- Email: [email protected]
Tu1937 ATG16L1 Genotype Is Associated With Response to Anti-TNF
specifically in early post-operative recurrence of Crohn's disease. The exact role of miR-196 in the inflammatory intestinal mucosa of CD patients needs to be further investigated.
Tu1935 miR-142-3p Directly Regulates Autophagy-Dependent Gene ATG16L1 in Crohn's Disease Frances Dang, David M. Rodrigues, Michal Sibony, Nicola L. Jones
AGA Abstracts
Tu1933
Numerous genome-wide association studies demonstrate that a variant in the autophagydependent gene ATG16L1 is associated with Crohn's Disease (CD), implicating autophagy in the development of inflammation. Emerging data show that altered regulation of pro and anti-inflammatory genes by miRNA is involved in many disease states including inflammatory bowel disease. miRNA are short non-coding RNA that bind to the complementary 3' untranslated region (UTR) of the target mRNA to repress their translation and promote their degradation. Bioinformatic target prediction tools were employed to identify miRNA predicted to target ATG16L1 3'UTR. miR-142-3p was then selected for further characterization based on homology across species and reported role in inflammatory states. Here we investigated the functional effect of miR-142-3p on ATG16L1 and autophagy. A dual luciferase reporter assay was used to determine whether miR-142-3p directly targets and suppresses ATG16L1. Delivery of miR-142-3p mimic suppresses ATG16L1-3'UTR luciferase activity. To characterize the functional effect of miR-142-3p, HeLa and HTC-116 cells were transfected with either a miR mimic or the anti-miR and quantitative real time PCR for ATG16L1 and western blotting was performed. In comparison to control cells, a decrease in ATG16L1 transcripts was detected in cells transfected with the miR-142-3p mimic. A reduction in ATG16L1 and LC3-II protein level was also seen in immunoblots. To assess the impact on autophagy, LC3 staining was performed on transfected cells and imaged by confocal microscopy to quantitate endogenous autophagic puncta. Consistently, a reduction in endogenous LC3 puncta reflecting a decrease in autophagosome abundance was detected in cells transfected with miR142-3p mimic, when compared to sham-transfected cells. In contrast, an increase in LC3 puncta was detected in cells treated with anti-miR-142-3p. Taken together, these results indicate that miR142-3p can directly regulate ATG16L1 and repress autophagy. We propose that miRNA regulation of the autophagy pathway could be critical in the pathogenesis of CD.
Expression Association Analysis of the ATG5, ATG12 and ATG16L1 Genes in Crohn's Disease (CD) Hu Zhang, Dunecan C. Massey, Miles Parkes Background: Autophagy has been implicated in the pathogenesis of CD. ATG12-ATG5ATG16L1 conjugate protein complex plays a critical role in autophagy pathway. We previously reported that rs26538 in ATG12 correlated with CD at a modest level, and genetic interaction between this SNP and the focal SNP rs2241880 in ATG16L1 could contribute to CD susceptibility. Our study aimed to explore expression association of the three genes in terminal ileum biopsies in an East Anglia CD cohort. Methods: TaqMan expression & genotyping assays were all ordered from Applied Biosystems. Both expression correlation regardless of genotype and eQTL experiments for a particular SNP were undertaken in a control panel. This panel comprised 97 healthy volunteers from Addenbrooke's hospital endoscopy centre. Blood DNA of normal controls was genotyped for the SNPs. mRNA was extracted from terminal ileum biopsies and then reverse-transcribed into cDNA, which was used to measure gene expression by qPCR (2-ddCt, normalised to GAPDH). A case-control study was designed to compare gene expression between control panel and case panel with non-inflamed (24) and inflamed (22) CD cases (Kruskal-Wallis test). Results: In this study ATG5, ATG12 and ATG16L1 gene expression significantly correlated one another in normal controls regardless of genotype (p<0.05). eQTL analysis found that ATG12 expression was not significantly different between minor (CC) and major (TT) genotype in normal terminal ileum biopsies (p=0.0684). Similarly, ATG16L1 expression was not significantly different between minor (AA) and major (GG) genotypes in normal terminal ileum biopsies (p= 0.4626). In the case-control study, ATG5 expression was significantly different among inflamed CD, non-inflamed CD and controls (p=0.0012). Its expression significantly decreased in inflamed CD compared with non-inflamed CD and controls (p<0.05 and p<0.001, respectively); but ATG5 expression was not significantly different between noninflamed CD and controls (P>0.05). ATG12 expression was not significantly different among inflamed CD, non-inflamed CD and controls (p=0.17395); ATG12 expression also was not significantly different for any pair (all p-values >0.05). ATG16L1 expression was significantly different among inflamed CD, non-inflamed CD and controls (p=0.0003); ATG16L1 expression was significantly different for any pair comparison (all p-values <0.05). Conclusion: ATG5, ATG12 and ATG16L1 gene expression levels significantly correlated with one another in normal controls. ATG5 and ATG16L1 expression significantly differed among noninflamed, inflamed and controls. Together with our previously reported genetic findings, it can be hypothesized that the three autophagy genes may work together to contribute to CD susceptibility.
