乙型慢加急性肝衰竭患者HBV基因突变的研究
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摘要
目的既往有研究提示HBV核苷酸突变可能和乙型肝炎患者的病情加重有关,但与慢性乙型肝炎患者慢加急性肝衰竭(ACLF)之间的关系还不明确,少数研究提示可能和前C区的突变有一定的关系,但仍没有定论。因此需要进一步研究以明确HBV突变与ACLF是否相关,并发现与ACLF相关的新的HBV核苷酸突变位点。
方法本试验采用回顾性研究,纵向及横向对比的研究方法。
(1)纵向对比研究：选取慢性HBV感染者6名。其中,4名为ACLF患者(P1-P4),2名为肝硬化(LC)患者(P5、P6)。P1一P4选取ACLF发生前和ACLF发生时血清标本各1份；P5、P6各留取慢性乙型肝炎(CHB)阶段和LC阶段血清标本各1份。对每位患者的2份标本进行HBV全基因组PCR扩增。PCR产物构建质粒,每份标本选取3-4个阳性克隆转化感受态细胞扩增后测序。
(2)横向对比研究：选取于慢性HBV感染者166例作横向对比研究。其中ACLF49人,CHB 45人,LC 45人,肝细胞癌(HCC)27人。对每位患者血清标本进行提取HBV DNA,进行BCP/前C区PCR扩增,扩增产物直接测序。通过Vector NTI Suite 9.0软件对序列测定结果进行比对,找出与ACLF有关的核苷酸突变位点。
结果(1)纵向研究提示：与LC患者比较,ACLF患者在疾病发展过程中出现更多的核苷酸突变。从突变的位置看,ACLF患者核苷酸突变一致且集中的区域主要位于前C基因内。4名ACLF患者中,有2名或2名以上患者可见的共同突变位点有：nt 53、nt1846和nt1896突变。C区1913位在P2也见有纵向突变,横向比较见P1、P2均见有此位点突变,而P5、P6均未发现。
(2)横向研究提示：nt1753、nt1762、nt1764、nt1846、nt1896和nt1913位核苷酸突变率在CHB、LC、ACLF和HCC四组间有统计学差异(P均<0.01)但在ACLF组与LC组之间,仅nt1846和nt1913突变有显著统计学意义(P均<0.01)；在ACLF组与HCC组之间,nt1762、nt1846和nt1913突变有统计学意义(P均<0.05)。联合突变分析提示：最常见的是A1762T/G1764A双突变,阳性率高达70.5%(117/166)；其它常见的两联突变还有A1846T/G1896A[25.9%(43/166)]、A1846T/C1913(A or G)[18.7%(31/166)]和A1752G/C1799G[10.8%(18/166)]等。在多位点联合突变中,上述突变组合经常一同出现,组成更为复杂的突变模式。突变数量与疾病进展有平行关系,ACLF患者可见更多的联合突变。突变对HBV DNA水平的影响：nt1753、nt1764、nt1846、nt1896和nt1899突变的患者HBV DNA水平要低于野生株患者；并且联合突变者HBV DNA水平更低。与HBeAg状态有关的突变位点有G1896A、G1899A、T1753(G或C)、A1846T、C1913(A或G)和G1915(A或C)。核苷酸突变与基因型的关系：nt1752、nt1799、nt1846三个位点的变异率在B型患者中高于C型；nt1753、nt1762、nt1764、nt1915的变异率在C型患者中高于B型。BCP/前C核苷酸突变率在以CHB为基础和以LC为基础的ACLF患者中无显著性差异。多因素分析结果提示nt1913(C→A or G)和nt1846(A→T)突变是预测ACLF的有效因素(比数比分别为3.875和3.151,P值分别为0.017和0.041)。
结论HBV BCP/前C区核苷酸突变与疾病进展密切相关。A1846T、G1896A和C1913(A或G)突变可能与ACLF密切相关,其中A1846T和C1913(A或G)突变是ACLF的独立预测因素。
Objective Previous studies suggested that HBV nucleotide mutations may be related to exacerbations of hepatitis B hepatitis, but the relationship between the occurrence of ACLF and HBV mutations in patients with chronic hepatitis B is not clear. The existing research suggested the mutations in pre-C region may be involved in occurrence of liver failure, but not conclusive. Hence the need for further research to clarify whether HBV mutations associated with ACLF and to identify new mutations associated with ACLF.
