Daiki Miki

A genome-wide association study of chronic hepatitis B identified novel risk locus in a Japanese population

Hepatitis B virus (HBV) infection is a major health issue worldwide which may lead to hepatic dysfunction, liver cirrhosis and hepatocellular carcinoma. To identify host genetic factors that are associated with chronic hepatitis B (CHB) susceptibility, we previously conducted a two-stage genome-wide association study (GWAS) and identified the association of HLA-DP variants with CHB in Asians; however, only 179 cases and 934 controls were genotyped using genome-wide single nucleotide polymorphism (SNP) arrays. Here, we performed a second GWAS of 519 747 SNPs in 458 Japanese CHB cases and 2056 controls. After adjustment with the previously identified variants in the HLA-DP locus (rs9277535), we detected strong associations at 16 loci with P-value of <5 × 10(-5). We analyzed these loci in three independent Japanese cohorts (2209 CHB cases and 4440 controls) and found significant association of two SNPs (rs2856718 and rs7453920) within the HLA-DQ locus (overall P-value of 5.98 × 10(-28) and 3.99 × 10(-37)). Association of CHB with SNPs rs2856718 and rs7453920 remains significant even after stratification with rs3077 and rs9277535, indicating independent effect of HLA-DQ variants on CHB susceptibility (P-value of 1.52 × 10(-21)- 2.38 × 10(-30)). Subsequent analyses revealed DQA1*0102-DQB1*0604 and DQA1*0101-DQB1*0501 [odds ratios (OR) =0.16, and 0.39, respectively] as protective haplotypes and DQA1*0102-DQB1*0303 and DQA1*0301-DQB1*0601 (OR = 19.03 and 5.02, respectively) as risk haplotypes. These findings indicated that variants in antigen-binding regions of HLA-DP and HLA-DQ contribute to the risk of persistent HBV infection.

Common variation of IL28 affects gamma-GTP levels and inflammation of the liver in chronically infected hepatitis C virus patients

BACKGROUND & AIMS A common genetic variation at the IL28 locus has been found to affect the response of peg-interferon and ribavirin combination therapy against chronic hepatitis C virus (HCV) infection. An allele associated with a favorable response (rs8099917 T), which is the major allele in the majority of Asian, American, and European populations, has also been found to be associated with spontaneous eradication of the virus. METHODS As no studies have yet analyzed the effect of the polymorphism on biochemical and inflammatory changes in chronic infection, we analyzed a cohort of patients with chronic hepatitis C (n=364) for the effect of the IL28 polymorphism on viral, biochemical, and histological findings. RESULTS We found that the proportion of HCV wild type core amino acids 70 and 91 was significantly greater (p=1.21 x 10(-4) and 0.034) and levels of gamma-GTP significantly lower (p=0.001) in patients homozygous for the IL28 major allele. We also found that inflammation activity and fibrosis of the liver were significantly more severe in patients homozygous for the IL28 major allele (p=0.025 and 0.036, respectively). Although the higher gamma-GTP levels were also associated with higher inflammatory activity and fibrosis, multivariate analysis showed that only the IL28 allele polymorphism, sex, alcohol consumption, and liver fibrosis were independently associated with gamma-GTP levels (p=0.001, 0.0003, 0.0013, and 0.0348, respectively). CONCLUSIONS These results suggest that different cytokine profiles induced by the IL28 polymorphism resulted in different biochemical and inflammatory conditions during chronic HCV infection and contribute to the progression of liver diseases.

IL28B But Not ITPA Polymorphism Is Predictive of Response to Pegylated Interferon, Ribavirin, and Telaprevir Triple Therapy in Patients With Genotype 1 Hepatitis C

BACKGROUND Pegylated interferon, ribavirin, and telaprevir triple therapy is a new strategy expected to eradicate the hepatitis C virus (HCV) even in patients infected with difficult-to-treat genotype 1 strains, although adverse effects, such as anemia and rash, are frequent. METHODS We assessed efficacy and predictive factors for sustained virological response (SVR) for triple therapy in 94 Japanese patients with HCV genotype 1. We included recently identified predictive factors, such as IL28B and ITPA polymorphism, and substitutions in the HCV core and NS5A proteins. RESULTS Patients treated with triple therapy achieved comparatively high SVR rates (73%), especially among treatment-naive patients (80%). Of note, however, patients who experienced relapse during prior pegylated interferon plus ribavirin combination therapy were highly likely to achieve SVR while receiving triple therapy (93%); conversely, prior nonresponders were much less likely to respond to triple therapy (32%). In addition to prior treatment response, IL28B SNP genotype and rapid viral response were significant independent predictors for SVR. Patients with the anemia-susceptible ITPA SNP rs1127354 genotype typically required ribavirin dose reduction earlier than did patients with other genotypes. CONCLUSIONS Analysis of predictive factors identified IL28B SNP, rapid viral response, and transient response to previous therapy as significant independent predictors of SVR after triple therapy.

