Colin L. Noble

A retrospective analysis of the efficacy and safety of infliximab as rescue therapy in acute severe ulcerative colitis

Backgroundu2002 Forty per cent of patients with acute severe ulcerative colitis will not respond to intravenous corticosteroids and require second‐line medical therapy or colectomy. A recent controlled trial has suggested that infliximab may be effective as rescue therapy.

Analysis of the influence of OCTN1/2 variants within the IBD5 locus on disease susceptibility and growth indices in early onset inflammatory bowel disease

Background and aims: The OCTN1 (SLC22A4 1672C→T) and OCTN2 (SLC22A5 −207G→C) variants within the IBD5 locus have been associated with susceptibility to adult onset Crohn’s disease (CD), but their contribution in children has not been examined. Methods: These OCTN1/2 variants and IBD5 marker single nucleotide polymorphisms (SNPs) (IGR2096a_1, IGR2198a_1, and IGR2230a_1) were examined in 299 Scottish children (200 with CD, 74 with ulcerative colitis (UC), and 25 with indeterminate colitis (IC)), together with 502 parents (for transmission disequilibrium testing) and 256 controls. Results: All SNPs were in strong linkage disequilibrium (D′ >0.94). TDT analysis showed association of the OCTN1 variant with inflammatory bowel disease (IBD) (pu200a=u200a0.01) and CD (pu200a=u200a0.04). Allele frequencies of the OCTN1/2 variants were significantly higher in IBD/CD cases (p<0.04). The homozygous mutant OCTN1/2 haplotype was increased in IBD (24.3% v 16.1%, pu200a=u200a0.02) and UC (28.2% v 16.1%, pu200a=u200a0.02) compared with controls. The OCTN1/2 variants were not independent of the background IBD5 risk haplotype in conferring disease susceptibility. Unifactorial analysis in CD patients showed that carriage of the TC haplotype was associated with lower weight, height, and BMI centile (<9th centile) at diagnosis (weight: 87.9% v 67.3% (pu200a=u200a0.002), odds ratio (OR)u200a=u200a3.52 (95% confidence interval, 1.51 to 8.22); height: 84.1% v 68.4% (p<0.05), ORu200a=u200a2.44 (1.00 to 5.99); BMI: 79.6% v 61.1% (pu200a=u200a0.02), ORu200a=u200a2.49 (1.14 to 5.44)), and lower weight centile at follow up (87.5% v 64.6% (pu200a=u200a0.03), ORu200a=u200a3.83 (1.03 to 14.24)). Multifactorial binary logistic regression analysis confirmed association of the TC haplotype with lower weight centile at diagnosis (pu200a=u200a0.02, ORu200a=u200a3.41 (1.20 to 9.66)). Conclusions: These data implicate variants within the IBD5 haplotype, as determinants of disease susceptibility and growth indices in early onset IBD. The OCTN1/2 variants remain potential positional candidate genes, but require further analysis.

Dysregulation of Human β-Defensin-2 Protein in Inflammatory Bowel Disease

Background Human β-defensin-2 (HBD2) is an antimicrobial peptide implicated in the pathogenesis of inflammatory bowel disease (IBD). Low copy number and concomitant low mRNA expression of the HBD2 gene have been implicated in susceptibility to colonic Crohns Disease (CD). We investigated the colonic distribution of HBD2 mRNA expression, and the contributions of genetic and environmental factors on HBD2 protein production. Methodology/Principal Findings We examined HBD2 mRNA expression at three colonic locations by microarray analysis of biopsies from 151 patients (53 CD, 67 ulcerative colitis [UC], 31 controls). We investigated environmental and genetic influences on HBD2 protein production using ex vivo cultured sigmoid colon biopsies from 69 patients (22 CD, 26 UC, 21 controls) stimulated with lipopolysaccharide (LPS) and/or nicotine for 24 hours. HBD2 and cytokines were measured in culture supernatants. Using DNA samples from these patients, regions in the HBD2 gene promoter were sequenced for NF-κB binding-sites and HBD2 gene copy number was determined. HBD2 mRNA expression was highest in inflamed (vs. uninflamed pu200a=u200a0.0122) ascending colon in CD and in inflamed (vs. uninflamed p<0.0001) sigmoid colon in UC. HBD2 protein production was increased in inflamed UC biopsies (pu200a=u200a0.0078). There was no difference in HBD2 protein production from unstimulated biopsies of CD, UC and controls. LPS-induced HBD2 production was significantly increased in CD (pu200a=u200a0.0375) but not UC (pu200a=u200a0.2017); this LPS-induced response was augmented by nicotine in UC (pu200a=u200a0.0308) but not CD (pu200a=u200a0.6872). Nicotine alone did not affect HBD2 production. HBD2 production correlated with IL8 production in UC (p<0.001) and with IL10 in CD (p<0.05). Variations in the HBD2 promoter and HBD2 gene copy number did not affect HBD2 production. Significance/Conclusions Colonic HBD2 was dysregulated at mRNA and protein level in IBD. Inflammatory status and stimulus but not germline variations influenced these changes.

