this study was conducted under institutional review board approval from wake forest university school of medicine .
identification , clinical characteristics , and recruitment of african american patients and controls have been previously described in detail ( 22 ) .
briefly , 1,033 unrelated african american patients with type 2 diabetes were recruited from dialysis facilities .
type 2 diabetes was diagnosed in african americans who reported developing type 2 diabetes after the age of 25 years and who did not receive only insulin therapy since diagnosis .
in addition , cases had to have at least one of the following three criteria for inclusion : 1 ) type 2 diabetes diagnosed at least 5 years before initiating renal replacement therapy , 2 ) background or greater diabetic retinopathy , and/or 3 ) 100 mg / dl proteinuria on urinalysis in the absence of other causes of nephropathy .
an additional 1,106 unrelated african americans without a current diagnosis of diabetes or renal disease were recruited from the community and internal medicine clinics as controls .
all type 2 diabetic cases and nondiabetic controls were born in north carolina , south carolina , georgia , tennessee , or virginia .
dna extraction was performed using the puregene system ( gentra systems , minneapolis , mn ) .
the dna screening panel was composed of 96 african american subjects : 48 type 2 diabetic cases and 48 controls .
dna sequencing reactions were performed using bigdye terminator v.1.1 cycle sequencing kits and analyzed on the applied biosystems 3730xl dna analyzer ( applied biosystems , foster city , ca ) .
a search of the region sequenced was performed at dbsnp ( www.ncbi.nlm.nih.gov ) to record all previously identified polymorphisms reported in the region .
genotyping of dg10s478 was performed by fragment length analysis on an abi prism dna analyzer 3700 with previously published primers ( 21 ) in a manner similar to that previously described ( 23 ) .
snp genotyping was performed on the iplex massarray genotyping platform ( sequenom , san diego , ca ) .
for all snps , the genotyping success rate was greater than 95% . in preliminary analyses ,
snps in the tcf7l2 gene region  10 kb ( c10:114689999114926060 ) were imputed using data from hapmap phase ii hybrid panel ( 1:1 , yri : ceu ; 108 snps ) and the 1000 genomes yri pilot 1 dataset ( 497 snps ) to circumvent limited coverage of the genetic diversity in this specific region by the affymetrix 6.0 array .
snps were imputed in 965 type 2 diabetic end - stage renal disease ( esrd ) cases and 1,029 controls with high - quality score ( rsq - hat 0.3 ) using the software mach ( www.sph.umich.edu/csg/abecasis ) ( 24 ) .
four common snps identified from direct sequence analysis had minor allele frequencies ( maf ) 0.05 and failed assay design on the sequenom platform . with the use of the resequencing genotype data obtained from the 96 african american samples as reference , these snps were imputed in the remaining 2,043 samples with high - quality score ( rsq - hat 0.5 ) using the software mach ( www.sph.umich.edu/csg/abecasis ) ( 24 ) .
snps were tested for departure from hardy - weinberg equilibrium ( hwe ) using an exact test of hwe proportions for the combined group of cases and controls and then for cases only and controls only ( 25 ) .
haplotype block structure was established using haploview 4.1 ( 26 ) , defining blocks using the method from gabriel et al .
unadjusted measures of ld and association were assessed using the software snpgwa ( http://www.phs.wfubmc.edu ) ( 28 ) .
snpgwa computes ld statistics , d and r , for each pair of tandem snps .
snpgwa also performs multiple tests of association including the overall two - degree of freedom test ( genotype ) , dominant model , recessive model , additive model ( cochran - armitage trend test ) , and the corresponding lack of fit to the additive model .
odds ratios , 95% confidence intervals , and p values were computed for each model of association .
assuming a log additive model , x = ( 1  f ) + 2f(1  f) + f where  is the estimated odds ratio ( or ) and f is the risk allele frequency .
ancestry estimates were determined from 70 biallelic admixture informative markers ( aims ) as previously described ( 16,29 ) .
