To investigate relationship between treatment efficiency and EEG background activity changes in absence epilepsy (AE) and juvenile myoclonic epilepsy (JME) patients.

Although approximately 10%-15% of patients with idiopathic generalized epilepsy (IGE)/genetic generalized epilepsy remain drug-resistant, there is no consensus or established concept regarding the underlying mechanisms and prevalence. This review summarizes the recent data and the current hypotheses on mechanisms that may contribute to drug-resistant IGE. A literature search was conducted in PubMed and Embase for studies on mechanisms of drug resistance published since 1980. The literature shows neither consensus on the definition nor a widely accepted model to explain drug resistance in IGE or one of its subsyndromes. Large-scale genetic studies have failed to identify distinct genetic causes or affected genes involved in pharmacokinetics. We found clinical and experimental evidence in support of four hypotheses: (1) "network hypothesis"-the degree of drug resistance in IGE reflects the severity of cortical network alterations, (2) "minor focal lesion in a predisposed brain hypothesis"-minor cortical lesions are important for drug resistance, (3) "interneuron hypothesis"-impaired functioning of γ-aminobutyric acidergic interneurons contributes to drug resistance, and (4) "changes in drug kinetics"-genetically impaired kinetics of antiseizure medication (ASM) reduce the effectiveness of available ASMs. In summary, the exact definition and cause of drug resistance in IGE is unknown. However, published evidence suggests four different mechanisms that may warrant further investigation.

Idiopathic generalized epilepsies (IGE) are a group of frequent age-related epilepsy syndromes. IGE are clinically characterized by generalized tonic-clonic, myoclonic and absence seizures. According to predominant seizure type and age of onset, IGE are divided in subsyndromes: childhood absence and juvenile absence epilepsy (AE), juvenile myoclonic epilepsy (JME) and generalized tonic-clonic seizures on awakening (GTCS). The limits between these subsyndromes are not well defined, supporting the existence of only one major syndrome. Visual assessment of routine magnetic resonance imaging (MRI) in patients with IGE is normal. MRI voxel-based morphometry (VBM) uses automatically segmented gray and white matter for comparisons, eliminating the investigator bias. We used VBM to study 120 individuals (47 controls, 44 with JME, 24 with AE and 15 with GTCS) to investigate the presence of subtle structural abnormalities in IGE subsyndromes. VBM was performed searching for abnormalities on gray matter concentration (GMC) between patients groups and controls. Compared to controls, JME presented increased GMC in frontobasal region and AE showed increased GMC in the superior mesiofrontal region. The GTCS group did not differ from controls. There were no areas of reduced GMC with the statistical level selected. Region of interest analysis showed increased GMC in the anterior portion of the thalamus in patients with absence seizures. Our results support subtle GMC abnormalities in patients with JME and AE when compared to controls. These findings suggest the existence of different patterns of cortical abnormalities in IGE subsyndromes.

Severe neonatal epilepsies with suppression-burst pattern are epileptic syndromes with either neonatal onset or onset during the first months of life. These disorders are characterized by a typical electroencephalogram pattern--namely, suppression burst, in which higher-voltage bursts of slow waves mixed with multifocal spikes alternate with isoelectric suppression phases. Here, we report the genetic mapping of an autosomal recessive form of this condition to chromosome 11p15.5 and the identification of a missense mutation (p.Pro206Leu) in the gene encoding one of the two mitochondrial glutamate/H(+) symporters (SLC25A22, also known as "GC1"). The mutation cosegregated with the disease and altered a highly conserved amino acid. Functional analyses showed that glutamate oxidation in cultured skin fibroblasts from patients was strongly defective. Further studies in reconstituted proteoliposomes showed defective [(14)C]glutamate uniport and [(14)C]glutamate/glutamate exchange by mutant protein. Moreover, expression studies showed that, during human development, SLC25A22 is specifically expressed in the brain, within territories proposed to contribute to the genesis and control of myoclonic seizures. These findings provide the first direct molecular link between glutamate mitochondrial metabolism and myoclonic epilepsy and suggest potential insights into the pathophysiological bases of severe neonatal epilepsies with suppression-burst pattern.

