【作者】 沈玥；

【导师】 米杰；

【作者基本信息】 北京协和医学院 ， 流行病与卫生统计学， 2013， 博士

【摘要】 背景：血脂异常是冠心病的重要的独立危险因子,血脂水平除了受环境因素影响,也和遗传相关。多种基因参与调节血脂代谢,基因的一般变异和罕见变异影响血脂浓度。GWAS相关基因的筛选验证分析,对于了解血脂代谢机制具有重要的意义。通过对遗传易感基因的分析,了解血脂代谢的遗传机制,获得有用的可干预靶点,可提供对脂代谢紊乱以及代谢综合征其他危险因子的预防和治疗策略。由于儿童期血脂水平与成人期密切相关,所以在儿童人群鉴别与血脂相关的基因多态性及其表达状态将增加对血脂代谢机制的了解。目的：探讨1.血脂易感基因单核苷酸多态性位点(Single nucleotide polymorphisms, SNP)(GALNT2:rs2144300,GCKR:rs1260333, rs1260326,LPL: rs10105606, TRIB1: rs2954029,ANGPTL3: rs1748195和APOA5-A4-C3-A1cluster-: rs964184)与学龄儿童血脂水平及脂代谢紊乱的关联性；2.血脂易感基因多态性对信使RNA (mRNA)表达的影响；3.基因一环境交互作用对学龄儿童脂代谢紊乱的影响。方法：1.研究对象：1)基因型与血脂水平的相关性研究：来源于2004年4-10月进行的以人群为基础的横断面研究：北京市儿童青少年代谢综合征(BCAMS)研究。BCAMS人群包括有代表性的北京市城乡地区6-18岁学龄儿童的19593人,其中男生占50%。进行人体测量,按照中国肥胖问题工作组(WGOC)推荐的“中国学龄儿童青少年超重、肥胖筛查体重指数值分类标准”评价体重状态,招募同意接受静脉采血的儿童总计3518人,其中肥胖组1229人,超重组655人,正常体重组1620人,其余14人因缺失部分数据未分组。采集空腹12小时静脉血,进行医学检查和生活行为因素问卷调查。2)mRNA表达实验：来源于2013年1-3月从首都儿科研究所附属医院病房入选50名5周岁以上儿童,经诊断无心、肺、肝、肾等重要脏器的慢性疾病、身体重要残疾(如脊柱和下肢残疾)、无内分泌类疾病以及药物性肥胖。测量身高、体重、腰围数据,评价体重状态,记录病历号。采集空腹血。2.基因型检测实验：采用盐析法或商品试剂盒从外周血白细胞中提取DNA。通过查阅文献和专家研讨,确定6个基因的7个多态性位点(SNP)(GALNT2: rs2144300, GCKR:rs1260333, rs1260326, LPL:rs10105606, TRIB1:rs2954029,ANGPTL3:rs1748195和APOA5-A4-C3-A1cluster:rs964184),采用TaqMan探针法检测基因型。3. mRNA表达实验：提取全血总RNA采用实时荧光定量聚合酶链反应(RTQ-PCR)技术检测上述6个血脂候选基因(GALNT2, GCKR, LPL, TRIB1, ANGPTL3和APOA5-A4-C3-A1cluster)的mRNA表达水平,看家基因选择GAPDHo4.统计分析：使用Access软件进行数据录入,统计分析采用SPSS13.0软件,主要统计方法包括ANCOVA,多元线性回归分析、多元logistic回归分析。结果：应用遗传加性模型,调整年龄、性别、体重指数和青春期,并进行FDR校正(标准为0.05)。6个SNP (rs2144300,rs1260333,rs1260326,rs10105606,rs748195和rs964184)与甘油三酯相关(p<0.05)。两个SNP (rs2954029口rs964184)与总胆固醇相关(p<0.05)。两个SNP (rs10105606和rs964184)与高密度脂蛋白胆固醇相关(p<0.05)。4个SNP (rs1260333, rs1260326, rs2954029和rs964184)与低密度脂蛋白胆固醇相关(p<0.05)。调整年龄、性别、BMI和青春期后,与儿童血脂代谢紊乱相关的SNP有3个,rs1260333(OR=1.22,95%CI1.09-1.36),rs1260326(OR=1.21,95%CI1.09-1.39)和rs964184(OR=1.37,95%CI1.20-1.55)。5个SNP与高甘油三酯血症相关,rs2144300, rs1260333, rs1260326, rs1748195和rs964184,OR值分别为1.42(95%CI1.12-1.80),1.34(95%CI1.13-1.59),1.31(95%CI1.11-1.55),1.31(95%CI1.07-1.61)和1.71(95%CI1.43-2.06)。仅一个SNP rs964184与低HDL-C血症相关,OR=1.34,95%CI1.12-1.61。随着遗传危险得分(GRSs)增加,TG, TC, HDL-C, LDL-C的水平呈线性相关,有统计学意义(p<0.05)每增加一个危险等位基因,可以引起脂代谢紊乱、高甘油三酯、高胆固醇、低高密度脂蛋白和高低密度脂蛋白血症的的风险分别增加17%,40%,9%,10%和11%。RTQ-PCR结果显示,GCKR mRNA随着rs1260333和rs1260326危险等位基因数目增加表达水平呈线性降低；ANGPTL3的mRNA表达是随着rs1748195危险等位基因数目增加而呈线性升高；APOA5-A4-C3-Al cluster上的rs964184无RR基因型,含有一个危险等位基因的杂合子CR型的APOA5-A4-C3-Al cluster的mRNA水平低于无危险等位基因的CC型。采用因子分析方法,共提取脂肪蛋白类食物、果蔬、烟酒、静态生活方式和业余体育活动5个公因子。脂代谢紊乱的基因—环境相互作用分析结果显示,GCKR位点rs1260333变异与烟酒、静态生活方式和运动三个因素存在交互作用,归因危险度百分比(AP)分别为2.61%、7.56%和3.89%。rs1260326与烟酒、静态生活方式和运动存在交互作用,AP分别为9.60%、6.95和0.35%。APOA5-A4-C3-A1cluster位点rs964184与脂肪蛋白类食物、蔬果、静态生活方式和运动存在交互作用,AP分别为15.73%、6.82%、2.15%和26.07%。血脂易感SNP与生活行为因素对于高甘油三酯血症的交互作用结果显示,rs2144300与烟酒、蔬果、静态生活方式和运动存在交互作用,AP分别为21.41%、18.88%、2.71%和25.58%；GCKR的两个SNPrs1260333和rs1260326仅与脂肪蛋白类食物存在交互作用,AP分别为8.30%和13.34%。