Brian Muchmore

From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

A variant upstream of IFNL3 ( IL28B ) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus

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.

IL-29 Is the Dominant Type III Interferon Produced by Hepatocytes During Acute Hepatitis C Virus Infection

Early, vigorous intrahepatic induction of interferon (IFN)‐stimulated gene (ISG) induction is a feature of hepatitis C virus (HCV) infection, even though HCV inhibits the induction of type I IFNs in vitro. To identify the cytokines and cells that drive ISG induction and mediate antiviral activity during acute HCV infection, type I and III IFN responses were studied in (1) serial liver biopsies and plasma samples obtained from 6 chimpanzees throughout acute HCV infection and (2) primary human hepatocyte (PHH) cultures upon HCV infection. Type I IFNs were minimally induced at the messenger RNA (mRNA) level in the liver and were undetectable at the protein level in plasma during acute HCV infection of chimpanzees. In contrast, type III IFNs, in particular, interleukin (IL)‐29 mRNA and protein, were strongly induced and these levels correlated with ISG expression and viremia. However, there was no association between intrahepatic or peripheral type III IFN levels and the outcome of acute HCV infection. Infection of PHH with HCV recapitulated strong type III and weak type I IFN responses. Supernatants from HCV‐infected PHH cultures mediated antiviral activity upon transfer to HCV‐replicon–containing cells. This effect was significantly reduced by neutralization of type III IFNs and less by neutralization of type I IFNs. Furthermore, IL‐29 production by HCV‐infected PHH occurred independently from type I IFN signaling and was not enhanced by the presence of plasmacytoid dendritic cells. Conclusion: Hepatocyte‐derived type III IFNs contribute to ISG induction and antiviral activity, but are not the principal determinant of the outcome of HCV infection. (HEPATOLOGY 2012;56:2060–2070)

Common genetic variants in the PSCA gene influence gene expression and bladder cancer risk

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.

The 19q12 bladder cancer GWAS signal: association with cyclin E function and aggressive disease.

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

IL28B rs12979860 Genotype and Spontaneous Clearance of Hepatitis C Virus in a Multi-Ethnic Cohort of Injection Drug Users: Evidence for a Supra-Additive Association

Among 1369 Urban Health Study participants, we evaluated genetic models for the association of IL28B genotype (rs12979860 and rs8099917) with hepatitis C virus (HCV) clearance. For rs12979860, adjusted odds ratios for spontaneous HCV clearance were as follows: IL28B-CC, 3.88 (P < .001); IL28B-CT, 1.48 (P = .08). On the basis of Akaike information criteria values and χ(2) tests, a supra-additive (quadratic) model fit these data best. Models based on rs8099917 provided poorer fit. Evidence that a supra-additive rs12979860-based model best fits the association of IL28B-genotype with HCV clearance may improve clinical prediction models and foster a better understanding of functional mechanisms underlying this association.

The 19q12 Bladder Cancer GWAS Signal: Association with Cyclin E Function and Aggressive Disease

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

The 19q12 bladder cancer GWAS signal

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

Abstract 2783: A novel functional variant is associated with regulation of the prostate stem cell antigen (PSCA) gene and bladder cancer risk in males

