By sequencing the putative regulatory region upstream of IL28B
, we identified a T deletion (previously described as rs67272382
NT_011109.16:g.12007372delT) adjacent to a T-to-G substitution (previously described as rs74597329
NT_011109.16:g.12007373T>G), located at position 39739154 and 39739155 in chromosome 19 (Genome Build 37.1). The presence of the TT/-G substitution, as well as the previously known rs12979860
, was explored in a cohort of 540 Caucasian patients with chronic HCV infection and in 93 patients with spontaneous HCV clearance (Table S1
). The rs12979860
and TT/-G polymorphisms were in strong LD (R2
= 0.91) and both had a minor allele frequency of 0.38. Despite such strong correlation, individuals who were discordant at both SNPs allowed us to explore which of the two SNPs is most strongly associated with the outcome and therefore more likely to be the functional variant.
We assessed whether IL28B
polymorphisms were associated with response to PEG-IFN-α/RBV therapy among chronically infected patients. When considering all viral genotypes together, both polymorphisms were associated with response to treatment (, , and Table S2
). The strongest and most significant association was found for TT/-G (OR = 0.38, 95% CI 0.28–0.51, P = 2.51−10
), compared with rs12979860
(0.46, 95% CI 0.35–0.62, P = 1.52−7
). TT/-G still provided the strongest and most significant association in a multivariate model, after adjustment for age, sex, HCV RNA level, fibrosis stage, and viral genotype (OR = 0.27, 95% Cl 0.17–0.45, P = 2.70−7
), compared with rs12979860
(OR = 0.37, 95% CI 0.23–0.59, P = 2.47−5
). When both polymorphisms were included in the same model, the mutant -G allele of TT/-G was still associated with reduced clearance, whereas the mutant T allele of rs12979860
was associated with increased clearance (OR>1, ). TT/-G had a better ability than rs12979860
to discriminate SVR rates (P = 0.04, ). The SVR proportion was 0.80, 0.55, and 0.41 for patients carrying the TT/TT, TT/-G, and -G/-G genotypes, compared with 0.77, 0.56, and 0.45 for those carrying rs12979860
CC, CT, and TT genotypes (P = 0.02, ). Altogether, this confirms that TT/-G is a better marker for HCV clearance than rs12979860
and that the mutant -G allele, but not the rs12979860
mutant T allele, is associated with reduced clearance. The rs12979860
mutant T allele likely represents a proxy for the effect of the mutant -G allele.
Figure 1. TT/-G is a better predictor of response to treatment than rs12979860. (A) Association of the mutant -G allele of TT/-G and the mutant T allele of rs12979860 with HCV clearance. * , P = 0.04. Error bars represent 95% confidence level. (B) TT/-G versus (more ...)
Association of IL28B polymorphisms with HCV clearance in chronic hepatitis C
Independent contribution of rs12979860 and TT/-G to HCV clearance
Similar observations were found after stratification by viral genotypes ( and ). In patients infected with HCV genotype 1/4, the strongest and most significant association was also found for TT/-G either in the univariate (OR = 0.25, 95% CI 0.16–0.40, P = 4.61−9 vs. OR = 0.31, 95% CI 0.20–0.49, P = 2.24−7 for rs12979860) or in the multivariate models (OR = 0.16, 95% CI 0.08–0.33, P = 2.75−7 vs. OR = 0.22, 95% CI 0.11–0.41, P = 3.46−6 for rs12979860). In patients infected with HCV genotype 2/3, TT/-G was the only SNP associated with response to treatment, but only in a recessive model (OR = 0.36, 95% CI 0.15–0.86, P = 2.13−2). Finally, TT/-G was also a better marker of spontaneous clearance ( and , P = 8.68−9), compared with rs12979860 (P = 1.66−8).
Because TT/-G is a better marker of HCV clearance, we speculated that it might correlate with IL28B mRNA expression. To address this issue, we measured IL28B mRNA expression in PBMCs from individuals carrying different allelic combinations of rs12979860 and TT/-G that were stimulated with the dsRNA analogue poly(I:C) (). Among individuals WT for rs12979860, PBMCs from those carrying one or two copies of the mutant allele -G of TT/-G had lower expression of IL28B mRNA compared with those from TT/-G WT individuals (P < 0.001). In contrast, among individuals WT for TT/-G, those carrying one or two copies of the mutant allele T of rs12979860 had similar expression of IL28B mRNA than those WT for rs12979860. Finally, among individuals carrying one or two copies of the mutant allele T of rs12979860, those carrying one or two copies of the mutant allele -G of TT/-G had lower expression of IL28B mRNA compared with those from TT/-G WT individuals (P = 0.007). The pattern of IL28B expression after stimulation was similar among patients with cured hepatitis C (SVR) and those with chronic infection (nonresponse) according to TT/-G, suggesting that the viremic state does not significantly influence acute response to stimulation in vitro (). Altogether, these data demonstrate that IL28B mRNA expression is driven by the presence of one or two mutant alleles of TT/-G but not by rs12979860. Because IL28B has a strong homology with IL28A due to ancestral gene duplication events, we sequenced previously cloned amplicons to unambiguously confirm that they were part of IL28B but not IL28A (unpublished data).
