Abstract
The WTCCC study identified 49 single nucleotide polymorphisms (SNPs) putatively associated with RA at p=1×10-4-1×10-5 (Tier 3). Here, we show that 3 of these SNPs, mapping to chromosome 10p15 (rs4750316), 12q13 (rs1678542) and 22q13 (rs3218253), are also associated (trend p = 4×10-5, p=4×10-4 and p=4×10-4, respectively) in a validation study of 4,106 RA cases and an expanded reference group of 11,238 subjects, confirming them as true susceptibility loci in Caucasians.
Rheumatoid arthritis (RA) is a common autoimmune disease, affecting an estimated 475,000 adults in the UK, in which inflammation of synovial joints is characteristic. The WTCCC genome wide association screen of 1860 RA case and 2938 healthy control Caucasian samples confirmed association of SNPs with previously identified loci within the HLA region and the PTPN22 gene (Tier1, p<5×10-7)1. Nine loci were associated in the next tier of significance (Tier 2, p=1×10-5-5×10-7) and association to one, on chromosome 6q23 has been replicated and confirmed in subsequent independent studies2, 3. Forty-nine SNPs were associated at p = 1 × 10-4 - 1 × 10-5 (Tier 3) and we now describe investigation of these SNPs in an independent cohort of 4,106 RA samples (supplementary methods). Causal variants within this group of SNPs are likely to confer small effect sizes, by virtue of the fact that the evidence for association with them in the original WTCCC study was weaker than for those SNPs in Tiers 1 and 2. In order to maximise power to replicate true associations, genotype data from an independent cohort of 3,599 healthy controls was combined with that from the 4 non-autoimmune disease case subjects (coronary artery disease, hypertension, type 2 diabetes and bipolar disorder) genotyped as part of the WTCCC study to create a large reference sample (total sample size of expanded reference group = 11,238)1. A Bonferroni correction of 49 was applied to account for the novel loci investigated, resulting in a p-value threshold of P<0.001 for claims of significance in this validation study. The validation cohort had 80% power to detect most of the effect sizes reported in the initial study at P<0.001(supplementary Table 1). In addition, we took the opportunity to re-evaluate the evidence for association with the Tier 2 SNPs using the expanded reference group for comparison.
Of the 49 novel Tier 3 SNPs tested, 3 showed association with RA: rs4750316 (C>G), mapping to chromosome 10p15 (OR minor allele =0.87, 95% CI 0.81-0.93, trend P = 4 × 10-5); rs1678542 (G>C) mapping to chromosome 12q13 (OR minor allele = 0.91, 95% CI 0.86-0.96, trend P = 4 × 10-4) and rs3218253 (C>T) mapping to chromosome 22 (OR minor allele = 1.11, 95% CI 1.05 - 1.17, trend P = 4 × 10-4) (Table 1, supplementary Tables 2 and 3). For all SNPs, the allele frequencies were similar across the control group tested in the WTCCC study and the expanded reference group tested here (rs4750316 minor allele frequency (MAF) 0.20 and 0.20; rs1678542 MAF 0.37 and 0.37; rs3218254 MAF 0.25 and 0.26, respectively) and all satisfied Hardy-Weinberg expectations. Both combined analysis and meta-analysis of the WTCCC data and the validation data provided strong statistical evidence for association between RA and all 3 SNPs (combined analysis: OR rs4750316 = 0.85 95% CI 0.80-0.90, trend P = 1.3×10-8; OR rs1678542 = 0.88, 95% CI 0.85-0.93, trend P = 1.3 × 10-7; OR rs3218253 = 1.13, 95% CI 1.07-1.18, trend P = 1.3 × 10-6) (Table 1 and supplementary Table 2).
Table 1.
