Identifying Methylation Patterns in Dental Pulp Aging: Application to Age-at-Death Estimation in Forensic Anthropology

Age-at-death estimation constitutes one of the key parameters for identification of human remains in forensic investigations. However, for applications in forensic anthropology, many current methods are not sufficiently accurate for adult individuals, leading to chronological age estimates erring by ±10 years. Based on recent trends in aging studies, DNA methylation has great potential as a solution to this problem. However, there are only a few studies that have been published utilizing DNA methylation to determine age from human remains. The aim of the present study was to expand the range of this work by analyzing DNA methylation in dental pulp from adult individuals. Healthy erupted third molars were extracted from individuals aged 22–70. DNA from pulp was isolated and bisulfite converted. Pyrosequencing was the chosen technique to assess DNA methylation. As noted in previous studies, we found that ELOVL2 and FHL2 CpGs played a role in age estimation. In addition, three new markers were evaluated—NPTX2, KLF14, and SCGN. A set of CpGs from these five loci was used in four different multivariate regression models, providing a Mean Absolute Error (MAE) between predicted and chronological age of 1.5–2.13 years. The findings from this research can improve age estimation, increasing the accuracy of identification in forensic anthropology.

Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on “molecular clocks” in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two “protein clocks”, the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the “D-Asp clock” loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The “Pen-clock” may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

Femoral histomorphometric age-at-death studies: The issue of sample size and standard error

Extant histomorphometric aging methods based on the analysis of the femoral cortex generally report small samples ( N<100) and highly variable standard error of the estimate (SEE) values (±1.51‒16.98 years). The present paper reviews the published literature on femoral histomorphometry for age-at-death estimation in order to examine the relationship between sample size and SEE values, and makes recommendations for minimum reporting requirements for age-at-death studies based on statistical data. The SEE from a total of 33 studies are analysed. Sample size and confidence intervals are explored using Hennig and Cooper’s simulation modelling. Analysis of effect size through a fixed-effect model is performed on 5/33 studies to examine the relationship between sample size and effect size. The pooled sex formulae from Nor et al., Martrille et al. and Thompson and the two sex-specific formulae of Pfeiffer are examined, as they report mean and standard deviation values for both chronological and estimated ages. The results of these analyses support sampling theory, specifically wide variation in SEE when N<100, narrowing as the sample size increases, and lower effect sizes in the larger of the five studies examined. The findings provide some support for a minimum threshold of 100‒150 individuals for histomorphometric age-at-death estimation. Analysis of effect size is suggested for future investigation in meta-analyses of forensic anthropological age-estimation studies. To ensure increased precision and meaningful comparison, large samples should be used for histomorphometry, and authors should report SEE and discrete statistics (e.g. n, mean, standard deviation) for both chronological age and estimated age.