Giuseppe Corritore

Investigation of Multiple Susceptibility Loci for Inflammatory Bowel Disease in an Italian Cohort of Patients

Background Recent GWAs and meta-analyses have outlined about 100 susceptibility genes/loci for inflammatory bowel diseases (IBD). In this study we aimed to investigate the influence of SNPs tagging the genes/loci PTGER4, TNFSF15, NKX2-3, ZNF365, IFNG, PTPN2, PSMG1, and HLA in a large pediatric- and adult-onset IBD Italian cohort. Methods Eight SNPs were assessed in 1,070 Crohns disease (CD), 1,213 ulcerative colitis (UC), 557 of whom being diagnosed at the age of ≤16 years, and 789 healthy controls. Correlations with sub-phenotypes and major variants of NOD2 gene were investigated. Results The SNPs tagging the TNFSF15, NKX2-3, ZNF365, and PTPN2 genes were associated with CD (P values ranging from 0.037 to 7×10−6). The SNPs tagging the PTGER4, NKX2-3, ZNF365, IFNG, PSMG1, and HLA area were associated with UC (P values 0.047 to 4×10−5). In the pediatric cohort the associations of TNFSF15, NKX2-3 with CD, and PTGER4, NKX2-3, ZNF365, IFNG, PSMG1 with UC, were confirmed. Association with TNFSF15 and pediatric UC was also reported. A correlation with NKX2-3 and need for surgery (P  =  0.038), and with HLA and steroid-responsiveness (P  =  0.024) in UC patients was observed. Moreover, significant association in our CD cohort with TNFSF15 SNP and colonic involvement (P  =  0.021), and with ZNF365 and ileal location (P  =  0.024) was demonstrated. Conclusions We confirmed in a large Italian cohort the associations with CD and UC of newly identified genes, both in adult and pediatric cohort of patients, with some influence on sub-phenotypes.

Associations between Genetic Polymorphisms in IL-33, IL1R1 and Risk for Inflammatory Bowel Disease

Background Recent evidence suggests that the IL-33/IL1RL1 axis plays a critical role in several autoimmune and inflammatory disorders; however, its mechanistic role in inflammatory bowel disease (IBD) has not been clearly defined. We investigated the contribution of IL-33 and IL1RL1 polymorphisms to IBD risk, and possible correlations with phenotype in an Italian cohort of adult and pediatric patients. Methods We evaluated the association of six SNPs in IL-33 and IL1RL1 genes, in 805 Crohn’s disease (CD), 816 ulcerative colitis (UC), and 752 controls, using Taqman. IL-33 and IL1RL1 mRNA expression was also analyzed. Results Significant allele and genotype associations with IL-33 rs3939286 were found in CD (P = 0.004; P = 0.035) and UC patients (P = 0.002; P = 0.038). After stratifying the cohort for age at diagnosis, the differences remained significant only in the IBD adult-onset. Significant associations were also obtained in CD patients with two IL1RL1 polymorphisms (rs13015714 and rs2058660, P<0.015). By combining homo- and heterozygous carriers of the rs13015714 risk allele, differences were still significant for both CD adult- and pediatric-onset. Upon genotype-phenotype evaluation, an increased frequency of extensive colitis in adult UC (P = 0.019) and in steroid-responsive pediatric patients (P = 0.024) carrying the IL-33 rs3939286 risk genotype, was observed. mRNA expression of IL-33 and IL1RL1 in inflamed IBD biopsy samples was significantly increased. Conclusions Common IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on sub-phenotypes.

Evaluating the role of the genetic variations of PTPN22, NFKB1, and FcGRIIIA genes in inflammatory bowel disease: A meta-analysis

Background: We tested several polymorphisms of genes involved in the mucosal immune system in a population of inflammatory bowel disease (IBD) patients to investigate their possible implication in disease predisposition. Methods: Polymorphisms of 3 candidate genes (PTPN22, NFkB1, and FcGRIIIA) were investigated in 649 IBD patients (343 with Crohns disease [CD] and 306 with ulcerative colitis [UC]), 176 unaffected relatives, and 256 healthy controls. Allele and genotype frequencies were correlated with clinical characteristics and major variants of the CARD15 gene. Our findings were pooled in a meta‐analysis with the available studies in the literature. Results: No significant difference for the PTPN22 and NFkB1 variants was found. In contrast, allele and genotype frequencies of the G559T allele of the FcGRIIIA gene were significantly different in CD patients compared to controls (allele T 12% versus 8%, odds ratio [OR] = 1.58, 95% confidence interval [CI] 1.06–2.35; GT genotype 23% versus 16%, OR = 1.64, 95% CI = 1.08–2.5). However, no significant overtransmission of the T allele was confirmed at the family‐based analysis. For all genes, neither an interaction with CARD15 gene, nor a significant difference at genotype/phenotype analysis was demonstrated, included response to medical therapy. Conclusions: Although involved in autoimmune diseases, the PTPN22 and NFkB1 genes do not seem involved in the IBD predisposition, also according to meta‐analysis results. The association with the G559T polymorphism of the FcGRIIIA gene in CD patients deserves further investigation. (Inflamm Bowel Dis 2007)

