the study sample consisted of 3,210 individuals ( 1,423 men and 1,787 women ) from the study on nutrition and health of aging population in china .
the study population , design , and protocols of this population - based cohort study have been previously described ( 19 ) .
briefly , all participants were unrelated chinese hans , aged 5070 years , with at least 20 years residence in beijing or shanghai . among them , 424 participants had type 2 diabetes ( 267 had previously diagnosed type 2 diabetes and 157 had screen - detected and treatment - naive type 2 diabetes ) , 878 participants had impaired fasting glucose ( ifg ) ( all 878 were screen detected and treatment naive ) , and 1,908 participants had normal fasting glucose ( nfg ) .
type 2 diabetes was defined by either 1999 world health organization criteria ( 20 ) or previously diagnosed type 2 diabetes .
nfg and ifg were defined as fasting glucose < 5.6 mmol / l ( 100 mg / dl ) and 5.6 mmol / l ( 100 mg / dl ) less than or equal to fasting glucose <
the study was conducted simultaneously in both beijing and shanghai from march to june 2005 .
the participants were recruited from two urban districts ( 400 participants for each district ) and one rural district ( 800 participants ) , representing people with high to low socioeconomic status , using a multistage sampling method in each city .
all participants were selected randomly from the eligible candidates listed in the residential registration record .
one person from each household was allowed to participate , and at least 40% of the total participants were men in each district .
individuals with the following conditions were excluded from the study : 1 ) severe psychological disorders , physical disabilities , cancer , cardiovascular disease , alzheimer 's disease , or dementia , within 6 months ; or 2 ) currently diagnosed with tuberculosis , aids , and other communicable diseases .
all participants attended a physical examination , during which standard anthropometric measurements and overnight fasting blood samples were collected .
glucose was measured enzymatically on an automatic analyzer ( hitachi 7080 ; hitachi , tokyo , japan ) with reagents purchased from wako pure chemical industries ( osaka , japan ) .
a1c concentrations were measured by turbidometric immunoassay in red blood cells on the hitachi 7080 analyzer using reagents from roche diagnostics ( indianapolis , in ) . homeostasis model assessment ( homa ) of insulin sensitivity ( homa - s ) and -cell function ( homa - b ) were estimated using levy 's computer model ( 21 ) .
written informed consent was obtained from all participants , and study protocols were approved by the institutional review board of the institute for nutritional sciences .
genomic dna was extracted from peripheral blood leukocytes by the salting - out procedure ( available at http://humgen.wustl.edu/hdk_lab_manual/dna/dna2.html ) .
snp genotyping was performed with the genomelab snpstream genotyping system ( beckman coulter ) , according to the manufacturer 's protocol .
seventeen snps previously reported to be associated with type 2 diabetes by at least one of the gwass ( 712 ) were successfully genotyped in our population .
these include snps near cdkal1 ( rs10946398 , rs7754840 , rs7756992 , and rs9465871 ) , hhex / ide ( rs1111875 , rs5015480 , and rs7923837 ) , ext2 ( rs1113132 , rs11037909 , and rs3740878 ) , cdkn2a / b ( rs10811661 and rs564398 ) , igf2bp2 ( rs4402960 and rs1470579 ) , slc30a8 ( rs13266634 ) ( r325w ) , loc387761 ( rs7480010 ) , and an intergenic snp ( rs9300039 ) in chromosome 11 .
the genotyping success rate was > 97.1% , and the concordance rate was > 99% based on 12% duplicate samples ( n = 384 ) . samples with ambiguous base calling were genotyped again .
genotype frequencies of all 17 snps were consistent with hardy - weinberg equilibrium ( p > 0.01 ) , and most of the minor allele frequencies observed in this study were comparable with those in the hapmap chb ( chinese han in beijing ) sample ( online appendix table 1 [ available at http://dx.doi.org/10.2337/db08-0047 ] ) .
genotypic distributions were similar in beijing and shanghai populations ( p > 0.05 ) , except for the three hhex snps ( p = 0.041 , 0.003 , and 0.005 for rs1111875 , rs5015480 , and rs7923837 , respectively ) .
