Rivkah Gonsky

Involvement of protein kinase Cepsilon (PKCepsilon) in thyroid cell death. A truncated chimeric PKCepsilon cloned from a thyroid cancer cell line protects thyroid cells from apoptosis.

The protein kinase C (PKC) family has been implicated in the regulation of apoptosis. However, the contribution of individual PKC isozymes to this process is not well understood. We reported amplification of the chromosome 2p21 locus in 28% of thyroid neoplasms, and in the WRO thyroid carcinoma cell line. By positional cloning we identified a rearrangement and amplification of the PKCε gene, that maps to 2p21, in WRO cells. This resulted in the overexpression of a chimeric/truncated PKCε (Tr-PKCε) mRNA, coding for N-terminal amino acids 1–116 of the isozyme fused to an unrelated sequence. Expression of the Tr-PKCε protein in PCCL3 cells inhibited activation-induced translocation of endogenous PKCε, but its kinase activity was unaffected, consistent with a dominant negative effect of the mutant protein on activation-induced translocation of wild-type PKCε and/or displacement of the isozyme to an aberrant subcellular location. Cell lines expressing Tr-PKCε grew to a higher saturation density than controls. Moreover, cells expressing Tr-PKCε were resistant to apoptosis, which was associated with higher Bcl-2 levels, a marked impairment in p53 stabilization, and dampened expression of Bax. These findings point to a role for PKCε in apoptosis-signaling pathways in thyroid cells, and indicate that a naturally occurring PKCε mutant that functions as a dominant negative can block cell death triggered by a variety of stimuli.

Tu1926 Functional Characterization of the CD40 IBD Risk SNP rs6074022: Integrated Gwas, eQTL, and mQTL Defines IBD Patient Subgroups With Altered Pathobiology and Distinct Natural History of Disease

Introduction: Genome-wide association studies suggest that there is dysregulation of distinct biological circuits involved in the maintenance of intestinal homeostasis in patients with inflammatory bowel diseases (IBD). Broadly, we seek to understand the relationship between patient genotype and disease-specific, tissue-dependent gene expression to elucidate critical pathways at play in health and disease, identify novel, clinically-applicable biomarkers, guide therapeutic discovery, and move towards personalized care for patients with IBD. Methods: Using the most-recent GWAS data for IBD, identifying 163 risk loci, a custom NanoString probeset of 683 IBD-associated genes and 15 housekeeping genes was designed; 25% of these genes were not assayed by conventional microarrays. Patients and controls were recruited at five IBD centers nationwide as part of the Sinai-Helmsley Alliance for Research Excellence (SHARE) consortium. To date, we have collected 352 specimens from 172 unique patients (Crohns disease, n=142, ulcerative colitis, n=28, indeterminate colitis, n=2). Total mRNA was isolated from 2-3 mucosal biopsies obtained at colonoscopy or taken from surgical specimens and used for downstream processing. A custom bioanalytic pipeline was designed for quality control and normalization to permit differential expression, data-driven clustering, and genotype-based expression quantitative trait loci (eQTL) analysis. Results: To serve as a basis for analysis in patients, we defined the expression patterns of these selected genes in healthy controls in multiple anatomic locations, including terminal ileum, ascending and descending colon, observing distinct expression patterns between ileum and colon and high correlation between controls. In the second phase of this project, we compared the expression patterns between diseased tissue versus healthy controls, inflamed versus uninflamed tissues from the same patient, and ulcerative colitis versus Crohns colitis. Preliminary analysis demonstrates a strong differential gene expression pattern with 120 upregulated genes and 8 down-regulated genes in the inflamed tissue as compared to the uninflamed tissue. Pathway analysis suggests differences in activity between inflamed and uninflamed tissues in pathways related to cell adhesion, immune system signaling, and biosynthetic processing. With targeted examination for eQTLs for four disease-related SNPs (NOD2, ATG16L1, IRGM, MST1), we have identified genotype-dependent, fold-change gene expression differences between paired inflamed and uninflamed specimens. Conclusions: Selected profiling of IBD-related genes in disease-relevant tissues by NanoString technology and in patients with known genotypes can robustly detect novel patterns of gene epistasis, identify eQTLs, and highlight dominant pathways perturbed in disease.

