| Summary: | 碩士 === 國立臺灣大學 === 臨床醫學研究所 === 91 === Introduction
The hepatitis B virus (HBV), discovered in 1965, infects more than 350 million people worldwide. HBV is the major cause of acute and chronic liver diseases, and persistent HBV infection is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC), accounting for 1 million deaths annually.
The natural history of chronic HBV infection could be divided into three sequential stages: (1) the immune tolerance stage, characterized by HBeAg reactivity in serum and only minor histological activity, active viral replication continues despite little or no elevation in the aminotransferase level and no symptoms of illness; (2) the immune clearance stage, during which serum is positive for HBeAg or anti-HBe, but serum HBV DNA levels drop as the number of infected cells declines and histological signs of chronic active hepatitis are prominent, the stage may persist for 10 or more years, leading to cirrhosis; and (3)the integrated stage, when the patient is anti-HBe positive and a marked decrease in viral DNA is observed, amintransferase levels become normal. However, patients remain positive for HBsAg, presumably because of the integration of the S gene into host’s hepatocyte genome.
Hepatitis B e antigen (HBeAg) is a reliable and sensitive marker of hepatitis B virus replication. Individuals negative for HBeAg were considered to have nonreplicative HBV infection, and their serum aminotransferase levels were normal or nearly normal.
In the early 1980s it became apparent that HBV could replicate in the absence of HBeAg. It is known that precore stop codon mutation (G1896A) that abolishes the synthesis of hepatitis B e antigen (HBeAg) plays a major role in patients with HBeAg-negative chronic hepatitis B. In addition, the double mutations in the basal core promoter (A1762T and G1764A) have been demonstrated to reduce the synthesis of HBeAg by suppressing the transcription of precore mRNA. Both mutations in the precore and basal core promoter regions have been reported to be associated with the occurrence of fulminant hepatitis, and are commonly observed in chronic hepatitis B. However, the clinical significance of precore and basal core promoter mutations remains controversial and needs to be established.
Although serological and genotypic classifications of HBV have been well recognized, the clinical significance of HBV genotypes in terms of clinical outcomes and therapeutic response to antiviral therapy in patients with chronic HBV infection remains largely unknown until recently. Previous studies have indicated that, genotype B and C were the predominant HBV strains in Taiwan and Japan, and genotype C is associated with more severe liver disease. In addition, HBV genotype C is associated with a higher frequency of basal core promoter mutation and a lower response rate to interferon alfa therapy as compared to genotype B in patients with HBeAg-positive chronic hepatitis B. However, whether such an association also holds true in those with HBeAg-negative chronic hepatitis B remains largely unknown. We thus investigated the association of HBV genotypes as well as precore/basal core promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative HBV related liver disease in Taiwan.
Materials and Methods
Patients. A total of 174 patients with chronic HBV infection who were regularly followed at the Taipei municipal Jen-Ai hospital were consecutively enrolled. They included 62 asymptomatic carriers with persistently normal serum alanine aminotransferase (ALT) levels at least for 2 years in periodic biochemical examination and 112 HBV related liver disease. Among them 49 had chronic hepatitis, 31 had cirrhosis and 32 had HCC. All of them were HBsAg-positive, HBeAg-negative and anti-HBe positive. They were negative for antibodies to hepatitis C virus (HCV), hepatitis D virus (HDV) or human immunodeficiency virus (HIV), and none had received antiviral treatment during the follow-up period. Serum samples were collected and stored at -70°C until use.
Serological testing. The biochemical tests were measured by using routine automated methods. The HBsAg, HBeAg, antibodies to HCV and HDV were assayed by commercially available kits (General Biological HBsAg RIA and HBeAg/Anti-HBe RIA, General Biological Cooperation, Taiwan. HCV EIA II and Anti-Delta. Abbot Laboratories, North Chicago, IL, USA).
Genotyping of HBV. The identification of HBV genotypes was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the surface gene of HBV as previously described. Briefly, DNA was extracted from 200 μL of serum samples by using the QIAamp blood kit (Qiagen, Chatsworth, CA, USA), and the fragment of the HBV genome between nucleotide positions 256 and 796 was then amplified. The PCR products were subsequently treated with restriction enzymes. After incubation, the samples were run on a 3% agarose gel and stained by ethidium bromide. Six genotypes (A-F) of HBV could be identified by the restriction patterns of DNA fragments. Unclassified genotype was defined as an unpredicted or atypical restriction pattern. To avoid false-positive results, instructions to prevent cross contaminations were strictly followed, and results were considered valid only when they were obtained in duplicate.
