C反应蛋白、血管生成素2遗传变异及其血浆浓度在脑卒中发病风险及复发中的作用研究

【作者】 于晖；

【导师】 惠汝太；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 研究背景：脑卒中是一种由环境因素及遗传因素共同作用造成的复杂性疾病,是国人重大的健康负担。脑卒中一旦发生,死亡率极高,而且存活后的残废率高达70%,脑卒中复发率也非常高,3年和5年的复发率分别为28%和51%。目前中国约有600万脑卒中残存者,每年新发病例150万,而且发病逐渐年轻化。脑卒中是一个症候群,包括缺血性脑卒中(动脉硬化血栓及栓塞)、出血性脑卒中(高血压动脉硬化性血管病变及血管畸形,动脉瘤)。脑卒中的发病机制十分复杂,动脉粥样硬化是其最主要的病理基础之一。目前认为动脉粥样硬化是复杂的血管性疾病,是血管对损伤的慢性炎症反应。C反应蛋白(C-reactive protein, CRP)作为系统性炎症的标志物,被认为在炎症反应中起着关键作用。血清CRP水平可以反映机体潜在的炎症活跃程度,并与脑卒中、冠心病等动脉硬化性疾病密切相关。无论是在机制研究还是观察性流行病学研究中,CRP在心脑血管发病中的作用,目前存在争议。一种观点倾向认为CRP直接参与、介导炎症反应并在心脑血管病的发生中起重要的作用。另一种观点认为CRP与心脑血管病的发生之间无直接的因果关系,CRP升高仅反映了动脉粥样硬化血管非特异性炎症反应的程度,CRP并未直接参与动脉硬化的发生、发展。本研究拟通过多中心病例对照关联研究探讨可影响血清CRP水平的CRP遗传变异和单体型与脑卒中易感性的关系；通过前瞻性研究探讨可影响血清CRP水平的CRP遗传变异和单体型与脑卒中复发的关系。研究目的：本研究拟揭示CRP遗传变异和单体型是否为脑卒中易感性和复发的遗传危险因素；研究方法：本研究采用病例对照研究,探讨可影响CRP水平的CRP的遗传变异与脑卒中易感性的关系；采用前瞻性研究,探讨可影响CRP水平的CRP的遗传变异与脑卒中复发的关系。研究对象包括1572例脑卒中病例(694例脑梗塞,437例腔隙性脑梗塞,441例脑出血)及1485例对照,来自中国的7个临床中心。与CRP水平相关的CRP遗传变异rs1205及rs2808630用限制性片段长度多态性法(RFLP)进行基因分型。血清CRP水平采用ELISA方法检测。对入选病例进行4.5年(中位数)随访,采用多元回归分析、Kaplan-Meier分析及Cox回归模型分析血清CRP水平及与CRP血清水平升高相关的CRP遗传变异与脑卒中的易感性和复发的关系。研究结果：1.无论是病例组总体还是脑卒中三个亚型中,血清CRP水平显著高于对照组。血清CRP水平分别为[中位数(全距)]：对照组0.7100(9.96)mg/L,脑卒中总体1.0113(9.97)mg/L(三个亚型分别为：脑梗0.9784(9.85)mg/L腔梗1.0094(9.97)mg/L、脑出血1.0381(9.95)mg/L),P<0.01。2.校正了年龄、性别、体重指数、收缩压、舒张压、总胆固醇、HDL-C、糖尿病及吸烟等传统心血管病危险因素后,无论是在病例组还是对照组,rs1205A及rs2808630G等位基因携带者,其血清CRP水平显著升高。rs1205基因型GG、GA及AA血清CRP水平[中位数(全距),mg/L]在病例组分别为：0.8106(7.76),1.0113(9.96),1.4400(9.84),P<0.001；对照组分别为：0.6294(9.95),0.7144(9.05),1.0100(9.28),P<0.001；rs2808630基因型AA、AG及GG血清CRP水平[中位数(全距),mg/L]在病例组分别为：0.9109(9.97),1.0769(9.71),1.8713(9.61),P<0.001；对照组分别为：0.6875(9.96),0.7119(9.39),3.6891(9.06),P=0.004。3.在校正了传统心血管病危险因素后,无论是显性模式、隐形模式还是加性模式下,CRP基因多态rs1205G/A、rs2808630A/G与脑卒中或脑卒中任一亚型无显著相关；rs1205(GA+AA vsGG)校正后的优势比(95%可信区间)分别为：脑卒中总体：0.97(0.81-1.15),脑梗组：1.01(0.81-1.26),腔梗组：1.00(0.78-1.27),脑出血组：1.01(0.77-1.34)。rs2808630(AG+GG vs AA)校正后的优势比(95%可信区间)分别为：脑卒中总体：0.96(0.80-1.15),脑梗组：0.85(0.67-1.07),腔梗组：1.14(0.89-1.47),脑出血组0.94(0.71-1.26)。4.在所有3个频率>5%的单体型(rs1205/rs2808630：GA、AA、AG)中,未发现单体型频率与脑卒中或脑卒中任一亚型显著相关,也未发现单体型频率与脑卒中复发相关。5.校正了年龄、性别、体重指数、收缩压、舒张压、总胆固醇、HDL-C、糖尿病及吸烟等传统心血管病危险因素后,rs1205G/A(相对危险度RR(95%CI)1.039(0.816-1.324)P=0.755)及rs2808630A/G(相对危险度RR(95%CI)1.094(0.853-1.403)P=0.479)与脑卒中复发仍为无显著相关。结论：CRP基因变异与CRP血清水平显著相关。CRP基因变异及单体型不是脑卒中发生及复发的遗传危险因素；CRP水平在脑卒中病例组显著升高,是脑卒中的标志物,但我们推测CRP不是脑卒中的致病因素。以CRP作为脑卒中发病及复发的预警指标应慎重考虑。研究背景：脑卒中是一种由环境因素及遗传因素共同作用造成的复杂性疾病,是国人重大的健康负担。脑卒中的病理过程包括动脉粥样硬化、血管炎症、血管渗透性改变等,其发病机制远未阐明。许多报道表明血管生成素(Angiopoeitin, Ang)及其受体酪氨酸激酶Tie2信号系统参与了许多与脑卒中密切相关的病理过程,不仅调控血管发生,而且参与调控内皮细胞炎症反应。血管生成素1(Angiopoietin1, Ang1)与血管生成素2(Angiopoietin2, Ang2)均为受体酪氨酸激酶Tie2(tyrosine kinase with immunoglobulin and epidermal growth factor homology domain2)的天然配体,与Tie2相互作用。Ang2和Ang1互为天然拮抗剂,Ang2特异结合于血管内皮Tie2受体后,并不引起受体的磷酸化,通过竞争性地抑制Ang1与Tie2受体结合,从而抑制Tie2磷酸化,起到破坏血管稳定,促使血管结构松解,解除血管周围细胞、细胞外基质对内皮的抑制作用。因此,Ang2被认为是Ang-Tie2系统的动态调节者,维持血管生长、退化的动态平衡。Ang2还参与调控炎症反应,在血管快速应答过程中与TNFa协同作用,促进TNFa诱导的炎症相关因子的表达,如ICAM1, VCAM1等。临床研究表明,Ang2在急性冠脉综合征及高血压患者中表达上调,而急性冠脉综合征及高血压均为脑卒中的危险因素。尽管目前尚不能明确Ang2水平增加是急性冠脉综合征及高血压的原因还是结果,但Ang2已被认为是高血压患者心血管病(心肌梗死、脑卒中和死亡)风险的生物标志物。考虑到Ang2在动脉粥样硬化、血管炎症反应及血管渗透性改变等病理、生理过程中发挥作用,这些过程与脑卒中的发生发展密切相关,我们提出假设：血浆Ang2水平及／或ANGPT2(编码Ang2的基因)的遗传变异与脑卒中的易感性和复发相关联。研究目的：本研究拟揭示ANGPT2遗传变异和单体型是否为脑卒中易感性和复发的遗传危险因素；血浆Ang2水平是否可以作为脑卒中易感性和复发的独立预测因子。