Basal Core Promoter Mutations of Hepatitis B Virus Increase the Risk of Hepatocellular Carcinoma in Hepatitis B Carriers

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1 GASTROENTEROLOGY 2003;124: Basal Core Promoter Mutations of Hepatitis B Virus Increase the Risk of Hepatocellular Carcinoma in Hepatitis B Carriers JIA HORNG KAO,*, PEI JER CHEN,*, MING YANG LAI,* and DING SHINN CHEN *Graduate Institute of Clinical Medicine, Division of Gastroenterology, Department of Internal Medicine, Hepatitis Research Center, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan Background & Aims: Hepatitis B viral (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC) compared with genotype B;however, the virologic factors contributing to the pathogenic differences remain unknown. We investigated the prevalence of T1762/A1764 basal core promoter mutant in a cohort of 250 genotype B- or C-infected HBV carriers with different stages of liver disease to clarify a possible role for this mutant in hepatocarcinogenesis. Methods: The sequences of basal core promoter of HBV genome were determined in 60 inactive HBV carriers and 190 patients with histologically verified chronic liver disease and HCC. Results: Genotype C has a higher prevalence of T1762/A1764 mutation than genotype B (odds ratio, 5.18;95% confidence interval [CI], ;P < 0.001). The likelihood of T1762/A1764 mutation parallels the progression of liver disease, from 3% in inactive carriers to 64% in HCC patients (odds ratio, 20.04;95% CI, ; P < 0.001). By multiple logistic regression analysis, patients with T1762/A1764 mutation were significantly associated with the development of HCC than those without (odds ratio, 10.60;95% CI, ; P < 0.001), and the risk was observed for both genotypes B and C. In addition, the prevalence of T1762/A1764 mutation in younger HCC patients was comparable with older HCC patients but was significantly higher than that in age-matched inactive carriers, irrespective of genotypes. Conclusions: Our data suggest that HBV carriers with T1762/A1764 basal core promoter mutant are at increased risk for HCC and that this mutant may contribute to the pathogenesis of HBV infection. Hepatitis B virus (HBV) infection is a global health problem, and more than 350 million people in the world are chronic carriers of the virus. 1 The clinical manifestations of HBV infection range from acute selflimiting infection or fulminant hepatic failure, inactive carrier state, chronic hepatitis with progression to cirrhosis, and hepatocellular carcinoma (HCC). 2 Pathogenesis of HBV infection is usually through the interaction between virus and host immune responses to HBVencoded antigens. 3 Currently, 7 HBV genotypes (A G) are identified based on the comparison of complete genomes, 4,5 and most of the genotypes have distinct geographic distributions. 4 6 Genotypes A and D are prevalent in Western countries, whereas genotypes B and C prevail in Asia. Genotype E is restricted to Africa and genotype F in Central America. Genotype G has been identified in France and North America. 5 Although the clinical significance of HBV genotypes remains to be firmly settled, it has been shown that genotype C is associated with the development of HCC 7 9 and has a lower response rate to interferon therapy as compared with genotype B. 10 However, the molecular virologic factors that contribute to these clinical and therapeutic differences among HBV genotypes remain largely unknown. Because of the spontaneous error rate of viral reverse transcriptase, HBV genome evolves during the course of infection under the antiviral pressure of the host immunity or specific therapy. 11 These HBV variants could display alteration of epitopes important in the host immune recognition, enhanced virulence with increased replication of HBV, resistance to antiviral therapies, or facilitated cell attachment/penetration. 12 Among these variants, isolates with an adenine (A) to thymine (T) transversion at nucleotide 1762 together with a guanine (G) to adenine (A) transition at nucleotide 1764 (T1762/ A1764) mutations in the basal core promoter (nucleotides ) are often present in hepatitis B carriers with chronic hepatitis, fulminant hepatitis, and HCC, and less often in inactive carriers and immunosuppressed patients. 12 In addition, the T1762/A1764 basal core promoter mutant has been claimed to be associated with Abbreviations used in this paper: Anti-HCV, antibody to HCV;anti- HDV, antibody to HDV;HBsAg, hepatitis B surface antigen;hbv, hepatitis B virus;hcc, hepatocellular carcinoma;hcv, hepatitis C virus; HDV, hepatitis D virus;pcr, polymerase chain reaction;rflp, restriction fragment length polymorphism by the American Gastroenterological Association /03/$35.00 doi: /gast

