You are here

Article Text

Article menu

This article has a correction. Please see:

Clinical and epidemiological research
Extended report
Association of CD40 with rheumatoid arthritis confirmed in a large UK case-control study
  1. Gisela Orozco1,
  2. Steve Eyre1,
  3. Anne Hinks1,
  4. Xiayi Ke1,
  5. Wellcome Trust Case Control consortium YEAR Consortium,
  6. Anthony G Wilson2,
  7. Deborah E Bax2,
  8. Ann W Morgan3,
  9. Paul Emery3,
  10. Sophia Steer4,
  11. Lynne Hocking5,
  12. David M Reid5,
  13. Paul Wordsworth6,
  14. Pille Harrison6,
  15. Wendy Thomson1,
  16. Anne Barton1,
  17. Jane Worthington1
  1. 1arc Epidemiology Unit, The University of Manchester, Manchester, UK
  2. 2School of Medicine and Biomedical Sciences, The University of Sheffield, Sheffield, UK
  3. 3NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK
  4. 4Clinical and Academic Rheumatology, Kings College Hospital NHS Foundation Trust, Denmark Hill, London, UK
  5. 5Bone Research Group, Department of Medicine and Therapeutics, University of Aberdeen, UK
  6. 6University of Oxford Institute of Musculoskeletal Sciences, Botnar Research Centre, Oxford, UK
  1. Correspondence to Dr Gisela Orozco, arc Epidemiology Unit, Faculty of Medical and Human Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK; gisela.orozco{at}manchester.ac.uk

Abstract

Objective A recent meta-analysis of published genome-wide association studies (GWAS) in populations of European descent reported novel associations of markers mapping to the CD40, CCL21 and CDK6 genes with rheumatoid arthritis (RA) susceptibility while a large-scale, case-control association study in a Japanese population identified association with multiple single nucleotide polymorphisms (SNPs) in the CD244 gene. The aim of the current study was to validate these potential RA susceptibility markers in a UK population.

Methods A total of 4 SNPs (rs4810485 in CD40, rs2812378 in CCL21, rs42041 in CDK6 and rs6682654 in CD244) were genotyped in a UK cohort comprising 3962 UK patients with RA and 3531 healthy controls using the Sequenom iPlex platform. Genotype counts in patients and controls were analysed with the χ2 test using Stata.

Results Association to the CD40 gene was robustly replicated (p=2×10−4, OR 0.86, 95% CI 0.79 to 0.93) and modest evidence was found for association with the CCL21 locus (p=0.04, OR 1.08, 95% CI 1.01 to 1.16). However, there was no evidence for association of rs42041 (CDK6) and rs6682654 (CD244) with RA susceptibility in this UK population. Following a meta-analysis including the original data, association to CD40 was confirmed (p=7.8×10−8, OR 0.87 (95% CI 0.83 to 0.92).

Conclusion In this large UK cohort, strong association of the CD40 gene with susceptibility to RA was found, and weaker evidence for association with RA in the CCL21 locus.

This paper is freely available online under the BMJ Journals unlocked scheme, see http://ard.bmj.com/info/unlocked.dtl

https://doi.org/10.1136/ard.2009.109579

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    Introduction

    Genome-wide association studies (GWAS) have led to a breakthrough in the search for complex disease susceptibility markers.1 Specifically for rheumatoid arthritis (RA), these studies have detected a number of new susceptibility loci, including the 6q23,2,–,4 TRAF1/C5,5,–,9 PRKCQ, KIF5A and IL2RB genetic regions.10 Recently, a meta-analysis of the Wellcome Trust Case Control Consortium (WTCCC) GWAS and a US GWAS4 5 confirmed association to PRKCQ, KIF5A and MMEL and identified additional RA-associated markers mapping to the CD40, CCL21 and CDK6 loci,11 that have yet to be confirmed in an independent UK population. In addition, a recent case-control association study in a Japanese population identified two functional variants mapping to the CD244 gene, rs3766379 and rs6682654, that were associated with RA, again yet to be tested for association in a UK population.12

    These studies therefore add to the recent explosion in the number of loci reported to be associated with RA susceptibility. In order for these loci to be confirmed as risk factors for RA, well powered validation studies are required to support these initial findings.

