The Research On Mutation And CSNP In The GABRG2 And SCN1B Of Patients With GEFS+

Epilepsy is a chronic encephalic disease caused by many factor, is a paroxysmal temporary brain disorder clinical manifestation caused by cranial nerves over-discharge, is a common central nervous system diseases. epilepsy is not a independent disease, rather than a group of disease and syndrome. In this large group of diseases, certain types can be viewed as independent, apart from its unique clinical features (including auxiliary examination data), the etiology, prognosis is also some specificity, and some type of seizure only as a syndrome. Generalized epilepsy with febrile seizures plus (GEFS+), is a new named epilepsy syndrome by International League Against Epilepsy in 2001.Generalized epilepsy with febrile seizures plus (GEFS+), is a common and representative genetic epilepsy syndrome, the clinical characteristics of GEFS+is that in febrile seizures family, children after the age of 6 are still associated with febrile seizures or seizures without fever, or combine other forms of epileptic seizures syndrome, side by side, apart from symptomatic epilepsy. Pedigree analysis of most GEFS+family indicate that this generalized epilepsy syndrome is autosomal dominant inheritance, has significant phenotypic heterogeneity and genetic heterogeneity.Member in the same family may be involved in different clinical phenotype, a large GEFS+family first reported by Scheffer, the core families in line with. autosomal dominant inheritance, penetrance rate was 89%. Singh on 9 GEFS+ family pedigree analysis also shows that for the autosomal dominant inheritance, Singh have analysis 9 GEFS+family also shows that there are autosomal dominant inheritance, penetrance rate is about 50%-60%. Because of incomplete penetrance leading to a variety of clinical phenotype and passive carriers that are not clinical symptoms, Singh and other advocates want to use complex multi-genetic inheritance to explain the phenotypic heterogeneity of GEFS+features. Onset form of GEFS+ has a rich heterogeneity, is a multi-shaped familial epilepsy syndrome, there is lack of epidemiological evidence, currently. The most common phenotype was febrile seizures, the followed was febrile seizures plus syndrome, GEFS+generally showed benign process, intellectual and movement developed normally, mostly stop attack before the age of 25 or children's later stage.In recent years, the research of GEFS+mutant gene has made great progress in abroad, found most caused by ion-channel gene encoding mutations, currently five genes associated with GEFS+have been reported in foreign countries-SCN1B(Voltage-gated sodium channelβ1 subunit gene), SCN1A (voltage-gated sodium channel al subunit gene), SCN2A (voltage-gated sodium channel a2 subunit gene), GABR G2 (A-type GABA receptor y-subunit gene) and GABRD (A-type GABA receptor 8 subunit gene).(1) SCN1B mutations and GEFS+:1998 Wallace, etc. Firstly take a large Australian GEFS+pedigrees to do linkage analysis, found a C121W missense mutation, Scheffer, etc. found SCN1B mutation in four epileptic families, these two new families mutation have been reported (C121W), the other two mutations are R85C and R85H. (2) SCN1A mutations and GEFS+:SCN1A which encoded by a-subunit is the most important GEFS+genes. Annesi, etc. take nine Italian GEFS+ pedigrees to do linkage analysis by SSCP screening, finally, found two abnormal SCN1A mutation in two of these families, the first mutation is A2336G; another mutation is T5522C. Spampanto, etc. studied on a Italian GEFS+family confirmed another SCN1A mutation D1866Y, Audenaert, etc. take a GEFS+pedigrees to do linkage analysis, located the disease-causing gene at 2p24. Pineda-Trujillo, etc. study on a South America GEFS+found the G1742D mutation, Nagao, also reported a GEFS+family, found a SCN1A missense mutation (G5569T) in patients. Barela, etc. in a Caucasus GEFS+family also prove another exception SCN1A mutation (R859C). (3) SCN2A mutations and GEFS+:2001 Sugawara, etc. first time screen sodium channel a2 subunit encoded by SCN2A in patients with GEFS+, found a very conservative sites, it is R187W mutation, a SCN2A truncate mutation has been confirmed in a refractory epilepsy which similar SMEI. (4) GABAA mutations and GEFS+:Baulac, etc. In a similar GEFS+pedigree, found a mutation inγ2 subunit(K289M).Wallace, etc. In a family with absence epilepsy (CAE) and febrile convulsions found anotherγ2 subunits mutation (R43Q). In 2006, GEFS+ pedigrees were found two new sites, E177A and R220H. In 2004, Dibbens in an Australian family found GABRD mutation, this mutant encode GABAA receptorδsubunit. Xi Shun HUANG, etc. collected seven GEFS+pedigrees to screen GABRA6, found C/G polymorphism in No.8 exon. Bonanni, etc. collected seven GEFS+family, in four of these families found single nucleotide polymorphism in the GABRG2 gene encoding, this study proved that GEFS+have genetic and phenotypic heterogeneity. Shou-shan Zhang sequenced GABRB2 coding region of all the 11 exons in a GEFS+family, finally discovered a new C/G polymorphism in the 133th base pairs of GABRB2 exon 2. Besides, Audenaert D, etc. reported a new position in the 2p24 in a Belgian four generations family, but its pathogenic gene remain unclear.Although genetic research has significant progress, but the known GEFS+ mutation only exists a small number of pedigrees, disease-causing gene of most GEFS+family remains to be more discoveries. For this reason, to better understand the pathogenesis of GEFS+, This study want to screen 8 families members total of 101 all GABRG2 gene exon mutation use direct sequencing PCR products, to find Chinese population GEFS+pathogenic genes, explore GABRG2 gene polymorphism in patients with GEFS+features, to clarify the molecular mechanism of pathogenesis of Chinese population GEFS+.