This is the first descriptive study to assess potential genetic determinants of PI-IBS. Genes that encode proteins involved in epithelial cell barrier function and the innate immune response to enteric bacteria are associated with development of IBS following acute gastroenteritis.

Four variants were associated with PI-IBS, although the association was not significant after correction for the total number of single nucleotide polymorphisms. Two were located in TLR9, which encodes a pattern recognition receptor (rs352139, P545P; P = .0059 and rs5743836, -T1237C; P = .0250; r 2 < 0.14); 1 was in CDH1, which encodes a tight junction protein (rs16260, -C160A; P = .0352); and 1 was in IL6, which encodes a cytokine (rs1800795, -G174C; P = .0420). Denser mapping of these 3 regions revealed 1 novel association in IL6 (rs2069861; P = .0069) and 14 associations that could be accounted for by linkage disequilibrium with the 4 original variants. The TLR9, IL6, and CDH1 variants all persisted as independent risk factors for PI-IBS when controlling for previously identified clinical risk factors.

We screened 79 functional variants of genes with products involved in serotoninergic pathways, intestinal epithelial barrier function, and innate immunity and performed fine mapping in regions of interest. We compared data from Walkerton residents who developed gastroenteritis and reported PI-IBS 2 to 3 years after the outbreak (n = 228, cases) with data from residents who developed gastroenteritis but did not develop PI-IBS (n = 581, controls).

Acute gastroenteritis is the strongest risk factor for irritable bowel syndrome (IBS). In May 2000, >2300 residents of Walkerton, Ontario, developed gastroenteritis from microbial contamination of the municipal water supply; a longitudinal study found that >36.2% of these developed IBS. We used this cohort to study genetic susceptibility to post-infectious (PI)-IBS.

Irritable bowel syndrome (IBS) is a chronic condition characterized by recurrent abdominal discomfort and disturbed defecation that cannot be explained by structural or biochemical abnormalities.Most people with IBS recall a gradual onset of symptoms, but some with previously normal bowel habit note an acute onset after an episode of gastroenteritis.After exclusion of known organic disorders, such patients are diagnosed with post-infectious irritable bowel syndrome (PI-IBS).A retrospective analysis reported that up to 30% of all IBS patients have an acute onset of symptoms.The annual costs of IBS in the United States include $1.7 billion to $10 billion in direct medical costs and $20 billion in indirect costs.

Although a link between chronic IBS-like symptoms and antecedent gastroenteritis was long suspected,the entity of PI-IBS was not formally recognized until prospective studies identified and quantified a strong association.Initial studies revealed that individuals were more likely to develop PI-IBS if they displayed a vigorous innate immune response to the infection, and inefficiently down-regulated this response after infection.In addition, it was shown that patients with PI-IBS had low-grade inflammation in the colon and increased intestinal permeability.Risk factors for developing PI-IBS include female gender, psychological factors, severity of the acute enteric illness, and microbial virulence factors.However, genetic risk factors for PI-IBS have never been evaluated.

Although familial aggregation and twin studies support the involvement of genetic risk factors in the pathogenesis of sporadic IBS, previous association studies have failed to identify clear and reproducible candidate genes. Limitations of these studies have included the heterogeneity of disease, ethnicity, small sample size, poor statistical analyses, and lack of clinical and epidemiological information regarding IBS patients.By studying a well-defined cohort of IBS patients with similar onset, etiology, and symptom profile, better insight into genetic risk and/or protective factors could be achieved. Ultimately, this may assist in the development of new biomarkers, preventive strategies, and treatments for this common condition.

Walkerton is a small rural town located in a prime agricultural area 180 km northwest of Toronto, Canada. In May 2000, heavy rainfall washed livestock fecal residue from nearby farms into an inadequately chlorinated shallow well. The municipal water supply was then contaminated with Escherichia coli 0157:H7, Campylobacter jejuni, and other pathogen species, leading to an outbreak of acute bacterial gastroenteritis that affected >2300 residents, with 27 recognized cases of the hemolytic uremic syndrome and 7 deaths.Approximately 36.2% of local residents exposed to gastroenteritis fulfilled Rome I diagnostic criteria for IBS when assessed 2 to 3 years after the outbreak. This unfortunate tragedy provided a unique opportunity to study the epidemiology, natural history, and genetic determinants of PI-IBS in a large, well-defined study cohort with simultaneous and well-characterized exposure.

