Predicting age from blood by droplet digital PCR using a set of three DNA methylation markers.

Evaluation of DNA methylation (DNAm) patterns is a promising tool for age estimation. The duplex droplet digital PCR (ddPCR) method has been recently investigated for DNAm evaluation, revealing to be a potential methodology for DNAm evaluation and molecular age estimation. In this study, we evaluated DNAm levels of CpGs located at the three age-associated genes ELOVL2, FHL2 and PDE4C using ddPCR to develop an age prediction model. Blood-derived DNA samples from 58 healthy individuals (42 women and 16 men; aged 1-93 years old) were submitted to bisulfite conversion followed by ddPCR using dual-labeled probes targeting methylated and unmethylated DNA sequences. Simple linear regression statistics revealed a strong correlation between DNAm levels and chronological age for FHL2 (R = 0.948; P = 1.472 × 10-29 ) and PDE4C (R = 0.819; P = 3.917 × 10-15 ), addressing only one CpG for each gene. For the ELOVL2 gene, evaluating five CpG sites in simultaneous, revealed a strong age correlation (R = 0.887; P = 2.099 × 10-20 ) in a simple linear regression statistics and very strong age correlation (R = 0.926; P = 2.202 × 10-25 ) when using quadratic regression statistics. The multivariable regression analysis, using methylation information captured on ELOVL2 (squared), FHL2 and PDE4C genes, revealed a very strong age correlation (R = 0.970; P = 5.356 ×10-33 ), explaining 93.7 % of age variance, displaying a mean absolute deviation (MAD) between chronological and predicted age of 4.657 years (RMSE = 6.044). We postulate that the ddPCR method should be further investigated for DNAm-based age prediction, because it is a relatively simple and an accurate method that can be routinely used in forensic laboratories for testing a few numbers of markers.