The polymorphism rs3024505 proximal to IL-10 is associated with risk of ulcerative colitis and Crohns disease in a Danish case-control study

  • Vibeke Andersen1Email author,

    Affiliated with

    • Anja Ernst3,

      Affiliated with

      • Jane Christensen2,

        Affiliated with

        • Mette Østergaard1, 8,

          Affiliated with

          • Bent A Jacobsen4,

            Affiliated with

            • Anne Tjønneland2,

              Affiliated with

              • Henrik B Krarup3 and

                Affiliated with

                • Ulla Vogel5, 6, 7

                  Affiliated with

                  BMC Medical Genetics201011:82

                  DOI: 10.1186/1471-2350-11-82

                  Received: 4 October 2009

                  Accepted: 28 May 2010

                  Published: 28 May 2010

                  Abstract

                  Background

                  Crohns disease (CD) and ulcerative colitis (UC) are characterized by a dysregulated inflammatory response to normal constituents of the intestinal flora in the genetically predisposed host. Heme oxygenase-1 (HO-1/HMOX1) is a powerful anti-inflammatory and anti-oxidant enzyme, whereas the pro-inflammatory interleukin 1β (IL-1β/IL1B) and anti-inflammatory interleukin 10 (IL-10/IL10) are key modulators for the initiation and maintenance of inflammation. We investigated whether single nucleotide polymorphisms (SNPs) in the IL-1β, IL-10, and HO-1 genes, together with smoking, were associated with risk of CD and UC.

                  Methods

                  Allele frequencies of the IL-1β T-31C (rs1143627), and IL-10 rs3024505, G-1082A (rs1800896), C-819T (rs1800871), and C-592A (rs1800872) and HO-1 A-413T (rs2071746) SNPs were assessed using a case-control design in a Danish cohort of 336 CD and 498 UC patients and 779 healthy controls. Odds ratio (OR) and 95% confidence interval (95% CI) were estimated by logistic regression models.

                  Results

                  Carriers of rs3024505, a marker polymorphism flanking the IL-10 gene, were at increased risk of CD (OR = 1.40, 95% CI: 1.06-1.85, P = 0.02) and UC (OR = 1.43, 95% CI: 1.12-1.82, P = 0.004) and, furthermore, with risk of a diagnosis of CD and UC at young age (OR = 1.47, 95% CI: 1.10-1.96) and OR = 1.35, 95% CI: 1.04-1.76), respectively). No association was found between the IL-1β, IL-10 G-1082A, C-819T, C-592A, and HO-1 gene polymorphisms and CD or UC. No consistent interactions between smoking status and CD or UC genotypes were demonstrated.

                  Conclusions

                  The rs3024505 marker polymorphism flanking the IL-10 gene was significantly associated with risk of UC and CD, whereas no association was found between IL-1β or HO-1 gene polymorphisms and risk of CD and UC in this Danish study, suggesting that IL-10, but not IL-1β or HO-1, has a role in IBD etiology in this population.

                  Background

                  The chronic inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), are complex diseases caused by an interplay between genetic and environmental factors [1].

                  The recent years have brought much progress regarding the genetics in IBD and the number of confirmed IBD associated loci and genes have risen dramatically [27]. Yet, still, only part of the genetic contribution to disease risk may be explained by the identified genes [8, 9]. Northern European populations, including the Danish, generally have low frequencies of the CD risk-associated variants of CARD15 [3, 10], and it is therefore of interest to search for more genetic determinants in these populations. Less progress has been achieved in the identification of environmental risk factors and gene-environmental interactions. Differences in environmental exposures and genetic heterogeneity between ethnic groups may have complicated the search for genetic and gene-environmental determinants.

                  The emerging picture of IBD pathogenesis is focused on the sequential occurrence of pivotal events leading to the initiation and subsequent perpetuation of inflammation [11, 12]. First, the initial interaction between luminal constituents and intestinal epithelial cells leads to activation of the innate immune system [11]. The recognition of highly conserved pathogen structures such as lipopolysaccharide (LPS), the main constituent of Gram-negative bacteria, by Toll-like receptors and other pattern recognition receptors on the epithelial and other immunologically active cells in the intestine, initiates the release of various cytokines and enzymes, including interleukins (IL) and heme oxygenase-1 [13, 14]. Second, the inflammation will eventually become chronic due to defective regulation of the immune response. Therefore, polymorphisms in genes encoding cytokines and other molecules involved in the innate immune system, may affect the course of the inflammatory cascade and thereby the risk of developing IBD.

                  Activation of the pro-inflammatory IL-1β leads to production of prostaglandin E2 (PGE2) and nitric oxide (NO) via the induction of cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) among others [15]. IL-1β knock-out mice have no spontaneous abnormalities, however, on challenge with LPS, a less pronounced acute phase response is observed, suggesting that IL-1β is required for an adequate immune response [15]. In both CD and UC patients, high levels of IL-1β are found in the intestinal mucosa [16] and stimulation by IL-1β leads to a more pronounced inflammatory response in CD immune cells compared to cells from healthy controls [17]. The variant alleles of two IL-1β promoter polymorphisms, IL-1β T-31C and IL-1β C-511T, have been found to be in almost complete linkage disequilibrium [18], and the haplotypes encompassing the IL-1β T-31C variant conferred higher transcription of IL-1β compared to the wild type haplotype [18]. The role of IL-1β polymorphisms in IBD has been explored in several studies [1924]. These studies did not find any association with IL-1β, however, the studies were rather small.

                  IL-10 is an anti-inflammatory cytokine, which leads to dampening of the activated immune system. IL-10 knock-out mice develop colitis if they are not kept in germ-free environment [25], and the administration of IL-10 ameliorates the inflammation in animal and in vitro models [26]. In patients, an impaired IL-10 production has been fund in severe cases of CD and UC [27, 28]. Recently, a strong association between the marker rs3024505 immediately downstream of the IL-10 gene and adult UC was found in a genome-wide association study [29]. This study also found a modest association between this marker and CD risk [29]. However, no association was found for rs3024493, a linked polymorphism located in an intron in the IL-10 gene, in a case-control study of paediatric onset CD [30]. The IL-10 promoter is polymorphic and genetic variation may account for different levels of cytokine production [31]. The IL-10 promotor polymorphisms G-1082A, C-819T, and C-592A have been most extensively studied. They are in tight linkage disequilibrium [32] and the haplotype encompassing these three polymorphisms is associated with low IL-10 protein production in lymphocytes in vitro [32] and low of levels of circulating IL-10 protein in Kenyan children [33] probably because the A allele of the IL-10 promoter polymorphism C-592A leads to the formation of a binding site for the ETS family of transcription factors [34]. Studies on the IL-10 promoter polymorphisms and IBD susceptibility have been inconsistent [22, 2931, 3540].

