【作者】 龙泓羽；

【导师】 肖波；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景：癫痫是最常见而且严重的慢性神经系统疾病之一,目前主要的治疗手段是服用抗癫痫药物。尽管近年来新的抗癫痫药物在癫痫治疗方面已取得长足的进步,但迄今仍有大约30%的癫痫患者对抗癫痫药物耐药。耐药性癫痫已经成为危害公共健康的一个主要疾病,研究表明耐药性癫痫的死亡率比一般的癫痫患者高4-7倍。GABA是中枢神经系统中最主要的抑制性神经递质,在神经网络的兴奋性调节中起关键作用,与癫痫的发生发展密切相关。GABA转运体(GAT)是一种主要存在于神经元和神经胶质细胞膜上的糖蛋白,参与GABA在中枢神经系统的转运与释放过程。一项在韩国人群中的研究发现,编码GAT3的SLC6A11基因与癫痫耐药性之间存在关联性。而在中国汉族人群中SLC6A11基因与癫痫耐药性之间的关系尚无相关研究报道。载脂蛋白E(APOE)是中枢神经系统中重要的脂质转运分子,在神经组织的损伤修复、轴索生长和突触形成中起重要作用。有研究表明APOE基因多态性与颞叶耐药癫痫有关,而APOE基因多态性是否与所有不同类型的耐药性癫痫相关尚不明确。因此针对中国汉族人群SLC6A11和APOE基因多态性与耐药性癫痫相关性的研究十分有必要。研究目的：分析编码GAT3的SLC6A11基因和编码载脂蛋白E的APOE基因的tagSNP的分型数据,探索中国汉族人群SLC6A11和APOE基因多态性与耐药性癫痫的相关性。研究方法：本研究采用基于人群的病例——对照研究设计。1、根据入选标准和排除标准收集2010年8月至2012年10月期间在湘雅医院门诊的480例癫痫患者(207例耐药组,273例敏感组)。2、酚氯仿法提取全血DNA。3、从国际人类基因组单体型图计划数据库在SLC6A11基因和APOE基因相应区段选取16个tagSNP。对研究对象应用Illumina Golden Gate定制芯片(X48-384)对所选的tagSNP进行基因型鉴定。4、拟和优度X2检验(Goodness-of-fit Chi-square test)分析对照组基因型频率的分布是否符合H-W平衡。5、分析单位点基因型频率的关联,评估单位点与耐药性癫痫的相关性。6、分析单体型的关联,评估单体型与耐药性癫痫发生风险的相关性。研究结果：本研究共检测了SLC6A11基因的14个tagSNP位点和APOE基因的2个tagSNP位点,其中SLC6A11基因的1个位点因染色体结构性变异导致聚类不好不适于本基因芯片检测方法。1、我们所研究的15个位点多态分布在癫痫患者人群中均符合Hardy-Weinberg平衡。2、敏感组与耐药组之间SLC6A11基因13个SNP位点的基因型频率无显著性差异(P>0.05),SLC6A11基因rs2304725和rsl881354之间存在强连锁,未发现具有统计学意义的单倍型。3、敏感组与耐药组之间APOE基因2个SNP位点的基因型频率无显著性差异(P>0.05)。结论：1、SLC6A11基因的13个候选SNP多态性可能与中国汉族人群的癫痫耐药性无关。2、APOE基因的2个候选SNP多态性可能与中国汉族人群的癫痫耐药性无关。3、SLC6A11基因rs2304725和rs1881354之间存在强连锁。更多还原

【Abstract】 Background:Epilepsy is one of the most common and serious chronic neurological diseases in human. Antiepileptic drugs (AEDs) is the main approach to the control of epileptic seizures. Although great progress has been made in drug treatment of epilepsy, but there are still about30%of epilepsy patients resistant to antiepileptic drugs. Drug-resistant epilepsy has become a big problem of public health, the mortality rate of drug-resistant epilepsy is4to7times higher than common epilepsy.GABA is one of the main inhibitory neurotransmitters in the central nervous system, plays a key role in mediation of the neural network excitatory and is closely related to epilepsy development. GABA transporter is a glycoprotein mainly located in neurons and glial cell membrane, participate in the transportation and release process of GABA in the central nervous system. A study in Korean found positive correlations between the SLC6A11rs2272400and drug resistant epilepsy. But there is no relevant reports about the correlations between the SLC6Alland drug resistant epilepsy in Chinese Han population. Apo lipoprotein E (APOE) is an important lipid transport molecules in central nervous system, plays a key role in axonal growth, synapse formation, impaired nerve tissue repair. Studies showed that APOE polymorphism was probably associated with drug resistant temporal lobe epilepsy. However, whether APOE polymorphism is associated with all types of drug resistant epilepsy is still unknown. So it is necessary to study the relationship between SLC6A11and APOE gene polymorphism and drug-resistant epilepsy in Chinese Han population.Objective:To investigate the association between the SLC6A11and APOE polymorphism and drug resistant epilepsy.Material and Methods:A population-based case-control design was applied in our study.1. The study consisted of480outpatients diagnosed with epilepsy who visited the Xiangya hospital central-south university from August2010to October2012.273patients were recruited in drug-responsive group and207patients were recruited in drug-resistant group.2. Genomic DNA was extracted from peripheral blood leukocytes of each subject by Phenol Chloroform Method.3.16Tag single nucleotide polymorphisms (tagSNP) in SLC6A11and APOE gene were selected through hapmap and National Center for Biotechnology Information (NCBI) databases. All the tagSNP were genotyped by VeraCode GoldenGate Genotyping Assay system and the data were analyzed by SPSS21. 4. To analysis the genotype frequency distribution of the control group whether meets Hardy-Weinberg equilibrium by Goodness-of-fit Chi-square test.5. All the data of15tagSNP were analyzed by the association study.Results:1. We detected16tagSNPs of SLC6A11and APOE genes, one SNPs which belongs to SLC6A11are not suitable for the gene chip detection for clusting caused by chromosome structural variation.2. The total15SNPs distribution in the population of patients with epilepsy accord with Hardy-Weinberg equilibrium3. Genotypes and alleles distributions in the13SNPs of SLC6A11showed no statistically difference between the drug-responsive and drug-resistant groups (P>0.05). Strong linkage was found between rs2304725and rs1881354. There is no statistically significant difference haplotype between the drug-responsive and drug-resistant groups (P>0.05).4. Genotypes and alleles distributions in the2SNPs of APOE showed no statistically difference between the drug-responsive and drug-resistant groups(P>0.05).Conclusion:1. The13SNPs of SLC6A11gene may not be related to drug-resistant epilepsy in Chinese Han population. 2. The2SNPs of APOE gene may not be related to drug-resistant epilepsy in Chinese Han population.3. Strong linkage was found between rs2304725and rs1881354in SLC6A11gene.更多还原