整合基因组学研究发现银屑病甲基化标记和遗传调控位点
发布时间：2018-07-16 15:52
【摘要】：银屑病是一类以界限清晰的红斑、银色鳞屑为主要表现的慢性免疫性皮肤病。因地理位置和种族差异不同,在世界范围内患病率差别较大,约为0.1-4%。总体而言,高加索人群患病率较亚洲人群高。我国银屑病患病率约为0.123%,患者总数将近1000万例,其分布趋势为城市高于农村,北方高于南方,男性高于女性。银屑病发病机制不清,普遍认为是遗传和环境因素共同作用的结果。既往研究显示其遗传度约为60-90%,然而当前基于DNA序列变异的研究仅能解释13%的疾病发生,提示表观学,环境因素,基因-基因相互作用等可能与疾病相关。以DNA甲基化和组蛋白修饰为主的表观遗传学是人类基因组学中重要的调控因素,甲基化能够解释部分DNA序列变异以外的疾病遗传度。DNA甲基化通过抑制转录因子与DNA序列的结合或者募集甲基化结合蛋白的方式控制基因的表达水平。甲基化结合蛋白与其他调控机制结合,如组蛋白乙酰化或去乙酰化,诱发染色体构象变化并控制基因的表达。DNA甲基化研究已在肿瘤等多种疾病和生物学通路中被广泛研究。现有银屑病的DNA甲基化研究在两个层面展开,即针对候选基因启动子和无偏倚的全基因水平。候选基因研究如p15和p21蛋白启动子基因在银屑病组织中甲基化过低；对比特应性皮炎患者和正常个体,发现SHP-1(PTPN6)基因启动子完全脱甲基化。全基因组甲基化分析病例对照研究发现一批与免疫、表皮分化复合体等疾病相关基因。然而既往研究存在样本量偏小,基因组覆盖度偏低,基因表达数据不完整等情况使得大量潜在位点或易感基因没有被发现。因此开展大范围的银屑病全基因组甲基化研究,整合高精度RNA表达数据将有助于发现疾病风险基因。目的：在全基因组范围内搜寻银屑病相关DNA甲基化位点,整合转录组表达数据,发现疾病风险易感基因。方法：采用IlluminaHuman450K芯片对114例患者皮损,41例非皮损,62例对照个体的皮肤组织DNA分析近450000个位点的CpG甲基化水平。位点甲基化差异分析采用Wilcox中位数秩和检验分析,并经Benjamini-Hochberg法矫正多重检验。Spearman相关性检验分析甲基化与基因表达相互关系。sequenom质谱系统验证差异性甲基化位点。结果：通过分析皮损/(非皮损+对照)DNA的甲基化高低,发现286个差异性甲基化位点,分布于188个基因。整合全基因组转录组数据,发现36个基因表达与甲基化相关,包括AIM2, TGFBR3, PHYHD1, PHOB, FOLR1, ZC3H12A, LYPD1和ZBP1等。1.比对41对病人皮损和非皮损发现1638个差异性位点,在皮损组织中过高和过低甲基化位点分别为954和684个。2.比对73例病人皮损和62个正常个体发现434个差异性甲基化位点。同时比较皮损/(非皮损+对照),发现286个差异甲基化位点,位点分布于188个基因上。3.分析286个位点在基因组中的分布情况,发现位点在特定CpG区域富集。近60%位点位于开放区域(Open Sea),42.4%位点位于基因体内(Gene body)4.DAVID基因功能注释显示,188个显著性基因中,显示甲基化过高的基因在蛋白磷酸化、RNA转录调节等方面富集。而低甲基化基因无明显富集(P0.05)5.相比既往欧美报道的1108个差异性位点,通过IlluminaHuman450K可获得776个位点数据,其中456个达到显著性水平,较随机出现的可能性有显著差异(Chi-square=276.5,P 2.2×10-16)。比较两项研究,456个位点中仅1个甲基化位点差异方向相反。6.通过转录组测序可分析188个基因当中178个基因的表达数据。发现19个(13.3%)DNA甲基化升高伴基因表达降低,16个(9.4%)甲基化降低伴基因表达升高。提示约有五分之一DNA甲基化差异与基因表达相关。呈现甲基化表达负相关的基因包括：AIM2, TGFBR3, PHYHD1, PHOB, FOLRl, ZC3H12A, LYPD1和ZBP1等。7.构建因果关系模型分析遗传标记(SNP),甲基化,疾病状态关系,发现3个CpG位点为潜在调节SNP作用于疾病的调控点。结论：通过非配对病例对照分析发现286个银屑病差异性甲基化位点,对应于188个基因。整合全基因组转录组表达数据,发现36个DNA甲基化/基因表达呈现负相关,包括AIM2, TGFBR3, PHYHD1, PHOB, FOLR1, ZC3H12A, LYPD1和ZBP1等,该批基因可能与银屑病发病机制相关；通过CIT因果分析发现C1orf106, DMBXl和SIK3基因可能在调节遗传变异位点对银屑病的过程中发挥作用。
[Abstract]:Psoriasis is a class of chronic immune dermatosis with distinct red spots and silver scales. The prevalence rate varies widely in the world because of different geographical location and racial differences. The prevalence rate of Caucasus in the Caucasus is higher than that of the Asian population. The prevalence rate of psoriasis in China is about 0.123%, and the total number of patients is nearly 0.123%. 10 million cases, its distribution trend is that the city is higher than the countryside, the north is higher than the south, and the male is higher than the female. The pathogenesis of psoriasis is not clear, and it is generally considered to be the result of the combination of genetic and environmental factors. The previous study shows that its heritability is about 60-90%, however, the current study based on DNA sequence variation can only explain the occurrence of 13% of the disease. Observation, environmental factors, gene gene interaction, etc. may be related to disease. Epigenetics, based on DNA methylation and histone modification, is an important regulatory factor in human genomics. Methylation can explain the combination of.DNA methylation of diseases other than partial DNA sequence variation by inhibiting the binding of transcription factors to DNA sequences. Or collect methylation binding proteins to control the level of gene expression. Methylated binding proteins are combined with other regulatory mechanisms, such as histone acetylation or deacetylation, induced chromosomal conformation changes and the control of gene expression.DNA methylation has