    The aims of this study were, therefore, first to validate the additional RA markers derived from the recent meta-analysis (CD40, CCL21 and CDK6) in an independent UK population and, second, to investigate a functional CD244 variant reported to be associated with RA in the Japanese study.11 12

    Methods

    Patients with RA (n=3962) were recruited from six centres (Manchester, Sheffield, Leeds, Aberdeen, Oxford and London) across the UK. All patients were Caucasian of North European ancestry and all fulfilled the 1987 American College of Rheumatology classification criteria modified for genetic studies.13 14 Clinical and demographic characteristics of the cohort are detailed in supplementary table 1. Briefly, 71.8% participants were women, 72.1% were rheumatoid factor positive and 67.9% carried anti-citrullinated protein antibodies (ACPA) as recognised by the anti-cyclic citrullinated protein (CCP) antibody test. This cohort is therefore representative of a hospital-based series of patients with RA. Autoantibody status data was available for 3723 and 2281 patients for rheumatoid factor (RF) and anti-CCP, respectively. Healthy controls (n=3531) were recruited from five of the six centres (patients only recruited from London). All participants were recruited after providing informed consent and the study was approved by the North West Research Ethics Committee (MREC 99/8/84).

    View this table:
    Table 1

    Validation study of novel rheumatoid arthritis (RA) susceptibility markers in a UK population

    Genotyping was performed using the Sequenom iPlex platform (http://www.sequenom.com) according to the manufacturer's instructions. All genotyping was performed at the arc Epidemiology Unit, Manchester. Only samples and single nucleotide polymorphisms (SNPs) exceeding a 90% success rate were used in the analysis.

    Genotype counts in patients and controls were analysed using Stata software (Stata, College Station, Texas, USA) with the χ2 test. For the meta-analysis, the Breslow–Day test for homogeneity and the Cochran Mantel–Haenszel test were performed using Stata. For all the tests performed, p values <0.0125 were considered statistically significant, after applying Bonferroni correction for four comparisons.

    Power calculations were performed using the Quanto V.0.5 software (Department of Preventive Medicine University of Southern California, California, USA), considering minor allele frequencies and ORs similar to those found in previous studies 4 5 11 12 at the 5% significance level.

    Results

    We selected four polymorphisms previously reported as RA susceptibility markers for testing in the current study:

    1. (1). rs4810485, located in the second intron of the CD40 gene;

    2. (2). rs2812378, mapping to the 5' untranslated region of CCL21;

    3. (3). rs42041, positioned in a CDK6 intron11;

    4. (4). rs6682654, located in CD244, which is an almost perfect proxy of the functional RA marker for the Japanese population within this gene, rs3766379 (D′=1, r2=0.97).12

    Genotype frequencies conformed to Hardy–Weinberg expectations in patients and controls for all the polymorphisms under study. The estimated power of the study was 86% for rs481048, 77% for rs2812378, 60% for rs42041 and 99% for rs6682654.

    Table 1 shows the results of this validation study. Allele frequencies were similar to those previously described in a US population for the CD40, CCL21 and CDK6 markers.11 We found that the minor T allele of CD40 rs4810485 was significantly increased in healthy controls compared to patients with RA (p=2×10−4, OR 0.86, 95% CI 0.79 to 0.93). This is in accordance with the previous study by Raychaudhuri et al.

    When we analysed CCL21 rs2812378, we observed a higher frequency of the minor C allele in patients with RA as previously described,11 although this skewing seemed to have a more modest effect in our population (p=0.04, OR 1.08, 95% CI 1.01 to 1.16). With regard to CDK6 rs42041, no evidence of association with RA was found in our UK population (table 1).

