重症乙型肝炎HBV DNA CP 区及前S区基因变异与河南地区HBV DNA 基因型的研究
摘要
乙型肝炎病毒(HBV)是一种DNA病毒，人类感染HBV后可以表现为不同的临床类型：无症状感染者、急性、慢性、重症乙型肝炎等。其中重症乙型肝炎患者病情凶险，是HBV感染后的重要转归之一，其病死率高，发病机制尚未清楚。现代研究认为重症乙型肝炎的发生，除了和宿主因素有关外，病原学因素是重要影响因素之一。HBV在感染过程中常发生基因变异，由此可导致感染的慢性化和重症化。此外，研究表明：病毒基因型反映了HBV自然感染史发生的变异特点，HBV的基因型可能与感染途径、疾病的进展等有一定的相关性。
目的 本文分两部分①以HBV DNA C基因启动子(CP)区及前S区为研究对象，研究重症乙型肝炎患者HBV DNA发生基因变异时有无位点特异性，探索HBV基因变异在重症乙型肝炎发病中所起的作用 ②应用巢式PCR技术，研究河南地区重症乙型肝炎和慢性乙型肝炎患者中HBV DNA基因型及各型比例，不同基因型与乙型肝炎重症化的相关性，进一步分析HBV DNA基因型、HBV基因变异与乙型肝炎重症化之间的相互关系。
方法
(1)自16例重症乙型肝炎患者的血清提取HBV DNA，聚合酶链反应(PCR)扩增，PCR产物聚丙烯酰胺凝胶电泳。随机选取可能存在变异的3份PCR扩增产物，
郑州大学医学花忆
硕士毕业论文(2004)
提纯后核昔酸序列测定，计算机软件分析基因序列的同源性、分析由基因序列推测的
氨基酸序列的同源性。
(2)自同一批16例重症乙型肝炎患者及50例慢性乙型肝炎患者血清提取HBV
DNA，应用特异性引物，巢式PCR扩增目的片段，琼脂糖电泳，分析不同组乙型肝
炎患者的HBV DNA基因型。
结果
(1)16份重症乙型肝炎患者的血清标本的HBV DNA CP区，各条带大小一致，
未发现基因缺失变异;前S区PCR扩增产物聚丙烯酞胺凝胶电泳，存在多个水平大
小不一的片段，选取3份标本进行同源性分析，其同源性>80%。由基因序列推测的
氨基酸序列的同源性为75.4%，未发现特殊突变位点和聚集性突变。
(2)重症乙型肝炎患者的血清标本中HBV均为B基因型，占100%，50例慢性
乙型肝炎患者有7例为B型，占14%(7/50)，其余为B、C混合型占86%(4 3/50)
结论
(l)基因变异在重症乙型肝炎患者的HBV DNA的前S区随机出现，主要集中
在前S;区下半段、前52区，但不具位点特异性和聚集性突变，而1侣V DNA CP区
未发现明显缺失突变，但在HBV DNA其他区域是否存在特征性变异不详。
(2)河南地区重症乙型肝炎患者HBV DNA以B基因型为主，而慢性乙型肝炎
患者以B、C混合型为主，HBV DNAB基因型和重症乙型肝炎关系密切。
(3)重症乙型肝炎患者所感染B基因型的HBV基因中，CP区和前S区基因变
异无位点特异性和聚集性突变。重症乙型肝炎患者感染的其他基因型别的HBv基因
变异是否具有相似突变特征和相关性，有待于进一步探索。
Hepatitis B is a kind of DNA virus. People who are infected with HBV could present with different clinical outcome: asymptomatic carriers, acute hepatitis, chronic hepatitis B, and fulminant hepatitis B. Patients with fulminant hepatitis B always develop severe liver disease and result in worse therapeutic effect and the mortality is very high. Yet, it's possible mechanisms are still unknown. Nowadays we think that the pathogeny plays an important role in the clinical course of fulminant hepatitis B besides the host's factors. HBV gene mutation is a kind of common phenomenon that could induce chronic and fulminant hepatitis B.Furthermore the genotype of HBV reflects the selection process behind gene variation.Furthermore ,it suggests that the genotypes of HBV have relation to the infection approach and the clinical course of hepatitis B.