We undertook a study to screen candidate gene variants in this cohort. These genes were selected a priori in 3 categories: (1) reported IBS genes and candidates in the serotoninergic pathways; (2) genes involved in intestinal epithelial barrier; and (3) genes encoding reported innate immunity risk variants. The purpose of the study was to identify polymorphisms that confer susceptibility or protection to PI-IBS and thereby to contribute to a better understanding of its pathogenesis.

We assessed univariate association between genetic variants and PI-IBS using mixed effects logistic regression models, in which a random intercept is included to account for the correlation between individuals belonging to the same family.Use of logistic mixed models allows inclusion of covariates and evaluation of multi-locus effects, in multivariate models. The genetic model used was based on log-additive effects of the alleles. Per-allele odds ratios (ORs) are reported for the SNPs. Analyses were done in R ( http://www.r-project.org/ ). Allelic frequencies that we report in cases and in controls are estimates that account for relatedness as described by McPeek et al,and have been calculated using MQLS software.Reported P values are not corrected (P) for the number of statistical tests performed, except when otherwise mentioned, in which case the corrected P value (P) is taken to be the product of the uncorrected P value and the specified number of tests (ie, Bonferroni correction).

For fine mapping of candidate genes showing significant association in univariate analysis, we first performed a thorough literature research to force include in our design any SNPs that had been previously reported to be associated with different inflammatory disorders, in order to facilitate comparisons. Additionally, we included all nonsynonymous SNPs reported in dbSNP (the SNP database of the National Center for Biotechnology Information) with a minor allele frequency >1%, as well as all SNPs within conserved motifs across species, SNPs disrupting putative transcription-factor, microRNA or enhancer binding sites that were all identified with the use of the University of California Santa Cruz (UCSC) Genome Browser Database ( http://genome.ucsc.edu ). We also included SNPs located in predicted regulatory regions using a computational method called Evolutionary and Sequence Pattern Extraction through Reduced Representations,which is also available from the Genome Browser Database under the name “7X Reg Potential” within the “Expression and Regulation” track group. To capture the remaining variations in the region of these 3 genes (defined as the DNA sequence ranging from 10 kb upstream of the coding sequence to 10 kb downstream), we used data from the HapMap Public Release #23 and selected tagging SNPs using a pairwise tagging approach(r≥ 0.8).

With the use of public databases, including PubMed and Online Mendelian Inheritance in Man, we selected 51 candidate genes that have been characterized and potentially associated with IBS, serotoninergic pathways (hypothesized to play a key role in IBS pathogenesis), intestinal epithelial barrier function (known to be altered in IBS and PI-IBS patients), or innate immune response, which may play key roles in the intestinal inflammatory process ( Supplementary Table 2 ). To select the final candidates, we proceeded on the basis of a comprehensive overview of the intestinal inflammatory system biology and with the hypothesis that PI-IBS might result from persistent local low-grade inflammation. We subsequently selected 79 polymorphisms in these genes that had been previously reported in the literature as being potentially “functional variants.” Most were located in promoter regions, exons, splice-donor or splice-acceptor sites in introns or coding regions of gene, where they were expected or were reported to cause changes in function or expression level of the encoded protein.

The significance levels that we report hereafter are calculated based upon these filtering rules, which consider all of the SNPs genotyped as part of this study, including the follow-up panels. Results that we obtained in the first exploratory phase of the study have thus been revisited once additional panels revealed more problematic samples that have been declared failed.

All first-degree relatives of family members involved in Mendelian errors were treated as having missing genotypes at the SNPs involved in the inconsistencies, or at all SNPs if the number of inconsistencies was ≥4.

DNA samples and SNPs were not included in the analysis if they did not reach high enough call rates. To be considered successful, a SNP had to have a call rate >95% among all genotyped samples, and show a distribution of genotypes consistent with Hardy–Weinberg equilibrium (HWE) at the level P > .001. For SNPs genotyped by the multiplex Sequenom assays, filtering of DNA samples based on missingness of genotype data was done on a panel-by-panel basis. For any given panel, a sample was considered failed and all its genotypes missing if it had a call rate <90% among the set of successful SNPs. Samples that failed all Sequenom panels have also been considered to have failed all TaqMan assays.

