慢性乙型肝炎患者HBV C基因启动子变异的临床意义

目的:探讨慢性乙型肝炎中C基因启动子(BCP)变异的临床意义。方法:采用错配PCR与限制性长度片段多态性分析(RFLP)相结合,检测35例慢性乙型肝炎患者BCP区核苷酸(nt)1762碱基A→T和1764G→A联合突变及前C区nt1896G→A终止变异。结果:在35例慢性乙型肝炎中检出BCP区T1762A1764变异8例(23％),其中6例血清HBeAg( ),2例抗HBe( ),而7例前C区A1896变异中HBeAg( )2例,抗HBe( )5例,未见T1762A1764变异和A1896变异同时出现者。结论:提示HBV毒株BCP区T1762A1764变异可能与前C区A1896变异不同,它的出现不足以导致HBeAg(—)型的慢性肝炎。     

乙型肝炎病毒YMDD及e抗原相关多重变异及其临床意义

目的 研究拉米夫定治疗慢性乙型肝炎期间HBVYMDD基序、影响HBeAg分泌的多重变异情况与临床的关系。方法 采用基因芯片技术对拉米夫定治疗9～30个月的慢性乙型肝炎患者进行YMDD基序、G1896A、A1814C、A1762T和G1764A(BCP双突变)单碱基变异检测。结果 102例慢性乙型肝炎患者拉米夫定平均治疗18个月时,22例发生YMDD变异,其中8例发生多重变异,包括G1896A3例、A1814C2例、G1896A A1814C、BCP双突变、BCP双突变 G1896A多重变异各1例,单纯YMDD变异和前5例联合变异均为HBeAg阳性,而后3例多重变异则为HBeAg阴性,其中1例多重变异继续治疗3个月后转变为单纯YMDD野生株阳性,同时伴有HBeAg的复阳。结论 拉米夫定治疗过程中存在YMDD及HBeAg相关多重变异的优势病毒株可能是HBVDNA复阳、同时伴有HBeAg阴转的原因之一,拉米夫定治疗过程中,HBeAg阴性时应监测其可能的相关变异。     

中药健脾益气汤联合拉米夫定治疗HBeAg阴性乙型肝炎

乙型肝炎在病程中或治疗后,部分病人出现HBeAg/抗HBe转换后,HBV-DNA阳性,可能是由于HBV-DNA出现前C区(1896位核苷G-A变异)或C区启动区突变(1762位核苷A—T变异、1764位核苷G—A变异)不能形成HBeAg,但病毒仍能复制及装配,病情不断进展,可以发展为肝硬化或肝癌。现将我们用中药健脾益气汤联合拉米夫定治疗HBeAg阴性乙型肝炎     

慢性乙型肝炎患者HBV前C区G1896A、核心启动子1762/1764变异与乙型肝炎自然史、血清HBsAg定量及疾病程度的相关性

目的研究HBV前C区G1896A、核心启动子1762/1764变异与HBV自然史、血清HBsAg水平间的相关性及对疾病严重程度的影响。方法分别各选取40例HBV野生株、前C区G1896A或核心启动子1762/1764变异以及两者联合变异的慢性乙型肝炎感染者为研究对象。采用PCR反向点杂交技术检测HBV前C区G1896A位点、核心启动子1762/1764变异及HBV基因分型。同时检测HBsAg定量、HBeAg、HBV DNA定量、肝脏生化指标等。结果(1)HBV前C区G1896A、核心启动子1762/1764变异患者的年龄、ALT高于野生株感染者(P0.001),Alb低于野生株感染者(P0.001)。(2)HBV自然史免疫耐受期以野生株感染为主(P0.001),免疫清除期、低(非)复制期野生株及PC G1896A或/和BCP1762/1764变异株差异无统计学意义(P0.05),再活动期以PC G1896A或/和BCP1762/1764变异株为主(P0.001)。(3)PC G1896A或/和BCP1762/1764变异株的HBsAg水平(lgIu/mL)、HBeAg阳性率较野生株组降低(P0.001),基因C型、HBV DNA水平(≥6 lg拷贝/mL)较野生株组差异无统计学意义(P0.05)。(4)HBV PC G1896A或/和BCP1762/1764变异中肝硬化患者多于野生株(P0.05)。结论慢性HBV感染者前C区G1896A、核心启动子1762/1764变异与乙型肝炎自然史、HBsAg水平、HBeAg的状态及疾病严重程度相关。     