2 328 KAO ET AL. GASTROENTEROLOGY Vol. 124, No. 2 a poor response to interferon therapy in patients with chronic hepatitis B. 13 Meantime, several recent studies have indicated that genotype C has a higher frequency of T1762/A1764 mutations as compared with genotype B. 10,14 These lines of evidence prompted us to investigate whether the different clinical outcomes between genotype B and genotype C infection correlate with the dinucleotide change in the basal core promoter of HBV genome. In the present study, the prevalence of T1762/A1764 mutation in the basal core promoter was examined in a cohort of genotype B- or C-infected hepatitis B carriers with different clinical stages of liver disease to clarify a possible role for T1762/A1764 mutant in hepatocarcinogenesis in this population. Materials and Methods Patients Serum samples were obtained from a cohort of 250 genotype B- or C-infected chronic HBV carriers (200 men, 50 women; 143 infected with genotype B and 107 with genotype C) with long-term follow-up at the gastroenterologic clinics of the National Taiwan University Hospital since They included 60 hepatitis B e antigen (HBeAg)-negative hepatitis B carriers (44 men, 16 women; 35 infected with genotype B and 25 with genotype C; mean age, years) with persistently normal serum alanine aminotransferase (ALT) levels for at least 3 years in periodic biochemical examinations (every 3 or 6 months) and were inferred as inactive HBsAg carriers and 190 HBsAg-positive patients with histologically verified chronic liver disease and HCC (Table 1). Among them, 35 (25 men, 10 women; 20 infected with genotype B and 15 with genotype C; mean age, 38 9 years) had chronic hepatitis, 28 (22 men, 6 women; 13 infected with genotype B and 15 with genotype C; mean age, years) had cirrhosis, and 127 (109 men, 18 women; 75 infected with genotype B and 52 with genotype C; mean age, years) had HCC. Of 127 patients with HCC, 48 (43 men, 5 women; 36 infected with genotype B and 12 with genotype C; mean age, 41 7 years) were younger than 50 years of age (younger HCC), and the remaining 79 (66 men, 13 women; 39 infected with genotype B and 40 with genotype C; mean age, 61 7 years) were 50 years of age or older (older HCC). 7 The mean age of genotype B- and genotype C-infected younger HCC patients was 38 7 years and 43 6 years, respectively. The histologic diagnosis of chronic liver disease was based on standard criteria. 15 None of them had coinfection with hepatitis C virus (HCV) or hepatitis D virus (HDV). Serum samples from each subject were stored at 70 C until use. For patients with histologically verified chronic liver disease, serum samples were collected at the time when liver biopsies were performed. Serologic Markers HBsAg, HBeAg, antibodies against HCV (anti-hcv), and antibodies against HDV (anti-hdv) were tested with commercially available kits (Ausria II, IMx HBe 2.0, HCV EIA II, Anti-Delta, Abbott Laboratories, North Chicago, IL). Genotyping of HBV HBV genotypes were determined by using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the surface gene of HBV as previously described. 6,7 Six genotypes (A to F) of HBV could be identified by the restriction patterns of DNA fragments. To avoid falsepositive results, instructions to prevent cross contaminations were strictly followed, 16 and results were considered valid only when they were consistently obtained in duplicate. Amplification and Sequencing of Precore (Nucleotides ), Enhancer II (Nucleotides ), and Basal Core Promoter (Nucleotides ) For sequence analysis, nested PCR for amplification of precore, enhancer II, and core promoter genes was performed as previously described. 