Tumour necrosis factor receptor II polymorphism and juvenile idiopathic arthritis

Paediatric Rheumatology/Series Editor: P. Woo

E. Zeggini, W. Thomson, A. Alansari, W. Ollier and R. Donn The British Paediatric Rheumatology Study Group*

ARC/EU, Stopford Building, Oxford Road, Manchester M13 9PT, UK

Abstract

Objectives. Juvenile idiopathic arthritis (JIA) is a complex polygenic disorder. The encouraging outcome of anti-tumour necrosis factor (TNF) treatment, as well as serological studies, has implicated TNF and its receptors (TNFRI and TNFRII, or TNFRSF1B) in the pathogenesis of JIA. The purpose of this study was to investigate the exon 6 TNFRII single nucleotide polymorphism (SNP) in a well-defined UK cohort of JIA patients, using case–control association analysis.

Methods. A total of 435 patients, spanning seven JIA subgroups, and 261 healthy individuals were screened for the polymorphism using the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method.

Results. No significant differences were observed between the SNP allelic or genotypic frequencies of patients and controls, or between JIA subgroups.

Conclusions. This TNFRII exon 6 SNP does not seem to be associated with susceptibility to JIA.

KEY WORDS: Complex genetic disease, Juvenile idiopathic arthritis, Single nucleotide polymorphism, Tumour necrosis factor receptor II.

Juvenile idiopathic arthritis (JIA) is the commonest arthritic condition of childhood and encompasses a heterogeneous group of syndromes, in which the onset of inflammatory arthritis occurs before the age of 16 yr with a minimum duration of 6 weeks [1]. JIA appears to be a polygenic disorder, with environmental factors playing a role in its aetiology [2, 3]. The attempt to dissect the genetic basis of JIA has primarily revealed disease associations with major histocompatibility complex (MHC) loci [4]. JIA phenotypes have well-documented HLA associations. Numerous reports have confirmed the involvement of diverse HLA-DRB1 alleles [5, 6]. Other molecules, however, have also been implicated in the aetiopathogenesis of JIA both through genetic and serological studies. The latter have demonstrated the involvement of tumour necrosis factor (TNF) and its receptors (TNFRs) in the aetiopathogenesis of the disease. The levels of both TNF and its receptors have been shown to fluctuate with disease in the serum and synovial fluid (SF) of JIA patients [79]. Systemic JIA patients exhibit the most pronounced elevations of some cytokines, followed by poly- and oligoarticular cases. The levels of expression of TNFRs have been found to be greater in SF than in serum, with TNFRII being a more reliable and sensitive marker of activity in oligoarticular arthritis. TNFR levels are generally shown to be elevated in all subgroups [10], hence emphasizing the need for more intensive studies into possible functional polymorphisms in the important TNF/TNFR system. The objective of this study was to examine the potential association of one such polymorphism in TNFRII with JIA.

TNF exerts its effects through two cell surface receptors of 55 and 75 kDa (TNFRI and TNFRII, respectively) [11]. When engaged, these receptors activate downstream pathways, leading to the induction of pro-inflammatory and immunomodulatory genes. A further role played by the TNFRs was discovered by cytotoxic assays, showing that soluble receptors may act as TNF inhibiting factors [12]. The soluble TNFRs (sTNFRs) neutralize the biological activity of their ligand in the fluid phase [6] by competing with their membrane-bound counterparts. TNF itself is a potent inducer of sTNFR production, both in vivo and in vitro, as are a variety of other stimuli [13].

TNFRII is a type I transmembrane protein. The TNFRII gene has been localized to chromosome 1p36.2 and has been found to span approximately 43 kbp, consisting of 10 exons and nine introns [14, 15]. The introduction of etanercept (p75:Fc fusion protein) as a treatment in JIA has rendered encouraging results [16], thereby driving the investigation of the receptor's possible involvement in disease processes. Although analysis of the locus has identified a number of possible polymorphic sites, only three of these variations in exons 4, 6, and 9 lead to a non-conservative amino acid change [15]. The exon 6 variation is a T to G single nucleotide polymorphism (SNP) that results in the substitution of methionine by arginine in codon 196. The altered amino acid is situated in the extracellular region of the receptor, responsible for its proteolytic cleavage and solubilization. This polymorphism has been found to be associated with systemic lupus erythematosus and familial rheumatoid arthritis, which are diseases of autoimmune pathology [17, 18]. The contribution of this polymorphism to JIA aetiology was the subject of investigation in this case–control association study.

