all studies participating in this meta - analysis are part of the international glaucoma genetics consortium . the discovery cohorts included 27,558 individuals of european ancestry from 14 studies ( alienor , bats , bmes , erf , framingham family study , ghs1 , ghs2 , orcades , raine ,   rs - i , rs - ii , rs - iii , test and twinsuk ) .
in addition , 7,738 individuals of asian ancestry from four cohorts ( bes , sces , simes , sindi , ) were included .
in addition four case - control population panels were used , all of european ancestry : anzrag , meei , neighbor and decode .
general methods , demographics and phenotyping of the study cohorts have previously been described extensively and are provided in the supplementary note and supplementary table 1 .
all studies were performed with the approval of their local medical ethics committees and written informed consent was obtained from all participants in accordance with the declaration of helsinki .
eligible participants underwent an ophthalmologic examination including measurements of iop and , for most but not all studies , measurements of central corneal thickness ( cct ) .
each participating cohort was phenotyped separately and iop measurement methods used by each are described in the supplementary table 1 .
the study samples were genotyped on either the illumina ( san diego , ca , usa ) or affymetrix ( santa clara , ca , usa ) platforms .
each study performed single nucleotide polymorphism ( snp ) imputation using the genotype data , together with the hapmap phase ii ethnically matched reference panels ( ceu , jpt+chb , or the 4 hapmap populations ) on the basis of build 36 databases ( release 22 or 24 ) .
the markov chain haplotyping software , impute or mach , were adopted for imputation . a detailed description regarding genotyping platforms and imputation procedures for each study is provided on supplementary table 1 .
samples with low call rates ( < 95% ) or with gender discrepancies were excluded .
cryptically related samples and outliers in population structure from principal component analyses were also excluded .
snps flagged with missingness > 5% , gross departure from hardy weinberg equilibrium ( p value < 10 ) and minor allele frequency ( maf ) <
1% were removed from further analyses . for each study , an allele - dosage regression model at each directly genotyped or imputed snp was conducted to determine its association with iop .
eyes with prior glaucoma surgery or laser were excluded . for subject receiving iop - lowering medication
, we added 25% to the measured iop levels to estimate pre - treatment iop , based on a reported average of 17% to 33% iop reduction caused by iop lowering medication in a meta - analysis of clinical trails .
when data from only one eye were available , the iop measurement from the available eye was used .
for the analyses , we assumed an additive genetic model where the dosage of each snp is a continuous variable ranging from 0 to 2 for minor alleles carried .
additional adjustment for principal components was carried out by a few participating cohorts to correct for subtle population substructure .
the per - snp meta - analyses were performed using the gwama software with weighted inverse - variance approach , assuming fixed effects , as for initial discovery purposes the fixed - effects model is preferred for increased statistical power .
a cochran s q test and i were used to assess heterogeneity across studies . for each participating cohort , only snps with sufficient imputation quality scores ( proper - info of impute or r of mach > 0.3 ) were included into the meta - analysis .
gene - based testing was conducted using vegas software on the european ancestry and asian ancestry meta - analysis results separately .
vegas incorporates information from the full set of markers within a gene and accounts for ld between markers by using simulations from the multivariate normal distribution . for samples of european descent
, we used the hapmap 2 ceu population as the reference to estimate patterns of ld . for asian ancestry groups
, we used the combined hapmap 2 jpt and chb populations as the reference population to approximate ld patterns . to include gene regulatory regions ,
we performed meta - analysis on the two sets of gene - based p - values using fisher s method .
pathway p - values were computed by summing  test statistics derived from vegas p - values .
empirical vegas - pathway p values for each pathway were computed by comparing the summed  test statistics from real data with those generated in 500,000 simulations where the relevant number ( according to the size of the pathway ) of randomly drawn  test statistics was summed . to ensure that clusters of genes did not adversely affect results , within each pathway
, gene sets were pruned such that each gene was > 500 kb away from all other genes in the pathway . where required , all but one of the clustered genes was dropped at random when genes were clustered .
