The causes of the common forms of idiopathic inflammatory

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1 GASTROENTEROLOGY 2001;121: The Interleukin 1 Receptor Antagonist Gene Allele 2 as a Predictor of Pouchitis Following Colectomy and IPAA in Ulcerative Colitis MARTYN J. CARTER,*, FRANCESCO S. DI GIOVINE, ANGELA COX, PETER GOODFELLOW, SIMON JONES,*, ANDREW J. SHORTHOUSE, GORDON W. DUFF, and ALAN J. LOBO*, *The Gastroenterology and Liver Unit; Division of Molecular and Genetic Medicine, University of Sheffield; and Department of Surgery, Royal Hallamshire Hospital, Sheffield, England Background & Aims: The interleukin 1 receptor antagonist gene allele 2 has been suggested as a determinant of both disease susceptibility and extent in ulcerative colitis. The aim of this study was to assess the allele as a predictor of both the indication for colectomy and the occurrence of pouchitis after ileal pouch anal anastomosis formation. Methods: Genotyping for the 2018 single nucleotide polymorphism in the interleukin 1 receptor antagonist gene was performed in 109 patients who had undergone colectomy, including 82 patients who had been followed prospectively after ileal pouch anal anastomosis formation. Results: Patients with pouchitis had a higher allele 2 carriage rate compared with those without pouchitis (72% vs. 45%) and Kaplan Meier survival analysis showed that allele 2 carriers had a significantly increased incidence of pouchitis compared with noncarriers (log-rank test, 6.5). After adjustment for confounding covariates in a Cox proportional hazards model, the relative hazard was 3.1 (95% confidence interval [CI], ; P 0.02). Although there was a higher allele 2 carriage rate in patients with chronic refractory compared with acute severe ulcerative colitis (63% vs. 48%), this difference was not significant (odds ratio, 1.9; 95% CI, ; P 0.1). Conclusions: The interleukin 1 receptor antagonist gene allele 2 predicts pouchitis after ileal pouch-anal anastomosis in ulcerative colitis. The causes of the common forms of idiopathic inflammatory bowel disease (IBD), ulcerative colitis (UC), and Crohn s disease (CD) remain unknown. Recent epidemiologic data, including family studies, 1 twin concordance studies, 2,3 and investigations in different ethnic populations, 4 suggests that UC and CD represent related, complex genetic diseases. Evidence from associations with genetic syndromes and diseases such as autoimmune disorders, which have a known genetic susceptibility, further support this concept. 5,6 More recently, genomewide linkage scans have identified a number of potential susceptibility loci, suggesting that several genes are associated with IBD Some of these loci are common to both CD and UC and others unique to either disease. Such genetic complexity is postulated to arise from epistasis (gene-gene interactions) and from gene-environment interactions. 11 This data also suggests that UC and CD are each unlikely to represent a single disease because different sets of genes may result in similar disease expression in different individuals. Such genetic heterogeneity may explain some of the variability observed in clinical phenotype but also increases the difficulty of identifying disease genes. Targeting select populations or defining uniform reproducible phenotypic classifications of disease may enable identification of genes that determine disease susceptibility and behavior by analysis of more homogeneous sets of patients. Patients who undergo colectomy for UC represent a subgroup with a severe disease phenotype in which the disease and extent of inflammation can be definitively confirmed by histologic examination of the resected colon. Pouchitis is the most frequent long-term complication of ileal pouch anal anastomosis (IPAA), occurring in approximately one third of patients. 12 Although the causes remain unknown, this complication is very unusual in patients who have IPAA for familial adenomatous polyposis, 13 suggesting that the genetic or immunologic susceptibility to UC is of paramount importance. 14 Cytokines have been implicated in the pathogenesis of many chronic inflammatory diseases and have a substantial regulatory and effector role in the mucosal immune and inflammatory response in IBD. 15 Interleukin (IL)-1 alpha and beta are major proinflammatory cytokines Abbreviations used in this paper: CI, confidence interval; IL, interleukin; IL-1ra, interleukin 1 receptor antagonist; IPAA, ileal pouch anal anastomosis; PCR, polymerase chain reaction; VNTR, variable number of tandem repeats by the American Gastroenterological Association /01/$35.00 doi: /gast

