the studies included in stage 1 were drawn from 11 epidemiological studies of prostate cancer among african american men .
these studies included : the multiethnic cohort ( mec ; 1,094 cases /1,096 controls ) , the southern community cohort study ( sccs , 212/419 ) , the prostate , lung , colorectal , and ovarian cancer screening trial ( plco , 286/269 ) , the cancer prevention study ii nutrition cohort ( cps - ii , 76/152 ) , prostate cancer case - control studies at md anderson ( mda , 543/474 ) , identifying prostate cancer genes ( ipcg , 368/172 ) , the los angeles study of aggressive prostate cancer ( laapc , 296/303 ) , prostate cancer genetics study ( cap genes , 75/85 ) , case - control study of prostate cancer among african americans in washington , dc ( dcpc , 292/359 ) , king county ( washington ) prostate cancer study ( kcpcs , 145/81 ) , and the gene - environment interaction in prostate cancer study ( gecap , 234/92 ) .
stage 2 included 1,396 cases and 2,383 controls from 7 studies : san francisco bay area prostate cancer study ( sfpcs , 86/37 ) , the flint men s health study ( fmhs , 135/353 ) , the multiethnic cohort / los angeles county ( mec - la , 554/557 ) , north carolina prostate cancer study ( ncpcs , 214/249 ) , wake forest university prostate cancer study ( wfpcs , 59/66 ) , washington university prostate cancer study ( wupcs , 75/153 ) , and the ghana men s health study ( ghs , 271/968 ) .
stage 3 included 484 cases and 947 controls from 3 studies : the study of clinical outcomes , risk and ethnicity ( score , 152/280 ) , prostate - genetique - recherche - senegal ( progrs , 86/414 ) and prostate cancer in a black population ( pcbp , 246/253 ) .
detailed information about the design and organization of each study is provided in the supplementary note .
genotyping in stage 1 ( 3,621 cases and 3,502 controls ) was conducted using the illumina infinium human1m - duo .
samples ( n=408 ) were removed based on the following exclusion criteria : 1 ) unknown replicates across studies , 2 ) call rates < 95% ; 3 ) > 10% mean heterozygosity on the x chromosome and/or < 10% mean intensity on the y chromosome , 4 ) ancestry outliers , and ; 5 ) samples that were related ( discussed below ) .
the concordance rate for 158 replicate samples was 99.99% . starting with 1,153,397 snps , we removed snps with < 95% call rate , mafs < 1% , or > 1 qc mismatch based on sample replicates ( n=105,411 ) .
we used plink to calculate the probabilities of sharing 0 , 1 , and 2 alleles ( z = z0 , z1 , z2 ) across all possible pairs of samples to determine individuals who were likely to be related to others within and across studies .
we identified 167 pairs of related subjects ( mz twin , parent - offspring , full and half - sibling pairs ) , based on the values of their observed probability vector z being within 1 sd of the expected values of z for their respective relationship .
the criterion for removal was such that individuals that were connected with a higher number of pairs were chosen for removal . in all other cases ,
the eigenstrat software was used to calculate eigenvectors that explained genetic differences in ancestry among samples in the study .
we included data from both hapmap populations ( ceph ( utah residents with ancestry from northern and western europe ) ( ceu ) , japanese in tokyo , japan ( jpt ) , yoruba in ibadan , nigeria ( yri ) , and african ancestry in southwestern u.s .
( asw ) ) and our study , so that comparisons to reference populations of known ethnicity could be made .
a total of 2,546 ancestry - informative snps from the illumina array were selected based on low inter - marker correlation and ability to differentiate between samples of african and european descent .
an individual was subject to filtering from the analysis if his value along eigenvector 1 or 2 was outside of 4 sds of the mean of each respective eigenvector .
eigenvector 1 was highly correlated ( =0.997 , p<1  10 ) with percentage of european ancestry , estimated in hapmix .
genotyping in the stage 2 and 3 studies was conducted using the taqman allelic discrimination assay . in stage 2 , we removed samples missing data for > 3 snps ( n=36 ) . to assess genotyping reproducibility each study included replicate samples ; the concordance was > 98% for each snp within each study .
snp rs13116912 deviated from hwe in all but one of the stage 2 studies and was removed from the stage 2 analysis .
no other snp deviated from hwe ( i.e. p<0.01 in > 2 studies ) in stage 1 or 2 .
