Abstract:
The present invention is intended to provide a method for predicting risk of hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected patients with a high accuracy. More specifically, the invention provides a method for detecting eight mutations of HBV genome associated with predisposition to HCC, comprising: C1653T, A1762T, G1764A, T1674C, T1753C, C3116T, T53C and A1846T mutations, and primers and probes sets used thereof consist of SEQ ID NO: 1-SEQ ID NO: 24.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    Infection with Hepatitis B virus (HBV) is a major public health problem, with more than 350 million HBV carriers estimated worldwide, and approximately 45% of the world&#39;s population lives in regions where HBV infection is endemic (Ganem D et al., N Eng J Med 2004; 350:1118-1129). Chronic HBV infection is one of the most important determinants of the occurrence of hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Principal risk factors of development of HCC are chronic infection with HBV, HCV and liver cirrhosis. It has been established that 80% of HCC worldwide is etiologically associated with HBV (Yu M C et al., Can J Gastroenterol. 2000; 14 (8): 703-709). 
         [0002]    HBV, a member of the family Hepadnaviridae, is a partially double-stranded DNA virus containing four overlapping open reading frames (ORFs) that encode the surface protein (S), pre-core/core, polymerase and a multifunction nonstructural protein called X. 
         [0003]    The PreS region that consists of the PreS1 (nucleotides 2848-3204) and the PreS2 (nucleotides 3205-154) domains overlaps a region encoding the polymerase gene. The enhancer II (nucleotides 1636-1744) and basal core promoter (nucleotides 1751-1769) regions overlap with the X gene (nucleotides 1374-1835) (Kay et al., 2007; 127: 164-176). 
         [0004]    The virus shows remarkable genetic variability and is currently classified into eight genotypes, designated A to H based on a sequence divergence greater than 8% in the entire HBV genome. Genotypes are further categorized into sub-genotypes based on nucleotide sequence divergence between 4% and 8%. HBV genotypes have distinct geographical distributions worldwide, and have been shown to differ with regard to clinical disease, prognosis and response to interferon treatment. Genotypes B and C are endemic in Asia, genotype D the Mediterranean area and Middle East, genotype E in middle Africa, genotype F in South American (Schaefer et al., J Viral Hepat. 2005; 12: 111-124; Chan et al. J Infect Dis 2005; 191:2022-2032.). 
         [0005]    Several studies have revealed that some nucleotide mutations in the HBV PreS and Precore regions were associated with the increase risk of HCC. Some representative references were present as follow. 
         [0006]    Laskus et al., (Laskus et al. Biochem Biophys Res Commun. 1998; 244: 812-814) reported earlier single nucleotide mutations at nucleotide A1762T/G1764A, G1896, G1899A. They studied 53 patients with HCC and 33 HBsAg positive controls. A functional part of HBV core promoter and whole precore region were sequenced directly and after cloning. HBV DNA was amplified from sera from 27 HCC patients and in all controls. As a result, fourteen (52%) patients and 12 (36%) controls were found to harbor an HBV strain with G to A transition mutation at position 1896 leading to HBeAg negative phenotype. Nine (33%) HCC patients and 2 (6%) controls harbored a mixture of wild type and HBV strains with deletions/insertions. 
         [0007]    Baptista et al. (Baptista et al., Hepatology. 1999; 29: 946-953.) noticed successively missense mutations for nucleotides G1809T, A1762T, T1764V and C1812T in the basic core promoter in his studies from southern African patients. They found that Nucleotide divergences were higher in the basic core promoter of hepatocellular carcinoma patients when compared with asymptomatic carriers (P&lt;0.0001). The prevalence of the nucleotides A1762T and T1764V missense mutations was 66% in patients with hepatocellular carcinoma compared with 11% in asymptomatic carriers (P&lt;0.0001). 
         [0008]    Ito et al. (Ito et al., Clin Infect Dis. 2006; 42: 1-7.) investigated a wide scope studies that across race and region in obtaining sample. In the study, the sera of 211 patients from different regional areas worldwide were obtained. As a result, the prevalence of T1653 was significantly higher among patients with hepatocellular carcinoma than among carriers of inactive virus who did not have hepatocellular carcinoma. Mutations in the basic core promoter region (T1762/A1764) were frequently found in all groups. 