Association Study of a Polymorphism in Clock Gene PERIOD3 and Risk of Inflammatory Bowel Disease

Altered body rhythmicity and deregulated clock gene expression may cause circadian disruption, which can lead to immune dysregulation and chronic inflammatory diseases. PERIOD3 (PER3) polymorphisms have been associated with circadian disruption and changed secretion of cytokines involved in chronic inflammation. Crohns disease (CD) and ulcerative colitis (UC) are multifactorial diseases resulting from complex interaction among environmental/microbial factors and the intestinal immune system, triggering an abnormal immune response in genetically susceptible individuals. We evaluated the influence of a polymorphism of the clock gene PER3 on susceptibility and behavior of these inflammatory bowel diseases. The rs2797685 variant of the PER3 gene was assessed in 1082 CD and 972 UC patients, 754 of whom had been diagnosed <18 yrs of age, and 1311 unrelated healthy controls. Allele and genotype frequencies of rs2797685 were significantly increased in both CD (p = 1.6 × 10−4, odds ratio [OR] = 1.38, 95% confidence interval [CI]: 1.17–1.63) and UC (p = .012, OR = 1.25, 95% CI: 1.05–1.48) patients. Difference between frequency distributions remained statistically significant after stratifying the cohort according to age at diagnosis for CD, but not for UC. Statistically significant association was found between PER3 polymorphism and use of immunosuppressive drugs in pediatric CD patients (p < .001) and with stricturing and fistulizing disease behavior in adult CD patients (p = .031). In conclusion, results of this association study suggest a possible role of PER3 polymorphism in determining susceptibility to CD and UC and phenotypic characteristics of CD. In particular, the rs2797685 variant of the PER3 gene is associated with a more aggressive form of CD, highlighted by higher use of immunosuppressants and more frequent stricturing and fistulizing disease behaviors, as well as early onset of CD. This is a descriptive study, and functional data are needed to prove a causal relationship; nonetheless, involvement of the clock gene machinery in the susceptibility and the behavior of inflammatory bowel diseases may suggest new pathophysiological mechanisms and new therapeutic approaches. (Author correspondence: g.mazzoccoli@ operapadrepio.it)

Variants at the 3p21 locus influence susceptibility and phenotype both in adults and early-onset patients with inflammatory bowel disease

Background: To date, a number of high‐profile studies have yielded over 50 inflammatory bowel disease (IBD) disease genes/loci. The polymorphisms rs9858542 (BSN) and rs3197999 (MST1), on 3p21 locus, have been found associated with susceptibility to IBD. We aimed to replicate these associations in adult and early‐onset cohorts of IBD Italian patients, by analyzing also potential gene–gene interactions with variants in NOD2/CARD15, IL23R, ATG16L1, and IRGM genes, and investigating genotype–phenotype correlation. Methods: In all, 1808 patients with IBD, 855 with Crohns disease (CD) and 953 with ulcerative colitis (UC), including 539 patients with their initial diagnosis <19 years of age, and 651 controls were analyzed for SNPs rs9858542 and rs3197999. Results: BSN and MST1 were significantly associated with either CD (Prs9858542 2.5 × 10−7; Prs3197999 3.9 × 10−7), and UC (Prs9858542 = 3.1 × 10−4; Prs3197999 = 8 × 10−4). Prevalence of these variants was significantly increased in both adult and early‐onset IBD patients. After stepwise logistic regression, the 2 variants were associated in adult UC with distal colitis (Prs9858542 = 0.013, odds ratio [OR] = 2.04, 95% confidence interval [CI] = 1.16–3.59; Prs3197999 = 0.018, OR 1.9, 95% CI 1.2–3.3), while the rs3197999 variant was inversely associated with occurrence of extraintestinal manifestations in adult CD(P = 0.017, OR 0.6, 95% CI 0.4–0.9). Conclusions: We confirmed the association of BSN and MST1 with IBD susceptibility, either in the adult or the early‐onset cohorts. These variants appeared to influence either the distal location of the disease in the UC cohort and extraintestinal manifestations in CD patients. (Inflamm Bowel Dis 2010)