ANGPTL3上的SNP rs1748195与脂肪蛋白类食物和静态生活方式存在交互作用,AP分别为4.04%和32.76%。rs964184与脂肪蛋白类食物、烟酒、蔬果、静态生活方式和运动都存在交互作用,AP分别为27.70%、5.39%、3.67%、8.35%和27.02%。结论：本研究首次在北京儿童人群中验证了血脂相关易感基因的多态性位点与血脂水平以及脂代谢紊乱存在关联。与血脂水平和脂代谢紊乱相关的基因多态性存在累积效应,随等位基因风险值增大,血脂水平升高,脂代谢紊乱和高甘油三酯血症患病率上升。血脂易感基因的mRNA表达水平在肥胖和非肥胖儿童中的差异表明,基因表达与肥胖可能存在关联；其次,基因型间的mRNA差异趋势提示,这些单核苷酸多态性位点可能通过影响基因的表达水平对血脂代谢进行调节。易感基因与环境之间存在交互作用,其中高脂高蛋白食物、低果蔬类摄入、运动较少和烟酒摄入均可影响易感基因与血脂紊乱的关系。更多还原

【Abstract】 Backgrounds:Epidemiological evidences indicated that abnormal lipids are strong independent risk factor for cardiovascular disease. Genetic and environmental factors contribute to the lipid levels. It is known that multiple genes participate into the regulation of lipid metabolism, common and rare variants in lipid-susceptibility genes may collectively determine blood lipid levels. Genome-wide association studies have identified association signals containing both classically established genes and previously unknown genomic regions as determinants of lipid levels, which is helpful to understand the mechanism of lipid metabolism, and get diagnosis goals from these identified locus. Levels of lipids variables in childhood were associated with levels in adulthood, identifying genes and genetic variants associated with blood lipids will enrich our understanding of blood lipid metabolism.Objective:1. In the current study, we aimed to test the associations between seven variants (GALNT2(rs2144300), GCKR (rs1260326, rs1260333), LPL (rs10105606), TRIB1(rs2954029), ANGPTL3(rs1748195), and APOA5-A4-C3-A1cluster (rs964184)) and levels of triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) as continuous traits in Chinese Han children population. Then we estimated the association of variants with risks of dyslipidemia as dichotomized traits.2. To test the relationship between genotypes of lipid-susceptibility loci and message RNA (mRNA).3. To investigate the effect of genes-environment interaction on dyslipidemia in Chinese Han children.Methods:1. Subjects:1) Association study:Subjects were recruited from the BCAMS study, a cross-sectional population-based survey carried out in2004. The survey included a questionnaire, medical examination, anthropometric measurement, and finger capillary blood tests among a representative sample (n=19593, boys accounted for50%of the sample) of Beijing school-age children aged6to18years from urban and rural districts. Anthropometric measurement included weight, height, waist circumference, and FMP by bioimpedance analysis. Birth weight was collected based on a self-report questionnaire, which was completed by parents or guardians. Within this large group of children,1229obese,655overweight and1620normal weight children