Association of the connexin36 gene with juvenile myoclonic epilepsy.
Association of the connexin36 gene with juvenile myoclonic epilepsy.
复制标题
connexin36 基因与青少年肌阵挛性癫痫的关联。
DOI：
10.1136/jmg.2003.017954
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发表时间：
2004
影响因子：
4
通讯作者：
Meda,P
中科院分区：
文献类型：
--
作者：
Mas,C;Taske,N;Deutsch,S;Guipponi,M;Thomas,P;Covanis,A;Friis,M;Kjeldsen,MJ;Pizzolato,GP;Villemure,J-G;Buresi,C;Rees,M;Malafosse,A;Gardiner,M;Antonarakis,SE;Meda,P
方法病例组和对照组共调查169例JME患者，其中29例来自15 q14易感基因家系，6例来自5所医院神经内科的RS患者，不考虑癫痫综合征是否影响一级亲属。[22]大多数15 q14连锁患者（2/3）在临床上被证实来自英国，其他来自其他五个欧洲国家（丹麦、法国、希腊、葡萄牙和瑞典）。根据国际抗癫痫联盟（ILAE）的分类进行诊断评估。对照组包括从人类多态性研究中心（CEPH）（n= 44）和瑞士人口（n= 79）的家庭中随机选择的无关个体。SNP C. 333T。在另外94个瑞士对照组上进一步对A进行基因分型。由于CEPH和瑞士对照组的基因型和单倍型的等位基因频率和分布相似，因此将这两个亚群合并为一个对照组，包括123名个体（50%为女性）。获得所有参与个体的知情同意书，并且该研究得到所有参与机构的伦理委员会（National Hospital for Neurology and Neurosurgery，伦敦;巴黎医院伦理委员会;和日内瓦大学医院伦理委员会）的批准。我们使用包含CX 36的人基因组序列（GenBank AC 012271）设计引物。选择引物对以扩增覆盖该基因的整个编码区、所有内含子-外显子连接和59个UTR的572 bp的片段。对于外显子1，引物序列为59 TAAAAGGAAAGGGGGATTCG 39和59 CT CAGTCCAGGTGTGAGAAGG 39;对于外显子2，引物序列为59 CAGCTCCCCA GTCAAAAGAC 39和59 GGTCACATAAATGAGGGTGGA 39;对于59 UTR，引物序列为59 ACGCAGGCGGAGACTACTTA 39和59 CCCGATCA TAGTGGAGTGCT 39。用100 ng总基因组DNA进行扩增反应。25 μ l PCR混合物含有250 μ M脱氧核苷酸三磷酸、200 nM每种引物、2.5 μ l 106 PCR缓冲液（Finnzymes）和1.25 U Dynazime Taq聚合酶（Finnzymes，埃斯波，芬兰）。所有样品在T梯度热循环仪96中扩增（Biometra，德国）在以下条件下进行：在94 ℃下初始变性4分钟，然后是35个循环的94 ℃变性30秒，60 ℃退火40秒，72 ℃延伸1分钟。纯化后，使用标准方案对PCR产物进行测序，ABI 377自动测序仪和外显子2的额外引物（59 CACCAGTCCGCCAAGCAG CGAG 39）。使用gap 4编辑器将来自受影响个体的扩增子的色谱图与CX 36基因的基因组序列进行比较，该编辑器可在英国HGMP资源中心（http：//www.hgp.org）获得。hgmp。MRC。AC.英国）。参照CX 36 cDNA序列（GenBank NM_020660）对5个SNP进行表征和命名。
METHODS Cases and control sample A total of 169 patients with JME were investigated: 29 of these were unrelated individuals from the families used to identify the 15q14 susceptibility locus, 6 whereas the other 140 patients were RS from the neurology departments of five hospitals, irrespective of whether an epileptic syndrome affected first degree relatives or not. 22 The majority of the 15q14-linked patients (2/3) were clinically ascertained from within the UK, the others originating from five other European countries (Denmark, France, Greece, Portugal, and Sweden). Diagnostic evaluation was made according to the classification of the International League Against Epilepsy (ILAE). The control group included unrelated individuals who were randomly selected from families of the Centre d’Etude du Polymorphisme Humain (CEPH)(n= 44), and of the Swiss population (n= 79). SNP c. 333T. A was further genotyped on 94 additional Swiss controls. Since allele frequency and distribution of both genotypes and haplotypes were similar in CEPH and Swiss controls, these two sub-populations were combined into a single control group, comprising 123 individuals (50% women). Informed consent was obtained from all participating individuals, and the study was approved by the ethic committees of all participating institutions (National Hospital for Neurology and Neurosurgery, London; Paris Hospital Ethics Committee; and Ethical Committee of the Geneva University Hospital).Mutation analysis and identification of SNPs Detection of mutation was performed by genomic PCR amplification and direct sequencing. We designed primers using the human genomic sequences (GenBank AC012271) which encompass CX36. Primer pairs were selected to amplify fragments covering the whole coding region of this gene, all intron–exon junctions, and 572 bp of the 59 UTR. Primers sequences were 59TAAAAGGAAAGGGGGATTCG39 and 59CT CAGTCCAGGTGTGAGAAGG39 for exon 1; 59CAGCTCCCCA GTCAAAAGAC39 and 59GGTCACATAAATGAGGGTGGA39 for exon 2; 59ACGCAGGCGGAGACTACTTA39 and 59CCCGATCA TAGTGGAGTGCT39 for the 59UTR. Amplification reaction was performed with 100 ng total genomic DNA. The 25 цl PCR mixture contained 250 цM deoxynucleotide triphosphates, 200nM each primer, 2.5 цl 106 PCR buffer (Finnzymes), and 1.25 U Dynazime Taq polymerase (Finnzymes, Espoo, Finland). All samples were amplified in a T Gradient Thermocycler 96 (Biometra, Germany), under the following conditions: initial denaturation at 94 C for 4 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 40 s, and extension at 72 C for 1 min. After purification, the PCR products were sequenced using standard protocols, an ABI 377 automated sequencer, and an additional primer for exon 2 (59CACCAGTCCGCCAAGCAG CGAG39). Chromatographs of amplicons from affected individuals were compared to the genomic sequence of the CX36 gene using the gap4 editor, which is available at the UK HGMP Resource Centre (http://www. hgmp. mrc. ac. uk). Five SNPs were characterised and named with reference to the CX36 cDNA sequence (GenBank NM_020660).