         [0009]    Yuan et al., (Yuan J et al., J Clin Virol. 2007; 39: 87-93) have realized much more nucleotide mutations associated with HCC development, which are nucleotide C1653T, T1762V, A1762T/G1764A, G1862T, G1888H, G1809A, and G1896. These mutations associated with HCC were V1753 and T1762/A1764; the prevalence of the V1753 was higher in HBV/C1 strains; A1898 was only found among HBV/C1, T1762/A1764 was frequently demonstrated in HBV. The T1858 (90%) and A1896 (40%) mutations were most frequent in HBV/C2. 
         [0010]    Sung et al., (Sung et al. J Virol. 2008; 7: 3604-3611) have observed genotype-specific difference in the frequency mutations associated with risk of HCC. Studies showed that in genotype B HBV, mutations C1165T, A1762T and G1764A, T2712C/A/G, and A/T2525C were associated with HCC. HCC-related mutations T31C, T53C, and A1499G were associated with HBV subgenotype Ce, and mutations G1613A, G1899A, T2170C/G, and T2441Cwere associated with HBV subgenotype Cs. 
         [0011]    Liu et al., (Liu et al. J Natl Cancer Inst 2009; 1066-1082.) have studied for the frequency of nucleotide mutations by statistic. They found that C1653T, T1753V, and A1762T/G1764A are associated with an increased risk of HCC. The frequencies of these mutations increase as chronic HBV infection progresses from the asymptomatic HBsAg carrier state to liver cirrhosis and HCC. Frequent examination of patients with chronic HBV infections for the presence of these mutations may be useful for identifying which patients require preventive antiviral treatment and for the prediction of HCC. 
         [0012]    However, most of these studies in investigated association between HBV mutations and the risk of HCC had a small number of patients and often examined only specific viral mutations. Thus, the genetic point mutations in HBV increase the risk for HCC, which contributed to certain SNPs to HCC remain to be identified definitively. 
       SUMMARY OF THE INVENTION 
       [0013]    The present invention provides an effective means and method of predicting risk for HCC with high accuracy and high predictability so as to contribute the prevention, diagnosis and treatment of HCC. 
         [0014]    In order to achieve the abovementioned objects, the inventors searched relevant literature from the database of the published medical articles. The 75 articles related to HBV nucleotide mutation associated with HCC were reviewed after identification and selection from the database. At last, 39 related articles were selected for results analysis. The most commonly reported HBV single nucleotide mutations associated with HCC risk were located in PreS region and basal core promoter region. 
         [0015]    The present inventors have extracted 14 mutation sites that are associated with HCC based on our literature review. Among them, eight mutation sites were evaluated further by our experiments, which are C1653T, A1762T, G1764A, T1674C, T1753C, C3116T, T53C, and A1846T. 
         [0016]    The literature review revealed 32 mutation sites associated with HCC, which are T31C, T53C, T1479C, C1485T, C1499H, G1613A, C1165T, C1653T, C1726A, T1727A, T1730C, A1752G, T1753V, A1762T, G1764A, T1765V, C1766T, C1773T, G1799C, G1809T, C1812T, T1856C, C1858T, G1862T, G1888H, G1896A, G1898A, G1899A, T2170C/G, T2441C, A/T2525C and T2712V. Seven of the mutation sites showed higher mutation frequency compared to the other 25 mutation sites. These are C1653T, T1753V, A1762T, G1764A, C1858T, G1896A, and G1899A. 
         [0017]    The further experimental findings verify the mutations of C1653T, T1753C, A1762T and G1764A were with the highest frequency mutations, which were essential for predictive risk of HCC. 
         [0018]    The present invention was made based on the abovementioned finding and provides the following configuration.
   1. The statistical results indicated that it is useful for prediction risk of HCC by determining nucleotides A1858T, G1896A, G1899A, C1485T, C1499A, G1613A, T1753C, T1674C, C1653T, A1762T, G1764A, C3116T, T53C, and A1846T.   2. The further experiments relevant to risk of HCC provide consensus mutation sites with literatures review, which are C1653T, T1753C, A1762T and G1764A.   3. The invention provides an effective means for prediction risk of development of HCC by determining mutation sites comprise: A1858T, G1896A, G1899A, C1485T, C1499A, G1613A, T1753C, T1674C, C1653T, A1762T, G1764A, C3116T, T53C, and A1846T.   4. The present invention discovered 14 mutations (A1858T, G1896A, G1899A, C1485T, C1499A, G1613A, T1753C, T1674C, C1653T, A1762T, G1764A, C3116T, T53C, and A1846T) are associated with susceptibility to development of HCC, based on statistical analysis and further experiment estimate. Specifically, C1653T, A1762T, G1764A, T1674C, T1753C, C3116T, T53C, and A1846T have been identified as major candidate determinants of the susceptibility to HCC in human.   
 
