【作者】 徐峰；

【导师】 陈玉国；

【作者基本信息】 山东大学 ， 急诊医学， 2012， 博士

【摘要】 背景急性冠脉综合征(acute coronary syndrome, ACS)是冠状动脉粥样硬化性心脏病(coronary atherosclerotic disease, CAD)的主要临床亚型,包括不稳定性心绞痛、非ST段抬高型心肌梗死(non-ST-segment elevation myocardial infarction, NSTEMI)和ST段抬高型心肌梗死(ST-segment elevation myocardial infarction, STEMI),该亚型的发生和发展是CAD致残和致死的主要原因之一。随着时代的发展,ACS的防治策略已经从最初的终末期治疗转向早期诊断和早期预防,相关研究也已经从改变生活方式和饮食习惯等整体层面发展到分子、基因水平。以往研究发现,乙醛脱氢酶2(aldehyde dehydrogenase2, ALDH2)是人体内酒精(乙醇)代谢的关键酶之一,酒精首先在乙醇脱氢酶作用下代谢为乙醛,再经过ALDH代谢为乙酸,排出体外。ALDH2基因第12外显子内存在一个单核苷酸多态(single nucleotide polymorphisms, SNPs)——rs671,出现碱基置换：鸟嘌呤(G)→腺嘌呤(A),故导致密码子改变：GAA→AAA,进而发生该酶第504个氨基酸的改变,谷氨酸(Glu)→赖氨酸(Lys)。所以,ALDH2有两个等位基因：野生型(ALDH2*1,*504Glu),突变型(ALDH2*2,*504Lys)。并且,研究也证实,这一SNPs导致ALDH2活性发生显著改变,且这一SNPs存在明显的种族差异,其在欧美白种人中少见,而在中、日、韩等东亚黄种人中突变率高达30%-50%,即全世界大约有6%的人携带ALDH2突变基因。早期关于ALDH2基因Glu504Lys多态与疾病的研究,往往关注该基因与肝炎、肝硬化、消化系统肿瘤、酒精性心肌病等酒精相关性疾病和恶性肿瘤等的关系。随着研究的进展,近年来,该多态与心血管疾病的关系开始引起了研究者们的注意。一项横断面研究表明,在日本人中ALDH2Glu504Lys多态与心肌梗死(myocardial infarction, MI)相关,我们早期的一项研究和一项韩国人群的研究也得到了相似的结果。并且,近期《science》的一篇研究提示,ALDH2在心肌缺血保护中发挥了重要作用,该研究发现,不同基因型ALDH2活性存在显著差异,表达不同活性ALDH2的心脏缺血后所造成的心肌损伤程度差异显著,提高ALDH2活性可明显降低心肌缺血后的损伤面积。该研究结论提示,携带突变型ALDH2的ACS患者心肌缺血坏死时损伤更为严重,可能预后不良。但是,另有一项日本人群的研究却提示,ALDH2基因突变型可能是动脉粥样硬化的保护因素。上述研究结果似乎矛盾,令人迷惑。因此,本课题拟在中国汉族人群中,通过较大样本的病例对照研究进一步明确ALDH2Glu504Lys多态性与ACS发生的相关性,并从多个角度探讨该多态影响ACS发生的可能机制,以及通过前瞻性队列研究探讨该多态对ACS患者预后的影响。至本课题完成前,国际上尚罕见上述三方面研究。在过去30年里,许多研究证实饮酒与MI、心绞痛发生密切相关,因此,ALDH2作为酒精代谢的关键酶之一,其Glu504Lys多态影响ACS发生的机制是否受饮酒影响?此外,近年研究证实,人类ALDH2不仅是乙醇代谢的关键酶,而且是硝酸甘油转化成一氧化氮的关键酶。动物实验也证实,抑制ALDH2活性可显著降低硝酸甘油引起的犬冠状动脉扩张。故有学者推测ALDH2突变基因影响ACS发生也可能与冠状动脉痉挛相关。而既往研究已证实,ACS发生的关键机制是炎症诱发的易损斑块破裂和血栓形成。最近有研究提示,ALDH2可显著影响氧化应激,而炎症又与氧化应激存在着十分密切的联系。另外,以往也有研究提示,内皮祖细胞(endothelial progenitor cells, EPCs)与ACS的发生发展密切相关。因此,本研究拟初步探讨,在ALDH2基因Glu504Lys多态影响ACS发生机制方面,饮酒、血管舒张功能、冠状动脉病变严重程度、炎症、EPCs等多种因素是否发挥作用。ACS是目前我国致残和致死率最高的疾病之一,基于中国汉族ALDH2基因*504Lys突变30%-50%的高发生率,开展该基因多态与ACS的相关性及其机制研究,将对我国ACS防治新策略的制订提供重要的依据,对促进我国ACS防治工作意义显著。目的本课题研究目的：①在山东汉族人群中,通过较大样本的病例对照研究进一步明确ALDH2Glu504Lys多态性与ACS发生的相关性；②探讨饮酒、血管舒张功能、冠状动脉病变严重程度、炎症、EPCs等多种因素是否参与ALDH2Glu504Lys多态影响汉族人群ACS发生的机制；③在山东汉族人群中,通过前瞻性队列研究探讨ALDH2Glu504Lys多态对ACS患者预后的影响。方法1.研究对象病例对照研究中,病例组选择2007年9月至2008年12月期间于本院行冠状动脉造影的山东汉族AC8患者为研究对象,对照组来自山东大学齐鲁医院健康体检中心以及与病例组同时期行冠状动脉造影结果正常者。病例组和对照组的研究对象均再根据ALDH2基因Glu504Lys多态性再次分组：野生型组和突变组。携带ALDH2*1/*1基因型者为野生型组,携带ALDH2*1/*2与*2/*2者归于一组,即突变组。前瞻性队列研究以2007年1月至2009年7月期间于山东大学齐鲁医院急诊内科病房住院行冠状动脉造影的山东汉族ACS患者为研究对象,依据ALDH2基因型分为野生型组和突变组。2.临床基本资料采用设计好的固定的调查表格记录所有研究对象的临床资料。包括年龄、性别、高血压、糖尿病、吸烟、心血管疾病家族史、血脂水平、既往MI史、既往血运重建史、入院诊断、造影结果、用药情况、出院诊断等项目。其中血脂包括总胆固醇(Total cholesterol, TC)、低密度脂蛋白(low density lipoprotein-cholesterol, LDL-C)高密度脂蛋白(high density lipoprotein-cholesterol, HDL-C)、甘油三酯(triglyceride, TG),各项生化指标均来自我院中心实验室检验结果。3.饮酒情况饮酒频率(平均每周饮酒的天数)和平均每日饮酒量的计算方法如参考文献所述。以半定量式问卷的形式记录研究对象自开始饮酒以来,饮酒频率和饮用普通啤酒、红酒和白酒的平均量。将平均每周饮酒频率≥1次定义为经常饮酒。将普通啤酒355ml、红酒118ml、白酒40ml定义为一单位’drink",因上述各规格酒精含量大致相同(约14g)。平均每日饮酒量由饮酒频率和每次饮酒量计算得出。4.基因型测定采集外周静脉血以测定ALDH2基因型。ALDH2基因多态性的测定同课题组以往采用的方法。简要来说,先从静脉血中提取出DNA,再用PCR扩增目的DNA片段,最后纯化PCR产物、测序。5.冠状动脉病变严重程度每例患者均接受了冠状动脉造影,每支主要血管至少2个投照体位。通过选择性冠状动脉血管造影确定冠状动脉的狭窄程度,狭窄≥50%被定义为有意义的血管狭窄。