2 806 CARTER ET AL. GASTROENTEROLOGY Vol. 121, No. 4 involved early in the inflammatory cascade. The interleukin 1 receptor antagonist (IL-1ra) is the natural inhibitor of these IL-1 agonists and acts by competitively binding to IL-1 receptors without eliciting signal transduction. 16 All 3 proteins are coded by genes in the IL-1 gene cluster on the long arm of chromosome Biological observations in both animal models of colitis 18 and patients with IBD suggest that an imbalance in the biologically important IL-1ra/IL-1 ratio may contribute to the chronic inflammatory response in UC. 19 Such evidence suggests that the IL-1 gene locus is an appropriate candidate region to study for determinants of both genetic susceptibility and disease phenotype in UC. The IL-1 gene cluster contains several genetic markers including a variable number of tandem repeats (VNTR) polymorphism in the IL-1ra gene (IL-1RN). 20,21 Allele 2 of this VNTR polymorphism (IL-1RN*2) was first reported to be associated with UC in Although not all subsequent studies in North European patients have confirmed the association, all these series lacked power to exclude a negative result. We have recently confirmed the genetic link in a large independent cohort and by meta-analysis of reported series in this ethnic population. 29 Investigations in Jewish and Hispanic populations have shown a stronger genetic link with this polymorphism, suggesting that the allele may have a greater contribution in particular ethnic groups, perhaps associated with specific environmental exposure, or that it reflects differing patterns of linkage disequilibrium. 30 The aims of this study were to determine whether the IL-1RN*2 is a predictor of clinical phenotype in UC patients requiring surgery, and whether the allele is a marker for the subsequent development of pouchitis. Materials and Methods Patients One hundred nine patients who had undergone colectomy for UC between 1988 and 1997 were studied, representing over 70% of individuals with UC who had undergone such surgery at our hospital during that time period. Diagnosis and extent of disease were confirmed by histologic assessment of the colectomy specimen. Patients were included only if histologic assessment was suggestive, highly suggestive, or diagnostic of UC according to the British Society of Gastroenterology guidelines. 31 Individuals with appearances of indeterminate chronic IBD were excluded. The histologic limit of inflammation was used to define disease extent, which was classified as either extensive or nonextensive depending on whether the inflammation extended beyond the splenic flexure. The indication for surgery was classified as either acute severe disease or chronic disease. Acute severe disease encompassed patients undergoing colectomy during in-patient treatment for severely active disease that failed to respond to medical therapy. Chronic disease encompassed patients undergoing elective colectomy for frequently relapsing, chronic active, or steroiddependant disease and those operated on for dysplasia or colorectal carcinoma. All patients were unrelated and of Northern European Caucasian ethnicity, and Jewish, Southern European, African, and Asian patients were not included in the study. All clinical details were collected prospectively at the time of surgery on a dedicated database. Data regarding sex, extent of colitis, and family history of UC and IBD, age at diagnosis, and surgery, time to surgery, indication for colectomy, and whether or not IPAA was undertaken are shown in Table 1. Eighty-two (75%) patients had undergone IPAA, all performed by the same consultant colorectal surgeon (A.J.S.). Prospectively collected data on pouch function and the occurrence of pouchitis were kept on these patients. Pouchitis was defined as the combination of characteristic symptoms of increased bowel frequency along with both evidence of endoscopic signs of inflammation at pouchoscopy and histologic acute inflammation in small bowel mucosa according to the St. Table 1. Demographic Characteristics of Patients With UC Undergoing Colectomy Total no. 109 Sex (M/F) 58/51 Extent Extensive 90 (83%) Nonextensive 19 (17%) FH of IBD 20 (18%) FH of UC 17 (16%) Age at diagnosis (yr) Range 4 72 Median 29 Age at surgery (yr) Range 7 82 Median 40 Time to surgery (mo) Range Median 61 Indication for colectomy Acute severe disease 46 (42.2%) Chronic refractory disease 63 (57.8%) IPAA 82 (75%) Pouchitis 29 (35%) NOTE. Acute severe disease encompassed patients undergoing colectomy during in-patient treatment for severely active disease that failed to respond to medical therapy. Chronic disease encompassed patients undergoing elective colectomy for frequently relapsing, chronic active, or steroid-dependent disease and those operated on for dysplasia or colorectal carcinoma. Extensive UC is defined as disease in which inflammation extends beyond the splenic flexure, whereas nonextensive disease refers to disease in which the inflammation is limited to the left colon and includes left-sided colitis, proctosigmoiditis, and proctitis. A family history was defined as presence of disease in a first- or second-degree relative. Age at diagnosis and at surgery is expressed in years. Time to surgery is expressed in months. Pouchitis was defined as the combination of characteristic symptoms of increased bowel frequency along with both evidence of endoscopic signs of inflammation at pouchoscopy and histologic acute inflammation in small bowel mucosa.

