【作者】 徐佳亮；

【导师】 何志义；

【作者基本信息】 中国医科大学 ， 神经病学， 2009， 博士

【摘要】 ALOX5AP基因和IL-1A基因多态性与缺血性卒中的相关性研究背景脑血管病是严重危害人类健康的常见病、多发病,是人类三大死亡原因之一;缺血性卒中(IS)的发病率占脑血管疾病发病率的80%。虽然其确切遗传机制尚不清楚,但近些年人们通过多态性关联分析等方法己经报告了一些与IS有关的新易感性基因,如ApoE、PDE4D、ALOX5AP、IL-1A等基因。但是针对这些候选基因在不同地区、针对不同种族的关联性研究结果均未得到有效的重复和肯定。我国北方地区为IS的高发区,有必要进行一项针对我国北方地区汉族人群IS易感基因的病例对照研究。动脉粥样硬化的形成和发展是IS的重要病理生理基础,而炎症反应在动脉粥样硬化的形成过程中具有重要的意义。因此本研究选取ALOX5AP基因和IL-1A基因这两个炎症相关基因,采用候选基因病例对照的研究方法,分析了由DECODE研究组命名的单倍型HapA的AOX5AP基因四个位点(SG13S25A/G、SG13S32A/C、SG13S89A/G、SG13S114A/T)及IL-1A基因-889位点的基因型、等位基因型在IS组、IS亚组及对照组中频率分布差异,从而探讨ALOX5AP基因、IL-1A基因多态性和中国北方汉族人群IS的相关性,为更详尽的了解IS发病潜在的遗传机制提供分子流行病学依据,并为早期诊断IS提供更多的分子生物学指标。研究方法采用病例-对照研究方法,IS组选取中国北方IS患者472例,男283例、女189例,年龄在40-80岁间,均为汉族,彼此无亲缘关系;选取性别、年龄与IS组相匹配的健康个体312例作为对照组,男183例、女129例;在此基础上将IS组按病因不同分为大血管组(血栓性脑梗死组)和小血管组(腔隙性脑梗死组)。应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)的方法检测AOX5AP基因SG13S32位点、SG13S89位点和IL-1A-889位点的多态性,应用基质辅助激光解吸附电离飞行时间质谱法(MALDI-TOF)检测SG13S114位点、SG13S25位点多态性;基因分型结果经双脱氧末端终止测序法进一步确认。用拟合优度x~2检验验证每个位点SNP基因型在抽样群体中的分布是否符合Hardy-Weinberg平衡。对于单位点的SNP数据,采用SPSS13.0统计软件x~2Fisher’s精确概率法检验其基因型、等位基因型在对照组与IS组及各亚组中的频率分布差异、风险度分析,最后结合临床资料,对相关数据进行条件Logistic回归方法排除传统混杂因素的干扰,以P＜0.05为检验水准。用Shesis在线软件计算ALOX5AP基因单倍型的频率。用UNPHASED软件分析ALOX5AP基因与IL-1A基因对IS的联合作用。研究结果1、所有研究对象的每个SNP基因型在抽样群体中的分布均符合Hardy-Weinberg平衡(P＞0.05),表示所选取的样本具有人群代表性适合做遗传学分析。2、ALOX5AP基因多态性在对照组与IS组及IS亚组中频率分布:SG13S32位点C等位基因频率IS组明显高于对照组(OR=1.271;95%CI:1.027-1.572;P=0.028);SG13S114位点AA基因型频率大血管组明显高于对照组(OR=1.987;95%CI:1.138-3.471;P=0.014),应用条件Logistic回归校正后仍有统计学差异(OR=1.479;95%CI:1.024-2.135:P=0.037)。3、ALOX5AP基因单倍型在对照组与IS组及IS亚组中频率分布:HapA单倍型频率分布在对照组、IS组及IS亚组中没有差异;GCGA单倍型的频率IS组明显高于对照组(OR＝1.683;95%CI:1.138-2.487;P=0.008),大血管组也高于对照组(OR=1.629;95%CI:1.047-2.532;P=0.029),小血管组与对照组差别更加明显(OR=1.806;95%CI:1.156-2.822;P=0.009);GAGA单倍型的频率IS组低于对照组(OR=0.763;95%CI:0.583-0.988;P=0.048),大血管组的频率也低于对照组(OR=0.719;95%CI:0.522-0.99;P=0.042)。4、不同性别的ALOX5AP基因单倍型的频率分布差异:所有单倍型在男、女性IS组与对照组中的频率分布未见到显著性差异。5、IL-1A基因多态性在对照组与IS组及IS亚组中频率分布:L-1A-889 TT基因型频率大血管组明显高于对照组(OR=2.473;95%CI:1.129-5.42;P=0.02),应用Logistic校正后仍有统计学意义(OR=1.581;95%CI:1.003-2.493;P=0.047);其等位基因T的频率大血管组也高于对照组(OR=1.540;95%CI:1.075-2.204;P=0.018)。IL-1A-889基因多态性在男、女性别中分布无差异。6、ALOX5AP基因与IL-1A基因的联合作用:同时携带ALOX5AP基因GCGA单倍型和IL-1A-889位点T等位基因的频率IS组高于对照组(OR＝1.608;95%CI:1.067-2.423:P=0.022)。结论1、ALOX5AP基因SG13S114位点AA基因型可能是中国北方汉族人群血栓性脑梗死的独立的风险因素,其风险性主要来源于A等位基因。2、SG13S32位点C等位基因可以增加患血栓性脑梗死的遗传易感性。3、ALOX5AP基因HapA单倍型与IS没有相关性。4、ALOX5AP基因GCGA单倍型可能是IS的风险单倍型,GAGA可能是IS的保护性单倍型。5、ALOX5AP基因多态性与IS的相关性与性别无关。6、IL-1A基因-889位点TT基因型可能是血栓性脑梗死的独立的风险因素,其遗传易感性主要来源于等位基因T。7、IL-1A-889基因多态性与IS的相关性与性别无关。8、ALOX5AP基因GCGA单倍型与IL-1A-889位点T等位基因的协同作用可以显著增加中国北方汉族人群患IS的遗传易感性。更多还原

【Abstract】 ObjectiveCerebrovascular disease(CVD)is one of the three major causes of death,and is harmful to human health.Ischemic stroke(IS)of incidence of cerebrovascular disease accounted for 80%.IS is a heterogeneous multifactorial disorder.Epidemiological data provide substantial evidence for a genetic component to the disorder,but the extent of predisposition is unknown.Genetic variance determines IS genetic predisposition. Although the exact mechanisms are not clarified,in recent years through polymorphisms linkage analysis some new