应用以下两个参数评价冠状动脉病变严重程度：(1)冠状动脉病变血管支数,(2)Gensini评分。6.血管舒张功能2007年9月至2008年1月,对166例ACS患者行高分辨率超声,测定内皮依赖性血管舒张功能(Flow-mediated dilation, FMD)及非内皮依赖性血管舒张功能(Non-Flow mediated dilation, NMD)。超声检查于冠状动脉造影后72小时内进行,检查前18小时停服血管扩张药。患者休息30min后行超声检查。具体步骤如下：患者取仰卧位,用高频探头(S12)分别测定研究对象在休息时(基线)、反应性充血及舌下含服药物如硝酸甘油后的肱动脉直径。每种状态重复测量三次,取平均值。FMD为血管直径从基线值到反应性充血的峰值的增长率,NMD为血管直径从基线值到硝酸甘油诱导血管扩张的峰值的增长率。7.病例组与对照组的炎症标记物所有病例组和对照组的研究对象通过高敏乳胶增强免疫比浊法来测定血清高敏C反应蛋白(High-sensitivity C-reactive protein, hs-CRP)。8.初发STEMI患者外周循环EPCs数对病例对照研究中所有初发STEMI患者,用FACS Calibur流式细胞仪测量外周循环CD34+KDR+EPCs数。首先,样品管血样中加入FITC标记的单克隆鼠抗人CD34抗体和PE标记的KDR,对照管血样中加入相应的鼠抗人同型对照抗体,避光条件下室温孵育20min。再将细胞进一步与红细胞裂解液一起孵育,用冷磷酸盐缓冲液(Phosphate-buffered saline, PBS)冲洗三次。最后样品管与对照管中分别加入PBS制成悬液,FACS Calibur流式细胞仪分析。对淋巴单核细胞群设门收集细胞数至少5万个,循环EPCs数表示为CD34+KDR+细胞与所有淋巴单核细胞的比值。9.随访以主要不良心血管事件(major adverse cardiac events, MACE)作为随访终点事件,包括心源性死亡、新发生的急性心肌梗死、再次血运重建、新发生的心力衰竭。10.统计学分析所有数值变量用均数±标准差或中位数及四分位数差表示；分类变量用频数和百分数表示。分析数值变量用t检验(hs-CRP值先做对数变换成为正态分布后再做分析)与Mann-Whitney非参数检验(用于甘油三酯、饮酒量、Gensini评分与EPCs),分析分类变量用χ2检验。logistic回归与线性回归分析用来检测ALDH2基因多态性与ACS、初发STEMI、冠状动脉病变严重程度、hs-CRP水平和循环EPCs数等的关系,校正年龄、性别、体重指数(BMI)、血脂水平(TC、 LDL-C、HDL-C、TG)、吸烟史、高血压、糖尿病和CAD家族史。呈偏态分布的数值变量先做对数变换再纳入回归分析。随访数据的生存分析采用Kaplan-Meier分析与Cox回归分析方法。双向检验P<0.05被认为有统计学意义,用SPSS软件进行统计学分析。结果1.基本临床特征病例对照研究共入选1092例研究对象,病例组546人,对照组546人。病例组中,122人为初发STEMI患者,以1：2选取对照进行初发STEMI亚组的分析。病例对照研究中,病例组BMI、高血压、糖尿病、CAD家族史比例均显著高于对照组,而TC、LDL-C、HDL-C显著低于对照组,经常饮酒的样本数和平均每日饮酒量均显著高于对照组。在初发STEMI亚组,高血压、CAD家族史比例均显著高于其对照组,而TC和HDL-C显著低于其对照组,经常饮酒的样本数和平均每日饮酒量较对照组有升高趋势,但未见统计学差异。前瞻性队列研究共入选377例ACS患者,野生型组229例,突变组148例,突变患者占全部患者的39.3%。两组各项基本临床指标如年龄、性别、BMI、血脂水平、吸烟史、高血压、糖尿病和CAD家族史、心肌梗死病史、冠状动脉血运重建史、本次入院时ACS诊断情况(STEMI、NSTEMI、不稳定性心绞痛)均无统计学差异。但是,突变组左主干病变例数显著高于野生型组,而其他造影及血运重建指标均无统计学差异,包括病变血管支数、行冠状动脉血运重建比例、PCI时处理血管支数、PCI后TIMI3级比例。2. ALDH2基因突变情况各研究人群的ALDH2基因型频率都符合Hardy-Weinberg遗传平衡。病例组携带ALDH2突变基因型者显著高于对照组(46.7%vs31.9%,P<0.001)。亚组分析显示,初发STEMI组携带ALDH2突变基因型者也显著高于其对照组(48.4%与32.9%,p=0.006)。3. ALDH2基因多态与饮酒在ACS患者及初发STEMI患者中,ALDH2基因野生型携带者的每日饮酒量和经常饮酒的例数(ACS:35.3%vs27,8%, P<0.001;初发STEMI:49.2%vs28.8%,P=0.034)均显著高于突变型携带者。4. ALDH2基因多态与ACS相关性二元logistic回归校正年龄、BMI、血脂、吸烟史、高血压、糖尿病和CAD家族史后显示,ALDH2基因突变是ACS的独立危险因素(比数比[odds ratio,OR]1.95,95%可信区间[confidence interval, CI]:1.31-2.92, P=0.001)。进一步将饮酒情况纳入分析模型后,二者相关性减弱但仍有统计学意义(OR1.82,95%CI：1.23-2.70,P=0.03)。初发STEMI亚组的logistic回归分析也得到了相似的结果：校正年龄、BMI、血脂、吸烟史、高血压、糖尿病和CAD家族史后显示,ALDH2基因突变是初发STEMI的独立危险因素(OR2.17,95%CI：1.30-3.62,P=0.003),纳入饮酒情况后,二者相关性减弱但仍有统计学意义(OR1.95,95%CI：1.18-3.22,P=0.009)。5. ALDH2基因多态与冠状动脉病变严重程度在546例ACS患者中,不同ALDH2基因型患者的Gensini评分与病变血管支数都没有明显差异；多元线性回归和logistic回归分析校正年龄、性别、BMI、脂质水平、吸烟史、高血压、糖尿病和CAD家族史后,ALDH2多态性与Gensini评分和病变血管支数均未见显著相关(P>0.05,P>0.05)。对全部ACS中326例无MI病史的不稳定性心绞痛患者的进一步分析也显示了相同的结果(P>0.05,P>0.05)。6. ALDH2基因多态与血管舒张功能在行超声检查的166例ACS患者中,安静状态下的肱动脉直径在不同基因型患者中没有显著差异；FMD也相似(突变型4.9±2.3%,野生型5.1±2.5%,P>0.05)。然而,ALDH2基因突变型患者的NMD比无突变患者显著降低(3.9±3.3%和10.5±3.8%,P<0.001)。7. ALDH2基因多态与hs-CRP水平病例组和对照组中,ALDH2基因突变型携带者比野生型携带者均有更高的hs-CRP水平。多元线性回归分析发现,校正年龄、性别、BMI、脂质水平、吸烟史、高血压和糖尿病后,病例组和对照组中,ALDH2基因突变与高水平hs-CRP均呈独立相关(P=0.002,P=0.009)。并且,在分析ALDH2基因多态性与ACS关系的logistic回归中,将hs-CRP水平作为独立变量纳入后降低了ALDH2基因突变型在回归模型中的重要性(OR1.17,95%CI：1.01-1.37,P=0.040)。