The contribution of OCTN1/2 variants within the IBD5 locus to disease susceptibility and severity in Crohn's disease

Background and Aims: Recent data suggest that polymorphisms in the organic cation transporter (OCTN) genes OCTN1 (SLC22A4) and OCTN2 (SLC22A5) represent disease-causing mutations within the IBD5 locus (chromosome 5q31). We investigated associations with disease susceptibility, phenotype, and evidence for epistasis with CARD15 in 679 patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: A total of 374 patients with CD, 305 patients with UC, and 294 healthy controls (HCs) were studied. Genotyping for single nucleotide polymorphisms IGR2096, IGR2198, and IGR2230, OCTN1 variant (SLC22A4 1672C→T), and OCTN2 variant (SLC22A5 −207G→C) was performed using the TaqMan system. Results: The IBD5 OCTN1 and OCTN2 polymorphisms were in strong linkage disequilibrium (D′, >0.959). IGR2198 variant allele frequency (49.1% vs 40.8%; P = .0046) and homozygosity (21% vs 14.8%; P = .044) were associated with CD versus HCs. Variant allelic frequency of OCTN1 (53.6% vs 43%; P = .0008) and OCTN2 (56.1% vs 48.4%; P = .0092) polymorphisms and homozygosity for the OCTN1/2-TC haplotype (28.4% vs 16%; P = .0042) were associated with CD versus HCs. IGR2198 homozygosity and TC homozygosity were associated with stricturing/penetrating disease at follow-up (P = .011 and P = .011, respectively) and disease progression (P = .038 and P = .049, respectively) on univariate analysis and with need for surgery on multivariate analysis (P = .016 and P = .004, respectively). In the absence of the IBD5 risk haplotype, no association of OCTN1/2 variants with CD was detected. No associations were seen with UC. Conclusions: The IBD5 locus influences susceptibility, progression, and need for surgery in CD. However, the contribution of OCTN1/2 variants is not independent of the IBD5 haplotype; a causative role for these genes remains plausible but is not yet proven. Further genetic, functional, and expression data are now required. </p

Serum Calprotectin - A novel diagnostic and prognostic marker in Inflammatory Bowel Diseases

OBJECTIVES: There is an unmet need for novel blood-based biomarkers that offer timely and accurate diagnostic and prognostic testing in inflammatory bowel diseases (IBD). We aimed to investigate the diagnostic and prognostic utility of serum calprotectin (SC) in IBD. METHODS: A total of 171 patients (n=96 IBD, n=75 non-IBD) were prospectively recruited. A multi-biomarker model was derived using multivariable logistic regression analysis. Cox proportional hazards model was derived to assess the contribution of each variable to disease outcomes. RESULTS: SC correlated strongly with current biomarkers, including fecal calprotectin (FC) (n=50, ρ=0.50, P=1.6 × 10−4). SC was the strongest individual predictor of IBD diagnosis (odds ratio (OR): 9.37 (95% confidence interval (CI): 2.82–34.68), P=4.00 × 10−4) compared with other markers (C-reactive protein (CRP): OR 8.52 (95% CI: 2.75–28.63), P=2.80 × 10−4); albumin: OR 6.12 (95% CI: 1.82–22.16), P=0.004). In a subset of 50 patients with paired SC and FC, the area under receiver operating characteristic discriminating IBD from controls was better for FC than for SC (0.99, (95% CI 0.87–1.00) and 0.87 (95% CI:0.78–0.97), respectively; P=0.01). At follow-up (median 342 days; interquartile range: 88–563), SC predicted treatment escalation and/or surgery in IBD (hazard ratio (HR) 2.7, 95% CI: 1.1–4.9), in particular Crohn’s disease (CD) (HR 4.2, 95% CI 1.2–15.3). A model incorporating SC and either CRP or albumin has a positive likelihood ratio of 24.14 for IBD. At 1 year, our prognostic model can predict treatment escalation in IBD in 65% of cases (95% CI: 43–79%) and 80% (95% CI: 31–94%) in CD if ≥2 blood marker criteria are met. CONCLUSIONS: A diagnostic and prognostic model that combines SC and other blood-based biomarkers accurately predicts the inflammatory burden in IBD and has the potential to predict disease and its outcomes. Our data warrant further detailed exploration and validation in large multicenter cohorts

