Hepatitis C virus (HCV) infection is a major global health problem affecting approximately 170 million people worldwide1. The prevalence varies notably from nearly 0.1%-1% in Europe until the 5% in Asia and Africa2,3. More than 20 % of these patients will progress to cirrhosis, hepatocellular carcinoma, liver transplantation or death4. Therefore, all patients are candidates for antiviral therapy. Dual PEGylated interferon α (PEG-IFN) ribavirin therapy has been the main HCV treatment of the last decade. However, the percentage of patients achieving a sustained virologic response (SVR), defined as undetectable HCV RNA 24 weeks after completion of treatment, is below target especially for the difficult to treat HCV genotypes 1 and 4 with dual therapy. Recently, a number of direct-acting antiviral agents (DAAs) have been developed for use with PEG-IFN/ribavirin as triple or IFN-free therapies. The first DAAs generation boceprevir and telaprevir in combination with PEG-IFN/ribavirin increased the SVR rates to 66%-75% in previously untreated HCV1 adults vs. 38% in the PEG-IFN/ribavirin group5,6. Although triple therapy has improved SVR rates, these combinations have some drawbacks, including drug-drug interactions, a low efficacy in patients with HCV 2, 3 and 4 genotypes and in those who have not responded to previous dual PEG-IFN/ribavirin therapy. In this respect, second-generation protease inhibitors, such as simeprevir, asunaprevir, and danoprevir, are currently being evaluated or used in combination with PEG-IFN/ribavirin in an effort to overcome the limited efficacy of the first-generation protease inhibitors7,9. In addition, new inhibitors of the NS5A/NS5B HCV polymerase ledipasvir and sofosbuvir are currently available as part of PEG-IFN/ribavirin triple therapy as well as in IFN-free dual therapy10.

Understanding the host and viral factors associated with viral clearance is necessary for individualizing therapy to maximize SVR rates, prevent progression to liver disease, and increase the overall benefits of therapy with respect to its prohibitive costs.

The SVR rate varies drastically among different races and ethnicities, with patients of African ancestry having much lower response rates as compared with Caucasians and Asians, supporting genetic predisposition to response11. Both host and viral factors have been previously reported to be associated with SVR. Predictors of SVR include HCV genotypes 2 and 3, lower baseline serum HCV RNA level, younger age, female sex, lower hepatic fibrosis stage, lack of insulin resistance, and lower body mass index12. Host genetics is increasingly recognized as important modulator of the therapy success. Genome-wide association studies have shown that single nucleotide polymorphisms (SNPs) in or near the interleukin-28B (IL-28B) gene are significantly associated with the treatment outcome for HCV-113. Therefore, the assessment of the host IL28B genotype is increasingly used among HCV genotype-1 patients to inform clinical prediction of treatment outcome14. More specifically, the rs12979860 and rs8099917 SNPs were the two most examined: they were found in linkage disequilibrium each other, and to be similarly informative as host predictors of sustained viral clearance. In a recent meta-analysis of results from nine studies regarding the correlation of IL28B genotype with PEG-IFN/ribavirin SVR in 3110 Caucasian HCV-1 patients, the favourable IL28B genotype CC was present in 41% of individuals, and 67% of them achieved SVR compared to 37% of those with the unfavourable genotypes15. Other pharmacogenetic studies have been performed in different populations, showing a broad number of SNPs located in different genes of the host immune response, inflammatory, fibrosis and virus response pathways to be associated with the SVR to PEG-IFN/ribavirin dual therapy16. Although genetic markers are used as predictors of PEG-IFN/ribavirin in clinics, these only explain the 60-70% of SVR in Caucasian population. Given the recent introduction of multiple new DAAs, HCV treatment options have increased in parallel to its elevated costs, making difficult the optimal treatment decision. Pharmacogenetic studies of specific populations together with the analysis of different clinical variables may be of potential value to appropriately select candidates for dual PEG-IFN/ribavirin therapy in the era of new DAAs.

The present study was designed to analyze the predictive value of different SNPs located at different host immune, inflammatory and liver fibrotic genes in a representative multicenter Spanish cohort of responders and non-responder HCV patients treated with PEG-IFN/ribavirin therapy.

Different DAAs have been approved recently for HCV infection. These compounds used in different combinations, including PEG-INF-based triple therapies to INF-free oral bitherapies, have increased SVR rates even above 90%18. In this scenario, host genetics might become appropriate to target patients who could more likely respond to conventional dual therapy with PEG-IFN and ribavirin which represents an efficient tool to individualize therapy.

The present work is a pharmacogenetic study conducted in a representative Spanish population of two cohorts of HCV infected patients who reached SVR and non-SVR to dual PEG-IFN and ribavirin therapy. In addition to well-known loci on the IL-28B gene, the previously unobserved SNPs located in the TNFRSF1B (rs1061622) and in different IL-10/ IL-4/ IL-6R/TNFα haplotypes were shown to have a significant association with the outcome of HCV treatment using PEG-IFNα and ribavirin. After multivariate analysis, the IL-28B rs12979860 CC genotype and TNFRSF1B/ IL-10/ IL-4 non-TTC haplotype were independently associated with SVR along with low baseline HCV RNA load and HCV genotypes 2 and 3 which may help to predict successful outcome in Spanish population.