Replication of Tier 3 WTCCC RA susceptibility loci
| SNP | Validation study a: n (%) | Combined analysisb: n (%) | Locus | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cases (n = 4,106) | Controls (n = 11,238) | OR (95% CI) |
p-trend | Cases (n = 5,968) | Controls (n = 14,176) | OR (95% CI) |
p-trend | ||||||||||
| 11 | 12 | 22 | 11 | 12 | 22 | 11 | 12 | 22 | 11 | 12 | 22 | ||||||
| rs4750316d | 2766 (68.3) |
1159 (28.6) |
124 (3.1) |
7283 (64.9) |
3511 (31.3) |
429 (3.8) |
0.87 (0.81- 0.93) |
3.9×10-5 | 4068 (68.9) |
1659 (28.1) |
180 (3.1) |
9171 (64.8) |
4445 (31.4) |
542 (3.8) |
0.85 (0.80- 0.90) |
1.3×10-8 | PRKCQ |
| rs1678542e | 1692 (42.7) |
1797 (45.3) |
478 (12.0) |
4487 (40.1) |
5128 (45.9) |
1566 (14.0) |
0.91 (0.86- 0.96) |
0.0004 | 2523 (43.3) |
2616 (44.9) |
684 (11.7) |
5661 (40.1) |
6455 (45.7) |
1999 (14.2) |
0.88 (0.85- 0.93) |
1.3×10-7 | KIF5A |
| rs3218253c,f | 2120 (52.1) |
1587 (39.0) |
360 (8.9) |
6053 (54.2) |
4365 (39.1) |
759 (6.8) |
1.11 (1.05- 1.17) |
0.0004 | 3071 (51.9) |
2336 (39.5) |
512 (8.7) |
7667 (54.4) |
5517 (39.1) |
912 (6.5) |
1.13 (1.07- 1.18) |
1.3×10-6 | IL2RB |
Genotype counts are shown, with percentages in parentheses. 1 = common allele, 2 = minor allele
OR (95% CI) - Minor allele odds ratio and 95% confidence intervals; p-trend - p value under additive model
The expanded reference group included 3,599 healthy controls, 1,868 bipolar disorder samples, 1,952 hypertension samples, 1,924 Type 2 diabetes samples and 1,894 Coronary heart disease samples.
WTCCC cases plus validation cases were compared with WTCCC controls plus validation expanded reference group.
p values under a recessive model equal to 1.6×10-5 and 4.4×10-8 in the validation study and in the combined analysis, respectively.
meta-analysis of validation and WTCCC data, OR 0.85 95%CI 0.80-0.90, p = 1.9 × 10-8
meta-analysis of validation and WTCCC data, OR 0.89 95%CI 0.85-0.93, p = 1.7 × 10-7
meta-analysis of validation and WTCCC data, OR 1.13 95%CI 1.08-1.19, p = 6.1 × 10-7
RA is characterised by the presence of autoantibodies in some but not all patients. Previous work has shown that established susceptibility variants, including HLA DRB1 and the chromosome 6q23 locus, have a stronger effect in the subgroup harbouring antibodies to citrullinated peptide antibodies (ACPA) recognised by the anti-CCP assay2, 4. Stratification analysis showed that, for all 3 loci, the direction and strength of effect size was similar in antibody positive and negative individuals although this did not always achieve statistical significance in the anti-CCP negative subgroup due to the smaller sample size (supplementary Table 4). Hence, these loci appear to be associated with RA per se rather than subgroups of the disease.
rs4750316 maps to an intergenic region of 10p15. Four SNPs genotyped by the HapMap Consortium (rs10752291, rs10796045, rs2146900 and rs1570527) in the same region have a pair-wise r2 >0.96 with rs4750316 and all 5 SNPs map within 12Kb of each other in a single linkage disequilibrium (LD) block containing no known genes or transcripts (supplementary Figure 1). The closest gene lies 76Kb from rs4750316 and is the PRKCQ gene, which encodes an atypical member of the protein kinase C family of proteins, PKCθ. It is thought to play a key role in T cell activation and PKCθ-deficient T cells in mice show a profound defect in IL-2 production and proliferation upon CD3/CD28 stimulation (reviewed in 5). As RA is thought to be primarily a T-cell mediated autoimmune disease, investigating the role of the associated SNPs in regulation of the PRCKQ gene will be a priority.
rs1678542 maps to intron 15 of the KIF5A gene (supplementary Figure 1). The gene encodes a kinesin heavy chain and mutations of the gene have been associated with hereditary spastic paraplegia 6. Hence, it is not an obvious candidate RA susceptibility gene. KIF5A maps between the dynactin2 (DCTN2) and the phosphatidylinositol 4 phosphate 5 kinase, type II, gamma (PIP5K2C) genes. The latter is expressed in B cells and is thought to play a role in B-cell receptor activation as well as development and function of B cells 7. It, therefore, represents a stronger candidate for a disease characterized by the presence of autoantibodies. There are now several examples where an associated variant mapping within one gene has subsequently been found to regulate a different gene but further investigation is required to determine whether this is true of the rs1678542 polymorphism 8, 9.
