Age estimation from DNA methylation markers has seen an exponential growth of interest, not in the least from forensic scientists. The current published assays, however, can still be improved by lowering the number of markers in the assay and by providing more accurate models to predict chronological age. From the published literature we selected 4 age-associated genes (ASPA, PDE4C, ELOVL2, and EDARADD) and determined CpG methylation levels from 206 blood samples of both deceased and living individuals (age range: 0-91 years). This data was subsequently used to compare prediction accuracy with both linear and non-linear regression models. A quadratic regression model in which the methylation levels of ELOVL2 were squared showed the highest accuracy with a Mean Absolute Deviation (MAD) between chronological age and predicted age of 3.75 years and an adjusted R 2 of 0.95. No difference in accuracy was observed for samples obtained either from living and deceased individuals or between the 2 genders. In addition, 29 teeth from different individuals (age range: 19-70 years) were analyzed using the same set of markers resulting in a MAD of 4.86 years and an adjusted R 2 of 0.74. Cross validation of the results obtained from blood samples demonstrated the robustness and reproducibility of the assay. In conclusion, the set of 4 CpG DNA methylation markers is capable of producing highly accurate age predictions for blood samples from deceased and living individuals

Anthropologic age estimation techniques are currently limited to teeth and bones and have significantly lowered accuracy when used for adult age estimation. We recently developed an epigenetic age estimation assay as an alternative forensic technique in which prediction models provided highly accurate age predictions for blood and dentin samples. Other matrices such as buccal swabs are equally relevant in forensic investigations. We demonstrate that the same set of 4 genes which we previously used for age determination of blood and dentin provides highly accurate age prediction results for buccal swabs too. A quadratic regression using DNA methylation values of specific CpG sites within the genes APSA, PDE4C, EDARADD and ELOVL2 provided age estimation accuracies with a Mean Absolute Deviation (MAD) value of 3.32 years for 50 buccal swabs.2015 Elsevier Ireland Ltd. All rights reserved.