Linda Smith

Genotype-phenotype analysis in childhood-onset Crohn's disease: NOD2/CARD15 variants consistently predict phenotypic characteristics of severe disease.

Introduction: The incidence of early‐onset CD in Scotland is among the highest worldwide. Three single nucleotide polymorphisms (SNPs) R702W, G908R and Leu1007finsC in the NOD2/CARD15 gene predispose to adult CD. We investigated the contribution of these variants to disease susceptibility and phenotype in the Scottish early‐onset IBD population. Patients and Methods: 906 individuals including 247 Scottish IBD patients aged <16 years at diagnosis, 414 parents and 245 controls were genotyped. Transmission disequilibrium testing (TDT), case‐control analysis and detailed genotype‐phenotype analysis were performed. Results: The Leu1007finsC variant was associated with susceptibility to CD by case‐control (4.2% versus. 1.4%, P = 0.01) and TDT analysis (P = 0.006). The Population Attributable Risk (PAR) for the 3 NOD2/CARD15 mutations was 7.9%. Carriage of NOD2/CARD15 variants was associated with, at diagnosis: decreased albumin (31.0% versus. 9.0%, P = 0.001) and raised CRP (25% versus. 9.5%, P = 0.04) and at follow up: need for surgery (39.5% versus. 12.8%, P = 0.0002) jejunal involvement (50% versus. 18.4%, P = 0.01) jejunal and ileal involvement (50% versus. 10.7%, P = 0.009), raised CRP (57.1% and 12.8%, P = 0.0009), lower weight/height centile (75.0% versus. 20.2%, P = 0.03, 50.0% versus. 16.0%, P = 0.001 respectively) and stricturing disease (45.5% versus. 19.4%, P < 0.05). Multifactorial analysis demonstrated carriage was associated with need for surgery (P = 0.004, OR 4.9 [1.5‐14.7]). Conclusions: These NOD2/CARD 15 variants in the Scottish early onset CD population have a definite, albeit relatively small contribution to CD susceptibility (PAR 7.9%) but a major impact on phenotype. In particular NOD2/CARD15 variants are strongly associated with several markers of disease severity in pediatric CD, notably need for surgery.

Does cigarette smoking influence the phenotype of Crohn's disease? Analysis using the Montreal classification.

OBJECTIVES:The clinical subclassification of Crohns disease by phenotype has recently been reevaluated. We have investigated the relationships between smoking habit, age at diagnosis, disease location, and progression to stricturing or penetrating complications using the Montreal classification.METHODS:408 patients (157 male, median age 29.4 yr) were assessed. Data were collected on smoking habit, age at diagnosis, anatomical distribution, and disease behavior. Follow-up data were available on all patients (median 10 yr).RESULTS:At diagnosis, ex-smokers (N = 53) were older than nonsmokers (N = 177) or current smokers (N = 178, medians 43.2 vs 28.3 or 28.9 yr, respectively, P < 0.001). Disease location differed according to smoking habit at diagnosis (χ2 = 24.1, P = 0.02) as current smokers had less colonic (L2) disease than nonsmokers or ex-smokers (30% vs 45%, 50%, respectively). In univariate Kaplan–Meier survival analysis, smoking habit at diagnosis was not associated with time to development of stricturing disease, internal penetrating disease, perianal penetrating disease, or time to first surgery. Patients with isolated colonic (L2) disease were slower to develop strictures (P < 0.001) or internal penetrating disease (P = 0.001) and to require surgery (P < 0.001). Cox models with smoking habit as time-dependent covariates showed that, relative to ileal (L1) location of disease, progression to stricturing disease was less rapid for patients with colonic (L2) disease (HR 0.140, P < 0.001), but not independently affected by smoking habit. Progression to surgery was also slower for colonic (L2) than ileal (L1) disease location (HR 0.273, P < 0.001), but was independent of smoking habit.CONCLUSIONS:Smoking habit was associated with age at diagnosis and disease location in Crohns disease, while disease location was associated with the rate of development of stricturing complications and requirement for surgery. The pathogenic basis of these observations needs to be explained.