10 Nucleotide sequences of the amplified products were directly determined by using fluorescence labeled primers with a 377 Automatic Sequencer (Applied Biosystems, Foster City, CA). Sequencing conditions were specified in the protocol for the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems). The inner primer pair was used as sequencing primers for both directions of the gene. Statistical Analysis Data were analyzed by Fisher exact test, 2 analysisof-contingency table with Bonferroni or Yates correction when appropriate. Logistic regression analysis was used to assess the likelihood of T1762/A1764 mutation in different clinical stages of chronic HBV infection and the influence of various factors on the risk of HCC development, after adjusting for potential confounders. A P value of 0.05 was considered statistically significant. Results The sequences of the enhancer II not overlapping with the basal core promoter (nucleotides ) were found to be identical among patients with genotype B or C infection. There was also no difference in this gene according to the clinical stages of chronic HBV infection. The prevalence of T1762/A1764 mutant in different clinical stages of 250 patients with chronic HBV infection is shown in Table 1 and Figure 1. Overall, the frequency of T1762/A1764 mutation increased with advancing clinical stages, from 3% in inactive carriers to 64% in HCC patients (P 0.001). For patients with genotype B infection, the frequency of T1762/A1764

3 February 2003 HBV CORE PROMOTER MUTATION AND HCC 329 Table 1. Prevalence of T1762/A1764 Basal Core Promoter Mutant in Different Clinical Stages of Patients With Chronic Hepatitis B Viral Genotype B or C Infection Prevalence of T1762/A1764 basal core promoter mutant Genotype Number Inactive carrier (%) Chronic hepatitis (%) Cirrhosis (%) Hepatocellular carcinoma (%) B 143 2/35 (6) 1/20 (5) 1/13 (8) 38/75 (51) a C 107 5/25 (20) 3/15 (20) 8/15 (53) 43/52 (83) a Total 250 7/60 (3) 4/35 (11) 9/28 (32) 81/127 (64) a T1762/A1764 basal core promoter mutant, changes of 2 nucleotides, an adenine (A) to thymine (T) transversion at nucleotide 1762 together with a guanine (G) to adenine (A) transition at nucleotide 1764, within the basal core promoter of hepatitis B viral genome. a P by 2 analysis-of-contingency table with Bonferroni correction. mutation was significantly higher in HCC patients compared with that in inactive carriers, chronic hepatitis, and cirrhotic patients (P 0.001). For those with genotype C infection, HCC patients also had a significantly higher prevalence of T1762/A1764 mutant than inactive carriers, chronic hepatitis, and cirrhotic patients (P 0.001). To determine whether the likelihood of T1762/A1764 mutant differed by different clinical stages of liver disease, multiple logistic regression analysis was used. The logistic regression model was adjusted for potential confounding factors (Table 2). For genotype B infection, only HCC patients had a significantly greater likelihood of T1762/A1764 mutant than inactive carriers (odds ratio, 17.79; 95% confidence interval [CI ], ; P 0.001). For genotype C infection, both cirrhotic and HCC patients had a significantly greater likelihood of T1762/A1764 mutant than inactive carriers (odds ratio, 4.91; 95% CI, ; P for cirrhosis and odds ratio, 18.70; 95% CI, ; P for HCC). The logistic regression analysis also showed that genotype C was significantly associated with the presence of T1762/A1764 mutation than genotype B (52% vs. 29%; odds ratio, 5.18; 95% CI, ; P 0.001) Figure 1. Prevalence of T1762/A1764 basal core promoter mutant in 250 patients with chronic hepatitis B virus infection stratified by clinical stages. The frequency of T1762/A1764 mutant increased with advancing clinical stages in both genotypes B (open column) and C(solid column) infected patients (6% and 20% in inactive carriers to 51% and 83% in hepatocellular carcinoma patients for genotypes B and C, respectively). IC, inactive carrier; CH, chronic hepatitis; LC, liver cirrhosis; HCC, hepatocellular carcinoma. after adjusting for sex, age, and