乙型肝炎病毒反转录酶编码区核苷酸变异与肝癌的相关性研究_高付敏

DOI: 10.3781/j.issn.1000-7431.2014.02.Copyright© 2014 by TUMOR乙型肝炎病毒反转录酶编码区核苷酸变异与肝癌的相关性研究高付敏1, 2，吴　燕2，赵芝梅2，金　晏2，钱耕荪2，陈陶阳3，张亚玮3，王金兵3，甘　愉2，项永兵4，屠　红21. 复旦大学公共卫生学院流行病学教研室，上海　200032；2. 上海交通大学附属仁济医院上海市肿瘤研究所癌基因及相关基因国家重点实验室，上海　200032；3. 启东市人民医院/启东肝癌防治研究所病因室，江苏 启东　226200；4. 上海交通大学附属仁济医院上海市肿瘤研究所流行病室，上海　200032[摘要]　目的：探讨乙型肝炎病毒（hepatitis B virus ，HBV ）-多聚酶（polymerase ，P ）基因反转录酶（reverse transcriptase ，RT ）区突变与肝癌发生的关系。

方法：收集202例肝癌患者及202例年龄、性别匹配的慢性HBV 携带者（对照组）的血清，采用病例-对照的方法进行研究；PCR 法扩增HBV -P 基因的RT 区，对扩增产物直接测序并进行序列分析，探讨该区域基因突变与肝癌发生的关系。

结果：HBV -P 基因RT 区共有3个位点发生突变T895A 、A904T 和C955T ，它们在肝癌组中的发生率均显著高于对照组，分别为47.5% vs 37.1%（P ＝0.034）、5.4% vs 1.0%（P ＝0.011）和5.4% vs 1.5%（P ＝0.030）。

Logistic 多因素分析结果显示，T895A 是肝癌的独立危险因素[比值比（odds ratio ，OR ）＝2.230，95%可信区间（confidence interval ，CI ）：1.230～4.044，P ＝0.008]。

A904T 和C955T 虽有增加肝癌风险的趋势（OR ＝6.523，95% CI ：0.838～50.733和OR ＝2.904，95% CI ：0.599～14.093），但差异无统计学意义。

然而，A904T 和C955T 中有一项为阳性的发生率在肝癌组和对照组中分别是9.4%和2.5% （P ＝0.003），经多因素调整后统计显示，与肝癌具有相关性（OR ＝4.145，95% CI ：1.170～14.681，P ＝0.028）。

结论：HBV -P 基因RT 区存在着与肝癌发生相关的突变。

[关键词]　肝肿瘤；肝炎病毒，乙型；DNA 突变分析；流行病学方法[中图分类号]　R735.7[文献标志码] A[文章编号]　1000-7431 (2014) 02-0141-06The association between mutations in the reverse transcriptase domain of hepatitis B virus polymerase gene with tumorigenesis of hepatocellular carcinomaGAO Fu-min 1, 2, WU Yan 2, ZHAO Zhi-mei 2, JIN Yan 2, QIAN Geng-sun 2, CHEN Tao-yang 3, ZHANG Ya-wei 3,WANG Jin-bing 3, GAN Yu 2, XIANG Yong-bing 4, TU Hong 21. Department of Epidemiology, School of Public Health, Fudan University, Shanghai 200032, China;2. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200032, China;3. Department of Etiology, Qidong People’s Hospital, Qidong Liver Cancer Institute, Qidong 226200, Jiangsu Province, China;4. Department of Epidemiology, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200032, China[ABSTRACT]　Objective: To explore the relationship between the mutations in the reverse transcriptase (RT) domain of hepatitis B virus polymerase (HBV -P ) gene and the occurrence and development of hepatocellular carcinoma (HCC). Methods: In this case-control study, the serum samples from 202 HCC patients and 202 chronic hepatitis B virus (CHB) carriers matching for age and gender were collected. The sequence of the RT domain of HBV -P gene was determined by direct sequencing following PCR ampli fi cation. The relationship between the mutations in the RT domain of HBV -P gene and the occurrence and development of HCC was analyzed. Results: The T895A, A904T and C955T mutations in the RT domain of HBV -P gene were all signi fi cantly associated with HCC compared to non-HCC controls (P = 0.034, 47.5% vs 37.1%; P = 0.011, 5.4% vs 1.0%; P = 0.030, 5.4% vs 1.5%). Furthermore, the Logistic multivariate analysis showed that T895A was an independent risk factor for HCC [odds ratio (OR ) = 2.230, 95% con fi dence interval (CI ): 1.230-4.044, P = 0.008]. A904T or C955T increased the risk of HCC occurrence either (OR = 6.523, 95% CI : 0.838-50.733; OR = 2.904,[基金项目] 传染病国家重大专项基金资助项目（编号：2012ZX10002-008-002）Correspondence to: TU Hong （屠　红） E-mail: tuhong@Received 2013-12-02 Accepted 2014-01-10007肿瘤2014年2月第34卷第2期　TUMOR Vol. 34, February 2014 141临床研究・Clinical Research95% CI: 0.599-14.093), but it did not reach statistical signifi cance. The mutation rate of combined mutation occurrence either in A904 or C955T was 9.4% in HCC group and 2.5% in non-HCC controls (P = 0.003). The adjusted OR was 4.145 (95% CI: 1.170-14.681), demonstrating its signifi cant association with HCC (P = 0.028). Conclusion: The mutations in the RT domain of HBV-P gene are associated with the tumorigenesis of HCC. [KEY WORDS]　Liver neoplasms; Hepatitis B virus; DNA mutational analysis; Epidemiologic methods[TUMOR, 2014, 34 (02): 141-146]乙型肝炎病毒（hepatitis B virus，HBV）慢性感染是导致我国肝癌（hepatocellular carcinoma，HCC）发生高的首要因素[1]。

由于HBV基因复制需要经过一特殊的反转录过程，而其反转录酶（reverse transcriptase，RT）又缺乏校读功能，因此HBV基因组常处于高突变状态。

一般认为，活动性乙肝患者体内每天HBV基因组的点突变频率可高达1010～1011[2]。

HBV基因组中的突变包括：前S区缺失突变 [3, 4]，基本核心启动/增强子Ⅱ区（basal core promoter/enhancer Ⅱ，BCP/EhnⅡ）中C1653T、T1753V和A1762T/ G1764A的双突变[5, 6]，前C/C区中G1896A、G1899A、A2159G和A2189C等基因的突变[7]，且以上突变均能增加肝癌发病的风险。

迄今为止，有关HBV-多聚酶（polymerase，P）基因区变异的报道多集中在核苷类似物抗病毒治疗诱导的耐药性突变，如在拉米夫定和替比夫定用药过程中产生的rtM204Ⅴ/Ⅰ突变，即酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸（tyrosine-methionine-aspartate-aspartate，YMDD）突变[8]等，而关于其在自然状态下的突变与肝癌发生关系的研究尚未见报道。