Methods The longitudinal and cross-sectional studies were employed. All serum samples were from serum bank of 302 hospital stored at-80℃.
Longitudinal comparison:Serum samples from the pre-ACLF stage, as well as during the occurrence of ACLF were obtained from 4 patients (P1-P4); we also collected serum samples from chronic hepatitis stage and liver cirrhosis stage from 2 patients (P5, P6) as control. HBV DNA was extracted and amplified and ligated into modified pCR2.1 plasmid. Of each individual,3-4 cloned HBV DNAs were sequenced and the sequences were aligned between pre-ACLF and post-ACLF and /or pre-Cirrhosis and post Cirrhosis.
Cross sectional comparison:HBV DNAs were extracted from serum samples from 166 patients including ACLF(n=46), chronic hepatitis (CHB n=45) 45, LC(n=65), HCC(n=27), and BCP/Pre-C and C region was sequenced and analyzed.
Results⑴Longitudinal analysis showed that there were more longitudinal nucleotide mutations in patients with ACLF than patients with LC. Longitudinal nucleotide mutations presented in more than 1 ACLF patients were nt53, nt1846 and nt1896. Longitudinal nucleotide 1913 mutation was also seen in P2, and C1913A mutation was seen in P1 and P2.
(2)Cross-sectional study showed that the rate of nt1753, nt1762, nt1764, nt1846, nt1896 and nt1913 mutations were significant different between 4 groups (P<0.01). Between group ACLF and LC, mutation rates of nt1846 and nt1913 had significant difference (P<0.01) and between group ACLF and HCC, nt1762,1846 and nt1913 had significant difference (P<0.05). A1762T/G1764A was the most common combined mutation with positive rate of 70.5% (117/166); other double mutations were A1846T/G1896A [25.9%(43/166)], A1846T/C1913(A or G) [18.7%(31/166)], A1752G/C1799G [10.8% (18/166)], etc. Also we found that the number of mutations had a relation to the progress of the disease, and HBV from ACLF patients presented more mutations. HBV DNA levels were lower in HBV with nt1753, nt1764, nt1846, nt1896 and nt1899 mutations compared to wild type. And the more nucleotide mutations, the lower HBV DNA level. Furthermore, we found that HBeAg negative patients had more G1896A, G1899A, T1753 (G or C) A1846T, C1913 (A or G) and G1915 (A or C) mutations than HBeAg positive patients. And mutations at ntl752, nt1799 and nt1846 were seen more frequently in HBV genotype B, while mutations at nt1753, nt1762, nt1764 and nt1915 were predominant in genotype C. There were no significant differences of BCP/Pre-C mutations between ACLF patients based on CHB with patients based on LC Multivariate analysis showed that nt1913(C→A or G) and nt1846(A→T) are independent factors for the ACLF [Exp(B)s=3.875 and 3.151, respectively; P=0.017 and 0.041, respectively].
Conclusion Nucleotide mutations of BCP/Pre-C are closely related to the progress of HBV-related liver diseases. And mutations at nt1846, nt1896 and nt1913 are possibly related to ACLF. A 1846T and C1913 (A or G) mutations are indenpendant factors for ACLF.