DLG5 variants do not influence susceptibility to inflammatory bowel disease in the Scottish population

Introduction: Recent data have suggested that specific haplotypic variants of the DLG5 gene on chromosome 10q23 may be associated with susceptibility to inflammatory bowel disease (IBD) in Germany. Haplotype D, notably characterised by the presence of a G→A substitution at nucleotide 113, was associated with susceptibility to Crohn’s disease (CD) whereas an extended haplotype A conferred protection. Aims: Association of DLG5 haplotypic variants with disease susceptibility, genotype-phenotype relationships, and epistasis with CARD15 was investigated in the Scottish population. Patients and methods: A total of 374 CD, 305 ulcerative colitis (UC), and 294 healthy controls (HC) were studied. Genotyping for the variants rs1248696 (113A, representing haplotype D) and the single nucleotide polymorphism tag rs2289311 (representing haplotype A) were typed using the Taqman system. Results: On analysis of the DLG5 variant 113A, there were no associations with IBD when allelic frequency (11.4% IBD v 13.2% HC; p = 0.30) and carrier frequency (19.2% IBD v 24.6% HC; p = 0.069) were analysed. No associations were observed between 113A variant allelic frequency (p = 0.37), carrier frequency (p = 0.057), and CD. In fact, 113A heterozygosity rates were lower in CD (16%) and IBD (16.9%) than in HC (23%) (p = 0.029 and p = 0.033, respectively). No associations between DLG5 and UC were observed. Haplotype A was not protective and there was no evidence of epistasis between DLG5 and CARD15. Conclusions: The present data contrast strongly with previous data from Germany. DLG5 113A is not associated with disease susceptibility and haplotype A does not confer resistance. Further work is required to evaluate the significance of DLG5 in other populations from geographically diverse regions.

Smoking Habit and Load Influence Age at Diagnosis and Disease Extent in Ulcerative Colitis

INTRODUCTION:Cigarette smoking affects susceptibility to ulcerative colitis (UC), but its effects on age at diagnosis, disease extent, and need for surgery are less well defined. We examined these parameters in a detailed retrospective analysis of a large cohort of well-characterized UC patients.METHODS:499 UC patients (254 male, median age 34.3 yr) were studied. Data were collected on smoking habits, smoking load (pack-years), age at recruitment, age at diagnosis, surgery, and disease extent. Colonoscopic and histological data at both diagnosis and follow-up (median follow-up time 4.6 yr) were available on 349 patients.RESULTS:Ex-smokers were older at diagnosis than current or nonsmokers, (46.5 yr vs 31.1 or 29.4 yr, respectively, P < 0.001). Before diagnosis, ex-smokers had a higher smoking load than current smokers (13.0 vs 6.94 pack-years, P < 0.001). A Cox model for age at diagnosis, with smoking as a time-dependent covariate, showed that at any age, ex-smokers were significantly more likely to develop UC than current smokers (hazard ratio 1.8, 95% CI 1.41–2.44, P < 0.001). For current smokers at latest colonoscopy, those with extensive disease were the lightest smokers (median 0.320 pack-years), whereas those with healthy colons were the heaviest smokers (median 9.18 pack-years, P = 0.006). At 5 yr, regression of extensive disease was more frequent in current than ex-smokers or nonsmokers (30% current smokers vs 8% nonsmokers and 5% ex-smokers, χ2 = 30.4, P < 0.001) but these differences were not maintained over a longer time period.CONCLUSIONS:Smoking habit influences the age at diagnosis and changes in disease extent in UC. Mechanisms are likely to be complex and require further investigation.

Autophagy Gene ATG16L1 Influences Susceptibility and Disease Location but Not Childhood-Onset in Crohn's Disease in Northern Europe

Background: The rs2241880A/G variant of the ATG16L1 gene has been associated with susceptibility to ileal Crohns disease (CD) in adults. Our aim was to assess whether germline variation of ATG16L1 acts as an independent determinant of susceptibility to childhood‐onset CD in the high‐incidence Scottish population. Methods: In all, 2195 subjects (361 children (inflammatory bowel disease [IBD] diagnosis <17 years), their parents (n = 634), 855 adult IBD patients, and 345 controls were genotyped. Case‐control analysis was powered to detect effect sizes with an odds ratio (OR) >1.39 in pediatric CD. Case‐control analysis, transmission disequilibrium testing (TDT), analysis of variance (ANOVA) of growth parameter z‐scores, Kruskal–Wallis test (age at diagnosis), and multifactorial genotype–phenotype analysis (Montreal classification) were performed. 7.8% of pediatric CD patients and 37.2% of adult CD patients had pure ileal disease. Results: We confirmed the association of the rs2241880G‐allele with adult‐onset CD (60.7% versus controls 53.9%, P = 0.01, OR 1.32, 95% confidence interval [CI] 1.07–1.63) in contrast to childhood‐onset CD (54.1% versus controls, P = 0.95, OR 1.01, 95% CI 0.80–1.26). TDT analysis was negative. Genotype–phenotype analysis demonstrated an association of pure ileal disease with the rs2241880G‐allele (P = 0.02, OR 1.34, 95% CI 1.03–1.74). Using binary logistic regression analysis we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (P = 0.03, OR 2.43, 95% CI 1.05–5.65). ATG16L1 genotype did not influence age at CD diagnosis. ANOVA of z‐scores of height, weight, and body mass index (BMI) at CD diagnosis in children showed no association with genotype. Conclusions: The ATG16L1 variant is associated with susceptibility to adult CD in Scotland, but not early‐onset disease. These contrasting effects are primarily driven by differences in disease location between early‐onset and adult‐onset disease.