                  Heme oxygenase-1 (HO-1) is involved in the degradation of heme, thereby reducing oxidative stress and protecting against acute and chronic inflammation [41]. Animal models of IBD have confirmed the anti-inflammatory effect of HO-1 [42]. Hence, blockade of HO-1 activity results in exacerbation of experimental colitis whereas increased HO-1 pathway activity ameliorates experimental murine colitis [4345]. Carbon monoxide (CO) is one of the main metabolite of the HO-1 pathway and CO administration has been shown to ameliorate chronic colitis in IL-10 deficient mice [42, 46]. In UC patients, HO-1 expression and protein levels have been reported to be increased in inflamed colon compared to normal mucosa from patients with UC [47]. Studies using luciferase reporter assays of a functional promoter polymorphism, HO-1 A-413T, indicated that the A allele promoter had significantly higher activity than the T allele promoter [48]. The AA genotype of this polymorphism has been associated with a reduced incidence of ischemic heart disease [48]. Another promoter polymorphism, the HO-1 (GT)N dinucleotide repeat polymorphism, was not associated with risk of inflammatory bowel disease [49]. Interestingly, smoking affects the risk of IBD differentially, increasing the risk of CD and reducing the risk of UC [50]. The mechanisms by which smoking affects risk of IBD is not clear and as tobacco smoke contains more than 3000 chemicals, several different mechanisms may be involved. Nicotine has been reported to modulate the immune balance in a Th1-dominant direction [51] in accordance with the beneficial effect of smoking on UC. Moreover, smoking increases the production of certain pro-inflammatory cytokines, but, on the other hand, smoking is a source of carbon monoxide (CO). Thus, another possible mechanisms may involve interactions between smoking and IL-1β, IL-10 and HO-1 activity in relation to intestinal inflammation [46].

                  In this study we wanted to assess the role of polymorphisms in IL-1β, IL-10, and HO-1 together with smoking in relation to risk of developing IBD in a Danish case-control study of 336 CD, 498 UC and 779 healthy controls, respectively.

                  Methods

                  Patients and controls

                  Patients with CD (n = 373) or UC (n = 541), and healthy controls (n = 796) were included. All information was available for 336 CD cases, 498 UC cases and 779 healthy controls. Diagnosis of CD or UC was based on clinical, radiological, endoscopic and histological examinations [52]. Infectious and other cases of inflammatory bowel diseases were excluded. IBD diagnoses were classified on basis of disease localization according to the Montreal classification; L1, L2, and L3 signifies ileal, colonic, and ileocolonic localisation in CD and E1, E2, and E3 signifies the extent of colon involvement in UC; proctitis (inflammation limited to rectum), left side (inflammation distal to the left flexure), and extensive colon (inflammation proximal to the left flexure) [53]. Patients were recruited from Viborg, Aalborg and Herning Regional Hospitals from January 2004 to March 2005. Healthy blood donors were recruited from Viborg Hospital. Subjects under the age of 18 and with ethnicity other than Caucasian were excluded from the study. Phenotypic data on age at diagnosis of disease, localisation, family disposition for IBD, and medical and surgical treatment of disease was collected. Information on smoking habits at the time of diagnosis (patients) and smoking habits at study entry (donors) was collected.

                  Genotyping

                  DNA was extracted from EDTA-stabilized peripheral blood samples from all patients and healthy controls by using either a PureGene (Gentra Systems, Minneapolis, MN, USA) or Wizard Genomic (Promega, Madison, WI, USA) DNA purification kit, according to the manufacturers' recommendations.

                  SNPs were chosen from literature studies. IL-1β T-31C (rs1143627), and IL-10 C-592T (rs1800872) were genotyped as previously described [54] Rs3024505, IL-10 G-1082A (rs1800896), C-819T (rs1800871) [29] were genotyped using the pre-developed allelic discrimination assays C_15983681 20, C_1747360_10 and C_1747362_10 (Applied Biosystems). The genotyping reaction was performed in a final volume of 6 μl consisting of 3 μl Universal PCR Master Mix, 0.075 μl 40 × assay-on-demand mix, 1.925 μl H2O, and 1 μl genomic DNA. HO-1 A-413T (rs2071746 was determined using the pre-developed allelic discrimination assay C_15869717_10 (Applied Biosystems). Genotyping was performed by TaqMan real-time PCR on an ABI7900HT (Applied Biosystems), using Allelic Discrimination. Twenty ng of DNA was genotyped in 5 μl containing 1 × Mastermix (Applied Biosystems, Nærum, Denmark), 100 nM probes, and 900 nM primers or as recommended by the manufacturer for predesigned assays.. Controls of known genotypes were included in each run, and repeated genotyping of a random 10% subset yielded 100% identical genotypes. Laboratory personnel were blinded to the case/control status of the study group [55].

                  Statistical analyses

                  We used logistic regression to analyse the relationship between the six polymorphisms and disease. The statistical analyses included only subjects where all information was available. Age was entered linear in the model after checking for linearity using a linear spline [56]. Subgroup analyses were done for the polymorphisms in relation to location of the disease (CD: L1, L2, L3, UC: E1, E2, E3), and age at diagnosis (above or not above 40 years of age) for the cases. The GENMOD procedure in SAS release 9.1 (SAS Institute, Inc., Cary, North Carolina, USA) was used for the statistical analyses.

                  Power analyses

                  We used Genetic Power Calculator for Case - control for discrete traits [57] for power analyses. This study has more than 80% power to detect a dominant effect with an OR of 1.5 in either CD or in UC or 1.4 if CD and UC were combined.

                  Ethical Considerations

                  All subjects received written and oral information and gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the local Scientific Ethical Committees at Viborg and Aalborg County (VN 2003/5).

