B型肝炎病毒核基因序列變異與肝細胞癌之關係：重疊病例對照研究

Abstract

背景：HBV core基因變異和慢性肝病的相關性研究尚相當稀少。本研究利用重疊病例對照研究設計探討HBV core基因區域核甘酸變異與肝細胞癌的關係，並分析core基因核甘酸變異與已知病毒因子(包括：病毒基因型、病毒量及HBeAg和anti-HBe狀態)的關係。 材料與方法：研究個案來自一個在1988-1992年間收案之男性HBV帶原者世代，病例組和對照組依進入研究時的年齡及血液檢體採集時間進行個別匹配。共計116名病例及153名對照個案進入本研究分析。Core基因序列變異是採直接定序分析獲得。以皮爾森相關係數和因素分析法探討和各種基因型叢聚的核甘酸變異特徵，利用Neighbor-Joining方法對HBV core基因序列進行演化樹分析，以非條件式邏輯斯迴歸計算HBV core基因序列變異之對比值(odds ratio, OR)及95%信賴區間(95% confidence interval, 95% CI)。 結果：HBV core基因序列之核甘酸平均變異率在病例組和對照組中分別為3.10% (95% CI= 2.50% -3.69%)和2.87% (95% CI= 2.40% -3.34%)，變異型頻率較高者大都集中於core基因核甘酸位置2059-2354。總共發現27個核甘酸位置和肝細胞癌有關，其中10個位置(包括2059、2104、2134、2170、2233、2251、2290、2293、2296和2354)和genotype呈現高度相關，而因素分析和演化樹分析的結果顯示，這10個變異點可作為分辨台灣族群HBV genotype的特徵SNP。針對其餘的17個變異點所對映之氨基酸位置分析，發現在控制HBV genotype以後，氨基酸位置13(多變項OR = 0.30, 95% CI=0.11-0.80)、21(多變項OR = 0.22, 95% CI =0.08-0.58)和113(多變項OR = 0.40, 95% CI=0.19-0.84)與HCC呈現顯著相關。由與genotype低度相關或不相關的12個核甘酸位置鑑定120種組合型態，其中原始型(prototype)在病例組和對照組各佔47.4%和24.2%。非原始型(non-prototype) 相較於原始型而言，其罹患肝細胞癌的危險性較低(多變項OR=0.43, 95%CI=0.24-0.77)。 結論：HBV core基因之核甘酸變異多集中在T細胞和B細胞的epitope處，且不同氨基酸的變異與肝細胞癌之發展具有顯著相關。

Background: The association between sequence variation in the core gene of hepatitis B virus (HBV) and the development of hepatocellular carcinoma (HCC) is largely unknown. Materials and Methods: Study subjects consisted of 116 cases and 153 controls nested within a cohort study of 4841 male, asymptomatic HBV carriers enrolled from 1988 through 1992. Controls were matched to the cases with respect to age at recruitment and blood collection time. Pearson correlation coefficient and factor analysis were employed to identify cosegregating nucleotide variant. HBV genotype was determined by phylogenetic analysis with the use of the neighbor-joining method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression model. Results: The mean percentages of nucleotide divergence were 3.10% (95% CI= 2.50% to 3.69%) in cases and 2.87% (95% CI= 2.40% to 3.34%) in controls. High nucleotide divergence clustered in the nucleotide region 2059-2354. Of the 27 single nucleotide polymorphisms (SNPs) statistically significantly associated with HCC, 10 (at positions 2059, 2104, 2134, 2170, 2233, 2251, 2290, 2293, 2296, and 2354) were highly correlated with genotype, showing a correlation coefficient of greater than 0.6. Results from factor analysis and phylogenetic analysis suggested that the 10 SNPs could be used as the signature SNPs to detect different genotypes in Taiwanese population. In analysis based on deduced amino acid (aa) sequence, we found aa substitution at three positions aa13, aa21, and aa113 was statistically significantly associated with the risk of HCC after adjusting for HBV genotype, showing OR of 0.30 (95% CI=0.11 to 0.80), 0.22 (95% CI =0.08 to 0.58), and 0.40 (95% CI=0.19 to 0.84), respectively. Based the combinations of 12 SNPs with poor or no correlation with genotype, prototype accounts for 47.4% of the HBV genomes isolated from cases and 24.2% of the HBV genomes isolated from controls. HBV carriers with non-prototype were at reduced risk (adjusted OR=0.43, 95% CI=0.24 to 0.77) of HCC compared with those who had the prototype. Conclusions: Nucleotide sequence variation in HBV core gene was majorly located at epitopes of T or B cell. Amino acid substitution in the core gene was significantly associated with the development of HCC.