severity of liver disease. The difference was most remarkable in the stages of cirrhosis and HCC (53% vs. 8%, P 0.01 and 83% vs. 51%, P 0.001, respectively, Figure 1). The attributable risk of multiple factors including sex, age, cirrhosis, T1762/A1764 mutation, and HBV genotype for HCC in hepatitis B carriers was determined by multiple logistic regression analysis (Table 3). Individuals with T1762/A1764 mutation were significantly associated with the development of HCC than those without this mutation (odds ratio, 10.60; 95% CI, ; P 0.001), and the risk was observed for both genotypes B and C. Statistically significant odds ratios were also obtained for men (P 0.033), older age (P 0.001), cirrhosis (P 0.001), and HBV genotype C (P 0.002). Of 127 patients with HCC, 76 (60%) were cirrhotic, and 54 (71%) of them had T1762/A1764 mutation, whereas only 27 (53%) of 51 noncirrhotic patients had T1762/A1764 mutation. Therefore, cirrhotic patients tended to have a higher frequency of T1762/A1764 mutant than noncirrhotic patients (71% vs. 53%, respectively, P 0.06). When stratified by age, the prevalence of T1762/A1764 mutant was comparable between younger and older HCC patients, irrespective of HBV genotypes (54% vs. 46% for genotype B and 92% vs. 80% for genotype C) (Figure 2). Nevertheless, the difference was significant between younger HCC patients and age-matched inactive carriers (54% vs. 6% for genotype B, P and 92% vs. 20% for genotype C, P 0.001). With a mean follow-up period of years (median, 6 years), 3 of 9 cirrhotic patients infected with T1762/A1764 mutant virus developed HCC as compared with none of 19 with non-t1762/a1764 mutant virus (33% vs. 0%, P 0.045). Of the 3 patients who developed HCC, 1 was infected with genotype B and 2 with genotype C. Other mutations in the basal core promoter were further analyzed. Mutation from A to G or Tat nucle-

4 330 KAO ET AL. GASTROENTEROLOGY Vol. 124, No. 2 Table 2. Association Between the Likelihood of T1762/A1764 Basal Core Promoter Mutation and Clinical Stages of Liver Disease in Patients With Chronic Hepatitis Viral Genotype B or C Infection Adjusted odds ratio (95% CI) Genotype Inactive carrier Chronic hepatitis Cirrhosis Hepatocellular carcinoma B a ( ) P C a ( ) P Total b ( ) P a Adjusted for age and sex. b Adjusted for age, sex, and genotype ( ) P ( ) P ( ) P ( ) P ( ) P ( ) P otide 1752 was found in 51 (20%) patients (45, G1752; 6, T1752) and was linked to genotype but not to the progression of liver disease. Mutation from Tto C at nucleotide 1753 was observed in 17 (7%) patients (all with genotype C) and always occurred together with T1762/A1764 mutation. A distinct genotype-specific polymorphism was noted from nucleotides 1726 to 1730: All 143 genotype B strains had CTGAG, whereas 90 (84%) of 107 genotype C strains had AAGAC, and the remaining 17 (16%) had AGGAC. Another polymorphism at nucleotide 1799 (G or C) was also genotype-related: 127 (89%) genotype B strains had G1799, whereas 86 (80%) genotype C strains had C1799. Less consistent genotype-associated mutation was seen at nucleotide Mutation from C to Tor A at this position was found in 13 (9%) genotype B strains and in 17 (16%) genotype C strains. No correlation was found between these mutations and the severity of liver disease or the development of HCC. The correlation between the precore stop codon mutant or A1896 mutant and the development of HCC was studied in inactive carriers and HCC patients (Table 4). The data indicated that HCC patients had a similar frequency of A1896 mutation to inactive carriers, irrespective of HBV genotypes. Discussion Having only 3200 base pairs in its genome, HBV is the smallest known DNA virus. 