Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies.

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.

Linkage and association analysis of CACNG3 in childhood absence epilepsy.

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy characterised by absence seizures manifested by transitory loss of awareness with 2.5-4 Hz spike-wave complexes on ictal EEG. A genetic component to aetiology is established but the mechanism of inheritance and the genes involved are not fully defined. Available evidence suggests that genes encoding brain expressed voltage-gated calcium channels, including CACNG3 on chromosome 16p12-p13.1, may represent susceptibility loci for CAE. The aim of this work was to further evaluate CACNG3 as a susceptibility locus by linkage and association analysis. Assuming locus heterogeneity, a significant HLOD score (HLOD = 3.54, alpha = 0.62) was obtained for markers encompassing CACNG3 in 65 nuclear families with a proband with CAE. The maximum non-parametric linkage score was 2.87 (P < 0.002). Re-sequencing of the coding exons in 59 patients did not identify any putative causal variants. A linkage disequilibrium (LD) map of CACNG3 was constructed using 23 single nucleotide polymorphisms (SNPs). Transmission disequilibrium was sought using individual SNPs and SNP-based haplotypes with the pedigree disequilibrium test in 217 CAE trios and the 65 nuclear pedigrees. Evidence for transmission disequilibrium (P < or = 0.01) was found for SNPs within a approximately 35 kb region of high LD encompassing the 5'UTR, exon 1 and part of intron 1 of CACNG3. Re-sequencing of this interval was undertaken in 24 affected individuals. Seventy-two variants were identified: 45 upstream; two 5'UTR; and 25 intronic SNPs. No coding sequence variants were identified, although four variants are predicted to affect exonic splicing. This evidence supports CACNG3 as a susceptibility locus in a subset of CAE patients.

Lack of evidence of an allelic association of a functional GABRB3 exon 1a promoter polymorphism with idiopathic generalized epilepsy.

PURPOSE: Mutation screening and linkage disequilibrium mapping of the gene encoding the GABA(A) beta(3) subunit (GABRB3) identified a common genetic variant in the exon 1a promoter region (C-allele of rs4906902) which displayed a reduced transcriptional activity and showed a strong allelic association with childhood absence epilepsy (CAE). The present population-based association study tested whether the C-allele of rs4906902 confers susceptibility to CAE or other common syndromes of idiopathic generalized epilepsy (IGE) in a German sample. METHODS: Seven hundred and eighty unrelated German IGE patients (250 CAE, 123 juvenile absence epilepsy, 303 juvenile myoclonic epilepsy (JME), 104 epilepsy with generalized tonic-clonic seizures on awakening) and 559 healthy population controls were genotyped for the single nucleotide polymorphism (SNP) rs4906902. RESULTS: The frequency of the risk-conferring C-allele did not differ significantly between CAE patients (f(C)=0.190) and controls (f(C)=0.183; P=0.376, one-tailed). Similarly, no evidence for an allelic association was found for 373 patients with idiopathic absence epilepsy, 303 JME patients, and the entire IGE sample (P>0.77, two-tailed). CONCLUSION: Our study failed to replicate an association of the common GABRB3 exon 1a promoter SNP rs4906902 with CAE. Moreover, the present results do not provide evidence that the common functional C-variant confers a substantial epileptogenic effect to a broad spectrum of IGE syndromes in the German population.

Linkage and mutational analysis of CLCN2 in childhood absence epilepsy.

In order to assess the chloride channel gene CLCN2 as a candidate susceptibility gene for childhood absence epilepsy, parametric and non-parametric linkage analysis was performed in 65 nuclear pedigrees. This provided suggestive evidence for linkage with heterogeneity: NPL score=2.3, p<0.009; HLOD=1.5, alpha=0.44. Mutational analysis of the entire genomic sequence of CLCN2 was performed in 24 unrelated patients from pedigrees consistent with linkage, identifying 45 sequence variants including the known non-synonymous polymorphism rs2228292 (G2154C, Glu718Asp) and a novel variant IVS4+12G>A. Intra-familial association analysis using the pedigrees and a further 308 parent-child trios showed suggestive evidence for transmission disequilibrium of the G2154C minor allele: AVE-PDT chi(1)2 = 5.17, p<0.03. Case-control analysis provided evidence for a protective effect of the IVS4+12G>A minor allele: chi(1)2 = 7.27, p<0.008. The 65 nuclear pedigrees were screened for three previously identified mutations shown to segregate with a variety of idiopathic generalised epilepsy phenotypes (597insG, IVS2-14del11 and G2144A) but none were found. We conclude that CLCN2 may be a susceptibility locus in a subset of cases of childhood absence epilepsy.