方法本试验采用回顾性研究,纵向及横向对比的研究方法。
(1)纵向对比研究：选取慢性HBV感染者6名。其中,4名为ACLF患者(P1-P4),2名为肝硬化(LC)患者(P5、P6)。P1一P4选取ACLF发生前和ACLF发生时血清标本各1份；P5、P6各留取慢性乙型肝炎(CHB)阶段和LC阶段血清标本各1份。对每位患者的2份标本进行HBV全基因组PCR扩增。PCR产物构建质粒,每份标本选取3-4个阳性克隆转化感受态细胞扩增后测序。
(2)横向对比研究：选取于慢性HBV感染者166例作横向对比研究。其中ACLF49人,CHB 45人,LC 45人,肝细胞癌(HCC)27人。对每位患者血清标本进行提取HBV DNA,进行BCP/前C区PCR扩增,扩增产物直接测序。通过Vector NTI Suite 9.0软件对序列测定结果进行比对,找出与ACLF有关的核苷酸突变位点。
结果(1)纵向研究提示：与LC患者比较,ACLF患者在疾病发展过程中出现更多的核苷酸突变。从突变的位置看,ACLF患者核苷酸突变一致且集中的区域主要位于前C基因内。4名ACLF患者中,有2名或2名以上患者可见的共同突变位点有：nt 53、nt1846和nt1896突变。C区1913位在P2也见有纵向突变,横向比较见P1、P2均见有此位点突变,而P5、P6均未发现。
(2)横向研究提示：nt1753、nt1762、nt1764、nt1846、nt1896和nt1913位核苷酸突变率在CHB、LC、ACLF和HCC四组间有统计学差异(P均<0.01)但在ACLF组与LC组之间,仅nt1846和nt1913突变有显著统计学意义(P均<0.01)；在ACLF组与HCC组之间,nt1762、nt1846和nt1913突变有统计学意义(P均<0.05)。联合突变分析提示：最常见的是A1762T/G1764A双突变,阳性率高达70.5%(117/166)；其它常见的两联突变还有A1846T/G1896A[25.9%(43/166)]、A1846T/C1913(A or G)[18.7%(31/166)]和A1752G/C1799G[10.8%(18/166)]等。在多位点联合突变中,上述突变组合经常一同出现,组成更为复杂的突变模式。突变数量与疾病进展有平行关系,ACLF患者可见更多的联合突变。突变对HBV DNA水平的影响：nt1753、nt1764、nt1846、nt1896和nt1899突变的患者HBV DNA水平要低于野生株患者；并且联合突变者HBV DNA水平更低。与HBeAg状态有关的突变位点有G1896A、G1899A、T1753(G或C)、A1846T、C1913(A或G)和G1915(A或C)。核苷酸突变与基因型的关系：nt1752、nt1799、nt1846三个位点的变异率在B型患者中高于C型；nt1753、nt1762、nt1764、nt1915的变异率在C型患者中高于B型。BCP/前C核苷酸突变率在以CHB为基础和以LC为基础的ACLF患者中无显著性差异。多因素分析结果提示nt1913(C→A or G)和nt1846(A→T)突变是预测ACLF的有效因素(比数比分别为3.875和3.151,P值分别为0.017和0.041)。
结论HBV BCP/前C区核苷酸突变与疾病进展密切相关。A1846T、G1896A和C1913(A或G)突变可能与ACLF密切相关,其中A1846T和C1913(A或G)突变是ACLF的独立预测因素。
Objective Previous studies suggested that HBV nucleotide mutations may be related to exacerbations of hepatitis B hepatitis, but the relationship between the occurrence of ACLF and HBV mutations in patients with chronic hepatitis B is not clear. The existing research suggested the mutations in pre-C region may be involved in occurrence of liver failure, but not conclusive. Hence the need for further research to clarify whether HBV mutations associated with ACLF and to identify new mutations associated with ACLF.
Methods The longitudinal and cross-sectional studies were employed. All serum samples were from serum bank of 302 hospital stored at-80℃.
Longitudinal comparison:Serum samples from the pre-ACLF stage, as well as during the occurrence of ACLF were obtained from 4 patients (P1-P4); we also collected serum samples from chronic hepatitis stage and liver cirrhosis stage from 2 patients (P5, P6) as control. HBV DNA was extracted and amplified and ligated into modified pCR2.1 plasmid. Of each individual,3-4 cloned HBV DNAs were sequenced and the sequences were aligned between pre-ACLF and post-ACLF and /or pre-Cirrhosis and post Cirrhosis.
