Skip to main content

Volume 3 Supplement 7

Genetic Analysis Workshop 16

Conditional analysis of the major histocompatibility complex in rheumatoid arthritis

Abstract

We performed a whole-genome association study of rheumatoid arthritis susceptibility using Illumina 550k single-nucleotide polymorphism (SNP) genotypes of 868 cases and 1194 controls from the North American Rheumatoid Arthritis Consortium (NARAC). Structured association analysis with adjustment for potential population stratification yielded 200 SNPs with p < 1 × 10-8 for association with RA, all of which were on chromosome 6 in a 2.7-Mb region of the major histocompatibility complex (MHC). Given the extensive linkage equilibrium in the region and known risk of HLA-DRB1 alleles, we then applied conditional analyses to ascertain independent signals for RA susceptibility among these 200 candidate SNPs. Conditional analyses incorporating risk categories of the HLA-DRB1 "shared epitope" revealed three SNPs having independent associations with RA (conditional p < 0.001). This supports the presence of significant effects on RA susceptibility in the MHC in addition to the shared epitope.

Background

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by damage to synovial joints as well as extraarticular manifestations. The strongest known genetic risk factor is the HLA-DRB1 gene on chromosome 6, namely a set of alleles sharing a common sequence known as the shared epitope (SE) [1]. Recent whole-genome association studies have revealed new risk genes outside of the HLA region [24], and some studies have also provided evidence of additional influences from the HLA class III and class I regions [5, 6]. In this analysis we sought first to identify and/or validate RA risk alleles throughout the genome, and then to identify independent associations with RA susceptibility in the major histocompatibility complex (MHC) in addition to the SE.

Methods

Illumina 550k genotyping data from a whole-genome association study by North American Rheumatoid Arthritis Consortium (NARAC) [3] was used for this study as part of the Genetic Analysis Workshop 16; duplicated and contaminated samples had been removed previously. Using the computer program PLINK [7], subjects were filtered who had less than 90% genotyping, and single-nucleotide polymorphisms (SNPs) were filtered that had less than 90% genotyping, Hardy-Weinberg equilibrium in controls p < 0.0001, or minor allele frequency (MAF) < 0.05. Using the computer program EIGENSTRAT [8], population outliers were removed who were >6 standard deviations from any of the first five principal components (PCs) identified in PC analysis.

First we analyzed the whole-genome data using structured association analyses of EIGENSTRAT; although the NARAC cases and controls are Caucasian, differences in intra-European ancestry [9] can produce false-positive associations. SNPs used for the PCA were filtered to remove regions of extended local associations (chr 8: 8-12 Mb, chr 6: 24-36 Mb, chr 11: 42-58 Mb, chr 5: 44-51.5 Mb, and chr 17: 40-43 Mb) and pruned to have r2 < 0.2 within a sliding window of 1 kb with a step size of 100, similar to methods of Fellay et al. [10] and Hom et al. [11]. We included correction for the first six PCs (see Results).

We used a conservative genome-wide significance threshold of p = 1 × 10-8. Because all SNPs exceeding this threshold (i.e., lower p-value) were in the MHC in a region of extended linkage disequilibrium (LD), we proceeded with conditional analyses to attempt to establish signals that were independent of the shared epitope and each other. We modeled the SE as a multi-allelic marker with values corresponding to negative, low-risk, or high-risk. SE alleles were considered high risk if they were one of DRB1*0401, 0404, 0405, 0408, or 0409. Table 1 shows the case-control ratios for each risk category.

Table 1 HLA-DRB1 risk levels. Definitions and case-control ratios for shared-epitope (SE) categories.

Our conditional analyses, using the computer program Whap [12], proceeded as follows. Starting with the SE as the top marker, we tested each two-SNP marker (SE plus each other SNP) for independence of the other SNP; in particular this uses a likelihood-ratio test to determine the significance of the difference between the two-SNP "alternate" model versus the one-SNP (SE in this case) "null" model. As long as the most significant SNP was <0.001, we added this best SNP to the list of independent SNPs, and proceeded to test all three-marker combinations compared to our best two-SNP model; and so on with larger haplotypes. In the final list, we also tested each locus for a significant addition to the model containing all other top SNPs.

