Association of specific interleukin 1 gene cluster polymorphisms with increased susceptibility for Behçet's disease

J. Karasneh, A. H. Hajeer, J. Barrett1, W. E. R. Ollier, M. Thornhill2 and A. Gul3

ARC Epidemiology Unit, University of Manchester, Manchester,
1 ICRF Genetic Epidemiology Laboratory, St James's University Hospital, Leeds,
2 Oral Disease Research Centre, Bart's and the London, Queen Mary's School of Medicine and Dentistry, UK and
3 Division of Rheumatology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. The aim of this study was to investigate if the inheritance of specific polymorphisms of interleukin 1 (IL-1) A, IL-1B and IL-1 receptor antagonist (IL-1RN) genes could affect the susceptibility to Behçet's disease (BD).

Methods. A total of 132 BD patients and 105 healthy controls were genotyped for IL-1A -889, IL-1B -511, -35, +5810, +5887, and IL-1RN +8006, +8061, +9589, +11100 single nucleotide polymorphisms, and IL-1RN 86-bp variable number of tandem repeat polymorphism. {chi}2-analysis was used to compare the allele and genotype frequencies of the cases and controls. IL-1A and IL-1B haplotypes were reconstructed using the Phase program.

Results. Inheritance of the C allele of the IL-1A -889 polymorphism was associated with BD (OR=2.0, P=0.01) and inheritance of the IL-1A -889C/IL-1B +5887T haplotype was identified as an increased risk for BD. The IL-1A -889 and IL-1B +5887 CC/TT combined genotype was significantly more observed in BD cases than in controls (57.5 vs 38.1%, OR=2.2, P=0.003). No association with BD was found for other investigated polymorphisms in the IL-1B and IL-1RN genes.

Conclusion. Susceptibility to BD is increased in individuals carrying both the IL-1A -889C and IL-1B +5887T haplotype. Individuals who are both homozygous CC at IL-1A -889 and TT at IL-1B +5887 appear to have twice the risk of developing BD as individuals having other IL-1A -889/IL-1B +5887 genotypes.

KEY WORDS: Behçet's disease, Interleukin 1, IL-1 receptor antagonist, Single nucleotide polymorphism, Haplotype.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Behçet's disease is an unclassified systemic vasculitis of unknown cause with recurrence of various symptoms and a chronic course. Behçet's disease was originally described as a triad of oral and genital ulcers and uveitis, but is now recognized as a systemic disease affecting all types and sizes of blood vessels, eyes, central nervous system, joints, lungs and intestines [13].

Behçet's disease has a worldwide distribution, but it is most common in Japan, the Middle East and the Mediterranean, i.e. countries along the ancient ‘Silk Road’. The prevalence of Behçet's disease in Turkey is particularly high at 0.8–3.7/1000 [4, 5]. Onset of the disease is usually in the third and fourth decade of life and the male to female sex ratio is approximately equal, although the disease often has a more severe course in males.

The cause of Behçet's disease remains to be elucidated. Environmental factors including streptococcal and viral infection, directly or by cross-reacting with the heat shock proteins have been suggested as triggering factors. Genetic factors are also implicated in the pathogenesis. There is a strong association with HLA-B51 [6, 7] and an increased incidence in close family members. Gul et al. [8] have calculated a sibling risk ratio ({lambda}s) of 11.4–52.5 in Turkish patients with Behçet's disease.

Immunological investigations have demonstrated the presence of immune dysregulation among Behçet's disease patients [9]. Lymphocytes from patients with Behçet's disease can generate supernatants with greater neutrophil-potentiating activity compared with control lymphocytes [10]. Chemotactic activity of polymorphonuclear leucocytes in patients with Behçet's disease has also been reported to be increased [11]. Since cytokines are involved in the regulation of functions of lymphocytes and phagocytes, they may play an important role in the pathogenesis of Behçet's disease [1215]. The serum concentrations of mainly TH1-type cytokines have been reported to be increased in Behçet's disease, including interleukin 1 (IL-1) [1315].

The IL-1 gene cluster on chromosome 2q12-q13 contains three related genes IL-1A, IL-1B and IL-1RN, encoding the pro-inflammatory cytokines IL-1{alpha}, IL-1ß and their endogenous receptor antagonist IL-1ra [16] (Fig. 1Go). IL-1{alpha} and IL-1ß are potent pro-inflammatory cytokines and play a central role in many inflammatory cascades. They also activate endothelial cells, and among other things, induce the expression of adhesion molecules that mediate the attachment and transendothelial migration of leucocytes at inflammatory sites [1720].



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FIG. 1. The polymorphic loci in the IL-1 gene cluster with approximate physical distances.