3 October 2001 THE IL 1 RECEPTOR ANTAGONIST AND POUCHITIS 807 Mark s criteria. 32 Median follow-up of IPAA patients was 48 months with a range from 2 to 131 months. This study was approved by the South Sheffield Research Ethics Committee in All patients participated in the study after giving informed, written consent. DNA Extraction and Genotyping Blood was taken into ethlyenediaminetetraacetic acid tubes (2 10 ml) and stored frozen until DNA was extracted by standard methods. 33 DNA was stored at 4 C. All patients were genotyped for the 2018 exon 2 SNP as described previously as a marker of the IL-1RN VNTR polymorphism. 33 The rarer allele of this exonic polymorphism is completely associated with allele 2 of the intronic VNTR polymorphism. 29,34 A 154 base pair fragment encompassing the polymorphic site of the template DNA was amplified using a forward primer 5 -CTA.TCT.GAG.GAA.CAA.CCA. ACT.AGT.AGC-3 adjoining the polymorphic site and a reverse primer 5 -TAG.GAC.ATT.GCA.CCT.AGG.GTT. TGT-3. The forward primer contains a single-base mismatch 2 bases from the 3 end to engineer an Alu I restriction site in allele 1 (AG CTGG). Allele 2 itself completes a restriction site for MspI (AGC CGG). Polymerase chain reaction (PCR) was performed with 1 cycle of 1 minute at 96, 35 cycles of 1 minute at 94, 1 minute at 57, and 2 minutes at 70, followed by 5 minutes at 70. Each PCR mixture was then divided into 2 aliquots and separately digested with AluI or MspI. PCR products were size fractionated by 9% polyacrylamide gel electrophoresis and visualized under UV light after staining with ethidium bromide. The 2 enzymes cut the 2 alleles differently. AluI cuts the 154 base pair fragment to produce 126 and 28 base pair fragments in allele 1, whereas it does not digest allele 2 (154 base pairs). MspI digestion produces 125 and 29 base pair fragments in allele 2 but does not digest allele 1 (154 base pairs). Hence, the 2 reactions (separated side by side on a gel) will give inverted patterns of digestion for homozygous individuals and identical patterns in heterozygotes. Statistical Methods Comparison within subgroups of UC patients undergoing colectomy defined by pouchitis status and indication for colectomy was undertaken for clinical characteristics including sex, extent of disease, family history of IBD, age at diagnosis, age at surgery, time to surgery, and time with IPAA. For categorical data, 2 2 contingency tables and 2 statistics were used, and Mann Whitney U test was used for continuous nonparametric data. The occurrence of pouchitis was compared between carriers and noncarriers of the IL-1RN*2 by the Kaplan Meier survival method 35 using the log-rank test for significance. A Cox proportional hazards model 36 was used to adjust for possible confounding covariates including age at diagnosis, sex, extent of disease, family history of IBD, time to surgery, indication for colectomy, and time with IPAA. Carriage rates of IL-1RN*2 were compared between disease phenotypes defined by indication for colectomy from 2 2 contingency tables using the 2 test for independence and odds ratios with 95% confidence intervals (CIs) were calculated. Because 2 disease phenotypes were studied with relationship to the IL-1RN*2, to correct for multiple comparisons using the Bonferroni correction, a P value should be taken as significant. Results Clinical Characteristics of Patient Subgroups Table 2 shows the clinical characteristics within the patient subgroups according to occurrence of pouchitis and indication for surgery. There was no statistical difference regarding sex, extent of disease, family history of IBD, age at diagnosis, or age at surgery between patients with and without pouchitis, and between those with acute severe and chronic disease. There was also no significant difference between patients with and without pouchitis regarding time from diagnosis to surgery. Patients with acute severe disease, however, had a significantly reduced time to surgery compared with those operated on for chronic disease (P ). Patients with pouchitis had significantly longer follow-up after IPAA formation compared with patients without pouchitis (P ). However, there was no significant difference in follow-up time comparing carriers and noncarriers of the IL-1RN*2. IL-1RN Genotypes Table 3 shows the IL-1RN genotypes for patients with UC undergoing colectomy and subgroups of patients defined by indication for surgery and occurrence of pouchitis. These genotypes gave an IL-1RN*2 carriage rate (proportion of individuals who have at least 1 copy of the allele) of 57% in UC patients that underwent colectomy. Thirty-five percent of the patients who underwent IPAA developed pouchitis. The median interval to pouchitis after formation of the pouch was 18 months with a range from 4 to 109 months. Those patients who developed pouchitis had a higher IL-1RN*2 carriage rate of 72% compared with 45% in those who did not develop pouchitis (Figure 1). The cumulative survival without pouchitis after formation of an IPAA was compared between carriers and noncarriers of the IL-1RN*2 using the Kaplan Meier survival method. This confirmed an increased incidence of pouchitis in carriers of the allele (log-rank test, 6.5) (Figure 2). After adjustment for covariates of sex, extent of disease, family history of IBD, age at diagnosis, time to surgery, indication for colec-