HBeAg阳性慢性乙型肝炎初治患者BCP区A1762T/G1764A变异株定量检测结果分析

目的探讨HBeAg阳性慢性乙型肝炎初治患者BCP区A1762T/G1764A变异株定量检测的临床意义。方法通过单标记探针联合选择性阻断实时荧光定量PCR法精确定量检测97例HBV DNA阳性慢性乙型肝炎初治患者BCP区A1762T/G1764A变异株,并进行统计学分析。结果 97例HBeAg阳性患者变异株的含量会随着HBV DNA水平的升高而降低(P0.01)。变异株含量同时与HBeAg水平呈显著相关,随着HBeAg水平的升高,变异株含量降低(P0.01)。结论对HBV感染者的BCP区A1762T/G1764A变异株进行定量检测,可对患者病情的可能发展进行预测,从而可以采取有效的防治措施。     

中药健脾益气汤联合拉米夫定治疗HBeAg阴性乙型肝炎

乙型肝炎在病程中或治疗后,部分病人出现HBeAg/抗HBe转换后,HBV-DNA阳性,可能是由于HBV-DNA出现前C区(1896位核苷G-A变异)或C区启动区突变(1762位核苷A—T变异、1764位核苷G—A变异)不能形成HBeAg,但病毒仍能复制及装配,病情不断进展,可以发展为肝硬化或肝癌。现将我们用中药健脾益气汤联合拉米夫定治疗HBeAg阴性乙型肝炎报道如下:     

乙型肝炎病毒前S基因变异与肝病进展的关系

目的探讨HBV前S基因变异与疾病进展的关系。方法收集无症状携带者（ASC）、慢性肝炎（CH）、肝炎肝硬化（LC）、肝细胞癌（HCC）患者血清138份，PCR扩增前S区基因．聚合酶链反应-限制性片段长度多态性检测前S2起始码变异，聚丙烯酰胺凝胶电泳（PAGE）分析前S缺失变异，直接测序确定前CA1896、基本核心启动子（BCP）T1762／A1764变异。数据行X^2检验。结果HCC、LC组前S缺失变异检出率分别为56．3％和42．9％，高于CH组的11．8％和ASC组的8．1％（P〈0．01）。前S2起始码变异检出率在HCC（50．0％）、LC（37．1％）组亦较CH（5．9％）、ASC（0）组高。前S缺失、前S2起始码变异在HBeAg阴性组的检出率分别为37．5％和36．1％，高于HBeAg阳性组的19．7％和7．6％（P〈0．01）。分析前S基因、T1762／A1764、A1896单独或联合变异在HCC、LC组和CH、ASC组的分布，Fisher精确检验表明，T1762／A1764和前S基因的联合变异是影响疾病进展、导致严重肝病的重要因素（P-0）。结论严重肝病患者前s基因变异发生率高，有T1762／A1764联合前S基因变异HBV感染者的肝病易进展。     

HBeAg阴性慢型乙型肝炎的临床分析及其与基因变异的关系

目的探讨HBeAg阴性慢性乙型肝炎(以下简称e-CHB)的临床特点及与前C区G1896A变异及基本核心启动子(BCP)区A1762T/G1764A双变异的关系。方法采用实时荧光定量PCR法检测HBVDNA,采用时间分辨荧光免疫分析法定量检测乙型肝炎病毒标志物,采用基因芯片检测基因变异。结果e-CHB患者其年龄、肝硬化及肝癌发生明显高于HBeAg阳性慢性乙型肝炎(以下简称e CHB)患者(P<0·05);e-CHB患者在HBVDNA定量,慢性轻度肝炎比例明显低于e CHB患者(P<0·05)。前C区G1896A变异及BCP区A1762T/G1764A双变异的总的变异率,e-CHB患者明显高于e CHB患者(χ2=6·66,P<0·01)。结论e-CHB患者年龄较大,肝硬化、肝癌发生率高,在慢性乙型肝炎自然史中处于较晚阶段。e-CHB病毒复制减少。e-CHB主要是基因变异的结果;血清不同e系统均可发生变异。     