Tu1936 Case-Control Study and Meta-Analysis of Glutathione S-Transferase Polymorphisms in Patients With Inflammatory Bowel Disease Mark M. Broekman, Rene te Morsche, Hennie M. Roelofs, Frank Hoentjen, Wilbert H. Peters, Geert Wanten, Dirk J. De Jong Background: Glutathione S-transferases (GSTs) are important in the detoxification of a wide variety of exogenous and endogenous compounds, including reactive oxygen species (ROS). Since ROS are involved in the aetiology of inflammatory bowel diseases (IBD), polymorphisms in GSTs might modulate the risk for Crohn's disease (CD) or ulcerative colitis (UC). In more detail, the null alleles of GST Mu (GSTM1*0) and GST Theta (GSTT1*0), which cannot be detected by immunochips used in genome wide association studies, lead to a failed protein synthesis. For GST Pi (GSTP), the single nucleotide substitutions A>G at codon 105 and C>T at codon 114, are considered as the most important polymorphisms and lead to a reduced enzyme function. Previous studies highlighting GST polymorphisms in IBD patients showed divergent results, therefore we conducted a case-control study and a meta-analysis of GST polymorphisms in GST Mu (GSTM1), GST Theta (GSTT1) and GSTP in IBD patients. Methods: Genomic DNA of 552 patients with CD, 223 patients with UC and 972 healthy controls from our centre was genotyped for polymorphisms in GSTM1, GSTP1 and GSTT1 using polymerase chain reaction techniques. Results of the case-control study were included in the meta-analysis. A search on Pubmed, EMBASE and Web of Science was conducted comprising the search terms: GST, glutathione S-transferase, IBD, Crohn's disease and ulcerative colitis. Case-control studies examining GST polymorphisms in patients with IBD compared to healthy controls were included, provided that absolute numbers of genotype distributions were given. With the most common homozygous GSTP genotype or homozygous + heterozygous GSTM1 or GSTT1 genotypes set as reference, data were pooled and weighed odds ratios (OR) were calculated with a random effect model using RevMan® V5.2 software. Results: The search for the meta-analysis identified 118 articles. After screening for title and abstract 10 articles remained. Three were excluded because of Chinese language (n=2) or overlapping data (n=1). A total of 1613 patients with CD, 1759 patients with UC and 4098 healthy controls, including our data, were used for further analysis. Pooled analysis showed an increased susceptibility for UC with the homozygous GSTT1*0 genotype (OR 2.27, 95%CI 1.31-3.92). In contrast to GSTT1, the GSTP 114 Ala/Val + Val/Val genotypes had lower risk for UC (OR 0.70, 95%CI 0.54-0.90). Overall, there was no significant association of GST polymorphisms with CD, however for the GSTP 114 Ala/Val + Val/Val genotype a trend towards a decreased susceptibility was seen (OR 0.80, 95%CI 0.64-1.01). Conclusion: The homozygous GSTT1*0 genotype, associated with a decreased detoxification of ROS, may increase the susceptibility for UC, whereas the GSTP 114 Val genotypes, associated with reduced enzyme activity, may result in a lower risk for UC.
Tu1934 Whole-Genome Sequencing and Transcriptome Analysis of Mice With Progressive Crohn's Disease-Like Ileitis Alexander Rodriguez-Palacios, Sheldon Bai, Fabio Cominelli Background & Aims: The SAMP1/YitFc (SAMP) mouse strain represents a spontaneous model of progressive Crohn's disease-like ileitis and originated from selective inbreeding of AKR/J mice, via the generation of senescent-accelerated-mice prone sublines. In previous microsatellite-linkage disequilibrium and consomic analyses, we identified candidate loci for ileitis, and showed that SAMP mice have a complex genetic background with inadvertent contamination with C57BL/6J mice. The aim of this study was to perform whole genomic and transcriptome analyses of SAMP mice and compare results to other relevant mouse strains. Methods: i) First, we resequenced the genome of an 11-wk-old SAMP mouse at a 40X coverage in collaboration with the Beijing Genomics Institute using HiSeq Illumina and SOAP bioinformatics, based on the reference mouse genome C57BL/6J mm10. ii) Following the identification of SNPs, the SAMP variants were compared to the catalogue of homozygous polymorphisms available for the parental AKR/J strain and the referent genome C57BL/6J. To determine the validity of our comparisons between our SNPs and that of published data, we compared the performance of our SOAP-based analysis and that of the publically available SNP analysis methods using the original sequencing reads from the first sequenced AKR/J mouse. iii) We then compared our SAMP-specific SNPs to that of other 16 unrelated sequenced mouse strains, after verifying with stereomicroscopy and histology the intestinal heath of representative and genetically-related sequenced strains (i.e., FVB/NJ, BALBC/J, C57BL/NJ, A/J). iv) To infer the relevance of SNPs in the exome, SAMP-specific SNPs were compared to the exome variants of other 9 related SAM mice. Sanger sequencing was used to validate novel SNPs. v) Lastly, full thickness transcriptomic experiments were conducted on SAMP mice to identify differentially expressed genes (DEG) in ileal tissues with respect to two genetic controls. Results: In total, 27 strains were used for comparative genomic analysis. Repeated genome assembly and SNP analysis of the raw AKR/J sequencing reads revealed that 97% of all our homozygous SNPs identified by our alignment and calling SOAP tools were contained in the published catalog of SNPs. Comparative genomic analysis revealed that at least 135 exomic SNPs (85 genes) are unique to SAMP, while transcriptomic analysis revealed 135 uniquely differentially expressed genes, some of which have no novel SNPs, but are clustered in a region of chromosome 8. Enrichment analysis indicated pathways (for SNPs and DEG) not previously associated with IBD in humans. Conclusion: Our findings in SAMP mice indicate that gene expression differences, and possibly DNA methylation, in addition to SNPs, play an additive role to help explain the clinical and genetic variability recognized in patients with IBD.