DNA from peripheral whole blood was extracted using the Gentra automated system Autopure (Qiagen, Ontario, Canada) according to manufacturer's protocols. All single nucleotide polymorphism (SNP) genotyping was performed using Sequenom iPLEX GOLD genotyping assays (MALDI-TOF mass spectrometry)or the TaqMan 5′ exonuclease assay(Applied Biosystems, Foster City, CA) (rs1800035: Assay ID C_7611968_30; rs2631367: Assay ID C_26479161_30). All primers and probes used for genotyping experiments are available in Supplementary Table 1

Eligible participants in the genetic study were divided into 4 groups for analysis: (1) those who did not experience acute gastroenteritis during the outbreak and did not develop IBS (n = 398); (2) those who experienced acute gastroenteritis but did not develop IBS (n = 581); (3) those with self-reported acute gastroenteritis who subsequently developed IBS (n = 142); and (4) those with confirmed gastroenteritis who subsequently developed IBS (n = 86) ( Table 1 ). Because of limited sample size, self-reported and confirmed gastroenteritis were combined for primary analysis (n = 228), although secondary analyses considered only confirmed gastroenteritis. All participants included in the analyses were white Caucasians of European descent; a total of 8 non-white Caucasian samples were excluded: 3 self-reported controls, 4 confirmed controls, and 1 self-reported case.

In 2006, participants returning for annual assessment were asked for consent to participate in the genetic study. Among the 4315 WHS participants who joined the study in 2002/2003, a total of 2002 individuals provided informed consent. Of these, 1253 were eligible to participate in the PI-IBS Genetic Study. Reasons for exclusion were: age younger than16 years at the time of the outbreak (n = 123), prior diagnosis of inflammatory bowel disease or IBS (n = 163), and nonpermanent residents of Walkerton (n = 463) ( Figure 1 ). All studies were approved by the Hamilton Health Sciences/McMaster University Faculty of Health Sciences and the McGill University Health Centre Research Ethics Boards and all participants gave written informed consent.

The WHS was initiated in 2002 to study the epidemiological and long-term health outcomes of municipal water contamination in May 2000, and to facilitate local residents' access to specialty clinical care. Details of the WHS cohort and study methodology and results of the original cohort have been reported previously.Briefly, in 2002–2003, a total of 4315 local residents were recruited ( Figure 1 ). Of these, 2069 subjects fulfilled the criteria for PI-IBS study cohort: (1) age at least 16 years at the time of the outbreak; (2) no diagnosis of IBS, inflammatory bowel disease, or celiac disease before the outbreak; and (3) permanent residency in Walkerton (as identified by postal code) at the time of the outbreak.A modified version of Talley's Bowel Disease Questionairewas administered to all eligible participants, and Rome I criteria were used to identify those with IBS.Exposure to acute gastroenteritis was classified as either self-reported or confirmed, if corroborated by review of health care records, stool cultures, or responses to a public health questionnaire administered during the outbreak. Participants provided blood samples for future genetic studies during the initial recruitment (2002/2003).

Sampling flowchart of the Walkerton Health Study post-infectious irritable bowel syndrome (PI-IBS) cohort. The flowchart describes the participants' distribution of the Walkerton Health Study cohort (WHS) and of the eligible PI-IBS cohort that were included or not in the genetic association study.

Figure 1 Sampling flowchart of the Walkerton Health Study post-infectious irritable bowel syndrome (PI-IBS) cohort. The flowchart describes the participants' distribution of the Walkerton Health Study cohort (WHS) and of the eligible PI-IBS cohort that were included or not in the genetic association study.

A previous analysis of 2069 subjects in the WHS cohort identified younger age, female gender, bloody stools, abdominal cramps, weight loss, and prolonged diarrhea as independent risk factors for developing PI-IBS.When this multiple logistic regression was repeated for the 1253 individuals from this cohort who provided DNA for genetic analysis, the same independent risk factors were observed ( Table 3 ). When 3 candidate polymorphisms from the TLR9, IL6, and CDH1 regions were added to the logistic model, the significance and effect size of the clinical covariates did not change, suggesting that confounding effects are unlikely ( Table 3 ). Moreover, the risk alleles suffer no meaningful loss of significance or effect size as compared to the uncontrolled analyses ( Table 3 Supplementary Table 3 ), indicating that we are not in the presence of confounding effects. Thus, the identified genetic risk factors in the TLR9, IL6, and CDH1 regions are independent risk factors for developing PI-IBS when controlling for previously identified clinical risk factors ( Table 3 ).

