Association of Hepatitis B Virus Pre-S Deletions with the Development of Hepatocellular Carcinoma in Qidong,China

Background/Aim

To 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.

Methods

We 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.

Results

After 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.

Conclusion

Pre-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.     

Variability in the precore and core promoter regions of HBV strains in morocco: characterization and impact on liver disease progression

Background

Hepatitis B virus (HBV) is one of the most common human pathogens that cause aggressive hepatitis and advanced liver disease (AdLD), including liver cirrhosis and Hepatocellular Carcinoma. The persistence of active HBV replication and liver damage after the loss of hepatitis B e antigen (HBeAg) has been frequently associated with mutations in the pre-core (pre-C) and core promoter (CP) regions of HBV genome that abolish or reduce HBeAg expression. The purpose of this study was to assess the prevalence of pre-C and CP mutations and their impact on the subsequent course of liver disease in Morocco.

Methods/Principal Findings

A cohort of 186 patients with HBeAg-negative chronic HBV infection was studied (81 inactive carriers, 69 with active chronic hepatitis, 36 with AdLD). Pre-C and CP mutations were analyzed by PCR-direct sequencing method. The pre-C stop codon G1896A mutation was the most frequent (83.9%) and was associated with a lower risk of AdLD development (OR, 0.4; 95% CI, 0.15–1.04; p = 0.04). HBV-DNA levels in patients with G1896A were not significantly different from the other patients carrying wild-type strains (p = 0.84). CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were associated with higher HBV-DNA level and increased liver disease severity. Multiple logistic regression analysis showed that older age (≥40 years), male sex, high viral load (>4.3 log₁₀ IU/mL) and CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were independent risk factors for AdLD development. Combination of these mutations was significantly associated with AdLD (OR, 7.52; 95% CI, 4.8–8; p<0.0001).

Conclusions

This study shows for the first time the association of HBV viral load and CP mutations with the severity of liver disease in Moroccan HBV chronic carriers. The examination of CP mutations alone or in combination could be helpful for prediction of the clinical outcome.     

The Influence of Hepatitis B Viral Load and Pre-S Deletion Mutations on Post-Operative Recurrence of Hepatocellular Carcinoma and the Tertiary Preventive Effects by Anti-Viral Therapy

Background

Whether or not hepatitis B virus (HBV) genotypes, mutations, and viral loads determine outcomes for patients with HBV-induced hepatocellular carcinoma (HCC) remains controversial.

Aims

To study the influence of HBV viral factors on prognoses for patients with HBV-induced HCC after resection surgery and investigate if antiviral therapy could counteract the adverse effects of viral factors.

Methods

A total of 333 HBV-related HCC patients who underwent tumor resection were enrolled retrospectively. Serum HBV DNA levels, mutations, anti-viral therapy, and other clinical variables were analyzed for their association with post-operative recurrence.

Results

After a median follow-up of 45.9 months, 208 patients had HCC recurrence after resection. The 5-year overall survival and recurrence-free survival rates were 55.4% and 35.3%, respectively. Multivariate analysis showed indocyanine green retention rate at 15 minutes >10%, gamma-glutamyltransferase (GGT) level >60 U/L, macroscopic and microscopic venous invasion, and the absence of anti-viral therapy were significant risk factors for recurrence. Anti-viral therapy could decrease recurrence in patients with early stage HCC, but the effect was less apparent in those with the Barcelona-Clinic Liver Cancer stage C HCC. For patients without antiviral therapy after resection, serum HBV DNA levels >10⁶ copies/mL, GGT >60 U/L, and macroscopic and microscopic venous invasion were significant risk factors predicting recurrence. Among the 216 patients without anti-viral therapy but with complete HBV surface gene mapping data, 73 were with pre-S deletion mutants. Among patients with higher serum HBV DNA levels, those with pre-S deletion had significantly higher rates of recurrence. Moreover, multivariate analysis showed multi-nodularity, macroscopic venous invasion, cirrhosis, advanced tumor cell differentiation, and pre-S deletion were significant risk factors predictive of recurrence.

