CARD15/NOD2 analysis in rheumatoid arthritis susceptibility

I. Ferreirós-Vidal, F. Barros1, J. L. Pablos2, A. Carracedo1, J. J. Gómez-Reino and A. Gonzalez

Research Laboratory 2 and the Rheumatology Unit, Hospital Clinico Universitario de Santiago, 1Molecular Medicine Unit-INGO-Universidad de Santiago de Compostela and 2Rheumatology Unit, Hospital 12 de Octubre, Madrid, Spain.

Correspondence to: A. Gonzalez, Laboratorio de Investigacion 2, Hospital Clinico Universitario de Santiago, 15706-Santiago de Compostela, Spain. E-mail: gon2906{at}globalum.com


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results and discussion
 Conflict of interest
 References
 
Objective. To determine if the mutations in the CARD15/NOD2 gene predisposing to Crohn's disease (CD) contribute also to the genetic susceptibility to rheumatoid arthritis (RA).

Methods. The frequencies of the three commonest mutations of CARD15/NOD2 predisposing to CD (2104C > T, 2722G>C and 3020insC) were determined in 210 RA patients and 227 controls.

Results. Allelic frequencies of the CARD15/NOD2 mutations in RA patients (2104C>T, 2.8%; 2722G>C, 0.9%; and 3020insC, 2.4%) did not differ significantly from the controls (2104C>T, 5.3%; 2722G>C, 0.7%; and 3020insC, 1.1%).

Conclusion. There was no evidence of association between the commonest CD CARD15/NOD2 mutations and RA susceptibility.

KEY WORDS: Rheumatoid arthritis, Crohn's disease, Genetic predisposition to disease, Autoimmune diseases.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results and discussion
 Conflict of interest
 References
 
Twin, family and HLA studies indicate that rheumatoid arthritis (RA) has a significant genetic component [1, 2]. The HLA genes account for 30–40% of RA genetic susceptibility [15]. The remaining 60–70% has an unknown molecular basis, although the involvement of some candidate genes has been suggested [2]. A fraction of the unexplained genetic susceptibility should be related to the loci linked to RA [36]. One of these loci, in chromosome 16q, has shown linkage in two genome-wide studies [3, 4] and overlaps with susceptibility loci to other diseases resulting from dysregulation of the immune system, including Crohn's disease (CD), psoriasis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus, ankylosing spondylitis and asthma. This coincidence is indicative of a common disease gene [79] and it suggests that the mutations on the CARD15/NOD2 gene located on the chromosome 16q locus, which cause susceptibility to CD [10, 11], could also contribute to RA predisposition.

The CARD15/NOD2 protein, expressed almost exclusively in monocytes, mediates the activation of NF-{kappa}B in response to bacterial products [11, 12]. Disruption of the regulatory region of CARD15/NOD2 by the CD mutations alters the magnitude of NF-{kappa}B activation leading to an uncontrolled inflammatory response [11]. These characteristics are compatible with current models of RA pathology [1315]. Therefore, both genetic and pathogenic evidence would argue for the participation of the CD CARD15/NOD2 mutations in RA susceptibility.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results and discussion
 Conflict of interest
 References
 
Patients
We studied 210 patients with RA, according to the American College of Rheumatology (ACR) criteria [16], and 227 controls of homogeneous Spanish ancestry. The regional ethics committee (Comité Ético de Investigación Clínica de Galicia) approved the study, and written informed consent was obtained from all patients.

Genotyping
DNA was extracted from peripheral blood by conventional techniques. Three polymorphisms of the CARD15/NOD2 gene (3020insC, 2722G>C, 2104C>T) were typed by analysis of the melting curve after hybridization with FRET probes on a LightCycler (Roche Diagnostics, Barcelona, Spain) polymerase chain reaction (PCR) system. Primers and FRET probes were synthesized by TIB MOLBIOL (Berlin, Germany) and have been reported previously [17]. PCR reactions were as described previously [17]. Selected samples were sequenced on the ABI PRISM 377 DNA Sequencer (Applied Biosystems, Madrid, Spain).

Statistical analysis
Allelic frequencies, odds ratios (OR), their confidence intervals (CI) and the {chi}2-test were calculated (http://home.clara.net/sisa/index.htm). The post-hoc power of the study was determined with the Gpower software (http://www.psycho.uni-duesseldorf.de/aap/projects/gpower/).