OBJECTIVE: (1)to study the CP and Pre-S gene variation in patients with fulminant hepatitis B and accordingly to investigate the relationship between HBV DNA gene variation and the occurrence of fulminant hepatitis B; (2) to investigate the proportion of the genotypes in chronic hepatitis B and fulminant hepatitis B in henan and to find the relationship between different genotypes, HBV gene mutation and the occurrence of fulminant hepatitis.
METHOD: (1)HBV DNA extracted from fulminant hepatitis B patients were
subjected to polymerase chain reaction and then polyacrylamide gel electrophoresis. Three different kinds of PCR products were analyzed by gene sequencing method and specific computer software was used to analyze the homology of these gene sequences ￤ith specific primers and HBV DNA extracted from the serum of the fulminant hepatitis B and chronic hepatitis B patients, we amplified the objective gene using nested-PCR and analyzed the genotypes of every samples through agarose electrophoresis.
RESULT: (1)we found the sequence hi the CP gene of 16 samples are of same length, but after polyacrylamide gel electrophoresis different length sequences in the Pre-S gene of 16 samples was detected. After analyzing the gene sequence, we found various kinds of gene variation apart from the apparent deletion mutation and the homology was more than 80% (2)All the samples from the fulminant hepatitis B were genotyped as B (100%). Among the samples from chronic hepatitis B, seven were genotyped as B (14%) and the other 43 samples were genotyped as B and C (86%).
CONCLUSION: (1) The gene variation was randomly occurred in Pre-S gene of HBV DNA getted from patients with fulminant hepatitis B , especially in the right end Pre-S i ^ Pre-S2;we didn't find deletion mutation in the CP gene. Whether there was other characteristic gene variation occurred in other sequences of HBV DNA was unknown.
(2) The most common viral genotype was B in the patients of henna with fulminant hepatitis B; B and C were the most prevalent genotypes in the patients with chronic hepatitis B. This suggested that the genotype B were associated with fulminant hepatitis B.
(3) In patients with fulminant hepatitis B, the gene mutation occurred hi CP and Pre-S sequences had no specificity and HBV DNA was all genotyped as B. Further investigation is needed on the characteristics of gene mutation of other genotypes hi the patients with fulminant hepatitis B.
目的 本文分两部分①以HBV DNA C基因启动子(CP)区及前S区为研究对象，研究重症乙型肝炎患者HBV DNA发生基因变异时有无位点特异性，探索HBV基因变异在重症乙型肝炎发病中所起的作用 ②应用巢式PCR技术，研究河南地区重症乙型肝炎和慢性乙型肝炎患者中HBV DNA基因型及各型比例，不同基因型与乙型肝炎重症化的相关性，进一步分析HBV DNA基因型、HBV基因变异与乙型肝炎重症化之间的相互关系。
方法
(1)自16例重症乙型肝炎患者的血清提取HBV DNA，聚合酶链反应(PCR)扩增，PCR产物聚丙烯酰胺凝胶电泳。随机选取可能存在变异的3份PCR扩增产物，
郑州大学医学花忆
硕士毕业论文(2004)
提纯后核昔酸序列测定，计算机软件分析基因序列的同源性、分析由基因序列推测的
氨基酸序列的同源性。
(2)自同一批16例重症乙型肝炎患者及50例慢性乙型肝炎患者血清提取HBV
DNA，应用特异性引物，巢式PCR扩增目的片段，琼脂糖电泳，分析不同组乙型肝
炎患者的HBV DNA基因型。
结果
(1)16份重症乙型肝炎患者的血清标本的HBV DNA CP区，各条带大小一致，
未发现基因缺失变异;前S区PCR扩增产物聚丙烯酞胺凝胶电泳，存在多个水平大
小不一的片段，选取3份标本进行同源性分析，其同源性>80%。由基因序列推测的
氨基酸序列的同源性为75.4%，未发现特殊突变位点和聚集性突变。
(2)重症乙型肝炎患者的血清标本中HBV均为B基因型，占100%，50例慢性