8. ALDH2基因多态与循环EPCs数量在初发STEMI患者中,突变型患者的循环EPCs数显著低于野生型患者。另外,在初发STEMI患者中,ALDH2基因为突变型者的年龄比野生型有更小的倾向(P=0.055),而且,ALDH2基因野生型携带者比突变型携带者有更高的HDL-C和更低的TG水平。多元线性回归校正年龄、性别、BMI、脂质水平、吸烟史、高血压和糖尿病后显示,ALDH2基因突变型是循环EPCs数的独立危险因素(P=0.032)。并且,二元logistic回归分析发现,在传统的心血管危险因素和饮酒史基础上,再将hs-CRP和循环EPCs数作为独立变量纳入后,ALDH2基因型和初发STEMI之间无显著相关(P=0.516)。9.随访结果前瞻性队列研究的随访时间为24-55个月,平均38个月,失访率为4.8%。随访期间两组患者治疗CAD的主要药物(阿司匹林、氯吡格雷、调脂药物、B-受体阻滞剂、血管紧张素转化酶抑制剂/血管紧张素Ⅱ受体拮抗剂)应用情况无显著差异。随访期间,该研究人群MACE发生率为13.8%,全因死亡率9%,其中心源性死亡率8%。突变组MACE和心源性死亡发生率均显著高于野生型组(21.0%vs9.2%,P=0.002；12.2%vs5.2%,P=0.026)。10.生存分析Kaplan-Meier分析显示,ALDH2基因突变型患者累计无MACE事件生存率和无心源性死亡生存率均显著低于野生型患者。Cox回归分析发现,ALDH2基因突变是ACS患者发生MACE事件和心源性死亡的独立危险因素(风险比[relative risk, RR]2.443,95%CI:1.390-4.296, P=0.002; RR2.727,95%CI:1.303-5.708, P=0.008)。结论1.在山东汉族人群中,健康人群ALDH2基因*504Lys突变(*1/*2和*2/*2)的发生率约为30%,而ACS患者ALDH2基因突变的发生率接近50%,两者存在显著差异。2.在山东汉族人群中,ALDH2基因*504Lys突变显著影响饮酒量和饮酒频率。3.在山东汉族人群中,ALDH2基因*504Lys突变是ACS的独立遗传危险因素,其机制与该突变对饮酒的影响有关,但也存在饮酒以外的机制。4.在山东汉族人群中,ALDH2基因*504Lys突变也是初发STEMI的独立遗传危险因素,其影响存在饮酒以外的机制。5.在汉族人群中,ALDH2基因*504Lys突变影响ACS的机制与饮酒显著减少、hs-CRP水平增高和循环EPCs数降低有关。6.在汉族人群中,ALDH2基因*504Lys突变影响ACS的机制与FMD所反映的血管舒张功能以及冠状动脉病变严重程度(以冠状动脉病变血管支数和Gensini评分评价)无明显相关。7.在山东汉族人群中,ALDH2基因*504Lys突变是ACS患者预后不良(发生MACE事件和心源性死亡)的独立预测因素。更多还原

【Abstract】 BackgroundAn association of alcohol consumption and coronary artery disease (CAD) has been suggested. Gene polymorphisms of key enzymes in alcohol metabolism, including alcohol dehydrogenase3(ADH3) and aldehyde dehydrogenase2(ALDH2), could be implicated in the association of alcohol consumption and CAD. The ADH3gene polymorphism, common in Caucasian populations, was found to be involved in the association of alcohol consumption and myocardial infarction (MI). The Glu504Lys single nucleotide polymorphism in the ALDH2gene exists mainly in East Asians. Between30%and50%of East Asians (or6%the world’s population) carry the mutant allele of ALDH2. Recently, cross-sectional studies in East Asia, including our pilot study, demonstrated a higher prevalence of the ALDH2Glu504Lys mutant in patients with MI than in control populations. An in vitro study showed that ALDH2derived from the wild type and mutant types had different activities and thus notably different effects on the extent of myocardial necrosis in acute MI. These findings suggest that the ALDH2Glu504Lys polymorphism may play a functional role in CAD. However, the association of ALDH2Glu504Lys polymorphism and acute coronary syndrome (ACS), and the potential mechanisms involved have not been examined in Han Chinese.The ALDH2enzyme is mainly located in mitochondria and is encoded by the ALDH2gene on chromosome12. The ALDH2gene is composed of13exons. Exon12contains a G-to-A missense mutation, whereby glutamate at position504is replaced by lysine (Glu504Lys); hence, two ALDH2alleles exist (Glu504and Lys504, also named ALDH2*1and ALDH2*2) with3genotypes, namely*1/*1(wild-type homozygote),*1/*2(heterozygote), and*2/*2(mutant homozygote). Studies suggest that ALDH2is the key enzyme for both alcohol and nitroglycerin