【作者】 曾艺；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 研究目的本研究旨在长沙地区汉族人群中,按多基因复杂疾病研究策略及方案,首次同时在散发性脑出血患者与健康对照人群及有家族聚集现象的脑出血家系中研究凝血因子Ⅰ(coagulation factorⅠ, F1)基因、凝血因子Ⅶ(coagulation factorⅦ, F7)基因、蛋白Z (protein Z, ProZ)基因、凝血因子Ⅻ(coagulation factorⅫ, F12)基因及血小板膜糖蛋白Ⅰa(glycoproteinⅠa, GPⅠa)基因等凝血系统基因相关单核苷酸多态(single nucleotide polymorphisms, SNPs)位点,比较其在不同群体中等位基因、基因型及单体型(haplotype)等遗传学分子标记物的差异并分析其与脑出血的关联,从分子水平探讨脑出血的可能发病机理,为预防和治疗脑出血提供坚实的理论依据和实践指导。研究方法收集2006年1月至2009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)；对照组来自同期湖南省长沙地区汉族健康体检人员。研究对象分为3组：(1)有家族聚集现象的脑出血家系(cerebral hemorrhage with family history, CHFH)组(65个家系共281例,包括脑出血患者65例,Ⅰ级亲属133例,Ⅱ级亲属36例、Ⅲ级亲属10例和健康无血缘关系亲属37例),(2)散发性脑出血(sporadic cerebral hemorrhage, SCH)组(195例),及(3)对照组(116例)。CHFH组、SCH组与对照组的年龄、性别具有可比性。采用多重单碱基延伸SNP (Multiplex SnaPshot)分型技术对F1基因相关SNPs位点(rs1800790、rs1800787、rs6050)、F7基因相关SNPs位点(rs510317、rs6046)、ProZ基因相关SNPs位点(rs2273971、rs3024718、rs3024731)、F12基因相关SNP位点(rs1801020)及GPⅠa基因相关SNP位点(rs1126643)进行基因分型。对不同人群的基因分型结果分两部分进行统计：(1)SCH组和对照组采用基于人群的病例-对照研究法,采用x2检验按Hardy-Weinberg平衡法检测样本的群体代表性。用x2检验及Logistic回归分析,研究等位基因及基因型分布规律,从而筛查出与散发性脑出血相关的基因；用haploview软件构建单体型块(haplotype block);用PHASE软件构建单体型,用x2检验及Logistic回归分析探讨单体型与脑出血的相关性,进一步筛查出与散发性脑出血相关的基因；(2) CHFH组采用基于家系的传递不平衡检验(transmission disequilibrium test, TDT)及关联分析法,用FBAT软件研究等位基因及单体型与脑出血的相关性,从而筛查出与有家族聚集现象的脑出血相关的基因。结果1.本研究结果显示在长沙地区汉族人群中存在F1基因rs1800790、rs1800787、rs6050, F7基因rs510317、rs6046, ProZ基因rs2273971、rs3024718、rs3024731, F12基因rs1801020及GPIa基因rs1126643此10种SNPs位点。2.本研究结果显示在长沙地区汉族人群中,GPIa基因rs1126643多态的T等位基因在SCH组中的频率为0.339,明显高于对照组(0.228, P=0.004)。Logistic回归分析显示T等位基因与散发性脑出血相关,是散发性脑出血的保护性因素(OR=0.577,95%CI:0.396-0.840,P=0.004)。但F1基因rs1800790、rs1800787、rs6050SNPs位点,F7基因rs510317、rs6046 SNPs位点,ProZ基因rs2273971、rs3024718、rs3024731 SNPs位点及F12基因rs1801020 SNP位点的等位基因频率在SCH组与对照组间比较均无显著性差异(P>O.05)。3.本研究结果显示在长沙地区汉族人群中,GPIa基因rs1l26643多态的CT及TT基因型在SCH组中的频率分别为0.455与0.111,明显高于对照组(分别为0.404与0.026,P=0.022)。Logistic回归分析显示CT及TT基因型均与散发性脑出血相关,且均是散发性脑出血的保护性因素(OR=0.180,95%CI：0.051-0.631,P=0.007；OR=0.267,95%CI：0.076-0.943,P=0.040)。但F1基因rs1800790、rs1800787、rs6050 SNPs位点,F7基因rs510317、rs6046 SNPs位点,ProZ基因rs2273971、rs3024718、rs3024731 SNPs位点及F12基因rs1801020SNP位点的基因型频率在SCH组与对照组间比较均无显著性差异(P>0.05)。4.本研究结果显示在长沙地区汉族人群SCH组及对照组中,F1基因的rs1800790、rs1800787、rs6050 SNPs位点位于同一单体型块(D’均＞0.7),其构成的单体型GCA (rs1800790+rs1800787+ rs6050)、单体型GA (rs1800790+rs6050)及单体型CA(rs1800787+rs6050)均可增加散发性脑出血的危险度近2倍(OR=1.738,95%CI：1.103-2.740,P=0.017)。其构成的其它所有主要单体型频率在SCH组及对照组间比较,结果无显著性差异(P>0.05)。蛋白Z基因rs2273971、rs3024718、rs3024731 SNPs位点位于同一单体型块(D’均>0.7),而F7基因rs510317及rs6046 SNPs位点可能位于同一单体型块(D’>0.5),F7基因rs6046及蛋白Z基因rs3024718亦可能位于同一单体型块(D’>0.5)。但上述3个单体型块中的SNPs构建出的所有主要单体型频率在SCH组及对照组间比较,结果亦均无显著性差异(P>0.05)。GPIa基因rs1126643及F12基因rs1801020 SNPs位点可能不在同一单体型块中(D’<0.5)。5.本研究结果显示在长沙地区汉族人群CHFH组中,ProZ基因rs2273971多态的G等位基因存在过度传递现象,与有家族聚集现象的脑出血相关(Z=1.882,P=0.049882)；而其A等位基因及rs3024718、rs3024731 SNPs位点的各等位基因无过度传递现象,与有家族聚集现象的脑出血无关(Z<0和／或P>0.05)。且F1基因rsl800790、rs1800787、rs6050 SNPs位点,F7基因rs510317 SNP位点,GPIa基因rs1126643及F12基因rs1801020 SNPs位点的各等位基因亦均无过度传递现象,与有家族聚集现象的脑出血无关(Z<0和／或P>0.05)。因本研究样本中携带F7基因rs6046 SNP位点的核心家系数不足FBAT软件的要求的最低10个,其等位基因TDT检验未能得到结果。6.