Ankylosing spondylitis (AS) is a common inflammatory disease that causes important structural and functional impairment in the axial skeleton.1 The aetiology of AS is determined by a strong genetic predisposition in combination with the interaction of certain environmental factors. At present, the contribution of the human leucocyte antigen (HLA) B27 allele to the genetics of AS is the best characterised. However, HLA-B27 constitutes only one-third of the overall estimated genetic risk for AS and it has been suggested that a substantial proportion of AS genetic susceptibility is encoded by non-HLA genes.2

As AS pathogenesis is characterised by an altered regulation of both innate and adaptive immune response, inflammatory mediators involved in the immunopathological cascade of this condition could be interesting functional candidate genes. Therefore, it has been suggested that the proinflammatory cytokine interleukin (IL)-1 is one of the non-HLA genetic factors that contributes to AS genetic susceptibility.2

Another important proinflammatory mediator of great relevance is IL-23.

IL-23 is primarily secreted by activated dendritic cells, monocytes and macrophages.3 IL-23 is a heterodimer composed of a subunit identical to IL-12 p40 and a novel IL-12 p35-related protein, p19. IL-23 activity is mediated by binding to the IL-23 receptor complex, which is composed of an IL-12Rβ1 and a unique cytokine receptor subunit termed IL-23R.4

Recent results have shown that the IL23R gene, coding for a subunit of the IL-23 receptor, is strongly associated with another autoimmune condition such as inflammatory bowel disease (IBD).5^–10 A coding variant (rs11209026, Arg381Gln) together with other genetic markers located in the IL23R gene and in its downstream intergenic region, were identified as having a potent protective effect against IBD susceptibility.6 The Arg381Gln non-synonymous polymorphism has also been involved in psoriasis genetic predisposition.11 Interestingly, in patients with AS the presence of skin involvement (psoriasis) and gut inflammation similar to that occurring in IBD is frequent.1 Therefore, it has been suggested that these autoimmune conditions might share common disease pathogenesis mechanisms and genetic factors.12 13

On this basis, the aim of the current study was to investigate the possible involvement of the IL23R gene as a new genetic factor for AS susceptibility.

MATERIALS AND METHODS

The study population consisted of 365 patients with AS independently recruited from three hospitals (Hospital Xeral-Calde, Lugo; Complejo Hospitalario Universitario Juan Canalejo, A Coruña; and Hospital Clínico San Carlos, Madrid) and 500 blood bank donors from the corresponding hospitals who were included as healthy controls. Patients with AS were diagnosed according to the New York modified criteria.14 Both the patient and control groups were of Spanish Caucasian origin. The control population was matched with the group of patients with AS by age and sex. All the participants of the study participants were included in the study after written informed consent.

Eight single nucleotide polymorphisms (SNPs) spanning the IL23R gene located in intronic, coding and 3′ untranslated (UTR) regions, previously described as genetic markers for IBD,5 6 were selected for our association study and were genotyped using a Taqman 5′ allelic discrimination assay as previously described (table 1).6 Only individuals with the genotype of the eight selected IL23R polymorphisms were included in the study (365 patients with AS and 500 controls).

Both allelic and genotypic frequencies were calculated and compared by χ² tests using Statcalc software (Epi Info 2002; Centers for Disease Control and Prevention, Atlanta, Georgia, USA). Significance was calculated by 2×2 contingency tables and Fisher’s exact test in order to obtain p values, odds ratios (OR) and 95% confidence intervals (CI). Statistical significance was considered when p<0.05. Haploview software was used to obtain linkage disequilibrium pairwise values. A conditional case–control test implemented in the unphased software was used as an overall test to analyse the independence of associations.

The estimation of the power of this study was performed using the Quanto v 0.5 software (Department of Preventive Medicine University of Southern California, California, USA). Considering the sample size included, a medium minor allele frequency of 0.35 and assuming an OR of 0.7 at the 5% significance level, the probability of detecting an association of IL23R and AS in our population is 80%. In addition, for the rs11209026 SNP that shows a significant lower minor allele frequency (0.06) the estimated power is 86% considering an OR of 0.5 at the 5% significance level.

RESULTS

The eight analysed IL23R genetic markers were found to be in Hardy–Weinberg equilibrium in both patients with AS and control populations. The distribution of genotypic frequencies of the eight studied IL23R polymorphisms is shown in table 1. Interestingly, two genetic variants showed statistically significant differences between patients with AS and controls by considering either allelic or genotypic frequencies. The most significant effect was observed for rs11209026 non-synonymous SNP showing that the A allele has a significant protective effect against AS development (OR 0.46 95% CI 0.2 to 0.7 p = 0.001) (table 2). Similarly, the rs1343151 A allele was associated with protection against AS (OR 0.68 95% CI 0.55 to 0.83 p = 0.0002) (table 2). In addition, we observed a borderline significant association of rs10889677 genetic variant with the same tendency observed for rs11209026 and rs1343151 polymorphisms (OR allele A 0.81 95% CI 0.65 to 0.99 p = 0. 0.039).

