Postmortem age estimation via DNA methylation analysis in buccal swabs from corpses in different stages of decomposition-a "proof of principle" study.

Age estimation based on the analysis of DNA methylation patterns has become a focus of forensic research within the past few years. However, there is little data available regarding postmortem DNA methylation analysis yet, and literature mainly encompasses analysis of blood from corpses without any signs of decomposition. It is not entirely clear yet which other types of specimen are suitable for postmortem epigenetic age estimation, and if advanced decomposition may affect methylation patterns of CpG sites. In living persons, buccal swabs are an easily accessible source of DNA for epigenetic age estimation. In this work, the applicability of this approach (buccal swabs as source of DNA) under different postmortem conditions was tested. Methylation levels of PDE4C were investigated in buccal swab samples collected from 73 corpses (0-90 years old; mean: 51.2) in different stages of decomposition. Moreover, buccal swab samples from 142 living individuals (0-89 years old; mean 41.2) were analysed. As expected, methylation levels exhibited a high correlation with age in living individuals (training set: r2 = 0.87, validation set: r2 = 0.85). This was also the case in postmortem samples (r2 = 0.90), independent of the state of decomposition. Only in advanced putrified cases with extremely low DNA amounts, epigenetic age estimation was not possible. In conclusion, buccal swabs are a suitable and easy to collect source for DNA methylation analysis as long as sufficient amounts of DNA are present.

Epigenetic clocks may come out of rhythm-implications for the estimation of chronological age in forensic casework.

There is a growing perception that DNA methylation may be influenced by exogenous and endogenous parameters. Knowledge of these factors is of great relevance for the interpretation of DNA-methylation data for the estimation of chronological age in forensic casework. We performed a literature review to identify parameters, which might be of relevance for the prediction of chronological age based on DNA methylation. The quality of age predictions might particularly be influenced by lifetime adversities (chronic stress, trauma/post-traumatic stress disorder (PTSD), violence, low socioeconomic status/education), cancer, obesity and related diseases, infectious diseases (especially HIV and Cytomegalovirus (CMV) infections), sex, ethnicity and exposure to toxins (alcohol, smoking, air pollution, pesticides). Such factors may alter the DNA methylation pattern and may explain the partly high deviations between epigenetic age and chronological age in single cases (despite of low mean absolute deviations) that can also be observed with "epigenetic clocks" comprising a high number of CpG sites. So far, only few publications dealing with forensic age estimation address these confounding factors. Future research should focus on the identification of further relevant confounding factors and the development of models that are "robust" against the influence of such biological factors by systematic investigations under targeted inclusion of diverse and defined cohorts.

New targeted approaches for epigenetic age predictions.

BACKGROUND: Age-associated DNA methylation changes provide a promising biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. RESULTS: In this study, we utilized 4647 Illumina BeadChip profiles of blood to select CpG sites that facilitate reliable age-predictions based on pyrosequencing. We demonstrate that the precision of DNA methylation measurements can be further increased with droplet digital PCR (ddPCR). In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower correlation between chronological age and DNA methylation at individual CpGs, while the age-predictions were overall relatively accurate. Furthermore, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often associated with a CTCF binding site. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified, but reveals a stochastic pattern. Based on this, we have developed a new approach for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. CONCLUSION: Targeted DNA methylation analysis at few age-associated CpGs by pyrosequencing, BBA-seq, and particularly ddPCR enables high precision of epigenetic age-predictions. Furthermore, we demonstrate that the stochastic evolution of age-associated DNA methylation patterns in BBA-seq data enables epigenetic clocks for individual DNA strands.