A functional SNP of interferon-gamma gene is important for interferon-alpha-induced and spontaneous recovery from hepatitis C virus infection

Abstract

Cytokine polymorphisms are associated with disease outcome and interferon (IFN) treatment response in hepatitis C virus (HCV) infection. We genotyped eight SNPs spanning the entire IFN-gamma gene in two cohorts and assessed the association between those polymorphisms and treatment response or spontaneous viral clearance. The first cohort was composed of 284 chronically HCV-infected patients who had received IFN-alpha-based therapy and the second was 251 i.v. drug users who had either spontaneously cleared HCV or become chronically infected. A SNP variant located in the proximal IFN-gamma promoter region next to the binding motif of heat shock transcription factor (HSF), -764G, was significantly associated with sustained virological response [P = 0.01, odds ratio (OR) = 2.66 [corrected] (confidence interval 1.3-5.6)[corrected]]. The association was independently significant in multiple logistic regression (P = 0.04) along with race, viral titer, and genotype. This variant was also significantly associated with spontaneous recovery [P = 0.04, OR = 3.51 (1.0-12.5)] in the second cohort. Functional analyses show that the G allele confers a two- to three-fold higher promoter activity and stronger binding affinity to HSF1 than the C allele. Our study suggests that the IFN-gamma promoter SNP -764G/C is functionally important in determining viral clearance and treatment response in HCV-infected patients and may be used as a genetic marker to predict sustained virological response in HCV-infected patients.

Fig. 1.

Functional analyses of SNP −764C/G in vitro. (A) The eight SNPs in the human IFN-γ gene genotyped in this study are shown with their IDs and the corresponding polymorphic bases. The four exons are represented as boxes. Sequence of the promoter and 5′ UTR region is presented with the position number relative to the transcription site as predicted by Gray and Goeddel (35). The open and shaded boxes in the sequence represent the transcription initiative site (TATA box) and the translation start codon (AUG), respectively. The previously reported transcription factor binding sites are underlined. Asterisk indicates the SNP rs2069707. (B) Jurkat cells were cotransfected with individual firefly luciferase (FLuc)-expressing plasmid (pGL3-control, pGL4-IFN(C)-Luc, or pGL4-IFN(G)-Luc) and pRL-CMV as described in Materials and Methods. Cells were harvested after incubation for indicated periods and relative luciferase activity (FLuc/Renilla luciferase) was determined. Bars and error bars indicate mean ± SD of three experiments. (C) The ³²P-labeled −764C, −764G, or NF-κB DNA oligonucleotide probe was used in the EMSA as indicated in the presence (lanes 2–10) of a HeLa cell nuclear extract in comparison with the absence of nuclear protein (lane 1). Antibodies against p65 (lanes 3, 6, and 9) and p50 (lanes 4, 7, and 10) were used for supershift assay. (D) EMSA was performed by using ³²P-labeled probe containing −764C with HeLa cell nuclear extract, with or without competition from unlabeled oligonucleotides containing −764C and −764G as indicated. Relative densities of bands are shown as mean ± SD. The first lane represents the absence of nuclear extract. (E) EMSA was performed by using biotin-labeled probe containing HSF binding motif with Jurkat cell nuclear extract, with or without competition from unlabeled oligonucleotides containing −764C, −764G, HSF probe itself, or anti-HSF1 antibody as indicated. The first lane represents the absence of nuclear extract.