慢性乙型肝炎病毒感染者病毒前C区和基本核心启动子区变异检测及意义

目的 探讨乙型肝炎病毒（HBV）前C区和基本核心启动子（BCP）区变异与基因型及疾病进展间的关系。方法 收集HBV携带者（ASC）、慢性乙型肝炎（CHB）、肝炎肝硬化（LC）、肝细胞肝癌（HCC）患者血清148份，用半巢式聚合酶链反应扩增HBV前C／C基因部分片段，产物纯化后直接测序，检测前C区A1896及BCP区T1762／A1764变异。用S基因聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP）方法确定HBV基因型。结果 有128份血清能够成功分型和测序，其中B基因型60份，C基因型68份。在B基因型感染者中前C区A1896变异检出率（48．33％）明显高于C基因型感染者（29．41％，X^2=4．83，P〈0．05）；而BCP区T1762／A1764变异检出率却明显低于C基因型感染者，差异亦有统计学意义（30．00％：73．54%，X^2=24．25。P〈0．05）。前C区A1896变异在CHB、LC、HCC中的阳性检出率分别为46．88％（15/32）、39．39％（13／33）、51．52％（17／33）。与ASC的13．33％（4／30）相比，P分别〈0．05，差异有统计学意义。BCP区T1762／A1764变异检出率在HCC、LC组分别为87．88％（29／33）和72．73％（24／33）．明显高于CHB组的37．50％（12／32）及ASC组10.00％（3／30）（P〈0．05）。结论 前C区A1896变异常见于B基因型感染者，而BCP区T1762/A1764变异C基因型感染者多见。除ASC外．前C区A1896变异与疾病进展关系不大．而BCP区T1762/A1764变异与乙型肝炎进展及顶后相关。     

HBeAg阴性慢性乙型肝炎患者的基因变异测定及意义

应用PCR方法检测45例HBeAg阴性慢性乙型肝炎(慢乙肝)患者(HBeAg阴性组)及15例慢乙肝并肝硬化患者(肝硬化组)的基因突变情况.结果 HBeAg阴性组1762位点变异3例,前(pre)-1896位点变异12例,1762 1764位点联合变异9例,1762 1764 pre-1896位点联合变异21例;肝硬化组分别为9例、3例、3例.认为HBeAg慢乙肝患者基因突变以1762 1764 pre-1896位点联合变异为主,其中pre-1896位点变异可能在肝硬化的发生中有重要意义.     

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

AIM: G1896A mutation in precore or A1762T/G1764A mutations in basal core promoter are suspected to be responsible for patients with detectable level of HBV DNA in serum after seroconversion from HBeAg to anti-HBe. However, G1896A variant has impaired, while A1762T/ G1764A variant may have intact replication ability. They themselves or their coexistence status may play different roles in such meaningless seroconversion. For these reasons, the significances of these two types of mutations were comparatively investigated in this study. METHODS: One hundred and sixty-five sera with positive anti-HBe and HBV DNA were collected from different patients. Mutations of G1896A and A1762T/G1764A among these serum samples were detected using competitively differentiated PCR. HBV DNA was demonstrated using real-time quantitative PCR. RESULTS: G1896A and/or A1762T/G1764A mutations were detected in 89.1% (147/165) out of patients with detectable HBV DNA in serum after HBeAg-to-anti-HBe seroconversion. The positive rate of G1896A variants was significantly higher than that of A1762T/G1764A mutations (77.6% vs 50.3%, X2= 26.61,P<0.01). The coexistence positive rate of these two types of mutations was 38.8% (64/165). Coexistence mutations were found in 77.1% (64/83) out of sera with A1762T/G1764A mutations, and in 50.0% (64/128) out of sera with G1896A mutation. Compared with variants with G1896A mutation only, the coexistence mutations were predominant in patients with high level of serum HBV DNA, and related to higher total bilirubin, lower serum albumin and progressive liver diseases. CONCLUSION: The coexistence of G1896A mutation and A1762T/G1764A mutations is very common, and responsible for the major cases with high level of HBV DNA in serum and progressive liver diseases after HBeAg-to-anti-HBe seroconversion. This coexistence mutation variant may have higher pathogenicity and replication ability.     

Clinical significance and evolution of core promoter and precore mutations in HBeAg-positive patients with HBV genotype B and C: a longitudinal study.

BACKGROUND/AIMS: The aims of this longitudinal study were to investigate whether the clinical outcome and evolution of core promoter and precore mutations were different during hepatitis B e antigen (HBeAg) seroconversion between hepatitis B virus (HBV) genotypes B and C in HBeAg-positive patients with chronic hepatitis B. PATIENTS AND METHODS: The core promoter and precore sequences were determined from serial sera of 156 HBeAg-positive patients with chronic HBV infection. RESULTS: In HBV genotype C, the T1762/A1764 mutant was detected earlier than the A1896 mutant, and the frequency was significantly higher than in HBV genotype Ba over the entire follow-up period. In HBV genotype Ba, A1896 was found earlier than the T1762/A1764 mutant, and the frequency was significantly higher than in genotype C only before HBeAg seroconversion, and the A1896 mutant played an important role in HBeAg seroconversion in HBV genotype Ba. In addition, the T1846 variant was an independent factor associated with HBeAg seroconversion. Furthermore, HBV genotype C was associated with the development of G or C1753 and T1766/A1768 mutations, and the reactivation of hepatitis after HBeAg seroconversion. Based on Cox's regression analysis, the significant risk factors of liver cirrhosis were older age at entry [hazard ratio (HR)=1.085, 95% confidence interval (CI)=1.036-1.136, P=0.001], alanine transaminase (ALT) >80 U/l (HR=3.48, 95% CI=1.37-8.86, P=0.009), and the T1762/A1764 mutant (HR=5.54, 95% CI=2.18-14.08, P<0.001). CONCLUSIONS: Our study showed that different HBV genotypes were associated with various mutations in the core promoter and precore regions during HBeAg seroconversion. T1762/A1764 mutation could be useful in predicting clinical outcomes in HBeAg-positive patients with HBV infection.     