                  Results

                  Characteristics of the Danish IBD patients and controls are shown in Table 1. A total of 834 Danish patients and 779 controls were included. 51% of the CD patients were current smokers at the time of diagnosis, whereas only 17% of the UC patients were current smokers. The genotype distributions among the controls did not deviate from Hardy-Weinberg equilibrium. The variant allele frequencies for IL-1β T-31C, IL-10 rs3024505, G-1082A, C-819T, C-592A and HO-1 A-413T were 0.35, 0.18, 0.45, 0.21, 0.21, and 0.42, respectively, in the control group.
                  Table 1

                  The basic descriptions of the Danish study subjects1.

                   

                  Crohns Disease

                  Ulcerative Colitis

                  Controls

                   

                  (n = 336)

                  (n = 498)

                  (n = 779)

                  Gender : n (%)

                     

                     male

                  131 (39)

                  241 (48)

                  397 (51)

                     female

                  205 (61)

                  257 (52)

                  382 (49)

                  Age :

                     

                     Median (5%-95%)

                  43 (23-77)

                  49 (24-76)

                  43 (23-60)

                  Age at diagnosis :

                     

                     Median (5%-95%)

                  30 (15-65)

                  35 (17-68)

                   

                  Smoking habits : n(%)

                     

                     Smokers

                  170 (51)

                  85 (17)

                  204 (26)

                     Never smokers

                  120 (36)

                  231 (46)

                  391 (50)

                     Former smokers

                  46 (14)

                  182 (37)

                  184 (24)

                  Location UC 2 :

                     

                     Proctitis (E1)

                   

                  211 (42)

                   

                     Left side (E2)

                   

                  183 (37)

                   

                     Extensive (E3)

                   

                  94 (19)

                   

                     Data not available

                   

                  10 (2)

                   

                  Location CD 2 : n (%)

                     

                     Colonic (L2)

                  75 (22)

                    

                     Ileal (L1)

                  155 (46)

                    

                     Ileocolonic (L3)

                  93 (28)

                    

                     Data not available

                  13 (4)

                    

                  Medication : n (%)

                     

                     Advanced3

                  142 (42)

                  104 (21)

                   

                     No advanced medication4

                  189 (56)

                  390 (78)

                   

                     Data not available

                  5 (1)

                  4 (1)

                   

                  Operation : n (%)

                     

                     Yes

                  153 (46)

                  15 (3)

                   

                     No

                  176 (52)

                  473 (95)

                   

                     Data not available

                  7 (2)

                  10 (2)

                   

                  1The analyses included subjects where all information was available,

                  2The location of CD and UC in accordance with the Montreal classification are shown.

                  3azathioprin, 6-mercaptopyrine, Tumor Necrosis Factor-inhibitors, or methrotrexate,

                  45-aminosalicylic acid, prednisolone

                  Associations between polymorphisms and disease

                  Carriers of the variant allele of rs3024505 flanking the IL-10 gene were at increased risk of both CD and UC. Homozygous variant allele carriers were at 2.48-fold (95% CI: 1.27-4.84) increased risk of CD and heterozygous carriers were at 1.31-fold (95% CI: 0.98-1.75) increased risk of CD after adjusting for age, gender and smoking status (Table 2). Homozygous variant allele carriers were at 2.31-fold (95% CI: 1.27-4.20) increased risk of UC and heterozygous carriers were at 1.34-fold (95% CI: 1.04-1.73) increased risk of UC after adjusting for age, gender and smoking status (Table 3). After correction for multiple testing the associations were borderline statistically significant. Minor allele frequencies of IL-10 gene polymorphisms in selected studies are shown in Table 4.
                  Table 2

                  Genotypes in Danish patients with Crohns Disease1.

                   

                  Ncase

                  Ncontrol

                  OR (95%CI)2

                  OR (95%CI)3

                  OR (95%CI)4

                  P-value5

                  IL1 β C-31T (rs1143627)

                           

                     TT

                  165

                  342

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     CT

                  139

                  342

                  0.84

                  (0.64-0.10)

                  0.85

                  (0.65-1.12)

                  0.86

                  (0.65-1.14)

                  0.29

                     CC

                  32

                  95

                  0.70

                  (0.45-1.09)

                  0.71

                  (0.45-1.10)

                  0.69

                  (0.44-1.09)

                  0.11

                     CT and CC

                  171

                  437

                  0.81

                  (0.63-1.05)

                  0.82

                  (0.63-1.07)

                  0.82

                  (0.63-1.07)

                  0.15

                  IL-10 C-592A (rs1800872)

                           

                     CC

                  214

                  483

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     AC

                  114

                  261

                  0.99

                  (0.75-1.29)

                  0.98

                  (0.75-1.30)

                  1.01

                  (0.76-1.34)

                  0.97

                     AA

                  8

                  35

                  0.52

                  (0.24-1.13)

                  0.53

                  (0.24-1.16)

                  0.54

                  (0.24-1.21)

                  0.13

                     AC and AA

                  122

                  296

                  0.93

                  (0.71-1.21)

                  0.93

                  (0.71-1.22)

                  0.95

                  (0.72-1.25)

                  0.72

                  IL-10 C-819T (rs1800871)

                           

                     CC

                  216

                  483

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     CT

                  111

                  259

                  0.96

                  (0.73-1.26)

                  0.95

                  (0.72-1.25)

                  0.97

                  (0.73-1.29)

                  0.83

                     TT

                  9

                  37

                  0.54

                  (0.26-1.15)

                  0.55

                  (0.26-1.18)

                  0.56

                  (0.26-1.22)

                  0.14

                     CT and TT

                  120

                  296

                  0.91

                  (0.69-1.18)

                  0.90

                  (0.69-1.18)

                  0.92

                  (0.70-1.21)

                  0.55

                  IL-10 G-1082A (rs1800896)

                           

                     GG

                  109

                  238

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     AG

                  171

                  374

                  1.00

                  (0.75-1.33)

                  0.99

                  (0.74-1.33)

                  0.97

                  (0.72-1.32)

                  0.87

                     AA

                  56

                  167

                  0.73

                  (0.50-1.07)

                  0.75

                  (0.51-1.10)

                  0.78

                  (0.53-1.16)

                  0.23

                     AG and AA

                  227

                  541

                  0.92

                  (0.70-1.21)

                  0.92

                  (0.69-1.21)