briefly , aims were selected to maximize european and african allele frequency differences and sample all non - acrocentric arms of the autosomal genome .
reference population allele frequencies were derived by genotyping 44 african ( yoruba from ibadan , nigeria ) and 39 european americans .
individual ancestral proportions were generated for each subject using frappe ( 30 ) , an expectation - maximization algorithm , under a two - population model .
the individual ancestral estimates were used as covariates in the association analyses . to test whether snps were independently associated with type 2 diabetes
, we performed an omnibus test for the haplotype association using plink ( 31 ) .
we further adjusted the omnibus test by controlling for one of the snps at a time .
an insignificant conditional test suggests that the conditioned snp is sufficient to explain the haplotype association and there is a single , rather than multiple , association signals at the haplotype .
this study was conducted under institutional review board approval from wake forest university school of medicine .
identification , clinical characteristics , and recruitment of african american patients and controls have been previously described in detail ( 22 ) .
briefly , 1,033 unrelated african american patients with type 2 diabetes were recruited from dialysis facilities .
type 2 diabetes was diagnosed in african americans who reported developing type 2 diabetes after the age of 25 years and who did not receive only insulin therapy since diagnosis .
in addition , cases had to have at least one of the following three criteria for inclusion : 1 ) type 2 diabetes diagnosed at least 5 years before initiating renal replacement therapy , 2 ) background or greater diabetic retinopathy , and/or 3 ) 100 mg / dl proteinuria on urinalysis in the absence of other causes of nephropathy .
an additional 1,106 unrelated african americans without a current diagnosis of diabetes or renal disease were recruited from the community and internal medicine clinics as controls .
all type 2 diabetic cases and nondiabetic controls were born in north carolina , south carolina , georgia , tennessee , or virginia .
dna extraction was performed using the puregene system ( gentra systems , minneapolis , mn ) .
the dna screening panel was composed of 96 african american subjects : 48 type 2 diabetic cases and 48 controls .
dna sequencing reactions were performed using bigdye terminator v.1.1 cycle sequencing kits and analyzed on the applied biosystems 3730xl dna analyzer ( applied biosystems , foster city , ca ) .
a search of the region sequenced was performed at dbsnp ( www.ncbi.nlm.nih.gov ) to record all previously identified polymorphisms reported in the region .
genotyping of dg10s478 was performed by fragment length analysis on an abi prism dna analyzer 3700 with previously published primers ( 21 ) in a manner similar to that previously described ( 23 ) .
snp genotyping was performed on the iplex massarray genotyping platform ( sequenom , san diego , ca ) .
in preliminary analyses , snps in the tcf7l2 gene region  10 kb ( c10:114689999114926060 ) were imputed using data from hapmap phase ii hybrid panel ( 1:1 , yri : ceu ; 108 snps ) and the 1000 genomes yri pilot 1 dataset ( 497 snps ) to circumvent limited coverage of the genetic diversity in this specific region by the affymetrix 6.0 array .
snps were imputed in 965 type 2 diabetic end - stage renal disease ( esrd ) cases and 1,029 controls with high - quality score ( rsq - hat 0.3 ) using the software mach ( www.sph.umich.edu/csg/abecasis ) ( 24 ) .
four common snps identified from direct sequence analysis had minor allele frequencies ( maf ) 0.05 and failed assay design on the sequenom platform . with the use of the resequencing genotype data obtained from the 96 african american samples as reference , these snps were imputed in the remaining 2,043 samples with high - quality score ( rsq - hat 0.5 ) using the software mach ( www.sph.umich.edu/csg/abecasis ) ( 24 ) .
snps were tested for departure from hardy - weinberg equilibrium ( hwe ) using an exact test of hwe proportions for the combined group of cases and controls and then for cases only and controls only ( 25 ) .
haplotype block structure was established using haploview 4.1 ( 26 ) , defining blocks using the method from gabriel et al .
