the stage 1 samples for the gwa scan included 1,999 t2d case subjects and 1,976 nondiabetic control subjects drawn from the nutrition and health of aging population in china ( nhapc ) study ( 312 case and 815 control subjects ) , the gut microbiota and obesity study ( gmos ) ( 82 case and 163 control subjects ) , the fudan - huashan study ( 807 case and 339 control subjects ) , and the beijing diabetes survey ( 798 case and 659 control subjects ) .
the stage 2 samples for replication testing of 96 single nucleotide polymorphisms ( snps ) consisted of 13,517 unrelated chinese hans ( 6,570 t2d case and 6,947 nondiabetic control subjects ) from the guizhou - bijie type 2 diabetes study ( gbtds ) , the beijing type 2 diabetes studies ( btds ) , and the hubei type 2 diabetes studies ( htds ) .
the stage 3 replication testing of 10 snps is carried out by de novo genotyping in 3,410 t2d patients and 3,412 nondiabetic control subjects from the shanghai diabetes inpatient database ( sdiid ) and the shanghai diabetes study ( sds ) and in silico replication in the agen - t2d gwas datasets ( 6,952 t2d case and 11,865 control subjects ) .
there was no overlap of participants among stage 1 , 2 , and 3 samples .
studies of t2d - related quantitative traits were performed among 3,614 participants from the nhapc and gmos , the two population - based studies .
all studies were approved by local ethics committees of each participating institution , and informed written consent was obtained from all participants .
a summary of the contributing studies , criteria for t2d case and nondiabetic control subjects in each study , and sample characteristics can be found in the supplementary materials and methods and supplementary table 1 .
the stage 1 dna samples were genotyped using the illumina human660w - quad beadchip ( illumina , inc . , san diego , ca ) .
quality control ( qc ) filters were applied at the individual and snp levels . at the individual level , we removed the samples that met any of the following criteria : 1 ) call rates < 97% ( n = 20 ) ; 2 ) with excessive heterozygosity ; 3 ) with sex mismatches between the reported and genetically inferred ( n = 69 ) ; and 4 ) with unexpected duplicates ( n = 19 ) or cryptic relatives ( n = 149 ) .
we also removed population outliers ( n = 6 ) detected by using principle component analysis ( 13 ) ( supplementary fig .
we excluded the copy number variation  related snps , the snps in y and mitochondrial chromosomes , and the snps if they had : 1 ) call rate < 95% ( n = 1,351 ) ; 2 ) minor allele frequency < 05% ( n = 63,263 ) ; and 3 ) hardy - weinberg equilibrium ( hwe ) p < 10 in control groups ( n = 1,047 ) .
the samples that passed all qc criteria were then used to impute for the ungenotyped or missing snps from the phase 2 hapmap chb+jpt ( release number 22 ) reference panel using impute ( version 2.1.2 ; http://mathgen.stats.ox.ac.uk/impute/impute.html ) software ( 14 ) .
we removed all imputed snps with an estimated call rate < 99% , minor allele frequency < 1% , hwe p < 10 , or the info measure 0.5 . for stage 2 replication , we genotyped 96 snps selected from stage 1 in three independent chinese han populations with 6,570 t2d case subjects and 6,947 control subjects from the gbtds , btds , and htds studies using taqman snp genotyping assays ( applied biosystems , foster city , ca ) in the fludigm ep1 platform .
we excluded the samples with call rate < 93% and the snps with call rate < 95% or deviation from hwe at p
< 5.2  10 ( bonferroni corrected p value for 96 tests ) in control groups .
the overall call rates were 99.7 , 99.4 , and 99.4% for samples from the gbtds , btds , and htds studies , respectively .
we genotyped > 5% duplicate samples for each study to assess genotyping reproducibility , and the concordance rates were 99.9% for 189 duplicate samples from the gbtds and 99.5% for 387 duplicate samples from btds and for 166 duplicate samples from htds .
we also performed cross - platform validation by genotyping the 96 snps in 135 stage 1 samples that had been run on the illumina assay using taqman snp genotyping assays in fludigm ep1 platform , and the concordance rate was 99.9% .
finally , 10 snps , selected based on results of meta - analysis that combined stage 1 and 2 data , were used for stage 3 replication , including a de novo genotyping in 3,410 t2d case and 3,412 control subjects from the sdiid / sds study and an in silico replication in 8 independent gwas datasets of 6,952 case and 11,865 control subjects from the agen consortium .
the de novo genotyping was performed using sequenom massarray ( sequenom , san diego , ca ) in the sdiid / sds study .
after quality control , 3,257 t2d case and 3,262 control subjects with genotyping call rate > 90% were used for association analyses .