Candidate gene analysis of the succinic semialdehyde dehydrogenase gene (ALDH5A1) in patients with idiopathic generalized epilepsy and photosensitivity.

Succinic semialdehyde dehydrogenase (SSADH) is involved in the degradation of the inhibitory neurotransmitter GABA and about 50% of patients with SSADH deficiency suffer from seizures. The gene encoding SSADH (gene symbol: ALDH5A1) maps in proximity to susceptibility loci for juvenile myoclonic epilepsy (JME) and photosensitivity on chromosome 6p22. The present study tested whether variation of the ALDH5A1 gene confers susceptibility to common syndromes of idiopathic generalized epilepsy (IGE) and an abnormal photoparoxysmal response (PPR). Mutation screening of the ALDH5A1 coding sequence of 35IGE/PPR patients and four healthy control subjects identified 17 sequence variants, of which three resulted in an exchange of amino acids (H180Y, P182L, A237S). Association analysis was carried out for six single nucleotide polymorphisms (SNPs) and one trinucleotide repeat polymorphism (TNR, intron 1), covering the genomic ALDH5A1 sequence. The study sample comprised 566 unrelated German IGE patients, including 218 JME and 95 photosensitive IGE patients, 78 PPR probands without IGE, and 662 German population controls. None of the investigated ALDH5H1 polymorphisms showed evidence for an allelic or genotypic association with either IGE, JME, or PPR, when corrected for multiple tests. A tentative haplotypic association of the two-marker haplotype (rs1883415-TNR) covering the 5'-regulatory region in IGE patients (chi2=11.65, d.f.=3, P=0.009) warrants further replication studies. The present results do not provide evidence that any ALDH5A1 missense variant itself contributes a common and substantial susceptibility effect (RR>2) to IGE syndromes or an increased liability to visually-induced cortical synchronization.

Confirmatory evidence for an association of the connexin-36 gene with juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is a genetically determined common subtype of idiopathic generalized epilepsy. Recently, linkage of JME to the chromosomal region 15q14, as well as an allelic and genotypic association between the synonymous coding single nucleotide polymorphism c.588C>T (dbSNP: rs3743123, S196S) of the positional candidate gene connexin-36 (CX36) and JME have been reported. The present replication study examined this tentative association in 247 German JME patients and 621 population controls. The frequency of the c.588T allele was significantly increased in the JME patients (35%) compared to controls (29.7%; P=0.016, one-tailed). Consistent to the original report, we also observed a significant increase of T/T homozygotes (13.4%) in the JME patients compared to controls (8.7%; P=0.019, one-tailed; OR(T/T+)=1.62; 95%-CI: 1.02-2.57). The present results provide confirmatory evidence for an allelic and genotypic association of the CX36 gene with JME.

Evaluation of CACNA1H in European patients with childhood absence epilepsy.

CACNA1H was evaluated in a resource of Caucasian European patients with childhood absence epilepsy by linkage analysis and typing of sequence variants previously identified in Chinese patients. Linkage analysis of 44 pedigrees provided no evidence for a locus in the CACNA1H region and none of the Chinese variants were found in 220 unrelated patients.

Association analysis of the Arg220His variation of the human gene encoding the GABA delta subunit with idiopathic generalized epilepsy.

PURPOSE: Mutation analysis of the gene encoding the GABA delta subunit (GABRD) identified a common missense variation (c.659G>A; Arg220His) of which the His220 allele displayed decreased GABA(A) alpha(1)beta(2)delta receptor current amplitudes. The present association study tested whether the functional GABRD His220 allele confers susceptibility to common syndromes of idiopathic generalized epilepsy (IGE). METHODS: Five hundred and sixty two unrelated German IGE patients and 664 healthy population controls were genotyped for the c.659G>A polymorphism in exon 6 of the GABRD gene. RESULTS: His220 allele frequencies did not differ significantly between IGE patients (2.3%) and the controls (2.8%; P=0.46). Likewise, no evidence for an allelic association was found with juvenile myoclonic epilepsy (n=218; 2.8%; P=0.97) or idiopathic absence epilepsy (n=260; 2.3%; P=0.56). CONCLUSION: Our results provide no evidence that the functional GABRD His220 allele mediates a substantial susceptibility effect to common IGE syndromes in the German population.

