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lichen sclerosus,18 psoriasis19 and IBD.13,20–23. Since this correlation between allele 2 and IBD has been observed in some studies,13,20–23 but could not ...
Genes and Immunity (2002) 3, 400–406 & 2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene

Interleukin-1 receptor antagonist VNTR-polymorphism in inflammatory bowel disease L Vijgen1, M Van Gysel1, A Rector1, I Thoelen1, N Esters2, T Ceelen1, E Vangoidsenhoven1, S Vermeire2, P Rutgeerts2 and M Van Ranst1 Laboratory of Clinical & Epidemiological Virology, Department of Microbiology & Immunology, Rega Institute for Medical Research; Department of Gastroenterology, UZ Gasthuisberg Leuven, Belgium

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Both genetic and environmental factors have been implicated in the etiology of inflammatory bowel diseases (IBD) i.e., Crohn’s disease (CD) and ulcerative colitis (UC). Polymorphisms in cytokine genes are likely to influence an individual’s predisposition to IBD. In intron 2 of the interleukin-1 receptor antagonist (IL-1ra) gene, a variable number of an 86-bp tandem repeat (VNTR) polymorphism leads to the existence of five different alleles. In order to analyze the association between certain IL-1ra VNTRalleles and IBD, we investigated the IL-1ra genotype and allele frequencies in 342 unrelated IBD patients and in 401 healthy control individuals. CD patients were also genotyped for the three main associated variants in the NOD2/CARD15 gene. In the IBD group, a significant decrease in the frequency of IL-1ra allele 1 (P ¼ 0.048) compared to controls was observed. The frequency of IL-1ra genotype 1/1 was significantly lower in the IBD population vs the control group (P ¼ 0.018). Analysis of the CD population without NOD2 homozygotes and compound heterozygotes revealed a more significant decrease in IL-1ra genotype 1/1 compared to controls (P ¼ 0.038). These results support the hypothesis that the IL-1ra VNTR-polymorphism could be among the genetic factors that are of importance in IBD susceptibility. Genes and Immunity (2002) 3, 400–406. doi:10.1038/sj.gene.6363888 Keywords: inflammatory bowel disease; Crohn’s disease; ulcerative colitis; interleukin-1; interleukin-1 receptor

antagonist

Introduction Ulcerative colitis (UC) and Crohn’s disease (CD), collectively termed inflammatory bowel disease (IBD), are a significant cause of morbidity in the developed world, with an actual combined prevalence of 150–200 per 100 000 in Europe and North America.1,2 Their pathology is characterized by an idiopathic and chronic uncontrolled inflammation of the intestines. Although IBD etiology remains poorly understood, several environmental and genetic factors have been implicated in the pathogenesis of IBD.3–5 Numerous infectious agents are believed to play a role, the most important ones being Mycobacterium paratuberculosis and Listeria monocytogenes.6,7 As no simple Mendelian pattern of inheritance applies to IBDs, they are thought to be polygenetic disorders, in which several susceptibility genes are considered to be important.8 Genes involved in the regulation of the immune response and in the maintenance of mucosal integrity have been target areas

Correspondence: M Van Ranst, Laboratory of Clinical and Epidemiological Virology, Department of Microbiology & Immunology, Rega Institute for Medical Research, Minderbroedersstraat 10, BE-3000 Leuven, Belgium. E-mail: [email protected]

for investigation using linkage analysis and association studies.5 The first gene underlying susceptibility to CD has recently been identified by two independent groups as the NOD2/CARD15 gene.9,10 This gene encodes a cytoplasmatic protein, belonging to the Apaf-1 superfamily of apoptosis regulators, with an expression restricted to monocytes. The NOD2 protein is composed of two caspase recruitment domains (CARD), a nucleotide-binding domain (NBD) and a carboxy-terminal leucine-rich repeat region (LRR). The LRR domain plays an important role in the recognition of bacterial lipopolysaccharides, which leads to activation of NFkB, a nuclear factor, regulating genes involved in the inflammatory response. Three mutations in the NOD2 gene, two amino acid changing SNPs (SNP 8: Arg702Trp; SNP 12: Gly908Arg) and one frameshift mutation (SNP 13: Leu1007fsinsC) causing a premature stop codon, have been found to be associated with CD.9–11 SNPs 12 and 8 cause non-conservative amino acid substitutions in the LRR domain and in the proximal adjacent region, respectively, while SNP 13 leads to a truncated LRR domain. Other less frequent polymorphisms in the NOD2 gene have been identified, and together with the SNP 8, 12 and 13 NOD2 mutations, they are considered to influence a person’s susceptibility to CD.9 However, many other susceptibility genes are likely to play a

