Steve Eyre

Genetics of rheumatoid arthritis contributes to biology and drug discovery

A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological data sets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA). Here we performed a genome-wide association study meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ∼10 million single-nucleotide polymorphisms. We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 101 (refs 2, 3, 4). We devised an in silico pipeline using established bioinformatics methods based on functional annotation, cis-acting expression quantitative trait loci and pathway analyses—as well as novel methods based on genetic overlap with human primary immunodeficiency, haematological cancer somatic mutations and knockout mouse phenotypes—to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery.

Rheumatoid arthritis association at 6q23

The Wellcome Trust Case Control Consortium (WTCCC) identified nine single SNPs putatively associated with rheumatoid arthritis at P = 1 × 10−5 − 5 × 10−7 in a genome-wide association screen. One, rs6920220, was unequivocally replicated (trend P = 1.1 × 10−8) in a validation study, as described here. This SNP maps to 6q23, between the genes oligodendrocyte lineage transcription factor 3 (OLIG3) and tumor necrosis factor-α–induced protein 3 (TNFAIP3).

High-density genetic mapping identifies new susceptibility loci for rheumatoid arthritis

Using the Immunochip custom SNP array, which was designed for dense genotyping of 186 loci identified through genome-wide association studies (GWAS), we analyzed 11,475 individuals with rheumatoid arthritis (cases) of European ancestry and 15,870 controls for 129,464 markers. We combined these data in a meta-analysis with GWAS data from additional independent cases (n = 2,363) and controls (n = 17,872). We identified 14 new susceptibility loci, 9 of which were associated with rheumatoid arthritis overall and five of which were specifically associated with disease that was positive for anticitrullinated peptide antibodies, bringing the number of confirmed rheumatoid arthritis risk loci in individuals of European ancestry to 46. We refined the peak of association to a single gene for 19 loci, identified secondary independent effects at 6 loci and identified association to low-frequency variants at 4 loci. Bioinformatic analyses generated strong hypotheses for the causal SNP at seven loci. This study illustrates the advantages of dense SNP mapping analysis to inform subsequent functional investigations.

Whole-Genome Scan, in a Complex Disease, Using 11,245 Single-Nucleotide Polymorphisms: Comparison with Microsatellites

Despite the theoretical evidence of the utility of single-nucleotide polymorphisms (SNPs) for linkage analysis, no whole-genome scans of a complex disease have yet been published to directly compare SNPs with microsatellites. Here, we describe a whole-genome screen of 157 families with multiple cases of rheumatoid arthritis (RA), performed using 11,245 genomewide SNPs. The results were compared with those from a 10-cM microsatellite scan in the same cohort. The SNP analysis detected HLA*DRB1, the major RA susceptibility locus (P=.00004), with a linkage interval of 31 cM, compared with a 50-cM linkage interval detected by the microsatellite scan. In addition, four loci were detected at a nominal significance level (P<.05) in the SNP linkage analysis; these were not observed in the microsatellite scan. We demonstrate that variation in information content was the main factor contributing to observed differences in the two scans, with the SNPs providing significantly higher information content than the microsatellites. Reducing the number of SNPs in the marker set to 3,300 (1-cM spacing) caused several loci to drop below nominal significance levels, suggesting that decreases in information content can have significant effects on linkage results. In contrast, differences in maps employed in the analysis, the low detectable rate of genotyping error, and the presence of moderate linkage disequilibrium between markers did not significantly affect the results. We have demonstrated the utility of a dense SNP map for performing linkage analysis in a late-age-at-onset disease, where DNA from parents is not always available. The high SNP density allows loci to be defined more precisely and provides a partial scaffold for association studies, substantially reducing the resource requirement for gene-mapping studies.

Rheumatoid arthritis susceptibility loci at chromosomes 10p15, 12q13 and 22q13.

The WTCCC study identified 49 SNPs putatively associated with rheumatoid arthritis at P = 1 × 10−4 − 1 × 10−5 (tier 3). Here we show that three 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 individuals with rheumatoid arthritis and an expanded reference group of 11,238 subjects, confirming them as true susceptibility loci in individuals of European ancestry.

