【作者】 陈良川；

【导师】 彭健；

【作者基本信息】 南方医科大学 ， 心血管内科， 2009， 硕士

【摘要】 一、研究背景心房颤动(房颤)是临床上最常见心律失常,常伴有脑卒中、心力衰竭等并发症发生,增加心血管疾病发病率和死亡率。因此在早期阶段诊断和治疗房颤,对预防顽固性房颤和心血管事件的发生有着重要意义。目前,我们对心房的早期病理生理变化不完全清楚,主要是由于对房颤分子生物学机制了解甚少,对房颤基因学的研究为房颤发病病因的了解提供了新的视野。少见家族性房颤主要由遗传性分子缺陷引起电生理异常所致,然而临床大部分房颤属非家族性房颤,与心房结构重构有关。队列比较研究显示肾素-血管紧张素-醛固酮系统及炎症相关基因基因单核苷酸多态性(SNP)房颤的发生有关,表明房颤形成复杂性。ABCA1基因是心血管疾病研究最常见的候选基因,它以ATP为能源,在胆固醇逆转运(reverse cholesterol transport,RCT)和高密度脂蛋白(high densitylipoprotein,HDL)生成起始步骤中起重要的作用。现有研究表明ABCA1基因可能通过减少HDL-C水平来增加氧化低密度脂蛋白(oxidized low-densitylipoprotein,OX-LDL)和炎症因子对血管壁的损伤,增加心血管疾病发生率,但是R219K位点SNP突变,可增加ABCA1基因的活性降低低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)水平、增加HDL-C水平的趋势减少心血管疾病发生。越来越多的证据表明ABCA1基因SNP与冠心病、高血压等疾病密切相关,而这些疾病都是房颤发病高危因素。因此ABCA1基因也可能通过对血脂和炎症因子的调节,对房颤的发病有所影响。二、目的我们研究小组对中国华南地区汉族人群205例房颤患者和205例非房颤者为研究对象,建立临床资料,检测CRP、血脂等血清水平,对目前可能涉及房颤发病的ABCA1基因R219K位点检测,从而了解ABCA1基因R219K位点SNP与中国华南地区房颤人群关联性情况;并且研究CRP、血脂与ABCA1基因R219K位点SNP的关系。从而从炎症的角度探索遗传因素在中国华南汉族人群房颤发病可能作用。三、方法我们观察房颤组和对照组两组人群。房颤组(排除孤立性房颤和甲亢引起的房颤)205例,男116例,女89例,年龄64.50±7.98岁。对照组根据心电图检查排除房颤205例,与房颤组进行性别、年龄、身高、体重指数、吸烟、左室功能、瓣膜病进行配对;高血压、冠心病、糖尿病与ABCA1基因型存在相关性,因此,这些因素的频率在对照组和房颤组也同样配对,男116例,女89例,平均64.11±8.15岁。所有的对象均获得知情同意,年龄50岁至70岁之间。合并感染、自身免疫性疾病、结核、肿瘤、严重肝肾功能不全、入选2周内口服降脂类药物者予以剔除。所有入选的研究对象进行血脂、血糖、CRP等生化指标的测定。用试剂盒提取静脉血白细胞基因组DNA。采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)测定ABCA1 R219K基因的多态性,并对PCR产物进行测序。比较两组之间不同基因型、等位基因频率的分布及CRP、血脂等相关指标的血清浓度的差异。两组均建立临床资料观察表,详细记录姓名、年龄、身高、体重、性别、吸烟、CRP、TC、TG、HDL-C、LDL-C、左房直径、左室射血分数(LVEF)等检查指标;以及高血压、冠心病、糖尿病等病史。四、结果1、经PCR-RFLP对ABCA1 R219K基因位点的检测,我们发现中国华南地区人群存在R219K位点SNP改变。2、PCR产物测序证实了ABCA1存在R219K改变,经序列分析,R219K多态为第7号外显子ABCA1基因第1051位核苷酸发生G碱基替换呈A碱基,导致精氨酸(Arg)→赖氨酸(Lys)替代。3、房颤组与对照组ABCA1 R219K位点RR、RK、KK基因型频数分别为89、94、22和71、87、47,差异有统计学意义(χ~2=11.354,P=0.003)。R等位基因、K等位基因分布频率为66.3%、33.7%和55.9%、44.1%,K等位基因分布频率在房颤组中较对照组降低,差异有统计学意义(χ~2=9.487,P=0.002)。4、ABCA1基因R219K位点基因多态性与房颤的关系,相对RR基因型而言,RK型和KK型,均能显著减少房颤发生的危险性,OR值(95%CI)分别为0.373(0.206～0.677)、0.433(0.242～0.777),P＜0.05。但RK+KK型相对RR型而言,OR值为0.691(0.463～1.029),P＞0.05。5、房颤组平均左房大小为43.76±8.06mm,对照组为41.94±7.27mm,两组比较有统计学差异(P=0.017)。6、房颤组CRP浓度明显高于对照组(P=0.001),血脂浓度无明显差异;R219K三种基因型间的CRP、HDL-C有统计学差异(P值分别为0.02、0.025)。两两基因型之间的比较采用LSD-t检验,RR基因型CRP水平高于RK、KK基因型CRP水平,存在统计学差异(P分别为0.011、0.037),而RK基因型、KK基因型之间CRP水平差异无显著性(P＞0.05)。RR基因型HDL-C水平低于RK、KK基因型HDL-C水平,存在统计学差异(P分别为0.032、0.017),RK基因型、KK基因型之间HDL-C水平差异无显著性(P＞0.05)。LDL-C、TG、TC水平在三种基因型间比较无统计学差异(P＞0.05)。五、结论1、ABCA1基因编码区外显子7中R219K SNP存在于中国华南地区汉族人群中。2、房颤患者CRP血清浓度明显高于对照人群,存在炎性反应。3、ABCA1 R219K K基因变异可能提高ABCA1的活性,引起HDL-C血清浓度的升高,从而降低炎性细胞因子CRP的释放。4、ABCA1 R219K位点SNP与中国人群房颤的发生有明显的相关性,K等位基因可能是房颤的保护因素,R等位基因可能是房颤发病的易患因素。5、R219K位点SNP影响房颤发生的可能机制:R等位基因降低HDL-C水平,升高CRP等炎性活性因子,并且增加心血管的疾病的发生,从而导致房颤的发生发展。6、他汀类药物、糖皮质激素可能通过抗炎作用由于房颤的治疗,具体机制有待于进一步证实。更多还原

【Abstract】 Background:Atrial fibrillation(AF) is the most common arrhythmia in clinical practice and is associated with an increase in cardiovascular mortality,as well as cardiovascular morbidity,including stroke and heart failure.It is therefore important to detect and treat AF at an early stage to prevent refractory AF and cardiovascular events in the future.Currently,our knowledge on the early pathophysiological changes in the atria is limited due to lacking understanding of molecular