unadjusted measures of ld and association were assessed using the software snpgwa ( http://www.phs.wfubmc.edu ) ( 28 ) .
snpgwa computes ld statistics , d and r , for each pair of tandem snps .
snpgwa also performs multiple tests of association including the overall two - degree of freedom test ( genotype ) , dominant model , recessive model , additive model ( cochran - armitage trend test ) , and the corresponding lack of fit to the additive model .
odds ratios , 95% confidence intervals , and p values were computed for each model of association .
assuming a log additive model , x = ( 1  f ) + 2f(1  f) + f where  is the estimated odds ratio ( or ) and f is the risk allele frequency .
ancestry estimates were determined from 70 biallelic admixture informative markers ( aims ) as previously described ( 16,29 ) .
briefly , aims were selected to maximize european and african allele frequency differences and sample all non - acrocentric arms of the autosomal genome .
reference population allele frequencies were derived by genotyping 44 african ( yoruba from ibadan , nigeria ) and 39 european americans .
individual ancestral proportions were generated for each subject using frappe ( 30 ) , an expectation - maximization algorithm , under a two - population model .
to test whether snps were independently associated with type 2 diabetes , we performed an omnibus test for the haplotype association using plink ( 31 ) .
we further adjusted the omnibus test by controlling for one of the snps at a time .
an insignificant conditional test suggests that the conditioned snp is sufficient to explain the haplotype association and there is a single , rather than multiple , association signals at the haplotype .
controls were significantly younger than type 2 diabetic cases ( p < 0.0001 ) , although they were significantly older than the mean age at type 2 diabetes diagnosis in the cases ( p < 0.0001 ) .
similar proportions of women were present in cases and controls ( 61% and 58% , respectively ) .
frappe ( 30 ) analysis of aim genotypes estimated the mean proportion of african ancestry overall was 0.79  0.12 and differed significantly ( p < 0.0001 ) between type 2 diabetic cases and controls ( 0.80  0.12 and 0.78  0.12 , respectively ) .
characteristics of african american study participants * number with data available . a preliminary analysis assessed association with type 2 diabetes for 59 snps across the entire 216-kb tcf7l2 gene
10 kb from the affymetrix 6.0 array - based analysis of 965 type 2 diabetic esrd cases and 1,029 controls ( fig .
1 ) . with the use of the tagger program of haploview ( 26 ) ,
43 of the 59 snps from the affy array were available in the hapmap yri dataset and captured common variation at 121 snps ( maf > 0.05 ; aggressive tagging algorithm ) with a mean r = 0.73 .
notably , the snp of greatest interest , rs7903146 , is not typed on the affymetrix 6.0 array .
this snp is located in a genomic interval that is not tagged well ( max r = 0.45 ) , resulting in only nominal evidence of association in the affymetrix 6.0 analysis . to circumvent limited coverage of the genetic diversity in this specific region ,
imputation was used . using data from hapmap phase ii hybrid panel ( 1:1 , yri : ceu ) ,
no variants were identified with the same magnitude of significance as rs7903146 . in a separate analysis ,
497 snps were imputed from the 1000 genomes yri pilot 1 dataset ( supplementary fig .
two variants were identified ( rs33998771 , chr10:114740378 ) proximal to our region with significant p values ( 3.58  10 and 3.29  10 , respectively ) similar to that of rs7903146 . to test
whether these snps ( rs33998771 , chr10:114740378 , rs7903146 ) were independently associated with type 2 diabetes , we performed an omnibus test for the haplotype association controlling for one snp at a time .
four common haplotypes ( act , att , ttt , and ttc ) accounted for 99.9% of all haplotypes .
