Kentaro Matsuura

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).

Pre-treatment prediction of response to pegylated-interferon plus ribavirin for chronic hepatitis C using genetic polymorphism in IL28B and viral factors

BACKGROUND & AIMS Pegylated interferon and ribavirin (PEG-IFN/RBV) therapy for chronic hepatitis C virus (HCV) genotype 1 infection is effective in 50% of patients. Recent studies revealed an association between the IL28B genotype and treatment response. We aimed to develop a model for the pre-treatment prediction of response using host and viral factors. METHODS Data were collected from 496 patients with HCV genotype 1 treated with PEG-IFN/RBV at five hospitals and universities in Japan. IL28B genotype and mutations in the core and IFN sensitivity determining region (ISDR) of HCV were analyzed to predict response to therapy. The decision model was generated by data mining analysis. RESULTS The IL28B polymorphism correlated with early virological response and predicted null virological response (NVR) (odds ratio=20.83, p<0.0001) and sustained virological response (SVR) (odds ratio=7.41, p<0.0001) independent of other covariates. Mutations in the ISDR predicted relapse and SVR independent of IL28B. The decision model revealed that patients with the minor IL28B allele and low platelet counts had the highest NVR (84%) and lowest SVR (7%), whereas those with the major IL28B allele and mutations in the ISDR or high platelet counts had the lowest NVR (0-17%) and highest SVR (61-90%). The model had high reproducibility and predicted SVR with 78% specificity and 70% sensitivity. CONCLUSIONS The IL28B polymorphism and mutations in the ISDR of HCV were significant pre-treatment predictors of response to PEG-IFN/RBV. The decision model, including these host and viral factors may support selection of optimum treatment strategy for individual patients.

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.

Distribution of Hepatitis B Virus Genotypes among Patients with Chronic Infection in Japan Shifting toward an Increase of Genotype A

ABSTRACT Acute hepatitis B virus (HBV) infection has been increasing through promiscuous sexual contacts, and HBV genotype A (HBV/A) is frequent in patients with acute hepatitis B (AHB) in Japan. To compare the geographic distribution of HBV genotypes in patients with chronic hepatitis B (CHB) in Japan between 2005 and 2006 and between 2000 and 2001, with special attention to changes in the proportion of HBV/A, a cohort study was performed to survey changes in genotypes of CHB patients at 16 hospitals throughout Japan. Furthermore, we investigated the clinical characteristics of each genotype and examined the genomic characteristics of HBV/A isolates by molecular evolutionary analyses. Of the 1,271 patients, 3.5%, 14.1%, and 82.3% were infected with HBV/A, -B, and -C, respectively. In comparison with our previous survey during 2000 and 2001, HBV/A was twice as frequent (3.5% versus 1.7%; P = 0.02). The mean age was lower in the patients with HBV/A than in those with HBV/B or -C. Based on phylogenetic analyses of 11 full-length genomes and 29 pre-S2/S region sequences from patients, HBV/A isolates were imported from Europe and the United States, as well as the Philippines and India. They clustered with HBV/A from AHB patients and have spread throughout Japan. HBV/A has been increasing in CHB patients in Japan as a consequence of AHB spreading in the younger generation through promiscuous sexual contacts, aided by a tendency of HBV/A to induce chronic hepatitis. The spread of HBV/A infection in Japan should be prevented by universal vaccination programs.

Genome-wide association study identified ITPA/DDRGK1 variants reflecting thrombocytopenia in pegylated interferon and ribavirin therapy for chronic hepatitis C

Hematologic abnormalities during current therapy with pegylated interferon and ribavirin (PEG-IFN/RBV) for chronic hepatitis C (CHC) often necessitate dose reduction and premature withdrawal from therapy. The aim of this study was to identify host factors associated with IFN-induced thrombocytopenia by genome-wide association study (GWAS). In the GWAS stage using 900K single-nucleotide polymorphism (SNP) microarrays, 303 Japanese CHC patients treated with PEG-IFN/RBV therapy were genotyped. One SNP (rs11697186) located on DDRGK1 gene on chromosome 20 showed strong associations in the minor-allele-dominant model with the decrease of platelet counts in response to PEG-IFN/RBV therapy [P = 8.17 × 10(-9); odds ratio (OR) = 4.6]. These associations were replicated in another sample set (n = 391) and the combined P-values reached 5.29 × 10(-17) (OR = 4.5). Fine mapping with 22 SNPs around DDRGK1 and ITPA genes showed that rs11697186 at the GWAS stage had a strong linkage disequilibrium with rs1127354, known as a functional variant in the ITPA gene. The ITPA-AA/CA genotype was independently associated with a higher degree of reduction in platelet counts at week 4 (P < 0.0001), as well as protection against the reduction in hemoglobin, whereas the CC genotype had significantly less reduction in the mean platelet counts compared with the AA/CA genotype (P < 0.0001 for weeks 2, 4, 8, 12), due to a reactive increase of the platelet count through weeks 1-4. Our present results may provide a valuable pharmacogenetic diagnostic tool for tailoring PEG-IFN/RBV dosing to minimize drug-induced adverse events.