Figure 2. Expression of IL28B and IP-10 mRNA relies on TT/-G but not rs12979860 polymorphism. PBMCs from healthy and HCV-infected individuals were stimulated with poly(I:C) for 4 h (black) and 8 h (dark gray). The mRNA expression of IL28B and IP-10 was measured (more ...)
Although the number of individuals carrying discrepant genotypes was low, the reduction in IL28B mRNA expression among samples from individuals carrying the mutant TT/-G allele was strongly significant. Recently, Prokunina-Olsson et al. (2013)
also identified TT/-G as a better predictor of response to treatment than rs12979860
among African Americans, whereas it did not improve this prediction among Caucasians. This can be explained by the lower level of LD between the two polymorphisms among African Americans (R2
= 0.71) compared with Caucasian (R2
= 0.92). Thus, the high proportion of rs12979860
and TT/-G discrepant individuals in the African American population would allow further validation of the functional role of TT/-G on IL28B mRNA expression.
Infection with HCV activates the endogenous IFN system, which leads to the induction of ISGs and contributes to viral clearance. Pretreatment plasmatic levels of ISGs, such as IFN-γ–inducible protein 10 (IP-10), are predictors of HCV clearance. Because the induction of IP-10 is thought to rely on both type I and type III IFNs, we analyzed IP-10 mRNA expression in PBMCs stimulated with poly(I:C) for 8 h (). As for IL28B, IP-10 expression was lower among individuals carrying the mutant allele of TT/-G but not in those carrying the mutant allele of rs12979860 (), indicating that Il28B expression could be a determinant for the induction of some ISGs. These observations suggest a strong link between the mutant -G allele of TT/-G, reduced expression of IL28B, lower induction of ISGs, and HCV treatment failure. However, the mechanisms by which IL28B TT/-G genotypes are related to ISG induction and clinical phenotypes remain to be elucidated. As a control, TNF mRNA expression after LPS stimulation was not influenced by both polymorphisms ().
Genetic polymorphisms can influence gene function through different mechanisms. For instance, they can disrupt or create DNA regulatory elements affecting gene expression. In addition, SNPs can influence the ability of DNA sequences to undergo methylation, thereby influencing gene expression. Both rs12979860 and TT/-G are located within a CpG island which has been identified on the UCSC human genome draft by the Miklem and Hillier method. As assessed by bisulfite sequencing, TT/-G polymorphism was found to promote the methylation of a cytosine residue which is unmethylated in WT DNA sequences (). Further studies are needed to determine the relationship between TT/-G and surrounding CpG methylation and IL28B and/or ISG expression.
Figure 3. The TT/-G creates a methylation site in the CpG region. The TT/-G substitution is associated with the methylation of the adjacent cytosine residue (indicated by arrow). As opposed to methylated cytosine residues (indicated by asterisks), unmethylated (more ...) Prokunina-Olsson et al. (2013)
showed that TT/-G introduces a frame shift in the DNA sequence, which transiently induces mRNA expression of an IFN analogue (IFNL4) in human hepatocytes stimulated with poly(I:C). Only patients carrying the mutant allele -G express IFNL4. The authors suggest that this protein could be a direct marker of HCV treatment failure and a new target for therapeutic intervention, raising major interest in the medical community. Nevertheless, issues regarding the molecular functions of IFNL4 remain to be clarified, such as the inability of recombinant IFNL4 to directly induce the Jak–STAT pathway in HepG2 cells, unless by transfection of an IFNL4 construct. Furthermore, the low secretion level of IFNL4 together with the lack of demonstration of its effective binding to the IFNL receptor and/or another specific receptor raises questions about its physiological function.
Using a large European cohort, we identified a new TT/-G polymorphism nearby IL28B that influences both IL28B and IP-10 mRNA expression and improves HCV clearance prediction in patients infected with HCV viral genotype 1/4 or 2/3. Because IL28B has antiviral properties, reduced IL28B expression may impair individual ability to clear HCV by itself. Whether this phenomenon is further influenced by another protein remains to be demonstrated. TT/-G genotyping may have an important impact on the management of both African American and Caucasian patients with chronic hepatitis C. Altogether, the identification of this TT/-G functional variant provides a new step in understanding the role of IL28B polymorphisms in the prediction of the response to chronic hepatitis C treatment.