A new EF-hand containing gene EFHC2 on Xp11.4: tentative evidence for association with juvenile myoclonic epilepsy.

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.

A splice-site mutation in GABRG2 associated with childhood absence epilepsy and febrile convulsions.

CONTEXT: Missense mutations in the GABRG2 gene, which encodes the gamma 2 subunit of central nervous gamma-aminobutyric acid (GABA)(A) receptors, have recently been described in 2 families with idiopathic epilepsy. In one of these families, the affected individuals predominantly exhibited childhood absence epilepsy and febrile convulsions. OBJECTIVE: To assess the role of GABRG2 in the genetic predisposition to idiopathic absence epilepsies. DESIGN: The GABRG2 gene was screened by single-strand conformation analysis for mutations. Furthermore, a population-based association study assessing a common exon 5 polymorphism (C588T) was carried out. PATIENTS: The sample was composed of 135 patients with idiopathic absence epilepsy and 154 unrelated and ethnically matched controls. RESULTS: A point mutation (IVS6 + 2T-->G) leading to a splice-donor site mutation in intron 6 was found. The mutation, which is predicted to lead to a nonfunctional protein, cosegregates with the disease status in a family with childhood absence epilepsy and febrile convulsions. The association study did not find any significant differences in the allele and genotype frequencies of the common exon 5 polymorphism (C588T) between patients with idiopathic absence epilepsy and controls (P>.35). CONCLUSIONS: Our study identified a splice-donor-site mutation that was probably causing a nonfunctional GABRG2 subunit. This mutation occurred in heterozygosity in the affected members of a single nuclear family, exhibiting a phenotypic spectrum of childhood absence epilepsy and febrile convulsions. The GABRG2 gene seems to confer a rare rather than a frequent major susceptibility effect to common idiopathic absence epilepsy syndromes.

Serotonergic gene transcriptional control regions: targets for antidepressant drug development?

The formation and integration of neural networks is dependent on the actions of the master control neurotransmitter serotonin (5-HT) acting through multiple 5-HT receptor subtypes. During brain development 5-HT regulates morphogenetic activities, such as neural differentiation, axon outgrowth, and configuration of synaptic connections. In the adult brain, midbrain raphe serotonergic neurons project to a variety of brain regions and modulate a wide range of physiological functions. Several lines of evidence indicate that genetically determined variability in serotonergic gene expression influences complex behaviour and may lead to conditions with increased anxiety, depression, and aggression. Investigation of the regulation of serotonergic gene transcription and its impact on neural development and plasticity will spur general interest to identify serotonergic gene-related molecular factors underlying disease states and to develop more effective treatment strategies. Gene targeting strategies have increasingly been integrated into investigations of brain function and along with the fading dogma of a limited capacity of neurons for regeneration, reproducibility, and plasticity, it is realized that gene transfer techniques using efficient viral vectors in conjunction with neuron-selective transcriptional control systems may also be applicable to complex disorders of the brain. Given the fact that the 5-HT system continues to be an important target for large-scale drug development and production, novel strategies aiming toward the modification of 5-HT function at the level of gene expression are likely to be exploited by enterprises participating actively in the introduction of alternative therapeutic approaches.

Association analysis between the human interleukin 1beta (-511) gene polymorphism and susceptibility to febrile convulsions.

Recently, an association between a regulatory polymorphism in the gene encoding the pro-inflammatory cytokine interleukin (IL)-1beta and febrile convulsions (FC) has been reported. In this study we attempted to confirm these findings in a sample consisting of 99 FC patients and 126 ethnically matched controls. Since about 3% of all FC patients experience unprovoked seizures (epilepsy) later during life we furthermore genotyped 43 patients with non-lesional temporal lobe epilepsy who reported a history of FC. In both samples we failed to show an association between the IL-1beta polymorphism and an increased risk for FC. We only found a trend towards an increased frequency and carriage of the putative IL-1beta susceptibility allele two in a sub-sample of 43 FC patients who reported a positive family history for seizures in first and/or second degree relatives. However, these trends did not reach statistical significance.

Failure to replicate an allelic association between an exon 8 polymorphism of the human alpha(1A) calcium channel gene and common syndromes of idiopathic generalized epilepsy.

