Background: Epigenome-wide association studies (EWAS) using measurements of blood DNA methylation are performed to identify associations of methylation changes with environmental and lifestyle exposures, and with risk of developing disease. However, little is known about the variation of methylation markers in the population and their stability over time, both important factors in the design and interpretation of EWAS. Methods: We estimated the intraclass correlation coefficient (ICC) for each probe on the Illumina 450K methylation array in paired samples collected approximately six years apart from 92 participants in the Breakthrough Generations Study. We also evaluated relationships with age, reproductive and hormonal history, weight, alcohol intake and smoking. Results: Approximately 17% of probes on the 450K array had an ICC>0.50 and were considered stable variable methylated probes (stable-VMPs). Stable-VMPs were enriched for probes located in "shores" bordering CpG islands, and at approximately 1.3kb downstream from the transcription start site in the transition between the unmethylated promoter and methylated gene body. Both cross-sectional and longitudinal data analyses provided strong evidence for associations between changes in methylation levels and ageing. Smoking-related probes at 2q37.1 and AHRR were stable-VMPs and, as previously reported, related to time since quitting smoking. We also observed an excess of associations between methylation and weight changes beyond those expected by chance. Conclusion: Our results provide support for the use of WBC DNA methylation as a biomarker of exposure in EWAS. Larger studies, preferably with repeated measures over time, will be required to establish associations between specific probes and exposures.
Overall design
The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across 485,577 CpGs . Samples included each sample taken at baseline and a second blood sample taken on average 6 years later, we include 4 pairs of duplicate samples and in vitro methylated (100%) and unmethylated (0%) control DNA samples. Bisulphite converted DNA was hybridized to the Illumina Infinium 450k Human Methylation Beadchip
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