慢性重型乙型肝炎前C区及BCP区基因突变的临床意义

应用基因芯片杂交技术检测慢性重型乙型肝炎（慢重肝）及非重型肝炎（包括慢乙肝及肝硬化）患者血清中HBV前C区A1896、A1899及BCP区T1762/A1764联合突变的发生率,比较慢重肝好转或治愈患者及恶化或死亡患者前C区及BCP区基因突变情况。结果慢重肝前C区A1896、A1899,BCP区T1762/A1764联合突变率及两区多位点突变率与慢乙肝或肝硬化患者比较均有显著统计学差异（P均〈0.01）;慢重肝好转或治愈患者A1896、A1899及多位点变异率与恶化或死亡患者比较均有统计学差异（P均〈0.05）。提示HBV前C区及BCP区基因变异为慢重肝的重要发病机制之一。     

慢性乙型肝炎患者HBe系统与HBV DNA前C区基因突变的分析

目的研究HBV DNA前C区基因突变与HBe系统的关系.方法对88例慢性乙型肝炎患者进行HBVDNA基因检测(PCR法)、分子克隆及序列分析.结果88例患者中,血清HBV DNA阴性22例,阳性66例.从阳性患者中检出野生株感染22例,占33.3%;突变株感染44例,占66.7%.44例突变株感染者中38例属1898位点及/或1901位点突变,占86.4%;其他形式突变6例,占13.6%;66例血清HBV DNA阳性中,CHB 49例,LC 17例,突变株感染检出率分别为69.3%、58.8%,两者经统计学处理无显著性差异(P＞0.05).在HBe系统中,HBV DNA阳性患者属HBeAg(+)者44例,其中突变株检出率为75.0%(33/44),属抗HBe(+)者20例,其中突变株检出率为50.0%(10/20),两者经统计学处理有显著性差异(P＜0.05).结论①在HBe系统中,HBeAg(+)患者的突变株检出率高于抗HBe(+)患者(P＜0.05),提示其位点突变株在HBeAg(+)中占优势,与以往文献报道中关于1896位点突变株主要存在于抗-HBe(+)患者中的结论不尽相同.因此,应考虑HBeAg(+)患者也存在病毒突变的可能.②HBV慢性感染过程中,CHB患者与LC患者的突变株感染检出率无显著性差异(P＞0.05),表明在1898及/或1901位点的突变株感染与肝脏损害程度无平行关系.     

慢性乙型肝炎患者乙型肝炎病毒前C区基因突变与临床关系

为了解慢性乙型肝炎患者是否有前Ｃ区基因突变及其与临床的关系，应用快速检测乙型肝炎病毒前Ｃ区基因突变方法，对５３例患者进行了研究。结果显示：２５例ＨＢＶＤＮＡ阳性患者中１３例有突变株感染，最常见的突变形式为１８９８位或伴有１９０１位突变（１０／１３例）。     

乙型肝炎和肝癌患者乙型肝炎病毒前C区1896位点突变的研究

为探索乙型肝炎病毒前Ｃ区突变是否与肝损程度及肝癌发生有关，对１３９例ＨＢｓＡｇ、ＨＢＶＤＮＡ和抗－ＨＢｅ阳性，ＨＢｅＡｇ阴性的慢性ＨＢＶ感染者和肝癌患者的血清标本，采用３＇碱基特异性聚合酶反应进行了第１８９６位核苷酸突变的检测分析。     

重型乙型肝炎患者肝组织和血清中乙型肝炎病毒前C区准种的比较

乙型肝炎病毒存在准种现象。为研究重型乙型肝炎患者血清和肝组织中乙型肝炎病毒（HBV）DNA前C区准种组成特点，本研究从3例重型乙型肝炎患者的血清和肝组织中提取HBV DNA，经巢式聚合酶链反应（PCR）扩增出HBV前C区，产物克隆后经单链构象多态性／异源双链分析（SSCP／HDA）筛选不同的克隆并测序，比较血清和肝组织中HBV前C区准种组成的异同。     

