【作者】 胡中扬；

【导师】 杨期东；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 研究背景和目的:脑卒中是当今世界公认的三大主要疾病死因之一,脑出血为其中的头号杀手,对人类生命与健康危害极大。脑出血在脑卒中所占的比例在不同的地域及种族存在差异。国外的流行病学资料表明脑出血在脑卒中所占的比例大约在6.5-19.6%之间。通过近20年的流行病学监测,我们发现1986～2000年长沙市地区人群脑卒中年平均发病率为236.6/10万,其中脑出血的发病率131.0/10万,占脑卒中的55.4%,长沙为世界脑出血的高发区之一,但该地区脑出血的原因尚不明确。为此我们进行了一系列的病因学研究。在研究中,我们发现长沙地区脑出血具有家族聚集现象,遗传因素可能是该地区脑出血高发的原因之一。候选基因筛查是目前研究脑出血这类复杂多基因病的主要手段。目前有相当多的证据表明与高血压及血管重构相关的激肽释放酶-激肽系统(kallikrein-kinin system,KKS)代谢通路在小动脉硬化、颅内动脉瘤形成乃至心脑血管疾病的“候选基因”研究中占有相当重要的地位,KKS系统可能在脑出血发病的病理生理机制中起着重要的作用。但KKS系统相关SNPs和单体型分子标志与有家族聚集现象的脑出血之间的关系研究,目前国内外迄今为止未见报道。本课题首次通过研究不同群体包括正常人群、散发性脑出血患者,有家族聚集现象的脑出血患者及其家系成员的相关基因,包括激肽原(KNG1)基因、组织型激肽释放酶(KLK1)基因和血管紧张素转化酶(ACE)基因的SNPs位点及单体型分子标志分布情况。从而比较不同群体的基因型的差异及其对脑出血的易感性,从分子水平探讨脑出血的发病机理,为预防和治疗脑出血提供理论依据。材料和方法:收集2006年1月至2008年10月在湘雅医院神经内科就诊的脑出血患者及部分家属的资料,所有病例均经CT和/或MRI确诊(依照第四届全国脑血管病学术会议诊断标准);对照组来自湖南省长沙地区汉族健康体检人员。对照组成员、脑出血患者及脑出血家系成员均为湖南省长沙地区汉族人,并签署知情同意书。所有的研究对象具体分组如下:(1)有家族聚集现象的脑出血家系(cerebralhemorrhage with family history,CHFH):38个家系共有200人,包括脑出血患者61例,Ⅰ级亲属95例,Ⅱ级亲属23例和健康无血缘关系亲属21例。(2)散发性脑出血组(sporadic cerebral hemorrhage,SCH):273例。(3)对照组:140例。病例组与对照组的年龄、性别具可比性。采用多重单碱基延伸SNP分型技术(Multiplex Snapshot)对组织型激肽释放酶(KLK1)基因rs3212855、rs5515、rs5516和rs5517位点,血管紧张素素转化酶(ACE)基因rs4291、rs4343位点和激肽原(KNG1)基因rs1656922、rs2304456等相关SNPs位点进行基因分型;采用聚合酶链式反应结合琼脂糖凝胶电泳分析方法检测血管紧张素转化酶(ACE)基因第16内含子的I/D多态位点(rs4646994)。在进一步对基因分型结果的统计分析中,首先在基于人群的病例对照研究中,通过卡方检验比较散发性脑出血组和对照组基因型分布和等位基因频率的差异,从而确定与疾病关联的基因;进一步在以家系为基础的关联研究中,用FBAT软件进行传递不平衡检验(TDT),确认与长沙汉族人群脑出血相关的SNPs及单体型分子标志。结果:1.组织型激肽释放酶基因(KLK1)rs3212855、rs5515、rs5516和rs5517位点基因分型结果的分析:①在本研究样本中未能证实rs5515是多态性位点(所有检测的样本均为GG型纯合子,未见GA及AA基因型)。②其它三个多态性位点在散发性脑出血组和对照组的比较:rs3212855和rs5516位点的基因型和等位基因频率在两组间分布无差异(P＞0.05);而散发性脑出血组rs5517多态位点基因型分布与对照组比较存在显著差异(P＜0.05),散发性脑出血组rs5517 A等位基因频率显著高于对照组(P＜0.05)。③家系关联研究:在以家系为基础的关联检验(FBAT)中,有两个多态性位点显示等位基因传递具有统计学显著性(rs5516的G:Z=2.422,P=0.015424;rs5517的A:Z=2.963,P=0.003049)。多态性位点间的连锁不平衡检验显示三个多态性位点(rs3212855、rs5516和rs5517)紧密连锁,在一个单体型块(haplotype block)中,可以构建三个多态性位点组成的单体型模式。对三位点组成的单体型传递的总体检验显示KLK1基因与脑出血存在关联(x~2=12.664,自由度=4,P=0.013042),单个单体型的分析也显示rs3212855、rs5516、rs5517三位点构成的单体型CGA在患者中过度传递,差异有显著性(Z=2.881,P=0.003969)。2.激肽原基因(KNG1)rs1656922和rs2304456位点基因分型结果的分析:①散发性脑出血组和对照组的比较:rs1656922的基因型分布和等位基因频率在两组间无差异(P＞0.05);rs2304456的基因型分布和等位基因频率在两组间无差异(P＞0.05)。②家系关联研究:rs1656922和rs2304456两SNPs位点在以家系为基础的关联检验(FBAT)中,显示等位基因传递没有统计学显著性(P＞0.05)。多态性位点间的连锁不平衡检验显示这两个多态性位点呈连锁不平衡,在一个单体型块(haplotype block)中,可以构建两个多态性位点组成的单体型模式。单体型传递的总体检验及单个单体型的分析均未显示单体型存在过度传递现象(P＞0.05)。3.血管紧张素转化酶基因(ACE)的rs4291、rs4646994和rs4343位点基因分型结果的分析:①在散发性脑出血组和对照组的比较:rs4291、rs4646994和rs4343三个多态性位点的基因型分布和等位基因频率在两组间无差异(P＞0.05)。②家系关联研究:以家系为基础的关联检验(FBAT)中,显示三个多态性位点的等位基因的传递均没有统计学显著性(P＞0.05)。多态性位点间的连锁不平衡检验显示三个多态性位点(rs4291、rs4646994和rs4343)紧密连锁,在一个单体型块(haplotype block)中,可以构建三个多态性位点组成的单体型模式。单体型传递的总体检验及单个单体型的分析均未显示单体型存在过度传递现象(P＞0.05)。结论:1.组织型激肽释放酶基因(KLK1)的rs5517位点A等位基因及rs3212855、rs5516、rs5517三位点构成的单体型CGA可能与湖南长沙汉族人群脑出血发病有关。2.激肽原基因(KNG1)的rs1656922和rs2304456多态性位点及其构成的单体型可能与湖南长沙汉族人群脑出血发病无关。3.血管紧张素转化酶基因(ACE)的rs4291、rs4646994和rs4343多态性位点及其构成的单体型可能与湖南长沙汉族人群脑出血发病无关。更多还原

【Abstract】 Background and objective:Cerebral hemorrhage(CH) is a major cause of death in the world and is characterized by high rates of cases fatality and disability.CH is a multifactorial disease and the prevalence of CH varies widely among different population.Epidemiology study showed that in Europe and North America,up to 6.5-19.6%of strokes are attributed to CH,but there is a tendency for a higher proportion of CH in China.Especially in the recent Changsha city study,the author concluded that Changsha was one of the areas with high incidence of CH reported and also had a high proportion