In summary, both the rs2069861 (IL6) and rs2010724 (CDH1) variants showed significant associations with PI-IBS that withstood correction for the numbers of tests performed.

In the CDH1 region, the minor allele of 8 SNPs (rs7186693, rs16260, rs4783681, rs1125557, rs12597188, rs2010724, rs10431923, and rs7186053) located between the promoter and intron 3, and all in high LD with promoter variant rs16260, were associated with increased risk of developing PI-IBS in the subset of confirmed gastroenteritis cases ( Figure 4 B, Supplementary Table 4 ). The strongest association signal was observed with variant in intron 2, rs2010724 (P= .000038; OR = 2.27; 95% CI: 1.54−3.34; frequency of C allele: 45% in cases, 24.0% in controls). This association withstood Bonferroni correction for the total number of SNPs tested (P=.005, accounting for 133 SNPs). A second cluster of associations with decreased risk of developing PI-IBS is observed with 6 SNPs (rs7188750, rs8061932, rs1801026, rs13689, rs7197744, and rs6499203) that are all in high LD and located between CDH1 intron 5 and 3′UTR region ( Figure 4 B, Supplementary Table 4 ). The best association signal is observed with rs13689 (P= .0046; OR = 0.48; 95% CI: 0.28−0.80; frequency of G allele: 14% in cases, 27% in controls), located in CDH1 3′ UTR.

In the IL6 region, the SNP rs2069861 (P= .000196; OR = 2.95; 95% CI: 1.67−5.22; frequency of T allele: 20% in cases, 7.2% in controls) ( Figure 3 B, Supplementary Table 4 ) showed the greatest increase in risk for developing PI-IBS when the analysis was restricted to subjects with exposure to gastroenteritis. This association withstood Bonferroni correction for the total number of SNPs tested (P= .026, accounting for 133 SNPs).

When association analyses in these 3 candidate gene regions are limited to successfully genotyped white Caucasian subjects with confirmed exposure to gastroenteritis (168 controls and 82 cases), variants in both the IL6 ( Figure 3 B) and the CDH1 ( Figure 4 B) regions are even more significantly associated than in the analysis that also included subjects with self-reported exposure ( Supplementary Table 4 ). The TLR9 variants did not show greater association levels in the confirmed sample set ( Figure 2 B) ( Supplementary Table 4 ).

The studied CDH1 region (147 kb) also comprised part of TMCO7 coding sequence, with 2 tagging SNPs located in TMCO7 intron 1 (rs7197744 and rs6499203) ( Figure 4 A). Among the 28 SNPs successfully genotyped in this region, 5 additional SNPs (1 located in CDH1 promoter [rs7186693] and 2 in intron 2 [rs4783681, rs2010724], and 2 in intron 3 [rs10431923, rs7186053]) were also associated with PI-IBS ( Supplementary Table 3 ). These associations can be explained by the level of LD between these SNPs and the promoter variant rs16260 from the exploratory phase. The minor allele of all 6 SNPs conferred increased risk to developing PI-IBS (rs7186053: P= .0217; Supplementary Table 3 ).

Compilation of association results for the CDH1 region. Refer to Figure 2 legend for more details. Note that rs5030625 is a 1 base pair deletion whose deleted base is symbolized by ×.

A window of 29 kb was screened in the IL6 region, which only included the coding sequence of IL6 ( Figure 3 A). Among the 24 successfully genotyped SNPs, the minor alleles of 4 SNPs (ie, rs1800797, rs1554606, rs2069845, rs2069861) were additionally associated with increased risk of developing PI-IBS ( Supplementary Table 3 ). Associations with 3 of these SNPs (rs1800797, rs1554606, rs2069845) (rs2069845: P= .0301) are explained by their level of LD with the promoter variant rs1800795 associated in the exploratory phase. SNP rs2069861, located 34 bp downstream of IL6 3′ UTR, was independently associated with increased risk to developing PI-IBS (P= .0069; OR = 1.63; 95% CI: 1.14−2.31; frequency of T allele: 14% in cases, 9% in controls). This latter SNP tagged only for itself in our design.

