briefly , 993 unrelated african american patients with type 2 diabetes born in virginia , tennessee , north carolina , south carolina , or georgia were recruited from dialysis clinics .
type 2 diabetes was diagnosed if patients reported an initial diagnosis of diabetes after 35 years of age , received dietary therapy or hypoglycemic agents in the absence of insulin alone for at least 1 year after initial diagnosis , and were currently receiving diabetes medications .
case subjects had severe diabetes accompanied by nephropathy , and type 2 diabetes was diagnosed > 5 years before the start of renal replacement therapy , with background or greater diabetic retinopathy and/or a score of 3 + proteinuria on dipstick urinalysis .
nondiabetic control subjects were recruited from the same geographic region : 1,054 were african american and 36 european descent  derived without prior diagnosis of type 2 diabetes or renal disease .
dna extraction was performed using the puregene system ( gentra systems , minneapolis , mn ) .
dna was obtained from 44 yoruba nigerians from the national institute of general medical sciences human variation collection ( coriell cell repositories , camden , nj ) .
this study was approved by the institutional review board of wake forest university school of medicine and was in accordance with the principles of the declaration of helsinki .
snps pkn2 rs6698181 ; igf2bp2 rs4402960 ; flj39370 rs17044137 ; cdkal1 rs10946398 and rs7754840 ; slc30a8 rs13266634 ; cdkn2a / cdkn2b rs564398 and rs10811661 ; hhex / ide / kif11 rs1111875 , rs5015480 , and rs7923837 ; ext2/alx4 rs3740878 , rs11037909 , and rs1113132 ; fto rs8050136 ; loc387761 rs7480010 ; intragenic rs9300039 ; and tcf7l2 rs7903146 were genotyped using iplex technology ( sequenom , san diego , ca ) ( 12 ) .
snp genotyping success rates were > 98.6% in case and > 98.4% in control subjects .
sixty - eight biallelic ancestry - informative markers were genotyped using iplex technology ( sequenom , san diego , ca ) in 993 african american case , 1,054 african american control , 44 yoruba nigerian , and 36 european - american subjects .
both the yoruba nigerian and european - american individuals were utilized only for admixture analyses .
genotyping success rates for ancestry - informative markers were > 96.9% in african american case and > 95.2% in african american control subjects .
hardy - weinberg equilibrium ( hwe ) values were determined by calculating a  statistic and corresponding p value .
structures of the haplotype blocks for the snps genotyped in cdkal1 , ext2/alx4 , and ide / kif11/hhex were ascertained using haploview 3.2 ( 13 ) using the criteria outlined by gabriel et al .
measures of linkage disequilibrium and association were calculated using the program snpgwa ( 15 ) . for each tandem pair of snps , the linkage disequilibrium statistics d and r were computed .
( genotypic ) , additive ( cochran - armitage trend test ) , and corresponding lack of fit to additivity were calculated .
initially , tests were performed using 1,000 permutations . where association tests indicated possible significance ( empirical p < 0.10 )
, permutations were increased to 10,000 . the quantitative trait ( bmi ) association analysis for fto
estimates of case and control haplotype frequency were obtained using dandelion 1.26 ( 16 ) . to provide context for these tests of association ,
power analyses were computed using quanto ( 17 ) . specifically , power estimates to detect the range of reported odds ratios ( ors ) from the european - derived samples were computed assuming a type 2 diabetic population prevalence of 0.15 ( consistent with african american populations ) and polymorphism - specific type 1 error rates of  = 0.05 and  = 0.10 .
power was calculated based on the risk allele frequency observed in our african american sample .
individual ancestral proportions were calculated using an expectation - maximization algorithm ( frappe ) ( 18 ) under a two - population model .
estimates of ancestral allele frequencies were obtained from the genotyped african american and european - derived samples .
logistic regression tests of additive , dominant , and recessive genetic models included adjustments for individual estimates of african ancestry ( 19 ) .
snps pkn2 rs6698181 ; igf2bp2 rs4402960 ; flj39370 rs17044137 ; cdkal1 rs10946398 and rs7754840 ; slc30a8 rs13266634 ; cdkn2a / cdkn2b rs564398 and rs10811661 ; hhex / ide / kif11 rs1111875 , rs5015480 , and rs7923837 ; ext2/alx4 rs3740878 , rs11037909 , and rs1113132 ; fto rs8050136 ; loc387761 rs7480010 ; intragenic rs9300039 ; and tcf7l2 rs7903146 were genotyped using iplex technology ( sequenom , san diego , ca ) ( 12 ) .
snp genotyping success rates were > 98.6% in case and > 98.4% in control subjects .