these haplotype frequencies were significantly different between type 2 diabetic cases and controls ( 0.042 , 0.019 , 0.268 , and 0.672 , respectively for cases ; 0.020 , 0.016 , 0.229 , and 0.735 , respectively for controls , omnibus test p = 5.4  10 ) , with the haplotypes ttc and act strongly associated with protection and risk for type 2 diabetes , respectively ( p < 0.0001 ) . omnibus analysis revealed that the haplotype association was significantly reduced after adjusting for rs7903146 ( p = 0.023 ) , whereas strong association remained by conditioning rs33998771 or chr10:114740378 ( p = 0.0006 and 0.002 , respectively ) , suggesting that rs7903146 alone explained most of the haplotype association . regional association plot for tcf7l2 10 kb ( c10:114689999114926060 ) . all snps genotyped on the affy 6.0 array are plotted with their log10 p values of association with type 2 diabetes versus the genomic position ( national center for biotechnology information build 36.1 ) .
the tcf7l2 gene position was taken from the university of california , santa cruz genome browser ( green ) , and the core region of association ( c10:114744078114748339 ) analyzed by direct sequence analysis is depicted in red .
the most significantly associated snp from the array is depicted as a blue diamond with its correlated proxies ( red = r  0.80 ; orange = 0.50  r > 0.80 ) .
snp rs7903146 and microsatellite dg10s478 , depicted as gray circles , were typed independently in the same set of samples .
estimated recombination rates from hapmap are plotted in the background to depict the ld structure in the region .
( a high - quality color representation of this figure is available in the online issue . )
fourteen additional snps were genotyped in an expanded set of type 2 diabetic cases and controls within this interval and analyzed for association as summarized in table 2 .
the core region of association was between snps rs4132115 and rs7903146 ( admixture - adjusted additive p values ranging from 0.012 to 2.38  10 ) .
this region encompasses a 4.3-kb ld block in the yri population ( hapmap phase ii yri data ) , which is bounded by the two snps rs7901695 and the previously associated rs7903146 ( 16 ) .
single snp genotypic association results for snps in the tcf7l2 gene showing association with type 2 diabetes esrd * inconsistent with hwe in cases .
in addition , the previously associated ( 21 ) microsatellite marker dg10s478 , which lies 38 kb distal to and outside of this ld block , was typed , and the results of the analysis are presented in table 3 .
allele sizes and frequencies were consistent with prior genotyping in samples from african populations ( 15 ) .
evidence of association was observed with the protective alleles 4 and 16 ( p = 0.043 and 5.02  10 , respectively ) and the risk allele 8 ( p = 0.022 ) .
dg10s478 allelic association with type 2 diabetes esrd in african americans the core region of association ( c10:114744078114748339 )  2 kb was analyzed by direct sequence analysis in 96 samples ( 48 type 2 diabetic cases and 48 controls ) .
a total of 46 snps were identified , of which 72% ( 33/46 ) were novel and 17% ( 8/46 ) had a maf > 5% . when genotyped in the expanded cohort , five of the novel snps
were found to be monomorphic and 10 could not be typed on the sequenom platform because of the repetitive nature of the region .
these 10 were genotyped via direct sequence analysis on a subset of 96 type 2 diabetic cases and 96 controls .
four snps were common , and these sequencing data were paired with existing data to be used as the known set of haplotypes for imputation in the remaining cohort . in addition , rs61875120 was common but yielded poor quality imputation ( rsq = 0.31 ) and was therefore genotyped by direct sequence analysis .
the remaining five snps had low maf ( maf < 0.05 ) and were not genotyped .
of the 36 snps typed or imputed , 15 snps were found to be nominally associated with type 2 diabetes ( admixture - adjusted additive p values ranging from 0.050 to 6.32  10 ) .
the most striking associations were observed at rs34872471 , rs35198068 ( imputed ) , and rs7903146 , which were correlated ( r > 0.74 ; fig .