[Object and Method]1. Object:Nine probands attended at the Guangdong Provincial People's Hospital pediatric epilepsy specialist clinics in January 2002 to December 2009, are Han nationality, all from Guangdong.9 probands were identified as GEFS+ according to the 2001 International League Against Epilepsy (ILAE) classification of epilepsy and epilepsy syndrome diagnosis criteria, we develop a unified inquiry form, visited the family members by means of clinics and home investigation, strictly according national laws and regulations to obtain the consent of patients and their families, complete clinical data collection and blood sample. Collected 9 families total of 109 members. Control group:randomly selected 200 cases of healthy people in hospital medical centers to the control group, are Han nationality, gender, age group were matched with patients, examinations were normal.2. Clinical data collection:All patients completed long-term video EEG, head MRI, established GEFS+patients databases, developed and adopted a uniform questionnaire and methods, carrying out family surveys, data collection and processing.3. GABRG2 and SCN1B mutation screening:(1)200μl samples were taken from the peripheral venous blood of every patient and 200 healthy control subjects, extracted genomic DNA.(2)According to the standard GABRG2 and SCN1B genome sequence, design amplified exons of PCR primers, all patients specimens using PCR specificly amplification GABRG2 gene 1 to 9th and SCN1B 1gene 1 to 5 exon fragment.(3)Purified the PCR products were, using ABI3730 sequencing instrument sequence. Applied Vector NTI 8.0 software to analysis of sequencing results, compared to standards GenBank sequence, after define possible mutations, Amplification 200 healthy control group of related gene fragments to compare, define new mutations, except polymorphism. If identified as mutation, then use the above method proband for mutation screening of family members.[Results]1. In this study were collected nine family members received a total of 109 surveys (4 have already died, the medical history provided by relatives), The results showed that 40 cases of epilepsy, idiopathic generalized epilepsy in 2 cases,3 cases can not be clearly classified. Consistent with GEFS+clinical phenotype in 35 patients (1 died), of which 24 males and 16 females, male to female ratio was about 1.5:1, GEFS+patients with FS in 22 cases (65.6%), the most common seizure types, Onset age 5 months to 3 years old, mostly termination age of onset of 3 to 6 years old, number of episodes ranging from 1 second to several times. FS+8 cases (18.8%), the remaining 5 cases (15.6%) from each of the 2 patients with focal seizures for the FS+, FS+and absence seizures and 1 with FS+associated with myoclonic seizures.2. In this study, total 109 cases of experimental group members all successfully completed GABRG2 and SCN1B gene mutation screening, the results showed that all the families and patients and family members were all not found the three hot spot mutations GABRG2 (K289M, R43Q, Q351X) and SCN1B hot spot mutations (C121W, I70_E74del, R85C, R85 H) have been reported.3. In this study, all of GEFS+patients found GABRG2cDNA nucleotide sequence of the first-14 No. A deletion variation, the variation before of the start codon.4. In this study, three generations of familyⅠ, a total of six members of the men were found SCN1B heterozygous missense mutation H223D, located in exon 3, the mutation let Histidine replaced by Aspartic acid, followed 200 healthy control results were negative.[Conclusion]1. Nine families in this study, the major clinical phenotype was FS and FS+ also includes a small number of FS+and absence seizures, FS+with myoclonic seizures, FS+with focal seizures and idiopathic generalized epilepsy. Fully reflects the GEFS+phenotype heterogeneity.2. We have PCR amplification and sequenced GABRG2 gene exons of all patients, have not found the reported GABRG2 hot spot mutations (K289M, R43Q, Q351X). Tip that GABRG2 is may not the major disease areas GEFS+genes in China's southern Guangdong. Further confirmed the GEFS+has obvious genetic heterogeneity.3. In GEFS+, have found an A deletion mutationthe in No.14 nucleotide of GABRG2cDNA nucleotide sequence. The variation in all the GEFS+patients and healthy controls in this study, We believe that the mutation frequency in Chinese population there is a single nucleotide, not the disease-causing potential for GEFS+.4. We have PCR amplification and sequenced SCN1B gene exons of all patients, have not found the reported SCNIB hot spot mutations (C121W,I70_E74del,R85C,R85 H). In three generations of family I, a total of six members of the men were found SCNIB heterozygous missense mutation H223D, located in exon 3, the mutation let Histidine replaced by Aspartic acid, but whether sodium channel receptor function impact with GEFS+morbidity correlation further study. Suggesting SCNIB probably southern China crowd GEFS+patients potential pathogenic gene, H223D not yet abroad reported probably China Southern crowd GEFS+new genetics etiology.