susceptible genes have been reported such as apolipoproteinE(ApoE),phosphodiesterase 4D(PDE4D),Interleukin-1A(IL-1A), and arachidonate 5-lipoxygenase-activating protein(ALOX5AP)gene.However the association results of these candidate genes in different regions and for different ethnic population have not been effectively reapeated and confirmed.In view of northern Chinese population with higher incidence of IS,it is necessary to carry out an association study of candidated susceptible genes aimed at northern China region Han population IS.Atherosclerosis is an important pathophysiological basis of IS,and inflammatory reaction process is of great significance in atherosclerosis formation,so our study selected inflammation-related ALOX5AP and IL-1A gene.By introducing candidate genes case-control association analysis,we mesured ALOX5AP gene haplotype HapA four SNPs(SG13S25A/G、SG13S32A/C、SG13S89A/G、SG13S114A/T)and IL-1A-889 C/T SNP genotype and allele frequencies between IS group,IS subgroups and control group,and evaluated the association between these two genes SNPs and IS.So we can provide IS molecular epidemiology data for comprehensively understanding IS potential genetic mechanisms and more early IS molecular biology diagnosis indicators.Materials and methods A case-control design was introduced.The subjects of IS group were consecutively recruited from northern China region patients with a total of 472 cases male 283,female 189,aged 40-80 years old,Han nationality,no consanguinity with each other.The controls matched by age,sex,and ethnic orign were recruited from the same patients geographic region,with a total of 312 cases,male 183,female 129. Accordting to the different causes the patients were divided into two subgroups:the large vessels group(thrombotic cerebral infarction group) and the small vessels group(lacunar infarction group).ALOX5AP gene SG13S32,SG13S89,and IL-1A-889 SNPs were genotyped by polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP) analysis.ALOX5AP SG13S114 and SG13S25 SNPs were genotyped by means of Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF) primer extension reaction.Genotyping results were confirmed by dideoxy termination squencing method.SPSS 13.0 statistical software was used for data processing.Continuous variables were expressed as mean±SE and were assessed by One-Way ANOVA.Genotype and allele frequencies were calculated for control group and each of the patient groups.Univariate comparisons of allele and genotype distributions were done using x~2 test.The x~2 goodness of fit test was used to test for deviation of genotype distribution from Hardy-Weinberg equilibrium. Conditional Logistic Regression adjusted for IS traditional risk factors including age, blood pressure,blood sugar,blood lipids and smoking.The influence of AlOX5AP and IL-1A gene polymorphisms on IS was indicated by odds ratio(OR) and 95% confidenc(CI) with P＜0.05 as significant criteria.Haplotype-based analysis was infered by using Shesis online-software.The synergistic action of ALOX5AP gene and IL-1A gene for IS was analysed by UNPHASED 3.0.7 version software.Results1.The genotype frequencies of all SNPs