【作者】 马明明；

【导师】 杨期东；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 背景与目的：脑出血(Cerebral hemorrhage, CH)作为脑血管疾病的主要类型之一,其病死率、致残率高,严重危害人类健康。对脑出血病因的研究成为脑血管防治上的重要课题。目前分子遗传学研究认为,脑出血和其他复杂性疾病一样,是由于多个易感的微效基因相互作用,并与环境因素共同作用的结果。因此,对脑出血候选基因单核苷酸多态性(single nucleotide polymorphisms, SNPs)的研究将有助于阐明脑出血的遗传本质,为脑出血易感患者的早期防治提供依据。高血压目前公认是脑出血的独立危险因素,动脉粥样硬化也是脑出血的危险因素之一。长沙是世界脑出血高发区之一,受国家“七五”“八五”、“九五”攻关课题资助,我们既往在长沙地区的多年临床流行病学研究和分子遗传学研究表明,高血压和动脉粥样硬化均是是长沙汉族人群脑出血发病的危险因素,其中高血压和与之有关的遗传因素更是起到至关重要的作用。而内皮素家族(Endothelin Family)及其内皮素家族基因(human endothelin family genes)目前认为与高血压、动脉硬化均有密切关系,可能导致高血压的发生及动脉粥样硬化,增加脑出血的发病风险。迄今,有关内皮素家族基因合成调节途径相关基因与脑出血的关系国内外未见报道。本研究旨在探讨该途径中内皮素-1基因(EDN1)、内皮素受体A基因(EDNRA)、内皮素受体B基因(EDNRB)、内皮素转换酶基因(ECE1)的SNPs和单倍型(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2009年12月在湘雅医院神经内科就诊的脑出血患者及部分家属的临床资料和外周血标本,所有病例均经CT和／或MRI确诊。研究对象均来自长沙地区汉族人群,包括：1.有家族聚集现象的脑出血家系组(cerebral hemorrhage with family history, CHFH):共65个家系,包括家系成员281例,男178例,女103例,分为以下亚组：①家系组脑出血患者组：65例,男46例,女19例,平均年龄(52.42±11.50)岁。②Ⅰ级亲属组(CHFH-I):133例(包括患者父母,子女及同胞兄弟姐妹),男69例,女64例,平均年龄(39.52±14.17)岁。③Ⅱ级亲属组(CHFH-II):36例,男26例,女10例,平均年龄(31.93±12.21)岁。④Ⅲ级亲属组(CHFH-II):10例,男7例,女3例,平均年龄(32.65±12.95)岁。⑤健康无血缘关系亲属(unaffected non-blood relatives)共37例,男30例,女7,平均年龄(47.68±7.64)岁。来自以上有家族聚集现象脑出血家系中与先证者无血缘关系的家庭成员,为先证者及其Ⅰ、Ⅱ、Ⅲ级亲属的配偶。2.散发性脑出血(Sporadic cerebral hemorrhage, SCH)组：195例,男133例,女62例,平均年龄(58.40±11.21)岁。3.正常对照(control)组：男75例,女41例,平均年龄(54.97±10.41)岁。血压的测量：对所有受试对象的血压进行测量。根据2004年《中国高血压防治指南》公布的诊断标准,按收缩压≥140mmHg,和／或舒张压≥90mmHg诊断为高血压。血脂的检测：采用氧化酶法测定甘油三脂(Triglyceride, TG)、总胆固醇(Total cholesterol,TC);测定高密度脂蛋白(High density lipoprotein, HDL)先用沉淀剂沉淀,再用氧化酶法测定上清液中的胆固醇；低密度脂蛋白(Low density lipoprotein, LDL)由Friedwald公式：LDL=(CHO-HDL)-TG/5计算。基因分型：利用多重单碱基延伸SNP分型技术(Multiplex Snapshot)与DNA测序结合相互印证的方法检测所有研究对象内皮素-1合成调节途径4个基因10个SNPs位点多态性,包括：内皮素-1基因的rs1800541、rs2070699、rs5370,内皮素受体A基因rs1801708、rs5333、rs5335,内皮素受体B基因的rs3818416、rs5351,内皮素转化酶-1基因的rs2212528、rs213045。统计：采用SPSS软件对散发性脑出血与正常对照组进行统计分析,两组间基因型与等位基因频率的差异用x2检验,不同基因型亚组间血压水平比较采用ANOVA方差分析。采用FBAT软件对有家族聚集现象脑出血家系的内皮素家族基因的多态性位点及其构建的单体型进行传递不平衡检验(Transmission disequilibrium test, TDT)。结果：1.基因多态性分布特征：长沙地区汉族人群存在EDN-1基因rs1800541、rs2070699、rs5370位点多态性,EDNRA基因rs1801708、rs5333、rs5335位点多态性,EDNRB基因rs3818416、rs5351位点多态性和ECE-1基因rs212528, rs213045多态性。这些多态位点基因型和等位基因分布频率均与世界其他地区人群的基因多态性分布存在不同程度种族差异性。2.散发性脑出血与正常对照人群的病例-对照研究(case-control study)结果：①散发性脑出血组与对照组EDNRA基因rs1801708、rs5333各基因型及等位基因频率比较有统计学意义(p<0.05)；②散发性脑出血组与对照组EDNRB基因rs5351等位基因频率比较差异有统计学意义,散发组脑出血的最低等位基因G的频率(minor allele frequency, MAF)明显低于对照组(x 2=4.83,P<0.05)。③散发性脑出血组EDN-1基因rs1800541、rs2070699、rs5370多态各基因型及等位基因频率分布与对照组比较差异无统计学意义(P>0.05)。④散发性脑出血组与对照组ECE-1基因rs212528,rs213045多态各基因型及等位基因频率分布与对照组比较差异无统计学意义(P>0.05)。3.有家族聚集现象脑出血家系的FBAT研究结果：①TDT分析发现EDNRA基因rs5335多态性位点的等位基因C向后代发生了过度传递现象(P<0.05)。EDNRA基因rs1801708、rs5333、rs5335多态位点构成的单体型G-T-C存在过度传递现象(P<0.05)；其中由EDNRA基因rs1801708和rs5335多态位点构成的单体型G-C、由EDNRA基因rs5333和rs5335多态位点构成的单体型T-C也存在过度传递现象(P<0.05)。