Precore and core promoter mutations,hepatitis B virus DNA levels and progressive liver injury in chronic hepatitis B

BACKGROUND/AIMS: To elucidate the viral factors responsible for progressive liver injury in chronic hepatitis B. METHODS: We analyzed 179 persistently infected patients (21 asymptomatic carriers, 126 with chronic hepatitis and 32 with cirrhosis) with genotype C hepatitis B virus (HBV). HBeAg/anti-HBe, levels of HBV DNA, mutations in the basic core promoter (BCP) region at nucleotides 1762/1764 and mutation in the precore (preC) region at nucleotide 1896 were determined. Serial samples from 18 patients also were analyzed. RESULTS: HBeAg/anti-HBe and HBV DNA levels per se were not related to liver fibrosis. The frequency of BCP mutations increased with progression of liver fibrosis. Although the preC mutation was detected more often among the LC group, the role of this mutation in progression of fibrosis seems less than that of the BCP mutations. Sequential analysis showed that (1) rapidly progressing cases were positive continuously for double mutations in the BCP with a wild-type precore sequence, and (2) asymptomatic cases with anti-HBe acquired the preC mutation during their clinical course. CONCLUSIONS: Double mutations in the BCP region at nucleotide 1762/1764 are closely related to progression of chronic liver disease. Acquisition of mutation in the preC region at nucleotide 1896 may contribute to inactivation of chronic liver disease.     

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.     

慢加急性乙型肝炎肝衰竭患者乙型肝炎病毒前C/CP区变异研究进展*

慢加急性肝衰竭（ACLF）是我国肝衰竭的主要类型,其病情发展迅速、预后不良、病死率较高。在我国,乙型肝炎病毒（HBV）感染是导致ACLF的主要病因,其中HBV前C（PC）/核心启动子（CP）区基因变异与ACLF的发生密切相关。本文重点介绍HBV PC/CP区基本结构和功能、PC/CP区变异后生物学特性的改变、ACLF的免疫病理损伤机制、PC/CP区变异与ACLF疾病进展的临床意义等方面的研究进展。     

Hepatitis B virus DNA prediction rules for hepatitis B e antigen-negative chronic hepatitis B

After hepatitis B e antigen (HBeAg) seroconversion, hepatitis B may become inactive or progress to HBeAg-negative hepatitis with persistent or intermittent alanine aminotransferase (ALT) elevation. The aim of this study was to prospectively identify factors predictive of the clinical course in HBeAg-negative chronic hepatitis B (CHB). Patients were stratified by ALT and HBeAg status and followed every 3 months for up to 5 years. Kaplan-Meier and Cox regression analysis using the change from normal ALT to elevated ALT as endpoints were performed to determine factors associated with ALT elevation/normalization. Seventy-four HBeAg-negative and 32 HBeAg-positive patients were prospectively evaluated. For HBeAg-negative patients, hepatitis B virus (HBV) DNA was predictive of future ALT. Only 1 patient with normal ALT and an HBV DNA value lower than 10,000 copies/mL developed an elevated ALT within the subsequent year, whereas 67% with an HBV DNA value greater than 100,000 copies/mL had a rise in ALT above normal within 1 year. Patients with a previous history of ALT elevation and longer follow-up at all levels of HBV DNA were more likely to experience ALT elevations. For HBeAg-negative patients with elevated ALT and all HBeAg-positive patients, HBV DNA did not predict future ALT. Other viral and host factors were not predictive of future ALT. CONCLUSION: HBeAg-negative CHB has a fluctuating course. HBV DNA values lower than 10,000 copies/mL predict persistently normal ALT for at least 1 year. Patients with HBV DNA values between 10,000 and 100,000 copies/mL can safely be followed at 6 monthly intervals, whereas HBV DNA values greater than 100,000 copies/mL are highly predictive of future ALT elevation and should prompt regular follow-up.     