been widely studied in a variety of diseases and biological pathways. The DNA methylation study of the chip disease was carried out at two levels, namely, the candidate gene promoter and the total gene level without bias. Candidate gene studies, such as the low methylation of the p15 and p21 promoter genes in the psoriasis tissues, were compared with the patients with atopic dermatitis and normal individuals, and the complete demethylation of the SHP-1 (PTPN6) gene promoter was found. A case control study of genomic methylation has found a number of related genes related to immune and epidermal differentiation complexes. However, previous studies have found that a large number of subpotential sites or susceptible genes have not been found in a large range of psoriasis, which have not been found in a large number of samples, low genome coverage and incomplete gene expression data. Genomic methylation studies and integration of high precision RNA expression data will help to detect the disease risk genes. Objective: to search for the DNA methylation sites in the whole genome, to integrate the transcriptional data and to find the susceptibility genes of the disease risk. Methods: the IlluminaHuman450K chip was used in 114 patients' skin lesions and 41 cases were non skin lesions. DNA analysis of the CpG methylation level of nearly 450000 loci in 62 cases of the skin tissue of the control individuals. The difference analysis of loci methylation was analyzed by the median rank sum test of Wilcox, and the correlation test of multiple test.Spearman was corrected by Benjamini-Hochberg method to analyze the relationship between methylation and gene expression by.Sequenom mass spectrometry system to verify the difference a Results: by analyzing the methylation of DNA, 286 different methylation sites were found to be distributed in 188 genes. Integrated whole genome transcriptome data, 36 genes were found to be associated with methylation, including AIM2, TGFBR3, PHYHD1, PHOB, FOLR1, ZC3H12A, LYPD1, and ZBP1, and 41 pairs of diseases. 1638 difference sites were found in the skin lesions and non skin lesions. The high and low methylation sites in the skin lesions were 954 and 684.2., respectively, and 434 differential methylation sites were found in 73 cases of skin lesions and 62 normal individuals. At the same time, the lesions / (non skin lesions + control) were compared, and 286 methylation sites were found to be distributed in 188 bases. Because of the.3. analysis of the distribution of 286 loci in the genome, the found site is enriched in a specific CpG region. Nearly 60% loci are located in the open region (Open Sea), the 42.4% site is located in the gene (Gene body) 4.DAVID gene function annotation, and 188 significant genes show the hypermethylation gene in protein phosphorylation and RNA transcriptional regulation. There was no obvious enrichment of the low methylation gene (P0.05) 5. compared with the 1108 difference sites reported in the past and the United States, and 776 loci data could be obtained through IlluminaHuman450K, of which 456 reached significant levels, compared with the probability of random occurrence (Chi-square=276.5, P 2.2 x 10-16). Two studies and 456 bits were compared. The difference of 1 methylation sites in the point was opposite to.6. through the transcriptional sequence to analyze the expression data of 178 genes in the 188 genes. 