2 ) and associated with disease susceptibility ( or = 1.301.37 ) under an additive model .
three common haplotypes ( cct , ccc , and ttc ) accounted for 99.6% of all haplotypes .
these haplotype frequencies were significantly different between type 2 diabetic cases and controls ( 0.342 , 0.056 , and 0.602 , respectively for cases ; 0.278 , 0.058 , and 0.664 , respectively for controls , omnibus test p = 3.7  10 ) .
omnibus analysis revealed that the haplotype association was lost after adjusting for rs7903146 ( p = 0.85 ) , whereas modest association remained by conditioning rs34872471 or rs35198068 ( p = 0.05 ) , suggesting that rs7903146 alone is sufficient to explain the overall haplotype association .
single snp genotypic association results for snps identified by direct sequence analysis in the tcf7l2 gene showing association with type 2 diabetes esrd * low maf in 96 samples resulting in poor imputation .
. a : haploview - generated ld map of the 40 snps identified by direct sequence analysis ( c10:114742846114750274 ) in african american controls ( n = 1,106 ) .
regions of high ld ( d = 1 and logarithm of the odds [ lod ] > 2 ) are shown in dark red .
markers with lower ld ( 0.45 < d < 1 and lod > 2 ) are shown in dark through light red , with the color intensity decreasing with decreasing d value .
regions of low ld and low lod scores ( lod < 2 ) are shown in white .
b : haploview - generated ld map of the 17 common snps ( maf > 0.05 ) identified by direct sequence analysis ( c10:114742846114750274 ) in african american controls ( n = 1,106 ) . regions of high ld ( d = 1 and lod > 2 ) are shown in dark red .
markers with lower ld ( 0.45 < d < 1 and lod > 2 ) are shown in light red , with the color intensity decreasing with decreasing d value .
regions of low ld and low lod scores ( lod < 2 ) are shown in white .
( a high - quality color representation of this figure is available in the online issue . )
controls were significantly younger than type 2 diabetic cases ( p < 0.0001 ) , although they were significantly older than the mean age at type 2 diabetes diagnosis in the cases ( p < 0.0001 ) .
similar proportions of women were present in cases and controls ( 61% and 58% , respectively ) .
frappe ( 30 ) analysis of aim genotypes estimated the mean proportion of african ancestry overall was 0.79  0.12 and differed significantly ( p < 0.0001 ) between type 2 diabetic cases and controls ( 0.80  0.12 and 0.78  0.12 , respectively ) .
a preliminary analysis assessed association with type 2 diabetes for 59 snps across the entire 216-kb tcf7l2 gene 10 kb from the affymetrix 6.0 array - based analysis of 965 type 2 diabetic esrd cases and 1,029 controls ( fig .
1 ) . with the use of the tagger program of haploview ( 26 ) ,
43 of the 59 snps from the affy array were available in the hapmap yri dataset and captured common variation at 121 snps ( maf > 0.05 ; aggressive tagging algorithm ) with a mean r = 0.73 .
notably , the snp of greatest interest , rs7903146 , is not typed on the affymetrix 6.0 array .
this snp is located in a genomic interval that is not tagged well ( max r = 0.45 ) , resulting in only nominal evidence of association in the affymetrix 6.0 analysis . to circumvent limited coverage of the genetic diversity in this specific region ,
imputation was used . using data from hapmap phase ii hybrid panel ( 1:1 , yri : ceu ) , 108
no variants were identified with the same magnitude of significance as rs7903146 . in a separate analysis ,
497 snps were imputed from the 1000 genomes yri pilot 1 dataset ( supplementary fig .
two variants were identified ( rs33998771 , chr10:114740378 ) proximal to our region with significant p values ( 3.58  10 and 3.29  10 , respectively ) similar to that of rs7903146 . to test
whether these snps ( rs33998771 , chr10:114740378 , rs7903146 ) were independently associated with type 2 diabetes , we performed an omnibus test for the haplotype association controlling for one snp at a time .
four common haplotypes ( act , att , ttt , and ttc ) accounted for 99.9% of all haplotypes .
