Around the 12th/thirteenth century, a burial method called “more teutonico” was used for the remains of high-ranking individuals who had died far from their homeland and had to be brought back for burial. Before transportation, the bodies or body parts were boiled (macerated) to remove all soft tissue. The question of whether a body was boiled postmortem, is of particular interest for historians (burial “more teutonico”?) and this question may also be relevant in a forensic context, even if only in rare cases. In 1989, Bada, Herrmann, Payan and Man (“Amino acid razemization in bone and the boiling of Emperor Lothar I.” in Applied Geochemistry) suggested using the D-aspartic acid content of bone as an objective indicator of a postmortem exposure to heat. This approach was never substantiated by further studies, and more recent findings call the concept into question. Therefore, we tested the “Bada approach” by analysing the D-aspartic acid content in boiled bone samples (pig, human) using different “boiling recipes” of procedures “more teutonico” (using water, wine, or vinegar as boiling media), and discussed it under consideration of the recent literature. Using the boiled human bone samples, we also investigated whether the question of “boiled or not boiled?” could be answered by another parameter, namely by analysing the pentosidine content of a sample. An accumulation of D-aspartic acid was observed during boiling in both pig and human bone samples. It was considerably lower in the human samples and significantly influenced by the medium used. By contrast, boiling did not result in pentosidine accumulation. These experimental results call into question the reliability of the “Bada approach” – not least because the type of boiling medium may not be recreated in each individual case. Moreover, this approach does not take the current knowledge on the intravital accumulation of D-aspartic acid in bone samples of different origins into account, nor the influence of postmortem conditions on the D-aspartic acid content in non-heated samples (used as controls by Bada et al.). Overall, the approach of Bada et al. should not be accepted uncritically, as proving a treatment “more teutonico” based solely on the D-aspartic acid content seems hardly possible – and if so, only under very strict conditions. The analysis of pentosidine is not suitable for demonstrating a burial “more teutonico”.

Age-at-death estimation is of great relevance for the identification of unknown deceased individuals. In skeletonised corpses, teeth and bones are theoretically available for age estimation, but in many cases, only single bones or even only bone fragments are available for examination. In these cases, conventional morphological methods may not be applicable, and the application of molecular methods may be considered. Protein-based molecular methods based on the D-aspartic acid (D-Asp) or pentosidine (Pen) content have already been successfully applied to bone samples. However, the impact of the analysed type of bone has not yet been systematically investigated, and it is still unclear whether data from samples of one skeletal region (e.g. skull) can also be used for age estimation for samples of other regions (e.g. femur). To address this question, D-Asp and Pen were analysed in bone samples from three skeletal regions (skull, clavicle, and rib), each from the same individual. Differences between the bone types were tested by t-test, and correlation coefficients (ρ) were calculated according to Spearman. In all types of bone, an age-dependent accumulation of D-Asp and Pen was observed. However, both parameters (D-Asp and Pen) exhibited significant differences between bone samples from different anatomical regions. These differences can be explained by differences in structure and metabolism in the examined bone types and have to be addressed in age estimation based on D-Asp and Pen. In future studies, bone type-specific training and test data have to be collected, and bone type-specific models have to be established.

Age estimation based on DNA methylation (DNAm) can be applied to children, adolescents and adults, but many CG dinucleotides (CpGs) exhibit different kinetics of age-associated DNAm across these age ranges. Furthermore, it is still unclear how growth disorders impact epigenetic age predictions, and this may be particularly relevant for a forensic application. In this study, we analyzed buccal mucosa samples from 95 healthy children and 104 children with different growth disorders. DNAm was analysed by pyrosequencing for 22 CpGs in the genes PDE4C, ELOVL2, RPA2, EDARADD and DDO. The relationship between DNAm and age in healthy children was tested by Spearman’s rank correlation. Differences in DNAm between the groups “healthy children” and the (sub-)groups of children with growth disorders were tested by ANCOVA. Models for age estimation were trained (1) based on the data from 11 CpGs with a close correlation between DNAm and age (R ≥ 0.75) and (2) on five CpGs that also did not present significant differences in DNAm between healthy and diseased children. Statistical analysis revealed significant differences between the healthy group and the group with growth disorders (11 CpGs), the subgroup with a short stature (12 CpGs) and the non-short stature subgroup (three CpGs). The results are in line with the assumption of an epigenetic regulation of height-influencing genes. Age predictors trained on 11 CpGs with high correlations between DNAm and age revealed higher mean absolute errors (MAEs) in the group of growth disorders (mean MAE 2.21 years versus MAE 1.79 in the healthy group) as well as in the short stature (sub-)groups; furthermore, there was a clear tendency for overestimation of ages in all growth disorder groups (mean age deviations: total growth disorder group 1.85 years, short stature group 1.99 years). Age estimates on samples from children with growth disorders were more precise when using a model containing only the five CpGs that did not present significant differences in DNAm between healthy and diseased children (mean age deviations: total growth disorder group 1.45 years, short stature group 1.66 years). The results suggest that CpGs in genes involved in processes relevant for growth and development should be avoided in age prediction models for children since they may be sensitive for alterations in the DNAm pattern in cases of growth disorders.

