FULL MANUSCRIPT JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/jir.2014.0088 Homozygosity for HLA Group 2 Alleles Predicts Treatment Failure with Interferon-a and Ribavirin in Chronic Hepatitis C Virus Genotype 1 Infection Meadhbh Collison,1 Jun Liong Chin,2 Ahmed Abu Shanab,2 Ross Mac Nicholas,2 Ricardo Segurado,3 Suzie Coughlan,1 Jeff Connell,1 Michael J. Carr,1 Raphael B. Merriman,2 P. Aiden McCormick,2 and William W. Hall1 Host genetic factors influence treatment responses to antiviral therapy in chronic hepatitis C virus (HCV) infection. We retrospectively investigated associations between host genetic markers and treatment-induced virologic responses to dual therapy with interferon-a and ribavirin in chronically infected HCV genotype 1 (g1)– and genotype 3 (g3)–infected individuals. A total of 171 patients (89 HCV g1 and 82 HCV g3 infected) were investigated for genetic markers influencing treatment-induced sustained virologic response (SVR). Overall, SVR was observed for 46/89 (52%) HCV g1- and 57/82 (70%) HCV g3-infected patients. Of the 4 interleukin 28B (IL28B) single-nucleotide polymorphisms (SNPs), rs12979860 was the host genetic marker most significantly associated with failure to achieve an SVR in HCV g1-infected individuals [P = 3.83 · 10 - 4; odds ratio (OR) = 5.61; confidence interval (CI) = 2.07–15.18] and gave a positive predictive value for treatment failure of 81.3% for minor homozygotes (TT). Using additive (P = 3.54 · 10 - 4) and dominant models (P = 3.83 · 10 - 4), a dosage effect of the T allele was observed, with the dominance term not significant for this SNP. Logistic regression showed an association between HLA-C1/C1 and rapid virologic response in HCV g1 infections with an OR relative to the heterozygote of 10.0 (95% CI: 1.6–62.5, P = 0.014). HLA-C2 homozygosity was a significant predictor of nonresponse to treatment in HCV g1–infected individuals (P = 0.023). and its transport within the cells is mediated primarily by the equilibrative nucleoside transporter (ENT), ENT1 (Fukuchi and others 2010). Morello and others (2010) identified an SNP, termed rs760370, within the ENT1 gene to be associated with rapid virologic response (RVR) in HIV/HCV-coinfected patients who had received peg-IFN-a with ribavirin. Cysteine-cysteine chemokine receptor 5 (CCR5) is expressed on the surface of various cell types, including monocytes, macrophages, memory T cells, Th1 cells, and immature dendritic cells (He and others 1997), and studies that investigated the role of CCR5D32 in HCV infection have reported conflicting results regarding spontaneous clearance of HCV and susceptibility to infection (Mascheretti and others 2004; Goulding and others 2005; Nattermann and others 2011). Significant fibrosis (Ishak stage > 2) has been associated with a reduced chance of achieving SVR, and has also been shown to have a negative impact on early viral kinetics during dual therapy (Guedj and others 2012). An association with the rs738409 SNP in the PNPLA3 gene and presence of cirrhosis and accelerated fibrosis progression has been reported, Introduction D ual therapy for chronic hepatitis C virus (HCV) infection has consisted of pegylated interferon-a in combination with ribavirin; however, this is only effective in 40%–60% of HCV genotype 1 (g1) infections (Chou and others 2013). Several host genetic factors are known to influence treatment response. The interleukin 28B (IL28B) single-nucleotide polymorphism (SNP) rs12979860 is currently employed, in conjunction with other laboratory and clinical parameters, as a pretreatment predictor of sustained virologic response (SVR). Evidence exists to suggest that typing for a number of different IL28B SNPs and identifying an IL28B haplotype associated with treatment response may more accurately predict treatment outcome than typing for rs12979860 alone (Smith and others 2011). Additional host markers predicting spontaneous HCV clearance and treatment response in chronic HCV (CHC)infected individuals have also been described. The uptake of the guanosine nucleotide analogue ribavirin by hepatocytes 1 2 3 National Virus Reference Laboratory, University College Dublin, Dublin, Ireland. Liver Unit, St Vincent’s University Hospital, Dublin, Ireland. Centre for Support and Training in Analysis and Research (CSTAR), University College Dublin, Dublin, Ireland. 