Although the genetic and clinical aspects of epilepsy with myoclonic-atonic seizures (MAE) and early onset absence epilepsy (EOAE) have been investigated thoroughly, other early childhood-onset generalized epilepsies that share clinical features with MAE and EOAE have not been characterized. In this study, we aimed to delineate the genetic and phenotypic spectrum of early childhood-onset generalized epilepsies, including MAE and EOAE.

To report longitudinal clinical, EEG, and MRI findings in 2 sisters carrying compound heterozygous ARV1 mutations and exhibiting a peculiar form of developmental and epileptic encephalopathy (DEE). Neuropathologic features are also described in one of the sisters.

Developmental epilepsies are age-dependent seizure disorders for which genetic causes have been increasingly identified. Here we report six unrelated individuals with mutations in salt-inducible kinase 1 (SIK1) in a series of 101 persons with early myoclonic encephalopathy, Ohtahara syndrome, and infantile spasms. Individuals with SIK1 mutations had short survival in cases with neonatal epilepsy onset, and an autism plus developmental syndrome after infantile spasms in others. All six mutations occurred outside the kinase domain of SIK1 and each of the mutants displayed autophosphorylation and kinase activity toward HDAC5. Three mutations generated truncated forms of SIK1 that were resistant to degradation and also showed changes in sub-cellular localization compared to wild-type SIK1. We also report the human neuropathologic examination of SIK1-related developmental epilepsy, with normal neuronal morphology and lamination but abnormal SIK1 protein cellular localization. Therefore, these results expand the genetic etiologies of developmental epilepsies by demonstrating SIK1 mutations as a cause of severe developmental epilepsy.

Juvenile myoclonic epilepsy (JME) is one of the most common generalized idiopathic epilepsies of childhood and adolescence. In some patients with JME, mathematical calculus and praxis may induce myoclonic seizures.

Idiopathic epilepsies (IEs) are a group of disorders characterized by recurrent seizures in the absence of detectable brain lesions or metabolic abnormalities. IEs include common disorders with a complex mode of inheritance and rare Mendelian traits suggesting the occurrence of several alleles with variable penetrance. We previously described a large family with a recessive form of idiopathic epilepsy, named familial infantile myoclonic epilepsy (FIME), and mapped the disease locus on chromosome 16p13.3 by linkage analysis. In the present study, we found that two compound heterozygous missense mutations (D147H and A509V) in TBC1D24, a gene of unknown function, are responsible for FIME. In situ hybridization analysis revealed that Tbc1d24 is mainly expressed at the level of the cerebral cortex and the hippocampus. By coimmunoprecipitation assay we found that TBC1D24 binds ARF6, a Ras-related family of small GTPases regulating exo-endocytosis dynamics. The main recognized function of ARF6 in the nervous system is the regulation of dendritic branching, spine formation, and axonal extension. TBC1D24 overexpression resulted in a significant increase in neurite length and arborization and the FIME mutations significantly reverted this phenotype. In this study we identified a gene mutation involved in autosomal-recessive idiopathic epilepsy, unveiled the involvement of ARF6-dependent molecular pathway in brain hyperexcitability and seizures, and confirmed the emerging role of subtle cytoarchitectural alterations in the etiology of this group of common epileptic disorders.

MRI studies of genetic generalized epilepsies have mainly described group-level changes between patients and healthy controls. To determine the endophenotypic potential of structural MRI in juvenile myoclonic epilepsy (JME), we examined MRI-based cortical morphologic markers in patients and their healthy siblings.

