Eiji Mita

Genome-wide association of IL28B with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis C

The recommended treatment for patients with chronic hepatitis C, pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 × 10−13, and rs8099917, 3.11 × 10−15). We replicated these associations in an independent cohort (combined P values, 2.84 × 10−27 (OR = 17.7; 95% CI = 10.0–31.3) and 2.68 × 10−32 (OR = 27.1; 95% CI = 14.6–50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 × 10−24, and rs8099917, P = 1.11 × 10−27). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 × 10−28–2.68 × 10−32; OR = 22.3–27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).

Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy

BACKGROUND Interferon alfa is effective for controlling disease activity in chronic hepatitis C. However, many responders suffer relapse after cessation of therapy. In the present study, the serum concentration of hepatitis C virus RNA was correlated with a sustained response to interferon therapy. METHODS Fifty-three patients with chronic hepatitis C received a 26-week course of interferon alfa. Hepatitis C virus RNA was quantitated in serum at the beginning and end of treatment using a competitive assay that combined reverse transcription and polymerase chain reaction. RESULTS In long-term responders, whose alanine aminotransferase levels remained within the normal range during the 24 weeks after therapy, the titer of viral RNA (logarithmic transformed copy numbers per milliliter of serum) before therapy (7.1 +/- 1.2) was significantly lower (P < 0.001) than that of short-term responders (8.3 +/- 0.5) who had a relapse within the 24 weeks after therapy and nonresponders (8.1 +/- 0.4). Multivariate multiple logistic regression showed that the titer of viral RNA before therapy was the strongest independent predictor of a sustained response to interferon-alfa therapy (P = 0.002). CONCLUSIONS The titer of hepatitis C virus RNA is the most important factor influencing the sustained response to interferon treatment.

Predicting interferon therapy efficacy from hepatitis C virus genotype and RNA titer

We classified 53 Japanese patients with chronic hepatitis C who were treated with natural interferon-α into genotypes and also tested the amounts of hepatitis C virus (HCV) RNA. The rate of the long-term complete response group, whose alanine aminotransferase levels remained within the normal range during the six months after therapy, was significantly higher (P<0.01) in the type-III patients (4/5, 80.0%) than in type-II patients (4/43, 9.3%). For these long-term complete responders, the amounts of HCV RNA was less than 107 copies/ml serum in type-II patients, whereas two type-III patients with relatively high amounts of HCV RNA responded completely. These results confirm that the genotype of HCV is an important factor for predicting the response to interferon therapy. The amounts of HCV RNA can also predict its efficacy in type-II patients.

Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy

BACKGROUND/AIMS Hepatitis C virus (HCV) genome heterogeneity by sequence analysis in association with interferon (IFN) inefficacy has been reported. This study was performed to establish a convenient method for detecting the HCV quasispecies complexity and to determine the correlation between the complexity and the responsiveness to IFN therapy in patients with chronic hepatitis C. METHODS The quasispecies complexity of HCV hypervariable region 1 in patients treated with IFN-alpha was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). RESULTS Seven of 25 patients (28%) with low complexity (SSCP band number of < or = 2) were HCV RNA negative after treatment, whereas in 24 patients with high complexity (SSCP band number of > or = 3), the response to IFN was almost insignificant because only 1 patient (4.5%) remained HCV RNA negative after treatment (P < 0.05). Among type 1b patients, IFN therapy was only effective for patients with low amounts of HCV RNA (< or = 10(7.5) copies/mL serum) and low complexity. In contrast, most type 2a patients tended to respond to the therapy with exceptions being those with high amounts of HCV RNA and high complexity. CONCLUSIONS The complexity of the hypervariable region 1 quasispecies may be a factor for predicting IFN inefficacy in patients with chronic hepatitis C.

Genome-Wide Association Study Confirming Association of HLA-DP with Protection against Chronic Hepatitis B and Viral Clearance in Japanese and Korean

Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85–90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the HLA-DPA1 and HLA-DPB1 genes with Pmeta = 1.89×10−12 for rs3077 and Pmeta = 9.69×10−10 for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (Pmeta = 4.40×10−19 for rs3077 and Pmeta = 1.28×10−15 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule.

Fas system and apoptosis in viral hepatitis

Hepatitis C virus (HCV) and hepatitis B virus (HBV) are the major causative agents of chronic liver disease. However, the mechanisms responsible for liver cell injury remain to be clarified. Playing crucial roles in the clearance of viral infection are cytotoxic T lymphocytes. Recently, it has been demonstrated that perforin‐ and Fas‐based mechanisms account for all T cell‐mediated cytotoxicity. Therefore, we examined the correlation between liver cell damage and the Fas system in the liver of patients with chronic hepatitis C. Fas is a cell surface protein that mediates apoptosis with treatment of the Fas ligand or the anti‐Fas antibody. To investigate the role of Fas in type C hepatitis, we examined the correlation between liver cell damage and Fas expression. Fas expression was found mainly in the cytoplasm of hepatocytes and these positive cells were found particularly among infiltrating lymphocytes. A high prevalence of Fas expression was shown in liver tissue with more severe inflammation. The Fas system‐mediated death signal requires the interaction of Fas ligand with Fas on target cells. We isolated a 1.9 kb cDNA clone for the human Fas ligand and examined the expression of the Fas ligand in liver‐infiltrating mononuclear cells obtained from patients with chronic hepatitis C. The open reading frame encodes 281 amino acids. Next, we examined the expression of the Fas ligand in liver‐infiltrating mononuclear cells obtained from patients with chronic hepatitis C. The amplified products (231 bp) derived from Fas ligand transcripts were detected in liver‐infiltrating mononuclear cells, whereas no signal was observed in liver tissues. In HCV infection, Fas expression in hepatocytes is up‐regulated in accordance with the severity of liver inflammation. When HCV‐specific T cells migrate into hepatocytes and recognize the viral antigen via the T cell receptor, they become activated and express Fas ligand that can transduce the apoptotic death signal to Fas‐bearing hepatocytes. Thus, the Fas system plays an important role in liver cell injury by HCV infection.

Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease

Hepatitis C virus genotype and the amounts of circulating HCV RNA are the most important factors in determining the efficacy of interferon therapy for chronic hepatitis C. To clarify the correlation of these two factors to the progression of liver disease, we classified 148 Japanese patients with type C chronic liver disease into genotypes and also measured their HCV RNA titers (logarithmic transformed copy number/ml serum) by competitive reverse transcription-polymerase chain reaction. We found type II in 23 (76.7%) of 30 patients with chronic persistent hepatitis, 34 (79.1%) of 43 with chronic active hepatitis, 29 (72.5%) of 40 with cirrhosis and 30 (85.7%) of 35 with hepatocellular carcinoma. Thus, there was no significant difference in the prevalence of type II among the various stages of chronic liver disease. We also found the RNA titer to be significantly higher in patients with chronic active hepatitis (8.0 +/- 0.8) than in those with chronic persistent hepatitis (7.0 +/- 1.0, p < 0.001), and also those with cirrhosis (7.6 +/- 0.8, p < 0.05) or hepatocellular carcinoma (7.7 +/- 0.8, p < 0.05). When the titers were compared among genotypes, there was no significant difference between type II and III at any stage (type II vs. type III: chronic persistent hepatitis, 7.2 +/- 1.0 vs. 6.7 +/- 0.8; chronic active hepatitis, 8.1 +/- 0.7 vs. 7.8 +/- 1.0; cirrhosis, 7.7 +/- 0.8 vs. 7.8 +/- 0.7; hepatocellular carcinoma, 7.7 +/- 0.8 vs. 7.8 +/- 0.5). In conclusion, although genotype affects interferon therapy efficacy, it seems to have little influence on serum RNA levels and the progression of type C chronic liver disease.

Involvement of Fas system-mediated apoptosis in pathogenesis of viral hepatitis.

Enhanced Fas system‐mediated hepatocyte apoptosis is observed in hepatitis C virus (HCV)‐ and hepatitis B virus (HBV)‐associated chronic liver diseases. In these forms of viral hepatitis, liver‐infiltrating lymphocytes that recognize the viral antigen on hepatocytes become activated and express cytolytic Fas ligand (FasL) molecules. In contrast, hepatocytes exhibit enhanced Fas expression and become susceptible to FasL‐mediated death. Augmentation of the Fas system has also been observed in other liver diseases and biliary disorders. Moreover, the Fas system is involved in removing aged hepatocytes. Thus, Fas‐mediated apoptosis plays an important role in several liver diseases and in maintaining normal liver homeostasis.

Detection of Hepatitis C Virus RNA in Chronic Non-A, Non-B Liver Disease

Serum samples were tested for detection of hepatitis C virus (HCV) RNA from 156 patients with chronic non-A, non-B liver disease. HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-C100-3 but was also found in 15 (55.6%) of 27 patients negative for anti-C100-3. The rate of positivity for HCV RNA was not significantly different among various stages of liver diseases. These results showed that HCV continues to replicate even in advanced liver disease and that it seems to be related to half of the cases of chronic non-A, non-B liver disease negative for anti-C100-3.

Ribavirin dose reduction raises relapse rate dose-dependently in genotype 1 patients with hepatitis C responding to pegylated interferon alpha-2b plus ribavirin

Summary.  The impact of ribavirin exposure on virologic relapse remains controversial in combination therapy with pegylated interferon (Peg‐IFN) and ribavirin for patients with chronic hepatitis C (CH‐C) genotype 1. The present study was conducted to investigate this. Nine hundred and eighty‐four patients with CH‐C genotype 1 were enrolled. The drug exposure of each medication was calculated by averaging the dose actually taken. For the 472 patients who were HCV RNA negative at week 24 and week 48, multivariate logistic regression analysis showed that the degree of fibrosis (P = 0.002), the timing of HCV RNA negativiation (P < 0.001) and the mean doses of ribavirin (P < 0.001) were significantly associated with relapse, but those of Peg‐IFN were not. Stepwise reduction of the ribavirin dose was associated with a stepwise increase in relapse rate from 11% to 60%. For patients with complete early virologic response (c‐EVR) defined as HCV RNA negativity at week 12, only 4% relapse was found in patients given ≥12 mg/kg/day of ribavirin and ribavirin exposure affected the relapse even after treatment week 12, while Peg‐IFN could be reduced to 0.6 μg/kg/week after week 12 without the increase of relapse rate. Ribavirin showed dose‐dependent correlation with the relapse. Maintaining as high a ribavirin dose as possible (≥12 mg/kg/day) during the full treatment period can lead to suppression of the relapse in HCV genotype 1 patients responding to Peg‐IFN alpha‐2b plus ribavirin, especially in c‐EVR patients.