    Additionally, we examined the functional CD244 variant rs6682654, which was previously shown to be associated with RA in a Japanese population.12 Linkage disequilibrium (LD) between this SNP and the other marker within CD244, rs3766379, also reported to be associated with RA in the Japanese study, is higher in the Caucasian population (r2=0.97, D′=1) than in the Japanese population (r2=0.87, D′=1).15 When we compared the distribution of allele and genotype frequencies of rs6682654 between patients and controls, we did not find statistically significant differences (table 1). Allele frequencies of the reference G allele were lower in the UK population than in the previously described Japanese population (0.43 and 0.44 in patients and controls, respectively, for the UK population vs 0.66 and 0.59 in patients and controls, respectively, for the Japanese population), which is in accordance with the data deposited in HapMap (http://www.hapmap.org).

    Stratification analysis of the four polymorphisms under study showed that the rs4810485 CD40 SNP was strongly associated with the presence of RF and anti-CCP antibodies (p=8.10×10−6, OR 0.82, 95% CI 0.75 to 0.89 and p=9.52×10−5, OR 0.81, 95% CI 0.73 to 0.90, respectively). CCL21 rs2812378 also showed this trend, but p values for the association with seropositive disease were modest (p=0.02, OR 1.10, 95% CI 1.01 to 1.18 and p=0.03, OR 1.10, 95% CI 1.01 to 1.21, for RF and anti-CCP RA, respectively) (supplementary table 2).

    View this table:
    Table 2

    Meta-analysis of newly identified rheumatoid arthritis (RA) markers in populations of European ancestry

    Meta-analysis of these markers was undertaken, pooling data from the previously published RA GWASs,4 5 11 together with data from a validation cohort from the US11 and our validation study in a UK cohort. p Values for the Breslow–Day test were 0.31 for rs4810485, 0.81 for rs2812378 and 0.02 for rs42041 (table 2). The meta-analysis revealed strong evidence of association with the CD40 variant and a more modest, but suggestive association with the CCL21 variant. The evidence for association with CDK6 was relatively weak.

    The rs6682654 SNP mapping to CD244 and showing evidence for association with RA in a Japanese population12 was not tested in the meta-analysis by Raychaudhri et al.11 In order to investigate whether a weak effect on susceptibility could be detected in a UK population, we performed a meta-analysis of the variant, combining imputed data from the WTCCC dataset with that derived from direct genotyping in the current validation study. Pooled analysis revealed no association with RA in the UK population (p=0.87, OR 1.004, 95% CI 0.95 to 1.06). Joint analysis of the Japanese and UK cohorts was not feasible, due to the high degree of genetic heterogeneity between the populations (pBreslow−Day=1.8×10−6).

    Discussion

    In this large UK cohort, we have found strong association of the rs4810485 CD40 variant with susceptibility to RA, and weaker evidence for association with RA in the CCL21 locus. We found no evidence for association with either the CDK6 locus or the CD244 variant, previously reported to be associated with RA in Caucasian and Japanese populations, respectively.

    The CD40 and CCL21 genes both encode proteins that are involved in immune regulation. CD40 is expressed in a wide variety of immune cells, including B cells, monocytes and dendritic cells, while its ligand, CD154, is expressed by activated CD4 T cells. Binding of CD154 to CD40 provides a critical stimulus for B cell proliferation, immunoglobulin production, isotype switching and upregulation of other costimulatory molecules including B7-related molecules.16 CD40–CD154 interaction appears to have an important role in RA, as expression of CD154 on CD4 T cells in RA promotes increased activation of CD4 T lymphocytes and is associated with active disease. Indeed, blockade of this pathway has been suggested as a potentially useful therapeutic approach in RA and in autoimmune disease in general.16 17 It is interesting to note that the CD40 marker was strongly associated with anti-CCP and RF-positive RA, which suggest that this polymorphism, or other/s in LD with it, could play a role in the regulation of B cell response and the production of autoantibodies. The CCL21 gene encodes a protein that is a homeostatic lymphoid chemokine instrumental in the recruitment and organisation of T cells and dendritic cells into secondary lymphoid organs, which participate in lymphoid tissue microanatomical organisation in RA.18 Both genes, therefore, are plausible candidates in conferring risk of RA. Although we found statistically significant evidence for association with the CCL21 locus, this would not have remained significant if a correction for multiple testing had been applied and, therefore, confirmation of this locus as an RA susceptibility region will require further investigation in other large data series.