the in silico replication results were obtained by directly searching the meta - analysis results of 8 independent gwas datasets with 6,952 case and 11,865 control subjects in total that participated in the discovery stage of the agen - t2d consortium . for stage 1
gwas , the genotyped snps ( 495,686 ) and imputed snps ( 1,738,508 ) were tested for their associations with t2d using plink ( version 1.07 ; http://pngu.mgh.harvard.edu/~purcell/plink/ ) ( 15 ) , and snptest ( version 2.2.0 ; https://mathgen.stats.ox.ac.uk/genetics_software/snptest/snptest.html ) ( 14 ) , respectively .
the odds ratios ( ors ) and 95% cis were estimated by logistic regression under an additive genetic model , adjusting for age , sex , and the first two principal components in model 1 .
for the x - chromosome snps , males were treated as homozygotes , in that genotypes were coded as 2 for the coded allele ( 16 ) .
the initial association analyses were performed by pooling the data from the beijing and shanghai participants and then stratified by geographical regions ( beijing and shanghai ) to test whether the pooling would introduce a bias due to potential population stratification .
the p values were adjusted for genomic control inflation factor ( gc ) . to find more consistent evidence for associations between each snps and t2d , we further stratified the association analyses by sex or tested the associations between the case subjects with family history and the nondiabetic control subjects . the snp selection for stage 2 replication
was largely based on the p values in the discovery stage , and one or two snps with the smallest p values were chosen for each significant genomic region , defined as that contained a set of snps in linkage disequilibrium ( ld ) at r  0.1 with the most associated snp . for stage 2 and 3 replication analyses ,
logistic regression analysis was applied to test association of each snp with t2d , assuming an additive genetic effect and adjusting for age and sex .
the effect sizes across studies or stages were combined using fixed - effect inverse variance weighted meta - analysis , and the p values were calculated using a fixed effects meta - analysis with sample size weighted z - score .
we also examined the effect of t2d - associated snps on t2d - related quantitative traits in participants from the nhapc and gmos studies , of which the individuals receiving glucose - lowering treatment and gmos participants with bmi 28 were excluded from analyses ( supplementary table 10 ) .
the nhapc samples were genotyped using the illumina human660w - quad beadchip ( illumina , inc . ) using the stage 1 qc criteria , and the gmos samples were genotyped using taqman snp genotyping assays in fludigm ep1 platform .
finally , a total of 3,614 samples ( 2,229 from shanghai and 1,385 from beijing ) that passed all qc criteria were used for the association analyses , which were performed in the shanghai and beijing samples separately using linear regression analysis assuming an additive genetic model , adjusting for age , sex , and study covariates ( nhapc and gmos for the shanghai samples only ) .
values of insulin and homeostasis model assessment of -cell function ( homa - b ) and insulin sensitivity ( homa - s ) were natural log - transformed before analysis , and results of each study were combined by fixed - effect inverse variance weighted meta - analysis .
homa - s and homa - b were estimated by the homa model using levy s computer model ( 18 ) . for cis - eqtl analysis , we first searched rs10814916 , rs10886471 , rs7403531 , and their proxy snps ( r > 0.5 ) in the genevar eqtl database , including 166 adipose tissues , 156 lymphoblastoid cell lines , and 160 skin tissues derived from a subset of healthy female twins of the muther resource ( 19 ) .
associations between each snp and mrna expression level of nearby genes were obtained by using spearman rank correlation for < 10,000 permutations as the default settings of genevar ( version 3.1.1 ; http://www.sanger.ac.uk/resources/software/genevar/ ) .
we performed gkr5 mrna expression analysis in blood samples of 64 unrelated chinese hans recruited from the peking union medical college hospital , chinese academy of medical sciences , in december 2011 .
all participants provided written informed consent , and study protocol was approved by the local ethics committee .
genomic dna and rna samples were extracted from peripheral blood by using a qiagen kit ( qiagen ) and rna pure blood kit ( cowin biotech co. ) , respectively .
the genotype of g - protein  coupled receptor ( gpcr ) kinase 5 ( grk5)-rs10886471 was determined by sequencing , using the following primers : 5-tcctacactggaacaagcc-3 and 5-acggactaatacagacggg-3. quantitative real - time rt - pcr for grk5-rs10886471 was performed using the goldstar taqman mixture kit ( cowin biotech co. ) on a bio - rad iq5 system ( bio - rad ) . grk5
we carried out statistical analysis for expression data using linear regressions and an unpaired student t test .
p value from linear regression was calculated by assuming a dominant genetic effect of minor allele of rs10886471 and adjusted for the status of t2d .
the genomic regions for the grk5 and rasgrp1 loci were selected based on their regional ld plots ( supplementary figs . 6 and 7 ) .