4 808 CARTER ET AL. GASTROENTEROLOGY Vol. 121, No. 4 Table 2. Clinical Characteristics of UC Patient Subgroups According to Occurrence of Pouchitis and Indication for Surgery Pouchitis Indication for colectomy Yes No Acute Chronic Number 29 (35%) 53 (65%) 46 (42%) 63 (58%) Sex Male 20 (42%) 28 (58%) 24 (41%) 34 (59%) Female 9 (26%) 25 (74%) 22 (43%) 29 (57%) Extent Extensive 25 (38%) 40 (62%) 40 (44%) 50 (56%) Nonextensive 4 (24%) 13 (76%) 6 (32%) 13 (68%) Family history IBD 3 (23%) 10 (77%) 9 (45%) 11 (55%) Age at diagnosis Range Median Age at surgery Range Median Time to surgery Range Median IPAA follow-up Range Median NOTE. Acute severe disease encompassed patients undergoing colectomy during in-patient treatment for severely active disease that failed to respond to medical therapy. Chronic disease encompassed patients undergoing elective colectomy for frequently relapsing, chronic active, or steroid-dependent disease and those operated on for dysplasia or colorectal carcinoma. Extensive UC is defined as disease in which inflammation extends beyond the splenic flexure, whereas nonextensive disease refers to disease in which the inflammation is limited to the left colon and includes left-sided colitis, proctosigmoiditis, and proctitis. A family history was defined as presence of disease in a first- or second-degree relative. Age at diagnosis and at surgery is expressed in years. Time to surgery is expressed in months. Pouchitis was defined as the combination of characteristic symptoms of increased bowel frequency along with both evidence of endoscopic signs of inflammation at pouchoscopy and histologic acute inflammation in small bowel mucosa. tomy, and time with IPAA in a Cox proportional hazards model, the relative hazard of pouchitis associated with IL-1RN*2 carriage was 3.1 (95% CI, ; P 0.02). Time with IPAA was also predictive of pouchitis with the relative hazard per month of 1.02 (95% CI, ; P 0.002). There was a higher IL-1RN*2 carriage rate of 63% in patients with chronic disease compared with 48% in patients with acute severe disease, although this did not reach statistical significance (odds ratio, 1.9; 95% CI, ; P 0.1) (Figure 1). Discussion The results of this study provide evidence implicating the IL-1RN*2 as a determinant of genetic susceptibility in the subsequent development of pouchitis in UC patients who have undergone IPAA. This data also Table 3. IL-1RN Genotypes in Patients With UC Undergoing Colectomy and in Phenotypic Patient Subgroups IL-1RN Genotype N 1,1 1,2 2,2 IL1RN*2 Carriage UC undergoing colectomy (43%) 53 (49%) 9 (8%) 62 (57%) Acute severe UC (52%) 20 (43%) 2 (4%) 22 (48%) 0.1 Chronic refractory UC (37%) 33 (52%) 7 (11%) 40 (63%) No pouchitis (55%) 21 (40%) 3 (6%) 24 (45%) Pouchitis 29 8 (28%) 17 (59%) 4 (14%) 21 (72%) NOTE. Acute severe disease encompassed patients undergoing colectomy during in-patient treatment for severely active disease that failed to respond to medical therapy. Chronic disease encompassed patients undergoing elective colectomy for frequently relapsing, chronic active, or steroid-dependent disease and those operated on for dysplasia or colorectal carcinoma. Pouchitis was defined as the combination of characteristic symptoms of increased bowel frequency along with evidence of endoscopic signs of inflammation at pouchoscopy and histologic acute inflammation in small bowel mucosa. P

5 October 2001 THE IL 1 RECEPTOR ANTAGONIST AND POUCHITIS 809 Figure 1. Carriage of IL-1RN*2 in the subgroups of patients with UC requiring colectomy according to indication for surgery (chronic refractory or acute severe disease) and subsequent development of pouchitis (pouchitis or no pouchitis). IL-1RN genotypes were determined by PCR using specific oligonucleotide primers flanking the 2018 single nucleotide polymorphism in exon 2 followed by restriction enzyme digestion with AluI and MspI. Results are reported as the carriage rate, which is the percentage of individuals with at least 1 copy of the IL-1RN*2. Carriage rate of IL-1RN*2 in the subgroup who developed pouchitis (72%) is higher compared with the subgroup who did not develop pouchitis (45%). There was a higher carriage rate in the subgroup of patients with chronic refractory disease (63%) compared with the subgroup with acute severe disease, although this difference was not significant (odds ratio, 1.9; 95% CI, ; P 0.1). suggests that the allele may have a role in determining clinical phenotype with respect to indication for colectomy. The overall incidence of pouchitis in this series of patients was in agreement with that of other large series. 