Profile of hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND: Although chronic hepatitis B occurs in hepatitis B e antigen (HBeAg)-negative patients, its prevalence and clinical significance are not known. AIM: To determine the prevalence and profile of HBeAg-negative chronic hepatitis B virus (HBV) infection. METHODS: A retrospective analysis of 363 consecutive patients (mean age 36 y; 288 men) with chronic HBV infection was performed. All patients were HBsAg-positive. Tests for liver profile, HBeAg and anti-HBe antibody were performed in all patients. Serum HBV DNA was tested using branched DNA assay in 245 patients. The patients were classified into three groups: no cirrhosis with normal ALT levels, no cirrhosis with elevated ALT levels, and clinical or histological evidence of cirrhosis. RESULTS: Of 363 patients, 141 (39%) were HBeAg-positive and 222 (61%) HBeAg-negative. Of HBeAg-negative patients, 120 (54%) had normal ALT, 45 (20%) had elevated ALT and 57 (26%) had evidence of cirrhosis; corresponding figures in the HBeAg-positive patients were 40 (28%), 66 (47%) and 35 (25%). HBV DNA was positive in 53 of 131 (40%) HBeAg-negative patients tested; of these 53 patients, 9 (17%) had normal ALT, 20 (38%) had elevated ALT and 24 (45%) had cirrhosis. Thus, 72% of HBeAg-positive and 46% of HBeAg-negative patients had elevated ALT and/or cirrhosis. Among the latter group, 83% of HBV DNA-positive patients had elevated ALT and/or cirrhosis. Overall, 18% of HBsAg-positive patients had HBeAg-negative, HBV DNA-positive liver disease. CONCLUSION: HBeAg-negative chronic hepatitis B is not an uncommon and benign entity and chronic liver disease develops in a significant proportion of such patients.     

Relationship between the development of precore and core promoter mutations and hepatitis B e antigen seroconversion in patients with chronic hepatitis B virus

Chinese patients with chronic hepatitis B virus (332 with and 44 without cirrhosis-related complications) were studied. Fifty percent of patients <30 years old had precore mutations. The prevalence of precore mutations among hepatitis B e antigen (HBeAg)-positive patients, although lower than that among anti-HBe-positive patients (P=.031), was already high (44.2%). Median HBV DNA level in anti-HBe-positive patients was 1.5 x 10(6)-1.55 x 10(6) copies/mL, irrespective of the presence or absence of precore mutations. There was no difference in the prevalence of precore mutations between patients with and without complications (P, not significant). However the prevalence of core promoter mutations was higher among patients with complications than among those without complications (90.5% vs. 69.3%, respectively; P=.003). In conclusion, precore mutations occurred in a large proportion of Chinese patients with chronic hepatitis B virus before HBeAg seroconversion. The development of complications was not related to precore mutations but was probably due to the persistence of significant viremia after HBeAg seroconversion.     

Efficacy of lamivudine in patients with hepatitis B e antigen-negative/hepatitis B virus DNA-positive (precore mutant) chronic hepatitis B. Lamivudine Precore Mutant Study Group

This placebo controlled, double-blind study evaluated the efficacy and safety of lamivudine in patients with hepatitis B e antigen (HBeAg)-negative/hepatitis B virus (HBV) DNA-positive chronic hepatitis B. Patients were randomized to receive 100 mg lamivudine orally once daily for 52 weeks (n = 60) or placebo for 26 weeks (n = 65). Patients who were HBV DNA positive at week 24 were withdrawn at week 26. The primary efficacy endpoint was loss of serum HBV DNA plus normalization of alanine transaminase (ALT) at week 24. A significantly higher proportion of patients receiving lamivudine (63%) had a complete response at week 24 compared with patients receiving placebo (6%) (P <.001). Secondary efficacy parameters included histological response from baseline to week 52 in the lamivudine-treated patients. At week 52, 60% of lamivudine-treated patients with liver biopsy specimens available showed histological improvement (>/=2-point reduction in Knodell necro-inflammatory score), 29% showed no change, and 12% worsened. In a ranked assessment of pretreatment and post-treatment biopsy pairs 11% improved, 86% showed no change, and 2% worsened in fibrosis. At week 52, 27% of patients receiving lamivudine had YMDD (tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase) variant HBV. The incidence of adverse events and laboratory abnormalities was similar in both groups. In conclusion, lamivudine treatment results in a significant virological and biochemical improvement compared with placebo, induces an improvement or no change in histology in most patients, and is well tolerated. The response to lamivudine therapy in HBeAg-negative patients is similar to the response reported in previous studies of patients with HBeAg-positive chronic hepatitis B.     