                  0.92

                  (0.69-1.22)

                  0.56

                  (rs3024505)

                           

                     CC

                  203

                  522

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     CT

                  114

                  235

                  1.25

                  (0.95-1.64)

                  1.27

                  (0.96-1.68)

                  1.31

                  (0.98-1.75)

                  0.07

                     TT

                  19

                  22

                  2.22

                  (1.18-4.19)

                  2.60

                  (1.36-4.96)

                  2.48

                  (1.27-4.84)

                  0.01

                     CT and TT

                  133

                  779

                  1.33

                  (1.02-1.73)

                  1.37

                  (1.05-1.80)

                  1.40

                  (1.06-1.85)

                  0.02

                  HO-1 A-413T (rs2071746)

                           

                     AA

                  110

                  267

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     AT

                  165

                  373

                  1.07

                  (0.81-1.43)

                  1.06

                  (0.79-1.42)

                  1.05

                  (0.78-1.42)

                  0.75

                     TT

                  61

                  139

                  1.07

                  (0.73-1.55)

                  1.11

                  (0.76-1.62)

                  1.04

                  (0.70-1.53)

                  0.86

                     AT and TT

                  226

                  512

                  1.07

                  (0.82-1.41)

                  1.08

                  (0.82-1.42)

                  1.05

                  (0.79-1.39)

                  0.75

                  1OR = Odds Ratio, 95%CI = 95% confidence interval,

                  2 Crude,

                  3 Adjusted for age and gender,

                  4 Adjusted for age, gender and smoking status.

                  5P for the fully adjusted estimate

                  Table 3

                  Genotypes in Danish patients with ulcerative colitis1.

                   

                  Ncase

                  Ncontrol

                  OR (95%CI)2

                  OR (95%CI)3

                  OR (95%CI)4

                  P-value5

                  IL1 β C-31T (rs1143627)

                           

                     TT

                  204

                  342

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     CT

                  238

                  342

                  1.17

                  (0.92-1.48)

                  1.15

                  (0.90-1.47)

                  1.15

                  (0.89-1.47)

                  0.28

                     CC

                  56

                  95

                  0.99

                  (0.68-1.43)

                  0.99

                  (0.68-1.46)

                  1.02

                  (0.69-1.49)

                  0.94

                     CT and CC

                  294

                  437

                  1.13

                  (0.90-1.42)

                  1.11

                  (0.88-1.41)

                  1.12

                  (0.88-1.42)

                  0.35

                  IL-10 C-592A (rs1800872)

                           

                     CC

                  328

                  483

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     AC

                  149

                  261

                  0.84

                  (0.66-1.07)

                  0.83

                  (0.65-1.07)

                  0.83

                  (0.64-1.07)

                  0.14

                     AA

                  21

                  35

                  0.88

                  (0.51-1.55)

                  0.99

                  (0.56-1.76)

                  1.00

                  (0.56-1.77)

                  0.99

                     AC and AA

                  170

                  296

                  0.85

                  (0.67-1.07)

                  0.85

                  (0.67-1.08)

                  0.85

                  (0.66-1.08)

                  0.18

                  IL-10 C-819T (rs1800871)

                           

                     CC

                  325

                  483

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     CT

                  151

                  259

                  0.87

                  (0.68-1.11)

                  0.86

                  (0.67-1.11)

                  0.85

                  (0.66-1.10)

                  0.22

                     TT

                  22

                  37

                  0.88

                  (0.51-1.53)

                  1.00

                  (0.57-1.74)

                  1.00

                  (0.57-1.75)

                  0.99

                     CT and TT

                  173

                  296

                  0.87

                  (0.69-1.10)

                  0.88

                  (0.69-1.12)

                  0.87

                  (0.68-1.11)

                  0.27

                  IL-10 G-1082A (rs1800896)

                           

                     GG

                  169

                  238

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     AG

                  239

                  374

                  0.90

                  (0.70-1.16)

                  0.91

                  (0.70-1.19)

                  0.93

                  (0.72-1.22)

                  0.61

                     AA

                  90

                  167

                  0.76

                  (0.55-1.05)

                  0.78

                  (0.56-1.08)

                  0.76

                  (0.54-1.06)

                  0.10

                     AG and AA

                  329

                  541

                  0.86

                  (0.67-1.09)

                  0.87

                  (0.68-1.12)

                  0.88

                  (0.68-1.13)

                  0.31

                  (rs3024505)

                           

                     CC

                  297

                  522

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     CT

                  172

                  235

                  1.29

                  (1.01-1.64)

                  1.35

                  (1.05-1.73)

                  1.34

                  (1.04-1.73)

                  0.02

                     TT

                  29

                  22

                  2.32

                  (1.31-4.11)

                  2.37

                  (1.31-4.29)

                  2.31

                  (1.27-4.20)

                  0.01

                     CT and TT

                  201

                  779

                  1.37

                  (1.09-1.74)

                  1.43

                  (1.13-1.83)

                  1.43

                  (1.12-1.82)

                  0.004

                  HO-1 A-413T (rs2071746)

                           

                     AA

                  162

                  267

                  1.00

                  -

                  1.00

                  -

                  1.00

                  -

                   

                     AT

                  251

                  373

                  1.11

                  (0.86-1.43)

                  1.10

                  (0.85-1.42)

                  1.11

                  (0.85-1.44)

                  0.45

                     TT

                  85

                  139

                  1.01

                  (0.72-1.41)

                  1.00

                  (0.71-1.41)

                  1.01

                  (0.71-1.42)

                  0.98

                     AT and TT

                  336

                  512

                  1.08

                  (0.85-1.37)

                  1.07

                  (0.84-1.37)

                  1.08

                  (0.84-1.38)

                  0.55

                  1OR = Odds Ratio, 95%CI = 95% confidence interval,

                  2 Crude,

                  3 Adjusted for age and gender,

                  4 Adjusted for age, gender and smoking status.

                  5P for the fully adjusted estimate

                  Table 4

                  Odds ratios and 95% confidence intervals (OR (CI)) for associations between IL-10 gene polymorphisms and ulcerative colitis (UC) or Crohns disease (CD) in selected case-control studies3.

