DNA Methylation of Five Core Circadian Genes Jointly Contributes to Glucose Metabolism: A Gene-Set Analysis in Monozygotic Twins

The timing of daily fluctuations in blood glucose is tightly controlled by the circadian rhythm. DNA methylation accompanies the circadian clock, and aberrant DNA methylation has been associated with circadian disruption and hyperglycemia. However, the precise role of circadian genes methylation in glucose metabolism is unknown. Using a gene-set approach in monozygotic (MZ) twin pairs, we examined the joint effect of 77 CpGs in five core circadian genes (CLOCK, BMAL1, PER1, PER2, PER3) on glucose-related traits in 138 middle-aged, male-male MZ twins (69 pairs). DNA methylation was quantified by bisulfite pyrosequencing. We first conducted matched twin pair analysis to examine the association of single CpG methylation with glucose metabolism. We then performed gene-based and gene-set analyses by the truncated product method to examine the combined effect of DNA methylation at multiple CpGs in a gene or all five circadian genes as a pathway on glucose metabolism. Of the 77 assayed CpGs, only one site was individually associated with insulin resistance at FDR < 0.05. However, the joint effect of DNA methylation in all five circadian genes together showed a significant association with glucose metabolism. Our results may unravel a biological mechanism through which circadian rhythm regulates blood glucose, and highlight the importance of testing the joint effect of multiple CpGs in epigenetic analysis.