1,215 turkish behet 's disease ( bd ) cases and 1,278 genetically matched controls used in previous gwas were studied .
individuals who also met the tel - hashomer clinical criteria for the diagnosis of familial mediterranean fever ( fmf ) were excluded ( n=6 ) . for replication ,
an additional similarly collected 838 turkish cases ( none fulfilled fmf criteria ) and 630 controls , and 612 japanese bd cases and 740 control samples enrolled in the previous gwas were included .
all japanese bd patients fulfilled the japanese bd diagnostic criteria and 496 of them also fulfilled the international study group criteria .
all study participants provided written informed consent , and the study was approved by the ethics committees of each investigative institution .
for a reference panel , we used 96 turkish healthy controls who participated in the previous bd gwas using the humanhap370cnv chip ( illumina ) and additionally genotyped on the human omni 1 m chip ( illumina , san diego , ca ) . for quality control , we excluded snps from the reference panel if they had a minor allele frequency less than 5% , deviated from hardy  weinberg equilibrium ( p<0.0001 ) , or had a call rate below 95% , yielding 814,474 snps for the imputation .
a total of 779,465 imputed snps were included in the genome - wide association analysis .
twenty - two snps from the novel genetic loci were selected for evaluation in the turkish replication samples .
three of these snps with p < 0.001 and one snp from the phenotypic subset analysis identified four regions for validation and fine - mapping by i - plex assays ( tof - ms , sequenom , san diego , ca ) using the original turkish gwas samples .
the il12a locus with two snps with p < 0.05 in the replication samples was also investigated , but failed to reach genome - wide significance . for variants that failed tof - ms design or reaction ,
genotyping was performed in an unbiased fashion by masking the phenotype of the samples . for the fine - mapping
, we used the tagger snp selection tool from hapmap to select snps to augment the coverage of the gwas snps with the intent to obtain 100% coverage of the hapmap phase iii snps with greater than 5% minor allele frequency in the ceu hapmap population with pairwise r > 0.8 .
although already tagged , additional snps with r > 0.8 with the most significantly associated snp of the region were also included .
after quality control , the resulting coverage for the ccr1 locus ( chr3:45441901 - 46908964 , hg18 ) was 92% , the stat4 locus ( chr2:191602386 - 191769025 ) was 84% , the klrk1-klrc1 locus ( chr12:10329925 - 10557292 ) was 92% , and the erap1 - 2 locus ( chr5:96026703 - 96305246 ) was 94% . the most significantly associated marker from each region was used for the replications .
ccr1 mrna expression data were extracted from the report by zeller and colleagues , which includes genome - wide snp data along with mrna expression array data from monocytes of n = 1490 european ancestry individuals .
snp rs7616215 showed association with ccr1 mrna expression ( p=9.5410 ) , whereas ccr3 data were not reported , indicating that the association of rs7616215 with ccr3 is less significant ( p>510 ) .
stat4 mrna expression data in lymphoblastoid samples were obtained from gene expression omnibus ( geo ) datasets .
primary human monocytes from unrelated healthy volunteers were isolated from peripheral blood mononuclear cells by macs human monocyte isolation kit ( miltenyi biotec , gladbach , germany ) .
rna was isolated from pbmcs by rneasy kit ( qiagen , valencia , ca ) and preserved at 80 c until used .
cdnas were prepared from dnase i ( invitrogen , carlsbad , ca)-pretreated - rna using superscript ii according to manufacturer 's protocol ( invitrogen ) .
q - pcr gene expression assay for human ccr1 ( hs00174298_m1 ) was purchased from applied biosystems ( foster city , ca ) .
multiplex pcr ( gapdh with ccr1 ) was performed in triplicate or quadruplicate as in the manufacturer 's protocol ( applied biosystems ) .
a monocyte migration assay was performed with 24-well transwell 5 m polycarbonate membrane chambers ( costar , corning , ny ) .