 
Given the involvement of IL-1 in Behçet's disease, the aim of this study was to investigate the possibility that inheritance of specific polymorphisms of IL-1A, IL-1B and IL-1RN genes might be associated with increased susceptibility to Behçet's disease.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
EDTA-anticoagulated blood samples were obtained from 132 unrelated Behçet's disease patients (82 male and 50 female) of Turkish origin, in compliance with the principles of the Declaration of Helsinki. The diagnosis of Behçet's disease met the criteria of the International Study Group in all cases [3]. Ethnically matched 106 healthy volunteers (61 male, 44 female) who had no Behçet's disease-related manifestations, including recurrent aphthous stomatitis or a family history for Behçet's disease, comprised the control group. Genomic DNA was extracted from blood samples using the DNAce maxiblood purification system (Bioline, London, UK).

Genotyping
Genotypes for previously described IL-1A, IL-1B and IL-1RN gene polymorphisms were assigned to patients and controls. These included single nucleotide polymorphisms of IL-1A -889 C/T [21], IL-1B -511 C/T [22], -35 T/C [23], +5810 G/A [23] and +5887 T/C [23] and IL-1RN +8006 T/C [23], +8061 C/T [23], +9589 A/T [23], +11100 T/C [23] and 86-bp variable number of tandem repeat (VNTR) polymorphism of IL-1RN [24]. DNA (100–150 ng) was amplified by polymerase chain reaction (PCR) using the forward and reverse oligonucleotide primers described in the references given above. PCR reactions for IL-1A, IL-1B and IL-1RN were performed in 25-µl volumes using 1x KCl or 1x NH4 buffer with optimized MgCl2 concentration, 100 µM dNTPs, 6 pmol primers, 1U Taq polymerase (Bioline), and for some reactions 1x 4% betain or 1x 50% glycerol were added. Amplifications were performed using a Hybaid Omnigene Thermal Cycler (Teddington Middlesex, UK) at the relevant annealing temperature and for 35 cycles. Genotypes were assigned by restriction enzyme digestion of the PCR products. Each product was digested with 4 U of the appropriate restriction enzyme in a 15-µl volume overnight. PCR-digested products were run out by electrophoresis on a 3% agarose gel stained with ethidium bromide and visualized under UV light. PCR products for the IL-1RN 86-bp VNTR were run directly on 2% agarose gels.

Statistical analysis
Each polymorphism was examined in the control population to confirm that the distribution of the genotypes conformed to Hardy–Weinberg expectations. The distribution of alleles and genotypes for each polymorphism was compared between cases and controls using a {chi}2-test. The Estimated haplotype (EH) program [25] was used to calculate the linkage disequilibrium between the polymorphic sites, and the EH and Phase [26] programs were used to reconstruct haplotypes. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to assess the risk associated with particular alleles, genotypes or haplotypes. Calculations were performed using Stata version 6 (Stata Corporation, USA).


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The distribution of IL-1A -889 alleles and genotype frequencies were significantly different between cases and controls. This was reflected by an increase in both the -889C allele and CC genotype frequencies in patients with Behçet's disease compared with controls (76.6 vs 42.9%, OR=1.7, P=0.01, and 76 vs 45%, OR=1.95, P=0.012, respectively) (Table 1Go).


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TABLE 1. Allele and genotype frequencies of IL-1A gene -889*C/T polymorphism in patients with Behçet's disease and controls

 
In the IL-1B gene, there was a specific increase in both +5887T allele and TT genotype frequencies in cases vs controls (83.5 vs 76.2%, OR=1.6, P=0.05, and 92 vs 60%, OR=1.8, P=0.03, respectively) (Table 2Go).


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TABLE 2. IL-1B polymorphisms in Behçet's disease patients (BD) and controls

 
Examination of other markers in the IL-1B and IL-1 RN genes revealed that the overall distribution of alleles and genotypes in patients with Behçet's disease and controls were not significantly different (Tables 2Go and 3Go).


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TABLE 3. IL-1 receptor antagonist gene (IL-1 RN) single nucleotide and variable number of tandem repeat polymorphisms in Behçet's disease (BD) patients and controls

 
As both the IL-1A -889C/-889C and IL-1B +5887T/+5887T genotypes were found to be individually associated with Behçet's disease, we tested the possibility of linkage disequilibrium between the two polymorphic sites. The EH program revealed a D value of 0.79 suggesting the presence of linkage disequilibrium between IL-1A -889 and IL-1B +5887 (P < 0.0001) (data not shown for other polymorphic sites). The frequencies of the reconstructed IL-1A -889 and IL-1B +5887 haplotypes using both the EH and Phase software packages are given in Table 4Go. The IL-1A -889C/ IL-1B +5887T haplotype was inherited more often in cases than controls. When the inheritance of combined IL-1A -889C/-889C and IL-1B +5887T/+5887T genotype (CC/TT) was compared with inheritance of any other IL-1A -889/ IL-1B 5887 genotypes, there was a significant difference between cases and controls. Of 127 cases, 73 had the CC/TT genotype compared with only 40 of 105 controls (OR=2.2, CI=1.3–3.7, P=0.003).