Results

After quality control filtering (above), 486,078 SNPs remained for the whole-genome analyses. All subjects had genotyping >90%, and eight controls were removed as outliers detected by EIGENSTRAT, leaving 868 cases and 1186 controls for the final analyses. In structured association analysis with EIGENSTRAT, we corrected for the first six PCs because the scree graph of eigenvalues levelled off at the sixth component. PCs one, two, four, and five were all highly significant in association tests with cases and controls (all p ≤ 10-8). In this whole-genome analysis we identified exactly 200 SNPs with p < 10-8, all between 30.38 Mb and 33.08 Mb in the MHC region. Table 2 shows the significance of associations in this dataset for known RA risk alleles outside of the MHC [2, 3, 1317]; for SNPs not in the Illumina 550k panel, there were perfect proxy SNPs (r2 = 1) in the HapMap CEU population [18]. Although not reaching our 10-8 threshold, we observed p-values from 10-5 to 5 × 10-6 for PTPN22 and TRAF1-C5 SNPs, and p = 0.03 for STAT4. This dataset was underpowered to detect any of these risk alleles at a genome-wide level for their published odds ratios (ORs); for example, we had approximately 70% power to detect the highest OR of 1.75 (PTPN22, MAF = 11%) at p = 10-8, and only 50% power to detect the lowest OR of 1.15 (CD40, MAF = 25%) at p = 0.05.

Table 2 Significance of associations with published RA risk alleles

Results of our conditional analyses are shown in Table 3. Loci are shown in the order added by the algorithm (see Methods), i.e., rs261946 has the lowest p-value conditional on the SE, rs2074488 has the lowest p-value conditional on both SE and rs261946, and so on. Out of the 200 SNPs, three independent signals were evident in addition to the SE risk levels. One signal is located in the classical HLA class II region between genes BTNL2 and HLA-DRA, and two signals are in the classical HLA class I region (see Figure 1) near TRIM39 and HLA-C.

Figure 1
figure 1

Region with LD of SNPs studied in conditional analyses. Haploview diagram displays increasing red with higher D'. Three independently significant RA SNPs and HLA-DRB1 locations are indicated.

Table 3 Independent SNPs in conditional analysis of RA susceptibility

Table 4 shows the case-control frequencies and ORs for the final haplotypes, with the most common haplotype (ACG-1) as the reference haplotype. The highest-risk haplotype of these SNPs and the SE level had OR = 27.2 (95% CI, 16.7-44.4) in comparison to OR = 7.3 (95% CI, 4.7-11.1) overall comparing the SE risk levels alone (data not shown).

Table 4 Frequencies and odds ratios for haplotypes of three SNPs and shared epitope proxy risk level

Conclusion

In our analysis of the Genetic Analysis Workshop 16 dataset, there was insufficient power to detect known associations with RA susceptibility at a genome-wide significance level outside of the MHC; the most significant association was p = 5.3 × 10-6 for PTPN22. Clearly, the MHC is the most influential genetic region in RA susceptibility, but extensive LD makes isolating the precise loci difficult. We have used conditional analyses as a tool to investigate the presence of multiple RA risk factors in the MHC region in addition to the SE. Out of 200 candidate SNPs having unconditional p-values < 10-8, we have identified an additional HLA class II marker and two HLA class I markers which have significant associations with RA susceptibility that are not fully explained by LD with HLA-DRB1. A better understanding of these genetic influences can be helpful in elucidating the complex genetic components of RA.