Patients and methods

The patient group studied consisted of 435 JIA cases from the British Paediatric Rheumatology Group National Repository for JIA. All cases were UK Caucasians and fulfilled the International League of Associations for Rheumatology (ILAR) classification criteria for JIA. According to the classification system proposed by ILAR, JIA can be divided into eight subgroups [1]: oligoarthritis affects four or less joints during the first 6 months of disease and is further distinguished into persistent oligoarthritis (remaining as four joints) and extended oligoarthritis (affecting a cumulative total of at least five joints after the first 6 months); polyarthritis affects five or more joints during the first 6 months of disease and is divided into rheumatoid factor (RF)-positive and RF-negative polyarthritis, depending on the presence of RF; systemic arthritis is accompanied by or succeeds daily fever enduring for at least 2 weeks, quotidian in nature, and presents with at least one of transient evanescent rash, lymphadenopathy, hepatomegaly, splenomegaly, or serositis; enthesitis-related arthritis and psoriatic arthritis constitute two further JIA subgroups; other arthritis denotes the final JIA category that includes unclassifiable cases due to the failure of their diagnosis to comply with one of the other JIA subsets or because of the fulfilment of more than one set of criteria [1]. Patients categorized as unclassifiable were omitted from this study. A set of 261 healthy and unrelated Caucasian individuals were used as controls. These subjects were derived from the Norfolk Arthritis Register control collection [19] and from a blood donor reference data set. As the T to G substitution in exon 6 abolishes a recognition site for the restriction enzyme NlaIII, the control and patient groups were screened for this SNP using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP)-based assay. A 242 bp long segment of the TNFRII gene was amplified, utilizing the primer sequences stated in [17] and a modification of the PCR conditions described in [20] (50 ng of genomic DNA were amplified using primer concentrations of 100 µM at an annealing temperature of 64°C over 40 PCR cycles). Seven microlitres of the 242 bp PCR product was then digested with 30 U of NlaIII enzyme at 37°C over 48 h. The digestion product was analysed on a 3% agarose gel stained with ethidium bromide. The presence of the T allele resulted in an uncut, full-length product, while the presence of the G allele was detected as fragments of 109 and 133 bp on the gel pattern obtained. In order to confirm genotype assignment, seven representative samples, derived from both the patient and the control groups, were sent for direct sequencing.

Results

The data were statistically analysed with the aid of the Stata computer program [21]. The control samples were tested and found to comply with the Hardy–Weinberg equilibrium law. Allele and genotype frequencies were generated for analysis, as shown in Table 1Go. These were subsequently compared between JIA ILAR subgroups by means of the {chi}2 test. The rationale behind this analysis was based on the fact that separate comparisons between each JIA subset and controls would be accompanied by the risk of type I error generation. Comparison of the allelic and genotypic frequencies between the ILAR-defined subgroups gave rise to non-significant differences (P=0.52 and P=0.48, respectively). Furthermore, neither allelic (P=0.09) nor genotypic frequencies (P=0.14) exhibited significant differences when JIA patients as a whole were compared with the controls. The DNA sequencing results confirmed the RFLP data. In addition, alignment of both the forward and the reverse sequences in Clustalw, which is a multiple sequence alignment bioinformatics tool, identified a further possible polymorphic position, 63 bp upstream of the SNP under study. This apparent C to T substitution was in linkage disequilibrium with the codon 196 polymorphism in the seven sequenced samples. It is situated in intron 5 of the TNFRII gene, just 27 bp upstream of the first expressed nucleotide of exon 6. Its SNP status has to be confirmed by typing higher numbers of samples.


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TABLE 1.  TNFRII exon 6 polymorphism allele and genotype frequencies in JIA subgroups and controls, expressed as percentages

 

Conclusions

Although the exact role of TNFRII in JIA aetiopathogenesis has not been elucidated, several lines of evidence indicate that it is involved in the inherent imbalance of cytokines observed in the disease [710, 16, 22, 23]. In this study, we employed a case–control association analysis approach for the exon 6 polymorphism. This SNP does not appear to be associated with JIA or any of its subgroups in UK Caucasian patients. This finding, however, does not preclude the contribution of other polymorphisms within the same gene to disease susceptibility. On the other hand, the increased levels of serum and SF TNFRII could be caused by defects in the ligand, TNF itself, or even by variations in regulatory molecules responsible for transcriptional, translational, or post-translational events. It is difficult to pinpoint the contributory molecule(s), as the TNF/TNFRs pathway is complex and includes numerous components that play parts in feedback loops and signalling cascades. The alleviating effects of etanercept, which binds TNF and thus inhibits its activity, may be due to the fact that it acts on the top of the pathway, preventing the expression or up-regulation of the true gene carrying the deleterious phenotype.

In conclusion, further research studying other TNFRII polymorphisms should be carried out, in order to establish whether it constitutes a susceptibility gene for JIA. Moreover, members of the TNF/TNFRs pathway have to be examined for functional aberrations that may culminate in the observed phenotypes. This study has shown that the TNFRII exon 6 polymorphism is not associated with susceptibility to JIA, a disease with complex clinical presentation, mirrored by the intricate genetics that govern it.

Acknowledgments

The contributors to the British Paediatric Rheumatology Study Group are: Dr M. Abinun, Dr M. Becker, Dr A. Bell, Professor A. Craft, Dr E. Crawley, Dr J. David, Dr J. Gardener-Medwin, Dr J. Griffin, Dr A. Hall, Dr M. Hall, Dr A. Herrick, Dr P. Hollingworth, Dr L. Holt, Dr S. Jones, Dr G. Pountain, Dr C. Ryder, Professor T. Southwood, Dr I. Stewart, Professor P. Woo, Dr S. Wyatt and Dr H. Venning.

Notes

Correspondence to: E. Zeggini. Back

* The contributors to the British Paediatric Rheumatology Study Group are listed in the Acknowledgements. Back

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Submitted 19 March 2001; Accepted 28 September 2001





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