Identification of novel genetic determinants in the high prevalence early-onset inflammatory bowel disease population in Scotland

Grant no. 072789/Z/03/ZBackground & aims: The inflammatory bowel diseases (IBD), Crohn‟s disease (CD) and ulcerative colitis (UC), are common causes of chronic gastrointestinal morbidity, affecting up to 1 in 250 of the general population in Northern Europe. Up to 25% of IBD is diagnosed during childhood or adolescence. The aims for this thesis were to study the epidemiology, natural history and novel genetic determinants of childhood onset IBD in Scotland. Methods: The existing repository of childhood onset and adult onset IBD patients, established at the Western General Hospital in Edinburgh, was used and expanded. Thus, anatomical location and behaviour of disease were assessed in 416 childhood onset (276 CD, 99 UC, 41 IBDU diagnosed before 17th birthday) and 1297 adult patients (596 CD, 701 UC) using the Montreal classification. Additional phenotypic (at diagnosis and at regular follow-up intervals) and epidemiological data were gathered. In this cohort, genotyping of germline variants in putative susceptibility genes (NOD1/CARD4, IL23R, ATG16L1, IRGM, FLG) was performed to enable single variant and haplotype-tagging association studies. Genotypic data of population-matched healthy controls were obtained locally (n=342) and from the Wellcome Trust Case Control Consortium (n=2937). Results: Compared with adults, childhood-onset CD was characterized by a more extensive, “panenteric” phenotype (ileocolonic plus upper GI; p<0.0001 OR23.3; 95% CI (13.4–40.6) with less isolated ileal (p<0.0001 OR 0.06 (0.03–0.1) or colonic disease (p<0.0001, OR 0.3 (0.2–0.5)). In 39%, the anatomic extent increased within 2 years. UC was also more extensive in children at diagnosis vs adults (p0.05 after Bonferroni correction). We found that the allelic frequency of rs11209026*A located within the IL23R gene, differed significantly between IBD / CD cases and controls (p=0.01 OR 0.51(0.3-0.9) and p=0.04 OR 0.5 (0.3-0.98)). Using a gene-wide haplotype-tagging strategy, we demonstrated that the multiple association signals of the IL23R locus are independent of rs11209026 in childhood onset IBD and CD. In Scottish children, the effect of germline variation of ATG16L1 and IRGM on CD susceptibility was relatively small (OR< 1.4), and appeared less than in adult disease. Genotype–phenotype analysis demonstrated an association of pure ileal disease with the ATG16L1 rs2241880G-allele (p=0.02 OR 1.3 (1.03–1.7)). Using binary logistic regression analysis, we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (p=0.03 OR 2.4 (1.05–5.6)). Null alleles of the epithelial barrier protein FLG have no important effect on IBD susceptibility (p>0.4), but contribute to the high prevalence of atopy, notably co-existent eczema and food allergy (p=0.0003 OR 3.3 (1.7–6.6) and p=0.0001 OR 4.5 (2.0–10.0), respectively). Conclusion: Childhood onset IBD is characterised by extensive intestinal involvement and progression of disease after diagnosis. Genetic association studies in childhood and adult IBD have provided evidence for a large number of new genomic loci. These loci encode genes involved in a number of homeostatic mechanisms: innate pattern recognition receptors, the differentiation of Th17-lymphocytes, autophagy, maintenance of epithelial barrier integrity and the orchestration of the secondary immune response

