pre-S2 Start Codon Research Articles

Meta-analysis: Association between hepatitis B virus preS mutation and hepatocellular carcinoma risk.

Previous observational studies suggested that hepatitis B virus (HBV) preS mutation plays an important role in the existence of HBV-related hepatocellular carcinoma (HCC). However, the results are still debatable. With an increasing number of studies about this topic, this study employed a meta-analysis to identify the association between HBV preS mutation and HCC risk. We searched for eligible studies from PubMed, ProQuest, CINAHL, ScienceDirect and Springer databases to assess the association between HBV mutation and HCC risk. This meta-analysis was conducted using RevMan 5.3 to provide pooled estimate for odds ratio (ORs) with 95% confidence intervals (95% CIs). Twenty-one clinical studies were included in this meta-analysis study which consisted of 1738 participants with HBV-related HCC and 3740 HBsAg-positive patients without HCC. All studies used samples of Asian population. PreS deletion was the most common mutation found in all studies. We found that ORs of HBV overall preS deletion was associated with HCC (OR=3.28; 95% CI=2.32-4.65; P<.00001; random-effects model). Each preS1 and preS2 deletion was associated with increased risk of HCC, with OR 2.42 (95% CI=1.25-4.68, P=.008) and 3.36 (95% CI=2.04-5.55, P<.00001), respectively. PreS2 start codon mutation was also significantly associated with HCC risk (OR=2.47; 95% CI: 1.15-5.27; P=.02; random-effect model). The result of this meta-analysis suggested that HBV preS deletion (all, preS1 and preS2) and preS2 start codon mutation might contribute to the increased risk of HBV-related HCC.

Molecular characterization of hepatitis B virus isolated from Black South African cancer patients, with and without hepatocellular carcinoma.

In South Africa (SA), hepatitis B virus (HBV) infection is strongly associated with hepatocellular carcinoma (HCC). As HBV genotypes/subgenotypes and mutations can influence disease manifestation and progression, our aim was to molecularly characterize HBV in Black cancer patients, with and without HCC. The basal core promoter/precore (BCP/PC) and complete surface (S) regions of HBV isolates were amplified and sequenced from 55 HCC cases and 22 non-HCC cancer controls. Phylogenetic analysis of 43 polymerase/complete S region amplicons showed that the majority (88.4%) clustered with subgenotype A1, 4.7% with A2, and 7% with A3. The following mutations were significantly more frequent in HCC cases than in controls (p < 0.05): in the BCP/PC1753C/G (22.5% vs. 0%), 1764A (69.4% vs. 38.1%), and T64C (51.5% vs. 20%) in the preS2, which results in a F22L substitution. PreS1 and preS2 start codon mutants were detected only in HCC cases, occurring in two and 16 isolates, respectively. PreS deletion mutants were isolated from 11 HCC cases, which had a HBV viral load > 10,000 IU/mL and were significantly younger than non-HCC controls (34 ± 7.1 vs. 41.2 ± 9.5 years, p = 0.05). The 1762T/1764A double mutation was detected in the majority (90.9%) of the isolates from HCC cases with preS deletions. Black HBV carriers were mainly infected with subgenotype A1, with HCC cases carrying BCP/PC and preS mutant strains that are associated with hepatocarcinogenesis. This is the first study to compare the molecular characteristics of HBV from HCC and non-HCC cancer patients in SA.

HBV preS deletion mapping using deep sequencing demonstrates a unique association with viral markers.

