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Inflammatory bowel disease
Original article
The NOD2insC polymorphism is associated with worse outcome following ileal pouch-anal anastomosis for ulcerative colitis
  1. Andrea D Tyler1,2,
  2. Raquel Milgrom2,
  3. Joanne M Stempak2,
  4. Wei Xu3,
  5. John Hunter Brumell1,4,5,
  6. Aleixo M Muise1,4,6,
  7. Rishabh Sehgal7,
  8. Zane Cohen1,2,
  9. Walter Koltun7,8,
  10. Bo Shen9,
  11. Mark S Silverberg1,2
  1. 1Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
  2. 2Zane Cohen Centre for Digestive Diseases, Mount Sinai Hospital IBD Group, Toronto, Ontario, Canada
  3. 3Department of Public Health Sciences, University of Toronto, Toronto, Ontario, Canada
  4. 4Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada
  5. 5Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
  6. 6Division of Gastroenterology, Hepatology and Nutrition, Department of Paediatrics, The Hospital for Sick Children, Toronto, Canada
  7. 7Division of Colon and Rectal Surgery, Department of Surgery, Penn State Milton S Hershey Medical Center, Hershey, Pennsylvania, USA
  8. 8Department of Cellular and Molecular Physiology, Penn State Milton S Hershey Medical Center, Hershey, Pennsylvania, USA
  9. 9Center for Inflammatory Bowel Disease, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio, USA
  1. Correspondence to Dr Mark S Silverberg, Mount Sinai Hospital, 600 University Ave, Room 441, Toronto, ON M5G 1X5, Canada; msilverberg{at}mtsinai.on.ca

Abstract

Background Inflammatory complications after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) are common.

Objective To investigate whether genetic factors are associated with adverse pouch outcomes such as chronic pouchitis (CP) and a Crohn's disease-like (CDL) phenotype.

Design 866 patients were recruited from three centres in North America: Mount Sinai Hospital (Toronto, Ontario, Canada), the Cleveland Clinic (Cleveland, Ohio, USA) and Penn State Milton S Hershey Medical Center (Hershey, Pennsylvania, USA). DNA and clinical and demographic information were collected. Subjects were classified into post-surgical outcome groups: no chronic pouchitis (NCP), CP and CDL phenotype.

Results Clinical and genetic data were available on 714 individuals. 487 (68.2%) were classified as NCP, 118 (16.5%) CP and 109 (15.3%) CDL. The presence of arthritis or arthropathy (p=0.02), primary sclerosing cholangitis (p=0.009) and duration of time from ileostomy closure to recruitment (p=0.001) were significantly associated with outcome. The NOD2insC (rs2066847) risk variant was the single nucleotide polymorphism (SNP) most significantly associated with pouch outcome (p=7.4×10−5). Specifically, it was associated with both CP and CDL in comparison with NCP (OR=3.2 and 4.3, respectively). Additionally, SNPs in NOX3 (rs6557421, rs12661812), DAGLB (rs836518) and NCF4 (rs8137602) were shown to be associated with pouch outcome with slightly weaker effects. A multivariable risk model combining previously identified clinical (smoking status, family history of inflammatory bowel disease), serological (anti-Saccharomyces cerevisiae antibody IgG, perinuclear antineutrophil cytoplasmic antibody and anti-CBir1) and genetic markers was constructed and resulted in an OR of 2.72 (p=8.89×10−7) for NCP versus CP/CDL and 3.22 (p=4.11×10−8) for NCP versus CDL, respectively.

Conclusion Genetic polymorphisms, in particular, the NOD2insC risk allele, are associated with chronic inflammatory pouch outcomes among patients with UC and IPAA.

  • Ileal pouch-anal anastomosis
  • NOD2
  • inflammatory bowel disease genetics
  • restorative proctocolectomy
  • ulcerative colitis

https://doi.org/10.1136/gutjnl-2011-301957

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    Significance of this study

    What is already known on this subject?

    • Several genetic factors have previously been associated with inflammatory pouch outcomes in small cohorts.