we performed meta - analysis on the two sets of pathway p - values using fisher s method . to investigate shared genetic background by using a large number of autosomal snps we made a systematic evaluation of the overlap between the iop and poag based on profile scores following approaches previously described .
we estimated the relative risk for each snp of interest based on a discovery set ( iop ) , with a profile score computed for every individual in a target set of interest ( poag ) .
for each target set individual the profile score was computed as the number of risk alleles weighted by the effect size estimated in the discovery set .
the discovery set comprised the european ancestry derived samples from our meta - analysis and the target set was was a set of 590 glaucoma cases and 3956 controls , as previously described . to ensure that there was not a high degree of dependence between the snps included in the profile score ,
we filtered the set of snps used in the profile score so that only a set of 149,571 snps in low linkage disequilibrium ( r<0.5 ) were used .
we constructed models including more snps progressively lowering the threshold of inclusion ( i.e. p<0.000001 , p<0.00001 , p<0.0001 , p<0.001 , p<0.01 , p<0.1 , p<0.5 ) .
profiles derived from iop snp effects were tested for association with the phenotype ( poag here ) using a logistic regression .
variance explained was assessed using nagelkerke s pseudor measure . to assess whether and to what degree iop levels confer poag risk , we performed a causal inference analysis using an instrumental variable framework as previously described . in brief , we obtained estimates of effect size ( betaiop ) for the association of a given snp with iop from the meta - analysis of the 18 discovery cohorts . for the association of a given snp with poag , we obtained estimates of the effect size ( betapoag ) from the four case - control panels as described above .
we selected the the strongest associated snp from each of the genome - wide significant iop loci that we identified .
to assess whether the strength of snps association with iop predicts the risk of poag , we conducted linear regression analysis using the effect sizes of each snp for iop ( betaiop ) as independent variables and the effect sizes of poag ( betapoag ) as dependent outcome variables .
a total of eight independent iop - associated snps were used for this analysis , including rs7555523 ( tmco1 ) , rs6445055 ( fndc3b ) , rs10258482 ( cav1 ) , rs2472493 ( abca1 ) , rs8176743 ( abo ) , rs747782 ( nup160-ptprj ) , and rs9913911 ( gas7 ) .
adult ocular samples were obtained from normal eyes of an 82-year - old european ancestry female from the north carolina eye bank , winston - salem , north carolina , usa .
all adult ocular samples were stored in qiagen s rnalater within 6.5 hours of collection and shipped on dry ice overnight to the lab .
rna was extracted from each tissue sample independently using the ambion mirvana total rna extraction kit .
the tissue samples were homogenized in ambion lysis buffer using an omni bead ruptor tissue homogenizer per protocol .
the expression of the identified genes were assessed by running 10 ul reactions with qiagen s pcr products consisting of 1.26 ul h2o , 1.0 ul 10x buffer , 1.0 ul dntps , 0.3 ul mgcl , 2.0 ul q - solution , 0.06 ul taq polymerase , 1.0 ul forward primer , 1.0 ul reverse primer and 1.5.0 ul cdna .
the reactions were run on a eppendorf mastercycler pro s thermocycler with touchdown pcr ramping down 1c per cycle from 72 c to 55 c followed by 50 cycles of 94 c for 30 seconds , 55 c for 30 seconds and 72 c for 30 seconds with a final elongation of 7 minutes at 72 c .
primer sets were run on a custom tissue panel including clontech s human mtc panel i , fetal mtc panel i and an ocular tissue panel .
all studies participating in this meta - analysis are part of the international glaucoma genetics consortium . the discovery cohorts included 27,558 individuals of european ancestry from 14 studies ( alienor , bats , bmes , erf , framingham family study , ghs1 , ghs2 , orcades , raine ,   rs - i , rs - ii , rs - iii , test and twinsuk ) .
in addition , 7,738 individuals of asian ancestry from four cohorts ( bes , sces , simes , sindi , ) were included .
in addition four case - control population panels were used , all of european ancestry : anzrag , meei , neighbor and decode .
