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Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease affecting up to 30% of psoriasis vulgaris (PsV) cases and approximately 0.25% to 1% of the general population. To identify common susceptibility loci, we performed a meta-analysis of three imputed genome-wide association studies (GWAS) on psoriasis, stratified for PsA. A total of 1,160,703 SNPs were analyzed in the discovery set consisting of 535 PsA cases and 3,432 controls from Germany, the United States and Canada. We followed up two SNPs in 1,931 PsA cases and 6,785 controls comprising six independent replication panels from Germany, Estonia, the United States and Canada. In the combined analysis, a genome-wide significant association was detected at 2p16 near the REL locus encoding c-Rel (rs13017599, P=1.18×10−8, OR=1.27, 95% CI=1.18–1.35). The rs13017599 polymorphism is known to associate with rheumatoid arthritis (RA), and another SNP near REL (rs702873) was recently implicated in PsV susceptibility. However, conditional analysis indicated that rs13017599, rather than rs702873, accounts for the PsA association at REL. We hypothesize that c-Rel, as a member of the Rel/NF-κB family, is associated with PsA in the context of disease pathways that involve other identified PsA and PsV susceptibility genes including TNIP1, TNFAIP3 and NFκBIA.
Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease that occurs in the context of psoriasis vulgaris (PsV) affecting up to 30% of psoriasis patients (Gladman et al., 2005). Apart from the skin manifestation of PsV, the clinical phenotype of PsA overlaps with ankylosing spondylitis and rheumatoid arthritis (RA) (Castelino and Barton, 2010). While PsV has a prevalence of up to 3% in populations of European ancestry (Bowcock and Barker, 2003; Griffiths and Barker, 2007), the estimated prevalence of PsA varies from 0.25% to 1% (Gladman et al., 2005). The strong genetic component of PsA is evidenced by the high sibling recurrence risk (λS) of 27–47 (Chandran et al., 2009; Gladman et al., 2003; Myers et al., 2005), which is much higher than the estimated λS=4–11 of PsV (Bhalerao and Bowcock, 1998; Elder et al., 1994), suggesting that PsA patients harbor additional susceptibility loci beside the loci that contribute to risk for PsV. In addition to the significant heritable component, environmental risk factors play an important role for both PsV and PsA. The onset of PsA generally occurs in the fourth and fifth decades of life. In nearly 80% of patients, the skin disease (PsV) precedes the arthritis (PsA). The clarification of similarities and differences of the genetic background in PsV and PsA offers chances for well-targeted interventions, i.e. specific therapies for both conditions. It would also provide an opportunity to identify PsV patients with increased risk of PsA and would allow treatment at the earliest signs of joint involvement (Bowes and Barton, 2010). Several susceptibility loci for PsV have been identified with genome-wide levels of statistical significance in populations of European origin, including HLA-Cw6, IL12B, IL23R, IL4-IL13, IL23A, TNIP1, TNFAIP3, LCE3B-LCE3C, RNF114, TRAF3IP2, NFκBIA, NOS2, FBXL19, TYK2, IFIH1, REL, IL28RA and ERAP1 (Duffin et al., 2010; Ellinghaus et al., 2010; Liu et al., 2008; Strange et al., 2010; Stuart et al., 2010). Many of the susceptibility loci for PsV tested so far are also genome-wide significantly associated with PsA such as HLA-Cw6, IL12B, TNIP1, FBXL19 and TRAF3IP2 or at the level of P < 0.05 such as IL23R, IL23A, TNFAIP3, NFκBIA, NOS2 and IL13 (Bowes and Barton, 2010; Eder et al., 2011; Ellinghaus et al., 2010; Huffmeier et al., 2010; Stuart et al., 2010). However, because the causative allele(s) have not been identified within most of these regions, their actual contribution to disease risk remains unknown.
