Nasheed Moqueet* 1,2, James Young 2, Connie Lisle 3, Ron Carter 3 and Marina B. Klein 1,2 1 McGill University, Montreal, QC Canada; 2 Montreal Chest Inst., McGill Univ. Health Ctr, Montreal, QC Canada; 3 McMaster University, Hamilton, ON Canada Nasheed Moqueet Dept of Epidemiology, Biostatistics and Occ Health 1020 Pine Avenue West Montreal, Quebec H3A 1A2 Phone: #32227 Abstract Background Homozygous recessive SNPs (rs CC, rs TT) in the human Interleukin 28B (IL28B) gene have been associated with spontaneous HCV clearance and better treatment response in various populations worldwide, possibly via functional variants at rs These relationships and genotypic distributions have not been studied in Canadian HIV/HCV co-infected populations where aboriginal people are overrepresented. Previous studies have also suggested that aboriginals (First Nations, Metis, Inuit) may have greater rates of spontaneous clearance. Methods A case-control study nested in a prospective multicentre Canadian Co-infection Cohort (n=1020). Only those who have never been treated for HCV with at least 2 consecutive RNA tests (n=436) were included. We matched on self- reported ethnicity and used incidence density sampling to select for each HCV negative case (n=62), 2 HCV+ controls within 2 years of when a case occurred. Conditional logistic regression was used with multiple imputation to account for missing data and adjusted for sex and HCV genotype (1 or 4 vs. 2 or 3). In addition, all aboriginal participants were genotyped (n=135). Results Overall, 73% were male with median HCV duration of 18 years; 15% reported aboriginal ancestry. 62 participants cleared spontaneously in person-years of risk (6.4/100 person-years, 95% CI 5-8.2/100 p-y). The adjusted ORs (95% CI) for spontaneous clearance for each SNP analysed were---rs : 3.44 (1.70, 6.95); female, 1.73 (0.82, 3.65); and HCV genotype 1 or 4, 0.64 (0.24, 1.69); at rs : 6.61(2.20, 19.89); female, 1.89 (0.90, 3.98); and HCV genotype, 0.68 (0.24, 1.93), and at rs : 4.19 (1.93, 9.08); female, 1.80 (0.82, 3.92), HCV genotype, 0.52 (0.20, 1.38). Ethnicity matching precluded analysis of the association of aboriginal ethnicity with spontaneous clearance. However, among aboriginals, at rs , 74.5% had the C allele, at rs , 81.4% had the T allele, and at rs , 75% had the T allele. The corresponding values among Canadian whites are 72.8%, 81.9%, and 75%, respectively. These allele frequencies are very similar to those previously reported among European populations, all lower than in East Asians. Conclusions Genotypes rs CC, rs TT, and rs TT in the IL28B gene are strongly linked to spontaneous clearance in HIV/HCV co-infected Canadians. These genotypes do not appear to be more common among Canadian aboriginals. Resolvers were also more likely to be female and infected with HCV genotypes 2 or 3, though these associations did not reach statistical significance. Results. Limitations  Because the study participants have been infected with HCV for a long time, impossible to say if clearance is first or repeat occurrence.  We did not have sufficient power to detect interactions between the SNPs and gender or SNPs and HCV genotype.  Functional assays and other studies are needed to verify the effect of the rs variant on the IL28B protein. Favourable IL28B Genotypes are Associated with Spontaneous Clearance but not Aboriginal Ancestry in HIV/Hepatitis C (HCV) Co-infected Individuals from the Canadian Co-infection Cohort Background Conclusions  Results indicate that, consistent with trends in other populations, the CC genotype at rs and TT genotype at rs are associated with over a two-fold increase in spontaneous HCV clearance.  The effect is also similar with TT genotype at the functional variant rs , which leads to an arginine-lysine substitution at position 70 in the IL28B protein.  Distribution of IL28B alleles is very similar in Canadian whites and aboriginals.  Being female is also associated with a higher rate of spontaneous clearance, though this effect lacked statistical significance, likely due to small sample size.  HCV genotype 1 or 4 is linked with lower clearance, though the effect did not reach statistical significance. Funded by: HIV and HCV co-infection affects over 10 million individuals worldwide. Adverse outcomes such as HCV treatment failure and viral persistence are higher in co-infected individuals compared to HCV monoinfected patients. Thus, there is a critical need to not only understand the pathogenesis of HCV in co-infection, but also identify factors that would aid in clinical decision-making.  Some factors independently associated with higher rates of spontaneous HCVclearance include female sex, aboriginal ethnicity and infection with HCV genotypes 2 and 3.  Certain homozygous recessive SNPs (rs CC, rs TT) in Interleukin 28B (IL28B) are also associated with spontaneous HCV clearance and/or better response to treatment.  These SNPs are tightly linked with the SNP at rs , which leads to a nonsynonymous amino acid substitution (K70R) in the IL28B protein.  