19 (13.3%) DNA methylation was elevated with the decrease of gene expression, 16 (9.4%) methylation decreased with the increase of gene expression. It suggested that about 1/5 DNA methylation was associated with gene expression. AIM2, TGFBR3, PHYHD1, PHOB, FOLRl, ZC3H12A, LYPD1, ZBP1 and other.7. construction causality models were constructed to analyze the genetic markers (SNP), methylation, and the relationship between disease status. The 3 CpG loci were found to be the regulatory point of the potential regulating SNP in the disease. Conclusion: 28 by non matched case control analysis. 6 psoriatic differential methylation sites, corresponding to 188 genes, integrated the whole genome transcriptome expression data, and found that 36 DNA methylation / gene expression showed negative correlation, including AIM2, TGFBR3, PHYHD1, PHOB, FOLR1, ZC3H12A, LYPD1 and ZBP1, which could be related to the pathogenesis of psoriasis, and found C1o through CIT causality analysis. Rf106, DMBXl and SIK3 genes may play a role in psoriasis regulation by regulating genetic variation sites.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R758.63
，
本文编号：2126877
[Abstract]:Psoriasis is a class of chronic immune dermatosis with distinct red spots and silver scales. The prevalence rate varies widely in the world because of different geographical location and racial differences. The prevalence rate of Caucasus in the Caucasus is higher than that of the Asian population. The prevalence rate of psoriasis in China is about 0.123%, and the total number of patients is nearly 0.123%. 10 million cases, its distribution trend is that the city is higher than the countryside, the north is higher than the south, and the male is higher than the female. The pathogenesis of psoriasis is not clear, and it is generally considered to be the result of the combination of genetic and environmental factors. The previous study shows that its heritability is about 60-90%, however, the current study based on DNA sequence variation can only explain the occurrence of 13% of the disease. Observation, environmental factors, gene gene interaction, etc. may be related to disease. Epigenetics, based on DNA methylation and histone modification, is an important regulatory factor in human genomics. Methylation can explain the combination of.DNA methylation of diseases other than partial DNA sequence variation by inhibiting the binding of transcription factors to DNA sequences. Or collect methylation binding proteins to control the level of gene expression. Methylated binding proteins are combined with other regulatory mechanisms, such as histone acetylation or deacetylation, induced chromosomal conformation changes and the control of gene expression.DNA methylation has been widely studied in a variety of diseases and biological pathways. The DNA methylation study of the chip disease was carried out at two levels, namely, the candidate gene promoter and the total gene level without bias. Candidate gene studies, such as the low methylation of the p15 and p21 promoter genes in the psoriasis tissues, were compared with the patients with atopic dermatitis and normal individuals, and the complete demethylation of the SHP-1 (PTPN6) gene promoter was found. A case control study of genomic methylation has found a number of related genes related to immune and epidermal differentiation complexes. However, previous studies have found that a large number of subpotential sites or susceptible genes have not been found in a large range of psoriasis, which have not been found in a large number of samples, low genome coverage and incomplete gene expression data. Genomic methylation studies and integration of high precision RNA expression data will help to detect the disease risk genes. Objective: to search for the DNA methylation sites in the whole genome, to integrate the transcriptional data and to find the susceptibility genes of the disease risk. Methods: the IlluminaHuman450K chip was used in 114 patients' skin lesions and 41 cases were non skin lesions. DNA analysis of the CpG methylation level of nearly 450000 loci in 62 cases of the skin tissue of the control individuals. The difference analysis of loci methylation was analyzed by the median rank sum test of Wilcox, and the correlation test of multiple test.Spearman was corrected by Benjamini-Hochberg method to analyze the relationship between methylation and gene expression by.Sequenom mass spectrometry system to verify the difference a Results: by analyzing the methylation of DNA, 286 different methylation sites were found to be distributed in 188 genes. Integrated whole genome transcriptome data, 36 genes were found to be associated with methylation, including AIM2, TGFBR3, PHYHD1, PHOB, FOLR1, ZC3H12A, LYPD1, and ZBP1, and 41 pairs of diseases. 1638 difference sites were found in the skin lesions and non skin lesions. The high and low methylation sites in the skin lesions were 954 and 684.2., respectively, and 434 differential methylation sites were found in 73 cases of skin lesions and 62 normal individuals. At the same time, the lesions / (non skin lesions + control) were compared, and 286 methylation sites were found to be distributed in 188 bases. Because of the.3. analysis of the distribution of 286 loci in the genome, the found site is enriched in a specific CpG region. Nearly 60% loci are located in the open region (Open Sea), the 42.4% site is located in the gene (Gene body) 4.DAVID gene function annotation, and 188 significant genes show the hypermethylation gene in protein phosphorylation and RNA transcriptional regulation. There was no obvious enrichment of the low methylation gene (P0.05) 5. compared with the 1108 difference sites reported in the past and the United States, and 776 loci data could be obtained through IlluminaHuman450K, of which 456 reached significant levels, compared with the probability of random occurrence (Chi-square=276.5, P 2.2 x 10-16). Two studies and 456 bits were compared. The difference of 1 methylation sites in the point was opposite to.6. through the transcriptional sequence to analyze the expression data of 178 genes in the 188 genes. 19 (13.3%) DNA methylation was elevated with the decrease of gene expression, 16 (9.4%) methylation decreased with the increase of gene expression. It suggested that about 1/5 DNA methylation was associated with gene expression. AIM2, TGFBR3, PHYHD1, PHOB, FOLRl, ZC3H12A, LYPD1, ZBP1 and other.7. construction causality models were constructed to analyze the genetic markers (SNP), methylation, and the relationship between disease status. The 3 CpG loci were found to be the regulatory point of the potential regulating SNP in the disease. Conclusion: 28 by non matched case control analysis. 6 psoriatic differential methylation sites, corresponding to 188 genes, integrated the whole genome transcriptome expression data, and found that 36 DNA methylation / gene expression showed negative correlation, including AIM2, TGFBR3, PHYHD1, PHOB, FOLR1, ZC3H12A, LYPD1 and ZBP1, which could be related to the pathogenesis of psoriasis, and found C1o through CIT causality analysis. Rf106, DMBXl and SIK3 genes may play a role in psoriasis regulation by regulating genetic variation sites.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R758.63
，
本文编号：2126877
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