AimDeletions are observed frequently in the preS1/S2 region of hepatitis B virus (HBV) genome, in association with liver disease advancement. However, the most significant preS1/S2 region and its influences on viral markers are unclear.MethodsThe preS1/S2 HBV regions of 90 patients without antiviral therapy were subjected to deep sequencing and deleted regions influencing viral markers were investigated.ResultsFrom the deletion frequency analysis in each patient, deletions were observed most frequently in the preS2 codon 132–141 region. When the patients were divided into three groups (0–0.1%: n = 27, 0.1%-10%: n = 34, 10–100%: n = 29), based on the deletion frequency, FIB-4 (p < 0.01), HBV DNA (p < 0.01), HBcrAg (p < 0.01) and preS1/S2 start codon mutations (p < 0.01, both) were significantly associated with the deletion. When clinical and viral markers were investigated by multivariate analysis for their association with the deletion, FIB-4 (p < 0.05), HBcrAg (p < 0.05), and preS1 start codon mutation (p < 0.01) were extracted as independent variables. When the influence of the preS codon 132-141deletions on HBsAg and HBcrAg, relative to HBV DNA, was investigated, the HBsAg/HBV DNA ratio was lower (0–10% vs. 10%-100%, p<0.05), while the HBcrAg/HBV DNA rati o was higher (0–0.1% vs. 10%-100%, p<0.05) in the presence of the preS codon 132-141deletions.ConclusionThe preS codon.132-141 deletions have a significant influence on the clinical characteristics and viral markers, even when present as a minor population. Importantly, the preS codon 132–141 deletions have a clear influence on the viral life cycle and pathogenesis.

Pre-S2 Start Codon Mutation of Hepatitis B Virus Subgenotype B3 Effects on NF-κB Expression and Activation in Huh7 Cell Lines.

A cross-sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation (M120 V) with cirrhosis and hepatocellular carcinoma (HCC), which was dissimilar from studies from other populations where pre-S2 deletion mutation was more prevalent. Different mutation patterns were attributed to different hepatitis B virus (HBV) subgenotypes in each population study. HBV surface proteins are reported to induce the activation of NF-κB, a transcriptional factor known to play an important role in the development of liver disease. This study aimed to see the effects of HBs variants in HBV subgenotype B3 on the expression and activation of NF-κB as one of the mechanisms in inducing advanced liver disease. HBV subgenotypes B3, each carrying wild-type (wt) HBs, M120 V, and pre-S2 deletion mutation were isolated from three HCC patients. HBs genes were amplified and cloned into pcDNA3.1 and were transfected using Lipofectamine into a Huh7 cell line. NF-κB activation was measured through IκB-α expression, which is regulated by NF-κB. RNA expressions for HBs, IκB-α, and NF-κB subunit (p50) were evaluated using real-time PCR. M120 V mutant had a significantly higher mRNA level compared with wt and pre-S2 deletion mutant; however, there were no significant differences in HBs protein expressions. The transcription level of p50 was higher in M120 V mutation compared with HBs wild-type and pre-S2 deletion mutant. NF-κB activation was higher in HBs wild-type compared with the two mutant variants. Pre-S2 mutations had no effect on the increment of NF-κB activation. However, M120 V mutation may utilize a different pathway in liver disease progression that involves high expression of NF-κB subunit, p50.

Three types of preS1 start codon deletion variants in the natural course of chronic hepatitis B infection.

Naturally occurring hepatitis B virus variants carrying a deletion in the preS1 start codon region may evolve during long-lasting virus-host interactions in chronic hepatitis B (CHB). The aim of this study was to determine the immune phase-specific prevalent patterns of preS1 start codon deletion variants and related factors during the natural course of CHB. A total of 399 CHB patients were enrolled. Genotypic analysis of three different preS1 start codon deletion variants (classified by deletion size: 15-base pair [bp], 18-bp, and 21-bp deletion variants) was performed. PreS1 start codon deletion variants were detected in 155 of 399 patients (38.8%). The predominant variant was a 15-bp deletion in the immune-tolerance phase (18/50, 36%) and an 18-bp deletion in the immune-clearance phase (69/183, 37.7%). A 21-bp deletion was the predominant variant in the low replicative phase (3/25, 12.0%) and reactivated hepatitis Be antigen (HBeAg)-negative phase (22/141, 15.6%). The 15-bp and 18-bp deletion variants were more frequently found in HBeAg-positive patients (P<0.010 and P<0.001, respectively), whereas the 21-bp deletion variant was more frequently found in HBeAg-negative patients (P<0.001). On multiple logistic regression analyses, the 21-bp deletion variant was independently associated with liver cirrhosis (P=0.006), and the 15-bp deletion variant was significantly related to an incomplete response to antiviral agents (P=0.012). The predominant type of preS1 start codon deletion variants changes according to the immune phases of CHB infection, and each variant type is associated with different clinical parameters. PreS1 start codon deletion variants might interact with the host immune response differently according to their variant types.

