gwas2 included 450 unrelated generalized vitiligo patients ( cases ) of non - hispanic / latino european ancestry ( eur ) from north america and europe , who met strict clinical criteria for generalized vitiligo .
controls for gwas2 were 3182 eur individuals not specifically known to have any autoimmune disease or malignant melanoma , for whom genome - wide genotypes were obtained from the database of genotypes and phenotypes ( dbgap ; phs000092v1 , phs000125v1 , phs000138v2 , phs000168v1 , and phs000206v3 ) , or from the illumina icontroldb . the replication study included 1440 unrelated eur generalized vitiligo cases and 1316 unrelated eur controls from north america and europe , principally spouses of vitiligo patients from the gwas2 study and the replication study itself .
there was no overlap of cases and controls between the gwas1 , gwas2 , and replication cohorts .
cases and controls provided clinical history regarding vitiligo and other autoimmune diseases as described previously for gwas1 , and controls having known relatives with vitiligo or reporting any known autoimmune diseases or melanoma were excluded .
this study was approved by each institutional review board and was conducted according to declaration of helsinki principles .
genomic dna was prepared from saliva specimens using a dna self - collection kit per the manufacturer 's instructions ( oragene , dna genotek ) . for genome - wide genotyping ,
dna concentrations were assayed by both fluorescence staining ( picogreen method , invitrogen ) and ultraviolet a260 spectrophotometry ( nanodrop , thermo scientific ) . for genotyping specific snps in the replication study , dna concentrations were assayed by nanodrop .
genome - wide genotyping for gwas2 cases was performed for 657,366 snps using illumina human660w - quad beadchips per the manufacturer 's instructions .
gwas2 controls obtained from dbgap and illumina icontroldb had been genotyped using illumina human 610-quad and human 1mv1 beadchips .
genotyping of 46 snps in the replication study was performed using a custom illumina goldengate assay per the manufacturer 's instructions .
quality control filtering of genome - wide genotype data in gwas2 was carried out as described for gwas1 using illumina genomestudio , version 3 and plink , version 1.07 .
cases were excluded on the basis of snp call rates < 98.5% ( n = 16 ) , discordance between reported and observed sex ( n = 0 ) , and/or inadvertent subject duplication ( n = 0 ) . beyond prior quality control procedures , controls were excluded on the basis of snp call rates < 95% ( n = 0 )
. additional cases ( n = 6 ) and controls ( n = 356 ) were excluded on the basis of cryptic relatedness based on pair - wise identity - by - descent estimation ( pi - hat > 0.05 ) of the entire ( gwas1 + gwas2 ) summary dataset , in which case the individual with lower snp call rate was excluded .
snps were excluded on the basis of genotype missing rate  2% overall ( n = 97,301 ) in cases plus controls , observed minor allele frequency < 0.01 ( n = 23,064 ) , significant deviation ( p < 10 ) from hardy - weinberg equilibrium in the control dataset ( n = 5,241 ) , and/or significant difference ( p < 10 ) in genotype missing rate in cases versus controls ( n = 35,939 ) . data for snps with p values < 10 were reviewed with respect to genotype clusters and allele calls in cases , and minor allele frequency in controls compared to data in public data sources , and were excluded if there were apparent data quality problems ( n = 2 ) . to control for population stratification , we performed principle components analysis using eigensoft , version 4.2 , and excluded as outliers cases ( n = 10 ) and controls ( n = 16 ) whose ancestry was  6 standard deviations from the mean on one of the top ten eigenvectors . in the replication study we successfully determined genotypes for 46 snps that showed suggestive ( p < 10 ) or significant p values in the genome - wide meta - analysis of gwas1 and gwas2 , using a custom illumina goldengate assay .
snp genotypes were subjected to quality control filters similar to those in gwas1 and gwas2 , as appropriate . for gwas2 , after quality control filtering of subjects and snp data and removal of genetic outliers , we compared allele frequencies of the remaining 495,821 snps in the final 418 cases and 2810 controls using the unadjusted cochran - armitage trend test implemented in plink and the adjusted cochran - armitage trend test implemented in eigensoft , in which both phenotypes and genotypes of subjects were adjusted for ancestry using the top ten eigenvectors .
the unadjusted cochran - armitage trend tests and the adjusted cochran - armitage trend tests yielded genomic inflation factors of 1.054 and 1.050 , respectively , indicating that residual population stratification was negligible .
odds ratios and 95% confidence limits were calculated by logistic regression analysis by use of plink . to fully assess association at the 24 novel suggestive loci , we imputed genotypes for each locus using mach , ver.1.0 based on patterns of haplotype variation in the 1000 genomes project european ancestry samples ( release aug 4 , 2010 ) .