Su1747 Combined Epigenetic and mRNA Expression Profiling Defines a Crohn's Disease (CD) Patient Population With Need for Early Surgical Intervention

Background: Studies have confirmed associations with TNFSF15 and CD in Caucasian and Asian populations, but these studies have been limited by the number of markers tested. Decoy Receptor 3 (DcR3), previously identified as a susceptibility gene from a Caucasian pediatric CD GWAS, is a decoy receptor that can neutralize pro-inflammatory ligands including, TL1A (TNFSF15). Aim: To perform transethnic fine mapping across TNFSF15 and DcR3 in Caucasian, Puerto Rican and Korean CD. Methods: Genotyping was performed in Non-Jewish Caucasians (NJ; 779 CD, 4182 controls), Ashkenazi-Jews (AJ; 492 CD, 395 controls), Puerto Ricans (PR; 300 CD, 235 controls), and South Koreans (SK; 729 CD, 469 controls) using the 200K SNP ImmunoChip genotyping array. Analysis of TNFSF15 (416 SNPs) and DcR3 (13 SNPs) was conducted. Principal components analysis controlled for population structure, and logistic regression models tested for association of each SNP with CD. Results: TNFSF15 analyses: 4 rare variants (MAF,1%) and 9 common variants (MAF= 27.7-34.9%) were significantly associated with NJ CD after correction for multiple-testing. Analyses in NJ CD confirmed that rare and common variants associations could each be tagged by a single SNP (rare: rs1322057, MAF=0.83%, p=3.02E-6, OR=6.97; common: rs7869487, MAF=22.6%, p=7.26E-5, OR=0.78). Further, the associations with CD of these SNPs were independent of each other. All 13 NJ CD SNP associations were replicated in SK CD (all P ,3.15E-10) but with higher MAF (0.13% vs 34.6% for rs1322057 and 32.4% vs 40.9% for rs7869487 in controls). The independence of the 2 SNPs seen in NJ was replicated in SK. The rare variants were also associated with PR CD but with higher MAF than in NJ (6.6% for rs1322057, p=3.19E-2, OR=1.62), but no association was seen at 9 common variants in PR. No significant SNP associations were observed with AJ CD. DcR3 analyses: rs6062496, located in intron 6 of DcR3, was significantly associated with NJ CD (p=5.83E-4, OR=1.22). This association was confirmed in SK CD (p=2.01E-2, OR=1.32) but not in AJ or PR CD. The associated SNP was also significantly associated with protection against stricturing CD (p=2.95E-2, OR=0.75). There were no significant gene-gene interactions between rs6062496 in DCR3 and the 2 independent SNPs from TNFSF15 in both NJ (p.0.18) and SK CD (p.0.61). Conclusion: We have identified novel associations of rare variants in TNFSF15 with NJ CD that are independent of previously reported common variants, and are also found in PR and SK. These variants are more common in SK and also associated with SK CD, perhaps explaining the ‘dominant role of this locus in Asian IBD susceptibility. DcR3 is significantly associated with NJ CD, a finding replicated in SK and variation at this locus modifies a stricturing phenotype.

Genetic Predictors of Cytomegalovirus Infection in Patients With Inflammatory Bowel Disease

A Functional Variant of the Farnesoid X Receptor (FXR) Predisposes to Ileocolonic Localization of Crohns Disease Rian M. Nijmeijer, Raffaella Maria Gadaleta, Karel J. Van Erpecum, Adriaan A. van Bodegraven, J. Bart A. Crusius, Gerard Dijkstra, Daniel W. Hommes, Dirk J. De Jong, Cyriel Ponsioen, Hein W. Verspaget, Rinse K. Weersma, Christien J. van der Woude, Marguerite E. Schipper, Cisca Wijmenga, Saskia W. van Mil, Bas Oldenburg

S1174 UC Patients With High Level Antibody Responses to CD-Associated Microbial Antigens Exhibit Aberrant IFNG DNA Methylation Patterns

AIM: Colonic polyps are causally related to the nearly 150,000 yearly incident cases of colon cancer in the USA. The study aim was to identify and quantitate variables that affect polyp size in a large sample of patients undergoing CS for indications of routine screening or family history of colorectal cancer. METHODS: 93,110 adult patients from 67 GI practices in 25 states undergoing CS who had colorectal polyps on exam were identified in the Clinical Outcomes Research Initiative (CORI) database. Polyp size of the largest polyp was the outcome variable; patient age, race, gender, type of practice, anatomic location, and indication for procedure were independent variables. Ten-year age bands were used to stratify patient age; the size of the largest polyp in each patient was stratified into four ordinal groups (05 mm, 5.1-9, 9.1-15, >15 mm). Ordinal logistic regression was used to obtain multivariateadjusted estimates of polyp size in relation to patient age. RESULTS: For patients 80 years. After multivariate adjustment, odds of having a larger polyp increased with each increasing age group, compared to those under 50 years (see Table). The odds ratio for polyp size in the oldest age group versus youngest age group was 1.61 (95% CI = 1.46-1.77, p = <0.0001). Other factors with multivariate-adjusted significant odds ratios were gender (males vs females 1.18, P = <0.0001), race (blacks versus whites 1.33, P = <0.0001), practice type (community vs academic 1.26, P = <0.0001), and polyp location (distal vs proximal colon 0.89, P = <0.0001). CONCLUSION: Size of the largest polyp detected at CS increases significantly with age, and among variables included in a multivariate model, age has the strongest association with polyp size.