研究方法：本研究采用病例对照研究,探讨血浆Ang2水平及ANGPT2的遗传变异与脑卒中的易感性的关系；采用前瞻性研究,探讨血浆Ang2水平及ANGPT2的遗传变异与脑卒中复发的关系。研究对象包括1513例脑卒中病例(674例脑梗塞,419例腔隙性脑梗塞,420例脑出血)及1582例对照,来自中国的7个临床中心。采用限制性片段长度多态性法(RFLP)对ANGPT2启动子区所有tagSNP(rs3739390,rs2515507及rs3739391)进行基因分型。前瞻性研究中,1735例脑卒中患者(794例脑梗塞,475例腔隙性脑梗塞,466例脑出血)采用ELISA方法检测血浆Ang2水平并进行4.5年(中位数)随访,采用多元回归分析、Kaplan-Meier分析及Cox回归模型分析血浆Ang2水平及ANGPT2的遗传变异与脑卒中的易感性和复发的关系。Haploview软件来计算遗传标记之间的连锁不平衡程度。为了研究其潜在的机制,本研究进一步运用荧光素酶报告系统研究ANGPT2基因启动子区变异对转录活性的影响。研究结果：1.校正了年龄、性别、体重指数、吸烟、饮酒等传统危险因素后,ANGPT2+442C等位基因携带者与野生型G等位基因携带者相比,腔梗易感性增加(显性模式：优势比(OR)1.42,95%可信区间(95%CI)(1.08-1.87)。在Bonferroni correction校正后,ANGPT2+442C仍与腔梗易感性相关(P=0.012)；荧光素酶报告系统显示,ANGPT2+442C基因型转录活性为+442G转录活性2.10倍(P=0.014)。2.血浆Ang2水平与腔梗复发相关联,校正了传统危险因素后,与Ang2水平最低四分位相比,第二四分位危险比(hazard ratio, HR)(95%CI)为1.48(0.74-2.95),第三四分位危险比(95%CI)2.56(1.35-4.86),第四四分位(危险比(95%CI)为2.15(1.11-4.17)。进一步校正ANGPT2启动子区多态(+442G/C、-220G/A、+398C/T),仍显示血浆Ang2水平增加与腔梗复发相关。3. ANGPT2基因启动子区+398C/T变异与血浆Ang2水平相关联,校正了年龄、性别、体重指数、吸烟、饮酒等传统危险因素后,+398T等位基因携带者(n=126),其血浆Ang2水平(平均浓度为4.26ng/m1,浓度范围：0.13-59.44ng/m1)高于+398C等位基因携带者(n=234,平均浓度为3.95ng/ml,浓度范围：0.12-49.41ng/ml,P=0.033)。4. ANGPT2启动子区+398C/T变异与腔梗复发相关,与脑梗及脑出血复发无关。与+398C相比,+398T腔梗复发危险比(95%CI)为1.67(1.06-2.63)；进一步校正血浆Ang2水平后,仍有显著关联。5. ANGPT2基因单体型G-G-T(-220/+442/+398)可增加脑梗(OR=1.54,95%CI1.15-2.08)和脑出血(OR=1.64,95%CI1.17-2.32)的发病风险；而携带+442C等位基因的单体型G-C-C(-220/+442/+398),与脑梗(OR=1.84,95%CI1.18-2.87)、腔梗(OR=2.16,95%CI1.30-3.57)和脑出血(OR=2.10,95%CI1.27-3.46)发病风险显著相关。结论：本研究提示ANGPT2启动子区变异及单体型与脑卒中发病风险相关；血浆Ang2水平及其启动子区基因多态可能是预测脑卒中(特别是腔隙性脑梗塞)复发的新的生物标志物；Ang2可能在脑血管疾病的发生发展中起到重要的调节作用,并可成为脑血管病新的诊治靶点。更多还原

【Abstract】 Background:Stroke is a complex disease caused by synergy between genetic and environmental factors. It is a major health burden in China. Mortality rate is high for stroke victims. The disability rate after stroke is up to70%in stroke survivors with very high recurrence rates (3and5-year recurrence rates were28%and51%, respectively). In China, there are about6-million stroke remnants and1.5-million of the new cases per year. This incidence gradually increases with younger age. Stroke is not one disease but rather a heterogeneous group of disorders, three subtypes of stroke were included:atherothrombotic, intracerebral hemorrhage and lacunar infarct. The mechanisms underlying stroke are multifactorial. One of the most important pathological basis of stroke is atherosclerosis. Currently, atherosclerosisis is a complex vascular disease with chronic inflammatory responses due to vascular injury. C-reactive protein (CRP), as one of systemic inflammation markers, plays a key role in the inflammatory responses. CRP levels in serum may reflect the potentially inflammatory activity in the body, and is closely related to stroke and other arteriosclerosis diseases, such as coronary heart disease. However, there are disputes concerning CRP in the pathogenesis of cerebrovascular diseases, from both the mechanism and epidemiological studies. Some believed that CRP directly mediates inflammatory responses and plays an important role in the occurrence of cardiovascular and cerebrovascular diseases. Others supported that no direct relationship was found between CRP levels and the occurrence of cardiovascular and cerebrovascular diseases. The increase of