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CARD8 gene variant is a risk factor for recurrent surgery in patients with Crohn's disease

Digestive and Liver Disease, In Press, Corrected Proof, Available online 29 July 2015, January 1970

Abstract

Background and aims

Post-operative recurrence is frequent in Crohn's disease. Genetic factors associated with post-operative recurrence remain poorly understood. Identification of genetic variants associated with repeat surgery would allow risk stratification of patients who may benefit from early aggressive therapy and/or post-operative prophylactic treatment.

Methods

Crohn's disease patients who had at least one bowel resection were retrospectively identified from the “Nancy IBD cohort”. Covariates and potential interactions were assessed using the Cox proportional hazard model. Kaplan–Meier curves for time to surgical recurrence were developed for 200 genetic variants and analyzed with the log-rank test.

Results

137 patients had at least 1 resection in our cohort: 38 had a surgical recurrence (28%). In multivariate analysis, current smoker status (OR 6.97, 95% CI 1.85–26.22, p = 0.004), post-operative complications after prior surgery (OR 2.72, 95% CI 1.02–7.22, p = 0.044), and Caspase recruitment domain-containing protein 8 (CARD8) homozygosity for the risk allele (OR 7.56, 95% CI 1.13–50.37, p = 0.036) remained significantly and independently associated with surgical recurrence.

Conclusion

Current smoker status was associated with increased risk of surgical recurrence. A novel association between CARD8 and increased risk of surgical recurrence in Crohn's disease was observed. CARD8 could be a new marker for risk stratification and prevention of recurrent surgery.

Keywords: CARD8, Crohn's disease, Surgical recurrence.

1. Introduction

Crohn's disease (CD) is a chronic disabling and progressive condition characterized by a relapsing-remitting disease course leading to bowel resection in approximately 80% of CD patients [1] and [2]. However, surgery is not a cure and surgical recurrence is common in patients with CD. Notably, up to 70% of patients require a second surgery in the 10 years following their initial surgery [3] . Post-operative recurrence is thought to result from a combination between genetic, environmental, bacterial and immune risk factors [4] . Several studies have investigated the risk factors for surgical recurrence in CD. Smoking status, prior intestinal resection, penetrating behaviour, perianal disease and extensive bowel disease (>50 cm) are well-established risk factors [5] .

Advances in the field of genetics have led to the identification of numerous susceptibility genes for inflammatory bowel disease (IBD). The association of NOD2/CARD15 polymorphism with predisposition to CD was first reported in 2001 [6] and [7]. A recent meta-analysis concluded that there are insufficient data to support a role for NOD2/CARD15 in predicting surgical recurrence, but did not exclude a role for it [8] . While Fowler et al. found that variant in the SMAD3-encoding gene was a risk factor for recurrent surgery [9] , the influence of the aforementioned IBD risk alleles on the postsurgical recurrence of CD remains poorly investigated. In their study, Sehgal et al. described that CD patients carrying an “at risk” allele for IRGM (rs4958847) had an overall more severe disease course with a greater number of surgeries in comparison with those who were “wild-type” [10] .

The identification of genetic risk factors associated with the surgical recurrence in CD could allow risk stratification of patients who may benefit from early aggressive therapy and/or post-operative prophylactic treatment and may also identify novel therapeutic targets. We herein report a genetic association study on CD surgical recurrence using a well-defined cohort of 857 CD patients, namely the “Nancy IBD cohort” [4] .

2. Methods

2.1. Study subjects

As previously described, the “Nancy IBD cohort” is a large observational cohort of patients with IBD with detailed clinical information on 1173 patients with IBD (857 CD, 316 ulcerative colitis) [4] . Patients were followed prospectively according to standard of care. Information about the “Nancy IBD cohort” is reported to the Commission Nationale de l’Informatique et des Libertés (no. 1404720), which supervises the implementation of the act regarding data processing, data files, and individual liberties that came into effect on January 6th 1978, and was amended on August 6th 2004 to protect the personal data of individuals. Available information retrospectively collected includes age at diagnosis, gender, disease duration, disease location and behaviour according to the Montreal classification [11] , extra-intestinal manifestations, smoking status, date, type and indication of surgery, and date of recurrence. Smoking status was defined as non-smoker or current smoker at time of surgery. Surgery for early complications of first surgery, for perianal disease, fistulotomy and/or faecal diversion were excluded.