metabolism. The mutant ALDH2*2is involved in the transformation from nitroglycerin to nitric oxide, which plays a key role in nitroglycerin tolerance among Asian patients with CAD. A study in a canine model also showed that different ALDH2genotypes could influence coronary artery dilatation triggered by nitroglycerin. These studies suggest that ALDH2could influence coronary spasm-associated ACS. In addition, level of high-sensitivity C-reactive protein (hs-CRP), a classical inflammatory biomarker, and number of circulating endothelial progenitor cells (EPCs) are associated with vulnerable plaque which has been recognized as a fundamental mechanism of ACS.Objectives1. To investigate the relationship between ALDH2Glu487Lys polymorphism and the risk of ACS in Han Chinese of Shandong Province.2. To reveal the underlying mechanisms of ALDH2Glu487Lys polymorphism influencing the occurrence of ACS in Han Chinese.3. To evaluate the effect of ALDH2Glu487Lys polymorphism on prognosis of ACS in Han Chinese of Shandong Province.Methods1. Study populationCase-control study enrolled Han Chinese patients with ACS who underwent coronary angiography in our hospital from September2007to December2008, and non-CAD controls. The control population was gender-and age (±1year)-matched Han Chinese subjects without CAD recruited from the Health Examination Center of Qilu Hospital, Shandong University and patients with a negative coronary angiographic finding. Confirmation of most non-CAD control subjects was based on negative history of coronary artery diseases, absence of chest pain symptoms and normal electrocardiographic results; confirmation for some was based on normal coronary angiographic results. ACS patients and their controls were separately divided into two groups according to the ALDH2gene polymorphism:group with wild genotype (*1/*1) and group with mutant genotypes (*1/*2and*2/*2). Prospective cohort study enrolled Han Chinese patients with ACS admitted in Emergency Internal Department of Qilu Hospital, Shandong University from January2007to July2009. These patients were also divided into two groups according to the ALDH2gene polymorphism:group with wild genotype and group with mutant genotypes.These studies were approved by the Ethics Committee of Qilu Hospital, Shandong University. All subjects provided their informed consent to the study. During the first24hours after admission, blood samples were collected from all patients.2. Clinical basic characteristicsThe clinical basic characteristics of all subjects were recorded with designed forms which had such contents as age, gendle, hypertension, diabetes mellitus, smoking, family history of CAD, lipids, histories of MI or revascularization, admission diagnosis, angiographic characteristics, medication use and so on.3. Alcohol consumptionThe frequency of drinking (drinking days per week) and the average daily alcohol consumption were evaluated as previously described. Namely, the usual frequency of drinking and average amount of beer, wine or liquor during the participant’s