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Background and Aims: Occult HBV infection (OBI) in children has proven to be associated with their immune response to hepatitis B vaccine (HepB). This study aimed to investigate the effect of a booster HepB on OBI, which is rarely investigated., Methods: This study enrolled 236 maternal HBsAg-positive children who were followed up annually until 8 years of age and were hepatitis B surface antigen (HBsAg) negative. Of those 100 received a booster HepB between 1 and 3 years of age (booster group), and 136 were never boosted (non-booster group). Serial follow-up data of children and baseline data of their mothers were collected and between-group differences were analyzed., Results: The incidence of OBI varied dynamically during follow-up, with 37.14% (78/210), 19.09% (42/220), 20.85% (44/211), 31.61% (61/193), 8.65% (18/208) and 12.71% (30/236) at 7 months, 1, 2, 3, 4, and 8 years of age. At 8 years of age, the negative conversion rate of HBV DNA in the booster group was significantly higher than that in non-booster group [57.89% (11/19) vs. 30.51% (18/59), p =0.032]. For children without OBI at 7 months old, the incidence of OBI in booster group was significantly lower than that in non-booster group [25.64% (10/39) vs. 67.74% (63/93), p <0.001]., Conclusions: The incidence of OBI in maternal HBsAg-positive children was high, serum HBV DNA in children with OBI was intermittently positive at low levels, and a booster HepB in infancy reduced the incidence of OBI in children with HBsAg-positive mothers., Competing Interests: The authors have no conflict of interests related to this publication., (© 2023 Authors.)

Background: The long-term protective effect of hepatitis B vaccine (HepB), the incidence of hepatitis B virus (HBV) vaccine breakthrough infections (VBIs), and whether a booster HepB is necessary remain to be clarified in children born to mothers with chronic HBV infection., Methods: Based on a long-term follow-up prospective cohort of 1177 hepatitis B surface antigen (HBsAg)-positive mothers and their paired infants which was established from 2009 to 2011, total 454 children with immunoprophylaxis success as determined by postvaccination serologic testing (PVST) at 7 months old were included in this study. Among the 454 children, 246 never had a booster HepB, and 208 children received a booster HepB from 1 to 5 years of age. Multivariate logistic regression analysis was used to analyse the risk factors for HBV VBIs., Results: The hepatitis B surface antibody (anti-HBs) levels declined sharply from 7 months to 2 years old, and the anti-HBs seronegative rate in the children increased significantly from 2 years old. A total of 31 (6.83%) of the 454 children experienced VBIs, of which 7 had overt and 7 had occult HBV infections. Notably, 14 (45.16%) of the 31 children with VBIs were diagnosed at 2 years old, and all of them had anti-HBs positivity (> 10 mIU/mL) at 1 year old. Maternal hepatitis B e antigen (HBeAg) positivity, higher HBV DNA and HBsAg levels, lower initial infant anti-HBs levels and not receiving a booster HepB were independent risk factors for VBIs. The incidence of VBIs was significantly lower in children with a booster HepB than in nonboosted children (0.50 vs. 11.90%, P < 0.001), and none of the boosted children developed overt or occult HBV infection. The anti-HBs levels of 76.67% for the children with VBIs in the nonboosted group indicated positivity before VBIs was detected., Conclusions: After the primary full immunization with HepB, children born to mothers with chronic HBV infection, especially the children with maternal HBeAg positivity, high HBV DNA levels, high HBsAg levels and/or low initial infant anti-HBs levels, were at a high risk of VBIs, and a booster HepB for these children before 2 years old, instead of when their anti-HBs level is < 10 mIU/mL, could reduce the incidence of VBIs., (© 2022. The Author(s).)

As a high-risk factor of perinatal HBV transmission, the potential role of maternal hepatitis B e antigen (HBeAg) to guide antiviral prophylaxis has not yet been fully reported. This large prospective cohort study enrolled 1177 hepatitis B surface antigen (HBsAg)-positive pregnant women without antiviral treatment and their newborns. HBeAg, HBsAg, and viral load in maternal serum collected before delivery were measured. All the newborns were given standard passive-active immunoprophylaxis within 12 h after birth, and post-vaccination serologic testing was performed at 7 (±7d) months of age. The results revealed that 20 of the 1177 infants (1.70%) were immunoprophylaxis failure, and all their mothers were HBeAg positive. Maternal quantitative HBeAg was positively correlated with viral load ( r  = 0.83; P  < .0001) and quantitative HBsAg ( r  = 0.68; P  < .0001). The area under the receiver operating characteristic curve (AUC) for predicting immunoprophylaxis failure by maternal HBeAg was comparable to that by maternal viral load (0.871 vs 0.893; P  = .441) and HBsAg (0.871 vs 0.871; P  = .965). The optimal cutoff value of maternal quantitative HBeAg to predict perinatal infection was 2.21 log 10 PEI U/mL, and the sensitivity and specificity was 100.0% and 74.5%, respectively. According to maternal viral load >2 × 10 5 IU/mL, the sensitivity and specificity of maternal qualitative HBeAg to identify the risk of HBV MTCT for pregnant women and determine the necessity for antiviral prophylaxis was 95.5% and 92.6%, respectively. This study showed that maternal HBeAg can be a surrogate marker of HBV DNA for monitoring and evaluating whether antiviral prophylaxis is necessary for preventing perinatal HBV transmission.

Background: Previous works have observed that younger infants with chronic hepatitis B virus (HBV) infection are more responsive to antiviral treatment. However, the underlying mechanism remains unclear. In this study, the dynamic changes of HBV quasispecies in infants with immunoprophylaxis failure were investigated to provide virological explanations for clinical management on infantile antiviral therapy., Methods: Thirteen 7-month-old infants with immunoprophylaxis failure and their mothers were enrolled from a prospective cohort, and 8 of them were followed up to 3 years old. The sequences of HBV quasispecies were analyzed by the full-length genome clone-based sequencing, and compared among mothers and their infants at different ages., Results: The results revealed that the complexity, mutation frequency and genetic distance of HBV quasispecies decreased significantly at full-length, partial open reading frames and regulatory regions of HBV genome at nucleotide level in 7-month-old infants comparing with their mothers, whereas increased significantly to near the maternal level when infants grew up to 3 years old. Furthermore, similar changes were also found in Core, PreS2, RT and P regions of HBV genome at amino acid level, especially for potential NAs-resistant mutants in RT region and immune-escape mutants in Core and PreS2 regions., Conclusions: This study uncovered the evolution of HBV quasispecies in infancy after mother-to-child transmission, which may provide the virological evidence for explaning that younger children are more responsive to antiviral therapy., (© 2021. The Author(s).)

Occult hepatitis B virus (HBV) infection (OBI) has been observed among infants born to hepatitis B surface antigen (HBsAg)-positive mothers despite successful immunoprophylaxis. This study enrolled 549 infants [349 infants received a 10μg/dose of hepatitis B vaccine (HepB), and 200 infants received 20μg/dose HepB] born to HBsAg-positive mothers with HBV DNA load >6log 10 IU/mL. The anti-HBs levels in the 10μg group were significantly lower than that in the 20μg group both at 7 [652.48 (564.05-754.82) vs. 1541.72 (1268.69-1873.51) mIU/mL, P <0.001] and 12 months old [257.44 (220.29-300.88) vs. 1073.41 (839.27-1372.78) mIU/mL, P <0.001]. The OBI incidence in the 10μg group was significantly higher than that in the 20μg group at both 7 [21.55% (25/116) vs. 7.56% (9/119), P =0.002] and 12 months old [17.07% (14/82) vs. 6.90% (6/87), P =0.041]. OBI incidence in infants with anti-HBs levels <100mIU/mL was higher than that of those with anti-HBs ≥100mIU/mL [35.71% (5/14) vs. 13.12% (29/221), P =0.036]. This study showed that increasing the immunisation dose from 10μg to 20μg significantly improved anti-HBs levels and decreased OBI incidence in infants with a high maternal viral load. We recommend 20μg HepB to treat this high-risk population.