Role of hepatitis B virus base core and precore/core promoter mutations on hepatocellular carcinoma in untreated older genotype C Chinese patients

The aim of the study was to investigate the prevalence of mutations of basal core promoter (BCP) and precore (PreC) region of hepatitis B virus (HBV) and their association with hepatocellular carcinoma. A total of 341 untreated older HBV patients were divided into three groups: chronic hepatitis B (CHB, 185), cirrhotic hepatocellular carcinoma (LC-HCC, 113) and non-cirrhotic hepatocellular carcinoma (non-LC-HCC, 43). HBV BCP and PreC mutations and genotypes were determined by direct sequencing. Using univariate analysis, age (≥ 45 years), single mutations including A1896 and A1899 and multiple mutations T1762/A1764 + A1896, T1762/A1764 + A1899 and T1762/A1764 + A1896 + A1899 were more frequently detected in LC-HCC and non-LC-HCC patients than in CHB patients. BCP T1762/A1764 mutations were highly detected in LC-HCC patients than in CHB patients. Multivariate logistic regression analysis (adjusted for age and gender) revealed that among HBeAg-positive patients, BCP T1762/A1764 mutations (OR, 5.975; P = 0.05), PreC A1899 mutation (OR, 4.180; P = 0.013) and multiple mutations T1762/A1764 + A1899 (OR, 6.408; P = 0.006) were independently associated with the development of LC-HCC; PreC A1899 mutation (OR, 7.347; P = 0.034) was also independently associated with the development of non-LC-HCC. On the other hand, among HBeAg-negative patients, PreC A1896 mutation (OR, 5.176; P = 0.002) and multiple mutations T1762/A1764 + A1896 (OR, 4.149; P = 0.007) were independently associated with the development of non-LC-HCC. These results indicated that older age (≥ 45 years) was associated with LC-HCC and non-LC-HCC development. BCP T1762/A1764 mutations and PreC A1899 mutation were associated with the LC-HCC development in HBeAg-positive patients. PreC A1896 mutation was associated with the non-LC-HCC development in HBeAg-negative patients.     

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.     

Real-time quantification of hepatitis B virus core-promoter and pre-core mutants during hepatitis E antigen seroconversion

BACKGROUND/AIMS: Detection of hepatitis B virus (HBV) core-promoter A(1762)T-G(1764)A and pre-core G(1896)A mutants has relied on qualitative assays. We tested the hypothesis that the quantity of A(1762)T-G(1764)A and G(1896)A mutants might have clinical impact, by quantifying these mutants before and after HBe antigen (HBeAg) seroconversion in 58 patients. METHODS: A real-time quantitative-polymerase chain reaction (Q-PCR) was developed, using minor groove binder (MGB)-conjugated TaqMan probes to impart reaction specificity for wildtype/mutant HBV populations. RESULTS: Significant quantities (>20%) of core-promoter A(1762)T-G(1764)A mutant existed in 65% of patients before and after HBeAg seroconversion, and were significantly changed (>20% increase/decrease) in 13% of patients after seroconversion. Quantity of A(1762)T-G(1764)A mutants was positively correlated with alanine aminotransferase (ALT) (P<0.001) and HBV DNA (P<0.001) levels, both before and after HBeAg seroconversion. Significant quantities of pre-core G(1896)A mutant existed in about 90% of patients before and after HBeAg seroconversion, and were changed in 16% of patients after seroconversion. Quantity of G(1896)A mutant was negatively correlated with ALT (P=0.044) and HBV DNA (P=0.007) levels. CONCLUSIONS: The A(1762)T-G(1764)A and G(1896)A mutants existed in a high proportion of patients before and were unaffected after HbeAg seroconversion. The quantities of A(1762)T-G(1764)A mutant were positively and G(1896)A mutant negatively correlated with liver inflammation and viral replication.     