Re-evaluation of putative rheumatoid arthritis susceptibility genes in the post-genome wide association study era and hypothesis of a key pathway underlying susceptibility

Rheumatoid arthritis (RA) is an archetypal, common, complex autoimmune disease with both genetic and environmental contributions to disease aetiology. Two novel RA susceptibility loci have been reported from recent genome-wide and candidate gene association studies. We, therefore, investigated the evidence for association of the STAT4 and TRAF1/C5 loci with RA using imputed data from the Wellcome Trust Case Control Consortium (WTCCC). No evidence for association of variants mapping to the TRAF1/C5 gene was detected in the 1860 RA cases and 2930 control samples tested in that study. Variants mapping to the STAT4 gene did show evidence for association (rs7574865, P = 0.04). Given the association of the TRAF1/C5 locus in two previous large case–control series from populations of European descent and the evidence for association of the STAT4 locus in the WTCCC study, single nucleotide polymorphisms mapping to these loci were tested for association with RA in an independent UK series comprising DNA from >3000 cases with disease and >3000 controls and a combined analysis including the WTCCC data was undertaken. We confirm association of the STAT4 and the TRAF1/C5 loci with RA bringing to 5 the number of confirmed susceptibility loci. The effect sizes are less than those reported previously but are likely to be a more accurate reflection of the true effect size given the larger size of the cohort investigated in the current study.

Fine Mapping Seronegative and Seropositive Rheumatoid Arthritis to Shared and Distinct HLA Alleles by Adjusting for the Effects of Heterogeneity

Despite progress in defining human leukocyte antigen (HLA) alleles for anti-citrullinated-protein-autoantibody-positive (ACPA(+)) rheumatoid arthritis (RA), identifying HLA alleles for ACPA-negative (ACPA(-)) RA has been challenging because of clinical heterogeneity within clinical cohorts. We imputed 8,961 classical HLA alleles, amino acids, and SNPs from Immunochip data in a discovery set of 2,406 ACPA(-) RA case and 13,930 control individuals. We developed a statistical approach to identify and adjust for clinical heterogeneity within ACPA(-) RA and observed independent associations for serine and leucine at position 11 in HLA-DRβ1 (p = 1.4 × 10(-13), odds ratio [OR] = 1.30) and for aspartate at position 9 in HLA-B (p = 2.7 × 10(-12), OR = 1.39) within the peptide binding grooves. These amino acid positions induced associations at HLA-DRB1(∗)03 (encoding serine at 11) and HLA-B(∗)08 (encoding aspartate at 9). We validated these findings in an independent set of 427 ACPA(-) case subjects, carefully phenotyped with a highly sensitive ACPA assay, and 1,691 control subjects (HLA-DRβ1 Ser11+Leu11: p = 5.8 × 10(-4), OR = 1.28; HLA-B Asp9: p = 2.6 × 10(-3), OR = 1.34). Although both amino acid sites drove risk of ACPA(+) and ACPA(-) disease, the effects of individual residues at HLA-DRβ1 position 11 were distinct (p < 2.9 × 10(-107)). We also identified an association with ACPA(+) RA at HLA-A position 77 (p = 2.7 × 10(-8), OR = 0.85) in 7,279 ACPA(+) RA case and 15,870 control subjects. These results contribute to mounting evidence that ACPA(+) and ACPA(-) RA are genetically distinct and potentially have separate autoantigens contributing to pathogenesis. We expect that our approach might have broad applications in analyzing clinical conditions with heterogeneity at both major histocompatibility complex (MHC) and non-MHC regions.

