There were 919 individuals with spontaneous resolution of HCV infection identified by detection in blood of HCV specific antibodies but no HCV RNA (Appendix ) and 1,482 individuals with chronic HCV infection identified by detection in blood of HCV specific antibodies and HCV RNA. After quality control measures were exercised there was information on 792,721 SNPs. Marked differences in allele frequency between persons with HCV clearance and persistence were evident on chromosomes 19q13.13 and 6p21.32 (). These associations were consistent when both adjusted and stratified by HIV status, sex, and ancestry (Appendix Figures 5–8).
On chromosome 19, significant differences in allele frequency were detected for 11 SNPs representing 55,000 base pairs (bp) spanning IL28B
, as previously described(5
). The most significant association was with rs12979860, located ~3kb upstream of the IL28B
start codon which has an allele frequency of 20–100% (C allele) in worldwide populations (5
). Overall, the favorable homozygous CC genotype was detected in 523 (56.9%) HCV spontaneous resolvers and 470 (38.9%) HCV persistent individuals compared to CT in 297 (32.3%) HCV spontaneous resolvers and 739 (61.1%) HCV persisters and TT 84 (9.1%)HCV spontaneous resolvers and 257 (21.3%) HCV persisters (per-allele ORmeta
= 0.45, P = 2.17 × 10−30
). Since this finding included rs12979860 that was previously reported to be associated with spontaneous clearance, no confirmatory testing was performed.
This finding was consistent in substrata classified by genetic ancestry, gender, HIV-infection, and mode of HCV infection. The linkage disequilibrium block marked by rs12979860 chiefly explained the association, as the other 7 SNPs were no longer significantly associated with clearance in analyses conditioned on rs12979860 (P > 5 × 10−8
, Appendix Figure 9). In contrast, rs12979860 remained strongly associated with HCV clearance in analyses conditioned on the other SNPs, even rs8099917, which was associated with spontaneous clearance in a previous Swiss study (Appendix Figure 10–11–14)(29
). Imputation to 1000 genomes sequence data revealed another strongly associated SNP, rs4803217 (odds ratiometa
= 0.44, p = 2.12 x10 −30
), located in the 3′ untranslated region of IL28B
(). However, the association of rs4803217 could not be assessed separately from rs12979860 because of strong pairwise linkage disequilibrium in persons of both European (CEU, r2
=0.91) and African (YRI, r2
=0.86) ancestry. We also typed the rs12979860 allele on all available members of the Baltimore, Maryland ALIVE cohort in whom the greater HCV persistence in persons of African ancestry was originally discovered (4
). In the same, fully-adjusted model of spontaneous resolution that was previously reported, by adding the results of rs12979860 testing, the fully-adjusted odds of persistence in persons of African compared to Caucasian ancestry was reduced from 4.53 (95% CI 2.16, 9.52) to 2.75 (95% CI 1.24, 6.12).
On chromosome 6, differences in allele frequencies between HCV spontaneous clearance and persistence were detected in 117 SNPs spanning 1,090,000 base pairs and encompassing genes for human leukocyte antigen (HLA). The strongest associations were detected for SNPs in the HLA class II region, such as the C allele at rs4273729 (, ). Overall, the homozygous GG genotype was detected in 429 (46.7%) HCV spontaneous resolvers and 495 (40.9%) HCV persisters compared to GC in 403 (43.9%) HCV spontaneous clearers and 697 (57.7%) HCV persisters and CC 84 (9.1%) HCV spontaneous clearers and 287 (23.7%) HCV persisters (per-allele ORmeta= 0.59, P = 1.71x 10−16). The finding was consistent in substrata classified by genetic ancestry, gender, HIV-infection, and mode of HCV infection. Using conditional analyses, a single SNP (rs4273729) explained the significant findings in this region (p>5.0 x10−8, Appendix Figure 12). Imputation to 1000 genomes refined the locus to the DQA1-DQB2 HLA region, although no imputed SNP was more significant than rs4273729. Additional analyses incorporating study site in the model confirmed the findings (Appendix Table 9). The significance of this region was confirmed in a replication dataset comprising 745 individuals from 3 cohorts including 284 individuals with spontaneous clearance and 461 individuals with chronic infection (, rs4273729 odds ratio replication = 0.77, p=0.015 and OR meta+rep= 0.63, p= 5.2 × 10−17).
Although strong, long-distance linkage disequilibrium made it impossible to pin point a single casual allele, classical HLA allelic typing available on 823 individuals from this study demonstrated that DQB1*03:01
contributes significantly to the association (OR=1.93, p=1.84 × 10−5
European ancestry, OR =1.82, p=0.016 African ancestry, Appendix Table 4–5). Since DQB1*03:01
exists in haplotypes with multiple DR
alleles, this finding may explain why other studies have reported DQB1*03:01
and/or diverse additional DR
). Interestingly, in similar GWAS, host control of other chronic viral infections has been mapped to different major histocompatibility (MHC) genes such as HLA-B*57
for HIV, HLA-DP
for HBV, and MHC class I polypeptide-related sequence B for dengue, underscoring differences in the roles of HLA
loci in the host control of chronic viral infections (33
The associations in chromosomes 19 and 6 with HCV clearance were independent and additive. Conditioning on the chromosome 19 SNP, rs12979860, did not alter the chromosome 6 associations, and conditioning on chromosome 6 did not alter chromosome 19 associations (). There was a stepwise increase in the percent spontaneous resolution with the presence of each protective allele, indicating an additive effect of the protective variants (). In a population with prevalence of spontaneous clearance of 30%, an overall estimate of 15% of the variance in spontaneous HCV resolution is explained by SNPs at chromosome 19 (8.4African, 10.1European%, rs12979860) and chromosome 6 (7.4African, 4.8European%, rs4273729). For individuals of European ancestry, the total variance explained is 14.9% (95% CI 8.5–22.6%) and for individuals of African ancestry the total variance explained is 15.8% (95% CI 4.4–31%) (Appendix Figure 13).
Conditional Analyses of HLA Class II and IL28B Regions
Additive Relationship for HCV clearance for HLA Class II and IL28B SNPs