本文選題：代謝綜合征 + 全基因組關聯(lián)分析��； 參考：《浙江大學》2017年博士論文

【摘要】：代謝綜合征是一組代謝指標異常癥候群,主要表現為中心性肥胖、高血壓和糖脂代謝紊亂。研究表明,代謝綜合征會增加心腦血管疾病、糖尿病的發(fā)病風險。按中國糖尿病協(xié)會定義,2010年我國居民代謝綜合征患病率達17.6%。雙生子研究表明,遺傳因素在代謝異常的發(fā)生中起了重要的作用。經典的全基因組關聯(lián)研究(genome-wide association study,GWAS)已發(fā)現了一些與代謝異常相關的遺傳變異,但仍存在以下兩大問題:(1)已發(fā)現的位點僅能解釋少量的遺傳度,即存在大量丟失的遺傳度(missing heritability)有待進一步探究;(2)以往研究主要關注表型相關的標簽SNP(tag SNP)及與之距離最近的基因,但后續(xù)研究發(fā)現,大部分的定位信號并不通過與之距離最近的基因起作用,如現已證明,定位在FTO上的肥胖相關位點主要通過遠程調控IRX3基因表達影響表型�？傊�,以往對GWAS定位信號的解析非常有限,多數關鍵基因和效應位點(causal variant)未被找到,不利于后續(xù)功能研究。近些年研究發(fā)現,大部分表型相關遺傳變異通過調控基因表達水平進而影響表型。若以基因表達調控作為注釋,可以將全基因組的篩選范圍縮小到基因表達數量性狀基因座(expression Quantitative Trait Loci,eQTLs)上,提高發(fā)現表型相關位點的效率。ENCODE、GTEx、Roadmap等研究項目陸續(xù)公布了大量可用于注釋遺傳變異對表型調控路徑中多個組學的信息,包括具有組織特異性的基因表達、DNA甲基化、組蛋白修飾、轉錄因子結合等。這些信息可用于進一步精確篩選表型相關遺傳變異,也可用于對已知信號的重新解析。以往的研究主要以歐裔人群為主,在我國漢族人群中的研究還很有限,由于不同人種間存在遺傳背景的差異,歐裔人群的研究結果不能直接外推至中國漢族人群。為了系統(tǒng)地探索代謝綜合征及代謝組分相關的遺傳易感位點,本研究以我國漢族人群為主要研究對象,通過基于多組學注釋的全基因組關聯(lián)分析,發(fā)現并解析代謝異常相關的遺傳變異。研究內容分為三個部分(圖1):第一部分采用經典的全基因組關聯(lián)分析策略,篩選與代謝綜合征及代謝組分關聯(lián)信號最強的遺傳變異位點并進行多階段驗證、功能分析和基因環(huán)境交互效應分析。第二部分在第一部分研究的基礎上,通過對轉錄調控的注釋,進一步篩選與糖脂代謝相關的遺傳變異,通過精細定位和功能實驗,解析基本調控路徑。第三部分針對已知的糖脂代謝關聯(lián)信號,利用最新的多組學注釋信息對信號區(qū)域進行系統(tǒng)化的重解析,探究關鍵的調控基因,為關聯(lián)分析的成果轉化提供支持。圖1研究內容示意圖左上側圖為課題組開展的代謝綜合征GWAS關聯(lián)信號(P值)的曼哈頓圖,圖下左側為第一部分技術路線,右側為第二部分技術路線。右側圖為NHGRI-EBI GWAS catalog記錄的表型相關的位點,為研究的第三部分,采用多組學注釋,對與代謝異常相關的位點進行系統(tǒng)化重解析。第一部分代謝綜合征的遺傳易感性研究一、研究目的:在中國漢族人群中篩選并驗證代謝綜合征相關的遺傳易感性位點。二、材料和方法:以單核苷酸多態(tài)性位點為遺傳變異標記,首先在杭州蕭山地區(qū)1742例樣本中采用全基因組關聯(lián)分析的方法,篩選出與代謝綜合征及代謝組分關聯(lián)信號最強的位點,然后對這些位點進行多階段獨立樣本驗證,驗證樣本來自我國東部、北部、東北部等多個地區(qū),共計10978例。合并多階段驗證結果后,對達到全基因組陽性水平的位點進行功能預測、基因環(huán)境交互效應分析。三、研究結果:通過代謝綜合征的全基因組關聯(lián)分析及多階段獨立樣本的驗證,研究發(fā)現位于APO45上的rs651821位點和位于ADF2上亞洲人特有的高頻錯義突變位點rs671的基因型與代謝綜合征的遺傳易感性相關。在控制了 APO4基因簇區(qū)域內最強信號rs651821后,位于BUD13上的rs180326位點仍然與血清甘油三酯(triglyceride,TG)水平相關(Pconbined=2.4E-08),是APO4基因簇內一個新的第二信號(secondary signal)。在整合了 遺傳變異位點 rs651821、rsl80326、血清 APOA5、BUD13的蛋白水平、TG水平后分析發(fā)現,除了APO外,BUD13也參與了血清TG水平的調控。此外,研究發(fā)現rs671位點的多態(tài)性不僅會通過影響乙醛代謝影響人們的飲酒行為,該位點還與飲酒行為之間存在對代謝綜合征及相關代謝表型的交互效應,其效應主要存在于飲酒人群中。四、小結:(1)在中國漢族人群中,rs651821(APOA5)和rs671(ALH2)位點的基因型與代謝綜合征的遺傳易感性相關;(2)rs180326(BUD13)基因型與血清TG水平相關,其效應獨立于已知位點rs651821(APOA5);(3)rs671(ALDH2)的基因型與飲酒行為存在交互效應。第二部分基于注釋信息的糖脂代謝相關遺傳變異篩選及精細定位一、研究目的:在第一部分研究的基礎上,結合多組學注釋信息,進一步篩選和驗證糖脂代謝指標相關的遺傳變異位點;對新發(fā)現的糖脂代謝相關遺傳變異位點進行精細定位,并通過功能實驗確認效應位點。二、材料和方法:首先,通過對1742例樣本(同第一部分)的糖脂代謝的全基因組關聯(lián)分析,得到與表型相關的但又未被第一部分驗證過的遺傳變異位點;然后分析這些位點與脂肪、肝臟、胰島和骨骼肌中基因表達水平的關聯(lián),并將這些位點的信號與表型關聯(lián)信號共定位,找出與糖脂代謝表型相關的eQTLs;進一步通過多階段獨立樣本驗證基因型與表型的關聯(lián);對驗證達到全基因組陽性水平的位點,通過以基因為單位的分析策略(gene-based analyses)推測其可能的調控基因;并通過ENCODE和Roadmap計劃提供的相關組織細胞中染色質狀態(tài)、組蛋白修飾、轉錄因子結合信號推測調控活性區(qū)域及其對應的效應位點;最后構建包含不同等位基因的載體,通過熒光素酶報告基因實驗確認其表達調控作用。三、研究結果:通過全基因組關聯(lián)分析及eQTL的注釋,發(fā)現了 22個與糖脂代謝相關的遺傳變異位點在特定的組織中影響基因表達,多階段獨立樣本驗證確認了 rsl880118位點與血清HDL-C水平的關聯(lián)(Pcombined= 1.4E-10)。該位點可以tag的區(qū)域主要包括DAGLB和RAC1兩個基因,在加性模型下,rsl880118的基因型可以解釋DAGLB(diacylglycerol lipase,beta)基因在皮下脂肪組織中表達水平變異的47.7%(P =5.9E-42)。同時,通過TWAS、SMR、Sherlock等以基因為單位的研究方法,我們發(fā)現D4GLB基因與血清HDL-C水平之間存在關聯(lián),關聯(lián)的P值分別為3.0E-08、1.1E-04和1.6E-06。