乙型肝炎患者有7例为B型，占14%(7/50)，其余为B、C混合型占86%(4 3/50)
结论
(l)基因变异在重症乙型肝炎患者的HBV DNA的前S区随机出现，主要集中
在前S;区下半段、前52区，但不具位点特异性和聚集性突变，而1侣V DNA CP区
未发现明显缺失突变，但在HBV DNA其他区域是否存在特征性变异不详。
(2)河南地区重症乙型肝炎患者HBV DNA以B基因型为主，而慢性乙型肝炎
患者以B、C混合型为主，HBV DNAB基因型和重症乙型肝炎关系密切。
(3)重症乙型肝炎患者所感染B基因型的HBV基因中，CP区和前S区基因变
异无位点特异性和聚集性突变。重症乙型肝炎患者感染的其他基因型别的HBv基因
变异是否具有相似突变特征和相关性，有待于进一步探索。
Hepatitis B is a kind of DNA virus. People who are infected with HBV could present with different clinical outcome: asymptomatic carriers, acute hepatitis, chronic hepatitis B, and fulminant hepatitis B. Patients with fulminant hepatitis B always develop severe liver disease and result in worse therapeutic effect and the mortality is very high. Yet, it's possible mechanisms are still unknown. Nowadays we think that the pathogeny plays an important role in the clinical course of fulminant hepatitis B besides the host's factors. HBV gene mutation is a kind of common phenomenon that could induce chronic and fulminant hepatitis B.Furthermore the genotype of HBV reflects the selection process behind gene variation.Furthermore ,it suggests that the genotypes of HBV have relation to the infection approach and the clinical course of hepatitis B.
OBJECTIVE: (1)to study the CP and Pre-S gene variation in patients with fulminant hepatitis B and accordingly to investigate the relationship between HBV DNA gene variation and the occurrence of fulminant hepatitis B; (2) to investigate the proportion of the genotypes in chronic hepatitis B and fulminant hepatitis B in henan and to find the relationship between different genotypes, HBV gene mutation and the occurrence of fulminant hepatitis.
METHOD: (1)HBV DNA extracted from fulminant hepatitis B patients were
subjected to polymerase chain reaction and then polyacrylamide gel electrophoresis. Three different kinds of PCR products were analyzed by gene sequencing method and specific computer software was used to analyze the homology of these gene sequences ￤ith specific primers and HBV DNA extracted from the serum of the fulminant hepatitis B and chronic hepatitis B patients, we amplified the objective gene using nested-PCR and analyzed the genotypes of every samples through agarose electrophoresis.
RESULT: (1)we found the sequence hi the CP gene of 16 samples are of same length, but after polyacrylamide gel electrophoresis different length sequences in the Pre-S gene of 16 samples was detected. After analyzing the gene sequence, we found various kinds of gene variation apart from the apparent deletion mutation and the homology was more than 80% (2)All the samples from the fulminant hepatitis B were genotyped as B (100%). Among the samples from chronic hepatitis B, seven were genotyped as B (14%) and the other 43 samples were genotyped as B and C (86%).
CONCLUSION: (1) The gene variation was randomly occurred in Pre-S gene of HBV DNA getted from patients with fulminant hepatitis B , especially in the right end Pre-S i ^ Pre-S2;we didn't find deletion mutation in the CP gene. Whether there was other characteristic gene variation occurred in other sequences of HBV DNA was unknown.