general methods , demographics and phenotyping of the study cohorts have previously been described extensively and are provided in the supplementary note and supplementary table 1 .
all studies were performed with the approval of their local medical ethics committees and written informed consent was obtained from all participants in accordance with the declaration of helsinki .
eligible participants underwent an ophthalmologic examination including measurements of iop and , for most but not all studies , measurements of central corneal thickness ( cct ) .
each participating cohort was phenotyped separately and iop measurement methods used by each are described in the supplementary table 1 .
the study samples were genotyped on either the illumina ( san diego , ca , usa ) or affymetrix ( santa clara , ca , usa ) platforms .
each study performed single nucleotide polymorphism ( snp ) imputation using the genotype data , together with the hapmap phase ii ethnically matched reference panels ( ceu , jpt+chb , or the 4 hapmap populations ) on the basis of build 36 databases ( release 22 or 24 ) .
the markov chain haplotyping software , impute or mach , were adopted for imputation . a detailed description regarding genotyping platforms and imputation procedures for each study is provided on supplementary table 1 .
samples with low call rates ( < 95% ) or with gender discrepancies were excluded .
cryptically related samples and outliers in population structure from principal component analyses were also excluded .
snps flagged with missingness > 5% , gross departure from hardy weinberg equilibrium ( p value < 10 ) and minor allele frequency ( maf ) <
for each study , an allele - dosage regression model at each directly genotyped or imputed snp was conducted to determine its association with iop
. eyes with prior glaucoma surgery or laser were excluded . for subject receiving iop - lowering medication
, we added 25% to the measured iop levels to estimate pre - treatment iop , based on a reported average of 17% to 33% iop reduction caused by iop lowering medication in a meta - analysis of clinical trails .
the mean of the right and left iop measurements was used . when data from only one eye were available , the iop measurement from the available eye was used . for the analyses
, we assumed an additive genetic model where the dosage of each snp is a continuous variable ranging from 0 to 2 for minor alleles carried .
additional adjustment for principal components was carried out by a few participating cohorts to correct for subtle population substructure .
the per - snp meta - analyses were performed using the gwama software with weighted inverse - variance approach , assuming fixed effects , as for initial discovery purposes the fixed - effects model is preferred for increased statistical power .
a cochran s q test and i were used to assess heterogeneity across studies . for each participating cohort , only snps with sufficient imputation quality scores ( proper - info of impute or r of mach > 0.3 ) were included into the meta - analysis .
gene - based testing was conducted using vegas software on the european ancestry and asian ancestry meta - analysis results separately .
vegas incorporates information from the full set of markers within a gene and accounts for ld between markers by using simulations from the multivariate normal distribution . for samples of european descent
, we used the hapmap 2 ceu population as the reference to estimate patterns of ld . for asian ancestry groups
, we used the combined hapmap 2 jpt and chb populations as the reference population to approximate ld patterns . to include gene regulatory regions ,
we performed meta - analysis on the two sets of gene - based p - values using fisher s method .
pathway p - values were computed by summing  test statistics derived from vegas p - values .
empirical vegas - pathway p values for each pathway were computed by comparing the summed  test statistics from real data with those generated in 500,000 simulations where the relevant number ( according to the size of the pathway ) of randomly drawn  test statistics was summed . to ensure that clusters of genes did not adversely affect results , within each pathway
, gene sets were pruned such that each gene was > 500 kb away from all other genes in the pathway . where required ,
all but one of the clustered genes was dropped at random when genes were clustered .
we performed meta - analysis on the two sets of pathway p - values using fisher s method .
to investigate shared genetic background by using a large number of autosomal snps we made a systematic evaluation of the overlap between the iop and poag based on profile scores following approaches previously described .
we estimated the relative risk for each snp of interest based on a discovery set ( iop ) , with a profile score computed for every individual in a target set of interest ( poag ) .
for each target set individual the profile score was computed as the number of risk alleles weighted by the effect size estimated in the discovery set .
