Inconsistent reproducibility has been a vexing problem for genetic association studies in complex diseases.6,7,21
False positive reports of association, false negative attempts at replication, and genetic heterogeneity often complicate the picture, and thus a true genetic association usually emerges only after carefully conducted, large-scale association studies confirm the original report.8
A limited number of common genetic variants meet that high standard in type 2 diabetes.22
In most cases, the genotypic risk is modest (1.15 to 1.25), requiring very large sample sizes for detection. The recent identification of a common allele in the TCF7L2
gene that increases the risk of type 2 diabetes by approximately 1.45 in heterozygotes and 2.41 in homozygotes4
is therefore quite provocative. Despite these significant results in a cross-sectional study,4
it was essential to replicate this genetic association in other cohorts and to do so prospectively. In addition, our evaluation of a potential mechanism by which the risk of diabetes is increased and the determination of whether these variants cause differential responses to validated preventive strategies represent important next steps in exploring the association.
The DPP is a unique study in which to carry out such analyses. The large sample size and the cohort of several races and ethnic groups, reflecting the diversity of the U.S. population with type 2 diabetes, make it possible to test the role of genetic variants in different races or ethnic groups, even if they confer only modest risk. The DPP study is different from other large observational studies23
because of its interventional design and exclusive enrollment of overweight or obese persons with elevated fasting plasma glucose concentrations and impaired glucose tolerance, which indicate a high risk of diabetes at baseline.
Our data indicate that the risk alleles in rs7903146 and rs12255372 predict the risk of diabetes prospectively, beyond that conferred by the clinical risk factors reflected by the DPP eligibility criteria. The genotypic relative risk may differ slightly from the odds ratio documented by Grant et al.4
owing to a different study design, an overestimate of the initial finding,6
population heterogeneity in the DPP, various degrees of linkage disequilibrium between the DG10S478 microsatellite and the single-nucleotide polymorphisms evaluated here, or our limited temporal window (three years on average) for the clinical transition from impaired glucose tolerance to diabetes. The size of the effect seems robust for a sample of this size; given the higher probability conferred by the initial report, our finding is a strong confirmation of the original genetic association.
Grant and coworkers4
exhaustively assessed coding variation in whites by a variety of deep re-sequencing methods in the region, suggesting that other functional variants in this gene are unlikely to have been missed. In addition to rs12255372 and rs7903146, other single-nucleotide polymorphisms in linkage disequilibrium with them were also strongly associated with diabetes. Which of these single-nucleotide polymorphisms is responsible for the observed association requires further study in adequately powered samples. In particular, the absence of linkage disequilibrium between rs7903146 and rs12255372 in African Americans may help distinguish whether one of the two single-nucleotide polymorphisms (or the haplotype formed by the risk alleles at both loci) is the sole source of the association signal; we could not make this distinction after initial exploratory analyses in our data set, perhaps because of inadequate sample size. Given their allele frequencies and assuming an overall genotypic relative risk of 1.54 () and diabetes prevalence of 10 percent among African Americans, we estimate that the enrollment of approximately 1400 persons of African ancestry would be necessary for the case–control study to have 80 percent power (with a P value of less than 0.05 considered to indicate statistical significance) to distinguish between rs7903146 and rs12255372 as the source of the association. Sample sizes at least twice as large would be needed if one intended to detect a signal arising solely from the haplotype formed by the minor alleles at both loci.
We studied the TCF7L2
single-nucleotide polymorphisms in nonwhite populations. Subgroup analysis according to race or ethnic group showed similar effect sizes at this locus, but these effect sizes were not individually significant, possibly because of inadequate sample size. However, we cannot rule out effects of genotype on risk that were specific to race or ethnic group; with the results we have obtained here, a cohort of more than 20,000 persons would be needed to detect an interaction between genotype and African-American ethnicity, at a nominal (unadjusted) P value of less than 0.05. Similarly, our sample size may not have been sufficient to detect a significant effect of the heterozygous genotype on the risk of diabetes. Nevertheless, the results of the report by Grant et al.4
and our findings of a specific effect of a single copy of the T allele on quantitative glycemic traits suggest that heterozygosity at this locus may have phenotypic consequences.
The original study speculated that genetic variation in TCF7L2
might impair the expression of glucagon-like peptide 1 in enteroendocrine cells, possibly by interfering with β-catenin–mediated transcriptional activation of its gene GCG
Our finding that insulin secretion is decreased in carriers of the risk-conferring genotype, which is consistent with an increased incidence of diabetes, lends indirect support to this model. However, it is not readily apparent how rs12255372 and rs7903146, which lie in short interspersed repeat elements approximately 41 kb upstream and 9 kb downstream, respectively, of exon 4 in TCF7L2,
might affect TCF7L2
expression or the function of its protein product. Fine mapping of the association signal and directed functional studies should help determine the molecular consequences of genetic variation at this locus.
The enhanced insulin sensitivity (and the lower BMI and smaller waist circumference) in carriers of the T allele at both single-nucleotide polymorphisms was unexpected and may be an artifact of our requirement that patients have high-risk characteristics for diabetes at enrollment. Specifically, if the T allele leads to decreased insulin secretion and a higher risk of diabetes, subjects with additional insulin resistance would be more likely to have diabetes at baseline (all other factors being equal), precluding their enrollment in this trial. Conversely, at-risk participants carrying the T allele may not have had diabetes at the time of enrollment because of enhanced insulin sensitivity owing to other genetic or environmental factors. In either case, our results strongly suggest that these variants do not cause insulin resistance in persons with impaired glucose tolerance and support the notion that variants in TCF7L2
lead to diabetes by means of defects in insulin secretion. Like KCNJ11, TCF7L2
seems to be a diabetes-associated gene in which common polymorphisms primarily affect the beta cell.23,25,26
Finally, we did not detect significant interactions between genotypes at either single-nucleotide polymorphism and the DPP interventions. The absence of an effect may not be surprising, since these interventions succeeded primarily by improving insulin sensitivity17
and these variants affect insulin secretion. However, we did not observe any effect of genotype at these loci in the lifestyle-intervention group, raising the possibility that a behavioral intervention can mitigate the risk conferred by genetic background. Conversely, the intervention groups may have been underpowered for these analyses. Whether the response to drugs designed to improve insulin secretion (e.g., sulfonylureas, meglitinides, or incretins) will be affected by these common variants requires specific testing in pharmacogenetic trials.
In summary, our results from this large prospective study confirm and extend the finding that the transcription factor gene TCF7L2 is associated with susceptibility to type 2 diabetes. Further understanding of the mechanisms by which variation in this gene affects glucose homeostasis may provide new insights into the molecular basis of diabetes and opportunities for more targeted interventions for prevention and therapy.