    It is becoming well established that there exists an ethnic heterogeneity of genetic susceptibility factors in RA, meaning that findings from one ethnic group may not be readily applicable to all. For example, rs2476601 in the PTPN22 gene, which is the most robustly validated RA marker in European populations after human leukocyte antigen (HLA), is not polymorphic in Asian populations.19 In contrast, polymorphisms in PADI4 are far more strongly associated with RA in Far East populations than European populations, despite having a comparable allele frequencies.19,–,21 Other RA susceptibility loci, such as STAT4, are common to European and Asian populations.9 22,–,24 More relevant to the current study, it has recently been reported that the CD40 and CCL21 variants tested in the present study, are not associated with RA in a Korean population.25 However, it is possible that the CD244 SNP tested in the current study is in LD with the true causative variant in the Japanese population, but not in the UK population, which could explain the lack of replication of the association of this marker.

    This is the first study to test the association of CD244 with RA in a European population, but we have found no evidence for association, despite it being a functional polymorphism with a regulatory effect on the transcription of CD244.12 Insufficient power is an unlikely cause of the lack of replication, since we achieved >98% power to detect an association with an effect size similar to that reported in the prior Japanese study12 at a 5% significance level. However, the ‘winner’s curse' means that effect sizes are often overestimated in the first study in which they are reported. It is possible that the effect size at this locus is smaller and that, by chance, it has not been detected in the current study. The lack of power of individual studies may be overcome by meta-analysis where published data is available and this is particularly pertinent to the CDK6 rs42041 SNP where the power to detect association in the sample set tested in the current study was low (approximately 60%). The meta-analysis presented here yielded a statistically significant, yet modest, association (p=0.002, OR 1.08, 95% CI 1.03 to 1.14) of the CDK6 rs42041 marker with RA susceptibility. It should be noted that we detected some heterogeneity in the effect sizes between studies (pBreslow−Day=0.02), so the pooled OR should be considered with caution and further validation in independent cohorts will be required in order to clarify the role of this variant in RA susceptibility. For the CD244 marker, by contrast, no evidence for association was detected even after pooled analysis of the UK WTCCC and validation datasets, which included approximately 5400 patients with RA and approximately 5800 healthy controls. In this case, genetic heterogeneity would appear to explain the lack of replication as allele frequencies for the rs6682654 SNP are significantly different between the Japanese and UK populations.

    In the past year, the first wave of GWAS has improved our understanding of the genetic basis of common, complex diseases, including RA. These studies have made possible the identification of new RA susceptibility loci, such as those mapping to the 6q23, TRAF1/C5, PRKCQ, KIF5A, STAT4 and now CD40. However, these initial GWAS have shown that the effect sizes resulting from associations with common SNPs are modest and, therefore, the remaining RA susceptibility genes may have even smaller effect sizes. Validation studies using large sample sizes and ethnically different populations as well as meta-analysis will be essential to robustly define new potential susceptibility loci. Only then will the investment in fine mapping and functional studies be justified to identify the aetiological variants within the validated loci. In conclusion, we have validated association of CD40 rs4810485 and detected weak evidence for association of CCL21 rs2812378 with RA in a UK population, laying the foundations for further studies of the functional and genetic effects of these variants in conferring risk of disease.