CRP levels just reflects the extent of non-specific inflammatory responses in atherosclerotic lesions. CRP is not directly involved in the development of atherosclerosis.In this study, the relationships between stroke susceptibility and stroke recurrence with serum CRP levels due to CRP genetic variations and haplotypes were investigated by multi-center case-control sdudy and prospective study, respectively.Objectives:This study intended to reveal whether CRP genetic variatnts and haplotypes are genetic risk factors for stroke susceptibility and recurrence, and whether CRP can be causal factor for stroke.Methods:A multi-center case-control study was used to investigate the association of serum levels of CRP (determined by using ELISA) and CRP variants with stroke susceptibility. A prospective study was used to explore the causal relation of CRP level and the CRP variants with stroke recurrence. Total1735patients with stroke (lacunar infarct (n=475), atherothrombotic (n=794) and intracerebral hemorrhage (n=466)) were followed-up for a period of4.5years (mean), the correlation was evaluated by using Kaplan-Meier analysis and the Cox regression models. The degree of linkage disequilibrium between genetic markers was calculated using Haploview software.Results:1. Serum CRP levels were significantly higher in patients and three stroke subtypes than in control. CRP levels were (median (range)):0.7100(9.96)mg/L for control group,1.0113(9.97)mg/L for overall stroke group,(for three subtypes:0.9784(9.85)mg/L for atherothrombotic stroke,1.0094(9.97)mg/L for lacunar infarct,1.0381(9.95)mg/L for intracerebral hemorrhage), P<0.01.2. After adjustment for traditional cardiovascular risk factors including age, sex, body mass index, systolic blood pressure, diastolic blood pressure, total cholesterol, HDL-C, diabetes and smoking, serum CRP levels in patients with rs1205A and rs2808630G alleles were significantly increased in both case and control group. CRP levels (median (range),mg/L) with rs1205allele were0.8106(7.76) for GG,1.0113(9.96) for GA,1.4400(9.84) for AA, respectively(P<0.001) in case and0.6294(9.95) for GG,0.7144(9.05) for GA,1.0100(9.28) for AA, respectively(P<0.001) in control. CRP levels (median (range),mg/L) with rs2808630allele were:0.9109(9.97)for AA,1.0769(9.71) for AG,1.8713(9.61) for GG in case group(P<0.001) and0.6875(9.96) for AA,0.7119(9.39) for AG,3.6891(9.06) for GG (P=0.004) in the control.3. After adjustment for traditional cardiovascular risk factors, whether it is in the dominant model, recessive model or additive model, there was no significant association of CRP polymorphisms rs1205G/A and rs2808630A/G with any one of the subtypes of stroke; the adjusted odds ratio of rs1205(GA+AA vs.GG)(95%confidence interval, CI) were:0.97(0.81-1.15) for overall stroke group,1.01(0.81-1.26) for atherothrombotic,1.00(0.78-1.27) for lacunar infarct,1.01(0.77-1.34) for intracerebral hemorrhage. The adjusted odds ratio of rs2808630(AG+GG vs. AA)(95%CI) were0.96(0.80-1.15) for stroke overall,0.85(0.67-1.07) for patients with atherothrombotic,1.14(0.89-1.47) for patients with lacunar infarct,0.94(0.71-1.26) for patients with intracerebral hemorrhage. There was no significant association between stroke and any subtypes of