In order to further our understanding of PsA etiology, we conducted a meta-analysis of the PsA subsets of three independent genome-wide association studies of PsV from Kiel, Germany (Ellinghaus et al., 2010), the Collaborative Association Study of Psoriasis (CASP) (Nair et al., 2009) and Canada (Genizon, unpublished data), consisting of 535 PsA patients and 3,432 healthy controls. Genotype imputation with HapMap phase 3 reference samples considerably increased the genomic coverage of the three GWAS datasets and facilitated the combined analysis. For replication we selected ten SNPs based on their P-value ranking in the meta-analysis and genotyped them in two independent replication sets from Germany and Estonia. The two most strongly associated SNPs were further genotyped in four additional replication cohorts from Michigan, the National Psoriasis Foundation (NPF), the CASP Deep Follow-up (CASP-DFU) and Estonia. Together, this replication set comprised 1,931 PsA cases and 6,785 controls (Table S1). We identified genome-wide significant association for SNP rs13017599, which is located between the genes REL and PUS10 and has been already reported to be associated with RA (Gregersen et al., 2009). Our results indicate that REL is an important candidate susceptibility locus not only for PsV and RA but also for PsA.
Before genotype imputation and meta-analysis we applied extensive quality control filters to the three GWAS datasets separately. We excluded all samples with low genotyping rate (<95%) as well as non-European outliers or cryptically related individuals. Further, we excluded SNPs with a minor allele frequency <1%, a genotyping success rate <95% or deviation of the genotype distribution from Hardy-Weinberg equilibrium in the controls (P<10−4). After applying these stringent quality control filters genotype imputation was performed with Beagle v.3.2.1 (Browning and Browning, 2009) using HapMap phase 3 reference haplotypes (Altshuler et al., 2010). Only SNPs imputed with high confidence (estimated r2 between imputed and true genotypes ≥ 0.3) in all three GWAS were considered for subsequent statistical analysis, leaving a total of 1,160,703 SNPs. A logistic regression procedure was applied to test both genotyped and imputed SNPs for association. To account for uncertainty in the imputation procedure we used allele dosages from the imputation. After combining the association results of all three scans, a low genomic control value (meta GWAS: λGC=1.02) indicated a minimal overall inflation of the test statistics due to population stratification (Figure S1). Our combined discovery panel had 80% power to detect variants conferring odds ratios (OR) of 1.40 or higher at the 5% significance level, assuming a frequency of the disease-associated risk allele of at least 15% in controls (Figure S2). In line with previous studies, we detected the strongest associations with PsA at SNPs in the HLA complex at chromosome 6p21 approximately 35 kb upstream of HLA-C (rs12212594, P=4.98×10−38, OR=3.60; rs12191877, P=3.95×10−35, OR=2.73 and rs10484554, P=3.03×10−34, OR=2.72) (Manhattan plot see Figure 1). SNP rs12191877 was previously identified as most strongly associated with PsV in a GWAS of individuals with genuine European ancestry (Nair et al., 2009), while rs10484554 has been described to be most significantly associated in a combined study of PsA and PsV (Liu et al., 2008). In our discovery panel, rs12212594 was in moderate LD with rs12191877 (r2=0.57), indicating that these signals are not independent. Additionally, we found evidence of association at the previously reported susceptibility loci TNIP1 (Nair et al., 2009) (rs17728338, P=6.75×10−10, OR=2.07) and IL12B (Liu et al., 2008) (rs3212227, P=3.82×10−7, OR=1.64). For selection of follow-up SNPs we excluded all SNPs within the extended MHC region (7,773 SNPs on chromosome 6 at 25–34Mb) as well as SNPs within the genes TNIP1 and IL12B since these loci have already been shown to be associated with PsA at a genome-wide significant level. Furthermore we employed only those SNPs that were available