IL-1ra VNTR-polymorphism in IBD L Vijgen et al

significant role in IBD, and identification of these loci remains an essential object of several studies. The interleukin-1 receptor antagonist (IL-1ra) is a unique naturally occurring cytokine that inhibits interleukin-1 (IL-1) activity by binding to the IL-1 receptors without signal transduction.12 Although IL-1 plays an important role in protecting the organism against internal and external threats, the margin between clinical benefit and toxicity in humans is exceedingly narrow. To reduce the destructive IL-1 effects during inflammation, its antagonist IL-1ra strictly regulates its action. In IBD and several other inflammatory diseases, an imbalance between IL-1 and IL-1ra production, which contributes to maintenance of the chronic inflammation, is observed.13,14 The relative deficiency of IL-1ra in IBD offers an opportunity for treatment of IBD through administration of recombinant IL-1ra, but this remains in an experimental stage.15 In intron 2 of the IL-1ra gene, a polymorphism due to the presence of variable numbers of an 86-bp tandem repeat (VNTR) has been described.16 This polymorphism leads to the existence of five alleles, each corresponding to a different number of repeats. Even though this IL-1ra VNTR-polymorphism is located in intron 2, a non-coding region of the IL-1ra gene, it is possible that it is responsible for an alteration in IL-1ra production and in that way could influence an individual’s susceptibility to IBD.13 Given the importance of IL-1ra in the pathogenesis of IBD, this VNTR-polymorphism and its functional consequences have been the object of various investigations. An association was found between IL-1ra allele 2 (two repeats) and several chronic inflammatory diseases including systemic lupus erythematosus (SLE),17 lichen sclerosus,18 psoriasis19 and IBD.13,20–23 Since this correlation between allele 2 and IBD has been observed in some studies,13,20–23 but could not be supported by others,24–26 the role of the IL-1ra polymorphism in IBD remains controversial. The number of repeats, present in intron 2 of the IL-1ra gene, may be of functional significance as it is believed that each repeat contains an amount of binding sites for transcription factors.16 Therefore, a greater number of repeats would lead to an increased transcriptional activity. Other studies suggest that the IL-1ra allele 2 has rather an effect on IL-1b production, as was demonstrated by an increased in vitro production of IL1b in mononuclear cells from carriers of the IL-1ra allele 2, regardless of the presence of IL-1b polymorphisms.27 There are two theories about the kind of association of IL-1ra allele 2 with IBD: according to the first theory allele 2 would be a direct susceptibility factor, while the second one rather determines allele 2 as a severity factor. This second theory is supported by studies in which allele 2 disease association is the most remarkable in patients with a more severe form of the disease.20 Although this hypothesis, according to which the VNTR-polymorphism itself has a functional importance, is an acceptable explanation for the influence of the polymorphism on IBD pathogenesis, another hypothesis can be postulated. It is possible that the IL-1ra allelic variance is in linkage disequilibrium with another mutation in the same gene or with a predisposition gene on the same chromosome. The human IL-1ra gene has been mapped to band q14-q21 on the long arm of chromosome 2, near the genes for IL-1a and IL-1b.28,29

The IL-1b gene has also been proven to be polymorphic and its correlation with several inflammatory disorders like IBD has been studied.30 It is therefore possible that the IL-1ra VNTR-polymorphism is not directly in association with IBD but is linked with the IL-1b allelic variance, which influences a person’s susceptibility to IBD. Our aim was to study the distribution of the IL-1ra VNTR-polymorphism in Belgian IBD patients and in unaffected controls in an attempt to determine whether an association between particular VNTR-alleles of the IL1ra gene and IBD could be found. Since the IL-1ra VNTRpolymorphism has previously been described as a disease severity factor in IBD,20 we also analyzed the clinical parameters of our IBD population in relation to their IL-1ra genotype. As the NOD2 gene has been proven to be a major CD susceptibility gene, we also subdivided the CD patient population according to their NOD2 SNP 8, 12 and 13 genotype.