the varld algorithm is used to compare difference in the regional ld patterns among phase 2 hapmap chb samples ( 45 chinese hans ) , ceu samples ( 60 white europeans ) , and jpt samples ( 45 japanese ) , and results were presented as monte carlo p values ( 20 ) .
the stage 1 samples for the gwa scan included 1,999 t2d case subjects and 1,976 nondiabetic control subjects drawn from the nutrition and health of aging population in china ( nhapc ) study ( 312 case and 815 control subjects ) , the gut microbiota and obesity study ( gmos ) ( 82 case and 163 control subjects ) , the fudan - huashan study ( 807 case and 339 control subjects ) , and the beijing diabetes survey ( 798 case and 659 control subjects ) .
the stage 2 samples for replication testing of 96 single nucleotide polymorphisms ( snps ) consisted of 13,517 unrelated chinese hans ( 6,570 t2d case and 6,947 nondiabetic control subjects ) from the guizhou - bijie type 2 diabetes study ( gbtds ) , the beijing type 2 diabetes studies ( btds ) , and the hubei type 2 diabetes studies ( htds ) .
the stage 3 replication testing of 10 snps is carried out by de novo genotyping in 3,410 t2d patients and 3,412 nondiabetic control subjects from the shanghai diabetes inpatient database ( sdiid ) and the shanghai diabetes study ( sds ) and in silico replication in the agen - t2d gwas datasets ( 6,952 t2d case and 11,865 control subjects ) .
there was no overlap of participants among stage 1 , 2 , and 3 samples .
studies of t2d - related quantitative traits were performed among 3,614 participants from the nhapc and gmos , the two population - based studies .
all studies were approved by local ethics committees of each participating institution , and informed written consent was obtained from all participants .
a summary of the contributing studies , criteria for t2d case and nondiabetic control subjects in each study , and sample characteristics can be found in the supplementary materials and methods and supplementary table 1 .
the stage 1 dna samples were genotyped using the illumina human660w - quad beadchip ( illumina , inc .
, san diego , ca ) . quality control ( qc ) filters were applied at the individual and snp levels . at the individual level , we removed the samples that met any of the following criteria : 1 ) call rates < 97% ( n = 20 ) ; 2 ) with excessive heterozygosity ; 3 ) with sex mismatches between the reported and genetically inferred ( n = 69 ) ; and 4 ) with unexpected duplicates ( n = 19 ) or cryptic relatives ( n = 149 ) .
we also removed population outliers ( n = 6 ) detected by using principle component analysis ( 13 ) ( supplementary fig .
1 ) . at the snp level , we excluded the copy number variation  related snps , the snps in y and mitochondrial chromosomes , and the snps if they had : 1 ) call rate < 95% ( n = 1,351 ) ; 2 ) minor allele frequency < 05% ( n = 63,263 ) ; and 3 ) hardy - weinberg equilibrium ( hwe ) p < 10 in control groups ( n = 1,047 ) .
the samples that passed all qc criteria were then used to impute for the ungenotyped or missing snps from the phase 2 hapmap chb+jpt ( release number 22 ) reference panel using impute ( version 2.1.2 ; http://mathgen.stats.ox.ac.uk/impute/impute.html ) software ( 14 ) .
we removed all imputed snps with an estimated call rate < 99% , minor allele frequency < 1% , hwe p < 10 , or the info measure 0.5 . for stage 2 replication
, we genotyped 96 snps selected from stage 1 in three independent chinese han populations with 6,570 t2d case subjects and 6,947 control subjects from the gbtds , btds , and htds studies using taqman snp genotyping assays ( applied biosystems , foster city , ca ) in the fludigm ep1 platform .
we excluded the samples with call rate < 93% and the snps with call rate < 95% or deviation from hwe at p
< 5.2  10 ( bonferroni corrected p value for 96 tests ) in control groups .
the overall call rates were 99.7 , 99.4 , and 99.4% for samples from the gbtds , btds , and htds studies , respectively .
we genotyped > 5% duplicate samples for each study to assess genotyping reproducibility , and the concordance rates were 99.9% for 189 duplicate samples from the gbtds and 99.5% for 387 duplicate samples from btds and for 166 duplicate samples from htds .
we also performed cross - platform validation by genotyping the 96 snps in 135 stage 1 samples that had been run on the illumina assay using taqman snp genotyping assays in fludigm ep1 platform , and the concordance rate was 99.9% .
finally , 10 snps , selected based on results of meta - analysis that combined stage 1 and 2 data , were used for stage 3 replication , including a de novo genotyping in 3,410 t2d case and 3,412 control subjects from the sdiid / sds study and an in silico replication in 8 independent gwas datasets of 6,952 case and 11,865 control subjects from the agen consortium .