進一步通過組蛋白信號H3K27ac、H3K4me3,H3K9ac及轉錄因子結合區(qū)域、DNA酶Ⅰ超敏位點的定位,找到了位于DAGLB基因5'區(qū)域的調控活性片段,熒光素酶報告基因的結果顯示,該活性片段中rs4724806位點(與rs1880118位點LD r2 = 0.77)可能是真正的效應位點,其最小等位基因會增加轉錄活性,與eQTL分析的結果一致。四、小結:rsl880118是一個在中國漢族人群中新發(fā)現的與血清HDL-C水平相關的遺傳變異位點,其效應位點rs4724806通過調控轉錄活性影響DAGLB基因表達,該部分研究提示了 A4GLB基因在脂代謝中的作用。第三部分利用多組學注釋系統(tǒng)解析糖脂代謝相關的遺傳變異一、研究目的:結合多組學注釋信息,對已知的糖脂代謝相關遺傳變異信號區(qū)域進行系統(tǒng)的解析,探究基因型到表型的調控路徑,為后續(xù)功能研究提供支持。二、研究方法:首先,整理已報道的與糖脂代謝相關的遺傳變異位點;然后,利用千人基因組計劃提供的位點間連鎖不平衡信息,填補出所有與lead SNP高度連鎖不平衡的位點用于后續(xù)的精細定位和功能預測;接著,對這些位點進行系統(tǒng)的多組學注釋,包括基因表達、染色質狀態(tài)、DNA甲基化、組蛋白修飾、轉錄因子結合等。對位于編碼區(qū)域的遺傳變異位點,再進行物種間保守性估計、翻譯后修飾等翻譯水平的注釋;,最后,整理推測出可能的基因型到表型調控路徑。三、研究結果:對經篩選過濾后得到的592個糖脂代謝相關遺傳變異位點進行填補后,共計17646個位點納入后續(xù)精細定位分析(LD r20.5)。在轉錄水平,通過遺傳變異與基因表達相關的注釋,發(fā)現了 104個在內臟脂肪、肝臟、骨骼肌或胰島細胞中與基因表達水平相關的位點。同時,發(fā)現了一些與特定環(huán)境刺激相關的eQTLs,如rs702485位點與DAGLB基因表達的關聯(lián)僅出現在LPS刺激后(Pbefore0.05,Pafter=2.52E-16)。133個糖脂代謝相關位點與脂肪組織或胰島細胞中的DNA甲基化相關,其中許多位點與多個CpG位點的甲基化水平相關。經過對遺傳變異位點的精細定位,我們發(fā)現有49個位點可以關聯(lián)(tag)到一個或以上的位于組蛋白修飾信號峰區(qū)域內的位點,且具有組織特異性。在翻譯水平,有122個(r20.5)或43個(r20.8)位點可以關聯(lián)到一個或以上的非同義突變位點,其中有16個位點經SIFT和Polyphen注釋均提示存在影響蛋白功能的可能。對糖代謝相關位點rsl535500精細定位和功能預測后發(fā)現,該位點G到T的變異與附近7個CpG位點的存在相關聯(lián),這些位點靠近KCNK17基因5'端的CpG島,存在影響DNA甲基化的可能。通過對胰島細胞中多個組學的信息整合,發(fā)現該位點確實可以通過影響附近位點甲基化水平進而影響KCNK17的表達,這不同于以往報道認為該信號主要通過KCNK16起作用。四、小結:(1)通過整合多個組學信息,對糖脂代謝GWAS信號進行重新解析后發(fā)現了許多可能參與到遺傳變異影響表型調控路徑中的基因及調控元件。與其他復雜表型類似,經過注釋后,三分之一的基因與以往對該位點報道一致。(2)僅有7%-20%的糖脂代謝表型相關遺傳變異位點可以關聯(lián)到一個或以上的非同義或無義突變,其余可能通過轉錄水平影響表型。(3)通過對糖代謝相關的位點rs1535500的精細定位,發(fā)現該位點G到T的變異與附近位點CpG位點的存在相關聯(lián),進而影響甲基化水平調控KCNK17的表達。結論:基于以上三部分內容,得出以下結論:(1)在中國漢族人群中,rs651821(APO45)和rs671(ALDH2)位點的基因型與代謝綜合征的遺傳易感性相關;新發(fā)現的rs180326(BUD13)位點與血清TG水平的關聯(lián)獨立于區(qū)域內已知位點rs651821;rs671與飲酒行為存在交互效應。(2)結合轉錄調控注釋信息可優(yōu)化GWAS的篩選策略;rs1880118是一個在中國漢族人群中新發(fā)現的與血清HDL-C水平相關的遺傳變異位點,與該位點高度連鎖不平衡的rs4724806位點多態(tài)性會通過調控DAGLB基因表達影響表型。(3)80%以上的糖脂代謝相關位點主要通過調控轉錄水平影響表型;經多組學注釋推測的調控基因中有三分之一與原GWAS報道的基因一致,如文獻報道糖代謝相關位點rs1535500可能的調控基因為KCNK16,但注釋信息提示該位點可能通過影響甲基化水平調控KCNK17的表達進而影響表型。
[Abstract]:Metabolic syndrome is a group of abnormal metabolic syndrome syndrome, mainly characterized by central obesity, hypertension and glucose and lipid metabolism disorder. Studies have shown that metabolic syndrome can increase the risk of cardiovascular and cerebrovascular diseases and diabetes. According to the definition of China Diabetes Association, the prevalence rate of metabolic syndrome in Chinese residents in 2010 was 17.6%. twins. Genetic factors play an important role in the occurrence of metabolic abnormalities. The classical genome-wide association study (GWAS) has found some genetic variations associated with metabolic abnormalities, but there are still two major problems: (1) the found loci can only explain a small number of heritability, that is, there is a large number of lost heredity. Missing heritability remains to be further explored; (2) previous studies mainly focus on phenotypic related label SNP (tag SNP) and the closest genes to it, but subsequent studies have found that most of the location signals do not play a role