Possible association of a silent polymorphism in the neuronal nicotinic acetylcholine receptor subunit alpha4 with common idiopathic generalized epilepsies.

The alpha4 subunit gene of the neuronal nicotinic acetylcholine receptor (CHRNA4) has recently been identified as the first gene underlying an idiopathic partial epilepsy syndrome in human, autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE). CHRNA4 is located in the candidate region for benign familial neonatal convulsions and low-voltage EEG on chromosome 20q. In the present study, we examined the possible role of CHRNA4 in common subtypes of idiopathic generalized epilepsy (IGE), comprising childhood and juvenile absence epilepsy and juvenile myoclonic epilepsy (JME), by systematically screening the coding region of the gene for sequence variants. We present here a population-based association study testing the hypothesis that variants of the CHRNA4 gene confer genetic susceptibility to common subtypes of IGE. The missense mutation (Ser248Phe), associated with ADNFLE, and four silent polymorphisms in the CHRNA4 gene were genotyped in 103 IGE patients and 92 controls by polymerase chain reaction and subsequent restriction analysis. Without correction for multiple testing, the frequency of the T-allele of the silent CfoI bp595 polymorphism was increased in the entire group of IGE patients (f(T) = 0.085) compared to that in the controls (f(T) = 0.027). The allelic association was not restricted to any subgroup of IGE with either JME or idiopathic absence epilepsies. This result suggests that variation of the CHRNA4 gene, or so-far-undetected sequence variants near the CHRNA4 locus, confer susceptibility to the common IGE syndromes.

The voltage gated potassium channel KCNQ2 and idiopathic generalized epilepsy.

Mutations in the voltage gated potassium channel gene KCNQ2 and the homologous gene KCNQ3 have been found to cause a rare monogenic subtype of idiopathic generalized epilepsy, the benign familial neonatal convulsions. Recently, the heteromeric KCNQ2/KCNQ3 channel was found to contribute to the native M-current, one of the most important regulators of neuronal excitability. By performing a systematic mutation scan of the coding region and an association study involving a frequent Thr752Asn substitution polymorphism, we, therefore, investigated whether allelic variation of the KCNQ2 gene confers susceptibility to common subtypes of idiopathic generalized epilepsy. Our results do not provide evidence that allelic variation of the KCNQ2 gene contributes a common and relevant effect to the pathogenesis of common subtypes of idiopathic generalized epilepsy.

Mutation screening of the CHRNA4 and CHRNB2 nicotinic cholinergic receptor genes in Alzheimer's disease.

Potential genomic changes leading to decreased nicotine binding, crucial for cognitive dysfunction in Alzheimer's disease (AD), have not yet been studied. A search for mutations of the genes coding for the most widely distributed nicotinic receptor subtype alpha4beta2 (CHRNA4/CHRNB2) has been performed in AD patients by screening the coding regions of both genes by single strand conformation analysis and heteroduplex analysis of fibroblast-derived genomic DNA. Polymorphisms in CHRNA4, none of which led to amino acid changes in the predicted sequence, were found in three patients. Although the other receptor subunits have yet to be screened, it appears likely that the reduction of nicotine binding sites in AD is not due to genomic changes.

Expression of nicotinic acetylcholine receptors in Alzheimer's disease: postmortem investigations and experimental approaches.

Nicotinic ligand binding studies have shown rather early that the cholinoceptive system is affected in Alzheimer's disease (AD). Today, molecular histochemistry enables one to study the nicotinic acetylcholine receptor (nAChR) subunit expression on the cellular level in human autopsy brains, in animal models and in in vitro approaches, thus deciphering the distribution of nAChRs and their role as potential therapeutic targets. The studies on the nAChR expression in the frontal and temporal cortex of AD patients and age-matched controls could demonstrate that both, the numbers of alpha4- and alpha7-immunoreactive neurons and the quantitative amount, in particular of the alpha4 protein, were markedly decreased in AD. Because the number of the corresponding mRNA expressing neurons was unchanged these findings point to a translational/posttranslational rather than a transcriptional event as an underlying cause. This assumption is supported by direct mutation screening of the CHRNA4 gene which showed no functionally important mutations. To get more insight into the underlying mechanisms, two model systems organotypic culture and primary hippocampal culture - have been established, both allowing to mimic nAChR expression in vitro. In ongoing studies the possible impact of beta-amyloid (Abeta) on nAChR expression is tested. Preliminary results obtained from primary cultures point to an impaired nAChR expression following Abeta exposure.

