[Show abstract][Hide abstract] ABSTRACT: IFN-l4 is a novel type-III interferon with strong clinical significance in humans. Only a subset of individuals— up to 10% of Asians, 50% of Europeans, and 90% of Africans—carry the DG allele of a genetic variant rs368234815-TT/DG and are genetically able to produce IFN-l4 protein. Carriers of the DG allele have impaired ability to clear infection with hepatitis C virus (HCV). IFN-l4 is also predicted to exist and be functionally important in several nonhuman mammals. In this study, we present the first comparative analysis of 12 mammalian IFN-l4 orthologs in a human hepatic cell line, HepG2, which supports signaling of the human IFN-l4. We show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman IFN-l4 proteins are comparable—they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. We show that several IFN-l4 orthologs can be detected by Western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human IFN-l4. Studies of IFN-l4 in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions.
Journal of Interferon & Cytokine Research 08/2015; DOI:10.1089/jir.2015.0096 · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon lambda 4 (IFN-l4) is a novel type-III interferon that can be generated only in individuals carrying a DG frame-shift allele of an exonic genetic variant (rs368234815-DG/TT). The rs368234815-DG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-l4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-l4 but also detected secreted IFN-l4 in the culture media of expressing cells. Secreted IFN-l4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-l4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-l4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-l4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-l4-induced phenotypes— activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-l4-specific antibody. Our study offers new insights into biology of IFN-l4 and its possible role in HCV clearance.
Journal of Interferon & Cytokine Research 07/2015; DOI:10.1089/jir.2014.0161 · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon lambda 4 gene (IFNL4) encodes IFN-λ4, a new member of the IFN-λ family with antiviral activity. In humans IFNL4 open reading frame is truncated by a polymorphic frame-shift insertion that eliminates IFN-λ4 and turns IFNL4 into a polymorphic pseudogene. Functional IFN-λ4 has antiviral activity but the elimination of IFN-λ4 through pseudogenization is strongly associated with improved clearance of hepatitis C virus (HCV) infection. We show that functional IFN-λ4 is conserved and evolutionarily constrained in mammals and thus functionally relevant. However, the pseudogene has reached moderately high frequency in Africa, America, and Europe, and near fixation in East Asia. In fact, the pseudogenizing variant is among the 0.8% most differentiated SNPs between Africa and East Asia genome-wide. Its raise in frequency is associated with additional evidence of positive selection, which is strongest in East Asia, where this variant falls in the 0.5% tail of SNPs with strongest signatures of recent positive selection genome-wide. Using a new Approximate Bayesian Computation (ABC) approach we infer that the pseudogenizing allele appeared just before the out-of-Africa migration and was immediately targeted by moderate positive selection; selection subsequently strengthened in European and Asian populations resulting in the high frequency observed today. This provides evidence for a changing adaptive process that, by favoring IFN-λ4 inactivation, has shaped present-day phenotypic diversity and susceptibility to disease.
[Show abstract][Hide abstract] ABSTRACT: Response to pegylated interferon-alpha and ribavirin (IFNα/RBV) treatment for chronic hepatitis C virus (HCV) infection is influenced by host genetic factors, but their role for IFNα-free directly acting antiviral (DAA) regimens is unclear. An exonic deletion allele (IFNL4-ΔG) bolsters the established association with IFNα/RBV therapy treatment outcome of another IFNL4 variant, rs12979860, located upstream of IFNL3 (IL28B). We report that in patients treated with the DAA sofosbuvir with RBV, IFNL4-ΔG is associated with slower early viral decay, due to slower loss of free virus (p=0.039) and decreased drug efficacy (p=0.048), suggesting functional relevance of IFN-λ4 in IFNα-free DAA therapies.
The Journal of Infectious Diseases 12/2013; 209(11). DOI:10.1093/infdis/jit827 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Candidate gene and genome-wide association studies (GWAS) have identified 11 independent susceptibility loci associated with bladder cancer risk. To discover additional risk variants, we conducted a new GWAS of 2,422 bladder cancer cases and 5,751 controls, followed by a meta-analysis with two independently published bladder cancer GWAS, resulting in a combined analysis of 6,911 cases and 11,814 controls of European descent. TaqMan genotyping of 13 promising SNPs with P< 1x10(-5) was pursued in a follow-up set of 801 cases and 1,307 controls. Two new loci achieved genome-wide statistical significance: rs10936599 on 3q26.2 (P=4.53×10(-9)) and rs907611 on 11p15.5 (P=4.11×10(-8)). Two notable loci were also identified that approached genome-wide statistical significance: rs6104690 on 20p12.2 (P=7.13×10(-7)) and rs4510656 on 6p22.3 (P=6.98×10(-7)); these require further studies for confirmation. In conclusion, our study has identified new susceptibility alleles for bladder cancer risk that require fine-mapping and laboratory investigation, which could further understanding into the biological underpinnings of bladder carcinogenesis.
