Interleukin-1ß and interleukin-1 receptor antagonist gene polymorphisms in ankylosing spondylitis
M. van der Paardt1,
J. B. A. Crusius2,
M. A. García-González2,
P. Baudoin1,
P. J. Kostense4,
B. Z. Alizadeh2,5,
B. A. C. Dijkmans1,3,
A. S. Peña2 and
I. E. van der Horst-Bruinsma3,
1 The Jan van Breemen Institute, Amsterdam,
2 Department of Gastroenterology and Laboratory for Gastrointestinal Immunogenetics and
3 Department of Rheumatology, Vrije Universiteit Medical Centre, Amsterdam,
4 Department of Clinical Epidemiology and Biostatistics, Vrije Universiteit, Amsterdam and
5 Genetic Epidemiology Unit, Department of Epidemiology & Biostatistics and Clinical Genetics, Erasmus Medical University, Rotterdam, The Netherlands
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Abstract
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Objective. Since ulcerative colitis and Crohn's disease, which are associated with ankylosing spondylitis (AS), have been found to be variably associated with the IL-1B and the IL-1RN genes encoding interleukin-1ß (IL-1ß) and the interleukin-1 receptor antagonist (IL-1ra), we have investigated whether these polymorphisms in IL-1B and IL-1RN are also involved in AS.
Methods. DNA was isolated from peripheral blood of 106 patients with AS and 104 healthy controls. All patients and controls were Dutch Caucasians. Bi-allelic polymorphisms at positions +3953 and -511 in the IL-1B gene, and a penta-allelic polymorphism in intron 2 of the IL-1RN gene were studied by polymerase chain reaction-based methods.
Results. Allele IL-1RN*2 was significantly increased in AS (odds ratio=1.60; 95% confidence interval=1.202.80; P=0.031) compared with healthy controls, and independent from the polymorphism in loci IL-1B511 and IL-1B+3953. No significant associations were found between AS and the IL-1B511 or IL-1B+3953 polymorphisms.
Conclusion. Similar to other chronic inflammatory diseases, AS is associated with the IL-1RN*2 allele. Further studies are necessary to determine the biological significance of these findings in relation to susceptibility or severity of the disease.
KEY WORDS: Ankylosing spondylitis, Gene polymorphism, Genetics, Interleukin-1ß, Interleukin-1 receptor antagonist.
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Introduction
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Ankylosing spondylitis (AS) is a common familial rheumatic disorder. HLA-B27 is the major genetic determinant identified. However, only a small fraction of B27-positive individuals develop the disease, and B27-positive first-degree relatives of AS patients are 5.615 times more likely to develop AS than B27-positive individuals without affected relatives. Therefore, other genes, possibly within the major histocompatibility complex (MHC) and in other chromosomes, might influence susceptibility to the disease [1, 2].
Recent genome-wide screening identified a total of six non-MHC regions with linkage with AS. These regions were located on chromosomes 1p, 2q, 9q, 10q, 16q and 19q. The maximum non-MHC logarithm of odds (LOD) score obtained was 4.7 (P=0.005) on chromosome 16q at 101 cM from the p-telomere. The MHC locus was found to be the largest component of susceptibility to AS, with a LOD score of 15.6, and the contribution of the MHC locus [
=5.2; 95% confidence interval (CI)=3.09.0] to the recurrence risk ratio in AS was <40% [3]. The locus identified on chromosome 2q14 (
=1.7; 95% CI=1.32.3) is of interest because the interleukin (IL)-1 gene family is encoded in this region. Recently, a study from Scotland reported an association between allele IL-RN*2 and AS [4].
The IL-1 gene family contains, within a 430-kb region, three related genes, IL-1A, IL-1B and IL-1RN, which encode the pro-inflammatory cytokines IL-1
, IL-1ß and their endogenous receptor antagonist IL-1ra, respectively [5].
Bi-allelic polymorphisms, at position +3953 in exon 5 [6] and position 511 in the promoter region [7] of the IL-1B gene, and a penta-allelic polymorphism in intron 2 of the IL-1RN gene, with combined frequencies of alleles IL-1RN*1 and IL-1RN*2 exceeding 96% [8, 9] have been described.
These polymorphic IL-1RN and IL-1B genes have been related to a number of chronic inflammatory diseases such as multiple sclerosis and inflammatory bowel disease (IBD) [10, 11]. Reports have shown that the less common allele IL-1RN*2 is associated with a more extended localization of the disease in ulcerative colitis (UC) in several populations [9, 12, 13]. Furthermore, in both UC and Crohn's disease (CD), carriers of allele IL-1RN*2 were found more often to be non-carriers of allele 2 of the IL-1B+3953 polymorphism than in healthy controls [11]. These findings indicate that these polymorphisms may have functional significance in the regulation of chronic inflammation in UC [6, 14, 15].
