the study protocol was approved by the institutional review boards of the vanderbilt university medical center , the shanghai cancer institute , the shanghai center for disease prevention and control , and meharry medical college .
informed consent was obtained from all participants . for detailed descriptions of the genotyping and quality control procedures
briefly , in stage i , the initial 300 subjects were genotyped using the affymetrix genechip mapping 500k array set and the remaining 2,776 samples were genotyped using the affymetrix genome - wide human snp array 6.0 . in each of the 96-well plates genotyped using the affymetrix
snp 6.0 array , three positive qc samples ( na15510 , na10851 , and na18505 ) purchased from coriell cell repositories ( http://ccr.coreill.org/ ) , and a negative qc sample ( water ) were included .
snp data obtained from the positive qc samples showed a very high concordance rate of called genotypes based on 79,764,872 comparisons ( mean , 99.87% ; median , 100% ) .
in addition , 742 snps were genotyped using the affymetrix target genotyping system , taqman or sequenom for a subset of subjects included in previous studies .
a high concordance rate was also observed between genotypes determined using these platforms and affymetrix snp array 6.0 based on 1,478,383 comparisons ( mean , 99.1% ; median , 99.8% ) .
samples with genotyping call rates less than 95% were excluded , and the remaining samples were recalled by using birdseed v2 .
the gender of all study samples was confirmed to be female . the identity - by - descent analysis based on identity - by - state
was performed to detect first degree cryptic relationships using the plink 1.04 25 ( http://pngu.mgh.harvard.edu/purcell/plink/ ) .
a final data set included 3,027 individuals and 607,728 markers in the affymetrix snp array 6.0 and 330,885 in the affymetrix 500k array set that met the following criteria : ( 1 ) genotype call rate  95% ; ( 2 ) maf  5% ; and ( 3 ) genotyping concordance rate in qc samples  95% . also excluded from the analyses were 21,223 snps that are on affymetrix 500k array set but not on the snp array 6.0 .
included in each 96-well plate as qc samples were two negative controls ( water ) , two blinded duplicates , and two samples included in the hapmap project .
the mean concordance rate was 99.7% for blind duplicates ( 2,572 comparisons ) and 99.2% for hapmap samples
the plink version 1.04 was used to analyze genome - wide data obtained in stage i. a set of 4,305 snps with maf  0.35 and  100 kb between two adjacent snps was selected to evaluate the population structure .
the inflation factor  was estimated to be 1.024 , suggesting that population substructure , if present , should not have any appreciable effect on the results .
odds ratios ( or ) and 95% confidence intervals ( ci ) were estimated from logistic regression analysis .
age was adjusted in the gwa analyses of stage i data to select promising snps .
additional adjustment was made for education in the analyses of stages ii and iii data .
finally , multivariate analyses including age , education , study stage , body mass index , age at menarche , and age at first live birth were performed in the pooled analyses of data from all stages .
none of the 29 markers genotyped in stage ii showed deviations from hardy - weinberg equilibrium ( p > 0.05 ) .
haplotype analyses methods : snps genotyped in stage i in a region of 100 kb containing rs2046210 were used to reconstruct haplotypes .
haplotype blocks were defined using the methods of gabriel et al16 and 9 blocks were observed ( supplementary figure s3 ) .
block 5 includes rs2046210 and seven other snps , rs7740686 , rs7763637 , rs9397436 , rs9397437 , rs6908732 , rs852004 , and rs865898 ( supplementary table s4 ) .
associations of haplotypes with breast cancer risk were analyzed with hapstat software27 based on additive models .
additional analyses were performed for haplotypes defined using snps included in blocks 4 and 5 with a total of 15 snps ( supplementary figure s3 , supplementary table s4 ) .
imputation analysis : we used plink to impute genotypes for autosomal snps that were present in hapmap phase ii release 23a but not genotyped in our gwa scan .
hapmap genotype data from the 90 asian hapmap subjects that have maf  1% and genotyping rate 
an information score was generated for each imputed snp that reflected how confidently genotypes were inferred .
values below 0.80 were taken to be indicative of poor quality and were not analyzed . besides the 0.61 million observed snps , an additional 1.14 million imputed snps were tested for association with breast cancer .
the study protocol was approved by the institutional review boards of the vanderbilt university medical center , the shanghai cancer institute , the shanghai center for disease prevention and control , and meharry medical college .
briefly , in stage i , the initial 300 subjects were genotyped using the affymetrix genechip mapping 500k array set and the remaining 2,776 samples were genotyped using the affymetrix genome - wide human snp array 6.0 . in each of the 96-well plates genotyped using the affymetrix
snp 6.0 array , three positive qc samples ( na15510 , na10851 , and na18505 ) purchased from coriell cell repositories ( http://ccr.coreill.org/ ) , and a negative qc sample ( water ) were included .