Nowadays most of the hepatitis B virus (HBV) infected population are adults, among which hepatitis B e antigen (HBeAg) negative infection occupied the largest proportion of HBV infection in China. HBeAg-negative patients are heterogeneous, and the corresponding interventions are different. Therefore, it is worth researching the infection characteristics of HBeAg-negative patients to help guide the interventions.A total of 11,738 treatment-naïve HBeAg-negative adult patients were randomly selected, and their demographic and medical history information were collected. The liver biochemistry, and HBV infection biomarkers including hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), HBeAg, hepatitis B e antibody (anti-HBe), hepatitis B core antibody (anti-HBc), and hepatitis B virus deoxyribonucleic acid (HBV-DNA) levels were tested. The infection characteristics and their influencing factors were explored.Sixty percent of the patients presented HBV-DNA-positive, of which 31.2% had HBV-DNA level higher than 2000 IU/mL, and 16.5% had HBV-DNA level higher than 20,000 IU/mL. HBV-DNA levels tended to increase along with the increasing of age, and the male patients had significant higher HBV-DNA levels than the female patients. Twenty-four percent of the patients had abnormal transaminase. The male patients were more vulnerable to abnormal transaminase (30.0%) than the female patients (18.4%). Fifty-five percent patients with HBV-DNA ≥20,000 IU/mL presented abnormal alanine aminotransferase (ALT) or aspartate transaminase (AST), which was significantly higher than that of patients with HBV-DNA levels below 20,000 IU/mL (19.0-21.7%). Multivariate logistic regression analyses revealed that the male patients and the patients with higher viral load had higher risk of having abnormal liver function.A considerable number of HBeAg-negative patients were virological active and had liver damage. It is necessary and urgent to carry out regular active interventions for the chronic HBV-infected patients.

Importance: Hepatitis B virus (HBV) has been identified as a major risk factor for hepatocellular carcinoma. However, the associations between HBV infection and other cancer types are not well understood., Objective: To assess the associations between chronic HBV infection and risk of all cancer types., Design, Setting, and Participants: This population-based study involved 3 cohorts in China. The China Kadoorie Biobank (CKB) prospective cohort study, conducted between June 2004 and July 2008, used a dipstick assay for detection of serum hepatitis B surface antigen (HBsAg) among 496 732 participants to determine the association between HBV infection and risk of all cancer types. Two cohort studies were used to validate the associations by applying more precise serum HBsAg detection assays: the Qidong cohort (37 336 participants enrolled from November 2007 to April 2011) and the Changzhou nested case-control study (17 723 participants enrolled from June 2004 to September 2005). A total of 97 samples of stomach cancer tissues, 10 samples of pancreatic cancer tissues, and 9 samples of lung cancer tissues were included to assess the presence of HBV replication and expression. Statistical analysis was performed from December 2016 to October 2018., Exposures: Serum HBsAg status in the population-based stage and HBV DNA status, the expression of hepatitis B X protein, and hepatitis B core antibody (anti-HBc) in the tissue-based stage., Main Outcomes and Measures: Incidence of all cancer types during follow-up., Results: In the CKB cohort, the mean (SD) age of the 496 732 participants was 51.5 (10.7) years; 59.0% of the participants were women. After 4.4 million person-years of follow-up, participants who were HBsAg seropositive (n = 15 355) had a higher risk of hepatocellular carcinoma (hazard ratio [HR], 15.77; 95% CI, 14.15-17.57), stomach cancer (HR, 1.41; 95% CI, 1.11-1.80), colorectal cancer (HR, 1.42; 95% CI, 1.12-1.81), oral cancer (HR, 1.58; 95% CI, 1.01-2.49), pancreatic cancer (HR, 1.65; 95% CI, 1.03-2.65), and lymphoma (HR, 2.10; 95% CI, 1.34-3.31) when compared with participants who were HBsAg seronegative (n = 481 377). Because of the limitation of sample size, only associations of HBV infection with hepatocellular carcinoma and stomach cancer were validated in the Qidong cohort (hepatocellular carcinoma: HR, 17.51; 95% CI, 13.86-22.11; stomach cancer: HR, 2.02; 95% CI, 1.24-3.29); the Changzhou nested case-control study validated only an association between HBV infection and stomach cancer (odds ratio, 1.76; 95% CI, 1.04-2.98). Moreover, among 22 participants with stomach cancer from the Qidong cohort who were anti-HBc seropositive, 12 samples (54.5%) of cancer tissues were HBV DNA positive, while among 25 participants with stomach cancer who were anti-HBc seronegative, no HBV DNA was detected. The same negative and positive rate was observed in the validation set from Zhejiang Tumor Hospital (19 of 35 samples [54.3%] were HBV DNA positive). Moreover, among the 8 patients with stomach cancer from the Qidong cohort who were anti-HBc seropositive, anti-HBc and hepatitis B X protein were expressed in all of their stomach cancer tissue samples. The same phenomenon was observed in the patients with pancreatic cancer but not in the patients with lung cancer, which was consistent with the population-based results of the CKB cohort., Conclusions and Relevance: This study found that HBV infection was also associated with the risk of nonliver cancer, especially digestive system cancers among adults in China.

Background & Aims: Although non-alcoholic fatty liver disease (NAFLD) has been studied extensively, the potential risk factors for NAFLD among chronic hepatitis B (CHB) patients have not been fully known., Methods: A population-based cohort of adult CHB patients without a history of alcohol drinking or NAFLD were recruited and followed up from October 2012 to January 2015 in Jiangsu province, China. Using Cox proportional hazards regression model, potential risk factors including viral and metabolic factors for NAFLD were evaluated., Results: Two thousand three hundred and ninety-three adult CHB patients (mean age 50.7 ± 13.2 years) were included in the cohort. With 4429 person-years of follow-up, 283 individuals progressed to NAFLD with an incidence rate of 63.89/1000 person-years. Overweight and obese CHB patients had an increased risk of NAFLD (overweight adjusted hazard ratio [HR], 3.10; 95% CI, 2.29-4.18; obese HR, 8.52; 95%CI, 5.93-12.25) compared to normal weight carriers. The incidence of NAFLD was associated with concurrent type 2 diabetes mellitus (DM) (HR, 1.88; 95%CI, 1.15-3.08). However, no associations between viral factors with NAFLD incidence rate were identified. In a subgroup of participants with concurrent type 2 DM, detectable HBV DNA levels were negatively associated with the development of NAFLD (HR, 0.37; 95%CI, 0.14-0.98). There was super-multiplicative interaction between BMI and gender with respect to incidence of NAFLD, with an ROR of 2.08 (95%CI, 1.02-4.23)., Conclusion: Metabolic factors play an important role in the presence of NAFLD among Chinese CHB patients. However, viral replication factors are not related to NAFLD except among those with concurrent type 2 DM., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Seroclearance of hepatitis B surface antigen (HBsAg) has been widely studied; however, seroconversion of HBsAg and characteristics of viral load among hepatitis B e antigen (HBeAg)-negative chronic infection patients after HBsAg lost is not clear. We performed a large-scale study in a HBeAg-negative chronic infection cohort to evaluate spontaneous HBsAg seroclearance incidence from October 2012 to April 2017 in Jiangsu province, China. We also elucidated the characteristics of HBsAg seroconversion and hepatitis B virus (HBV) DNA detectability among patients who cleared HBsAg. A total of 2997 HBeAg-negative chronic infection patients (mean age 52.3 ± 12.9 years at baseline) were included. With 10 519 person-years of follow-up, 348 patients successfully spontaneously cleared HBsAg, with an incidence rate of 3.31 per 100 person-years. Patients with HBV DNA detectable ~1999 IU/mL at baseline had a lower probability of HBsAg seroclearance relative to those with undetectable HBV DNA, with a hazard ratio of 0.31 (95% CI = 0.23, 0.41). HBsAg seroconversion occurred in 37.3% of those patients who cleared HBsAg. The geometric mean of anti-HBs among those with HBsAg conversion was 79.4 mIU/mL. Female had a higher HBsAg seroconversion rate (P = 0.011). Among those with HBsAg seroclearance, 11.2% still had HBV DNA levels of higher than 100 IU/mL. Patients with higher HBV DNA at baseline had a higher risk of detectable HBV DNA levels even after HBsAg seroclearance (P < 0.001). This study reveals HBsAg seroconversion rates and HBV DNA undetectability epidemiological characteristics of patients with HBsAg seroclearance and suggests that monitoring HBV DNA is needed when managing HBeAg-negative chronic patients, even after clearing HBsAg., (© 2018 John Wiley & Sons Ltd.)