Conclusions

Ongoing HBV viral replication and pre-S deletion are crucial for determining post-operative tumor recurrence. Anti-viral therapy can help reduce recurrence and improve prognosis, especially for those with early stage HCC.     

Novel Point and Combo-Mutations in the Genome of Hepatitis B Virus-Genotype D: Characterization and Impact on Liver Disease Progression to Hepatocellular Carcinoma

Background

The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.

Methods

The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.

Results

Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.

Conclusions

Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.     

Genome replication,virion secretion,and e antigen expression of naturally occurring hepatitis B virus core promoter mutants

The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg(+) individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 x 10(9) to 5.7 x 10(9) copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 x 10(7) to 4.6 x 10(7) copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.     

乙型肝炎病毒变异与肝细胞癌发生关系

乙型肝炎病毒（hepatitisBvirus,HBV）基因组复制时,以病毒前基因组RNA作为模板合成子代病毒DNA,催化该过程的逆转录酶缺乏校对功能,所以HBV易出现变异。近年来,各国学者通过比较肝细胞癌（hepatocellular carcinoma,HCC）患者和非HCC患者的HBV基因序列,发现HBV基本核心启动子区的A1762T／G1764A变异或T1753V变异、增强子Ⅰ区的G1053A或G1229A变异、前S蛋白的F141L变异、前s2区基因缺失变异和x基因的截短变异,分别是HCC的易患因素,而前c区常见的G1896A变异,与HCC的发生无关。增强子Ⅱ区的C1653T变异在c基因HBV感染中可能与发生HCC有关,而在A基因型可能无关。     

Evolutionary Changes of Hepatitis B Virus Pre-S Mutations Prior to Development of Hepatocellular Carcinoma

Background and Aims

Deletions/mutations in the hepatitis B virus (HBV) pre-S region have been associated with hepatocellular carcinoma (HCC). We aimed to study the evolutionary changes of pre-S mutations prior to HCC development.

Methods

We studied the HBV pre-S sequences at 1 to 10 years preceding diagnosis of HCC in 74 patients with HBV-related HCC (HCC group). 148 chronic hepatitis B patients matched for sex and age in 2:1 ratio, who had been followed up for at least 3 years without HCC (HCC-free group) were recruited as controls. 56 and 47 patients of HCC and HCC-free groups respectively had serially stored sera for longitudinally examination at 1–3 years, 4–6 years, 7–9 years and ≥10 years prior to the recruitment of the study.

Results

Compared to the HCC-free group, higher frequencies of pre-S deletions and point mutations (at 11 codons) were observed in the HCC group (p<0.05). Multiple logistic regression analysis showed that pre-S deletions, point mutations at codon 51 and 167 were independent factors associated with HCC. Longitudinal observation showed that pre-S deletions and most of the 11 HCC-associated pre-S point mutations existed at least 10 years before HCC development, and were more prevalent preceding HCC development in patients from HCC groups than HCC-free group. The number of HCC-associated pre-S point mutations increased over time preceding HCC development, and correlated positively with the time to HCC diagnosis (r = 0.220, p = 0.005).

Conclusions

High prevalence and cumulative evolution of pre-S mutations preceding HCC development suggested a possible carcinogenic role of pre-S mutations and their potential application in HCC risk prediction.     