These relationships have not been characterized in Canadian HIV/HCV co-infected populations, where aboriginal populations are over represented. Objectives:  To evaluate the association of IL-28B Single Nucleotide Polymorphisms (rs , rs , rs ) with spontaneous HCV clearance in individuals enrolled in the Canadian Co-infection Cohort Study (CCC)  To measure and compare the distribution of IL28B alleles among Canadian whites and aboriginals enrolled in the CCC Methods Table1 Most people have been HCV infected for a long time. Majority of the participants are male, older (mean age=45 yrs) and have a history of drug use. Very few had ever been treated for HCV at first visit. Table 1: Baseline Characteristics of CCC, the source population, n=1,020 Table 2: Characteristics of matched case control sets Table 2: Clearance is rare, at a rate of 6.4/100 person-years ( /100 p-y). Cases more likely to be female compared to controls (37% vs. 27%). Cases less likely to be infected with HCV genotype 1 than the others. Cases are more likely to have the favourable genotype ** at all SNPs than controls: CC at rs , TT at rs , and TT at rs Table 3: Univariate analysis of factors associated with spontaneous HCV clearance Table 3: All SNPs are in Hardy-Weinberg Equilibrium in white and aboriginal controls (p>0.05). o Favourable genotypes at all SNPs are linked with higher odds of clearance o These effects are statistically significant o Females have 54% higher odds of clearing than males and transgenders o This effect was not statistically significant. o Infection with HCV genotype 1 or 4 is linked with a 39% lower odds of clearance than genotypes 2 or 3 Table 4: Multivariate analysis of factors associated with spontaneous HCV clearance Table 4: o Because the SNPs are tightly linked, they were analysed separately. o All IL28B genotypes ** are linked with spontaneous clearance, with effects of similar magnitude. o Effects of all variables are very similar as in univariate analyses o Indicating that effect of the SNP is independent of gender and HCV genotype. o Not enough power to detect any gender or HCV genotype specific differences in the effect of IL28B. Figure 1. IL28B Favourable Allele Frequency in Whites and Aboriginals from the CCC Figure 1: o Prevalence of all beneficial alleles is fairly common in both populations o IL28B allele frequencies are almost identical among Canadian whites and aboriginals o Higher clearance rates among aboriginals are not due to differential frequency of IL28B alleles Study design: Nested case control Setting/Source population: The Canadian Co-infection Cohort Study (CCC, n=1,020) is a prospective cohort of HIV/HCV co- infected individuals recruited from 16 centres across Canada, representing 20% of the co-infected population under care. Data and samples are collected at visits every 6 months. Median follow-up is 2 years with an IQR of years. Study population: Only individuals who have never been treated for HCV (n=845) with at least 2 HCV RNA tests available were included. Visits after HCV treatment initiation were censored. Of the 845 individuals, we excluded  3 due to missing HCV duration  16 due to missing ethnicity  390 for < 2 HCV RNA measures The final study population was 436 individuals contributing person-years of risk. Case definition: A case was HCV-RNA negative on 2 consecutive PCR tests, while a control was HCV-RNA positive on 2 successive tests within two years of when cases occur. Control selection and time axis: We matched on self-reported ethnicity and used incidence density sampling to select 2 controls for each case.  HCV duration, which is the time axis, was based on date of HCV seroconversion, if known, or on the year of first injection drug use or blood product exposure. Exposure: IL28B genotypes at SNPs rs , rs , and rs from stored plasma, analyzed as dichotomous (having the favourable genotype vs. not) Analysis: Data were analyzed using conditional logistic regression Software:All analyses were conducted using Stata v11.2 (College Station, TX: StataCorp LP. 2009). Variable Summary Median follow-up time, years (IQR) 2 (0.7-3) Mean age at baseline, yrs (SD) 45 (8) Male, n(%) 744 (73%) Ethnicity, n (%) White 782 (78%) Black 38 (4%) Aboriginal 155 (15%) Other 29 (3%) Median HCV duration, yrs (IQR) 18 (11-25) Injection drug user, n (%) 822 (81%) HCV genotype 1, n (%) 556 (72%) Ever treated for HCV, n (%) 175 (17%) Ethnicity-matched Controls (n=97) Cases (n=62) Female31 (27%)23 (37%) Aboriginal20 (17%)12 (19%) HCV genotype, n (%) 178 (79 %)9 (53%) 27 (7%)1 (6%) 314 (14%)7 (41%) Event rate (95% CI) 6.4 per 100 person-years ( ) IL28B genotypes rs **CC, n (%)49 (45%) 43 (71%) CT, n (%)50 (45%)10 (16%) TT, n (%)11 (10%)2 (3%) rs **TT, n (%)71 (63%)56 (92%) GT, n (%)32 (28%)5 (8%) GG, n (%)10 (9%)0 (0) rs **TT, n (%)51 (46%)49 (80%) CT, n (%)51(46%)10 (16%) CC, n (%)8 (7%)2 (3%) OR (95% CI) rs CC3.4 (1.73, 6.69) rs TT6.18 (2.13, 17.96) rs TT3.92(1.90, 8.11) female1.54 (0.79, 3.02) HCV genotype (0.24, 1.58) rs CCrs TTrs TT Adjusted OR (95% CI) 3.44 (1.70, 6.95) **6.61(2.20,19.89) **4.19 (1.93, 9.08) ** Female1.73 (0.82, 3.65)1.89 (0.90, 3.98)1.80 (0.82, 3.92) HCV genotype (1/4 vs. 2/3) 0.64 (0.24, 1.69)0.68 (0.24, 1.93)0.52 (0.20, 1.38) We acknowledge: Rhyan Pineda, Laurence Brunet, Kathleen Rollet, Claire Infante-Rivard, and the participants, co-investigators & coordinators of the HIV-HCV Canadian Cohort (CTN 222).