         [0023]    The present invention also provides a method for detecting eight mutations of HBV associated with susceptibility to cause of HCC, including C1653T, A1762T, G1764A, T1753C, T1674C, C3116T, T53C and A1846T, the composition comprising as follows: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 DNA 
                 5 
                 μL 
               
               
                   
                 Primers 
                 1-5 
                 pmol 
               
               
                   
                 probes 
                 1-5 
                 pmol 
               
               
                   
                 Taq DNA polymerase 
                 1-3 
                 U 
               
               
                   
                 Buffer 
                 5-20 
                 μL 
               
               
                   
                 dNTP 
                 100-300 
                 μM 
               
               
                   
                 MgCL 2   
                 1-10 
                 mM 
               
               
                   
                 H 2 O 
                 20-60 
                 μL 
               
               
                   
                   
               
             
          
         
       
     
         [0024]    The present invention also provides the primers and probes for detecting eight mutations of HBV, comprising mutations C1653T, A1762T, G1764A, T1753C, T1674C, C3116T, T53C and A1846T, the primers and probes sequence consist of SEQ ID NO: 1-SEQ ID NO: 24. 
         [0025]    The present invention also provides kits for detecting HBV eight mutations to cause HCC. In one embodiment, the kits comprise primers and probes consist of SEQ ID NO: 1-SEQ ID NO: 24 and following composition. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 DNA 
                 5 
                 μL 
               
               
                   
                 Primers 
                 1-5 
                 pmol 
               
               
                   
                 probes 
                 1-5 
                 pmol 
               
               
                   
                 Taq DNA polymerase 
                 1-3 
                 U 
               
               
                   
                 Buffer 
                 5-20 
                 μL 
               
               
                   
                 dNTP 
                 100-300 
                 μM 
               
               
                   
                 MgCL 2   
                 1-10 
                 mM 
               
               
                   
                 H 2 O 
                 20-60 
                 μL 
               
               
                   
                   
               
             
          
         
       
     
         [0026]    The invention provides specific primers and probes neclueotide sequence and substantically identical nucleotide sequence thereto, which allow accurate detection of eight mutations, comprising C1653T, A1762T, G1764A, T1753C, T1674C, C3116T, T53C and A1846T. 
         [0027]    “Identical nucleotide sequence” in the primer and probe sequence context as used herein, means hybridizaion to a target under stringent condidation, and also that the nucleotide sequence segments, when compared, are the same when properly aligned, with the appropriate nucleotide insertion and deletions, in at least 70% of the nucleotides, typically, usually, at least 90% of nucleotides. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0028]      FIG. 1  is a flow chart showing a strategy for selected nucleotide mutations sites: HBV=hepatitis B virus; HCC=hepatocellular carcinoma; 
           [0029]      FIG. 2  is a graph showing statistical mutation frequency of all mutation sites in HBV all genotypes. 
           [0030]      FIG. 3  is a graph showing statistical mutation frequency of all mutation sites in HBV B/C genotypes. 
           [0031]      FIG. 4A  is a graph illustrating mutation of PCR detection for T1753C from HCC sample. 
           [0032]      FIG. 4B  is a graph illustrating wildtype of PCR detection for T1753C from control sample. 
           [0033]      FIG. 5A  is a graph illustrating mutation of PCR detection for A1846T from HCC sample. 
           [0034]      FIG. 5B  is a graph illustrating wildtype of PCR detection for A1846T from control sample. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0035]    The present invention provides an effective means to predict which HBV-infected individuals are more likely to develop HCC, as well as a method for early diagnosis of HCC, so as to allow early treatment before HCC becomes advanced. 
         [0036]    The inventors have identified Single Nucleotide Polymorphisms (SNPs) in the genome of HBV that are associated with risk for HCC. A SNP is the smallest unit of genetic variation, which represents a position in a genome where individuals of the same species may have different nucleotides inserted into their DNA sequences. 
         [0037]    SNPs in the genome of HBV have been noticed previously in the peer reviewed scientific literatures. However, there is no consensus about the link between HBV SNPs and the development of HCC. The present inventors searched databases using terms “hepatitis B Virus,” “Nucleotide mutation” and “Hepatocellular Carcinoma” to find related studies. 
         [0038]    The present inventors searched and identified 1255 potentially relevant articles from databases of the published literatures, then 1001 literatures were excluded after abstract review, among 253 abstract of the literatures related to HBV nucleotide mutations associated with HCC and liver cirrhosis were reviewed, and 75 full manuscripts review relevant to HBV single nucleotide polymorphism. At last, 39 articles were selected for analysis. These 39 studies included 14 from mainland China, seven from Japan, five from Taiwan, four from the United States, two each from Korea, Vietnam and South Africa, and five from two or more countries or regions. The most commonly reported HBV single nucleotide mutations associated with HCC risk were located in PreS region and basal core promoter region. In the selecting process, the studies that were not published as full reports were excluded, The work flow chart of work idea was showed in  FIG. 1 . 
         [0039]    The inventors identified 32 distinct sites from total 176 repeatable count mutation sites selected from the 39 abovementioned literatures, and then these 32 mutation sites were analyzed statistically, based on frequency of occurrences for each mutation sites out of the total number of 176 mutation sits. The mutation sites from references statistic was present in  FIG. 2 . As a result, seven mutations was found with higher mutation frequency, which are C1653T, T1753V, A1762T, G1764A, C1858T, G1896A, G1899A, A1762T, G1764A and G1896A were with the highest mutation frequency among them. It was shown same mutation frequency (2.28%) for C1858T and G1899A. the mutation sites C1653T and T1753C showed higher mutation frequency, which were 7.34% and 8.47%, respectively. 
         [0040]    The occurrence frequency of others 25 single mutation sites are between 0.56% and 1.13%. The total percent of the seven mutation sites covered 77.8% out of total mutation sites, as shown in Table 3. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Mutation Sites 
                 C1653T 
                 T1753C 
                 A1762T 
                 G1764A 
                 C1858T 
                 G1896A 
                 G1899A 
               