the de novo genotyping was performed using sequenom massarray ( sequenom , san diego , ca ) in the sdiid / sds study .
after quality control , 3,257 t2d case and 3,262 control subjects with genotyping call rate > 90% were used for association analyses .
the in silico replication results were obtained by directly searching the meta - analysis results of 8 independent gwas datasets with 6,952 case and 11,865 control subjects in total that participated in the discovery stage of the agen - t2d consortium .
for stage 1 gwas , the genotyped snps ( 495,686 ) and imputed snps ( 1,738,508 ) were tested for their associations with t2d using plink ( version 1.07 ; http://pngu.mgh.harvard.edu/~purcell/plink/ ) ( 15 ) , and snptest ( version 2.2.0 ; https://mathgen.stats.ox.ac.uk/genetics_software/snptest/snptest.html ) ( 14 ) , respectively .
the odds ratios ( ors ) and 95% cis were estimated by logistic regression under an additive genetic model , adjusting for age , sex , and the first two principal components in model 1 .
bmi was further included as a covariate in model 2 . for the x - chromosome snps ,
males were treated as homozygotes , in that genotypes were coded as 2 for the coded allele ( 16 ) .
the initial association analyses were performed by pooling the data from the beijing and shanghai participants and then stratified by geographical regions ( beijing and shanghai ) to test whether the pooling would introduce a bias due to potential population stratification .
the p values were adjusted for genomic control inflation factor ( gc ) . to find more consistent evidence for associations between each snps and t2d , we further stratified the association analyses by sex or tested the associations between the case subjects with family history and the nondiabetic control subjects . the snp selection for stage 2 replication
was largely based on the p values in the discovery stage , and one or two snps with the smallest p values were chosen for each significant genomic region , defined as that contained a set of snps in linkage disequilibrium ( ld ) at r  0.1 with the most associated snp . for stage 2 and 3 replication analyses ,
logistic regression analysis was applied to test association of each snp with t2d , assuming an additive genetic effect and adjusting for age and sex .
the effect sizes across studies or stages were combined using fixed - effect inverse variance weighted meta - analysis , and the p values were calculated using a fixed effects meta - analysis with sample size weighted z - score .
we also examined the effect of t2d - associated snps on t2d - related quantitative traits in participants from the nhapc and gmos studies , of which the individuals receiving glucose - lowering treatment and gmos participants with bmi 28 were excluded from analyses ( supplementary table 10 ) .
the nhapc samples were genotyped using the illumina human660w - quad beadchip ( illumina , inc . ) using the stage 1 qc criteria , and the gmos samples were genotyped using taqman snp genotyping assays in fludigm ep1 platform .
finally , a total of 3,614 samples ( 2,229 from shanghai and 1,385 from beijing ) that passed all qc criteria were used for the association analyses , which were performed in the shanghai and beijing samples separately using linear regression analysis assuming an additive genetic model , adjusting for age , sex , and study covariates ( nhapc and gmos for the shanghai samples only ) .
values of insulin and homeostasis model assessment of -cell function ( homa - b ) and insulin sensitivity ( homa - s ) were natural log - transformed before analysis , and results of each study were combined by fixed - effect inverse variance weighted meta - analysis .
homa - s and homa - b were estimated by the homa model using levy s computer model ( 18 ) .
for cis - eqtl analysis , we first searched rs10814916 , rs10886471 , rs7403531 , and their proxy snps ( r > 0.5 ) in the genevar eqtl database , including 166 adipose tissues , 156 lymphoblastoid cell lines , and 160 skin tissues derived from a subset of healthy female twins of the muther resource ( 19 ) .
associations between each snp and mrna expression level of nearby genes were obtained by using spearman rank correlation for < 10,000 permutations as the default settings of genevar ( version 3.1.1 ; http://www.sanger.ac.uk/resources/software/genevar/ ) .
we performed gkr5 mrna expression analysis in blood samples of 64 unrelated chinese hans recruited from the peking union medical college hospital , chinese academy of medical sciences , in december 2011 .
all participants provided written informed consent , and study protocol was approved by the local ethics committee .
genomic dna and rna samples were extracted from peripheral blood by using a qiagen kit ( qiagen ) and rna pure blood kit ( cowin biotech co. ) , respectively .
the genotype of g - protein  coupled receptor ( gpcr ) kinase 5 ( grk5)-rs10886471 was determined by sequencing , using the following primers : 5-tcctacactggaacaagcc-3 and 5-acggactaatacagacggg-3. quantitative real - time rt - pcr for grk5-rs10886471 was performed using the goldstar taqman mixture kit ( cowin biotech co. ) on a bio - rad iq5 system ( bio - rad ) . grk5
we carried out statistical analysis for expression data using linear regressions and an unpaired student t test .