Previously, we identified UBE2L3 as a susceptibility gene for chronic hepatitis B virus (HBV) infection through genome-wide association study. Here, we analysed the association between genetic variants of UBE2L3 and the susceptibility to HBV-related hepatocellular carcinoma (HCC) and further explored its role in HCC. This case-control study included 1344 subjects who cleared HBV, 1560 HBV carriers and 1057 HBV-related HCC patients. Two single nucleotide polymorphisms (SNPs) were genotyped, including rs2266959 and rs4821116. Logistic regression analysis was performed to compute the odds ratio (OR) and 95% confidence interval (CI). We further analysed the expression of UBE2L3 and its association with pathological features based on The Cancer Genome Atlas (TCGA) data and our tissue microarray. Proliferation and migration assays were performed in hepatoma cell lines with or without UBE2L3 knockdown. Further RNA-seq analysis was performed to explore the underlying oncogenic mechanism. The variant genotypes of rs4821116 in UBE2L3 were associated with decreased risk for HCC and chronic HBV infection. Moreover, based on both TCGA and our tissue microarray data, higher levels of UBE2L3 expression were correlated with higher tumour grade, advanced tumour stage and poor survival. In vitro analysis revealed that UBE2L3 may promote hepatocyte proliferation and migration. RNA-seq analysis showed that UBE2L3 was inversely correlated with CDKN2B, a negative regulator of cell cycle, and CLDN1, loss of which may promote cancer metastasis. In conclusion, UBE2L3 may also be a susceptibility gene in HBV-related HCC, and it may promote HCC proliferation and migration by negatively regulating CDKN2B and CLDN1., (© 2018 John Wiley & Sons Ltd.)

Individuals fail to elicit protective antibody after hepatitis B vaccination remain at risk for hepatitis B virus infection. Analysis of the transcriptome of peripheral blood mononuclear cells (PBMCs) is essential to elucidate the characteristics of gene expression in non-responders. In this study, we enrolled seven responders who had received three injections and seven non-responders who had six injections of hepatitis B vaccine before. All the participants were then vaccinated with a three-dose boost regimen. Microarray analysis and Luminex assay were applied to examine mRNA expression and Th1/Th2/Th9/Th17/Th22/Treg cytokine and chemokine profiles in non-responders and responders. Differentially expressed genes in PBMCs of non-responders at 5 time points, i.e. pre-vaccination, 3 rd , 7 th , 28 th day post the first dose vaccination and 7 th day post the second dose vaccination indicated a dense network trend. Compared with responders, nine coding genes (BPI, DEFA1B, DEFA4, CEACAM8, MMP8, FOLR3, LTF, TCN1 and TKTL1) were significantly up-regulated in non-responders at all 5 time points, which could probably be the characteristic genes in hepatitis B vaccine non-responsiveness. Gene ontology analysis revealed that most of the DEGs were related with immune responses. Validation results of these 9 genes using quantitative real-time polymerase chain reaction were mostly consistent with the results of microarray. Cytokine analysis demonstrated that IL-27 and CXCL12 concentrations in responders were significantly higher than non-responders on the 3 rd day after the first dose and 7 th day after the second dose of vaccination, respectively. No significant difference was observed in other cytokine and chemokine signatures between the two groups. In conclusion, our results revealed characteristic transcriptome and cytokine changes in hepatitis B vaccine non-responders after boost immunization.

Background: Observational studies show an association between telomere length and Hepatocellular carcinoma (HCC) risk, but the relationship is controversial. Particularly, it remains unclear whether the association is due to confounding or biases inherent in conventional epidemiological studies. Here, we applied Mendelian randomization approach to evaluate whether telomere length is causally associated with HCC risk., Methods: Individual-level data were from HBV-related HCC Genome-wide association studies (1,538 HBV positive HCC patients and 1,465 HBV positive controls). Genetic risk score, as proxy for actual measured telomere length, derived from nine telomere length-associated genetic variants was used to evaluate the effect of telomere length on HCC risk., Results: We observed a significant risk signal between genetically increased telomere length and HBV-related HCC risk (OR=2.09, 95% CI 1.32-3.31, P=0.002). Furthermore, a U-shaped curve was fitted by the restricted cubic spline curve, which indicated that either short or long telomere length would increase HCC risk (P=0.0022 for non-linearity test). Subgroup analysis did not reveal significant heterogeneity between different age, gender, smoking status and drinking status groups., Conclusions: Our results indicated that a genetic background that favors longer or shorter telomere length may increase HBV-related HCC risk-a U-shaped association., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Previously we identified that HBV(Hepatitis B virus) sequence variation, which may interact with host human leukocyte antigen (HLA) genetic variation, could influence host risk of hepatocellular carcinoma (HCC). More HBV-host interactions need to be identified. Protein tyrosine phosphatase nonreceptor type 12 (PTPN12), serves as an antagonist to tyrosine kinase signaling, may play integral roles in immune response against HBV infection and the development of HCC. Rs11485985 was an expression quantitative trait loci (eQTL) for PTPN12 by bioinformatics analyses. In this study, we genotyped the PTPN12 eQTL and sequenced the HBV region EnhII/BCP/PC in a case-control cohort including 1507 HBV-related HCC cases and 1560 HBV persistent carriers as controls. The variant genotype GG of rs11489585 increased HCC risk compared to the HBV persistent carriers (adjusted OR = 2.03, 95% confidence interval [CIs] = 1.30-3.18). We also detected borderline significant associations of PTPN12 eQTL rs11489585 with HBV mutations (P = 0.05 for G1799C). Taken together, PTPN12 may influence HCC risk accompanied by HBV mutations., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Several genome-wide association studies (GWAS) have demonstrated the association between genetic variants in the major histocompatibility complex (MHC) region and chronic hepatitis B (CHB) virus infection, but it is still unknown about the disease-causing loci and potential mechanisms owing to the complicated linkage disequilibrium for this region. To systematically characterize the MHC variations in relation to the CHB infection, we fine mapped the MHC region on our existing GWAS data with SNP2HLA taken the Pan-Asian panel as reference and finally identified four independent associations. The HLA-DPβ1 amino acid positions 84-87, which drove the effect of reported single nucleotide polymorphisms rs9277535 and rs3077, showed the most significant association (OR = 0.65, P = 2.03 × 10(-8)). The Leu-15 of HLA-C, conferring the effect of rs3130542, increased the risk of CHB infection independently (OR = 1.61, P = 3.42 × 10(-7)). The HLA-DRβ1*13, in perfect LD with glutamic at site 71, and rs400488, an expression quantitative trait locus for HLA-J, were newly identified to be associated with CHB infection independently (OR = 1.84, P = 3.84 × 10(-9); OR = 0.28, P = 6.27 × 10(-7), respectively). HLA-DPβ1 positions 84-87 and HLA-DRβ1 position 71 implicated the P1 and P4 in the antigen-binding groove, whereas HLA-C position 15 affected the signal peptide. These four independent loci together can explain ∼ 6% of the phenotypic variance for CHB infection, accounting for 72.94% of that explained by known genetic variations. We fine mapped the MHC region and identified four loci that independently drove the chronic HBV infection. The results provided a deeper understanding of the GWAS signals and identified additional susceptibility loci which were missed in previous association studies., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Tumor necrosis factor receptor superfamily member 10 (TNFRSF10) is a death domain-containing receptor for the apoptotic ligand TNFSF10, which involves multiple processes, including hepatocarcinogenesis and immune response against HBV infection. Several single nucleotide polymorphisms (SNPs) were identified as expression quantitative trait loci (eQTLs) for TNFRSF10. To assess the association of TNFRSF10 eQTL SNPs with the risk of hepatocellular carcinoma (HCC) and chronic HBV infection, we designed a case-control study that included 1,300 HBV-related HCC patients, 1,344 chronic HBV carriers, and 1,344 subjects with HBV natural clearance, and then genotyped two TNFRSF10 eQTL SNPs (rs79037040 and rs2055822). We found that rs79037040 GT/TT genotypes were associated with a decreased HCC risk (adjusted odds ratio [OR] = 0.83, 95% confidence intervals [CIs] = 0.71-0.97, P = 0.021) but an increased chronic HBV infection risk of borderline significance (adjusted OR = 1.14, 95%CIs = 0.98-1.33, P = 0.085). In contrast, the rs2055822 G allele was a risk factor for HCC (adjusted OR = 1.12, 95%CIs = 1.00-1.25, P = 0.041) but a protective factor for chronic HBV infection (adjusted OR = 0.89, 95%CIs = 0.80-0.99, P = 0.038). Furthermore, we observed a dose-dependent relationship between the number of alleles (rs79037040-T and rs2055822-A) and the risk of HCC and chronic HBV infection. In comparison with "0" alleles, having "1-4" alleles was significantly associated with decreased HCC risk and increased HBV infection risk. These findings suggest that eQTL SNPs for TNFRSF10 may be susceptibility markers for HCC and chronic HBV infection., (© 2015 Wiley Periodicals, Inc.)