               
                   
               
             
             
               
                 Frequency 
                 12 
                 15 
                 37 
                 37 
                 5 
                 26 
                 5 
               
               
                 Percentage (%) 
                 6.82% 
                 8.52% 
                 21.02% 
                 21.02% 
                 2.84% 
                 14.77% 
                 2.84% 
               
               
                   
               
             
          
         
       
     
         [0041]    It was reported that over 70% belonged to C genotype and over 20% was B genotype in HCC patients in China study. (Meng et al., Chin Clin Oncol; 2007, 12, 435-440). Thus, the mutation rates of B/C genotypes of HBV were investigated separately. The inventors identified 26 different mutation sites from total 92 repeatable count mutation sits, and then these 26 mutation sites were analyzed statistically. Mutation sites C1653T, T1753C, A1762T, G1764A and G1896A showed the highest frequency, and total frequency covered 67% out of all mutations. Mutation rates of G1613A, T1856C and G1899A were 2.17% respectively. Other mutation rates were lower than 1.2%. The statistical results were present in  FIG. 3 . 
         [0042]    At last, 11 mutation sites were identified for prediction risk of HCC by selection based on mutation frequency in HBV B/C genotypes and HBV all genotypes, which were C1653T, G1613A, T1753C, A1762T, G1764A, C1858T, G1896A, G1899A, C1485T, C1499A and T53C. The Chi-square tests at conventional level of 0.05 were performed to examine significance of these 11 mutations in HBV B/C genotypes and HBV all genotypes. The SPSS 13.0 software was used in the statistical tests, and the statistical results indicated that these 11 mutations were no significance differences in HBV B/C genotypes and HBV all genotypes. The results were shown in Table 5. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                   
                 11 
                 Other 
                   
               
               
                   
                   
                 mutations 
                 mutations 
               
               
                   
                 HBV 
                 frequency 
                 frequency 
                 P value 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 HBV all 
                 143 
                 32 
                 0.710 
               
               
                   
                 genotypes 
               
               
                   
                 HBV 
                 73 
                 19 
               
               
                   
                 B/C genotypes 
               
               
                   
                   
               
             
          
         
       