in the nearest gene, for example, the obesity related loci on FTO have been mainly passed through. Long distance regulation of IRX3 gene expression affects phenotypes. In a word, the previous analysis of GWAS localization signals is very limited. Most of the key genes and effect sites (causal variant) have not been found, which is not conducive to the follow-up function study. In recent years, most of the phenotypic related genetic variations have passed the regulation gene expression level and then affect the phenotype. As a note of expression regulation, the screening range of the whole genome can be narrowed to the gene expression quantitative trait loci (expression Quantitative Trait Loci, eQTLs) to improve the efficiency of the detection of the phenotypic related sites,.ENCODE, GTEx, Roadmap and other research projects have been published in a large number of studies that can be used to annotate genetic variation in the pathway of phenotypic regulation. The information of multiple histones, including tissue specific gene expression, DNA methylation, histone modification, transcription factor binding, and so on. These information can be used to further accurately screen phenotypic related genetic variations and can be used to reinterpret known signals. Because of the differences in genetic background between different human species, the results of the European population can not be directly extrapolated to the Chinese Han population. In order to systematically explore the metabolic syndrome and the genetic susceptibility loci related to the metabolic components, the main object of this study is to study the whole base of the Chinese Han population based on the multi group annotation. The genetic variation related to metabolic abnormalities was found and resolved by group association analysis. The study was divided into three parts (Figure 1): in the first part, the classical whole genome association analysis strategy was used to screen the strongest genetic variation loci of metabolic syndrome and metabolic components and carry out multistage validation, functional analysis and genetic environment interaction. The second part, on the basis of the first part of the study, further screened the genetic variation related to glycolipid metabolism through the annotation of transcriptional regulation, and analyzed the basic regulation path through fine location and functional experiments. The third part aimed at the known glycolipid metabolic signals, using the latest multi group annotation information to the signal. The region carries out systematic reanalysis, explores key regulatory genes, and provides support for the transformation of correlation analysis. The upper left side map of Figure 1 is the Manhattan map of the metabolic syndrome associated signal (P value) carried out by the project group. The left side is the first part of the technical route and the right side is the second part of the technical route. The right side map is the right map. 1 The third part of the phenotypic related loci recorded by NHGRI-EBI GWAS catalog, which