World War Mass Graves Research Articles

A recommended sampling strategy for genetic identification of Second World War victims in Slovenia

Skeletonized human remains from Second World War mass graves in Slovenia are a major challenge in genetic identification, and bones with a high DNA yield must be selected for successful identification. The goal of this study was to construct skeletal sampling strategy recommendations through comparison of the most appropriate groups of skeletal elements. Altogether, 566 bones and teeth from the same mass grave were compared, half analyzed in this study and half in previous studies performed by our group. After anthropological examination, mechanical and chemical cleaning was performed, followed by bone and tooth powdering. Total demineralization of 0.5 g of bone and tooth was followed by extraction and purification of DNA with a Biorobot EZ1 device (Qiagen). The qPCR PowerQuant kit (Promega) was used to measure the amount of DNA, and statistical analysis was performed. Skeletal elements were selected according to known better preservation of DNA in the human body, and they were arranged in seven groups: petrous bone, long bones (femur and tibia), torso bones (first rib and 12th vertebra), metacarpals, metatarsals, short and sesamoid bones (talus, navicular, medial cuneiform, cuboid, calcaneus, and patella), and teeth. Sampling strategy recommendations were constructed based on DNA quantity and quality results. The petrous bone group, metacarpal group, torso bone group, and short and sesamoid bone group produced the highest DNA yields. Accordingly, in addition to standard sampling of long bones (femurs and tibias) and teeth, those additional bone types should be collected for Slovenian Second World War victim identification.

Intra-bone nuclear DNA variability and STR typing success in Second World War 12th thoracic vertebrae

Bones are an important source of DNA for identification in forensic medicine, especially when the remains are skeletonized, which is the case when dealing with victims of the Second World War. Often the amount of bone available for sampling is limited, and therefore it is crucial to sample the bone segment with the highest adequate DNA quantity for identification. Studies performed on all representative skeletal element types of the human body showed that the amount of DNA obtained from different skeletal elements of different body regions varies greatly. When bones from torso were analyzed, thoracic vertebrae outperformed other vertebrae (cervical and lumbar) and, alongside the first ribs, were among the most appropriate bone elements for identification purposes. It was also shown that the quantity of DNA varies significantly within a single bone type. This study focused on exploring intra-bone DNA variability between five parts of 12th thoracic vertebrae (laminae + spinous process, pedicles + transverse processes, and corpus right, left, and middle). The research was based on the theory that the distribution of body weight and consequently bone remodeling, as well as the ratio between cancellous and cortical bone, contribute to different quantities of DNA in different parts of vertebra sampled. The vertebrae were cleaned and cut into five parts, and each part was completely ground to obtain homogenous bone powder. Half a gram of powder from each part was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 machine (Qiagen). DNA quantity and quality were determined using the PowerQuant System (Promega) and autosomal STR typing success using the GlobalFiler Amplification Kit (Applied Biosystems). Thirty-five 12th thoracic vertebrae were sampled from a single Second World War mass grave. The best results with the highest DNA quantity were found in laminae and the spinous process, and among them all vertebrae analyzed yielded full STR profiles except three, where only a few dropouts occurred. The second-ranked bone part was the pedicles and transverse processes. The comparison of DNA degradation in the vertebral segments analyzed does not show statistically significant differences. Considering our research, when only the torso is available for identification, the 12th thoracic vertebra should be collected and the vertebral arch should be sampled for genetic analyses.

Comparison of nuclear DNA yield and STR typing success in Second World War petrous bones and metacarpals III

DNA yield and STR typing success differ among skeletal element types and within individual bones. Consequently, it is necessary to identify the skeletal elements and their intra-skeletal parts that will most likely yield utilizable and informative endogenous DNA for human identification of skeletal remains. The petrous portion of the temporal bone has been shown to be the most suitable skeletal part for sampling archaeological skeletons, and it has also been used successfully in some forensic cases. When all representative bone types were analyzed for three complete Second World War skeletons, metatarsals and metacarpals yielded more DNA than petrous bones (which generated full profiles even if the DNA yield was lower) and, among almost 200 Second World War metatarsals and metacarpals analyzed, metacarpals III were found to be the highest-yielding bones. To further improve the sampling strategy in DNA analysis of aged skeletal remains, a comparison between 26 petrous bones and 30 metacarpals III from Second World War skeletons was made considering intra-bone DNA yield variability. In metacarpals III only epiphyses were sampled, and in petrous bones only the dense part within the otic capsule was used. To exclude the influence of taphonomic issues as much as possible, petrous bones and metacarpals III from a single Second World War mass grave were examined. The difference between petrous bones and metacarpals III was explored by measuring nuclear DNA yield and success of STR typing. After cleaning the samples, full demineralization extraction was used to decalcify 0.5 g of powdered bone. PowerQuant (Promega) was used to determine DNA content and DNA degradation rates, and STR typing was performed using the PowerPlex ESI 17 Fast System (Promega). Metacarpals III produced the same DNA yields and STR typing success as petrous bones with no intra-individual difference observed in concentration of DNA, degradation rate, percentage of successfully amplified alleles, and intensity of electrophoretic signals. Sampling and processing of metacarpal III epiphyses is consequently recommended for genetic identification of highly degraded skeletal remains in routine forensic casework and in buried non-commingled aged skeletal remains whenever metacarpals III are preserved. Metacarpals III are easy to sample and less prone to contamination with modern DNA because no saw is needed for sampling in comparison to the petrous portion of the temporal bone. The data obtained in this study further improve the sampling strategy for genetic identification of Second World War skeletal remains in Slovenia.