drinking lifetime was recorded by use of a semiquantitative questionnaire;355ml of beer,118ml of wine, or40ml of liquor were defined as one drink and considered to contain the same amount of alcohol (approximately14g). The average daily alcohol consumption was calculated by the frequency of consumption and the amount of each beverage:No patients who frequently (more than once per week) drank hard liquor were enrolled.4. ALDH2genotypingThe ALDH2polymorphism was detected as we described previously. In brief, DNA was extracted from200μL of venous blood; genomic DNA underwent PCR amplification; finally, PCR products were purified and directly sequenced.5. Severity of CADAll ACS patients underwent coronary angiography by the Judkins’method. For each major epicardial coronary artery, at least two views were observed. The degree of coronary stenosis was assessed by quantitative coronary angiography and luminal diameter stenosis≥50%was defined as vessels with significant disease. The following two parameters were used to assess the severity of CAD:1) the number of coronary arteries with significant stenosis and2) Gensini score. The methods of evaluation have been described previously.6. Brachial artery vasodilatationFrom September2007to January2008, a subset of consecutive ACS patients (n=166) underwent high-resolution ultrasonography (HP Sonos7500, USA) to detect flow-mediated dilation (FMD) and non-flow mediated dilation (NMD) of the brachial arteries. The ultrasonography was performed within72hr after coronary angiography, and vasodilative medications was stopped18hr before scanning. After patients rested for30min, ultrasonography was performed by a physician blinded to the study protocol using previously reported methods. With the patient in a supine position, the diameters of the brachial arteries at rest (baseline) and in response to reactive hyperemia (FMD) and sublingual administration of nitroglycerin (NMD) were measured using a high frequency probe (S12). The measurements were repeated for three times under each experimental condition and the values were averaged. The percentage increase of the brachial arterial diameters from the baseline value to the value measured at the peak of reactive hyperemia and to the value measured at the peak of nitroglycerin-induced vasodilation was taken as FMD and NMD, respectively.7. Inflammatory biomarkers in ACS and control subjectsIn all ACS and control subjects, serum hs-CRP levels were measured by experts blinded to the study protocol using high-sensitivity latex-enhanced immunonephelometry with a commercial kit as described previously.8. Number of circulating EPCs in patients with primary ST-segment elevation MI (STEMI)For all patients with primary STEMI in the case-control study, the number of peripheral circulating CD34+KDR+EPCs was assessed by use of FACSCalibur cytometry (Becton Dickinson, USA). First, blood samples were incubated with FITC-labeled monoclonal mouse anti-human CD34antibody and PE-labeled KDR for 20min at room temperature away from the light or with the