in all three scans, and that showed no heterogeneity between the studies (I2 statistic=0). We selected ten SNPs for replication (see Manhattan plot, Figure 1) in two independent samples from Germany and Estonia (replication panel 1 and 2) comprising 252 PsA cases and 1,740 controls. All SNPs passed quality control measures which means that they had a high call rate (>95% in cases or controls), were not monomorphic (minor allele frequency >1% in cases or controls) and did not deviate from Hardy-Weinberg equilibrium in the control population (HWE P >10−4). Taking into account the minor differing genetic backgrounds of the two replication study groups, we used the Cochran-Mantel-Haenszel test for the combined analysis (Table 1). Of the ten follow-up SNPs, two SNPs were nominally associated with PsA in the replication dataset (rs13017599 and rs2829866 with P<0.05) and were therefore genotyped in four additional replication panels from Michigan, the NPF, the CASP-DFU and Estonia (replication panels 3 through 6), yielding a replication sample consisting of 1,931 PsA cases and 6,785 controls. In the combined replication analysis rs13017599 - located 15 kb downstream of REL and 5 kb downstream of PUS10 - remained significantly associated with PsA, yielding P=4.24×10−5 (OR=1.23, 95% CI=1.14–1.33, no heterogeneity: I2=0). In contrast, rs2829866 showed no evidence for association in the extended replication sample (α=0.005, calculated as 0.05/10). The combined analysis of the discovery and the six replication panels (2,466 PsA cases and 10,217 controls) yielded a genome-wide significant P-value of 1.18×10−8 (OR=1.27, 95% CI=1.18–1.35, I2=0) for the lead SNP rs13017599 (Table 2). To fine map a region of 500 kb around the lead SNP rs13017599, we performed imputation based on CEU haplotypes generated by the 1000 Genomes Project for the three GWAS panels used for discovery phase (The 1000 Genomes Project Consortium, 2010). The regional plot of the combined P-values confirmed the results of the HapMap phase 3 imputed meta-analysis, with rs13017599 displaying the strongest association (P=1.88×10−5) at this locus (Figure 2). Additionally, we genotyped the SNP rs702873, which was reported to be associated with PsV by Strange et al., in the replication panels 1 through 6 (1,931 PsA cases and 6,785 controls). Meta-analysis of the three GWAS and the six replication study groups (2,466 PsA cases and 10,217 controls) yielded P=1.93×10−7 (OR=1.20, 95% CI=1.13–1.28), confirming the newly identified association of this locus with PsA (Table 1 and and2).2). The lead SNP rs13017599 and the PsV-associated variant rs702873 are about 83 kb apart and are in moderate linkage disequilibrium (LD in 566 CEU haplotypes generated by the 1000 Genomes Project (The 1000 Genomes Project Consortium, 2010); r2=0.625). To test for the independence of these two markers, we performed a logistic regression analysis. When conditioning for the lead SNP rs13017599, rs702873 was not significantly associated with PsA (P=0.20), whereas rs13017599 was still associated (P=2.90×10−2) in the reverse conditioning, indicating that the two SNPs are not completely independent, but that rs13017599 accounts for the association of PsA at the REL gene locus rather than rs702873. In order to investigate whether the REL locus was more strongly associated with PsA than with PsV, we additionally analyzed the two REL SNPs rs13017599 and rs702873 in PsV cases without PsA from the same three GWAS and six replication panels (5,912 PsV cases without PsA and 10,217 controls). This analysis yielded much weaker association signals compared to the PsA-cohort (rs13017599, PPsV=3.48×10−5, OR=1.13, 95% CI=1.08–1.19; rs702873, PPsV=4.70×10−6, OR=1.14, 95% CI=1.09–1.19), highlighting that the REL-association is stronger in PsA but nevertheless confirming the previously reported association of rs702873 with PsV. For each of the two associations at rs13017599 and rs702873 there is an overlap in confidence intervals for PsA and PsV which further proves that the REL gene represents a shared susceptibility locus for PsA and PsV.