Potential Susceptibility Mutations in C Gene for Hepatitis B-Related Hepatocellular Carcinoma Identified by a Two-Stage Study in Qidong, China.

A two stage study was conducted to explore new potential mutations in the full genome of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China. In stage 1, full genomes of HBV were compared between 30 HCC cases and 30 controls. In stage 2, an independent case–control study including 100 HCC cases and 100 controls was enrolled to verify the relationship between hot-spot mutations and HCC development. Furthermore, a longitudinal study was conducted on 11 HCC cases with serial serum samples available before HCC diagnosis. A total of 10 mutations (including pre-S2 start codon mutation and pre-S deletion in pre-S gene, G1613A, C1653T, A1762T, and G1764A mutations in X gene, A2159G, A2189Y, G2203W, and C2288R mutations in C gene) showed an increased risk of HCC. In the validation study, pre-S deletion, C1653T, A1762T/G1764A, A2159G, A2189Y, G2203W, and C2288R mutations were associated with increased HCC risk in univariate analysis. Multivariate analysis indicated that pre-S deletion, A1762T/G1764A, A2159G, and A2189Y mutations were independently related with HCC development. Moreover, a significant biological gradient of HCC risk by number of mutations in the C gene was observed. Longitudinal observation demonstrated a gradual combination of the above mutations accumulated during the progression of HCC.

Quantitative evaluation of hepatitis B virus mutations and hepatocellular carcinoma risk: a meta-analysis of prospective studies.

The temporal relationship between hepatitis B virus (HBV) mutations and hepatocellular carcinoma (HCC) remains unclear. We conducted a meta-analysis including cohort and nested case-control studies to prospectively examine the HCC risk associated with common variants of HBV in the PreS, Enhancer II, basal core promoter (BCP) and precore regions. Pertinent studies were identified by searching PubMed, Web of Science and the Chinese Biological Medicine databases through to November 2014. Study-specific risk estimates were combined using fixed or random effects models depending on whether significant heterogeneity was detected. Twenty prospective studies were identified, which included 8 cohort and 12 nested case-control studies. There was an increased risk of HCC associated with any PreS mutations with a pooled relative risk (RR) of 3.82 [95% confidence interval (CI): 2.59-5.61]. The pooled-RR for PreS deletion was 3.98 (95% CI: 2.28-6.95), which was higher than that of PreS2 start codon mutation (pooled-RR=2.63, 95% CI: 1.30-5.34). C1653T in Enhancer II was significantly associated with HCC risk (pooled-RR=1.83; 95% CI: 1.21-2.76). For mutations in BCP, statistically significant pooled-RRs of HCC were obtained for T1753V (pooled-RR=2.09; 95% CI: 1.49-2.94) and A1762T/G1764A double mutations (pooled-RR=3.11; 95% CI: 2.08-4.64). No statistically significant association with HCC risk was observed for G1896A in the precore region (pooled-RR=0.77; 95% CI: 0.47-1.26). This study demonstrated that PreS mutations, C1653T, T1753V, and A1762T/G1764A, were associated with an increased risk of HCC. Clinical practices concerning the HCC risk prediction and diagnosis may wish to focus on patients with these mutations.

