【作者】 王心；

【导师】 沈岩； 刘英；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 第一部分内侧颞叶癫痫的遗传易感性研究研究背景癫痫是最常见的神经系统疾病之一,发达国家的患病率达到5～10‰,中国的患病率为3.5～4.8‰。作为临床上主要的药物难治性癫痫,内侧颞叶癫痫(mesialtemporal lobe epilepsy,MTLE)是发病率最高的成人发作的局灶性癫痫。散发的MTLE是一类遗传因素和环境因素共同作用的复杂性疾病。基因水平变异可能通过影响抽搐发作易感性、药物反应及临床特征等多个方面对MTLE的发生、发展及预后产生效果。环境因素包括头部外伤、中枢神经系统感染、产伤、热性惊厥或颅脑肿瘤等。目前的研究提示,包括MTLE在内的癫痫等发作性疾病是一种离子通道病,但MTLE的遗传易感基因方面各种群间还没有发现一致的证据。本文应用病例对照的研究方法,以γ-氨基丁酸B型受体2亚基(γ-aminobutyric acid type B receptor 2,GABBR2)基因、γ-氨基丁酸B型受体1亚基(γ-aminobutyric acid type B receptor 1,GABBR1)基因、朊蛋白(prion protein,PRNP)基因和大电导钙离子激活钾离子通道M亚系β4亚基(potassium large conductance calcium-activated channel,subfamily M,beta member 4,KCNMB4)基因为候选基因,分析遗传因素与中国汉族人群MTLE的相关性。研究方法以MTLE患者和非癫痫受试者为研究对象,应用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法、测序方法检测中国汉族人群标签SNPs(tag SNPs),结合功能SNPs,分析这些SNPs与MTLE的相关性并探讨基因型/单倍型与癫痫临床表型的关联。研究结果1.GABBR2基因多态性与MTLE的相关性在315名MTLE患者和318名非癫痫对照受试者中,经logistic回归分析调整性别混杂因素影响后,GABBR2基因rs967932的A等位基因在MTLE组的频率显著高于非癫痫对照组频率(48.43%vs.41.59%;递加模型下P=0.018,OR=1.305,95%CI 1.048-1.624,显性模型下P=0.003,OR=1.667,95%CI 1.186-2.343)。Bonferroni多重比较校正后,显性模型下rs967932的基因型频率组间分布仍可见显著性差异(P=0.036)。另外,rsl999501和rs944688的基因型频率在显性模型下也可见MTLE组和非癫痫对照组间的显著性差异(P分别为0.033和0.025),但这些差异在Bonferroni校正后消失。GABBR2基因rs3780428、rs1999501、rs967932和rs944688组成的单倍型G-C-A-C在MTLE组的频率显著高于非癫痫对照组频率(12.26%vs.6.5 1%,P=0.0004)。Bonferroni校正后单倍型G-C-A-C仍为MTLE的风险因素(P=0.002,OR=2.028,95%CI 1.359-3.024)。携带此单倍型MTLE患者的发病时间较不携带患者显著提前(14岁vs.19岁,P=0.028)。2.GABBRl基因多态性与MTLE的相关性在315名MTLE患者和318名非癫痫对照受试者中,rs29259的C等位基因频率在非癫痫对照组的频率显著高于MTLE组(显性模型下P=0.046,OR=0.697,95%C1 0.488—0.994),但Bonferroni校正后显著性差异消失(P=0.276)。rs29261的各基因型/等位基因频率在递加模型、显性模型和隐性模型下均未见MTLE组和非癫痫对照组间的分布差异。G1465A位点则未检测出多态性。由rs29259和rs29261组成的单倍型频率组间分布分析中,未见各单倍型频率在MTLE组与非癫痫对照组间的显著分布差异。将MTLE患者按照是否合并海马硬化进行分组,比较有/无海马硬化患者组间rs29295和rs29261的基因型及等位基因频率,仍未见显著的组间差异。3.PRNP基因多态性与MTLE的相关性在320名MTLE患者和558名非癫痫对照受试者中,没有发现PRNP基因M129V的VV基因型。M129V的MM和MV基因型/等位基因频率在MTLE组和非癫痫对照组间的频率分布没有统计学差异(基因型:P=0.240;等位基因:P=0.240)。将受试者按照性别分组分析时,该位点的各基因型/等位基因频率未见显著的组间差异。将受试者按照小于50岁和不小于50岁分组分析时,该位点的各基因型/等位基因频率仍无组间差异。临床表型分析中未见携带该位点MV基因型MTLE患者的显著异常。4.KCNMB4基因多态性与MTLE的相关性在合计321名MTLE患者和496名非癫痫对照受试者中,KCNMB4基因的rs787931、rs9634299和rs10784846的各基因型/等位基因频率在递加模型、显性模型和隐性模型下均未见MTLE组和非癫痫对照组间的频率分布差异。这三个位点组成的单倍型频率分布分析中,未见MTLE组和非癫痫对照受试者组间频率大于3%常见单倍型的频率分布差异。将MTLE患者按照是否合并海马硬化进行分组,比较有/无海马硬化患者组间3个位点的基因型及等位基因频率,仍未见显著的组间差异。研究结论综合上述,本研究为以GABBR2基因、GABBR1基因、PRNP基因和KCNMB4基因为候选基因、以中国汉族MTLE患者为病例、以非癫痫受试者为对照开展的病例对照研究,可得出如下结论:携带GABBR2基因rs967932-A等位基因是MTLE发生的危险因素,GABBR2基因是中国汉族MTLE的遗传易感基因;而GABBR1基因、PRNP基因和KCNMB4基因对MTLE易感性的贡献可能不大。更多还原

【Abstract】 BackgroundsEpilepsy is one of the most common neurological disorders with 5～10‰of morbidity in developed countries.Prevalence rate of epilepsy in China is 3.5～4.8‰and mesial temporal lobe epilepsy(MTLE) possesses the highest incidence of adult onset focal seizures. Sporadic MTLE is regarded as a complex disease attributed to the interaction of genetic factors and environmental factors.Genetic variations can affect the epileptogenesis, development