Systematic analysis of circadian genes using genome-wide cDNA microarrays in the inflammatory bowel disease transcriptome

Simultaneous analysis of the transcripts of thousands of genes by cDNA microarrays allows the identification of genetic regulatory mechanisms involved in disease pathophysiology. The circadian clock circuitry controls essential cell processes and the functioning of organ systems, which are characterized by rhythmic variations with 24-hour periodicity. The derangement of these processes is involved in the basic mechanisms of inflammatory, metabolic, degenerative and neoplastic diseases. We evaluated by genome-wide cDNA microarray analysis the transcriptome of endoscopic mucosal biopsies of patients with inflammatory bowel diseases (IBD) focusing on the expression of circadian genes in Crohn’s disease (CD) and ulcerative colitis (UC). Twenty-nine IBD patients (15 with CD and 14 with UC) were enrolled and mucosal biopsies were sampled at either inflamed or adjacent non-inflamed areas of the colon. A total of 150 circadian genes involved in pathways controlling crucial cell processes and tissue functions were investigated. In CD specimens 50 genes were differentially expressed, and 21 genes resulted up-regulated when compared to healthy colonic mucosa. In UC specimens 50 genes were differentially expressed, and 27 genes resulted up-regulated when compared to healthy colonic mucosa. Among the core clock genes ARNTL2 and RORA were up-regulated, while CSNK2B, NPAS2, PER1 and PER3 were down-regulated in CD specimens. Conversely, ARNTL2, CRY1, CSNK1E, RORA and TIPIN were up-regulated, while NR1D2 and PER3 were down-regulated in UC. In conclusion, in CD and UC patients there are differences in the expression of circadian genes between normal and diseased intestinal mucosa. The deregulated genes evidenced by transcriptome analysis in the major IBDs may play a crucial role in the pathophysiological mechanisms and may suggest novel therapeutic approaches.

Variation in genes encoding for interferon λ‐3 and λ‐4 in the prediction of HCV‐1 treatment‐induced viral clearance

In patients with chronic HCV‐1 infection, recent evidences indicate that determination of a dinucleotide polymorphism (ss469415590, ΔG/TT) of a new gene, designated IFN λ‐4, might be more accurate than the 12979860CC type of the IL28B locus in predicting sustained virological response (SVR) following peg‐interferon and ribavirin. In addition, combined genotyping of different SNPs of the IL28B locus was shown to help dissect patients most prone to SVR among those with rs12979860CT.

Genome-wide Pathway Analysis Using Gene Expression Data of Colonic Mucosa in Patients with Inflammatory Bowel Disease

Background:Ulcerative colitis (UC) and Crohns disease (CD) share some pathogenetic features. To provide new steps on the role of altered gene expression, and the involvement of gene networks, in the pathogenesis of these diseases, we performed a genome-wide analysis in 15 patients with CD and 14 patients with UC by comparing the RNA from inflamed and noninflamed colonic mucosa. Methods:Two hundred ninety-eight differentially expressed genes in CD and 520 genes in UC were identified. By bioinformatic analyses, 34 pathways for CD, 6 of them enriched in noninflamed and 28 in inflamed tissues, and 19 pathways for UC, 17 in noninflamed and 2 in inflamed tissues, were also highlighted. Results:In CD, the pathways included genes associated with cytokines and cytokine receptors connection, response to external stimuli, activation of cell proliferation or differentiation, cell migration, apoptosis, and immune regulation. In UC, the pathways were associated with genes related to metabolic and catabolic processes, biosynthesis and interconversion processes, leukocyte migration, regulation of cell proliferation, and epithelial-to-mesenchymal transition. Conclusions:In UC, the pattern of inflammation of colonic mucosa is due to a complex interaction network between host, gut microbiome, and diet, suggesting that bacterial products or endogenous synthetic/catabolic molecules contribute to impairment of the immune response, to breakdown of epithelial barrier, and to enhance the inflammatory process. In patients with CD, genes encoding a large variety of proteins, growth factors, cytokines, chemokines, and adhesion molecules may lead to uncontrolled inflammation with ensuing destruction of epithelial cells, inappropriate stimulation of antimicrobial and T cells differentiation, and inflammasome events.