TUBA1A tubulinopathy is a rare neurodevelopmental disorder associated with brain malformations as well as early-onset and intractable epilepsy. As pathomechanisms and genotype-phenotype correlations are not completely understood, we aimed to provide further insights into the phenotypic and genetic spectrum. We here present a multicenter case series of ten unrelated individuals from four European countries using systematic MRI re-evaluation, protein structure analysis, and prediction score modeling. In two cases, pregnancy was terminated due to brain malformations. Amongst the eight living individuals, the phenotypic range showed various severity. Global developmental delay and severe motor impairment with tetraparesis was present in 63% and 50% of the subjects, respectively. Epilepsy was observed in 75% of the cases, which showed infantile onset in 83% and a refractory course in 50%. One individual presented a novel TUBA1A-associated electroclinical phenotype with evolvement from early myoclonic encephalopathy to continuous spike-and-wave during sleep. Neuroradiological features comprised a heterogeneous spectrum of cortical and extracortical malformations including rare findings such as cobblestone lissencephaly and subcortical band heterotopia. Two individuals developed hydrocephalus with subsequent posterior infarction. We report four novel and five previously published TUBA1A missense variants whose resulting amino acid substitutions likely affect longitudinal, lateral, and motor protein interactions as well as GTP binding. Assessment of pathogenic and benign variant distributions in synopsis with prediction scores revealed sections of variant enrichment and intolerance to missense variation. We here extend the clinical, neuroradiological, and genetic spectrum of TUBA1A tubulinopathy and provide insights into residue-specific pathomechanisms and genotype-phenotype correlations.

Genetic generalized epilepsies (GGEs) include well-established epilepsy syndromes with generalized onset seizures: childhood absence epilepsy, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy (JAE), myoclonic absence epilepsy, epilepsy with eyelid myoclonia (Jeavons syndrome), generalized tonic-clonic seizures, and generalized tonic-clonic seizures alone. Genome-wide association studies (GWASs) and exome sequencing have identified 48 single-nucleotide polymorphisms (SNPs) associated with GGE. However, these studies were mainly based on non-admixed, European, and Asian populations. Thus, it remains unclear whether these results apply to patients of other origins. This study aims to evaluate whether these previous results could be replicated in a cohort of admixed Brazilian patients with GGE. We obtained SNP-array data from 87 patients with GGE, compared with 340 controls from the BIPMed public dataset. We could directly access genotypes of 17 candidate SNPs, available in the SNP array, and the remaining 31 SNPs were imputed using the BEAGLE v5.1 software. We performed an association test by logistic regression analysis, including the first five principal components as covariates. Furthermore, to expand the analysis of the candidate regions, we also interrogated 14,047 SNPs that flank the candidate SNPs (1 Mb). The statistical power was evaluated in terms of odds ratio and minor allele frequency (MAF) by the genpwr package. Differences in SNP frequencies between Brazilian and Europeans, sub-Saharan African, and Native Americans were evaluated by a two-proportion Z-test. We identified nine flanking SNPs, located on eight candidate regions, which presented association signals that passed the Bonferroni correction (rs12726617; rs9428842; rs1915992; rs1464634; rs6459526; rs2510087; rs9551042; rs9888879; and rs8133217; p-values <3.55e-06). In addition, the two-proportion Z-test indicates that the lack of association of the remaining candidate SNPs could be due to different genomic backgrounds observed in admixed Brazilians. This is the first time that candidate SNPs for GGE are analyzed in an admixed Brazilian population, and we could successfully replicate the association signals in eight candidate regions. In addition, our results provide new insights on how we can account for population structure to improve risk stratification estimation in admixed individuals.

The progressive myoclonic epilepsies (PMEs) are a group of rare neurodegenerative diseases characterized by myoclonus, epileptic seizures, and progressive neurological deterioration with cerebellar involvement. They include storage diseases like Gaucher disease, Lafora disease, and forms of neuronal ceroid lipofuscinosis (NCL). To date, 13 NCLs have been reported (CLN1-CLN8, CLN10-CLN14), associated with mutations in different genes. These forms, which affect both children and adults, are characterized by seizures, cognitive and motor impairments, and in most cases visual loss. In NCLs, as in other PMEs, central nervous system (CNS) neurodegeneration is widespread and involves different subpopulations of neurons. One of the most affected regions is the cerebellar cortex, where motor and non-motor information is processed and transmitted to deep cerebellar nuclei through the axons of Purkinje cells (PCs). PCs, being GABAergic, have an inhibitory effect on their target neurons, and provide the only inhibitory output of the cerebellum. Degeneration of PCs has been linked to motor impairments and epileptic seizures. Seizures occur when some insult upsets the normal balance in the CNS between excitatory and inhibitory impulses, causing hyperexcitability. Here we review the role of PCs in epilepsy onset and progression following their PME-related loss. In particular, we focus on the involvement of PCs in seizure phenotype in NCLs, highlighting findings from case reports and studies of animal models in which epilepsy can be linked to PC loss.