     
         [0043]    It was termed as wild-type nucleotides when a nucleotide appeared with the highest frequency in HBV after the alignment from the asymptomatic hepatitis B surface antigen carriers. It was termed as mutations that a nucleotide was substituted with three other nucleotides and deletion at each site. 
         [0044]    In order to further investigate the frequency of abovementioned mutation sites associated with risk of HCC, an example experiment was performed by inventors. 
       Example 
       [0045]    In this example, eight mutations which are C1653T, A1762T, G1764A, T1753C, T1674C, C3116T, T53C and A1846T were analyzed for negative or positive association with prediction of HCC from HBV DNA sequences. 
       Patients 
       [0046]    Serum samples were collected from 85 patients with HBV-related hepatocellular carcinoma (HCC) and age-matched 85 HBV-infected patients who had not been diagnosed with HCC as control group. 
       Extraction of DNA 
       [0047]    Serum viral HBV DNA was extracted from 200 μl of serum using the AmoyDx Diagnostic Kit (AmoyDx, Xiamen, China) according to the manufacturer&#39;s instructions. Real-time PCR Amplification. 
         [0048]    The primers and probes used in real-time fluorescence PCR were designed according to the eight mutation sites (C1653T, A1762T, G1764A, T1753C, T1674C, C3116T, T53C and A1846T) in HBV gene, and PCR detecting method was performed to detect the mutations. The specific primers for single mutation were designed, and only the corresponding mutation could be amplified. The PCR products could bind to probes, which issued a detectable fluorescent signal to identify the mutation type precisely. The primers and probes sequences were presented in Table 1. The compositions of real-time fluorescence PCR amplification as following: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 DNA 
                 5 
                 μL 
               
               
                   
                 Primers 
                 1-5 
                 pmol 
               
               
                   
                 Probes 
                 1-5 
                 pmol 
               
               
                   
                 Taq DNA polymerase 
                 1-3 
                 U 
               
               
                   
                 Buffer 
                 5-20 
                 μL 
               
               
                   
                 dNTP 
                 100-300 
                 μM 
               
               
                   
                 MgCL 2   
                 1-10 
                 mM 
               
               
                   
                 H 2 O 
                 20-60 
                 μL 
               
               
                   
                   
               
             
          
         
       
     
         [0049]    Real-time PCR was carried out with a 3 min initial denaturation at 95° C., followed by 10 cycles of amplification (95° C. for 20 s, 65° C. for 20 s, and 72° C. for 20 s), then 35 cycles of amplification (95° C. for 20 s, 60° C. for 35 s, and 72° C. for 20s). The final 35 cycles are detected the FAM signal in anneal steps. Fluorescence PCR detection was performed using an MX3000P real-time PCR (SRATAGENE). 
         [0050]    All the serum samples were conducted with direct DNA sequencing for detecting the eight mutations to confirm real-time PCR detected results. The results indicated our PCR detection shown high agreement with direct sequencing, the results was present in Table 6. 
         [0051]    The experimental results indicated that the eight mutations showed significantly higher mutation rates in HCC patients than in control patients (p&lt;0.0001), as shown in Table 6. The mutation rates of A1762T and G1764A were 74%, whereas they were 27% in the control group. The mutation rates of C3116T, C1653T and A1846T were 54%, 49% and 59%, respectively in HCC patients, whereas they were 11%, 8% and 14% in the control group. The mutation T1674C, T1753C and T53C were 41%, 38% and 42%, whereas they were 8%, 14% and 11% in the control group. The total odd ratio of eight mutations in HCC patients was 5.48 compared with control patients. The statistical significance was examined at the conventional level of 0.05 by Chi-square test. The statistical tests were performed by using the SPSS 13.0 software. The results of test were shown in Table 6. 
         [0052]    The statistical results and experiment support the claim that the 14 mutations (A1858T, G1896A, G1899A, C1485T, C1499A, G1613A, T1753C, T1674C, C1653T, A1762T, G1764A, C3116T, T53C, and A1846T) are associated with susceptibility to development of HCC, Specifically, mutations C1653T, A1762T, G1764A, T1753C, T1674C, C3116T, T53C and A1846T have been identified as major candidate determinants of the susceptibility to HCC in human. 
         [0053]    It is understood that the examples described herein are to illustrate the purpose only and that any changes and modifications in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. 
         [0000]    
       
         
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Name 
                 Nucleotide sequence (5′-3′) 
                 Nt positions 
                 Direction 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Primers and probes for C1653T 
               
             
          
           
               
                 C1653T-F 
                 AGGAACCTGCCCAAGGTCTTGT 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 1 
                   
               
               
                 C1653T-R 
                 CACCAACTCCTCCCACTCAGTA 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 2 
                   
               
               
                 C1653T-P 
                 FAM- 
                 SEQ ID 
                 Sense 
               
               
                   
                 TCTTGGACTTTCAGCAATGTCAAC- 
                 NO: 3 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for A1762T 
               