③TDT分析未发现EDN-1基因rs1800541、rs2070699、rs5370多态性位点的等位基因存在传递不平衡现象(P>0.05),这三个位点构建单体型后进行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)。④TDT分析未发现EDNRB基因rs3818416、rs5351多态位点从亲代到患病子代的过度传递现象(P>0.05),对他们构建的单体型分析也未发现过度传递现象(P>O.05)。⑤TDT分析未发现ECE-1基因rs212528,rs213045位点多态位点从亲代到患病子代的过度传递现象(P>0.05),对他们进行单体型构建后TDT分析未发现单个特异单体型存在过度传递现象(P>0.05)。4.长沙汉族人群内皮素家族基因多态性对血压水平的影响：①散发性脑出血组EDNRA基因rs5335的GG基因型亚组与GC/CC基因型亚组间舒张压和平均动脉压之间存在显著性差异(P<0.05)。②散发性脑出血组ECE-1基因rs212528的AA与AG/GG基因型亚组之间的收缩压水平存在显著性差异(P<0.05)。③散发性脑出血组及对照组EDN-1基因rs1800541的TT基因型亚组与GT/GG基因型亚组之间、rs2070699位点GG基因型亚组与GT/TT基因型亚组之间、rs5370位点的GG基因型亚组与GT/TT基因型亚组之间的收缩压、舒张压和平均动脉压水平均无显著性差异(P>0.05)。④散发性脑出血组和对照组EDNRB基因rs5351各多态位点的基因型亚组之间的收缩压、舒张压和平均动脉压水平无显著性差异(P>0.05)。结论：1. EDNRA基因rs1801708、rs5333、rs5335, EDNRB基因rs5351位点多态位点可能与长沙汉族人群脑出血发病有关。2. EDNRA基因rs1801708、rs5333、rs5335多态位点构成的单体型G-T-C、由EDNRA基因rs1801708和rs5335多态位点构成的单体型G-C、由EDNRA基因rs5333和rs5335多态位点构成的单体型T-C可能是长沙汉族人群脑出血的遗传易感因素之一。3. EDNRA基因rs5335、ECE-1基因rs212528多态位点可能与长沙汉族脑出血易感人群的血压水平有关,其中EDNRA基因rs5335可能通过影响血压而增加了长沙汉族人群脑出血的发病风险。4.EDN-1基因rs1800541、rs2070699、rs5370, EDNRB基因rs3818416, ECE-1基因rs213045位点多态性可能与长沙汉族人群脑出血和血压水平无关；ECE-1基因rs212528位点与与长沙汉族脑出血易感人群的血压水平有关但与脑出血的易感性无关。更多还原

【Abstract】 Background and objective:Cerebrovascular disease is the second commonest cause of death and leading cause of adult disability worldwide, of which cerebral hemorrhage (CH) represents a main subtype with high morbidity and disability. The etiology of CH is increasingly becoming an important concern in prevention and cure of cerebrovascular disease. Current studies on the clinical and molecular genetics stress this heterogeneous disease caused by the interactions among many minor genes and environmental factors. The study on candidate genes SNPs(single nucleotide polymorphisms) about CH will help us to understand the molecular nature of this disease and suggest a theory for prevention and treatment of CH.Hypertension is an independent risk factor of CH and atherosclerosis is an important risk factor of CH, too. Our clinical epidemiology study on CH in the past dozen years has verified the conclusion that hypertension and atherosclerosis are risk factors related to CH, in which hypertension is the most important one. The human endothelin family gene is thought to play an important role in the development of atherosclerosis, hypertension and may increase the risk of CH. Up to now, there are few studies on the relationship between the polymorphism of Endothelin family gene and CH. Identification of gene polymorphisms of endothelins that confer susceptibility to CH and the development of genetic risk diagnosis systems may contribute to the personalized prevention of these conditions. Our aim is to investigate the correlations of the polymorphism of Endothelin family gene with CH in the Changsha Han ethnic group.Methods:All subjects came from Changsha Han population which were composed of 3 groups:(1) 281 members