Approximately 30 percent of individuals spontaneously clear acute hepatitis C infection. Host genetic variation is assumed to explain the heterogeneity in HCV clearance across individuals because such differences occur even after exposure to the same HCV inoculum and because there are ethnic differences in clearance frequency6,7
. Variation in genes involved in the immune response has already been linked to outcome of acute HCV infection8,9
, presumably due to alteration in the strength and quality of the immune response. However, most variability in spontaneous HCV clearance remains unexplained.
A recent genome wide association study (GWAS) of >1600 individuals chronically infected with hepatitis C participating in a clinical treatment trial with pegylated interferon alpha and ribavirin identified a single nucleotide polymorphism (SNP) on chromosome 19q13, rs12979860, that was strongly associated with sustained virological response (SVR)5,10
. This SNP maps 3 Kb upstream of the IL28B
gene which encodes the type III interferon IFN-λ3. The C/C genotype was associated with 2.5 or greater rate (depending on ethnicity) of SVR compared to the T/T genotype and the C allele was over-represented in a random multi-ethnic population as compared to the chronically-infected study cohort, raising the possibility that the C allele may favor spontaneous clearance of HCV.
In order to address directly the role of the rs12979860 SNP in HCV clearance, we genotyped 1008 individuals from 6 independent HCV cohorts composed of individuals who cleared virus (N = 388) and individuals with persistent infection (N = 620). Genotypes were in Hardy-Weinberg equilibrium in both blacks and whites (p = 0.47 and 0.77, respectively). The frequency of the C allele was significantly greater among whites than blacks in both the clearance (p = 3×10−10) and persistence groups (p = 1×10−21) (). In both blacks and whites, however, there were significant differences in allele frequencies (C vs. T) between the clearance and persistence groups, where the C allele showed greater frequencies in the clearance group than in the persistence group (80.3% vs. 66.7% respectively in whites, p = 7×10−8; 56.2% vs. 37% respectively in blacks, p = 1×10−5).
Characteristics of study subjectsa
More striking differences were observed in an analysis of genotype frequencies where patients with the C/C genotype were three times more likely to clear HCV relative to patients with the C/T and T/T genotypes combined (OR = 0.33, p < 10−12
for combined ethnic groups; and ). Stratification of this analysis by ethnicity indicated that the strength of the protective C/C effect was similar in blacks and whites (OR = 0.32, p = 1 × 10−4
and OR = 0.38, p = 1 × 10−7
, respectively). However, a comparison of C/C to the T/T group alone suggested stronger protection conferred by C/C in blacks (OR = 0.21, p = 3 × 10−5
) relative to that in whites (OR = 0.50, p = 0.04), though our power to detect a true difference is limited due to small sample sizes in some groups. Overall, the protective effect of C appears to be primarily recessive, as no significant difference was observed between the C/T and T/T genotypes in blacks, whites, or combined ethnic groups for clearance of HCV (data not shown), and C/C was consistently protective relative to C/T and/or T/T ( and ). The proportion of individuals with C/T or T/T genotypes who cleared the virus (28% for combined genotypes) was similar to a ‘general population’ expectation, since HCV clearance occurs in approximately 30% of those infected. In contrast, HCV clearance was observed much more frequently than expected (53%) in the C/C group. These results nicely mirror the protective effect of the C/C genotype on SVR after HCV treatment observed previously5
, where the protection conferred by the C allele also appeared to be recessive in both their Caucasian and African American patients.
Effect of IL28B rs12979860 genotype on clearance of hepatitis C virus
Percentage of HCV clearance by rs12979860 genotype. Data are shown for all patients, as well as whites and blacks separately.
Some individuals used in this study of HCV were co-infected with hepatitis B virus (HBV) and/or HIV. In order to eliminate the possibility that co-infection with these viruses may confound the effect of rs12979860 on HCV outcome, analyses were performed using a multivariate model that included hepatitis B surface antigen status as a co-variate or stratifying by HIV status. Neither of these two chronic viral infections altered the effect of this locus on outcome of an acute HCV infection (Suppl. Table 1 and 2
). We also tested whether there were any differences in the effect of the protective rs12979860 C allele as a function of the route of HCV acquisition (plasma products vs. injection drug use), but found no significant differences between the two groups (data not shown). Finally, adjusting by other host genetic factors that associate with clearance of HCV did not alter the protection conferred by the CC genotype (Supplementary information I
Patients with lower baseline HCV viral load (VL) respond more favorably to interferon alpha treatment11,12
. However, little is known regarding the impact of VL during acute infection on spontaneous HCV clearance because very few HCV-infected individuals are identified and studied at this early phase13
. We reasoned that the mechanism of protection of the CC genotype might also extend to greater control of VL in the chronic phase, but there was no correlation between rs12979860 genotype and VL (Supplementary Information II
and Supplementary Figure 1
). However, differences in viral load assays used in the various cohorts may mask a small correlation, even after conversion to international units.