                  Ncases/control

                  Trs3024505C1

                  Grs3024493T1

                  C-819T2

                  (rs1800871)

                  C-592A2

                  (rs1800872)

                  G-1082A2

                  (rs1800896)

                  Crs2222202T

                   

                  UC

                         

                  1855/3091

                  1.46 (1.31-1.62)

                   

                  Neg

                  Neg

                  neg

                   

                  [29]

                  203/391

                    

                  neg

                   

                  1.66 (1.30-2.14)

                   

                  [36]

                  CD

                         

                  1848/1804

                  1.17 (1.01-1.34)

                       

                  [29]

                  270/336

                   

                  neg

                  0.77 (0.58-1.00)

                    

                  1.29 (1.01-1.64)

                  [30]

                  234-6/188-231

                     

                  neg

                  neg

                   

                  [35]

                  1rs3024505 and rs3024493 are in complete linkage disequilibrium [30]

                  2IL-10 G-1082A, C-819T, and C-592A are in linkage disequilibrium [32] C-819T, and C-592A are in complete linkage disequilibrium [34]

                  3Negative associations are indicated by "Neg"

                  No association was found between the IL-1β, IL-10 G-1082A, C-819T, C-592A,, and HO-1 polymorphisms and risk of CD or UC (Table 2 and 3). The three IL-10 promoter polymorphisms were found to be in almost complete linkage as previously described for Caucasians [32]. Therefore, no haplotype analyses were performed.

                  No significant difference in the genotype distribution between CD and UC was found (data not shown). When combining UC and CD data to increase the statistical power there were still no associations between the IL-1β, the three IL-10 promoter polymorphisms, and HO-1 gene polymorphisms and risk of IBD (results not shown).

                  Subgroup analyses showed that variant allele carriers of rs3024505 were at 1.47-fold (95% CI: 1.10-1.96) and 1.35-fold (95% CI: 1.04-1.76) higher risk of a diagnosis of CD and UC, respectively, before the age of 40 years than the homozygous wildtype carriers (results not shown). No associations between rs3024505 genotype and disease localisation, or between IL-1β, the three IL-10 promoter polymorphisms, and HO-1 polymorphisms and age at diagnosis or disease localisation were found.

                  Gene-smoking interaction analyses

                  The effect of smoking habits at diagnosis on the genotype associations was investigated for CD and UC, respectively (Additional file 1 : Interaction between the studied polymorphisms and smoking status in relation to risk of Crohns Disease and Additional file 2 : Interaction between the studied polymorphisms and smoking status in relation to risk of ulcerative colitis). No consistent interactions between smoking status and any of the genotypes were found.

                  Discussion

                  The present case-control study showed that the rs3024505 marker polymorphism flanking the IL-10 gene was significantly associated with risk of CD and UC, and, furthermore, with risk of a diagnosis of CD and UC at young age. None of the polymorphisms IL-1β T-31C, IL-10 G-1082A, C-819T, C-592A, or HO-1 A-413T were associated with risk of CD, UC, or UC and CD combined. No consistent interactions between smoking status and genotypes were found.

                  Our results replicate the findings by Franke et al. [29] (Table 4). In addition, we found that the association was carried by a stronger association in the younger age group. Franke et al found that the variant allele of rs3024505 was associated with increased risk of UC with OR of 1.46 (95% CI: 1.31-1.62) and with CD with OR of 1.17 (95% CI: 1.01-1.34). Furthermore, they found no association between the three IL-10 promoter polymorphisms and risk of UC (results regarding CD were not reported). Previous studies were unable to find association between IBD and the IL-10 promoter polymorphisms [22, 31, 35, 39, 40] whereas other studies have found associations between paediatric onset of CD and IL-10 C-819T wildtype allele [30], Crs2222202T variant allele [30] and between the IL-10 G-1082A variant allele and risk of UC [58] (Table 4).

                  The biological significance of rs3024505 in IBD remains unclear [29]. The polymorphism is is located in an intergenic region proximal to the 3'UTR end of the IL-10 gene. The region has a high potential for containing regulatory sequences, and may thus regulate IL-10 gene expression [29]. Furthermore, rs3024505 is in perfect linkage with other polymorphisms located within the IL-10 gene [29]. On the other hand, since no associations were found between risk of UC or CD and the IL-10 promoter polymorphisms with proven functional effects on the IL-10 gene expression, this may suggest either that the rs3024505 has a much stronger regulatory effect on IL-10 levels than the promoter polymorphisms or that the effect of the polymorphism on disease risk is unrelated to IL-10 expression.

                  IL-1β, IL-10 and HO-1 are key players in the homeostasis of the intestinal immune system. Due to their pro-inflammatory and anti-inflammatory effects they are of significance for the development and maintenance of chronic inflammation. IL-1β has pro-inflammatory effects, whereas IL-10 and HO-1 have anti-inflammatory effects. A substantial number of studies document the roles of the interleukins, including IL-1β and IL-10, and HO-1 in intestinal inflammation in various animal IBD models and in IBD patients [15, 25, 26, 4347]. Therefore, genetic variations in these genes may cause imbalance in intestinal homeostasis and thereby contribute to chronic inflammation. On this background, IL-1β, IL-10 and HO-1 are relevant candidates for IBD susceptibility genes.

                  Our results are in accordance with previous studies which were unable to find association between IBD and IL-1β T-31C [24], taqI [19, 23] or C-511T [20, 21]. The HO-1 A-413T polymorphism has not previously been studied in relation to IBD, whereas no association was found between IBD and HO-1 (GT)N [49]. However, all these studies were small, the largest studies included 500 participants, and thus with limited statistical power to exclude an association. The polymorphisms analysed in the present study, IL-1β T-31C, and HO-1 A-413T have been shown to have biological effect [18, 48, 59], and the SNPs have previously been associated to risk of various disease entities [48, 60].

                  We found no consistent interactions between the studied polymorphisms and smoking in relation to risk of CD or UC. Although both smoking and nicotine administration lower the exaggerated IL-1β response in IBD patients [61, 62], the present study does not indicate that smoking at the time of diagnosis influences IBD risk by pathways involving IL-1β, IL-10 or HO-1, since the polymorphisms had no effect among present smokers. Cigarette smoke has been reported to act differentially on inflammation in the small and large intestine, thus worsening small intestinal inflammation, but ameliorating colitis [63]. We were not able to perform subgroup analyses to target this question due to limited statistical power.