1 2 suggesting this SNP as a marker for treatment response (Trepo and others 2011; Valenti and others 2011). The natural killer (NK) cell receptor family, killer immunoglobulin-like receptor (KIR), and its ligand, HLAC, are known to play an important role in the immune response to many viral infections, including HCV [reviewed in Rajagopalan and Long (2005)]. While some combinations of KIRs and HLA-C appear to have a protective effect against HCV infection or are associated with an SVR, other combinations have been shown to have a detrimental effect and contribute to chronic infection, disease progression, or nonresponse to therapy (Khakoo and others 2004; Knapp and others 2010). A relationship between KIRs, HLA-C, and IL28B polymorphisms has also been identified (Dring and others 2011; Suppiah and others 2011). Based upon these data, we have investigated whether a combination of innate immune genes and other loci may better predict the response to therapy and improve the selection of patients for treatment, rather than 1 genetic biomarker alone. In this retrospective, single-center, observational study, we analyzed a suite of genetic markers in a cohort of HCVmonoinfected patients to evaluate the association of host genetic markers, viral genotype, and early viral load kinetics on treatment response in patients receiving interferon-based therapy with ribavirin. Materials and Methods Study population One hundred seventy-one individuals chronically infected with HCV were included in the present study. All were monoinfected with either HCV g1 or g3. The HCV g1-infected cohort comprised 89 individuals (46 responders and 43 nonresponders) and the HCV g3-infected cohort consisted of 82 individuals (57 responders and 25 nonresponders). HCV viral load (IU/mL) was determined for individuals at baseline (pretreatment) and at 4 and 12 weeks after starting treatment. Treatment responders were defined as those with HCV RNA decline ‡ 2-log10 IU/mL from pretreatment HCV viral load at week 12 and who had undetectable levels of HCV RNA 24 weeks after treatment cessation. Treatment nonresponders were defined as those individuals who did not achieve an on-treatment RNA decline ‡ 2-log10 IU/mL from baseline levels at 12 weeks or who had detectable HCV RNA levels 24 weeks after treatment was discontinued. Plasma and serum samples were obtained from the National Virus Reference Laboratory (NVRL), University College Dublin (UCD). Samples had been collected between 2002 and 2012 and stored at - 20C. Ethical approval for this study was obtained from the ethics and medical research committee of St. Vincent’s University Hospital. Treatment schedule Peginterferon alpha 2a (Pegasys) was administered subcutaneously at a dose of 180 mg per week. Ribavirin was also given at a dose of 1,000 mg/day for < 75 kg and 1,200 mg/day for > 75 kg in the HCV g1-infected individuals and at 800 mg/day for HCV g3-infected individuals. Alternatively, peginterferon alpha 2b (pegIntron) was administered subcutaneously at a dose of 1.5 mg per week and ribavirin was given at a dose of 800–1,400 mg/day for HCV g1- and HCV g3-infected individuals. Length of treatment was 48 weeks for HCV g1 and 24 weeks for g3, with treatment cessation COLLISON ET AL. after 12 weeks if there was not a > 2 log10 decline in HCV viral RNA for HCV g1 and g3. Virological testing Measurements of HCV viral load were made using either the Cobas TaqMan HCV 1.0 assay (Roche Diagnostics, Basel, Switzerland) or the VERSANT HCV 3.0 RNA (bDNA) assay (Siemens Healthcare, Erlangen, Germany). Data were analyzed in IU/mL using previously determined conversion factors of 2.56 and 5.2 for the Roche and Siemens assays, respectively. HCV genotyping data were generated using the Innogenetics VERSANT HCV genotype 2.0 assay (Siemnes Healthcare). DNA isolation and genotyping Genomic DNA was extracted manually from serum and plasma using the viral RNA minikit as described by the manufacturer (Qiagen, Crawley, United Kingdom). Each gene of interest was amplified by polymerase chain reaction sequencespecific primers (PCR-SSP) using HotStarTaq Master Mix (Qiagen). Oligonucleotide primers (Metabion, Martinsried, Germany) used to type the HLA-C genes (HLA-C1 and HLAC2) and the 4 KIR genes (KIR2DL3, KIR2DL2, KIR2DS3, and KIR2DS2) were as previously described (Tajik and others 2010). For HLA typing, the final optimized concentration of HLA-C1 and -C2 primers in each reaction was 0.4 mM, and for the human growth hormone (HGH) endogenous control primers was 0.2 mM. For KIR typing, the final optimized concentration of KIR primers was 1 mM, and the HGH primers was 0.4 mM. End-point PCR for KIR and HLA typing was carried out on a Veriti thermocycler (Applied Biosystems, Foster City, CA) under the following conditions: 95C for 15 min; 40 cycles of 94C for 1 min, 65C for 1 min, and 72C for 1 min; and 72C for 10 min. External quality control panels from the International Histocompatability Working Group (IHWG) in the Fred Hutchinson Cancer Research Center (Seattle, WA) were used to validate the genotyping methodologies and optimize the specificity and sensitivity of the assays prior to screening. For CCR5 genotyping, oligonucleotide primer sequences used for end-point PCR across the 32-bp deletion within CCR5 (rs333) were designed using Primer3Plus software (Primer3Plus.com) and produced amplicons of 202 and 170 bp for wild-type and mutant alleles, respectively. Primers were used at a final concentration of 1 mM (F: 5¢GTCTTCATTACACCTGCAGCTC TC3¢, R: 5¢ATGGTGAAGATAAGCCTCACAGC3¢). Assay-on-demand SNP genotyping assays (Applied Biosystems) were used for allelic discrimination of IL28B rs12979860, rs8099917, rs7248668, ENT1 rs760370, and PNPLA3 rs738409. The assays-by-design SNP genotyping assay (Applied Biosystems) was employed for allelic discrimination of rs4803221. A detection mix consisting of a 40 · mix of unlabelled PCR primers and TaqMan minor-grovebinding probes labeled with a 5¢ VIC (2¢-chloro-7¢-phenyl-1, 4dichloro-6-carboxyfluorescein) for the major alleles and FAM (6-carboxyfluorescein) for the minor alleles for each SNP was diluted to 25 · in nuclease-free water. Allelic discrimination assays were performed on the ABI7300 SDS Real-Time PCR System (Applied Biosystems) with fluorescence acquisition occurring during the annealing/extension stage. Pre- and postassay plate reads were performed for 1 min at 60C to determine the change in fluorescence during the real-time PCR. HLA-C INFLUENCE ON TREATMENT RESPONSE IN CHRONIC HCV Statistical analysis Hardy–Weinberg equilibrium tests were performed for SNPs investigated in the present study for association with treatment response with a 1 degree of freedom (d.f.) chisquare goodness-of-fit test in the R statistical package, version 2.15.0. Genetic association tests were performed in PASW Statistics version 18. Single markers were tested for association with nonresponse using a 2 d.f. chi-square test on the 3 genotype categories, or a 1 d.f. chi-square test assuming a dominant mode of inheritance on a reference homozygote versus collapsed heterozygous and ‘‘risk’’ homozygous genotypes. Presence or absence of the KIR genes was tested for association with nonresponse with a 1 d.f. chisquare test. Linear regression was applied to investigate the additive (dosage) effect of alleles on baseline log10-transformed HCV viral load. The dynamics of change in viral load from baseline through to week-4 and -12 follow-up time-points were examined using repeated-measures analysis of variance (ANOVA). Genotypes of each genetic marker were examined as a categorical between-subject factor, time as a withinsubject factor, and a genotype · time interaction term, to model genotype-mediated differences in the pattern of change in viral load over time. Results Correlation of sustained virologic response with IL28B SNPs In HCV g1-infected individuals, a strong association was found between all 4 IL28B SNPs (rs12979860, rs8099917, rs4803221, and rs7248668) and SVRs (Fig. 1). No signifi- FIG. 1. Percentage frequency of interleukin 28B (IL28B) single-nucleotide polymorphisms (SNPs) in hepatitis C virus (HCV) genotype 1 (g1)–infected treatment responders versus nonresponders. The frequency of the 3 allelic groups— minor homozygous, heterozygous, and major homozygous— is shown for the 4 IL28B SNPs: rs12979860, rs8099917, rs4803221, and rs7248668. For each SNP, allele frequencies are compared between HCV g1-infected responders and nonresponders. 