conformed to the expectations of Hardy-Weinberg equilibrium(P>0.05).It was suggested that the selected samples could represent the population and were suitable for genetic analysis.2.ALOX5AP gene SNPs frequencies distributions among the patients and the controls:SG13S32 C allele frequencies of the IS group were higher than the control group(OR=1.271;95%CI:1.027-1.572;P=0.028);SG13 S 114 AA genotyp frequencies of the large vessels group were higher than the control group(OR=1.987;95%CI: 1.138-3.471;P=0.014),and after conditional logistic regression the differece was still significant(OR=1.479;95%CI:1.024-2.135;P=0.037).3.ALOX5AP gene haplotype frequencies among the patients and the controls: ALOX5AP gene HapA haplotype frequencies were normal among the patients and the controls;GCGA haplotype frequencies of the IS group were higher than the control group(OR=1.683;95%CI:1.138-2.487;P=0.008),the frequencies of the large vessels were higher than the control group(OR=1.629;95%CI:1.047-2.532;P=0.029),and the frequencies of the small vessels group were remarkably higher than the control group(OR=1.806;95%CI:1.156-2.822;P=0.009);GAGA haplotype frequencies of the IS group were lower than the control group(OR=0.763;95%CI:0.583-0.988; P=0.048),and the frequencies of the large vessels group were lower than the control group(OR=0.719;95%CI:0.522-0.99;P=0.042)4.ALOX5AP gene haplotype gender-specific frequencies distributions: Significant differences of all the haplotypes were not observed in the male and female IS group and control group.5.L-1A-889 SNPs frequencies distributions among the patients and the controls: IL-1A-889 TT frequencies of the IS group were higher than the control group (OR=2.473;95%CI:1.129-5.42;P=0.02),and after conditional logistic regression the difference was still significant(OR=1.581;95%CI:1.003-2.493;P=0.047); IL-1A-889 T allele frequencies of the large vessels group were higher than the control group(OR=1.540;95%CI:1.075-2.204;P=0.018).IL-1A-889 gene polymorphisms distributions were not different among male and female.6.ALOX5AP gene and IL-1A gene synergistic reaction:The individuals who carried the haplotype GCGA and IL-1A-889 C allele suffered IS with the 1.608 folds risk(OR=1.608;95%CI:1.067-2.423;P=0.022).Conclusions1.ALOX5AP gene SG13S114 AA genotype might be the independent risk factors of northern China region han population thrombotic cerebral infarction,and its risk mainly came from T allele.2.SG13S32 C allele can increase genetic susceptibilty of thrombotic cerebral infaction.3.The association ofALOX5AP gene HapA haplotype with IS was not identified.4.ALOX5AP gene GCGA haplotype might be at-risk haplotype of IS,and GAGA haplotype might be protective haplotype of IS.5.The association of ALOX5AP gene with IS was not related with male and female sex.6.IL-1A-889 TT genotype could be independent risk factor of thrombotic cerebral infarction,and its genetic susceptibility mainly came from T allele.7.The association of IL-1A-889 with IS was not related with male and female sex.8.ALOX5AP and IL-1A gene polymorphisms can synergistically confer higher risk for IS of northern China region population.更多还原