                  It is important to stress the strengths and limitations of the study. The present study included 1600 participants and power analyses showed that this study has more than 80% power to detect a dominant effect with an OR of 1.5 in relation to either CD or UC and or 1.4 if CD and UC were combined. Moreover, genetic determinants may be stronger among patients with extensive disease and ileal disease [64, 65] and disease onset at low age. The effects of the polymorphisms might thus be below the detection level of our study.

                  Conclusions

                  In conclusion, the rs3024505 marker polymorphism flanking the IL-10 gene was associated with risk of UC and CD in the present Danish case-cohort study, and, furthermore, with risk of a diagnosis of CD and UC at young age. None of the polymorphisms IL-1β T-31C, IL-10 G-1082A, C-819T, C-592A, or HO-1 A-413T were associated with risk of CD or UC. No consistent interactions between smoking status and genotypes were found. The study suggests that IL-10, but not IL-1β or HO-1, play a role in IBD etiology.

                  Abbreviations

                  CD: 

                  Crohns disease

                  CI: 

                  confidence interval

                  CO: 

                  carbon monoxide

                  COX-2: 

                  cyclooxygenase 2

                  HO-1: 

                  heme oxygenase 1

                  IBD: 

                  inflammatory bowel disease

                  IL-1β: 

                  interleukin 1β

                  IL-10: 

                  interleukin 10

                  iNOS: 

                  inducible nitric oxide synthase

                  NO: 

                  nitric oxide

                  OR: 

                  odds ratio

                  RQ-PCR: 

                  real-time quantitative RT-PCR

                  SNP: 

                  single nucleotide polymorphism

                  UC: 

                  ulcerative colitis

                  PGE2

                  prostaglandin E2

                  Declarations

                  Acknowledgements

                  Lars Bentzen is thanked for excellent technical assistance. Patients and donors are kindly thanked for participation. We thank Dr. Andre Franke for the suggestion to include rs3024505 in this study. We thank the staff at the Library, Viborg Regional Hospital. The project has been supported by the "Familien Erichsen Mindefond", the Lundbeck Foundation, the Danish Research Council, the Western Danish Research Forum for Health Science, the County of Viborg, the Danish Colitis-Crohn Association, "John M Klein og hustrus mindelegat" and "The A.P. Møller Foundation for the Advancement of Medical Science".

                  Authors’ Affiliations

                  (1)
                  Medical Department, Viborg Regional Hospital
                  (2)
                  Institute of Cancer Epidemiology, Danish Cancer Society
                  (3)
                  Department of Clinical Biochemistry, Aarhus University Hospital
                  (4)
                  Department of Medical Gastroenterology, Aarhus University Hospital
                  (5)
                  National Food Institute, Technical University of Denmark
                  (6)
                  Institute for Science, Systems and Models, University of Roskilde
                  (7)
                  National Research Centre for the Working Environment
                  (8)
                  Department of Clinical Biochemistry, Viborg Regional Hospital

