DNA methylation levels at individual age-associated CpG sites can be indicative for life expectancy

Abstract

DNA-methylation (DNAm) levels at age-associated CpG sites can be combined into epigenetic aging signatures to estimate donor age. It has been demonstrated that the difference between such epigenetic age-predictions and chronological age is indicative for of all-cause mortality in later life. In this study, we tested alternative epigenetic signatures and followed the hypothesis that even individual age-associated CpG sites might be indicative for life-expectancy. Using a 99-CpG aging model, a five-year higher age-prediction was associated with 11% greater mortality risk in DNAm profiles of the Lothian Birth Cohort 1921 study. However, models based on three CpGs, or even individual CpGs, generally revealed very high offsets in age-predictions if applied to independent microarray datasets. On the other hand, we demonstrate that DNAm levels at several individual age-associated CpGs seem to be associated with life expectancy - e.g., at CpGs associated with the genesPDE4C and CLCN6. Our results support the notion that small aging signatures should rather be analysed by more quantitative methods, such as site-specific pyrosequencing, as the precision of age-predictions is rather low on independent microarray datasets. Nevertheless, the results hold the perspective that simple epigenetic biomarkers, based on few or individual age-associated CpGs, could assist the estimation of biological age.

Keywords: CLCN6; DNA-methylation; PDE4C; age; aging; epigenetic; mortality; prediction; predictor; survival.

Figure 1. Epigenetic age-predictions correlate with mortality

(A) Scheme for the study design. Epigenetic age was estimated based on our previously published models with 99 CpGs, 3 CpGs, or individual CpGs thereof [8]. The offset of predicted age and chronological age was subsequently correlated with all-cause mortality in the Lothian Birth Cohort 1921 (LBC1921) and LBC1936. (B) The 99-CpG model and (C) the 3-CpG model were specifically trained for Illumina HumanMethylation450 BeadChip data using Hannum dataset (GSE40279) and subsequently validated using additional 12 DNAm datasets of blood samples. (D) The 99-CpG model was then applied on DNAm profiles of LBC1921 and the deviation of predicted and chronological age (Δ_age) was determined for each sample. Samples in the lowest (Q1) and highest quartiles (Q4) of Δ_age are depicted in navy and red, respectively. (E) Kaplan-Meier plots (K-M) of LBC1921 participants classified by respective quartiles indicate lower mortality for those with increased Δ_age.. (F, G) In analogy the same analysis was performed for the 3-CpG model, but there was no significant association between these age-predictions and mortality. (H, I) Alternatively, we tested association of DNAm at individual age-associated CpGs with mortality. To this end, linear models were trained for each CpG site using Hannum dataset and then applied to the LBC1921 cohort. cg17861230 (associated with PDE4C) reveals a significant association with mortality. The calculated Δ_age is subject to survival analysis with adjustment for chorological age and gender.

Figure 2. A CpG site in CLCN6 is indicative for survival in LBC1921 and LBC1936

(A) A CpG site associated with the gene for the chloride transport protein 6 (CLCN6; cg05228408) was used to estimate age in the LBC1921 cohort. (B) Participants in the lowest and highest quartiles of Δ_age were subsequently analysed in Kaplan-Meier plots (K-M). Hazard ratio and cox-regression analysis of survival were subsequently tested with adjustment for chorological age and gender. (C, D) In analogy, the same analysis was performed for this CpG site using the LBC1936 cohort.