these haplotype frequencies were significantly different between type 2 diabetic cases and controls ( 0.042 , 0.019 , 0.268 , and 0.672 , respectively for cases ; 0.020 , 0.016 , 0.229 , and 0.735 , respectively for controls , omnibus test p = 5.4  10 ) , with the haplotypes ttc and act strongly associated with protection and risk for type 2 diabetes , respectively ( p < 0.0001 ) .
omnibus analysis revealed that the haplotype association was significantly reduced after adjusting for rs7903146 ( p = 0.023 ) , whereas strong association remained by conditioning rs33998771 or chr10:114740378 ( p = 0.0006 and 0.002 , respectively ) , suggesting that rs7903146 alone explained most of the haplotype association . regional association plot for tcf7l2 10 kb ( c10:114689999114926060 ) .
all snps genotyped on the affy 6.0 array are plotted with their log10 p values of association with type 2 diabetes versus the genomic position ( national center for biotechnology information build 36.1 ) .
the tcf7l2 gene position was taken from the university of california , santa cruz genome browser ( green ) , and the core region of association ( c10:114744078114748339 ) analyzed by direct sequence analysis is depicted in red .
the most significantly associated snp from the array is depicted as a blue diamond with its correlated proxies ( red = r  0.80 ; orange = 0.50  r > 0.80 ) .
snp rs7903146 and microsatellite dg10s478 , depicted as gray circles , were typed independently in the same set of samples .
estimated recombination rates from hapmap are plotted in the background to depict the ld structure in the region .
( a high - quality color representation of this figure is available in the online issue . )
fourteen additional snps were genotyped in an expanded set of type 2 diabetic cases and controls within this interval and analyzed for association as summarized in table 2 .
the core region of association was between snps rs4132115 and rs7903146 ( admixture - adjusted additive p values ranging from 0.012 to 2.38  10 ) .
this region encompasses a 4.3-kb ld block in the yri population ( hapmap phase ii yri data ) , which is bounded by the two snps rs7901695 and the previously associated rs7903146 ( 16 ) .
single snp genotypic association results for snps in the tcf7l2 gene showing association with type 2 diabetes esrd * inconsistent with hwe in cases .
in addition , the previously associated ( 21 ) microsatellite marker dg10s478 , which lies 38 kb distal to and outside of this ld block , was typed , and the results of the analysis are presented in table 3 .
allele sizes and frequencies were consistent with prior genotyping in samples from african populations ( 15 ) .
evidence of association was observed with the protective alleles 4 and 16 ( p = 0.043 and 5.02  10 , respectively ) and the risk allele 8 ( p = 0.022 ) .
the core region of association ( c10:114744078114748339 )  2 kb was analyzed by direct sequence analysis in 96 samples ( 48 type 2 diabetic cases and 48 controls ) .
a total of 46 snps were identified , of which 72% ( 33/46 ) were novel and 17% ( 8/46 ) had a maf > 5% . when genotyped in the expanded cohort , five of the novel snps
were found to be monomorphic and 10 could not be typed on the sequenom platform because of the repetitive nature of the region .
these 10 were genotyped via direct sequence analysis on a subset of 96 type 2 diabetic cases and 96 controls .
four snps were common , and these sequencing data were paired with existing data to be used as the known set of haplotypes for imputation in the remaining cohort . in addition , rs61875120 was common but yielded poor quality imputation ( rsq = 0.31 ) and was therefore genotyped by direct sequence analysis .
the remaining five snps had low maf ( maf < 0.05 ) and were not genotyped .
of the 36 snps typed or imputed , 15 snps were found to be nominally associated with type 2 diabetes ( admixture - adjusted additive p values ranging from 0.050 to 6.32  10 ) .
the most striking associations were observed at rs34872471 , rs35198068 ( imputed ) , and rs7903146 , which were correlated ( r > 0.74 ; fig .