The present replication study tested the validity of a previously reported allelic association between a single nucleotide polymorphism in exon 8 (SNP8) of the gene encoding the alpha(1A)-calcium channel subunit (CACNA1A) and common subtypes of idiopathic generalized epilepsy (IGE). Pyrosequencing was applied to assess the SNP8 genotypes in 354 unrelated German IGE probands, both parents of 118 IGE probands, and 186 healthy control subjects of German descent. Our population-based association analysis did not provide evidence for an allelic association of SNP8 with either IGE or two phenotypically more homogeneous IGE subtypes, consisting of either 139 probands with juvenile myoclonic epilepsy or 207 probands whose IGE started with typical absence seizures (P>0.72). In addition, the transmission disequilibrium test did not indicate a preferential transmission of SNP8 alleles in 97 informative parent-child transmissions (McNemar chi(2)=0.093, df=1, P=0.76). Accordingly, we failed to confirm previous evidence that genetic variation of the CACNA1A gene confers susceptibility to common IGE syndromes.

Association of the 867Asp variant of the human anion exchanger 3 gene with common subtypes of idiopathic generalized epilepsy.

Genetic factors play a major role in the etiology of idiopathic generalized epilepsies (IGE). Our recent genome-wide search revealed suggestive evidence for a susceptibility locus for common IGE syndromes in the chromosomal region 2q36. The gene encoding the anion exchanger isoform 3 (AE3; gene symbol: SLC4A3) has been mapped to this candidate region. AE3 is prominently expressed in the brain and performs an electroneutral exchange of chloride and bicarbonate. To study the potential role of AE3 in the epileptogenesis of IGE, we performed a mutation analysis of the AE3 coding region, including the adjacent exon/intron boundaries, and the 5'-untranslated region in 16 IGE probands of families linked to chromosome 2q36 (cumulative two-point lod score: Z=5.32 at D2S371). Three exonic sequence variants were found: exon 17: 2600C/A, Ala867Asp; exon 21: 3391C/T, Leu1131Leu; exon 23: 3771G/A, 3'-UTR. Our subsequent population-based association study of the Ala867Asp substitution polymorphism revealed a significant increase of the 867Asp variant in 366 unrelated German IGE patients compared with 183 German control subjects (chi(2)=5.37, df=1, P=0.021). Consistently, the transmission disequilibrium test (TDT) of 121 parent-child trios showed a significant preferential transmission of the 867Asp allele (McNemar chi(2)=5.81, df=1, P=0.016). Our results support the hypothesis that variation of the AE3 gene confers a common but small susceptibility effect to the etiology of a broad spectrum of IGE syndromes.

Linkage analysis between childhood absence epilepsy and genes encoding GABAA and GABAB receptors, voltage-dependent calcium channels, and the ECA1 region on chromosome 8q.

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy (IGE) characterised by onset of typical absence seizures in otherwise normal children of school age. A genetic component to aetiology is well established but the mechanism of inheritance and the genes involved are unknown. Available evidence suggests that mutations in genes encoding GABA receptors or brain expressed voltage-dependent calcium channels (VDCCs) may underlie CAE. The aim of this work was to test this hypothesis by linkage analysis using microsatellite loci spanning theses genes in 33 nuclear families each with two or more individuals with CAE. Seventeen VDCC subunit genes, ten GABA(A)R subunit genes, two GABA(B) receptor genes and the ECA1 locus on 8q24 were investigated using 35 microsatellite loci. Assuming locus homogeneity, all loci gave statistically significant negative LOD scores, excluding these genes as major loci in the majority of these families. Positive HLOD scores assuming locus heterogeneity were observed for CACNG3 on chromosome 16p12-p13.1 and the GABRA5, GABRB3, GABRG3 cluster on chromosome 15q11-q13. Association studies are required to determine whether these loci are the site of susceptibility alleles in a subset of patients with CAE.

Allelic association of a truncation mutation of the KCNMB3 gene with idiopathic generalized epilepsy.