and prognosis of MTLE by multiple facets including the susceptibility of seizure on-set,medication response and seizure features.Head trauma,brain infection, birth injury,and febrile convultion are environmental factors of MTLE.Although the view that the paroxysmal disorder including epilepsy is a channolapathy disease is accepted,the gene involved in genetic susceotibility of MTLE is still unknown.This study was then planned to test the hypothesis that one or more variants inγ-aminobutyric acid type B receptor 2(GABBR2),γ-aminobutyric acid type B receptor 1(GABBR1),prion protein (PRNP) and potassium large conductance calcium-activated channel subfamily M beta member 4(KCNMB4) genes are associated with sporadic MTLE using tag SNPs method in a Han Chinese MTLE patients and non-epilepsy control subjects design.MethodsEligible MTLE patients and non-epilepsy control subjects were recruited in this study. Polymorphisms including tag SNPs of 4 candidate genes were genotyped by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),and then the relationship between allele/gentotype distribution and MTLE presence were analyzed, the effect to disease phenotype was also discussed. Results1.Association of tag SNPs in GABBR2 gene with MTLEWe assessed the association of each genotype by logistic regression analyses adjusting for sex in additive,dominant and recessive genetic model in a 315-case-and-318-control sample set.Three tag SNPs of GABBR2 were found to be associated with the risk of MTLE. rs967932 A-allele showed an increased risk of MTLE when applying an additive and dominant genetic model(P = 0.018,OR = 1.305,95%CI 1.048-1.624 and P = 0.003,OR = 1.667,95%CI 1.186-2.343).rs1999501 of GABBR2 gene showed an increased risk of MTLE with a dominant model(P = 0.033,OR = 1.469,95%CI 1.032-2.090),while rs944688 conferred a reduced risk of disease with a dominant model(P = 0.025,OR = 0.608,95%CI 0.393-0.939).After a Bonferroni correction,only rs967932 A-allele significantly increased the risk of MTLE in a dominant model(P = 0.036).There was no significant difference in genotype frequencies between cases and controls for rs3780428. Comparison analysis of haplotype frequencies demonstrated that the frequency of haplotype G-C-A-C(rs3780428-rs1999501-rs967932-rs944688) was significantly increased in MTLE patients compared to controls(12.26%vs.6.51%,P = 0.0004).After a Bonferroni correction was applied,haplotype G-C-A-C was still the significant risk factor (P = 0.0024,OR = 2.028,95%CI 1.359-3.024).Analyses of the relationship between haplotype G-C-A-C and clinical phenotypes revealed that haplotype G-C-A-C was associated with an earlier onset age compared to other haplotype(14 years vs.19 years,P = 0.028)2.Association of tag SNPs in GABBR1 gene with MTLEWe assessed the association of each genotype by logistic regression analyses adjusting for sex in additive,dominant and recessive genetic model in a 315-case-and-318-control sample set,and one tag SNPs of