For the five remaining polymorphisms (rs1004819, rs7517847, rs10489629, rs11209032 and rs1495965), we did not observe statistically significant differences between patients with AS and controls considering either allelic or genotypic frequencies (table 2).

We next investigated whether the effect of the two strongest associated genetic variants, the rs11209026 non-synonymous and the rs1343151 SNPs, was independent or due to linkage disequilibrium. A low degree of linkage disequilibrium between these two IL23R genetic markers was observed (r² = 0.07). In addition, the conditional case–control analysis showed that the effect of the Arg381Gln is independent of the rs1343151 polymorphism and vice versa (conditional case–control extended p value 0.02 respectively).

DISCUSSION

Various candidate gene association studies have been carried out in order to identify the non-HLA genes involved in AS susceptibility.2 In this study we selected the IL23R gene, an excellent functional candidate, which is involved in the IL-23 proinflammatory pathway. Interestingly, the analysis of eight IL23R genetic markers revealed that the Arg681Gln non-synonymous polymorphism and the rs1343151 genetic variant are implicated in AS genetic predisposition in our population with independent effects.

A strong protective effect against AS development was conferred by the uncommon Gln allele of the Arg681Gln genetic variant. This polymorphism is located in the initial portion of the IL23R intracytoplasmic region, very close to the first putative tyrosine phosphorylation site at position 399.4 By changing the highly conserved Arg381 for Gln381 at this position, the interaction between IL23R and its associated Jack2 kinase may be modified.4 This change may have functional consequences in the IL23R transducing pathway leading to a reduction in cellular response to IL-23 and providing a possible explanation for the protective effect attributed to the infrequent Gln381 allele.

IL-23 is one of the master regulators of innate and adaptive immunity.15 The proinflammatory activities of IL-23 have been partially attributed to its ability to support the development of a novel subset of CD4+ inflammatory T cells known as Th17 cells. This subset of T cells are characterised by IL-17 production and are associated with strong proinflammatory responses and the induction of severe autoimmunity.16 Together, these data are very suggestive and point to a possible implication of the IL-23 pathway in AS pathogenesis that should be further investigated.

We tested IL23R alleles on the basis of previous evidence obtained from a reported association in other autoimmune diseases. Thus, multiple testing may not be applicable to our study. Nevertheless, the rs11209026 and rs1343151 association would still be significant for our AS study if an eightfold Bonferroni correction for multiple hypothesis testing was used.

During the course of this work, a similar study reported an initial association of IL23R gene with AS in a British population that was additionally confirmed in a replication North American cohort.17 In accordance with our results, both rs112209026 and rs1343151 showed a strong association with AS. However, in this study, five additional IL23R genetic variants that were also included in our work (rs1004819, rs10489629, rs10889677, rs11209032, rs1495965), showed a statistically significant association with AS. This apparent lack of concordance probably resides in the higher sensitivity of the study promoted by the Wellcome Trust Case Control Consortium and Australo-Anglo-American Spondylitis Consortium that included a much larger case–control set (1000 patients with AS and 1000 controls).

In the same way as that observed for AS, recent studies have found that the IL23R gene is a risk factor for IBD and psoriasis.5–11,14 The uncommon 381Gln allele together with other IL23R variants, including rs1343151, have been associated with a strong protective effect against IBD development in independent populations.5 7 In addition, the Arg381Gln coding variant have shown a significant association with psoriasis.11 However, the involvement of IL23R in the genetic basis of other autoimmune disorders is unclear. In rheumatoid arthritis two independent studies analysing the role of IL23R in disease susceptibility show controversial results, whereas in systemic lupus erythematosus the IL23R gene does not to play a major part in disease susceptibility.18^–20 These results are intriguing and resemble those obtained with the R360W PTPN22 polymorphism, which has been described as a genetic marker for rheumatoid arthritis, systemic lupus erythematosus or T1D but not for IBD, AS, coeliac disease or multiple sclerosis.21^–28 On this basis, it has been speculated that the R360W PTPN22 genetic variant defines a subgroup of autoimmune disorders with an important humoral component.29 In the same way, the IL23R Arg381Gln polymorphism could be a genetic risk factor for a subgroup of autoimmune conditions where the local but not systemic inflammation has great relevance, such as AS, IBD or psoriasis. This hypothesis is additionally supported by recent studies of animal models that have shown that IL-23 is essential for local tissue inflammation but not for systemic inflammation in mice.30

To summarise, our results together with very recent findings strongly suggest that the IL23R gene seems to be one of the genetic markers involved in AS susceptibility. These findings, together with the well established association of IL23R with IBD, provide new evidence to support the hypothesis of a common genetic background for AS and IBD.31