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.

The last flight of F/O Tadeusz Stabrowski. Identification of the polish pilot.

The paper presents the process of identifying an unnamed soldier of the Polish armed forces in the west, whose remains were found in a nameless grave at the municipal cemetery in Le Crotoy in France. The Polish Genetic Database of Victims of Totalitarianism team carried out the research in cooperation with the Ministry of Culture and National Heritage. A comprehensive analysis of autosomal and Y-STR markers was performed. Historical, anthropological, and forensic examinations of the remains were also carried out. The items found with the remains were also examined. Identification based on DNA analysis made it possible to restore the identity of the Polish pilot who died on 11 March 1943 near the French coast, F/O Tadeusz Stabrowski. The airman regained his name in 2018, he was about 26 years old at the time of his death and left behind a grieving wife and son in the United Kingdom. The success of identifying the NN remains was guaranteed by the appointment of an interdisciplinary team consisting of specialists in archaeology, anthropology, history, forensic medicine and forensic genetics. The analysis of historical sources allowed to determine 4 missing airmen whose remains could have been buried in the cemetery in Le Crotoy. An interesting aspect of the research was the cooperation with history enthusiasts and fans of Polish aviation, thanks to which it was finally possible to narrow down the group of pilots sought and reach the family of Tadeusz Stabrowski, who submitted comparative material for research. This is the first case of establishing the identity of a Polish pilot killed in France. Many institutions have been involved in the project, including Polish Ministry of Culture and National Heritage (MDiKN), which partially funded the research.

Skeletal evidence of the ethnic cleansing actions in the Free City of Danzig (1939-1942) based on the KL Stutthof victims analysis.

In the early days of World War II, many of the prominent and influential people of Polish nationality from the Free City of Danzig were arrested by the Germans and sent to the nearby concentration camp KL Stutthof. Nearly a hundred of them died within the next seven months upon their arrival, and were buried in a clandestine mass grave in a nearby forest. However, the exact nature of their death is unknown, as it is unclear what the attitude of the aggressors was toward the victims. We do not know whether there was only one executioner or there were several assassins, nor if the killing methodology was consistent with the other state-regulated executions. The studied material represents the commingled remains of a minimum thirty-four people, possibly all male, aged from under eighteen to over sixty at the time of death. Perimortem traumatic lesions are shown mainly on the skull bones. We asked whether the perimortem trauma lesions visible on the victims' skeletons could be informative on the cause and manner of their death. Our results show the prevalence of the perimortem trauma inflicted by a blunt object are on the parietal bones above the Hat Brim Line (HBL), which is commonly associated with a violent attack. The gunshot trauma was usually localized on the occipital bone or posterior parietal, which could indicate a shot to the back of the head, and this was commonly encountered during executions. No signs of defensive injuries can be explained either by restraining of the hands or by a surprise attack. The abundance and variability of the trauma type can be evident of multiple assailants. Moreover, the multiple impact points detected on several crania prove unnecessary overkill and brutality, which reflects the personal attitudes of the executioners towards the victims.

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.