Analysis of germline GLI1 variation implicates hedgehog signalling in the regulation of intestinal inflammatory pathways.

Background Ulcerative colitis (UC) and Crohns disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis. Methods and Findings Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C→G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09–1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1+/lacZ mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1+/lacZ mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model. Conclusions HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

Gender-stratified analysis of DLG5 R30Q in 4707 patients with Crohn disease and 4973 controls from 12 Caucasian cohorts

Background: DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case–control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male–female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD. Methods: DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male–female frequency differences in controls and for case–control frequency differences in men and in women. Results: The data showed no male–female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (pu200a=u200a0.049, ORu200a=u200a0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (pu200a=u200a0.058, ORu200a=u200a0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (pu200a=u200a0.010, ORu200a=u200a0.86, 95% CI 0.76 to 0.97), but not in men. Conclusion: DLG5 30Q is associated with a small reduction in risk of CD in women.

PepT1 oligopeptide transporter (SLC15A1) gene polymorphism in inflammatory bowel disease

Background: Human polymorphisms affecting gut epithelial barrier and interactions with bacteria predispose to the inflammatory bowel diseases (IBD) Crohns disease (CD) and ulcerative colitis (UC). The intestinal transporter PepT1, encoded by the SLC15A1 gene, mediates intracellular uptake of bacterial products that can induce inflammation and NF‐&kgr;B activation upon binding to NOD2, a protein often mutated in CD. Hence, we tested SLC15A1 polymorphisms for association with IBD. Methods: Twelve SLC15A1 single nucleotide polymorphisms (SNPs) were genotyped in 1783 individuals from 2 cohorts of Swedish and Finnish IBD patients and controls. An in vitro system was set up to evaluate the potential impact of SLC15A1 polymorphism on PepT1 transporter function by quantification of NOD2‐mediated activation of NF‐&kgr;B. Results: The common allele (C) of a coding polymorphism (rs2297322, Ser117Asn) was associated with CD susceptibility both in Sweden and in Finland, but with genetic effects in opposite directions (risk and protection, respectively). The best evidence of association was found in both populations when the analysis was performed on individuals not carrying NOD2 common risk alleles (Sweden allelic P = 0.0007, OR 1.97, 95% confidence interval [CI] 1.34–2.92; Finland genotype P = 0.0013, OR 0.63, 95% CI 0.44–0.90). The PepT1 variant encoded by the C allele (PepT1‐Ser117) was associated with reduced signaling downstream of NOD2 (P < 0.0001 compared to Pept1‐Asn117). Conclusions: A functional polymorphism in the SLC15A1 gene might be of relevance to inflammation and antibacterial responses in IBD. Whether this polymorphism truly contributes to disease susceptibility needs to be further addressed, and should stimulate additional studies in other populations. Inflamm Bowel Dis 2009

A trial of mercaptopurine is a safe strategy in patients with inflammatory bowel disease intolerant to azathioprine: an observational study, systematic review and meta-analysis

Thiopurines maintain remission and modify disease course in inflammatory bowel disease. Use is limited by intolerance and subsequent drug withdrawal in approximately 17% of patients treated with azathioprine. Previous case series have addressed the success rates of re‐treatment with mercaptopurine in these individuals.

Two-stage Genome-wide Methylation Profiling in Childhood-onset Crohn's Disease Implicates Epigenetic Alterations at the VMP1/MIR21 and HLA Loci

Background:As a result of technological and analytical advances, genome-wide characterization of key epigenetic alterations is now feasible in complex diseases. We hypothesized that this may provide important insights into gene-environmental interactions in Crohns disease (CD) and is especially pertinent to early onset disease. Methods:The Illumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leukocyte DNA in discovery and replication pediatric CD cohorts and controls. Data were corrected for differential leukocyte proportions. Targeted replication was performed in adults using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa. Results:We identified 65 individual CpG sites with methylation alterations achieving epigenome-wide significance after Bonferroni correction (P < 1.1 × 10−7), and 19 differently methylated regions displaying unidirectional methylation change. There was a highly significant enrichment of methylation changes around GWAS single nucleotide polymorphisms (P = 3.7 × 10−7), notably the HLA region and MIR21. Two-locus discriminant analysis in the discovery cohort predicted disease in the pediatric replication cohort with high accuracy (area under the curve, 0.98). The findings strongly implicate the transcriptional start site of MIR21 as a region of extended epigenetic alteration, containing the most significant individual probes (P = 1.97 × 10−15) within a GWAS risk locus. In extension studies, we confirmed hypomethylation of MIR21 in adults (P = 6.6 × 10−5, n = 172) and show increased mRNA expression in leukocytes (P < 0.005, n = 66) and in the inflamed intestine (P = 1.4 × 10−6, n = 99). Conclusions:We demonstrate highly significant and replicable differences in DNA methylation in CD, defining the disease-associated epigenome. The data strongly implicate known GWAS loci, with compelling evidence implicating MIR21 and the HLA region.