2.2. Genotyping

Genomic DNA was isolated from blood samples taken from the first 280 consecutive adult CD patients from the “Nancy IBD Cohort”. 200 CD-associated single nucleotide polymorphisms (SNPs) from previous studies were analyzed [12] (Supplementary Table S1). In this study, 31 new distinct susceptibility loci for CD were identified and data from three studies were combined (a total of 3230 cases and 4829 controls) and the authors performed replication in 3664 independent cases [12] . All patients signed a consent form for genetic analysis. SNPs were genotyped using the Applied Biosystems (Foster City, CA) SNPlex technology, which is based on the Oligonucleotide Ligation Assay combined with multiplex PCR amplification. Experimental methods were as per the manufacturer's instructions. Genotype assignment was performed by capillary electrophoresis analysis using an ABI 3730xl DNA Analyzer and ABI GeneMapper v3.7 software. Genotyping was considered to be successful if the overall success rate for the SNP was >80% and the Hardy–Weinberg equilibrium did not depart significantly from the expected distribution (p > 0.05 for Chi2-test between expected and actual values).

2.3. Statistical analysis

Our primary outcome measure was repeat CD-related bowel resection. Continuous variables were summarized using means and standard deviations. Polymorphisms were tested for Hardy–Weinberg equilibrium before using the Fisher exact test (or χ2 test) to compare allelic and genotype frequencies. Categorical variables were expressed as proportions. The t test was used to compare continuous variables while the chi-square test (or Fisher's exact test) was used for categorical variables. Cox proportional hazards models were constructed to examine the effect of the various genetic polymorphisms adjusting for potential confounders. Time from first surgery to surgical recurrence (or to end of follow-up if one single surgery) was used as the time variable in the Cox proportional hazards model. Variables reaching a p-value of ≤0.10 in the univariate analysis were entered into the multivariate analysis where a p-value <0.05 indicated independent statistical significance. Kaplan–Meier curves for time to repeat surgery were developed for each genetic variant and compared using the log-rank test.

3. Results

3.1. Study population

One hundred and thirty-seven patients of the “Nancy IBD cohort” required a CD-related bowel resection. Mean age at diagnosis was 23 years (range, 3–82), with a mean disease duration of 5 years (range, 0–32).

One hundred eight patients were males (39%), 84 had ileitis (30%), 57 had colitis (20%), 131 had ileo-colonic disease (47%), 8 had upper gastrointestinal involvement (3%); 103 patients had purely inflammatory disease (37%), 95 had stricturing disease (34%), and 82 had penetrating disease according to the Montreal classification (29%). The mean duration of follow-up was 13.5 years (range 0–34).

Indication for first surgery is shown in Fig. 1 .

gr1

Fig. 1 Indication for first surgery in 280 genotyped Crohn's disease patients.

3.2. Factors associated with surgical recurrence

Of 137 patients requiring an intestinal resection, 38 patients (28%) required a second surgery during follow-up. Clinical characteristics of CD patients according to surgical recurrence are shown in Table 1 .

Table 1 Clinical characteristics of Crohn's disease patients according to surgical recurrence.

  No surgical recurrence Surgical recurrence p-value
  N (%) N (%)  
N 99 38  
Female gender 65 (65.6) 17 (44.7) 0.02
Mean age at diagnosis (years) 23 ± 13 21 ± 9 0.92
Mean disease duration to first surgery (years) 7 ± 7 4 ± 3 0.05
Mean time to second surgery (years) NA 9.2 ± 1.0  
Mean duration of follow-up (years) 14.1 ± 0.8 15.3 ± 0.6 0.11
Location     0.65
 Ileal 53 (53.5) 17 (44.7)  
 Colonic 17 (17.1) 8 (21.1)  
 Ileocolonic 29 (29.2) 13 (34.2)  
 Upper gastrointestinal 5 (0.5) 2 (0.5) 0.96
Behaviour      
 Inflammatory 21 (21.2) 5 (13.1) 0.13
 Stricturing 46 (46.5) 25 (65.8)  
 Penetrating 32 (32.3) 8 (21.1)  
Type of surgery (n, (%))      
 Ileocaecal resection 59 (59.6) 19 (50.0) 0.07
 Small bowel resection 5 (5.0) 7 (18.4)  
 Colonic resection 29 (29.3) 11 (28.9)  
 Small bowell and colic resection 5 (5.0) 0  
 Defunctioning loop stoma 1 (0.1) 1 (2.6)  
Perianal 49 (49.5) 15 (39.5) 0.29
Current smoker 54 (54.5) 34 (89.5) 0.001
Post-operative complications 18 (18.2) 16 (42.1) 0.004

NA, not applicable.