Homology search
An extensive search of CARD15/NOD2 (GenBank: NP_071445) homologues located in chromosome 16q was done with BLAST, FASTA3 and PSI-BLAST search algorithms. Homologue sequences were located on the NCBI sequence of the Human Genome (build 29).


    Results and discussion
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 Abstract
 Introduction
 Patients and methods
 Results and discussion
 Conflict of interest
 References
 
The possibility of the participation of genes predisposing to several autoimmune diseases in the aetiology of RA has been a recurrent theme in the genome-wide linkage studies [35]. Coincidences have been signalled with IDDM, SLE, ankylosing spondylitis, multiple sclerosis, asthma and CD. One of the RA loci, on chromosome 16q, found in two genome scans undertaken in American [4] and European [3] families, respectively, could be related to the mutations on the CARD15/NOD2 genes predisposing to CD. However, our study did not find any evidence of association between the three commonest CD CARD15/NOD2 mutations and RA susceptibility, taken individually or collectively (Table 1). Analysis of several disease features (gender, age of onset, rheumatoid factor, sensitivity to disease-modifying anti-rheumatic drugs) showed no correlation with the genotypes. We did not find any homozygote for 2104C>T or 2722G>C, nor any compound heterozygote, and only one homozygote for 3020insC among the RA patients. This patient, a 54-yr-old man, had no distinctive features.


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TABLE 1. Allelic frequencies of the three commonest CD CARD15/NOD2 mutations (3020insC, 2722G>C, 2104C>T) in RA patients and controls

 
The negative result found in this study was associated with a post-hoc power to detect association at the 0.05 level of 99.98% and 80% for attributable risks of 2 and 1.5, respectively. In this analysis, the three mutations were considered together owing to their independent origin and inheritance and because they seem to act through the same pathogenic mechanisms [10, 1821]. Additionally, the frequencies of the three alleles in the control population were very similar to the frequencies that have been found in different European populations [10, 1821], and the genotype frequencies of the three polymorphisms were distributed according to the Hardy–Weinberg equilibrium. This result confirms the lack of association reported by Steer et al. [21] between two of the mutations studied here (2104C>T and 3020insC) and RA in the British population and a smaller study published previously with French RA patients [22]. These results should be considered together with similar studies exploring the involvement of the CD CARD15/NOD2 mutations in susceptibility to other autoimmune diseases linked to chromosome 16q—psoriasis [17, 22, 24], and SLE (I. Ferreiros-Vidal et al., unpublished). All of them have failed to find evidence of association. Therefore, it seems that the CD CARD15/NOD2 mutations have an effect restricted to CD. This result was unexpected and it provokes more questions about the function of the CARD15/NOD2 protein and how the mutated forms contribute to CD pathology.

A possible alternative to a single gene underlying all the autoimmune diseases linked to a given region is a cluster of functionally related genes, each of them underlying susceptibility to a different disease [9]. There are many examples of this kind of gene cluster, some of them central in immune responses, such as the MHC, immunoglobulin and T-cell receptor gene complexes. Therefore, we have searched for CARD15/NOD2 homologues in other regions of the same chromosome. This analysis excluded a cluster of CARD15/NOD2-related genes on chromosome 16q as it found only a putative protein, derived from cDNA libraries, that is a distant homologue of CARD15/NOD2 and maps more than 7 Mb telomeric to it. Therefore, no candidate for the RA susceptibility locus on chromosome 16q can be proposed.


    Conflict of interest
 Top
 Abstract
 Introduction
 Patients and methods
 Results and discussion
 Conflict of interest
 References
 
The authors have declared no conflicts of interest.


    Acknowledgments
 
We thank the patients and the staff of the Rheumatology Unit for their contribution. We thank also Lorena Fernandez-Blanco for her help in processing samples. This work has been supported by grant 01/3138 of the Fondo de Investigaciones Sanitarias (Spain).


