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Association Between the Various Mutations in Viral Core Promoter Region to Different Stages of Hepatitis B, Ranging of Asymptomatic Carrier State to Hepatocellular Carcinoma - pdf atached
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Am J Gastroenterol Jan 2011
"Hepatitis B virus (HBV) infection remains a major public health problem globally. More than two billion people have been infected worldwide, and of these, >350 million suffer from chronic HBV infection (1). Chronic HBV infection results in approximately one third of all cases of liver cirrhosis (LC) and more than three quarters of all cases of hepatocellular carcinoma (HCC) worldwide (2). Chronic hepatitis B (CHB), LC, and HCC are progressive stages of chronic HBV infection."......."In conclusion, this study systemically investigated the associations of HBV core promoter mutations with CHB, LC, or HCC. We found that C1673T, A1726C, A1727T, C1730G, T1768A, C1773T, and C1799G were specific for LC. A1846T and T1674C/G were novel factors independently associated with cirrhosis and HCC, respectively. Haplotypic carriages with two or more HBV mutations, rather than single one, were significantly associated with HCC. The frequencies of C1653T, T1753V, and A1762T/G1764A, rather than T1674C/G, consecutively increased with increasing age in ASCs and the patients with CHB, but not in the patients with HCC. Average frequencies of T1674C/G, C1653T, T1753V, and A1762T/G1764A were >30% in the patients with HCC. Sequential examination of these HBV mutations may be helpful in classifying the HBV-infected subjects who will develop HCC in future and need active antiviral treatments to postpone the occurrence of HCC. Although some mutations statistically associated with LC or HCC were found in this study, further study is needed to see if these mutations predict the development of LC or HCC in those who carry the mutation but do not yet have the associated disease.".....
"Factors independently associated with HCC as compared with those without HCC: The multiple factors, containing age (years), sex (male vs. female), HBV DNA level (≥104 vs. <104 copies/ml), HBV genotype (C vs. B), ALT (abnormal vs. normal), HBeAg status (positive vs. negative), and the presence of C1653T, T1674C/G, G1719T, A1762T/G1764A, T1753V, A1846T, and C1913A were introduced into the multivariate regression model. Old age, abnormal ALT, HBV DNA (≥104 copies/ml), genotype C, C1653T, T1674C/G, T1753V, and A1762T/G1764A were independently associated with HCC, as compared with the subjects without HCC (Table 4)."
Jianhua Yin MD, PhD1,10, Jiaxin Xie PhD1,10, Shijian Liu MD, PhD1, Hongwei Zhang MD, PhD1, Lei Han MS1, Wenying Lu MS1, Qiuxia Shen MS1, Guozhang Xu MS2, Hongjun Dong MS2, Jie Shen MD3, Jun Zhang MS3, Jiankang Han MD4, Lin Wang MD5, Yan Liu MS1, Fan Wang MS1, Jun Zhao MD6, Qian Zhang MD7, Wu Ni MD8, Hongyang Wang MD, PhD9 and Guangwen Cao MD, PhD1
1Department of Epidemiology, Second Military Medical University, Shanghai, China, 2Department of Infectious Disease, Municipal Center for Disease Control and Prevention of Ningbo, Ningbo, China, 3Department of Infectious Disease, Municipal Center for Disease Control and Prevention of Soochow, Soochow, China, 4Department of Infectious Disease, Municipal Center for Disease Control and Prevention of Huzhou, Huzhou, China, 5Department of Infectious Disease, District Center for Disease Control and Prevention of Yangpu, Shanghai, China, 6Department of Hepatobiliary Surgery, The Third Affiliated Hospital, Second Military Medical University, Shanghai, China, 7Department of Infectious Disease, The First Affiliated Hospital, Second Military Medical University, Shanghai, China, 8Department of Infectious Disease, The Second Affiliated Hospital, Second Military Medical University, Shanghai, China, 9Laboratory for Signal Transduction, The Third Affiliated Hospital, Second Military Medical University, Shanghai, China,10These authors contributed equally to this work
Correspondence: Guangwen Cao, MD, PhD, Department of Epidemiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. E-mail: gcao@smmu.edu.cn
Abstract
OBJECTIVES: The objective of this study was to determine the association of 19 mutations with frequencies ≥10% in the core promoter region of hepatitis B virus (HBV) with chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma (HCC).