of CH among all subtypes of stroke (accounted for 55.4%of all strokes).The prevalence risk factors of stroke and CH cannot give a perfect explanation for the reason of the high incidence and high proportion of CH in this area,so we carried out a series of etiology researches about CH.We find the phenomenon of familial aggregation of CH,which strongly indicate that genetic influence in the pathogenesis of CH.Genetic study may play an important role for uncovering the etiology of CH.Results from pathologic studies of CH showed that most CHs originate from the rupture of small,deep arteries with diameters between 50 and 200μm and remodeling(including hyalinosis and aneurysm formation) of these arterioles is the underlying pathologic change resulting in CH.It is well known that vascular remodeling and aneurysm formation are partially caused by gene defect.The kallikrein-kinin system(KKS) is an endogenous metabolic cascade,triggering of which results in the release of vasoactive kinins.The pharmacologically active kinins are implicated in many physiological and pathological processes such as hypertension,vascular remodeling and aneurysm formation, which are involved in the pathogenesis of CH.In this study,we observed the relationship between CH and SNPs in the genes coding for the key enzymes(KLK1 and ACE) and precursors(KNG1) involved in the kinins metabolism pathway.The aim is to determine whether genetic variation in KKS system is associated with increased risk of CH in Changsha Han Chinese.Methods:Collecting the CH patients who were in the department of Neurology,Xiangya Hospital of Central South University from Jan 2006 to Oct 2008 and part of their family members.All cases were diagnosed by CT and/or MRI and fulfilled the diagnose criteria of the fourth national annual meeting of cerebrovascular disease.The subjects of the control group were eliminated cerebrovascular disease by clinical examination,CT and/or MRI,and were made sure without family history of cerebrovascular disease.All subjects were Han Chinese in Changsha area and given informed consent,which were composed of three groups:①38 Chinese Han pedigree of CH including 200 subjects which consisted of 61 CH patients,95 first degree relatives and 23 second degree relatives and 21 non-blood relationships spouse of the family members.②273 sporadic cerebral hemorrhage(SCH) patients③140 healthy control subjects.The age and sex between SCH and control group were not significant.We examined single-nucleotide polymorphisms (SNPs) of genes involved in the kinins metabolism pathway in attempting to examine whether SNPs or haplotype molecular markers would confer a significant susceptibility to CH.The SNPs present in the genes coding for KLK1(rs3212855、rs5515、rs5516 and rs5517),ACE(rs4291 and rs4343) and KNG1(rs1656922 and rs2304456) were analyzed by Multiplex Snapshot method;The ACE I/D polymorphism(rs4646994) was analyzed by PCR and agarose gel electrophoresis.For the genotyping data,we performed a two-step analysis.The first step was to carry out the polymorphism screening for a genetic association between SNPs and phenotypes in 273 SCH patients and in 140 healthy subjects.The second step was to validate the initial findings resulting from the above case-control study by using the family-based association tests(FBAT) in 38 pedigree of CH,aiming to determine weather the SNPs or haplotype is associated with CH in Changsha Han Chinese.Result:1.We completed an association analysis with four SNPs(rs3212855, rs5515,rs5516 and rs5517) located within the KLK1 gene using the case control association tests,the results