As a contribution to the discussion about the possible effects of ethnicity/ancestry on age estimation based on DNA methylation (DNAm) patterns, we directly compared age-associated DNAm in German and Japanese donors in one laboratory under identical conditions. DNAm was analyzed by pyrosequencing for 22 CpG sites (CpGs) in the genes PDE4C , RPA2 , ELOVL2 , DDO , and EDARADD in buccal mucosa samples from German and Japanese donors ( N = 368 and N = 89, respectively). Twenty of these CpGs revealed a very high correlation with age and were subsequently tested for differences between German and Japanese donors aged between 10 and 65 years ( N = 287 and N = 83, respectively). ANCOVA was performed by testing the Japanese samples against age- and sex-matched German subsamples ( N = 83 each; extracted 500 times from the German total sample). The median p values suggest a strong evidence for significant differences ( p < 0.05) at least for two CpGs ( EDARADD , CpG 2, and PDE4C , CpG 2) and no differences for 11 CpGs ( p > 0.3). Age prediction models based on DNAm data from all 20 CpGs from German training data did not reveal relevant differences between the Japanese test samples and German subsamples. Obviously, the high number of included “robust CpGs” prevented relevant effects of differences in DNAm at two CpGs. Nevertheless, the presented data demonstrates the need for further research regarding the impact of confounding factors on DNAm in the context of ethnicity/ancestry to ensure a high quality of age estimation. One approach may be the search for “robust” CpG markers—which requires the targeted investigation of different populations, at best by collaborative research with coordinated research strategies.

With the increase of globalization and migration, the topic of age estimation has become more and more important in diverse fields of application, especially for age estimation in living individuals as well as for age estimation in the identification of unknown deceased and of unknown donors of a trace. Especially in the last decade, the traditional spectrum of morphological methods has been expanded to numerous new approaches based on the use of age-dependent molecular changes. Articles in this field have been and are being published in quick succession but not all approaches can (already) meet the demands of forensic practice. It may be a challenge for the forensic practitioner to keep track of suitable methods and to find the optimal method for a single case with its specific questions, conditions and requirements. This overview is intended to provide orientation on the question of which molecular approaches can already be used or will be applicable in the foreseeable future in different application fields. The focus is on the accumulation of D‑aspartic acid and pentosidine, DNA methylation and the use of the bomb pulse-derived carbon-14 (14C).

The identification and description of "molecular clocks" (posttranslational protein modifications, DNA methylation) offer new possibilities for the development of methods for postmortem age estimation; however, so far these approaches have only been used independently. Their combination promises a better recording of highly complex aging processes and thus the possibility of developing optimized age estimation procedures for a wide variety of scenarios in forensic practice. In preparation for large-scale research to test this hypothesis, different molecular clocks (accumulation of D-aspartic acid, accumulation of pentosidine and the DNA methylation markers RPA2, ZYG11A, F5, HOXC4, NKIRAS2, TRIM59, ELOVL2, DDO, KLF14 and PDE4C) were examined in 4 decay-resistant tissues (bone, tendon, intervertebral disc, epiglottis) from 15 individuals. In all tissues examined both protein markers as well as several DNA methylation markers showed a strong correlation with age. Thereby, the examined parameters showed tissue-specific changes with age. The results of the pilot study demonstrate the potential of combining molecular methods for postmortem age estimation. Further studies will show how accurate postmortem age estimates might be if age information from posttranslational protein modifications and DNA methylation from different tissues are combined in multivariate models.

ZusammenfassungMit der Identifikation und Beschreibung „molekularer Uhren“ (posttranslationale Proteinmodifikationen, DNA-Methylierung) eröffnen sich neue Möglichkeiten zur Entwicklung von Verfahren zur postmortalen Lebensaltersschätzung. Bislang werden diese Ansätze aber nur unabhängig voneinander eingesetzt. Ihre Verknüpfung verspricht eine bessere Erfassung hochkomplexer Alterungsprozesse und damit die Möglichkeit zur Entwicklung optimierter Verfahren zur Altersschätzung für verschiedenste Szenarien der forensischen Praxis.In Vorbereitung umfangreicher Untersuchungen zur Überprüfung dieser Hypothese wurden verschiedene molekulare Uhren (Akkumulation von D‑Asparaginsäure, Akkumulation von Pentosidin und DNA-Methylierungsmarker [RPA2, ZYG11A, F5, HOXC4, NKIRAS2, TRIM59, ELOVL2, DDO, KLF14 und PDE4C]) in 4 fäulnisresistenten Geweben (Knochen, Sehne, Bandscheibe, Epiglottis) von 15 Individuen untersucht.In allen untersuchten Geweben fand sich eine starke Korrelation beider Proteinmarker sowie jeweils mehrerer DNA-Methylierungsmarker mit dem Lebensalter. Dabei zeigten die untersuchten Parameter gewebsspezifische Veränderungen mit dem Alter.Die Ergebnisse der Pilotstudie belegen das Potenzial der Verknüpfung molekularer Verfahren für die postmortale Altersschätzung. Weitere Untersuchungen werden zeigen, wie genau postmortale Altersschätzungen sein können, wenn Altersinformationen aus posttranslationalen Proteinmodifikationen und DNA-Methylierung aus verschiedenen Geweben in multivariaten Modellen verknüpft werden.

Genetic analyses such as STR-typing are routinely used for identification purposes in forensic casework. Although genotyping techniques only require a minimum amount of DNA to provide a genetic profile, DNA quality differs not only between but also within tissues during ongoing decomposition. Initiated by a recent case where, due to the constitution of the body, preferred tissue was not available or only resulted in a partial and not usable DNA profile, the analysis of intervertebral discs as a source of DNA was considered. As the analysis of this tissue resulted in a high quality DNA profile a further study was performed in which thirty intervertebral discs dissected from bodies in different stages of decay were analyzed. All samples yielded good quality DNA in quantities suitable for STR-based amplification with no or only low degradation indices, resulting in complete genetic profiles. These results demonstrate the robustness of human intervertebral disc tissue as a source of DNA for molecular identification purposes.

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.

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: r 2 = 0.87, validation set: r 2 = 0.85). This was also the case in postmortem samples ( r 2 = 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.

Several molecular modifications accumulate in the human organism with increasing age. Some of these “molecular clocks” in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of d-aspartic acid (d-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5–11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, d-Asp) and expand our set of tissues.