In addition to the recently reported association of SNP rs13017599 with RA (Gregersen et al., 2009) and SNP rs702873 - located 27kb upstream of the REL gene - with PsV (Strange et al., 2010), several other SNPs downstream of REL have been identified to be associated with ulcerative colitis (McGovern et al., 2010), Crohn’s Disease (Franke et al., 2010), celiac disease (Dubois et al., 2010) and primary sclerosing cholangitis (Janse et al., 2011), demonstrating that REL is a shared disease locus of several complex diseases and thereby highlighting its role in inflammatory barrier disease susceptibility. REL encodes one of four subunits found in NF-κB dimers and belongs to the NF-κB (REL) family of transcription factors which is highly important in the innate immune system. The above mentioned association findings are therefore consistent with the importance of the NF-κB pathway in these inflammatory diseases. c-Rel contains trans-activation domains that are a prerequisite for gene transcription, and T cell-mediated immune responses were markedly impaired in c-Rel−/− animals. The confirmed PsA/PsV susceptibility locus TNFAIP3 and a variety of other genes are regulated by c-Rel in T cells (Bunting et al., 2007). Notably, c-REL mediates differentiation of CD4+ FoxP3+ regulatory T cells which have the capacity to develop into IL-17 producing cells in psoriasis (Bovenschen et al., 2011). It also promotes IL-12B and IL-23 expression, by which TH-1 and TH-17 types of immune response might be evoked (Mise-Omata et al., 2007; Reinhard et al., 2011). Altogether these findings further support the hypothesis that REL is an important integrator of inflammatory signaling pathways observed in the complex emerging landscape of genetic susceptibility for PsA. In conclusion, we were able to provide - to our knowledge previously unreported - evidence at genome-wide significance for involvement of the REL locus in PsA susceptibility, and we could confirm the previously reported association with PsV. The current results demand not only a thorough analysis of all genes involved in this pathway to search for additional risk alleles but also overlap analyses between complex disorders sharing a certain genetic background like PsA, PsV and RA. It should be noted however, that because most of the patients develop PsA following the onset of PsV, PsA might be considered as disease within a disease with psoriasis as the parent disease (Eder et al., 2011), so that any association study on PsA is bound to identify PsV susceptibility genes such as the established PsV/PsA risk loci HLA-Cw6, IL12B, IL23R, TNIP1 and FBXL19 which do not allow for genetic discrimination between the two conditions. On the other hand, the genetic component of PsA is much higher than that of PsV and only about one third of PsV patients additionally develop PsA, implying that differences in the genetic backgrounds do exist. Genes involved in innate (such as REL) and adaptive immunity are expectedly more often shared between different immune-mediated diseases, while genes of skin barrier function are rather restricted to PsV. So far, no PsA-specific genes have been identified with genome-wide significance, but then again, only three GWAS on PsA have been conducted until now, our study included (Huffmeier et al., 2010; Liu et al., 2008). Lack of power to find genetic variants with weaker effects, and rare variants that contribute to disease risk might explain the sparse results, thereby emphasizing the need for larger studies and targeted next-generation sequencing of candidate regions.
All German PsA (219) and PsV (733) cases in the GWAS panel and the replication panel 1 were recruited either at the Department of Dermatology of the Christian-Albrechts-University Kiel or the Department of Dermatology and Allergy of the Technical University Munich through local outpatient services. PsA was diagnosed by a clinical finding of joint complaints and radiological and rheumatologic confirmation of criteria according to Moll and Wright (Moll and Wright, 1973), or more recently, to the Classification Criteria for Psoriatic Arthritis (CASPAR) (Taylor et al., 2006). 63 PsA cases from Munich were diagnosed by a physician, with negative diagnosis of RA. Individuals were considered to be affected by PsV if chronic plaque or guttate psoriasis lesions covered more than 1% of the total body surface area or if at least two skin, scalp, nail or joint lesions were clinically diagnosed of psoriasis by a dermatologist.