Amplification and sequencing of precore/basal core promoter gene. For the first stage PCR, 25μl of reaction mixture containing 2μl of the DNA sample, 1X PCR buffer (10 mM tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin and 0.1% triton X-100), 10mM of each dNTP, 100 ng of each outer primer and 1unit of Taq DNA polymerase was amplified in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) for 35 cycles. Each cycle entailed denaturation at 95°C for 60s, primer annealing at 52°C for 30s and extension at 72°C for 60s with final extension step at 72°C for 10 min. After the first amplification, 1μl of the PCR products was reamplified for another 35 cycles with 100 ng of each inner primer. For the precore region, the outer sense primer was 5''-CTGGGAGGAGTTGGGGGA-3'', nucleotide positions 1730-1747; the outer antisense primer was 5''-CAATGCTCAGGAGACTCTAAGGC -3'', nucleotide positions 2043-2021; the inner sense primer was 5''-GGTCTTTGTACTCGGAGGCTG -3'', nucleotide positions 1763-1783; the inner antisense primer was 5''-GTCAGAAGGCAAAAAAGAGAG-3'', nucleotide positions 1966-1946. For the basal core promoter region, the outer sense primer was 5''-CTAGCCGCTTGTTTTGCTCG-3'', nucleotide positions 1282-1301; the outer antisense primer was 5''-CACAGCTTGGAGGCTTGAAC-3'', nucleotide positions 1881-1862; the inner sense primer was 5''-CTCATCTGCCGGACCGTGTG-3'', nucleotide positions 1562-1581; the inner antisense primer was 5''-TAGGACATGAACAAGAGATG-3'', nucleotide positions 1859-1840. Nucleotide sequences of the amplified products were directly determined by using fluorescence labeled primers with a 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing conditions were specified in the protocol for Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems). The inner primers were used as sequencing primers for both directions of each region.
Quantitation of serum HBV DNA level. Serum HBV DNA level was quantitated by a polymerase chain reaction assay with a lower limit of detection of 200 copies/ml (Cobas Amplicor HBV Monitor, Roche Diagnostic Systems Inc.)
Statistical analysis. Data were analyzed by chi-square test, Fisher''s exact test, logistic regression, Student’s t test or Pearson correlation where appropriate. All of the tests of significance were two-tailed and a p value of less than 0.05 was considered statistically significant.
Results
Patient Characteristics. The baseline clinical and virological characteristics of the 174 HBeAg negative patients with chronic HBV infection are shown in Table 2. There was a male predominance and the mean age of the patients was 47 years (range, 17 to 81 years). The genotype distribution in the 174 patients as follows: A, 1(0.6%); B, 111(63.8%); C, 44(25.3%); D, 4(2.3%); E, 0; F, 6(3.4%) and unclassified, 8(4.6%). The overall prevalence of precore stop codon mutant and basal core promoter 1762/1764 mutants was 79% and 64%, respectively.
Comparison Between Asymptomatic Carriers and HBV Related Liver Disease. The male-to-female ratio and mean age were significantly higher in HBV related liver disease patients (P=0.004 and P<0.001). Genotype B was more prevalent in asymptomatic carriers (50 in 62, 81%), and genotype C significantly increased in patients with HBV related liver disease (39 in 112, 35%) (P=0.001). The prevalence of precore stop codon mutant was comparable between asymptomatic carriers and HBV related liver disease (79% vs. 79%). Patients with HBV related liver disease had significantly higher prevalence of basal core promoter 1762/1764 mutants than asymptomatic carriers (72% vs. 50%, P=0.014).
Comparison Between Genotype B and C Patients. The baseline clinical features in patients with genotype B and C infection were comparable in terms of mean age, sex ratio, mean serum ALT level, and prevalence of precore stop codon mutant (Table 3). Patients with genotype C infection had a significantly higher mean age (52 years vs. 46 years, P=0.012), prevalence of basal core promoter 1762/1764 mutants (79% vs.59%, P=0.002) and ratio of HBV related liver disease (89% vs.55%, P=0.0001).
Factor Associated With HBV Related Liver Disease. The prevalence of basal core promoter 1762/1764 mutants in different clinical stage of chronic HBV infection was shown in Table 4. The frequency of basal core promoter 1762/1764 mutants increased with advanced clinical stages, from 50% in asymptomatic carriers to 88% in HCC patients. To determine whether the likelihood of basal core promoter 1762/1764 mutants differed by different clinical stages of liver disease, logistic regression analysis was used. Liver cirrhosis and HCC had a significantly greater likelihood of basal core promoter 1762/1764 mutants than asymptomatic carriers (odds ratio, 4.8; 95% confidence interval [CI] 1.83-12.58 for liver cirrhosis and odds ratio, 7.33; 95% confidence interval [CI] 2.19-24.5 for HCC, score test for the trend of odds P=0.0001).