    • Patients with familial adenomatous polyposis and a pelvic pouch do not typically develop inflammatory pouch complications, suggesting that genetic factors are important in pouchitis and a Crohn's disease-like phenotype among individuals with ulcerative colitis.

    What are the new findings?

    • The NOD2insC polymorphism, which is important in innate immune recognition of microbial muramyl dipeptide, is associated with inflammatory pouch outcomes.

    • Additional genes which are mediators in the generation of reactive oxygen species may also have a role in pouch inflammation.

    How might it impact on clinical practice in the foreseeable future?

    • These polymorphisms may be useful for predicting which patients are at an increased risk of developing pouch inflammatory complications after surgery for ulcerative colitis.

    • A combination of clinical, serological and genetic factors may be a useful clinical tool for assisting in decision making for patients considering a colectomy and pelvic pouch for ulcerative colitis.

    Introduction

    Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. About 20% of affected individuals will require colectomy at some point in their disease.1–4 Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the preferred surgical procedure in fulminant or chronic, treatment-refractory UC and UC-associated colonic neoplasia.5 ,6 However, de novo inflammation of the ileal reservoir (pouchitis) is a common post-surgical complication with a prevalence ranging from 12% to >50%.5 ,7–9 An additional subset of patients (up to 17%) will develop a Crohn's disease-like (CDL) phenotype characterised by abdominal or perianal fistulae and/or inflammation of the small bowel proximal to the pouch (afferent limb).10

    The causes of ileal inflammation in the pouch in patients with a preoperative diagnosis of UC are unknown. Clearly, evolution of the microbiome of the pouch after closure of a diverting ileostomy plays a role, as patients rarely develop pouchitis before restoration of continuity of the fecal stream. The decreased risk of inflammation among those with familial adenomatous polyposis and reports by some groups that a family history of CD may increase the risk for the CDL phenotype in the pouch, suggests that heritable susceptibility may be a critical factor in disease pathogenesis.11 ,12 However, previous genetic studies have been small in scale and have not yielded consistently reproducible results.13 ,14 Among others, variants in IL1RN15 ,16 and TLR117 have been associated with pouch inflammation. Additional studies have tentatively linked NOD2 variants with chronic pouchitis.18–20 The aim of this study was to evaluate whether inflammatory bowel disease (IBD) susceptibility polymorphisms which have been previously implicated in CD, UC or pouchitis are associated with chronic pouch inflammatory outcomes after surgery for UC in a large IPAA cohort.

    Methods

    Study population

    All study protocols were carried out in accordance with the research ethics boards at each centre and informed consent was obtained from all patients before their enrolment. Patients who had undergone colectomy with IPAA at Mount Sinai Hospital (MSH), Cleveland Clinic and the Penn State Milton S Hershey Medical Center (HMC) were contacted by research staff between 2007 and 2010. The demographic and clinical features of subjects from MSH have been previously reported and described in detail,21 and the NOD2 genotype status has been previously reported for the HMC cohort.20 Any patients with a confirmed precolectomy diagnosis of UC and who had had their ileostomy closed a minimum of 1 year before study enrolment were included in the study. UC diagnosis was confirmed based on clinical, endoscopic and pathological evidence—particularly from the colectomy specimen. Those who had undergone their procedure outside any of these institutions were included only if the medical documentation was available. Patients with CD, IBD of the colon-type unclassified or indeterminate colitis based on precolectomy medical chart review or colectomy surgical pathology were excluded.

    Clinical data collection

    Study data were obtained through a detailed retrospective chart review and patient questionnaire with investigators blinded to patients' genetic results. To ensure uniformity between sites, rigorous definitions were agreed upon and applied to classify patients into outcome groups. Data collected directly by patient interview included gender, age, family history of IBD, smoking status and clinical symptoms of pouch function after surgery (daily number of bowel movements, incontinence, presence or absence of blood in stool). UC diagnosis date, surgical history and dates, presence of any extraintestinal manifestations of IBD such as arthritis or arthropathy, osteoporosis/osteopenia, erythema nodosum, pyoderma gangrenosum, primary sclerosing cholangitis (PSC) or ocular inflammation, as well as the post-surgical outcome were confirmed through medical chart review. The extent of precolectomy disease was classified based on the Montreal classification.22 The presence of backwash ileitis (inflammation of the terminal ileum in individuals with pan-colitis) precolectomy was documented and patients were included only if all other findings were consistent with UC.