A1762T/G1764A mutations of hepatitis B virus, associated with the increased risk of hepatocellular carcinoma, reduce basal core promoter activities

Hepatitis B virus (HBV) infection is a major global health problem that causes over one million deaths annually. A1762T and G1764A mutations in the basal core promoter are often present in HBV patients but seldom in asymptomatic carriers, and are highly correlated with the increased risk of HBV-associated hepatocellular carcinoma (HCC). In this study, for the first time, we show that the basal core promoter activity of HBV strains isolated from asymptomatic carriers is decreased when 1762A is mutated to 1762T or 1764G is mutated to 1764A by site directed mutagenesis. By contrast, the promoter activity of HBV strains isolated from HCC patients is increased when 1762T and 1764A are reversely mutated into 1762A and 1764G, respectively. 1764G contributes more promoter activity than 1762T. We also show that T1762A and G1764A double mutations synergize the reduction of the promoter activity. A mechanism of HBV evasion from host immunoresponse that may facilitate disease development is also discussed.     

Associations of pri-miR-34b/c and pre-miR-196a2 Polymorphisms and Their Multiplicative Interactions with Hepatitis B Virus Mutations with Hepatocellular Carcinoma Risk

Background

Genetic polymorphisms of pri-miR-34b/c and pre-miR-196a2 have been reported to be associated with the susceptibility to cancers. However, the effect of these polymorphisms and their interactions with hepatitis B virus (HBV) mutations on the development of hepatocellular carcinoma (HCC) remains largely unknown. We hypothesized that these polymorphisms might interact with the HBV mutations and play a role in hepatocarcinogenesis.

Methods

Pri-miR-34b/c rs4938723 (T>C) and pre-miR-196a2 rs11614913 (T>C) were genotyped in 3,325 subjects including 1,021 HBV-HCC patients using quantitative PCR. HBV mutations were determined by direct sequencing. Contributions of the polymorphisms and their multiplicative interactions with gender or HCC-related HBV mutations to HCC risk were assessed using multivariate regression analyses.

Results

rs4938723 CC genotype was significantly associated with HCC risk compared to HBV natural clearance subjects, adjusted for age and gender (adjusted odds ratio [AOR] = 2.01, 95% confidence interval [CI] = 1.16–3.49). rs4938723 variant genotypes in dominant model significantly increased HCC risk in women, compared to female healthy controls (AOR = 1.85, 95% CI = 1.20–2.84) or female HCC-free subjects (AOR = 1.62, 95% CI = 1.14–2.31). rs4938723 CC genotype and rs11614913 TC genotype were significantly associated with increased frequencies of the HCC-related HBV mutations T1674C/G and G1896A, respectively. rs11614913 was not significantly associated with HCC risk, but its CC genotype significantly enhanced the effect of rs4938723 in women. In multivariate regression analyses, rs4938723 in dominant model increased HCC risk (AOR = 1.62, 95% CI = 1.05–2.49), whereas its multiplicative interaction with C1730G, a HBV mutation inversely associated with HCC risk, reduced HCC risk (AOR = 0.34, 95% CI = 0.15–0.81); rs11614913 strengthened the G1896A effect but attenuated the A3120G/T effect on HCC risk.

Conclusions

rs4938723 might be a genetic risk factor of HCC but its effect on HCC is significantly affected by the HBV mutations. rs11614913 might not be a HCC susceptible factor but it might affect the effects of the HBV mutations or rs4938723 on HCC risk.     

Induction of Bcl-2 expression by hepatitis B virus pre-S2 mutant large surface protein resistance to 5-fluorouracil treatment in Huh-7 cells

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear.

Methodology/Principal Findings

Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p = 0.05) and protein (3.2±0.3-fold vs. vehicle, p = 0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo.

Conclusion/Significance

Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor.     

Association of Single Nucleotide Polymorphisms in VDR and DBP Genes with HBV-Related Hepatocellular Carcinoma Risk in a Chinese Population

Background

Polymorphisms of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (DBP) have been widely investigated because of the complex role played by vitamin D in cancer tumorogenesis. In this study, we investigated the association between VDR and DBP gene polymorphisms and HBV-related HCC risk in a Chinese population.