本研究结果显示在长沙地区汉族人群CHFH组中,F7基因rs510317、rs6046与蛋白Z基因rs2273971、rs3024718、rs3024731SNPs位点在同一个单体型块中(0<D’≤1),其构成的单体型CGA(rs6046+rs2273971+rs3024731)、单体型GA(rs2273971+rs3024731)及单体型CG(rs6046+rs2273971)均存在过度传递现象,与有家族聚集现象的脑出血相关(Z=1.977,P=0.048076；Z=1.922,P=0.044644；Z=1.826,P=0.047838)；其构成的其余所有单体型均无过度传递现象,与有家族聚集现象的脑出血无关(Z<0和／或P>0.05)。F1基因rs1800790、rs1800787、rs6050 SNPs位点在同一个单体型块中(0<D’≤1), GPIa基因rs1126643与F12基因rs1801020 SNPs位点亦在同一个单体型块中(0<D’≤1),但它们各自构成的所有单体型均无过度传递现象,与有家族聚集现象的脑出血无关(Z<0和／或P＞0.05)。结论1.本研究首次发现长沙地区汉族人群中凝血系统基因存在遗传异质性,为遗传因素可能是长沙地区汉族人群脑出血的发病机理之一提供了进一步的证据。2.本研究在国内外首次发现F1基因单体型GCA(rs1800790+rs1800787+rs6050)、单体型GA(rs1800790+rs6050)及单体型CA(rs1800787+rs6050)均是散发性脑出血的遗传易感因素,但与有家族聚集现象的脑出血无关；且F1基因rs6050位点可能是散发性脑出血的重要遗传易感因素。而GPIa基因rs1126643多态的T等位基因及CT、TT基因型则均是散发性脑出血的保护性因素,但与有家族聚集现象的脑出血无关；且T等位基因可能是散发性脑出血的重要遗传保护性因素。3.本研究在国内外首次发现ProZ基因rs2273971多态的G等位基因、单体型GA (ProZ基因rs2273971+rs3024731)、单体型CG(F7基因rs6046+ProZ基因rs2273971)及单体型CGA(F7基因rs6046+ProZ基因rs2273971、rs3024731)均与有家族聚集现象的脑出血相关,但与散发性脑出血无关。且ProZ基因rs2273971 SNP位点G等位基因可能是有家族聚集现象脑出血的重要相关因素。4.本研究首次发现在长沙地区汉族人群中,F7基因rs510317多态、ProZ基因rs3024718多态及F12基因rs1801020多态与散发性脑出血及有家族聚集现象的脑出血均无关。更多还原

【Abstract】 Objective According to the multigene complex diseases analyzing strategy and precept, this study was to analyze the molecular markers like alleles、genotypes and haplotypes of single nucleotide polymorphisms (SNPs) of genes involved in the coagulation system and determine their relationships with cerebral hemorrhage in different groups of Changsha Han Chinese at the same time and for the first time. The genes and SNPs mentioned above included coagulation factorⅠ(F1) gene’s rs1800790、rs 1800787 and rs6050, coagulatin factorⅦ(F7) gene’s rs510317 and rs6046, Protein Z (ProZ) gene’s rs2273971、rs3024718 and rs3024731, coagulation factorⅫ(F12) gene’s rs1801020 and platelet membrane glycoproteinⅠa (GPⅠa) gene’s rs1126643. And the different groups included sporadic cerebral hemorrhage patients, healthy controls and cerebral hemorrhages families with family history.Methods We collected the cerebral hemorrhage patients, who were in Department of Neurology, Xiangya hospital, Central South University from January 2006 to October 2009, and part of their families. All cerebral hemorrhage cases were confirmed by CT and/or MRI (according to the diagnosis criterion of the fourth National Congress of cerebrovascular diseases). We both collected healthy Changsha Han Chinese as controls of the same time. All subjects were divided into three groups:(1) cerebral hemorrhage with family history (CHFH) group (281 subjects from 65 pedigrees, including 65 cerebral hemorrhage patients, 133 first-degree relatives,36 second-degree relatives,10 third-degree relatives, and 37 healthy family members with no blood relationship with the proband), (2) sporadic cerebral hemorrhage (SCH) group (195 subjects) and (3) control group (116 subjects). The age and sex of these groups were comparable. We used Multiplex Snapshot technique to genotyping the SNPs of the genes involved in the coagulation system, including F1 gene’s rs1800790、rs1800787 and rs6050, F7 gene’s rs510317 and rs6046, ProZ gene’s rs2273971、rs3024718 and rs3024731, F12 