    Post-surgical classification

    Patients were classified into one of three outcome groups based on clinical, endoscopic and histological factors. The ‘no chronic pouchitis’ (NCP) group included individuals without an episode of pouchitis, and those who had had fewer than four clinical acute pouchitis episodes per year, each responding to 2 weeks or less of antibiotics (ciprofloxacin, metronidazole, or combination). This clinical definition of acute pouchitis has been previously described.23 Typically, these subjects may not have had a full clinical assessment but clearly represent a distinct category as demonstrated by their immediate response to antibiotics. The ‘chronic pouchitis’ (CP) group included antibiotic-dependent and antibiotic-refractory patients who required prolonged (>1 month) antibiotic treatment, or medical intervention for pouchitis more than three times a year, or the use of second- or third-line drugs (5-acetylsalicylic acid, steroids, immunomodulators, biological agents). All such subjects had endoscopic assessment at some point during their disease.23 The final group was the CDL phenotype. Individuals classified into this group met at least one of the following criteria: (a) development of a perianal fistula more than 1 year after ileostomy closure documented through physical examination, examination under anaesthesia or imaging; (b) development of a stricture proximal to the pouch which was not related to a surgical complication and was confirmed by endoscopy or small-bowel imaging; (c) evidence of inflammation (ulceration, erythema, friability) extending above the pouch inlet and into the afferent limb/pre-pouch ileum or more proximal small intestine detected on pouchoscopy or upper endoscopy. Anastomotic ulceration or ulceration around the pouch inlet alone was not sufficient to classify patients into the CDL outcome group. Additional post-surgical outcomes, including mechanical or surgical complications or cuffitis, were also documented.23 Time to diagnosis of pouchitis was defined as the time from ileostomy closure to the time of onset of symptoms and diagnosis.

    DNA collection and genotyping

    Whole blood (3–6 ml) was obtained by venepuncture using standard EDTA collection tubes at both MSH and HMC. DNA was extracted using the QIAGEN Gentra Puregene blood kit (Qiagen, CA, USA). DNA samples from the Cleveland Clinic were extracted from clotted blood using the Maxwell 16 Tissue DNA purification kit (Promega, WI, USA), according to manufacturer's protocol. DNA from all study sites was then stored in sealed Matrix screw top tubes at 4°C before a spectrophotometric quality and quantity check using the NanoDrop 1000 (Thermo Scientific, IL, USA). Most DNA samples had concentrations ranging from 100 to 300 ng/μl with all concentrations >20 ng/μl. Those falling below 18 ng/μl were whole-genome amplified using the Illustra Genomiphi HY DNA Amplification Kit (GE Healthcare, NJ, USA). Single nucleotide polymorphism (SNP) genotyping was performed using the Illumina Goldengate custom SNP assay on the Illumina BeadStation500G (San Diego, California, USA) at The Center for Applied Genomics (TCAG, Toronto, Canada) and the Sequenom iPLEX platform (Génome Québec, Montreal, Canada). NOD2insC genotyping was performed using the TaqMan SNP genotyping platform (TCAG).

    Statistical analysis and quality control

    Descriptive statistics were reported as mean and range for continuous variables and frequencies and proportions for categorical variables. For phenotypic results, Fisher’s exact test and Pearson's χ2 test were used to compare proportions; non-parametric tests were used to compare the continuous variables. PLINK version 1.0624 was used to obtain descriptive statistics of the SNPs such as the allele frequency, genotype distribution and to test for Hardy–Weinberg equilibrium (HWE) for each marker based on Pearson's χ2 test. All SNPs which were successfully genotyped in at least 95% of the study cohort, satisfied HWE criteria (p HWE >0.001) and had a minor allele frequency >0.01, were included in the analysis. To ensure that results were not due to population stratification, only Caucasian individuals were included in the genetic analysis. Logistic regression models were applied for the association analysis. Although an additive genetic model was used for the primary analysis,25 we also explored dominant and recessive genetic models. Throughout this report the p values are those obtained from the additive genetic model unless otherwise stated. ORs and 95% CIs were calculated. Two-sided statistical tests were applied and all analyses were performed with SAS V.9.2 (SAS Institute).

