阿德福韦酯治疗前后乙型肝炎病毒Pre-S区基因突变分析
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摘要
乙型肝炎病毒（hepatitis B virus，HBV）感染是全球性的重要公共卫生问题，严重影响人类健康。HBV逆转录酶缺乏校正功能，在选择压力作用下，HBV在复制过程中会产生大量变异株。Pre-S区是HBVDNA中变异最大的区段，容易出现缺失突变。Pre-S区缺失热点区域为Pre-S1的3'末端和Pre-S2的5'末端，可伴有Pre-S2起始密码子的缺失或点突变，而且在B和C基因型中发生频率高。大量研究证实，Pre-S区缺失突变可作为一个独立危险因素，与肝细胞癌发生具有密切关系。因此有效抑制HBV DNA尤其是Pre-S区缺失突变毒株的复制可能有利于减少HCC的发生。核苷（酸）类似物是临床上抗HBV的常用药物，能够抑制HBV DNA复制，降低血清ALT水平，改善肝组织学。但是有研究表明，Pre-S区缺失突变也可在使用核苷（酸）类似物治疗是出现，而且Pre-S区缺失突变毒株对于这类药物的反应性并不清楚。因此，解决这一疑问是关系到临床用药的安全性问题。
目的：观测阿德福韦酯（ADV）治疗慢性HBV患者基线和48W时血清中HBV DNA Pre-S区突变情况。
方法：提取61位患者基线和48W血清中HBV DNA，用半巢式PCR扩增Pre-S区。PCR产物纯化后，与pMD19-T Simple Vector连接进行TA克隆，随机挑取30个阳性克隆测序。采用我室建立的荧光定量分型PCR方法测定HBV DNA病毒载量。HBsAg、HBeAg、HBeAb采用美国雅培（Abbott）公司和上海科华公司相应试剂盒检测。ALT采用Roche Modular DDP全自动生化分析仪检测。
结果：1、基线时，55.7%（34/61）的患者血清中可检测到Pre-S突变体；ADV治疗48W时，37.7%（23/61）的患者血清中可检测到Pre-S突变体。在有Pre-S区突变感染患者中，除了患者X39在基线时只有突变型外，其余患者在基线和48W时均为突变和野生型混合感染。根据基线时是否有Pre-S突变型感染，将患者分成野生型感染组（W-0）和突变型感染组（M-0）。基线时，2组在性别、年龄、基因型、血清HBV DNA载量和ALT水平上均无统计学差异。ADV治疗48W，M-0组血清HBV DNA病毒载量和ALT水平明显降低，HBeAg阴转率明显升高，有统计学差异。
2、患者X37在ADV治疗48W时，第7号克隆在nt2978和nt2979之间出现一个长度45bp插入片段。此片段与该克隆序列的nt2934-2978完全相同。
3、基线时，61名患者均为HBV B和/或C型感染。ADV治疗48W，C型和B/C混合型感染患者的基因型发生变化。20名C型感染患者，有5名患者的基因型转变为单一的B型，6名患者的基因型转变为B/C混合型。3名B、C混合感染的患者，1名转变为单一C型；1名转变为单一B型；1名仍然是混合型感染，但是B型占阳性克隆比例增加。用药后48W，C型和B/C混合型患者中出现B基因型（即C型减少的患者），HBV病毒载量下降≥2log10的人数比率明显高于48W时单一C型感染的患者，差异具有显著性意义。
4、5位B/C基因型混合感染的患者中出现B/C重组现象，其中一位出现在基线，其余出现在用药48W时。结论：Pre-S区缺失突变的频率较高，使用ADV治疗能够有效抑制缺失突变株。与野生株相比，Pre-S缺失突变株对ADV的治疗可能更敏感。HBV混合基因型感染的比例较高，ADV治疗过程中出现患者基因型变化，结合病毒载量变化，提示基因型C对于ADV治疗更敏感。
Hepatitis B virus (HBV) infection is a major public health problemworldwide. HBV reverse transcriptase lacks a proofreading function whichleads to the emergence of a great deal of genetic variants undermulti-selective pressures. Pre-S region is the most variable part of the HBVgenome. HBV isolates with Pre-S deletion mutations are often found inhepatitis B carriers. Hot spots of mutation include the3’ half of the Pre-S1and the5’ half of the pre-S2, and Pre-S2start codon deletion/pointmutation. The prevalence of Pre-S deletions is higher in genotype B and C.Pre-S deletions, as an independent factor, have been documented to play apotential role in hepatocarcinogenesis. Therefore, effectively suppressingHBV replication, especially the isolates with Pre-S deletions, may help toreduce the incidence of HCC. Nucleot(s)ide analogues (NAs) used to treatHBV infection have been demonstrated clinically to significantly suppressHBV replication, decrease serum ALT level and improve liver histology.Recent studies showed that Pre-S deletions were found during the treatment of NAs. The effects of NAs therapy on Pre-S deletion strains are largelyunknown. These questions related to drug safety require to be resolved.