rs3218253 maps to the interleukin 2 receptor B (IL2RB) gene (supplementary Figure 1). We have previously reported borderline evidence for association with another SNP in the gene (rs743777)2. Combining the data from the validation cases, the expanded reference group and original WTCCC data, the evidence for association of the rs743777 SNP was strengthened (trend P = 4.6 × 10-8) (Table 2). For both SNPs, the evidence for association was even stronger under a recessive model (rs3218253 P = 4.4 × 10-8 and rs743777 P = 5.2 × 10-9, respectively). rs743777 maps 6Kb upstream of the transcription start site of IL2RB whilst rs3218253 maps to intron 1 but both SNPs lie within a single LD block spanning 6.8Kb and show reasonable correlation with each other (r2 = 0.77). Therefore, haplotype analysis was undertaken. Haplotype frequencies were significantly different between cases and controls but the effect of the haplotype of minor alleles at each locus was not significantly increased over that of the minor allele at rs743777 alone (data not shown). Furthermore, logistic regression analysis suggested that the effect at rs3218253 could be fully accounted for by the genotype at rs743777, suggesting that the latter SNP is primarily driving the association and the association with rs3218253 is arising as a result of its LD with rs743777 (p value for rs3218253 after conditioning on rs743777 = 0.60). The IL2RB gene is an attractive candidate for an autoimmune disease because of the key role played by IL2 in T cell activation and regulation. The IL2RB gene encodes the beta unit of the IL2R, which is present in the moderate and high affinity forms of the receptor required for signal transduction from IL2 10.
Table 2.
Tier2 SNP re-analysis including WTCCC non-autoimmune disease samples as expanded control panel
| SNP | Validation study a: n (%) | Combined analysisb: n (%) | Locus | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cases (n =5,063) | Controls (n =11,487) | OR (95% CI) |
p-trend | Cases (n=6,923) | Controls (n=14,425) | OR (95% CI) |
p-trend | ||||||||||
| 11 | 12 | 22 | 11 | 12 | 22 | 11 | 12 | 22 | 11 | 12 | 22 | ||||||
| rs10910099c,f | 495 (10.6) |
2020 (43.2) |
2159 (46.2) |
1279 (11.5) |
4913 (44.2) |
4922 (44.3) |
0.94 (0.89- 0.99) |
0.02 | 646 (9.9) |
2817 (43.3) |
3042 (46.8) |
1609 (11.5) |
6262 (44.7) |
6148 (43.8) |
0.90 (0.86- 0.94) |
8.2×10-6 | In LD with rs6684865 MMEL1 |
| rs3816587d | 698 (17.3) |
1969 (48.7) |
1372 (34.0) |
1977 (17.6) |
5387 (48.0) |
3863 (34.4) |
1.03 (0.97- 1.09) |
NS | 1086 (18.4) |
2806 (47.6) |
2005 (34.0) |
2437 (17.2) |
6853 (48.4) |
4873 (34.4) |
1.05 (1.01- 1.10) |
0.03 | |
| rs11162922 | 18 (0.4) |
525 (11.3) |
4123 (88.4) |
36 (0.3) |
1331 (11.9) |
9786 (87.7) |
0.96 (0.86- 1.06) |
NS | 22 (0.3) |
694 (10.7) |
5800 (89.0) |
48 (0.3) |
1729 (12.3) |
12306 (87.4) |
0.87 (0.80- 0.95) |
0.002 | |
| rs6920220g | 272 (5.8) |
1769 (37.8) |
2639 (56.4) |
499 (4.4) |
4005 (35.6) |
6761 (60.0) |
1.15 (1.09- 1.22) |
9.3×10-7 | 399 (6.1) |
2492 (38.1) |
3646 (55.8) |
628 (4.4) |
5054 (35.6) |
8518 (60.0) |
1.18 (1.12- 1.24) |
2.3×10-11 | OLIG3- TNFAIP3 |
| rs11761231h | 561 (12.0) |
2118 (45.3) |
2001 (42.8) |
1456 (12.9) |
5193 (46.1) |
4620 (41.0) |
0.94 (0.90- 0.99) |
0.02 | 750 (11.5) |
2954 (45.2) |
2832 (43.3) |
1891 (13.3) |
6528 (46.0) |
5787 (40.7) |
0.91 (0.87- 0.95) |
1.2×10-5 | PODXL |
| rs2104286i | 312 (6.7) |
1740 (37.3) |
2608 (56.0) |
790 (7.0) |
4464 (39.6) |
6006 (53.3) |
0.93 (0.88- 0.98) |
0.007 | 415 (6.4) |
2442 (37.5) |
3660 (56.2) |
1036 (7.3) |
3652 (39.8) |
7506 (52.9) |
0.90 (0.86- 0.94) |
6.4×10-6 | IL2RA |
| rs9550642 | 47 (1.0) |
835 (17.8) |
3796 (81.1) |
113 (1.0) |
1985 (17.6) |
9162 (81.4) |
1.01 (0.93- 1.10) |
NS | 71 (1.1) |
1202 (18.4) |
5261 (80.5) |
131 (0.9) |
2444 (17.2) |
11616 (81.9) |
1.09 (1.02- 1.17) |
0.02 | CRYL1 |
| rs2837960 | 153 (3.3) |
1332 (28.5) |
3194 (68.3) |
352 (3.1) |
3206 (28.5) |
7703 (68.4) |
1.01 (0.95 - 1.08) |
NS | 238 (3.7) |
1849 (28.4) |
4415 (67.9) |
412 (2.9) |
4091 (28.8) |
9689 (68.3) |
1.04 (0.98- 1.10) |
NS | |
| rs743777e,j | 532 (11.4) |
2031 (43.4) |
2117 (45.2) |
1040 (9.3) |
4832 (43.1) |
5328 (47.6) |
1.11 (1.05- 1.17) |
0.0001 | 753 (11.5) |
2829 (43.3) |
2946 (45.1) |
1262 (8.9) |
6093 (43.2) |
6761 (47.9) |
1.13 (1.08- 1.18) |
4.6×10-8 | IL2RB |
p-trend - p value under additive model
The expanded reference group included 3,599 healthy controls, 1,868 bipolar disorder samples, 1,952 hypertension samples, 1,924 Type 2 diabetes samples and 1,894 Coronary heart disease samples.