AGA Abstracts
Tu1937 ATG16L1 Genotype Is Associated With Response to Anti-TNF Manon E. Wildenberg, Alon D. Levin, Johannan F. Brandse, Jessica R. de Bruyn, Geert R. D'Haens, Gijs R. van den Brink We have previously shown that infliximab (IFX) treatment of mixed lymphocyte reactions results in the induction of macrophages with immunosuppressive and wound healing properties. In patients who respond to IFX treatment, the number of these macrophages increases significantly in the intestine. In contrast, no increase is seen in non-responders, indicating the clinical importance of this cell population. Genetic studies have shown an association between various autophagy related genes and the development of Crohn's disease. The aim of this study was to determine whether autophagy is involved in the induction of regulatory macrophages by IFX and whether autophagy related polymorphisms influence the response to IFX. Methods Peripheral blood was isolated from 29 healthy volunteers and rs_2241880 genotype was determined by pcr. After isolation of PBMC mixed lymphocyte reactions containing cells from 2 donors were established for 150 separate donor combinations.
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regarding patients with UC was found. Conclusions: HIF1α 1772TT genotype is associated with presence of UC (compared with CD) and with lower risk of extraintestinal manifestation in IBD. HIF1α 1772TT and VEGFA 634CC polymorphisms are associated with disease location and behaviour in CD but not in UC. Table 1. Genotype frequencies.
AGA Abstracts
Cultures were treated with IFX or control IgG and incubated for 6-7 days. Cells were analyzed by gene array, immunofluorescence, flow cytometry and thymidine incorporation. Results Anti-TNF induced regulatory macrophages displayed increased numbers of autophagosomes as well as an increased expression of autophagy related transcripts including atg5, atg7, atg9 and atg16l2, suggesting induction of autophagy by IFX treatment. Of all donors, 7 were homozygously carrying the CD associated risk allele, 14 were heterozygous and 7 were homozygous for the WT allele. The number of CD14+ regulatory macrophages correlated significantly with the number of WT alleles present in each individual culture, with the largest number of macrophages found in cultures containing 3 or 4 WT alleles (2 WT donors or 1 WT and 1 heterozygous). Similarly, expression of CD206, which is associated with the immunosuppressive function of macrophages, positively correlated with the number of WT alleles present. To confirm the functional consequences of these findings, IFX mediated suppression of T cell proliferation was determined. Again, the level of suppression correlated significantly with the number of WT alleles present in the respective donor combinations. Conclusion Induction of regulatory macrophages by IFX is associated with induction of autophagy. In vitro, the number and function of IFX induced macrophages correlated with the number of WT alleles for the autophagy related gene ATG16L1. This suggests that an intact autophagy pathway is important for effectiveness of anti-TNF therapy. Tu1938
Table 2. OR analysis for allelic frequencies.
Identification of New Genetic Variants Related to Thiopurine-Induced Myelotoxicity in Inflammatory Bowel Disease (IBD) Patients With Normal Thiopurines-Methyltransferase (TPMT): A Genome-Wide Association Study Maria Chaparro, Anna González-Neira, Manuel Román, Guillermo Pita, Teresa Cabaleiro, Daniel Herrero, Belen Herraez, Rosario Alonso, Carlos Taxonera, Pilar Lopez-Serrano, Pilar Martínez-Montiel, Isabel Vera, Fernando Bermejo, Antonio López-SanRomán, Francisco Abad-Santos, Javier P. Gisbert Background: Only a minority of patients with thiopurine-induced myelotoxicity are carriers of a mutant TPMT allele. Aim: To identify genetic variants associated with thiopurineinduced myelotoxicity in IBD patients with TPMT wild-type alleles and normal activity of this enzyme. Methods: 93 IBD patients with normal TPMT activity and wild-type genotype treated with thiopurines were included. Case group: patients with thiopurine-induced myelotoxicity and without other thiopurine-induced adverse effect. Control group: patients without thiopurine-induced side effects consecutively included. TPMT activity over 13.7 UI/mL was considered normal. DNA was extracted from peripheral blood nucleated cells. TPMT genotype was determined by sequencing (TPMT*2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, *9, *10, *15, *16, *19 and *22 alleles). Myelotoxicity was defined as <3,000/ml leucocytes, <1,500/ ml neutrophils, or <100,000/ml platelets. Genomic DNA was analysed using Illumina OmniExpress Exome BeadChip genotyping array. This array interrogates a total of 951,117 single nucleotide polymorphisms (SNPs) (660K common and 200k rare coding variants). Genotype calls were generated using Illumina GenomeStudio. After standard QC control three samples were excluded. Logistic regression analysis for each SNP using PLINK package and gene-based analysis using SKAT software was performed. Results: The distribution of gender, type of IBD, mean age, mean TPMT activity and mean dose of mercaptopurine were similar between cases and controls (34 cases and 56 controls). The percentage of patients with mercaptopurine was higher among cases (32.4 vs. 12.5%, p=0.02), while mean azathioprine dose was slightly higher among controls (2.2 vs. 2.5 mg/kg, p=0.03). Five SNPs with p<10-4 were found: SNP1 (p=6.2 x 10-5, OR=0.1); SNP2 (p=7.6 x 10-5, OR=10.2); SNP3 (p=8 x 10-5, OR= 7.3), SNP4 (p=9.4 x 10-5, OR=4.2) and SNP5 (p=7.8 x 10-5, OR=6.5). The last SNP is located close to a gene that encodes a relevant enzyme of the thiopurine metabolic pathway. Thought gene-based analysis a gene, GENE1, that encodes an enzyme that catabolizes a mediator of inflammation present in the human bone marrow, was associated with mielotoxicity development (p=1.7 x 10-4). Conclusion: Using a genome-wide approach in patients without TPMT deficiency 5 new SNPs and genetic variants in a gene have been found to be associated with thiopurine-induced myelotoxicity. The genetic variants in this gene could alter the levels of a mediator of leukocyte functions, platelet aggregation and inflammation, that could explain the susceptibility differences to suffer myelotoxicity. These promising results need to be validated.