Methods

Study subjects were divided into three groups: 184 HBV patients with HCC, 296 HBV patients without HCC, and 180 healthy controls. The VDR rs2228570, and rs3782905 and the DBP rs7041 polymorphisms were genotyped using PCR-RFLP and the VDR rs11568820 polymorphism was genotyped by PCR-SSP, respectively. DNA sequencing was performed to validate the genotype results.

Results

We found that there were significant differences in the genotype and allele frequencies of the VDR rs2228570 and DBP rs7041 polymorphisms between HBV patients with HCC and healthy controls. The rs2228570 T allele was associated with a significant increased HBV-related HCC risk as compared with the C allele. The rs2228570 TT and TT/TC genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type CC homozygote. Similarly, the rs7041 G allele was associated with a significant increased HBV-related HCC risk as compared with the T allele. The rs7041 GG and GG/TG genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type TT homozygote. However, we did not observe any significant effect of VDR rs11568820, and rs3782905 polymorphisms on HBV-related HCC risk in this population. In haplotype analysis, we also did not find any significant differences in haplotype frequencies of the VDR gene between HBV patients with HCC and the healthy controls.

Conclusions

We conclude that the VDR rs2228570 and DBP rs7041 polymorphisms may contribute to increased susceptibility to HBV-related HCC in the Chinese population. Due to the marginal significance, further large and well-designed studies in diverse ethnic populations are needed to confirm our results.     

Occult Hepatitis B Virus Infection in Anti-HBs-Positive Infants Born to HBsAg-Positive Mothers in China

Objective

To investigate the prevalence of occult HBV infection (OBI) among children and to characterize virology of occult HBV, we conducted an epidemiological survey.

Methods

186 HB-vaccinated infants born to HBsAg-positive mothers were included in the study. Serological tests for HBV markers were performed using commercial ELISA kits. Real-time quantitative PCR and nested PCR were used to detect HBV DNA. PCR products of the C and pre-S/S regions were sequenced and analyzed.

Results

1.61% (3/186) infants were HBsAg positive, and 4.92% (9/183) infants were considered as occult infection. The viral load of mothers was associated with occult infection (P = 0.020). Incomplete three-dose injections of HB vaccine was associated with HBV infection (P = 0.022). Six OBI infants were positive for anti-HBs, but their titers were not greater than 100 mIU/mL. Seven isolated HBV pre-S/S sequences were obtained from nine OBI infants. Three of the sequences were genotype C, and four of the sequences were genotype C/D. Escape mutation S143L was found in the four sequences of genotype C/D. All seven sequences lacked G145R and other escape mutation in S region.

Conclusions

Occult HBV infection was detected in anti-HBs positive infants born to HBsAg-positive mothers in China. Occult infection was associated with absent anti-HBs or with low anti-HBs level, high maternal viral loads and escape mutations in the S gene.     

Association Analysis between SNPs in IL-28B Gene and the Progress of Hepatitis B Infection in Han Chinese

Objective

As a candidate gene association study, we investigated the genetic association of SNPs in IL-28B genes with different outcomes of HBV infection, including LC and HCC occurrence.

Methods

Chinese Han subjects were categorized into two groups: 406 LC caused by CHB and 406 HCC caused by CHB. Genomic DNA was isolated from whole blood samples, SNPs were detected using high resolution melting curve (HRM) method. PCR amplification was carried out under the same conditions in a 96-well plate in Real-Time PCR System. Then 341 LC and 356 HCC patients caused by HBV infection were analyzed as a verification by independent sample. 393 CHB patients and 244 health subjects were included as control.

Results

CHB patients who progress to LC or HCC showed a significant different frequency in rs12979860 (p = 0.046). Patients with HCC carried more frequently the T alleles in rs12979860 comparison to LC. Same results were found in the independent sample.

Conclusion

IL-28B rs12979860 C/T polymorphism T allele appears to be more prevalent in patients with HCC than in LC. Carriage of this allele seems to enhance the risk for developing HCC. Gene polymorphism of IL-28B may confer symptomatic specificity in progress and extent of hepatitis B infection.     

