D. Scimeca

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 u200a=u200a 0.038), and with HLA and steroid-responsiveness (P u200a=u200a 0.024) in UC patients was observed. Moreover, significant association in our CD cohort with TNFSF15 SNP and colonic involvement (P u200a=u200a 0.021), and with ZNF365 and ileal location (P u200a=u200a 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.

Early post-operative endoscopic recurrence in Crohn's disease patients: Data from an Italian Group for the study of inflammatory bowel disease (IG-IBD) study on a large prospective multicenter cohort

INTRODUCTIONnThe incidence of endoscopic recurrence (ER) in Crohns disease following curative resection is up to 75% at 1 year. Endoscopy is the most sensitive method to detect the earliest mucosal changes and the severe ER at 1 year seems to predict a clinical relapse.nnnMETHODSnThe aim of this prospective study was to evaluate the incidence of early ER 6 months after curative resection. Secondary outcome was to evaluate the role of 5-aminosalicylic acid (5-ASA) in the prevention of ER at 6 months. A total of 170 patients were included in the study. They were carried-out from the evaluation of the appearance of ER during a trial performed to assess the role of azathioprine vs. 5-ASA as early treatment of severe ER. All the patients started 5-ASA treatment 2 weeks after surgery.nnnRESULTSnSix months after surgery ER was observed in 105 patients (62%). The endoscopic score was reported as severe in 78.1% of them (82 out of 105). At univariable analysis only ileo-colonic disease influenced the final outcome associating to a lower risk of severe ER (p=0.04; OR 0.52, 95% CI 0.277-0.974).nnnCONCLUSIONnIn this prospective Italian multicenter IG-IBD study a great proportion of ER occur within 6 months from ileo-colonic resection, with a significant rate of severe ER. Furthermore this study confirms the marginal role of 5-ASA in the prevention of ER. This suggests that post-surgical endoscopic evaluation should be performed at 6 months instead of 1 year to allow an adequate early treatment.

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 (pu2009=u20091.6u2009×u200910−4, odds ratio [OR]u2009=u20091.38, 95% confidence interval [CI]: 1.17–1.63) and UC (pu2009=u2009.012, ORu2009=u20091.25, 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 (pu2009<u2009.001) and with stricturing and fistulizing disease behavior in adult CD patients (pu2009=u2009.031). 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)

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.

Role of interleukin polymorphisms in gastric cancer: “Pros and cons”

Helicobacter pylori (H. pylori) infection is the leading cause of gastric cancer worldwide. Infection with this bacterium causes a chronic active immune response that persists for the life of the host. The combination of bacterial factors, environmental insults, and the host immune response drives the initiation and progression of mucosal atrophy, metaplasia, and dysplasia toward GC. Among the host factors, IL-1 gene cluster polymorphisms (IL-1B encoding IL-1β and IL-1RN encoding IL-1ra, its naturally occurring receptor antagonist) play a decisive role in modulating the risk of developing hypochlorhydria, gastric atrophy and GC in the presence of H. pylori infection. In particular, one single nucleotide polymorphism in the IL-1B promoter (IL-1B-511C⁄T), and the short allele of a 86-bp variable number of tandem repeats polymorphism in the IL-1RN second intron (IL-1RN*2) are associated with an increased risk for GC. However this hypothesis is still to be fully confirmed. This review focuses on the divergent results obtained by several epidemiological and functional in vitro and in vivo studies and show that IL-1 genotyping has still no role in the clinical management of patients with H. pylori infection.

Can Endoscopic Ultrasound Distinguish Between Mediastinal Benign Lymph Nodes and Those Involved by Sarcoidosis, Lymphoma, or Metastasis?