the association between each snp and the risk of type 2 diabetes and ifg was examined using logistic regression .
generalized linear regression was applied to study the associations between each snp and type 2 diabetes  related quantitative traits .
participants with known diabetes or receiving glucose - lowering treatment ( n = 267 ) were excluded from the type 2 diabetes  related quantitative trait analyses .
all association analyses assumed an additive effect of the risk allele and were adjusted for sex , age , bmi ( where appropriate ) , and geographical region ( shanghai versus beijing ) .
bmi , insulin , and homa - s were log transformed before analyses , and the data were presented as geometric means .
because of a significant difference in genotype distribution of the three hhex / ide snps ( p < 0.05 ) and in diabetes prevalence between the shanghai and beijing participants ( p < 0.0001 ) , analyses for these snps were performed for shanghai and beijing separately .
gene - gene interactions were assessed by including the respective interaction terms of pairwise snps in logistic regressions using the maximum likelihood estimation .
the combined effect of multiple snps on the risk of type 2 diabetes and/or ifg was determined by logistic regression after categorizing the participants into groups according to the number of the risk alleles they carried .
association analyses were performed with sas version 9.1 ( sas institute , cary , nc ) .
meta - analyses were conducted with stata ( version 9.2 ; stata , college station , tx ) .
power calculations were performed using quanto software ( available at http://hydra.usc.edu/gxe/ ) , and the power shown in online appendix table 1 was calculated for association between each snp and type 2 diabetes using the odds ratios ( 712 ) reported in the original studies and sample size and minor allele frequencies in our own study .
genomic dna was extracted from peripheral blood leukocytes by the salting - out procedure ( available at http://humgen.wustl.edu/hdk_lab_manual/dna/dna2.html ) .
snp genotyping was performed with the genomelab snpstream genotyping system ( beckman coulter ) , according to the manufacturer 's protocol .
seventeen snps previously reported to be associated with type 2 diabetes by at least one of the gwass ( 712 ) were successfully genotyped in our population .
these include snps near cdkal1 ( rs10946398 , rs7754840 , rs7756992 , and rs9465871 ) , hhex / ide ( rs1111875 , rs5015480 , and rs7923837 ) , ext2 ( rs1113132 , rs11037909 , and rs3740878 ) , cdkn2a / b ( rs10811661 and rs564398 ) , igf2bp2 ( rs4402960 and rs1470579 ) , slc30a8 ( rs13266634 ) ( r325w ) , loc387761 ( rs7480010 ) , and an intergenic snp ( rs9300039 ) in chromosome 11 .
the genotyping success rate was > 97.1% , and the concordance rate was > 99% based on 12% duplicate samples ( n = 384 ) . samples with ambiguous base calling were genotyped again .
genotype frequencies of all 17 snps were consistent with hardy - weinberg equilibrium ( p > 0.01 ) , and most of the minor allele frequencies observed in this study were comparable with those in the hapmap chb ( chinese han in beijing ) sample ( online appendix table 1 [ available at http://dx.doi.org/10.2337/db08-0047 ] ) .
genotypic distributions were similar in beijing and shanghai populations ( p > 0.05 ) , except for the three hhex snps ( p = 0.041 , 0.003 , and 0.005 for rs1111875 , rs5015480 , and rs7923837 , respectively ) .
the association between each snp and the risk of type 2 diabetes and ifg was examined using logistic regression .
generalized linear regression was applied to study the associations between each snp and type 2 diabetes  related quantitative traits .
participants with known diabetes or receiving glucose - lowering treatment ( n = 267 ) were excluded from the type 2 diabetes  related quantitative trait analyses .
all association analyses assumed an additive effect of the risk allele and were adjusted for sex , age , bmi ( where appropriate ) , and geographical region ( shanghai versus beijing ) .
bmi , insulin , and homa - s were log transformed before analyses , and the data were presented as geometric means .