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Results IL-1ra VNTR-polymorphism in the unaffected control population The IL-1ra genotype and allele frequencies in the unaffected Flemish control population are given in Table 1. The IL-1ra allele frequencies were 0.743, 0.238 and 0.019 for allele 1 (four repeats), 2 (two repeats) and 3 (five repeats), respectively, while allele 4 (three repeats) and 5 (six repeats) were not observed in the control population. IL-1ra VNTR-polymorphism in IBD patients IL-1ra genotype and allele distributions in IBD patients, and its subgroups UC and CD patients are shown in Table 1. In the total group of IBD patients the allele frequencies were 0.676, 0.291 and 0.031 for allele 1, 2 and 3, respectively, while allele 4 and 5 both showed a frequency of 0.001. We observed several differences between IBD patients and the control population, as well in allele as in genotype frequencies. The frequency of genotype 1/1 was lower in the IBD population compared to the control group, while the IBD group also showed a lower frequency of allele 1 compared to the healthy controls; this is in favor of allele 2 and 3. Statistical analysis of these results revealed a significant decrease in frequency of genotype 1/1 in the IBD population, as well as in the UC and CD subpopulations, compared to the control group (P ¼ 0.0009, 0.0113 and 0.0049, respectively). After Bonferroni correction for multiple comparisons, only the decrease in genotype 1/1 frequency in the whole IBD group compared to controls remained significant (P ¼ 0.018, 0.226 and 0.098, respectively). Genotype 1/2 was significantly increased in the IBD and CD group, in comparison with healthy controls (P ¼ 0.0076 and 0.0091, respectively), which was no longer significant after Bonferroni correction (P ¼ 0.152 and 0.182, respectively). UC patients had a significantly higher frequency of genotype 2/3, compared to controls (P ¼ 0.0146). Application of the Bonferroni correction led to a non-significant P-value (P ¼ 0.292). Allele 1 was significantly decreased in frequency in IBD, UC and CD patients compared Genes and Immunity

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to healthy controls (P ¼ 0.004, 0.0117 and 0.0312, respectively). After Bonferroni correction for multiple comparisons, the allele 1 frequency only remained significantly decreased in IBD patients vs the control group (P ¼ 0.048, 0.1404 and 0.3744, respectively). IBD patients in general had a significantly higher frequency of allele 2 (P ¼ 0.0211) while a significant increase in allele 3 frequency was observed in the UC population (P ¼ 0.0230), both in comparison with controls. Both these P-values were no longer significant after Bonferroni correction (P ¼ 0.253 and 0.276, respectively). No significant differences in genotype, nor in allele frequencies, could be detected between UC and CD patients. Clinical features of IBD patients according to IL-1ra genotype The clinical parameters of the IBD patients in relation to their IL-1ra genotype are shown in Table 2(a) and (b). There was no correlation between IL-1ra variance and the age at IBD diagnosis or disease localization. A total of 114 CD patients needed surgical intervention. After subdividing our CD population into surgically treated patients and patients without surgery, an IL-1ra genotype distribution was determined, as is shown in Table 3. A significant association between the IL-1ra polymorph-