2 ) and associated with disease susceptibility ( or = 1.301.37 ) under an additive model .
three common haplotypes ( cct , ccc , and ttc ) accounted for 99.6% of all haplotypes .
these haplotype frequencies were significantly different between type 2 diabetic cases and controls ( 0.342 , 0.056 , and 0.602 , respectively for cases ; 0.278 , 0.058 , and 0.664 , respectively for controls , omnibus test p = 3.7  10 ) .
omnibus analysis revealed that the haplotype association was lost after adjusting for rs7903146 ( p = 0.85 ) , whereas modest association remained by conditioning rs34872471 or rs35198068 ( p = 0.05 ) , suggesting that rs7903146 alone is sufficient to explain the overall haplotype association .
single snp genotypic association results for snps identified by direct sequence analysis in the tcf7l2 gene showing association with type 2 diabetes esrd * low maf in 96 samples resulting in poor imputation .
. a : haploview - generated ld map of the 40 snps identified by direct sequence analysis ( c10:114742846114750274 ) in african american controls ( n = 1,106 ) .
regions of high ld ( d = 1 and logarithm of the odds [ lod ] >
markers with lower ld ( 0.45 < d < 1 and lod > 2 ) are shown in dark through light red , with the color intensity decreasing with decreasing d value .
regions of low ld and low lod scores ( lod < 2 ) are shown in white .
b : haploview - generated ld map of the 17 common snps ( maf > 0.05 ) identified by direct sequence analysis ( c10:114742846114750274 ) in african american controls ( n = 1,106 ) .
regions of high ld ( d = 1 and lod > 2 ) are shown in dark red .
markers with lower ld ( 0.45 < d < 1 and lod > 2 ) are shown in light red , with the color intensity decreasing with decreasing d value .
regions of low ld and low lod scores ( lod < 2 ) are shown in white .
( a high - quality color representation of this figure is available in the online issue . )
this study illustrates the power of genetic analyses in african - derived populations to facilitate identification of trait - defining variants .
tcf7l2 has been identified as one of the strongest type 2 diabetes susceptibility genes to date with associations across multiple ethnically diverse populations ( 12,15,16,21,32 ) .
our study is consistent with the initial association of snp rs7903146 in an african american type 2 diabetic case - control population . by taking advantage of reduced ld in the african american population
this 4.3-kb region , flanked by snps rs4132115 and rs7903146 , was the focus of resequencing in an effort to infer which sequence variant(s ) are causally associated with type 2 diabetes .
of the 46 snps identified by resequencing , 15 snps were nominally associated with type 2 diabetes with the most significant associations observed at rs34872471 , rs35198068 ( imputed ) , and rs7903146 , which were highly correlated ( r > 0.74 ; fig . 1 ) and associated with disease susceptibility ( or = 1.301.37 ) .
this analysis suggests that association at rs34872471 and rs35198068 was the result of correlation with the true signal from rs7903146 .
although this study has eliminated the possibility of additional common variants ( maf > 0.01 ) contributing to type 2 diabetes susceptibility within the fine - mapped interval ( c10:114744078114748339  2 kb ) of the tcf7l2 locus , four variants ( ivs3 + 42245 , ivs3 + 42428 , ivs3 + 43487 , and ivs4 43007 ) were found to have maf < 0.01 .
to date these variants have not been identified by other ongoing studies , i.e. , the 1000 genomes project , suggesting they are private mutations .
additionally , given the low maf , these variants are not likely to explain the association observed at the tcf7l2 locus , but we can not rule out the possibility that these and other unidentified rare variants contribute to disease susceptibility .
if this were so , effect sizes of such rare variants would have to be in a range unprecedented for noncoding variants . as a result of fine - mapping the tcf7l2 locus to determine the region most likely to harbor susceptibility variants ,
dg10s478 is located 41 kb proximal to the critical interval defined in the african american population and is in weak ld with rs7903146 ( d = 0.35 , r = 0.07 ) .
only a single common allele of dg10s478 is nominally associated with type 2 diabetes , with the strongest association , which is protective , seen with low maf variants .