sixty - eight biallelic ancestry - informative markers were genotyped using iplex technology ( sequenom , san diego , ca ) in 993 african american case , 1,054 african american control , 44 yoruba nigerian , and 36 european - american subjects .
both the yoruba nigerian and european - american individuals were utilized only for admixture analyses .
were > 96.9% in african american case and > 95.2% in african american control subjects .
hardy - weinberg equilibrium ( hwe ) values were determined by calculating a  statistic and corresponding p value .
structures of the haplotype blocks for the snps genotyped in cdkal1 , ext2/alx4 , and ide / kif11/hhex were ascertained using haploview 3.2 ( 13 ) using the criteria outlined by gabriel et al .
measures of linkage disequilibrium and association were calculated using the program snpgwa ( 15 ) . for each tandem pair of snps , the linkage disequilibrium statistics d and r were computed .
( genotypic ) , additive ( cochran - armitage trend test ) , and corresponding lack of fit to additivity were calculated .
initially , tests were performed using 1,000 permutations . where association tests indicated possible significance ( empirical p < 0.10 )
, permutations were increased to 10,000 . the quantitative trait ( bmi ) association analysis for fto
estimates of case and control haplotype frequency were obtained using dandelion 1.26 ( 16 ) . to provide context for these tests of association ,
specifically , power estimates to detect the range of reported odds ratios ( ors ) from the european - derived samples were computed assuming a type 2 diabetic population prevalence of 0.15 ( consistent with african american populations ) and polymorphism - specific type 1 error rates of  = 0.05 and  = 0.10 .
power was calculated based on the risk allele frequency observed in our african american sample .
individual ancestral proportions were calculated using an expectation - maximization algorithm ( frappe ) ( 18 ) under a two - population model .
estimates of ancestral allele frequencies were obtained from the genotyped african american and european - derived samples .
logistic regression tests of additive , dominant , and recessive genetic models included adjustments for individual estimates of african ancestry ( 19 ) .
eighteen snps in 12 type 2 diabetes loci previously associated in european - derived populations were genotyped in a sample of 993 african american type 2 diabetic case subjects enriched for diabetic nephropathy and in 1,054 african american control subjects .
, we observed a greater percentage of female subjects in the case ( 63% ) compared with the control subjects ( 55% ) .
control subjects were younger than case subjects but older than the mean age at which type 2 diabetes was diagnosed .
the mean age of end - stage renal disease diagnosis was 58.4  10.5 years . in the case subjects , the estimated mean proportion of african ancestry was 0.821 , whereas in the control subjects it was 0.795 .
all snps were consistent with hwe proportions in the entire population ( supplementary table 1 [ available in an online appendix at http://dx.doi.org/10.2337/db07-1319 ] ) .
rs9300039 , which is located in an intragenic region of chromosome 11p12 , was inconsistent with hwe in the case population ( p = 0.028 ) because of an excess of homozygotes .
high levels of linkage disequilibrium were observed between the two cdkal1 snps rs10946398 and rs7754840 ( d = 1.000 ; r = 0.996 ) .
the three snps within the ide / kif11/hhex region were in high - to - moderate linkage disequilibrium with pairwise d values of 0.679 ( r = 0.244 ) between rs1111875 and rs5015480 , 0.945 ( r = 0.140 ) between rs5015480 and rs7923837 , and 0.894 ( r = 0.236 ) between rs1111875 and rs7923837 .
likewise , high linkage disequilibrium was also observed within the three snps in the ext2/alx4 region , with pairwise d values of 1.000 ( r = 0.569 ) between rs1113132 and rs11037909 , 0.988 ( r = 0.601 ) between rs11037909 and rs3740878 , and 1.000 ( r = 0.935 ) between rs1113132 and rs3740878 .
evidence of association with type 2 diabetes was observed with rs10946398 ( or 1.15 ; additive p [ pa ] = 0.029 ) and rs7754840 ( or 1.14 ; pa = 0.039 ) , both of which are located in intron five of cdkal1 .
a significant association with type 2 diabetes was also seen with loc387761 snp rs7480010 ( or 1.33 ; pa = 0.002 ) , but this marker also did not show evidence of association after adjusting for admixture ( pa = 0.084 ) .
rs9300039 , located in an intragenic region of chromosome 11p12 , was also associated under the admixture - adjusted dominant model ( pd = 0.029 ) but deviated from hwe proportions in the case population ( hwe p = 0.028 ) .
in addition , the minor allele homozygote count for rs9300039 is small ( 21 in the case and 9 in the control subjects , respectively [ supplementary table 1 ] ) , so association values for this marker should be interpreted with caution .
