轻度认知障碍候选基因的DNA甲基化研究
发布时间：2018-07-25 10:12
【摘要】：目的:OPRK1与OPRM1受体属于阿片受体家族,在神经系统中的分布广泛,其与AD疾病密切相关,课题前期研究提示基因甲基化与AD疾病的关联性,本研究将继续探索其在AD的早期阶段即轻度认知障碍(MCI)中的DNA甲基化改变。此外,利用高通量芯片检测方法识别新的疾病风险甲基化基因,为后续的研究提供新的依据。方法:在新疆地区的流行病学调查研究得到的5398例样本中,按照年龄、性别、民族匹配的原则随机抽取MCI和对照各96例,利用磁珠法进行基因组DNA的提取,亚硫酸氢盐转化后利用焦磷酸测序方法定量检测OPRK1、OPRM1基因的甲基化水平,并且应用双荧光素酶报告系统进行启动子活性检测。此外,来自宁波市康宁医院6对性别年龄匹配的AD与对照样本,利用450K芯片进行全基因的甲基化水平测定。结果:第一部分:病例对照组比较分析结果:维族和汉族中OPRK1基因甲基化与MCI没有关联(p0.05),性别分层结果显示OPRK1基因高甲基化与汉族女性MCI患病相关(p=0.015);OPRM1基因,CpG1位点高甲基化与维族MCI患病相关(总体p=5.16E-05,男性p=0.015,女性p=1.20E-03),CpG2-4低甲基化与汉族MCI患病相关(总体p=0.002,男性p=0.018,女性p=4.98E-02)。不同民族间的甲基化比较结果:两个民族中基本资料存在差异,应用logistic回归对其进行校正,OPRK1甲基化在MCI与对照族中均没有民族差异(p0.05),OPRM1基因中CpG1位点甲基化在病例组中存在民族差异(CpG1 p=0.002,adjust p=0.009)CpG2-4位点甲基化在对照组中存在民族差异(CpG2-4 p=0.005,adjust p=0.009)。此外,结合前期的研究,将不同地域(新疆和浙江)汉族对照人群的基因甲基化结果进行比较,OPRK1甲基化在总体及男性分组中存在地域差异,而在女性中没有地域差异(总体p=0.006,男性p=2.31E-04,女性p0.05),OPRM1基因在新疆汉族人群中甲基化水平显著高于浙江汉族人群中甲基化水平(CpG1总体p=2.99E-05,男性p=0.006,女性p=0.001;CpG2-4总体p=1.32E-13,男性p=1.71E-08,女性p=2.74E-05)。偏相关分析(控制组别、性别、BMI、血糖、甘油三酯、胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇因素)显示维族中OPRM1 CpG2-4甲基化与年龄存在正相关关系(r=0.220,p=0.039)。双荧光素酶实验结果进一步提示,相较于阴性对照pGL3-Basic,OPRK1与OPRM1重组质粒均能促进下游萤火虫荧光素酶的表达,OPRK1使表达水平增加2.248倍(p=0.008),OPRM1增加2.616倍(p=0.003)。第二部分:应用450K检测6对样本之间的甲基化水平差异,AD与对照组间甘油三酯水平差异有统计学意义(p=0.025),后续的差异分析中利用线性模型对其进行校正,以|delta beta|0.2且p0.05为标准,共得到41个差异甲基化位点对应于33个基因,其中18个位点高甲基化,23个位点低甲基化。结论:OPRK1高甲基化为汉族女性MCI的风险因素,并且该片段具有启动子活性,可能存在低表达水平,环境因素对于汉族男性OPRK1甲基化存在影响,对汉族女性没有影响。OPRM1特定位点甲基化与不同民族的MCI风险相关,CpG1位点高甲基化是维族MCI患病的风险因素,CpG2-4位点低甲基化是汉族MCI患病的风险因素。此外,CpG1位点甲基化在MCI中存在民族差异,而CpG2-4位点甲基化在对照中存在民族差异。环境因素能够影响OPRM1甲基化水平,表现为新疆汉族OPRM1甲基化水平高于浙江汉族;年龄是MCI重要的危险因素,维族人群中OPRM1 CpG2-4基因甲基化水平随年龄增长而增长,OPRM1检测片段具有启动子活性。高通量芯片研究为后续提供新的研究依据,其中近启动子区域的差异甲基化位点的应在扩大样本中进行验证,尤其应对MCI到AD的进展过程中甲基化如何改变进行研究。
[Abstract]:Objective: OPRK1 and OPRM1 receptors belong to the opioid receptor family, which are widely distributed in the nervous system and are closely related to the AD disease. Earlier studies suggest the association between gene methylation and AD disease. This study will continue to explore the changes in DNA methylation in the early stage of AD, the mild cognitive impairment (MCI). In addition, high throughput core will be used. This method identifies new disease risk methylation genes and provides a new basis for subsequent research. Methods: in 5398 samples of epidemiological studies in Xinjiang, 96 cases of MCI and 96 cases were randomly selected according to the principle of age, sex and national match, and the extraction of genomic DNA by magnetic beads and hydrogen sulfite salt After conversion, the methylation level of OPRK1, OPRM1 gene was detected by pyrosequencing method, and the promoter activity was detected by using the dual luciferase reporter system. In addition, 6 sex matched AD and control samples from Corelle hospital in Ningbo were used to determine the level of methylation of all genes by 450K chip. Results: first Part: comparison and analysis of the case control group: the OPRK1 gene methylation in the Uygur and Han nationalities was not associated with MCI (P0.05). The results of sex stratification showed that the hypermethylation of the OPRK1 gene was associated with the MCI disease of the Han women (p=0.015); the OPRM1 gene, the high methylation of the CpG1 loci was associated with the Uygur MCI disease (general p=5.16E-05, male p=0.015, female p=1.20E-03). CpG2-4 hypomethylation is associated with the prevalence of MCI in the Han nationality (overall p=0.002, male p=0.018, female p=4.98E-02). Comparison results of methylation among different ethnic groups: the basic data of the two nationalities are different, the logistic regression is used to correct them. There is no national difference (P0.05) in OPRK1 methylation in MCI and the control group, and CpG1 loci in the OPRM1 gene. Methylation in the case group has ethnic differences (CpG1 p=0.002, adjust p=0.009) CpG2-4 locus methylation (CpG2-4 p=0.005, adjust p=0.009) in the control group (CpG2-4 p=0.005, adjust p=0.009). In addition, the results of the gene methylation of the Han control group in different regions (Xinjiang and Zhejiang) were