建立DNA甲基化分析平台並應用於人類甲基化異常疾病與紅斑性狼瘡之臨床研究

Abstract

隨著人類基因圖譜的解密，科學家發現基因體調控序列的甲基化與基因的表現與否相關，DNA的甲基化調控著許多基因的活化與否，並控制許多基因的表現。DNA的甲基化，尤其是在基因啟動子區域中的 CpG島嶼上的胞嘧啶被DNA甲基轉移酶催化，形成5-甲基胞嘧啶，會阻礙轉錄因子與 DNA結合，進而導致基因靜默 (silencing)。當甲基化程度低下(hypomethylation)時，轉錄的工作就會被活化。目前已知基因體異常的甲基化可能會導致許多疾病的發生，例如癌症、免疫力缺乏以及自體免疫疾病等等。 我們的研究分為兩部分，第一部分是建立DNA甲基化分析平台，分析人類甲基化異常疾病(包含小胖威利症、天使症候群、貝克威斯魏德曼症候群以及西弗羅素症候群)的甲基化變異及基因劑量。我們建立了許多分析技術檢測DNA的甲基化，包括重亞硫酸鹽序列分析、甲基化特異性聚合酶連鎖反應、敏感性甲基化高解析熔解分析、即時定量聚合酶連鎖反應併熔解分析、專一性甲基化多重聚合酶連鎖反應以及專一性甲基化多重連接探針擴增技術。其中，由我們自行研發設計的「專一性甲基化多重聚合酶連鎖反應」及「即時定量聚合酶連鎖反應併熔解分析」可以在單一試管中同時分析DNA劑量及DNA甲基化程度，不但準確且可以提高檢測的效率、輸出率、方便性並避免檢體汙染。 研究的第二部分是利用全基因體甲基化晶片探討紅斑性狼瘡與DNA甲基化的關係。紅斑性狼瘡是一種複雜性的疾病，截至目前為止，真正的致病因子並不清楚，已知具有相當程度的遺傳傾向，但沒有單一基因突變可以解釋紅斑性狼瘡的發生。目前已經有許多研究證據認為DNA的甲基化低下在紅斑性狼瘡的發生扮演當重要的角色，因此，我們假設異常的DNA甲基化會活化免疫反應進而誘發紅斑性狼瘡的疾病活性。在我們第二部分的研究發現12名紅斑性狼瘡患者DNA甲基化的平均值確實比12名健康人的甲基化程度低(β值分別為0.048與0.054, p=0.0001)，而且藉由生物知識資料庫軟分析發現到這些甲基化異常的基因可能牽涉到免疫細胞遷徙的生理網絡中。由此網絡，我們特別針對IL10與IL1R2基因進行分析，結果顯示這兩個基因在66名紅斑性狼瘡患者血中DNA甲基化的程度的確比102健康人顯著地低下(兩組IL10基因甲基化的平均值分別為0.272與0.428, p<0.0001，IL1R2則分別為0.043與0.103, p<0.0001)，而且，愈嚴重的紅斑性狼瘡患者其IL10與IL1R2基因DNA甲基化低下的程度愈明顯，且比例愈高。透過線性迴歸分析，我們發現當個體具有IL10基因甲基化低下時，罹患紅斑性狼瘡的勝算比是22.061(95% 信賴區間為9.643-50.473)，而具有IL1R2基因甲基化低下時，罹患紅斑性狼瘡的勝算比是10.533 (95% 信賴區間為4.960-22.365)。若是具有IL10或IL1R2基因一股甲基化低下，則罹病的勝算比為2.476 (95% 信賴區間為0.903-113.904)，若同時兩個基因皆發生甲基化低下的現象，則罹病的勝算比為40.857(95% 信賴區間為14.655-113.904，p for trend <0.0001)，換言之，我們的研究顯示當個體具有IL10與IL1R2基因DNA甲基化低下時，就較容易得到紅斑性狼瘡或者紅斑性狼瘡的疾病活性就愈強。此外，我們還發現到類風濕性關節炎患者體內IL10與IL1R2基因DNA甲基化也有低下的情形，但是IL1R2基因的變化又比IL10的變化更為顯著。藉由此研究，未來IL10與IL1R2基因甲基化低下將可能可以成為自體免疫疾病臨床的生物外遺傳指標。

Epigenetics is defined as the investigation of “heritable changes in gene expression that occur without a change in DNA sequence”, with the methylation of DNA and acetylation of histones being involved in such changes. Cytosine methylation of the regulatory sequences of DNA is an epigenetic mechanism that is associated with transcriptional inactivation of genes, while hypomethylation contributes to the activation of transcription. Recent epigenetic investigations have contributed to the pathogenesis of various disorders, including cancer, immunodeficiency and autoimmunity. There are two parts of our research. The first part is to establish the analytic platforms for DNA methylation. We used human DNA methylation diseases such as Prader-Willi syndrome, Angelman syndrome, Beckwith Wiedemann syndrome and Silver Russell syndrome as our disease models to analyze the genetic dosage and DNA methylation pattern. We have set up severeal analytic technologies to examine the DNA methylation, including Methylation-specific PCR, Methylation-sensitive high resolution melting ananlysis, Quantitative and qualitative real-time PCR with melting curve analysis, Methylation-specific multiplex PCR and Methylation-specific multiplex ligation-dependent probe amplification. The noval methods, 「quantitative and qualitative real-time PCR with melting curve analysis」and 「methylation-specific multiplex PCR」 are developed by us. With these two methods, we can analyze both allelic copy numbers and DNA methylation patterns within one single tube, providing accurate, high-throughput, efficient results. After establishing the analytic platforms for DNA methylation, we would like to apply the technologies to study complex human disease further. The second part of our research is to investigate epigetic changes over systemic lupus erythematosus(SLE) by whole genome methylation array. The etiology of systemic lupus erythematosus involves a complex interaction of genetic and environmental factors. Investigations have shown that environmentally driven epigenetic changes contribute to the etiology of SLE. Here, we hypothesized that aberrant DNA methylation may contribute to the activation of the immune machinery and trigger lupus disease activity. A whole genome methylation array was applied to investigate the DNA methylation changes between 12 pairs of active SLE patients and healthy controls. The results were further confirmed in 66 SLE patients and 102 healthy controls. The methylation statuses of the IL10 and IL1R2 genes were significantly reduced in the SLE patient samples relative to the healthy controls (age-adjusted odds ratios, 64.2 and 16.9, respectively, P<0.0001). There was a trend toward SLE patients having hypomethylated IL10 and IL1R2 genes accompanied by greater disease activity. We observed the methylation degree of IL10 and IL1R2 genes were reduced in the rheumatoid arthritis(RA) patients as well but the hypomethylation change was more significant in IL1R2 genes than in the IL10 genes in RA patients. This study demonstrated that DNA hypomethylation might be associated with SLE. Hypomethylated IL10 and IL1R2 genes may provide potential epigenetic markers as clinical predictors for autoimmune diseases.