DNA Methylation-Based Forensic Age Estimation from Degraded Biological Samples
DOI:
https://doi.org/10.5281/zenodo.22760934Keywords:
DNA methylation, epigenetic age, forensic genetics, ELOVL2, pyrosequencing, degraded DNA, bisulfite sequencing, age predictionAbstract
Age-associated DNA methylation changes at a small number of CpG sites provide a molecular basis for estimating
chronological age from a biological sample, a capability of substantial forensic interest, but the bisulfite conversion
and amplification steps underlying most methylation assays are sensitive to the DNA fragmentation and chemical
damage typical of forensic evidence, and the robustness of methylation-based age estimation to this degradation
has not been systematically characterized. We selected five age-informative CpG loci within ELOVL2, FHL2,
KLF14, TRIM59 and EDARADD, previously validated in blood, and developed short bisulfite amplicon assays
(59-91 bp post-conversion) designed specifically to remain amplifiable from fragmented template, then trained an
elastic-net regression model for age prediction on 312 venous blood samples from donors aged 3-89 years. We
experimentally degraded aliquots of 60 samples spanning the training age range by controlled UV irradiation, dry
heat incubation and extended room-temperature storage to simulate casework-relevant degradation, and quantified
methylation at each locus in both intact and degraded aliquots by pyrosequencing. The intact-sample model
achieved a mean absolute prediction error of 3.4 years by leave-one-out cross-validation. Prediction error in
degraded aliquots increased significantly only in samples degraded to a mean fragment size below 150 bp, at which
the mean absolute error rose to 7.8 years, driven predominantly by reduced amplification efficiency at the FHL2 and
TRIM59 loci rather than by a systematic shift in methylation level at successfully amplified sites. A simplified
three-locus model restricted to ELOVL2, KLF14 and EDARADD, the loci least affected by fragmentation, retained a
mean absolute error of 5.1 years even in the below-150 bp category, at a modest cost in intact-sample accuracy
(4.1 years). These results indicate that short-amplicon methylation-based age estimation remains usable in severely
degraded forensic samples provided that amplicon selection is designed around fragmentation robustness.
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