The evidential value of dental calculus in the identification process.

DNA analysis-based identification is by far the gold standard in forensic genetics and it should be performed in every case involving human remains or unidentified bodies. Bones and teeth are the preferred source of human DNA for genetic analysis. However, there are cases where the nature of the proceedings and historical significance prevent the disruption of skeletal structure. The remains may also be heavily degraded. In such situations, forensic geneticists seek alternative sources of human DNA. Teeth calculus has proven to be a viable source of DNA for identification purposes. The aim of this study was to assess the concentration of human DNA in teeth calculus and evaluate the usefulness of teeth calculus as a DNA source in the identification process. Teeth calculus was collected from skeletons exhumed between 2021 and 2022 by the PBGOT (Polish Genetic Database of Victims of Totalitarianism) team from the former Stalag IID prisoner-of-war camp in Stargard. Genetic analyses included the determination of autosomal and Y-STR markers. The total concentration of human DNA was also evaluated in samples from teeth calculus and teeth taken from the same individuals. The pilot study included 22 skeletons with a sufficient amount of calculus for isolation (specified in the protocol). Samples were taken from the largest areas of calculus deposited on lingual surfaces of mandibular incisors. The prepared samples underwent DNA extraction. Our study demonstrated that teeth calculus is a source of human DNA for remains from the World War II period. The obtained DNA concentration allowed for the determination of STR markers. It was shown that teeth calculus contains human DNA in an amount suitable for preliminary identification analyses.

Black Devils in Normandy-Identification of an Unknown Soldier Found in the Polish War Cemetery of Urville-Langannerie (France).

A paper dedicated to the identification of a Polish soldier from the 1st Armoured Division under the command of General Stanislaw Maczek, who fell in 1944 in Normandy, during World War II. The remains were found at the Urville-Langannerie Polish War Cemetery. A team from the Department of Forensic Genetics at the Pomeranian Medical University in Szczecin, commissioned by the Ministry of Culture Heritage and Sport, exhumed the remains in order to carry out genetic identification tests. A comprehensive anthropological analysis of the heavily degraded remains was carried out, and biological samples were secured for genetic testing. The identification of Jan Dusza is the first case of restoring the identity of an active combatant from the First Armoured Division. In the case analysis, the analysis of mitochondrial DNA in highly degraded biological material proved crucial. Genetic studies decided to reject the original historical hypothesis No. I at their preliminary stage. Regarding hypothesis No. II, a comprehensive genetic analysis of mitochondrial and autosomal DNA was carried out. Comparative material was obtained from the alleged victim's sister. Thanks to the analysis of kinship in the maternal line based on the mtDNA haplotype, it was possible to establish that the remains belong to Jan Dusza, who served in the Podhale Rifle Battalion, part of the Polish 1st Armoured Division. The research was co-financed by the Polish Ministry of Heritage and National Culture.

Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks.

BACKGROUND: DNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity. RESULTS: The protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging. CONCLUSIONS: We propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.