We further analyzed the risk factor associated with HBV related liver disease. The risk factors including sex, age, precore stop mutant, basal core promoter 1762/1764 mutant and genotype for HBV related liver disease in chronic HBV infection were determined by multiple logistic regression analysis (Table 5). The independent factors associated with the progression of HBV related liver disease included male (odds ratio, 3.81; 95% CI, 1.58-9.12, P=0.003), old age (>50 years) (odds ratio, 3.64; 95% CI, 1.48-8.93, P=0.005) and genotype C infection (odds ratio, 6.56; 95% CI, 2.41-17.86, P<0.001).
Liver cirrhosis and HCC are the leading causes of death for chronic HBV infection, thus we emphasized the risk factors associated with the development of liver cirrhosis and HCC. Individuals with old age (>50 years) (odds ratio, 9.71; 95% CI, 4.22-22.22, P<0.001) and basal core promoter 1762/1764 mutants (odds ratio, 4.24; 95% CI, 1.63-11.04, P=0.003) were significantly associated with the development of liver cirrhosis and HCC (Table 6). When stratified by age, the prevalence of basal core promoter 1762/1764 mutants was comparable between younger and older liver cirrhosis and HCC patients (88% vs. 84%). Nevertheless, the difference was significant between younger liver cirrhosis and HCC patients and age-matched asymptomatic carriers (88% vs. 45%, P=0.002) (Figure 3).
Comparison Between male and female patients. The baseline clinical features between male and female patients were comparable in terms of mean age, mean serum ALT level, the prevalence of precore stop codon/basal core promoter 1762/1764 mutant and the prevalence of genotype B and C. Male patients had a significantly higher ratio of HBV related liver disease (71% vs.47%, P=0.004) than female patients (Table 7).
We found the different risk factors associated with the development of liver cirrhosis and HCC in male and female. Old age (>50 years) was significantly associated with the development of liver cirrhosis and HCC in both male and female patients (odds ratio, 4.69; 95% CI, 1.55-14.28, P=0.006 and odds ratio, 81.96; 95% CI, 5.58-1203, P<0.001, respectively). But basal core promoter 1762/1764 mutants (odds ratio, 4.34; 95% CI, 1.30-14.52, P=0.02) was significantly associated with the development of liver cirrhosis and HCC only in male patients (Table 8).
The Serum HBV DNA Levels of Chronic Hepatitis. Of the 46 patients with HBeAg-negative chronic hepatitis (Table 9), all of them had detectable serum HBV DNA (mean, 1.1×108 copies/ml; range, 4.9×103-1.2×109 copies/ml). Ten of the 46 patients had acute exacerbation, the mean serum HBV DNA levels was significantly higher than the patients without acute exacerbation (3.32×108 copies/ml vs. 5.33×107 copies/ml, P=0.0002). The serum HBV DNA level was significant correlation with serum ALT level by the Pearson correlation test (r = 0.333, P=0.024). We further analyzed the association of serum HBV DNA with HBV genotype and precore/basal core promoter mutants. There was no difference between patients with genotype B and genotype C infection (Table 10). In patients with both precore and basal core promoter 1762/1764 mutations, there was a lower risk of high detectable serum HBV DNA level (>10 pg/ml) than in patients with either precore stop codon mutation or the basal core promoter mutation alone, although the difference was not significant (P=0.32)(Table 11).
Discussion
HBV mutant strains selected during HBeAg seroconversion, which allow the persistence of viral replication after loss of HBeAg have been reported. Among these mutants, precore stop codon mutation (G1896A) that abolishes the synthesis of HBeAg has been extensively studied and may play a role in HBeAg-negative chronic hepatitis B. In addition, double mutations in the basal core promoter (A1762T and G1764A) are also demonstrated to reduce the synthesis of HBeAg by suppressing the transcription of precore mRNA in both HBeAg —positive and —negative patients.
It is known that the prevalence of the precore stop codon mutation differs with geographic location and is genotype-dependent, that is less frequent in genotype A (which prevails in North America and Europe). The prevalence of such a mutation in HBeAg-negative chronic hepatitis was 12%-27% in the United States and Northern Europe, but up to 90% in the Mediterranean region and in some Asian regions. In the present study, our result consistently indicated that HBV genotype A was rare in Taiwan, and we found that the prevalence of the precore stop codon mutation in Taiwanese patients with HBeAg-negative chronic HBV infection was 79%. In addition, we firstly found that the prevalence of double mutations in the basal core promoter was 67% in patients with HBeAg-negative patients in Taiwan, and this may contribute to the hepatitis activity in them. However, the influence of precore stop codon and basal core promoter mutations in the virulence of HBV infection as well as the pathogenesis of liver cell damage remains controversial.
In this study, there was no different prevalence of precore stop codon mutation between the different clinical stages of HBeAg-negative chronic HBV infection. Thus the precore stop codon mutation alone appears to have no direct pathogenic role in chronic HBV infection. In contrast, the frequency as well as likelihood of basal core promoter 1762/1764 mutants increased with advanced clinical stages. It seems that the mutant is more pathogenic than wild-type virus and may serve as a marker for progression of liver disease.