stroke with haplotype frequency in all three haplotypes with frequency>5%(rs1205/rs2808630:GA, AA and AG).4. After adjustment for traditional cardiovascular risk factors, including age, sex, body mass index, systolic blood pressure, diastolic blood pressure, total cholesterol, HDL-C, diabetes and smoking, there is still no significant correlations of stoke recurrence with rs1205G/A (relative risk (95%CI)1.039(0.816-1.324), P=0.755) and rs2808630A/G (relative risk(95%CI)1.094(0.853-1.403), P=0.479).Conclusions:Although these variants and corresponding hyplotypes in the CRP gene are associated with serum CRP concentrations, our study does not support that variants and corresponding hyplotypes studied here have a major influence on risk of stroke and stroke recurrence.Therefore, we speculate that CRP is not a causal factor for stroke. Backgroud Stroke is a complex disease caused by synergy between environmental and genetic factors. It is a major health burden in China. Although the pathological processes of stroke included atherosclerosis, vascular inflammation, vascular permeability changes, its underlying pathogenesis is not clear.Previous reports indicated that angiogenic factors and its receptor involve in a number of closely related pathological process of stroke. This signal system regulates not only angiogenesis, but also endothelial cell inflammatory responses. Both angiopoietin1(Angl) and angiopoietin2(Ang2) are natural ligands of tyrosine kinase receptor Tie2(tyrosine kinase with immunoglobulin and EGFR growth factor homology domain2). They can interact with Tie2. Ang2and Angl is natural antagonist each other. Ang2can bind to the vascular endothelial Tie2receptor, prevent the binding of Ang1to Tie2and inhibit phosphorylation of Tie2. It can destabilize the blood vessels, loose the vascular structure, and release the inhibition of the perivascular cells and endothelial extracellular matrix to endothelial cells. Therefore, Ang2is the dynamic regulater of Ang-Tie2system and maintain the dynamic equilibrium of blood vessel growth and degradation.Ang2also involved in the regulation of inflammation responses. It, with the synergized with TNFα during vascular rapid response, can promote TNFa-induced expression of inflammation-related factors, such as ICAM1and VCAM1. Clinical studies have shown that Ang2is upregulated in patients with acute coronary syndrome and hypertension. Both acute coronary syndrome and hypertension are risk factors for stroke. Althought it is not clear that increased serum levels of Ang2are the cause or result of the acute coronary syndrome and hypertension, Ang2has been considered as the risk biomarker for high blood pressure in patients with cardiovascular disease (myocardial infarction, stroke and death).In summary, we proposed a hypothesis: there is association of susceptibility and recurrence of stroke with the plasma Ang2levels and/or genetic variants of ANGPT2(gene coding Ang2).Objectives:The purpose of this study was to test the hypothesis that plasma levels of Ang2and variants of ANGPT2will confer susceptibility to stroke and stroke recurrence. Methods:Amulti-center case-control study was used to investigate the association of ANGPT2variants with stroke susceptibility. A prospective