Objective. To investigate HBV Pre-S mutants in patients with chronichepatitis B infection (CHB) before and after adefovir dipivoxil (ADV)treatment.
Methods. HBV DNA was extracted from the sera of61CHB patientsat baseline and at week48of ADV treatment. Semi-nested PCR wasperformed to amplify HBV Pre-S region. PCR products were purified andligated with pMD19-T Simple Vector at16℃overnight, then transformedinto an E. coli strain DH5α. PCR amplifications of insert-DNA werepurified and sequenced. Quantification of HBV DNA was performed byreal-time genotyping and quantitative PCR. Serological markers includingHBsAg、 HBeAg and HBeAb were determined using commercial kits(Abbott and Kehua). ALT levels were tested using Roche Modular DDPanalyzer.
Results.1. Pre-S mutants were detected in55.7%(34/61) of CHBpatients at baseline, and37.7(23/61) of CHB patients at week48of ADVtreatment. In most patients with Pre-S mutants infection, pre-S mutantscoexisted with the wild-type HBV strain except patient X39. Based on thepresence of Pre-S mutants at baseline, Patients were divided2groups: W-0(wild-type only) and M-0(Pre-S mutants existing). No significance wasdetermined in gender, age, genotype, HBV Viral load, and ALT levels between the2groups. At week48of ADV treatment, HBV Viral load andALT levels were decreased markedly in group M-0compared with groupW-0. Also, HBeAg loss were significantly elevated in group M-0.2. A45bp insertion mutation was investigated in one colony producenamed No.7from patient X37at week48. The inserted site was between nt2978and2979. The inserted segment was same as the nt2934-2978in thisPre-S sequence.3.61CHB patients were infected with genotype B and/or C. Genotypes inCHB patients which infected genotype C and B/C mixture were changed atweek48of ADV treatment. Of20patients infected genotype C,5patientspresented genotype B infection,6patients presented B/C mixture attreatment week48. Of3patients infected B/C mixture, one was changed togenotype C infection; the other was changed to genotype B infection; theremainder presented B/C mixture infection with increased genotype B attreatment week48.4. Recombinations based on Pre-S between genotypes B and C weredetermined in5patients with B/C mixture infection. One patient was foundrecombinations at baseline; the other4were found recombinations attreatment week48.
Conclusion. The prevalence of Pre-S mutants was high in CHBpatients. ADV can effectively suppress Pre-S mutants replication. Pre-Smutation strains may be more sensitive for ADV therapy compared with wild-type strain. Mixed genotype infections were common in CHB patients.Genotype C may be more sensitive for ADV therapy than genotype B.