Juvenile myoclonic epilepsy (JME), accounting for approximately 25% of idiopathic generalized epilepsies, is genetically heterogeneous. Mutations in the alpha-1 subunit of the GABAA receptor (GABRA1) and EFHC1 genes have been reported in a few families with autosomal dominant (AD) JME. We have investigated the contribution of these two genes to familial JME in our cohort of 54 JME Caucasian families. Syndromic classification of JME was based on previously published criteria. We considered kindreds with at least one affected first-degree relative and the evidence of a vertical transmission as definite AD JME, and families with at least one affected second-degree relative as probable AD JME. We included 33 families meeting criteria for definitive AD JME and 21 that were classified as probable AD JME. None of these families were considered informative enough to analyze candidate loci for JME using linkage analysis. We have systematically screened coding exons of these two genes using temperature gradient capillary electrophoresis. Every heteroduplex with an abnormal mobility was sequenced. No disease-causing mutations in the GABRA1 gene were identified. Analysis of EFHC1 gene found one putative disease-causing mutation R221H that was previously reported as a tandem mutation. Several synonymous and non-synonymous coding polymorphisms were identified but the allelic frequency did not differ between controls and affected individuals. Our data suggests that the majority of familial AD JME is not caused by mutations in the GABRA1 and EFHC1 genes.

Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies characterized by refractory seizures and developmental impairment. Sequencing approaches have identified causal genetic variants in only about 50% of individuals with DEEs.1-3 This suggests that unknown genetic etiologies exist, potentially in the ∼98% of human genomes not covered by exome sequencing (ES). Here we describe seven likely pathogenic variants in regions outside of the annotated coding exons of the most frequently implicated epilepsy gene, SCN1A, encoding the alpha-1 sodium channel subunit. We provide evidence that five of these variants promote inclusion of a "poison" exon that leads to reduced amounts of full-length SCN1A protein. This mechanism is likely to be broadly relevant to human disease; transcriptome studies have revealed hundreds of poison exons,4,5 including some present within genes encoding other sodium channels and in genes involved in neurodevelopment more broadly.6 Future research on the mechanisms that govern neuronal-specific splicing behavior might allow researchers to co-opt this system for RNA therapeutics.

Despite expanding next generation sequencing technologies and increasing clinical interest into complex neurologic phenotypes associating epilepsies and developmental/epileptic encephalopathies (DE/EE) with movement disorders (MD), these monogenic conditions have been less extensively investigated in the neonatal period compared to infancy. We reviewed the medical literature in the study period 2000-2020 to report on monogenic conditions characterized by neonatal onset epilepsy and/or DE/EE and development of an MD, and described their electroclinical, genetic and neuroimaging spectra. In accordance with a PRISMA statement, we created a data collection sheet and a protocol specifying inclusion and exclusion criteria. A total of 28 different genes (from 49 papers) leading to neonatal-onset DE/EE with multiple seizure types, mainly featuring tonic and myoclonic, but also focal motor seizures and a hyperkinetic MD in 89% of conditions, with neonatal onset in 22%, were identified. Neonatal seizure semiology, or MD age of onset, were not always available. The rate of hypokinetic MD was low, and was described from the neonatal period only, with WW domain containing oxidoreductase (WWOX) pathogenic variants. The outcome is characterized by high rates of associated neurodevelopmental disorders and microcephaly. Brain MRI findings are either normal or nonspecific in most conditions, but serial imaging can be necessary in order to detect progressive abnormalities. We found high genetic heterogeneity and low numbers of described patients. Neurological phenotypes are complex, reflecting the involvement of genes necessary for early brain development. Future studies should focus on accurate neonatal epileptic phenotyping, and detailed description of semiology and time-course, of the associated MD, especially for the rarest conditions.