When accompanied by arthritis or spondylitis, UC and CD are considered to belong to the spondylarthropathies [16]. Beside these enteropathic forms of spondylarthropathy, this group of disorders includes AS, psoriatic arthritis, reactive arthritis and forms of juvenile chronic arthritis. All spondylarthropathies are to some extent associated with HLA-B27; the frequency of HLA-B27 approaches 100% in Caucasian patients with AS, and approximately half the group of IBD patients with associated spondylitis are B27 positive [17].
This study was performed to assess whether AS shares with IBD the association with polymorphisms in the IL-1B and IL-1RN genes.
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Patients and methods
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Subjects
This study was approved by the ethical committees of the Slotervaart Ziekenhuis and Jan van Breemen Institute and Bovenij Ziekenhuis. After informed consent, a total of 106 AS patients was recruited from the institute's outpatient Department of Rheumatology. Controls were 104 randomly selected healthy blood donors from the Amsterdam region. All the subjects were unrelated Dutch Caucasians. A rheumatologist defined the diagnosis of AS, according to the Modified New York Criteria [18]. Clinical data were gathered by examining the patient record forms.
Methods
Genomic DNA was extracted from EDTA anticoagulated peripheral blood according to a standard proteinase K digestion and phenol/chloroform extraction method. The bi-allelic transition polymorphisms at position 511 in the promoter region and position +3953 in exon 5 of the IL-1B gene, and a penta-allelic variable numbers of tandem repeat polymorphism in intron 2 of the IL-1RN gene were genotyped according to previously described methods [7, 11]. The IL-1B511 C (allele 1)
T (allele 2) transition polymorphism is in almost absolute linkage disequilibrium with a potentially functionally significant polymorphism in the TATA-box of the IL-1B promoter [19].
Statistical analysis
Allele and genotype frequencies were tested for HardyWeinberg equilibrium (HWE) using the
2 test. Carriership of alleles was defined as any subject inheriting at least one copy of allele 2 of the three polymorphisms (the rare allele 3 of the penta-allelic IL-1RN gene polymorphism was pooled with allele 1) [20]. To compare frequencies, the
2 test was used. The magnitude of associations was expressed as an odds ratio (OR).
Allele and haplotype frequencies were calculated using log-linear modelling embedded within an expectation and maximation (EM) algorithm. The EM algorithm handles the phase uncertainty between cases and controls, and the log-linear modelling allows testing for linkage disequilibrium and disease association [21]. Maximum likelihood based
2-test was used to compare the log-linear model constructed under the assumption of linkage among the three loci and the disease status, and the model was constructed under the assumption of random inheritance of IL1 gene cluster polymorphisms independent of disease status. Intermediate models were fitted to test for linkage between each locus and the disease status, which was both unconditional (separately for each locus) and conditional on the polymorphism of the other two loci. The confidence intervals for the odds ratios estimated using the profile likelihood were calculated by estimating the log-likelihood of a series of constrained log-linear models using the results of an IPF (iterative proportional fitting) algorithm. The details of the method were implemented as described by others [22]. A two-tailed P value
0.05 was considered significant. Linkage analysis was performed using Stata 7.0 for Windows.
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Results
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Characteristics of the 106 AS patients are summarized in Table 1
. The distributions of the IL-1B and IL-1RN genotypes and allele frequencies in AS patients and controls are shown in Table 2
. The alleles and genotype frequencies were consistent with the HWE equation (IL1-RN:
2=4.64, P=0.91; IL-1B+3953:
2=1.48, P=0.22; IL-1B511:
2=0.04, P=0.84). The rare allele IL-1RN*3 was present in four AS patients and three controls (1.0 and 0.7% of total cases, respectively). None of the subjects was homozygous for allele IL-1RN*3. The frequency of allele IL-1RN*2 in AS patients was significantly increased in comparison with the controls (17.1 and 12.3%, respectively; OR=1.60; 95% CI=1.202.80; P=0.03). No significant differences were observed between AS patients and controls in the frequencies of the alleles IL-1 B511*2 and IL-1B+3953*2.
The likelihood ratio test showed a significant linkage between the alleles of the IL1 cluster in patients (P<0.0001) and controls (P<0.0001) (Table 3
). In both controls and cases, the haplotypes with the highest differences between observed and expected frequencies are: alleles IL-1RN*1/IL-1B+3953*1/IL-1B511*2, IL-1RN*1/IL-1B+3953*2/IL-1B511*1, IL-1RN*2/IL-1B+3953*2/IL-1B511*1, IL-1RN*2/IL-1B+3953*1/IL-1B511*2, IL-1RN*2/IL-1B+3953*1/IL-1B511*1 and IL-1RN*3/IL-1B+3953*2/IL-1B511*1 (Table 3
, stratified analysis). In a pooled analysis (Table 3
, total cohort), the last three haplotypes showed higher and lower observed frequencies in cases and controls, respectively, than expected. These differences did not reach significance (P=0.34).