*N.S. Not significant. Tu1940 CD24 Polymorphisms and Susceptibility to Inflammatory Bowel Disease Victoria Lisiansky, Sarah Kraus, Inna Naumov, Diana Kazanov, Ilana Naboichtchikov, Ohad Toledano, Moshe Leshno, Doran Avivi, Menachem Moshkowitz, Nadir Arber, Iris Dotan Background: Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are chronic inflammatory disorders of the GI tract probably resulting from an aberrant immune response to luminal microbial antigens in a genetically predisposed host. Despite significant progress in the understanding and treatment of IBD, patients have an increased risk of colorectal cancer. We have previously shown that CD24 plays an important role in the multistep process of colorectal carcinogenesis and that it may be a target for chemoprevention and anti-tumor therapy (Sagiv, Gastroenterol, 2006; Sagiv, Can Res, 2008; Shapira, Gastroenterol 2011). However, the role of CD24 in mediating colitis has not been elucidated. Recently, single nucleotide polymorphisms (SNPs) in the CD24 gene have been associated with the development of several autoimmune diseases. Aim: Evaluate whether CD24 SNPs are associated with a risk for IBD. Methods: The CD24 polymorphisms: C170T (rs8734), TG1527del (rs3838646), A1626G (rs1058881) and A1056G (rs1058818) were assessed in a case-control study of an Israeli cohort comprising 138 IBD patients and 105 age- and gender-matched healthy controls. Restriction fragment length polymorphism (RFLP) analysis was performed using BstX1, Bsr1, Mfe1, and BstU1 restriction enzymes, respectively. Odds ratio (OR) and 95% confidence interval (CI) were estimated by logistic regression models. Results: C170T carriers had more IBD (OR=3.022, 95% CI: 1.748-5.223, p=0.001): UC (OR=3.002, 95% CI: 1.661-5.427, p=0.001) and CD (OR=3.077, 95% CI: 1.334-7.095 p=0.008). Carrying the A1626G and A1056G SNPs was a risk factor for IBD: OR=2.460, 95% CI: 1.420-4.259, p=0.001 and OR=1.856, 95%: 1.0113.405, p=0.01, respectively, specifically UC: OR=2.218, 95% CI: 1.207-4.075, p=0.01 and OR=1.944, 95% CI: 0.995-3.798, p=0.01, respectively, but not for CD (p=0.086, p=0.299). A1626G and TG1527del were associated with a younger age of IBD onset (p=0.022, p= 0.027, respectively). Conclusions: 1. The CD24 C170T polymorphism is associated with IBD risk. 2. A1626G and A1056G SNPs might be specifically associated with UC risk. 3. CD24 SNPs associated with an earlier onset of IBD (A1626G and TG1527del) and may have a negative prognostic effect. 4. CD24 may be a new IBD genetic susceptibility factor, with clinical implications in the prediction of IBD phenotype, and the disease course.
Tu1939 Association of Hif1α Pathway Genes With Relevant Clinical Subphenotypes in Inflammatory Bowel Disease (IBD) Pablo M. Linares, Maria Encarnacion Fernandez Contreras, Maria Chaparro, Antonio Julià, Raul Tortosa, Sara Marsal, Javier P. Gisbert Background: The hypoxia-inducible HIF1α-activated genes include VEGFA, which is an angiogenesis promoter. Both factors are highly polymorphic and important regulators of the angiogenic development and inflammation during IBD pathogenesis. Aims: To evaluate the association of HIF1α 1772C/T and VEGFA 2578A/C and VEGFA 634G/C genotype polymorphisms with Crohn's disease (CD) and ulcerative colitis (UC). Methods: Patiens with CD and patients with UC were included. DNA samples from peripheral blood were extracted and genotyped for one HIF1α single nucleotide polymorphism (SNP) (1772C/T) and two VEGFA SNPs (2578A/C and 634G/C). The genotype data from all three polymorphisms was obtained using the Illumina bead array genotyping platform Quad610. Results: One hundred and seventy-one patients with IBD (103 UC, 68 CD) were included. The mean age was 45±21 years, and 53% were male. The mean disease duration was 12±7 years. Genotype frequencies are shown in table 1. The results of the comparison between wild type homozygous versus carriers of variant alleles by Odds-ratio (OR) analysis are shown in table 2. The homozygous 1772TT variant genotype was associated with risk of suffering UC, compared with CD (table 2). The allelic frequencies of the studied genotypes and the rate of carriers of variant alleles for 2578CC and 634CC were similar between patients with CD and UC. 1772TT SNP was associated with low risk of ileal location (OR 0.65, 95% CI 0.58-0.73; p<0.0001) and stricturing behaviour (OR 0.89, 95% CI 0.85-0.95; p<0.0001) in patients with CD, and extraintestinal manifestations (OR 0.82, 95% CI 0.76-0.89; p<0.0001) in patients with IBD. SNP 634CC was associated with low risk of stricturing behaviour (OR 0.891, 95% CI 0.840-0.944; p<0.0001) in patients with CD. No association
Tu1941 FCγ Receptor Type Iiia Polymorphisms and Their Correlation With Clinical Outcome in Patients With Inflammatory Bowel Disease During a Long Term Follow Up Giorgia Bodini, Vincenzo Savarino, Pietro Dulbecco, Isabella Baldissarro, Edoardo Savarino Introduction: A total of 20-30% of patients with active Crohn's disease (CD) do not respond to anti-TNF-α treatments and up to 40% of patients in chronic therapy experience a loss of response. Furthermore about 50% of patients with ulcerative colitis (UC) experience a
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AGA Abstracts