目的：观测阿德福韦酯（ADV）治疗慢性HBV患者基线和48W时血清中HBV DNA Pre-S区突变情况。
方法：提取61位患者基线和48W血清中HBV DNA，用半巢式PCR扩增Pre-S区。PCR产物纯化后，与pMD19-T Simple Vector连接进行TA克隆，随机挑取30个阳性克隆测序。采用我室建立的荧光定量分型PCR方法测定HBV DNA病毒载量。HBsAg、HBeAg、HBeAb采用美国雅培（Abbott）公司和上海科华公司相应试剂盒检测。ALT采用Roche Modular DDP全自动生化分析仪检测。
结果：1、基线时，55.7%（34/61）的患者血清中可检测到Pre-S突变体；ADV治疗48W时，37.7%（23/61）的患者血清中可检测到Pre-S突变体。在有Pre-S区突变感染患者中，除了患者X39在基线时只有突变型外，其余患者在基线和48W时均为突变和野生型混合感染。根据基线时是否有Pre-S突变型感染，将患者分成野生型感染组（W-0）和突变型感染组（M-0）。基线时，2组在性别、年龄、基因型、血清HBV DNA载量和ALT水平上均无统计学差异。ADV治疗48W，M-0组血清HBV DNA病毒载量和ALT水平明显降低，HBeAg阴转率明显升高，有统计学差异。
2、患者X37在ADV治疗48W时，第7号克隆在nt2978和nt2979之间出现一个长度45bp插入片段。此片段与该克隆序列的nt2934-2978完全相同。
3、基线时，61名患者均为HBV B和/或C型感染。ADV治疗48W，C型和B/C混合型感染患者的基因型发生变化。20名C型感染患者，有5名患者的基因型转变为单一的B型，6名患者的基因型转变为B/C混合型。3名B、C混合感染的患者，1名转变为单一C型；1名转变为单一B型；1名仍然是混合型感染，但是B型占阳性克隆比例增加。用药后48W，C型和B/C混合型患者中出现B基因型（即C型减少的患者），HBV病毒载量下降≥2log10的人数比率明显高于48W时单一C型感染的患者，差异具有显著性意义。
4、5位B/C基因型混合感染的患者中出现B/C重组现象，其中一位出现在基线，其余出现在用药48W时。结论：Pre-S区缺失突变的频率较高，使用ADV治疗能够有效抑制缺失突变株。与野生株相比，Pre-S缺失突变株对ADV的治疗可能更敏感。HBV混合基因型感染的比例较高，ADV治疗过程中出现患者基因型变化，结合病毒载量变化，提示基因型C对于ADV治疗更敏感。
Hepatitis B virus (HBV) infection is a major public health problemworldwide. HBV reverse transcriptase lacks a proofreading function whichleads to the emergence of a great deal of genetic variants undermulti-selective pressures. Pre-S region is the most variable part of the HBVgenome. HBV isolates with Pre-S deletion mutations are often found inhepatitis B carriers. Hot spots of mutation include the3’ half of the Pre-S1and the5’ half of the pre-S2, and Pre-S2start codon deletion/pointmutation. The prevalence of Pre-S deletions is higher in genotype B and C.Pre-S deletions, as an independent factor, have been documented to play apotential role in hepatocarcinogenesis. Therefore, effectively suppressingHBV replication, especially the isolates with Pre-S deletions, may help toreduce the incidence of HCC. Nucleot(s)ide analogues (NAs) used to treatHBV infection have been demonstrated clinically to significantly suppressHBV replication, decrease serum ALT level and improve liver histology.Recent studies showed that Pre-S deletions were found during the treatment of NAs. The effects of NAs therapy on Pre-S deletion strains are largelyunknown. These questions related to drug safety require to be resolved.
Objective. To investigate HBV Pre-S mutants in patients with chronichepatitis B infection (CHB) before and after adefovir dipivoxil (ADV)treatment.
Methods. HBV DNA was extracted from the sera of61CHB patientsat baseline and at week48of ADV treatment. Semi-nested PCR wasperformed to amplify HBV Pre-S region. PCR products were purified andligated with pMD19-T Simple Vector at16℃overnight, then transformedinto an E. coli strain DH5α. PCR amplifications of insert-DNA werepurified and sequenced. Quantification of HBV DNA was performed byreal-time genotyping and quantitative PCR. Serological markers includingHBsAg、 HBeAg and HBeAb were determined using commercial kits(Abbott and Kehua). ALT levels were tested using Roche Modular DDPanalyzer.
