【作者】 王红星；

【导师】 杨期东；

【作者基本信息】 中南大学 ， 神经病学， 2012， 博士

【摘要】 背景与目的：脑卒中(Stroke)为世界第二大常见死亡原因及成人致残的首要原因,脑出血(Intracerebral hemorrhage, ICH)是其最主要的亚型之一,具有高死亡率、高致残率的特点。由于脑出血缺乏有效的治疗手段,因此如何早期识别其相关危险因素并进行有效的干预成为了减轻疾病负担的主要手段。目前普遍认为,脑出血是一种受多个易感微效基因和环境多因素综合作用的复杂疾病。因此,对脑出血候选基因单核苷酸多态性(Single nucleotide polymorphisms, SNPs)的研究将有助于从分子水平阐明脑出血的本质,为脑出血高危人群的筛查及防治提供坚实的理论依据和实践指导。既往研究表明,长沙地区是世界脑出血高发区,脑出血发病存在明显的家族聚集现象,而高血压及动脉粥样硬化等是长沙地区脑出血高发的主要危险因素。大量研究发现,弹性蛋白(Elastin, ELN)及与其代谢相关的基质金属蛋白酶-9(Matrix metalloproteinases-9,MMP-9)、组织型基质金属蛋白酶抑制剂-1(Tissue inhibitors of metalloproteinase-1, TIMP-1)不仅与原发性高血压、动脉粥样硬化、颅内微小动脉瘤形成等多个脑出血的高危因素和病理过程密切相关,还可直接通过过度降解血管壁成分,破坏血脑屏障,诱发脑出血。目前,有关ELN及其代谢通路相关基因SNPs与脑出血,尤其是有家族聚集现象的脑出血之间的关联研究,国内外少见相关报道。本研究拟探讨ELN、MMP-9及TIMP-1基因SNPs及其构成的单体型(Haplotype)与长沙地区汉族人群脑出血的关系。方法：收集2006年1月至2011年4月在湘雅医院神经内科就诊的部分脑出血患者及其家属的临床资料和外周血标本。对照组为中南大学湘雅医院健康体检人员。研究对象共分为3组：1.有家族聚集现象的脑出血家系组(Intracerebral hemorrhage with family history, ICHFH):共82个家系,339例成员,男189例,女150例(包括脑出血患者82例,Ⅰ级亲属165例,Ⅱ级亲属30,Ⅲ级亲属12例,健康无血缘关系亲属50例)。2.散发性脑出血(Sporadic intracerebral hemorrhage, SICH)组：418例,男280例,女138例。3.正常对照组(Contro1):206例,男130例,女76例。ICHFH组、SICH组与对照组在年龄、性别方面具有可比性。候选基因SNPs的选择策略及分型方法：我们根据以下原则挑选目标SNPs:①该SNPs在高血压、动脉粥样硬化及颅内动脉瘤等其他血管疾病中已有阳性关联研究报道；②尽量选择位于外显子的错意突变SNPs或者位于转录调节区域的SNPs;③使用Haploview软件,选择标签SNPs (tag SNPs);④SNPs最小等位基因频率大于5%。基于以上原则,我们采用多重单碱基延伸分型技术(Multiplex SNaPshot)与DNA测序相互印证的方法对所有研究对象ELN及其代谢通路相关基因的8个SNPs位点进行分型,包括：ELN基因的rs2071307、rs34208922、rs28-56728、rs41350445,MMP-9基因的rs17576、rs2250889及TIMP-1基因的rs4898、rs2070584。统计学分析：根据研究人群的结构差异,将分型结果分两部分进行统计：①对SICH组和对照组采用基于人群的病例-对照研究法,使用Hardy-Weinberg平衡法检测样本的群体代表性。采用SPSS16.0软件对SICH组与对照组基因型及等位基因频率差异行卡方检验,不同基因型(或等位基因)亚组间血压比较采用ANOVA方差分析(或t检验)；②ICHFH组：使用FBAT软件对有家族聚集现象的脑出血家系各SNPs位点及其构建的单体型行传递不平衡检验(transmission disequilibrium test, TDT),寻找其相关的易感基因。结果：1.各基因SNPs位点分布特征：长沙地区汉族人群中存在ELN基因rs2071307、rs34208922、rs2856728、rs41350445位点多态性,MMP-9基因rs17576、rs2250889位点多态性及TIMP-1基因rs4898、rs2070584位点多态性。这些SNPs位点基因型和/或等位基因频率分布与世界其它地区、种族人群比较存在不同程度的差异性。2. SICH组与正常对照组病例-对照研究发现：①ELN基因rs2856728多态基因型分布在SICH组与对照组之间比较有显著性差异(x2=15.414,P<0.001),SICH组突变型C等位基因频率显著高于对照组(x2=13.123,P<0.001)；ELN基因rs41350445多态各基因型分布在SICH组与对照组之间有显著性差异(x2=4.241,P=0.039)；而ELN基因rs2071307及rs34208922多态基因型及等位基因频率在SICH组与对照组间比较均无统计学差异(P>0.05)。②MMP-9基因rs17576多态各基因型分布在SICH组与对照组之间比较存在显著性差异(x2=7.522,P=0.023),而MMP-9基因rs2250889多态基因型及等位基因频率分布在SICH组与对照组中比较均无统计学差异(P>0.05)。③TIMP-1基因rs2070584多态等位基因频率在男性SICH患者与男性健康对照者之间比较有显著性差异,男性SICH患者突变型G等位基因频率显著高于对照组(x2=5.814,P=0.016)。TIMP-1基因rs4898多态基因型及等位基因频率分布在不同性别SICH组与对照组中比较差异均无统计学意义(P>0.05)。3.有家族聚集现象的脑出血家系FBAT研究结果：①TDT分析显示ELN基因rs41350445多态的T等位基因向后代发生了过度传递现象(Z=2.180,P=0.029),但对其参与构成的单体型分析并未发现单个特异单体型存在过度传递现象(P>0.05)；TDT分析未发现ELN基因rs2071037、rs34208922及rs2856728多态各等位基因向后代发生了过度传递现象(P>0.05),对这三个SNPs位点参与构成的单体型行TDT分析也未发现单个特异单体型存在过度传递现象(P>0.05)。②TDT分析未发现MMP-9基因rs17576、rs2250889多态位点各等位基因向后代发生了过度传递现象(P>0.05),对他们构建的单体型分析也未发现单个特异单体型存在过度传递现象(P>0.05)。