BackgroundLymph nodes (LNs) echofeatures on endoscopic ultrasound (EUS) and concurrent fine needle aspiration (FNA) are alternatives to highly invasive approaches for etiologic diagnosis of mediastinal lymphadenopathy (MLAD).AimsTo evaluate the efficacy of LNs echofeatures and FNA via EUS to distinguish benign LNs from LNs involved by sarcoidosis, lymphoma, and metastasis in non-lung cancer patients.MethodsA retrospective review of patients who underwent EUS–FNA for MLAD was performed. Echofeatures of LNs including echogenicity, margins, shape, and LN size were recorded. Final diagnosis was made based on surgical sampling or clinical diagnosis with long-term follow-up. Only patients diagnosed as benign MLAD, sarcoidosis, lymphoma, and metastasis included. Diagnostic value of echofeatures and FNA was evaluated.ResultsIncluded were 162 patients with final diagnosis of benign (68), sarcoidosis (33), lymphoma (20), and metastasis (41). The median LN along axis in the benign group [20.5xa0mm (6–76)] was significantly shorter than in the metastasis [28xa0mm (9–82)] and sarcoidosis [27xa0mm (17–50)] groups (pxa0<xa00.05). The median LN short axis in the benign group [11xa0mm (2–50)] was significantly shorter than in the metastasis [17xa0mm (5–44)] and lymphoma [16xa0mm (7–47)] groups (pxa0<xa00.05). No other echofeatures showed a discriminant value among the groups. When performing FNA, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of EUS–FNA were 73.7, 100, 100, 72.2, and 84.4xa0%, respectively.ConclusionAlthough benign MLAD tend to be smaller than other etiologies, echofeatures of LNs are not reliable etiologic diagnostic approach to MLAD. Therefore, FNA is suggested when feasible. However, due to relatively low sensitivity, LNs with benign FNA results should be subjected to further work-up if they are clinically suspicious.

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.

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

Crohn’s Disease Localization Displays Different Predisposing Genetic Variants

Background Crohn’s disease (CD) is a pathologic condition with different clinical expressions that may reflect an interplay between genetics and environmental factors. Recently, it has been highlighted that three genetic markers, NOD2, MHC and MST1, were associated to distinct CD sites, supporting the concept that genetic variations may contribute to localize CD. Genetic markers, previously shown to be associated with inflammatory bowel disease (IBD), were tested in CD patients with the aim to better dissect the genetic relationship between ileal, ileocolonic and colonic CD and ascertain whether a different genetic background would support the three disease sites as independent entities. Methods A panel of 29 SNPs of 19 IBD loci were analyzed by TaqMan SNP allelic discrimination method both evaluating their distinct contribute and analyzing all markers jointly. Results Seven hundred and eight CD patients and 537 healthy controls were included in the study. Of the overall population of patients, 237 patients had an ileal involvement (L1), 171 a colonic localization (L2), and the 300 remaining an ileocolon location (L3). We confirmed the association for 23 of 29 variations (P < 0.05). Compared to healthy controls, 16 variations emerged as associated to an ileum disease, 7 with a colonic disease and 14 with an ileocolonic site (P < 0.05). Comparing ileum to colonic CD, 5 SNPs (17%) were differentially associated (P < 0.05). A genetic model score that aggregated the risks of 23 SNPs and their odds ratios (ORs), yielded an Area Under the Curve (AUC) of 0.70 for the overall CD patients. By analyzing each CD location, the AUC remained at the same level for the ileal and ileocolonic sites (0.73 and 0.72, respectively), but dropped to a 0,66 value in patients with colon localization. Conclusions Our findings reaffirm the existence of at least three different subgroups of CD patients, with a genetic signature distinctive for the three main CD sites.

Functional Implications of MicroRNAs in Crohn’s Disease Revealed by Integrating MicroRNA and Messenger RNA Expression Profiling

Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that emerges due to the influence of genetic and environmental factors. microRNAs (miRNAs) have been identified in the tissue and sera of IBD patients and may play an important role in the induction of IBD. Our study aimed to identify differentially expressed miRNAs and miRNAs with the ability to alter transcriptome activity by comparing inflamed tissue samples with their non-inflamed counterparts. We studied changes in miRNA–mRNA interactions associated with CD by examining their differential co-expression relative to normal mucosa from the same patients. Correlation changes between the two conditions were incorporated into scores of predefined gene sets to identify biological processes with altered miRNA-mediated control. Our study identified 28 miRNAs differentially expressed (p-values < 0.01), of which 14 are up-regulated. Notably, our differential co-expression analysis highlights microRNAs (i.e., miR-4284, miR-3194 and miR-21) that have known functional interactions with key mechanisms implicated in IBD. Most of these miRNAs cannot be detected by differential expression analysis that do not take into account miRNA–mRNA interactions. The identification of differential miRNA–mRNA co-expression patterns will facilitate the investigation of the miRNA-mediated molecular mechanisms underlying CD pathogenesis and could suggest novel drug targets for validation.