Precore/core region mutations of hepatitis B virus related to clinical severity

Despite the availability of an effective vaccine, hepatitis B virus(HBV) infection remains a major health problem, with more than 350 million chronically infected people worldwide and over 1 million annual deaths due to cirrhosis and liver cancer. HBV mutations are primarily generated due both to a lack of proofreading capacity by HBV polymerase and to host immune pressure, which is a very important factor for predicting disease progression and therapeutic outcomes. Several types of HBV precore/core(preC/C) mutations have been described to date. The host immune response against T cells drives mutation in the pre C/C region. Specifically, pre C/C mutations in the MHC class Ⅱ restricted region are more common than in other regions and are significantly related to hepatocellular carcinoma. Certain mutations, including preC G1896 A, are also significantly related to HBe Ag-negative chronic infection. This review article mainly focuses on the HBV pre C/C mutations that are related to disease severity and on the HBe Ag serostatus of chronically infected patients.     

Evolution of hepatitis B virus polymerase gene mutations in hepatitis B e antigen-negative patients receiving lamivudine therapy

Lamivudine has been shown to be effective in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, but its long-term efficacy and the rate of resistant mutations in patients with HBeAg-negative chronic hepatitis B is less clear. Twenty-nine patients with HBeAg-negative chronic hepatitis B, who have received lamivudine for at least 1 year were studied to determine the antiviral response, the rate and pattern of lamivudine-resistant mutations, and the effect of lamivudine-resistant mutations on HBeAg status. The mean duration of treatment was 21 +/- 7 months. Before treatment, core promoter variant was detected in 16 (55%) patients and precore stop codon variant in 18 (62%) patients. Serum hepatitis B virus (HBV) DNA was detected by solution hybridization assay in 62%, 4%, and 24% and by polymerase chain reaction (PCR) assay in 100%, 31%, and 40% at months 0, 6, and 24, respectively. The cumulative rates of detection of lamivudine-resistant mutations after 1 and 2 years of treatment were 10% and 56%, respectively. In addition to the duration of treatment, core promoter mutation was associated with the selection of lamivudine-resistant mutants. Three patients with lamivudine-resistant mutations had reversion of the precore stop codon mutation; in 2 patients this was accompanied by the reappearance of HBeAg. We found that lamivudine-resistant mutants were detected at similar rates in patients with HBeAg-negative as in patients with HBeAg-positive chronic hepatitis B. Additional changes in other parts of the HBV genome may restore the replication fitness of lamivudine-resistant mutants.     

Hepatitis B e antigen-negative chronic hepatitis B in Hong Kong

Hepatitis B e antigen-negative chronic hepatitis B (e-CHB) has been reported in Asia but its prevalence and clinical significance have not been determined. The aims of this study were to determine the prevalence of e-CHB in Hong Kong and the frequency of precore and core promoter mutations in these patients. A cross-sectional study was performed in 350 consecutive Chinese patients (230 men and 120 women; mean age +/-SD, 42 +/- 13 years) with chronic hepatitis B virus infection. A total of 243 (69%) patients were hepatitis B e antigen (HBeAg)-negative of whom 15% had clinical cirrhosis. In the remaining 85% of patients, 63% had normal and 22% had elevated transaminases. Serum hepatitis B virus (HBV) DNA was detectable using branched DNA assay in 46% of HBeAg-negative patients with clinical cirrhosis/elevated transaminases. Forty-five percent of the patients with e-CHB had the precore stop codon mutation, and an additional 41% had core promoter changes. There was no correlation between the presence of precore/core promoter mutations and liver disease or HBV-DNA levels. Overall, 17% of HBeAg-negative patients were viremic and had evidence of chronic liver disease (e-CHB) with mean HBV-DNA levels comparable with that in HBeAg-positive patients. In summary, we found that e-CHB may be present in up to 17% of HBeAg-negative patients seen in a tertiary referral center in Hong Kong. e-CHB may be a heterogeneous condition and is not invariably associated with the precore HBV mutant. Population studies are needed to determine the true prevalence of e-CHB in Asia and to assess its natural course and response to treatment.     