4.ELN基因、MMP-9基因及TIMP-1基因SNPs对血压水平的影响：①SICH组与对照组ELN基因rs2856728多态各基因型亚组之间收缩压、舒张压及平均动脉压水平均存在显著性差异(P<0.05)；SICH组与对照组ELN基因rs2071307、rs34208922、rs41350445多态位点各基因型亚组之间血压水平无显著性差异(P>0.05)。②对照组MMP-9基因rs17576多态各基因型亚组之间收缩压及平均动脉压水平存在显著性差异(P<0.05)；SICH组与对照组MMP-9基因rs2250889多态各基因型亚组之间收缩压、舒张压及平均动脉压水平均无显著性差异(P>0.05)。③女性SICH患者中rs2070584多态TG基因型与GG基因型亚组之间收缩压及平均动脉压水平存在显著性差异(P<0.05)。SICH组与对照组rs4898多态各等位基因(或基因型)亚组之间收缩压、舒张压及平均动脉压在男性及女性研究对象中均无统计学差异(P>0.05)。结论：1.ELN基因rs2856728、rs41350445,MMP-9基因rs17576,TIMP-1基因rs2070584多态位点可能与长沙地区汉族人群脑出血发病有关。2.ELN基因rs2071307、rs34208922,MMP-9基因rs2250889,TIMP-1基因rs4898多态位点可能与长沙地区汉族人群脑出血无关。3.ELN基因rs2071307、rs34208922、rs2856728、rs41350445,MMP-9基因rs17576、rs2250889多态位点构成的单体型可能与长沙地区汉族人群有家族聚集现象的脑出血无关。4.ELN基因rs2856728、MMP-9基因rs17576及TIMP-1基因rs2070584多态位点可能与长沙地区汉族人群的血压水平相关,其中rs2856728多态位点很可能是通过影响血压来增加长沙汉族人群脑出血的发病风险。而ELN基因rs2071307、rs34208922、rs41350445,MMP-9基因rs2250889,TIMP-1基因rs4898多态位点与长沙地区汉族人群血压水平无关。更多还原

【Abstract】 Background:Stroke is the second most common cause of death and a leading cause of adult disability worldwide. Intracerebral hemorrhage (ICH) is one of the most serious types of stroke with extraordinary high morbidity and mortality rate. Due to the lack of effective alternative therapies, accurate risk factors identification and preventive interventions have recently become the key issues in addressing the public health burden of ICH. ICH is a late-onset multifactorial disease that results from the contributions of environmental and a large number of minor genetic risk factors. Studies on candidate gene single nuclear polymorphisms (SNPs) will help us to uncover the genetic architectures of ICH and provide valid scientific basis for prevention and treatment of ICH.Our epidemiological study conducted on ICH has shown that Changsha had an extraordinarily high proportion of ICH with obvious family aggregation. In addition, hypertension and atherosclerosis were the major risk factors for ICH. Moreover, studies suggested that elastin family played a crucial role in the development of hypertension, atherosclerosis and micro-intracranial aneurysms, which further increased the susceptibility for ICH. However, there is dearth of information concerning the relationship between these genes’SNPs and ICH, especially ICH with family history. The aim of this study was to investigate the relationship between ELN and its metabolic pathway related MMP-9, TIMP-1genes’SNPs in Han people with ICH in Changsha, Hunan.Methods: This study enrolled ICH patients and relatives of Chinese Han in Changsha who were consecutively admitted to the Neurology Department of Xiangya Hospital, Central South University, between January2006and January2011. The control group was recruited from healthy Changsha Han Chinese for check-up in Xiangya Hospital. All subjects were divided into three groups:1. Intracerebral hemorrhage with family history group (ICHFH):339members from82pedigrees,189men