Analysis Of Genetic Etiology Of Nocturnal Frontal Lobe Epilepsy

Background and ObjectivesThe prevalence of epilepsy is estimated at 0.5% -1.5% around the world. It is estimated that this disease affects approximately 50 million people in the world. The repeated epilepsy seizures do great harm to patients'health and lives. Epilepsy brings huge disease burden to the patients, their families and the whole society. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is genetically transmitted idiopathic partial epilepsy. ADNFLE is characterized by clustered attacks of brief motor seizures with hyperkinetic or tonic manifestations originating from the frontal lobe mostly during the non-rapid eye motion phase sleep. ADNFLE is partly associated with mutations in the genes encoding theα4,β2, andα2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR), which is a member of the Cys-loop receptor family. The most abundant form of heteromeric nAChR in the brain contains a4 and (32 subunits. To date, all the mutations in CHRNA4, CHRNB2, and CHRNA2 identified in ADNFLE kindreds affect the first, second, or third transmembrane domains. But in Chinese ADNFLE families, none mutation of CHRNA4, CHRNB2, and CHRNA2 has been identified. Clinically, sporadic nocturnal frontal lobe epilepsy (NFLE) cases are more common than familial NFLE. Both types of disease have similar clinical feature; thus, it is supposed that the genetic basis of non-familial NFLE may resemble that of ADNFLE. In fact, de novo mutations in CHRNA4 have been found in a Lebanese and a Chinese sporadic NFLE patient, respectively. However, no mutations in CHRNB2 or CHRNA2 in non-familial NFLE cases have been reported. The purpose of the present study was to screen mutations in CHRNA4, CHRNB2, and CHRNA2, in Chinese sporadic NFLE patients and ADNFLE families and did the preliminary discussion about the structure and function change of nAChR subunit protein causing by mutation.MethodsWe recruited one hundred and five sporadic Chinese patients, who were diagnosed as NFLE based on the clinical history, EEG/VEEG and cranial magnetic resonance imaging (MRI). Control DNA samples were obtained from 200 Chinese unrelated healthy individuals. Genomic DNA was prepared from peripheral venous blood of each subject. Mutational screening was performed by sequencing two or three polymerase chain reactions (PCR) amplified DNA fragments from each gene, spanning exon 5 of CHRNA4, exon 5 of CHRNB2, and exon 6 of CHRNA2. These exons encode the M 1-3 regions that contribute to the ion pore of the three nAChR proteins. The purified PCR fragments were sequenced by DNA Analyzer. Any mutations identified in the patients were screened in the controls by PCR amplification and sequencing. The hydrophobicities and secondary structures of the wild-type and mutated proteins were analyzed by bioinformatics software. The change of protein function was predicted based on the results of analysis.ResultsWe discovered a missense mutation, V337G, in CHRNB2 in a 19 year-old female sporadic NFLE patient. The mutation causes a Val-to-Gly substitution at amino acid position 337, located in the M3-M4 intracellular loop. An alignment of the amino acid sequences of CHRNB2 from various species showed that the V337G mutation is located in an evolutionary conserved region. The hydrophobicity of the protein at the mutation site and the neighboring regions is decreased when compared to the wild-type protein. Secondary structure prediction indicated that the V337G substitution significantly altered the structure of the surrounding region.We found a missense mutation in CHRNA4 in a 7 years old male NFLE patient. The mutation caused a Ser-to-Phe substitution at amino acid position 405 in the M3-M4 intracellular loop. The mutation increased the hydrophobicity of the protein and changed the protein secondary structures. No mutations were found in the analyzed regions of CHRNA2 in these non-familial NFLE patients.ConclusionsThe missensse mutation V337G of CHRNB2 and S405F of CHRNA4 in Chinese sporadic NFLE patient may be a genetic etiology in the pathogenesis of sporadic NFLE. The mutations in M3-M4 intracellular loop of nAChR subunit may be associated with NFLE. This is the first study to establish that CHRNB2 is potentially associated with sporadic NFLE, which proved that the genetic basis of non-familial NFLE may resemble that of ADNFLE. Because no mutation in CHRNA2 was found in this study, we speculate that CHRNA2 mutations are rare in NFLE populations. Background and ObjectivesEpilepsy is the second common neurological disorder after cerebrovascular disease. The latest epidemiology data shows that there are about 9 million epilepsy patients in China and 300-400 thousands new diagnosis patients every year. Epilepsy seizures do great harm to patients'health and quality of life. It brings huge disease burden to the patients'families and the whole society. Frontal lobe epilepsy (FLE) is the second most partial epilepsy. FLE usually onsets during childhood and affects the physical and psycological health of child. FLE can be inherited as a Mendelian autosomal dominant or as a non-Mendelian complex genetic trait. The penetrance of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is 70%-80%. In 1995, a missense mutation in the a4 subunit gene of the neuronal nicotinic acetylcholine receptor (nAChR) was identified in one Australian ADNFLE pedigree. It is the first mutation described in idiopathic epilepsy. Since then, it has been shown that ADNFLE can be caused by mutations in CHRNA4, CHRNB2 and CHRNA2 and all the mutations identified in ADNFLE kindreds affect the first, second, or third transmembrane domains of nAChR.Clinically, the number of non-familial frontal lobe epilepsy (FLE) is larger than familial patients. Because the sporadic NFLE share a phenotype similar to those of familiar epilepsy, the pathophysiological mechanisms are probably very similar in the sporadic and the familial forms. Sporadic FLE is accepted as a complex disorder attributed to the interaction of genetic and environmental factors. The genetic factors play an important role in induction and maintainance of FLE. With the development of Human Genome Project, the relationship of single nucleotide polymorphisms (SNPs) and human diseases draws much concern and is studied deeply and widely. The mutations of CHRNA4 and CHRNB2 have been identified in three sporadic NFLE patients, but the influence of single nucleotide polymorphisms (SNPs) in sporadic NFLE has not been established. In all the mutations identified in familial and non-familial FLE patients, the CHRNA4 gene mutations take more than 50% part, which were proved to play important roles in the function of ion channel and sensitivity of acceptor in vitro and vivo experiments. We speculate that CHRNA4 may be a susceptibility gene of FLE, and in this study, we examine the association between the coding region polymorphisms of CHRNA4 gene and FLE.MethodsIn this study, we performed an independent case-control association study to analyze the genotype and allele distributions of the CHRNA4 coding region polymorphisms in a Chinese FLE population, and to investigate the association between polymorphisms and clinical variables of FLE patients. This study also focuses on if the SNPs have a synergistic effect on the development of FLE. We choose the SNPs of CHRNA4 from the SNP database and former literatures. After the preliminary test in Chinese health people, SNPs which are suitable for analyses were finally chosen. We genotyped the polymorphisms by polymerase chain reaction (PCR) and directly sequencing. Statistics software was used to calculate the association of SNP alleles and FLE. We used the Haploview software to analyses the haplotype. In addition,10% of the samples were randomly selected for repeated assays.ResultsAfter the preliminary test in Chinese health people, four SNPs, rs2229959, rs1044393, rs1044396, and rs1044397, were picked up. The four SNPs did not show allelic or genotypic association with FLE.ConclusionsBased on the results of the case-control association study we did in Chinese sporadic FLE patients and healthy group, the coding region polymorphisms of CHRNA4 do not contribute to the development of FLE.