Effect of functional nuclear factor-kappaB genetic polymorphisms on hepatitis B virus persistence and their interactions with viral mutations on the risk of hepatocellular carcinoma

Nonresolving inflammation and viral mutations are important in hepatitis B virus (HBV)-induced hepatocarcinogenesis. However, the effects of genetic polymorphisms affecting nuclear factor-kappaB (NF-κB) on HBV persistence and generation of hepatocellular carcinoma (HCC)-related HBV mutations remain unknown. rs28362491 (NFKB1 -94Ins > Del), rs2233406 (NFKBIA -826C > T), rs3138053 (NFKBIA -881A > G), and rs696 (NFKBIA +2758G > A) were genotyped in 1342 healthy controls, 327 HBV-clearance subjects, and 3976 HBV-positive subjects including 1495 HCC patients, using quantitative PCR. HBV mutations were determined by sequencing. The NFKBIA promoter activity was assessed by transient transfection. Multiplicative interactions of the polymorphisms and viral mutations were assessed by multivariate logistic regression. Compared with HBV-clearance subjects, rs2233406 (CT versus CC) and rs3138053 (AG or AG + GG versus AA) significantly decreased HBV persistence, especially in the genotype B HBV-infected subjects. In the genotype C HBV-infected subjects, rs2233406 variant genotypes were significantly associated with an increased risk of HCC [CT versus CC: age-, gender-adjusted odds ratio (AOR), 1.33; 95% confidence interval (CI) 1.01-1.75 in training set and AOR, 1.59; 95% CI 1.01-2.52 in validation set] compared with HCC-free HBV-infected subjects and significantly increased the frequencies of HCC-related HBV mutations (A1762T/G1764A, T1753V, preS1 start codon mutation, and preS deletion); rs28362491 (Del/Del or Ins/Del + Del/Del versus Ins/Ins) significantly increased the frequency of A1762T/G1764A and reduced the frequency of preS2 start codon mutation. The variant genotypes impaired NFKBIA promoter activity in hepatic cells. The interaction of rs2233406 variant genotypes (CT + TT versus CC) with A1762T/G1764A significantly increased HCC risk in genotype C HBV-infected subjects, with AOR of 2.61 (95% CI 1.09-6.26). Genetic polymorphisms improving NF-κB activity contribute to genotype B HBV clearance. The rs2233406 variant genotypes significantly increase HCC risk, possibly via facilitating immune selection of the HBV mutations. The host-virus interactions are important in identifying HBV-infected subjects who are more likely to develop HCC.

Impacts of human leukocyte antigen DQ genetic polymorphisms and their interactions with hepatitis B virus mutations on the risks of viral persistence, liver cirrhosis, and hepatocellular carcinoma

Human leukocyte antigen (HLA)-DQ genetic polymorphisms have been associated with chronic hepatitis B virus (HBV) outcomes. We aimed to determine impacts of HLA-DQ polymorphisms and their interactions with HBV mutations on the risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). rs2856718 (A>G) and rs9275319 (A>G) were genotyped in 1342 healthy controls, 327 HBV surface antigen (HBsAg) seroclearance subjects, 611 asymptomatic HBsAg carriers (ASCs), 1144 chronic hepatitis B (CHB) patients, 734 LC patients, and 1531 HCC patients using quantitative PCR. HBV mutations were detected by direct sequencing. Logistic regression analyses were utilized to assess the factors and/or multiplicative interactions significantly associated with liver diseases. rs9275319 variant genotypes were inversely associated with HBV persistence compared to HBV natural clearance subjects. rs2856718 variant genotypes significantly increased LC risk compared to ASCs plus CHB patients (GG vs. AA: odds ratio [OR], 1.52, 95% confidence interval [CI], 1.17–1.97 and AG+GG vs. AA: OR, 1.27; 95% CI, 1.04–1.54) and decreased HCC risk compared to HCC-free HBV-infected subjects (AG vs. AA: OR, 0.76; 95% CI, 0.65–0.89 and AG+GG vs. AA: OR, 0.78, 95% CI, 0.68–0.90). rs2856718 variant genotypes were significantly associated with an increased frequency of HBV A1726C mutation, a LC-risk, HCC-protective mutation, in genotype C. A rs9275319 variant genotype (GG) was significantly associated with an increased frequency of preS1 start codon mutation, an HCC-risk mutation, in genotype C. The interaction of rs2856718 AG+GG genotype with T1753V, a HCC-risk mutation, significantly reduced LC risk, with an OR of 0.26 (95% CI, 0.09–0.78); whereas the interaction of rs2856718 AG genotype with C1673T, a LC-risk mutation, significantly increased HCC risk, with an OR of 2.80 (95% CI, 1.02–7.66) in genotype C HBV-infected subjects. Conclusively, the HLA-DQ polymorphisms affect the risks of LC and HCC differently in chronic HBV-infected subjects, possibly via interacting with the HBV mutations.

Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in Qidong, China.

Background/AimTo investigate the roles of mutations in pre-S and S regions of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China.MethodsWe conducted an age matched case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence in pre-S/S regions was successfully determined in 96 HCC cases and 97 control subjects. In addition, a consecutive series of samples from 11 HCC cases were employed to evaluate the pre-S deletion patterns before and after the occurrence of HCC.ResultsAfter adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positivity, pre-S deletions, pre-S2 start codon mutations, and T53C mutation were significantly associated with HCC, showing adjusted odds ratios (ORs) from 1.914 to 3.199. HCC patients also had a lower frequency of T31C mutation in pre-S2 gene, compared with control subjects (0.524; 95% CI 0.280-0.982). HBV pre-S deletions were clustered mainly in the 5′ end of pre-S2 region. Multivariate analysis showed that pre-S deletions and pre-S2 start codon mutations were independent risk factors for HCC. The OR (95% CI) were 2.434 (1.063–5.573) and 3.065 (1.099–8.547), respectively. The longitudinal observation indicated that the pre-S deletion mutations were not acquired at the beginning of HBV infection, but that the mutations occurred during the long course of liver disease.ConclusionPre-S deletions and pre-S2 start codon mutations were independently associated with the development of HCC. The results also provided direct evidence that pre-S deletion mutations were not acquired from the beginning of infection but arose de novo during the progression of liver disease.

Association of a potential functional pre‐miR‐218 polymorphism and its interaction with hepatitis B virus mutations with hepatocellular carcinoma risk

MicroRNA-218 (miR-218) can function as a tumour suppressor and inactivate cancer-promoting inflammation. However, role of miR-218 on hepatocellular carcinoma (HCC) remains unclear. To determine the contribution of miR-218 genetic predisposition and its interaction with hepatitis B virus (HBV) mutations to HCC risk. rs11134527 located at putative promoter region of pre-miR-218 was genotyped in 1012 healthy controls, 302 hepatitis B surface antigen (HBsAg) seroclearance subjects and 2011 subjects with chronic HBV infection (1021 with HCC) using quantitative PCR. HBV mutation was determined by sequencing. rs11134527 variant genotypes in dominant model was associated with HCC risk compared with all HCC-free subjects [odds ratio (OR) = 1.22, 95% confidence interval (CI) = 1.04-1.43], HCC-free HBsAg-positive subjects (OR = 1.23, 95% CI = 1.02-1.50) and HBsAg seroclearance subjects (OR = 1.45, 95% CI = 1.08-1.96), adjusting for age and gender, and also associated with the generation of HBV preS deletion in men (adjusted OR = 1.85, 95% CI = 1.23-2.76). In multivariate regression analyses, rs11134527 in dominant model was associated with HCC risk (OR = 1.50, 95% CI = 1.05-2.13), whereas its multiplicative interaction with viral mutation T1674C/G was inversely associated with HCC risk (OR = 0.44, 95% CI = 0.21-0.96), adjusting for covariates including HBV mutations in the enhancer II-precore region; its interaction with HBV preS1 start codon mutation was associated with HCC risk (OR = 4.44, 95% CI = 1.27-15.55), adjusting for covariates including HBV mutations in the preS region. rs11134527 may be a novel genetic risk factor of HCC in HBV-exposed subjects, can facilitate HBV preS deletion generation and predispose the host to the effect of T1674C/G and preS1 start codon mutation in hepatocarcinogenesis.