mechanisms.Studies in genetics of AF may provide some new insights in the etiology of AF.The familial form of AF is uncommon and AF as electrical disease is more often related to molecular genetic defects,However,most non-AF relate to structural remodeling.Cohort studies of the genetic single nucletide polymorphism(SNP) in the renin angiotensin system genes and inflammatory genes are associated with AF indicating the complexity of the development of AF.Genetic variations in the ATP-binding cassette transporter A1(ABCA1) gene are also interesting candidates to play an important role in cardiovascular disease.ABCA1 is considerd to play an important role in the process of reserve cholesterol transport(RCT) and the primary formation of high density lipoprotein cholesterol(HDL-C) by using ATP as a source of energy.An decreased ABCA1 activity might increase oxidation of LDL-C(ox-LDL) and inflammation of the vascular wall through its effect on HDL-C.But,a high ABCA1 R219K SNP may be a determinant of decreased cardiovascular disease by icreasing ABCA1 activity and thereby reducing the cholesterol content of HDL-C relative to low density lipoprotein cholesterol(LDL-C).Accumulating evidence suggests that ABCA1 SNP may be linked to coronary artery disease,as well as hypertension,which are both strongly linked to the incidence of AF.Therefore,ABCA1 gene may also have an impact on AF through its effect on blood lipids and inflammatory factors.Objectives:The purpose of the present study is to analyze the association of ABCA1 genotype polymorphism with 205 AF subjects and 205 controls in Han Nationality of Southern China;detect the levels of CRP,lipid.As well as to clarify whether polymorphism of the ABCA1 gene are relate to AF and its relationship with plasma lipid and CRP in Han Nationality of Southern China patients.Methods:Individuals in two groups were studied.The AF groug included 205 subjects (116 males,and 89 females,64.50±7.98 years).The control group included 205 healthy individuals(116 males,and 89 females,64.11±8.15 years).The study included 205 patients with a history of AF.Patients with hyperthyroidism and lone AF were excluded.None of the AF was familial.Case and control patients were individually matched with regard to their gender,age,presence of left ventricular dysfunction,and presence of significant valvular heart disease.Hypertension, coronary artery disease and diabetes are known phenotypes associated with polymorphisms with in ABCA1 genes.Therefore,case and control patients were also matched as to these variables by frequency(group matching).After the cases and controls were matched,the clinical variables of the cases were comparable to those of the controls.The age of cases was 50 years old to 70 years old,all patients agreed to participate in the study,and verbal informed consent was obtained from each patient. Subjects complicated with infection,autoimmune diseases,tuberculosis,tumor, severe liver and kidney dysfunction,oral statins drugs with in two selected weeks were to be removed.All subjects Lipids、glucose,CRP and other biochemical parameters were determined in all subjects.Genomic DNA in white blood cell from venous blood was abstracted