Mutation analysis of the potassium chloride cotransporter KCC3 (SLC12A6) in rolandic and idiopathic generalized epilepsy.

Genetic predisposition plays a major role in the etiology of idiopathic epilepsies. The common epilepsy syndromes display a complex pattern of inheritance, with an unknown number of genes contributing to seizure susceptibility. During the last decade linkage studies have narrowed down several candidate regions for susceptibility loci of idiopathic epilepsies. Several lines of evidence point to the existence of an epilepsy susceptibility gene on chromosome 15q14. Evidence for linkage to this region has thus been reported for juvenile myoclonic epilepsy, common subtypes of idiopathic generalized epilepsy (IGE), in addition to the EEG trait 'centrotemporal spikes' in families with rolandic epilepsy. The chromosomal region 15q14 harbours several candidate genes that are involved in the regulation of neuronal excitability. One of the most promising candidate genes is the brain-expressed potassium chloride cotransporter KCC3, given that this class of ion transporter has been implicated in the regulation of neuronal chloride activity. We therefore performed a mutation analysis of KCC3 in the index patients of 23 IGE-families as well as of 16 families with rolandic epilepsy which where selected by positive evidence for linkage to D15S165. Four novel single nucleotide exchanges (SNPs) were identified, none of which change the coding sequence. These results do not support a major role for KCC3 in the etiology of rolandic epilepsy or common subtypes of IGE.

Exon-intron structure of the human neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4).

The human neuronal nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idiopathic epilepsies.

Independent occurrence of the CHRNA4 Ser248Phe mutation in a Norwegian family with nocturnal frontal lobe epilepsy.

PURPOSE: To describe the clinical features of a family from Northern Norway in which autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is associated with a Ser248Phe amino acid exchange in the second transmembrane domain of the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4). We also tested for evidence of a de novo mutation or founder effect by comparing haplotypes with the original Australian family where the Ser248Phe mutation was first described. METHODS: Clinical details were obtained from 19 family members. Personal interviews and genetic analysis were carried out in 17. Parts of the coding region of CHRNA4 were sequenced, and two known polymorphisms (bp555/FokI, bp594/CfoI) were typed by restriction analysis. RESULTS: Eleven individuals had ADNFLE. The haplotypes of the mutation-carrying alleles of affected individuals from the Northern Norwegian and the Australian ADNFLE family are different. The phenotypic expressions are remarkably similar. CONCLUSIONS: The Ser248Phe mutation occurred independently in both families. Given the rarity of the disease, this suggests that not only the position of a mutation in the coding sequence but also the type of an amino acid exchange is important for the etiology of ADNFLE. The phenotypic similarity of these two families with different genetic backgrounds suggests that the Ser248Phe mutation largely determines the phenotype, with relatively little influence of other background genes.

An insertion mutation of the CHRNA4 gene in a family with autosomal dominant nocturnal frontal lobe epilepsy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is the first, and to date only, idiopathic epilepsy for which a specific mutation has been found. A missense mutation in the critical M2 domain of the alpha4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently identified in one large Australian pedigree. Here we describe a novel mutation in the M2 domain of the CHRNA4 gene in a Norwegian family. Three nucleotides (GCT) were inserted at nucleotide position 776 into the coding region for the C-terminal end of the M2 domain. Physiological investigations of the receptor reconstituted with the mutated CHRNA4 subunit reveal that this insertion does not prevent the receptor function but increases its apparent affinity for ACh. In addition, this mutant receptor shows a significantly lower calcium permeability that, at the cellular level, may correspond to a loss of function. Comparison of the two mutations identified so far in families with ADNFLE illustrates that different mutations can result in similar phenotypes.