METHODS: Eight hundred forty-six asymptomatic hepatitis B surface antigen carriers (ASCs), 235 CHB patients, 188 cirrhosis patients, and 190 HCC patients with intact data of HBV genotyping, DNA sequencing, and serological parameters were studied. Nucleotides with the highest frequencies in HBV genotypes B and C from all ASCs were treated as wild-type nucleotides.
RESULTS: Mutations at nt.1674, nt.1719, nt.1762, nt.1764, nt.1846, nt.1896, and nt.1913 in genotype C were significantly associated with CHB, cirrhosis, and HCC, as compared with ASCs. C1673T, A1726C, A1727T, C1730G, C1766T, T1768A, C1773T, and C1799G in genotype C were significantly associated with cirrhosis compared with the CHB patients, whereas these mutations were inversely associated with HCC compared with the cirrhosis patients. Multivariate regression analyses showed that age, male, abnormal alanine aminotransferase (ALT), T1768A, A1762T/G1764A, and A1846T were independently associated with cirrhosis compared with ASCs and the patients with CHB. Age, abnormal ALT, HBV DNA (≥104 copies/ml), genotype C, C1653T, T1674C/G, T1753V, and A1762T/G1764A were independently associated with HCC compared with those without HCC. Haplotypic carriages with two or more HBV mutations were significantly associated with HCC. T1674C/G, C1653T, and T1753V were specific for HCC. A1762T/G1764A had a moderate sensitivity and specificity for HCC.
CONCLUSIONS: C1673T, A1726C, A1727T, C1730G, C1766T, T1768A, C1773T, and C1799G in genotype C are specific for cirrhosis. A1846T and T1674C/G are novel factors independently associated with cirrhosis and HCC, respectively.
INTRODUCTION
Hepatitis B virus (HBV) infection remains a major public health problem globally. More than two billion people have been infected worldwide, and of these, >350 million suffer from chronic HBV infection (1). Chronic HBV infection results in approximately one third of all cases of liver cirrhosis (LC) and more than three quarters of all cases of hepatocellular carcinoma (HCC) worldwide (2). Chronic hepatitis B (CHB), LC, and HCC are progressive stages of chronic HBV infection.
HBV belongs to the hepadnaviridae, a family of enveloped viruses with an incomplete double-stranded DNA genome of 3.2 kb. HBV genome contains four overlapping open reading frames that encode the surface, core, X, and polymerase proteins. HBV core promoter region including the basal core promoter (nucleotides 1751-1769) and the enhancer II (nucleotides 1636-1744) overlaps with the X open reading frame (nucleotides 1374-1835). The basal core promoter, which is regulated by the enhancer II and to some extent by the enhancer I, controls the transcription of precore mRNA and pregenomic RNA (3). The precore region encodes the precore protein, which is processed in the endoplasmic reticulum to produce secreted hepatitis B e antigen (HBeAg). HBeAg expression indicates active viral replication. HBeAg expression and high viral load are associated with an increased risk of HCC ((4,5,6)). According to a sequence divergence >8% in the entire HBV genome, 10 genotypes (A-J) have been identified so far ((7,8,9,10)). HBV genotypes have distinct geographical distributions worldwide, and have been shown to differ with regard to clinical diseases, prognosis, and response to interferon treatment ((10,11,12,13,14,15,16,17,18,19)). Genotypes B and C are endemic in Asia. Infection with genotype C is often associated with the risks of LC and HCC at older age compared with infection with genotype B ((10,11,12,13,14,15,16)). HBV genotypes have distinct patterns of the core promoter mutations that have been shown to be associated with the risk of HCC ((20,21,22,23,24,25,26,27,28,29,30,31)). Our recent meta-analysis has shown that C1653T, T1753V, and A1762T/G1764A are each associated with an increased risk of HCC, and these mutations are increasingly more prevalent as chronic HBV infection progress from asymptomatic hepatitis B surface antigen (HBsAg) carrier (ASC) state to LC or HCC (32). A1762T/G1764A has been shown to be a valuable biomarker for identifying a subset of HBV-infected subjects who are at extremely high risk of HCC in prospective studies ((26,29,30)).
