Evidence for differences in DNA methylation between Germans and Japanese.

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.

Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

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.