showed that the rs5515 is not a polymorphic site in Changsha Han Chinese.As for other three SNPs,the results showed that the frequecy of A allele in rs5517 were significantly higher in SCH patients than control subjects(P＜0.05);while genotye and allele frequecy in rs3212855 and rs5516 were found not different between SCH patients and control subjects(P＞0.05).Furthermore,we completed a transmission disequilibrium test with three SNPs(rs3212855,rs5516 and rs5517) located within the KLK1 region using the family-based association tests(FBAT) in 38 CH pedigree.We found significant transmission desequilibrium between CH and two of the SNPs markers (rs5516 G:Z=2.422,P=0.015424 and rs5517 A:Z=2.963,P=0.003049, repectively).Three SNPs(rs3212855,rs5516 and rs5517) were in strong linkage disequilibrium by linkage disequilibrium analysis.They were in the same haplotype block.The global chi-square test for the haplotype transmission also revealed a strong association(x~2=12.664,df=4,P= 0.013042).Haplotype CGA showed the excess transmission to affected individuals(Z=2.881,P=0.003969). 2.We completed an association analysis with two SNP(rs1656922 and rs2304456) located within the KNG1 gene using the case control association tests,the results showed that genotye and allele frequecy in the two SNPs were foud not different between SCH patients and control subjects(P＞0.05).Furthermore,we completed a transmission disequilibrium test with two SNPs(rs1656922 and rs2304456) located within the KNG1 gene using the family-based association tests(FBAT) in 38 CH pedigree.We found no evidence for transmission disequilibrium with CH(P＞0.05).Two SNPs(rs1656922 and rs2304456) were in strong linkage disequilibrium by linkage disequilibrium analysis.They were in the same haplotype block.The haplotype analysis also revealed no evidence for transmission disequilibrium with CH(P＞0.05).3.We completed an association analysis with three SNP(rs4291, rs4646994 and rs4343) located within the ACE gene using the case control association tests,the results showed that genotye and allele frequecy in the three SNPs were found not different between SCH patients and control subjects(P＞0.05).Furthermore,we completed an transmission disequilibrium test with three SNPs(rs4291,rs4646994 and rs4343) located within the ACE gene using the family-based association tests(FBAT) in 38 CH pedigree.We found no evidence for transmission disequilibrium with the SNP markers(P＞0.05).Three SNPs(rs4291, rs4646994 and rs4343) were in strong linkage disequilibrium by linkage disequilibrium analysis.They were in the same haplotype block.The haplotype analysis also revealed no evidence for transmission disequilibrium with CH(P＞0.05). Conclusion:1.Our preliminary results provide evidence that the rs5517 A alelle of KLK1 gene may be important risk factors for the development of CH in the population.The haplotype CGA which composed of the rs3212855 C,rs5516 G and rs5517 A alleles significantly increased the susceptibility of CH.2.Our results do not support the association between these SNPs (rs1656922 and rs2304456) of KNG1 gene and CH susceptibility in the population.The results suggest that haploype composed of alleles of these SNPs of KNG1 gene do not contribute significantly to the predisposition to develop CH in our sample.3.Our results do not support the association between these SNPs (rs4291,rs4646994 and rs4343) of ACE gene and CH susceptibility in the population.The results suggest that haploype composed of alleles of these SNPs of ACE gene do not contribute significantly to the predisposition to develop CH in our sample.更多还原