1 The partially doublestranded circular HBV DNA consists of 4 overlapping genes encoding the viral envelope (pre-s and S), nucleocapsid (precore and core), polymerase, and X protein. It is known that the enhancer II and core promoter are the regulatory sequences located at the downstream end of the X gene. 17 They control the transcription not only of the X gene but also of both precore messenger RNA (mrna) and pregenomic RNA in the replication cycle Table 3. Association of Selected Factors With the Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Virus Infection Total (n 250) Genotype B (n 143) Genotype C (n 107) Factor Odds ratio(95% CI) P value Odds ratio(95% CI) P value Odds ratio(95% CI) P value Sex F M 2.35 ( ) ( ) ( ) 0.3 Age 50 years years 3.97 ( ) ( ) ( ) Cirrhosis Absence Presence 3.43 ( ) ( ) ( ) T1762/A1764 mutant Absence Presence ( ) ( ) ( ) Genotype B 1 C 3.30 ( ) CI, confidence interval; T1762/A1764 mutant, changes of 2 nucleotides, an adenine (A) to thymine (T) transversion at nucleotide 1762 together with a guanine (G) to adenine (A) transition at nucleotide 1764 within the basal core promoter of hepatitis B viral genome.

5 February 2003 HBV CORE PROMOTER MUTATION AND HCC 331 Figure 2. Prevalence of T1762/A1764 basal core promoter mutant in 127 patients with hepatocellular carcinoma (HCC) stratified by age. The frequency of T1762/A1764 mutant was comparable between younger ( 50 years) and older ( 50 years) HCC patients, irrespective of HBV genotypes (54% vs. 46% for genotype B and 92% vs. 80% for genotype C). of HBV. 18 Accordingly, the completely conserved enhancer II region not overlapping with basal core promoter when aligned to respective sequences of HBV genotype B or C observed in our study is not unanticipated. Nevertheless, several core promoter variants have been reported to produce HBV virions that do not produce HBeAg, 11 and the most well-defined basal core promoter mutant has a dual change of T1762 and A1764 that diminishes production of HBeAg and a resultant increased host immune response. 12,19 Although cases of fulminant hepatitis, severe chronic hepatitis, and HCC associated with T1762/A1764 mutant have been described, the overall pathogenic significance of this mutant remains controversial. 13 Previous studies have indicated that HBV genotype C correlates with advanced liver disease and has a higher frequency of T1762/A1764 mutation than genotype B. 7 10,14,20 Thus, this mutation may serve as a useful marker for progressive liver damage. By using the logistic regression analysis, our results consistently showed that genotype C patients had a significantly higher overall frequency of T1762/A1764 mutation than genotype B patients who were controlled for sex, age, and severity of liver disease (52% vs. 29%; P 0.001). The difference was most remarkable in the stages of cirrhosis and HCC (Figure 1). These data indicated an association of T1762/A1764 mutant with the progression of chronic hepatitis B, and this fact may explain, at least in part, the different clinical outcomes between genotypes B and genotype C infection as previously reported. 7 9,14 It is proposed that changes in the secondary structure of pregenome, giving rise to T1762/A1764 mutation, may increase viral replication. 21 This mutation can enhance the synthesis of core protein by 15-fold in an expression system 22 and create a binding site for HNF1 (hepatocyte nuclear factor) transcription factor, which may increase the transcription of pregenomic RNA. 23 Whether these observations are relevant to the enhanced virulence of T1762/A1764 mutant of HBV awaits further studies. The X gene of HBV genome encodes 2 proteins that have potent transactivation activities on HBV enhancer in aiding viral replication. In addition, the X protein is considered a transactivator of viral as well as cellular genes. 24 This property thus makes the X gene a candidate for a role in the development of HCC in patients with chronic HBV infection. Several studies have indicated a direct interaction of X protein with p53 as well as DNA repair factor, suggesting a direct link to cellular DNA repair, cell growth, and apoptosis. 25 Taken together, these lines of evidence suggested that the X protein may be important in hepatocarcinogenesis. Because its coding sequence overlaps regions of crucial importance for virus replication such as enhancer II and core promoter, mutations including T1762/A1764 in this region may therefore induce not only an amino acid change in the X protein but also an alteration of HBV gene expression. 