Cross-sectional and longitudinal changes in DNA methylation with age: an epigenome-wide analysis revealing over 60 novel age-associated CpG sites

Abstract

Understanding the role of epigenetic modifications, e.g. DNA methylation, in the process of aging requires the characterization of methylation patterns in large cohorts. We analysed >480 000 CpG sites using Infinium HumanMethylation450 BeadChip (Illumina) in whole blood DNA of 965 participants of a population-based cohort study aged between 50 and 75 years. In an exploratory analysis in 400 individuals, 200 CpG sites with the highest Spearman correlation coefficients for the association between methylation and age were identified. Of these 200 CpGs, 162 were significantly associated with age, which was verified in an independent cohort of 498 individuals using mixed linear regression models adjusted for gender, smoking behaviour, age-related diseases and random batch effect and corrected for multiple testing by Bonferroni. In another independent cohort of 67 individuals without history of major age-related diseases and with a follow-up of 8 years, we observed a gain in methylation at 96% (52%, significant) of the positively age-associated CpGs and a loss at all (89%, significant) of the negatively age-associated CpGs in each individual while getting 8 years older. A regression model for age prediction based on 17 CpGs as predicting variables explained 71% of the variance in age with an average accuracy of 2.6 years. In comparison with cord blood samples obtained from the Ulm Birth Cohort Study, we observed a more than 2-fold change in mean methylation levels from birth to older age at 86 CpGs. We were able to identify 65 novel CpG sites with significant association of methylation with age.

Figure 1.

Distribution of Spearman correlation coefficients between age and DNA methylation levels at 480 675 CpG sites calculated in phase 1, ‘Exploratory analysis’. The distribution of correlation coefficients is shown on a larger scale in the left window for correlation coefficients below −0.25 and in the right window for correlation coefficients of >0.25.

Figure 2.

Gene ontology enrichment for genes with significantly age-related differentially methylation.

Figure 3.

Mean difference in DNA methylation between 8-year follow-up (8-y FU) and baseline determined in the longitudinal analysis (‘confirmatory analysis II’) in relation to the regression coefficient for age determined by mixed models in the cross-sectional (‘confirmatory analysis I, sensitivity analysis’) for 94 significant age-associated CpG sites.

Figure 4.

Distribution of mean methylation level for age-associated CpG sites located in CpG islands (A), in CpG shores or shelves (B) or ‘open sea’ (C). In parentheses, the proportion of CpG sites with methylation negatively associated with age is presented.

Figure 5.

Spearman correlation coefficient in relation to mean methylation level for the significant age-associated CpG sites.

Figure 6.

Methylation levels measured in newborn and in 50-, 60- and 70-year-old adults sorted by increasing methylation levels at birth. For CpG sites with large differences between the methylation levels in newborn and older adults, the gene location was provided.