             
          
           
               
                 A1762T-F 
                 CGGAGGAGGTTAGGTTAAT 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 4 
                   
               
               
                 A1762T-R 
                 GGACATGAACATGAGATGATTAGGC 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 5 
                   
               
               
                 A1762T-P 
                 FAM- 
                 SEQ ID 
                 antisense 
               
               
                   
                 GTGAAAAAGTTGCATGGTGCTGGT- 
                 NO: 6 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for G1764A 
               
             
          
           
               
                 G1764A-F 
                 CGGAGGAGGTTAGGTTAAAGA 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO:7 
                   
               
               
                 G1764A-R 
                 GGACATGAACATGAGATGATTAGGC 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 8 
                   
               
               
                 G1764A-P 
                 FAM- 
                 SEQ ID 
                 antisense 
               
               
                   
                 GTGAAAAAGTTGCATGGTGCTGGT- 
                 NO: 9 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for T1753C 
               
             
          
           
               
                 T1753C-F 
                 ACCTGCCCAAGGTCTTGCATAAGAG 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 10 
                   
               
               
                 T1753C-R 
                 CCTCCTAGTACAAAGACCTTTAAC 
                 SEQ ID 
                 Antisense 
               
               
                   
                 CTAG 
                 NO: 11 
                   
               
               
                 T1753C-P 
                 FAM- 
                 SEQ ID 
                 Sense 
               
               
                   
                 GCAATGTCAACGACCGACCTTGAG- 
                 NO: 12 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for A1846T 
               
             
          
           
               
                 A1846T-F 
                 CCTCTGCCTAATCATCTCT 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 13 
                   
               
               
                 A1846T-R 
                 CACAGAAGCTCCAAAT 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 14 
                   
               
               
                 A1846T-P 
                 FAM- 
                 SEQ ID 
                 sense 
               
               
                   
                 ACTGTTCAAGCCTCCAAGCTG- 
                 NO: 15 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for T1674C 
               
             
          
           
               
                 T1674C-F 
                 AAGAGGACTCTTGGACTTC 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 16 
                   
               
               
                 T1674C-R 
                 TCCACCAACTCCTCCCACTC 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 17 
                   
               
               
                 T1674C-P 
                 FAM- 
                 SEQ ID 
                 sense 
               
               
                   
                 CAGCAATGTCAACGACCGACCT- 
                 NO: 18 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for C3116T 
               
             
          
           
               
                 C3116T-F 
                 TACTCACAACTGTGCCAGT 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 19 
                   
               
               
                 C3116T-R 
                 AGGTGGAGATAAGGGAGTA 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 20 
                   
               
               
                 C3116T-P 
                 FAM- 
                 SEQ ID 
                 sense 
               
               
                   
                 GCCTCCACCAATCGGCAGTCA- 
                 NO: 21 
                   
               
               
                   
                 BHQ1 
                   
                   
               
               
                   
               
             
          
           
               
                 Primers and probes for T53C 
               
             
          
           
               
                 T53C-F 
                 AGGAAGGCAGCCTACTCC 
                 SEQ ID 
                 sense 
               
               
                   
                   
                 NO: 22 
                   
               
               
                 T53C-R 
                 TGGAGCCACCAGCAGGAAG 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 23 
                   
               
               
                 T53C-P 
                 TCATCCTCAGGCCATGCAGT 
                 SEQ ID 
                 antisense 
               
               
                   
                   
                 NO: 24 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
             
             
               
                   
                   
               
               
                   
                 HCC 
                 Control 
                   
                 Con- 
               
             
          
           
               
                   
                 Muta- 
                   
                 Muta- 
                   
                   
                   
                 sis- 
               
               
                 Position 
                 tion 
                 Wild 
                 tion 
                 Wild 
                 p value 
                 OR 
                 tency 
               
               
                   
               
             
          
           
               
                 T1753C 
                 32 
                 53 
                 12 
                 73 
                 P &lt; 0.0001 
                 5.48 
                  94% 
               
               
                 T1674C 
                 35 
                 50 
                 7 
                 78 
                 P &lt; 0.0001 
                   
                  98% 
               
               
                 C1653T 
                 42 
                 43 
                 7 
                 78 
                 P &lt; 0.0001 
                   
                  95% 
               
               
                 C3116T 
                 46 
                 39 
                 10 
                 75 
                 P &lt; 0.0001 
                   