were recruited and diagnosed by the Chinese age-and sex-specific BMI cutoffs, there were14un-grouped by the diagnosis criteria for missing data. Venipuncture blood samples were collected after a12-hour overnight fast.2) mRNA expression study: Children aged5to15years were recruited from patient wards in Capital Institute of Pediatrics from January to March in2013. The participants was diagnosed without chronic diseases of heart, lung, liver, kidney and other vital organs, without major physical disabilities, such as spinal and lower limb disability, and without endocrine diseases and drug-induced obesity. Anthropometric measurement included weight, height, waist circumference. The medical identification numbers were recorded and venipuncture blood samples were collected after a12-hour overnight fast.2. Genotyping:Genomic DNA was isolated from peripheral blood white cells using the salt fractionation method or commercial kit. Seven variants (GALNT2(rs2144300), GCKR (rs1260326, rs1260333), LPL (rs10105606), TRIB1(rs2954029), ANGPTL3(rs1748195), and APOA5-A4-C3-A1cluster (rs964184)) were selected from previous GWAS studies. All genotyping were performed by TaqMan probes Allelic Discrimination Assays with the GeneAmp7900Sequence Detection System.3. mRNA expression:Total RNA from venipuncture blood was extracted, and mRNA levels of GALNT2, GCKR, LPL, TRIB1, ANGPTL3and APOA5-A4-C3-A1cluster were detected by real time quantitative polymerase chain reaction (RTQ-PCR). The house-keeping gene is GAPDH.4. Statistical analysis:Access2003was used for data entry and SPSS, version13.0(SPSS Inc., Chicago, Illinois) was used for data analysis. The statistical methods included ANCOVA, multiple linear regression, multiple logistic regression.Results:With genetic additive model, after age, sex, BMI, and puberty stage, and correction with FDR (p<0.05), six SNPs(rs2144300,rs1260333,rs1260326,rs10105606, rs1748195and rs964184) associated with triglycerides (p<0.05). Two SNPs (rs2954029and rs964184) associated with total cholesterol (p<0.05). Two SNP (rs10105606and rs964184) associated with HDL-C(p<0.05). Four SNPs (rs1260333, rs1260326, rs2954029and rs964184) associated with LDL-C (p<0.05)).After adjustment for age, sex, BMI, and puberty stage, there were significant associations between rs1260333(OR=1.22,95%CI1.09-1.36), rs1260326(OR=1.21,95%CI1.09-1.39), rs964184(OR=1.37,95%CI1.20-1.55) and dyslipidemia. Five SNPs, rs2144300(OR=1.42,95%C11.12-1.80), rs1260333(OR=1.34,95%CI1.13-1.59), rs1260326(OR=1.31,95%CI1.11-1.55), rs1748195(OR=1.31,95%CI1.07-1.61), and rs964184(OR=1.71,95%CI1.43-2.06) associated with hypertriglyceridemia. Only rs964184associated with low HDL-C (OR=1.34,95%CI1.12-1.61).With the increased genetic risk scores (GRSs), there were a