p value from linear regression was calculated by assuming a dominant genetic effect of minor allele of rs10886471 and adjusted for the status of t2d .
the genomic regions for the grk5 and rasgrp1 loci were selected based on their regional ld plots ( supplementary figs . 6 and 7 ) .
the varld algorithm is used to compare difference in the regional ld patterns among phase 2 hapmap chb samples ( 45 chinese hans ) , ceu samples ( 60 white europeans ) , and jpt samples ( 45 japanese ) , and results were presented as monte carlo p values ( 20 ) .
in stage 1 , we tested the association with t2d of 2,234,194 genotyped and imputed snps that passed all qc criteria in 3,712 chinese hans from beijing and shanghai , of which 1,839 were case subjects , and 1,873 were control subjects .
no significant population stratification was observed between case and control subjects using principle component analysis ( supplementary fig .
2 ) , and the genomic control factor was also quite close to 1.0 (  = 1.03 ) , suggesting there is no genome - wide inflation due to population stratification for stage 1 samples .
the quantile  quantile plot showed substantial deviation from the expected at the lower p values , suggesting that some associations were more significant than expected by chance ( supplementary fig .
3 ) . snps in four previously reported t2d loci ( cdkal1 , cdkn2a / b , kcnq1 , and dusp9 ) reached genome - wide significance ( p < 5  10 ) ( fig . 2 and supplementary fig .
4 ) , with directional consistency as previously reported ( 2125 ) . for the other previously reported t2d loci ,
apart from three loci that were monomorphic in chinese hans , 48 of 51 loci showed directionally consistent association with t2d ( binomial test , p = 9.8  10 ) , of which 19 were associated with t2d at p < 0.05 ( supplementary table 3 ) . of these ,
only the rs7754840 in cdkal1 showed a significantly larger effect on t2d in chinese hans than in white europeans ( p for heterogeneity = 3.33  10 ) , consistent with our previous findings ( 26 ) .
manhattan plot for genome - wide association analysis of 495,686 genotyped snps in stage 1 .
the log10 
 p values were from pooled analysis , adjusting for age , sex , and the first two principle components .
the red dots at each locus indicate the signals with p < 10 . for follow - up
, we chose one or two snps per locus that had the smallest p values in stage 1 , except for the two strongest association signals at cdkal1 and kcnq1 ( p  4.24  10 , or = 1.37 ) , for which association with t2d has been repeatedly confirmed in east asian populations . as such , the 96 most significant snps representing 84 independent loci were selected for stage 2 replication ( supplementary tables 2 and 4 ) , which consisted of de novo genotyping of the snps in three independent chinese han populations with 6,570 t2d case and 6,947 control subjects ( supplementary materials and methods and supplementary table 1 ) .
all snps , except for slc30a8-rs13266634 that showed significant deviation from hwe , passed the qc criteria and were included in the replication analyses .
a total of eight snps in or near rasgrp1 , glis3 , cdkn2b , cdc123 , hhex , hnf1b , fam58a , and dusp9 were significantly associated with t2d after bonferroni correction for multiple tests in the stage 2 samples , and the p value cutoff for bonferroni correction was 5.26  10 ( 0.05/95 ) ( supplementary table 5 ) . in meta - analyses combining stage 1 and 2 data ,
the snps in or near rasgrp1 , cdkn2b , cdc123 , hhex , hnf1b , fam58a , and dusp9 reached the genome - wide significance ( supplementary table 5 ) , of which cdkn2b ( 24,25,27 ) , hhex ( 24,25,27 ) , cdc123 ( 28 ) , hnf1b ( 29 ) , and dusp9 ( 29 ) are established t2d loci .
rs10814916 ( p = 5.29  10 ) at glis3 and rs10886471 ( p = 5.92  10 ) at grk5 were close to the genome - wide significance threshold ( supplementary table 5 ) .
the association between glis3 variant ( rs7034200 ) and t2d risk had been observed in a previous candidate gene association study ( 30 ) , but the evidence was inconclusive .
the snp rs12010175 in fam58a is in modest ld with rs5945326 near dusp9 ( r = 0.35 ) .
conditional analyses by including the two snps in one logistic regression model revealed that these two snps represent the same locus ( p adjusted for rs5945326 = 0.02 ) .
apart from the snps that represent previously established t2d loci , 10 snps ( each representing independent loci ) showed association with t2d at p < 5  10 in a meta - analysis that combined stage 1 and 2 data , and no additional novel signal ( p < 5  10 ) was observed when the association analyses were stratified by sex ( supplementary table 6 ) .