compared with the previous studies, and OPRK1 methylation was in general and in general. There were regional differences in the male group, while there was no regional difference among women (total p=0.006, male p=2.31E-04, female P0.05). The methylation level of OPRM1 gene in Xinjiang Han population was significantly higher than that in the Han population of Zhejiang (CpG1 overall p=2.99E-05, male P =0.006, female p=0.001; CpG2-4 p=1.32E-13, male p=1.71E-08). P=2.74E-05. Partial correlation analysis (control group, sex, BMI, blood sugar, triglyceride, cholesterol, HDL cholesterol, low density lipoprotein cholesterol) showed that there was a positive correlation between OPRM1 CpG2-4 methylation and age in the Uygur group (r=0.220, p= 0.039). The results of double luciferase experiment were further suggested, compared with negative control PGL3-Basic, OPRK1 and OPRM1 recombinant plasmids could promote the expression of luciferase in the lower reaches of the firefly. OPRK1 increased the expression level by 2.248 times (p=0.008) and OPRM1 increased by 2.616 times (p=0.003). The second part: the difference of the methylation level between the samples was detected with 450K, and the difference between AD and the level of triglyceride between the groups was statistically significant (p=0.025). In the subsequent difference analysis, a linear model was used to correct it. With |delta beta|0.2 and P0.05 as the standard, 41 different methylation sites were matched to 33 genes, of which 18 loci were methylation and 23 loci were low methylation. Conclusion: OPRK1 hypermethylation is the risk factor for the female MCI of the Han nationality, and the fragment has the promoter activity. Sex, may have low level of expression, environmental factors have influence on OPRK1 methylation in Han men, and there is no effect on the risk of MCI risk in the.OPRM1 specific location methylation of Han women. High methylation at the CpG1 locus is a risk factor for the MCI disease of the Uygur nationality, and the low methylation of CpG2-4 loci is the risk factor of the Han nationality in the Han nationality. In addition, C There are ethnic differences in the methylation of pG1 loci in MCI, and there are ethnic differences in the methylation of CpG2-4 loci. Environmental factors can affect the level of OPRM1 methylation, which shows that the level of OPRM1 methylation in Xinjiang Han is higher than that of the Han people in Zhejiang; age is an important risk factor for MCI, and the methylation level of OPRM1 CpG2-4 gene in the Uygur population varies with age. The OPRM1 detection fragment has the promoter activity. High throughput chip research provides a new research basis for follow-up, in which the differential methylation sites in the near promoter region should be verified in the expanded sample, especially in response to the change of methylation in the progress of MCI to AD.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.1
[Abstract]:Objective: OPRK1 and OPRM1 receptors belong to the opioid receptor family, which are widely distributed in the nervous system and are closely related to the AD disease. Earlier studies suggest the association between gene methylation and AD disease. This study will continue to explore the changes in DNA methylation in the early stage of AD, the mild cognitive impairment (MCI). In addition, high throughput core will be used. This method identifies new disease risk methylation genes and provides a new basis for subsequent research. Methods: in 5398 samples of epidemiological studies in Xinjiang, 96 cases of MCI and 96 cases were randomly selected according to the principle of age, sex and national match, and the extraction of genomic DNA by magnetic beads and hydrogen sulfite salt After conversion, the methylation level of OPRK1, OPRM1 gene was detected by pyrosequencing method, and the promoter activity was detected by using the dual luciferase reporter system. In addition, 6 sex matched AD and control samples from Corelle hospital in Ningbo were used to determine the level of methylation of all genes by 450K chip. Results: first Part: comparison and analysis of the case control