is the third part of the study, uses a multi - group annotation to systematically re - analyze the sites associated with metabolic disorders. Susceptibility loci. Two, materials and methods: using single nucleotide polymorphic loci as genetic variation markers, the best loci with metabolic syndrome and metabolic components were screened in 1742 samples of Xiaoshan, Hangzhou, with the method of full genome association analysis. The sample is from the eastern, northern, northeastern and other regions of China, with a total of 10978 cases. After the combined multistage validation results, the functional prediction of the positive levels of the whole genome, the analysis of the interaction effect of the gene environment. Three, the results of the study: through the whole genome association analysis of the metabolic syndrome and the verification of the multi stage independent samples, The study found that the genotype of the rs651821 site on APO45 and the genotype rs671 of the high frequency missense mutation site specific to Asians on ADF2 is related to the genetic susceptibility to metabolic syndrome. After controlling the strongest signal rs651821 in the APO4 gene cluster region, the rs180326 site on BUD13 is still with the serum triglyceride (triglyceride, TG) water. Flat correlation (Pconbined=2.4E-08) is a new second signal (secondary signal) in the APO4 gene cluster. After integrating the genetic variation loci rs651821, rsl80326, serum APOA5, BUD13 protein level and TG level after analysis, it is found that BUD13 also participates in the regulation of serum TG levels except APO. Furthermore, the polymorphism of the locus is found not. Only by influencing the metabolism of acetaldehyde affects people's drinking behavior, and the interaction effect on metabolic syndrome and related metabolic phenotypes exists between the site and drinking behavior. The effect is mainly in the drinking crowd. Four, (1) the genotype and metabolic syndrome of rs651821 (APOA5) and rs671 (ALH2) loci in Chinese Han population Genetic susceptibility is related; (2) rs180326 (BUD13) genotype is related to serum TG level, its effect is independent of the known site rs651821 (APOA5); (3) the genotype of rs671 (ALDH2) has interaction effects with drinking behavior. The second part is based on the annotated information of glycolipid Xie Xiang pass genetic mutation screening and fine location one. The purpose of this study is to study the first part of the study On the basis of the study, the genetic variation loci related to glycolipid metabolic indices were further screened and verified with multi group annotation information, and the newly discovered genetic mutation sites related to glycolipid metabolism were fine located, and the functional loci were confirmed by functional experiments. Two, materials and methods: first, the glycolipid of the 1742 samples (the same part) was obtained. Genetic variation sites associated with phenotypic but not verified by the first part of metabolism; then the association of these sites with the gene expression levels in the