11,26,27 In terms of the clinical situations, Takahashi et al. 28 suggested that the frequency of T1762/A1764 mutant increases with disease progression in genotype C-infected Japanese HBsAg carriers and further indicated a high frequency (90%) of this mutant in patients with HCC. 29 Similarly, a higher prevalence of T1762/A1764 mutant in black Africans with HCC as compared with asymptomatic carriers (66% vs. 11%, P ) has been recently reported in South Africa where HBV genotype E is prevalent. 27 Although these data implied that these mutations and resulting alternation of the amino acid Table 4. Frequency of Precore Stop Codon Mutant in Inactive Carriers and Hepatocellular Carcinoma Patients With Chronic Hepatitis B Viral Genotype B or C Infection Frequency of precore stop codon mutant Genotype Inactive carrier (%) Hepatocellular carcinoma (%) B 24/35 (69) 49/75 (65) C 15/25 (60) 29/52 (56) Total 39/60 (65) 78/127 (61) Precore stop codon mutant, a guanine (G) to adenine (A) transition at nucleotide 1896 in the precore gene of hepatitis B viral genome, which creates a premature stop codon.

6 332 KAO ET AL. GASTROENTEROLOGY Vol. 124, No. 2 sequences of the X protein may play a role in hepatocarcinogenesis, the possibility of cohort effect cannot be excluded. In addition, whether this association also holds true for other HBV genotypes remains largely unknown. In the present study, we, therefore, studied the prevalence of T1762/A1764 mutant in a cohort of Taiwanese hepatitis B carriers with different clinical stages, including inactive carrier state, chronic hepatitis, cirrhosis, and HCC. Our data showed an appreciable stepwise increase in the prevalence of T1762/A1764 mutation from inactive carriers to HCC patients with genotype B or C infection (Table 1 and Figure 1). By the multiple logistic regression analysis after adjusting for sex, age, and genotype, cirrhotic and HCC patients showed a significantly greater likelihood of T1762/A1764 mutation than inactive carriers (Table 2). Risk factors associated with the development of HCC include chronic infection with either HBV or HCV, the presence of cirrhosis, carcinogen exposure, alcohol abuse, genetic factors, male gender, and advanced age. 30 Among these risk factors, chronic hepatitis virus infection, particularly when accompanied by cirrhosis, shows the strongest association with the development of HCC. 31 In this study, the attributable risk of multiple factors including sex, age, cirrhosis, T1762/A1764 mutation, and HBV genotype for HCC in hepatitis B carriers was determined by multiple logistic regression analysis (Table 3). Our results consistently showed that male patients with advanced age, cirrhosis, or HBV genotype C were at increased risk for the development of HCC. 30,31 Furthermore, we also documented that patients with T1762/A1764 mutation were similarly at increased risk for HCC, and this was observed for both genotypes B and C infection. In addition, cirrhotic patients infected with T1762/A1764 mutant virus had a higher incidence of HCC development compared with those with non- T1762/A1764 mutant virus after a median follow-up of 6 years. Taken together, these lines of evidence suggested that the T1762/A1764 mutant may play a role in HBVrelated hepatocarcinogenesis of diverse HBV genotypes, and, thus, could serve as a molecular marker in predicting the clinical outcomes of patients with chronic HBV infection. This speculation was further strengthened by the fact that the prevalence of T1762/A1764 mutation in younger HCC patients was comparable with older HCC patients (Figure 2) but was significantly higher than that in age-matched inactive carriers. These findings also excluded the possibility of cohort effect that patients with chronic HBV infection are prone to have T1762/ A1764 mutation in advanced age, as is the situation of precore stop codon mutation. 12 Contrary to reports from China and Japan, 8,9 more than 50% of HBV-related HCC patients in Taiwan are infected with HBV genotype B. 7 Accordingly, the genotype B strains in Taiwan are proposed to divide into 3 subtypes based on the rate of liver disease progression. 