gene’s rs1801020 and GPIa gene’s rs1126643. Then we chose a two-step method to analyze the genotyping results of different groups:(1) to the SCH group and the control group, based on case-control study, we firstly used variance test and Logistic regression analysis to study the distribution rules of the alleles and genotypes of these genes’SNPs, and to find out the genes and SNPs associated with sporadic cerebral hemorrhage; secondly we used haploview software to find out the haplotype blocks; and thirdly we used PHASE software to compose the haplotypes, and then used variance test and Logistic regression analysis to screen out the genes and SNPs associated with sporadic cerebral hemorrhage. (2) to CHFH group, based on familial association study, we used Transmission Disequilibrium Test (TDT) and association analysis by FBAT software to to screen out the genes and SNPs associated with familial cerebral hemorrhage.Results 1. This study showed that in Changsha Han Chinese, F1 gene had rs1800790、rs1800787 and rs6050 SNPs, F7 gene had rs510317 and rs6046 SNPs, ProZ gene had rs2273971、rs3024718 and rs3024731 SNPs, F12 gene had rs1801020 SNP, and GPIa gene had rs1126643 SNP.2.This study showed that in Changsha Han Chinese, the frequency of allele T of GPIa gene rs1126643 was 0.339 in the SCH group, which was obviously higher than that in the control group (0.228, P=0.004) Logistic regression analysis showed allele T was associated with SCH, and was one of its protective factors (OR=0.577,95%CI:0.396-0.840, P=0.004).But the frequencies of alleles of F1 gene rs 1800790、rs1800787 and rs6050, F7 gene rs510317 and rs6046, ProZ gene rs2273971、rs3024718 and rs3024731, and F12 gene rs1801020 had no obvious difference between the SCH group and the control group (P>0.05)3. This study showed that in Changsha Han Chinese, the frequencies of genotype CT and TT of GPIa gene rs1126643 were 0.455 and 0.111 in the SCH group, which were obviously higher than those in the control group (0.404 and 0.026 respectively, P=0.022). Logistic regression analysis showed genotype CT and TT were associated with SCH, and were its protective factors (OR=0.180,95%CI:0.051-0.631, P=0.007; OR=0.267,95%CI:0.076-0.943, P=0.040). But the frequencies of genotypes of F1 gene rs1800790、rs1800787 and rs6050, F7 gene rs510317 and rs6046, ProZ gene rs2273971、rs3024718 and rs3024731, and F12 gene rs 1801020 had no obvious difference between the SCH group and the control group (P>0.05).4. This study showed that in the SCH group and the control group in Changsha Han Chinese, F1 gene rs1800790、rs1800787 and rs6050 were in the same haplotype block (D’all>0.7), and the haplotype GCA (rs1800790+rs1800787+rs6050)、haplotype GA (rs1800790+rs6050) and haplotype CA (rs1800787+rs6050) all could double the sensibility of SCH(OR=1.738,95%CI:1.103-2.740, P=0.017).But the frequencies of all the other haplotypes composed by them showed no obvious difference between the SCH group and the control group (P>0.05). ProZ gene rs2273971、rs3024718 and rs3024731 were in the same haplotype block (D’all>0.7), while F7 gene rs510317 and rs6046 might be in the same