Results.1. Pre-S mutants were detected in55.7%(34/61) of CHBpatients at baseline, and37.7(23/61) of CHB patients at week48of ADVtreatment. In most patients with Pre-S mutants infection, pre-S mutantscoexisted with the wild-type HBV strain except patient X39. Based on thepresence of Pre-S mutants at baseline, Patients were divided2groups: W-0(wild-type only) and M-0(Pre-S mutants existing). No significance wasdetermined in gender, age, genotype, HBV Viral load, and ALT levels between the2groups. At week48of ADV treatment, HBV Viral load andALT levels were decreased markedly in group M-0compared with groupW-0. Also, HBeAg loss were significantly elevated in group M-0.2. A45bp insertion mutation was investigated in one colony producenamed No.7from patient X37at week48. The inserted site was between nt2978and2979. The inserted segment was same as the nt2934-2978in thisPre-S sequence.3.61CHB patients were infected with genotype B and/or C. Genotypes inCHB patients which infected genotype C and B/C mixture were changed atweek48of ADV treatment. Of20patients infected genotype C,5patientspresented genotype B infection,6patients presented B/C mixture attreatment week48. Of3patients infected B/C mixture, one was changed togenotype C infection; the other was changed to genotype B infection; theremainder presented B/C mixture infection with increased genotype B attreatment week48.4. Recombinations based on Pre-S between genotypes B and C weredetermined in5patients with B/C mixture infection. One patient was foundrecombinations at baseline; the other4were found recombinations attreatment week48.
Conclusion. The prevalence of Pre-S mutants was high in CHBpatients. ADV can effectively suppress Pre-S mutants replication. Pre-Smutation strains may be more sensitive for ADV therapy compared with wild-type strain. Mixed genotype infections were common in CHB patients.Genotype C may be more sensitive for ADV therapy than genotype B.
引文
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[5] Glebe D, Urban S. Viral and cellular determinants involved in hepadnaviral entry[J].World J Gastroenterol.2007,13(1):22-38.
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[1] Carey WD. The prevalence and natural history of hepatitis B in the21st century[J].Cleve Clin J Med.2009,76Suppl3:S2-5.
[2]Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and currentand emerging prevention and control measures[J]. J Viral Hepat.2004,11(2):97-107.
[3] Beasley RP, Hwang L-Y, Lin C-C, et al. Hepatocellular carcinoma and hepatitis Bvirus: a prospective study of22,707men in Taiwan[J]. Lancet.1981,2(8256):1129-1133
[4] Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virusinfection[J]. Nat Rev Immunol.2005,5(3):215-229.
[5] Glebe D, Urban S. Viral and cellular determinants involved in hepadnaviral entry[J].World J Gastroenterol.2007,13(1):22-38.
[6] Chen BF, Liu CJ, Jow GM, et al. High prevalence and mapping of pre-S deletion inhepatitis B virus carriers with progressive liver diseases[J]. Gastroenterology.2006,130(4):1153-1168.
[7] Zoulim F. Mechanism of viral persistence and resistance to nucleoside andnucleotide analogs in chronic hepatitis B virus infection[J]. Antiviral Res.2004,64(1):1-15.
[8] Bowyer SM, Sim JG. Relationships within and between genotypes of hepatitis Bvirus at points across the genome: footprints of recombination in certain isolates[J].J Gen Virol.2000;81(Pt2):379-392.
[9] Shen FC, Su IJ, Wu HC, et al. A pre-S gene chip to detect pre-S deletions in hepatitisB virus large surface antigen as a predictive marker for hepatoma risk in chronichepatitis B virus carriers[J]. J Biomed Sci.2009,16:84.
[10] Fan YF, Lu CC, Chen WC, et al. Prevalence and significance of hepatitis B virus(HBV) pre-S mutants in serum and liver at different replicative stages of chronicHBV infection[J]. Hepatology.2001,33(1):277-286.
[11] Fang ZL, Sabin CA, Dong BQ, et al. Hepatitis B virus pre-S deletion mutations area risk factor for hepatocellular carcinoma: a matched nested case-control study[J]. JGen Virol,2008,89(11):2882-2890.
[12] Chen BF, Liu CJ, Jow GM, et al. High prevalence and mapping of pre-S deletion inhepatitis B virus carriers with progressive liver diseases[J]. Gastroenterology.2006,130(4):1153-1168.