and150women, including:82ICH patients,165first-degree relatives,30second-degree relatives,12third-degree relatives and50relatives with no kinship with proband.2. Sporadic intracerebral hemorrhage (SICH) group:418patients with sporadic intracerebral hemorrhage,280men and138women.3. Control group:206healthy controls,130men and76women. ICHFH group, SICH group and control group were comparable in age and sex.Selection of candidate gene’s SNPs were based on the following criteria:①SNPs that have been proved to be associated with artery diseases, especially hypertension, atherosclerosis and aneurysms;②SNPs located at exons that cause amino acid changes or located at transcriptional regulation regions;③SNPs with minor allele frequency>5%to improve genetic calculator power;④The use of Haploview software to screen tag SNPs. With respect to the above basis, Multiplex SNaPshot technique and DNA sequencing were used to genotype8SNPs of3genes associated with ELN metabolism of all subjects, including: rs2071307, rs34208922, rs2856728and rs41350445of ELN gene; rs17576and rs2250889of MMP-9gene; rs4898and rs2070584of TIMP-1gene.We took a two-step method to analyze the genotyping results:①In step one, we analyzed the SICH and control group based on case-control study. The frequencies of alleles and genotypes between the two groups were analyzed using χ2test. Blood pressure levels were compared among genotypes using ANOVA analysis (t test for alleles). We performed all these analyses with SPSS software (Version16.0);②In step two, based on familial association study, we carried out transmission disequilibrium test (TDT) and association analysis by using FBAT software to screen out the causative mutation for familial intracerebral hemorrhage.Results:1. The distribution features of the candidate genes’SNPs:There were ELN gene rs207137、rs34208922、rs2856728、rs41350445polymorphisms, MMP-9gene rs17576、rs2250889polymorphisms and TIMP-1gene rs4898、rs2070584polymorphisms in Changsha Han population. The distributions of these SNPs’genotypes and/or alleles showed obvious racial and geographic differences.2. The results of the case-control study in SICH and normal controls were as follows:①There was significant difference of ELN gene rs2856728genotypes distribution between SICH group and control group (χ2=15.414, p<0.001). The frequency of rs2856728T allele was significantly higher in SICH group than in controls (χ2=13.123, p<0.001). There was significant difference of ELN gene rs41350445genotypes’distribution between SICHs and controls (χ2=4.241, p=0.039). However, there were no differences in genotype and allele frequencies of ELN gene rs2071307and rs34208922between SICH and control groups.②There was significant difference of MMP-9gene rs17576genotypes distribution between SICHs and controls (χ2=7.522, p=0.023), while no significant differences were observed in MMP-9gene rs2250889distribution (P>0.05).