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TABLE 3. Linkage disequilibrium between IL-1RN, IL-1B+3953 and IL-1B511 polymorphisms in controls and patients, and in total
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In addition, the immediately available clinical characteristics of the AS patients were analysed for possible associations with the carriership of allele 2 of the studied polymorphisms. No significant associations were found between carriage of each of the alleles IL-1B511*2, IL-1B+3953*2 or IL-1RN*2 and sex, existence (either past or present) of peripheral arthritis or acute anterior uveitis, age at first complaint, years between first complaint and actual diagnosis of AS, and the number of patients with at least one family member with AS.
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Discussion
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A significant association between AS and allele IL-1RN*2 was observed, independently from the alleles of the IL-1B511 and IL-1B+3953 polymorphisms (P=0.03) (Table 2
). The strong linkage disequilibrium found between the alleles of the IL1 gene cluster was independent from AS (Table 3
). Our results are in accordance with the findings of a study from Scotland [4]. Some statisticians argue that in this study significance is only reached when P<0.017 according to Bonferroni's correction for multiple testing. This is disputable according to others. The fact that our findings strongly point towards an involvement of allele IL-1RN*2 in the pathogenesis of AS (P=0.03) supports our hypothesis based on the association observed in other spondylarthropathies. An increased frequency of IL-1RN*2 has also been found in UC, a disorder interrelated with AS. In UC, allele IL-1RN*2 seems to be associated with extension of the disease [9, 12, 13]. Secondly, recent studies have shown the association between IL-1RN*2 homozygotes and an increased risk of gastric cancer [19]. In UC and CD, an association with the allelic combination IL-1RN*2+/IL-1B+3953*1 has been reported [11, 13]. In multiple sclerosis, the allelic combination IL-1RN*2+/IL-1B+3953*1 has been found to be related to disease progression [10]. In this study, no significant association between specific haplotypes and AS could be found (Table 3
).
The possible mechanisms of the association of allele IL-1RN*2 with AS remain unclear. An imbalance between IL-1ra and IL-1ß may be involved. Carriage of IL-1RN*2 was associated with a decreased production of total IL-1ra protein by cultured peripheral blood mononuclear cells from both UC patients and normal donors [15]. However, monocytes from normal donors carrying allele IL-1RN*2 revealed an increased IL-1ra secretion [14]. Thus, the effects of IL-1RN*2 on IL-1ra production are contradictory. In a Finnish population, Santtila et al. found allele IL-1RN*2 to be associated with enhanced IL-1ß production in vitro, irrespective of the presence or absence of IL-1B alleles [23].
Recently, within the IL-1 gene cluster (less abundantly) and in human tissues, variably expressed paralogues of the IL-1RN gene IL-1HY1 (IL-1H3, IL-1RP3, IL-1
, FIL1
, IL-1L1) [24], IL-1HY2 [25], IL-1H1 (IL-1RP2, IL-1
) [26], FIL1
, FIL1
, and FIL1
(IL-1H4, IL-1RP1) [27] have been found. Given the strong linkage disequilibrium in the British Caucasian population between distinct markers in this region, these genes may contribute or may even themselves be causative to the disease [28].
Additional research is necessary to confirm the association between AS and polymorphisms in the IL-1RN gene and genes in the region, in larger case-control and family-based studies in different ethnic groups. Further, it needs to be determined whether IL-1RN*2 and/or allelic combinations of the IL-1RN and IL-1B gene polymorphisms are associated with disease severity in AS, as measured by the appropriate indices.
In animal models, gene therapy with IL-1ra has proven to be effective against arthritis [29]. Randomized clinical trials in rheumatoid arthritis with recombinant human IL-1ra demonstrated modest anti-inflammatory effects and significant slowing in the rate of joint damage [30]. This may suggest that in certain patients the lack of production of IL-1ra, the natural antagonist of IL-1ß, contributes to the chronicity of the inflammation.
At present, the therapeutic options in AS are limited. Treatment of AS patients with recombinant human IL-1ra or gene therapy with IL-1ra might broaden these options. Therefore, future research into the therapeutic intervention of AS with recombinant human IL-1ra and IL-1ra gene therapy is necessary. Due to the costs and limited availability of these interventions, eligible patients should be selected for the presence of polymorphic genes of the IL-1 family.
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Acknowledgments
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The authors thank Ms P. Nosti-Escanilla for typing control subjects for the IL-1B511 polymorphism.
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Notes
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Correspondence to: I. E. van der Horst-Bruinsma, Vrije Universiteit Medical Centre, room 4A-U2, PO Box 7057, 1007 MB Amsterdam, The Netherlands. 
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Submitted 27 March 2002;
Accepted 16 May 2002