Age prediction in living: Forensic epigenetic age estimation based on blood samples

DNA methylation analysis in a variety of genes has brought promising results in age estimation. The main aim of this study was to evaluate DNA methylation levels from four age-correlated genes, ELOVL2, FHL2, EDARADD and PDE4C, in blood samples of healthy Portuguese individuals. Fifty-three samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for CpG dinucleotide methylation status. Linear regression models were used to analyze relationships between methylation levels and chronological age. The highest age-associated CpG in each locus was chosen to build a multi-locus age prediction model (APM), allowing to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 5.35 years, explaining 94.1% of age variation. Validation approaches demonstrated the accuracy and reproducibility of the proposed multi-locus APM. Testing the APM in 51 blood samples from deceased individuals a MAD of 9.72 years was obtained. Potential differences in methylation status between samples from living and deceased individuals could exist since the highest age-correlated CpGs were different in some genes between both groups. In conclusion, our study using the bisulfite PCR sequencing method is in accordance with the high age prediction accuracy of DNA methylation levels in four previously reported age-associated genes. DNA methylation pattern differences between blood samples from living and deceased individuals should be taken into account in forensic contexts.