Contribution of CARD15 variants in determining susceptibility to Crohn's disease in Sweden.

Objective. Crohns disease (CD) is a chronic inflammatory bowel disorder caused by environmental and genetic factors. Mutations in the CARD15 gene have been associated with CD. No previous case-control CARD15 study has been performed in the Swedish population. Material and methods. The study comprised of 321 individuals: 178 with CD and 143 healthy controls (HCs), all from Stockholm County. All were genotyped for the three main CD-associated CARD15 variants (R702W, G908R and 1007fs) and phenotypic associations were investigated. Results. The allele frequencies of the R702W variant (4.5% CD versus 0.7% HC, p=0.008, ORu200a=u200a6.8) and the G908R variant (2.0% CD versus 0% HC, p=0.045) were more common in CD patients than in controls. No significant difference in1007fs variant allele frequency was found between CD patients and controls (2.0% CD versus 1.7% HC, pu200a=u200a0.8, ORu200a=u200a1.1). Carriage of CARD15 variants was more common in the CD patients than in controls (15.2% CD versus 4.2% HC, pu200a=u200a0.001, ORu200a=u200a4.1, population attributable risk (PAR)u200a=u200a11.4%). Genotype–phenotype analysis demonstrated that CARD15 variants were associated with ileal disease (p=0.0006, ORu200a=u200a9.3, CIu200a=u200a2.2–34) and protective for colonic CD (pu200a=u200a0.01, ORu200a=u200a0.18). An association between CARD15 variants and ileal CD (p=0.004, ORu200a=u200a6.6) was confirmed by multivariate analyses. Conclusions. The CARD15 variants R702W and G908R, but not 1007fs, are associated with susceptibility to CD in Stockholm County. Genotype–phenotype analysis shows an association with ileal CD. The contribution of these CARD15 mutations in Swedish CD patients overall is low in relation to studies elsewhere in Central Europe and North America, but is consistent with emerging data from elsewhere in Scandinavia and in Northern Europe.

Contribution of the IBD5 locus to Crohn's disease in the Swedish population

Objective. Recent data have controversially suggested that variants of the organic cation transport genes SLC22A4 (OCTN1) and SLC22A5 (OCTN2) are responsible for the contribution of IBD5 to disease susceptibility in Crohns disease (CD). The objective of this study was to assess the contribution of the SLC22A4 variant (1672C→T) and SLC22A5 variant (−207G→C) together with three IBD5 haplotype markers in the previously uninvestigated Swedish CD population. Material and methods. The study comprised 178 CD patients and 143 healthy controls (HC). Genotyping for IBD5 single nucleotide polymorphisms (SNPs) IGR2096a_1, IGR2198a_1, IGR2230a_1, SLC22A4 1672C→T and SLC22A5 −207G→C was carried out using the TaqMan® system. Associations with disease susceptibility and disease phenotype were investigated. Results. Strong linkage disequilibrium was observed between the investigated SNPs (D prime >0.92). IGR2096a_1 allelic frequency and homozygosity rates were associated with CD (44% CD versus 33.8% HC, p =0.008, OR =1.55 and 20% CD versus 12% HC, p =0.04, OR =1.93, respectively). Variant allelic frequency of SLC22A4, 1672C→T (44% versus 36%, p =0.03, OR =1.4) and homozygosity for the SLC22A4, SLC22A5 TC haplotype (1672C→T, −207G→C) (21.3% versus 12%, p =0.03, OR =1.78, population attributable risk (PAR) =11%) were associated with CD. There was no association between the allelic frequency of SLC22A5 and CD (46.6% CD versus 41.5% HC, p =0.82). The association of the TC haplotype with CD was not independent of the SNPs representing the extended IBD5 linkage interval. Conclusions. The IBD5 locus is associated with CD in the Swedish population. The strongest association is with the marker SNP IGR2096a_1, lying p-telomeric to SLC22A4 and SLC22A5. The effect of the TC haplotype was not an independent determinant in this population.