Change in Hepatitis B Virus Large Surface Antigen Variant Prevalence 13 Years after Implementation of a Universal Vaccination Program in China

A nationwide hepatitis B virus (HBV) vaccination program was implemented in China starting in 1992. To study the change in HBV variant prevalence with massive immunization, large HBV surface protein (LHBs) genes from HBV surface antigen (HBsAg)-positive sera were amplified and sequenced. The prevalences of LHBs mutants were compared between the 1992 and 2005 surveys in child and adult groups. The prevalence of "α" determinant mutants in the children increased from 6.5% in 1992 to 14.8% in 2005, where the G145R mutant occurred most frequently. In contrast, mutation frequencies showed little difference between 1992 (9.4%) and 2005 (9.9%) in adults. Moreover, compared to the 1992 survey, the child group surface (S) protein mutation frequency specifically increased (P = 0.005) in the 2005 survey, but the pre-S region mutation frequency did not show a significant difference (P > 0.05). However, the mutation frequency in the adult group increased in both the pre-S and S regions. Furthermore, the frequencies of the disease-related pre-S2 deletion and start codon mutations were significantly higher in the adult groups than in the child groups in both the 1992 and 2005 surveys (P < 0.01). Massive immunization enhances the HBV S protein mutation; the prevalence of LHBs mutants, particularly disease-related mutants, tends to increase with patient age.

Interaction of signal transducer and activator of transcription 3 polymorphisms with hepatitis B virus mutations in hepatocellular carcinoma

Hepatitis B virus (HBV) mutations and signal transducer and activator of transcription 3 (STAT3) activation are closely associated with hepatocellular carcinoma (HCC). However, single nucleotide polymorphisms (SNPs) of STAT3 have not been implicated in HCC susceptibility. This study was designed to evaluate the effect of STAT3 SNPs and their interactions with HBV mutations on HCC risk. A total of 2,011 HBV-infected subjects (including 1,021 HCC patients) and 1,012 healthy controls were involved in this study. SNPs rs4796793 (-1697, C>G), rs2293152 (intron 11, C>G), and rs1053004 (3' untranslated region, T>C) were genotyped using quantitative polymerase chain reaction. HBV mutations were determined via direct sequencing. It was found that rs2293152 (GG versus CC) was significantly associated with HCC risk compared with the subjects without HCC, adjusting for age and sex (adjusted odds ratio [AOR], 1.30; 95% confidence interval [CI], 1.04-1.62). The impact of rs2293152 was greater in women compared with men. Compared with HCC-free HBV-infected subjects, rs2293152 GG was solely associated with HCC in women (AOR, 2.04; 95% CI, 1.15-3.61). rs2293152 GG was significantly associated with high viral load (≥1 × 10(4) copies/mL) (AOR, 1.37; 95%, CI 1.01-1.88) and increased frequencies of T1674C/G (AOR, 1.61; 95% CI, 1.06-2.46) and A1762T/G1764A (AOR, 1.64; 95% CI, 1.14-2.35). In multivariate regression analyses, multiplicative interaction of rs1053004 with T1674C/G significantly increased HCC risk, whereas rs2293152 and A1726C interaction reduced it, adjusting for covariates including HBV mutations in the enhancer II/basal core promoter/precore region; the interaction of rs4796793 with preS2 start codon mutation significantly increased HCC risk, adjusting for covariates including HBV mutations in the preS region. STAT3 SNPs appear to predispose the host with HBV mutations to hepatocarcinogenesis, and this effect may differ in men versus women. STAT3 SNPs may have applicability in future HCC surveillance algorithms. (Hepatology 2013;57:2369-2377).