Genetic Variation in Efflux Transporters Influences Outcome to Methotrexate Therapy in Patients with Psoriasis

Methotrexate, an inexpensive first-line systemic therapy for moderate-to-severe psoriasis, is limited in its use by unpredictable efficacy and toxicity. This study was designed to test the hypothesis that single-nucleotide polymorphisms (SNPs) in methotrexate transmembrane transporters and adenosine receptors are associated with efficacy and/or toxicity of the drug. DNA was collected from 374 patients with chronic plaque psoriasis who had been treated with methotrexate. Phenotypic data on efficacy and toxicity were available. Haplotype tagging SNPs (r(2)>0.8) across the relevant genes, with a minor allele frequency of >5%, were selected from the HAPMAP phase II data. SNPs within the efflux transporter genes ABCC1 (ATP-binding cassette, subfamily C, member 1) and ABCG2 (ATP-binding cassette, subfamily G, member 2) are associated with good response to methotrexate therapy in patients with psoriasis; the former gene was also associated with the onset of toxicity. With one SNP in ABCC1, rs246240, the carriage of two copies of allele 1 gives an odds ratio of 2.2 (95% confidence interval: 1.3-3.6; P=0.001) for developing toxicity to methotrexate. These data indicate that knowledge of SNPs in genes relevant to methotrexate efflux may be important in selecting patients suitable for this therapy.

Study of the common genetic background for rheumatoid arthritis and systemic lupus erythematosus

Background Evidence is beginning to emerge that there may be susceptibility loci for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) that are common to both diseases. Objective To investigate single nucleotide polymorphisms that have been reported to be associated with SLE in a UK cohort of patients with RA and controls. Methods 3962 patients with RA and 9275 controls were included in the study. Eleven SNPs mapping to confirmed SLE loci were investigated. These mapped to the TNFSF4, BANK1, TNIP1, PTTG1, UHRF1BP1, ATG5, JAZF1, BLK, KIAA1542, ITGAM and UBE2L3 loci. Genotype frequencies were compared between patients with RA and controls using the trend test. Results The SNPs mapping to the BLK and UBE2L3 loci showed significant evidence for association with RA. Two other SNPs, mapping to ATG5 and KIAA1542, showed nominal evidence for association with RA (p=0.02 and p=0.02, respectively) but these were not significant after applying a Bonferroni correction. Additionally, a significant global enrichment in carriage of SLE alleles in patients with RA compared with controls (p=9.1×10−7) was found. Meta-analysis of this and previous studies confirmed the association of the BLK and UBE2L3 gene with RA at genome-wide significance levels (p<5×10−8). Together, the authors estimate that the SLE and RA overlapping loci, excluding HLA-DRB1 alleles, identified so far explain ∼5.8% of the genetic susceptibility to RA as a whole. Conclusion The findings confirm the association of the BLK and UBE2L3 loci with RA, thus adding to the list of loci showing overlap between RA and SLE.

Combined effects of three independent SNPs greatly increase the risk estimate for RA at 6q23

The most consistent finding derived from the WTCCC GWAS for rheumatoid arthritis (RA) was association to a SNP at 6q23. We performed a fine-mapping of the region in order to search the 6q23 region for additional disease variants. 3962 RA patients and 3531 healthy controls were included in the study. We found 18 SNPs associated with RA. The SNP showing the strongest association was rs6920220 [P = 2.6 × 10−6, OR (95% CI) 1.22 (1.13–1.33)]. The next most strongly associated SNP was rs13207033 [P = 0.0001, OR (95% CI) 0.86 (0.8–0.93)] which was perfectly correlated with rs10499194, a SNP previously associated with RA in a US/European series. Additionally, we found a number of new potential RA markers, including rs5029937, located in the intron 2 of TNFAIP3. Of the 18 associated SNPs, three polymorphisms, rs6920220, rs13207033 and rs5029937, remained significant after conditional logistic regression analysis. The combination of the carriage of both risk alleles of rs6920220 and rs5029937 together with the absence of the protective allele of rs13207033 was strongly associated with RA when compared with carriage of none [OR of 1.86 (95% CI) (1.51–2.29)]. This equates to an effect size of 1.50 (95% CI 1.21–1.85) compared with controls and is higher than that obtained for any SNP individually. This is the first study to show that the confirmed loci from the GWA studies, that confer only a modest effect size, could harbour a significantly greater effect once the effect of additional risk variants are accounted for.