血脂易感基因多态性及mRNA水平与北京市儿童脂代谢的相关性研究

发布时间：2018-06-02 16:23

本文选题：易感基因 + 单核苷酸多态性　；参考：《北京协和医学院》2013年博士论文

【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GAPDHo 4.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(p0.05)。两个SNP (rs2954029口rs964184)与总胆固醇相关(p0.05)。两个SNP (rs10105606和rs964184)与高密度脂蛋白胆固醇相关(p0.05)。4个SNP (rs1260333, rs1260326, rs2954029和rs964184)与低密度脂蛋白胆固醇相关(p0.05)。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(p0.05)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。
[Abstract]:Background : Abnormal blood lipid is an important independent risk factor for coronary heart disease . The level of blood lipid is related to genetic factors . It is important to understand the mechanism of blood lipid metabolism , the general variation of genes and the rare variation .

Objective : To investigate the relationship between blood lipid level and lipid metabolism disorder in school - age children by single nucleotide polymorphisms ( SNPs ) ( GALNT2 : rs21443, GCKR : rs1260333 , rs1260326 , RANGPTL3 : rs10105606 , TRIB1 : rs294029 , ANGPTL3 : rs1748195 and APOA5 - A4 - C3 - A1cluster - rs964184 ) .

2 . The effect of polymorphism of lipid - susceptible gene on the expression of messenger RNA ( mRNA ) ;

3 . Effects of gene - environment interaction on lipid metabolism disorders in school - age children .

Method :

1 . Study object :

1 ) Correlation between genotype and blood lipid level : A cross - sectional study based on population from April to October 2004 : The BCAMS population includes 19593 people aged 6 - 18 years of age in urban and rural areas of Beijing .

2 ) mRNA expression experiment : From January to March 2013 , 50 children above the age of 5 were enrolled from the Affiliated Hospital of the Capital Medical Institute . It was diagnosed without heart , lung , liver , kidney and other important organs such as chronic diseases , non - endocrine diseases and drug obesity . The height , body weight , waist circumference data , weight status were measured , and the medical record number was recorded . Blood was collected .

2 . Genotypic test : DNA was extracted from peripheral blood leukocytes by salting - out method or commercial kit . Seven polymorphic loci ( SNPs ) of 6 genes ( GALNT2 : rs21443, GCKR : rs1260333 , rs1260326 , RANGPTL3 : rs10105606 , TRIB1 : rs294029 , ANGPTL3 : rs1748195 and APOA5 - A4 - C3 - A1cluster : rs964184 ) were determined by means of a review of literature and experts .

3 . mRNA expression assay : The mRNA expression level of the above six blood lipid candidate genes ( GALNT2 , GCKR , LPS , TRIB1 , ANGPTL3 and APOA5 - A4 - C3 - A1cluster ) was detected by real - time fluorescence quantitative polymerase chain reaction ( RTQ - PCR ) using real - time fluorescence quantitative polymerase chain reaction ( RTQ - PCR ) technique .

4 . Statistical analysis : Using Access software for data entry , SPSS 13.0 software was used for statistical analysis . The main statistical methods included ANCOVA , multivariate linear regression analysis and multivariate logistic regression analysis .

Results :

There were three SNPs ( rs1260333 , rs1260333 , rs1260326 , rs10105606 , rs1260326 , rs1260326 , rs10105606 , rs748195 and rs964184 ) related to triglyceride ( p . 05 ) .

The levels of TG , TC , HDL - C , LDL - C were linearly correlated with the increase of genetic risk score ( GGS ) . There was a statistical significance ( p < 0.05 ) . The risk of lipid metabolism disorder , triglyceride , high cholesterol , low density lipoprotein and low density lipoprotein were increased by 17 % , 40 % , 9 % , 10 % and 11 % , respectively .

RTQ - PCR showed that GCKR mRNA decreased linearly with the number of rs1260333 and rs1260326 dangerous alleles .
The mRNA expression of ANGPTL3 increased linearly with the number of rs1748195 dangerous alleles .
There is no RR genotype of rs964184 on APOA5 - A4 - C3 - Al cluster , and the mRNA level of apoA5 - A4 - C3 - Al cluster containing a dangerous allele is lower than CC type without dangerous allele .

The results of genetic - environmental interaction analysis showed that the variation of rs1260333 was 2.61 % , 7.56 % and 3.89 % , respectively . The results showed that the percentage of risk was 2.61 % , 7.56 % and 3.89 % , respectively , and the AP was 9.60 % , 6.95 and 0.35 % , respectively . The APOA5 - A4 - C3 - A1cluster locus rs964184 had interaction with fat protein food , vegetable and fruit , static lifestyle and movement . The AP was 15.73 % , 6.82 % , 2.15 % and 26.07 % , respectively .

The interaction of SNP and lifestyle factors with high triglyceride was found in the interaction between SNP and lifestyle factors . The interaction of rs21443s with tobacco wine , vegetable and fruit , static life style and movement showed that AP was 21.41 % , 18.88 % , 2.71 % and 25.58 % , respectively .
The two SNPrs1260333 and rs1260326 of GCKR interacted with fat protein food only , AP was 8.30 % and 13.34 % respectively . SNP rs1748195 on ANGPTL3 had interaction with fat protein food and static lifestyle , AP was 4.04 % and 32.76 % respectively . The AP was 27.70 % , 5.39 % , 3.67 % , 8.35 % and 27.02 % , respectively .

Conclusion : The polymorphism sites of lipid - related susceptibility genes are correlated with lipid levels and lipid metabolism disorders for the first time in Beijing children population .

The cumulative effects of gene polymorphisms associated with lipid levels and lipid metabolism disorders increased with increasing allele risk values , increased lipid levels , lipid metabolism disorders , and high triglyceride levels .

The mRNA expression level of lipid - susceptible genes in obese and non - obese children suggests that gene expression may be associated with obesity ;
Second , the mRNA differential trend between genotypes suggests that these single nucleotide polymorphism sites may regulate blood lipid metabolism by influencing the level of expression of the gene .

There is interaction between susceptible gene and environment , among which high - fat and high - protein food , low fruit and vegetable intake , less exercise and alcohol intake can affect the relationship between susceptible gene and lipid disorder .
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R589.2

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