fat, liver, islets, and skeletal muscles and the co location of signals from these sites with phenotypic correlation signals to identify eQ related to the glycolipid metabolic phenotype. TLs; further verify the association between genotypes and phenotypes by multistage independent samples; to verify the locus of the positive level of the whole genome and to speculate on the possible regulatory genes by the analysis strategy (gene-based analyses) based on the basis of the unit; and the chromatin state in the related tissue cells provided by the ENCODE and Roadmap plan, and the group of eggs White modification, the transcription factor binding signal conjectured the active region and its corresponding effect loci; finally, the vector containing the different alleles was constructed, and the expression regulation was confirmed by the luciferase reporter gene experiment. Three, the results were found to be related to the metabolism of glycolipid by whole genome association analysis and eQTL annotation. Genetic variation loci affect gene expression in specific tissues. Multi stage independent sample validation confirms the association of rsl880118 loci with serum HDL-C levels (Pcombined= 1.4E-10). The loci can mainly include two genes of DAGLB and RAC1. Under the additive model, the genotype of rsl880118 can explain DAGLB (diacylglycerol Li). Pase, beta) gene expression in subcutaneous adipose tissue is 47.7% (P =5.9E-42). At the same time, we find that there is a association between the D4GLB gene and the serum HDL-C level by TWAS, SMR, Sherlock and so on. The P values of the associated P are 3.0E-08,1.1E-04 and 1.6E-06. further through the histone signals. E3, H3K9ac and transcription factor binding region, the location of DNA enzyme I hypersensitivity loci, found a regulatory active fragment located in the 5'region of the DAGLB gene. The results of the luciferase reporter gene show that the rs4724806 locus (and rs1880118 LD R2 = 0.77) in the active fragment may be a true effect site, and its minimum allele will increase the transcriptional activity. Sex, in accordance with the results of eQTL analysis. Four, summary: rsl880118 is a newly discovered genetic locus of variation associated with serum HDL-C levels in Chinese Han population. Its effect locus rs4724806 affects the expression of DAGLB gene by regulating transcriptional activity. This part of the study suggests the role of A4GLB gene in lipid metabolism. The third part is used in this study. The multi group annotation system parses the genetic variation related to glycollipid metabolism. The purpose of this study is to systematically analyze the known region of genetic variation signals related to glycolipid metabolism by combining multi group annotation information to explore the regulation path of genotype to phenotype to provide support for subsequent functional studies. Two, research methods: first, the report has been reported. Genetic variation loci associated with glycolipid metabolism; then, using interloci linkage disequilibrium information provided by the Millennium