Genome-wide association studies of HbA1c levels in cohorts of white individuals of European ancestry revealed a combination of glycemic and non-glycemic biological influences on HbA1c, with three loci associated with HbA1c in or near genes likely involved in glycemic control pathways and seven loci associated with HbA1c in or near genes likely to be involved in erythrocyte biology . In this study we found that in the nationally representative NHANES III sample of US adults, heterogeneity in risk allele frequencies exists across race-ethnic groups for six of these HbA1c-associated SNPs. Five SNP risk allele frequencies in NHB were significantly lower or higher than the other two groups. Risk allele frequencies observed in NHANES III were generally consistent with frequencies of comparable populations available in HapMap, suggesting that HapMap and NHANES III can be considered representative of each other at these SNPs at least with respect to white, African American and Mexican American race-ethnic populations. An 11-HbA1c- associated SNP genotype score was subtly different by race-ethnicity and was associated with increase in HbA1c in NHW and MA but not NHB. The 11-SNP genotype score was not significantly associated with mortality in any group.
There are several potential sources for the inter-race-ethnic heterogeneity of SNP and genotype risk score associations with HbA1c that we observed. One potential source of heterogeneity is race-specific selection acting on erythrocyte-related loci that influence HbA1c. Variants in the β- hemoglobin gene (HBB), for example, produce abnormal erythrocytes that can affect HbA1c levels  but are protective against malaria and are thus maintained in populations and found at highest frequencies in regions historically exposed to this disease like Africa and India . Rare mutations in many loci associated with HbA1c (SPTA1
TMPRSS6) are known to cause hereditary red blood cell disorders  and common variants at several loci (SPTA1, HFE, ANK1
TMPRSS6) are associated with hematological traits like hemoglobin concentration and mean corpuscular volume [32–34]. Adjustment of models of these common variants predicting HbA1c levels for levels of hemoglobin concentration or mean corpuscular volume attenuate SNP-HbA1c relationships, suggesting mediation of HbA1c varation by elements of erythrocyte biology . Further, a recent genetic association study showed some differences in the genetic regulation of hematological traits in Europeans compared with Africans . Our analyses of differentiation and selection suggest that there may be some selection pressure at the ANK1
ATP11A, TMPRSS6 and ABC11/G6PC2 loci, the first four of which are erythrocyte-related loci. However, in the present study, race-ethnic differences in association with HbA1c by SNP were observed at only two of these loci (ANK1 [rs4737009] and TMPRSS6). We also examined inter-population allele frequency differences of trait-associated SNPs which may indicate that selection is operating on the trait . While frequencies of some disease-associated alleles have been reported as largely heterogeneous between race-ethnicities [36–39], other data suggest no greater differentiation than would be expected from a random set of SNPs . We found heterogeneous inter-race-ethnic risk allele frequencies at six of the HbA1c-associated SNPs and three of these (SNPs near ANK1 [both SNPs] and TMPRSS6) showed inter-race heterogeneity in SNP association with HbA1c.
We found modest race-ethnic differences in the association of individual or collective HbA1c-associated SNPs and levels of HbA1c. We found nominally significant associations with an HbA1c-associated SNP genotype score and levels of HbA1c in NHW, as expected, and also in MA, but not in NHB individuals. Ancestral variation in LD probably accounts for some of this difference in association. LD is more fine-grained in genomes of African individuals , so some of the HbA1c-associated SNPs may be more tightly linked to putative functional alleles in NHW and MA than in NHB. Modest power given the relatively small sample size of NHANES III could also account for the relatively weak association of HbA1c SNPs with HbA1c in each race-ethnic group ( Additional file 1: Table S3). No significant interactions were observed, also possibly due to low power. T2D diagnosis was based on fasting glucose with no OGTT, which may have introduced misclassification in T2D status of study subjects. Furthermore, greater heterogeneity exists in NHB, and this heterogeneity may have influenced variability in HbA1c levels. Since there are no ancestry markers available in NHANES to evaluate genetic heterogeneity within populations, we were unable to evaluate substructure within ethnic groups and, for the purposes of this study, assumed little to no intra-population substructure.
Despite previous epidemiological associations of HbA1c levels with mortality or cardiovascular disease [12–19] and race-ethnic variation in mortality rates in NHANES III, we did not see any evidence of an association of HbA1c-associated loci with mortality in any race-ethnic group. If HbA1c is associated with mortality, it is likely to be mediated through HbA1c’s association with hyperglycemia and insulin resistance, but many HbA1c-associated loci are associated with erythrocyte biology and not hyperglycemia. A lack of association of the HbA1c-associated SNPs studied here and cardiovascular disease events has also been shown previously in white cohorts . This unlinking of hyperglycemia from HbA1c biology also has bearing on diabetes screening and diagnosis. Another explanation for a lack of association of the HbA1c genetic risk score with mortality is the lack of statistical power due to small sample size within each ethnicity ( Additional file 1: Table S5). When pooling the entire sample and carrying out an interaction model we also observed no significant genetic risk score x ethnicity interaction on mortality.
Race-ethnic differences in HbA1c levels were observed in the present study and have been shown previously [41–46]. Population differences in HbA1c levels are partly attributable to variability in non-biological factors including race-ethnic differences in lifestyle, socioeconomics, health insurance access or screening intensity [41, 44]. Further, there are likely race ethnic differences in non-glycemic biological factors including glycemic level, hemoglobinopathies [30, 47–49], iron deficiency anemias [21, 48, 50–54], and erythrocyte survival [48, 55, 56]. The data suggest that glycemic control is not the only root cause of inter-race-ethnic differences in HbA1c. Although the clinical impact of HbA1c genetics on diabetes detection appears to be modest in whites, at least , whether race-ethnic heterogeneity in HbA1c genetics influences diabetes diagnosis in other race-ethnic groups requires further investigation.
The major strengths of this study include genotyping of all 11 known HbA1c-associated SNPs in the nationally representative, multi-race-ethnic NHANES III cohort. The heterogeneity of HbA1c–associated SNP frequencies across race-ethnic groups and the limited impact of these SNPs on HbA1c level in NHB individuals underscore the importance of extending association studies and the discovery of causal variants to diverse populations for a comprehensive understanding of HbA1c genetic architecture. As diverse populations become increasingly incorporated into genetic studies for variant detection, inter-race-ethnic variation will likely continue to be revealed, necessitating careful investigation of its sources and significance.