③There was significant difference of TIMP-1gene rs2070584allele distribution between male SICHs and male controls. The frequency of the G allele of rs2070584was significantly higher in male SICHs than in male controls (χ2=5.814, P=0.016). There were no significant differences in genotype or allele frequencies of TIMP-1gene rs2070584between the two groups (P>0.05).3. The results of FBAT of ICHFH pedigrees showed the following characteristics:①TDT analysis revealed that C alleles of ELN gene rs41350445was over-transmitted, and was associated with ICHFH susceptibility (Z=2.180, P=0.029). Both single SNP per test and haplotype analysis showed that ELN gene rs2071307, rs34208922and rs2856728were not over-transmitted in ICHFH pedigrees (P>0.05).②MMP-9gene rs17576、rs2250889revealed no evidence of transmission disequilibrium with ICHFH (P>0.05). Haplotyping and Haplotype Frequency Estimation of the two sites didn’t reveal any significant results, either (P>0.05).4. Analysis of ENL, MMP-9and TINP-1genes’polymorphisms with blood pressure levels in Changsha Chinese Han:①The levels of systolic pressure, diastolic pressure and mean arterial blood pressure were statistically different among genotypes of ELN gene rs2856728in both SICH and control groups. However, there were no significant differences of blood pressure levels among genotypes of rs2071307, rs34208922and rs41350445polymorphisms of ELN gene either in SICHs or in control groups.②The levels of systolic pressure and mean arterial blood pressure were different among genotypes of MMP-9gene rs17576in controls while there were no significant differences of blood levels among genotypes of MMP-9gene rs2250889polymorphisms.③The levels of systolic pressure and mean arterial blood pressure of TIMP-1gene rs2070584TG genotype were significantly higher than those of the GG genotype in female SICHs. There were no significant differences of systolic blood pressure, diastolic pressure and mean arterial blood pressure levels among rs4898genotypes of TIMP-1gene (P>0.05). Conclusions:1. The ELN gene rs2856728、rs41350445, MMP-9gene rs17576and TIMP-1gene rs2070584polymorphism could be associated with increased susceptibility of ICH in Changsha Han Chinese population.2. There is no association of ELN gene rs207137、rs34208922, MMP-9gene rs2250889and TIMP-1gene rs4898polymorphisms with ICH in Changsha Han Chinese population.3. There is no association between haplotypes comprised by ELN gene rs2071307、rs34208922、rs2856728、rs41350445, MMP-9gene rs17576、rs2250889polymorphisms and ICHFH in Changsha Han Chinese population.4. The polymorphisms of ELN gene rs2856728, MMP-9gene rs17576and TIMP-1rs2070584could be associated with high blood pressure in Changsha Han population. Furthermore, rs2856728polymorphism may increase the susceptibility of ICH by affecting blood pressure.更多还原