snp data obtained from the positive qc samples showed a very high concordance rate of called genotypes based on 79,764,872 comparisons ( mean , 99.87% ; median , 100% ) .
in addition , 742 snps were genotyped using the affymetrix target genotyping system , taqman or sequenom for a subset of subjects included in previous studies .
a high concordance rate was also observed between genotypes determined using these platforms and affymetrix snp array 6.0 based on 1,478,383 comparisons ( mean , 99.1% ; median , 99.8% ) .
samples with genotyping call rates less than 95% were excluded , and the remaining samples were recalled by using birdseed v2 .
the gender of all study samples was confirmed to be female . the identity - by - descent analysis based on identity - by - state
was performed to detect first degree cryptic relationships using the plink 1.04 25 ( http://pngu.mgh.harvard.edu/purcell/plink/ ) .
a final data set included 3,027 individuals and 607,728 markers in the affymetrix snp array 6.0 and 330,885 in the affymetrix 500k array set that met the following criteria : ( 1 ) genotype call rate  95% ; ( 2 ) maf  5% ; and ( 3 ) genotyping concordance rate in qc samples  95% . also excluded from the analyses were 21,223 snps that are on affymetrix 500k array set but not on the snp array 6.0 .
included in each 96-well plate as qc samples were two negative controls ( water ) , two blinded duplicates , and two samples included in the hapmap project .
the mean concordance rate was 99.7% for blind duplicates ( 2,572 comparisons ) and 99.2% for hapmap samples
the plink version 1.04 was used to analyze genome - wide data obtained in stage i. a set of 4,305 snps with maf  0.35 and  100 kb between two adjacent snps was selected to evaluate the population structure .
the inflation factor  was estimated to be 1.024 , suggesting that population substructure , if present , should not have any appreciable effect on the results .
odds ratios ( or ) and 95% confidence intervals ( ci ) were estimated from logistic regression analysis .
age was adjusted in the gwa analyses of stage i data to select promising snps .
additional adjustment was made for education in the analyses of stages ii and iii data .
finally , multivariate analyses including age , education , study stage , body mass index , age at menarche , and age at first live birth were performed in the pooled analyses of data from all stages .
none of the 29 markers genotyped in stage ii showed deviations from hardy - weinberg equilibrium ( p > 0.05 ) .
haplotype analyses methods : snps genotyped in stage i in a region of 100 kb containing rs2046210 were used to reconstruct haplotypes .
haplotype blocks were defined using the methods of gabriel et al16 and 9 blocks were observed ( supplementary figure s3 ) .
block 5 includes rs2046210 and seven other snps , rs7740686 , rs7763637 , rs9397436 , rs9397437 , rs6908732 , rs852004 , and rs865898 ( supplementary table s4 ) .
associations of haplotypes with breast cancer risk were analyzed with hapstat software27 based on additive models .
additional analyses were performed for haplotypes defined using snps included in blocks 4 and 5 with a total of 15 snps ( supplementary figure s3 , supplementary table s4 ) .
imputation analysis : we used plink to impute genotypes for autosomal snps that were present in hapmap phase ii release 23a but not genotyped in our gwa scan .
hapmap genotype data from the 90 asian hapmap subjects that have maf  1% and genotyping rate  95% were used as reference . during the imputation ,
an information score was generated for each imputed snp that reflected how confidently genotypes were inferred .
values below 0.80 were taken to be indicative of poor quality and were not analyzed .
besides the 0.61 million observed snps , an additional 1.14 million imputed snps were tested for association with breast cancer .
 a genome - wide association study was conducted among chinese women to identify risk variants for breast cancer . by analyzing 607,728 snps in 1505 cases and 1522 controls , we selected 29 promising snps for a fast - track replication in an independent set of 1554 cases and 1576 controls . 
 four replicated loci were further investigated in a third set of samples including 3472 cases and 900 controls . 
 snp rs2046210 at 6q25.1 , located upstream of the estrogen receptor 1 gene ( esr1 ) , exhibited strong and consistent association with breast cancer across all three stages . adjusted odds ratio ( 95% ci ) were 1.36 ( 1.241.49 ) and 1.59 ( 1.401.82 ) , respectively , for genotypes a / g and a / a versus g / g ( p for trend , 2.01015 ) in the pooled analysis of samples from all three stages . a similar , 
 although weaker , association was also found in an independent study including 1591 cases and 1466 controls of european ancestry ( ptrend , 0.01 ) . 
 these results provide strong evidence implicating 6q25.1 as a susceptibility locus for breast cancer . 
METHODS
Study population
Genotyping and quality control procedures
Statistical analyses
Supplementary Material