the call rate for rs7210100 was very high in stage 1 ( 99.9% ) and similar in cases ( 99.9% ) and controls ( 99.9% ) .
the call rate for this snp was also very high in stages 2 ( 99.8% overall , 99.9% in cases and 99.8% in controls ) and 3 ( 96.1% overall , 97.3% in cases and 95.5% in controls ) .
bi - directional sequencing of rs7210100 and the 5 coding exons of znf652 was performed in 48 subjects ( 20 homozygous for the risk variant , 20 heterozygous for the risk variant and 8 homozygous for the wild - type allele . )
primers were designed at least 50 bases upstream and downstream from each exon . in stage 1 , we tested the association of each snp and prostate cancer risk using a 1-d.f .
 likelihood ratio test from a logistic regression analysis adjusted for age , study and the first 10 eigenvectors estimated by principal components analysis . over - inflation of the test statistic was examined with and without adjustment for ancestry and visualized with quantile - quantile plots .
lambdas were estimated as the median of the test statistics divided by 0.456 ( the median of the 1-d.f .
age - adjusted odds ratios ( or ) and 95% confidence intervals ( 95% ci ) for each snp were estimated from the same logistic regression model . at each locus and for each participant ,
local ancestry was defined as the estimated number of european chromosomes ( continuous between 02 ) carried by the participant , estimated via the hapmix program .
local ancestry at the 17q21 locus was evaluated as a confounder in the analysis of rs7210100 . phased haplotype data from the founders of the ceu and
yri hapmap phase 2 samples were used to infer ld patterns in order to impute untyped markers .
the rsq metric was used as a threshold in determining which snps to filter from analysis ( rsq<0.3 ) .
imputed snps in the 17q21 risk region , as shown in figure 2 , were examined in association with prostate cancer risk as described for typed snps above . in stage 2 , the snps were analyzed using logistic regression controlling for age and study ( in the pooled analysis ) .
information regarding european ancestry was available for 7 studies included in stages 2 and 3 .
as observed in stage 1 ( supplementary table 2 ) the or for rs7210100 was similar with and without adjustment for estimated european ancestry in these studies ( supplementary table 4 ) .
the results for rs7210100 in stage 2 , stage 3 and stages 1 + 2 + 3 are presented without adjustment for ancestry .
association testing in the stage 2 and stage 3 studies was performed using logistic regression , adjusting for age and study . for seven of the replication studies , information about global european ancestry was available and examined as a confounding factor for variant rs7210100 . for rs7210100 ,
effect modification by age and first - degree family history of prostate cancer was assessed in stratified analyses , and significance determined comparing the model with and without the cross - product term using a likelihood ratio test .
we also examined the association of rs7210100 genotype with stage , gleason score as well as the combination of stage and grade , with advanced disease defined as gleason score8 or stage 2 ( non - localized disease ) and non - advanced disease defined as gleason score<8 and stage=1 ( localized disease ) .
case - only analysis was used to test for differences in the association of rs7210100 with disease phenotypes .
the association of rs7210100 with least - squares geometric mean psa levels was examined using multiple linear regression adjusting for age , body mass index and study .
we estimated the risk ratio between populations of different ancestral origin ( african / european ) due to rs7210100 as rr= [ ( 1-pa)+2pa(1-pa)rr1+parr2]/(1-pe)+2pe(1-pe)rr1+perr2 ] . here
pa is the risk allele frequency in african origin populations , pe is the risk allele frequency in european populations and rr1 is the relative risk associated with carrying 1 copy of the risk allele ( compared to none ) and rr2 is the relative risk associated with carrying 2 copies of the risk allele .
we used values pa = 0.05 , pe = 0 , rr1 = 1.5 , and rr2 = 1.5 so that the risk ratio between populations due to the influence of this risk allele was estimated to be equal to 1.050625 . using the seer incidence rates of prostate cancer in african americans ( 234.6 per 100,000 ) and non - hispanic whites ( 150.4 cases per 100,000 )
the percentage of greater risk to african americans that may be associated with rs7210100 was estimated as 1-[(1.561.050625)/(1.56 - 1 ) ]  100 .
the studies included in stage 1 were drawn from 11 epidemiological studies of prostate cancer among african american men .