Tu1935 miR-142-3p Directly Regulates Autophagy-Dependent Gene ATG16L1 in Crohn's Disease Frances Dang, David M. Rodrigues, Michal Sibony, Nicola L. Jones
AGA Abstracts
Tu1933
Numerous genome-wide association studies demonstrate that a variant in the autophagydependent gene ATG16L1 is associated with Crohn's Disease (CD), implicating autophagy in the development of inflammation. Emerging data show that altered regulation of pro and anti-inflammatory genes by miRNA is involved in many disease states including inflammatory bowel disease. miRNA are short non-coding RNA that bind to the complementary 3' untranslated region (UTR) of the target mRNA to repress their translation and promote their degradation. Bioinformatic target prediction tools were employed to identify miRNA predicted to target ATG16L1 3'UTR. miR-142-3p was then selected for further characterization based on homology across species and reported role in inflammatory states. Here we investigated the functional effect of miR-142-3p on ATG16L1 and autophagy. A dual luciferase reporter assay was used to determine whether miR-142-3p directly targets and suppresses ATG16L1. Delivery of miR-142-3p mimic suppresses ATG16L1-3'UTR luciferase activity. To characterize the functional effect of miR-142-3p, HeLa and HTC-116 cells were transfected with either a miR mimic or the anti-miR and quantitative real time PCR for ATG16L1 and western blotting was performed. In comparison to control cells, a decrease in ATG16L1 transcripts was detected in cells transfected with the miR-142-3p mimic. A reduction in ATG16L1 and LC3-II protein level was also seen in immunoblots. To assess the impact on autophagy, LC3 staining was performed on transfected cells and imaged by confocal microscopy to quantitate endogenous autophagic puncta. Consistently, a reduction in endogenous LC3 puncta reflecting a decrease in autophagosome abundance was detected in cells transfected with miR142-3p mimic, when compared to sham-transfected cells. In contrast, an increase in LC3 puncta was detected in cells treated with anti-miR-142-3p. Taken together, these results indicate that miR142-3p can directly regulate ATG16L1 and repress autophagy. We propose that miRNA regulation of the autophagy pathway could be critical in the pathogenesis of CD.
Expression Association Analysis of the ATG5, ATG12 and ATG16L1 Genes in Crohn's Disease (CD) Hu Zhang, Dunecan C. Massey, Miles Parkes Background: Autophagy has been implicated in the pathogenesis of CD. ATG12-ATG5ATG16L1 conjugate protein complex plays a critical role in autophagy pathway. We previously reported that rs26538 in ATG12 correlated with CD at a modest level, and genetic interaction between this SNP and the focal SNP rs2241880 in ATG16L1 could contribute to CD susceptibility. Our study aimed to explore expression association of the three genes in terminal ileum biopsies in an East Anglia CD cohort. Methods: TaqMan expression & genotyping assays were all ordered from Applied Biosystems. Both expression correlation regardless of genotype and eQTL experiments for a particular SNP were undertaken in a control panel. This panel comprised 97 healthy volunteers from Addenbrooke's hospital endoscopy centre. Blood DNA of normal controls was genotyped for the SNPs. mRNA was extracted from terminal ileum biopsies and then reverse-transcribed into cDNA, which was used to measure gene expression by qPCR (2-ddCt, normalised to GAPDH). A case-control study was designed to compare gene expression between control panel and case panel with non-inflamed (24) and inflamed (22) CD cases (Kruskal-Wallis test). Results: In this study ATG5, ATG12 and ATG16L1 gene expression significantly correlated one another in normal controls regardless of genotype (p<0.05). eQTL analysis found that ATG12 expression was not significantly different between minor (CC) and major (TT) genotype in normal terminal ileum biopsies (p=0.0684). Similarly, ATG16L1 expression was not significantly different between minor (AA) and major (GG) genotypes in normal terminal ileum biopsies (p= 0.4626). In the case-control study, ATG5 expression was significantly different among inflamed CD, non-inflamed CD and controls (p=0.0012). Its expression significantly decreased in inflamed CD compared with non-inflamed CD and controls (p<0.05 and p<0.001, respectively); but ATG5 expression was not significantly different between noninflamed CD and controls (P>0.05). ATG12 expression was not significantly different among inflamed CD, non-inflamed CD and controls (p=0.17395); ATG12 expression also was not significantly different for any pair (all p-values >0.05). ATG16L1 expression was significantly different among inflamed CD, non-inflamed CD and controls (p=0.0003); ATG16L1 expression was significantly different for any pair comparison (all p-values <0.05). Conclusion: ATG5, ATG12 and ATG16L1 gene expression levels significantly correlated with one another in normal controls. ATG5 and ATG16L1 expression significantly differed among noninflamed, inflamed and controls. Together with our previously reported genetic findings, it can be hypothesized that the three autophagy genes may work together to contribute to CD susceptibility.