234例慢性乙型肝炎患者HBV前C/BCP区突变分析

目的调查慢性乙型肝炎患者血清HBV前C/基本核心启动子（BCP）区的突变情况,分析各种突变对HBeAg表达的影响。方法抽提患者血清DNA,采用改进的巢式PCR技术,扩增HBV前C/BCP区基因,对PCR产物进行DNA测序。用NTI软件比对结果,根据文献报道,选取1753、1762、1764、1862、1896和1899共6个位点进行突变分析,并重点分析不同突变对患者血清HBeAg阳性率和病情的影响。结果在234例中6个位点均无突变者为74例（31.6%）,其血清HBeAg阳性率为74.3%（55/74）。在234例中6个位点检出至少1种突变的病例为160例（68.4%）,突变形式包括4种单一位点突变和21种组合形式的突变;检出G1896A突变73例（31.2%）,其中36例检出有共存未突变序列,37例仅检出突变序列,后者血清HBeAg的阳性率为18.9%（7/37）。检出G1896A以外其他形式突变的有87例,HBeAg阳性率为63.2%（55/87）;其中以A1762T＋G1764A最为常见,HBeAg阳性率为69.4%（34/49）。在1753、1862位点上检出4种特殊碱基突变,前C区有基因片段插入或缺失的有2例。结论多数慢性乙型肝炎患者在HBV前C/BCP区可检出突变,突变形式多样,其中G1896A突变样本血清HBeAg阳性率显著下降,而其他突变对其影响较小。应用DNA序列测定法分析HBV前C/BCP区基因突变,获得的信息全面,对临床评估病情进展和实施抗病毒治疗有参考价值。     

乙型重型肝炎患者血清HBV前C/BCP变异检测及临床意义

目的探索乙型重型肝炎患者HBV前C/BCP区变异与临床病情关系。方法用荧光定量PCR法检测血清HBVDNA载量;PCR扩增前C/BCP区基因后进行序列测定。结果30例乙型重型肝炎患者中BCP变异有18例,HB-VDNA平均水平为2.71×107copies/mL,未发生BCP变异者有12例,HBVDNA平均水平为5.35×106copies/mL,两者间有显著差异。测序结果显示HBV前C区nt1896G→A终止变异和BCP变异(nt1762A→T和1764G→A)在乙型重型肝炎患者中发生率分别为43.3%(13/30)和60%(18/30),在慢性乙型肝炎患者则分别为40%(12/30)和30%(9/30),两组BCP变异(nt1762A→T和1764G→A)相比有显著差异(P=0.02)。结论BCP变异可影响病毒复制,BCP变异可能与肝病的严重程度存在着相关性。     

前C区基因变异对慢性重型乙型肝炎免疫应答的影响

目的：分析乙型肝炎病毒(HBV)前C区G1896A变异对慢性重型乙型肝炎(慢重肝)发病及免疫应答的影响．方法：流式细胞仪检测31例慢重肝患者外周血细胞胞内细胞因子IFN-γ,IL-4的百分率;测定Th1,Th2,Tc1,Tc2;固相夹心法酶联免疫吸附试验检测其血清IL-4,IFN-γ的水平;套式聚合酶链反应扩增HBV DNA前C/C基因并作序列分析．结果：在31例患者中8例(25．8%)发生G1896A变异,8例G1896A变异株中4例(50%)出现1913位联合变异,而23例野毒株仅1例(4．3%)出现C1913A变异;G1896A变异株Tcl水平显著高于野毒株(P=0．023,t=2．407);G1896A变异株与野毒株相比IL-4,IFN-γ水平无显著性差异(P＞0．05);变异株与野毒株2组死亡率无显著性差异(P＞0．05)．结论：G1896A变异能显著增强Tcl水平,后者在慢重肝发病中起重要作用．     