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results and discussion
 Conflict of interest
 References
 

  1. Seldin MF, Amos CI, Ward R, Gregersen PK. The genetics revolution and the assault on rheumatoid arthritis. Arthritis Rheum 1999;42:1071–9.[CrossRef][ISI][Medline]
  2. Barton A, Ollier W. Genetic approaches to the investigation of rheumatoid arthritis. Curr Opin Rheumatol 2002;14:260–9.[CrossRef][ISI][Medline]
  3. Cornelis F, Faure S, Martinez M et al. New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study. Proc Natl Acad Sci USA 1998;95:10746–50.[Abstract/Free Full Text]
  4. Jawaheer D, Seldin MF, Amos CI et al. A genomewide screen in multiplex rheumatoid arthritis families suggests genetic overlap with other autoimmune diseases. Am J Hum Genet 2001;68:927–36.[CrossRef][ISI][Medline]
  5. MacKay K, Eyre S, Myerscough A et al. Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom. Arthritis Rheum 2002;46:632–9.[CrossRef][ISI][Medline]
  6. Shiozawa S, Hayashi S, Tsukamoto Y et al. Identification of the gene loci that predispose to rheumatoid arthritis. Int Immunol 1998;10:1891–5.[Abstract]
  7. Vyse TJ, Todd JA. Genetic analysis of autoimmune disease. Cell 1996;85:311–8.[ISI][Medline]
  8. Becker KG, Simon RM, Bailey-Wilson JE et al. Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases. Proc Natl Acad Sci USA 1998;95:9979–84.[Abstract/Free Full Text]
  9. Wanstrat A, Wakeland E. The genetics of complex autoimmune diseases: non-MHC susceptibility genes. Nat Immunol 2001;2:802–9.[CrossRef][ISI][Medline]
  10. Hugot JP, Chamaillard M, Zouali H et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001;411:599–603.[CrossRef][ISI][Medline]
  11. Ogura Y, Bonen DK, Inohara N et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001;411:603–6.[CrossRef][ISI][Medline]
  12. Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem 2001;276:4812–8.[Abstract/Free Full Text]
  13. Silman AJ, Pearson JE. Epidemiology and genetics of rheumatoid arthritis. Arthritis Res 2002;4(Suppl. 3):S265–72.[CrossRef][Medline]
  14. Kinne RW, Brauer R, Stuhlmuller B, Palombo-Kinne E, Burmester GR. Macrophages in rheumatoid arthritis. Arthritis Res 2000;2:189–202.[CrossRef][ISI][Medline]
  15. Makarov SS. NF-kappa B in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res 2001;3:200–6.[CrossRef][ISI][Medline]
  16. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  17. Ferreirós-Vidal I, Amarelo J, Barros F, Carracedo A, Gómez-Reino JJ, Gonzalez A. Lack of association of ankylosing spondylitis with the commonest NOD2 susceptibility alleles to Crohn's disease. J Rheumatol 2003;30:102–4.[ISI][Medline]
  18. Lesage S, Zouali H, Cezard JP et al. CARD15/NOD2 mutational analysis and genotype–phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002;70:845–57.[CrossRef][ISI][Medline]
  19. Ahmad T, Armuzzi A, Bunce M et al. The molecular classification of the clinical manifestations of Crohn's disease. Gastroenterology 2002;122:854–66.[ISI][Medline]
  20. Cuthbert AP, Fisher SA, Mirza MM et al. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. Gastroenterology 2002;122:867–74.[ISI][Medline]
  21. Steer S, Fisher SA, Fife M et al. Development of rheumatoid arthritis is not associated with two polymorphisms in the Crohn's disease gene CARD15. Rheumatology 2003;42:304–7.[Abstract/Free Full Text]
  22. Miceli-Richard C, Zouali H, Lesage S et al. CARD15/NOD2 analyses in spondylarthropathy. Arthritis Rheum 2002;46:1405–6.[CrossRef][ISI][Medline]
  23. Nair RP, Stuart P, Ogura Y et al. Lack of association between NOD2 3020InsC frameshift mutation and psoriasis. J Invest Dermatol 2001;117:1671–2.[Free Full Text]
  24. Crane AM, Bradbury L, Van Heel DA et al. Role of NOD2 variants in spondylarthritis. Arthritis Rheum 2002;46:1629–33.[CrossRef][ISI][Medline]
Submitted 23 October 2002; Accepted 19 March 2003





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