At univariate analysis, variables associated with surgical recurrence were female gender (p = 0.02), preoperative disease duration (p = 0.05), current smoker status (p = 0.001), and postoperative complications after prior surgery (p = 0.004). There was no association between surgical recurrence and other clinical variables such as disease location and behaviour (p = 0.65 and p = 0.13 respectively). Genetic variables associated with surgical recurrence on univariate analysis were Caspase recruitment domain-containing protein 8 (CARD8) (rs2043211) homozygosity for the risk allele (p = 0.0007) and N-acetyltransferase 2 (NAT2) (rs1799930) homozygosity for the risk allele (p = 0.001). No other genetics variants were associated with recurrent surgery ( Table 1 ). These two SNPs were in Hardy–Weinberg equilibrium ( Table 2 ).

Table 2 CARD8 and NAT2 allele frequency distribution.

SNP Wild type allele (homozygote) Heterozygote Risk allele (homozygote) Minor allele frequency p *
CARD8 (rs2043211) 124 (AA) 127 (AT) 20 (TT) 0.31 0.1
NAT2 (rs1799930) 128 (GG) 114 (AG) 18 (AA) 0.28 0.2

* Pearson's chi-squared test for deviation from Hardy–Weinberg equilibrium.

All variables reaching a p-value of ≤0.10 in the univariate analysis were entered into the multivariate analysis. In the multivariate analysis, current smoker status (OR 6.97, 95% CI 1.85–26.22, p = 0.004), post-operative complications after prior surgery (OR 2.72, 95% CI 1.02–7.22, p = 0.04), and CARD8 homozygosity for the risk allele (OR 7.56, 95% CI 1.13–50.37, p = 0.04) remained significantly and independently associated with surgical recurrence ( Table 3 ). Disease duration at first surgery, female gender and NAT2 homozygosity for the risk allele were no longer statistically significant. The Kaplan–Meier survival curves for repeat surgery according to CARD8 risk allele status is depicted on Fig. 2 No other genetic variants were associated with recurrent surgery.

Table 3 Multivariable logistic regression analysis of risk of surgical recurrence.

  Odds ratio 95% Confidence interval p-value
Current smoker status 6.97 1.85–26.22 0.004
Post-operative complications after prior surgery 2.72 1.02–7.22 0.04
CARD8 a homozygosity for the risk allele 7.56 1.13–50.37 0.04

a Caspase recruitment domain-containing protein 8.

gr2

Fig. 2 Survival curve for time to surgical recurrence according to CARD8 genotype.

4. Discussion

Numerous studies have focused on the identification of genetic determinants on CD progression, but very few have focused on the impact of genetic factors on the risk of surgical recurrence [8], [9], [13], [14], [15], [16], and [17]. Our series is the first one that studied the influence of 200 CD-associated SNPs on surgical recurrence in CD, in a well-defined cohort namely the “Nancy IBD cohort” with a mean follow-up of 13.5 years. We reported the association of CARD8 and NAT2 genes with the risk of surgical recurrence in CD. A correlation between genotype and clinical phenotype shall allow us to envisage some substantial improvements in CD management by identifying patients at high risk of surgical recurrence. This may lead to the development of targeted strategies aim at changing the natural course of CD.

The role of genetics factors in surgical recurrence has not been clearly defined. To date, 7 studies have examined the association between genetic variants and surgical recurrence. Six of these series have focused on NOD2/CARD15 variants [14], [15], [16], [17], [18], and [19]. A meta-analysis concluded that there are insufficient data to support a role for NOD2/CARD15 in predicting surgical recurrence, but did not exclude a role for it [8] . In our study population, the predictive value for surgical recurrence of 200 SNPs, including NOD2/CARD15, along with other clinical characteristics was studied. While NOD2/CARD15 variants were not predictor of surgical recurrence in our population, we herein described that homozygosity for the CARD8 and the NAT2 risk alleles were associated with increased risk of recurrent surgery in CD in univariate analysis.