likelihood ratio tests were used to examine genotype distribution in beijing and shanghai . because of a significant difference in genotype distribution of the three hhex / ide snps ( p < 0.05 ) and in diabetes prevalence between the shanghai and beijing participants ( p < 0.0001 ) , analyses for these snps were performed for shanghai and beijing separately .
gene - gene interactions were assessed by including the respective interaction terms of pairwise snps in logistic regressions using the maximum likelihood estimation .
the combined effect of multiple snps on the risk of type 2 diabetes and/or ifg was determined by logistic regression after categorizing the participants into groups according to the number of the risk alleles they carried .
association analyses were performed with sas version 9.1 ( sas institute , cary , nc ) .
meta - analyses were conducted with stata ( version 9.2 ; stata , college station , tx ) .
power calculations were performed using quanto software ( available at http://hydra.usc.edu/gxe/ ) , and the power shown in online appendix table 1 was calculated for association between each snp and type 2 diabetes using the odds ratios ( 712 ) reported in the original studies and sample size and minor allele frequencies in our own study .
we first examined the association with the risk of type 2 diabetes and ifg ( table 2 ) .
the four cdkal1 snps spanned two ld blocks ( r = 1.0 for rs7754840 and rs10946398 and 0.96 for rs7756992 and rs9465871 ) and were each significantly associated with type 2 diabetes ( odds ratios ranged between 1.38 and 1.49 ; p < 1.9  10 ) and with combined ifg / type 2 diabetes ( between 1.20 and 1.22 ; p < 0.0013 ) .
the cdkn2a / b rs10811661 variant was also associated with type 2 diabetes ( odds ratio 1.31 [ 95% ci 1.121.54 ] ; p = 0.001 ) and combined ifg / type 2 diabetes ( 1.26 [ 1.131.41 ] ; p = 2.76  10 ) .
the second cdkn2a / b snp ( rs564398 ) , which was not in ld with rs10811661 ( r = 0 ) , was not associated with type 2 diabetes or combined type 2 diabetes / ifg .
the two snps in igf2bp2 ( r = 0.83 ) and the slc30a8 snp ( rs13266634 ) showed modest association with combined ifg / type 2 diabetes ( odds ratios between 1.12 and 1.17 ; p = 0.0130.033 ) but not with type 2 diabetes alone .
the three ext2 variants were in complete ld ( r = 1.0 ) and occurred less frequently in our population ( 58% ) than in european populations ( 70% ) .
these variants , as well as those in chromosome 11 ( rs7480010 and rs9300039 , r = 0.037 ) , were not associated with type 2 diabetes or ifg .
analyses for the three snps in the hhex / ide ld block were performed separately in shanghai and beijing populations , as the difference in genotype distribution and prevalence of type 2 diabetes and ifg could lead to spurious associations due to population stratification ( table 2 ) .
all three hhex / ide snps were significantly associated with type 2 diabetes in shanghai participants , with rs5015480 and rs7923837 also associated with combined ifg / type 2 diabetes .
meta - analyses suggested that the associations exhibited significant heterogeneity for snps rs1111875 ( p = 0.006 ) and rs5015480 ( p = 0.028 ) between beijing and shanghai populations .
we next examined the association between genetic variants and type 2 diabetes  related quantitative traits ( glucose , a1c , insulin , homa - b , homa - s , and bmi ) to investigate whether these variants conferred risk of type 2 diabetes through their effects on any of these intermediate traits ( table 3 ) .
consistent with the case - control analyses , the snps that showed significant evidence for association with diabetes - related phenotypes were those that were also associated with type 2 diabetes or ifg , except for cdkn2a / b rs10811661 and loc387761 rs7480010 .
all four cdkal1 snps were significantly associated with a1c ( p values 0.0360.0096 ) and homa - b ( p values 0.0240.0009 ) .
the snps ( rs7756992 and rs9465871 ) in the second ld block of this locus also showed significant association with fasting glucose levels ( p < 0.04 ) .
interestingly , the allele of slc30a8 snp rs13266634 that increases the risk of combined ifg / type 2 diabetes was significantly associated with lower bmi ( p = 0.0087 ) and marginally associated with decreased homa - b ( p = 0.05 ) . only the associations of cdkal1-rs10946398 , rs7754840 , and igf2bp2-rs4402960 with homa - b remained significant after bonferroni correction for multiple testing ( p = 0.0014 , 0.05/36 tests ) . to examine whether the associations for the cdkal1 variants were independent , we performed additional multiple regression analyses that included all four cdkal1 snps in one model .
results showed that none of the four snps remained significant ( p  0.17 ) .