3 cant association between the 4 IL28B polymorphisms and HCV g3-infected patients was observed (Fig. 2). Both dominant and genotypic models were used to identify the most appropriate model to predict treatment failure for the HCV g1-infected cohort. Assuming a dominant model, the minor alleles of all 4 IL28B SNPs were found to be significantly associated with treatment nonresponse (P = 3.83 · 10 - 4, P = 0.004, P = 0.0019, and P = 0.014), respectively. Rs12979860 was the most significant, with the T risk allele the best independent genetic predictor of nonresponse to treatment using the dominant model. With the genotypic ‘‘additive’’ model, a dosage effect of the minor T allele was observed, with heterozygotes less likely to respond to treatment than major homozygotes (CC), and minor homozygotes (TT) less likely to respond to treatment than heterozygotes (CT) with only 1 copy of the risk allele. The rs12979860 minor homozygote (TT) gave a positive predictive value for treatment failure of 81.3%, with the presence of the major allele homozygote (CC) giving a negative predictive value of 77.4% for treatment failure (Table 1). In our cohort, while the IL28B haplotype described by Smith and others (2011) was a significant predictor of treatment outcome in HCV g1-infected individuals (P = 0.0053), the association with treatment nonresponse was not as strong as rs12979860 alone. As rs12979860 was found to be the most significantly associated with virologic response, this SNP was used in all subsequent analyses. While the IL28B SNPs were tested for association with nonresponse with a 2 d.f. genotypic model, and with a 1 d.f. dominant model for the minor allele, the pattern of the genotypic odds ratios (ORs) did not indicate obvious dominance. A logistic regression model was fitted with an additive ( - 1, 0, 1) term and a dominance ( - 0.5, 0.5, - 0.5) genotype parameterization to determine whether any significant dominance variance was present. The dominance term was not significant (P = 0.881; OR = 1.077) in the presence of the additive term (P = 4.67 · 10 - 4; OR = 3.85), for rs12979860 (in HCV g1). Therefore, only the additive term was used in subsequent analyses. In HCV g3-infected individuals, no FIG. 2. Percentage frequency of IL28B SNPs in HCV genotype 3 (g3)–infected treatment responders versus nonresponders. 4 COLLISON ET AL. Table 1. Predictive Values of the IL28B Genetic Variants in HCV g1- and HCV g3-Infected Individuals Receiving Interferon/Ribavirin Therapy HCV genotype g1 Host marker Reference variant Risk variant PPV % NPV % rs12979860 Ca CC Ca CC Ta TT Ga GG Ca CC Ca CC Ta TT Ga GG TT Ta GG Ga GG Ga AA Aa TT Ta GG Ga GG Ga AA Aa 81.3 62.1 75.0 63.8 75.0 63.0 75.0 60.9 16.7 30.2 0.0 33.3 0.0 33.3 50.0 34.4 58.9 77.4 54.3 69.0 54.3 67.4 54.3 65.1 68.4 69.2 69.1 71.4 69.1 71.4 70.0 72.0 rs4803221 rs8099917 rs7248668 g3 rs12979860 rs4803221 rs8099917 rs7248668 Prediction measures were calculated using nonresponse as the outcome of interest, and each genetic variant as the ‘‘test’’ for nonresponse. a Refers to all genotypes containing that allele. Bold values refer to the alleles with the highest positive predictive and negative predictive values. g1, Genotype 1; g3, genotype 3; HCV, hepatitis C virus; IL28B, interleukin 28B; PPV, positive predictive value: the proportion of test positive (risk variant carriers) who were nonresponders; NPV, negative predictive value: the proportion of test negative (reference variant carriers) who were responders. FIG. 3. Frequency of HLA-C1 and -C2 alleles in HCV g1 and g3 treatment responders versus nonresponders. in HCV g1 (n = 89) and g3 (n = 82) responder and nonresponder groups. No significant difference was observed between either HCV g1-infected group (P = 0.575) or HCV g3-infected group (P = 0.557). The frequency of the PNPLA3 rs738409 SNP was also examined between groups and no significant difference was found between responders and nonresponders in either HCV genotypes. Influence of host genetics on early viral kinetics association