白血球DNA甲基化、多重環境暴露與B型肝炎相關之肝細胞癌罹病風險

Leukocyte DNA Methylation, Multiple Environmental Exposures, and Hepatitis B-Related Hepatocellular Carcinoma Risk

楊舒涵(Shu-Han Yang)

指導教授 ： 于明輝

國立臺灣大學/公共衛生學院/流行病學與預防醫學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11371

摘要

背景與目的：肝細胞癌為高致死率癌症，發展風險評估和早期偵測的生物標記有必要性。過去研究已發現表基因的改變可能是危險因子引發肝細胞癌的中介因子，但尚無利用前瞻型研究和血液樣本探討甲基化和肝細胞癌發展的關係。本篇研究目的主要有二：一為利用pyrosequencing驗證先前全基因體晶片分析結果，再由選定的候選標記探討白血球的甲基化、多重危險因子、與B型肝炎相關之肝細胞癌罹病風險。
樣本與方法：本研究個案來自一B型肝炎研究世代，利用巢式病例對照研究設計。第一階段為實驗驗證，包括48組病例和對照配對，病例均為早發型肝細胞癌，由pyrosequencing分析38個探針標的CpG位點。第二階段聚焦於第一階段選出的7個實驗驗證成功的特定探針，利用一個獨立樣本，包含149位病例和184位對照個案，進行晚發型肝細胞癌病例的研究。
結果：第一階段，發現23個特定探針在兩實驗平台相對應的CpG位點呈現高度相關(spearman correlation coefficients= 0.44-0.93)，在甲基化的數值上則會有所落差(intra-class correlation coefficients= 0.00-0.86)。第二階段，7個特定探針中僅有一個被發現和第一階段產生一致的結果，隨甲基化程度四分位數上升（Q1-Q4），最顯著的CpG位點其多變項調整後相對危險性（95%信賴區間）各為0.33（0.14-0.76）、0.32（0.13-0.81）、0.24（0.10-0.58）；此CpG位點甲基化程度亦和喝酒習慣顯著相關，隨甲基化程度四分位數上升（Q1-Q4），多變項調整後相對危險性（95%信賴區間）各為0.63（0.30-1.29）、0.63（0.13-1.32）、0.44（0.21-0.93）。雖然兩支探針標的DNA序列甲基化被發現和病毒量及基因型有關，但在第二階段研究，均未被發現和肝細胞癌罹病風險呈現顯著相關。
結論：通過兩階段的實驗分析，我們發現pyrosequencing是定量甲基化程度的穩定實驗平台。白血球甲基化程度的改變，能夠作為評估B型肝炎相關之肝細胞癌罹病風險的生物標記。未來，有機會能夠將其發展為早期診斷的工具。

關鍵字

肝細胞癌 ； B型肝炎 ； 白血球DNA甲基化 ； 焦磷酸測序

並列摘要

Background and Aims: Hepatocellular carcinoma is a high mortality rate of cancer. It is necessary todevelop the risk assessment and early detection biomarkers. Previous studies have found that epigenetic alternations may be the mediator of risk factors and HCC. However, none of prospective study using blood samples explores the relationship between DNA methylation and HCC progression. In this study, the first aim performs experimental validation on array results by pyrosequencing analysis. The second aim uses the candidate markers to examine the association between leukocyte DNA methylation, multiple risk factors, and Hepatitis B-related hepatocellular carcinoma risk.
Patients and Methods: Study populations in the nested case-control study come from the established hepatitis B cohort. In the first-stage analysis, we particularly focused on early-onset HCC including 48 youngest patients and their individual match. Thirty-eight target probes from the array-based analysis were validated by pyrosequencing. In the second-stage analysis, 7 selected target probes, which were successfully validated from the first-stage, were used to examine the association between DNA methylation, risk factors, and HCC in the independent data including 149 eldest patients and 184 controls.
Results: Stage I study, the corresponding CpG sites of 23 target probes showed high correlation between two platforms (spearman correlation coefficients= 0.44-0.93); however, the methylation values were almost different (intra-class correlation coefficients= 0.00-0.86). Stage II study, only one target probes had the consist result from the first-stage among 7 selected target probes. Multivariate logistic regression model showed an association between HCC risk and increasing quartile levels of DNA methylation at the second CpG site of cg17 (AORs for Q2-Q4 of 0.33, 95%CI=0.14-0.76; 0.32, 95%CI=0.13-0.81; 0.24, 95%CI=0.10-0.58, respectively). This site also showed an association between alcohol consumption and increasing quartile levels of DNA methylation (AORs for Q2-Q4 of 0.33, 95%CI=0.14-0.76; 0.32, 95%CI=0.13-0.81; 0.24, 95%CI=0.10-0.58, respectively). Although DNA methylation of 2 selected target probes were associated with HBV DNA and HBV genotype, those were not significantly associated with HCC in the second-stage analysis.
Conclusion: Through our two-stage study, we found that pyrosequencing is a gold method for quantification of DNA methylation levels. Measurements of leukocyte DNA methylation alternations may help to assess the subsequent risk of HBV-related HCC. In the future, leukocyte methylation signatures have the potential applications in early detection of HCC.

並列關鍵字

Hepatocellular carcinoma ； Hepatitis B ； Leukocyte DNA methylation ； Pyrosequencing

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