The screened TLR9 region (44.9kb) included a cluster of other coding genes comprising ALAS1, TWF2, PPM1M, and WDR82 ( Figure 2 A). Among the 11 SNPs successfully genotyped in this region, 6 were additionally associated with PI-IBS status. These associations are divided into 2 clusters of associations and are explained by their level of LD with the 2 SNPs from the exploratory phase ( Figure 2 A). The first cluster includes SNPs in LD with rs352139 (rs352163, rs352140, rs1060330), where the minor allele of all 4 SNPs is associated with increased risk for developing PI-IBS (rs352163: P= .0022; Supplementary Table 3 ). The second cluster includes SNPs in LD with rs5743836 (rs352143, rs11717574, rs4082828), where the associated allele of all 4 SNPs is associated with decreased risk of developing PI-IBS (rs4082828: P= .0158; Supplementary Table 3 ).

Compilation of association results for the Toll-like receptor 9 (TLR9) region. In the upper panel are the single nucleotide polymorphisms (SNPs) with their positions in the genes and the linkage disequilibrium (LD) structure between them. The upper left portion is D′ and the lower right portion is r 2 . In the lower panel are the results from association analysis of combined self-reported and confirmed (A) and of solely confirmed (B) eligible white Caucasian post-infectious irritable bowel syndrome (PI-IBS) cases and controls affected with gastroenteritis. P values for individual alleles are reported in a logarithmic scale on the Y-axis, with symbols representing the associated allele (▴ = A, ● = C, ▾ = T, ♦ = G). The color spectrum is used to represent the strength of LD and the frequency of the associated alleles.

Figure 2 Compilation of association results for the Toll-like receptor 9 (TLR9) region. In the upper panel are the single nucleotide polymorphisms (SNPs) with their positions in the genes and the linkage disequilibrium (LD) structure between them. The upper left portion is D′ and the lower right portion is r 2 . In the lower panel are the results from association analysis of combined self-reported and confirmed (A) and of solely confirmed (B) eligible white Caucasian post-infectious irritable bowel syndrome (PI-IBS) cases and controls affected with gastroenteritis. P values for individual alleles are reported in a logarithmic scale on the Y-axis, with symbols representing the associated allele (▴ = A, ● = C, ▾ = T, ♦ = G). The color spectrum is used to represent the strength of LD and the frequency of the associated alleles.

Four candidates were associated with PI-IBS in the first stage of the study. Two are located in the Toll-like receptor 9 (TLR9) regions, which plays a role in the innate immune system ( Supplementary Table 2 ) by encoding a human pattern recognition receptor for bacteria that detects unmethylated CpG dinucleotide.They are coding SNP rs352139 (P545P) (P= .0059; OR = 1.38; 95% confidence interval [CI]: 1.10−1.73; frequency of allele A: 48% in cases, 41% in controls) and promoter SNP rs5743836 (-T1237C) (P= .025; OR = 1.45; 95% CI: 1.05−2.00; frequency of allele T: 87% in cases, 82% in controls) ( Supplementary Table 2 ). These 2 variants are 2.4 kb apart and are in low linkage disequilibrium (LD) with each other (r< 0.14). The third candidate, rs1800795 (-G174C) (P= .042; OR = 1.28; 95% CI: 1.01−1.64; frequency of C allele: 44% in cases, 39% in controls), is located in the promoter region of IL6, which also plays a role in the innate immune system ( Supplementary Table 2 ) by encoding a pleiotropic cytokine and main mediator of acute inflammatory responses. The fourth variant showing trends of association, rs16260 (-C160A) (P= .062; OR = 1.26; 95% CI: 0.99−1.61; frequency of A allele: 31% in cases, 26% in controls), is located in CDH1 promoter region and plays a role in intestinal epithelial barrier function ( Supplementary Table 2 ) by encoding a tight junction that enables epithelial cell adhesion. The variant rs16260 was initially significantly associated upon the exploratory association analyses, suggesting CDH1 as a PI-IBS candidate gene, but we report here the result of the final analysis that combines the fine mapping experiment results (Methods). Although the significance level of these results did not withstand correction for the total number of SNPs tested, these 3 associated regions were evaluated further in subsequent fine mapping experiments using a tagging strategy (Methods).

From all the SNPs that were considered successfully genotyped, we identified 224 Mendelian inconsistencies, a large majority of which (n = 216) clustered in 13 nuclear families, likely the result of mispaternity, contamination, or unresolved sample switch. After removal of these nuclear families, only 8 errors remained, all involving different families.