Predictability of Liver-Related Seromarkers for the Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Patients

Background

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is a major global health problem. A few risk calculators have been developed using mainly HBV seromarkers as predictors. However, serum HBV DNA level, HBV genotype, and mutants are not routinely checked in regular health examinations. This study aimed to assess the predictability of HCC risk in chronic hepatitis B patients, using a combination of liver-related seromarkers combined with or without HBV seromarkers.

Methods

A prospective cohort of 1,822 anti-HCV-seronegative chronic HBV carriers was included in this study. Liver-related seromarkers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alpha-fetoprotein (AFP), gamma-glutamyltransferase (GGT), total bilirubin, total protein, albumin, serum globulins, apolipoprotein A1, and apolipoprotein B were examined. Hazard ratios of HCC with 95% confidence intervals were estimated using Cox proportional hazards regression models. Regression coefficients of seromarkers significantly associated with HCC risk in multivariate analyses were used to create integer risk scores. The predictability of various risk models were assessed by area under receiver operating characteristic curves (AUROCs).

Results

During a median follow-up of 5.9 years, 48 newly-developed HCC cases were ascertained. Elevated serum levels of ALT (≥28 U/L), AFP (≥5 ng/mL), and GGT (≥41 U/L), an increased AST/ALT ratio (AAR, ≥1), and lowered serum levels of albumin (≤4.1 g/dL) and alpha-1 globulin (≤0.2 g/dL) were significantly associated with an increased HCC risk (P<0.05) in multivariate analysis. The risk model incorporating age, gender, AAR, and serum levels of ALT, AFP, GGT, albumin, and alpha-1 globulin had an AUROC of 0.89 for predicting 6-year HCC incidence. The AUROC was 0.91 after the addition of HBV seromarkers into the model, and 0.83 for the model without liver-related seromarkers, with the exception of ALT.

Conclusion

Liver-related seromarkers may be combined into useful risk models for predicting HBV-related HCC risk.     

The Characteristic Changes in Hepatitis B Virus X Region for Hepatocellular Carcinoma: A Comprehensive Analysis Based on Global Data

Objectives

Mutations in hepatitis B virus (HBV) X region (HBx) play important roles in hepatocarcinogenesis while the results remain controversial. We sought to clarify potential hepatocellular carcinoma (HCC)-characteristic mutations in HBx from HBV genotype C-infected patients and the distribution of those mutations in different disease phases and genotypes.

Methods

HBx sequences downloaded from an online global HBV database were screened and then classified into Non-HCC or HCC group by diagnosis information. Patients'' data of patient age, gender, country or area, and viral genotype were also extracted. Logistic regression was performed to evaluate the effects of mutations on HCC risk.

Results

1) Full length HBx sequences (HCC: 161; Non-HCC: 954) originated from 1115 human sera across 29 countries/areas were extracted from the downloaded 5956 HBx sequences. Genotype C occupied 40.6% of Non-HCC (387/954) and 89.4% of HCC (144/161). 2) Sixteen nucleotide positions showed significantly different distributions between genotype C HCC and Non-HCC groups. 3) Logistic regression showed that mutations A1383C (OR: 2.32, 95% CI: 1.34-4.01), R1479C/T (OR: 1.96, 95% CI: 1.05-3.64; OR: 5.15, 95% CI: 2.53-10.48), C1485T (OR: 2.40, 95% CI: 1.41-4.08), C1631T (OR: 4.09, 95% CI: 1.41-11.85), C1653T (OR: 2.58, 95% CI: 1.59-4.19), G1719T (OR: 2.11, 95% CI: 1.19-3.73), and T1800C (OR: 23.59, 95% CI: 2.25-247.65) were independent risk factors for genotype C HBV-related HCC, presenting different trends among individual disease phases. 4) Several genotype C HCC risk mutations pre-existed, even as major types, in early disease phases with other genotypes.