We found that CD patients carrying the CARD8 allele risk (rs2043211) were positively and independently associated with surgical recurrence in CD. The CARD8 gene, which is highly expressed in both monocytes and the gut epithelium, is a potential candidate risk gene for IBD, based on gene location (in the IBD6 locus) and biological function [20], [21], [22], and [23]. CARD8 is a negative regulator of nuclear factor-kappa B (NF-κB) and is a suppressor of apoptosis. Recent studies found that CARD8 interacts with NLRP3-coupled inflammasome and members of the NOD-like receptor family thus implying a role for CARD8 in the regulation of innate and adaptative immunity [20], [21], [22], and [23]. Activation of NLRP3 by a diverse panel of ligands leads to the formation of an inflammasome complex consisting of caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD), which mediates the production and secretion of IL-1β in response to microbial challenge. Recently attention has focused on the inflammasome and its role in maintaining gut homeostasis and mediating host immune defenses against pathogenic bacteria and stress signals. The dysregulation of the inflammasome complex is thought to contribute to chronic inflammation [24] . We could speculate that the high level risk of surgical recurrence may result from the failure of homozygous patients for the CARD8 allele risk to finely tuned NF-κB activation. It may be explained by the presence of a dysfunctional NLRP3 inflammasome caused by the expression of mutated forms of NLRP3 in combination with a CARD8, not being able to act as a suppressor of NF-κB activation.

CARD8 was an independent predictor of the risk of surgical recurrence. Indeed the association was not influenced by other factors known to increase the risk such as smoking. Among them, postoperative complications could have a detrimental influence on the long-term outcome following intestinal resections, leading to increased number of repeat resection surgery. Our study confirms a high rate of repeat resection with 27.7% of patients requiring a second surgery [25] and [26]. In our series, there was a significant difference regarding surgical recurrence rate between smokers and non-smokers (p = 0.001). Smoking is known to be associated with a more aggressive disease course and increased postoperative recurrence [13] . Recently, a meta-analysis described a 2.5-fold increase in the risk of second surgery in smokers compared with non-smokers [27] . Unfortunately, the potential confounding effect of smoking could not be analyzed as only 11.5% of patients were non-smokers (n = 4) among patients with surgical recurrence. Patients with postoperative complications were more likely to require repeat surgical intervention for CD recurrence. Two studies described surgical complications as an independent predictor for surgical recurrence [28] and [29]. In line with our report, the aforementioned studies showed that development of postoperative complications is a risk factor for early recurrence in CD. Postoperative complications may have a detrimental influence on the long-term outcome following intestinal resections, leading to repeated resection surgeries.

Our results need to be confirmed in a replication cohort, which a clear limitation of our study. If this association can be confirmed, it would allow for early risk classification of patients who may benefit from pre- and post-operative aggressive treatment. In our retrospective study it was not possible to account for the effects of all IBD-related medications.

Our study is the first to have studied the influence of 200 CD-associated SNPs on surgical recurrence in CD, in a well-defined cohort namely the “Nancy IBD cohort” and with a mean follow-up of 13.5 years. We evaluated surgical recurrence, which is an objective outcome measure. We had too missing data for evaluating the association between genetic factors and endoscopic recurrence. Also, due to the retrospective nature of the study, there were missing data regarding the use of postoperative medications and this parameter could not be analyzed. It is noteworthy that the patients enrolled in the present study were included in the international IBD genetics consortium [12] ; it is the reason why the entire Nancy IBD cohort could not be genotyped.

In conclusion, an independent association between CARD8 risk alleles and increased risk of surgical recurrence in CD was identified. In addition, current smoker status and post-operative complications were confirmed as two main clinical predictors for surgical recurrence. The identification of CARD8 as a risk allele for surgical recurrence is of particular interest as it may be a new marker for the treatment of CD in the postoperative setting.

Conflict of interest

None declared.

Appendix A. Supplementary data

The following are the supplementary data to this article:

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Footnotes

a Department of Digestive Surgery, University Hospital of Nancy-Brabois, Vandoeuvre-les-Nancy, France

b INSERM U954, Nutrition-Génétique et Exposition aux risques environnementaux, Faculté de Médecine, Université de Lorraine, Vandoeuvre-les-Nancy, France

c Institut Pasteur de Lille, Center for infection and immunity of Lille, Lille, France

d INSERM U1019, Team 7, Equipe FRM, Lille, France

e Department of Gastroenterology, University Hospital of Nancy-Brabois, Vandoeuvre-les-Nancy, France

Corresponding author at: Department of Digestive Surgery, University Hospital of Nancy-Brabois, Vandoeuvre-les-Nancy, France. Tel.: +33 83153120; fax: +33 83153119.