Three genome-wide association studies (GWAS) have been conducted on the genetic susceptibility of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), two of which consistently identified tagging single nucleotide polymorphisms (SNPs) around HLA-DQ/DR. In contrast, large multi-centre association studies between HBV genotype, mutations and the risk of HCC are relatively rare, and their interactions with host variants are even less. We performed a multi-centre study of 1,507 HBV-related HCC cases and 1,560 HBV persistent carriers as controls to evaluate the effects of HBV genotype, mutations, GWAS-identified HLA-DQ/DR SNPs (rs9272105 and rs9275319) and their interactions on HCC risk. We found HBV genotype C was more frequent in HBV-related HCC. And 11 HBV hotspot mutations were independently and significantly associated with HCC risk. We also detected significant interactions of rs9272105 with both the HBV genotype and mutations. Through stepwise regression analysis, HBV genotype, the 11 mutations, HLA-DQ/DR SNPs, and the interaction of rs9272105 with mutation A1752G were all entered into the HCC prediction model, and the area under the curve for the panel including the HLA-DQ/DR SNPs, HBV genotype and mutations was 0.840. The HBV genotype, the mutations and the HLA-DQ/DR SNPs may serve as biomarkers for the surveillance of HBV persistent carriers.

Zinc ribbon domain containing 1 (ZNRD1), cloned from human leukocyte antigen (HLA) region, may play integral roles in diverse processes including immune response against HBV infection and hepatocarcinogenesis. ZNRD1-AS1 (ZNRD1 antisense RNA 1) may be an important regulator of ZNRD1. By bioinformatics analyses, we identified that several single nucleotide polymorphisms (SNPs) in ZNRD1-AS1 may be expression quantitative trait loci (eQTLs) for ZNRD1. In this study, we hypothesized that these eQTLs SNPs in ZNRD1-AS1 may influence both chronic HBV infection and hepatocellular carcinoma (HCC) development. We designed a case-control study of 1300 HBV-positive HCC patients, 1344 HBV persistent carriers and, 1344 HBV natural clearance subjects to test the associations of three ZNRD1 eQTLs SNPs (rs3757328, rs6940552 and, rs9261204) in ZNRD1-AS1 with the risk of both chronic HBV infection and HCC. Logistic regression analyses in additive genetic model showed that variant alleles of all the three SNPs increased host HCC risk, whereas variant allele of rs3757328 was associated with HBV clearance. Moreover, the haplotype containing variant alleles of the three SNPs was significantly associated with both HCC development (adjusted OR = 1.18, 95% CI = 1.01-1.38, P = 0.035) and HBV clearance (adjusted OR = 0.83, 95% CI = 0.71-0.96, P = 0.013), when compared with the most frequent haplotype. In vitro experiments showed that ZNRD1 knockdown inhibited the expression of HBV mRNA and promoted proliferation of HepG2.2.15 cells. These findings suggest that ZNRD1 regulatory SNPs may be susceptibility makers for risk of both chronic HBV infection and HCC., (© 2014 Wiley Periodicals, Inc.)

Caspase recruitment domain family, member 8 (CARD8) can coordinate innate and adaptive immune responses and sensitize cells to apoptosis, which may participate in tumorigenesis of virus-induced hepatocellular carcinoma (HCC) and cervical cancer. By bioinformatics analyses, we identified several single nucleotide polymorphisms (SNPs) within a new identified long non-coding RNA (lncRNA) as expression quantitative trait loci (eQTLs) for CARD8. In this study, we therefore hypothesized that CARD8 eQTLs SNPs within lncRNA may influence the risk of HCC and cervical cancer. We performed two independent case-control studies of 1,300 cases with HBV-positive HCC and 1,344 normal controls, together with 1,486 cervical cancer patients and 1,536 control subjects to test the association between eQTLs SNP (rs7248320) for CARD8 and the risk of HCC and cervical cancer. The variant genotype of rs7248320 was significantly associated with increased risk of HCC and cervical cancer [GG vs. AA/GA: adjusted odds ratio (OR) = 1.28, 95% confidence interval (CI) = 1.03-1.61, P = 0.028 for HCC; adjusted OR = 1.34, 95% CI = 1.09-1.66, P = 0.006 for cervical cancer]. Moreover, the effect of rs7248320 on cervical cancer risk was more prominent in premenopausal women. Further interactive analysis detected a significantly multiplicative interaction between rs7248320 and menopausal status on cervical cancer risk (P = 0.018). These findings suggest that CARD8 eQTLs SNP may serve as a susceptibility marker for virus-related HCC and cervical cancer.