However, the association of the HBV mutations in the core promoter region with HCC remains controversial because of conflicting data ((33,34)). Most of the studies only had a limited number of participants, and often examined only specific viral mutations and rarely analyzed other mutations that might have important roles. To elucidate the association of HBV mutations with the progressive liver diseases, it is essential to clarify "wild-type" patterns of given HBV genotypes from the subjects without clinical liver diseases. However, no epidemiological study with sufficient participants has shown the "wild-type" pattern of a given HBV genotype and the genotype-specific mutation pattern in the core promoter region of HBV as well. Although prospective study is more reliable than cross-sectional study in elucidating causal relationship between HBV mutations and HCC, the number of persons who developed HCC during follow-up was very limited, and some confounding factors like unavoidably receiving antiviral treatment and exposure to other risk factors might be introduced during the long-term follow-up. Thus, cross-sectional case-control study, which compares the frequencies of HBV mutations in ASCs with those in the patients with CHB, in the patients with LC, and in those with HCC, should be reliable for analyzing the association of HBV mutation with HCC. In this study, we initially identified the wild-type nucleotides in the core promoter region of HBV genotypes B and C from 846 ASCs, and then evaluated the association of all mutations with the frequencies >10% in this region with CHB, LC, or HCC.
DISCUSSION
In this study, we excluded the subjects who had received antiviral treatment. As the presence of A1762T/G1764A is associated with a high rate of antiviral response in HBeAg-positive patients (39), antiviral therapy could decrease the ability to detect mutations. Mutations examined in this study are of naturally occurring HBV core promoter mutations during chronic HBV infection. We successfully sequenced 87.5%, 71.9%, 90.0%, and 69.1% HBV samples from ASCs, the CHB patients, the LC patients, and the HCC patients, respectively. Failure to amplify the core promoter region due to low viral concentration in sera or HBV mutations in the primer binding sites is the major reason for unsuccessfully sequencing the samples.
In this study, 19 hotspots in HBV core promoter region were evaluated. We first determined wild-type nucleotides in this region of genotypes B and C from 846 ASCs, and found that wild-type nucleotides were the same as those from ASCs seropositive for HBeAg. The frequencies of 78.9% mutations in genotype B and 63.2% mutations in genotype C were significantly higher in HBeAg-negative than in HBeAg-positive ASCs infected with the corresponding HBV genotype. In the HBeAg-positive period, serum viral load is high and immune selection is week. Immune selection may increase during HBeAg seroconversion. HBeAg negativity has been associated with the presence of mutations in the basal core promoter and/or in the precore regions (40). Six of the 19 hotspots had different "wild-type" nucleotides between genotype B and genotype C. These evidences support that our definition of HBV wild-type nucleotides in the core promoter region is reasonable and the wild-type nucleotide in each hotspot may serve as a reference to evaluate the association of HBV mutations with the progressive liver diseases.
Some of the HBV mutations consecutively increase with aging and the advanced liver diseases are more frequent in men than in women ((10,26)). Age and sex may be the major confounders. We adjusted age and sex in evaluating the association of HBV mutations with CHB, LC, or HCC. In the subjects with genotype C, mutations at nt.1674, nt.1719, nt.1762, nt.1764, nt.1846, nt.1896, and nt.1913 were each associated with CHB, LC, and HCC, as compared with ASCs; whereas mutations at nt.1674, nt.1753, and nt.1762 were each associated with HCC compared with the CHB patients or the LC patients. In the subjects with genotype B, mutations at nt.1674, nt.1762, nt.1764, and nt.1896 were associated with HCC compared with the CHB patients (Figure 1). These mutations may indicate different stages of hepatitis B.