Tu1936 Case-Control Study and Meta-Analysis of Glutathione S-Transferase Polymorphisms in Patients With Inflammatory Bowel Disease Mark M. Broekman, Rene te Morsche, Hennie M. Roelofs, Frank Hoentjen, Wilbert H. Peters, Geert Wanten, Dirk J. De Jong Background: Glutathione S-transferases (GSTs) are important in the detoxification of a wide variety of exogenous and endogenous compounds, including reactive oxygen species (ROS). Since ROS are involved in the aetiology of inflammatory bowel diseases (IBD), polymorphisms in GSTs might modulate the risk for Crohn's disease (CD) or ulcerative colitis (UC). In more detail, the null alleles of GST Mu (GSTM1*0) and GST Theta (GSTT1*0), which cannot be detected by immunochips used in genome wide association studies, lead to a failed protein synthesis. For GST Pi (GSTP), the single nucleotide substitutions A>G at codon 105 and C>T at codon 114, are considered as the most important polymorphisms and lead to a reduced enzyme function. Previous studies highlighting GST polymorphisms in IBD patients showed divergent results, therefore we conducted a case-control study and a meta-analysis of GST polymorphisms in GST Mu (GSTM1), GST Theta (GSTT1) and GSTP in IBD patients. Methods: Genomic DNA of 552 patients with CD, 223 patients with UC and 972 healthy controls from our centre was genotyped for polymorphisms in GSTM1, GSTP1 and GSTT1 using polymerase chain reaction techniques. Results of the case-control study were included in the meta-analysis. A search on Pubmed, EMBASE and Web of Science was conducted comprising the search terms: GST, glutathione S-transferase, IBD, Crohn's disease and ulcerative colitis. Case-control studies examining GST polymorphisms in patients with IBD compared to healthy controls were included, provided that absolute numbers of genotype distributions were given. With the most common homozygous GSTP genotype or homozygous + heterozygous GSTM1 or GSTT1 genotypes set as reference, data were pooled and weighed odds ratios (OR) were calculated with a random effect model using RevMan® V5.2 software. Results: The search for the meta-analysis identified 118 articles. After screening for title and abstract 10 articles remained. Three were excluded because of Chinese language (n=2) or overlapping data (n=1). A total of 1613 patients with CD, 1759 patients with UC and 4098 healthy controls, including our data, were used for further analysis. Pooled analysis showed an increased susceptibility for UC with the homozygous GSTT1*0 genotype (OR 2.27, 95%CI 1.31-3.92). In contrast to GSTT1, the GSTP 114 Ala/Val + Val/Val genotypes had lower risk for UC (OR 0.70, 95%CI 0.54-0.90). Overall, there was no significant association of GST polymorphisms with CD, however for the GSTP 114 Ala/Val + Val/Val genotype a trend towards a decreased susceptibility was seen (OR 0.80, 95%CI 0.64-1.01). Conclusion: The homozygous GSTT1*0 genotype, associated with a decreased detoxification of ROS, may increase the susceptibility for UC, whereas the GSTP 114 Val genotypes, associated with reduced enzyme activity, may result in a lower risk for UC.
Tu1934 Whole-Genome Sequencing and Transcriptome Analysis of Mice With Progressive Crohn's Disease-Like Ileitis Alexander Rodriguez-Palacios, Sheldon Bai, Fabio Cominelli Background & Aims: The SAMP1/YitFc (SAMP) mouse strain represents a spontaneous model of progressive Crohn's disease-like ileitis and originated from selective inbreeding of AKR/J mice, via the generation of senescent-accelerated-mice prone sublines. In previous microsatellite-linkage disequilibrium and consomic analyses, we identified candidate loci for ileitis, and showed that SAMP mice have a complex genetic background with inadvertent contamination with C57BL/6J mice. The aim of this study was to perform whole genomic and transcriptome analyses of SAMP mice and compare results to other relevant mouse strains. Methods: i) First, we resequenced the genome of an 11-wk-old SAMP mouse at a 40X coverage in collaboration with the Beijing Genomics Institute using HiSeq Illumina and SOAP bioinformatics, based on the reference mouse genome C57BL/6J mm10. ii) Following the identification of SNPs, the SAMP variants were compared to the catalogue of homozygous polymorphisms available for the parental AKR/J strain and the referent genome C57BL/6J. To determine the validity of our comparisons between our SNPs and that of published data, we compared the performance of our SOAP-based analysis and that of the publically available SNP analysis methods using the original sequencing reads from the first sequenced AKR/J mouse. iii) We then compared our SAMP-specific SNPs to that of other 16 unrelated sequenced mouse strains, after verifying with stereomicroscopy and histology the intestinal heath of representative and genetically-related sequenced strains (i.e., FVB/NJ, BALBC/J, C57BL/NJ, A/J). iv) To infer the relevance of SNPs in the exome, SAMP-specific SNPs were compared to the exome variants of other 9 related SAM mice. Sanger sequencing was used to validate novel SNPs. v) Lastly, full thickness transcriptomic experiments were conducted on SAMP mice to identify differentially expressed genes (DEG) in ileal tissues with respect to two genetic controls. Results: In total, 27 strains were used for comparative genomic analysis. Repeated genome assembly and SNP analysis of the raw AKR/J sequencing reads revealed that 97% of all our homozygous SNPs identified by our alignment and calling SOAP tools were contained in the published catalog of SNPs. Comparative genomic analysis revealed that at least 135 exomic SNPs (85 genes) are unique to SAMP, while transcriptomic analysis revealed 135 uniquely differentially expressed genes, some of which have no novel SNPs, but are clustered in a region of chromosome 8. Enrichment analysis indicated pathways (for SNPs and DEG) not previously associated with IBD in humans. Conclusion: Our findings in SAMP mice indicate that gene expression differences, and possibly DNA methylation, in addition to SNPs, play an additive role to help explain the clinical and genetic variability recognized in patients with IBD.