[13] Huy TT, Ushijima H, Win KM, et al. High prevalence of hepatitis B virus pre-smutant in countries where it is endemic and its relationship with genotype andchronicity. J Clin Microbiol.2003,41(12):5449-5455.
[14] Sugauchi F, Ohno T, Orito E, et al. Influence of hepatitis B virus genotypes on thedevelopment of preS deletions and advanced liver disease[J]. J Med Virol.2003,70(4):537-44.
[15] Zoulim F. Mechanism of viral persistence and resistance to nucleoside andnucleotide analogs in chronic hepatitis B virus infection[J]. Antiviral Res.2004,64(1):1-15.
[16] Yeung P, Wong DK, Lai CL, et al. Association of hepatitis B virus pre-S deletionswith the development of hepatocellular carcinoma in chronic hepatitis B[J]. J InfectDis.2011,203(5):646-654.
[17] Lin CL, Liu CH, Chen W, et al. Association of pre-S deletion mutant of hepatitis Bvirus with risk of hepatocellular carcinoma[J]. J Gastroenterol Hepatol.2007,22(7):1098-1103.
[18] Huang HP, Hsu HY, Chen CL, et al. Pre-S2deletions of hepatitis B virus andhepatocellular carcinoma in children[J]. Pediatr Res.2010,67(1):90-94.
[19] Choi MS, Kim DY, Lee DH, et al. Clinical significance of pre-S mutations inpatients with genotype C hepatitis B virus infection[J]. J Viral Hepat.2007,14(3):161-168.
[20] Cao Z, Bai X, Guo X, et al. High prevalence of hepatitis B virus pre-S mutationand its association with hepatocellular carcinoma in Qidong, China[J]. Arch Virol.2008,153(10):1807-1812.
[21] Lin CM, Wang GM, Jow GM, et al. Functional analysis of hepatitis B virus pre-Sdeletion variants associated with hepatocellular carcinoma[J]. J Biomed Sci.2012,19(1):17.
[22] Pollicino T, Amaddeo G, Restuccia A, et al. Impact of hepatitis B virus (HBV)preS/S genomic variability on HBV surface antigen and HBV DNA serum levels[J].Hepatology.2012Jan23. doi:10.1002/hep.25592.
[23] Di Bisceglie AM. Hepatitis B and hepatocellular carcinoma[J]. Hepatology.2009,49(5Suppl):S56-60.
[24] Mun HS, Lee SA, Jee Y, et al. The prevalence of hepatitis B virus preS deletionsoccurring naturally in Korean patients infected chronically with genotype C[J]. JMed Virol.2008,80(7):1189-1194.
[25] Su IJ, Wang HC, Wu HC, et al. Ground glass hepatocytes contain pre-S mutantsand represent preneoplastic lesions in chronic hepatitis B virus infection[J]. JGastroenterol Hepatol.2008Aug;23(8Pt1):1169-74.
[26] Wang HC, Wu HC, Chen CF, et al. Different types of ground glass hepatocytes inchronic hepatitis B virus infection contain specific pre-S mutants that may induceendoplasmic reticulum stress[J]. Am J Pathol.2003,163(6):2441-2449.
[27] Bl ckberg J, Kidd-Ljunggren K. Mutations within the hepatitis B virus genomeamong chronic hepatitis B patients with hepatocellular carcinoma[J]. J Med Virol.2003,71(1):18-23.
[28] Hsieh YH, Su IJ, Wang HC, et al. Pre-S mutant surface antigens in chronichepatitis B virus infection induce oxidative stress and DNA damage [J].Carcinogenesis.2004,25(10):2023-2032.
[29] Yang JC, Teng CF, Wu HC, et al. Enhanced expression of vascular endothelialgrowth factor-A in ground glass hepatocytes and its implication in hepatitis B virushepatocarcinogenesis[J]. Hepatology.2009,49(6):1962-1971.
[30] Hsieh YH, Su IJ, Wang HC, et al. Hepatitis B virus pre-S2mutant surface antigeninduces degradation of cyclin-dependent kinase inhibitor p27Kip1through c-Junactivation domain-binding protein1[J]. Mol Cancer Res.2007,5(10):1063-1072.
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