these 10 snps were taken forward for replication in stage 3 , which included a de novo genotyping replication in 3,410 t2d case and 3,412 control subjects of chinese hans and an in silico replication in a previously published meta - analysis of gwas of 6,952 t2d case and 11,865 control subjects ( including 4,026 case and 4,654 control subjects from chinese hans and the remaining 2,926 case and 7,211 control subjects from korean , japanese , malay , and filipino populations ) of the agen - t2d consortium ( 5 ) .
a total of three snps at glis3 , grk5 , and rasgrp1 , respectively , exceeded the genome - wide significance threshold ( combined p values : 7.1  10 to 6.0  10 ; or : 1.101.12 ) when we combined the data across all three stages ( table 1 and supplementary table 7 ) .
we found no evidence of heterogeneity for the associations at these three loci across all study populations ( p for heterogeneity  0.26 ) or between chinese hans and other east asians ( p for heterogeneity  0.14 ) ( supplementary table 8) .
while our in silico replication was ongoing , glis3 was reported as a t2d locus by the agen - t2d consortium ( 5 ) .
the association signal ( rs10814916 ) at glis3 in our study is in high ld ( r = 0.93 ) with the snp ( rs7041847 ) identified in the agen - t2d meta - analysis . as such
, we only consider grk5 and rasgrp1 as the novel t2d loci identified by our study .
snps reaching genome - wide significance in a meta - analysis of combined stages 1 , 2 , and 3 the association between rasgrp1-rs7403531 and t2d was confirmed by results from diagram plus gwas meta - analysis ( 22 ) ( 8,130 t2d case and 38,987 control subjects of european origin ) , with comparable effect size and consistent direction ( p = 0.023 ; or = 1.06 ; p for heterogeneity between east asians and europeans = 0.22 ) , and two imputed snps ( rs8043085 and rs12593201 , r  0.8 with rs7403531 ) at this locus even showed a stronger association ( p  3.82  10 ; or = 1.07 ) in european populations from the diagram consortium ( supplementary tables 8 and 13 ) .
however , the t2d association for the grk5 locus was not replicated in the diagram plus gwas datasets ( rs10886471 : p = 0.352 , or = 0.98 ; p for heterogeneity between east asians and europeans = 4.42  10 ) ( supplementary tables 8 and 13 ) . to examine whether adiposity was a potential mediator in the association of the two novel loci and t2d , we tested the association with further adjustment for bmi in populations across all stages .
the effect sizes for each snp remained largely unchanged ( p adjusted for bmi = 1.73  10 to 2.87  10 ; or = 1.101.12 ) ( supplementary table 9 ) , suggesting that their associations with t2d were not mediated through adiposity .
we further examined associations for t2d - related quantitative traits in 3,614 participants ( normal fasting glucose : 2,142 ; impaired fasting glucose : 1,211 ; and t2d : 261 ) from the nhapc and the gmos studies , two population - based studies of chinese hans .
risk allele of the grk5-rs10886471 was also associated with higher fasting plasma insulin levels ( p = 0.0204 ) , but not with fasting plasma glucose , whereas risk allele of the rasgrp1-rs7403531 was also significantly associated higher fasting plasma glucose levels ( p = 0.0213 ) and lower homa - b ( p = 0.0027 ) ( supplementary table 11 ) .
similar results were observed when the association analyses were performed in normal fasting glucose individuals only ( supplementary table 11 ) .
the cis - eqtl ( cis - expression quantitative loci ) analysis using the genevar eqtl database ( 19 ) ( http://www.sanger.ac.uk/resources/software/genevar/ ) showed suggestive evidence of association between an intronic snp ( rs4752300 , r = 0.79 with rs10886471 ) in grk5 and grk5 mrna expression levels at p = 6  10 ( r = 0.361 between the risk allele and grk5 expression levels ) in adipose tissue .
however , no evidence was found for associations between snps at rasgrp1 and mrna expression levels of their nearby genes . to further confirm whether rs10886471 genotypes modify grk5 mrna expression , the grk5 expression levels were estimated using quantitative real - time rt - pcr in peripheral blood mrna samples from 64 unrelated chinese hans , including 30 t2d case subjects and 34 nondiabetic control subjects .
the t2d risk - increasing c - allele of rs10886471 was significantly associated with increased mrna expression levels of grk5 ( p[dom ] = 0.02 ) with the same direction of effect in both t2d case subjects and control subjects , consistent with the finding in the genevar eqtl database .
moreover , the grk5 expression levels in t2d case subjects increased by 40% compared with their nondiabetic counterparts ( p = 0.0048 ) ( figs . 3 and 4 and supplementary table 12 ) .
