1. ¹The Jan van Breemen Institute, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands
2. ²The Laboratory of Immunogenetics, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands
3. ³The Department of Gastroenterology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands
4. ⁴The Department of Rheumatology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands

Correspondence: IE van der Horst-Bruinsma, MD, PhD, Vrije Universiteit Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: secr.reumatologie@vumc.nl

Abstract

An insertion mutation at nucleotide 3020 (3020insC) and a missense mutation G2722C in the CARD15 gene on chromosome 16p have been reported to be associated with Crohn's disease (CD). The protein encoded by the CARD15 gene is expressed in peripheral monocytes and regulates apoptosis and NF-B activation, factors which play an important role in inflammation. Since CD and ankylosing spondylitis (AS) are interrelated disorders, we have investigated whether these mutations in the CARD15 gene are also associated with AS. We studied 113 unrelated AS patients and 152 unrelated healthy controls. No significant differences were found between patients and controls in the prevalence of the insertion 3020insC mutation and the G2722C missense mutation, OR = 1.36, 95% CI: 0.27–6.84, P = 0.70 and OR = 0.58; 95% CI: 0.18–1.94; P = 0.38, respectively. We conclude that the insertion 3020insC mutation and the G2722C missense mutation in the CARD15 gene are not involved in the susceptibility to AS.

Ankylosing spondylitis (AS) is a rheumatic disorder that primarily affects the axial skeleton, but peripheral arthritis and acute anterior uveitis frequently occur as complicating features. The etiology of this chronic, familial disease is unknown. Heritability does play a major role. Twin studies suggest that up to 97% of the susceptibility to AS can be attributed to genetic factors.¹ HLA-B27 is the major genetic determinant identified. However, genome scanning has shown that the major histocompatibility complex (MHC), including HLA-B27, contributes less than 40% to the recurrence risk ratio in AS.² Apart from the MHC, the same study identified another six loci on chromosomes 1p, 2q, 9q, 10q, 16q, and 19q to be linked to AS. The maximum likelihood of odds (LOD) score obtained was 4.7 (P < 0.005) on chromosome 16q at 101 cM from the 16p-telomere. Therefore, other genes beside the MHC influence the susceptibility to AS.

An insertion mutation at nucleotide 3020 (3020insC) in the CARD15 gene, located on chromosome 16p12, has been described as a susceptibility factor for Crohn's disease (CD).^3,4,5,6 This mutation in the distal leucine-rich repeat region (LRR) results in a 33 amino acid shorter version of a 1040-amino-acid protein also characterised by two amino-terminal caspase recruitment domains (CARD) and a nucleotide (ATP or GTP) binding domain. The low-frequency G2722C missense mutation is also located in the LRR domain of CARD15 and is associated with CD.^3,4 The protein encoded by the CARD15 gene is expressed in peripheral monocytes and regulates apoptosis and NF-B activation, factors which play an important role in inflammation.

The spondylarthropathies are a group of interrelated disorders, as based on clinical, genetic and radiologic features.⁷ Beside AS, the spondylarthropathies include psoriatic arthritis, reactive arthritis and the chronic inflammatory bowel diseases (IBD), encompassing CD and ulcerative colitis. All spondylarthropathies are to some extent associated with HLA-B27, but the frequency varies among the several diseases. 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.⁸

This study was aimed to assess whether AS shares with CD the association with the 3020insC and G2722C mutations in the CARD15 gene, compared with healthy controls.

After informed consent, 113 AS patients from our outpatient clinic and 152 healthy controls were included. All subjects were unrelated Dutch Caucasians. A rheumatologist defined the diagnosis of AS, according to the Modified New York Criteria.⁹ Clinical data were gathered by examining the patient record forms. The CARD15 3020insC was genotyped by allele specific multiplex PCR,⁵ and the CARD15 G2722C polymorphism (NCBI SNP Id rs2066845) by PCR-RFLP with genomic DNA isolated from blood. Frequencies were compared by means of 2 statistics or Fisher's exact test, when appropriate. The strength of association was estimated by odds ratios (OR) with 95% confidence interval. Carrier status was considered if any subject inherited at least one copy of the mutant allele. A two-tailed P value <0.05 was considered significant. Statistical analysis was performed using SPSS version 10.07 for windows (SPSS Inc., Chicago, Il, USA). The genotype frequencies in patients and controls were in Hardy–Weinberg equilibrium.

The AS patients were older than the controls and less often female (Table 1). Table 2 shows the allele and carrier frequencies of the CARD15 3020insC and CARD15 2722C mutations in patients and controls. No significant differences were found between patients and controls in carrier rate of the CARD15 3020insC (OR = 1.36, 95% CI: 0.27–6.84, P = 0.70) or the CARD15 2722C mutation (OR = 0.58; 95% CI: 0.18–1.94; P = 0.38). None of the patients or controls were homozygous for the insertion or missense mutation and none was carrier of both mutations.

Table 1 - Demographic and clinical characteristics of patients with ankylosing spondylitis (AS) (n=113) and healthy controls (n=152).

Full table

Table 2 - Allele and carrier frequencies of the CARD15 3020insC and G2722C mutations in ankylosing spondylitis (AS) patients and healthy controls.

Full table

These data indicate that the CARD15 3020insC and CARD15 G2722C mutations are not involved in the pathogenesis of primary AS. However, a larger study is needed to confirm these results, especially concerning the CARD15 3020insC mutation in view of the 95% CI that extends to an OR of 6.8. The conclusion that the CARD15 3020insC and CARD15 G2722C mutations are not involved in the pathogenesis of primary AS is supported by genome screening, in which no linkage was found between marker D16S3068 on chromosome 16p12 and AS.² In future studies, it would be of interest to assess whether the CARD15 mutations are associated with the subgroup of CD patients in which arthritis and/or spondylitis are complicating features.