IL28B expression depends on a novel TT/-G polymorphism which improves HCV clearance prediction

Abstract

Approximately 3% of the world population is chronically infected with the hepatitis C virus (HCV), with potential development of cirrhosis and hepatocellular carcinoma. Despite the availability of new antiviral agents, treatment remains suboptimal. Genome-wide association studies (GWAS) identified rs12979860, a polymorphism nearby IL28B, as an important predictor of HCV clearance. We report the identification of a novel TT/-G polymorphism in the CpG region upstream of IL28B, which is a better predictor of HCV clearance than rs12979860. By using peripheral blood mononuclear cells (PBMCs) from individuals carrying different allelic combinations of the TT/-G and rs12979860 polymorphisms, we show that induction of IL28B and IFN-γ-inducible protein 10 (IP-10) mRNA relies on TT/-G, but not rs12979860, making TT/-G the only functional variant identified so far. This novel step in understanding the genetic regulation of IL28B may have important implications for clinical practice, as the use of TT/G genotyping instead of rs12979860 would improve patient management.

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 rs12979860 in SVR to treatment in chronically HCV-infected patients. **, P = 0.02. Analyses were performed in 540 Caucasian patients from the Swiss Hepatitis C Cohort Study. Numbers of patients in each group are described in Table 1. All genotypes were validated by an independent laboratory as described in the materials and methods. Odds ratios are for an additive model, accounting for the effect of one or two copies of the mutant allele. Multivariate models are adjusted for age and sex, HCV RNA level, fibrosis stage, and viral genotype. P-values were calculated using the integrated discrimination improvement (IDI) test.

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 by RT-PCR. Results grouped according to different allelic combinations of the mutant T allele of rs12979860 and the mutant -G allele of TT/-G are shown in A (IL28B) and B (IP-10). Individual results are shown in C. As a control, TNF expression was measured after stimulation with LPS (C, bottom). AU, arbitrary units; mut, mutant allele; NA, not assessed; NR, nonresponse to treatment. For each individual, stimulation was performed in triplicate. P-values are calculated by linear regression. * , P < 0.001; **, P = 0.007; ⁺, P < 0.001; ⁺⁺, P = 0.006. N stands for the number of individuals. Error bars represent standard error. #, unstimulated cells.

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 cytosine residues, C, are replaced by thymine, T, after bisulfite treatment and subsequent PCR amplification.