the most significant association with type 2 diabetes was observed with tcf7l2 snp rs7903146 ( or 1.39 ; pa = 1.73  10 ) , and this snp remained statistically significant after admixture adjustment ( pa = 1.59  10 ) .
all other snps examined failed to show evidence of association with type 2 diabetes in this african american cohort under an additive model ( table 2 ) .
tests of association under the dominant and recessive genetic models are shown in supplementary table 2 .
we also tested whether fto snp rs8050136 was associated with bmi as previously reported in european- derived populations ( 4,6 ) .
we found no evidence of association with this polymorphism and bmi ( pa = 0.480 ) .
association with type 2 diabetes adjusted for age or sex was calculated for each snp ( data not shown ) but did not meaningfully change the results of this study .
it is interesting to note that 5 of the 18 snps had ors in the opposite direction , inconsistent with the previously reported or .
in fact , the 95% ci for 7 of the 18 snps did not include the previously reported or from the european - american samples .
with this very limited evidence of association in our african american population , the question arises whether adequate power was available in this sample to detect association .
this has been addressed by calculating the power to detect association for each snp based on the estimated effect size from studies in european - derived populations and the observed allele frequency of the risk allele in the african american population .
power was calculated for  = 0.05 ( nominal evidence of association ) and  = 0.10 ( for the ability to detect loci that are trending toward association ) .
the power estimates using an intermediate measure of effect size ( between the highest and lowest reported in european - derived populations ) under the additive model are shown in table 2 .
more extensive power estimates are shown in supplementary table 3 , which also provides estimates for high and low observed effect sizes .
for some snps , the power is quite low ( e.g. , rs7480010 ) but for other snps ( rs3740878 , rs11037909 , and rs8050136 ) power is 70% for  = 0.05 .
all three snps examined in the ext2/alx4 region reside in a single linkage disequilibrium block but showed no haplotypic association with type 2 diabetes ( data not shown ) .
of the three snps genotyped in the ide / kif11/hhex region , two ( rs5015480 and rs7923837 ) were located in an linkage disequilibrium block but also showed no evidence of association .
haplotype analysis of the two cdkal1 snps ( rs10946398 and rs7754840 ) show significant association with type 2 diabetes ( global empirical p = 0.036 ; data not shown ) but provides little information beyond that obtained with single - snp analysis as a result of high measures of linkage disequilibrium ( d = 1.000 ; r = 0.996 ) .
we examined 18 snps in 12 loci identified in recent genome - wide association studies in european - derived populations that were associated with type 2 diabetes or bmi ( fto ) for association with type 2 diabetes in a large african american population .
there is very limited evidence that any of these european - derived genome - wide association study snps contribute to diabetes in african americans .
in contrast , significant association with type 2 diabetes was observed with the previously described tcf7l2 snp rs7903146 .
tcf7l2 has been the focus of multiple investigations and is one of the most highly replicated type 2 diabetes candidate genes in several populations ( 15 ) .
few publications have examined the influence of tcf7l2 on type 2 diabetes risk in african americans . in this study , we expand on the previous investigation from our center by sale et al .
( 8) , which described compelling evidence of association with type 2 diabetes for polymorphisms in this gene . from the results of these studies and
the convincing replication of association with type 2 diabetes in european - derived populations , a more comprehensive study of tcf7l2 polymorphisms in african americans is warranted .
parenthetically , this observation of strong association gives us confidence that the ascertainment and diagnoses used to recruit these african american subjects are accurate and the analyses of the european - derived type 2 diabetes loci presented here are valid .
of all the other loci examined , the only nominally significant association with type 2 diabetes in african americans before admixture adjustment was observed with the snps located in cdkal1 and loc387761 . if one takes into consideration that multiple snps were genotyped , any standard multiple - comparisons correction suggests that the evidence for association is very modest .
both rs10946398 and rs7754840 have been previously associated with type 2 diabetes in european - derived populations ( 3,4,6 ) .
in addition , other publications have also reported evidence of association in this region , making cdkal1 one of the most highly replicated genes identified from recent wga studies ( 2,5 ) . measures of linkage disequilibrium between rs10946398 and rs7754840 in african americans ( d = 1.000 ; r = 0.996 ) were similar to those reported in european americans ( 6 ) . in contrast
, the frequencies of the rs10946398 and rs7754840 risk allele ( c for both ) differed substantially between african american ( 0.60 , 0.60 ) , yoruba in ibaden , nigeria ( yri ) ( 0.67 , 0.67 ) , and utah residents with ancestry from northern and western europe ( ceu ) ( 0.31 , 031 ) populations .
in addition , fto snp rs8050136 was previously associated with bmi ( 7 ) ; however , we found no evidence of association with this trait in any genetic model .