Role of IL28B for chronic hepatitis C treatment toward personalized medicine

Genome‐wide association studies recently revealed that certain interleukin‐28B (IL28B) polymorphisms are strongly associated with responses to pegylated interferon (PEG‐IFN) and ribavirin (RBV) therapy in patients chronically infected with hepatitis C virus (HCV) genotype 1, as well as with spontaneous clearance of HCV. Subsequent reports revealed that IL28B genotypes also affect treatment efficacy in chronic infection with other HCV genotypes. Furthermore, there have been several reports that implicate IL28B genotypes in inflammatory status, progression of fibrosis and adverse clinical outcomes in chronic hepatitis C (CHC). Therapy of CHC recently entered a new era with the deployment of direct‐acting antivirals. These include nonstructural 3/4A protease inhibitors which have shown promise in combination with PEG‐IFN/RBV in several clinical trials. IFN‐free therapy is expected to be useful especially in IFN‐resistant patients and may become the standard of care in the future. Several clinical trials have revealed an association between IL28B genotype and treatment efficacy in triple therapy or IFN‐free regimens. On the other hand the mechanism of the effect of IL28B on HCV infection has not yet been elucidated. Recently, it was shown that the polymorphism of IFN‐lambda 4 (IFNL4) is in high linkage disequilibrium with that of near IL28B, and more strongly associated with spontaneous or treatment‐induced HCV clearance than IL28B genotypes, especially in individuals of African ancestry. This finding provides new insights into the genetic regulation of HCV clearance and its clinical management. IL28B genotyping will be also useful for personalized CHC treatment in the forthcoming era of direct‐acting antivirals.

Virological and clinical characteristics on reactivation of occult hepatitis B in patients with hematological malignancy

The virological characteristics of hepatitis B virus (HBV) implicated in the reactivation of occult hepatitis B in patients who have received hematopoietic stem‐cell transplantation or chemotherapy for the hematological malignancy are not well defined. Twenty‐eight HBsAg‐negative patients who received hematopoietic stem‐cell transplantation and 138 HBsAg‐negative patients treated for malignant lymphoma with chemotherapy including rituximab were enrolled. Three of the 28 patients (10.7%) received hematopoietic stem‐cell transplantation and one of the 138 (0.72%) patients treated for malignant lymphoma with chemotherapy developed de novo HBV hepatitis. Anti‐HBc was detected in four and anti‐HBs in two patients. Genotype Bj was detected in two and C in two of they all possessed wild‐type sequences in the core promoter region. A precore stop mutation (A1896) was detected in a patient with genotype Bj who developed fulminant hepatic failure. HBV DNA was detected in pretreatment HBsAg‐negative samples in two of four patients, and the HBV genome sequence identified from sera before chemotherapy and at the time of de novo HBV hepatitis showed 100% homology. In an in vitro replication model, genotype Bj with the A1896 clone obtained from a fulminant case had a replication level much higher than clones obtained from de novo hepatitis B patients with genotype Bj or C with G1896. In conclusion, this is the first report demonstrating de novo hepatitis B from the reactivation of occult HBV infection confirmed by molecular evolutional analysis. The fulminant outcome of HBV reactivation can be associated with genotype Bj exhibiting high replication due to the A1896 mutation. J. Med. Virol. 83:412–418, 2011.

Relationship between polymorphisms of the inosine triphosphatase gene and anaemia or outcome after treatment with pegylated interferon and ribavirin.

