Genetic and Inflammatory Biomarkers Classify Small Intestine Inflammation in Asymptomatic First-degree Relatives of Patients With Crohn's Disease.

BACKGROUND & AIMS: Relatives of individuals with Crohn's disease (CD) carry CD-associated genetic variants and are often exposed to environmental factors that increase their risk for this disease. We aimed to estimate the utility of genotype, smoking status, family history, and biomarkers can calculate risk in asymptomatic first-degree relatives of patients with CD. METHODS: We recruited 480 healthy first-degree relatives (full siblings, offspring or parents) of patients with CD through the Guy's and St Thomas' NHS Foundation Trust and from members of Crohn's and Colitis, United Kingdom. DNA samples were genotyped using the Immunochip. We calculated a risk score for 454 participants, based on 72 genetic variants associated with CD, family history, and smoking history. Participants were assigned to highest and lowest risk score quartiles. We assessed pre-symptomatic inflammation by capsule endoscopy and measured 22 markers of inflammation in stool and serum samples (reference standard). Two machine-learning classifiers (elastic net and random forest) were used to assess the ability of the risk factors and biomarkers to identify participants with small intestinal inflammation in the same dataset. RESULTS: The machine-learning classifiers identified participants with pre-symptomatic intestinal inflammation: elastic net (area under the curve, 0.80; 95% CI, 0.62-0.98) and random forest (area under the curve, 0.87; 95% CI, 0.75-1.00). The elastic net method identified 3 variables that can be used to calculate odds for intestinal inflammation: combined family history of CD (odds ratio, 1.31), genetic risk score (odds ratio, 1.14), and fecal calprotectin (odds ratio, 1.04). These same 3 variables were among the 5 factors associated with intestinal inflammation in the random forest model. CONCLUSION: Using machine learning classifiers, we found that genetic variants associated with CD, family history, and fecal calprotectin together identify individuals with pre-symptomatic intestinal inflammation who are therefore at risk for CD. A tool for detecting people at risk for CD before they develop symptoms would help identify the individuals most likely to benefit from early intervention.

Association of genetic variants in CHEK2 with oesophageal squamous cell carcinoma in the South African Black population.

Oesophageal squamous cell carcinoma (OSCC) has a high incidence in southern Africa and a poor prognosis. Limited information is available on the contribution of genetic variants in susceptibility to OSCC in this region. However, recent genome-wide association studies have identified multiple susceptibility loci in Asian and European populations. In this study, we investigated genetic variants from seven OSCC risk loci identified in non-African populations for association with OSCC in the South African Black population. We performed association studies in a total of 1471 cases and 1791 controls from two study sample groups, which included 591 cases and 852 controls from the Western Cape and 880 cases and 939 controls from the Johannesburg region in the Gauteng province. Thereafter, we performed a meta-analysis for 11 variants which had been genotyped in both studies. A single nucleotide polymorphism in the CHEK2 gene, rs1033667, was significantly associated with OSCC [P = 0.002; odds ratio (OR) = 1.176; 95% confidence interval (CI): 1.06-1.30]. However, single nucleotide polymorphisms in the CASP8/ALS2CR12, TMEM173, PLCE1, ALDH2, ATP1B2/TP53 and RUNX1 loci were not associated with the disease (P > 0.05). The lack of association of six of these loci with OSCC in South African populations may reflect different genetic risk factors in non-African and African populations or differences in the genetic architecture of African genomes. The association at CHEK2, a gene with key roles in cell cycle regulation and DNA repair, in an African population provides further support for the contribution of common genetic variants at this locus to the risk of oesophageal cancer.

Pooled sequencing of 531 genes in inflammatory bowel disease identifies an associated rare variant in BTNL2 and implicates other immune related genes.

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn's disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10-10, OR = 2.3[95% CI = 1.75-3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (<1%) and low frequency (1-5%) variants in 3 additional genes showed suggestive association (p<0.005) with either an increased risk (ARIH2 c.338-6C>T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis.

A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.

We performed a genome-wide association study of 19,779 nonsynonymous SNPs in 735 individuals with Crohn disease and 368 controls. A total of 7,159 of these SNPs were informative. We followed up on all 72 SNPs with P 0.4), these data suggest that the underlying biological process may be specific to Crohn disease.

Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility.

A genome-wide association scan in individuals with Crohn's disease by the Wellcome Trust Case Control Consortium detected strong association at four novel loci. We tested 37 SNPs from these and other loci for association in an independent case-control sample. We obtained replication for the autophagy-inducing IRGM gene on chromosome 5q33.1 (replication P = 6.6 x 10(-4), combined P = 2.1 x 10(-10)) and for nine other loci, including NKX2-3, PTPN2 and gene deserts on chromosomes 1q and 5p13.

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

BACKGROUND: Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. METHODS: Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. RESULTS: E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. CONCLUSION: In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Altered intestinal microbiota and blood T cell phenotype are shared by patients with Crohn's disease and their unaffected siblings.

OBJECTIVE: Crohn's disease (CD) is associated with intestinal dysbiosis, altered blood T cell populations, elevated faecal calprotectin (FC) and increased intestinal permeability (IP). CD-associated features present in siblings (increased risk of CD) but not in healthy controls, provide insight into early CD pathogenesis. We aimed to (1) Delineate the genetic, immune and microbiological profile of patients with CD, their siblings and controls and (2) Determine which factors discriminate between groups. DESIGN: Faecal microbiology was analysed by quantitative PCR targeting 16S ribosomal RNA, FC by ELISA, blood T cell phenotype by flow cytometry and IP by differential lactulose-rhamnose absorption in 22 patients with inactive CD, 21 of their healthy siblings and 25 controls. Subject's genotype relative risk was determined by Illumina Immuno BeadChip. RESULTS: Strikingly, siblings shared aspects of intestinal dysbiosis with patients with CD (lower concentrations of Faecalibacterium prausnitzii (p=0.048), Clostridia cluster IV (p=0.003) and Roseburia spp. (p=0.09) compared with controls). As in CD, siblings demonstrated a predominance of memory T cells (p=0.002) and elevated naïve CD4 T cell ß7 integrin expression (p=0.01) compared with controls. FC was elevated (>50 µg/g) in 8/21 (38%) siblings compared with 2/25 (8%) controls (p=0.028); whereas IP did not differ between siblings and controls. Discriminant function analysis determined that combinations of these factors significantly discriminated between groups (χ(2)=80.4, df=20, p<0.001). Siblings were separated from controls by immunological and microbiological variables. CONCLUSIONS: Healthy siblings of patients with CD manifest immune and microbiological abnormalities associated with CD distinct from their genotype-related risk and provide an excellent model in which to investigate early CD pathogenesis.

Defining predictors of responsiveness to advanced therapies in Crohn's disease and ulcerative colitis: protocol for the IBD-RESPONSE and nested CD-metaRESPONSE prospective, multicentre, observational cohort study in precision medicine.

INTRODUCTION: Characterised by chronic inflammation of the gastrointestinal tract, inflammatory bowel disease (IBD) symptoms including diarrhoea, abdominal pain and fatigue can significantly impact patient's quality of life. Therapeutic developments in the last 20 years have revolutionised treatment. However, clinical trials and real-world data show primary non-response rates up to 40%. A significant challenge is an inability to predict which treatment will benefit individual patients.Current understanding of IBD pathogenesis implicates complex interactions between host genetics and the gut microbiome. Most cohorts studying the gut microbiota to date have been underpowered, examined single treatments and produced heterogeneous results. Lack of cross-treatment comparisons and well-powered independent replication cohorts hampers the ability to infer real-world utility of predictive signatures.IBD-RESPONSE will use multi-omic data to create a predictive tool for treatment response. Future patient benefit may include development of biomarker-based treatment stratification or manipulation of intestinal microbial targets. IBD-RESPONSE and downstream studies have the potential to improve quality of life, reduce patient risk and reduce expenditure on ineffective treatments. METHODS AND ANALYSIS: This prospective, multicentre, observational study will identify and validate a predictive model for response to advanced IBD therapies, incorporating gut microbiome, metabolome, single-cell transcriptome, human genome, dietary and clinical data. 1325 participants commencing advanced therapies will be recruited from ~40 UK sites. Data will be collected at baseline, week 14 and week 54. The primary outcome is week 14 clinical response. Secondary outcomes include clinical remission, loss of response in week 14 responders, corticosteroid-free response/remission, time to treatment escalation and change in patient-reported outcome measures. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Wales Research Ethics Committee 5 (ref: 21/WA/0228). Recruitment is ongoing. Following study completion, results will be submitted for publication in peer-reviewed journals and presented at scientific meetings. Publications will be summarised at www.ibd-response.co.uk. TRIAL REGISTRATION NUMBER: ISRCTN96296121.