                  References

                  1. Xavier RJ, Podolsky DK: Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007, 448: 427–434.View ArticlePubMed
                  2. Franke A, Balschun T, Karlsen TH, Hedderich J, May S, Lu T, et al.: Replication of signals from recent studies of Crohn's disease identifies previously unknown disease loci for ulcerative colitis. Nat Genet 2008, 40: 713–715.View ArticlePubMed
                  3. Ernst A, Jacobsen B, Ostergaard M, Okkels H, Andersen V, Dagiliene E, et al.: Mutations in CARD15 and smoking confer susceptibility to Crohn's disease in the Danish population. Scand J Gastroenterol 2007, 42: 1445–1451.View ArticlePubMed
                  4. A Catalog of Published Genome-Wide Association Studies, Hindorff LA, Junkins HA, Manolio TA: A Catalog of Published Genome-Wide Association Studies. [http://​www.​genome.​gov/​26525384] 2008.
                  5. Ostergaard M, Ernst A, Labouriau R, Dagiliene E, Krarup HB, Christensen M, et al.: Cyclooxygenase-2, multidrug resistance 1, and breast cancer resistance protein gene polymorphisms and inflammatory bowel disease in the Danish population. Scand J Gastroenterol 2009, 44: 65–73.View ArticlePubMed
                  6. Fisher SA, Tremelling M, Anderson CA, Gwilliam R, Bumpstead S, Prescott NJ, et al.: Genetic determinants of ulcerative colitis include the ECM1 locus and five loci implicated in Crohn's disease. Nat Genet 2008, 40: 710–712.View ArticlePubMed
                  7. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, et al.: Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet 2008, 40: 955–962.View ArticlePubMed
                  8. Duerr RH: Update on the genetics of inflammatory bowel disease. J Clin Gastroenterol 2003, 37: 358–367.View ArticlePubMed
                  9. Brant SR, Shugart YY: Inflammatory bowel disease gene hunting by linkage analysis: rationale, methodology, and present status of the field. Inflamm Bowel Dis 2004, 10: 300–311.View ArticlePubMed
                  10. Hugot JP, Zaccaria I, Cavanaugh J, Yang H, Vermeire S, Lappalainen M, et al.: Prevalence of CARD15/NOD2 mutations in Caucasian healthy people. Am J Gastroenterol 2007, 102: 1259–1267.View ArticlePubMed
                  11. Baumgart DC, Carding SR: Inflammatory bowel disease: cause and immunobiology. Lancet 2007, 369: 1627–1640.View ArticlePubMed
                  12. Strober W, Fuss I, Mannon P: The fundamental basis of inflammatory bowel disease. J Clin Invest 2007, 117: 514–521.View ArticlePubMed
                  13. Fukata M, Abreu MT: What Are Toll-like Receptors and what Role May They Have in IBD. Inflamm Bowel Dis 2008, 14: S90-S91.View ArticlePubMed
                  14. Werner T, Haller D: Intestinal epithelial cell signalling and chronic inflammation: From the proteome to specific molecular mechanisms. Mutat Res 2007, 622: 42–57.PubMed
                  15. Dinarello CA: Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 2009, 27 (519–50) : 519–550.View ArticlePubMed
                  16. Guimbaud R, Bertrand V, Chauvelot-Moachon L, Quartier G, Vidon N, Giroud JP, et al.: Network of inflammatory cytokines and correlation with disease activity in ulcerative colitis. Am J Gastroenterol 1998, 93: 2397–2404.View ArticlePubMed
                  17. Kidd M, Gustafsson BI, Drozdov I, Modlin IM: IL1beta- and LPS-induced serotonin secretion is increased in EC cells derived from Crohn's disease. Neurogastroenterol Motil 2009, 21: 439–450.View ArticlePubMed
                  18. Chen H, Wilkins LM, Aziz N, Cannings C, Wyllie DH, Bingle C, et al.: Single nucleotide polymorphisms in the human interleukin-1B gene affect transcription according to haplotype context. Hum Mol Genet 2006, 15: 519–529.View ArticlePubMed
                  19. Hacker UT, Bidlingmaier C, Gomolka M, Keller E, Eigler A, Hartmann G, et al.: Inflammatory bowel disease: no association between allele combinations of the interleukin (IL) I beta and IL-I receptor antagonist gene polymorphisms. Eur J Clin Invest 1998, 28: 214–219.View ArticlePubMed
                  20. Corleto VD, Pagnini C, Margagnoni G, Guagnozzi D, Torre MS, Martorelli M, et al.: IL-1beta-511 and IL-1RN*2 polymorphisms in inflammatory bowel disease: An Italian population study and meta-analysis of European studies. Dig Liver Dis 2010, 42: 179–84.View ArticlePubMed
                  21. Ferreira AC, Almeida S, Tavares M, Canedo P, Pereira F, Regalo G, et al.: NOD2/CARD15 and TNFA, but not IL1B and IL1RN, are associated with Crohn's disease. Inflamm Bowel Dis 2005, 11: 331–339.View ArticlePubMed
                  22. Balding J, Livingstone WJ, Conroy J, Mynett-Johnson L, Weir DG, Mahmud N, et al.: Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms. Mediators Inflamm 2004, 13: 181–187.View ArticlePubMed
                  23. Stokkers PC, van Aken BE, Basoski N, Reitsma PH, Tytgat GN, van Deventer SJ: Five genetic markers in the interleukin 1 family in relation to inflammatory bowel disease. Gut 1998, 43: 33–39.View ArticlePubMed
                  24. Queiroz DM, Oliveira AG, Saraiva IE, Rocha GA, Rocha AM, das Gracas Pimenta SM, et al.: Immune response and gene polymorphism profiles in Crohn's disease and ulcerative colitis. Inflamm Bowel Dis 2009, 15: 353–358.View ArticlePubMed
                  25. Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W: Interleukin-10-deficient mice develop chronic enterocolitis. Cell 1993, 75: 263–274.View ArticlePubMed
                  26. Ishizuka K, Sugimura K, Homma T, Matsuzawa J, Mochizuki T, Kobayashi M, et al.: Influence of interleukin-10 on the interleukin-1 receptor antagonist/interleukin-1 beta ratio in the colonic mucosa of ulcerative colitis. Digestion 2001, 63 (Suppl 1) : 22–7-22–27.PubMed
                  27. Correa I, Veny M, Esteller M, Pique JM, Yague J, Panes J, et al.: Defective IL-10 production in severe phenotypes of Crohn's disease. J Leukoc Biol 2009, 85: 896–903.View ArticlePubMed
                  28. Schreiber S, Heinig T, Thiele HG, Raedler A: Immunoregulatory role of interleukin 10 in patients with inflammatory bowel disease. Gastroenterology 1995, 108: 1434–1444.View ArticlePubMed
                  29. Franke A, Balschun T, Karlsen TH, Sventoraityte J, Nikolaus S, Mayr G, et al.: Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 2008, 40: 1319–1323.View ArticlePubMed
                  30. Amre DK, Mack DR, Morgan K, Israel D, Lambrette P, Costea I, et al.: Interleukin 10 (IL-10) gene variants and susceptibility for paediatric onset Crohn's disease. Aliment Pharmacol Ther 2009, 29: 1025–1031.View ArticlePubMed
                  31. Koss K, Satsangi J, Fanning GC, Welsh KI, Jewell DP: Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies. Genes Immun 2000, 1: 185–190.View ArticlePubMed
                  32. Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV: An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 1997, 24: 1–8.View ArticlePubMed
                  33. Ouma C, Davenport GC, Were T, Otieno MF, Hittner JB, Vulule JM, et al.: Haplotypes of IL-10 promoter variants are associated with susceptibility to severe malarial anemia and functional changes in IL-10 production. Hum Genet 2008, 124: 515–524.View ArticlePubMed
