Royal National Hospital for Rheumatic Diseases and Department of Medical Sciences, University of Bath, Bath and 1National Heart and Lung Institute, Imperial College of Science and Medicine, 1B Manresa Road, London, UK.
Correspondence to: N. J. McHugh, Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath BA1 1RL, UK. E-mail: N.J.McHugh{at}bath.ac.uk
Abstract
Objectives. To investigate polymorphisms of interleukin (IL) 1, IL-1ß and IL-1 receptor R1 genes in patients with psoriatic arthritis (PsA), their relationship to the age of onset of psoriasis and the pattern of joint involvement.
Methods. One hundred and forty well-characterized patients with PsA were studied. One hundred healthy controls were recruited from primary care. All were genotyped for single-nucleotide polymorphisms in the genes for IL-1 (position 889), IL-1ß (position +3953) and IL-1R1 (position +970). The frequencies of the respective variants were compared between patients and controls and in relation to age of onset of psoriasis, to clinical subsets of the disease and to the presence of erosions.
Results. All three polymorphisms were in HardyWeinberg equilibrium in both patients and controls. The frequency of IL-1 889 CC homozygotes was significantly increased in PsA patients compared with normal controls [58 vs 40%, odds ratio (OR) 2.06, 95%, confidence interval (CI) 1.223.47]. The frequency of the IL-1
889 C allele was significantly increased in PsA patients compared with controls (75 vs 65%, OR 1.65, 95% CI 1.112.45). In subset analysis there were no other significant differences in allelic frequencies for the IL-1
889 C/T, IL-1ß +3953 C/T and IL-1R1 +970 C/T polymorphisms.
Conclusions. The IL-1 gene complex may play a role in the development of PsA and/or psoriasis or act as a marker for other genes on chromosome 2q12 to 2q13.
KEY WORDS: Psoriatic arthritis, Cytokine gene, Interleukin 1.
Psoriatic arthritis (PsA) is an inflammatory disorder that occurs in 742% of patients with psoriasis [1]. This skin disease has been classified into type 1 or early-onset psoriasis (age of onset before 40 yr), which is associated with a family history of psoriasis, and type 2 or late-onset psoriasis (age of onset after 40 yr), which is thought to be sporadic [2]. The joint disease is characterized by diverse pathological processes, including synovitis, enthesitis, bone and cartilage resorption and bone and periosteal proliferation. Long-term follow-up studies support the concept of at most two major subgroups, based on axial vs peripheral joint disease involvement [3]. However, frequent distal interphalangeal joint involvement of the hands and feet help distinguish PsA from other forms of chronic inflammatory arthritis.
The aetiology of PsA and psoriasis is unknown, although familial aggregation and twin studies suggest that genetic factors play an important role in the development of both diseases. The prevalence of PsA among first-degree relatives of patients with PsA is 5.5% compared with an estimated 0.3% in the normal population [4], and in psoriasis there is a higher concordance rate in monozygotic twins (6572%) than in dizygotic twins (1530%) [5]. In psoriasis, the strongest association occurs with the PSORS1 gene locus on chromosome 6 [6], which may explain associations between type 1 psoriasis and HLA-Cw6 [7]. However, the percentage of patients with psoriasis who are Cw6-positive ranges between only 10 (Japanese) and 45 (Caucasian) and the percentage of patients with PsA who are Cw6-positive is only 17 [810]. There are weaker associations between PsA and MHC class II genes, for example HLA-DR7 and HLA-DR4 [11, 12]. Genome-wide mapping in psoriasis has demonstrated susceptibility loci on several chromosomes, including 1, 2, 3, 4, 6, 15 and 17, implying a significant contribution from non-MHC genes in psoriasis and possibly PsA [1316].
Cytokines are thought to play a pivotal role in both psoriasis and PsA, and the genes that encode them have been investigated as potential candidate genetic markers for disease susceptibility and severity in both conditions. Interleukin (IL) 1 is a cytokine with potent proinflammatory and immunomodulatory effects that is secreted by monocytes/macrophages. IL-1 occurs in two forms, IL-1 and IL-1ß, IL-1ß being the major extracellular agonist [17]. The biological action of IL-1
and IL-1ß is signalled by combining with the type I IL-1 receptor (IL-1R1) [18]. The type II IL-1 receptor (IL-1R2) does not transmit any signal and functions as a cell bound inhibitor of IL-1. The action of IL-1 is also inhibited by an endogenous inhibitor, the IL-1 receptor antagonist (IL-1ra). IL-1ra shares a high degree of homology with IL-1
and IL-1ß and binds both receptors, but again does not transmit any signal [17]. IL-1 has been shown to have a role in bone and cartilage resorption and synovial inflammation in murine models of arthritis [19]. The binding of IL-1 by IL-1ra reduces joint erosion in patients with rheumatoid arthritis [20]. Increased expression of IL-1
and IL-1ß has been demonstrated in the serum, synovial fluid, synovium and skin of patients with PsA [21, 22].