Conserved γδ T cell selection by BTNL proteins limits progression of human inflammatory bowel disease.

Murine intraepithelial γδ T cells include distinct tissue-protective cells selected by epithelial butyrophilin-like (BTNL) heteromers. To determine whether this biology is conserved in humans, we characterized the colonic γδ T cell compartment, identifying a diverse repertoire that includes a phenotypically distinct subset coexpressing T cell receptor Vγ4 and the epithelium-binding integrin CD103. This subset was disproportionately diminished and dysregulated in inflammatory bowel disease, whereas on-treatment CD103+γδ T cell restoration was associated with sustained inflammatory bowel disease remission. Moreover, CD103+Vγ4+cell dysregulation and loss were also displayed by humans with germline BTNL3/BTNL8 hypomorphism, which we identified as a risk factor for penetrating Crohn's disease (CD). Thus, BTNL-dependent selection and/or maintenance of distinct tissue-intrinsic γδ T cells appears to be an evolutionarily conserved axis limiting the progression of a complex, multifactorial, tissue-damaging disease of increasing global incidence.

Distinct genetic association at the PLCE1 locus with oesophageal squamous cell carcinoma in the South African population.

Oesophageal squamous cell carcinoma (OSCC) has a high prevalence in the Black and Mixed Ancestry populations of South Africa. Recently, three genome-wide association studies in Chinese populations identified five new OSCC susceptibility loci, including variants at PLCE1, C20orf54, PDE4D, RUNX1 and UNC5CL, but their contribution to disease risk in other populations is unknown. In this study, we report testing variants from these five loci for association with OSCC in the South African Black (407 cases and 849 controls) and Mixed Ancestry (257 cases and 860 controls) populations. The RUNX1 variant rs2014300, which reduced risk in the Chinese population, was associated with an increased risk of OSCC in the Mixed Ancestry population [odds ratio (OR) = 1.33, 95% confidence interval (CI) = 1.09-1.63, P = 0.0055], and none of the five loci were associated in the Black population. Since PLCE1 variants increased the risk of OSCC in all three Chinese studies, this gene was investigated further by sequencing in 46 Black South Africans. This revealed 48 variants, 10 of which resulted in amino acid substitutions, and much lower linkage disequilibrium across the PLCE1 locus than in the Chinese population. We genotyped five PLCE1 variants in cases and controls, and found association of Arg548Leu (rs17417407) with a reduced risk of OSCC (OR = 0.74, 95% CI = 0.60-0.93, P = 0.008) in the Black population. These findings indicate several differences in the genetic contribution to OSCC between the South African and Chinese populations that may be related to differences in their genetic architecture.

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease.

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case-control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case-control studies.

Independent and population-specific association of risk variants at the IRGM locus with Crohn's disease.

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohn's disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5' untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 x 10(-5) to 1.40 x 10(-9)), and that the CNV and the 5'-untranslated region variant -308(GTTT)(5) contribute independently to CD susceptibility (P = 2.6 x 10(-7) and P = 2 x 10(-5), respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10(-12)) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case-control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene-gene or gene-environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European-Asian split.

Searching for genotype-phenotype structure: using hierarchical log-linear models in Crohn disease.