these results suggested that rs10886471 risk c - allele might contribute to t2d risk through increased grk5 expression , but should be interpreted cautiously because that blood is not a very likely tissue to be involved in t2d .
regional plots of two novel t2d loci . a and b : imputed snps were estimated by mach software ( http://www.sph.umich.edu/csg/abecasis/mach/ ) using ld information from 194 asians ( including 68 chb , 25 chs , 84 jpt , and 17 mxl ) in 1000 genome 201008 release as references .
p values were from pooled analysis , adjusting for age , sex , and the first two principle components in stage 1 samples .
the regional plots for the 500-kb region centered on index snps were generated by using locuszoom ( http://csg.sph.umich.edu/locuszoom/ ) .
the log10 
 p values of snps were plotted against their genomic position ( national center for biotechnology information build 37 ) .
the positions of genes were annotated from the university of california santa cruz genome browser by using grch37 assembly .
other snps are colored according to their ld ( r ) with the index snp from 1000 genome asn .
the recombination rate is shown as a light blue line to reflect the local ld structure .
the relative expression levels of grk5 were measured in blood samples from 30 t2d case subjects ( rs10886471 genotype : tt+tc / n = 11 ; cc / n = 19 ) and 34 nondiabetic control subjects ( rs10886471 genotype : tt+tc / n = 13 ; cc /
we compared the ld structure of the two novel t2d loci between chinese hans and europeans and japanese ( supplementary figs . 6 and 7a ) , respectively .
different pairwise ld patterns were observed between chinese hans and europeans at both grk5 ( p = 0.0432 ) and rasgrp1 ( p = 0.0043 ) loci , but not between chinese hans and japanese ( p  0.101 ) ( supplementary table 14 ) . the risk allele frequencies of both grk5-rs10886471 and rasgrp1-rs7403531 in east asians ( 0.79 and 0.33 in the hapmap chb population and 0.74 and 0.45 in the hapmap jpt population for rs10886471 and rs7403531 , respectively ) were also higher than those in europeans ( 0.48 and 0.28 for rs10886471 and rs7403531 in hapmap ceu population , respectively ) ( supplementary table 13 ) .
in this study , we not only confirmed more than 20 previously reported loci , but also identified two novel t2d loci : grk5 ( rs10886471 ) and rasgrp1 ( rs7403531 ) .
the snp rs10886471 maps to an ld block within intron 3 of the grk5 gene , the only gene in this ld block ( supplementary fig .
regional plots showed that rs10886471 represents the strongest association signals at the grk5 locus ( fig .
grk5 belongs to the gpcr kinase family and plays a crucial role in phosphorylation of multiple gpcrs and non - gpcr substrates , such as glucagon receptor ( 31 ) , 2-adrenergic receptor ( 32,33 ) , hsp70-interacting protein ( 34 ) , and nuclear factor-b1/p105 ( 35 ) , which are either key regulators of glucose homeostasis or inflammation .
disruption of grk5 leads to decreased production of multiple inflammatory cytokines / chemokines and decreased nuclear factor-b activation both in vivo and in vitro ( 36 ) .
the t2d risk - increasing allele of rs10886471 was also associated with higher grk5 mrna expression levels , higher fasting insulin , but not with fasting glucose , suggesting that it might impair insulin sensitivity by increasing inflammatory response and consequently contributes to t2d risk in chinese hans or east asians .
notably , grk5 is a population - specific t2d locus and the t2d association of grk5-rs10886471 was not replicated in populations of european origin .
the risk allele frequency of grk5-rs10886471 in chinese hans was also much higher than in europeans ( 0.79 and 0.48 in hapmap chb and ceu populations , respectively ) , and the corresponding population attributable risk in chinese hans was estimated to be 8.66% .
moreover , there are significant different ld structure for grk5 ( p = 0.0432 ) locus between chinese hans and europeans .
the second novel association signal ( rs7403531 ) is located at chr15q14 and in intron 2 of the rasgrp1 gene ( supplementary fig .
7a ) , which has also been replicated in europeans from the diagram consortium ( supplementary tables 8 and 13 ) .
although no heterogeneity of effect was observed between chinese hans and europeans , the risk allele frequency of rs7403531 in chinese hans was moderately higher than in europeans ( 0.33 and 0.28 in hapmap chb and ceu populations , respectively ) .
regional plots showed that the strongest association signal at this locus was observed with an imputed snp rs12593201 ( r = 0.81 with rs7403531 ) ( fig . 3b and supplementary fig .