Human Molecular Genetics 10/2013; DOI:10.1093/hmg/ddt519 · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon lambda 4 protein can be generated in IFNL4-ΔG carriers, but not IFNL4-TT homozygotes. We studied 890 anti-hepatitis C virus (HCV) positive participants in the Women's Interagency HIV Study. Among African Americans (n=555), HCV was more often cleared for those with genotype IFNL4-TT/TT (32.6%; odds ratio (OR), 3.59; p=3.3x10(-5)) than IFNL4-TT/ΔG (11.3%; OR, 0.95; p=0.86) or IFNL4-ΔG/ΔG (11.9%; referent). Pooling these data with published results in African Americans (n=1,678), ORs were: IFNL4-TT/TT, 3.84 (p=8.6x10(-14)); IFNL4-TT/ΔG, 1.44 (p=0.03); area under the curve was 0.64 for IFNL4-ΔG genotype and 0.61 for rs12979860 ('IL28B'). IFNL4-ΔG is strongly associated with impaired spontaneous HCV clearance.
The Journal of Infectious Diseases 08/2013; 209(3). DOI:10.1093/infdis/jit433 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.
[Show abstract][Hide abstract] ABSTRACT: A monoclonal antibody against prostate stem cell antigen (PSCA) has emerged as a novel cancer therapy currently being tested in clinical trials for prostate and pancreatic cancers, but this treatment is likely to be efficient only in patients with PSCA-expressing tumors. The present study demonstrates that a genetic variant (rs2294008) discovered by bladder cancer genome-wide association studies is a strong predictor of PSCA protein expression in bladder tumors, as measured by two-sided multivariable linear regression (P = 6.46×10(-11); n = 278). The association pattern is similar in non-muscle-invasive tumors, stages Ta (P = 3.10×10(-5); n = 173) and T1 (P = 2.64×10(-5); n = 60), and muscle-invasive tumors, stages T2 (P =.01; n = 23) and T3/4 (P =.03; n = 22). The study suggests that anti-PSCA immunotherapy might be beneficial for bladder cancer patients with high tumor PSCA expression, which is statistically significantly associated with the presence of CT and TT genotypes of a common genetic variant, rs2294008. Future clinical studies will be needed to validate PSCA as a therapeutic target for bladder cancer.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies have identified a SNP, rs2294008, on 8q24.3 within the prostate stem cell antigen (PSCA) gene, as a risk factor for bladder cancer. To fine-map this region, we imputed 642 SNPs within 100 Kb of rs2294008 in addition
to 33 markers genotyped in one of the reported genome-wide association study in 8,652 subjects. A multivariable logistic regression
model adjusted for rs2294008 revealed a unique signal, rs2978974 (r2 = 0.02, D′ = 0.19 with rs2294008). In the combined analysis of 5,393 cases and 7,324 controls, we detected a per-allele odds
ratio (OR) = 1.11 [95% confidence interval (CI) = 1.06–1.17, P = 5.8 × 10−5] for rs2294008 and OR = 1.07 (95% CI = 1.02–1.13, P = 9.7 × 10−3) for rs2978974. The effect was stronger in carriers of both risk variants (OR = 1.24, 95% CI = 1.08–1.41, P = 1.8 × 10−3) and there was a significant multiplicative interaction (P = 0.035) between these two SNPs, which requires replication in future studies. The T risk allele of rs2294008 was associated
with increased PSCA mRNA expression in two sets of bladder tumor samples (n = 36, P = 0.0007 and n = 34, P = 0.0054) and in normal bladder samples (n = 35, P = 0.0155), but rs2978974 was not associated with PSCA expression. SNP rs2978974 is located 10 Kb upstream of rs2294008, within an alternative untranslated first exon of PSCA. The non-risk allele G of rs2978974 showed strong interaction with nuclear proteins from five cell lines tested, implying
a regulatory function. In conclusion, a joint effect of two PSCA SNPs, rs2294008 and rs2978974, suggests that both variants may be important for bladder cancer susceptibility, possibly through
different mechanisms that influence the control of mRNA expression and interaction with regulatory factors.
Proceedings of the National Academy of Sciences 03/2012; 109(13):4974-4979. DOI:10.1073/pnas.1202189109 · 9.81 Impact Factor