Variation in the DEPDC5 locus is associated with progression to hepatocellular carcinoma in chronic hepatitis C virus carriers

Chronic viral hepatitis is the most important risk factor for progression to hepatocellular carcinoma (HCC). To identify genetic risk factors for progression to HCC in individuals with chronic hepatitis C virus (HCV), we analyzed 467,538 SNPs in 212 Japanese individuals with chronic HCV with HCC and 765 individuals with chronic HCV without HCC. We identified one intronic SNP in the DEPDC5 locus on chromosome 22 associated with HCC risk and confirmed the association using an independent case-control population (710 cases and 1,625 controls). The association was highly significant when we analyzed the stages separately as well as together (rs1012068, Pcombined = 1.27 × 10−13, odds ratio = 1.75). The significance level of the association further increased after adjustment for gender, age and platelet count (P = 1.35 × 10−14, odds ratio = 1.96). Our findings suggest that common variants within the DEPDC5 locus affect susceptibility to HCC in Japanese individuals with chronic HCV infection.

HCV substitutions and IL28B polymorphisms on outcome of peg-interferon plus ribavirin combination therapy

Background and aims A number of recent studies have shown that human polymorphisms near the IL28B type III interferon (IFNλ) gene influence the response to peg-interferon plus ribavirin combination therapy for infection with chronic hepatitis C virus (HCV). Viral polymorphisms, including substitutions within the HCV core and NS5A proteins, have also been shown to influence treatment outcome, but it is not known whether these factors act independently of the IL28B polymorphism or if they reflect the same or a different underlying mechanism. Multiple logistic regression was used to determine whether host and viral polymorphisms independently predict sustained virological response (SVR). Methods Two single nucleotide polymorphisms were genotyped in the IL28B locus (rs12979860 and rs8099917) from 817 patients with chronic HCV infection, and substitutions at amino acids 70 and 91 of the HCV core protein and within the NS5A interferon sensitivity-determining region (ISDR) were analysed. Results It was found that independent predictors of an SVR included IL28B rs12979860 CC genotype (OR=4.98; p=4.00E-08), core amino acid 70 substitutions (OR=0.53; p=0.016), age and baseline viral load. For non-virological response, the IL28B rs12979860 CT/TT genotype (OR=0.23; p=1.96E-8) and age were independent predictors. IL28B rs12979860 genotype (p=1.4E-8), core amino acid 70 substitutions (p=0.0013), ISDR substitutions (p=0.0019), baseline viral load, γ-glutamyltranspeptidase, alanine aminotransferase and platelet count were independent predictors for change in viral load by week 4 of treatment. Conclusions IL28B polymorphisms and HCV core amino acid 70 substitutions contribute independently to an SVR to peg-interferon plus ribavirin combination therapy.

Involvement of Epithelial Cell Transforming Sequence-2 Oncoantigen in Lung and Esophageal Cancer Progression

Purpose: This study aims to isolate potential molecular targets for diagnosis, treatment, and/or prevention of lung and esophageal carcinomas. Experimental Design: We screened for genes that were frequently overexpressed in the tumors through gene expression profile analyses of 101 lung cancers and 19 esophageal squamous cell carcinomas (ESCC) by cDNA microarray consisting of 27,648 genes or expressed sequence tags. In this process, we identified epithelial cell transforming sequence 2 (ECT2) as a candidate. Tumor tissue microarray was applied to examine the expression of ECT2 protein in 242 archived non–small-cell lung cancers (NSCLC) and 240 ESCC specimens and to investigate its prognostic value. A role of ECT2 in lung and esophageal cancer cell growth and/or survival was examined by small interfering RNA experiments. Cellular invasive activity of ECT2 in mammalian cells was examined using Matrigel assays. Results: Northern blot and immunohistochemical analyses detected expression of ECT2 only in testis among 23 normal tissues. Immunohistochemical staining showed that a high level of ECT2 expression was associated with poor prognosis for patients with NSCLC (P = 0.0004) as well as ESCC (P = 0.0088). Multivariate analysis indicated it to be an independent prognostic factor for NSCLC (P = 0.0005). Knockdown of ECT2 expression by small interfering RNAs effectively suppressed lung and esophageal cancer cell growth. In addition, induction of exogenous expression of ECT2 in mammalian cells promoted cellular invasive activity. Conclusions: ECT2 cancer-testis antigen is likely to be a prognostic biomarker in clinic and a potential therapeutic target for the development of anticancer drugs and cancer vaccines for lung and esophageal cancers.

HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice.

The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patients serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log(10) copies ml(-1) and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections.

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA-SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA-SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA-SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8weeks after injection) was similar in both TK-NOG mice and uPA-SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.

Pretreatment predictor of response, time to progression, and survival to intraarterial 5-fluorouracil/interferon combination therapy in patients with advanced hepatocellular carcinoma

BackgroundSeveral studies have reported survival benefits of combination therapy with intraarterial 5-fluorouracil (5-FU) and subcutaneous interferon (IFN) α for advanced hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT). We investigated the pretreatment predictive factors of early response, time to progression (TTP), and survival in response to intraarterial 5-FU/IFN combination therapy.MethodsPatients with nonresectable HCC and variable PVTT grades (without PVTT to PVTT in the trunk) received intraarterial 5-FU/IFN combination therapy (n = 55).ResultsAfter two courses of the combination therapy, 1 (2%), 15 (27%), 16 (29%), 12 (22%), and 11 (20%) of 55 patients showed complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), or had dropped out (DO), respectively, when their early response to treatment was assessed. Univariate analysis identified only hepatitis C virus (HCV) antibody positivity as having significantly influenced the early response (P = 0.028) and TTP (P = 0.021). Multivariate analysis identified performance status (P = 0.003) and HCV antibody positivity (P = 0.007) as significant and independent determinants of survival. PVTT grade did not influence early response, TTP, or survival. The survival rate was significantly higher in patients who achieved CR or PR than in those that assessed as SD or PD, or DO (P < 0.0001, each).ConclusionsHCV antibody positivity may be a significant pretreatment predictor of early response, TTP, and survival of patients with advanced HCC treated with 5-FU/IFN. CR or PR as the early response to the combination therapy might indicate a more favorable prognosis in patients with advanced HCC. PVTT grade did not seem to influence the efficacy of combination therapy.

Circulating microRNA‐22 correlates with microRNA‐122 and represents viral replication and liver injury in patients with chronic hepatitis B

Hepatitis B virus (HBV) infection is associated with increased expression of microRNA‐122. Serum microRNA‐122 and microRNA‐22 levels were analyzed in 198 patients with chronic HBV who underwent liver biopsy and were compared with quantitative measurements of HBsAg, HBeAg, HBV DNA, and other clinical and histological findings. Levels of serum microRNA‐122 and microRNA‐22 were determined by reverse transcription‐TaqMan PCR. Serum levels of microRNA‐122 and microRNA‐22 were correlated (R2 = 0.576; P < 0.001), and both were elevated in chronic HBV patients. Significant linear correlations were found between microRNA‐122 or microRNA‐22 and HBsAg levels (R2 = 0.824, P < 0.001 and R2 = 0.394, P < 0.001, respectively) and ALT levels (R2 = 0.498, P < 0.001 and R2 = 0.528, P < 0.001, respectively). MicroRNA‐122 levels were also correlated with HBV DNA titers (R2 = 0.694, P < 0.001 and R2 = 0.421, P < 0.001). Levels of these microRNAs were significantly higher in HBeAg‐positive patients compared to HBeAg‐negative patients (P < 0.001 and P < 0.001). MicroRNA‐122 levels were also lower in patients with advanced liver fibrosis (P < 0.001) and lower inflammatory activity (P < 0.025). These results suggest that serum micro‐RNA levels are significantly associated with multiple aspects of HBV infection. The biological meaning of the correlation between microRNA‐122 and HBsAg and should be investigated further. J. Med. Virol. 85:789–798, 2013.