Single-nucleotide polymorphisms in GALNT8 are associated with the response to interferon therapy for chronic hepatitis C.

New anti-hepatitis C virus (HCV) therapeutics developed recently are more effective and lead to improvements in sustained viral response. However, interferon (IFN) monotherapy is still used to a limited extent for fear of adverse effects. This study investigated host genetic factors affecting the IFN response in patients with chronic hepatitis C (CHC). Using a two-step design, a large-scale association screening including 1088 Japanese CHC patients treated with IFN was performed employing ~70 000 gene-based single-nucleotide polymorphisms (SNPs). Replication was tested in an independent Japanese cohort of 328 patients. Fine-mapping and functional analyses were also performed. Through two-step screening, it was found that rs2286580 in intron 6 of the gene encoding N-acetylgalactosaminyltransferase 8 (GALNT8) on chromosome 12 was significantly associated with a sustained viral response (combined P = 3.9×10(-6), odds ratio 1.52, 95 % confidence interval 1.29-1.82). The association was replicated in an additional cohort of 328 Japanese patients. In subgroup analysis, GALNT8 variants were associated with treatment outcome independently of HCV genotype. By contrast, the outcome of pegylated IFN and ribavirin combined therapy was not affected by the SNP. Fine-mapping analysis revealed that the association peak was at rs10849138 in intron 6 of GALNT8. Allele-specific transcription analysis demonstrated that GALNT8 expression was upregulated by an unfavourable allele of the variant. A luciferase reporter assay demonstrated that overexpression of GALNT8 attenuated IFN-α-induced gene transcription via the IFN-stimulated response element. These results suggest that GALNT8 variants contribute to the response to IFN therapy against CHC, providing a new insight into antiviral mechanisms of IFN.

IL-28B predicts response to chronic hepatitis C therapy--fine-mapping and replication study in Asian populations.

Type I interferon (IFN) is used for the treatment of chronic hepatitis C virus (HCV) infection. Despite advances in antiviral therapy, a large proportion of patients remain infected following current therapies. Through a genome-wide scan, we found two variants (rs8099917 and rs12979860) in the IL-28B locus that affect the outcome of PEG-IFN and ribavirin combination therapy, consistent with recent studies (P = 6.52×10(-8); odds ratio 2.46 and P = 8.63×10(-8), odds ratio 2.40, respectively). Significant associations were also observed in the case of IFN monotherapy for HCV genotypes 1b and 2a. With rs8099917, HCV genotype 1b patients had a significantly lower frequency of the favourable genotype (86.6 %) compared with healthy controls (91.7 %), and HCV genotype 2a patients had an intermediate frequency (89.9 %). Similar results were found for rs12979860. Fine-mapping analysis revealed that rs8099917 had the strongest association with treatment outcome and 14 others, including four novel single nucleotide polymorphisms, had comparable associations. Haplotype analysis revealed that none of the haplotypes showed stronger association than any single marker. Early non-responders who could not achieve 2 log viral decline during the first 12 weeks of treatment had higher odds ratios for these two variants. The favourable allele of rs8099917 is also associated with initial viral decline at 2 and 4 weeks following the start of therapy. Multivariate analysis of PEG-IFN and ribavirin-treated patients showed that rs8099917 genotype, viral load, fibrosis and age were significant predictors of response to therapy. Common variation at the IL-28B locus is predictive of various IFN-based therapies for HCV independent of regimen or HCV genotype.

ITPA polymorphism affects ribavirin-induced anemia and outcomes of therapy--a genome-wide study of Japanese HCV virus patients.

BACKGROUND & AIMS: Ribavirin-induced anemia is one of the major causes of discontinuation and dose reduction during anti-hepatitis C virus therapy. Factors influencing this anemia, especially host genetic factors, are poorly understood. In this study we investigated predictive factors in hepatitis C virus patients treated with combination therapy. METHODS: We performed a 2-step genome-wide screening followed by replication analysis and fine-mapping using a total of 923 Japanese hepatitis C virus 1b-infected patients treated with pegylated-interferon plus ribavirin. We also applied logistic regression analysis to search for possible independent associations of clinical parameters and genetic variants with treatment-induced hemoglobin (Hb) decline as well as treatment outcomes. RESULTS: We identified a variant, located upstream of the inosine triphosphate pyrophosphatase gene on chromosome 20p13 that is significantly associated with treatment-induced anemia (combined P = 6.0 × 10(-14)). Resequencing and fine-mapping revealed several single nucleotide polymorphisms (SNPs) strongly associated with Hb decline, including the nonsynonymous SNP rs1127354 (P = 3.5 × 10(-44)), which was recently reported for other ethnic groups. Another reported SNP, the splicing variant-related SNP rs7270101, was not polymorphic in the Japanese population. Stratified analysis based on rs1127354 genotype revealed that inosine triphosphate pyrophosphatase expression is not correlated with Hb decline, suggesting that rs1127354 is a direct causal variant in the Japanese population. Multivariate analysis demonstrated that age, baseline Hb, baseline platelet count, and rs1127354 were independently associated with severe anemia (Hb <10 g/dL). CONCLUSIONS: A missense substitution in inosine triphosphate pyrophosphatase gene affects ribavirin-induced anemia in hepatitis C virus-infected Japanese patients.

Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus.

The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was 55 degrees C for Bst-RNase HIII and 35 degrees C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 degrees C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.

Importance of an N-terminal extension in ribonuclease HII from Bacillus stearothermophilus for substrate binding.

The gene encoding ribonuclease HII from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The overproduced protein, Bst-RNase HII, was purified and biochemically characterized. Bst-RNase HII, which consists of 259 amino acid residues, showed the highest amino acid sequence identity (50.2%) to Bacillus subtilis RNase HII. Like B. subtilis RNase HII, it exhibited Mn2+-dependent RNase H activity. It was, however, more thermostable than B. subtilis RNase HII. When the Bst-RNase HII amino acid sequence is compared with that of Thermococcus kodakaraensis RNase HII, to which it shows 29.8% identity, 30 residues are observed to be truncated from the C-terminus and there is an extension of 71 residues at the N-terminus. The C-terminal truncation results in the loss of the alpha9 helix, which is rich in basic amino acid residues and is therefore important for substrate binding. A truncated protein, Delta59-Bst-RNase HII, in which most of the N-terminal extension was removed, completely lost its RNase H activity. Surface plasmon resonance analysis indicated that this truncated protein did not bind to the substrate. These results suggest that the N-terminal extension of Bst-RNase HII is important for substrate binding. Because B. subtilis RNase HII has an N-terminal extension of the same length and these extensions contain a region in which basic amino acid residues are clustered, the Bacillus enzymes may represent a novel type of RNase H which possesses a substrate-binding domain at the N-terminus.