we retained imputed snps with r > 0.3 and minor allele frequency > 0.01 for further analyses .
for the replication study , after quality control filtering , we compared allele frequencies for genotyped snps in the remaining 1377 patients and 1284 controls using the cochran - armitage trend test . odds ratios and 95% confidence limits were calculated by logistic regression analysis . to obtain combined ors and p values for gwas1 and gwas2 , and for the combined gwas1 , gwas2 , and replication studies , we performed meta - analysis using a cochran - mantel - haenszel test .
a breslow - day test was used to test for heterogeneity of ors of the same snp in different study cohorts .
calculation of linkage disequilibrium between snps in regions of association was carried out using haploview , version 4.2 . as a test of the independent effect of a given locus conditioned on the effect of another locus , we compared the fit of a model containing both loci to a model containing only the conditioning locus , assuming a multiplicative genotypic effect for the high - risk allele of each locus .
we estimated the contribution of known vitiligo susceptibility loci to the total variance in vitiligo liability as the difference between the variance accounted for by all snps genome - wide and the variance remaining after removing snps in known vitiligo susceptibility loci , using gcta to estimate the two components of variance .
we estimated heritability of vitiligo liability assuming a vitiligo prevalence in the eur population of 0.0038 and sibling risk 0.06 . to gain insights into potential functional relationships among proteins encoded by vitiligo risk loci
, we carried out functional interaction network analysis using the search tool for the retrieval of interacting genes ( string ) 9.0 .
input data were all known vitiligo susceptibility loci ( including ticam1 , and selecting btnl2 to represent the mhc class ii gene region ) .
in addition , we iteratively tested inclusion of all proteins encoded by genes in the 16q24.3 and 22q13.2 association regions as well as fam76b as potentially representing the 11q21 association region as a means of possible gene identification .
gwas2 included 450 unrelated generalized vitiligo patients ( cases ) of non - hispanic / latino european ancestry ( eur ) from north america and europe , who met strict clinical criteria for generalized vitiligo .
controls for gwas2 were 3182 eur individuals not specifically known to have any autoimmune disease or malignant melanoma , for whom genome - wide genotypes were obtained from the database of genotypes and phenotypes ( dbgap ; phs000092v1 , phs000125v1 , phs000138v2 , phs000168v1 , and phs000206v3 ) , or from the illumina icontroldb . the replication study included 1440 unrelated eur generalized vitiligo cases and 1316 unrelated eur controls from north america and europe , principally spouses of vitiligo patients from the gwas2 study and the replication study itself .
there was no overlap of cases and controls between the gwas1 , gwas2 , and replication cohorts .
cases and controls provided clinical history regarding vitiligo and other autoimmune diseases as described previously for gwas1 , and controls having known relatives with vitiligo or reporting any known autoimmune diseases or melanoma were excluded .
this study was approved by each institutional review board and was conducted according to declaration of helsinki principles .
genomic dna was prepared from saliva specimens using a dna self - collection kit per the manufacturer 's instructions ( oragene , dna genotek ) . for genome - wide genotyping ,
dna concentrations were assayed by both fluorescence staining ( picogreen method , invitrogen ) and ultraviolet a260 spectrophotometry ( nanodrop , thermo scientific ) . for genotyping specific snps in the replication study ,
genome - wide genotyping for gwas2 cases was performed for 657,366 snps using illumina human660w - quad beadchips per the manufacturer 's instructions .
gwas2 controls obtained from dbgap and illumina icontroldb had been genotyped using illumina human 610-quad and human 1mv1 beadchips .
genotyping of 46 snps in the replication study was performed using a custom illumina goldengate assay per the manufacturer 's instructions .
quality control filtering of genome - wide genotype data in gwas2 was carried out as described for gwas1 using illumina genomestudio , version 3 and plink , version 1.07 .
cases were excluded on the basis of snp call rates < 98.5% ( n = 16 ) , discordance between reported and observed sex ( n = 0 ) , and/or inadvertent subject duplication ( n = 0 ) . beyond prior quality control procedures , controls were excluded on the basis of snp call rates < 95% ( n = 0 )
. additional cases ( n = 6 ) and controls ( n = 356 ) were excluded on the basis of cryptic relatedness based on pair - wise identity - by - descent estimation ( pi - hat > 0.05 ) of the entire ( gwas1 + gwas2 ) summary dataset , in which case the individual with lower snp call rate was excluded .