of 65 family with CH aggregation, including:①65 CH patients with family history:46 men and 19 women, averagely aged 52.42±11.50 years;②133 first-degree relatives:69 men and 64 women, averagely aged 39.52±14.17 years;③36 second-degree relatives:26 men and 10 women, averagely aged 31.93±12.21 years;④10 third-degree relatives:7 men and 3 women, averagely aged 32.65±12.95 years;⑤37 relatives with no kinship with proband:30 men and 7 women, averagely aged 47.68±7.64 years, who are spouses of the proband or the first-degree and second-degree relatives. (2)Sporadic cerebral hemorrhage (SCH) group:195 patients with sporadic cerebral hemorrhage,133 men and 62 women, averagely aged 58.40±11.21 years. (3) Control group:116 healthy controls,41 men and 75 women, averagely aged 54.97±10.41 years.The measures of blood pressure:The blood pressure of every subject was measured and the hypertension was diagnosed according 2005 guidelines for The prevention and treatment of hypertension in China.Serum lipid levels were tested:Total cholesterol (TC), High density lipoprotein (HDL) and triglycerides (TG) were measured by standard enzymatic methods. LDL cholesterol levels were calculated with the Friedewald formula (LDL=TC-HDL-TG/5).Multiplex SNaPshot genotyping analysis and DNA sequencing were used to detect 10 SNPs in 4 genes associated with endogenous endothelins synthesis of some subjects, including:rs 1800541、rs2070699、rs5370 of endthelin-1 gene, rs1801708、rs5333、rs5335 of endothelin receptor A gene, rs3818416、rs5351 of endothelin receptor B gene and rs2212528、rs213045 of Endothelin-converting enzyme 1 gene.The frequencies of the alleles and genotypes between the SCH group and control group were analyzed byχ2 analysis, and blood pressure levels were compared among genotypes by ANOVA using SPSS. The SNP and haplotype transmissions of the CH pedigrees were analyzed by transmission disequilibrium test (TDT) using FBAT computer program. Results:1. The distribution of Endothelin family gene polymorphism in Changsha Hans population:There are EDN-1gene rs 1800541、rs2070699、rs5370 polymorphisms, EDNRA gene rs 1801708、rs5333、rs5335 polymorphisms, EDNRB gene rs3818416、rs5351 polymorphisms and ECE-1 gene rs3818416、rs5351 polymorphisms in Changsha Han population. These gene polymorphisms in Changsha Han population revealed obvious racial difference comparing with those of other regions in the world in different extents.2. The results of the case-control study in SCH and normal controls were as follows:①There is significant difference of genotype and allele frequencies of EDNRA gene rs180178、rs5333 between SCH group and control group(p<0.05).②The frequencies of G allele (MAF) of EDNRB gene rs5351 was significantly lower in SCH group than those in control group (p< 0.05).③There is no significant difference of genotype and alleles frequencies of EDN-lgene rs1800541、rs2070699、rs5370 between SCH group and control group (P>0.05).