Impact of genetic polymorphisms on the pathogenesis of idiopathic achalasia: Association with IL33 gene variant

AIM To investigate the association of single nucleotide polymorphisms (SNPs) of genes involved in the regulation of immune responses, IL33, IL1RL1, IL23R, and IL10, with idiopathic achalasia in an Italian cohort of patients. MATERIALS AND METHODS A panel of eleven polymorphisms were genotyped in 116 unrelated idiopathic achalasic patients and 371 healthy subjects, by using TaqMan genotyping assays. RESULTS Significant differences of allele (P=0.0065, OR=1.59, CI=1.14-2.22) and genotype (P=0.0097, OR=1.74, CI=1.14-2.65) frequencies of the IL33 rs3939286 variant were found between achalasic patients and controls. No association of the other investigated SNPs was detected. No differences in genotype and allele distribution were found with respect to clinical characteristics of patients. CONCLUSION We provide for the first time an association between the risk of developing idiopathic achalasia and IL-33 variant, underling the role of cytokines and inflammatory mediators on the pathogenesis of the disease.

IL23R, ATG16L1, IRGM, OCTN1, and OCTN2 mRNA expression in inflamed and noninflamed mucosa of IBD patients

The inflammatory bowel diseases (IBDs), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic intestinal inflammatory disorders caused by a disruptive interaction between the mucosal immune system and some gut luminal/ environmental factor(s). Several genomewide association (GWA) meta-analyses have identified more than 50 risk-conferring loci, with more than 20 of them being shared between the two diseases. However, functional studies that evaluate mucosal expression of candidate genes are scarce. This study was designed to investigate the expression of IL23R, ATG16L1, IRGM, OCTN1 (SLC22A4), and OCTN2 (SLC22A5) mRNAs in the colonic mucosa of patients with IBD during an active phase of disease. In 62 consecutive IBD patients, 31 CD (22 male, mean age at diagnosis 26 6 12 years) and 31 UC (20 male, mean age at diagnosis 35 6 12 years), mucosal biopsies were taken at either an inflamed or adjacent noninflamed area of the gut during the active phase of the disease. Informed consent was obtained from each subject. Biopsy specimens were rapidly transferred in RNAlater (Qiagen, Valencia, CA). For RNA isolation each specimen was extracted with the RNeasy Mini Kit (Qiagen). The realtime polymerase chain reaction (PCR) reactions were performed with ABI7700TaqMan (Applied Biosystems, Foster City, CA) using TaqMan Gene Expression Assays in the presence of GAPDH (Applied Biosystems). Samples were analyzed with ABI 2.3 software and the relative gene expression was normalized to GAPDH using the comparative 2 DDCT method. The variants of the investigated gene were genotyped by Taqman single nucleotide polymorphism (SNP) genotyping assays following the manufacturer’s instructions: rs7517847 and rs11209026 (IL23R), rs1000113 and rs4958847 (IRGM), rs2241880 (ATG16L1), rs2631367 (OCTN2), and rs1050152 (OCTN1). Data are expressed both as mean and medians, with Q1 and Q3 interquartiles. The Mann–Whitney U-test or Wilcoxon test were used where appropriate. Whole mucosal mRNAs expression of IL23R, ATG16L1, IRGM, and OCTN1 was not significantly different between inflamed and noninflamed mucosa, both in CD and UC patients (Fig. 1); the expression pattern did not differ even after stratifying carriers of the risk genotypes. In addition, no correlation with disease activity index was found (Harvey– Bradshaw index [HBI] for CD and the Ulcerative Colitis Disease Activity Index [UCDAI] for UC patients). In contrast, the whole expression level of OCTN2 mRNA at the inflamed mucosa was significantly reduced compared to noninflamed areas, both in CD (Fig. 1A) and UC patients (Fig. 1B), (CD: median 1⁄4 0.34, Q1–Q3 1⁄4 0.17–0.74, P 1⁄4 0.001; UC: median 1⁄4 0.47, Q1–Q31⁄4 0.24–0.77, P 1⁄4 0.0004) (Fig. 1A) irrespective of the risk genotypes for the rs2631367 variant. OCTN2 actively facilitates the transport of L-carnitine and several other cationic drugs through membranes and has been considered to play a physiological role in regulating the absorption of xenobiotics. The reduced OCTN2 expression may in turn lead to reduced carnitine absorption. A number of observations have underlined the importance of L-carnitine in intestinal homeostasis, since it plays a key role in cell metabolism by regulating the mitochondrial transport of long-chain free fatty acids and the generation of adenosine triphosphate by b-oxidation. Accordingly, the OCTN2 / mice develop colonic atrophy and spontaneous inflammation due to the abnormal stricture and morphology of intestinal epithelial cells. In addition, carnitine deficiency results in T-cell hyperactivation, which is implicated in the mounting and perpetuation of inflammation and dysregulation both of the innate and adaptive arms of the immune response. The decreased OCTN2 mRNA levels linked to the inflammatory status of the affected tissue might become particularly relevant in metabolically ‘‘challenged’’ intestinal epithelial cells in the context