High prevalence of hepatitis B virus genotype C/C1 in the Minangkabau ethnic group in Indonesia

BackgroundThe Minangkabau is one of the major ethnic groups in Indonesia. Previous studies with a limited number of samples have shown a different prevalence of HBV/C in the Minangkabau compared to the Indonesian population in general. The aim of this study was to assess the HBV genotype distribution pattern and the prevalence of pre-S, T1753V and A1762T/G1764A mutations among the Minangkabau HBV carriers. The samples were collected from Padang, West Sumatera and from western Java. Mixed primers for specific genotypes were used to determine the HBV genotype. Pre-S or S genes were amplified, sequenced and aligned with reference sequences from GenBank to derive a phylogenetic tree for subgenotyping. Pre-S genes were also analyzed for mutations. The basal core promoter (BCP) region was amplified and directly sequenced to analyze T1753V and A1762T/G1764A mutations.ResultsThe predominant HBV genotype among the Minangkabau HBV carriers (n=117) was C (72.6%) followed by B (24.8%) and co-infection with B and C (2.6%). The prevalence of pre-S mutations, including both the pre-S deletion and pre-S2 start codon mutation, was 41.0%, and the T1753V and A1762T/G1764A mutations were found in 51.9% and 71.2% respectively. HBV/C1 was the predominant HBV subgenotype in the Minangkabau HBV carriers, and was found in 66.2%, followed by B3, B7, C8, B2, B9, C2, and C10 (18.3%, 7.0%, 2.8%, 1.4%, 1.4%, 1.4%, and 1.4% respectively). From samples that were found to be co-infected with HBV B and C, two samples were successfully cloned and subgenotyped, including one with mixed subgenotypes of B3 and C1, and another one with mixed subgenotypes of B7, C1, putative intergenotypic of B/A, and C/A. Furthermore, three samples from donors of non-Minangkabau ethnicity from Padang were found to be infected with an intragenotypic recombination form, including a putative recombinant of B8/B3 and B9/B7.ConclusionHBV/C with subgenotype C1 was the predominant HBV genotype among HBV carriers of Minangkabau ethnicity. The prevalence of pre-S, A1762T/G1764A, and T1753V mutations was higher among the Minangkabau compared to Indonesian HBV carriers in general.

Lack of association between hepatitis B virus pre‐S mutations and recurrence after surgical resection in hepatocellular carcinoma

Pre-S mutation of hepatitis B virus (HBV) is known to be a risk factor for hepatocarcinogenesis. A previous study suggested that pre-S mutation(s) may associate with increased recurrence after surgical resection. In the present study, 64 patients with HBV-related hepatocellular carcinoma (HCC) were categorized into two groups according to the presence or absence of pre-S mutation(s). The clinicopathological variables of the two groups were analyzed to assess the relationship between pre-S mutations and postoperative recurrence. Nineteen patients (29.7%) had pre-S mutations;13 had a pre-S deletion, three had a pre-S2 start codon mutation, two patients had both a pre-S deletion, and a pre-S2 start codon mutation, and one patient had a pre-S2 insertion. The two groups did not differ in terms of baseline clinicopathological parameters. Cirrhosis and satellite lesion(s) were predictive factors for postoperative recurrence and poor overall survival. Recurrence-free survival (P = 0.320) and overall survival (P = 0.238) did not differ significantly when pre-S mutations were present. In conclusion, this study did not find evidence supporting the notion that pre-S mutation(s) are associated with postoperative recurrence after surgical resection.

Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India

To molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection. Sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student's t test. The χ² test or Fisher's exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05. Irrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D. Kerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.

Impact of hepatitis B virus (HBV) preS/S genomic variability on HBV surface antigen and HBV DNA serum levels

To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells. In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection.