( rs7754840 and rs7756992 were chosen to represent each of the pairs ) were independent from each other for the associations with type 2 diabetes or related quantitative traits in multiple regression models with both rs7754840 and rs7756992 genotypes in the model , with age , sex , region , and bmi ( where appropriate ) as covariates .
the results revealed that the association seems to be driven by rs7754840 , for the associations with type 2 diabetes , bmi , and homa - b , or by rs7756992 , for the association with a1c , but interestingly , rs7754840 and rs7756992 seem to have independent effects on the associations with homa - s or insulin ( online appendix table 2 ) .
we also performed a meta - analysis with the data from the previously published studies ( 1012 ) , including those from japanese , korean , and hong kong chinese populations ( 2225 ) , to assess the heterogeneity between caucasians and asians for the cdkal1 and cdkn2a / b loci ( rs7754840 and rs10811661 were chosen to represent each of them , respectively ) .
the results showed that for the cdkn2a / b loci ( rs10811661 ) , the heterogeneity between caucasians and asians did not reach significance ( p = 0.059 ) , while a significant heterogeneity was observed between caucasians and asians ( p = 8.872  10 ) for the cdkal1 loci ( rs7754840 ) ( online appendix fig .
1 ) , and this is consistent with the recent finding reported by ng et al . ( 25 ) .
although we did not observe the association among the loc387761 snp rs7480010 and type 2 diabetes or ifg , we found that the allele that increased the diabetes risk in european populations was modestly associated ( p < 0.03 ) with increased insulin sensitivity ( homa - s ) and lower fasting insulin levels . furthermore , despite a strong association between the cdkn2a / b snp rs10811661 and type 2 diabetes , no association was observed with any of the diabetes - related quantitative traits .
the intergenic snp rs9300039 and the three ext2 snps ( rs3740878 , rs11037909 , and rs1113132 ) were not associated with any of the diabetes - related quantitative traits .
we found no evidence of multiplicative gene - gene interactions among the main snps ( rs9465871 , rs10811661 , rs4402960 , and rs13266634 ) in each of the cdkal1 , cdkn2a / b , igf2bp2 , and slc30a8 genes . a significantly higher proportion of participants with type 2 diabetes carry increasing numbers of risk alleles , compared with participants with nfg ( fig .
each additional risk allele increased the risk of type 2 diabetes by 1.24-fold ( p = 2.85  10 ) ( fig .
1b ) and combined ifg / type 2 diabetes by 1.21-fold ( p = 6.31  10 ) ( fig .
participants harboring seven or all eight risk alleles had a 4.44-fold increased risk for type 2 diabetes ( p = 5  10 ) compared with those with one or no risk alleles ( fig .
consistently , participants with increasing numbers of risk alleles tended to have increased fasting levels of plasma glucose ( p = 0.013 ) ( fig .
1e ) , as well as decreased homa - b values ( p = 3.34  10 ) ( fig .
, participants with increasing numbers of risk alleles tended to have significantly lower bmi ( p = 5.3  10 ) ( fig .
1f ) , which is consistent with previous results found for the cdkal1 and slc30a8 polymorphisms ( table 3 ) .
in this study of chinese hans , we replicated associations with several diabetes susceptibility variants recently identified through gwass in white europeans ( 712 ) . variants in cdkal1 , cdkn2a / b , igf2bp2 , slc30a8 , and hhex loci were significantly associated with the risk of type 2 diabetes or combined ifg / type 2 diabetes .
furthermore , variants in cdkal1 and igf2bp2 were strongly associated with -cell function estimated by homa - b .
these associations were stronger than those observed in individuals of european ancestry ( 810,12 ) ( online appendix table 1 ) , and cdkal1 risk allele frequencies are also substantially higher in chinese ( 4355% ) than europeans ( 1531% ) .
moreover , significant heterogeneity between caucasians and asians was found for the cdkal1 loci ( rs7754840 ) in the meta - analysis that combined the data from the previous studies in white europeans , japanese , korean , hong kong chinese , and our study ( p = 8.872  10 ) ( online appendix fig .