group: the OPRK1 gene methylation in the Uygur and Han nationalities was not associated with MCI (P0.05). The results of sex stratification showed that the hypermethylation of the OPRK1 gene was associated with the MCI disease of the Han women (p=0.015); the OPRM1 gene, the high methylation of the CpG1 loci was associated with the Uygur MCI disease (general p=5.16E-05, male p=0.015, female p=1.20E-03). CpG2-4 hypomethylation is associated with the prevalence of MCI in the Han nationality (overall p=0.002, male p=0.018, female p=4.98E-02). Comparison results of methylation among different ethnic groups: the basic data of the two nationalities are different, the logistic regression is used to correct them. There is no national difference (P0.05) in OPRK1 methylation in MCI and the control group, and CpG1 loci in the OPRM1 gene. Methylation in the case group has ethnic differences (CpG1 p=0.002, adjust p=0.009) CpG2-4 locus methylation (CpG2-4 p=0.005, adjust p=0.009) in the control group (CpG2-4 p=0.005, adjust p=0.009). In addition, the results of the gene methylation of the Han control group in different regions (Xinjiang and Zhejiang) were compared with the previous studies, and OPRK1 methylation was in general and in general. There were regional differences in the male group, while there was no regional difference among women (total p=0.006, male p=2.31E-04, female P0.05). The methylation level of OPRM1 gene in Xinjiang Han population was significantly higher than that in the Han population of Zhejiang (CpG1 overall p=2.99E-05, male P =0.006, female p=0.001; CpG2-4 p=1.32E-13, male p=1.71E-08). P=2.74E-05. Partial correlation analysis (control group, sex, BMI, blood sugar, triglyceride, cholesterol, HDL cholesterol, low density lipoprotein cholesterol) showed that there was a positive correlation between OPRM1 CpG2-4 methylation and age in the Uygur group (r=0.220, p= 0.039). The results of double luciferase experiment were further suggested, compared with negative control PGL3-Basic, OPRK1 and OPRM1 recombinant plasmids could promote the expression of luciferase in the lower reaches of the firefly. OPRK1 increased the expression level by 2.248 times (p=0.008) and OPRM1 increased by 2.616 times (p=0.003). The second part: the difference of the methylation level between the samples was detected with 450K, and the difference between AD and the level of triglyceride between the groups was statistically significant (p=0.025). In the subsequent difference analysis, a linear model was used to correct it. With |delta beta|0.2 and P0.05 as the standard, 41 different methylation sites were matched to 33 genes, of which 18 loci were methylation and 23 loci were low methylation. Conclusion: OPRK1 hypermethylation is the risk factor for the female MCI of the Han nationality, and the fragment has the promoter activity. Sex, may have low level of expression, environmental factors have influence on OPRK1 methylation in Han men, and there is no effect on the risk of MCI risk in the.OPRM1 specific location methylation of Han women. High methylation at the CpG1 locus is a risk factor for the MCI disease of the Uygur nationality, and the low methylation of CpG2-4 loci is the risk factor of the Han nationality in the Han nationality. In addition, C There are ethnic differences in the methylation of pG1 loci in MCI, and there are ethnic differences in the methylation of CpG2-4 loci. Environmental factors can affect the level of OPRM1 methylation, which shows that the level of OPRM1 methylation in Xinjiang Han is higher than that of the Han people in Zhejiang; age is an important risk factor for MCI, and the methylation level of OPRM1 CpG2-4 gene in the Uygur population varies with age. The OPRM1 detection fragment has the promoter activity. High throughput chip research provides a new research basis for follow-up, in which the differential methylation sites in the near promoter region should be verified in the expanded sample, especially in response to the change of methylation in the progress of MCI to AD.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.1
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