these studies included : the multiethnic cohort ( mec ; 1,094 cases /1,096 controls ) , the southern community cohort study ( sccs , 212/419 ) , the prostate , lung , colorectal , and ovarian cancer screening trial ( plco , 286/269 ) , the cancer prevention study ii nutrition cohort ( cps - ii , 76/152 ) , prostate cancer case - control studies at md anderson ( mda , 543/474 ) , identifying prostate cancer genes ( ipcg , 368/172 ) , the los angeles study of aggressive prostate cancer ( laapc , 296/303 ) , prostate cancer genetics study ( cap genes , 75/85 ) , case - control study of prostate cancer among african americans in washington , dc ( dcpc , 292/359 ) , king county ( washington ) prostate cancer study ( kcpcs , 145/81 ) , and the gene - environment interaction in prostate cancer study ( gecap , 234/92 ) .
stage 2 included 1,396 cases and 2,383 controls from 7 studies : san francisco bay area prostate cancer study ( sfpcs , 86/37 ) , the flint men s health study ( fmhs , 135/353 ) , the multiethnic cohort / los angeles county ( mec - la , 554/557 ) , north carolina prostate cancer study ( ncpcs , 214/249 ) , wake forest university prostate cancer study ( wfpcs , 59/66 ) , washington university prostate cancer study ( wupcs , 75/153 ) , and the ghana men s health study ( ghs , 271/968 ) .
stage 3 included 484 cases and 947 controls from 3 studies : the study of clinical outcomes , risk and ethnicity ( score , 152/280 ) , prostate - genetique - recherche - senegal ( progrs , 86/414 ) and prostate cancer in a black population ( pcbp , 246/253 ) .
detailed information about the design and organization of each study is provided in the supplementary note .
genotyping in stage 1 ( 3,621 cases and 3,502 controls ) was conducted using the illumina infinium human1m - duo .
samples ( n=408 ) were removed based on the following exclusion criteria : 1 ) unknown replicates across studies , 2 ) call rates < 95% ; 3 ) > 10% mean heterozygosity on the x chromosome and/or < 10% mean intensity on the y chromosome , 4 ) ancestry outliers , and ; 5 ) samples that were related ( discussed below ) .
the concordance rate for 158 replicate samples was 99.99% . starting with 1,153,397 snps , we removed snps with < 95% call rate , mafs < 1% , or > 1 qc mismatch based on sample replicates ( n=105,411 ) .
we used plink to calculate the probabilities of sharing 0 , 1 , and 2 alleles ( z = z0 , z1 , z2 ) across all possible pairs of samples to determine individuals who were likely to be related to others within and across studies .
we identified 167 pairs of related subjects ( mz twin , parent - offspring , full and half - sibling pairs ) , based on the values of their observed probability vector z being within 1 sd of the expected values of z for their respective relationship .
the criterion for removal was such that individuals that were connected with a higher number of pairs were chosen for removal . in all other cases ,
the eigenstrat software was used to calculate eigenvectors that explained genetic differences in ancestry among samples in the study .
we included data from both hapmap populations ( ceph ( utah residents with ancestry from northern and western europe ) ( ceu ) , japanese in tokyo , japan ( jpt ) , yoruba in ibadan , nigeria ( yri ) , and african ancestry in southwestern u.s .
( asw ) ) and our study , so that comparisons to reference populations of known ethnicity could be made .
a total of 2,546 ancestry - informative snps from the illumina array were selected based on low inter - marker correlation and ability to differentiate between samples of african and european descent .
an individual was subject to filtering from the analysis if his value along eigenvector 1 or 2 was outside of 4 sds of the mean of each respective eigenvector .
eigenvector 1 was highly correlated ( =0.997 , p<1  10 ) with percentage of european ancestry , estimated in hapmix .
genotyping in the stage 2 and 3 studies was conducted using the taqman allelic discrimination assay . in stage 2 , we removed samples missing data for > 3 snps ( n=36 ) . to assess genotyping reproducibility each study included replicate samples ; the concordance was > 98% for each snp within each study .
snp rs13116912 deviated from hwe in all but one of the stage 2 studies and was removed from the stage 2 analysis .
no other snp deviated from hwe ( i.e. p<0.01 in > 2 studies ) in stage 1 or 2 .
the call rate for rs7210100 was very high in stage 1 ( 99.9% ) and similar in cases ( 99.9% ) and controls ( 99.9% ) .
the call rate for this snp was also very high in stages 2 ( 99.8% overall , 99.9% in cases and 99.8% in controls ) and 3 ( 96.1% overall , 97.3% in cases and 95.5% in controls ) .