Of the 2115 samples genotyped, 4 were considered to have failed all SNPs due to low call rate in all Sequenom panels. A number of samples that were considered failed for at least 1 of the 4 panels, but not failed for at least another ranged from 14 to 65 (average of 39).

In both the exploratory and follow-up phases, 79 unique SNPs were evaluated. In the exploratory phase, 4 failed due to low call rates, 4 had minor allele frequency <1% and were excluded, and none of the remaining SNPs failed HWE at the level 0.001. In the follow-up phase, these numbers of excluded SNPs are 9, 7, and 1, respectively.

There were more females among the 227 eligible white Caucasian cases than among the 574 eligible white Caucasian controls (64.8% vs 53.7%), but their mean ages were similar (44.3 vs 48.1 years) ( Table 2 Table 3 ). Twice as many cases as controls reported bloody stools (33.3% vs 17.6%) and weight loss of at least 10 pounds (31.4% vs 14.6%) ( Table 2 Table 3 ). Rates of fever, duration of diarrhea during 7 days, and >10 loose stools per day were all higher among cases ( Table 2 Table 3 ).

The primary analysis compared subjects who experienced gastroenteritis but did not develop PI-IBS (581 controls; 570 successfully genotyped) to those who experienced gastroenteritis and reported PI-IBS 2 to 3 years after the outbreak (228 cases; 220 successfully genotyped) ( Table 1 ). Among the 790 successfully genotyped cases and controls, 533 (149 cases, 384 controls) did not have known relationship with any other individual. The remaining 257 samples (71 cases, 186 controls) belonged to 90 families.

Discussion

13 Marshall J.K.

Thabane M.

Garg A.X.

et al. Incidence and epidemiology of irritable bowel syndrome after a large waterborne outbreak of bacterial dysentery. This is the first study looking at genetic risk factors for PI-IBS. Using an exploratory panel of 71 functional variants that were successfully genotyped, we identified 3 promoter (ie, rs16260, rs1800795, and rs5743836) and 1 coding (ie, rs352139) variants associated with susceptibility to PI-IBS. Although the significance of these associations did not withstand corrections for multiple testing, they led to fine mapping experiments in 3 gene regions of interest, ie, CDH1, IL6, and TLR9. All proved to be independent risk factors for developing PI-IBS when controlling for previously reported PI-IBS clinical risk factors in this unique study cohort.The observation of clusters of SNPs in LD showing similar level of associations in all three genes, and the enhanced associations seen in analyses restricted to subjects with confirmed exposure to gastroenteritis further support and strengthen these findings.

27 Li L.C.

Chui R.M.

Sasaki M.

et al. A single nucleotide polymorphism in the E-cadherin gene promoter alters transcriptional activities. 28 Fishman D.

Faulds G.

Jeffery R.

et al. The effect of novel polymorphisms in the interleukin-6 (IL6) gene on IL6 transcription and plasma IL6 levels, and an association with systemic-onset juvenile chronic arthritis. 29 Heesen M.

Bloemeke B.

Heussen N.

et al. Can the interleukin-6 response to endotoxin be predicted? Studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines. , 30 Qi L.

van Dam R.M.

Meigs J.B.

et al. Genetic variation in IL6 gene and type 2 diabetes: tagging-SNP haplotype analysis in large-scale case-control study and meta-analysis. 31 Lazarus R.

Klimecki W.T.

Raby B.A.

et al. Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three US ethnic groups and exploratory case-control disease association studies. The CDH1 promoter variant rs16260 (-C160A) was included in the exploratory panel because it had previously been reported to influence CDH1 transcriptional activity. More specifically, the “A” allele associated with PI-IBS susceptibility has been reported to decrease transcription efficiency by 68% compared to the “C” allele.The IL6 the promoter variant rs1800795 (-G174C) was included in the exploratory phase because the “G” allele has been reported to achieve an overall higher rate of transcription,although numerous inconsistencies within and between in vitro studies have been reported in the effect of that promoter variant on gene expression.Finally, the coding variant P545P (rs352139) and promoter variant -T1237C (rs5743836) of TLR9 were originally selected because they had been reported to distinguish the 4 common TLR9 haplotypes, accounting for >75% of the chromosome.Upon denser mapping of these 3 regions, we observed only 1 novel independent association located 34 bp from IL6 3′UTR and 14 additional associations that could all be accounted for by the level of LD with the 4 original variants from the exploratory phase. Two of these associations (ie, rs2010724 in CDH1 and rs2069861 in IL6) resisted correction for multiple testing.