Cross sectional comparison:HBV DNAs were extracted from serum samples from 166 patients including ACLF(n=46), chronic hepatitis (CHB n=45) 45, LC(n=65), HCC(n=27), and BCP/Pre-C and C region was sequenced and analyzed.
Results⑴Longitudinal analysis showed that there were more longitudinal nucleotide mutations in patients with ACLF than patients with LC. Longitudinal nucleotide mutations presented in more than 1 ACLF patients were nt53, nt1846 and nt1896. Longitudinal nucleotide 1913 mutation was also seen in P2, and C1913A mutation was seen in P1 and P2.
(2)Cross-sectional study showed that the rate of nt1753, nt1762, nt1764, nt1846, nt1896 and nt1913 mutations were significant different between 4 groups (P<0.01). Between group ACLF and LC, mutation rates of nt1846 and nt1913 had significant difference (P<0.01) and between group ACLF and HCC, nt1762,1846 and nt1913 had significant difference (P<0.05). A1762T/G1764A was the most common combined mutation with positive rate of 70.5% (117/166); other double mutations were A1846T/G1896A [25.9%(43/166)], A1846T/C1913(A or G) [18.7%(31/166)], A1752G/C1799G [10.8% (18/166)], etc. Also we found that the number of mutations had a relation to the progress of the disease, and HBV from ACLF patients presented more mutations. HBV DNA levels were lower in HBV with nt1753, nt1764, nt1846, nt1896 and nt1899 mutations compared to wild type. And the more nucleotide mutations, the lower HBV DNA level. Furthermore, we found that HBeAg negative patients had more G1896A, G1899A, T1753 (G or C) A1846T, C1913 (A or G) and G1915 (A or C) mutations than HBeAg positive patients. And mutations at ntl752, nt1799 and nt1846 were seen more frequently in HBV genotype B, while mutations at nt1753, nt1762, nt1764 and nt1915 were predominant in genotype C. There were no significant differences of BCP/Pre-C mutations between ACLF patients based on CHB with patients based on LC Multivariate analysis showed that nt1913(C→A or G) and nt1846(A→T) are independent factors for the ACLF [Exp(B)s=3.875 and 3.151, respectively; P=0.017 and 0.041, respectively].
Conclusion Nucleotide mutations of BCP/Pre-C are closely related to the progress of HBV-related liver diseases. And mutations at nt1846, nt1896 and nt1913 are possibly related to ACLF. A 1846T and C1913 (A or G) mutations are indenpendant factors for ACLF.
引文
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[54]Utama A, Purwantomo S, Siburian MD, et al. Hepatitis B virus subgenotypes and basal core promoter mutations in Indonesia. World J Gastroenterol, 2009,15(32):4028-36.
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[58]Peng XM, Huang GM, Li JG, et al. High level of hepatitis B virus DNA after HBeAg-to-anti-HBe seroconversion is related to coexistence of mutations in its precore and basal core promoter. World J Gastroenterol, 2005,11(20):3131-4.
[59]Yoo BC, Park JW, Kim HJ, et al. Precore and core promoter mutations of hepatitis B virus and hepatitis B e antigen-negative chronic hepatitis B in Korea. J Hepatol,2003,38(1):98-103.
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[65]Tsai A, Kawai S, Kwei K, et al. Chimeric constructs between two hepatitis B virus genomes confirm transcriptional impact of core promoter mutations and reveal multiple effects of core gene mutations. Virology,2009,387(2):364-72.
[66]Buckwold VE, Xu Z, Chen M, et al. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol,1996,70(9):5845-51.
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[65]Tsai A, Kawai S, Kwei K, et al. Chimeric constructs between two hepatitis B virus genomes confirm transcriptional impact of core promoter mutations and reveal multiple effects of core gene mutations. Virology,2009,387(2):364-72.
[66]Buckwold VE, Xu Z, Chen M, et al. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol,1996,70(9):5845-51.