Genome-wide association studies (GWAS) identified allele T of SNP rs2294008 within the prostate stem cell antigen (PSCA) gene as a risk factor for diffuse gastric cancer (OR=1.67, p=2.2×10-15) (The Study Group of Millennium Genome Project for Cancer, Nat Genet, 2008) and bladder cancer (OR=1.13, p=4.4×10-11) (Rothman et al, Nat Genet, 2010). In the present study we used data from the Stage 1 bladder cancer GWAS, which was performed on 8,801 individuals of European ancestry (3,529 cases and 5,115 controls), and information from the 1000 Genomes and HapMap 3 projects to impute all other SNPs within +/- 50kb region surrounding PSCA. Association analysis on the combined set of all genotyped and imputed SNPs (n=375) revealed a novel bladder cancer association signal for a variant genotyped in all samples. The association was found only in males (n=7,359, crude OR =1.14, p=1.15×10-4; adjusted for all covariates and rs2294008, OR=1.13, p=1.32×10-3), while no association was observed in females (n=1,442, crude OR=0.98, p=0.81; adjusted for all covariates and rs2294008, OR=0.98, p=0.82). The low linkage disequilibrium between these two SNPs (D’=0.202 and r2=0.019 in 8801 GWAS samples) suggests these variants are independent. A joint analysis showed that these two SNPs have a compound association with bladder cancer dependent on the number of risk genotypes (0, 1 or 2) in males (adjOR=1.18, p=3.93×10-6) but not in females (adjOR=1.01, p=0.95). The risk allele T of rs2294008 is functional as it introduces a novel translation start site that creates a PSCA protein with a leader peptide extended by 11 amino acids. The novel SNP that was found 10 Kb upstream of rs2294008, lies within an alternative untranslated first exon of PSCA that is marked by a H3K4me3 signal and is in a vicinity of an androgen receptor binding site, suggesting a possible role for this variant in regulation of PSCA promoter activity. In conclusion, a novel independent functional variant associated with bladder cancer risk was found within the PSCA gene, which might be important for male-specific regulation of PSCA expression and carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2783. doi:10.1158/1538-7445.AM2011-2783

Abstract 3751: Expression analysis of the IL28A, IL28B, IL29 and IL28L genes in primary human peripheral blood mononuclear cells and hepatocytes: Effects of activation mode, time-course and genotypes

Expression analysis of the IL28A, IL28B, IL29 and IL28L genes in primary human peripheral blood mononuclear cells and hepatocytes: Effects of activation mode, time-course and genotypes. Chronic hepatitis C virus (HCV) infection is the leading cause of liver cancer and liver transplantation in the United States. Recently, several genome-wide association studies (GWAS) identified genetic variants within the IL28A/B locus on chromosome 19q13.2 as the strongest predictors of outcome of HCV infection and response to treatment (e.g. rs12980275, p=2.84×10-27, OR=17.7; 95% CI=10.0-31.3, Tanaka, Nat. Gen. 2009). This region harbors the highly homologous IL29, IL28A, IL28B genes and a pseudogene IL28-like (IL28L), also known as interferon-lambda-1, interferon-lambda-2, interferon-lambda-3 and interferon-lambda-4-pseudo, respectively. We hypothesized that the associated genetic variants might affect mRNA expression of one or more of these genes. We performed mRNA expression analysis of IL28A, IL28B, IL29 and IL28L in human peripheral blood mononuclear cells (PBMCs) and primary hepatocytes using highly specific custom-designed assays to overcome the up to 97% sequence homology between these genes. In PBMCs and hepatocytes, mRNA transcripts were not expressed under baseline conditions, but activation of these cells with IFN-alpha or IFN-lambda did moderately induce expression of some transcripts after 24 hours of activation. However, when IL28A, IL28B, IL29 and IL28L were activated with the immunostimulant poly I:C, a Toll-like receptor-3 agonist that mimics infection by RNA viruses, expression of all transcripts was strongly increased. Gene expression was measured at 0, 1, 2, 4, 8 and 24 hours and was compared to the 2-hour point, at which expression was first stably seen. We observed a suggestive association between time-specific activation of all transcripts and genetic variants correlated with HCV infection and treatment outcome. In hepatocytes, mRNA levels of all transcripts were increased in carriers of risk alleles associated with poor treatment outcome at 24 hours compared to 2 hours (60-fold for IL28A, 85-fold for IL28B, 222-fold for IL29 and 5-fold for IL28L) suggesting that under certain circumstances increased baseline interferon production in carriers of risk variants could be deleterious. The clinical relevance of our in vitro findings should be validated by similar studies in primary liver and blood samples derived from HCV-infected individuals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3751. doi:10.1158/1538-7445.AM2011-3751