    A list of 646 SNPs was generated and chosen for the analysis in the initial cohort based on their previous known associations with CD, UC or pouchitis. To accommodate the Sequenom platform, the top 46 hits from the initial analysis as well as 20 previously described important CD/UC/pouchitis-associated SNPs were selected for genotyping in the replication cohort (online supplementary table 1). Owing to the large number of tests performed, nominal significance was defined in both the preliminary analysis and the combined cohort as p<0.01. Stringent Bonferroni correction was applied to the final p value to adjust for multiple comparisons with p<7.5×10−4 required to declare significance.

    Finally, multivariate analysis was performed with factors previously shown by our group and others to be associated with pouch outcome. These included the serological markers anti-Saccharomyces cerevisiae antibody (ASCA) IgG, perinuclear antineutrophil cytoplasmic antibody (pANCA) and anti-CBir121; clinical factors included smoking and family history of IBD; and the SNPs which were significantly associated with outcome in our Caucasian cohort. This analysis was performed in a subset of 341 patients in whom all of the preceding data was available. A stepwise procedure was performed to generate a risk score based on the significant risk factors from the multivariate analysis. The risk score was based on the weighted combination of the risk factors with the standardised logistic regression coefficient as the weight.26 Receiver operating characteristic curves were generated to calculate the area under the curve and the sensitivity and specificity of the risk score.26

    Results

    Study cohort

    The initial cohort consisted of 399 patients recruited from MSH and the second included 467 patients recruited from MSH, Cleveland Clinic and HMC, for a total cohort of 866. Within both the first and second cohorts, 339 and 401, respectively had both genotype and phenotype information available for a total genetic cohort of 740. Among these individuals 714 (96.5%) were Caucasian and only these subjects were subsequently analysed.

    Clinical variables associated with pouch outcome

    Clinical and phenotypic characteristics of the study population are shown in table 1. A description of the pouch outcome groups among the combined Caucasian cohort is shown in figure 1. Of the 714 individuals, 487 (68.2%) were classified as NCP, 118 (16.5%) CP and 109 (15.3%) CDL. The mean age at UC diagnosis (29, range 2–59 years) and at IPAA (37, range 7–61 years) was the same between groups. Within the CDL group, 56.6% were diagnosed based on the presence of a fistula or abscess developing more than 1 year after surgery, 36.4% based on inflammation extending into the afferent limb and 7.0% met both criteria. Factors previously related to pouch outcome such as smoking and family history of IBD10 ,12 were not found to be significantly associated in this cohort. However, the presence of large-joint arthritis (p=0.02) and PSC (p=0.009) were significantly associated with both the CP and CDL outcomes. A longer duration of time from ileostomy closure to study enrolment was associated with a worse pouch outcome (p=0.001). The mean time to diagnosis of CP and CDL was 2.4 and 4.4 years, respectively, well below the mean post-surgical follow-up time for any of the outcome groups.

    View this table:
    Table 1

    Clinical and phenotypic characteristics of the study population

    Figure 1
    Figure 1

    Proportion of individuals in each specific outcome group.