32 Wu S.

Rhee K.J.

Zhang M.

et al. Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and gamma-secretase-dependent E-cadherin cleavage. , 33 Frank C.F.

Hostetter M.K. Cleavage of E-cadherin: a mechanism for disruption of the intestinal epithelial barrier by Candida albicans. , 34 Barbara G. Mucosal barrier defects in irritable bowel syndrome Who left the door open?. 7 Spiller R.C.

Jenkins D.

Thornley J.P.

et al. Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome. , 35 Marshall J.K.

Thabane M.

Garg A.X.

et al. Intestinal permeability in patients with irritable bowel syndrome after a waterborne outbreak of acute gastroenteritis in Walkerton, Ontario. , 36 Dunlop S.P.

Hebden J.

Campbell E.

et al. Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndromes. 35 Marshall J.K.

Thabane M.

Garg A.X.

et al. Intestinal permeability in patients with irritable bowel syndrome after a waterborne outbreak of acute gastroenteritis in Walkerton, Ontario. 37 Stemmler M.P.

Hecht A.

Kemler R. E-cadherin intron 2 contains cis-regulatory elements essential for gene expression. 38 COGENT Study

Meta-analysis of genome-wide association data identifies four new susceptibility loci for colorectal cancer. 39 The UK IBD Genetics Consortium & the Wellcome Trust Case Control Consortium 2

Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region. 40 Muise A.M.

Walters T.D.

Glowacka W.K.

et al. Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is associated with Crohn's disease. 38 COGENT Study

Meta-analysis of genome-wide association data identifies four new susceptibility loci for colorectal cancer. 39 The UK IBD Genetics Consortium & the Wellcome Trust Case Control Consortium 2

Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region. 40 Muise A.M.

Walters T.D.

Glowacka W.K.

et al. Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is associated with Crohn's disease. 40 Muise A.M.

Walters T.D.

Glowacka W.K.

et al. Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is associated with Crohn's disease. The 3 candidate genes described may play key roles in the regulation of intestinal mucosal immune response. CDH1 encodes for E-cadherin. This transmembrane glycoprotein is a tight junction that is found in adherent junctions and that forms, together with adherent junctions, the apical junctional complex, which is responsible for normal intestinal barrier function by acting as a key mediator of intracellular adhesion in the intestinal epithelium. Enteric pathogens, such as E coli and Campylobacter species, both of which were implicated in the Walkerton outbreak, can increase mucosal permeability during the acute phase of infection, mainly through disruption of epithelial tight junctions.It is therefore of particular interest that PI-IBS patients have been found to have increased small intestinal permeability.In a substudy of the WHS cohort, intestinal permeability was increased in 35% of subjects with IBS vs 13% without IBS.The CDH1 variant rs2010724 that resisted multiple testing correction, along with 3 other association signals, mapped to CDH1 intron 2 region, which has previously been reported to contain cis-regulatory elements and to be required for both the initiation and maintenance of CDH1 transcription.More recently, a meta-analysis of 2 genome-wide,1 genome-wide,and 1 candidateassociation studies all reported associations between variants located in this intron 2, and variants in LD with that region, and susceptibility to colorectal cancer,ulcerative colitis,and Crohn's disease(CD). The presence of CD-associated SNPs resulted in a truncated E-cadherin protein in vivo, which accumulated in the cytoplasm and led to disorganized epithelial architecture. This latter functional alteration could potentially contribute to the increased intestinal permeability observed in CD patients.These results further emphasize the importance of CDH1 intron 2 region, which, together with the -C160A promoter variant, may contribute to CDH1 regulation in colonic epithelial cells. Overall, defects in CDH1 may contribute to PI-IBS symptoms by increasing intestinal permeability.

35 Marshall J.K.

Thabane M.

Garg A.X.

et al. Intestinal permeability in patients with irritable bowel syndrome after a waterborne outbreak of acute gastroenteritis in Walkerton, Ontario. 41 Bisping G.

Lügering N.

Lütke-Brintrup S.

et al. Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells. 42 Cario E.

Becker A.

Sturm A.

et al. Peripheral blood mononuclear cells promote intestinal epithelial restitution in vitro through an interleukin-2/interferon-gamma-dependent pathway. 43 Holtkamp W.