The majority of HBV-associated HCC (73.2% in this study) has LC. LC is an important end-stage liver disease of chronic HBV infection, and is common in HBeAg-negative patients. Old age, reactivation of hepatitis B, high ALT level, and genotype C have been shown to be risk factors of LC from the subjects chronically infected with HBV ((41,42,43,44,45)). Serum viral load has been found to be significantly associated with LC in the HBeAg-negative HBV-infected subjects ((44,45)). In this study, we found that mutations at nt.1653, nt.1674, nt.1719, nt.1753, nt.1762, and nt.1846 in genotype C were associated with HCC compared with the LC patients, and these associations were only found in HBeAg-negative, rather than in HBeAg-positive subjects (see Supplementary Table 5). Interestingly, C1673T, A1726C, A1727T, C1730G, T1768A, C1773T, and C1799G were significantly associated with LC compared with the patients with CHB, whereas these mutations were inversely associated with HCC compared with the patients with LC. This result indicates that C1673T, A1726C, A1727T, C1730G, T1768A, C1773T, and C1799G are specific for LC in those infected with genotype C. These mutations might be associated with the natural wound healing process to necroinflammation, rather than carcinogenesis. The multivariate analysis showed that old age, male sex, abnormal ALT, T1768A, A1762T/G1764A, and A1846T were independently associated with LC (Table 3). T1768A and A1762T/G1764A have been shown to be associated with LC ((40,44)). In this study, we added A1846T as a novel factor independently associated with LC. Thus, continuing liver damage in older, male subjects infected with HBV genotype C with mutations T1768A, A1762T/G1764A, and A1846T are apt to develop LC, especially for the HBV-infected subjects seronegative for HBeAg.
Multivariate regression analysis showed that C1653T, T1674C/G, A1762T/G1764A, and T1753V were independently associated with HCC compared with the subjects without HCC (Table 4). C1653T, T1753V, and A1762T/G1764A have been demonstrated to be significantly associated with HCC (32). In our study for analyzing another group of study subjects mostly recruited from May to October 2009, T1753V and A1762T/G1764A have been demonstrated to be independently associated with HCC, as compared with the age, sex-matched HBV-infected subjects without HCC (37). The results of this study are quite consistent with the previous findings. We demonstrated, for the first time, that T1674C/G was a novel factor independently associated with HCC. The HCC-associated HBV mutants in the core promoter region, for example, A1762T/G1764A mutants, may not transmit via mother-to-child vertical transmission because the children whose mothers carrying HBV mutants were mostly found to be infected with wild-type form of the same viruses ((46,47)). In this study, we found that although the frequencies of C1653T, T1753V, and A1762T/G1764A, rather than T1674C/G, consecutively increased with increasing age in ASCs and the patients with CHB, the frequencies of the six HCC-associated mutations went up to high levels in the patients with HCC even at relative young age (Figure 2). We therefore hypothesize that the HCC-associated HBV mutations are likely selected at one or several key steps of hepatocarcinogenesis.
Average frequencies of T1674C/G, C1653T, T1753V, and A1762T/G1764A were >30% in the patients with HCC, which make clinical examination feasible and clearly discriminate the HBV-infected patients with and without HCC. To elucidate the association of combined mutations in a given HBV genome with the risk of HCC, we innovatively investigated the estimated haplotype frequency of HBV mutations in combination and its association with HCC. Of the four HBV mutations independently associated with HCC, haplotypic carriages with two or more HBV mutations, rather than single one, were significantly associated with HCC. In the analysis of the sensitivity and specificity of these mutations, we found that T1674C/G, C1653T, and T1753V were specific for HCC, whereas A1762T/G1764A had moderate sensitivity and specificity for HCC (Table 6). These mutations, alone or in combination, may be reasonably arranged as molecular markers for HCC in the HBV-infected subjects.