the genotyping data have been evaluated for association with type 2 diabetes taking into consideration the european - african admixture of african americans .
we have adjusted association statistics using estimates of individual proportions of european and african ancestry for each subject .
this approach may under- or overestimate the true association at each locus because the difference in allele frequencies between the ancestral populations might vary at each marker , and , thus , the adjustment for an individual genome may overestimate or underestimate admixture at a specific site .
thus , it is possible in this dataset that stronger evidence of association might be revealed by a locus - specific analysis . given the magnitude of the great majority of the p values observed here ( table 2 ) , this influence would have to be very substantial to adjust the p values into the range suggesting association .
even though all of the snps examined in this study showed highly significant association with type 2 diabetes in european - derived populations , we found limited evidence of association with these markers in african americans .
for some of these snps , we have limited power to detect evidence of association given the sample sizes , allele frequencies , and effect sizes ( table 2 ) ( supplementary table 3 ) . there is , however , reasonable power to detect association for a number of the snps .
it is important to note that these conventional power analyses reflect the power to detect association at a single locus .
if one takes into consideration the overall study , we would have expected to see evidence of association with four to five loci at  = 0.05 .
another caveat to this study is that we have tested only one to three snps per gene that were highly associated in european - derived populations .
ongoing extensive analyses of individual genes , a pending genome - wide association study in these samples , and genotyping in additional samples of case and control subjects will further clarify the role of these loci in the african american diabetic population .
the absence of association that was observed in this study may , however , be accountable to differences in genetic risk factors that exist between african and european - derived populations .
moreover , multiple other type 2 diabetes susceptibility genes reported in populations with european descent fail to show association in african - derived samples ( 9 ) .
disparities in allele frequencies between different ethnic groups may impair our ability to observe true associations if they exist in african americans .
for example , the type 2 diabetes risk allele reported by recent wga studies for cdkn2a / cdkn2b snps rs10811661 and rs564398 , ide / kif11/hhex snp rs7923837 , and loc387761 snp rs7480010 have frequencies of 1.00 , i.e. , are monomorphic , in the yri population of the hapmap project ( 20 ) ( table 3 ) .
we have compared these sequences with chimpanzee and other available primate sequences , but no common pattern of sequence variation is apparent .
presumably , the non - african alleles for these loci are the result of european admixture , which is not great enough to provide an observable level of protection from type 2 diabetes .
the observation that these loci are nonpolymorphic in africans and that solely africans have the risk allele suggests that these loci may , in part , contribute to the increased overall prevalence of type 2 diabetes in african - derived populations compared with that in european - derived populations .
thus , the appropriate conclusion from this study is that these european type 2 diabetes susceptibility loci do not measurably contribute to differential susceptibility in the african american population .
 objective several whole - genome association studies have reported identification of type 2 diabetes susceptibility genes in various european - derived study populations . 
 little investigation of these loci has been reported in other ethnic groups , specifically african americans . 
 striking differences exist between these populations , suggesting they may not share identical genetic risk factors . 
 our objective was to examine the influence of type 2 diabetes genes identified in whole - genome association studies in a large african american case - control population.research design and methods single nucleotide polymorphisms ( snps ) in 12 loci ( e.g. , tcf7l2 , ide / kif11/hhex , slc30a8 , cdkal1 , pkn2 , igf2bp2 , flj39370 , and ext2/alx4 ) associated with type 2 diabetes in european - derived populations were genotyped in 993 african american type 2 diabetic and 1,054 african american control subjects . 
 additionally , 68 ancestry - informative markers were genotyped to account for the impact of admixture on association results.results little evidence of association was observed between snps , with the exception of those in tcf7l2 , and type 2 diabetes in african americans . 
 one tcf7l2 snp ( rs7903146 ) showed compelling evidence of association with type 2 diabetes ( admixture - adjusted additive p [ pa ] = 1.59  106 ) . 
 only the intragenic snp on 11p12 ( rs9300039 , dominant 
 p [ pd ] = 0.029 ) was also associated with type 2 diabetes after admixture adjustments . 
 interestingly , four of the snps are monomorphic in the yoruba population of the hapmap project , with only the risk allele from the populations of european descent present.conclusions results suggest that these variants do not significantly contribute to interindividual susceptibility to type 2 diabetes in african americans . 
 consequently , genes contributing to type 2 diabetes in african americans may , in part , be different from those in european - derived study populations . 
 high frequency of risk alleles in several of these genes may , however , contribute to the increased prevalence of type 2 diabetes in african americans . 
RESEARCH DESIGN AND METHODS
Candidate gene genotyping.
Admixture analyses genotyping.
Statistical analyses.
RESULTS
DISCUSSION