Many studies over the last three decades have evaluated genetic predictors of HCV treatment response. Although the IL-28B genotype has emerged as the strongest predictor of treatment response, a recent systematic review found that more than thirty genes have been associated with treatment response to INF-based therapies16. In this study we found an independent SVR predictive value for the IL-28B rs12979860 CC genotype but not for the rs7248668, rs8105790 or rs8099917 SNPs mainly because they are in linkage disequilibrium with rs12979860. It has been commented before the importance of the ethnicity to predict SVR. In fact, IL-28B frequency in each different SNP varies depending of the ethnicity which explains different responses to dual PEG-IFN/ribavirin treatment. Thus, for example, the Asian population shows a favorable 77% rs12979860 CC genotype distribution vs. 38% in Caucasian and 15% in African population which reflects the highest SVR of the Asian population13,19,20. In addition, the most powerful IL-28B rs8099917 genotype predicts near to 90% of SVR in Asian population13 while in Caucasian population the best predictor was rs12979860 with a 60-70% of predictive value. Although Spanish population belongs to Caucasian ethnicity, to our knowledge there are no studies with a representative Spanish population from different regions which may add some variation to the current data. In this work we observed a 38.2% of rs12979860 CC genotype distribution in the general population studied, similar to the previously described in Caucasian population. Patients with SVR showed a 71.8% of rs12979860 CC genotype vs. 28.2% of non-responders, slightly lower than in general Caucasian population of the original studies13. Furthermore, the rs8099917 favorable TT genotype was the worst predictive IL-28B SNP. In terms of clinical use, the haplotype analysis revealed that combining all IL-28B SNPs results did not improve the strength of the association between the IL-28B genotype and SVR as compared with the results using rs12979860 genotype alone as recently was observed in other populations21. Cytokines and chemokines which are secreted by immune cells contribute to viral control, or liver damage. Examples are INFα/β, INFγ, TNFα, IL-6, IL-10, IL-4 and transforming growth factor beta (TGFβ) between others22. They are able to render uninfected cells resistant to infection and cure the infected ones from the virus by stopping viral replication. In fact, PEG-INF-based therapy mimics the effect of host defense against HCV, increasing the expression of interferon stimulated genes (ISG) to kill HCV. In this regard, recent works have shown an association of different SNPs on TNFα (rs1800629), TGFβ (rs1800469), IL-6 (rs1800797), IL-10 (rs1800896) and IL-4 (rs2070874) with SVR to PEG-INF ribavirin therapy16,23 which highlight the importance of an optimal host genetic anti-viral response for success of dual therapy.

TNF-α can bind to two different receptors: TNFRSF1A (tumour necrosis factor receptor superfamily 1A) and TN-FRSF1B (tumour necrosis factor receptor superfamily 1B); which activates NF-κB and triggers several inflammatory pathways with anti-viral properties24,25. Rs1061622 is located in the exon 6 of TNFRSF1B, and the change of T to G causes a functional amino acid substitution at codon 196 from methionine (Met) to arginine (Arg) in the translated protein TNF receptor 2 (TNFR2). This variant G is supposed to produce a change of biological function of the TNFR2 protein affecting TNF-a binding and NF-κB activation which consequently modifies INF I and III production25. Interestingly, in some drug sensitivity studies, rs1061622 was found to be associated with a favorable prognosis of non-small cell lung cancer patients treated with chemoradiotherapy26 and with a beneficial response to infliximab in Crohn’s disease27. In this work, it was observed for the first time an association of the TNFR-SF1B rs1061622 G allele with a SVR. In the same way, different haplotypes combining IL-10, IL-6R, TNFα and IL-4 cytokines increased the predictive value of TNFRS-F1B rs1061622, indicating that anti-viral inflammatory response is a complex molecular process which encompasses a broad number of inflammatory cells and molecular pathways. In fact, TNFRSF1B/ IL-10/ IL-4 non-TTC hplotype reached significant association with SVR in the multivariate analysis.

As part of the different inflammatory pathways involved in HCV liver inflammation, GNB3 rs5443 CC genotype, which encodes a splice variant of the β3 subunit of heterotrimeric G proteins, has been associated with an inactivation of GNB3, thus reducing the activation of broad number of inflammatory receptors coupled to GNB3. This fact, may explain rapid normalization of AST and ALT hepatic enzymes in GNB3 CC carriers observed in this work, although no association with SVR was observed.

However, as we commented before, host genetics cannot explain the 100% of SVR. There are other clinical and viral factors that influence the response to PEG-IFN/ribavirin treatment. In this work we observed an association of lower age, lower basal HCV-RNA load, HCV2 and HCV3 genotypes, lower fibrosis stage F0-F2, and elevated LDL cholesterol with a SVR, which confirm previous reports28,29. However, the multivariate analysis independently associated lower basal HCV-RNA load, HCV 2 and 3 genotypes as predictors of SVR, probably because the study sample did not reach enough size.

In summary, we found genetic, viral an clinical parameters associated with SVR, particularly IL-28B rs12979860 CC, TNFRSF1B/ IL-10/ IL-4 non-TTC haplotype, low baseline HCV RNA load and HCV genotypes 2 and 3 which may help to predict successful outcome to PEG-IFN/ribavirin therapy in Spanish population. Further analysis would be required to confirm these associations with triple therapy including new DAAs.