(2) The most common viral genotype was B in the patients of henna with fulminant hepatitis B; B and C were the most prevalent genotypes in the patients with chronic hepatitis B. This suggested that the genotype B were associated with fulminant hepatitis B.
(3) In patients with fulminant hepatitis B, the gene mutation occurred hi CP and Pre-S sequences had no specificity and HBV DNA was all genotyped as B. Further investigation is needed on the characteristics of gene mutation of other genotypes hi the patients with fulminant hepatitis B.
引文
1.Jin lin hou,Yulong Lin,Jenny Waters et al.Detection and "significance of a G1862T variant of hepatitis B virus in Chinese patients with fulminant hepatitis .J of General Virology,2002,83:2291-2298.
2.K.Takahashi ,K.Aoyama,N.Ohno et al.The precore/core promoter mutant(T1762A1764) of hepatitis B virus :Clinical significance and an easy method for detection .J of general virology, 1995,76:3159-3164.
3.Li W, Ikematsu H, Yamaji TK, et al.Hepatitis B virus genomes of chronic hepatitis patients do not contain specific mutations related to acute exacerbation.Dig Dis Sci, 2001;46:2104-12.
4.Komatsu H, Inui A, Morinishi Y, et al.Sequence analysis of hepatitis B virus genomes from an infant with acute severe hepatitis and a hepatitis B e antigen-positive carrier mother.J Med Virol,2001 ;65:457-62.
5.Pollicino T, Zanetti AR ,Cacciola I.Pre-S_2 defective hepatitis B virus infection in patients with fulminant hepatitis .Hepatology,1997, 26:495-499.
6.Sterneck M, Kalinina T, Otto S, et al.Neonatal fulminant hepatitis B: structural and functional analysis of complete hepatitis B virus genomes from mother and infant.J Infect
Dis,1998;177:1378-1381.
7．阎丽。乙型肝炎病毒基因型研究及其临床意义。国外医学·流行病传染病学分册，2000：27：254-258。
8.Huy TT, Ushijima H, Win KM,et al.High prevalence of hepatitis B virus pre-s mutant in countries where it is endemic and its relationship with genotype and chronicity.J Clin Microbiol.2003;41(12):5449-5455.
9.Friedt M, Gemer P, Lausch E,et al .Mutation in the basic core promoter and precore region of hepatitis B virus and their selection in children with fulminant and chronic hepatitis B.Hepatology,1999; 29:1252-1258.
10.Mayerat C,Mantegani A,Frei PC,et al.Does hepatitis B virus (HBV)genotypes influence the clinical outcome of HBV infection .J Viral Hepat ,1999; 6:299-304.
11.Pult L Chouard T Wieland Set al.A hepatitis B virus mutant with a new hepatocyte nuclear factor 1 binding site emerging in transplant-transmitted fulminant hepatitis B.Hepatology, 1997;25:1507-1515.
12．骆抗先。乙型肝炎基础和临床。北京：人民卫生出版社。2001，第二版。
13.Chen IH,Huang CJ,Ting LROverlapping initiator and TATA box functions in the basal core promoter ofhepatits B vires .J virol ,1995; 69:3647-3657.
14.Luo kang xian.Hot spot mutations of hepatitis B virus pre-c/c gene and its promoter in Chinese patients and the clinical implications.Chinese medical journal,1999;112(2): 182-184.
15．董菁 成军 王勤环等。乙型肝炎病毒C基因启动子区准种与变异特点的研究。中华实验和临床病毒学杂志，2002；16：264-266。
16.Li J, Buckwold VE, Hon MW ,et al.Mechanism of suppession of hepatitis B virus precore RNA transcription by a frequent double mutation .J virol, 1999;73:1239-1244.
17.Lenhoff RJ, Luscombe CA,Summers J,et al.Acute liver injury following infection.with a cytopathic strain of duck hepatitis B .Hepatology, 1999;29(2):563-571.