[67]Li J, Buckwold VE, Hon MW, et al. Mechanism of suppression of hepatitis B virus precore RNA transcription by a frequent double mutation. J Virol, 1999,73(2):1239-44.
[68]Chauhan R, Kazim SN, Bhattacharjee J, et al. Basal core promoter, precore region mutations of HBV and their association with e antigen, genotype, and severity of liver disease in patients with chronic hepatitis B in India. J Med Virol,2006,78(8):1047-54.
[69]Du H, Li T, Zhang HY, et al. Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection. Liver Int,2007,27(2):240-6.
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[1]. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev.2000, 64(1):51-68.
[2]. A. Lisotti, C. Grenci, A. Caponi, E. Roda. Chronic hepatitis B in 2008. Digestive and Liver Disease.2008, Supplements 2:3-6.
[3]. Locarnini S. Molecular virology of hepatitis B virus. Semin Liver Dis.2004,24 Suppl 1:3-10.
[4]. Block TM, Guo H, Guo JT. Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis.2007,11(4):685-706, vii.
[5]. Zhang YY, Zhang BH, Theele D, Litwin S, Toll E, Summers J. Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. Proc Natl Acad Sci U S A.2003,100(21):12372-7.
[6]. Harrison TJ. Hepatitis B virus:molecular virology and common mutants. Semin Liver Dis.2006,26(2):87-96.
[7]. Bartholomeusz A, Locarnini S. Hepatitis B virus mutants and fulminant hepatitis B:fitness plus phenotype. Hepatology.2001,34(2):432-5.
[8]. Ozasa A, Tanaka Y, Orito E, Sugiyama M, Kang JH, Hige S, et al. Influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis B virus infection. Hepatology.2006,44(2):326-34.
[9]. Sugiyama M, Tanaka Y, Kurbanov F, Nakayama N, Mochida S, Mizokami M. Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene. Virology.2007,365(2):285-91.
[10].Chen MT, Billaud JN, Sallberg M, Guidotti LG, Chisari FV, Jones J, et al. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A.2004,101(41):14913-8.
[11].Sainokami S, Abe K, Sato A, Endo R, Takikawa Y, Suzuki K, et al. Initial load of hepatitis B virus (HBV), its changing profile, and precore/core promoter mutations correlate with the severity and outcome of acute HBV infection. J Gastroenterol.2007,42(3):241-9.
[12].Parekh S, Zoulim F, Ahn SH, Tsai A, Li J, Kawai S, et al. Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants. J Virol.2003,77(12):6601-12.
[13].Jammeh S, Tavner F, Watson R, Thomas HC, Karayiannis P. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol.2008,89(Pt 4):901-9.
[14].Chan HL, Hussain M, Lok AS. Different hepatitis B virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology.1999,29(3):976-84.
[15].Li J, Xu Z, Zheng Y, Johnson DL, Ou JH. Regulation of hepatocyte nuclear factor 1 activity by wild-type and mutant hepatitis B virus X proteins. J Virol. 2002,76(12):5875-81.
[16].Bergametti F, Sitterlin D, Transy C. Turnover of hepatitis B virus X protein is regulated by damaged DNA-binding complex. J Virol.2002,76(13):6495-501.
[17].Leupin O, Bontron S, Strubin M. Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities. J Virol.2003,77(11):6274-83.
[18].Bouchard MJ, Wang LH, Schneider RJ. Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science.2001,294(5550):2376-8.
[19].Dienstag JL. Hepatitis B virus infection. N Engl J Med 2008;359(14):1486-500.
[20]. Anna SF LOK.乙型肝炎病毒耐药性临床需知.肝脏.2007,12(1)：43-45.
[21].Kay A, Zoulim F. Hepatitis B virus genetic variability and evolution. Virus Res. 2007,127(2):164-76.
[22].Baumert TF, Thimme R, von Weizsacker F. Pathogenesis of hepatitis B virus infection. World J Gastroenterol JT-World journal of gastroenterology WJG.2007,13(1):82-90.