this study represents the first comprehensive evaluation of variation within the tcf7l2 gene in a large african american population . taking advantage of the ld structure in our african - derived sample of african americans
, we were able to reduce the genomic interval of association to 4.3 kb and exclude the possible contribution of the previously identified microsatellite marker to type 2 diabetes susceptibility .
our analysis identified three snps , rs34872471 , rs35198068 ( imputed ) , and rs7903146 , which were highly associated with type 2 diabetes ; all had p values that were two orders of magnitude stronger than other snps .
snp rs7903146 remains the most significantly associated variant within the tcf7l2 gene with a calculated par of 17.4% .
we can not exclude this possibility with total confidence , but the assessment of markers in tcf7l2 by direct genotyping , imputation , and then through the use of the 1000 genomes data using conditional analysis suggests the likelihood that such a common variant exists is low .
evaluation of long range ld on chromosome 10 shows little evidence for a remote variant ( data not shown ) .
an alternative is the possibility that a rare variant of large effect in ld with rs7903146 is the actual functional variant .
although theoretically possible ( 33 ) , we have recently shown empirically that it is easy to differentiate between a rare functional variant with large effect and a common variant in ld ( 34 ) . thus , fine - mapping at the tcf7l2 locus using an african ancestry population has statistically implicated rs7903146 as the causal variant .
( 20 ) have implicated rs7903146 as a functional variant by mapping sequence variants to open chromatin sites .
they found that rs7903146 is located in islet - selective open chromatin , and human islet samples heterozygous for rs7903146 showed allelic imbalance in islet enhancer activity .
thus genetic and functional studies make a consistent case for a functional role for rs7903146 .
 objectivevariation in the transcription factor 7-like 2 ( tcf7l2 ) locus is associated with type 2 diabetes across multiple ethnicities . 
 the aim of this study was to elucidate which variant in tcf7l2 confers diabetes susceptibility in african americans.research design and methodsthrough the evaluation of tagging single nucleotide polymorphisms ( snps ) , type 2 diabetes susceptibility was limited to a 4.3-kb interval , which contains the yri ( african ) linkage disequilibrium ( ld ) block containing rs7903146 . to better define the relationship between type 2 diabetes risk and genetic variation we resequenced this 4.3-kb region in 96 african american dnas . 
 thirty - three novel and 13 known snps were identified : 20 with minor allele frequencies ( maf ) > 0.05 and 12 with maf > 0.10 . 
 these polymorphisms and the previously identified dg10s478 microsatellite were evaluated in african american type 2 diabetic cases ( n = 1,033 ) and controls ( n = 1,106).resultsvariants identified from direct sequencing and databases were genotyped or imputed . 
 fifteen snps showed association with type 2 diabetes ( p < 0.05 ) with rs7903146 being the most significant ( p = 6.32  106 ) . 
 results of imputation , haplotype , and conditional analysis of snps were consistent with rs7903146 being the trait - defining snp . 
 analysis of the dg10s478 microsatellite , which is outside the 4.3-kb ld block , revealed consistent association of risk allele 8 with type 2 diabetes ( odds ratio [ or ] = 1.33 ; p = 0.022 ) as reported in european populations ; however , allele 16 ( maf = 0.016 cases and 0.032 controls ) was strongly associated with reduced risk ( or = 0.39 ; p = 5.02  105 ) in contrast with previous studies.conclusionsin african americans , these observations suggest that rs7903146 is the trait - defining polymorphism associated with type 2 diabetes risk . 
 collectively , these results support ethnic differences in type 2 diabetes associations . 
RESEARCH DESIGN AND METHODS
Study subjects.
Sequencing.
Genotyping.
Imputation.
Analysis.
Conditional haplotype analysis.
RESULTS
Population characteristics.
Refinement of the type 2 diabetes associated genomic interval.
Direct sequence analysis and association analysis of the associated genomic interval.
DISCUSSION