Genotypes B and C hepatocellular carcinoma–associated hepatitis B virus pre‐S mutants: their detection among F1b and A2 – but not F4 – isolates from Argentina

Prevalence rates of hepatocellular carcinoma (HCC)-associated hepatitis B virus (HBV) pre-S mutants among most genotypes are still lacking. In this study, viral (sub)genotypes of 70 Argentine nucleotide sequences (33 newly obtained) were determined by phylogenetic analysis, and the presence of such mutants was assessed in the American continent for the first time. Nucleotide substitutions of the pre-S2 start codon were observed in 10% of the HBV/A2 sequences. Ten per cent of the HBV/A2 and 12.5% of the HBV/F1b - but none of HBV/F4 - exhibited a deletion in the pre-S1/pre-S2 region. The contribution of these variants to liver cirrhosis (LC) and/or HCC development among HBV/F and HBV/A isolates deserves further prospective clinical studies.

Original Article. Hepatitis B Virus S Promoter Deletion in Hepatocellular Carcinoma

Abstract Objective To identify the difference and significance of dominant types of hepatitis B virus (HBV) pre-S mutation between liver tumor tissues and paired adjacent non-tumor tissues and to test if the mutations were tumor tissue specific. Methods HBV DNA isolated from 34 paired intratumoral and peritumoral tissues of hepatocellular carcinoma (HCC) patients were screened by PCR and direct sequencing. All patients carried HBV with genotype C, except for one B/C heterozygote. The expression, localization and excretion of LHBs mutant carrying pre-S deletions were characterized in vitro. The expression of endoplasmic reticulum (ER) GRP78 mRNA was assayed. Results Four patterns of pre-S mutations were identified: pre-S1 in-frame deletion involving the first start codon; pre-S2 in-frame deletion; pre-S2 start codon mutation with or without in-frame deletion; and S promoter in-frame deletion (ΔSP). The first two types were evenly found in both tumor and non-tumor tissues. They were rarely present as dominant strains. The last two types were frequently found in the dominant strains in tumor tissues. The overall prevalence of HBV carrying ΔSP was 17.64% (6/34) in tumor tissues, but none were dominant in nontumor tissues. HBV carrying ΔSP was unable to produce S protein in vitro. Immunocytofluorescence assay showed that the ΔSP LHBs mutant aggregated in the cytoplasm, accumulating mainly in the ER. Transient transfection and expression of ΔSP mutant caused GRP78 up-regulation in vitro. Conclusions HBV S promoter deletion was found dominantly in HCC tumor tissue. The aggregation of mutant large surface proteins in the ER possibly involved in HBV-related HCC.

Differential pattern of pre-S mutations/deletions and its association with hepatitis B virus genotypes in Eastern India

The presence of three different HBV genotypes (A, C and D) in Eastern India provided us a unique opportunity to study HBV pre-S mutants in these genotypes and subtypes among the same ethnic population. Furthermore, we also aimed to investigate the association of the HBV pre-S mutation with clinical outcome. Pre-S1-S2 and S gene was amplified and sequenced from 86 HBsAg positive study subjects with varying clinical manifestation. The genetic variability in the pre-S region (mutations) was studied with respect to different HBV genotypes, subtypes and different clinical categories. Six different types of HBV pre-S mutations were detected in 25 cases (29.07%), among which pre-S2 start codon mutation (28.0%) and pre-S2 deletion (24.0%) were most common. Pre-S mutation was highest in HBV/C (7/18; 38.89%) followed by HBV/A (9/27; 33.33%) and HBV/D (9/40; 22.50%). Pre-S1 deletion is common in HBV/D, whereas pre-S2 start codon mutation and pre-S2 deletions are frequent among HBV/A and HBV/C, respectively. Interestingly, in HBV/A and HBV/C the tendency of mutation/deletion increases from pre-S1 to pre-S2 region while in HBV/D the opposite tendency was observed. A significantly higher association of pre-S mutation (p=0.013) and pre-S2 deletion/ablation (p=0.016) was found among the HBeAg negative cases. Pre-S1 deletion and pre-S2 deletion were common among the ASC and CLD cases respectively, while pre-S2 start codon mutation was significantly associated with cirrhosis (p<0.05). The study underscores the association of types of pre-S mutations with particular HBV genotype and clinical outcome in the study population.