There has been considerable recent success in the detection of gene-disease associations. We consider here the development of tools that facilitate the more detailed characterization of the effect of a genetic variant on disease. We replace the simplistic classification of individuals according to a single binary disease indicator with classification according to a number of subphenotypes. This more accurately reflects the underlying biological complexity of the disease process, but it poses additional analytical difficulties. Notably, the subphenotypes that make up a particular disease are typically highly associated, and it becomes difficult to distinguish which genes might be causing which subphenotypes. Such problems arise in many complex diseases. Here, we concentrate on an application to Crohn disease (CD). We consider this problem as one of model selection based upon log-linear models, fitted in a Bayesian framework via reversible-jump Metropolis-Hastings approach. We evaluate the performance of our suggested approach with a simple simulation study and then apply the method to a real data example in CD, revealing a sparse disease structure. Most notably, the associated NOD2.908G-->R mutation appears to be directly related to more severe disease behaviors, whereas the other two associated NOD2 variants, 1007L-->FS and 702R-->W, are more generally related to disease in the small bowel (ileum and jejenum). The ATG16L1.300T-->A variant appears to be directly associated with only disease of the small bowel.

Population-specific genetic associations with oesophageal squamous cell carcinoma in South Africa.

Genetic variants in multiple cellular pathways have been associated with an altered risk of oesophageal cancer. In this study, eight genes previously associated with an altered risk of oesophageal squamous cell carcinoma (OSCC) in European or Asian populations were investigated in two South African populations. We genotyped 12 single-nucleotide polymorphisms and one insertion/deletion variant in 1463 individuals from the Black and Mixed Ancestry populations. No polymorphisms were associated with OSCC in the Black population. In the Mixed Ancestry population, ALDH2 +82 G > A (rs886205) was significantly associated with a reduced risk of OSCC (odds ratio = 0.70, 95% confidence interval = 0.55-0.89; P = 0.0038). Several other polymorphisms showed a suggestive association (P < 0.05), including ADH1B Arg48His (rs1229984), COX-2 -1195G > A (rs689466), CASP8 Asp302His (rs1045485) and MGMT Leu84Phe (rs12917). Haplotype analysis indicated that the FAS polymorphisms -670 A > G (rs1800682) and -1377 G > A (rs2234767) were both associated with OSCC in the Mixed Ancestry population (P = 0.006 and P = 0.004, respectively), as well as the CASP8 (-652 6Ndel:302His) haplotype (P = 0.0013). This study indicates several instances of population-specific differences in the genetic etiology of OSCC between these two South African populations and between them and other high-risk populations, which may reflect differences in their ancestry and environmental exposures.

Investigation of Crohn's disease risk loci in ulcerative colitis further defines their molecular relationship.

BACKGROUND & AIMS: Identifying shared and disease-specific susceptibility loci for Crohn's disease (CD) and ulcerative colitis (UC) would help define the biologic relationship between the inflammatory bowel diseases. More than 30 CD susceptibility loci have been identified. These represent important candidate susceptibility loci for UC. Loci discovered by the index genome scans in CD have previously been tested for association with UC, but those identified in the recent meta-analysis await such investigation. Furthermore, the recently identified UC locus at ECM1 requires formal testing for association with CD. METHODS: We analyzed 45 single nucleotide polymorphisms, tagging 29 of the loci recently associated with CD in 2527 UC cases and 4070 population controls. We also genotyped the UC-associated ECM1 variant rs11205387 in 1560 CD patients and 3028 controls. RESULTS: Nine regions showed association with UC at a threshold corrected for the 29 loci tested (P < .0017). The strongest association (P = 4.13 x 10(-8); odds ratio = 1.27) was identified with a 170-kilobase region on chromosome 1q32 that contains 3 genes. We also found association with JAK2 and replicated a recently reported association with STAT3, further implicating the role of this signaling pathway in inflammatory bowel disease. Additional novel UC susceptibility genes were LYRM4 and CDKAL1. Twenty of the loci were not associated with UC, and several appear to be specific to CD. ECM1 variation was not associated with CD. CONCLUSIONS: Collectively, these data help define the genetic relationship between CD and UC and characterize common, as well as disease-specific mechanisms of pathogenesis.

Streptococcus Salivarius: A Potential Salivary Biomarker for Orofacial Granulomatosis and Crohn's Disease?

