Epigenetic age in blood and bones from deceased individuals: assessment of DNA methylation at the FHL2 gene by droplet digital PCR

Blood and bones are essential DNA resources in forensic contexts. We examined DNA methylation (DNAm) levels at the <i>FHL2</i> CpG site cg06639320 for age estimation purposes using droplet digital PCR (ddPCR) on DNA extracts from bones and blood samples of deceased Portuguese individuals. Blood samples from 56 individuals (11 women and 45 men, aged 24 to 91 years) and bone samples from 26 individuals (four women and 22 men, aged 26 to 80 years) were collected. The DNA was subjected to bisulphite conversion followed by ddPCR using dual-labelled probes targeting both methylated and unmethylated DNA sequences. Simple linear regression allowed us to obtain a strong correlation between DNAm levels and chronological age in blood samples (<i>R</i> = 0.789; <i>p</i> = 5.14 × 10⁻¹³), explaining 62% of age variance, as well as in bones (<i>R</i> = 0.849; <i>p</i> = 4.136 × 10⁻⁸), explaining 71% of age variance. The achieved mean absolute deviation (MAD) between chronological and predicted ages was 8.42 years (RMSE = 10.22) for blood and 6.09 years (RMSE = 7.07) for bones. Using ddPCR methodology, this study emphasizes the <i>FHL2</i> CpG site cg06639320 as well as blood and bone samples of deceased individuals for age estimation in forensic contexts.