significant increasing in the lipid traits (TG, TC, HDL-C and LDL-C levels) with a significant stepwise manner (p<0.05). And with risk scores increased, the risks of dislipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL-C and high LDL-C is increasing with17%,40%,9%,10%and11%.Under the genetic additive model of the loci genotyping, mRNA of GCKR gene decreased with the number of risk allele of rs1260333and rs1260326; mRNA of ANGPTL3increased with the number of risk allele of rs1748195; we didn’t found the RR homozygotes for the risk allele for APOA5-A4-C3-A1cluster SNP rs964184, mRNA level of heterozygotes with one risk allele (CR) was lower than non-risk allele homozygotes (CC).Using factor analysis methodology, five main factors, including lipid-protein, fruits&vegetable, smoke&drinking, sedentary behavior and physical exercise in spare time were extracted. We analyzed the interaction between gene and environment for dyslipidemia. The SNP rs1260333in GCKR interacted with smoke&drinking, sedentary behavior and physical exercise in spare time, and the attributable percent (AP) is2.61%,7.56%and3.89%, respectively. rs1260326interacted with smoke&drinking, sedentary behavior and physical exercise in spare time,too. And the AP is9.60%,6.95%and0.35%, respectively. APOA5-A4-C3-A1cluster SNP rs964184interacted with lipid-protein, vegetables&fruits, sedentary behavior and physical exercise in spare time, the AP is15.73%,6.82%,2.15%and26.07%.For hypertriglyceridemia, GALNT2SNP rs2144300interacted with smoke&drinking, fruits&vegetable, sedentary behavior and physical exercise in spare time, the AP is21.41%,18.88%,2.71%and25.58%, respectively. GCKR SNP rs1260333and rs1260326interacted with lipid-protein, the AP is8.30%and13.34%, respectively. ANGPTL3SNP rs1748195interacted with lipid-protein and sedentary behavior. The AP is4.04%and32.76%, respectively. APOA5-A4-C3-A1cluster SNP rs964184interacted with all five main factors, the AP is27.70%、5.39%、3.67%、8.35%and27.02%for lipid-protein, fruits&vegetable, smoke&drinking, sedentary behavior and physical exercise in spare time, respectively.Conclusions:Our study firstly validated the association of seven variants with blood lipids and risk of dyslipidemia among the Chinese Beijing children and adolescents.There were accumulative risk effects of these seven variants on lipid traits, risks of dislipidemia and hypertriglyceridemia.The trends between genotype and mRNA levels, implied that the genes expression might be influenced by the lipid-susceptibility SNPs.There were interactions between lipid-susceptible SNPs and environmental factors. Low vegetables&fruits, less physical exercise and smoke&drinking might co-mordify the effects of lipid-susceptibility genes on the risk of dyslipidemia.更多还原