32 The first is the slowly progressive subtype that is associated with a tendency for early disappearance of HBeAg during a carrier s lifetime or in the course of chronic hepatitis and subsequently leads to the low death rate from HCC as has been observed in Okinawa of Japan. 33 The second is the rapidly progressive subtype that is associated with the development of HCC in younger hepatitis B carriers before their fourth decade even in the absence of cirrhosis, suggesting that an oncogenic potential of certain particular HBV strains may exist as is in the case of HCC in woodchucks. 34 The third is the intermediately progressive subtype that runs a typical natural course of chronic HBV infection with the development of HCC, usually in their sixth decade, as does genotype C. Although the hypothesis needs to be proven, a dramatic step-up in incidence of T1762/A1764 mutant from genotype B-related cirrhosis (8%) to HCC (51%) in the present study indeed provide important molecular virologic evidence to differentiate these genotypic subtypes. Accordingly, patients infected with genotype B harboring T1762/A1764 mutant may be at higher risk for the early development of HCC than those infected with non-t1762/a1764 mutant genotype B. Further analysis in large-scale, longitudinal studies is warranted to solve this important issue. In agreement with a previous report, 20 our data indicated that mutations other than T1762/A1764 in the basal core promoter were more rare. Among these mutations, C1753 was only seen in a small proportion of patients with genotype C infection and always occurred together with T1762/A1764 mutation, whereas variation at several other nucleotide positions was usually genotype related. Whether interactions occur between these variants, particularly the effect of C1753 mutation, and T1762/A1764 mutant in the basal core promoter awaits further studies. Although HBeAg-negative chronic hepatitis B because of precore gene mutants is considered to have a relatively aggressive course with frequent development of cirrhosis and HCC, 35 precore gene mutations are also detected in inactive HBsAg carriers. 36,37 Among these mutations, precore stop codon mutation is most frequently detected. 38 In this study, we, therefore, investigated the correlation between the precore stop codon mutation and the development of HCC in inactive carriers and HCC patients; our results indicated that HCC

7 February 2003 HBV CORE PROMOTER MUTATION AND HCC 333 patients had a similar frequency of precore stop codon mutation to inactive carriers, irrespective of HBV genotypes (Table 4). Thus, the precore stop codon mutation may be an innocent bystander, 12 and additional mutations outside the precore gene such as basal core promoter mutation, which usually accompany precore mutations, 38 may act together to enhance the virulence of HBV and probably contribute to the aggressiveness of this special clinical setting. In summary, our results indicate that the occurrence of T1762/A1764 basal core promoter mutation of HBV is more frequent in genotype C than B patients, and the likelihood of T1762/A1764 mutation parallels the progression of chronic liver disease. Meanwhile, patients with T1762/A1764 mutant are at increased risk for HCC. These data suggest a possible role for T1762/ A1764 mutant in the pathogenesis and carcinogenesis of chronic HBV infection. This mutant may serve as a useful molecular marker in predicting the clinical outcomes of patients with chronic hepatitis B. In addition, the higher frequency of T1762/A1764 mutation in certain genotypic strains may account for the pathogenic differences among HBV genotypes. Further studies in other parts of the world, particularly the Western countries where genotypes A and D prevail, are needed to confirm these observations. References 1. Kao JH, Chen DS. Global control of hepatitis B virus infection. Lancet Infect Dis 2002;2: Chen DS. From hepatitis to hepatoma: lessons from type B viral hepatitis. Science 1993;262: Chisari FV, Ferrari C. Hepatitis B immunopathogenesis. Annu Rev Immunol 1995;13: Magnius LO, Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 1995;38: Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R. 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