ism and the necessity of surgery could be found: compared to controls, there was a significantly lower frequency of genotype 1/1 (P ¼ 0.003) and a significantly higher frequency of genotype 1/2 (P ¼ 0.017) in the group of surgically treated CD patients, which is in correspondence with the results of comparing the whole CD group to controls. After Bonferroni correction for multiple comparisons, only the decrease in genotype 1/1 frequency remained significant (P ¼ 0.03 and 0.17, respectively). When compared to CD patients without surgery, a significant decrease in the frequency of genotype 1/1 (P ¼ 0.042) and a significant increase in genotype 2/2 frequency (P ¼ 0.043) were observed in CD patients with surgery. However, application of the Bonferroni correction converted these P-values into non-significant values (P ¼ 0.42 and 0.43, respectively). No significant differences in genotype frequencies could be detected between UC patients with surgery and healthy controls, nor between UC patients with surgery and those without. NOD2 genotyping of the CD patient population A total of 204 CD patients were genotyped for the NOD2 SNP 8, SNP 12 and SNP 13 mutations. The allele frequencies of the analyzed mutations in the Belgian

Table 1 IL-1ra genotype and allele frequencies IBD (n=342)

CD (n=218)

UC (n=124)

Controls (n=401)

n

%

n

%

n

%

n

%

Genotype frequency 1/1 1/2 1/3 1/4 1/5 2/2 2/3 3/3

157 136 11 1 1 26 10 0

45.9 39.8 3.2 0.3 0.3 7.6 2.9 0.0

101 89 6 1 1 16 4 0

46.3 40.8 2.8 0.5 0.5 7.3 1.8 0.0

56 47 5 0 0 10 6 0

45.2 37.9 4.0 0.0 0.0 8.1 4.8 0.0

233 122 8 0 0 32 5 1

58.1 30.4 2.0 0.0 0.0 8.0 1.2 0.3

Allele frequency Allele 1 Allele 2 Allele 3 Allele 4 Allele 5

462 199 21 1 1

67.6 29.1 3.1 0.1 0.1

299 125 10 1 1

68.6 28.7 2.3 0.2 0.2

164 73 11 0 0

66.1 29.5 4.4 0.0 0.0

596 191 15 0 0

74.3 23.8 1.9 0.0 0.0

Table 2 Clinical features of (a) CD and (b) UC patients according to IL-1ra genotype IL-1ra genotype (a) Age at diagnosis (range) Surgery-neccessitating CD(%) Disease localization: ileitis (%) ileocolitis (%) colitis (%) Anal involvement (%)

1/1

1/2

1/3

1/4

1/5

2/2

2/3

Total

n=101 24.7 (11–52) 48/99 (48.5) 32/99 (32.3) 49/99 (49.5) 18/99 (18.2) 31/99 (31.3)

n=89 26.1 (12–62) 48/86 (55.8) 31/86 (36.0) 38/86 (44.2) 17/86 (19.8) 26/86 (30.2)

n=6 18.0 (8–24) 3/6 (50.0) 3/6 (50.0) 1/6 (16.7) 2/6 (33.3) 2/6 (33.3)

n=1 33.0 1/1 (100.0) 1/1 (100.0) 0/1 (0.0) 0/1 (0.0) 0/1 (0.0)

n=1 21.0 0/1 (0.0) 0/1 (0.0) 1/1 (100.0) 0/1 (0.0) 1/1 (100.0)

n=16 27.4 (14–55) 12/16 (75.0) 6/16 (37.5) 9/16 (56.2) 1/16 (6.3) 5/16 (31.3)

n=4 22.3 (7–40) 2/4 (50.0) 0/4 (0.0) 4/4 (100.0) 0/4 (0.0) 1/4 (25.0)

n=218 25.2 (7–62) 114/213 (53.5) 73/213 (34.3) 102/213 (47.9) 38/213 (17.8) 66/213 (31.0)

n=47 28.3 (8–55) 3/47 (6.4)

n=5 n=0 43.4 (26–45) F 0/5 (0.0) F

n=0 F F

n=10 n=6 n=124 29.9 (18–68) 30.2 (12–57) 29.5 (8–70) 2/10 (20.0) 0/6 (0.0) 12/123 (9.8)

(b) n=56 Age at diagnosis (range) 29.5 (8–70) Surgery-neccessitating UC (%) 7/55 (12.7)

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Table 3 IL-1ra genotypes in CD patients with and without surgery IL1-ra genotype

CD patients with surgery (n=114)