bi - directional sequencing of rs7210100 and the 5 coding exons of znf652 was performed in 48 subjects ( 20 homozygous for the risk variant , 20 heterozygous for the risk variant and 8 homozygous for the wild - type allele . )
in stage 1 , we tested the association of each snp and prostate cancer risk using a 1-d.f .
 likelihood ratio test from a logistic regression analysis adjusted for age , study and the first 10 eigenvectors estimated by principal components analysis . over - inflation of the test statistic was examined with and without adjustment for ancestry and visualized with quantile - quantile plots .
lambdas were estimated as the median of the test statistics divided by 0.456 ( the median of the 1-d.f .
age - adjusted odds ratios ( or ) and 95% confidence intervals ( 95% ci ) for each snp were estimated from the same logistic regression model . at each locus and for each participant ,
local ancestry was defined as the estimated number of european chromosomes ( continuous between 02 ) carried by the participant , estimated via the hapmix program .
local ancestry at the 17q21 locus was evaluated as a confounder in the analysis of rs7210100 . phased haplotype data from the founders of the ceu and
yri hapmap phase 2 samples were used to infer ld patterns in order to impute untyped markers .
the rsq metric was used as a threshold in determining which snps to filter from analysis ( rsq<0.3 ) .
imputed snps in the 17q21 risk region , as shown in figure 2 , were examined in association with prostate cancer risk as described for typed snps above . in stage 2
, the snps were analyzed using logistic regression controlling for age and study ( in the pooled analysis ) .
information regarding european ancestry was available for 7 studies included in stages 2 and 3 .
as observed in stage 1 ( supplementary table 2 ) the or for rs7210100 was similar with and without adjustment for estimated european ancestry in these studies ( supplementary table 4 ) .
the results for rs7210100 in stage 2 , stage 3 and stages 1 + 2 + 3 are presented without adjustment for ancestry .
association testing in the stage 2 and stage 3 studies was performed using logistic regression , adjusting for age and study . for seven of the replication studies , information about global european ancestry was available and examined as a confounding factor for variant rs7210100 . for rs7210100 ,
effect modification by age and first - degree family history of prostate cancer was assessed in stratified analyses , and significance determined comparing the model with and without the cross - product term using a likelihood ratio test .
we also examined the association of rs7210100 genotype with stage , gleason score as well as the combination of stage and grade , with advanced disease defined as gleason score8 or stage 2 ( non - localized disease ) and non - advanced disease defined as gleason score<8 and stage=1 ( localized disease ) .
case - only analysis was used to test for differences in the association of rs7210100 with disease phenotypes .
the association of rs7210100 with least - squares geometric mean psa levels was examined using multiple linear regression adjusting for age , body mass index and study .
we estimated the risk ratio between populations of different ancestral origin ( african / european ) due to rs7210100 as rr= [ ( 1-pa)+2pa(1-pa)rr1+parr2]/(1-pe)+2pe(1-pe)rr1+perr2 ] . here
pa is the risk allele frequency in african origin populations , pe is the risk allele frequency in european populations and rr1 is the relative risk associated with carrying 1 copy of the risk allele ( compared to none ) and rr2 is the relative risk associated with carrying 2 copies of the risk allele .
we used values pa = 0.05 , pe = 0 , rr1 = 1.5 , and rr2 = 1.5 so that the risk ratio between populations due to the influence of this risk allele was estimated to be equal to 1.050625 . using the seer incidence rates of prostate cancer in african americans ( 234.6 per 100,000 ) and non - hispanic whites ( 150.4 cases per 100,000 )
the percentage of greater risk to african americans that may be associated with rs7210100 was estimated as 1-[(1.561.050625)/(1.56 - 1 ) ]  100 .
 in search of common risk alleles for prostate cancer that could contribute to high rates of the disease in men of african ancestry , we conducted a genome - wide association study ( gwas ) , with 1,047,986 single nucleotide polymorphism ( snp ) markers examined in 3,425 african american prostate cancer cases and 3,290 african american male controls . 
 the most significant 17 novel associations in stage 1 were followed - up in 1,844 cases and 3,269 controls of african ancestry . 
 we identified a novel risk variant on chromosome 17q21 ( rs7210100 ; odds ratio per allele=1.51 ; p=3.41013 ) . 
 the frequency of the risk allele is ~5% in men of african descent while it is rare in other populations ( < 1% ) . 
 further studies are needed to investigate the biological contribution of this allele to prostate cancer risk . 
 these findings emphasize the importance of conducting gwas in diverse populations . 