1 ) , while no significant heterogeneity was observed among the asians ( p = 0.369 ) .
these observations suggest that these cdkal1 variants might play an even more important role in diabetes susceptibility in chinese .
the risk allele of the first pair of cdkal1 variants was strongly associated with reduced -cell function ( homa - b ) and increased a1c levels , while the second pair of cdkal1 variants showed an association with impaired -cell function ( homa - b ) and higher glucose levels , as well as with increased a1c .
the results from additional multiple regression analyses suggest that the four snps most likely represent the effects of a single cdkal1 locus .
however , none of these four snps stands out as being the variant driving the association .
therefore , we assume that none of them is likely to be the causal variant , but presumably they are in moderate to high ld with the causal snp and are therefore less consistently associated with the traits of interest .
this region would benefit from a detailed fine mapping to identify possible causal variants in future studies .
these results support previous findings ( 9,13,26 ) that the four cdkal1 snps confer the risk of type 2 diabetes through reduced insulin secretion , although the causal snp is yet to be identified .
we also observed significant association between cdkn2a / b rs10811661 and type 2 diabetes and ifg with a slightly higher odds ratio ( 1.3 ) than that observed in europeans ( 1.20 ) ( 1012 ) .
the risk allele is twice as prevalent in chinese hans ( 46% ) as in europeans ( 21% ) .
however , we did not observed significant heterogeneity between caucasians and asians in the meta - analysis with data from the previously published studies ( p = 0.059 ) .
interestingly , none of the diabetes - related traits showed an association with cdkn2a / b rs10811661 .
the second cdkn2a / b variant , rs564398 , which is less frequent in chinese hans ( 13% ) than in europeans ( 38% ) , was not associated with type 2 diabetes or any related traits .
the association between variants in igf2bp2 and type 2 diabetes was not significant , although the odds ratios were similar to those observed in european populations ( 1.15 ) , suggesting that our study may not have been sufficiently powered .
indeed , assuming an additive model and a minor allele frequency of 25% , we had < 50% power to detect previously reported odds ratios at p < 0.05 .
we did , however , find a significant association with combined ifg / type 2 diabetes .
the associations with homa - b suggest that igf2bp2 confer type 2 diabetes risk through a reduced -cell function .
similarly , we found no association between the slc30a8 rs13266634 variant and type 2 diabetes , while an association with combined ifg / type 2 diabetes reached borderline significance .
interestingly , the risk allele that increased diabetes risk in europeans was also associated with a lower bmi in this population .
we also failed to find any evidence for association between type 2 diabetes and the snps in ext2 ( rs3740878 , rs11037909 , and rs1113132 ) and the intergenic snp rs9300039 , despite 80% power to detect previously reported effect estimates ( 7 ) .
although these snps exhibited marginal associations with type 2 diabetes in the original study ( 7 ) , they were largely negative in the subsequent four gwass and other replication studies in samples from u.k .
( 8,12 ) , finnish ( 10,11 ) , swedish ( 10 ) , icelandic ( 9 ) , german ( 27 ) , and japanese ( 23 ) populations .
therefore , the original associations for these snps were either population specific or overestimated due to the  winner 's curse 
( 28,29 ) , but the consistent lack of replication suggests that these findings were more likely false - positives .
meta - analyses or studies with larger sample sizes will be required to draw definitive conclusions .
although there was no association between rs7480010 ( loc387761 ) and type 2 diabetes or ifg , the allele conferring risk of diabetes in europeans was associated with increased insulin sensitivity and showed a tendency toward a reduced -cell function as well .