Serum cytokine profile in patients with hepatitis B e antigen-negative chronic active hepatitis B and inactive hepatitis B virus carriers

An insufficient cellular immune response seems to be critical for the immunopathogenesis of chronic hepatitis B virus infection.We have previously demonstrated no differences of T-lymphocyte subsets in blood between inactive hepatitis B s antigen(HBsAg) carriers and patients with HBeAg-negative chronic active hepatitis B.This study investigated the peripheral blood cytokine profile in patients with HBeAg-negative chronic active hepatitis B infection(Group A,n = 21) and inactive HBsAg carriers(Group B,n = 13).Serum cytokines [interferon(IFN)-γ,tumor necrosis factor-α,interleukin(IL)-1b,IL-4,IL-12,IL-10,IL-2,IL-5,IL-8] were analyzed by using flow cytometry.Patients with chronic active disease presented with significantly decreased levels of IFN-γ and IL-10 compared to inac-tive carriers(P = 0.048 and P = 0.008,respectively).In HBeAg-negative chronic active hepatitis B patients,a significant negative correlation of IFN-γ levels with serum hepatitis B viral load was noted(P = 0.021).In conclusion,patients with HBeAg-negative chronic active hepatitis B and HBsAg inactive carriers display a different cytokine profile.Decreased Th1 response observed in patients with chronic active hepatitis B could be implicated in the persistence of virus replication and ongoing progression of liver disease.     

Basal core-promoter mutant of hepatitis B virus and progression of liver disease in hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND/AIMS: The long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are distinct from those in HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in this special clinical setting remain largely unknown. We thus investigated the association of hepatitis B virus (HBV) genotypes as well as precore/basal core-promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative chronic hepatitis B in Taiwan. METHODS: HBV genotypes and sequences of precore and basal core-promoter regions of the HBV genome were determined in 174 HBeAg-negative chronic HBV infection patients including 62 inactive carriers and 112 with different stages of liver disease. RESULTS: HBV carriers with older age (> 50 years) (odds ratio, 9.09; 95% confidence interval (CI), 3.22-25, P < 0.001) and basal core-promoter mutant of HBV (odds ratio, 4.12; 95% CI, 1.41-12.03, P = 0.01) were associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). The gender-related risk factors associated with the development of liver cirrhosis and HCC were further analyzed, and basal core-promoter mutant was only associated with the development of liver cirrhosis and HCC in male carriers (odds ratio, 4.35; 95% CI, 1.30-14.52, P = 0.02). CONCLUSIONS: The risk of development of liver cirrhosis and HCC is significantly increased in patients with advanced age as well as with basal core-promoter mutant of HBV. In addition, basal core-promoter mutant might contribute to the gender difference of the progression of liver disease in HBeAg-negative chronic hepatitis B in Taiwan.     

Effect of multiple mutations in the core promoter and pre-core/core region of hepatitis B virus genome on the response to interferon in e antigen-positive chronic hepatitis B

BACKGROUND AND AIMS: Hepatitis B virus (HBV) genomic mutations may be one of the factors that influence the efficacy of interferon (IFN) therapy. The aim of this study was to investigate the effects of mutations in different parts of the HBV genome on IFN therapy. METHODS: We studied the baseline clinical, biochemical, serologic and virologic parameters in 17 patients with e antigen-positive chronic hepatitis B. The DNA sequence of the X gene and pre-core/core gene in serum samples of these patients was analyzed before the initiation of IFN therapy. RESULTS: All five patients with the T1762-A1764 mutation were IFN responsive, while among the 12 remaining patients, only two responded to therapy. Among five patients with both a pre-core A1896 mutation and a mutation in the epitope aa 107-118 of the core region, four were non-responders whereas the fifth responded to therapy. In three other patients with A1896 mutations, one with simultaneous mutations in five lymphocytic epitopes did not respond to therapy; the two remaining patients with concomitant mutations in one or two epitopes were responsive. Serum HBV-DNA levels were lower and titers of antibody to hepatitis B virus core antigen-immunoglobin M (anti-HBc-IgM) were higher in the responders than in the non-responders. Hepatitis B virus genotypes B and C were found to be in all these Chinese patients. CONCLUSIONS: These results suggest that HBV genomic mutations, serum viral loads and titers of anti-HBc-IgM might be predictive of the efficacy of IFN therapy. These clinical findings should be further investigated by in vivo and in vitro experiments.     