HBV genotype-associated mutations in the core promoter region might be selected by HBV-host interaction during HBV chronic infection and in turn promote host hepatocarcinogenesis. It is also possible that the mutated X protein transactivates host oncogenes responsible for the development of HCC or that transactivators encoded by some oncogenes select the specific HBV mutations during hepatocarcinogenesis. HBV genotype C is independently associated with HCC, so are the core promoter mutations. We therefore hypothesized that the mutations in the core promoter region of genotype C might alter the binding sites of some nuclear factors related to hepatocarcinogenesis. To test this hypothesis, we determined "wild-type" HBV sequence from nt.1651 to nt.1920 by using the sequences of HBV genotype C from 562 ASCs, and then created a paired "mutated" sequence by replacing the "wild-type" nucleotides with the corresponding mutations that were significantly associated with the risk of HCC compared with ASCs after the adjustment (see Supplementary Table 2). With the use of TFSEARCH software online (http://www.cbrc.jp/research/db/TFSEARCH.html), we found different transcriptional factor binding sites between the "wild-type" sequence and the "mutated" sequence (see Supplementary Figure 2). Generation of HFH-2-, HNF-3b-, Skn-1-, Cdx-1-, cap-, and Oct-1-binding sites in the "mutated" sequence might represent novel virus-host interactions related to the development, viral replication, protooncogene activation, and carcinogenesis ((48,49,50)). The core promoter region encodes the C-terminal of HBV X protein. C1653T, T1674G/C, T1753V, A1762T, and G1764A mutations leaded to the amino acid (aa) transitions at aa94 (H-to-T), aa101 (S-to-A/P), aa127 (I-to-T/N/S), aa130 (K-to-M), and aa131 (V-to-I) sites. In addition, A1762T introduced a translation initiation site ATG at the C-terminal of HBx protein. HBV X mutations, especially the C-terminal deletions, have been frequently found in tumor tissues of HCC ((51,52)). Most of the C-terminally truncated HBx proteins lose their inhibitory effects on cell proliferation and transformation, retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis, which may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to hepatocarcinogenesis (53). However, the role of the amino acids substitutions at the C-terminal of HBx protein on hepatocarcinogenesis is rarely elucidated. It has been demonstrated that the core promoter mutations other than those at positions 1762 and 1764 have major impact on viral DNA replication and high viral load is the most important risk factor for HCC occurrence and recurrence after resection ((5,6,11,12,54,55)). High viral load due to HBV core promoter mutations may be one of the important causes of HBV-associated HCC.
In conclusion, this study systemically investigated the associations of HBV core promoter mutations with CHB, LC, or HCC. We found that C1673T, A1726C, A1727T, C1730G, T1768A, C1773T, and C1799G were specific for LC. A1846T and T1674C/G were novel factors independently associated with cirrhosis and HCC, respectively. Haplotypic carriages with two or more HBV mutations, rather than single one, were significantly associated with HCC. The frequencies of C1653T, T1753V, and A1762T/G1764A, rather than T1674C/G, consecutively increased with increasing age in ASCs and the patients with CHB, but not in the patients with HCC. Average frequencies of T1674C/G, C1653T, T1753V, and A1762T/G1764A were >30% in the patients with HCC. Sequential examination of these HBV mutations may be helpful in classifying the HBV-infected subjects who will develop HCC in future and need active antiviral treatments to postpone the occurrence of HCC. Although some mutations statistically associated with LC or HCC were found in this study, further study is needed to see if these mutations predict the development of LC or HCC in those who carry the mutation but do not yet have the associated disease.
RESULTS
Clinical and virological characteristics of study subjects
Eight hundred forty-six ASCs, 235 CHB patients, 188 LC patients, and 190 HCC patients who had intact data of HBV genotyping and HBV DNA sequencing were included in the analysis. Clinical and virological characteristics of the study subjects are listed in Table 1. Of the HCC patients, 139 (73.2%) had LC. The patients with HCC were significantly older, and had a significantly higher proportion of men than ASCs and the patients with CHB. There were no significant differences in ALT levels and the compositions of age and sex between the patients with LC and those with HCC. There was no difference in viral load between the patients with HCC and those with CHB. The frequency of HBeAg expression in the patients with LC was significantly lower than that in those with CHB and those with HCC after the adjustment for age and sex (adjusted odds ratio=0.35, 95% confidence interval=0.21-0.57; adjusted odds ratio=0.44, 95% confidence interval=0.27-0.71, respectively).