was observed between any of the IL28B SNPs and virologic response. Analysis of host genetic markers and sustained virologic response HLA-C1 and -C2 typing was performed on 138 patients (71 HCV g1 and 67 HCV g3) to evaluate the influence of HLA-C on virological response and the frequency of HLA-C in responders and nonresponders in HCV g1- and g3infected individuals (Fig. 3). Within the HCV g1-infected group, HLA-C was found to be associated with nonresponse to treatment (P = 0.023), and notably the frequency of HLAC2 homozygotes was higher in treatment nonresponders compared with responders (7/37; 18.9% vs. 0/34; 0%). In the HCV g3-infected group, no significant association was observed between HLA-C type and nonresponse to treatment (Fig. 3). There was a higher frequency of HLA-C1 homozygotes in nonresponders when compared with responders (13/23; 56.5% and 13/44; 29.5%, respectively) and a lower frequency of C1/C2 heterozygotes in nonresponders (9/23; 39.1%) when compared with responders (26/44; 59.1%) but this was not significant (P = 0.09). The frequencies of 4 KIR genes (KIR2DL3, KIR2DL2, KIR2DS3, and KIR2DS2) in the study were compared between responders and nonresponders in both HCV g1- (n = 87) and g3-infected groups (n = 80; Fig. 4). No significant differences were found between frequencies of the genes in responders and nonresponders in either genotype groups. The allele frequency of the CCR5D32 mutation was examined Early viral load kinetics (log10 IU/mL) were examined for association with the following genetic markers: IL28B rs12979860, the IL28B haplotype, ENT1 rs760370, PNPLA3 rs738409, and CCR5D32. HCV RNA levels (IU/mL) were obtained for patients at 3 different time-points: baseline (pretreatment), 4, and 12 weeks after start of treatment. All markers were examined to determine any difference in baseline HCV viral load between allelic genotypes. The markers ENT1 rs760370, PNPLA3 rs738409, CCR5D32, and IL28B haplotype showed no association. IL28B rs12979860 FIG. 4. Comparison of the frequencies of the KIR genes in HCV g1 and g3 treatment responders and nonresponders. HLA-C INFLUENCE ON TREATMENT RESPONSE IN CHRONIC HCV showed no association in the HCV g1-infected group (P = 0.124), but was found to be associated with baseline viral load in the HCV g3-infected group (P = 0.024). Baseline viral load was highest in major homozygotes (CC), and lower with each additional copy of the T allele. The pattern of allelic groups compared to changes in HCV RNA over time was also examined. ENT1 rs760370 and CCR5D32 showed no significant association in either HCV g1 or g3-infected groups. However, the IL28B SNP rs12979860 was associated with a difference in viral load over time between major homozygotes (CC), heterozygotes (CT), and minor homozygotes (TT) in both HCV g1-infected (n = 68; P = < 0.001) and g3-infected (n = 70; P = 0.004) groups. The association of rs12979860 was observed in both responders (n = 35; P = 0.048) and nonresponders (n = 33; P = < 0.001) of HCV g1-infected individuals, and in responders of HCV g3-infected individuals (n = 49; P = 0.028) but not nonresponders (n = 21). The effect of HLA-C1 and -C2 alleles on RVR (defined as undetectable viral RNA at week 4 post-treatment) in individuals who achieved an SVR was investigated in HCV g1 cases. Due to the high RVR and SVR in the HCV g3infected cohort, too few individuals were present for statistical analysis. Logistic regression showed an association between HLA-C1/C1 and RVR, with an OR relative to the heterozygote of 10.0 [95% confidence interval (CI): 1.6–62.5, P = 0.014]. Discussion Since the identification of an association between polymorphisms near the IL28B gene and treatment-induced clearance of HCV by independent genome-wide association studies conducted in 2009 (Ge and others 2009; Suppiah and others 2009; Tanaka and others 2009), IL28B SNPs and HCV infection have been studied extensively. However, the best genetic model to represent the association between IL28B genotype and treatment-induced HCV clearance is still unclear. Some authors report a dominant effect of the minor risk allele, with no significant differences observed between heterozygotes and minor homozygotes in