BACKGROUND A genome-wide association study revealed an association between variants of the inosine triphosphatase (ITPA) gene and ribavirin (RBV)-induced anaemia. The aim of this study was to replicate this finding in an independent Japanese cohort and to define a method to allow pretreatment prediction of anaemia in combination with other factors. METHODS Genotype 1b chronic hepatitis C patients (n=132) treated with pegylated interferon (PEG-IFN)-α and RBV for 48 weeks were genotyped for ITPA rs1127354 and examined for anaemia and treatment outcome. RESULTS Variants of the ITPA gene protected against severe anaemia throughout the 48-week treatment period and were associated with lower incidence of anaemia-related RBV dose reduction. A combination of the ITPA genotype with baseline haemoglobin (Hb) and creatinine clearance (CLcr) levels predicted severe anaemia with high accuracy (90% sensitivity and 62% specificity). Among a subset of patients with the IL28B genotype of TT at rs8099917, patients with variants of the ITPA gene were associated with a higher rate of receiving >80% of the expected RBV dose, a higher rate of sustained virological response (SVR), and a lower rate of relapse. CONCLUSIONS The variants of the ITPA gene, which could protect against haemolytic anaemia and RBV dose reduction, were associated with a high rate of SVR by standard PEG-IFN and RBV therapy in a subset of Japanese patients with the favourable TT genotype at rs8099917 of IL28B. A combination of ITPA genetic polymorphisms with baseline Hb and CLcr levels further improves the predictive accuracy of severe anaemia.

Investigation of residual hepatitis C virus in presumed recovered subjects

Recent studies have found hepatitis C virus (HCV) RNA in peripheral blood mononuclear cells (PBMCs) of the majority of presumed recovered subjects. We investigated this unexpected finding using samples from patients whose HCV RNA and anti‐HCV status had been serially confirmed. HCV RNA was detected in PBMCs from 66 of 67 chronic HCV carriers. Subpopulation analysis revealed that the viral load (log copies/106 cells) in B cells (4.14 ± 0.71) was higher than in total PBMCs (3.62 ± 0.71; P < 0.05), T cells (1.67 ± 0.88; P < 0.05), and non‐B/T cells (2.48 ± 1.15; P < 0.05). HCV negative‐strand RNA was not detected in PBMCs from any of 25 chronically infected patients. No residual viral RNA was detected in total PBMCs or plasma of 59 presumed recovered subjects (11 spontaneous and 48 treatment induced) using nested real‐time polymerase chain reaction with a detection limit of 2 copies/μg RNA (from ∼1 × 106 cells). PBMCs from 2 healthy HCV‐negative blood donors became HCV RNA positive, with B‐cell predominance, when mixed in vitro with HCV RNA–positive plasma, thus passively mimicking cells from chronic HCV carriers. No residual HCV was detected in liver or other tissues from 2 spontaneously recovered chimpanzees. Conclusion: (1) HCV RNA was detected in PBMCs of most chronic HCV carriers and was predominant in the B‐cell subpopulation; (2) HCV detected in PBMCs was in a nonreplicative form; (3) HCV passively adsorbed to PBMCs of healthy controls in vitro, becoming indistinguishable from PBMCs of chronic HCV carriers; and (4) residual HCV was not detected in plasma or PBMCs of any spontaneous or treatment‐recovered subjects or in chimpanzee liver, suggesting that the classic pattern of recovery from HCV infection is generally equivalent to viral eradication. (HEPATOLOGY 2013)

Positive Selection of Core 70Q Variant Genotype 1b Hepatitis C Virus Strains Induced by Pegylated Interferon and Ribavirin

BACKGROUND Approximately 20% of patients with hepatitis C virus (HCV) genotype 1b infection have nonresponse to the most current treatment, pegylated interferon with ribavirin. Mutations in the HCV core region were recently proposed to be associated with nonresponse. Our aim was to evaluate the viral factors associated with treatment failure. METHODS HCV variants were determined directly and after cloning in 66 HCV-1b-infected Japanese patients and in 5 urokinase-type plasminogen activator transgenic severe combined immunodeficiency mice with human hepatocytes (chimeric mice), at baseline, during treatment, and after treatment. RESULTS At baseline, glutamine at position 70 of the HCV core protein (70Q) was detected by direct sequencing in 20% of patients with virologic response and in 43.8% of patients with nonresponse. Among patients with nonresponse, who were examined during and after treatment, the prevalence of the 70Q substitution increased to 56.3%, which indicates that treatment-induced selection occurred in all patients with nonresponse who had 70Q quasispecies detectable by cloning. This observation was reinforced by the results from experimentally infected chimeric mice. Logistic regression analysis indicated that detection of 70Q quasispecies was associated with a statistically significantly increased risk of nonresponse (odds ratio, 15.1; P = .004) in the studied cohort. CONCLUSION Presence of the 70Q quasispecies at baseline was associated with an increased risk of treatment failure, as indicated by the positive selection of the 70Q clones induced by treatment with pegylated interferon with ribavirin. These results urge further investigation of the mechanisms of this association.