study was used to explore the the association of plasma Ang2and the variants in ANGPT2promoter with stroke recurrence.Total1513patients with stroke of three subtypes(lacunar infarct (n=419), atherothrombotic(n=674) and intracerebral hemorrhage (n=420))and1485control in case-control study were genotyped by restriction fragment length polymorphism(RFLP)); in the prospective study, the association of plasma Ang2(determined by using ELISA) and the variants in ANGPT2promoter with stroke recurrence was tested in1735patients with stroke (lacunar infarct (n=475), atherothrombotic (n=794) and hemorrhage (n=466)). The patients were followed-up for a period of4.5years (mean); the association was evaluated by using Kaplan-Meier analysis and the Cox regression models. The degree of linkage disequilibrium between genetic markers was calculated using Haploview software. To further probe the potential mechanism, a luciferase reporter system was used to evaluation the effect of the variation in the promoter region of ANGPT2gene on its transcriptional activity.Results:1. Allele C of rs3739390conferred a1.42-fold risk for lacunar infarction{adjusted odds ratio [OR],1.42(95%CI,1.08-1.87); P=0.012)and a2.10-fold higher transcriptional activity as compared with the corresponding G allele (P=0.014)2. Elevated Ang2levels were strongly associated with risk of stroke recurrence only in the patients with lacunar infarct and the association remained after adjustment for age, sex, hypertension, or diabetes, BMI, cigarette smoking, alcohol consuming, glucose, TG and TC. As compared with the lowest quartile (the first), the hazard ratio (HR)(95%CI) for recurrence of stroke was1.48(0.74-2.95) for the second quartile (middle level),2.56(1.35-4.86) for the third quartile,2.15(1.11-4.17) for the fourth quartile (the highest level). Additional adjustment for variants in the promoter of ANGPT2did not substantially change the results.3. Allele T of rs3739391was associated with elevated Ang2levels. After adjustment for traditional risk factors, plasma Ang2level in the+398T allele carriers (n=126, the average concentration was4.26ng/ml, range:0.13-59.44ng/ml) is higher than the+398C allele carriers(n=234, the average concentration of3.95ng/ml, range:0.12-49.41ng/ml,P=0.033).4. Allele T of rs3739391was associated with risk of stroke recurrence in the patients with lacunar infarct. Compared with+398C,+398T allele led a risk ratio of1.67(95%CI) (1.06-2.63) for lacunar infarction recurrence; After further adjustment for plasma Ang2levels, the result remains similar.5. The haplotype G-G-T conferred a1.54-fold risk for atherothrombotic stroke and a1.64-fold risk for hemorrhagic stroke, while the haplotype G-C-C conferred a1.84-fold risk for atherothrombotic stroke,2.10fold risk for hemorrhagic stroke and2.16fold risk for lacunar infarction. Our results indicate that haplotypes in the promoter of ANGPT2gene conferred high risk of stroke in a Chinese population.Conclusions:In this study, we found that haplotypes in the promoter of the ANGPT2conferred high risk of stroke in a Chinese population. The plasma levels of Ang2and genetic polymorphism in ANGPT2promoter region may be used as a new predicting maker for recurrence of stroke (especially lacunar stroke).更多还原