snps were excluded on the basis of genotype missing rate  2% overall ( n = 97,301 ) in cases plus controls , observed minor allele frequency < 0.01 ( n = 23,064 ) , significant deviation ( p < 10 ) from hardy - weinberg equilibrium in the control dataset ( n = 5,241 ) , and/or significant difference ( p < 10 ) in genotype missing rate in cases versus controls ( n = 35,939 ) . data for snps with p values < 10 were reviewed with respect to genotype clusters and allele calls in cases , and minor allele frequency in controls compared to data in public data sources , and were excluded if there were apparent data quality problems ( n = 2 ) . to control for population stratification , we performed principle components analysis using eigensoft , version 4.2 , and excluded as outliers cases ( n = 10 ) and controls ( n = 16 ) whose ancestry was  6 standard deviations from the mean on one of the top ten eigenvectors . in the replication study we successfully determined genotypes for 46 snps that showed suggestive ( p < 10 ) or significant p values in the genome - wide meta - analysis of gwas1 and gwas2 , using a custom illumina goldengate assay .
snp genotypes were subjected to quality control filters similar to those in gwas1 and gwas2 , as appropriate .
for gwas2 , after quality control filtering of subjects and snp data and removal of genetic outliers , we compared allele frequencies of the remaining 495,821 snps in the final 418 cases and 2810 controls using the unadjusted cochran - armitage trend test implemented in plink and the adjusted cochran - armitage trend test implemented in eigensoft , in which both phenotypes and genotypes of subjects were adjusted for ancestry using the top ten eigenvectors . the unadjusted cochran - armitage trend tests and the adjusted cochran - armitage trend tests yielded genomic inflation factors of 1.054 and 1.050 , respectively , indicating that residual population stratification was negligible .
odds ratios and 95% confidence limits were calculated by logistic regression analysis by use of plink . to fully assess association at the 24 novel suggestive loci , we imputed genotypes for each locus using mach , ver.1.0 based on patterns of haplotype variation in the 1000 genomes project european ancestry samples ( release aug 4 , 2010 ) .
we retained imputed snps with r > 0.3 and minor allele frequency > 0.01 for further analyses .
for the replication study , after quality control filtering , we compared allele frequencies for genotyped snps in the remaining 1377 patients and 1284 controls using the cochran - armitage trend test . odds ratios and 95% confidence limits were calculated by logistic regression analysis . to obtain combined ors and p values for gwas1 and gwas2 , and for the combined gwas1 , gwas2 , and replication studies , we performed meta - analysis using a cochran - mantel - haenszel test .
a breslow - day test was used to test for heterogeneity of ors of the same snp in different study cohorts .
calculation of linkage disequilibrium between snps in regions of association was carried out using haploview , version 4.2 . as a test of the independent effect of a given locus conditioned on the effect of another locus , we compared the fit of a model containing both loci to a model containing only the conditioning locus , assuming a multiplicative genotypic effect for the high - risk allele of each locus .
we estimated the contribution of known vitiligo susceptibility loci to the total variance in vitiligo liability as the difference between the variance accounted for by all snps genome - wide and the variance remaining after removing snps in known vitiligo susceptibility loci , using gcta to estimate the two components of variance .
we estimated heritability of vitiligo liability assuming a vitiligo prevalence in the eur population of 0.0038 and sibling risk 0.06 .
to gain insights into potential functional relationships among proteins encoded by vitiligo risk loci , we carried out functional interaction network analysis using the search tool for the retrieval of interacting genes ( string ) 9.0 .
input data were all known vitiligo susceptibility loci ( including ticam1 , and selecting btnl2 to represent the mhc class ii gene region ) .
in addition , we iteratively tested inclusion of all proteins encoded by genes in the 16q24.3 and 22q13.2 association regions as well as fam76b as potentially representing the 11q21 association region as a means of possible gene identification .
 in previous linkage and genome - wide association studies we identified 17 susceptibility loci for generalized vitiligo . by a second genome - wide association study , meta - analysis , and independent replication study , 
 we have now identified 13 additional vitiligo - associated loci , including oca2-herc2 , a region of 16q24.3 containing mc1r , a region of chromosome 11q21 near tyr , several immunoregulatory loci including ifih1 , cd80 , clnk , bach2 , sla , casp7 , cd44 , ikzf4 , sh2b3 , and a region of 22q13.2 where the causal gene remains uncertain . 
 functional pathway analysis shows that most vitiligo susceptibility loci encode immunoregulatory proteins or melanocyte components that likely mediate immune targeting and genetic relationships among vitiligo , malignant melanoma , and normal variation of eye , skin , and hair color . 
METHODS
Subjects
Genotyping and quality control
Statistical analyses
Functional network analysis
Supplementary Material