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TABLE 4. The frequencies of IL-1A -889 and IL-1B +5887 haplotypes reconstructed using the EH and Phase programs

 


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Although the aetiology of Behçet's disease is not yet known, genetic predisposition and immune dysregulation seem to be important factors in the pathogenesis. Serum levels of pro-inflammatory and TH1-type cytokines, including IL-1, have been reported to be elevated in Behçet's disease [1215]. In addition, peripheral blood monocytes from patients with Behçet's disease produce significantly more IL-1 when stimulated with bacterial lipopolysaccharide than monocytes from normal controls or from patients with other inflammatory diseases [12, 27]. Hence, we have hypothesized that increased IL-1 production in Behçet's disease could be due to functional polymorphisms encoding differential gene regulation and expression.

This study demonstrates a significant association between the IL-1A -889 polymorphism and Behçet's disease. This polymorphism lies upstream in the 5' regulatory region in a sequence involved in transcriptional silencing of the IL-1A gene [21]. The IL-1A -889 polymorphism has been reported to be associated with a number of inflammatory diseases including juvenile rheumatoid arthritis [21] and Alzheimer's disease [28]. However, in both studies the disease was associated with increased frequency of the IL-1A -889T allele. In contrast, we found an increase in the -889C allele and CC genotype in patients with Behçet's disease compared with controls. One possible explanation is that the over-representation of the -889C allele could be secondary to another functional polymorphism associated with Behçet's disease that is in linkage disequilibrium with the -889C allele. This putative polymorphism in linkage disequilibrium with the C, but not with the T allele, might be specific to Behçet's disease. Alternatively, this linked putative polymorphism might be the same one as in juvenile rheumatoid arthritis or Alzheimer's disease, but it might be in linkage disequilibrium with different IL-1A -889 alleles in different populations. Although, a G->T polymorphism at position +4845 of the IL-1A gene has been reported to be in linkage disequilibrium with the -889 polymorphism, this polymorphism causes a conservative amino acid substitution (alanine to serine) at position 114, without any known functional significance [29]. Hence, a putative functional polymorphism in linkage disequilibrium with IL-1A -889 has yet to be identified.

Of four single nucleotide polymorphisms within the IL-1B gene investigated, only the +5887 polymorphism was found to be associated with Behçet's disease. In previous studies the IL-1B +5887 polymorphism has been associated with a number of inflammatory conditions such as ulcerative colitis [30] and diabetic nephropathy [31], where the +5887T allele was increased in patients compared with controls. In this study, there was no significant difference in the overall distribution of genotype frequencies for the IL-1B +5887 polymorphism in patients with Behçet's disease. However, there was a significant increase in the specific frequency of the IL-1B +5887T allele and (TT) genotype in Behçet's disease patients compared with controls. Although this polymorphism occurs in exon 5 of the gene and represents a conservative substitution, it has been reported that the allele dosage of T at +5887 is directly related to IL-1ß expression with yet unknown mechanisms, i.e. TT>CT>CC [32].

The combined risk of carrying both homozygous IL-1A -889CC and IL-1B +5889TT genotypes (OR=2.2, CI=1.3–3.7) was higher than the individual risks of having one copy of IL-1A -889C (OR=1.7, CI 1.1–2.5) and IL-1B +5889T (OR=1.6, CI 1.0–2.5) or IL-1A -889CC (OR=1.95, CI 1.2–3.3) and IL-1B +5889TT (OR=1.8, CI 1.1–3.1) genotypes. The functional consequences of these IL-1 polymorphisms are not yet known, and the putative functional polymorphic site on the IL-1A -889C/IL-1B +5889T haplotype and the characteristics of its biological effects remains to be elucidated.

The P values reported in this study were not corrected for multiple testing and thus should be interpreted with caution. It is unclear what level of correction should be applied; i.e. whether each polymorphism should be viewed as a separate test (corrected by multiplying by 10), or if each gene should be considered separately (correction of IL-1A by multiplying by 1, IL-1B by multiplying by 4 and IL-1 RA by multiplying by 5). Given the evidence of strong linkage disequilibrium between the polymorphic sites, it is very difficult to analyse these polymorphisms independently.

In conclusion, this study suggests that IL-1 gene cluster polymorphisms are associated with an increased risk for Behçet's disease. These polymorphisms may contribute to the enhanced inflammatory reactivity observed in patients with Behçet's disease. Association of IL-1 gene cluster polymorphisms with Behçet's disease in other ethnic groups or its relationship with specific manifestations or severity of Behçet's disease needs to be further studied.


    Notes
 
Correspondence to: W. E. R. Ollier, The Centre for Integrated Genomic Medical Research, Manchester University, Medical School, Stopford Building, Oxford Road, Manchester M13 9PT, UK. E-mail: bill{at}fs1.ser.man.ac.uk Back


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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 22 July 2002; Accepted 3 December 2002





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