WTCCC cases plus validation cases were compared with WTCCC controls plus validation expanded reference group
The marker genotyped and found to be associated with RA in the WTCCC was rs6684865 but the marker genotyped in the current study, rs10910099, shows high correlation (r2 = 0.95) and serves as a proxy for it.
Not included in original analysis due to low genotyping success rate (<80%), now re-genotyped in 3,595 cases and 3,214 controls
p values under a recessive model equal to 6.3×10-5 and 5.2×10-9 in the validation study and in the combined analysis, respectively.
meta-analysis of validation and WTCCC data, OR 0.91 95%CI 0.87-0.95, p = 1.2 × 10-5
meta-analysis of validation and WTCCC data, OR 1.17 95%CI 1.12-1.23, p = 9.4 × 10-11
meta-analysis of validation and WTCCC data, OR 0.91 95%CI 0.87-0.95, p = 1.0 × 10-5
meta-analysis of validation and WTCCC data, OR 0.89 95%CI 0.85-0.94, p = 3.4 × 10-6
meta-analysis of validation and WTCCC data, OR 1.14 95%CI 1.09-1.19, p = 2.5 × 10-8
Given that the evidence for association with IL2RB was strengthened by analysing all the available data including the expanded reference cohort, we were interested to investigate whether evidence for association with any of the other SNPs tested previously from Tier 2 was increased. In the expanded validation samples, 5 of the 9 Tier 2 SNPs showed evidence for association (p < 0.05) and, in a combined analysis of 6,923 RA cases and 14,425 combined reference samples, the strength of evidence for association for 4 of these 5 loci was increased over that seen in the original WTCCC study (Table 2). These included the SNP mapping to 6q23, which has previously been confirmed independently as being associated with RA susceptibility and the IL2RB SNP (rs743777) already discussed 2, 3. In addition, the evidence for association to SNPs mapping to the membrane metallo-endopeptidase-like 1 (MMEL1) and IL2RA genes (OR 0.90, 95% CI 0.86-0.94, p = 8.2 ×10-6 and OR 0.90 95% CI 0.86-0.94, p = 6.4 ×10-6, respectively) was enhanced. Interestingly, in analysis of an independent cohort of RA cases and controls from Europe and the United States published in this issue of Nature Genetics, evidence for association of a SNP mapping to the MMEL1 gene region has also been reported (P = 0.01). Furthermore, that study also independently replicates the findings for the PRKCQ and KIF5A loci reported here (P = 0.03 and 0.009, respectively) suggesting that these regions harbour true RA susceptibility variants with modest but important effects11.
In conclusion, novel RA susceptibility loci, mapping close to the PRKCQ and KIF5A genes have been identified in Caucasian samples and evidence for a recessive effect at a third, the IL2RB gene, is compelling. In addition, evidence for association to the MMEL1 and IL2RA gene regions is mounting. The challenge will now be to identify and characterise the aetiological variants of the confirmed RA susceptibility genes in order to determine how they predispose to RA12.
Supplementary Material
Acknowledgements
We thank the Arthritis Research Campaign for their support. arc grant reference no.: 17552. We would also like to acknowledge the NHS Research and Development Support Fund for Guy’s and St Thomas’ and Lewisham NHS Trusts. 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. We thank the YEAR consortium for allowing us access to their DNA collection. DP is supported by the European Community’s Sixth Framework Programme Autocure funding. IT support was provided by Phil Steer and technical support by Paul Gilbert, Bhaneeta Lad and Stephen G Martin. Nursing support was provided by Janet Grumley, Julie Shotton and Claire Farrar.
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