response to treatment (Tanaka and others 2009). This dominant model suggests that the protective effect of the major allele of the IL28B SNPs is primarily recessive and therefore only effective in individuals homozygous for the major allele. Other studies suggest an additive model, with a dosage effect of the risk allele such that heterozygotes with only 1 copy of the risk allele respond better to treatment than minor homozygotes with 2 copies of the risk allele (Mangia and others 2010). In the present study, all SNPs were examined using both dominant and additive models to investigate the best model for predicting treatment outcome. The pattern of the genotypic OR did not indicate obvious dominance of the minor allele. The nonsignificance of the dominance term in the IL28B SNP rs12979860 (P = 0.881) in the presence of the additive term (P = 4.67 · 10 - 4) indicates a dosage effect of the minor T allele in the cohort. A study by McFarland and others (2014) identified a functional polymorphism (rs4803217) in the 3¢ untranslated region of the IL28B (IFNL3) mRNA that influences the stability of the transcript by targeting inhibition of HCV-induced microRNAs. The group found the IL28B SNP rs12979860 to be in linkage disequilibrium with the functional rs4803217 SNP and propose that the latter SNP is responsible for the association of rs12979860 with clearance of HCV. 5 Prokunina-Olsson and others (2013) identified a novel IFN-l family member, IFNL4, which is orthologous to but distinct from known IFNs and other class 2 cytokines. The IFNL4-creating polymorphism ss469415590[DG] allele is perfectly correlated with the unfavorable rs12979860[T] allele in Asians and well correlated in Europeans; however, it is only moderately correlated in African populations. The IL28B markers rs12979860 and rs8099917 are located 367bp downstream and 4-kb upstream of ss469415590, respectively. The ss469415590 variant and rs12979860 have been shown to predict equivalently the virologic response to dual therapy interferon-a and ribavirin in Caucasian HCV g1/g4-infected patients (Real and others 2014) and in Asian HCV g1-infected patients (Akkarathamrongsin and others 2014). However, typing of the former IFNL4 polymorphism may be more informative for prediction of treatment response rates in chronic-HCV-infected African patients (Prokunina-Olsson and others 2013). Pretreatment assessment of ss469415590 is also predictive in Asian CHC patients receiving triple therapy with the protease inhibitor telaprevir in combination with IFN-a with ribavirin (Nagaoki and others 2014). Several studies that examined the association of IL28B SNPs on baseline viral load have found an association between the major homozygotes (CC) and higher baseline viral loads (Ge and others 2009). In our cohort, IL28B rs12979860 CC homozygotes had a higher baseline viral load than CT heterozygotes, whom in turn had a higher baseline viral load than TT homozygotes. However, after transforming HCV viral load using log10, this association was only significant in the g3-infected group (P = 0.004). Lower baseline viral load for TT homozygotes has previously been shown in an HCV g3-infected cohort by Stenkvist and others (2013) and it is possible that this lower baseline level may play a role in the detrimental effect of the allele in treatment response. The IL28B SNP rs12979860 was found to be significantly associated with early viral kinetics in both HCV g1- and g3infected groups. The average viral load at the 3 time-points (baseline, 4, and 12 weeks after start of treatment combined) was significantly different between major homozygotes (CC), heterozygotes (CT), and minor homozygotes (TT; P = < 0.001 and P = 0.048, respectively). In the HCV g1infected group, rs12979860 CC major homozygotes had a significantly lower estimated measure of mean viral load than either heterozygotes (CT) or minor homozygotes (TT) at week 4. As rs12979860 CC major homozygotes also had a significantly increased chance of achieving SVR, this would suggest that an early decline of viral load is a predictor of treatment response. Analyzing HCV g1-infected responders and nonresponders separately, viral loads in all 3 genotypes of rs12979860 were reduced