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 . , the strong genetic component of psa is evidenced by the high sibling recurrence risk ( s ) of 2747 ( chandran et al . , 2009 ; gladman et al . , 2003 ; myers et al . , 2005 ) , which is much higher than the estimated s=411 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 , nfbia , nos2 , fbxl19 , tyk2 , ifih1 , rel , il28ra and erap1 ( duffin et al . 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 , nfbia , 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 ) . 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 r 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 : gc=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=4.9810 , or=3.60 ; rs12191877 , p=3.9510 , or=2.73 and rs10484554 , p=3.0310 , 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 ( r=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=6.7510 , or=2.07 ) and il12b ( liu et al . , 2008 ) ( rs3212227 , p=3.8210 , 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 2534 mb ) 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 ( i 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 ) . 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=4.2410 ( or=1.23 , 95% ci=1.141.33 , no heterogeneity : i=0 ) . in contrast , rs2829866 showed no evidence for association in the extended replication sample ( =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 1.1810 ( or=1.27 , 95% ci=1.181.35 , i=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=1.8810 ) 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=1.9310 ( or=1.20 , 95% ci=1.131.28 ) , confirming the newly identified association of this locus with psa ( table 1 and 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 ) ; r=0.625 ) . to test for the independence of these two markers when conditioning for the lead snp rs13017599 , rs702873 was not significantly associated with psa ( p=0.20 ) , whereas rs13017599 was still associated ( p=2.9010 ) 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=3.4810 , or=1.13 , 95% ci=1.081.19 ; rs702873 , ppsv=4.7010 , or=1.14 , 95% ci=1.091.19 ) , 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 27 kb 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-b dimers and belongs to the nf-b ( 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-b 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 . , 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 . 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 qubec 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 qubec , 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 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 ) . 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 , 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 . 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 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 illumina human 1 m 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 : gc=1.01 , casp gwas : gc=1.02 and canadian gwas : gc=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 ) . 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 . 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 qubec 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 qubec , 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 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 ) . 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 , 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 . 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 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 illumina human 1 m 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 : gc=1.01 , casp gwas : gc=1.02 and canadian gwas : gc=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 ) . 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 .