2,127 German healthy control individuals in the GWAS panel and the replication panel 1 were obtained from the PopGen biobank (Krawczak et al., 2006). 465 German healthy controls were selected from the KORA S4 survey, an independent population-based sample from the general population living in the region of Augsburg, southern Germany (Wichmann et al., 2005).
The CASP GWAS (for details see (Nair et al., 2009; Stuart et al., 2010)) consisted of 335 PsA cases, 915 PsV cases and 1,322 controls after quality control measures. The datasets used for the analyses described in this manuscript were obtained from the database of Genotype and Phenotype (dbGaP, http://www.ncbi.nlm.nih.gov/gap).
The Canadian GWAS sample (from Genizon BioSciences) consisted of 139 PsA cases, 614 PsV cases and 987 controls sampled from the Québec founder population (QFP). Membership in the QFP was defined as having four grandparents with French-Canadian family names who were born in the Province of Québec, Canada or in adjacent areas in the provinces of New Brunswick and Ontario or in New England or New York State. This criterion assured that all subjects were descendants of French-Canadians living before the 1960s, after which time admixture with non-French-Canadians became more common. Inclusion criteria were the presence of plaque forming psoriasis determined by a dermatologist, and disease onset between 18 and 40 years of age. Exclusion criteria were other forms of psoriasis, such as pustular, guttate, inverse, erythrodermic and isolated site psoriasis and late onset. The presence of psoriatic arthritis was noted but not an ascertainment criterion.
The samples of the Estonian replication panel 2 were unrelated Estonian Caucasian patients with a clear clinical diagnosis of psoriasis vulgaris, collected at the Department of Dermatology and Venerology and at the Department of Physiology and Centre of Translational Medicine at the University of Tartu. Patients were classified to have psoriatic arthritis if this diagnosis had been established by an experienced rheumatologist. The Estonian replication panel 6 consisted of samples provided by the population-based biobank of the Estonian Genome Center, University of Tartu (EGCUT). Subjects were recruited by general practitioners (GP) and physicians in the hospitals. Physicians in the hospitals were randomly selected from individuals visiting GP offices or hospitals. Diagnosis of PsV and/or PsA based on clinical symptoms was posed by a general practitioner and confirmed by a dermatologist. At the moment of recruitment, the controls did not report diagnosis of osteoarthritis, psoriasis or autoimmune diseases.
Replication panels 3 through 5 (Michigan, NPF and CASP-DFU) consisted of 1,649 PsA cases, 2,349 PsV cases and 4,061 controls of white European ancestry from the United States and Canada. Diagnosis of psoriasis was always confirmed by a dermatologist. All PsA patients were diagnosed by a rheumatologist and/or fulfilled CASPAR criteria for the classification of PsA. All Candadian PsV patients underwent a rheumatological examination to rule out the presence of inflammatory arthritis. Patients with other forms of inflammatory arthritis were excluded. Written, informed consent was obtained from all study participants, and all protocols were approved by the respective institutional ethical review committees of the participating centers. The investigations were conducted according to the Declaration of Helsinki Principles. The samples were organized in panels which corresponded to the successive steps of the present study and all panels (GWAS panels and replication panels 1 through 6) were independent from each other.
The genotyping for the German GWAS, which was part of the German GWAS initiative funded by the National Genome Research Network (NGFN), was performed by Illumina’s service facility using the Illumina HumanHap 550K v1 with 561,466 SNP markers. All experimental steps were carried out according to standard protocols. For genome-wide genotyping of the CASP samples the Perlegen 600k array was used. The genotyping was provided through the Genetic Association Information Network (GAIN). The Illumina Human 1M BeadChip was used for generating the Canadian genome-wide data (Genizon GWAS) as well as for genotypes of SNPs rs13017599 and rs2829866 in replication panel 5 (CASP-DFU). Functionally tested TaqMan SNP Genotyping Assays (Applied Biosystems) were used to genotype variants in replication panels 1 through 6.