at week 4 in responders when compared with nonresponders. An association between early decline in viral load and treatment response has been shown in previous studies (Ferenci 2004) and this study and others suggest its potential therapeutic use in predicting treatment outcome in HCV g1-infected individuals. Interestingly, in the HCV g1-infected responder group, the 1 individual who was IL28B rs12979860 minor homozygote (TT) achieved RVR, with no detectable HCV RNA at week 4. A significant finding of the present study is the absence of significant associations of the 4 IL28B SNPs influencing dual-therapy, treatment-induced response rates in HCV 6 g3-infected individual, in support of findings by Rauch and others (2010) who have reported that rs8099917 had no significant impact on SVR in a Swiss cohort infected with HCV g2 or g3. In contrast, in an Italian cohort, Mangia who analyzed the rs12979860 SNP identified an association with SVR in patients infected with HCV g2 and g3 who did not achieve an RVR (Mangia and others 2010). Sarazzin and others (2011) in a German study of HCV g2- and g3-infected individuals found that rs12979860, but not rs8099917, was significantly associated with RVR and SVR. Morello and others (2010) found the SNP rs760370 at the ENT1 gene to be significantly associated with RVR in HIV/ HCV-coinfected individuals, and speculated that the A > G polymorphism affects the expression or activity of the ENT1 protein, which in turn modulates uptake of ribavirin in hepatocytes. No significant association was observed in the present study between the rs760370 SNP and baseline viral load, average viral load between 0 and 12 weeks, or pattern of change of viral load between 0 and 12 weeks, in either HCV g1- or g3-infected groups. Studies that investigated a role of the CCR5D32 allele in HCV acquisition report conflicting data with regard to spontaneous clearance of the virus and susceptibility to infection (Mascheretti and others 2004; Goulding and others 2005; Nattermann and others 2011). CCR5D32 heterozygosity has a frequency of 9% in the Irish population (Martinson and others 1997), and consequently the statistical power is limited in our study due to the low frequency of the allele. Numbers of individuals within a study would need to be very large in order to find a significant association between CCR5D32 and treatment outcome. In this regard, a large study conducted by Suppiah and others (2013) to clarify the role of CCR5D32 in treatment response to dual therapy in CHC-infected individuals found that CCR5D32 did not influence treatment response, or improve the power of the IL28B SNP rs12979860 in predicting treatment response. The NK cell receptor family, KIR, and its ligand, HLA-C, are known to play an important role in the immune response to many viral infections, including HCV. In HCV g1infected individuals in this study, all HLA-C2 homozygotes failed to respond to treatment. These data suggest an important role for HLA-C2 in determining HCV treatment outcome, perhaps through a suppression of NK cytotoxicity, resulting in a reduced killing of infected hepatocytes. Suppiah and others (2011) hypothesized that as HLA-C1 is known to activate NK cells more rapidly and aggressively than HLA-C2 in the natural course of infection, upregulation of HLA-C by IFN-a during treatment would also cause a stronger activation of NK cells by HLA-C1 than HLA-C2. NK cells are known to influence adaptive immune responses to infection. The production of cytokines, such as IFN-g, by NK cells influences T cell responses in lymph nodes, and NK-cell-mediated killing of target cells also impacts T cell responses, possibly by decreasing the antigenic load of the T cells or by promoting antigen cross-presentation to cytotoxic T cells (Krebs and others 2009). HLA-C phenotype may influence the interaction between NK cells and the adaptive immune system, which could explain the absence of HLAC2 homozygotes in HCV g1-infected individuals responding to treatment in our present study (Andrews and others 2010). In this study, no association was observed between any of the 4 KIR genes studied—KIR2DL3, KIR2DL2, KIR2DS3, and KIR2DS2—with treatment response. Strikingly, the COLLISON