Mutations in hotspots of HBV genotypes B and C and their association with HBeAg expression
The hotspots were observed at nt.1652, nt.1653, nt.1673, nt.1674, nt.1703, nt.1719, nt.1726, nt.1727, nt.1730, nt.1753, nt.1762, nt.1764, nt.1766, nt.1768, nt.1773, nt.1799, nt.1846, nt.1896, and nt.1913. The frequencies of mutations at nt.1653, nt.1674, nt.1703, nt.1719, nt.1727, nt.1753, nt.1762, nt.1764, nt.1766, and nt.1768 were significantly higher in genotype C than genotype B, and the opposite was true for nt.1652, nt.1726, nt.1896, and nt.1913 in all study subjects. Wild-type nucleotides determined by using the sequences from all ASCs were the same as those from ASCs seropositive for HBeAg. Among these hotspots, wild-type nucleotides at nt.1652, nt.1673, nt.1726, nt.1727, nt.1730, and nt.1799 were different between genotype B and genotype C in all ASCs. The frequencies of 78.9% (15/19) mutations in genotype B and 63.2% (12/19) mutations in genotype C were significantly higher in the HBeAg-negative than in the HBeAg-positive ASCs infected with the corresponding genotype (see Supplementary Table 1 online).
Association of mutations in the core promoter region of HBV genotypes C and B with progressive liver diseases
To investigate the associations of mutations in the core promoter region of HBV genotypes B and C with CHB, LC, and HCC, we initially treated ASCs as controls. Mutations at nt.1674, nt.1719, nt.1762, nt.1764, nt.1846, nt.1896, and nt.1913 in genotype C were significantly associated with CHB, LC, and HCC, after the adjustment for age and sex, respectively (see Supplementary Table 2). As compared with the patients with CHB, mutations at nt.1673, nt.1727, nt.1730, nt.1764, nt.1766, nt.1768, nt.1773, nt.1799, and nt.1896 in both genotypes were significantly associated with LC after the adjustment (see Supplementary Table 3). Interestingly, mutation at nt.1719 was significantly associated with LC in genotype B, whereas it was inversely associated with LC in genotype C. As compared with the patients with LC, mutations at nt.1653, nt.1674, nt.1719, nt.1753, and nt.1762 in genotype C were significantly associated with HCC, whereas mutations at nt.1673, nt.1703, nt.1726, nt.1727, nt.1730, nt.1766, nt.1768, nt.1773, and nt.1799 in genotype C were inversely associated with HCC after the adjustment (see Supplementary Table 4). The associations of mutations at nt.1653, nt.1674, nt.1719, nt.1753, and nt.1762 in genotype C with HCC were only found in the HBeAg-negative patients, as compared with the patients with LC. Mutation at nt.1846 was significantly associated with HCC in the HBeAg-negative patients, whereas it was inversely associated with HCC in the HBeAg-positive patients after the adjustment (see Supplementary Table 5). Figure 1 presents mutations significantly associated with CHB, LC, or HCC after the adjustment.
C1653T, T1674C, G1719T, A1762T, and A1846T were significantly associated with HCC, whereas A1652G, C1673T, A1726C, A1727T, C1730G, T1768A, C1773T, and C1799G were inversely associated with HCC, as compared with the patients with LC in the genotype C HBV-infected patients seronegative for HBeAg. C1673T, A1726C, A1727T, C1730G, C1766T, T1768A, C1773T, and C1799G were significantly associated with LC, as compared with the patients with CHB, whereas these mutations were inversely associated with HCC, as compared with the patients with LC (Table 2). As compared with ASCs and the patients with CHB, C1673T, T1674C, A1703G, A1726C, C1730G, C1799G, A1846T, and C1913A in genotype C and C1653T, G1719T, A1727G, T1753A/C, A1762T, G1764A, C1766T, T1768A, C1773T, and G1896A in both genotypes were significantly associated with LC (see Supplementary Table 6). As compared with those without HCC, C1653T, T1674C/G, G1719T, A1727T, T1753V, C1913A in genotype C and T1753G, A1762T, G1764A, A1846T, and G1896A in both genotypes were significantly associated with HCC (see Supplementary Table 7).