    Association of SNPs with pouch outcome

    Of the 646 SNPs selected for analysis in the initial cohort of subjects (n=369), 66 SNPs which met initial criteria for significance or were SNPs of importance in prior IBD genetic studies were genotyped in the additional cohort (n=345). Of these, 12 had a low call rate and were not included in the analysis. Of the remaining 54 SNPs, five were significantly associated with outcome at a p value threshold of p<0.01 in a three-way comparison. However, only the NOD2 insertion variant (NOD2insC, rs2066847) remained significantly associated with pouch outcome after stringent Bonferroni correction (p=7.4×10−5; pcorr=4.9×10−3) (table 2). Specifically, the NOD2insC variant was detected significantly more frequently among individuals with CP and CDL than among those with NCP, with an OR of 3.21 (CI=1.38 to 7.47) and 4.30 (CI=1.90 to 9.77), respectively (table 3). This association remained significant even when the previously reported samples obtained from HMC were excluded.

    View this table:
    Table 2

    Single nucleotide polymorphisms (SNPs) associated with outcome at a nominal significance threshold of p<0.01

    View this table:
    Table 3

    ORs and CIs for the five single nucleotide polymorphisms (SNPs) which are associated with outcome

    Given the low minor allele frequency of the NOD2insC allele in our cohort, to fully assess the risk associated with variants in NOD2 and pouch-related inflammatory complications, we examined the risk associated with compound heterozygosity or homozygosity for combinations of markers (rs2066847, rs2066845 and rs2066844). We found that increased numbers of variants at this locus were associated with increased risk of CDL in comparison with NCP, (p=0.002; OR=2.08; CI=1.31 to 3.50); however, no increased risk was seen when NCP was compared with CP.

    Additional SNPs associated with outcome in the three-way analysis (p<0.01) were variants in NOX3 (rs6557421) and DAGLB (rs836518); however, these did not remain significant after correction for multiple testing (table 2). In the pairwise analysis, rs6557421 showed a trend towards being protective for both of the inflammatory outcomes, and rs836518 was protective against only the CDL phenotype (table 3). Additional variants in NOX3 (rs12661812) and NCF4 (rs8137602) were found to be associated with outcome (p<0.01) using the dominant genetic model, with variants at both loci more common in individuals with inflammatory pouch phenotypes. However, these polymorphisms did not remain significantly associated with outcome after correction for multiple testing.

    We next conducted analyses in which we compared the NCP group with the combined CP/CDL cohort (ncomb=227). We found that both rs2066847 (NOD2insC) and rs6557421 (NOX3) were associated with outcome with the minor allele of rs2066847 (p=3.3×10−4) increasing disease risk and that of rs6557421 (p=2.8×10−4) protective against inflammatory phenotypes (table 3).

    Variants which had been previously associated with pouch complications, including those in TLR1,17 CD14, TLR914 and other NOD2 variants, were not found to be associated with outcome among our cohort (table 4). IL1RN16 showed a trend towards significance in our initial analysis, but did not remain significant after correction for multiple testing. Additionally, many of the other well known IBD-associated genetic polymorphisms including those specific to CD (ie, ATG16L1) and those associated with both CD and UC (ie, IL23R), were not associated with any of the outcome groups.

    View this table:
    Table 4

    Single nucleotide polymorphisms (SNPs) previously associated with pouch outcome. IL1RN did not meet specified quality criteria for combined analysis (successfully genotyped in <95% of the cohort)

    Multivariate analysis and risk score

    In the preliminary multivariate analysis, five factors were found to be independently associated with outcome and were included in the subsequent multivariate analysis, including rs2066847 (NOD2insC), rs6557421 (NOX3), rs836518 (DAGLB), anti-CBir1 and smoking (table 5). These factors were used to generate a weighted risk score, with the comparison of both NCP with CDL (p=4.11×10−8; OR=3.22, CI=2.12 to 4.89) and NCP with combined CP and CDL (p=8.89×10−7; OR=2.72; CI=1.89 to 3.91) resulting in highly significant associations. The sensitivity and specificity were assessed by generating receiver operating characteristic curves. The best model was that comparing CDL with NCP and containing all three genetic markers, as well as the serological and clinical factors (sensitivity = 80.0%; specificity = 70.3%). This model performed significantly better than those generated using smoking alone, or smoking and anti-CBir1 (figure 2).

    View this table:
    Table 5

    Factors significantly associated with outcome by multivariate analysis

    Figure 2
    Figure 2

    Receceiver operating characteristic curves generated for the risk score analysis. NOD2insC—rs2066847; NOX3—rs6557421; DAGLB—rs836518. AUC, area under the curve.