Stollberg T.

Reis H.E. Serum interleukin-6 is related to disease activity but not disease specificity in inflammatory bowel disease. 44 Liebregts T.

Adam B.

Bredack C.

et al. Immune activation in patients with irritable bowel syndrome. 44 Liebregts T.

Adam B.

Bredack C.

et al. Immune activation in patients with irritable bowel syndrome. Although the immune activation observed in PI-IBS could result from increased intestinal permeability, it is equally plausible that inflammation and immune activation could disrupt the mucosal barrier.Peripheral immune cells are known to significantly interact with intestinal epithelial cells.Cytokines released from PBMCs, such as interleukin (IL)-6, may modulate intestinal epithelial barrier function.IL-6 has been reported to reflect both the disease activityand severity of intestinal inflammation.A previous report showed that PBMCs isolated from IBS patients secrete a higher baseline and lipopolysaccharide-induced IL-6 levels compared to healthy controls.These latter observations, together with our 2 independent IL6 association results, further support the involvement of this cytokine in PI-IBS susceptibility.

26 Wagner H. Bacterial CpG DNA activates immune cells to signal infectious danger. 45 Krieg A.M. CpG motifs in bacterial DNA and their immune effects. 46 Ewaschuk J.B.

Backer J.L.

Churchill T.A.

et al. Surface expression of Toll-like receptor 9 is upregulated on intestinal epithelial cells in response to pathogenic bacterial DNA. 9 Gwee K.A.

Collins S.M.

Read N.W.

et al. Increased rectal mucosal expression of interleukin 1beta in recently acquired post-infectious irritable bowel syndrome. The only evidence supporting a role for TLR9 in PI-IBS is the observation of clusters of SNPs in LD that show levels of association similar to the 2 variants identified in the exploratory phase. However, unlike the CDH1 and IL6 regions, TLR9 associations did not resist multiple testing corrections and did not increase when the analysis was restricted to clinically confirmed cases of gastroenteritis. TLR9 was an interesting candidate because it is the only human pattern recognition receptor for bacteria that detects unmethylated CpG dinucleotide.In the Walkerton outbreak, such a candidate may have played a critical role in recognizing the microbial threat and initiating the intestinal innate immune response. Recognition of unmethylated CpG dinucleotide activates transcription factor signaling cascades and stimulates the proliferation of B cells and secretion of proinflammatory cytokines (eg, IL-6, IL-12, and tumor necrosis factor–α).A previous study also reported that TLR9 surface localization and expression are up-regulated on intestinal epithelial cells in response to pathogenic bacteria.Because the epithelial inflammatory response to pathogenic DNA is mediated by TLR9, any defect in recognition of the initial threat may contribute to a poorly controlled innate response.However, our current results do not conclusively support the role of TLR9 in PI-IBS susceptibility.

Considering that the Walkerton PI-IBS population cohort is unique in its size, duration, and access to DNA samples, there currently exist no similar cohort in which we could replicate our findings. An important strength of our study is that the WHS cohort is more homogeneous, arising from a single community with a common environmental trigger and access to a range of clinical and epidemiological data. A limitation of our study regards the approach used to select candidate SNPs, as we cannot fully exclude the involvement of all other candidate genes (aside from TLR9, IL6 and CDH1) without performing thorough fine-mapping experiments in all these regions. Another is that only a minority of patients submitted stool specimens for culture during the Walkerton outbreak because of constraints on local resources. This reduces the proportion of WHS subjects with confirmed exposure to gastroenteritis in our sample set, and could dilute genetic association signals in analyses that include subjects with self-reported exposure.

Results from the multiple logistic regression analysis suggest that both clinical and genetic risk factors contribute independently to PI-IBS pathophysiology. PI-IBS is likely to be a complex trait wherein variability in clinical presentation is partially explained by heterogeneity in underlying genetic and environmental risk factors. This descriptive study identified potential genetic factors involved in PI-IBS susceptibility and provides new insight into its pathophysiology. The identified genetic risk factors, which encode epithelial barrier function and innate immune response to enteric bacteria, are consistent with pathophysiologic disturbances observed in patients with PI-IBS, such as increased intestinal permeability and low-grade immune activation. Altogether, these observations indicate that genetic risk factors are likely to be involved in the development of IBS following acute gastroenteritis.