the discovery set comprised the european ancestry derived samples from our meta - analysis and the target set was was a set of 590 glaucoma cases and 3956 controls , as previously described . to ensure that there was not a high degree of dependence between the snps included in the profile score , we filtered the set of snps used in the profile score so that only a set of 149,571 snps in low linkage disequilibrium ( r<0.5 ) were used .
we constructed models including more snps progressively lowering the threshold of inclusion ( i.e. p<0.000001 , p<0.00001 , p<0.0001 , p<0.001 , p<0.01 , p<0.1 , p<0.5 ) .
profiles derived from iop snp effects were tested for association with the phenotype ( poag here ) using a logistic regression .
variance explained was assessed using nagelkerke s pseudor measure . to assess whether and to what degree iop levels confer poag risk , we performed a causal inference analysis using an instrumental variable framework as previously described . in brief , we obtained estimates of effect size ( betaiop ) for the association of a given snp with iop from the meta - analysis of the 18 discovery cohorts .
for the association of a given snp with poag , we obtained estimates of the effect size ( betapoag ) from the four case - control panels as described above .
we selected the the strongest associated snp from each of the genome - wide significant iop loci that we identified .
to assess whether the strength of snps association with iop predicts the risk of poag , we conducted linear regression analysis using the effect sizes of each snp for iop ( betaiop ) as independent variables and the effect sizes of poag ( betapoag ) as dependent outcome variables .
a total of eight independent iop - associated snps were used for this analysis , including rs7555523 ( tmco1 ) , rs6445055 ( fndc3b ) , rs10258482 ( cav1 ) , rs2472493 ( abca1 ) , rs8176743 ( abo ) , rs747782 ( nup160-ptprj ) , and rs9913911 ( gas7 ) .
adult ocular samples were obtained from normal eyes of an 82-year - old european ancestry female from the north carolina eye bank , winston - salem , north carolina , usa . all adult ocular samples were stored in qiagen s rnalater within 6.5 hours of collection and shipped on dry ice overnight to the lab .
rna was extracted from each tissue sample independently using the ambion mirvana total rna extraction kit .
the tissue samples were homogenized in ambion lysis buffer using an omni bead ruptor tissue homogenizer per protocol .
the expression of the identified genes were assessed by running 10 ul reactions with qiagen s pcr products consisting of 1.26 ul h2o , 1.0 ul 10x buffer , 1.0 ul dntps , 0.3 ul mgcl , 2.0 ul q - solution , 0.06 ul taq polymerase , 1.0 ul forward primer , 1.0 ul reverse primer and 1.5.0 ul cdna .
the reactions were run on a eppendorf mastercycler pro s thermocycler with touchdown pcr ramping down 1c per cycle from 72 c to 55 c followed by 50 cycles of 94 c for 30 seconds , 55 c for 30 seconds and 72 c for 30 seconds with a final elongation of 7 minutes at 72 c .
primer sets were run on a custom tissue panel including clontech s human mtc panel i , fetal mtc panel i and an ocular tissue panel .
 elevated intraocular pressure ( iop ) is an important risk factor in developing glaucoma and iop variability may herald glaucomatous development or progression . 
 we report the results of a genome - wide association study meta - analysis of 18 population cohorts from the international glaucoma genetics consortium ( iggc ) , comprising 35,296 multiethnic participants for iop . 
 we confirm genetic association of known loci for iop and primary open angle glaucoma ( poag ) and identify four new iop loci located on chromosome 3q25.31 within the fndc3b gene ( p=4.191008 for rs6445055 ) , two on chromosome 9 ( p=2.801011 for rs2472493 near abca1 and p=6.391011 for rs8176693 within abo ) and one on chromosome 11p11.2 ( best p=1.041011 for rs747782 ) . 
 separate meta - analyses of four independent poag cohorts , totaling 4,284 cases and 95,560 controls , show that three of these iop loci are also associated with poag . 
Online Methods
IGGC participants
Phenotype measurements
Genotyping & imputation
Statistical Analysis
Gene expression in human tissues
Supplementary Material