Analysis of Germline GLI1 Variation Implicates Hedgehog Signalling in the Regulation of Intestinal Inflammatory Pathways

Charlie Lees and colleagues identify a reduced-function variant of the hedgehog signaling pathway protein GLI1 that associates with inflammatory bowel disease, and investigate its role in a mouse model of colitis

Genetic Variation in the Familial Mediterranean Fever Gene (MEFV) and Risk for Crohn's Disease and Ulcerative Colitis

BACKGROUND AND AIMS: The familial Mediterranean fever (FMF) gene (MEFV) encodes pyrin, a major regulator of the inflammasome platform controlling caspase-1 activation and IL-1beta processing. Pyrin has been shown to interact with the gene product of NLRP3, NALP3/cryopyrin, also an important active member of the inflammasome. The NLRP3 region was recently reported to be associated with Crohn's disease (CD) susceptibility. We therefore sought to evaluate MEFV as an inflammatory bowel disease (IBD) susceptibility gene. METHODOLOGY AND RESULTS: MEFV colonic mucosal gene expression was significantly increased in experimental colitis mice models (TNBS p<0.0003; DSS p<0.006), in biopsies from CD (p<0.02) and severe ulcerative colitis (UC) patients (p<0.008). Comprehensive genetic screening of the MEFV region in the Belgian exploratory sample set (440 CD trios, 137 UC trios, 239 CD cases, 96 UC cases, and 107 healthy controls) identified SNPs located in the MEFV 5' haplotype block that were significantly associated with UC (rs224217; p = 0.003; A allele frequency: 56% cases, 45% controls), while no CD associations were observed. Sequencing and subsequent genotyping of variants located in this associated haplotype block identified three synonymous variants (D102D/rs224225, G138G/rs224224, A165A/rs224223) and one non-synonymous variant (R202Q/rs224222) located in MEFV exon 2 that were significantly associated with UC (rs224222: p = 0.0005; A allele frequency: 32% in cases, 23% in controls). No consistent associations were observed in additional Canadian (256 CD trios, 91 UC trios) and Scottish (495 UC, 370 controls) sample sets. We note that rs224222 showed marginal association (p = 0.012; G allele frequency: 82% in cases, 70% in controls) in the Canadian sample, but with a different risk allele. None of the NLRP3 common variants were associated with UC in the Belgian-Canadian UC samples and no significant interactions were observed between NLRP3 and MEFV that could explain the observed flip-flop of the rs224222 risk allele. CONCLUSION: The differences in association levels observed between the sample sets may be a consequence of distinct founder effects or of the relative small sample size of the cohorts evaluated in this study. However, the results suggest that common variants in the MEFV region do not contribute to CD and UC susceptibility.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease

Genetic association studies have identified 215 risk loci for inflammatory bowel disease, thereby uncovering fundamental aspects of its molecular biology. We performed a genome-wide association study of 25,305 individuals and conducted a meta-analysis with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 25 new susceptibility loci, 3 of which contain integrin genes that encode proteins in pathways that have been identified as important therapeutic targets in inflammatory bowel disease. The associated variants are correlated with expression changes in response to immune stimulus at two of these genes ($\textit{ITGA4 }$ and $\textit{ITGB8}$) and at previously implicated loci ($\textit{ITGAL }$and $\textit{ICAM1}$). In all four cases, the expression-increasing allele also increases disease risk. We also identified likely causal missense variants in a gene implicated in primary immune deficiency, $\textit{PLCG2}$, and a negative regulator of inflammation, $\textit{SLAMF8}$. Our results demonstrate that new associations at common variants continue to identify genes relevant to therapeutic target identification and prioritization.This work was co-funded by the Wellcome Trust [098051] and the Medical Research Council, UK [MR/J00314X/1]. Case collections were supported by Crohn’s and Colitis UK. KMdL, LM, CAL, YL, DR, JG-A, NJP, CAA and JCB are supported by the Wellcome Trust [098051; 093885/Z/10/Z; 094491/Z/10/Z]. KMdL is supported by a Woolf Fisher Trust scholarship. CAL is a clinical lecturer funded by the NIHR. We thank Anna Stanton for co-ordinating the Guy’s and St Thomas’ patient recruitment. We acknowledge support from the Department of Health via the NIHR comprehensive Biomedical Research Centre awards to Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London and to Addenbrooke’s Hospital, Cambridge in partnership with the University of Cambridge. This research was also supported by the NIHR Newcastle Biomedical Research Centre. The UK Household Longitudinal Study is led by the Institute for Social and Economic Research at the University of Essex and funded by the Economic and Social Research Council