                  34. Shin HD, Winkler C, Stephens JC, Bream J, Young H, Goedert JJ, et al.: Genetic restriction of HIV-1 pathogenesis to AIDS by promoter alleles of IL10. Proc Natl Acad Sci USA 2000, 19 (97) : 14467–14472.View Article
                  35. Fowler EV, Eri R, Hume G, Johnstone S, Pandeya N, Lincoln D, et al.: TNFalpha and IL10 SNPs act together to predict disease behaviour in Crohn's disease. J Med Genet 2005, 42: 523–528.View ArticlePubMed
                  36. Tedde A, Laura PA, Bagnoli S, Congregati C, Milla M, Sorbi S, et al.: Interleukin-10 promoter polymorphisms influence susceptibility to ulcerative colitis in a gender-specific manner. Scand J Gastroenterol 2008, 43: 712–718.View ArticlePubMed
                  37. Tagore A, Gonsalkorale WM, Pravica V, Hajeer AH, McMahon R, Whorwell PJ, et al.: Interleukin-10 (IL-10) genotypes in inflammatory bowel disease. Tissue Antigens 1999, 54: 386–390.View ArticlePubMed
                  38. Fernandez L, Martinez A, Mendoza JL, Urcelay E, Fernandez-Arquero M, Garcia-Paredes J, et al.: Interleukin-10 polymorphisms in Spanish patients with IBD. Inflamm Bowel Dis 2005, 11: 739–743.View ArticlePubMed
                  39. Parkes M, Satsangi J, Jewell D: Contribution of the IL-2 and IL-10 genes to inflammatory bowel disease (IBD) susceptibility. Clin Exp Immunol 1998, 113: 28–32.View ArticlePubMed
                  40. Klein W, Tromm A, Griga T, Fricke H, Folwaczny C, Hocke M, et al.: The IL-10 gene is not involved in the predisposition to inflammatory bowel disease. Electrophoresis 2000, 21: 3578–3582.View ArticlePubMed
                  41. Pae HO, Kim EC, Chung HT: Integrative survival response evoked by heme oxygenase-1 and heme metabolites. J Clin Biochem Nutr 2008, 42: 197–203.View ArticlePubMed
                  42. Lee TS, Chau LY: Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med 2002, 8: 240–246.View ArticlePubMed
                  43. Berberat PO, Rahim YI, Yamashita K, Warny MM, Csizmadia E, Robson SC, et al.: Heme oxygenase-1-generated biliverdin ameliorates experimental murine colitis. Inflamm Bowel Dis 2005, 11: 350–359.View ArticlePubMed
                  44. Paul G, Bataille F, Obermeier F, Bock J, Klebl F, Strauch U, et al.: Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis. Clin Exp Immunol 2005, 140: 547–555.View ArticlePubMed
                  45. Wang WP, Guo X, Koo MW, Wong BC, Lam SK, Ye YN, et al.: Protective role of heme oxygenase-1 on trinitrobenzene sulfonic acid-induced colitis in rats. Am J Physiol Gastrointest Liver Physiol 2001, 281: G586-G594.PubMed
                  46. Hegazi RA, Rao KN, Mayle A, Sepulveda AR, Otterbein LE, Plevy SE: Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway. J Exp Med 2005, 19 (202) : 1703–1713.View Article
                  47. Takagi T, Naito Y, Mizushima K, Nukigi Y, Okada H, Suzuki T, et al.: Increased intestinal expression of heme oxygenase-1 and its localization in patients with ulcerative colitis. J Gastroenterol Hepatol 2008, 23 (Suppl 2) : S229-S233.View ArticlePubMed
                  48. Ono K, Goto Y, Takagi S, Baba S, Tago N, Nonogi H, et al.: A promoter variant of the heme oxygenase-1 gene may reduce the incidence of ischemic heart disease in Japanese. Atherosclerosis 2004, 173: 315–319.View ArticlePubMed
                  49. Hausmann M, Paul G, Kellermeier S, Frey I, Scholmerich J, Falk W, et al.: (GT)N dinucleotide repeat polymorphism of haem oxygenase-1 promotor region is not associated with inflammatory bowel disease risk or disease course. Clin Exp Immunol 2008, 153: 81–85.View ArticlePubMed
                  50. Mahid SS, Minor KS, Soto RE, Hornung CA, Galandiuk S: Smoking and inflammatory bowel disease: a meta-analysis. Mayo Clin Proc 2006, 81: 1462–1471.View ArticlePubMed
                  51. Kikuchi H, Itoh J, Fukuda S: Chronic nicotine stimulation modulates the immune response of mucosal T cells to Th1-dominant pattern via nAChR by upregulation of Th1-specific transcriptional factor. Neurosci Lett 2008, 432: 217–221.View ArticlePubMed
                  52. Podolsky DK: Inflammatory bowel disease. N Engl J Med 2002, 347: 417–429.View ArticlePubMed
                  53. Satsangi J, Silverberg MS, Vermeire S, Colombel JF: The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut 2006, 55: 749–753.View ArticlePubMed
                  54. Macarthur M, Sharp L, Hold GL, Little J, El-Omar EM: The role of cytokine gene polymorphisms in colorectal cancer and their interaction with aspirin use in the northeast of Scotland. Cancer Epidemiol Biomarkers Prev 2005, 14: 1613–1618.View ArticlePubMed
                  55. Vogel U, Christensen J, Wallin H, Friis S, Nexo BA, Tjonneland A: Polymorphisms in COX-2, NSAID use and risk of basal cell carcinoma in a prospective study of Danes. Mutat Res 2007, 617: 138–146.PubMed
                  56. Greenland S: Dose-response and trend analysis in epidemiology: alternatives to categorical analysis. Epidemiology 1995, 6: 356–365.View ArticlePubMed
                  57. Genetic Power Calculator [http://​pngu.​mgh.​harvard.​edu/​~purcell/​gpc/​cc2.​html] 2009.
                  58. Tedde A, Laura PA, Bagnoli S, Congregati C, Milla M, Sorbi S, et al.: Interleukin-10 promoter polymorphisms influence susceptibility to ulcerative colitis in a gender-specific manner. Scand J Gastroenterol 2008, 43: 712–718.View ArticlePubMed
                  59. El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, et al.: Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000, 404: 398–402.View ArticlePubMed
                  60. Vogel U, Christensen J, Wallin H, Friis S, Nexo BA, Raaschou-Nielsen O, et al.: Polymorphisms in genes involved in the inflammatory response and interaction with NSAID use or smoking in relation to lung cancer risk in a prospective study. Mutat Res 2008, 639: 89–100.PubMed
                  61. Birrenbach T, Bocker U: Inflammatory bowel disease and smoking: a review of epidemiology, pathophysiology, and therapeutic implications. Inflamm Bowel Dis 2004, 10: 848–859.View ArticlePubMed
                  62. Aldhous MC, Prescott RJ, Roberts S, Samuel K, Waterfall M, Satsangi J: Does nicotine influence cytokine profile and subsequent cell cycling/apoptotic responses in inflammatory bowel disease? Inflamm Bowel Dis 2008, 14: 1469–1482.View ArticlePubMed
                  63. Karban A, Eliakim R: Effect of smoking on inflammatory bowel disease: Is it disease or organ specific? World J Gastroenterol 2007, 13: 2150–2152.PubMed
                  64. Fowler EV, Doecke J, Simms LA, Zhao ZZ, Webb PM, Hayward NK, et al.: ATG16L1 T300A shows strong associations with disease subgroups in a large Australian IBD population: further support for significant disease heterogeneity. Am J Gastroenterol 2008, 103: 2519–2526.View ArticlePubMed
                  65. Achkar JP, Dassopoulos T, Silverberg MS, Tuvlin JA, Duerr RH, Brant SR, et al.: Phenotype-stratified genetic linkage study demonstrates that IBD2 is an extensive ulcerative colitis locus. Am J Gastroenterol 2006, 101: 572–580.View ArticlePubMed
                  66. Pre-publication history

                    1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2350/​11/​82/​prepub

                  Copyright

                  © Andersen et al. 2010

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.