by reagent box.R219K locus allele in exon domain 7 of ABCA1 gene were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in both groups.Distribution of R219 locus enotypes and frequency of R219 locus allele were compared between the two groups.Lipids and CRP levels was compared in three genotypes.Both groups were set up observation of clinical data form,including name height,weight,sex,smoking,CRP,TC,TG,HDL-C,LDL-C,left atrial diameter, LVEF,and high blood pressure,coronary heart disease,diabetes and other medical history.Results:1.R219K locus SNP exists in the Han Nationality of Southern China.2.Genotypes were determined by PCR product sequencing.The G(1051 nucleotides) of R219K locus allele in exon domain 7 of ABCA1 gene was replaced by A, resulting in arginine(Arg)→lysine(Lys) substitution.3.The frequencies of genotype of RR,RK and KK were 89,94,22;71,87,47 in two groups,there was a significant difference in the distributions of the genotypes (RR,RK,KK) between AF subjects and controls(χ~2 = 11.354,P = 0.003).The frequency of the allele R,allele K genotype of ABCA1 in AF group and control group was 66.3%,33.7%,55.9%,44.1%;allele K genotype frequency were significantly different between AF group and control group(χ~2 =9.487,P = 0.002).4.The presence of K allele of ABCA1 gene was found to be a greater protective factor in AF subjects than in normal control.The odds ratio(OR) of RK and KK were 0.373(0.206～0.677),0.433(0.242～0.777),respectively,when compared with RK+KK genotype,the P＞0.05.5.The average left atrial size of AF group was 43.76±8.06mm,the control group was 41.94±7.27mm,there was significant difference between two groups(P = 0.017).6.The serum concentrations of CRP in AF group was higher than those in control group(P =0.001),but there was no difference between two groups in plasma lipid;The serum concentrations of CRP of patients with RR genotype were higher than those with RK genotype and KK genotype(P = 0.011,0.037),and HDL-C with RR genotype less than RK genotype and KK genotype(P= 0.032,0.017).There was no significant difference between two groups in LDL-C,TG,TC(P＞0.05).Conclusion:1.R219K locus SNP exists in the Han Nationality of Southern China.2.The serum concentrations of CRP in AF group was higher than those in control group,presenceing inflammatory response.3.Allele K genotype of ABCA1 may enhance the activity decrease the serum concentrations of CRP through its effect on HDL.4.R219K polymophic site is associated with susceptibility of AF in Han Nationality of Southern China.The K allele may be protective,while the allele may be susceptibility gene for AF.5.The underlying mechanism of AF caused by R219K locus SNP:Allele R genotype decrease ABCA1 activity and thereby reducing the cholesterol content of HDL relative to LDL and,increasing cardiovascular disease.A logical explanation would be that the widely acknowledged association between ABCA1 gene variations resulting in lower HDL-C and higher LDL-C,CRP levels.This mechanism is very likely increasing the cardiovascular disease.6.Statins,glucocorticoid may treat AF through its effect on anti -inflammatory, the specific mechanism remains to be confirmed in the further study.更多还原