Objective: To investigate the hepatitis C virus (HCV) infection rate among general population in Jiangsu province and to analyze the main risk factors for HCV infection., Methods: Subjects in 3 counties were surveyed by stratified cluster random sampling in the National Major Science and Technology Projects demonstration area in Jiangsu province. Interview was carried out with uniformly- designed questionnaires. Blood samples were collected and anti-HCV tested. Data were analysed under SPSS 13.0. Case-control study was conducted on two groups with subjects under 1:4 matching by sex, age, village of residence and status of HCV infection. Cox's proportional hazards regression analysis was then performed to analyze the risk factors for HCV infection., Results: The positive rate of anti-HCV was higher in females (0.965%, 95%CI:0.899%-1.035%) than in males (0.572%, 95%CI:0.517%-0.632%). Significant differences were noticed among the positive rates of anti-HCV in the three counties (0.131%, 95%CI:0.103%-0.164%;0.316%, 95%CI: 0.268%-0.370%; 2.173% , 95% CI:2.039%-2.315% , respectively). Peak prevalence of anti-HCV (1.577%, 95%CI:1.425%-1.740%) was observed among persons at 50-59 years of age. Bottom rate (0.161%, 95%CI:0.125%-0.204%) was observed below 30 years of age. High anti-HCV positive rate was detected in people with lower education background or belonged to 'floating population'. Factors as having histories of hospitalization (OR = 3.049, 95% CI:1.322-7.036), blood transfusion (OR = 14.319, 95%CI:2.318-88.459) or sharing razor (OR = 3.604, 95%CI:1.096-11.851) were risk factors of HCV infection in the area with the lowest anti-HCV positive rate. In another county, factor as having histories of 'blood products transfusion' (OR = 7.202, 95% CI:1.170-44.310) appeared important while in the third county, having history of 'blood donation' (OR = 7.496, 95%CI:6.121- 9.180); 'blood transfusion' (OR = 2.305, 95%CI:1.578-3.369) and 'invasive physical examination' (OR = 1.258, 95% CI:1.021-1.549) appeared to be important. Age seemed a risk factor for HCV infection. "Sharing razor" was a specifically important risk factor among the 30- year-olds. "Having received acupuncture" was noticed a risk factor only among people at 50-59 years of age while "Having received invasive physical examination" was important in the 50-59 and 60-69 year-olds., Conclusion: High anti-HCV positive rate was observed in people at middle of aged population, as well as in those with poor education or under 'floating'. Unsafe blood donation or having received blood-product transfusion might be the risk factors in the last decades. Factors as sharing razor, having received acupuncture or invasive examination might be specifically risky in different populations.

Hepatitis B immunization programs for newborns, children, and adolescents in China have shown remarkable results. To establish whether there would be any benefit in extending the program to cover older individuals, we examined both the epidemiology of hepatitis B virus (HBV) infection and the coverage of hepatitis B vaccinations among adults born before routine vaccinations were implemented. We then evaluated the impact of hepatitis B vaccination in adults aged 20-59 years. A large-scale cross-sectional epidemiological survey of HBV infection was performed in the province of Jiangsu, south-east China, between September 2009 and March 2010. A total of 86,732 adults aged 20-59 years were included, of which 8,615 (9.9%, 95% CI = 9.7-10.1%) were HBsAg sero-positive. Self-reported vaccination status suggested that the coverage was approximately 23.7% (95% CI = 23.4-24.0%). It was shown that higher HBV vaccination coverage was associated with a lower rate of HBsAg seropositivity among adults. There was a negative correlation between hepatitis B vaccination coverage and HBsAg prevalence (correlation coefficient = -0.805, p = 0.016), which might demonstrate the combined effects of vaccination and pre-vaccination HBsAg screening. In the unvaccinated group, the HBsAg-positive rate had an obvious upward trend with age growing among 20-39 year-olds (Trend χ2 = 22.605, P<0.001), while the vaccinated group showed no such trend (Trend χ2 = 3.462, P = 0.063). Overall, hepatitis B vaccination in adults might reduce the rate of HBsAg positivity. Therefore, routine immunization of adults aged 20-39 years should be seriously considered.

The hepatitis C virus (HCV) alternate reading frame protein (ARFP or F protein) of the HCV 1b genotype is a double-frameshift product of the HCV core protein (Core). The discovery of HCV F protein challenges various biological functions attributed to Core. However, the specific characteristics of the host cellular immune response to F protein during HCV infection have yet to be fully elucidated. Therefore, the present study investigated the cytokine response to HCV Core or F protein in peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) from patients with chronic HCV and healthy donors in vitro. The results demonstrated that the levels of interferon (IFN)-α, analyzed by an enzyme-linked immunosorbent assay, secreted by PBMCs in patients positive for the anti-F protein antibody, were lower than those of patients negative for the anti-F protein antibody. Moreover, the frequency of PDCs in patients negative for the anti-F protein antibody, were higher than in the group positive for the anti-F protein antibody. Furthermore, HCV F protein and Core not only inhibited specific unmethylated CpG oligonucleotide sequences of type A (CpG‑A)-induced IFN-α production by PBMCs and PDCs, but also upregulated the production of interleukin (IL)-10 by PBMCs in patients with chronic HCV and healthy controls. Notably, following neutralization of IL-10 in the media and in vitro Core or F protein stimulation, levels of IFN-α were increased. Moreover, the results revealed that the roles of F protein and Core were similar with regard to the induction of apoptosis of PDCs in patients with chronic HCV. These findings suggest that F protein may inhibit PBMC IFN-α secretion by regulating the production of IL-10, and may contribute to an increase in the rates of apoptosis in PDCs. In conclusion, the results have revealed a potential involvement of F protein in the mechanisms of chronic hepatitis C.

Chronic hepatitis B virus (HBV) infection is a challenging global health problem. To identify genetic loci involved in chronic HBV infection, we designed a three-phase genome-wide association study in Han Chinese populations. The discovery phase included 951 HBV carriers (cases) and 937 individuals who had naturally cleared HBV infection (controls) and was followed by independent replications with a total of 2,248 cases and 3,051 controls and additional replications with 1,982 HBV carriers and 2,622 controls from the general population. We identified two new loci associated with chronic HBV infection: rs3130542 at 6p21.33 (near HLA-C, odds ratio (OR) = 1.33, P = 9.49 × 10(-14)) and rs4821116 at 22q11.21 (in UBE2L3, OR = 0.82, P = 1.71 × 10(-12)). Additionally, we replicated the previously identified associations of HLA-DP and HLA-DQ variants at 6p21.32 with chronic HBV infection. These findings highlight the importance of HLA-C and UBE2L3 in the clearance of HBV infection in addition to HLA-DP and HLA-DQ.

Background: The let-7 family plays a vital role in the normal cellular activity of liver cells and the carcinogenesis of hepatocellular carcinoma (HCC). In the previous study, we have detected the association between single nucleotide polymorphisms (SNPs) in the promoter region of let-7 and susceptibility to HCC. However, it is still unknown whether these polymorphisms are associated with HCC prognosis., Methods: We investigated the effect of two potentially functional SNPs in the promoter region of let-7 family, rs10877887 (T>C) and rs13293512 (T>C), on the overall survival of 331 HCC patients. Log-rank test and Cox proportional hazard models were used for the survival analyses., Results: We found that HCC patients carrying the C allele of rs10877887 had a significantly increased death risk (adjusted HR=1.22, 95%CI=1.02-1.47, P=0.03 in the additive model), compared to those with T allele. In the stratified analysis, the risk effect was evident in HCC patients with Barcelona Clinic Liver Cancer (BCLC) stage B (adjusted HR=1.24, 95%CI=1.02-1.51, P=0.03) and in those who received chemotherapy or intervention (adjusted HR=1.25, 95%CI=1.02-1.53, P=0.04)., Conclusions: Our results suggest that rs10877887 in the promoter region of let-7 may be a prognostic biomarker for HCC patients, which need the validation from other larger studies in different populations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Genetic variations in miRNA processing genes may affect the biogenesis of miRNA, hence risk of HBV infection and hepatocellular carcinoma (HCC) development. In the present study, we hypothesized that potentially functional polymorphisms in 3'-untranslated region (UTR) of miRNA processing genes might contribute to susceptibility of HBV infection and HCC development. To test the hypothesis, we genotyped three selected SNPs (rs1057035 in DICER1, rs3803012 in RAN, and rs10773771 in PIWIL1) in a case-control study of 1300 HBV-positive HCC cancer cases, 1344 HBV persistent carriers, and 1344 HBV natural clearance subjects in Chinese. We observed that DICER1 rs1057035 CT/CC variant genotypes were associated with a significant decreased risk of HCC (adjusted OR = 0.79, 95% CI = 0.64-0.96) compared with wild-type TT and RAN rs3803012 AG/GG variant genotypes increased the risk of HBV persistent infection compared with AA genotype (adjusted OR = 1.35, 95% CI = 1.03-1.77). However, PIWIL1 rs10773771 CT/CC variant genotypes were associated with an approaching decreased risk of HCC (adjusted OR = 0.86, 95% CI = 0.73-1.01) and similar with RAN rs3803012 AG/GG (adjusted OR = 0.80, 95% CI = 0.61-1.06). Furthermore, reporter gene assays indicated that the three SNPs (rs1057035, rs3803012, and rs10773771) might change the binding ability of miRNAs to the 3'UTR of the three genes (DICER1, RAN, and PIWIL1), respectively. These findings indicated that DICER1 rs1057035, RAN rs3803012, and PIWIL1 rs10773771 might contribute to the risk of HBV-related HCC., (© 2013 Wiley Periodicals, Inc.)