genome project to fill out all the highly linked unbalanced sites with lead SNP for subsequent fine location and functional prediction; and then systematically annotate these loci, including gene expression, chromatin State, DNA methylation, histone modification, transcription factor binding, etc., annotations to the genetic variation loci in the coding region, interspecies conservatism estimation, post translation modification and other translation levels; finally, the possible genotype to phenotypic regulation path was conjectured. Three, research results: 592 glycolipids obtained after screening and filtering. After the metabolism related genetic mutation sites were filled, a total of 17646 loci were included in the follow-up fine location analysis (LD r20.5). At the transcriptional level, 104 loci related to the gene expression level in visceral fat, liver, skeletal muscle, or islet cells were found by the annotation related to genetic variation and gene expression. EQTLs related to specific environmental stimuli, such as the association of rs702485 loci with DAGLB gene expression, only occurs after LPS stimulation (Pbefore0.05, Pafter=2.52E-16).133 glycolipid related sites are associated with DNA methylation in adipose tissue or islet cells, many of which are related to the level of methylation at multiple CpG sites. We found that 49 loci can associate (tag) to one or more loci in the peak region of the histone modified signal, and have tissue specificity. At the translation level, 122 (r20.5) or 43 (r20.8) loci can be associated with one or more of the non synonymous mutation sites, of which 16 loci are in SI. FT and Polyphen notes suggest the possibility of affecting protein function. After the fine localization and function prediction of the glucose metabolism related site rsl535500, the mutation of the site G to T is associated with the existence of the 7 CpG loci near the KCNK17 gene, which is near the CpG island of the 5'terminal of the KCNK17 gene, and the possibility of the DNA methylation is possible. It is found that this site can affect the expression of KCNK17 by influencing the methylation level of the nearby loci, which is different from the previous reports that the signal is mainly mediated by KCNK16. Four, a summary: (1) a number of GWAS signals are reinterpreted by integrating a number of Informatics, and many can be found. Genes and regulatory elements that can participate in genetic variation affecting the pathway of phenotypic regulation. Similar to other complex phenotypes, after annotation, 1/3 of the genes are consistent with the previous reports. (2) only 7%-20%'s glycolipid phenotype related genetic variation loci can be associated with one or more unsynonymous or nonsense mutations. The phenotype may be affected by transcription level. (3) by mapping the site of rs1535500 related to glycometabolism, it is found that the locus G to T

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R589

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相關期刊論文 前1條

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