                 100% 
               
               
                 A1846T 
                 50 
                 35 
                 23 
                 62 
                 P &lt; 0.0001 
                   
                 100% 
               
               
                 T53C 
                 36 
                 49 
                 18 
                 67 
                 P &lt; 0.0001 
                   
                  98% 
               
               
                 A1762T/ 
                 63 
                 22 
                 23 
                 62 
                 P &lt; 0.0001 
                   
                 100% 
               
               
                 G1764A 
               
               
                   
               
               
                 OR: Total odd ratio 
               
               
                 Consistency: Consistency compared with direct sequencing 
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Study 
                 Country 
                 Mutation Sites 
                 HBV genotype 
               
               
                   
               
             
             
               
                 Kim et al., 2008 
                 Korea 
                 C1653T T1753V A1762T G1764A 
                 C 
               
               
                 Sakamoto et al., 2006 
                 Japan, 
                 C1653T T1858HV A1762T G1764A C1862T 
                 A, B, C 
               
               
                   
                 Philippines 
                 G1888H G1809T C1812T G1896A 
               
               
                 Yuan et al., 2007 
                 China 
                 C1653T T1753V A1762T G1764A T1856C 
                 B, C 
               
               
                   
                   
                 G1898A C1858T G1896A G1899A 
               
               
                 Zhang et al., 2006 
                 China 
                 A1762T G1764A 
                 NA 
               
               
                 Zhou et al., 2007 
                 China 
                 A1762T G1764A G1896A 
                 NA 
               
               
                 Baptista et al., 1999 
                 South Africa 
                 T1753V A1762T G1764A G1809T C1812T 
                 NA 
               
               
                 Blackberg et 
                 Sweden, others 
                 T1753V A1762T G1764A G1896A 
                 A, B, C, D 
               
               
                 al., 2003 
               
               
                 Chen et al., 2006 
                 Taiwan 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Deng et al., 2004 
                 China 
                 A1762T G1764A 
                 NA 
               
               
                 Ding et al., 2006 
                 China 
                 A1762T G1764A 
                 B, C 
               
               
                 fang et al., 2002 
                 China 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Ito et al., 2006 
                 Japan, 
                 C1653T T1753V A1762T G1764A G1896A 
                 NA 
               
               
                   
                 United States 
               
               
                 Laskus et al., 1998 
                 Gambia 
                 A1762T G1764A G1896A G1899A 
                 NA 
               
               
                 Liu et al., 2006 
                 Taiwan 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Livingston et 
                 United States 
                 A1762T G1764A G1896A 
                 A, C, D, F 
               
               
                 al., 2007 
               
               
                 Muroyama et 
                 Japan 
                 C1485T A1762T G1764A G1896A 
                 C 
               
               
                 al., 2006 
               
               
                 Mendy et al., 2008 
                 Gambia 
                 A1762T G1764A G1896A 
                 NA 
               
               
                 Ni et al., 2003 
                 Taiwan 
                 G1896A 
                 NA 
               
               
                 Shinkai et al., 2007 
                 Japan 
                 C1653T T1753V A1762T G1764A C1485T 
                 C2 
               
               
                   
                   
                 T1479C C1499H G1896A G1613A 
               
               
                 Song et al., 2005 
                 Vietnam 
                 A1762T G1764A G1766A C1773T C1858T 
                 NA 
               
               
                 Sung et al., 2008 
                 Hong Kong 
                 G1613A C1165T A1762T G1764A T31C 
                 B, C 
               
               
                   
                   
                 T53C 
               
               
                   
                   
                 C1499H T2170C/G G1899A T2441C 
               
               
                   
                   
                 A/T2525C T2712V 
               
               
                 Tanaka et al., 2006 
                 Japan, 
                 C1653T T1753V A1762T G1764A T1765V 
                 NA 
               
               
                   
                 Hong Kong 
                 G1896A G1899A 
               
               
                 Tanakashi et 
                 Japan 
                 C1653T T1753V A1762T G1764A 
                 A, B, C, D 
               
               
                 al., 1999 
               
               
                 Tong et al., 2007 
                 United States 
                 A1762T G1764A G1896A 
                 A, B, C, D 
               
               
                 Truong et al., 2007 
                 Japan, Vietnam 
                 C1653T T1753V A1762T G1764A C1858T 
                 C 
               
               
                   
                   
                 G1896A 
               
               
                 Wang et al., 2007 
                 China 
                 C1653T T1753V A1762T G1764A T1856C 
                 B, C 
               
               
                   