corresponding mouse anti-human isotype-matched control antibodies. Second, cells were further incubated with FACS lysing solution and washed with cold phosphate-buffered saline (PBS)3times. Finally, the samples and isotype controls were suspended with PBS and measured by FACSCalibur. At least50,000events were acquired in the lymphomonocytic gate; the number of circulating EPCs was expressed as proportion of CD34+KDR+cells to all lymphomonocytic cells.9. Follow-upFor the evaluation of long-term clinical outcome, Major adverse cardiac events (MACE), including cardiac death, new acute MI, new revascularization, and new congestive heart failure, were considered as end points.10. Statistical analysisQuantitative data are expressed as mean±SD, or median and interquartile range; categorical data are presented as numbers and percentages. Students’t test (the values of hs-CRP were compared after log transformation to achieve normal distribution) and Mann-Whitney nonparametric test (for triglycerides, alcohol consumption, Gensini score, EPCs) were used for analysis of quantitative data. Chi-square test was used for analysis of categorical data. Numerical variables of abnormal distribution were first log transformed before they were included in regression analyses. Multivariate logistic and linear regression analyses were performed to examine the association of ALDH2polymorphism with ACS, primary STEMI, severity of CAD, level of hs-CRP and number of circulating EPCs. We used multivariate regression analysis to control for age, gender, body mass index (BMI), lipid levels (total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol), history of smoking, hypertension, diabetes meliitus and family history of CAD. Kaplan-Meier analysis and Cox regression analysis were conducted for the prognosis evaluation. A two-tailed P<0.05was considered statistically significant. All data analysis involved use of SPSS v10.0.Results1. Demographic characteristics In the case-control study, a total of1,092subjects were enrolled, including546patients with ACS and546non-CAD controls. Among ACS patients,122were diagnosed as having primary STEMI. The subgroup analysis of primary STEMI was designed as a1:2control;243non-CAD control subjects were enrolled, including122who were from the initial matching control group and another121who were identified from the remaining424of all546gender-and age-matched controls (we could not identify a control who met the matching criteria for only1case). ACS patients had higher BMI, TC, LDL-C, rates of hypertension and diabetes mellitus and history of CAD and lower HDL-C level than its controls, furthermore, the rate of drinking frequently and daily alcohol consumption were both higher in ACS patients as compared with its controls.In the prospective cohort study,377patients with ACS were enrolled, including229cases with wild genotype and148with mutant genotypes. The clinical basic characteristics were similar between the two groups with different ALDH2genotypes. Furthermore, the angiographic characteristics were also