④There is no significant difference of genotype and alleles frequencies of ECE-1 gene rs3818416、rs5351 between SCH group and control group (P>0.05).3. The results of FBAT in those member of CIFH pedigrees showed the following characteristics:①TDT analysis revealed that the frequency of transmission (from parent to the offspring with CH) of allele C of rs5335 polymorphism in CH increased significantly (P<0.05).②Haplotype analysis of EDNRA gene rs 1801708、rs5333、rs5335 revealed there were significant evidence for transmission disequilibrium with CH (P>0.05) in these haplotype:EDNRA rs1801708 rs5335 G-C, EDNRA rs5333 rs5335 T-C.③EDN1 gene rs1800541、rs2070699、rs5370 revealed no evidence for transmission disequilibrium with CH (P >0.05). Haplotyping and Haplotype Frequency Estimation of the 3 sites hasn’t revealed significant results too(P>0.05).④Application of TDT to genotype data from EDNRB gene rs3818416、rs5351 polymorphisms and their Haplotype Frequency Estimation haven’t got any positive results too.4. Analysis of Endothelin family gene polymorphism and blood pressure level in Changsha Hans CH and control population:①In SCH group, the levels of diastolic pressure and mean arterial blood pressure of the GG gene type of rs5335 were significantly higher than those of the GC/CC gene type (p<0.05)。②In SCH group, the levels of systolic blood pressure of the AA gene type of ECE-1 gene rs5335 was significantly higher than that of the AG/GG gene type (p<0.05)。③There was no significant difference of levels of systolic blood pressure, diastolic pressure and mean arterial blood pressure between TT and GT/GG gene type of EDN1 gene rs1800541, between GG and GT/TT gene type of EDN1 gene rs2070699 and between GG and GT/TT gene type of EDN1 gene rs5370 (P>0.05)。④There was no significant difference of levels of systolic blood pressure, diastolic pressure and mean arterial blood pressure between AA and AG/GG gene type of EDNRB gene (P>0.05)。Conclusions:1. The EDNRA gene rs1801708、rs5333、rs5335 and EDNRB gene polymorphisms are associated with risks of CH in Changsha Han population.2. The haplotypes of EDNRA rs1801708 rs5333 rs5335 G-T-C, EDNRA rs1801708 rs5335 G-C and rs5333 rs5335T-C may be related to with CH in Changsha Han population.3. The polymorphism of EDNRA gene rs5335, ECE-1 gene rs212528 may have relation with the higher levels of blood pressure in the CH susceptible population in Changsha Han population, in which EDNRA gene rs5335 polymorphism may increase CH risk by influencing hypertension.4. There may be no correlation between the polymorphism EDN-1 gene rs 1800541、rs2070699、rs5370,EDNRB gene rs3818416 and ECE-1 gene rs213045 and CH in Changsha Han population; The polymorphism of ECE-1 gene rs212528 may be related to the levels of blood pressure but not related to the susceptibility for CH.更多还原