AGA Abstracts
Tu1937 ATG16L1 Genotype Is Associated With Response to Anti-TNF Manon E. Wildenberg, Alon D. Levin, Johannan F. Brandse, Jessica R. de Bruyn, Geert R. D'Haens, Gijs R. van den Brink We have previously shown that infliximab (IFX) treatment of mixed lymphocyte reactions results in the induction of macrophages with immunosuppressive and wound healing properties. In patients who respond to IFX treatment, the number of these macrophages increases significantly in the intestine. In contrast, no increase is seen in non-responders, indicating the clinical importance of this cell population. Genetic studies have shown an association between various autophagy related genes and the development of Crohn's disease. The aim of this study was to determine whether autophagy is involved in the induction of regulatory macrophages by IFX and whether autophagy related polymorphisms influence the response to IFX. Methods Peripheral blood was isolated from 29 healthy volunteers and rs_2241880 genotype was determined by pcr. After isolation of PBMC mixed lymphocyte reactions containing cells from 2 donors were established for 150 separate donor combinations.
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regarding patients with UC was found. Conclusions: HIF1α 1772TT genotype is associated with presence of UC (compared with CD) and with lower risk of extraintestinal manifestation in IBD. HIF1α 1772TT and VEGFA 634CC polymorphisms are associated with disease location and behaviour in CD but not in UC. Table 1. Genotype frequencies.
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Cultures were treated with IFX or control IgG and incubated for 6-7 days. Cells were analyzed by gene array, immunofluorescence, flow cytometry and thymidine incorporation. Results Anti-TNF induced regulatory macrophages displayed increased numbers of autophagosomes as well as an increased expression of autophagy related transcripts including atg5, atg7, atg9 and atg16l2, suggesting induction of autophagy by IFX treatment. Of all donors, 7 were homozygously carrying the CD associated risk allele, 14 were heterozygous and 7 were homozygous for the WT allele. The number of CD14+ regulatory macrophages correlated significantly with the number of WT alleles present in each individual culture, with the largest number of macrophages found in cultures containing 3 or 4 WT alleles (2 WT donors or 1 WT and 1 heterozygous). Similarly, expression of CD206, which is associated with the immunosuppressive function of macrophages, positively correlated with the number of WT alleles present. To confirm the functional consequences of these findings, IFX mediated suppression of T cell proliferation was determined. Again, the level of suppression correlated significantly with the number of WT alleles present in the respective donor combinations. Conclusion Induction of regulatory macrophages by IFX is associated with induction of autophagy. In vitro, the number and function of IFX induced macrophages correlated with the number of WT alleles for the autophagy related gene ATG16L1. This suggests that an intact autophagy pathway is important for effectiveness of anti-TNF therapy. Tu1938
Table 2. OR analysis for allelic frequencies.
Identification of New Genetic Variants Related to Thiopurine-Induced Myelotoxicity in Inflammatory Bowel Disease (IBD) Patients With Normal Thiopurines-Methyltransferase (TPMT): A Genome-Wide Association Study Maria Chaparro, Anna González-Neira, Manuel Román, Guillermo Pita, Teresa Cabaleiro, Daniel Herrero, Belen Herraez, Rosario Alonso, Carlos Taxonera, Pilar Lopez-Serrano, Pilar Martínez-Montiel, Isabel Vera, Fernando Bermejo, Antonio López-SanRomán, Francisco Abad-Santos, Javier P. Gisbert Background: Only a minority of patients with thiopurine-induced myelotoxicity are carriers of a mutant TPMT allele. Aim: To identify genetic variants associated with thiopurineinduced myelotoxicity in IBD patients with TPMT wild-type alleles and normal activity of this enzyme. Methods: 93 IBD patients with normal TPMT activity and wild-type genotype treated with thiopurines were included. Case group: patients with thiopurine-induced myelotoxicity and without other thiopurine-induced adverse effect. Control group: patients without thiopurine-induced side effects consecutively included. TPMT activity over 13.7 UI/mL was considered normal. DNA was extracted from peripheral blood nucleated cells. TPMT genotype was determined by sequencing (TPMT*2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, *9, *10, *15, *16, *19 and *22 alleles). Myelotoxicity was defined as <3,000/ml leucocytes, <1,500/ ml neutrophils, or <100,000/ml platelets. Genomic DNA was analysed using Illumina OmniExpress Exome BeadChip genotyping array. This array interrogates a total of 951,117 single nucleotide polymorphisms (SNPs) (660K common and 200k rare coding variants). Genotype calls were generated using Illumina GenomeStudio. After standard QC control three samples were excluded. Logistic regression analysis for each SNP using PLINK package and gene-based analysis using SKAT software was performed. Results: The distribution of gender, type of IBD, mean age, mean TPMT activity and mean dose of mercaptopurine were similar between cases and controls (34 cases and 56 controls). The percentage of patients with mercaptopurine was higher among cases (32.4 vs. 12.5%, p=0.02), while mean azathioprine dose was slightly higher among controls (2.2 vs. 2.5 mg/kg, p=0.03). Five SNPs with p<10-4 were found: SNP1 (p=6.2 x 10-5, OR=0.1); SNP2 (p=7.6 x 10-5, OR=10.2); SNP3 (p=8 x 10-5, OR= 7.3), SNP4 (p=9.4 x 10-5, OR=4.2) and SNP5 (p=7.8 x 10-5, OR=6.5). The last SNP is located close to a gene that encodes a relevant enzyme of the thiopurine metabolic pathway. Thought gene-based analysis a gene, GENE1, that encodes an enzyme that catabolizes a mediator of inflammation present in the human bone marrow, was associated with mielotoxicity development (p=1.7 x 10-4). Conclusion: Using a genome-wide approach in patients without TPMT deficiency 5 new SNPs and genetic variants in a gene have been found to be associated with thiopurine-induced myelotoxicity. The genetic variants in this gene could alter the levels of a mediator of leukocyte functions, platelet aggregation and inflammation, that could explain the susceptibility differences to suffer myelotoxicity. These promising results need to be validated.