慢性重型乙型肝炎患者T细胞免疫状态与HBV前C区基因变异的关系

ＨＢＶ前Ｃ区最常见的基因变异是第 1896位核苷酸Ｇ→Ａ突变 ,使第 2 8位密码子形成终止密码 ,导致ＨＢｅＡｇ合成终止。前Ｃ区变异被认为是免疫选择的结果 ,并与暴发性乙型肝炎有密切的关系 ,但尚没有直接证据表明前Ｃ区变异株病毒毒力增强 ,而且也不是所有感染前Ｃ区变异株的患者都会出现病情加重。我们对慢性重型乙型肝炎患者杀伤性Ｔ细胞 (Ｔｃ)和抑制性Ｔ细胞 (Ｔｓ)与ＨＢＶ前Ｃ区基因变异的关系进行了研究 ,现报道如下。材料与方法一、研究对象42例慢性重型乙型肝炎患者为武汉协和医院 1998年10月至 1999年 5月间的住院患者 ,男 39例…     

Hepatitis B genotypes and precore/basal core promoter mutants in HBeAg-negative chronic hepatitis B

Background. Mutations in the precore stop codon (G1896A) and the basal core promoter (A1762T and G1764A) are frequently found in hepatitis B envelope antigen (HBeAg)-negative chronic hepatitis B. However, the clinical significance of these mutations remains controversial. We therefore investigated the influence of hepatitis B virus (HBV) genotypes, as well as precore/basal core promoter mutants, on the clinical and virological features of patients with HBeAg-negative chronic hepatitis B. Methods. Serum samples from 37 patients with HBeAg-negative chronic hepatitis B were collected for serological and molecular assays. The precore and basal core promoter regions were amplified by polymerase chain reaction and the amplicons were directly sequenced and analyzed. HBV geno-type was determined by polymerase chain reaction-restriction fragment length polymorphism. Results. Most of the patients had detectable serum HBV DNA, and genotypes B and C were the predominant strains. The overall prevalence of the precore stop codon mutant and basal core promoter mutant was 67% and 60%, respectively. The baseline clinical and virological features of patients with genotype B and genotype C infection were comparable. However, in the patients with precore/basal core promoter dual mutations there was a significantly lower proportion of individuals with a high detectable serum HBV DNA level (>100 pg/ml) than in the patients with either the precore stop codon mutation alone or the basal core promoter mutation alone (P = 0.04 by the logistic regression test for the trend). Conclusions. Our data suggest a high prevalence of precore stop codon and basal core promoter mutation in Taiwanese patients with HBeAg-negative chronic hepatitis B, and the influence of the basal core promoter mutation on HBV replication is modulated by the emergence of the precore stop codon mutation. Received: May 14, 2001 / Accepted: September 14, 2001     

慢性HBV感染者抗病毒治疗前HBV基本核心启动子及前C区突变的检测及意义

目的研究慢性乙型肝炎病毒（HBV）感染者抗病毒治疗前HBV基本核心启动子（BCP）突变和前C区（PreC）突变与HBeAg、HBV DNA水平和慢性肝病进展的关系。方法收集283例慢性HBV感染者抗病毒治疗前的血清标本,其中慢性乙型肝炎（CHB）185例,肝硬化（LC）98例。采用PCR后直接测序法检测HBV BCP和PreC区突变,同时确定基因型。结果在HBeAg阴性和HBeAg阳性CHB患者中,前C区A1896变异率分别为44.6%（37/83）和21.6%（22/102）（χ2=11.154,P=0.001）,LC患者分别为43.4%（23/53）和17.0%（8/47）（χ2=8.101,P=0.004）。在HBeAg阳性患者中,BCP T1762/A1764双突变率LC组和CHB组分别为89.4%（42/47）和70.6%（72/102）（χ2=6.310,P=0.012）。在单变量分析中,只有年龄（≥45岁）（χ2=27.861,P〈0.001）、BCP T1762/A1764双突变（χ2=8.675,P=0.003）和HBV DNA（≥105拷贝/ml）（χ2=20.499,P〈0.001）与LC进展有关。多因素Logistic回归分析（匹配年龄和性别）发现,BCP T1762/A1764双突变（OR=3.260,95%CI：1.401～7.586;wald=7.517,P=0.006）和HBV DNA（≥105拷贝/ml）（OR=4.640,95%CI：2.331～9.237;wald=19.089,P〈0.001）是LC进展的危险因素。结论前C区A1896突变与HBeAg的消失有关;年龄（≥45岁）、BCP T1762/A1764双突变和HBV DNA高载量（≥105拷贝/ml）与肝硬化进展有关。     

