急性冠脉综合征全基因组甲基化差异及其功能的生物信息学分析

发布时间：2018-04-10 22:39

本文选题：DNA甲基化 + 甲基化芯片　；参考：《南京大学》2015年硕士论文

【摘要】：冠心病(coronary artery disease, CAD):具有多基因遗传性,其发生过程往往是多因素、多疾病协同的结果,存在众多危险因素,被认为是受表观遗传学规律控制的人类重要疾病。DNA甲基化是最早发现的表观遗传修饰方式之一,也是目前该领域研究的热点。甲基化修饰虽不改变DNA序列,但对基因的表达与沉默起重要的调节作用。急性冠脉综合征(acute coronary syndrome, ACS)包括不稳定型心绞痛(unstable angina, UA)、急性心肌梗死(acute myocardial infarction, AMI)等各种急性心肌缺血引起的临床综合征,发病突然,致死率高,是造成猝死的重要原因。DNA甲基化在ACS的发生发展过程中扮演着怎样的角色,以及在病理生理机制中起到什么样的作用有待进一步的探索与研究。目的：(1)应用高密度基因芯片(450K Infinium Methylation BeadChip)技术分析ACS患者与正常人外周血全基因组DNA甲基化差异水平,寻找ACS患者DNA甲基化水平显著变化的基因,并进行功能富集分析以探索其在ACS疾病发生、发展过程中所发挥的作用。(2)应用甲基化特异性PCR(methylation-specific PCR, MS-PCR/MSP)检测CAD患者与正常对照组中C型1类尼曼-匹克蛋白(Niemann-Pick C1 protein, NPC1)基因启动子区甲基化水平的差异,为冠心病早期诊断、治疗提供的新靶标和实验依据。方法：(1)收集ACS患者和正常人(作为对照)全血标本各3份,分别提取DNA并进行亚硫酸盐转化,与Illumina HD 450K Infinium Methylation BeadChip芯片杂交,对450,000多个甲基化位点进行检测,覆盖96%的CpG岛；针对Illumine 450K甲基化芯片中的探针甲基化位点,进行数据标准化(Control normalization)预处理,计算探针水平的甲基化程度的水平(Beta score);对高甲基化基因进行GO功能分析(Gene Ontology Analysis, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of Genes and Genomes, KEGG)功能分析。(2)收集64例正常人、37例稳定型冠心病(stable coronary artery disease, SCAD)患者和55例ACS患者的全血标本,分别提取DNA并进行亚硫酸盐转化,用MS-PCR法检测NPC1启动子区甲基化水平,分析患者与正常人的甲基化水平差异。结果：(1)ACS患者与正常人全血基因组间共发现770个基因存在DNA甲基化水平的差异(Beta score0.2, p0.05),其中甲基化程度降低的基因有640个,升高的基因有130个。GO和KEGG功能分析结果显示甲基化差异的基因功能主要集中在细胞信号通路、糖脂代谢、细胞粘附分子和细胞周期细胞凋亡,这些基因的DNA甲基化水平升高可能是导致ACS的原因之一。(2)SCAD患者全血DNA的NPC1启动子区甲基化水平明显高于正常对照组和ACS患者,而ACS患者NPC1启动子区甲基化水平较正常对照组无明显差异。结论：(1)ACS患者全基因组甲基化水平较正常对照组发生显著变化,这可能是导致ACS发生、发展的重要因素之一。(2)冠心病患者NPC1甲基化水平明显升高,可能与AS的斑块形成和AS逐渐严重时斑块稳定相关。对这些基因的甲基化水平进行检测将有益于冠心病,特别是ACS的早期诊断和病情监控。
[Abstract]:Coronary heart disease (coronary artery disease, CAD): with polygenic inheritance, the process is often multi factors, multi disease collaborative results, there are many risk factors, was found to be affected by the apparent human diseases.DNA methylation genetics control law is the most early one of epigenetic modification, is currently the focus in this field. Methylation does not alter the sequence of DNA, but the expression of gene silencing and plays an important regulatory role. Acute coronary syndrome (acute coronary, syndrome, ACS) including unstable angina (unstable, angina, UA), acute myocardial infarction (acute myocardial infarction, AMI) by a variety of acute myocardial ischemia in clinical syndrome, sudden onset, high fatality rate, is the important cause of sudden death caused by.DNA methylation plays a role in what happened in the process of ACS development, as well as the pathologist To further explore and study what kind of role play mechanism. Objective: (1) the application of high density gene chip (450K Infinium Methylation BeadChip) technology, different levels of DNA methylation in peripheral blood of ACS patients and normal people, looking for ACS DNA in patients with significant changes in methylation level and gene. The function enrichment analysis to explore its occurrence in ACS disease, which play a role in the process of development. (2) by methylation specific PCR (methylation-specific, PCR, MS-PCR/MSP) detection of CAD patients and normal control group C type 1 - Niemann PEAK (Niemann-Pick C1 protein, NPC1 protein) gene promoter hypermethylation differences the level of the early diagnosis of coronary heart disease, and provide experimental basis for new target treatment. Methods: (1) collected from ACS patients and normal people (as control) whole blood specimens were collected from 3, DNA were extracted and sulfite Salt transformation, hybridization and Illumina HD 450K Infinium Methylation BeadChip chip was used to detect the methylation sites of the more than 450000, covering 96% of the CpG island methylation sites for the probe; Illumine 450K methylation chip, data standardization (Control normalization) pretreatment, the methylation level computing probe level level (Beta score GO); functional analysis of high methylation gene (Gene Ontology Analysis, GO) and Kyoto Encyclopedia of genes and genomes (Kyoto Encyclopedia of Genes and Genomes, KEGG) functional analysis. (2) collected from 64 normal subjects, 37 patients with stable coronary heart disease (stable coronary artery disease, SCAD) and 55 patients with whole blood samples ACS patients, DNA were extracted and the sulfite conversion, detection of NPC1 promoter methylation level by MS-PCR method, analysis of methylation level in patients with normal differences. Results: (1) ACS patients and normal human blood genome were found between the 770 gene methylation level of DNA (Beta score0.2 P0.05), the decrease of methylation of 640 genes, 130 genes were increased.GO and KEGG function analysis showed that the gene methylation difference in the main function focus on cell signaling pathways, lipid metabolism, cell adhesion molecules, cell cycle and apoptosis, increased the level of DNA methylation of these genes may be the cause of ACS. (2) SCAD patients blood DNA NPC1 promoter methylation was significantly higher than the normal control group and ACS patients, and ACS patients NPC1 methylation levels compared with normal control group, no significant difference. Conclusion: (1) genomic DNA methylation levels in ACS patients compared with normal control group changed significantly, which may lead to the occurrence of ACS, the development of one of the important factors. (2) coronary heart The level of NPC1 methylation is significantly higher in patients with disease. It may be related to plaque formation in AS and plaque in AS gradually. Detection of methylation level of these genes will be beneficial for early diagnosis and monitoring of coronary heart disease, especially ACS.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.4

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