    Discussion

    Our results demonstrate that phenotypic characteristics, including PSC and arthritis, as well as several SNPs are associated with pouch outcome after IPAA in individuals with UC. PSC has previously been associated with chronic pouchitis,27 as have general autoimmune disorders including arthritis.28 The co-occurrence of additional inflammatory disorders previously documented in CD and UC, such as those described above, with pouch complications suggest overlapping disease mechanisms may be important in the onset and propagation of inflammatory outcomes. Interestingly, we did not observe any association between a family history of IBD or smoking and pouch outcome.11 ,21 The sample size of this study, which was approximately double that of previous studies, provided us with increased power to detect true associations. Our data describing no correlation between IBD family history or smoking and outcome suggest that these previously described associations may have been the result of a type I error.

    We have shown that several genetic polymorphisms are associated with pouch inflammation, confirming that host genetic factors are critical in the aetiology of both pouchitis and the CDL phenotype. The well established NOD2insC polymorphism, which has previously been implicated in ileal CD29 ,30 and which results in a truncation of the leucine-rich repeat region of the NOD2 protein,19 is associated with CP and a CDL outcome in our large cohort. While the precise effect of this polymorphism is unknown, gene knockout studies have demonstrated that loss of NOD2 is associated with reduced ability to detect microbial pathogens.31 ,32 Further, NOD2 variants have been implicated in intestinal allograft rejection and graft-versus-host disease after allogeneic stem cell transplantation.33 ,34 Our findings demonstrating the association between NOD2 and pouch complications, confirm results seen in several smaller cohorts,20 ,35 and suggest that studies failing to detect significant associations were likely underpowered.17 This growing body of evidence demonstrates that pathways which are important in ileal CD may also be critical in inflammation of the ileal pouch after surgery.

    It is interesting to note that the NOD2insC variant is typically only rarely found in UC. In the MSH UC patient population, for example, the NOD2insC allele frequency is 0.014 which is comparable to the reported allele frequency in this study for those without inflammatory pouch complications.36 However, in the 227 subjects with CP or CDL, the NOD2insC allele frequency is >0.05. These patients were all carefully chart reviewed to exclude any clinical or pathological evidence of CD or IBD of the colon-type unclassified before colectomy. Additionally, the colectomy pathology was all carefully reviewed and no features of CD were seen in these subjects. These data therefore suggest that the utility of traditional clinical and histological diagnostic classification and phenotyping is not sufficient to categorise subjects for prognosis after pelvic pouch surgery. Thus, a diagnosis of clinical UC may not imply that pouch complications are less likely to arise but rather that the subject's genotype may be a more important determinant of outcome.

    Other variants which demonstrated modest association with outcome include those which are located in the non-coding regions of NOX3 and DAGLB or adjacent to NCF4. NCF4, encoding p40phox which is a component of the NADPH oxidase complex, was recently identified in a genome-wide association study as an ileal CD susceptibility gene.37 Mechanistic studies have demonstrated that patients with CD who are carriers of risk alleles at this locus have significantly reduced amounts of reactive oxygen species—important for host innate immunity and defence from microbial pathogens—generated from granulocyte-macrophage colony-stimulating factor-primed neutrophils compared with patients not carrying these mutations.38 NOX3, another component of this complex in various tissue types, is also important in the production of reactive oxygen species.39 ,40 Together, these findings suggest an important role for this pathway in the pathogenesis of pouch inflammatory outcomes and IBD, in general. The function of DAGLB and a potential role for it in IBD pathogenesis remains unclear; however, the SNP in this gene is in a region of high linkage disequilibrium with RAC1—a gene involved in host immune defence and which has been previously associated with UC.41