Detection of HBV core promoter and precore mutations helps distinguish flares of chronic hepatitis from acute hepatitis B

Background and Aim: Acute exacerbation of chronic hepatitis B has to be distinguished from acute hepatitis, because treatment strategies differ between them. Methods: Mutations in the core promoter and precore region of hepatitis B virus (HBV) were determined in 36 patients with acute exacerbation of chronic hepatitis B, in whom alanine aminotransferase (ALT) increased above 500 IU/L, as well as the 36 patients with acute hepatitis. Results: Mutations in the core promoter (A1762T/G1764A) and precore region (G1896A) were more frequent in patients with acute exacerbation of chronic hepatitis than acute hepatitis (81% vs 19%; P < 0.0001 and 58% vs 6%; P < 0.0001, respectively). Of the 19 patients with mutations in both the core promoter and precore region, 17 (89%) had acute exacerbation of chronic hepatitis. In contrast, among the 32 patients with the wild‐type for both the core promoter and precore region, 29 (89%) developed acute hepatitis. By multivariate analysis, the double mutation in the core promoter was predictive of acute exacerbation in chronic hepatitis with the highest odds ratio at 26.4. Conclusions: In patients with hepatitis B having ALT levels >500 IU/L, mutations in the core promoter and precore region are useful in distinguishing acute exacerbation of chronic from acute HBV infection. Detection of these mutations would be useful for commencing prompt antiviral treatments on patients with acute exacerbation of chronic hepatitis for a better prognosis.     

Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection.

BACKGROUND/AIMS: To investigate the correlation of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations in patients with chronic hepatitis B. METHODS: HBV genotyping, nucleotide mutation, serum HBV DNA level and serological markers were analyzed in 121 patients with chronic HBV infection using INNO-LiPA HBV genotyping, polymerase chain reaction (PCR) product-based sequencing, fluorescence quantitative PCR and enzyme-linked immunosorbent assays respectively. RESULTS: Forty (33.0%), 77 (63.6%), two (1.7%) and two (1.7%) patients had genotypes B, C, B/C and D infections respectively. Significant differences were found in serum HBV DNA levels (log10 copies/ml: 6.18 vs. 5.61, P=0.042) and mutations at nucleotide (nt) 1762/1764 (71.4% vs. 42.5%, P=0.002) between genotypes C- and B-infected patients. There were significant differences in the mean age, serum biochemical parameter levels and mutation rates in BCP/PC among hepatitis e antigen (HBeAg)-positive and -negative chronic hepatitis B (CHB) and liver cirrhosis (LC) groups. CONCLUSION: Genotypes C and B are predominant in China, and the frequent nt 1762/1764 mutation, which occurs commonly in HBeAg-negative CHB, especially in genotype C patients, may be associated with the progress of chronic HBV infection.     

Association of core promoter/precore mutations and viral load in e antigen-negative chronic hepatitis B patients

Apart from core promoter A1762T/G1764A and precore G1896A mutations, other hepatitis B virus (HBV) mutants are detected in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). The aim of this study was to determine the effects of those mutants on clinical manifestation and viral loads of genotypes B and C HBV. Seventy-nine HBeAg-negative CHB patients with hepatitis flare were enrolled in this study and their HBV precore/core region were sequenced. Serial biochemical profiles and viral loads were assessed and compared. Fifty-three patients (67%) were infected by genotype B HBV and 26 (33%) were infected by genotype C HBV. The clinical manifestation and HBV viral loads were comparable between the two groups. However, genotype B was significantly associated with precore G1896A mutation (92.5%), and more mutations within nucleotide 1809-1817 were detected in patients infected by genotype B as compared with those infected by genotype C (18.9%vs 3.8%). Most of the cases had mutations at the -2, -3 or -5 position from the precore AUG initiation codon. Triple core promoter mutations T1753C/A1762T/G1764A [corrected] appeared to be linked to genotype C rather than genotype B HBV (19.2%vs 1.9%; P = 0.013). In multivariate analysis, the presence of either triple core promoter 1753/1762/1764 mutation or nucleotide 1809-1817 mutation was the only factor associated with lower HBV viral load (<70 Meq/mL) (odds ratio = 9.01; 95% CI 1.11-71.43; P = 0.04). In conclusion, minor HBV variants with mutations in the core promoter and precore region were detectable in genotypes B and C. Such HBV variants are genotype specific and related to viraemia levels.     