Conclusions

Mutations associated with HCC risk were mainly located in HBx transactivation domain, viral promoter, protein/miRNA binding sites, and the area for immune epitopes. Furthermore, the signatures of these mutations were unique to disease phases leading to HCC, suggesting molecular counteractions between the virus and host during hepatocarcinogenesis.     

Distinct Distribution Pattern of Hepatitis B Virus Genotype C and D in Liver Tissue and Serum of Dual Genotype Infected Liver Cirrhosis and Hepatocellular Carcinoma Patients

Aims

The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.

Methods

The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.

Results

HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.

Conclusions

Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.     

ABO blood group and the risk of hepatocellular carcinoma: a case-control study in patients with chronic hepatitis B

Background

Studies have observed an association between the ABO blood group and risk of certain malignancies. However, no studies of the association with hepatocellular carcinoma (HCC) risk are available. We conducted this hospital-based case-control study to examine the association with HCC in patients with chronic hepatitis B (CHB).

Methods

From January 2004 to December 2008, a total of 6275 consecutive eligible patients with chronic hepatitis B virus (HBV) infection were recruited. 1105 of them were patients with HBV-related HCC and 5,170 patients were CHB without HCC. Multivariate logistic regression models were used to investigate the association between the ABO blood group and HCC risk.

Results

Compared with subjects with blood type O, the adjusted odds ratio (AOR) for the association of those with blood type A and HCC risk was 1.39 [95% confidence interval (CI), 1.05–1.83] after adjusting for age, sex, type 2 diabetes, cirrhosis, hepatitis B e antigen, and HBV DNA. The associations were only statistically significant [AOR (95%CI) = 1.56(1.14–2.13)] for men, for being hepatitis B e antigen positive [AOR (95%CI) = 4.92(2.83–8.57)], for those with cirrhosis [AOR (95%CI), 1.57(1.12–2.20)], and for those with HBV DNA≤10⁵copies/mL [AOR (95%CI), 1.58(1.04–2.42)]. Stratified analysis by sex indicated that compared with those with blood type O, those with blood type B also had a significantly high risk of HCC among men, whereas, those with blood type AB or B had a low risk of HCC among women.

Conclusions

The ABO blood type was associated with the risk of HCC in Chinese patients with CHB. The association was gender-related.     

A genetic variant in the promoter region of miR-106b-25 cluster and risk of HBV infection and hepatocellular carcinoma

Background

MiR-106b-25 cluster, hosted in intron 13 of MCM7, may play integral roles in diverse processes including immune response and tumorigenesis. A single nucleotide polymorphism (SNP), rs999885, is located in the promoter region of MCM7.

Methods

We performed a case-control study including 1300 HBV-positive hepatocellular carcinoma (HCC) cases, 1344 HBV persistent carriers and 1344 subjects with HBV natural clearance to test the association between rs999885 and the risk of HBV persistent infection and HCC. We also investigated the genotype-expression correlation between rs999885 and miR-106b-25 cluster in 25 pairs of HCC and adjacent non-tumor liver tissues.

Results

Compared with the HBV natural clearance subjects carrying rs999885 AA genotype, those with AG/GG genotypes had a decreased risk of chronic HBV infection with an adjusted odds ratio (OR) of 0.79 [95% confidence intervals (CIs) = 0.67–0.93]. However, the AG/GG genotypes were significantly associated with an increased HCC risk in HBV persistent carriers (adjusted OR = 1.25, 95% CIs = 1.06–1.47). Expression analysis revealed that the expression level of miR-106b-25 cluster was significantly higher in AG/GG carriers than those in AA carriers in non-tumor liver tissues.

Conclusions

These findings indicate that the A to G base change of rs999885 may provide a protective effect against chronic HBV infection but an increased risk for HCC in HBV persistent carriers by altering the expression of the miR-106b-25 cluster.