    Acknowledgments

    We acknowledge use of DNA from the British 1958 Birth Cohort collection, funded by the Medical Research Council grant G0000934 and the Wellcome Trust grant 068545/Z/02. IT support was provided by Mark Lay, statistical advice from Mark Lunt and technical support by Paul Gilbert, Bhaneeta Lad and Stephen G Martin. Nursing support was provided by Janet Grumley, Julie Shotton and Claire Farrar. This work was supported by the Arthritis Research Campaign (arc grant reference number 17552). We would also like to acknowledge the National Health Service (NHS) Research and Development Support Fund for Guy's and St Thomas' and Lewisham NHS Trusts. We acknowledge the arc, Leeds Teaching Hospitals Charitable Trustees and NIHR Leeds Musculoskeletal Biomedical Research Unit for supporting the establishment of the Leeds cohorts. We thank the Manchester Biomedical Research Centre for support.

    References

    1. ↵
      1. McCarthy MI,
      2. Abecasis GR,
      3. Cardon LR,
      4. et al
      . Genome-wide association studies for complex traits: consensus, uncertainty and challenges. Nat Rev Genet 2008;9:356–69.
      OpenUrlCrossRefPubMedWeb of Science
    2. ↵
      1. Thomson W,
      2. Barton A,
      3. Ke X,
      4. et al
      . Rheumatoid arthritis association at 6q23. Nat Genet 2007;39:1431–3.
      OpenUrlCrossRefPubMedWeb of Science
    3. ↵
      1. Plenge RM,
      2. Cotsapas C,
      3. Davies L,
      4. et al
      . Two independent alleles at 6q23 associated with risk of rheumatoid arthritis. Nat Genet 2007;39:1477–82.
      OpenUrlCrossRefPubMedWeb of Science
    4. ↵
      Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 2007;447:661–78.
      OpenUrlCrossRefPubMedWeb of Science
    5. ↵
      1. Plenge RM,
      2. Seielstad M,
      3. Padyukov L,
      4. et al
      . TRAF1-C5 as a risk locus for rheumatoid arthritis – a genomewide study. N Engl J Med 2007;357:1199–209.
      OpenUrlCrossRefPubMed
    6. ↵
      1. Kurreeman FA,
      2. Padyukov L,
      3. Marques RB,
      4. et al
      . A candidate gene approach identifies the TRAF1/C5 region as a risk factor for rheumatoid arthritis. PLoS Med 2007;4:e278.
      OpenUrlCrossRefPubMed
    7. ↵
      1. Zervou MI,
      2. Sidiropoulos P,
      3. Petraki E,
      4. et al
      . Association of a TRAF1 and a STAT4 gene polymorphism with increased risk for rheumatoid arthritis in a genetically homogeneous population. Hum Immunol 2008;69:567–71.
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Kurreeman FA,
      2. Rocha D,
      3. Houwing-Duistermaat J,
      4. et al
      . Replication of the tumor necrosis factor receptor-associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study. Arthritis Rheum 2008;58:2670–4.
      OpenUrlCrossRefPubMed
    9. ↵
      1. Barton A,
      2. Thomson W,
      3. Ke X,
      4. et al
      . Re-evaluation of putative rheumatoid arthritis susceptibility genes in the post-genome wide association study era and hypothesis of a key pathway underlying susceptibility. Hum Mol Genet 2008;17:2274–9.
      OpenUrlAbstract/FREE Full Text
    10. ↵
      1. Barton A,
      2. Thomson W,
      3. Ke X,
      4. et al
      . Rheumatoid arthritis susceptibility loci at chromosomes 10p15, 12q13 and 22q13. Nat Genet 2008;40:1156–9.
      OpenUrlCrossRefPubMedWeb of Science
    11. ↵
      1. Raychaudhuri S,
      2. Remmers EF,
      3. Lee AT,
      4. et al
      . Common variants at CD40 and other loci confer risk of rheumatoid arthritis. Nat Genet 2008;40:1216–23.
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      1. Suzuki A,
      2. Yamada R,
      3. Kochi Y,
      4. et al
      . Functional SNPs in CD244 increase the risk of rheumatoid arthritis in a Japanese population. Nat Genet 2008;40:1224–9.
