胆固醇吸收关键基因Numb与冠心病及血脂水平的关联分析研究

发布时间：2018-05-01 19:54

  本文选题：胆固醇 + NPC1L1　； 参考：《新疆医科大学》2016年博士论文

【摘要】：目的:(1)识别胆固醇关键基因变异与疾病的关联研究,为新疆地区汉族、维族、哈族人群心血管疾病高危人群制定和防止疾病最佳治疗方案提供帮助。(2)探讨Numb基因多态性与胆固醇吸收的分子机制研究。(3)探讨新疆汉族、维吾尔族人群Numb基因多态性与冠心病及血脂的相关性研究。方法:(1)收集民族、性别、年龄均匹配的健康患者共349例并根据低密度脂蛋白-胆固醇水平(LDL-C),分极高、极低组,采用多重酶联免疫法PCR的方法,对32条胆固醇相关基因,共635个外显子片段进行高通量二代测序,分析基因变异位点与血脂的关系。(2)采用Numb基因的所有外显子和外显子-内含子连接区域测序的方法,对671例血脂LDL-C极高,极低组患者进行分析,发现Numb基因新的突变位点,利用细胞实验对Numb变异位点与胆固醇吸收的分子机制进行研究。(3)采用病例-对照研究,选择经冠状动脉造影确诊的冠心病患者890例和造影阴性的健康人群对照组786例,利用Taqman技术,对Numb基因多态性进行基因分型以及单体型构建,分析Numb基因标签SNPs和突变位点与冠心病及血脂的关系。结果:(1)本研究利用人类胆固醇关键基因外显子测序的方法对新疆汉族、维吾尔族、哈萨克族人群进行差异性筛选并筛查获得胆固醇基因表达差异32条。汉族相较于其他两个民族独有的1条差异基因;维吾尔族相较于其他两个民族独有的胆固醇相关基因差异有6条;哈萨克族相较于其他两个民族独有的1条差异基因,说明民族间基因差异表达,对于防治心血管疾病提供了一定的基础。(2)对新疆地区筛选的671例患者中进行Numb基因外显子测序,发现基因编码区的两个非同义突变和八个同义突变,两个非同义突变(A587T和Q533K)均分布在血浆低密度脂蛋白-胆固醇水平极低组。(3)对Numb变异位点进行胆固醇响应、蛋白定位、亲和力及稳定性等实验,进一步在细胞水平上验证两个突变体在胆固醇循环中的作用,发现与野生型相比,Numb A587T和Q533K变异均对胆固醇响应和稳定性检测无明显差别,而细胞定位较少和相关蛋白亲和力的作用减弱。(4)Numb基因标签SNP的位点(rs2108552)与冠心病的发病具有相关性。在新疆汉族总体和男性人群中Numb基因rs2108552的显性模型与冠心病具有关联性,在调整了体质指数(BMI)、甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白-胆固醇(LDL-C)、高血压、糖尿病及吸烟等危险因素的影响,rs2108552的显性模型在总体和男性人群中仍存在差异并有统计学意义(总:OR:1.876,95%CI:1.482~1.979,P=0.004;男:OR:1.498,95%CI:1.305~1.815,P=0.006),而在女性人群中差别无统计学意义。在维吾尔族人群中未发现此位点与冠心病具有相关性。另外,Numb基因两个突变位点(A587T和Q533K)在汉族、维吾尔族人群中未发现与冠心病具有相关性。(5)Numb基因3个标签SNPs位点在汉族和维吾尔族人群中共建立5个单体型。在汉族总体、男性人群中,G-G-T单体型对照组中的频率显著高于CAD组(P=0.007,OR=0.616;P=0.013,OR=0.701),而T-C-T单体型在病例组频率显著高于对照组(P=0.002,OR=1.482;P=0.004,OR=1.334),而在女性人群中差别无统计学意义。在维吾尔族人群中,冠心病组和对照组单体型频率未发现差别有统计学意义。(6)在调整性别、年龄、民族、肥胖、吸烟等因素前后rs12435797基因型均与TC水平,rs2108552基因型与TC、LDL-C水平,rs1019075与TC水平具有关联性且差别有统计学意义(均P0.05)。另外,Numb基因两个突变位点(A587T和Q533K)等位基因与TC、LDL-C、HDL-C具有关联性且差别有统计学意义(均P=0.000)。结论:(1)基因外显子测序结果表明血脂相关基因在民族间有差异表达变化,特别是维吾尔族和哈萨克族;发现与胆固醇吸收关键基因NPC1L1,Numb,SOAT2及基因突变位点。(2)新发现的Numb基因的两个非同义突变A587T和Q533K可能对Numb蛋白胆固醇吸收的功能有影响。(3)Numb基因rs2108552的CC基因型以及三个单核苷酸多态性的单体型T-C-T可能是汉族男性人群中冠心病发病的危险因素,G-G-T单体型是冠心病发病的保护因素。(4)Numb基因3个SNPs基因型和2个突变位点与一个或两个以上的血脂指标具有关联性且突变型的TC和LDL-C水平较低,而HDL-C水平较高。
[Abstract]:Objective: (1) to identify the correlation between the key gene mutation of cholesterol and the disease, and to provide help for the formulation and prevention of the best treatment for the high-risk population of the Han, Uygur and Kazakh people in Xinjiang. (2) to explore the molecular mechanism of Numb gene polymorphism and cholesterol absorption. (3) to explore the Num of the Han and Uygur people in Xinjiang. Study on the correlation between B gene polymorphism and coronary heart disease and blood lipid. Methods: (1) a total of 349 healthy patients with matched national, sex and age were collected and the low density lipoprotein cholesterol level (LDL-C) was very high and very low. The multiple ELISA PCR method was used for 32 cholesterol related genes and a total of 635 exons were high. Two generations of fluxes were sequenced to analyze the relationship between gene mutation sites and blood lipids. (2) the method of sequencing of all exons and exons and introns of the Numb gene was used to analyze 671 patients with extremely high blood lipid LDL-C and extremely low groups, and to find a new mutation site of the Numb gene and use cell experiments to absorb the Numb heterotopic point and cholesterol. Molecular mechanism was studied. (3) a case control study was used to select 890 cases of coronary heart disease diagnosed by coronary angiography and 786 cases of contrast negative healthy population control group. Using Taqman technique, the Numb gene polymorphisms were genotyping and haplotype construction, and Numb gene label SNPs and mutation sites were analyzed with coronary heart disease and blood. Results: (1) in this study, we screened and screened the Han, Uygur and Kazak population of Xinjiang, Uygur and Kazakh people by using the exons of human cholesterol key genes to screen and screen 32 differences in cholesterol gene expression. Compared to the other two ethnic groups, the Han