孕妇中乙型肝炎病毒前C区和核心启动子区热点变异研究

目的研究乙型肝炎病毒（HBV）携带产妇体内HBV前C区和核心启动子（CP）区热点变异情况。方法82例无症状慢性HBV携带临产孕妇采用HBV核酸扩增荧光定量检测试剂盒抽提孕妇血清HBVDNA,血清HBsAg、HBeAg采用EIA法检测,半巢式PCR法扩增产妇HBV前C区、CP区核苷酸片段,ABI3730型DNA自动荧光测序仪对PCR产物直接测序。结果82例无症状HBV携带临产孕妇中HBsAg单阳性52例,HBsAg、HBeAg双阳性30例;单阳性孕妇中1896G→A变异的检出率为21．2％（11／52）,仅测到1例双阳性孕妇存在1896G→A变异,1899G→A变异仅发生在1例单阳性孕妇,且与1896G→A变异连锁出现。1762A→／1764G→A变异总是连锁出现,且仅发生于单阳性孕妇,单阳性孕妇中双变异的检出率为17．3％（9／52）。结论HBV前C区、CP区1896G→A及1762A→T／1764G→A热点变异在无症状HBV携带单阳性孕妇中有较高的检出率,HBV前C区和核心启动子区的热点变异可能与肝损伤的严重程度无关。     

Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B

In chronic replicative hepatitis B the significance of mutations in the basic core promoter (BCP), core upstream regulatory sequences (CURS) and negative regulatory element (NRE) for response to interferon (IFN) is unknown. A sequence analysis of the NRE, CURS, BCP, and precore region was performed from sera of 96 patients with chronic replicative hepatitis B (64 hepatitis B e antigen [HBeAg]-positive patients and 32 HBeAg-negative patients) treated with alfa-IFN (IFN-alpha). The overall sustained response (SR) rate to IFN was 30% with no significant difference between HBeAg-positive and HBeAg-negative patients. IFN responsiveness correlated to hepatitis B virus (HBV)-DNA levels, hepatitis B surface antigen (HBsAg) levels, the number of mutations in the complete BCP, especially nucleotide (nt) region 1753 to 1766 and mutations at nt 1762 and 1764. In HBeAg-positive hepatitis, SR to IFN was associated with a high number of mutations in the BCP (P <.04) and nucleotide region 1753 to 1766 (P <.015) as well as mutations at nucleotide 1764 (P <.007). In HBeAg-negative hepatitis, SR to IFN correlated with a low number of mutations in the BCP (P <.04) and nucleotide region 1753 to 1766 (P <.02) and a wild-type sequence at nt 1764 (P <.003). Prediction of IFN response was possible on the basis of nt 1764 in 77% of HBeAg-positive patients and 78% of HBeAg-negative patients. IFN response did not correlate with the occurrence of the 1896 mutation, mutations in the CURS or NRE, disease duration, ethnic origin of the patient, alanine transaminase (ALT) levels and HBV genotype. Our data suggest that HBV genome mutations located within the BCP are determinants of a response to IFN therapy.     

乙型肝炎病毒基因型、BCP及PC区变异与拉米夫定治疗后HBV DNA反弹的关系

目的:探讨HBV基因型、C区基本核心启动子(BcP)及前C(PC)区变异与拉米夫定抗病毒治疗后HBV DNA反弹的关系.方法:应用多引物对巢式PCR法,PCR-序列分析法,检测拉米夫定治疗27例乙型肝炎患者(治疗组),以及19例从未用过抗病毒治疗患者(对照组)的HBV基因型PC区,BCP的突变位点.结果:27例HBV DNA反弹的患者9例检出G1896A变异率高于对照组(33.33% vs 5.26%,P<0.05),4例检出C1856T变异(14.81%).治疗组4份治疗前标本未检出G1896A、C1856T和BCP变异.与对照组比较,治疗组PC(G1896A)及BCP(A1762T G1764A)双变异的患者中B基因型的构成比增高,分别为75%和50%,C基因型的构成比下降,分别为25%和50%.其中在BCP(A1762T G1764A)变异患者中B、C基因型构成比与对照组比较有显著性差异(P<0.05).4例HBV DNA反弹患者治疗前未检出有基因变异,治疗后有2例检出变异,BCP变异1例,BCP PC变异1例.27例HBV DNA反弹患者BCP变异4例,PC变异2例,BCP PC变异8例.结论:BCP(T1762/A1764)变异、PC区(G1896A)变异可能与拉米夫定治疗后HBV DNA反弹有关.病毒变异导致的HBV DNA反弹可以是单基因变异引起,也可以是多个基因联合变异引起,拉米夫定治疗后B基因型患者更易发生A1762T G1764A变异.