Contribution of the NOD1/CARD4 insertion/deletion polymorphism +32656 to inflammatory bowel disease in Northern Europe

Background: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern‐recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations. Methods: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case‐control, Transmission Disequilibrium Testing (TDT) and detailed genotype–phenotype (Montreal) analyses were performed. The case‐control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD. Results: In case‐control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohns disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative. Conclusions: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene‐wide haplotype‐based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD. (Inflamm Bowel Dis 2007)

Detailed Haplotype- Tagging Study of Germline Variation of MUC19 in Inflammatory Bowel Disease

To the Editor:Since the discovery of NOD2 as aCrohn’s disease (CD) susceptibilitygene, more recent genomewide associa-tion scans (GWAS), detecting loci con-ferring odds ratios (OR) of above 1.3–1.5, have uncovered a large number offurther susceptibility loci and genes,including IL23R in the TH17 pathway,and the autophagy genes ATG16L1 andIRGM.

Surgery in Crohn's Disease: Analysis of 15000 Patient-Years Experience in Eastern Scotland

G A A b st ra ct s clinical depression was not significant. Using corticosteroids was associated with less fatigue (OR 0.12; 95% CI 0.03-0.52). The different hospitals settings (academic vs general), age and other medication use were not associated with higher fatigue scores. In group B, 545 pts were included (64% female, 77.4% CD, 22.6% UC, mean age 42 (SD 14)) and this group verified the results of group A. However, overall only 60% experienced fatigue and mean CIS score was lower (mean 37 (range 8-56) compared with group A. Furthermore, the use of anti-TNF and male gender were associated with lower percentage of fatigue in CD pts. Conclusion: A high percentage of IBD pts experience severe fatigue. Depression and disease activity were associated with fatigue in these patients. The use of anti-TNF and male gender in CD pts showed the opposite effect and were associated with less fatigue. In UC pts the use of corticosteroids showed less fatigue.

W1740 Loss-of-Function Variants of the Epithelial Barrier Protein Filaggrin Predispose to Marked Atopic Co-Morbidity in Paediatric Inflammatory Bowel Disease

Background: The gastric epithelial cell layer is a pivotal physical defence mechanism necessary for the integrity of gastric mucosa. Disruption of cell junctions and cell cytoskeleton, key elements in the efficiency of this barrier, leads to the initiation of gastric mucosal injury. Isoflavones, selective estrogen receptor modulators, exert their effects by selectively binding to estrogen receptors (ER), particularly ERβ, and are involved in the control of gastric mucosal homeostasis. ERβ are expressed mainly in the human gastric antrum and their role in the gastric physiological functions is still unknown. Aim: In this study we evaluated the effects of isoflavones, administrated as soy germ pasta, on gastric mucosal gene expression through an extensive microarray analysis in healthy individuals. Materials and Methods: Biologically active isoflavones (aglycones) were administrated using a 2% soy-germ pasta (SP) (Aliveris srl, Perugia, Italy). Equivalent conventional pasta (CP) was used as a control. 5 healthy volunteers (3 F, age: 30±3 years) consumed a standard diet containing CP (80gr/ daily) for 1 week followed by SP (80gr/daily; approx. 33mg of isoflavones) for 1 week. An upper endoscopy with biopsy was performed at the end of each week. Gastric samples were collected for microarray analysis and qRT-PCR. Biotin-labeled cRNA was obtained and hybridized to an Affymetrix HG-U133 Plus2.0 GeneChipArray according to standard procedures. Results: Microarray analysis highlighted a dramatic difference in gene expression after consumption of SP compared to CP. The gene list obtained was clearly efficient in separating the two groups at the end of the two different diets. Functional gene classification was performed based on different sources (GeneOntology, KEGG, SOURCE) with integrated information from the literature and 58 genes were included in 8 different gene categories: cytoskeleton, organization and biogenesis, cell-cell communication, contractility, signal transduction, stress response, transcription and other/unknown. Interestingly, one of these genes, PGTDS, which encodes an enzyme involved in prostaglandin synthesis, was 5-fold upregulated after SP administration compared to CP. Of note, while the expression of ERα and ERβ was not affected, 10 out of the 58 modulated genes contained one estrogen responsive element. Conclusion: This is the first evidence that isoflavones modulate the expression of genes involved in cell-cell and cell-extra cellular matrix adhesion process in human gastric mucosa, suggesting a potential role of isoflavones in gastric mucosal defense.