nationality was compared with the other two genes, and the Uygur ethnic group was compared to the other two. There are 6 unique differences in cholesterol related genes in ethnic groups, and Kazak is compared to 1 different genes in other two ethnic groups, indicating the difference in gene expression between ethnic groups, which provides a certain basis for the prevention and control of cardiovascular disease. (2) the Numb gene exon sequencing was carried out in 671 patients screened in Xinjiang, and the gene coding region was found. Two unsynonymous and eight synonymous mutations, two unsynonymous mutations (A587T and Q533K) were distributed in a very low level of plasma low density lipoprotein cholesterol level. (3) the cholesterol response, protein localization, affinity and stability of the Numb heterotopic points were carried out to further verify the two mutant in the cholesterol cycle at the cell level. It was found that the Numb A587T and Q533K variations were not significantly different from those of the wild type, but there was less cell location and associated protein affinity. (4) the locus (rs2108552) of the Numb gene label SNP (rs2108552) was related to the incidence of coronary heart disease. In the population of Xinjiang Han population and the male population, Numb The dominant model of the gene rs2108552 is associated with coronary heart disease, and the dominant model of rs2108552 still exists in the overall and male population and has statistical significance in adjusting the influence of the body mass index (BMI), triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), hypertension, diabetes and smoking. Total: OR:1.876,95%CI:1.482~1.979, P=0.004, men: OR:1.498,95%CI:1.305~1.815, P=0.006), and there is no statistically significant difference in the female population. In Uygur people, this site is not found to be associated with coronary heart disease. In addition, two mutation sites (A587T and Q533K) of the Numb gene are not found in the Uygur population with coronary heart disease. (5) the 3 labelling SNPs loci of the Numb gene set up 5 haplotypes in the Han and Uygur population. In the Han population, the frequency of G-G-T haplotype control group was significantly higher than that of the CAD group (P=0.007, OR=0.616; P=0.013, OR=0.701), and the frequency of T-C-T monotype in the case group was significantly higher than that of the control group (P=0.002, OR=1.482) In the Uygur population, there was no significant difference in haplotype frequency between the CHD group and the control group in the Uygur population. (6) the rs12435797 genotype of the rs12435797 and the TC, the rs2108552 genotypes and TC, the LDL-C level, and the rs101 in the sex, age, nationality, obesity, smoking and other factors were adjusted. 9075 and TC levels were correlated and statistically significant (P0.05). In addition, the two mutation sites (A587T and Q533K) alleles of the Numb gene were associated with TC, LDL-C, and HDL-C (all P=0.000). Conclusion: (1) the results of exons of the gene showed that the blood lipid related genes were differentially expressed among ethnic groups. Especially the Uygur and Kazakh people; found the key genes of cholesterol absorption NPC1L1, Numb, SOAT2 and gene mutation. (2) the two non synonymous mutations of the newly discovered Numb gene, A587T and Q533K, may have an effect on the function of the Numb protein cholesterol absorption. (3) the CC genotypes of the Numb gene rs2108552 and the three single nucleotide polymorphisms Haplotype T-C-T may be a risk factor for coronary heart disease in the Han male population. G-G-T haplotype is a protective factor for coronary heart disease. (4) the 3 SNPs genotypes and 2 mutation sites of the Numb gene are associated with one or more than two blood lipids, and the level of TC and LDL-C is lower and the level of HDL-C is higher.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.4
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本文编号：1830824