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Updated: May 20, 2026

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
Temporal stability of epigenetic markers: sequence characteristics and predictors of short-term DNA methylation
Hyang-Min Byun1, Francesco Nordio, Brent A Coull
1Laboratory of Environmental Epigenetics, Exposure Epidemiology and Risk Program, Harvard School of Public Health, Boston, Massachusetts, United States of America. hmbyun@hsph.harvard.edu
Background:
DNA methylation is an epigenetic mechanism that has been increasingly investigated in observational human studies, particularly on blood leukocyte DNA. Characterizing the degree and determinants of DNA methylation stability can provide critical information for the design and conduction of human epigenetic studies.
Methods:
We measured DNA methylation in 12 gene-promoter regions (APC, p16, p53, RASSF1A, CDH13, eNOS, ET-1, IFNγ, IL-6, TNFα, iNOS, and hTERT) and 2 of non-long terminal repeat elements, i.e., L1 and Alu in blood samples obtained from 63 healthy individuals at baseline (Day 1) and after three days (Day 4). DNA methylation was measured by bisulfite-PCR-Pyrosequencing. We calculated intraclass correlation coefficients (ICCs) to measure the within-individual stability of DNA methylation between Day 1 and 4, subtracted of pyrosequencing error and adjusted for multiple covariates.
Results:
Methylation markers showed different temporal behaviors ranging from high (IL-6, ICC = 0.89) to low stability (APC, ICC = 0.08) between Day 1 and 4. Multiple sequence and marker characteristics were associated with the degree of variation. Density of CpG dinucleotides nearby the sequence analyzed (measured as CpG(o/e) or G+C content within ±200 bp) was positively associated with DNA methylation stability. The 3' proximity to repeat elements and range of DNA methylation on Day 1 were also positively associated with methylation stability. An inverted U-shaped correlation was observed between mean DNA methylation on Day 1 and stability.
Conclusions:
The degree of short-term DNA methylation stability is marker-dependent and associated with sequence characteristics and methylation levels.
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