                   
                 G1898A C1858T G1896A G1899A 
               
               
                 Yuen et al., 2008 
                 Hong Kong 
                 C1653T T1753V A1762T G1764A G1896A 
                 B, C 
               
               
                 Zhang et al., 2007 
                 China 
                 A1762T G1764A 
                 B, C, D 
               
               
                 Chou et al., 2008 
                 China 
                 C1726A T1753V A1762T G1764A G1799C 
                 A, B, C 
               
               
                   
                   
                 T1727A T1730C G1896A 
               
               
                 Guo et al., 2008 
                 China 
                 C1653T T1753V A1762T G1764A C1766T 
                 B, C 
               
               
                 Kao et al., 2003 
                 Taiwan 
                 A1752T T1753V A1762T G1764A C1773T 
                 B, C 
               
               
                   
                   
                 G1799C G1896A 
               
               
                 Zhang et al., 2007 
                 China 
                 A1762T G1764A 
                 NA 
               
               
                 Zhu et al., 2008 
                 China 
                 A1752G C1726A A1762T G1764A T1730C 
                 C 
               
               
                 Tong et al., 2007 
                 United states 
                 A1762T G1764A G1896A 
                 A, B, C 
               
               
                 Jang et al., 2007 
                 Korea 
                 A1762T G1764A G1896A 
                 C 
               
               
                 Yang et al., 2008 
                 Taiwan 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Liu et al., 2009 
                 China, Others 
                 C1653T T1753V A1762T G1764A 
                 A, B, C, D, F 
               
               
                 Pisit et al., 2010 
                 Thailand 
                 A1762T G1764A G1899A 
                 NA 
               
               
                 Zhang et al., 2010 
                 China 
                 G1896A 
                 NA 
               
               
                   
               
               
                 NA: genotype undetermined. 
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Study 
                 Country 
                 Mutation Sites 
                 HBV genotype 
               
               
                   
               
             
             
               
                 Kim et al., 2008 
                 Korea 
                 C1653T T1753V A1762T G1764A 
                 C 
               
               
                 Yuan et al., 2007 
                 China 
                 C1653T T1753V A1762T G1764A T1856C 
                 B, C 
               
               
                   
                   
                 G1898A C1858T G1896A G1899A 
               
               
                 Chen et al., 2006 
                 Taiwan 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Ding et al., 2006 
                 China 
                 A1762T G1764A 
                 B, C 
               
               
                 fang et al., 2002 
                 China 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Liu et al., 2006 
                 Taiwan 
                 A1762T G1764A G1896A 
                 B, C 
               
               
                 Muroyama et 
                 Japan 
                 C1485T A1762T G1764A G1896A 
                 C 
               
               
                 al., 2006 
               
               
                 Shinkai et al., 2007 
                 Japan 
                 C1653T T1753V A1762T G1764A C1485T 
                 C 
               
               
                   
                   
                 T1479C C1499H G1896A G1613A 
               
               
                 Sung et al., 2008 
                 Hong Kong 
                 G1613A C1165T A1762T G1764A T31C T53C 
                 B, C 
               
               
                   
                   
                 C1499H T2170C/G G1899A T2441C A/T2525C 
               
               
                   
                   
                 T2712V 
               
               
                 Truong et al., 2007 
                 Japan, 
                 C1653T T1753V A1762T G1764A C1858T 
                 C 
               
               
                   
                 Vietnam 
                 G1896A 
               
               
                 Wang et al., 2007 
                 China 
                 C1653T T1753V A1762T G1764A T1856C 
                 B, C 
               
               
                   
                   
                 G1898A C1858T G1896A G1899A 
               
               
                 Yuen et al., 2008 
                 Hong Kong 
                 C1653T T1753V A1762T G1764A G1896A 
                 B, C 
               
               
                 Guo et al., 2008 
                 China 
                 C1653T T1753V A1762T G1764A C1766T 
                 B, C 
               
               
                 Kao et al., 2003 
                 Taiwan 
                 A1752T T1753V A1762T G1764A C1773T 
                 B, C 
               
               
                   
                   
                 G1799C G1896A 
               
               
                 Zhu et al., 2008 
                 China 
                 A1752G C1726A A1762T G1764A T1730C 
                 C 
               
               
                 Jang et al., 2007 
                 Korea 
                 A1762T G1764A G1896A 
                 C 
               
               
                 Yang et al., 2008 
                 Taiwan 
                 A1762T G1764A G1896A 
                 B, C