    Other variants which have been previously associated with pouch outcome among smaller cohorts were not associated with outcome in our group. A possible explanation for the difference between our results and others is that this study has a much larger sample size, which would help to reduce type I error. Additionally, population stratification may account for different results as some previous studies did not control for ethnicity.17 ,42 To limit these effects, only Caucasian individuals were analysed in our study. We also applied very rigorous statistical correction to our data in order to reduce the likelihood of reporting false positive results, which may have been too stringent to allow detection of weaker associations. The four variants discussed in this paper which did not remain significant after multiple testing correction, may therefore warrant further investigation in an additional cohort to definitively assess their importance in pouchitis pathogenesis. This would ideally be attempted in the setting of the large International IBD Genetics Consortium Cohort but would require detailed phenotyping of precolectomy data as well as of pouch outcomes, both of which may not be readily available in existing databases.

    We hypothesise that patients with NOD2insC may be more likely to develop inflammatory pouch complications owing to alteration in the microbial composition of the pouch mucosa. This hypothesis is supported by our data showing that patients with UC with a CDL phenotype after pelvic pouch surgery are more likely to be positive for ASCA and anti-CBir1,21 serological markers more typically associated with CD, and by the data from other groups demonstrating a strong association between the common UC-associated serum marker, pANCA and pouch outcome.43 ,44 Additional evidence that NOD2 variants affect the composition of the ileal flora suggests that there may be a relationship between IBD-associated genetic polymorphisms and the presence of serum antibodies.32 ,45 Our findings of the presence of both serological markers of CD and genetic polymorphisms which have been previously associated with ileal CD showing association with pouch outcome, suggest that the mechanism of ileal inflammation which leads to a CP or CDL outcome in the pouch may be similar to those which lead to the development of inflammation within the ileum. Additionally, the inclusion of individuals with non-chronic pouchitis in the NCP group and the lack of evidence for any associations between genotype and this outcome, suggests that the chronic inflammatory phenotypes proceed via diverging mechanistic pathways from those which lead to antibiotic-responsive pouchitis. Furthermore, the overlap in genetic susceptibility between CP and CDL suggests that distinction between these outcomes may be of limited consequence compared with the more clinically relevant phenotype of chronic, medication-refractory inflammation which is characteristic of both disorders and results from a common aetiology.

    While it is conceivable that these patients with CDL phenotype were misclassified before colectomy, the stringent phenotypic classification which was used to determine which patients were included in this analysis would suggest that this is unlikely to be the case. Rather, our results combined with recent data published by Waterman et al showing few differences in the prevalence of several IBD-associated SNPs between those with UC and those with CD,36 support the concept of IBD as a mosaic of inflammatory disorders. The varying phenotypes associated with overlapping genetic susceptibility loci suggest that while genetic predisposition may be a key inflammatory mediator, other factors such as bacteria or as yet unknown environmental stimuli are necessary for immune dysregulation and inflammation to occur.

    The aetiology of pouch complications remains unknown. However, the data presented in this paper emphasise the similarities between pouch inflammation and IBD in general and suggest that the pelvic pouch model may be useful for evaluating factors which contribute to de novo inflammation. It will be important to evaluate the impact of the pouch microbiome and the impact of host genotype on the composition of the microbiome to fully understand the mechanisms of pouch inflammation. Our data also demonstrate that a model for assessing risk of pouch complications which encompasses genetic and serological factors may be a more useful tool than current clinical assessment.

    Acknowledgments

    The authors thank Diane Verbeeten and Lucy T Zhang for their assistance with the study and Harden Huang and Brenda O'Connor for maintaining and providing access to the Mount Sinai Hospital Pelvic Pouch Database.

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    Supplementary materials

    • Supplementary Data

      This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

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    Footnotes

    • Funding This study was supported by a grant from the Crohn's and Colitis Foundation of Canada. MSS is partially supported by the Gale and Graham Wright Research Chair in Digestive Disease. Partial funding for genotyping was supported by a CIHR operating grant (MOP97756) to AMM and JHB.

    • Competing interests MSS receives speaker fees, consulting fees and research support from Prometheus Laboratories.

    • Ethics approval Research ethics boards of Mount Sinai Hospital, Cleveland Clinic and Milton S Hershey Medical Center.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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