*N.S. Not significant. Tu1940 CD24 Polymorphisms and Susceptibility to Inflammatory Bowel Disease Victoria Lisiansky, Sarah Kraus, Inna Naumov, Diana Kazanov, Ilana Naboichtchikov, Ohad Toledano, Moshe Leshno, Doran Avivi, Menachem Moshkowitz, Nadir Arber, Iris Dotan Background: Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are chronic inflammatory disorders of the GI tract probably resulting from an aberrant immune response to luminal microbial antigens in a genetically predisposed host. Despite significant progress in the understanding and treatment of IBD, patients have an increased risk of colorectal cancer. We have previously shown that CD24 plays an important role in the multistep process of colorectal carcinogenesis and that it may be a target for chemoprevention and anti-tumor therapy (Sagiv, Gastroenterol, 2006; Sagiv, Can Res, 2008; Shapira, Gastroenterol 2011). However, the role of CD24 in mediating colitis has not been elucidated. Recently, single nucleotide polymorphisms (SNPs) in the CD24 gene have been associated with the development of several autoimmune diseases. Aim: Evaluate whether CD24 SNPs are associated with a risk for IBD. Methods: The CD24 polymorphisms: C170T (rs8734), TG1527del (rs3838646), A1626G (rs1058881) and A1056G (rs1058818) were assessed in a case-control study of an Israeli cohort comprising 138 IBD patients and 105 age- and gender-matched healthy controls. Restriction fragment length polymorphism (RFLP) analysis was performed using BstX1, Bsr1, Mfe1, and BstU1 restriction enzymes, respectively. Odds ratio (OR) and 95% confidence interval (CI) were estimated by logistic regression models. Results: C170T carriers had more IBD (OR=3.022, 95% CI: 1.748-5.223, p=0.001): UC (OR=3.002, 95% CI: 1.661-5.427, p=0.001) and CD (OR=3.077, 95% CI: 1.334-7.095 p=0.008). Carrying the A1626G and A1056G SNPs was a risk factor for IBD: OR=2.460, 95% CI: 1.420-4.259, p=0.001 and OR=1.856, 95%: 1.0113.405, p=0.01, respectively, specifically UC: OR=2.218, 95% CI: 1.207-4.075, p=0.01 and OR=1.944, 95% CI: 0.995-3.798, p=0.01, respectively, but not for CD (p=0.086, p=0.299). A1626G and TG1527del were associated with a younger age of IBD onset (p=0.022, p= 0.027, respectively). Conclusions: 1. The CD24 C170T polymorphism is associated with IBD risk. 2. A1626G and A1056G SNPs might be specifically associated with UC risk. 3. CD24 SNPs associated with an earlier onset of IBD (A1626G and TG1527del) and may have a negative prognostic effect. 4. CD24 may be a new IBD genetic susceptibility factor, with clinical implications in the prediction of IBD phenotype, and the disease course.
Tu1939 Association of Hif1α Pathway Genes With Relevant Clinical Subphenotypes in Inflammatory Bowel Disease (IBD) Pablo M. Linares, Maria Encarnacion Fernandez Contreras, Maria Chaparro, Antonio Julià, Raul Tortosa, Sara Marsal, Javier P. Gisbert Background: The hypoxia-inducible HIF1α-activated genes include VEGFA, which is an angiogenesis promoter. Both factors are highly polymorphic and important regulators of the angiogenic development and inflammation during IBD pathogenesis. Aims: To evaluate the association of HIF1α 1772C/T and VEGFA 2578A/C and VEGFA 634G/C genotype polymorphisms with Crohn's disease (CD) and ulcerative colitis (UC). Methods: Patiens with CD and patients with UC were included. DNA samples from peripheral blood were extracted and genotyped for one HIF1α single nucleotide polymorphism (SNP) (1772C/T) and two VEGFA SNPs (2578A/C and 634G/C). The genotype data from all three polymorphisms was obtained using the Illumina bead array genotyping platform Quad610. Results: One hundred and seventy-one patients with IBD (103 UC, 68 CD) were included. The mean age was 45±21 years, and 53% were male. The mean disease duration was 12±7 years. Genotype frequencies are shown in table 1. The results of the comparison between wild type homozygous versus carriers of variant alleles by Odds-ratio (OR) analysis are shown in table 2. The homozygous 1772TT variant genotype was associated with risk of suffering UC, compared with CD (table 2). The allelic frequencies of the studied genotypes and the rate of carriers of variant alleles for 2578CC and 634CC were similar between patients with CD and UC. 1772TT SNP was associated with low risk of ileal location (OR 0.65, 95% CI 0.58-0.73; p<0.0001) and stricturing behaviour (OR 0.89, 95% CI 0.85-0.95; p<0.0001) in patients with CD, and extraintestinal manifestations (OR 0.82, 95% CI 0.76-0.89; p<0.0001) in patients with IBD. SNP 634CC was associated with low risk of stricturing behaviour (OR 0.891, 95% CI 0.840-0.944; p<0.0001) in patients with CD. No association
Tu1941 FCγ Receptor Type Iiia Polymorphisms and Their Correlation With Clinical Outcome in Patients With Inflammatory Bowel Disease During a Long Term Follow Up Giorgia Bodini, Vincenzo Savarino, Pietro Dulbecco, Isabella Baldissarro, Edoardo Savarino Introduction: A total of 20-30% of patients with active Crohn's disease (CD) do not respond to anti-TNF-α treatments and up to 40% of patients in chronic therapy experience a loss of response. Furthermore about 50% of patients with ulcerative colitis (UC) experience a
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