1/1 1=2 1/3 1=4 1/5 2/2 2/3

CD patients without surgery (n=99)

n

%

n

%

48 48 3 1 0 12 2

42.1 42.1 2.6 0.9 0.0 10.5 1.8

51 38 3 0 1 4 2

51.5 38.4 3.0 0.0 1.0 4.1 2.0

CD population were 0.08 (SNP 8), 0.06 (SNP 12) and 0.06 (SNP 13), respectively, with the total frequency of mutated alleles equal to 0.20. Out of the 204 CD patients, 69 patients carried at least one of the three mutations in the NOD2 gene, which corresponds to a global frequency of 33.8%. Five patients were homozygous for the SNP 8 mutation, and six patients were carriers of two of the described mutations in the NOD2 gene (compound heterozygous). Fifty-eight simple heterozygotes were found in our CD population, out of which 18 were heterozygous for the SNP 8 mutation, 19 for SNP 12, and 21 for SNP 13. NOD2 genotypes were also analyzed in relation to the IL-1ra genotypes of the 204 CD patients, as is shown in Table 4. In an attempt to rule out any association between IL-1ra genotypes and CD, which would actually be a result of a NOD2-CD association, we reduced our CD population by taking out the NOD2 homozygotes and compound heterozygotes, as they have been proven to have the highest relative risk for CD.9 We observed a more significant decrease in IL-1ra genotype 1/1 when we compared the non-NOD2-associated CD subgroup with a control population (P ¼ 0.0019), than when we compared the complete CD population. Also after Bonferroni correction, this association remained significant (P ¼ 0.038).

Discussion As there were no previous reports on Il-1ra allele and genotype frequencies in a Flemish population, we defined them as a first part of our study and compared them with the frequencies determined in other popula-

tion groups. Allele 1 has a frequency of 0.743 in our Flemish control population, which is in accordance with frequencies previously determined in smaller study groups from other countries (0.756 in the Netherlands, 0.736 in the UK and 0.710 in a white South-African population).16,20,31 The allele frequency of allele 2 in our Flemish group is 0.238, which is comparable to a frequency of 0.238 in the Netherlands, 0.214 in the UK and 0.250 in white South Africa. Allele 3 shows a frequency of 0.019 in our population, slightly higher than that in the Dutch population (0.006) and lower than that in the British (0.036) and white South-African populations (0.030). Alleles 4 and 5 were not observed in our Flemish group nor in the studied Dutch population, while in the UK the determined allele frequency of allele 4 and 5 was 0.007, and in the white South-African group allele 4 had a frequency of 0.010 while allele 5 was not observed. Although the pathogenesis of IBD remains unclear, it is believed that a dysregulation of the immune system plays an important role. Cytokines, produced by mucosal T lymphocytes, are thought to modulate the intestinal immune system and can be classified into proinflammatory (IL-1, IL-6, TNF-a, etc.) and anti-inflammatory or immunoregulatory (IL-2, IL-4, IL-1ra, TNF-b, etc.) mediators. In IBD an imbalance in these pro- and anti-inflammatory cytokines is thought to be present, which corresponds to the observed abnormal ratio of IL1 and IL-1ra in IBD patients.13,14 Therefore, the IL-1ra VNTR-polymorphism could be an important contributing factor in a person’s susceptibility to IBD. Statistical analysis of the IL-1ra allele and genotype frequencies in the IBD patient group in comparison to the unaffected control population confirms the importance of the IL-1ra VNTR-polymorphism in IBD. In this study, DNA from the IBD patients was extracted from peripheral blood lymphocytes. On the other hand, healthy controls were genotyped using epithelial cells from mouthwashes, since a less invasive, painless method was preferred. As there is no significant difference between genotyping from blood or from non-blood specimens, comparisons between the IBD group and the control population could be made.32 A significant decrease in the frequency of allele 1 and a concomitant increase in the frequency of allele 2 are seen in the IBD group compared to healthy controls. Genotype 1/1 is less frequent in the total IBD group and in its subgroups UC and CD, while an increase in frequency of genotype 1/2 in IBD and CD, and of genotype 2/3 in the UC group is observed,