The loci for human IL-1, IL-1ß, IL-1R1 and IL-1ra gene are found as a cluster on chromosome 2q12 to 2q13 [23]. Biallelic polymorphisms at positions IL-1
889, IL-1ß +3953 and IL-1R1 +970 have been described. Polymorphisms of the IL-1 gene complex, including IL-1
889 and IL-1ß +3953, are thought to influence IL-1 expression [24, 25]. Positive associations have been observed between alleles from this gene cluster and rheumatoid arthritis [26], psoriasis [27], ankylosing spondylitis [28] and ulcerative colitis [29]. A recent study using multigene microarray analysis and reverse transcriptionpolymerase chain reaction to analyse peripheral blood mononuclear cells suggested that there was increased IL-1ß gene expression in PsA patients compared with normal controls [30]. Given the putative role of IL-1 in PsA and the observed associations between the IL-1 gene cluster and related diseases, it is plausible that IL-1 genes may act as markers for disease susceptibility, pattern and severity in PsA.
The objective of this study was to determine whether known polymorphisms within the IL-1, IL-1ß and IL-1 receptor gene loci influence susceptibility to PsA or are associated with particular disease subtypes, joint erosions or the onset of psoriasis.
Materials and methods
Patients and controls
One hundred and forty unselected Caucasian patients with PsA were studied. All had been referred to a PsA clinic at the Royal National Hospital for Rheumatic Diseases. All patients were negative for rheumatoid factor and fulfilled the Moll and Wright criteria for the diagnosis of PsA [31]. One hundred healthy Caucasian control subjects from a general practice were used as controls for the IL-1, IL-1ß and IL-1 receptor gene polymorphisms. With these numbers there is 80% power to detect an increase from 15 to 30% in the frequency of any given genotype at the 5% level of significance.
Clinical data were collected prospectively on patients with PsA at each clinic visit according to a standard protocol. Information included the age of onset of skin psoriasis and arthritis, the presence, site and number of inflamed joints and the presence or absence of radiologically determined joint erosions. For the purpose of this study, patients were divided into two main subgroups: spinal disease or spondylitis and peripheral arthritis (non-spinal disease). Spondylitis was defined as a history of inflammatory back pain and radiological evidence of sacroiliitis (grade 2 or greater) or syndesmophytes.
Ethical approval for the study was given by the Bath Regional Ethics Committee and informed written consent was obtained from all participants.
Genetic analysis
Genomic DNA was prepared from whole peripheral blood collected into EDTA (ethylenediamine tetraacetate) tubes using a standard salting-out method. The genomic DNA extracted was used to type individuals for single-nucleotide polymorphisms at IL-1 (889 C/T, 5' flanking region), IL-1ß (+3953 C/T, exon 5) and IL-1R1 (+970C/T, 5' uTR) using the polymerase chain reaction with sequence-specific primers (PCR-SSP), as described previously [32, 33]. The PCR products were analysed on 2% agarose gel stained with ethidium bromide.
Statistical analysis
All data were recorded on a Microsoft Excel spreadsheet. Allele and genotype frequencies were tested for HardyWeinberg equilibrium using the 2 test. Comparisons of allelic and genotype frequencies between patients with PsA and controls were made using 2 x 2 contingency tables with
2 analysis. If the numbers of patients and controls were less than 10, Fisher's exact test was used. Comparisons of genotype distribution between patients with PsA and controls were made using 2 x 3 contingency tables with
2 analysis. The P values were corrected by the Bonferroni method, Pcorr = 1 (1 Puncorr)n, where n = number of loci [33]. Odds ratios (OR) and their 95% confidence intervals (CI) were also calculated. In subset analysis, allele frequencies were compared between type 1 and type 2 psoriasis, between spinal disease and non-spinal disease, and between erosive and non-erosive arthritis. Linkage disequilibrium between pairs of polymorphic loci was estimated by calculating the relative linkage disequilibrium value (Drel) where Drel = Dij/Dmax [34].
Results
Of the 140 patients with PsA studied, 69 were males and 71 females (mean age 55 yr, range 2893 yr). The mean disease duration of arthritis was 18 yr (range 558 yr). Eighty-four patients had early onset of psoriasis. Forty patients had spinal disease. Erosions of peripheral joints were present in 82 patients.
Genetic results
All three polymorphisms were in HardyWeinberg equilibrium in both patients and controls. Allele frequencies for the genes studied in PsA patients and controls are shown in Table 1.
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Discussion
In this study we observed a significant increase in the frequency of carriage of the 889 C homozygous genotype of the IL-1 gene in patients with PsA, which has not been reported previously. The OR for this observation, however, suggests that any association between PsA and the IL-1 gene cluster is likely to be modest and may be explained in part by minor but non-significant deviation of the control population from HardyWeinberg equilibrium. Nevertheless, links between psoriasis and the IL-1 gene complex have been reported. Tarlow et al. [27] found a positive association between an intronic polymorphism of the gene coding IL-1ra (IL-1RN) and early-onset psoriasis. Reich et al. [35] observed no association of the IL-1ß +3953 C/T polymorphism with psoriasis. In contrast, we found a trend towards increased IL-1ß +3953 CC homozygotes in late-onset psoriasis. It may be argued that our observations and the above studies reflect a link between the IL-1 gene complex and skin disease only, rather than joint disease. However, in a study of gene expression profiles in patients with psoriasis, the IL-1 gene was not differentially expressed in psoriatic skin compared with normal skin [36]. Another study using a 588-gene microarray analysis of peripheral blood mononuclear cells of patients with inflammatory arthritis including PsA showed that the IL-1ß gene was more highly expressed in PsA patients compared with normal controls, suggesting a possible role for the IL-1 gene complex in PsA [30]. Genome-wide searches in multiplex families have also failed to identify a susceptibility locus for skin psoriasis alone on chromosome 2q12 to 2q13.