Interferon alpha treatment and retreatment of hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND AND AIMS: In hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHBe-), interferon (IFN) achieves very low biochemical sustained response rates. No information exists on retreatment. METHODS: Two hundred sixteen CHBe- patients treated for 5 or 12 months with 3 MU IFN-alpha2b thrice weekly and retreated (51 patients, 60 courses) because of no response or relapse were retrospectively analyzed. RESULTS: After 7.0 years of median follow-up, 39 naive patients (18.1%) were still in biochemical and virologic remission after a single IFN course. Longer treatment and a biochemical response within 4 months were significant predictors, inversely related to relapse by multivariate analysis (relative hazard [RH], 0.611; 95% confidence interval [CI], 0.448-0.834 and RH, 0.290; 95% CI, 0.192-0.438, respectively). Retreatment resulted in 18.4% sustained response by intention-to-treat (18 of 98 patients). Patients with sustained response had persistently normal alanine aminotransferase levels, undetectable hepatitis B virus (HBV) DNA by molecular hybridization, and significant improvement of histologic grade, and 32% of them lost hepatitis B surface antigen. In sustained responders, serum HBV DNA was undetectable or very low at the end of treatment and at the end of follow-up (median 3934 and 903 copies/mL, respectively) by quantitative polymerase chain reaction. CONCLUSIONS: IFN induced long-term biochemical and virologic remission in approximately 18% of naive or retreated patients with CHBe-. Sustained responders exhibited significant histologic improvement and a high rate of HBsAg loss.     

Epidemiological study of hepatitis B virus genotypes, core promoter and precore mutations of chronic hepatitis B infection in Hong Kong

BACKGROUND/AIMS: We conducted a population study to document the prevalence of hepatitis B virus (HBV) genotypes in Hong Kong. METHODS: HBV genotypes, core promoter (CP) and precore mutations were determined in 776 asymptomatic patients. RESULTS: 92.6% patients had single genotype [B (32.5%), C (62.5%)]. 99.1% of genotype B was subtype Ba. Patients with age <50 years had a lower prevalence of genotype B than patients with age >51 years (32.5% vs. 41%, respectively, P=0.028). Compared to patients with genotype C, patients with genotype B had a higher cumulative rate (P=0.018) and younger age (40.1 vs. 34.2 years, respectively, P=0.018) of HBeAg seroconversion. There were no differences in the HBV DNA levels between patients with genotypes B and C, and with wild-type and mutants of CP and precore regions. By multivariate analysis, patients with genotype C and with CP mutations had higher alanine aminotransferase (ALT) levels. CONCLUSIONS: B and C were the two most common HBV genotypes in Hong Kong. The former had a higher chance of earlier HBeAg seroconversion and lower ALT levels. The prevalence of genotype B was lower in patients with age <50, probably related to influx of immigrants from China since 1949.     

Hepatitis B e antigen-negative chronic hepatitis B: natural history and treatment

Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B evolves in the natural history of chronic hepatitis B virus (HBV) infection linked with selection of nonproducing HBeAg but replication-competent HBV mutants, and may have a potentially severe and progressive course. Effective suppression of HBV replication is the main therapeutic target. Sustained off-therapy responses are rare with treatment of finite duration, except perhaps for interferon-based therapies, which induce such responses in a sizeable, yet small proportion of patients. Eventually, the majority of patients will be treated with long-term oral antiviral therapy, which improves patients' outcome but is associated with progressively increasing rates of viral resistance. The long-term resistance profile of adefovir is significantly better than that of lamivudine (LMV), whereas data for entecavir currently are limited to 2 years, with resistance developing in LMV-resistant but not in treatment-na?ve patients. Combination therapy with adefovir added to LMV in LMV-resistant patients is extremely effective; cases of adefovir-resistance have not been reported to date.