      OpenUrlCrossRefPubMedWeb of Science
    13. ↵
      1. Arnett FC,
      2. Edworthy SM,
      3. Bloch DA,
      4. et al
      . The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.
      OpenUrlPubMedWeb of Science
    14. ↵
      1. MacGregor AJ,
      2. Bamber S,
      3. Silman AJ
      . A comparison of the performance of different methods of disease classification for rheumatoid arthritis. Results of an analysis from a nationwide twin study. J Rheumatol 1994;21:1420–6.
      OpenUrlPubMedWeb of Science
    15. ↵
      1. Frazer KA,
      2. Ballinger DG,
      3. Cox DR,
      4. et al
      . A second generation human haplotype map of over 3.1 million SNPs. Nature 2007;449:851–61.
      OpenUrlCrossRefPubMed
    16. ↵
      1. Toubi E,
      2. Shoenfeld Y
      . The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity 2004;37:457–64.
      OpenUrlCrossRefPubMedWeb of Science
    17. ↵
      1. Liu MF,
      2. Chao SC,
      3. Wang CR,
      4. et al
      . Expression of CD40 and CD40 ligand among cell populations within rheumatoid synovial compartment. Autoimmunity 2001;34:107–13.
      OpenUrlCrossRefPubMedWeb of Science
    18. ↵
      1. Manzo A,
      2. Paoletti S,
      3. Carulli M,
      4. et al
      . Systematic microanatomical analysis of CXCL13 and CCL21 in situ production and progressive lymphoid organization in rheumatoid synovitis. Eur J Immunol 2005;35:1347–59.
      OpenUrlCrossRefPubMedWeb of Science
    19. ↵
      1. Yamada R,
      2. Yamamoto K
      . Mechanisms of disease: genetics of rheumatoid arthritis--ethnic differences in disease-associated genes. Nat Clin Pract Rheumatol 2007;3:644–50.
      OpenUrlCrossRefPubMedWeb of Science
    20. ↵
      1. Barton A,
      2. Bowes J,
      3. Eyre S,
      4. et al
      . Investigation of polymorphisms in the PADI4 gene in determining severity of inflammatory polyarthritis. Ann Rheum Dis 2005;64:1311–15.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      1. Iwamoto T,
      2. Ikari K,
      3. Nakamura T,
      4. et al
      . Association between PADI4 and rheumatoid arthritis: a meta-analysis. Rheumatology (Oxford) 2006;45:804–7.
      OpenUrlAbstract/FREE Full Text
    22. ↵
      1. Kobayashi S,
      2. Ikari K,
      3. Kaneko H,
      4. et al
      . Association of STAT4 with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Japanese population. Arthritis Rheum 2008;58:1940–6.
      OpenUrlCrossRefPubMedWeb of Science
    23. ↵
      1. Lee HS,
      2. Remmers EF,
      3. Le JM,
      4. et al
      . Association of STAT4 with rheumatoid arthritis in the Korean population. Mol Med 2007;13:455–60.
      OpenUrlPubMed
    24. ↵
      1. Orozco G,
      2. Alizadeh BZ,
      3. Delgado-Vega AM,
      4. et al
      . Association of STAT4 with rheumatoid arthritis: a replication study in three European populations. Arthritis Rheum 2008;58:1974–80.
      OpenUrlCrossRefPubMedWeb of Science
    25. ↵
      1. Lee HS,
      2. Korman BD,
      3. Le JM,
      4. et al
      . Genetic risk factors for rheumatoid arthritis differ in Caucasian and Korean populations. Arthritis Rheum 2009;60:364–71.
      OpenUrlCrossRefPubMedWeb of Science

    Supplementary materials

    • Web Only Data ard.2009.109579

      Files in this Data Supplement:

    Footnotes

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the North West Research Ethics Committee (MREC 99/8/84).

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • For more information on the WTCCC and YEAR Consortiums, see supplementary material.

    Linked Articles

    • Miscellaneous
      Corrections
      BMJ Publishing Group Ltd and European League Against Rheumatism
      Annals of the Rheumatic Diseases 2011; 70 1519-1519 Published Online First: 28 Jun 2011. doi: 10.1136/annrheumdis-2011-70-08