ET AL. frequencies of all 4 genes were almost identical between responders and nonresponders in both HCV g1- and g3infected groups and suggest that they may not be of as great importance as genetic predictors of treatment response in the context of HCV as others have shown (Suppiah and others 2011); however, this could be due to the limited power of our study. Overall, the results of the study suggest that in HCV g1-infected individuals receiving dual therapy, HLA-C alone determines treatment outcome, regardless of KIR genotype. In the present study, IL28B SNPs and HLA-C2/C2 were found to independently predict treatment nonresponse in HCV g1-infected individuals. Forty-two of the 89 HCV g1infected individuals were CT for IL28B rs12979860 (47%). Of these heterozygotes, almost half (19/42; 44%) responded to treatment. Of note, all HLA-C2/C2 individuals (7/71) were rs12979860 heterozygous, and none achieved an SVR. These data suggest that homozygosity for HLA-C2 predicts nonresponse to treatment, regardless of the fact that the IL28B heterozygous genotype would predict a response rate of almost 50%. In the multivariate analysis, an additive model was used for both IL28B rs12979860 and HLA-C, and no interaction was observed between the 2 loci. As a detrimental effect of HLA-C2 has been observed in HLA-C2 homozygotes but not HLA-C1/C2 heterozygotes, this may account for an absence of an interaction between these 2 innate immune genes. Potential limitations of this study include the cohort size and the investigation of genetic influence on treatment outcome specifically in individuals receiving interferon-a and ribavirin therapy. Larger studies are required to investigate potential associations of IL28B rs12979860, HLA-C2, and other genetic markers with treatment outcome in antiHCV protease and polymerase-based therapies due to the prohibitive cost of the newer therapeutic options and the cost benefit of identifying individuals who may respond to dual therapy. The findings in the present study demonstrate that IL28B rs12979860 is the most significant predictor of treatment response in HCV g1- but not g3-infected individuals and that the HLA-C2 locus is an important pretreatment genetic predictor of treatment failure also in HCV g1- but not g3infected individuals receiving dual IFN/RBV therapy. The inclusion of HLA-C1 and HLA-C2 typing in a clinical algorithm requires further investigation for determining treatment response rates in HCV g1-infected individuals and also for determining the influence of these loci on treatmentinduced clearance with direct-acting antiviral therapy. These findings are of particular relevance in resource-limited settings where the prohibitive cost of the direct-acting antivirals for treatment of CHC-infected individuals may preclude their introduction and dual-therapy, interferon-containing regimens may remain the only treatment option available. Author Disclosure Statement Meadhbh Collison: No relationships/conditions/circumstances that present potential conflict of interest. Jun Liong Chin: No relationships/conditions/circumstances that present potential conflict of interest. Ahmed Abu Shanab: Meeting expenses—AASL meeting 2013 sponsored by Janssen and Janssen. Ross Mac Nicholas: No relationships/ conditions/circumstances that present potential conflict of HLA-C INFLUENCE ON TREATMENT RESPONSE IN CHRONIC HCV interest. Ricardo Segurado: Payment of consulting fee for article preparation as part of CSTAR, which is a not-forprofit Academic Research Centre in University College Dublin, which provides statistical support for academic and industry researchers for an hourly fee. Coauthorship is independent of the fee, and assessed on the basis of ICMJE guidelines (see cstar.ie). Suzie Coughlan: No relationships/ conditions/circumstances that present potential conflict of interest. Jeff Connell: No relationships/conditions/circumstances that present potential conflict of interest. Michael J. Carr: No relationships/conditions/circumstances that present potential conflict of interest. Raphael B. Merriman: No relationships/conditions/circumstances that present potential conflict of interest. P. 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