We used GWAS datasets that passed stringent quality control filters for genotype imputation with Beagle (Browning and Browning, 2009) using HapMap phase 3 reference samples from the CEU, TSI, MEX and GJT collections, as well as for the subsequent statistical analyses. Low genomic control values for all three scans (German GWAS: λGC=1.01, CASP GWAS: λGC=1.02 and Canadian GWAS: λGC=1.03) indicated a minimal overall inflation of the test statistics due to population stratification.
Power calculations were carried out using PS Power and Sample Size v3.0.12 (Dupont and Plummer, 1997) (Figure S2). GWAS data were analyzed using R statistical environment version 2.10.0 and gPLINK v2.049 in combination with PLINK v1.05 (Purcell et al., 2007).
We selected ten SNPs based on their P-value ranking for replication in two independent samples from Germany and Estonia comprising 252 PsA cases and 1,740 controls. All SNPs passed quality control measures which means that they had a high call rate (>95% in cases or controls), were not monomorphic (minor allele frequency >1% in cases or controls) and did not deviate from Hardy-Weinberg equilibrium in the control population (exact HWE P >10−4). Two of the ten follow-up SNPs were significantly associated with PsA and were therefore genotyped in four additional replication panels from the United States, Canada and Estonia.
The Cochran-Mantel-Haenszel test was used for combined replication analysis of replication panel 1 and 2 from Germany and Estonia, in order to take the possibility of minor differing genetic backgrounds into account. The initial GWAS meta-analysis was performed using PLINK’s meta-analysis function with its standard error of odds ratio weighting option. Association results of replication panel 5 (CASP-DFU) were obtained with the statistical association test “Efficient Mixed-Model Association eXpedited” (EMMAX) to account for sample structure (Kang et al., 2010) and the combined replication analysis of all six replication panels as well as the combined GWAS-replication analysis was performed with METAL (Willer et al., 2010). Logistic regression analysis to test for the independence of the two REL SNPs rs13017599 and rs702873 was performed with PLINK using the conditional analysis commands --logistic and --condition to test one SNP but adding the allelic dosage for the other SNP as a covariate. The conditional analysis was carried out for each cohort separately and results were subsequently combined by means of meta-analysis.
We thank all individuals with psoriasis and/or psoriatic arthritis, their families and physicians for their cooperation. We acknowledge the cooperation of Genizon Biosciences. We wish to thank Tanja Wesse, Tanja Henke, Catharina Fürstenau and Susan Ehlers for expert technical help. This study was supported by the German Ministry of Education and Research (BMBF) through the National Genome Research Network (NGFN) and the Popgen biobank. The project received infrastructure support through the DFG cluster of excellence “Inflammation at Interfaces”. This research was also supported by grants R01-AR42742, R01-AR050511, AR050266 and R01-AR054966 from the National Institutes of Health, the Psoriasis and Psoriatic arthritis New Emerging Team grant from the Canadian Institutes of Health Research and the Krembil Foundation. S.W. is supported by a Heisenberg Professorship of the German Research council DFG (WE 2678/4-1). The KORA (Kooperative Gesundheitsforschung in der Region Augsburg) study was initiated and financed by the Helmholtz Zentrum München - National Research Center for Environmental Health, which is funded by the German Federal Ministry of Education, Science, Research and Technology and by the State of Bavaria. Part of this work was financed by the German National Genome Research Network (NGFNPlus: 01GS0823) and was supported within the Munich Center of Health Sciences (MC Health) as part of LMUinnovativ. EGCUT received financing by FP7 grants (201413, 245536), also received targeted financing from Estonian Government SF0180142s08, from University of Tartu in the frame of Center of Translational Genomics and from the European Union through the European Regional Development Fund, in the frame of. We acknowledge EGCUT and Estonian Biocentre personnel, especially Ms. M. Hass and Mr. V. Soo.
Conflict of Interest
The authors declare no conflict of interest