Capture And Identification Of New Pathogenic Genes In Genetic Epilepsy With Febrile Seizures Plus

Febrile seizures(FS) is a most common seizure disorder in human childhood, The prevalence of FS in children under 6 years old was up to 3%～4%. With the deepening of the research, many FS families were found in certain familial transmissibility and few of the FS children would develop to epilepsy in high-risk. The severity of their afebrile seizures is differ in a variety of phenotypes. In 1997, Scheffer first reported a new epilepsy syndrome——general epilepsy with febrile seizures plus (GEFS+).GEFS+ was diagnosed as an epilepsy syndrome based on the whole of the family. In the same family, family members of the spectrum could be made of a variety of phenotypes and a variety of clinical seizures of form. More and more studies show that the phenotypes of general seizures and partial seizures were coexist in some family. Therefore the name of GEFS+ comes to a challenge. In 2009, Scheffer proposed the name of "generalized epilepsy with febrile seizures plus(GEFS+)" should be transform to "genetic epilepsy with febrile seizures plus(GEFS+)". This new concept includes more genetic epilepsy associated with FS, which helps to resolve some question about naming of disorder syndrome in the past related to FS.Currently this name of "genetic epilepsy with febrile seizures plus" has been widely recognized by the international researchers. The common characteristic of GEFS+ family was with the family history of febrile seizure. But family members associated with other epileptic seizure types are different, from single to complex, mild to severe. Unlike other epilepsy syndrome, the diagnosis of GEFS+ is not confined to the individual. The diagnosis was based on the several members with clinical phenotype of spectrum belongs to GEFS+. Febrile seizure(FS) is the most common phenotype, which performance for aged 3 months to 6 years old children showed typical seizure when their temperature more than 38℃,and intracranial infection, and other organic cause convulsions and metabolic disease should be ruled out. Febrile seizures plus is the next most frequent clinical phenotype, in which FS continue past the typical age of 6 years and/or afebrile convulsions occurgeneralized tonic clonic seizures.Other phenotypes including FS/FS+ with other generalized seizures, such as the FS/FS+ with absence seizures, FS/FS+ with myoclonic seizures, FS/FS+ with loss of tension seizures, and FS/FS+ with partial seizures. The most serious and rare phenotypes were epileptic encephalopathy, including myoclonic-astatic epilepsy and infant evere myoclonic epilepsy in infancy, also known as Dravet syndrome, and other refractory epilepsy, or other idiopathic generalized epilepsy phenotype.As more and more GEFS+ family were reported continually, many new clinical phenotypes were accepted gradually.The pathogenesis of GEFS+ is complex, including genetic factors, and environmental factors. Molecular change induced genetic factors plays an important role in the cause of excessive brain neurons abnormal discharge. With he development of molecular biology genetics research have laid a good foundation of the disease. After nearly 10+ years of international research, most of the analysis showed that GEFS+ family is in autosomal dominant inheritance with incomplete penetrance. The penetrance rate was about 50%-89%. Family members have different clinical phenotype severity due to the incomplete penetrance. GEFS+ was associated with 5 ion channel gene, including coding voltage-gated sodiumα1、α2、β1 subunit gene (SCN1A, SCN2A,SCN1B) and chlorine ion channel coding ligand gating GABAA receptory2,δsubunit gene (GABRG2,GABRD). In 1998, Wallaee etc first confirmed SCN1B gene mutation in the patients with GEFS+. In 2000, Escayg then found SCN1A gene missense mutation in the GEFS+ patients. In 2001, Sugawar and Baulac found missense mutation in SCN2A and GABRG2 respectively.2004 Dibbens found GABRD genes associated with GEFS+. In above all, SCN1A gene is in the most closely relationship with GEFS+.Although a lot of GEFS+ family have been reported, but gene mutations founded in the proportion is less than 20% of the total GEFS+ family. The pathogenic genes in most GEFS+ family are still unclear and the mechanism has not yet been clarified. In recent years some domestic GEFS+ family had been reported, however, only few mutations of SCN1A and GABRG2 gene were found. GEFS+ is related to genetic factors with obvious genetic heterogeneity, which are ethnic and regional differences. Therefore, our country is lack of more details about genetic analysis. It is great importance to finish the study the relationship between genotype and clinical phenotypes in GEFS+. At the same time, It is urgently to use a new generation of sequencing technology to find out new pathogenic genes in our Chinese GEFS+ family efficiently. It is not only help to reveal the pathogenesis of the disease, but also offer a new method for the diagnosis and treatment of the disease.Whole exome sequencing technology developed gradually in recent years.The high-throughput sequencing technology is used for sequencing the exome to discover the vast majority of variations associated with disease, common variants and low frequency(frequency< 5%) mutation. Due to these advantages, exome sequencing has become a powerful and efficient strategy which required less samples and low cost to identify the genes responsible for mendelian disorders and complex diseases, gradually understood and applied by the researchers.In this study we collected 23 GEFS+ families and decided to detect mutations by using Sanger sequencing of PCR product in all the exons of genes(SCNlA, SCN2A, SCN1B, GABRG2,GABRD) to search the pathogenic gene and mutation feature in those prabands. Then we plan to select a GEFS+ family without all known mutations to capture and identify new pathogenic genes by using whole exome sequencing.The study may be useful to identify GEFS+ new disease genes of Chinese population background,and investigate the etiology and mechanism of GEFS+, thus GEFS+ early diagnosis, treatment and provides the theory basis for the development of new drugs and new treatment targets.