Previous studies of MHC effects on RA susceptibility beyond the SE have identified additional independent signals but have been largely inconsistent, due at least in part to the difficulty of narrowing down regions of association in the presence of extended LD [1, 13]. Multiple studies have implicated the TNF-lymphotoxin locus in class III [1], which were not significant in our conditional analysis. Other studies also using NARAC cases have observed signals in class I [5, 14], including HLA-C, our second SNP added in conditional analysis. Our first SNP is in the gene TRIM39, also in class I but not previously implicated. Our third SNP, in class II, is 150 kb upstream from HLA-DRB1 between the BTNL2 and HLA-DRA genes. BTNL2 has been associated with RA, systemic lupus erythematosus, and type 1 diabetes [15]; this is attributed to its association with predisposing HLA DQB1-DRB1 haplotypes, which may explain its presence in our data as well.

It is important to note that the NARAC population is primarily Caucasian. Other populations could have quite different distributions of these haplotypes as well as other haplotypes and allele frequencies. A similar analysis in other ethnic groups could be very informative.

Abbreviations

LD:

Linkage disequilibrium

MAF:

Minor allele frequency

MHC:

Major histocompatibility complex

NARAC:

North American Rheumatoid Arthritis Consortium

OR:

Odds ratio

PCA:

Principal components analysis

RA:

Rheumatoid arthritis

SE:

Shared epitope

SNP:

Single-nucleotide polymorphism

References

  1. Newton JL, Harney SM, Wordsworth BP, Brown MA: A review of the MHC genetics of rheumatoid arthritis. Genes Immun. 2004, 5: 151-157. 10.1038/sj.gene.6364045.

    Article  CAS  PubMed  Google Scholar 

  2. Remmers EF, Plenge RM, Lee AT, Graham RR, Hom G, Behrens TW, de Bakker PI, Le JM, Lee HS, Batliwalla F, Li W, Masters SL, Booty MG, Carulli JP, Padyukov L, Alfredsson L, Klareskog L, Chen WV, Amos CI, Criswell LA, Seldin MF, Kastner DL, Gregersen PK: STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus. N Engl J Med. 2007, 357: 977-986. 10.1056/NEJMoa073003.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Plenge RM, Seielstad M, Padyukov L, Lee AT, Remmers EF, Ding B, Liew A, Khalili H, Chandrasekaran A, Davies LR, Li W, Tan AK, Bonnard C, Ong RT, Thalamuthu A, Pettersson S, Liu C, Tian C, Chen WV, Carulli JP, Beckman EM, Altshuler D, Alfredsson L, Criswell LA, Amos CI, Seldin MF, Kastner DL, Klareskog L, Gregersen PK: TRAF1-C5 as a risk locus for rheumatoid arthritis--a genomewide study. N Engl J Med. 2007, 357: 1199-1209. 10.1056/NEJMoa073491.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Plenge RM, Cotsapas C, Davies L, Price AL, de Bakker PI, Maller J, Pe'er I, Burtt NP, Blumenstiel B, DeFelice M, Parkin M, Barry R, Winslow W, Healy C, Graham RR, Neale BM, Izmailova E, Roubenoff R, Parker AN, Glass R, Karlson EW, Maher N, Hafler DA, Lee DM, Seldin MF, Remmers EF, Lee AT, Padyukov L, Alfredsson L, Coblyn J, Weinblatt ME, Gabriel SB, Purcell S, Klareskog L, Gregersen PK, Shadick NA, Daly MJ, Altshuler D: Two independent alleles at 6q23 associated with risk of rheumatoid arthritis. Nat Genet. 2007, 39: 1477-1482. 10.1038/ng.2007.27.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Jawaheer D, Li W, Graham RR, Chen W, Damle A, Xiao X, Monteiro J, Khalili H, Lee A, Lundsten R, Begovich A, Bugawan T, Erlich H, Elder JT, Criswell LA, Seldin MF, Amos CI, Behrens TW, Gregersen PK: Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis. Am J Hum Genet. 2002, 71: 585-594. 10.1086/342407.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Vignal C, Bansal AT, Balding DJ, Binks MH, Dickson MC, Montgomery DS, Wilson AG: Genetic association of the major histocompatibility complex with rheumatoid arthritis implicates two non-DRB1 loci. Arthritis Rheum. 2009, 60: 53-62. 10.1002/art.24138.

