本文選題：易感基因 + 單核苷酸多態(tài)性��； 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：背景：血脂異常是冠心病的重要的獨立危險因子,血脂水平除了受環(huán)境因素影響,也和遺傳相關(guān)。多種基因參與調(diào)節(jié)血脂代謝,基因的一般變異和罕見變異影響血脂濃度。GWAS相關(guān)基因的篩選驗證分析,對于了解血脂代謝機制具有重要的意義。通過對遺傳易感基因的分析,了解血脂代謝的遺傳機制,獲得有用的可干預(yù)靶點,可提供對脂代謝紊亂以及代謝綜合征其他危險因子的預(yù)防和治療策略。由于兒童期血脂水平與成人期密切相關(guān),所以在兒童人群鑒別與血脂相關(guān)的基因多態(tài)性及其表達狀態(tài)將增加對血脂代謝機制的了解。 目的：探討1.血脂易感基因單核苷酸多態(tài)性位點(Single nucleotide polymorphisms, SNP)(GALNT2:rs2144300,GCKR:rs1260333, rs1260326,LPL: rs10105606, TRIB1: rs2954029,ANGPTL3: rs1748195和APOA5-A4-C3-A1cluster-: rs964184)與學(xué)齡兒童血脂水平及脂代謝紊亂的關(guān)聯(lián)性； 2.血脂易感基因多態(tài)性對信使RNA (mRNA)表達的影響； 3.基因一環(huán)境交互作用對學(xué)齡兒童脂代謝紊亂的影響。 方法： 1.研究對象： 1)基因型與血脂水平的相關(guān)性研究：來源于2004年4-10月進行的以人群為基礎(chǔ)的橫斷面研究：北京市兒童青少年代謝綜合征(BCAMS)研究。BCAMS人群包括有代表性的北京市城鄉(xiāng)地區(qū)6-18歲學(xué)齡兒童的19593人,其中男生占50%。進行人體測量,按照中國肥胖問題工作組(WGOC)推薦的“中國學(xué)齡兒童青少年超重、肥胖篩查體重指數(shù)值分類標(biāo)準(zhǔn)”評價體重狀態(tài),招募同意接受靜脈采血的兒童總計3518人,其中肥胖組1229人,超重組655人,正常體重組1620人,其余14人因缺失部分數(shù)據(jù)未分組。采集空腹12小時靜脈血,進行醫(yī)學(xué)檢查和生活行為因素問卷調(diào)查。 2)mRNA表達實驗：來源于2013年1-3月從首都兒科研究所附屬醫(yī)院病房入選50名5周歲以上兒童,經(jīng)診斷無心、肺、肝、腎等重要臟器的慢性疾病、身體重要殘疾(如脊柱和下肢殘疾)、無內(nèi)分泌類疾病以及藥物性肥胖。測量身高、體重、腰圍數(shù)據(jù),評價體重狀態(tài),記錄病歷號。采集空腹血。 2.基因型檢測實驗：采用鹽析法或商品試劑盒從外周血白細胞中提取DNA。通過查閱文獻和專家研討,確定6個基因的7個多態(tài)性位點(SNP)(GALNT2: rs2144300, GCKR:rs1260333, rs1260326, LPL:rs10105606, TRIB1:rs2954029,ANGPTL3:rs1748195和APOA5-A4-C3-A1cluster:rs964184),采用TaqMan探針法檢測基因型。 3. mRNA表達實驗：提取全血總RNA采用實時熒光定量聚合酶鏈反應(yīng)(RTQ-PCR)技術(shù)檢測上述6個血脂候選基因(GALNT2, GCKR, LPL, TRIB1, ANGPTL3和APOA5-A4-C3-A1cluster)的mRNA表達水平,看家基因選擇GAPDHo 4.統(tǒng)計分析：使用Access軟件進行數(shù)據(jù)錄入,統(tǒng)計分析采用SPSS13.0軟件,主要統(tǒng)計方法包括ANCOVA,多元線性回歸分析、多元logistic回歸分析。 結(jié)果： 應(yīng)用遺傳加性模型,調(diào)整年齡、性別、體重指數(shù)和青春期,并進行FDR校正(標(biāo)準(zhǔn)為0.05)。6個SNP (rs2144300,rs1260333,rs1260326,rs10105606,rs748195和rs964184)與甘油三酯相關(guān)(p0.05)。兩個SNP (rs2954029口rs964184)與總膽固醇相關(guān)(p0.05)。兩個SNP (rs10105606和rs964184)與高密度脂蛋白膽固醇相關(guān)(p0.05)。