The frequency of HCV clearance varies markedly across ethnic groups9
and differences in allele frequencies for the rs12979860 SNP were observed in the present study () and in Ge et al5
. Indeed, the observation that the C allele is less frequent among individuals of African descent relative to those of European descent might explain in part the observed discrepancy in the frequency of viral clearance in these two ethnic groups, where clearance occurs in 36.4% of HCV infections in nonblacks, but only 9.3% in blacks7
. In order to gain greater insight into the geographic frequency distribution of the protective C allele, we genotyped 2371 individuals from 51 populations worldwide ( and ).
rs12979860 C allele frequency in worldwide populations
Figure 2 Sampling locations, allele frequencies and degree of regional differentiation of the rs12979860 C allele. (A) The numbers identifying populations are given in . The pie charts show the frequency of the C (green) and T (blue) alleles in each population (more ...)
The global pattern of allele frequencies shows a striking pattern in which the allele leading to greater natural HCV clearance is nearly fixed throughout East Asia, has an intermediate frequency in Europe, and is the minor allele in Africa (). A comparison of the rs12979860 allele frequency diversity across 32 world populations (as measured by FST
) with that for 1062 SNPs typed in these same samples shows that the rs12979860 polymorphism has a greater differential frequency (FST
= 0.23) than most of the other polymorphisms (mean FST
= 0.12, standard deviation = 0.1), falling within the upper 12.5 percentile of the distribution of FST
values (). Notably, the high frequencies of the C allele found in North and Eastern Asian populations are not reflected in correspondingly high frequencies in their American relatives. Thus, if this locus has been under selection pressure, changes in the selective force that may be dependent on geographical locale, likely occurred after the colonization of the New World. That a common variant has such a strong impact on hepatitis C may indicate that it has actually been under selection, adding to an emerging interpretation of genome-wide association studies whereby common variants rarely have large effects unless they were selected to do so14
The rs12979860 SNP is only 3 Kb upstream of the IL28B
gene, which encodes the type III interferon IFN-λ3, and this SNP is in strong linkage disequilibrium (r2
>0.85) with a nonsynonymous coding variant in the IL28B
gene (213A>G, K70R; rs8103142)5
. Thus, it is possible that this 213A>G change alters the function of IFN-λ3 and explains the genetic data described herein, but functional data will be essential to defining the precise biological mechanism. Type III interferons include three members, IFN-λ1, IFN-λ2 and IFN-λ3, and the genes encoding these molecules are clustered on human chromosome 19q1315,16
. They are structurally related to the IL-10 superfamily of cytokines, but share functional characteristics with the type I interferons (IFN-α and IFN-β) in that they are induced by viral infections, signal through the JAK-STAT pathway, and exhibit anti-viral activity in vitro15,16
. IFN-λ1 has been shown to exhibit dose- and time-dependent HCV inhibition, induce increases in levels of interferon stimulated genes, and enhance the antiviral efficacy of IFN-α17
. It is possible that IFN-λ3 works through a similar mechanism. In vitro, IFN-λ3 is at least as potent as IFN-λ1 in terms of protecting HepG2 cells from lysis after infection with encephalomyocarditis virus18
. Severe side effects in HCV treatment have been observed with IFN-α therapy19
, whereas IFN-λ treatment may exhibit less “interferon-like” adverse effects because their receptor is only expressed on a limited number of cell types20
. Whether IFN-λ may serve as an alternative treatment modality for HCV infection is under investigation.
In conclusion, we have shown that the rs12979860 polymorphism upstream of IL28B which was previously associated with HCV treatment response also has a dramatic impact on natural clearance of HCV and may have been under selection in human history. It is now a priority to determine the mechanisms through which IL28B promotes viral defense, and the full range of viruses affected by these mechanisms.