similar, except that the rate of left main lesion in the patients with mutant genotype was markedly higher than that in those patients with wild-type genotypes.2. ALDH2polymorphism in various groups In the case-control study, more patients with ACS and primary STEMI showed ALDH2mutant genotypes than did their controls (46.7%vs.31.9%, P<0.001;48.4%vs.32.9%, P=0.006; respectively).3. ALDH2polymorphism and drinking In the case-control study, patients with ACS with wild-type ALDH2showed significantly higher daily alcohol consumption and frequency of drinking than did those with mutant genotypes (35.3%vs27,8%, P<0.001), and similar results were found among the patients primary STEMI (49.2%vs28.8%, P=0.034).4. ALDH2polymorphism and ACS or primary STEMI On binary logistic regression, ALDH2mutant was an independent risk factor for ACS (odds ratio [OR]1.95,95%confidence interval [CI]:1.31-2.92, P=0.001) after controlling for age, BMI, lipid levels, history of smoking, hypertension, diabetes mellitus and family history of CAD. The association was weakened but still statistically significant when alcohol consumption was entered into the analytical model (OR1.82,95%CI:1.23-2.70, P=0.003); similar results were shown in the subgroup analysis of primary STEMI (OR2.17,95%CI:1.30-3.62, P=0.003; OR1.95,95%CI:1.18-3.22, P=0.009, after adjusting for alcohol consumption; respectively).5. ALDH2polymorphism and severity of CADThe546ACS patients did not differ by ALDH2genotypes in Gensini score or number of significantly diseased coronary arteries. Multivariate linear and logistic regression analyses demonstrated no association of ALDH2polymorphism and Gensini score or number of significantly diseased coronary arteries after adjusting for age, gender, BMI, lipid levels, history of smoking, hypertension, diabetes mellitus and family history of CAD (all P>0.05). Further analysis in the subset of patients with unstable angina with no history of MI (n=326) also did not show any association(P>0.05, respectively).6. ALDH2polymorphism and artery vasodilatationIn these ACS patients, the diameter of the brachial artery at rest did not differ by genotype; FMD was also similar (4.9±2.3%for the mutant genotype vs.5.1±2.5%for the wild type, P>0.05). However, NMD in patients with mutant genotypes was significantly less in patients with than without the mutant ALDH2genotype (3.9±3.3%vs.10.5±3.8%, P<0.001).7. ALDH2polymorphism and hs-CRP levelSubjects with the ALDH2mutant genotypes had higher hs-CRP levels than did those with the wild-type genotype, whether ACS patients or controls. Moreover, multivariate linear regression revealed the ALDH2mutation independently associated with high hs-CRP level in both ACS and control subjects (P=0.002and P=0.009, respectively), with age, gender, BMI, lipid levels, history of smoking, hypertension and diabetes mellitus adjusted in the model. Furthermore, on analyzing the association of ALDH2polymorphism and ACS, inclusion of hs-CRP level as an independent variable weakened the importance of the ALDH2mutant genotype in the