12 The most striking finding in this study was the significant association between carriage of the IL-1RN*2 and the subsequent development of pouchitis. Fortyseven percent of patients who carried the allele developed pouchitis compared with only 22% of those who did not. The survival analysis shows that IL-1RN*2 carriage and duration of time with IPAA were significant independent factors associated with the development of pouchitis. Moreover, the IL-1RN*2 carriage rate in UC patients that had had a colectomy and had not developed pouchitis was similar to that of healthy controls. 29 This suggests that the allele 2 association with UC overall may be accounted for by the particular dysregulation that makes UC patients following IPAA at risk for pouchitis. Thus, specific biologic variances caused by the IL- 1RN*2 seem to be specific for the pouchitis phenotype. The results of this study show how genetic polymorphisms may be used to stratify patients with different clinical phenotypes and may have important implications in further understanding the pathogenic mechanisms of pouchitis. This current data confirms provisional findings in a smaller series of patients from the United Kingdom (IL-1RN*2 carriage rates 68% vs. 50%), although this latter study took no account of duration of follow-up after IPAA. 37 A further study from the United Kingdom did not confirm this association, although full data were not reported 38 and the information on pouchitis was collected retrospectively from analysis of case records and was therefore less rigorous than the prospective data used in this current series. The causes of pouchitis remain unknown. Uncontrolled trials demonstrating a response to antibiotics and the uncommon occurrence of pouchitis in defunctioned pouches suggest bacterial factors are likely to be involved. Stasis and chronic obstruction also seem to be important because inflammation in conventional ileostomies is very rare. Other possible causes include abnormal mucus, depletion of short-chain fatty acids in the pouch lumen, excessive release of inflammatory mediators, immunologic or hormonal imbalances, and pouch ischemia. However, the most striking feature of pouchitis is that it rarely occurs in patients who undergo IPAA formation for other reasons, such as familial adenomatous polyposis, suggesting that the genetic or immunologic susceptibility to UC is critical for its development. 14 Recent observations have suggested that the IL- 1RN*2 is associated with reduced levels of IL-1ra in the colonic mucosa of patients with IBD. 39,40 This may Figure 2. Kaplan Meier survival plot for time without pouchitis from ileal pouch-anal anastomosis comparing carriers and noncarriers of the IL-1RN*2. IL-RN genotypes were determined by PCR using specific oligonucleotide primers flanking the 2018 single nucleotide polymorphism in exon 2 followed by restriction enzyme digestion with AluI and MspI. Carriers of the IL-1RN*2 are significantly more likely to develop pouchitis (log-rank test, 6.5). After adjustment for covariates of sex, extent of disease, family history of IBD, age at diagnosis, time to surgery, indication for colectomy, and time with IPAA in the Cox proportional hazards model, the relative hazard was 3.1 (95% CI, ; P 0.02).

6 810 CARTER ET AL. GASTROENTEROLOGY Vol. 121, No. 4 prevent adequate control of mucosal inflammation and provides a biologically plausible explanation for the genetic association between the allele and the disease. The association of pouchitis with this allele may be caused by an unsuppressed inflammatory response to internal or external stimuli, such as bacterial overgrowth. It is likely that pouchitis develops as a result of a complex interaction between a susceptible patient and a pouch that has undergone functional adaptation to a colon-like morphology in response to fecal stasis. The VNTR polymorphism is in a noncoding region of the gene in intron 2 and the exon 2 polymorphism, with which it is in complete linkage disequilibrium, is a conservative base pair change that does not alter the protein amino acid sequence. It is difficult to envisage how either polymorphism has a direct functional effect in terms of IL-1ra production. It is therefore likely that both polymorphisms are in linkage disequilibrium with a functional polymorphism, as yet undefined. This functional genetic change is perhaps most likely to be either in the gene promoter region or in the 3 untranslated region, affecting gene transcription or post-transcriptional events, respectively. Alternatively, although less likely, is the possibility that the demonstrated association is caused by linkage disequilibrium with a genetic variant in another gene within the gene cluster, because of the presence of linkage disequilibrium. There was a higher carriage rate of the IL-1RN*2 in those patients with chronic refractory disease compared with those who had acute severe disease. However, because of the small numbers of patients, this did not reach statistical significance. Additional, larger studies are required to investigate this further. 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