Juvenile myoclonic epilepsy (JME) is the most common idiopathic generalized epilepsies (IGEs), affecting 12-30% of all epilepsies in medical centers. To date genetic linkage studies have revealed putative loci on different chromosomes, but these findings are still inconclusive about which gene precisely is responsible for the disease. Here, we report the genetic and clinical analysis of a (JME) consanguineous Tunisian family with four affected children out of eight. A genome-wide search was carried out by using the Affymetrix GeneChip Mapping 500K NspI chip. Pairewise logarithm of the odds (LOD) scores were calculated with MERLIN (1.1) assuming an autosomal recessive model, and a complementary homozygous mapping analysis was performed with AutoSNPa software. The genome-wide parametric linkage analysis showed suggestive linkage to chromosome 2q. Interactive visual analysis of SNP data using AutoSNPa revealed two large regions of shared homozygosity by descent on 2q23.3 and on 2q24.1. We decided to sequence the exons of the two genes coding for such proteins located in 2q23.3, CACNB4 and 2q24.1, KCNJ3. No nucleotide variation--comprising the previously reported mutations--was detected.

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements.

HCN channels are highly expressed and functionally relevant in neurons and increasing evidence demonstrates their involvement in the etiology of human epilepsies. Among HCN isoforms, HCN4 is important in cardiac tissue, where it underlies pacemaker activity. Despite being expressed also in deep structures of the brain, mutations of this channel functionally shown to be associated with epilepsy have not been reported yet. Using Next Generation Sequencing for the screening of patients with idiopathic epilepsy, we identified the p.Arg550Cys (c.1648C>T) heterozygous mutation on HCN4 in two brothers affected by benign myoclonic epilepsy of infancy. Functional characterization in heterologous expression system and in neurons showed that the mutation determines a loss of function of HCN4 contribution to activity and an increase of neuronal discharge, potentially predisposing to epilepsy. Expressed in cardiomyocytes, mutant channels activate at slightly more negative voltages than wild-type (WT), in accordance with borderline bradycardia. While HCN4 variants have been frequently associated with cardiac arrhythmias, these data represent the first experimental evidence that functional alteration of HCN4 can also be involved in human epilepsy through a loss-of-function effect and associated increased neuronal excitability. Since HCN4 appears to be highly expressed in deep brain structures only early during development, our data provide a potential explanation for a link between dysfunctional HCN4 and infantile epilepsy. These findings suggest that it may be useful to include HCN4 screening to extend the knowledge of the genetic causes of infantile epilepsies, potentially paving the way for the identification of innovative therapeutic strategies.

Genetic factors play a major role in the etiology of idiopathic generalized epilepsies (IGE). An oligogenic or polygenic predisposition is suspected in the majority of families with common IGE syndromes. It has been hypothesized that some IGE genes might increase the general level of neuronal excitability while others specify the age of onset and the seizure type. The EFHC1 gene on 6p12-p11 was previously described as the first susceptibility gene for juvenile myoclonic epilepsy (JME). EFHC1 codes for a protein of unknown function that is characterized by Ca2+-binding EF-hand motifs and DM10 domains. We have now cloned the brain-expressed paralog EFHC2 (Xp11.3) and carried out an association study of six single nucleotide polymorphisms (SNPs) in a large sample of 654 German IGE patients and 662 population controls. A tentative association was found between the amino acid exchange S430Y in exon 9 of EFHC2 and 97 male JME patients (chi2=4.705, d.f.=1, P=0.030; OR=2.17; 95-CI: 1.06-4.43). The allelic association was even stronger for the 81 males with "classical" JME (JME without absence seizures) (chi2=6.06, d.f.=1, P=0.014; OR=2.46; 95-CI: 1.18-5.13). An association with the gonosomal gene EFHC2 would be in accordance with the observed preponderance of maternal inheritance in JME maternal inheritance of JME. Independent replication studies are needed to further analyse the tentative association described here.