Chronically elevated FPG is a primary indicator of diabetes, making it an important barometer of the progression of impaired glucose metabolism. In this paper, we attempted to replicate, in nearly 1,000 African-Americans, significant GWAS loci for FPG in populations of predominantly European ancestry. In light of well-reported increased genetic diversity in populations with African ancestry [9, 10], our replication strategy not only focused on the reported SNPs but also included querying variants in LD with the reported SNPs.
We focused our replication analysis on the MAGIC study of over 122,000 participants to identify FPG-associated SNPs shared across ethnically diverse populations. In addition, we included SNPs from prior GWASs of FPG to add breadth to our replication pool, keeping in mind that potential differences in susceptibility loci between populations may exist . Of the 12 SNPs reported by the MAGIC study that were directly testable in our African-American cohort, we replicated three SNPs within DGKB-TMEM195, TCF7L2 and SLC30A8. We also replicated SNPs in ZMAT4, which encodes a zinc finger, matrin type 4 protein identified in previous GWASs but not replicated in the MAGIC meta-analysis. Using the local (LD-based) replication strategy, we replicated additional SNPs in or near previously reported genes, including the insulin receptor substrate 1 gene.
Interestingly, comparison of the LD structure in HUFS to HapMap reference samples CEU and YRI supports the utility of African-American population samples in refining association loci. For example, the covariance matrix generated for the local replication of rs625643 spans 40 kb and includes 16 SNPs. In HapMap CEU, nearly the entire region is in moderate LD, whereas in HapMap YRI two distinct LD blocks are observed (ESM Fig. 2) and lower (on average) r
2 values are observed between rs625643 and downstream SNPs (0.78 for CEU and 0.5 for YRI). As expected, African-American samples (i.e. HapMap African Ancestry in Southwest, USA [ASW] and this study HUFS) show an LD structure intermediate to CEU and YRI (ESM Fig. 3 and ESM Fig. 2). Furthermore, given the association signals in HUFS, a case can be made for a narrowing of the region of interest from 40 kb to 3 kb between the locally replicated SNP rs671431 and the original discovery SNP (ESM Fig. 3).
We acknowledge the fluid interpretation of r
2 values within the context of establishing variants in LD with each other as well as the concern of being overly conservative in our correction for multiple comparisons. At an r
2 ≥ 0.3, we attempted to first capture a significant portion of SNPs in LD within the reference sample while maintaining confidence in the ability of related SNPs to serve as proxies . In addition, a blanket search window of 500 kb would allow for capture of some unique characteristics of LD and long-range LD associated with admixed populations such as African-Americans . To address the burden of potentially overcorrecting for multiple comparisons, our Bonferroni correction strategy was based on the estimation of effective degrees of freedom , which provides an analysis of covariance among HUFS SNPs in the extracted LD block that was based on CEU HapMap samples. We feel this approach better describes the relationship of SNPs in LD within the queried window instead of assuming the very conservative approach of independent effects for all tested SNPs.
We also acknowledge the limitation of our study of about 1,000 participants to detect some of the very small effect sizes reported by the MAGIC study, which included more than 122,000 participants. However, this study had over 80% power to replicate similar effect sizes for 10 of the 14 SNPs reported by the MAGIC study (ESM Table 3); this is evident in this study’s ability to replicate several of the published GWAS variants for FPG. We caution that lack of replication in this study may be due to limited sample size, differences in effect sizes calculated for SNPs and difference in allele frequency between populations of European and African ancestries.
The need for understanding differential susceptibility to diseases at the population level makes the identification of risk factors for diabetes and its indicators, including FPG, particularly important in the African-American community and other ethnic groups, given their disproportionate rate of morbidity and mortality from diabetes and associated complications. Unfortunately and for multiple reasons, the majority of GWASs aimed at identifying genetic variants associated with FPG and diabetes have so far focused predominantly on individuals of European ancestry. While the results from these studies provided tremendous insight into the genetic architecture of the disease, recent studies of non-European populations have shown utility in expanding the breadth of populations studied. Specifically, studies in East Asians allowed for a ‘wider net’ to be cast in the identification of type 2 diabetes susceptibility variants [4, 11]. The present study’s focus on individuals self-identified as African-Americans not only widens the net but also underscores the need for directed investigation of under-represented populations.