block (D’>0.5), and F7 gene rs6046 and ProZ gene rs3024718 might be also (D’>0.5).But the frequencies of all the other main haplotypes composed by them showed no obvious difference between the SCH group and the control group (P>0.05). GPIa gene rs1126643 and F12 gene rs 1801020 might not be in the same haplotype block (D’<0.5).5. This study showed that in the CHFH group in Changsha Han Chinese, allele G of ProZ gene rs2273971 showed over-transmitted phenomenon, and was associated with CHFH (Z=1.882, P=0.049882),while its allele A and the alleles of rs3024718 and rs3024731 showed no over-transmitted phenomenon, and were not associated with CHFH (Z<0 and/or P>0.05). The alleles of F1 gene rs1800790、rs1800787 and rs6050, F7 gene rs510317, GPIa gene rs 1126643 and F12 gene rs 1801020 all showed no over-transmitted phenomenon, and were not associated with CHFH (Z<0 and/or P>0.05). And because the core family number didn’t reach 10, the TDT of allele of F7 gene rs6046 showed no result.6. This study showed that in the CHFH group in Changsha Han Chinese, F7 gene rs510317, rs6046 and ProZ gene rs2273971,rs3024718, rs3024731 were in the same haplotype block (0<D’≤1), and their haplotype CGA (rs6046+rs2273971+rs3024731)、haplotype GA (rs2273971+rs3024731) and haplotype CG (rs6046+rs2273971) all showed over-transmitted phenomenon, and were associated with CHFH (Z=1.977, P=0.048076; Z=1.922, P=0.044644; Z=1.826, P=0.047838); and all their other haplotypes showed no over-transmitted phenomenon, and were not associated with CHFH (Z<0 and/or P>0.05).F1 gene rs1800790、rs1800787、rs6050 were in the same haplotype block (0<D’≤1), GPIa gene rs 1126643 and F12 gene rs 1801020 were in the same haplotype block too (0<D’≤1), but all their haplotypes showed no over-transmitted phenomenon, and were not associated with CHFH (Z<0 and/or P>0.05). Conclusions 1. This study found for the first time that the coagulation system genes had genetic heterogeneity in Changsha Han Chinese, which provided farther evidence to that genetic factors might play an role in the onset of cerebral hemorrhage in Changsha Han Chinese.2. This study found for the first time that F1 gene haplotype GCA (rs1800790+rs1800787+rs6050)、haplotype GA (rs1800790+rs6050) and haplotype CA (rs1800787+rs6050) might be the sensitive factors of SCH, but were not associated with CHFH; and F1 gene rs6050 might be an important sensitive factor of SCH. While allele T and genotype CT and TT of GPIa gene were all strong protective factors of SCH, but were not associated with CHFH, and allele T might be an important one.3. This study found for the first time that allele G of ProZ gene, haplotype GA (ProZ gene rs2273971+rs3024731)、haplotype CG (F7 gene rs6046+ProZ gene rs2273971) and haplotype CGA (F7 gene rs6046+ProZ gene rs2273971、rs3024731) were all associated with CHFH, but were not associated with SCH. And allele G of ProZ gene rs2273971 might be an important association factor of CHFH.4. This study found for the first time that in Changsha Han Chinese, F7 gene rs510317、ProZ gene rs3024718 and F12 gene rs1801020 were not associated with both SCH and CHFH.更多还原