5 ) , which also showed a stronger t2d association in the diagram plus gwas meta - analysis ( p = 2.4  10 ) ( supplementary table 13 ) .
conditional analysis in stage 1 samples suggested that neither of the snps were likely to be the snp driving the association ( p  0.11 ) .
notably , rs7403531 was in modest ld ( r = 0.46 in hapmap ceu population ) with a variant ( rs7171171 ) previously shown to be associated with type 1 diabetes ( t1d ) in populations of european ancestry ( 37 ) , but they are not in ld in chinese hans ( r = 0.03 in hapmap chb population ) ( supplementary fig .
7b ) , suggesting that the t2d association at rasgrp1 is unlikely to be driven by the t1d - associated snp .
rasgrp1 encodes the ras guanyl releasing protein 1 ( rasgrp1 ) that functions as a guanine nucleotide exchange factor , which is required for the activation of ras / mitogen - activated protein kinase pathways ( 38 ) and critically mediates the development and function of both t and b lymphocytes ( 3942 ) .
rasgrp1-deficient mice exhibit defects in lymphocyte proliferation ( 40,43,44 ) , inflammatory cytokine production ( 43,45,46 ) , and apoptosis ( 38 ) .
rasgrp1 is highly expressed in lymphocytes but also in various other cells , including pancreatic -cells ( 47,48 ) .
its dysfunction in -cells may lead to islet inflammation and impaired -cell function , which are considered as major factors involved in t2d pathogenesis ( 49,50 ) . in accordance with this ,
the t2d risk - increasing allele of rasgrp1-rs7403531 was also associated higher plasma glucose and lower homa - b , suggesting that the t2d risk conferred by rs7403531 is likely mediated through an impaired -cell function . however
, more functional studies are required to draw a firm conclusion . to test possible misclassification of t1d in our study
, we also examined associations of all known t1d - associated variants with t2d in our stage 1 samples .
in contrast to the multiple replicated t2d loci , very few of the t1d - associated variants showed a trend toward association with t2d ( p values : 0.010.05 , p for binomial test = 0.24 ) ( supplementary table 15 ) , suggesting a low misclassification rate in our stage 1 sample .
moreover , we observed a similar or ( combined p = 1  10 ; or = 1.09 ) when we performed secondary association analyses for rasgrp1-rs7403531 in 5,678 t2d case and 8,438 control subjects from several hospital - based case - control studies , in which all t2d case subjects were negative for glutamic acid decarboxylase and insulin autoantibody tests . all of these results , together with the facts that all genotyped t2d case subjects in our study were > 30 years of age and had no prior history of t1d , suggested that the observed t2d association signals at rasgrp1 and grk5 were unlikely to be driven by t1d - associated variants or by case misclassification in our study . taken together , this study is the largest gwas of t2d performed in chinese hans thus far .
we have identified two novel loci ( grk5 and rasgrp1 ) that are associated with t2d at genome - wide significant levels . in particular , the association signal at grk5 seems to be specific to east asians , but this finding needs to be confirmed in further studies .
we have also confirmed the known t2d loci at kcnq1 , cdkal1 , cdkn2b , cdc123 , hnf1b , glis3 , and dusp9 at genome - wide significant levels .
our findings not only provide new insights into the pathophysiology of t2d , but may also shed light on the ethnic differences in t2d susceptibility .
 substantial progress has been made in identification of type 2 diabetes ( t2d ) risk loci in the past few years , but our understanding of the genetic basis of t2d in ethnically diverse populations remains limited . 
 we performed a genome - wide association study and a replication study in chinese hans comprising 8,569 t2d case subjects and 8,923 control subjects in total , from which 10 single nucleotide polymorphisms were selected for further follow - up in a de novo replication sample of 3,410 t2d case and 3,412 control subjects and an in silico replication sample of 6,952 t2d case and 11,865 control subjects . besides confirming seven 
 established t2d loci ( cdkal1 , cdkn2a / b , kcnq1 , cdc123 , glis3 , hnf1b , and dusp9 ) at genome - wide significance , we identified two novel t2d loci , including g - protein  coupled receptor kinase 5 ( grk5 ) ( rs10886471 : p = 7.1  109 ) and rasgrp1 ( rs7403531 : p = 3.9  109 ) , of which the association signal at grk5 seems to be specific to east asians . in nondiabetic individuals , 
 the t2d risk - increasing allele of rasgrp1-rs7403531 was also associated with higher hba1c and lower homeostasis model assessment of -cell function ( p = 0.03 and 0.0209 , respectively ) , whereas the t2d risk - increasing allele of grk5-rs10886471 was also associated with higher fasting insulin ( p = 0.0169 ) but not with fasting glucose . 
 our findings not only provide new insights into the pathophysiology of t2d , but may also shed light on the ethnic differences in t2d susceptibility . 