510 cells were seeded in the upper chamber and the ccr1 chemokine mip1- ( 0 or 10ng / ml ) ( r&d , minneapolis , mn ) was placed in lower chamber .
after incubation for 2 hours , cells that migrated into the membrane were fixed and stained by diffquik ( siemens , newark , de ) .
cells were counted in 5 high power fields on membranes from each of two duplicate wells .
the relative change in cell migration in response to the chemokine was determined by dividing the migrated cell count obtained with mip1- by the cell count obtained from the same sample without mip1- ( n=61 ) .
genome - wide snp association tests were performed based on allelic tests by comparing the allele frequencies between bd cases and controls , using golden helix svs 7.5.2 software ( golden helix , bozeman , mt ) .
p<510 was considered genome - wide significance . for the uveitis subset analysis , as well as the basic allelic test , we applied dominant and recessive genetic model tests and therefore employed 1.6710 for genome - wide significance .
conditional analyses were performed by fitting the logistic regression model with snps rs2617170 ( for klrk1-klrc1 ) or rs7616215 ( for ccr1-ccr3 ) as covariates . for meta - analysis ,
cochran - mantel - haenszel tests were performed . to test for the interactive effects , we fit the log likelihood of the full model , including additive terms of the main effects and a multiplicative term of the interaction effect , versus a reduced model of the additive terms of the main effects only .
p value was calculated by likelihood ratio tests between full and reduced models based on 1 degree of freedom chi - square test statistics . for comparisons in expression and chemotaxis data ,
the kruskal - wallis rank sum test as a non - parametric version of one - way anova and spearman 's rank correlation test were performed .
1,215 turkish behet 's disease ( bd ) cases and 1,278 genetically matched controls used in previous gwas were studied .
individuals who also met the tel - hashomer clinical criteria for the diagnosis of familial mediterranean fever ( fmf ) were excluded ( n=6 ) . for replication ,
an additional similarly collected 838 turkish cases ( none fulfilled fmf criteria ) and 630 controls , and 612 japanese bd cases and 740 control samples enrolled in the previous gwas were included .
all japanese bd patients fulfilled the japanese bd diagnostic criteria and 496 of them also fulfilled the international study group criteria .
all study participants provided written informed consent , and the study was approved by the ethics committees of each investigative institution .
for a reference panel , we used 96 turkish healthy controls who participated in the previous bd gwas using the humanhap370cnv chip ( illumina ) and additionally genotyped on the human omni 1 m chip ( illumina , san diego , ca ) . for quality control , we excluded snps from the reference panel if they had a minor allele frequency less than 5% , deviated from hardy  weinberg equilibrium ( p<0.0001 ) , or had a call rate below 95% , yielding 814,474 snps for the imputation .
a total of 779,465 imputed snps were included in the genome - wide association analysis .
twenty - two snps from the novel genetic loci were selected for evaluation in the turkish replication samples .
three of these snps with p < 0.001 and one snp from the phenotypic subset analysis identified four regions for validation and fine - mapping by i - plex assays ( tof - ms , sequenom , san diego , ca ) using the original turkish gwas samples .
the il12a locus with two snps with p < 0.05 in the replication samples was also investigated , but failed to reach genome - wide significance . for variants that failed tof - ms design or reaction ,
for the fine - mapping , we used the tagger snp selection tool from hapmap to select snps to augment the coverage of the gwas snps with the intent to obtain 100% coverage of the hapmap phase iii snps with greater than 5% minor allele frequency in the ceu hapmap population with pairwise r > 0.8 .
although already tagged , additional snps with r > 0.8 with the most significantly associated snp of the region were also included .
after quality control , the resulting coverage for the ccr1 locus ( chr3:45441901 - 46908964 , hg18 ) was 92% , the stat4 locus ( chr2:191602386 - 191769025 ) was 84% , the klrk1-klrc1 locus ( chr12:10329925 - 10557292 ) was 92% , and the erap1 - 2 locus ( chr5:96026703 - 96305246 ) was 94% .
ccr1 mrna expression data were extracted from the report by zeller and colleagues , which includes genome - wide snp data along with mrna expression array data from monocytes of n = 1490 european ancestry individuals .