A susceptibility locus for ankylosing spondylitis, however, has been found 0.3 centimorgans from the IL-1 gene complex [37] and may explain some of the trends we found between the IL-1 889 CC and IL-1ß +3953 CC genotypes and spinal disease in PsA. The association of the IL-1
889 CC and IL-1ß +3953 CC genotypes also reflects the tight linkage disequilibrium that we found between these two loci, which has been documented previously [33]. There have been no previous reports of an association of IL-1 genes with spinal disease in PsA. Indeed, no association was observed between IL-1
889 and IL-1ß +3953 polymorphisms and ankylosing spondylitis in two separate studies, although both groups found a positive association of the IL-1RN gene with ankylosing spondylitis [28, 38]. Phenotypic differences between the spinal disease of PsA and ankylosing spondylitis and, in particular, the prevalence of concomitant peripheral disease (85% in our PsA group vs 30% in the ankylosing spondylitis group) may explain the genotype differences. Nevertheless, the above trends in spinal disease in our study were not statistically significant and associations of the IL-1RN gene observed in ankylosing spondylitis were small, implying a modest role for this locus in the development of spinal disease.
We found no difference between PsA and controls in the allelic frequencies of the IL-1R1 +970 C/T polymorphism and no differences in the subset analysis. There have been no previous studies of this polymorphism in psoriasis, PsA or any of the other spondyloarthropathies. It may be that our study was powered to detect large genetic effects and minor effects may have been missed.
Increased amounts of IL-1 have been reported in psoriatic skin, synovial fluid and synovium [21, 22]. IL-1 and IL-1ß production shows interindividual variation, with high and low producers. Polymorphisms of the IL-1 gene complex are of considerable interest as they are thought to influence levels of IL-1 secretion. Most studies of IL-1 production have measured levels of IL-1ß (the main extracellular agonist) and IL-1ra (the main IL-1 antagonist). The influence of IL-1
promoter gene polymorphisms on IL-1 secretion has not been studied in psoriasis and PsA. However, healthy individuals homozygous for the IL-1
889 T allele had increased IL-1ß plasma levels compared with other genotypes [24]. The IL-1ß +3953 C/T polymorphism has been studied in psoriasis and been shown not to influence IL-1ß or IL-1ra secretion levels in an in vitro model [35]. However, in rheumatoid arthritis, carriage of the IL-1ß +3953 T allele was associated with lower IL-1ra plasma levels [25]. The above studies seem to be counterintuitive to the findings in our study, in which the C allele of the IL-1
889 polymorphism and the C allele of the IL-1ß +3953 polymorphism appear to be associated with PsA and/or psoriasis. However, more recent studies by Vamvakopoulos et al. [39] found that an intronic polymorphism encoding IL-1ra was the principal regulator of both constitutive and stimulated IL-1ra and IL-1ß. The second intron of the IL-1RN gene has a variable-number tandem repeat with up to five variants depending on the number of repeats of the 86-base-pair fragment. Allele 2 of the IL-1RN gene (IL-1RN*2), which has two repeat sequences, was associated with higher IL-1ra and lower IL-1ß release. Given the associations of the IL-1RN gene with ankylosing spondylitis and psoriasis, further studies of the IL-1RN gene in PsA would be of interest.
The determination of the genetic contribution to the development of PsA is challenging on account of a number of confounding variables. There is strong evidence for a genetic predisposition to psoriasis alone that may mask those genetic factors important for the development of PsA, and few studies have compared patients with skin disease alone and those with PsA. Although most would accept PsA as a distinct disease entity, there can be phenotypic overlap with rheumatoid arthritis, ankylosing spondylitis, reactive arthritis, Reiter's disease and arthritis associated with inflammatory bowel disease. Although not all these confounding factors have been overcome in the present study, we have concentrated on investigating candidate genes in two broad categories of patients with PsA that cover the disease spectrum. We have observed non-MHC associations between the IL-1 gene complex and PsA and/or psoriasis, implying either that this region has a direct role in disease susceptibility and severity or that these genes may act as markers for other genes between 2q12 and 2q13. Further studies will need to be done to confirm these observations and also to investigate the role of the IL-1RN gene in PsA.
Conflict of interest
The authors have declared no conflicts of interest.
Acknowledgments
The authors are grateful to the Royal National Hospital for Rheumatic Diseases, the NHS Executive South West Research and Development Directorate, and Remedi Trust UK for funding the work.
References
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