4個SNP (rs1260333, rs1260326, rs2954029和rs964184)與低密度脂蛋白膽固醇相關(guān)(p0.05)。調(diào)整年齡、性別、BMI和青春期后,與兒童血脂代謝紊亂相關(guān)的SNP有3個,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個SNP與高甘油三酯血癥相關(guān),rs2144300, rs1260333, rs1260326, rs1748195和rs964184,OR值分別為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)。僅一個SNP rs964184與低HDL-C血癥相關(guān),OR=1.34,95%CI1.12-1.61。 隨著遺傳危險得分(GRSs)增加,TG, TC, HDL-C, LDL-C的水平呈線性相關(guān),有統(tǒng)計學(xué)意義(p0.05)每增加一個危險等位基因,可以引起脂代謝紊亂、高甘油三酯、高膽固醇、低高密度脂蛋白和高低密度脂蛋白血癥的的風(fēng)險分別增加17%,40%,9%,10%和11%。 RTQ-PCR結(jié)果顯示,GCKR mRNA隨著rs1260333和rs1260326危險等位基因數(shù)目增加表達水平呈線性降低；ANGPTL3的mRNA表達是隨著rs1748195危險等位基因數(shù)目增加而呈線性升高；APOA5-A4-C3-Al cluster上的rs964184無RR基因型,含有一個危險等位基因的雜合子CR型的APOA5-A4-C3-Al cluster的mRNA水平低于無危險等位基因的CC型。 采用因子分析方法,共提取脂肪蛋白類食物、果蔬、煙酒、靜態(tài)生活方式和業(yè)余體育活動5個公因子。脂代謝紊亂的基因—環(huán)境相互作用分析結(jié)果顯示,GCKR位點rs1260333變異與煙酒、靜態(tài)生活方式和運動三個因素存在交互作用,歸因危險度百分比(AP)分別為2.61%、7.56%和3.89%。rs1260326與煙酒、靜態(tài)生活方式和運動存在交互作用,AP分別為9.60%、6.95和0.35%。APOA5-A4-C3-A1cluster位點rs964184與脂肪蛋白類食物、蔬果、靜態(tài)生活方式和運動存在交互作用,AP分別為15.73%、6.82%、2.15%和26.07%。 血脂易感SNP與生活行為因素對于高甘油三酯血癥的交互作用結(jié)果顯示,rs2144300與煙酒、蔬果、靜態(tài)生活方式和運動存在交互作用,AP分別為21.41%、18.88%、2.71%和25.58%；GCKR的兩個SNPrs1260333和rs1260326僅與脂肪蛋白類食物存在交互作用,AP分別為8.30%和13.34%。ANGPTL3上的SNP rs1748195與脂肪蛋白類食物和靜態(tài)生活方式存在交互作用,AP分別為4.04%和32.76%。rs964184與脂肪蛋白類食物、煙酒、蔬果、靜態(tài)生活方式和運動都存在交互作用,AP分別為27.70%、5.39%、3.67%、8.35%和27.02%。 結(jié)論：本研究首次在北京兒童人群中驗證了血脂相關(guān)易感基因的多態(tài)性位點與血脂水平以及脂代謝紊亂存在關(guān)聯(lián)。 與血脂水平和脂代謝紊亂相關(guān)的基因多態(tài)性存在累積效應(yīng),隨等位基因風(fēng)險值增大,血脂水平升高,脂代謝紊亂和高甘油三酯血癥患病率上升。 血脂易感基因的mRNA表達水平在肥胖和非肥胖兒童中的差異表明,基因表達與肥胖可能存在關(guān)聯(lián)；其次,基因型間的mRNA差異趨勢提示,這些單核苷酸多態(tài)性位點可能通過影響基因的表達水平對血脂代謝進行調(diào)節(jié)。 易感基因與環(huán)境之間存在交互作用,其中高脂高蛋白食物、低果蔬類攝入、運動較少和煙酒攝入均可影響易感基因與血脂紊亂的關(guān)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 .
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R589.2

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