GABBR1 were found to be associated with the risk of MTLE.rs29259 C-allele showed an reduced risk of MTLE when applying an dominant genetic model(P = 0.046,OR = 0.697,95%CI 0.488-0.994),but did not reach a significant level when Bonferroni correction was applied(P = 0.276).There was no significant difference in genotype frequencies between cases and controls for rs29261. The G1465A genotype of all subjects was GG.Comparison analysis of haplotype frequencies demonstrated that no haplotype of rs29259-rs29261 was significantly different in MTLE patients compared to controls.When MTLE patients were subgrouped according to the presence of hippocampal sclerosis(HS),still no differences were detected between subgroups at the genotypes/alleles of rs29259 and rs29261.3.Association of polymorphisms in PRNP gene with MTLEThe frequency of V allele was 0.94%in 320 MTLE cases and 1.61%in 558 controls. No significant differences in the genotype(P = 0.24) or allele(P = 0.24) frequencies of M129V polymorphism in MTLE patients and controls were observed.Even when the samples were stratified by gender,no significant association was noted between the cases and controls.By sub-grouping control subjects in 2 age groups,i.e.,individuals aged 13-49 years(34.8%) and those aged 50-89 years(65.2%),additional statistical analysis of the relation of the genotype and M129V allele to age was obtained.No significant difference was observed between MTLE patients and controls with regard to genotype distribution(P = 0.76 and 0.43 respectively) or allelic frequency(P = 0.06 and 0.43 respectively).We also analyzed clinical features according to the presence of 129V allele in MTLE patients.No apparent differences were found between 6 patients heterozygous for M129V and others with the MM genotype with respect to age,ratio of males,age at onset,aura,family history,antecedent FC,MRI signs,and response to medication.4.Association of tag SNPs in KCNMB4 with MTLEWe assessed the association of each genotype by logistic regression analyses adjusting for sex in additive,dominant and recessive genetic model in a total 321-cases-and-496-control sample set,and none of three tag SNPs KCNMB4 were found to be associated with the risk of MTLE.Comparison analysis of haplotype frequencies demonstrated that no haplotype of rs787931-rs9634299-rs10784846 was significantly different in MTLE patients compared to controls.When MTLE patients were subgrouped according to the presence of hippocampal sclerosis(HS),still no differences were detected between subgroups at the genotypes/alleles of rs787931,rs9634299 or rs10784846.ConclusionsIn summary,GABBR2,GABBR1,PRNP and KCNMB4 were selected as candidate genes to explore their genetic roles to MTLE by a tag-SNP-based method in a Han Chinese MTLE patients and non-epileptic control subjects design.It suggested that A-allele of GABBR2 rs967932 is the at-risk factor to MTLE,and the role of GABBR1, PRNP and KCNMB4 in the aeteology of MTLE may be small or modest.更多还原