Factors independently associated with LC as compared with ASCs and the CHB patients
The multiple factors, containing age (years), sex (male vs. female), HBV DNA level (≥104 vs. <104 copies/ml), HBV genotype (C vs. B), ALT (abnormal vs. normal), HBeAg status (positive vs. negative), and the presence of C1673T, A1703G, A1726C, C1727G, A1762T/G1764A, C1766T, T1768A, C1773T, A1846T, and G1896A were introduced into the multivariate regression model. Old age, male, abnormal ALT, T1768A, A1762T/G1764A, and A1846T were found to be independently associated with LC, as compared with ASCs and the patients with CHB (Table 3).
Factors independently associated with HCC as compared with those without HCC
The multiple factors, containing age (years), sex (male vs. female), HBV DNA level (≥104 vs. <104 copies/ml), HBV genotype (C vs. B), ALT (abnormal vs. normal), HBeAg status (positive vs. negative), and the presence of C1653T, T1674C/G, G1719T, A1762T/G1764A, T1753V, A1846T, and C1913A were introduced into the multivariate regression model. Old age, abnormal ALT, HBV DNA (≥104 copies/ml), genotype C, C1653T, T1674C/G, T1753V, and A1762T/G1764A were independently associated with HCC, as compared with the subjects without HCC (Table 4).
Changes in the frequencies of mutations with increasing age of the subjects at different disease states
All participants were divided into five age groups. The proportion of ASCs consecutively decreased with increasing age (Ptrend<0.001), the proportions of the LC patients and the HCC patients consecutively increased with increasing age (Ptrend<0.001 for each) (see Supplementary Figure 1). Figure 2 presents changes in the frequencies of the six mutations independently associated with LC or HCC with increasing age of the subjects at different disease states. In the HBV-infected subjects without the end-stage liver diseases, the frequencies of C1653T, T1753V, T1768A, A1846T, and A1762T/G1764A, rather than T1674C/G, consecutively increased with increasing age (Ptrend<0.05 for each). The frequencies of the six mutations did not increase with increasing age in the patients with HCC. The frequencies of C1653T, T1674C/G, T1768A, A1846T, and A1762T/G1764A, rather than T1753V, did not increase with increasing age in the patients with LC. Average frequencies of C1653T, T1674C/G, T1753V, and A1762T/G1764A in the HCC patients were 30.3%, 34.6%, 33.0%, and 79.3% and significantly higher than those (8.8%, 11.8%, 9.3%, and 27.0%) in the HBV-infected subjects without HCC, respectively (P<0.001 for each). Average frequencies of T1768A, A1846T, and A1762T/G1764A in the LC patients were 24.3%, 29.0%, and 61.9% and significantly higher than those (5.3%, 9.8%, and 21.0%) in ASCs and the CHB patients, respectively (P<0.001 for each).
Association of estimated haplotype frequencies of HBV mutations in combination with HCC
Table 5 shows the estimated haplotype frequencies of C1653T, T1674C/G, T1753V, and A1762T/G1764A in combination and their association with HCC. Haplotypic carriage with wild-type forms of the four polymorphisms was inversely associated with HCC. In the absence of other three mutations, haplotypic carriage with A1762T/G1764A alone was not significantly associated with HCC. The same was true for C1653T, T1674C/G, and T1753V. Of the four HBV mutations, haplotypic carriages with two or more HBV mutations were significantly associated with HCC.
Sensitivity and specificity of HBV mutations for HCC
We then evaluated the potential value of the presence of HBV mutations that were independently associated with HCC for the indication of HCC. As shown in Table 6, T1674C/G, C1653T, and T1753V were specific for HCC. A1762T/G1764A had a moderate sensitivity and specificity for HCC. A1762T/G1764A or T1753V, A1762T/G1764A or C1653T, and A1762T/G1764A or T1674C/G were sensitive for HCC.
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