mutations in epileptic encephalopathies. We bring statistical evidence that mutations in DNM1 cause epileptic encephalopathy, find suggestive evidence for a role of three additional genes, and show that at least 12% of analyzed individuals have an identifiable causal de novo mutation. Strikingly, 75% of mutations in these probands are predicted to disrupt a protein involved in regulating synaptic transmission, and there is a significant enrichment of de novo mutations in genes in this pathway in the entire cohort as well. These findings emphasize an important role for synaptic dysregulation in epileptic encephalopathies, above and beyond that caused by ion channel dysfunction.

Rolandic epilepsy (RE) and its atypical variants (atypical rolandic epilepsy, ARE) along the spectrum of epilepsy-aphasia disorders are characterized by a strong but largely unknown genetic basis. Two genes with a putative ( ELP4) or a proven ( SRPX2) function in neuronal migration were postulated to confer susceptibility to parts of the disease spectrum: the ELP4 gene to centrotemporal spikes and SRPX2 to ARE. To reexamine these findings, we investigated a cohort of 280 patients of European ancestry with RE/ARE for the etiological contribution of these genes and their close interaction partners. We performed next-generation sequencing and single-nucleotide polymorphism (SNP)-array based genotyping to screen for sequence and structural variants. In comparison to European controls we could not detect an enrichment of rare deleterious variants of ELP4, SRPX2, or their interaction partners in affected individuals. The previously described functional p.N327S variant in the X chromosomal SRPX2 gene was detected in two affected individuals (0.81%) and also in controls (0.26%), with some preponderance of male patients. We did not detect an association of SNPs in the ELP4 gene with centrotemporal spikes as previously reported. In conclusion our data do not support a major role of ELP4 and SRPX2 in the etiology of RE/ARE. A PowerPoint slide summarizing this article is available for download in the Supporting Information section . [ABSTRACT FROM AUTHOR]

Objective: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation We identified 8 known missense mutations, previously reported as pathogenic, in a total of 17 unrelated epilepsy patients (17/448; 3.80%). Our re-evaluation indicates that 7 out of these 8 variants (p.R27T; p.R28C; p.R542Q; p.R604H; p.T1250M; p.E1308D; p.R1928G; NP_001159435.1) are not pathogenic. Only the p.T1174S mutation may be considered as a genetic risk factor for epilepsy of small effect size based on the enrichment in patients (P = 6.60 x 10−4; OR = 0.32, fishers exact test), previous functional studies but incomplete penetrance. Thus, incorporation of previous studies in genetic counseling of SCN1A sequencing results is challenging and may produce incorrect conclusions.