snp rs7616215 showed association with ccr1 mrna expression ( p=9.5410 ) , whereas ccr3 data were not reported , indicating that the association of rs7616215 with ccr3 is less significant ( p>510 ) .
stat4 mrna expression data in lymphoblastoid samples were obtained from gene expression omnibus ( geo ) datasets .
primary human monocytes from unrelated healthy volunteers were isolated from peripheral blood mononuclear cells by macs human monocyte isolation kit ( miltenyi biotec , gladbach , germany ) .
rna was isolated from pbmcs by rneasy kit ( qiagen , valencia , ca ) and preserved at 80 c until used .
cdnas were prepared from dnase i ( invitrogen , carlsbad , ca)-pretreated - rna using superscript ii according to manufacturer 's protocol ( invitrogen ) .
q - pcr gene expression assay for human ccr1 ( hs00174298_m1 ) was purchased from applied biosystems ( foster city , ca ) .
multiplex pcr ( gapdh with ccr1 ) was performed in triplicate or quadruplicate as in the manufacturer 's protocol ( applied biosystems ) .
a monocyte migration assay was performed with 24-well transwell 5 m polycarbonate membrane chambers ( costar , corning , ny ) .
510 cells were seeded in the upper chamber and the ccr1 chemokine mip1- ( 0 or 10ng / ml ) ( r&d , minneapolis , mn ) was placed in lower chamber .
after incubation for 2 hours , cells that migrated into the membrane were fixed and stained by diffquik ( siemens , newark , de ) .
cells were counted in 5 high power fields on membranes from each of two duplicate wells .
the relative change in cell migration in response to the chemokine was determined by dividing the migrated cell count obtained with mip1- by the cell count obtained from the same sample without mip1- ( n=61 ) .
genome - wide snp association tests were performed based on allelic tests by comparing the allele frequencies between bd cases and controls , using golden helix svs 7.5.2 software ( golden helix , bozeman , mt ) .
p<510 was considered genome - wide significance . for the uveitis subset analysis , as well as the basic allelic test , we applied dominant and recessive genetic model tests and therefore employed 1.6710 for genome - wide significance .
conditional analyses were performed by fitting the logistic regression model with snps rs2617170 ( for klrk1-klrc1 ) or rs7616215 ( for ccr1-ccr3 ) as covariates . for meta - analysis , cochran - mantel - haenszel tests were performed . to test for the interactive effects , we fit the log likelihood of the full model , including additive terms of the main effects and a multiplicative term of the interaction effect , versus a reduced model of the additive terms of the main effects only .
p value was calculated by likelihood ratio tests between full and reduced models based on 1 degree of freedom chi - square test statistics . for comparisons in expression and chemotaxis data , the kruskal - wallis rank sum test as a non - parametric version of one - way anova and spearman 's rank correlation test were performed .
 patients with behet 's disease ( bd ) suffer from episodic inflammation often affecting the orogenital mucosa , skin , and eyes . to discover new bd - susceptibility loci 
 , we performed a genome - wide association study ( gwas ) of 779,465 snps with imputed genotypes in 1,209 turkish bd patients and 1,278 controls . 
 we identified novel associations at ccr1 , stat4 , and klrc4 . 
 additionally , two snps in erap1 , encoding erap1 p.asp575asn and p.arg725gln , recessively conferred disease risk . 
 these findings replicated in 1,468 independent turkish and/or 1,352 japanese samples ( combined meta - analysis p < 2  109 ) . 
 we also found evidence for interaction between hla - b*51 and erap1 ( p = 9  104 ) . 
 the ccr1 and stat4 variants were associated with gene expression differences . 
 three risk loci shared with ankylosing spondylitis and psoriasis ( mhc - i , erap1 , and il23r , and the mhc - i - erap1 interaction ) , as well as two loci shared with inflammatory bowel disease ( il23r and il10 ) implicate shared pathogenic pathways in the spondyloarthritides and bd . 
Online methods
Patients
Genotype imputation
Validation and fine-mapping
None
Statistical analysis
Supplementary Material