18．张瑞 顾长海 张绪清。HBV前S_1／S_2基因变异所致大、中、小蛋白比例变化与肝损伤的关系。中华肝脏病杂志，1999；7：146-148。
19.Huangfu J, Dong J, Deng H,et al.A preliminary study on the heterogeneity of PreS_2
region in hepatitis B virus .Zhonghua Nei Ke Za Zhi.2002; 41(4):233-6.
2.K.Takahashi ,K.Aoyama,N.Ohno et al.The precore/core promoter mutant(T1762A1764) of hepatitis B virus :Clinical significance and an easy method for detection .J of general virology, 1995,76:3159-3164.
3.Li W, Ikematsu H, Yamaji TK, et al.Hepatitis B virus genomes of chronic hepatitis patients do not contain specific mutations related to acute exacerbation.Dig Dis Sci, 2001;46:2104-12.
4.Komatsu H, Inui A, Morinishi Y, et al.Sequence analysis of hepatitis B virus genomes from an infant with acute severe hepatitis and a hepatitis B e antigen-positive carrier mother.J Med Virol,2001 ;65:457-62.
5.Pollicino T, Zanetti AR ,Cacciola I.Pre-S_2 defective hepatitis B virus infection in patients with fulminant hepatitis .Hepatology,1997, 26:495-499.
6.Sterneck M, Kalinina T, Otto S, et al.Neonatal fulminant hepatitis B: structural and functional analysis of complete hepatitis B virus genomes from mother and infant.J Infect
Dis,1998;177:1378-1381.
7．阎丽。乙型肝炎病毒基因型研究及其临床意义。国外医学·流行病传染病学分册，2000：27：254-258。
8.Huy TT, Ushijima H, Win KM,et al.High prevalence of hepatitis B virus pre-s mutant in countries where it is endemic and its relationship with genotype and chronicity.J Clin Microbiol.2003;41(12):5449-5455.
9.Friedt M, Gemer P, Lausch E,et al .Mutation in the basic core promoter and precore region of hepatitis B virus and their selection in children with fulminant and chronic hepatitis B.Hepatology,1999; 29:1252-1258.
10.Mayerat C,Mantegani A,Frei PC,et al.Does hepatitis B virus (HBV)genotypes influence the clinical outcome of HBV infection .J Viral Hepat ,1999; 6:299-304.
11.Pult L Chouard T Wieland Set al.A hepatitis B virus mutant with a new hepatocyte nuclear factor 1 binding site emerging in transplant-transmitted fulminant hepatitis B.Hepatology, 1997;25:1507-1515.
12．骆抗先。乙型肝炎基础和临床。北京：人民卫生出版社。2001，第二版。
13.Chen IH,Huang CJ,Ting LROverlapping initiator and TATA box functions in the basal core promoter ofhepatits B vires .J virol ,1995; 69:3647-3657.
14.Luo kang xian.Hot spot mutations of hepatitis B virus pre-c/c gene and its promoter in Chinese patients and the clinical implications.Chinese medical journal,1999;112(2): 182-184.
15．董菁 成军 王勤环等。乙型肝炎病毒C基因启动子区准种与变异特点的研究。中华实验和临床病毒学杂志，2002；16：264-266。
16.Li J, Buckwold VE, Hon MW ,et al.Mechanism of suppession of hepatitis B virus precore RNA transcription by a frequent double mutation .J virol, 1999;73:1239-1244.
17.Lenhoff RJ, Luscombe CA,Summers J,et al.Acute liver injury following infection.with a cytopathic strain of duck hepatitis B .Hepatology, 1999;29(2):563-571.
18．张瑞 顾长海 张绪清。HBV前S_1／S_2基因变异所致大、中、小蛋白比例变化与肝损伤的关系。中华肝脏病杂志，1999；7：146-148。
19.Huangfu J, Dong J, Deng H,et al.A preliminary study on the heterogeneity of PreS_2
region in hepatitis B virus .Zhonghua Nei Ke Za Zhi.2002; 41(4):233-6.