for the three snps in hhex / ide gene region , the associations with type 2 diabetes or ifg were observed only in shanghai individuals in whom each risk allele resulted in 1.45- to 1.79-fold increased diabetes risk , suggesting that geographical stratification may exist in our population for these snps and their roles in type 2 diabetes susceptibility .
however , given the relatively small sample size , we can not rule out sampling bias .
we found no evidence of pairwise synergistic gene - gene interactions on type 2 diabetes and the related phenotypes among cdkal1-rs9465871 , cdkn2a / b - rs10811661 , igf2bp2-rs4402960 , and slc30a8-rs13266634 . in joint analyses ,
the risk of type 2 diabetes was increased by 1.24-fold for each additional risk allele , and participants with seven or all eight risk alleles ( 3.8% ) had a 4.44-fold increased risk of type 2 diabetes ( p = 5  10 ) compared with those with one or no risk allele .
( 11 ) , who examined combined effects of 10 risk variants in a gwas of european populations . compared with scott 's study ,
the advantage of our study is that our data are based on the general population .
however , a replication in larger population is required to examine whether combinations of risk alleles from these variants have good predictive and diagnostic potential in chinese hans . in conclusion
, we replicated the association of type 2 diabetes with the snps in cdkal1 and cdkn2a / b genes and confirmed that the snps in slc30a8 and igf2bp2 were associated with the risk of combined ifg / type 2 diabetes .
importantly , the risk variants in cdkal1 , cdkn2a / b , igf2bp2 , and slc30a8 appear to act in an additive manner to increase the risk of type 2 diabetes and related phenotypes .
these results provide solid evidence for the notion that these variants individually or collectively contribute to the risk of type 2 diabetes in the chinese han population , possibly by impairing -cell function or reducing insulin secretion .
 objective genome - wide association studies have identified common variants in cdkal1 , cdkn2a / b , igf2bp2 , slc30a8 , hhex / ide , ext2 , and loc387761 loci that significantly increase the risk of type 2 diabetes . 
 we aimed to replicate these observations in a population - based cohort of chinese hans and examine the associations of these variants with type 2 diabetes and diabetes - related phenotypes.research design and methods we genotyped 17 single nucleotide polymorhisms ( snps ) in 3,210 unrelated chinese hans , including 424 participants with type 2 diabetes , 878 with impaired fasting glucose ( ifg ) , and 1,908 with normal fasting glucose.results we confirmed the associations between type 2 diabetes and variants near cdkal1 ( odds ratio 1.49 [ 95% ci 1.271.75 ] ; p = 8.91  107 ) and cdkn2a / b ( 1.31 [ 1.121.54 ] ; p = 1.0  103 ) . 
 we observed significant association of snps in igf2bp2 ( 1.17 [ 1.031.32 ] ; p = 0.014 ) and slc30a8 ( 1.12 [ 1.011.25 ] ; p = 0.033 ) with combined ifg / type 2 diabetes . 
 the snps in cdkal1 , igf2bp2 , and slc30a8 were also associated with impaired -cell function estimated by homeostasis model assessment of -cell function . when combined , each additional risk allele from cdkal1-rs9465871 , cdkn2a / b - rs10811661 , igf2bp2-rs4402960 , and slc30a8-rs13266634 increased the risk for type 2 diabetes by 1.24-fold ( p = 2.85  107 ) or for combined ifg / type 2 diabetes by 1.21-fold ( p = 6.31  1011 ) . 
 none of the snps in ext2 or loc387761 exhibited significant association with type 2 diabetes or ifg . 
 significant association was observed between the hhex / ide snps and type 2 diabetes in individuals from shanghai only ( p < 0.013 ) but not in those from beijing ( p > 0.33).conclusions our results indicate that in chinese hans , common variants in cdkal1 , cdkn2a / b , igf2bp2 , and slc30a8 loci independently or additively contribute to type 2 diabetes risk , likely mediated through -cell dysfunction . 
RESEARCH DESIGN AND METHODS
Genotyping.
Statistical analyses.
RESULTS
DISCUSSION
Supplementary Material