[Subjects and Methods]1. Subjects:We collected 23 GEFS+ families with from the Guanngdong general hospital pediatric department between 2011-2014. Two hundred healthy samples were used as controls. The study was approved by the Ethics Review Committee of Guangdong general hospital. Parents of children should providinformed consent before the children participated the study.The control group did not have any history of febrile seizures, seizure or seizure-like episodes, or any neurological disease.2. Clinical data:All participants were evaluated including the clinical syndrome, seizure types、video-electroencephalography(VEEG) and and neuroimaging (MRI), The seizure types of patients were diagnosed by the classification of seizure types according to the epilepsy syndrome diagnostic criteria of the ILAE(2010).3. Mutation sequencing in known pathogenic gene(1) The blood samples of 118 members of GEFS+ families and 200 healthy cotroll group were obtained. Genomic DNA was extracted from the blood samples.(2) The primers were designed for the exons and exon-intron boundaries of 5 genes(SCN1A,SCN2A,SCN1B,GABRG2,GABRD) using Primer 3.0 software online with the UCSC Genome Browser website and NCBI/Primer-Blat. The candidate genes were amplified by PCR.(3) The PCR products were purified,then sequenced using the ABI 3730. The sequencing results were analyzed using the Vector NTI8.0 software, and compares with the standard in the GenBank sequence. Then the possible mutations were compared with those in 200 cases of healthy control group except gene polymorphisms. If mutations were identified, the family members of should also be screened4. Whole exome sequencing searching for the pathogenic variant(1) capture of candidate genes in GEFS+ familyExome sequencing was conducted in two affected individuals and two unaffected individuals from a GEFS+ family in which the known gene mutations was excluded. High throughput sequencing platform for whole genome and exons data results combined with biological information analysis technology to find out a background in Chinese population GEFS+ patients new pathogenic candidate gene mutation.(2) Verified mutation by Sanger sequencingAt first we verified the candidate variations harboring in two affected individuals. Data were analyzed by the chromatogram file editor software Chromas 2.22, and compared with the standard sequence of the gene in order to rule out false positive variants, positive variants for further verification① Validation in the family:The positive variants in seven members of family D (including 3 patients and four members) were verified by sequencing. We excluded the variant which don’t exist in patients because GEFS+ is an autosomal dominant genetic disease, the validation criteria should conform to the phenotype and genotype were separate.3.3 We sequenced all exons of the gene got from step 3.2 in all cases. If a mutation was found, we verified it by comparing with NCBI SNP database and 500 healthy controls.② Validation in the normal cases:The positive variants were verified by PCR sequencing method in 200 normal to exclude the SNP③ validation outside the family:The positive variants were tested in the rest of the GEFS+ family by sanger sequencing.[Results]1. In this study we collected 23 GEFS+ pedigrees, with a total 96 patients(male 52 cases and female 44 cases). The genetic patterns belong to autosomal dominant inheritance. Clinical phenotypes of FS 42 cases and FS+ 31 cases account for about 43.8% and 32.3% respectively.2. Screening results in known pathogenic gene mutation:(1) Family A and B(2/23,8.70%) were found carried two novel SCN1A gene mutations(R500Q, C1274Y) presenting heterozygous forms. The two mutaions located in sodium channel alpha subunit protein structure domain D1-2 link and D3 S2 area.(2) The clinical phenotype of the proband in Family A was Dravet syndrome. The clinical phenotype of the proband in Family B was FS+. The common characteristics of probands A and B were earlier onset age, both in 6 months after birth. The two missense mutations (R500Q, C1274Y) were all traced in family heredity. But the clinical phenotype of their elder generation is mild presenting FS.(3) A novel SCN2A gene missense mutation(D1823G) was found firstly in Family C,which was carried in the proband and his father. The proband had a history of FS+ with absence seizures and his father only had a history of FS.(4) None of SCN1B, GABRG2, GABRD gene mutation has been found in all families.3. Capture and verify the result of Whole exome sequencing(1) We obtained SNP and Indels of 4 samples (including two affected individuals and two unaffected individuals) by whole exome sequencing;(2) We tested 3 affected members and 4 unaffected individual of family D and identified a novel missense mutation in the tenth exon of KCNAB3 gene(H258R). comparing with the NCBI database. This mutation existed in highly conserved region. SIFT predicts the score is 0.00, this result means that the mutations have great effect on protein structure. This mutation is absent in the control group.(3) We failed to discover KCNAB3 mutation in other 19 families.[Conclusions]1. Genomic DNA from 17 GEFS+ families were screened for mutations in all exons of SCNIA, SCNIB, GABRG2 and GABRD using PCR methodology followed by Sanger sequencing. Two novel SCNIA missense mutations(R500Q, C1274Y) were found. Missense mutations of SCNIA were most common in GEFS+. SCNIA genotypes and clinical phenotype of GEFS+ was in a certain correlation.2. A novel SCN2A gene mutation was found in a GEFS+ family for the first time and the heterozygous missense mutation (D1823G) has never been reported.3. None of SCNIB, GABRG2, GABRD gene mutation has been found in all families.4. The mutation H258R in KCNAB3 gene was discovered first time in Chinese GEFS+ pedigree and it never been reported.5. We speculated that the KCNAB3 gene may be a new pathogenic gene in Chinese population with GEFS+.