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Table 4 NOD2-IL-1ra genotypes in the CD population (n=204) NOD2 genotype Wild type IL-1ra genotype

n

%

1/1 1/2 1/3 1/4 1/5 2/2 2/3

94 82 6 1 1 16 4

46.1 40.2 2.9 0.5 0.5 7.8 2.0

SNP 8 65 51 5 0 0 11 3

6 8 0 0 1 3 0

Simple heterozygous SNP 12 SNP 13 7 8 1 0 0 2 1

8 12 0 1 0 0 0

Homozygous SNP 8

Compound heterozygous

3 2 0 0 0 0 0

5 1 0 0 0 0 0

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compared to the control population. After Bonferroni correction for multiple comparisons, the decrease in genotype 1/1 and in allele 1 frequency in IBD patients compared to controls remains significant. These results clearly illustrate the genetic heterogeneity in the IBD pathology. Although UC and CD are characterized by a similar clinical–pathological pattern, they do not show the same trends in distribution of IL-1ra genotypes and alleles. This confirms the findings of other studies that have found a difference in genetic background for both diseases.33,34 Our data only partly correspond to the findings of previous IL-1ra-IBD-association studies, which revealed a significant increased frequency of allele 2 in UC patients compared to controls.13,20–23 We did see a significantly higher frequency of allele 2 in the whole IBD population, in comparison with unaffected controls, but not in the UC patient population, when analyzed separately. Application of the Bonferroni correction rendered this increase in allele 2 frequency to a nonsignificant P-value. However, our data do confirm a role for IL-1ra polymorphisms in the pathogenesis of IBD, as the observed significant decrease in allele 1 in IBD patients vs controls remained significant after Bonferroni correction. The hypotheses, that IL-1ra allele 2 would lead to an insufficient production of IL-1ra protein or to an overproduction of IL-1b in response to immune and/ or inflammatory stimuli, could explain why carriage of IL-1ra allele 2 could influence an individual’s susceptibility to IBD.13,27 Analysis of the IL-1ra genotype frequencies in function of the patients’ clinical features revealed some results supporting the theory of allele 2 as a disease severity factor, but only in Crohn patients. Genotype 1/1 is significantly decreased in CD patients with surgery in favor of a significant increase of genotype 1/2, compared to healthy controls. When being compared to CD patients without surgery, a significant decrease in the frequency of genotype 1/1 and increase in genotype 2/2 frequency were observed. After Bonferroni correction, only the decrease in genotype 1/1 frequency in operated CD patients compared to controls remained significant. The CD patient population was subdivided according to their NOD2 genotype, by taking out NOD2 homozygotes and compound heterozygotes, as they have the highest relative risk for CD.9 Compared to the complete CD population, analysis of the non-NOD2-associated CD subgroup revealed a more significant decrease in IL-1ra genotype 1/1 vs the control population. In this study we demonstrate that the IL-1ra VNTRpolymorphism belongs to the large group of genetic factors that are of importance in the pathogenesis of IBD. Since the functional significance of this polymorphism remains unclear, further investigation into a possible correlation between a certain IL-1ra genotype and IL-1ra production quantity is necessary, with a view to a possible new therapeutical approach in inflammatory diseases.

Materials and methods Subjects A total number of 124 unrelated patients with UC and 218 unrelated patients with CD, treated at the Gastroenterology Unit of the University Hospital Gasthuisberg Genes and Immunity