Sequential analysis of hepatitis B virus core promoter and precore regions in cancer survivor patients with chronic hepatitis B before,during and after interferon treatment

We analysed the hepatitis B virus (HBV) core-promoter (CP) and precore (PC) regions before, during and after interferon treatment in young Caucasian cancer survivors who had acquired HBV infection during chemotherapy for malignancies. Fourteen patients with chronic hepatitis B [hepatitis B e antigen (HBeAg) /HBV-DNA positive] received α-2a interferon (IFN), 5  M U/m² t.i.w. for 12 months. HBV CP and PC region sequences were analysed following polymerase chain reaction (PCR) amplification. Sera from responders were studied at: T₀ (before starting IFN), T₁ [at alanine aminotransferase (ALT) peak preceding HBeAg seroconversion], T₂ (at ALT normalization), T₃ (at end of IFN) and T₄ (at one year after IFN) and in nonresponders at time points T₀, T₃ and T₄. Amplified HBV-DNA was cloned and sequenced automatically. Six of 14 patients (43%) responded to IFN treatment. Five of the six (83%) responders displayed the double CP mutation A1762T/G1764A always in association with a T1753C change. None of the nonresponders showed these mutations at any time point. The G1896A change creating the PC stop codon mutation was never detected in any of the patients. In our cancer survivors, IFN-induced HBeAg/anti-HBe seroconversion appeared to correlate with CP mutations and was not influenced by previous chemotherapy. These mutations in addition to low HBV DNA levels and elevated ALT can be considered favourable factors of response to IFN-induced anti-HBe seroconversion.     

Clinical features of hepatitis B patients at immune‐tolerance phase with basal core promoter and/or precore mutations

Little data exist on basal core promoter/precore (BCP/PC) mutations in chronic hepatitis B (CHB) patients at the immune‐tolerance (IT) phase. We studied consecutive treatment‐naïve, CHBe‐antigen (HBeAg)‐positive patients who had undergone liver biopsy and genotyping. Those in the IT phase or immune‐clearance (IC) phase were enrolled for comparison of the frequency of BCP/PC mutations and their clinical presentations. Subgroup analyses for the IT group were also performed between patients with and without mutations, and IC patients between fibrosis stages ≤2 vs fibrosis >2. Among 301 patients enrolled, 88/301 (29.24%) and 213/301 (70.76%) were at the IT and IC phase, respectively. The frequency of BCP/PC mutations in IT phase was significantly lower than those in IC phase (15.91% vs 64.79%, P < .001). The BCP mutation only was significantly more frequent than the PC mutation in both groups and also in all IC subgroups. IT patients with BCP/PC mutations had significantly higher quantitative anti‐HBc levels compared with those of patients with wild‐type virus (P < .05). They also had significantly lower mean levels of alanine transaminase, aspartate transaminase, total bilirubin and qAnti‐HBc compared with those of IC patients (all P < .05). Additionally, they were significantly younger in mean age, had higher platelet count, higher levels of HBV DNA and surface antigen, as well as higher frequency of genotype B than those of IC patients with fibrosis >2 (all P < .05). BCP/PC mutations were found in IT patients with CHB. They had distinct clinical characteristics when compared with patients with wild‐type or at IC phase. Further studies are needed to understand their natural history and treatment outcomes.