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 Crohn's 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 &#60; 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 &#60; 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.</p

Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A

The major histocompatibility complex (MHC) on chromosome 6 is associated with susceptibility to more common diseases than any other region of the human genome, including almost all disorders classified as autoimmune. In type 1 diabetes the major genetic susceptibility determinants have been mapped to the MHC class II genes HLA-DQB1 and HLA-DRB1 (refs 1-3), but these genes cannot completely explain the association between type 1 diabetes and the MHC region. Owing to the region's extreme gene density, the multiplicity of disease-associated alleles, strong associations between alleles, limited genotyping capability, and inadequate statistical approaches and sample sizes, which, and how many, loci within the MHC determine susceptibility remains unclear. Here, in several large type 1 diabetes data sets, we analyse a combined total of 1,729 polymorphisms, and apply statistical methods - recursive partitioning and regression - to pinpoint disease susceptibility to the MHC class I genes HLA-B and HLA-A (risk ratios >1.5; Pcombined = 2.01 × 10-19 and 2.35 × 10-13, respectively) in addition to the established associations of the MHC class II genes. Other loci with smaller and/or rarer effects might also be involved, but to find these, future searches must take into account both the HLA class II and class I genes and use even larger samples. Taken together with previous studies, we conclude that MHC-class-I-mediated events, principally involving HLA-B*39, contribute to the aetiology of type 1 diabetes. ©2007 Nature Publishing Group

Changes to serum sample tube and processing methodology does not cause inter-individual variation in automated whole serum N-glycan profiling in health and disease

Serum N-glycans have been identified as putative biomarkers for numerous diseases. The impact of different serum sample tubes and processing methods on N-glycan analysis has received relatively little attention. This study aimed to determine the effect of different sample tubes and processing methods on the whole serum N-glycan profile in both health and disease. A secondary objective was to describe a robot automated N-glycan release, labeling and cleanup process for use in a biomarker discovery system.25 patients with active and quiescent inflammatory bowel disease and controls had three different serum sample tubes taken at the same draw. Two different processing methods were used for three types of tube (with and without gel-separation medium). Samples were randomised and processed in a blinded fashion. Whole serum N-glycan release, 2-aminobenzamide labeling and cleanup was automated using a Hamilton Microlab STARlet Liquid Handling robot. Samples were analysed using a hydrophilic interaction liquid chromatography/ethylene bridged hybrid(BEH) column on an ultra-high performance liquid chromatography instrument. Data were analysed quantitatively by pairwise correlation and hierarchical clustering using the area under each chromatogram peak. Qualitatively, a blinded assessor attempted to match chromatograms to each individual.There was small intra-individual variation in serum N-glycan profiles from samples collected using different sample processing methods. Intra-individual correlation coefficients were between 0.99 and 1. Unsupervised hierarchical clustering and principal coordinate analyses accurately matched samples from the same individual. Qualitative analysis demonstrated good chromatogram overlay and a blinded assessor was able to accurately match individuals based on chromatogram profile, regardless of disease status.The three different serum sample tubes processed using the described methods cause minimal inter-individual variation in serum whole N-glycan profile when processed using an automated workstream. This has important implications for N-glycan biomarker discovery studies using different serum processing standard operating procedures