Leuven, were studied. The diagnosis of CD and UC was based on the Lennard-Jones criteria.35 Full clinical details on age at diagnosis, surgery and localization of disease were obtained for 123/124 UC patients and for 213/218 CD patients. The control group consisted of 401 unrelated healthy volunteers, without any familial history of IBD or other immune-mediated diseases. All patients and controls were Caucasians from the region of Flanders, the northern part of Belgium. Informed consent was obtained from all participants and the study had the approval, based upon the guidelines from the World Medical Association’s Declaration of Helsinki (www.wma.net),36 of the Ethics Board of the University of Leuven. DNA extraction Genomic DNA from IBD patients was extracted from whole venous blood samples using a salting-out procedure and was stored at 801C.37 DNA samples of the control population were collected through a non-invasive ‘swish-and-spit’ technique. In this method genomic DNA was acquired from oral epithelial cells, by rinsing the oral cavity with a 0.9% saline solution, after which DNA was extracted using an alkaline lysis procedure.38 IL-1ra VNTR-polymorphism screening In intron 2 of the IL-1ra gene a variable number of identical tandem repeats (VNTR) of an 86-bp length of DNA has been described, which gives rise to five different alleles.16 Each allele corresponds to a different number of repeats (allele 1: four repeats; allele 2: two repeats; allele 3: five repeats; allele 4: three repeats; allele 5: six repeats). Detection of the different alleles of the IL1ra gene was performed using polymerase chain reaction (PCR) amplification of the VNTR-containing region. By using a set of primers, flanking the VNTR-containing fragment, amplicons of different length, relating to a different number of repeats, were generated. The forward primer used was 50 -CTCAGCAACACTCCTAT30 , while 50 -TCCTGGTCTGCAGGTAA-30 was used as reverse primer (position 8868–8884 and 9263–9279 in the IL-1ra gene, Genbank accession number X64532).16 Samples were amplified in a volume of 50 ml reaction mix, with a concentration of 0.2 mM of forward and reverse primer, 0.2 mM of nucleotides, 2.5 mM of MgCl2 and 1 unit of Taq polymerase (Applied Biosystems/ Roche Molecular System, Belgium) at pH 9. PCR conditions were composed as follows: an initial denaturation at 951C for 5 min, then 40 cycles of 30 s at 951C, 30 s at 561C and 35 s at 721C, and a final elongation at 721C for 7 min. The amplification reaction was performed in a Geneamps PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). Resulting PCR products were analyzed using polyacrylamide gel electrophoresis and ethidium bromide staining. The IL1ra genotypes could easily be distinguished on the basis of the difference in length of their PCR products, due to a different number of repeats. Genotyping NOD2 Arg702Trp, Gly908Arg and Leu1007fsinsC A total of 204 CD patients were genotyped for the Arg702Trp, Gly908Arg and Leu1007fsinsC variants in the NOD2/CARD15 gene (National Center For Biotechnology Information LocusLink ID 64127), as previously

IL-1ra VNTR-polymorphism in IBD L Vijgen et al

defined by Hugot et al.9 The missense mutation Arg702Trp (also referred to as SNP 8; GenBank accession number G67950) was genotyped by amplification refractory mutation system (ARMS) with primers 50 -ATCTGAGAAGGCCCTGCTCC-30 (wild type, forward) 50 ATCTGAGAAGGCCCTGCTCT-30 (mutated, forward) and 50 -CCCACACTTAGCCTTGATG-30 (reverse). The missense mutation Gly908Arg (SNP 12; GenBank accession number G67951) creates a restriction site for HhaI and was genotyped by an RFLP-PCR technique (50 -CCCAGCTCCTCCCTCTTC-30 and 50 -AAGTCTGTAATGTAAAGCCAC-30 ). The presence of a wild-type allele results in an intact 380-bp band, whereas the profile of the Gly908Arg variant is characterized by two bands of 138 and 242 bp on a 2% agarose gel, The ARMS technique as described by Ogura et al was used to genotype the Leu1007fsinsC (SNP 13; GenBank accession number G67955).10 Statistical analysis To analyze the comparison of the frequency of discrete variables between unrelated IBD patients and healthy controls, Pearson’s goodness of fit w2 tests or, when appropriate, Fisher’s exact test were used. Bonferroni correction for multiple comparisons was applied when necessary. A P-value of o0.05 was considered to indicate statistical significance.

Acknowledgements We would like to thank all the colleagues of the laboratory of Clinical & Epidemiological Virology, Department of Microbiology & Immunology, Rega Institute for Medical Research, University of Leuven, Belgium for helpful comments and discussion. This work was supported by a grant of the Fund for Scientific Research (FWO), Brussels, Belgium.

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