The gene encoding the beta3-subunit regulatory subunit (KCNMB3) of large conductance calcium-sensitive potassium (BK) channels represents a positional and functional candidate gene for idiopathic generalized epilepsy (IGE). A single base pair deletion in exon 4 of KCNMB3 (delA750) alters/truncates the terminal 21 amino acids of the 3-subunit and affects channel inactivation of the beta3b-isoform. The present association study tested whether the KCNMB3 delA750 mutation confers susceptibility to common IGE syndromes. In total, 592 unrelated German IGE patients and 462 healthy population controls were genotyped for the delA750 truncation mutation. The frequency of the delA750 mutation was significantly increased in the IGE patients (7.9%) compared to that in the controls (5.5%; P = 0.016, one-sided; OR = 1.52; 95%-CI: 1.05-2.21). The increase of the delA750 frequency was accentuated in 312 patients exhibiting typical absence seizures (8.8%, P = 0.005, one-sided; OR = 1.72; 95%-CI: 1.13-2.62) relatively to that observed in the 237 patients with myoclonic seizures on awakening (7.2%; P = 0.11, one-sided; OR = 1.36; 95%-CI: 0.85-2.19), when compared with controls. The present results suggest that the functional KCNMB3 beta3b-truncation confers a common epileptogenic effect preferentially to the ictogenesis of typical absence seizures.

A multicenter study of BRD2 as a risk factor for juvenile myoclonic epilepsy.

PURPOSE: Although complex idiopathic generalized epilepsies (IGEs) are recognized to have a significant genetic component, as yet there are no known common susceptibility variants. It has recently been suggested that variation in the BRD2 gene confers increased risk of juvenile myoclonic epilepsy (JME), which accounts for around a quarter of all IGE. Here we examine the association between the candidate causal SNP (the promoter variant rs3918149) and JME in five independent cohorts comprising in total 531 JME cases and 1,390 healthy controls. METHODS: The strongest candidate causal variant from the original report (rs3918149) was genotyped across all five cohorts. In an effort to identify novel candidate causal polymorphisms, previously unscreened regions of UTR were resequenced. RESULTS: We observed a significant effect in a small sample recruited in Britain (genotype p = 0.001, allele p = 0.001), a borderline significant effect in a sample recruited in Ireland and no association in larger samples of German, Australian, and Indian populations. There was no association with other common forms of epilepsy or any other clear candidate casual variants in or near the BRD2 region. CONCLUSIONS: The replication of an effect in the British cohort and suggestive evidence from that recruited in Ireland but lack of replication from the larger German, Australian, and Indian cohorts is surprising and difficult to explain. Further replication in carefully matched populations is required. Results presented here do not, however, support a strong effect for susceptibility to JME across populations of European descent.

A mutation in the GABA(A) receptor alpha(1)-subunit is associated with absence epilepsy.

OBJECTIVE: To detect mutations in GABRA1 in idiopathic generalized epilepsy. METHODS: GABRA1 was sequenced in 98 unrelated idiopathic generalized epilepsy patients. Patch clamping and confocal imaging was performed in transfected mammalian cells. RESULTS: We identified the first GABRA1 mutation in a patient with childhood absence epilepsy. Functional studies showed no detectable GABA-evoked currents for the mutant, truncated receptor, which was not integrated into the surface membrane. INTERPRETATION: We conclude that this de novo mutation can contribute to the cause of "sporadic" childhood absence epilepsy by a loss of function and haploinsufficiency of the GABA(A) receptor alpha(1)-subunit, and that GABRA1 mutations rarely are associated with idiopathic generalized epilepsy.

Exploration of the genetic architecture of idiopathic generalized epilepsies.

PURPOSE: Idiopathic generalized epilepsy (IGE) accounts for approximately 20% of all epilepsies and affects about 0.2% of the general population. The etiology of IGE is genetically determined, but the complex pattern of inheritance suggests an involvement of a large number of susceptibility genes. The objective of the present study was to explore the genetic architecture of common IGE syndromes and to dissect out susceptibility loci predisposing to absence or myoclonic seizures. METHODS: Genome-wide linkage scans were performed in 126 IGE-multiplex families of European origin ascertained through a proband with idiopathic absence epilepsy or juvenile myoclonic epilepsy. Each family had at least two siblings affected by IGE. To search for seizure type-related susceptibility loci, linkage analyses were carried out in family subgroups segregating either typical absence seizures or myoclonic and generalized tonic-clonic seizures on awakening. RESULTS: Nonparametric linkage scans revealed evidence for complex and heterogeneous genetic architectures involving linkage signals at 5q34, 6p12, 11q13, 13q22-q31, and 19q13. The signal patterns differed in their composition, depending on the predominant seizure type in the families. CONCLUSIONS: Our results are consistent with heterogeneous configurations of susceptibility loci associated with different IGE subtypes. Genetic determinants on 11q13 and 13q22-q31 seem to predispose preferentially to absence seizures, whereas loci on 5q34, 6p12, and 19q13 confer susceptibility to myoclonic and generalized tonic-clonic seizures on awakening.