BACKGROUND: Orofacial granulomatosis (OFG) is a rare disease characterised by chronic, noncaseating, granulomatous inflammation primarily affecting the oral cavity. Histologically, it is similar to Crohn's disease (CD), and a proportion of patients have both OFG and CD. The cause of OFG remains elusive, but it has been suggested that microbial interactions may be involved. The aim of this study was to compare the salivary microbial composition of subjects with OFG and/or CD and healthy controls. METHODS: Two hundred sixty-one subjects were recruited, of whom 78 had OFG only, 40 had both OFG and CD, 97 had CD only with no oral symptoms, and 46 were healthy controls. Bacterial community profiles were obtained by sequencing the V1-V3 region of the 16S rRNA gene. RESULTS: There were no differences in richness or diversity of the salivary bacterial communities between patient groups and controls. The relative abundance of the Streptococcus salivarius group was raised in patients with OFG or CD only compared with controls, whereas that of the Streptococcus mitis group was lower in CD compared with both OFG and controls. One S. salivarius oligotype made the major contribution to the increased proportions seen in patients with OFG and CD. CONCLUSIONS: The salivary microbiome of individuals with OFG and CD was similar to that found in health, although the proportions of S. salivarius, a common oral Streptococcus, were raised. One specific strain-level oligotype was found to be primarily responsible for the increased levels seen.

Novel isoforms of the CARD8 (TUCAN) gene evade a nonsense mutation.

CARD8 (TUCAN) is implicated in the regulation of apoptosis and inflammation, and is a positional and functional candidate gene for inflammatory bowel disease (IBD). Recent investigations have reported conflicting results of association between a CARD8 nonsynonymous SNP, rs2043211, and IBD. SNP rs2043211 results in an A>T transversion in the CARD8 template strand, which introduces a stop codon polymorphism (Cys10Stop), and genotyping of the Cys10Stop variant revealed that 9% of the control population was homozygous for the 'Stop' allele. The effect of the Stop allele on mRNA and protein expression of the two known isoforms of this gene was investigated. IBD patients homozygous for the Stop allele showed somewhat reduced expression of CARD8 mRNA, but, contrary to expectation, expressed a 48 kDa protein isoform. A search of the EST database and reverse transcription-PCR analysis revealed a novel coding exon and three novel CARD8 mRNA isoforms that are conserved in primates. The isoforms of CARD8 differ in their N-termini, resulting in diverse predicted molecular weights (47, 48, 51, 54 and 60 kDa) and multiple outcomes for the variant including Cys10Stop, Cys34Stop, Phe52Ile and Phe102Ile; one isoform may arise through transcription and translation initiated downstream of rs2043211 to yield a novel protein isoform of approximately 47 kDa. The multiple isoforms and differing consequences for a predicted stop codon polymorphism underline the importance of detailed analysis of the effects of proposed functional variants on gene expression.

Diverse effects of the CARD15 and IBD5 loci on clinical phenotype in 630 patients with Crohn's disease.

OBJECTIVES: Genetic variants at the CARD15 and IBD5 loci are strongly associated with Crohn's disease (CD), but evidence of the effect of these variants on the clinical expression of CD is conflicting and has often been hampered by small sample sizes. We studied 630 well-characterized patients to clarify the genotype/phenotype relationship in CD. METHODS: Patients and healthy controls were genotyped for three common mutations in CARD15 and a marker of the IBD5 risk haplotype. Allele frequencies were compared between phenotypic subgroups using chi2 or Fisher's exact tests. Genotype/phenotype analysis was carried out using multinomial logistic regression modelling allowing for adjustment for correlated or confounding factors. RESULTS: The mean age at diagnosis was significantly lower in carriers of the CARD15 or IBD5 risk alleles. After correction for age and smoking, CARD15 mutations were strongly associated with both ileal disease (P=8.8 x 10(-6)) and stenotic disease (P=0.003), but the association with stenotic disease appeared to be due to a confounding effect with ileal disease. CARD15 mutations were also associated with the presence of granulomas (P=5.7 x 10(-5)), which remained significant after adjustment for age at diagnosis and disease location (P=0.0047). The IBD5 risk haplotype frequency was significantly elevated in cases with perianal disease (P=0.028) and axial spondyloarthropathy (P=0.012). CONCLUSION: Genetic variants at the CARD15 and IBD5 loci have diverse effects on clinical expression in CD. CARD15 mutations are significantly correlated with the presence of granulomas.