Th1 and Th2 cytokine response has been confirmed to be correlated with the pathogenesis of HCV infection. The aim of the study is to investigate the Th1 and Th2 cytokine profiles induced by HCV alternate reading frame protein (F protein) in chronic hepatitis C patients. We assessed the immune responses specific to HCV F protein in 55 chronic HCV patients. IFN-γ, IL-2, IL-4 and IL-5 secretion by peripheral blood mononuclear cells (PBMC) post F protein stimulation were compared among HCV patients and healthy donors. Finally, the associations between HCV F protein and HLA class II alleles were explored. We found that the seroprevalence of anti-F antibodies in HCV-related hepatocellular carcinoma (HCC) patients was significantly higher than that of patients without HCC, but such a significant difference in humoral immune responses to F protein was not observed in HCV 1b-infected- and non-HCV 1b-infected-patients. Additionally, the PBMC proliferation of HCC patients was significantly lower than that of patients without HCC. Furthermore, F protein stimulation of PBMCs from F-seropositive patients resulted in Th2 biased cytokine responses (significantly decreased IFN-γ and/or IL-2 and significantly increased IL-4 and/or IL-5 levels) that reportedly may contribute to HCC progression and pathogenesis. However, no significant difference in the association between HCV F protein and HLA-DRB1*0201, 0301, 0405, 1001 and HLA-DQB1*0201, 0401, 0502, 0602 was observed in this study. These findings suggest that F protein may contribute to the HCV-associated bias in Th1/Th2 responses of chronic hepatitis C patients including the progress of HCC pathogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Recent studies showed that pseudogenes can regulate the expression of their coding gene partners by competing for miRNAs. The E2F family plays a crucial role in the control of cell cycle checkpoint. E2F3P1 is a pseudogene of E2F3. Few studies focused on genetic variations on pseudogenes. In this study, we performed a case-control study to assess the association between single nucleotide polymorphisms (SNPs) in E2F3P1 and hepatocellular carcinoma (HCC) risk in 1050 hepatitis B virus (HBV)-positive HCC cases and 1050 chronic HBV carriers. Logistic regression analysis was applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between genotypes and HCC risk. We found that the variant CT/TT genotypes of rs1838149 were associated with a significantly decreased risk of HCC (adjusted OR = 0.66, 95% CIs = 0.51-0.86, P = 0.002) compared to those with wildtype CC homozygote. Furthermore, the AA genotype of rs9909601 had an increased HCC risk with an adjusted OR of 1.41 (95% CIs = 1.07-1.86), and the A allele of rs9909601 was significantly associated with HCC risk compared to those with the G allele (adjusted OR = 1.17, 95% CIs = 1.03-1.33, P = 0.017). These results indicate that genetic variations in the pseudogene E2F3P1 may confer HCC risk.

Background: The interleukin-23 receptor (IL-23R) plays an important role in the T-helper 17 cell-mediated inflammatory process and is also involved in tumor immune surveillance, which may be linked to carcinogenesis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). In this study, we hypothesized that potentially functional genetic variants of the IL-23R gene may modify HCC risk., Methods: We genotyped two single-nucleotide polymorphisms (SNPs) of IL-23R, rs6682925 and rs1884444, in a case-control study of 837 HCC cases, 899 HBV surface antigen (HBsAg)-positive controls, and 743 HBsAg-negative controls. A reporter gene assay was performed to evaluate the functional relevance of the rs6682925 SNP located at the promoter region of the IL-23R gene., Results: We found that the two SNPs were associated with the risk of HCC when compared with both the HBsAg-positive and -negative controls. When compared with all controls, IL-23R rs6682925 and rs1884444 both increased the HCC risk in a recessive genetic model [rs6682925 CC vs. TT/TC: odds ratio (OR) 1.35, 95 % confidence interval (CI) 1.07-1.70; rs1884444 GG vs. TT/TG: OR 1.36, 95 % CI 1.05-1.77]. Furthermore, the variant C allele of rs6682925 in the promoter region of IL-23R was associated with increased reporter gene activity., Conclusions: These findings indicate that genetic variants in IL-23R may contribute to HCC development.

Recent genome-wide association studies showed that four single-nucleotide polymorphisms (SNPs) in human leukocyte antigen (HLA)-DP (rs3077 and rs9277535) and HLA-DQ (rs2856718 and rs7453920) were associated with chronic hepatitis B virus (HBV) infection in Japanese populations. More than 75% of hepatocellular carcinoma (HCC) patients are attributable to persistent infection of hepatitis B virus (HBV), especially in China. We genotyped these four SNPs in 1,300 HBV-positive HCC patients, 1,344 persistent HBV carriers, and 1,344 persons with HBV natural clearance from Southeast China to further test the associations of HLA-DP/DQ variants and with risk of both HBV clearance and HCC development. Logistic regression analyses showed that HLA-DQ rs2856718 significantly decreased host HCC risk, whereas three SNPs were associated with HBV clearance (HLA-DP rs9277535 as well as HLA-DQ rs7453920 and rs2856718). In addition, HLA-DP rs3077 showed an approaching significant effect on susceptibility to HBV persistent infection and HCC development when considering multiple testing adjustments. Taken together, we report, for the first time, that genetic variants in the HLA-DP and HLA-DQ loci may be marker SNPs for risk of both HBV clearance and HCC development., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Background: Recently, several studies have demonstrated that two long non-coding RNAs (lncRNAs), HULC and MALAT1, may participate in hepatocellular carcinoma (HCC) development and progression. However, genetic variations in the two lncRNAs and their associations with HCC susceptibility have not been reported. In this study, we hypothesized that single nucleotide polymorphisms (SNPs) in HULC and MALAT1 may contribute to HCC risk., Methods: We conducted a case-control study and genotyped two SNPs, rs7763881 in HULC and rs619586 in MALAT1, in 1300 HBV positive HCC patients, 1344 HBV persistent carriers and 1344 subjects with HBV natural clearance to test the associations between the two SNPs and susceptibility to HCC and HBV chronic infection., Results: The variant genotypes of rs7763881 were significantly associated with decreased HCC risk in a dominant genetic model [AC/CC vs. AA: adjusted odds ration (OR)  =  0.81, 95% confidence intervals (CIs)  =  0.68-0.97, P  =  0.022]. Furthermore, the variant genotypes of rs619586 was associated with decreased HCC risk with a borderline significance (AG/GG vs. AA: adjusted OR  =  0.81, 95% CIs  =  0.65-1.01, P  =  0.057). However, no significant association was found between the two SNPs and HBV clearance., Conclusions: The variant genotypes of rs7763881 in HULC may contribute to decreased susceptibility to HCC in HBV persistent carriers.

