B型肝炎病毒Surface基因和重疊Polymerase基因區域之突變頻譜和肝細胞癌危險性：重疊病例對照研究

Abstract

背景：HBV病毒因子與肝細胞癌的關係近年來著重在HBV DNA與基因型層面。本研究目的在利用重疊病例對照研究分析病例和對照組在PreS/S及其和P基因重疊區域各點核苷酸變異分佈的情形，同時考慮基因型的影響。方法：研究個案來自一個1988-1992年間收案之男性HBV帶原者世代，對照組是依照病例進入研究年齡及血液採檢時間進行個別匹配，共計86名病例和125名對照進入分析。對於追蹤期間的血液檢體分析，是選擇病例發病兩年內之血液檢體，對照則取與病例檢體距離最近時點之血液檢體分析，共計17名病例與27名對照個案。結果：以adw為參考序列和對照組基線血液檢體分析結果進行比對，發現8個頻率在20%以上的變異型，包括PreS1區的2989C、3050T、3097A、3174T、PreS2區35A、S區的285A、529G、586C。B基因型序列大多數位置之核苷酸型態和adw序列一致，只有在A2989C、C3050T、C3174T與S區的A529G處具有盛行率在20%以上的變異型。基因型C在整段序列的許多位置，均具有盛行率在20%以上的變異型，尤其在PreS2和S基因區域nt 85-nt 598，變異型頻率可達50%以上，病例組和對照組在頻率分佈上具顯著差異的位置到處可見，高差異區主要分布在PreS2的nt 76-nt 147處，顯著差異位置包括A76C、G85A、G87A、C93T、A96C、A99C 、T105C、T109A和G110C，變異型頻率差異在15.3%-30.40%。這些病例和對照組具顯著差異的位置對B基因型者而言，變異型的頻率均在6.3%以下，但對C基因型者而言，變異型頻率均介於37.1%-62.9%，因此再單就C基因型者分析這些核苷酸位置變異和HCC的關係，結果發現在nt 76-nt 110及nt 619處與HCC仍舊具有邊緣性統計相關。分析兩時點間序列的差異，發現大部分位置均處穩定狀態，只有nt 2898、nt 3174和nt 529具有大於20%以上的突變率。結論：B基因型大致反映adw序列，而C基因型病毒序列在各核苷酸位置和adw序列比較具較多變異。HBV PreS/S及與其重疊P基因區，各核苷酸位置在追蹤期間大致處於相當穩定狀態，以adw為參考序列，病例與對照組比較具變異型頻率較高，且分散在全區域，但高差異區集中在PreS2 區nt 76-nt 110處，和已知〝a〞determinant區域重疊。

Background and Aim: Variations in the viral genome of hepatitis B virus (HBV) has been associated with the clinical outcome of chronic HBV infection, but the significance of these variations in the development of hepatocellular carcinoma (HCC) remains largely unknown. The aim of this study was to longitudinally analyze the nucleotide variations in the PreS/S and overlapping P gene regions of the HBV genome and HCC risk. Materials and Methods: Direct sequencing of HBV gene was performed on plasma samples from 86 cases and 125 controls nested within a cohort of HBV male carriers recruited between 1988-1992. Controls were matched with cases on the age of entering the study and the time of collection of blood samples at baseline. We also performed sequencing of the HBV gene in plasma samples collected from a total of 17 cases and 27 controls during follow-up. At the second time point, for cases, blood samples collected within two years before the onset of HCC were used. For controls, measuring time was selected according to the nearest blood collecting time of cases. Results: Compared with adw as the reference sequence, 8 variants were found to have a frequency of ≧ 20% including 2989C, 3050T, 3097A, 3174T in PreS1 region, 35A in PreS2 region, and 285A, 529G, 586C in S region, at baseline samples of controls, the sequence of genotype B was mostly consistent with the adw sequence, except A2989C, C3050T, C3174T and A529G, which occurred at frequency of ≧ 20%. For genotype C, many positions in S region had variant types with prevalence ≧ 20%, especially at nt 85-nt 598 in PreS/S region, the frequency of variant type was ≧ 50%. Difference in nucleotide substitution between case and control groups was everywhere, high variant region was mainly at nt 76-nt 147 in PreS2, which contained 9 variants (including A76C, G85A, G87A, C93T, A96C, A99C, T105C, T109A and G110C) with frequency differences of 15.3%-30.40%. Differences in positions between case and control groups were lower than 6.3% for genotype B. The corresponding figure for genotype C was 37.1%-62.9%. When analysis was limited to these variant positions were still marginally statistically significantly associated with HCC subjects with genotype C. Comparing blood samples at baseline and those at follow-up, higher rate (defined as ≧ 20%) for nucleotide change during follow-up was only observed at three positions nt 2898,nt 3174 and nt 529. Conclusions: Genotype B was mostly consistent with adw sequence, while genotype C had much more variations compared with adw sequence. In most of the PreS/S and overlapping P gene region, nucleotide change was infrequent during long-term follow-up period. Compared with controls, cases had more variant types and high variant region was mainly at nt 76-nt 110 in PreS2 region, where lies within 〝a〞 determinant of HBsAg.