冠狀動脈瘤樣擴張疾病之全基因體甲基化研究

Abstract

冠狀動脈瘤樣擴張疾病（CAE）的定義為病患的冠狀動脈血管管徑有異常擴張的現象。冠狀動脈瘤樣擴張疾病在全世界的發生率約為5%，然而其詳細致病機轉目前仍不清楚，有待後續研究。截至目前為止，過去的研究顯示發炎反應、吸煙以及高脂血症與冠狀動脈瘤樣擴張疾病有關，並且這些危險因子皆與甲基化的變異息息相關。因此，本研究的目的是找出冠狀動脈瘤樣擴張疾病之病患其甲基化有顯著變異的基因，並藉此探討甲基化變異與疾病之間的相關性，以利了解疾病可能的致病機轉以及後續作為早期診斷生物標記的可行性。 本研究首先透過全基因體甲基化微陣列晶片針對共12對冠狀動脈瘤樣擴張疾病患者及其經過傾向分數配對後的正常控制組進行分析。在單一探針上，我們針對每個CpG位點進行Wilcoxon rank sum test，後續篩選出p <0.05和Δβ> | 0.1 |的CpG位點進行下一步的生物功能及傳導途徑分析(N=165)。針對篩選出來的甲基化變異基因以及全基因體的甲基化變異圖譜，我們分別使用兩種演算法進行分析。在篩選後的89個甲基化變異基因上，分析結果顯示甲基化變異基因主要參與在免疫和發炎反應中，而全基因體的甲基化變異圖譜分析亦指出與免疫反應高度相關的TGF-beta signaling pathway具有變異。在根據基因的甲基化變異程度及生物功能，挑選6個標的基因進行焦磷酸化實驗驗證，結果顯示TLR6和NOTCH4的甲基化變異具有顯著差異，且酵素連結免疫吸附法進一步確認TLR6的甲基化變異能影響到下游的蛋白質表現量。因此，冠狀動脈瘤樣擴張疾病與甲基化變異存在相關性，尤其是在免疫反應相關的基因(TLR6和NOTCH4)之上，可能具有高度相關性，後續研究應致力於探索這些甲基化變異之基因在致病機轉上扮演的角色。

Coronary artery ectasia (CAE) represents the abnormal dilation of the coronary artery. Till now, the underlying mechanisms of CAE are not yet fully understood and the incidence rate of CAE is approximately 5% in the whole world. Previous studies showed that inflammatory response, smoking and hyperlipidemia are associated with CAE, and interestingly all of them are related to methylation changes. Although methylation changes have been shown as an important player in coronary artery disease, its role in CAE remains unclear. Therefore, we aimed to perform a genome-wide methylation analysis in CAE patients to identify genes with significantly changed methylation. Therefore, we can investigate correlation between significantly changed methylation level and CAE in order to understanding the etiology of CAE and serving as biomarkers for early diagnosis in the future. A total of 12 CAE patients and 12 controls were analyzed using the Illumina 450K to identify significantly dysregulated methylation genes. For each probe, a Wilcoxon rank sum test was performed. Only the probes having a P value < 0.05 and Δβ > |0.1| remained for characterizing the biological functions and signaling pathways (N=165). For dysregulated methylation genes and genome-wide methylation change profile, two bioinformatics algorithms were analyzed respectively. The microarray analysis reported 89 genes were with significant methylation changes. Functional characterization of the 89 genes using DAVID showed that their major pathways were involved in immune and inflammatory response. Similar analysis results were observed in GSEA, revealing the genes related to TGF-beta signaling pathway had the most significant changes in methylation levels in CAE patients. According to dysregulated methylation level and biological functions, six target genes were selected for validation by pyrosequencing. The validated results showed that the methylation levels of TLR6 and NOTCH4 were significantly changed. Subsequently, enzyme-linked immunosorbent assay (ELISA) was used to confirm that changes in methylation levels of TLR6 would influence the expression levels of downstream proteins. Therefore, CAE and genes with dysregulated methylation had a correlation between each other, especially on the immune-related genes (TLR6 and NOTCH4). Follow-up studies should be developed to investigate those genes with changed methylation levels, which may play an importance role on the pathogenic of CAE.