    Article  CAS  PubMed  Google Scholar 

  7. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: a toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet. 2007, 81: 559-575. 10.1086/519795.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D: Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet. 2006, 38: 904-909. 10.1038/ng1847.

    Article  CAS  PubMed  Google Scholar 

  9. Seldin MF, Shigeta R, Villoslada P, Selmi C, Tuomilehto J, Silva G, Belmont JW, Klareskog L, Gregersen PK: European population substructure: clustering of northern and southern populations. PLoS Genet. 2006, 2: 1-13. 10.1371/journal.pgen.0020143.

    Article  Google Scholar 

  10. Fellay J, Shianna KV, Ge D, Colombo S, Ledergerber B, Weale M, Zhang K, Gumbs C, Castagna A, Cossarizza A, Cozzi-Lepri A, De Luca A, Easterbrook P, Francioli P, Mallal S, Martinez-Picado J, Miro JM, Obel N, Smith JP, Wyniger J, Descombes P, Antonarakis SE, Letvin NL, McMichael AJ, Haynes BF, Telenti A, Goldstein DB: A whole-genome association study of major determinants for host control of HIV-1. Science. 2007, 317: 944-947. 10.1126/science.1143767.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Hom G, Graham RR, Modrek B, Taylor KE, Ortmann W, Garnier S, Lee AT, Chung SA, Ferreira RC, Pant PV, Ballinger DG, Kosoy R, Demirci FY, Kamboh MI, Kao AH, Tian C, Gunnarsson I, Bengtsson AA, Rantapää-Dahlqvist S, Petri M, Manzi S, Seldin MF, Rönnblom L, Syvänen AC, Criswell LA, Gregersen PK, Behrens TW: Association of systemic lupus erythematosus with C8orf13-BLK and ITGAM-ITGAX. N Engl J Med. 2008, 358: 900-909. 10.1056/NEJMoa0707865.

    Article  CAS  PubMed  Google Scholar 

  12. Purcell S, Daly M, Sham P: WHAP: haplotype-based association analysis. Bioinformatics. 2007, 23: 255-256. 10.1093/bioinformatics/btl580.

    Article  CAS  PubMed  Google Scholar 

  13. Gregersen PK, Olsson LM: Recent advances in the genetics of autoimmune disease. Annu Rev Immunol. 2009, 27: 363-391. 10.1146/annurev.immunol.021908.132653.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Lee HS, Lee AT, Criswell LA, Seldin MF, Amos CI, Carulli JP, Navarrete C, Remmers EF, Kastner DL, Plenge RM, Li W, Gregersen PK: Several regions in the major histocompatibility complex confer risk for anti-CCP-antibody positive rheumatoid arthritis, independent of the DRB1 locus. Mol Med. 2008, 14: 293-300. 10.2119/2007-00123.Lee.

    Article  PubMed Central  PubMed  Google Scholar 

  15. Orozco G, Eerligh P, Sánchez E, Zhernakova S, Roep BO, González-Gay MA, López-Nevot MA, Callejas JL, Hidalgo C, Pascual-Salcedo D, Balsa A, González-Escribano MF, Koeleman BP, Martín J: Analysis of a functional BTNL2 polymorphism in type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Hum Immunol. 2005, 66: 1235-1241. 10.1016/j.humimm.2006.02.003.