Genome-wide association studies (GWAS) have recently identified KIF1B as susceptibility locus for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). To further identify novel susceptibility loci associated with HBV-related HCC and replicate the previously reported association, we performed a large three-stage GWAS in the Han Chinese population. 523,663 autosomal SNPs in 1,538 HBV-positive HCC patients and 1,465 chronic HBV carriers were genotyped for the discovery stage. Top candidate SNPs were genotyped in the initial validation samples of 2,112 HBV-positive HCC cases and 2,208 HBV carriers and then in the second validation samples of 1,021 cases and 1,491 HBV carriers. We discovered two novel associations at rs9272105 (HLA-DQA1/DRB1) on 6p21.32 (OR = 1.30, P = 1.13×10⁻¹⁹) and rs455804 (GRIK1) on 21q21.3 (OR = 0.84, P = 1.86×10⁻⁸), which were further replicated in the fourth independent sample of 1,298 cases and 1,026 controls (rs9272105: OR = 1.25, P = 1.71×10⁻⁴; rs455804: OR = 0.84, P = 6.92×10⁻³). We also revealed the associations of HLA-DRB1*0405 and 0901*0602, which could partially account for the association at rs9272105. The association at rs455804 implicates GRIK1 as a novel susceptibility gene for HBV-related HCC, suggesting the involvement of glutamate signaling in the development of HBV-related HCC., Competing Interests: The authors have declared that no competing interests exist.

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.

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.

The PreS1 region of the L protein is important in cell attachment and consequently in hepatitis B virus (HBV) infectivity. To identify novel PreS1 interacting protein, we performed Glutathione-S-transferase (GST) pull-down, two-dimensional gel electrophoresis (2-DE) and mass spectrometry assays. Glucose-regulated proteins (GRP) 78 and 75 were found to bind PreS1. The interactions between PreS1 and GRP75 were confirmed by a co-immunoprecipitation experiment. GRP78 and GRP75 may play important roles in mediating HBV virion entering into hepatocyte and regulating proper folding of the L protein due to their critical functions in protein folding and trafficking. The finding of novel PreS1 binding protein enriches our knowledge about molecular mechanism of HBV infection.

Matrix metallopeptidase-9 (MMP-9) plays a pivotal role in vascular remodeling and development of atherosclerotic lesion. The potentially functional MMP-9 polymorphisms may contribute to the susceptibility of coronary artery disease (CAD). A case-control study composed of 762 CAD cases and 555 CAD-free controls was conducted in a Chinese population to investigate the association between the MMP-9 -1562 C>T, R279Q, P574R and R668Q polymorphisms and CAD risk. It was found that the variant genotypes of R279Q, P574R and R668Q were associated with a non-significant decreased risk of CAD when compared with their wild-type genotypes, respectively, Furthermore, compared with those without any variant genotypes for these four nonsynonymouse loci, individuals carrying all four variant genotypes (-1562 CT/TT, 279 RQ/QQ, 574 PR/RR and 668 RQ/QQ) had a 51% decreased risk of CAD (adjusted OR = 0.49; 95% CI = 0.26-0.95, P = 0.033). Although no significant main effects were observed for MMP-9 -1562 C>T locus on CAD risk, variant genotypes of -1562 C>T were associated with a 2.53 increased risk of CAD in subjects with diabetes mellitus (DM) (95% CI = 1.18-5.45, P = 0.018). In CAD cases, variant genotypes of -1562 C>T were associated with a significantly increased risk of MI (adjusted OR, 1.48, 95% CI, 1.01-2.20, P = 0.048). These findings suggest that MMP-9 R279Q, P574R and R668Q may have combined effect in the occurrence of CAD and -1562 CT/TT genotypes may contribute to CAD in diabetics and MI in CAD patients.

The BRCA1 Associated RING Domain (BARD1) gene has been identified as a high penetrance gene for breast cancer, whose germline and somatic mutations were reported in both non-BRCA1/2 hereditary site-specific and sporadic breast cancer cases. BARD1 plays a crucial role in tumor repression, along with its heterodimeric partner BRCA1. In the current study, we tested the hypothesis that common non-synonymous polymorphisms in BARD1 are associated with breast cancer susceptibility in a case-control study of 507 patients with incident breast cancer and 539 frequency-matched cancer-free controls in Chinese women. We genotyped all three common (minor allele frequency (MAF)>0.10) non-synonymous polymorphisms (Pro24Ser, Arg378Ser, and Val507Met) in BARD1. We found that the BARD1 Pro24Ser variant genotypes (24Pro/Ser and 24Ser/Ser) and Arg378Ser variant homozygote 378Ser/Ser were associated with a significantly decreased breast cancer risk, compared with their wild-type homozygotes, respectively. Furthermore, a significant locus-locus interaction was evident between Pro24Ser and Arg378Ser (P(int )= 0.032). Among the 378Ser variant allele carriers, the 24Pro/Pro wild-type homozygote was associated with a significantly increased breast cancer risk (adjusted OR=1.81, 95% CI=1.11-2.95), but the subjects having 24Pro/Ser or Ser/Ser variant genotypes had a significantly decreased risk (adjusted OR=0.74, 95% CI=0.56-0.99). In stratified analysis, this locus-locus interaction was more evident among subjects without family cancer history, those with positive estrogen receptor (ER) and individuals with negative progesterone receptor (PR). These findings indicate that the potentially functional polymorphisms Pro24Ser and Arg378Ser in BARD1 may jointly contribute to the susceptibility of breast cancer.

p21(Cip1) and p27(Kip1) are cyclin-dependent kinase inhibitors, which can arrest cell proliferation and serve as tumor suppressors. Reduced protein expression of p21(Cip1) and p27(Kip1) was frequently observed in a subset of cancers, including breast cancer. In this study, we hypothesized that genetic variants in CDKN1A (encode for p21(Cip1)) and CDKN1B (encode for p27(Kip1)) may modulate the risk of breast cancer. To test this hypothesis, we evaluated the associations of the polymorphisms of Ser31Arg and C+20T in CDKN1A and C-79T and Gly109Val in CDKN1B, as well as their combinations, with breast cancer risk in a case-control study of 368 breast cancer cases and 467 cancer-free controls in a Chinese population. We found that a significantly increased risk of breast cancer was associated with the variant genotypes of CDKN1B C-79T [adjusted OR = 1.43 (95% CI = 1.03-1.98) for -79TC/TT], compared with the -79CC genotype, but no associations were observed for other variant genotypes. However, the combined variant genotypes of the 4 loci were associated with a significantly increased breast cancer risk (adjusted OR = 1.49, 95% CI = 1.11-2.01 among subjects carrying 3 or more variant alleles), especially among premenopausal women (adjusted OR= 2.30, 95% CI = 1.45-3.66). Furthermore, in premenopausal women, this significant association remained unchanged, after including other individual risk factors in the multivariate logistic regression model, suggesting an independent role of CDKN1A and CDKN1B variants in breast cancer risk. Although the exact biological mechanism remains to be explored, our findings suggest possible involvement of CDKN1A and CDKN1B variants in the etiology of breast cancer. Further large and functional studies are needed to confirm our findings.