The region of basal core promoter (nucleotides 1742 to 1849), overlaps with the X gene (nucleotide 1685-1849), controls the transcription of both precore messenger RNA and pregenomic RNA. The region also encodes the carboxyl-terminus of the X protein, which has transactivating function. Mutation in this region could influence HBeAg production and viral replication as well as the amino acid sequence of the X protein. The diminishes production of HBeAg and increases viral replication result in increased host immune response and enhanced liver cell damage. In present study, the basal core promoter 1762/1764 mutants was significantly associated with the development of liver cirrhosis and HCC (odds ratio, 4.24, P=0.003). The data indicated an association of basal core promoter 1762/1764 mutant with progression of chronic hepatitis, and may be caused liver cirrhosis and malignant transformation of liver cell after a long period of chronic hepatitis. In addition, the alternation of the amino acid sequences of the X protein may play a role in hepatocarcinogenesis. The effect of basal core promoter 1762/1764 mutant was remarkable in male. Reasons for the difference between male and female patients remain unknown. Many lines of evidence have demonstrated the modulatory effect of X protein in cell cycle progression and in the inactivation of tumor suppressors. Therefore, further cohort study to observe the effect of basal core promoter 1762/1764 mutant and the functional consequences of mutated X protein in male and females patients are needed to clarify this important issue.
The clinical, virological and therapeutic implications of HBV genotypes have been partially clarified. Previous reports shown genotype C is associated with more severe liver disease and genotype B is associated with the development of HCC in young noncirrhotic patients in Taiwan. By contrast, genotype B has a relatively good prognosis in Japan and Hong Kong. Similar to other Asian region, genotype B and C were the predominant strains in Taiwan. But we found the prevalence of genotype B is higher than genotype C in Taiwan. It is different from Japan and Hong Kong that the majority of HBeAg-negative chronic hepatitis infected with genotype C.
Several studies implied that the patients with genotype B infection experience earlier HBeAg seroconversion and genotype C seems to stay longer in the immune clearance phase and shifts to severe liver inflammation. In consistency with our study, the mean age of patients with genotype C infection was significantly higher than genotype B infection (52 years vs. 46 years, respectively, P=0.012). In addition, the proportion of HBV related liver disease including chronic hepatitis, liver cirrhosis and HCC was also significantly higher than patients with genotype B infection (89% vs. 55%, respectively, P=0.0001). Nevertheless, the pathogenic-link between the genotypes and the progression of liver disease remains largely unknown. In our study, the patients with genotype C had greater prevalence of basal core promoter 1762/1764 mutants than the patients with genotype B (79% vs.59%, P=0.002). This may contribute to the different clinical outcomes between genotype B and genotype C infection.
Of particular note is the role of genotype B and C in the progression of chronic liver disease. Genotype C was associated with progression of HBV related liver disease (odds ratio, 6.56, P<0.001). But genotype B and genotype C were at similar risk for the development of liver cirrhosis and HCC. Our result suggests that, although the patients with genotype B infection had earlier HBeAg seroconversion, but a proportion of patients progressed slowly and slower development of liver cirrhosis and HCC. The life-long risk of progression to advanced liver disease is similar among genotype B and C related liver disease.
Currently it is accepted that pathogenetic mechanisms of liver cell damage in HBeAg-negative chronic hepatitis B stem from immune response against replicating HBV. Ongoing HBV replication triggers strong immune responses against the virus. These mechanisms are most likely similar to those operating in HBeAg-positive chronic hepatitis B. So it is not unanticipated that the serum HBV DNA level was significant correlation with serum ALT level (P=0.024).
The potential impact of precore stop codon mutation and basal core promoter mutation on the replication of HBV remains controversial. In the treatment of HBeAg-positive chronic hepatitis B, low serum HBV DNA level (< 10 pg/ml) is one of the predictors for better response to interferon therapy. Thus we defined serum HBV DNA concentration > 10 pg/ml as a high HBV DNA level in the present study. Our data showed that patients with both mutations had a lower proportion of high detectable serum HBV DNA level than those with either precore stop codon mutation or basal core promoter mutation alone. These findings suggested that the impact of basal core promoter mutation on the replication of HBV is influenced by the emergence of precore stop codon mutation.
In conclusions, our results indicated that HBV genotype B and C were predominant in Taiwan. The risk of progression of HBV related liver disease is greater in patients infected with HBV genotype C and basal core promoter 1762/1764 mutants. In addition, basal core promoter 1762/1764 mutants may contribute to the different pathogenicity of different HBV genotype. The impact of mutations in the precore/basal core promoter on viral replication should also be further analyzed.
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