    Article  CAS  PubMed  Google Scholar 

  16. Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, Alexander HC, Ardlie KG, Huang Q, Smith AM, Spoerke JM, Conn MT, Chang M, Chang SY, Saiki RK, Catanese JJ, Leong DU, Garcia VE, McAllister LB, Jeffery DA, Lee AT, Batliwalla F, Remmers E, Criswell LA, Seldin MF, Kastner DL, Amos CI, Sninsky JJ, Gregersen PK: A missense single-nucleotide polymorphism in the protein tyrosine phosphatase PTPN22 is associated with rheumatoid arthritis. Am J Hum Genet. 2004, 75: 330-337. 10.1086/422827.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Kurreeman FA, Padyukov L, Marques RB, Schrodi SJ, Seddighzadeh M, Stoeken-Rijsbergen G, Helm-van Mil van der AH, Allaart CF, Verduyn W, Houwing-Duistermaat J, Alfredsson L, Begovich AB, Klareskog L, Huizinga TW, Toes RE: A candidate gene approach identifies the TRAF1/C5 region as a risk factor for rheumatoid arthritis. PLoS Med. 2007, 4: e278-10.1371/journal.pmed.0040278.

    Article  PubMed Central  PubMed  Google Scholar 

  18. Plenge RM, Cotsapas C, Davies L, Price AL, de Bakker PI, Maller J, Pe'er I, Burtt NP, Blumenstiel B, DeFelice M, Parkin M, Barry R, Winslow W, Healy C, Graham RR, Neale BM, Izmailova E, Roubenoff R, Parker AN, Glass R, Karlson EW, Maher N, Hafler DA, Lee DM, Seldin MF, Remmers EF, Lee AT, Padyukov L, Alfredsson L, Coblyn J, Weinblatt ME, Gabriel SB, Purcell S, Klareskog L, Gregersen PK, Shadick NA, Daly MJ, Altshuler D: Two independent alleles at 6q23 associated with risk of rheumatoid arthritis. Nat Genet. 2007, 39: 1477-1482. 10.1038/ng.2007.27.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. 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: 661-678. 10.1038/nature05911.

    Article  Google Scholar 

  20. Raychaudhuri S, Remmers EF, Lee AT, Hackett R, Guiducci C, Burtt NP, Gianniny L, Korman BD, Padyukov L, Kurreeman FA, Chang M, Catanese JJ, Ding B, Wong S, Helm-van Mil van der AH, Neale BM, Coblyn J, Cui J, Tak PP, Wolbink GJ, Crusius JB, Horst-Bruinsma van der IE, Criswell LA, Amos CI, Seldin MF, Kastner DL, Ardlie KG, Alfredsson L, Costenbader KH, Altshuler D, Huizinga TW, Shadick NA, Weinblatt ME, de Vries N, Worthington J, Seielstad M, Toes RE, Karlson EW, Begovich AB, Klareskog L, Gregersen PK, Daly MJ, Plenge RM: Common variants at CD40 and other loci confer risk of rheumatoid arthritis. Nat Genet. 2008, 40: 1216-1223. 10.1038/ng.233.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The Genetic Analysis Workshops are supported by NIH grant R01 GM031575 from the National Institute of General Medical Sciences. The authors are grateful for the support of Dr. Peter K. Gregersen and the NARAC investigative team.

This work was supported by grants from the National Institutes of Health: N01-AR-72232, R01 AI 065841, K24 AR02175, National Center for Research Resources M01 RR-00079 the Rosalind Russell Medical Research Center for Arthritis at UCSF, and a Kirkland Scholar Award to LAC.

This article has been published as part of BMC Proceedings Volume 3 Supplement 7, 2009: Genetic Analysis Workshop 16. The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/3?issue=S7.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Lindsey A Criswell.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

KET performed statistical analyses and drafted the manuscript. LAC recruited patients as part of the NARAC collaboration. LAC and KET designed the study, revised the manuscript, and read and approved the final manuscript.

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Taylor, K.E., Criswell, L.A. Conditional analysis of the major histocompatibility complex in rheumatoid arthritis. BMC Proc 3 (Suppl 7), S36 (2009). https://doi.org/10.1186/1753-6561-3-S7-S36

Download citation

  • Published:

  • DOI: https://doi.org/10.1186/1753-6561-3-S7-S36

Keywords