MDM2 is a phosphoprotein that interacts with P53 and inhibits its activity. Recently, a T/G substitution (SNP309) in the promoter of MDM2 was identified and has been demonstrated to be associated with an increased MDM2 expression and a significantly earlier age of onset of several tumors, including breast cancer. To test the hypothesis that this functional variant in the MDM2 promoter is associated with risk of breast cancer, we conducted a molecular epidemiological study of 366 breast cancer cases (BC), 263 patients with benign breast diseases (BBD) and 605 cancer-free controls in China, in which we genotyped this T/G variant and another common insertion/deletion polymorphism (Del1518) in the MDM2 promoter and evaluated the associations between these two polymorphisms and breast cancer risk. We found that the variant allele frequencies of these two polymorphisms were not statistically different between the cases and controls (SNP309G: 0.500, 0.542, and 0.506 in BC, BBD, and controls, respectively, and Del1518-: 0.296, 0.308, and 0.297 in BC, BBD, and controls, respectively). Logistic regression analyses revealed that the variant genotypes of both MDM2 SNP309 and Del1518 polymorphisms were not significantly associated with risk of breast cancer (adjusted OR, 1.03; 95% CI, 0.74-1.42 for SNP309 TG and GG; and adjusted OR, 1.09; 95% CI, 0.83-1.43 for Del1518 +/- and -/-). These findings suggest that these two MDM2 promoter variants may not play a major role in the etiology of breast cancer.

Background: Accumulative evidence suggests that low folate intake is associated with increased risk of breast cancer. Polymorphisms in genes involved in folate metabolism may influence DNA methylation, nucleotide synthesis, and thus individual susceptibility to cancer. Thymidylate synthase (TYMS) is a key enzyme that participates in folate metabolism and catalyzes the conversion of dUMP to dTMP in the process of DNA synthesis. Two potentially functional polymorphisms [a 28-bp tandem repeat in the TYMS 5'-untranslated enhanced region (TSER) and a 6-bp deletion/insertion in the TYMS 3'-untranslated region (TS 3'-UTR)] were suggested to be correlated with alteration of thymidylate synthase expression and associated with cancer risk., Methods: To test the hypothesis that polymorphisms of the TYMS gene are associated with risk of breast cancer, we genotyped these two polymorphisms in a case-control study of 432 incident cases with invasive breast cancer and 473 cancer-free controls in a Chinese population., Results: We found that the distribution of TS3'-UTR (1494del6) genotype frequencies were significantly different between the cases and controls (P = 0.026). Compared with the TS3'-UTR del6/del6 wild-type genotype, a significantly reduced risk was associated with the ins6/ins6 homozygous variant genotype (adjusted OR = 0.58, 95% CI = 0.35-0.97) but not the del6/ins6 genotype (OR = 1.09, 95% CI = 0.82-1.46). Furthermore, breast cancer risks associated with the TS3'-UTR del6/del6 genotype were more evident in older women, postmenopausal subjects, individuals with a younger age at first-live birth and individuals with an older age at menarche. However, there was no evidence for an association between the TSER polymorphism and breast cancer risks., Conclusion: These findings suggest that the TS3'-UTR del6 polymorphism may play a role in the etiology of breast cancer. Further larger population-based studies as well as functional evaluation of the variants are warranted to confirm our findings.

Interleukin 1beta (IL-1beta) is a multifunctional cytokine that upregulates the inflammatory response, and participates in carcinogenesis, malignant transformation, tumor growth, invasion and metastasis. Two potentially functional polymorphisms (T-31C and C-511T) in the IL-1beta gene promoter were suggested to be correlated with alteration of IL-1beta expression and therefore may be associated with cancer risk. To test the hypothesis that these 2 polymorphisms are associated with risk of breast cancer, we performed a case-control study of 365 breast cancer cases, 270 patients with benign breast diseases (BBD) and 631 cancer-free controls in a Chinese population. Multivariate logistic regression analyses revealed that increased risk of breast cancer was associated with IL-1beta-31C variant genotypes [adjusted odds ratio (OR)=1.28 and 95% confidence interval (CI)=0.91-1.80 for -31CT and 1.72 (95% CI=1.16-2.54) for -31CC], compared with the -31TT genotype. Similarly, IL-1beta-511T variant genotypes were also associated with increased risk of breast cancer (adjusted OR=1.20, 95% CI=0.86-1.67 for -511CT and adjusted OR=1.74, 95% CI=1.18-2.56 for -511TT), compared with the -511CC genotype. Furthermore, cancer risks associated with IL-1betaT-31C variant genotypes were more evident in older women, postmenopausal women and individuals with a later menarche age. Interestingly, although we did not find significant associations of these 2 variants with cancer risk when compared with the BBD patients, a 1.27-fold (95% CI=1.01-1.60) increased risk was observed with the -31C-511T common haplotype. These findings indicate that these 2 IL-1beta promoter variants may contribute to risk of developing breast cancer in the Chinese population., (Copyright (c) 2005 Wiley-Liss, Inc.)

p53-binding protein 1 (P53BP1), a central transducer of DNA-damage signals to p53, is required for both intra-S-phase and G2-M checkpoints, suggesting that these two proteins may work together in the p53-mediated transcriptional activation and DNA damage-repair signaling pathways. Because the p53-binding region of 53BP1 maps to the C-terminal BRCT domains, which are homologous to those found in the breast cancer protein BRCA1, we hypothesized that genetic variation in P53BP1 and p53 may contribute to breast cancer predisposition. To test this hypothesis, we simultaneously genotyped single nucleotide polymorphisms of T-885G, Glu353Asp, and Gln1136Lys in P53BP1 and Arg72Pro in p53 in a case-control study of 404 breast cancer cases and 472 cancer-free controls. We found that the P53BP1 variant genotypes (alleles) of T-885G and Gln1136Lys were associated with a significantly increased risk of breast cancer among p53 Pro/Pro carriers (OR=2.36, 95% CI 1.16-4.83 for -885TG/GG; OR=2.24, 95% CI 1.15-4.37 for 1136Gln/Lys+Lys/Lys and OR=2.82, 95% CI 1.15-6.94 for >4 variant alleles of these 3 loci). In addition, the variant genotypes of above 3 loci of P53BP1 were significantly associated with elevated risk of progesterone receptor (PR) negative breast cancer, and the T-885G and Gln1136Lys with estrogen receptor (ER) negative breast cancer. Furthermore, we found a significant gene-gene interaction between P53BP1 Gln1136Lys and p53 Arg72Pro variants in relation to breast cancer, and the OR of interaction for the presence of both P53BP1 1136Gln/Lys+Lys/Lys and p53 72Arg/Pro+Pro/Pro genotypes was 1.93 (95% CI 1.06-3.52) (P=0.031 for interaction). These findings indicate that the SNPs in P53BP1 and p53 jointly contribute to breast cancer risk, particularly ER (-) or PR (-) breast cancer, and the p53 Arg72Pro polymorphism may serve as a risk modifier. Further functional studies are needed to confirm our findings.

Adenosine diphosphate ribosyl transferase (ADPRT) and X-ray repair cross-complementing 1 (XRCC1) are two major base excision repair (BER) proteins and act cooperatively in the BER processes. Polymorphisms of ADPRT Val762Ala and XRCC1 Arg399Gln may alter their protein functions and BER activity, and were therefore hypothesized to be associated with breast cancer susceptibility. We examined the contributions of these two polymorphisms to breast cancer susceptibility in a case-control study of 302 breast cancer cases, 221 patients with benign breast disease (BBD) and 639 cancer-free controls in a Chinese population. We found that the variant genotypes of both ADPRT Val762Ala and XRCC1 Arg399Gln were not significantly associated with the risk of breast cancer (adjusted OR 0.87, 95% CI 0.64 to 1.19 for ADPRT Val/Ala + Ala/Ala; adjusted OR 0.82, 95% CI 0.61 to 1.11 for XRCC1 Arg/Gln + Gln/Gln; and adjusted OR 0.70, 95% CI 0.45 to 1.10 for these two combined variant genotypes. Similarly, we did not find any significant associations of these two genotypes with BBD risk. These findings suggest that the ADPRT Val762Ala and XRCC1 Arg399Gln polymorphisms may not play a role in the etiology of breast cancer.