regression model (OR1.17,95%CI:1.01-1.37, P=0.040).8. ALDH2polymorphism and number of circulating EPCsThe primary STEMI patients with wild-type tended to be older than those subjects with mutant genotypes (P=0.055), furthermore, the wild-type ALDH2was associated with higher HDL-C level and lower TG level. In patients with primary STEMI, the number of circulating EPCs was significantly lower in subjects with ALDH2mutant genotypes than in those with the wild-type genotype. On multivariate linear regression, ALDH2mutant genotype was an independent risk factor for a low number of circulating EPCs after controlling for age, gender, BMI, lipid levels, history of smoking, hypertension and diabetes mellitus (P=0.032). However, binary logistic regression analysis revealed no association of ALDH2genotypes and primary STEMI when hs-CRP level and number of circulating EPCs were included as independent variables as compared with traditional cardiovascular risk factors and alcohol consumption (P=0.516).9. Follow-upThe time of follow-up was from24to55months (mean period was38months). The follow-up was completed in95.2%of all patients. The use of medication, including aspirin, clopidogrel, statins, β-blockers, ACEI/ARB, was similar between the wild-type and mutant groups. The incidence of MACE was13.8%, moreover, the rate of death and cardiac death were9%and8%, respectively. The incidences of MACE and cardiac death were both higher in the group with mutant genotypes as compared with the group with wild genotype (21.0%vs9.2%,=0.002;12.2%vs5.2%, P=0.026).10. Survival analysisKaplan-Meier analysis for the MACE-free and cardiac-death-free cumulative survival in patients with ALDH2mutant genotypes demonstrated worse long-term outcomes for these patients. Furthermore, Cox regression analysis confirmed the ALDH2mutant genotypes as an independent predictor for MACE and cardiac death ([relative risk, RR]2.443,95%CI:1.390-4.296, P=0.002; RR2.727,95%CI:1.303-5.708, P=0.008). Conclusions1. In Han Chinese of Shandong Province, more patients with ACS carry ALDH2mutant genotypes (*1/*2and*2/*2) than do its non-CAD controls.2. In Han Chinese of Shandong Province, the people with wild-type ALDH2(*1/*1) show significantly higher daily alcohol consumption and frequency of drinking than do those with mutant genotypes.3. ALDH2mutation is a genetic risk marker for ACS in Han Chinese of Shandong Province, which is explained in part by alcohol consumption.4. ALDH2mutation is a genetic risk marker for primary STEMI in Han Chinese of Shandong Province, which is explained in part by alcohol consumption.5. The association between ALDH2Glu504Lys polymorphism and the risk of ACS in Han Chinese is explained in part by alcohol consumption, inflammation and circulating EPCs.6. In Han Chinese, ALDH2mutation is not associated with vasodilatation and severity of CAD as measured by FMD, the number of significantly diseased vessels and Gensini score respectively.7. ALDH2mutation is an independent predictor for cardiac death and MACE in Han Chinese patients with ACS.更多还原