Intra-bone nuclear DNA variability and STR typing success in Second World War first ribs

DNA sampling and typing are used for identifying missing persons or war victims. In recent forensic studies, little focus has been placed on determining intra-bone variability within a single skeletal element. When dealing with aged human bones, complete skeletal remains are rarely present. In cases in which only the torso is available, studies have shown that ribs are one of the most appropriate samples, but intra-bone variability has not yet been studied. A higher degree of remodeling was found to contribute to higher DNA yield in the parts of the skeletal element where the most strain is concentrated. This study explores intra-bone variability in proximal, middle, and distal parts of the first human rib by determining the quantity and quality of DNA using the PowerQuant System (Promega) and autosomal STR typing success using the PowerPlex ESI 17 Fast System (Promega). Thirty first ribs from a single Second World War mass grave were sampled. No variation in DNA degradation was observed across the individual rib. The highest quantity of DNA was measured in the proximal part of the first rib, and in all ribs except three, full or almost full genetic profiles were obtained. Thus, when only the torso is present in archaeological or medico-legal cases, first ribs are recommended to be collected if possible, and the proximal or vertebral ends should be sampled for genetic analysis.

Dealing with minor differences in bone matrix: can spectra follow the DNA preservation?

ABSTRACT Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is a relatively fast and inexpensive but reliable method that can be used as a rough screening tool for DNA preservation. Although it is an excellent analytical tool, it only provides relative information on the chemical structure of heterogeneous materials such as bone and its usefulness and accuracy for predicting the preservation of DNA in samples originating from different body regions of minimally altered remains was never tested before. 144 bone samples were collected from different body regions of three individuals discovered in a Second World War mass grave. DNA was extracted and quantified, followed by STR typing. Samples were analysed with ATR-FTIR spectroscopy. Comparing the preservation state of DNA and chemometric indices, the usefulness of the method was tested. Correlations between the DNA preservation and chemometric indices were highly variable, especially when considering different body regions. Best DNA preservation was correlated with a balanced ratio between carbonates and phosphates, and with high collagen quality and quantity. It seems that, when dealing with relatively young and well-preserved remains, more attention should be paid to collagen quality. In addition, the results indicate that DNA quality and DNA quantity correlate differently with chemometric indices.

Intra-bone nuclear DNA variability in Second World War metatarsal and metacarpal bones.

DNA analysis of Second World War skeletal remains is challenging because of the limited yield of DNA that is usually recovered. Recent forensic research has focused on determining which skeletal elements are superior in their preservation of DNA, and little focus has been placed on measuring intra-bone variability. Metatarsals and metacarpals outperformed all the other bones in DNA yield when analyzing all representative skeletal elements of three Second World War victims, and intra-bone variability was not studied. Soft-tissue remnants were found to contribute to higher DNA yield in trabecular bone tissue. Because metatarsals and metacarpals are composed of trabecular epiphyses and a dense diaphysis, the goal of this study was to explore intra-bone variability in DNA content by measuring nuclear DNA quantity and quality using the PowerQuant System (Promega). A total of 193 bones from a single Second World War mass grave were examined. From each bone, DNA was extracted from the compact diaphysis and from both spongy epiphyses combined. This study confirms higher DNA quantity in epiphyses than diaphyses among all the bones analyzed, and more DNA was obtained from metacarpal epiphyses than from metatarsal epiphyses. Therefore, whenever the possibility for sampling both metacarpals and metatarsals from skeletal remains exists, collecting metacarpals is recommended. In cases in which the hands are missing, metatarsals should be sampled. In any case, epiphyses are a richer source of DNA than diaphyses.

High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains

DNA yield varies by anatomical region, and the selection of bone types that yield maximum recovery of DNA is important to maximize the success of human identification of skeletal remains. The goal of our study was to explore inter- and intra-individual variation in DNA content by measuring nuclear DNA quantity and quality and autosomal STR typing success to determine the most promising skeletal elements for bone sampling. To exclude the influence of taphonomic issues as much as possible, three complete male skeletons from a single Second World War mass grave were examined and all representative skeletal element types of the human body were analyzed. Forty-eight different types of bones from the head, torso, arm, leg, hand, and foot were sampled from each skeleton, 144 bones altogether. The samples were cleaned, and half a gram of bone powder was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen). DNA content and rates of DNA degradation were determined with the PowerQuant (Promega), and the Investigator ESSplex SE QS (Qiagen) was used for STR typing. The highest-yielding bones mostly produced the most complete STR profiles. Among the skeletal elements containing on average the most DNA and producing the most complete profiles in all three skeletons examined were metacarpals, metatarsals, and the petrous portion of the temporal bone. Metatarsals and metacarpals can easily be sampled without using a saw, thus reducing potential DNA contamination. Skeletons from the Second World War can be used as a model for poorly preserved skeletal remains, and the results of the investigation can be applied for genetic identification of highly degraded skeletal remains in routine forensic casework. Although the research was limited to only three skeletons found in a unique mass grave, the data obtained could contribute to sampling strategies for identifying old skeletal remains. More Second World War skeletons will be analyzed in the future to investigate inter-bone variation in the preservation of DNA.

Strategy for STR typing of bones from the Second World War combining CE and NGS technology: A pilot study

The genetic identification of skeletal remains found in Second World War mass graves is complicated because of the poor quality of the samples. The aim of this study was to set up a workflow for STR typing of such samples combining PCR/CE and PCR/NGS technologies. To this end, 57 DNA samples from an equal number of 75-year-old femurs were studied. After a first round of PCR typing using GlobalFiler CE, 42 samples yielded a full profile and were therefore submitted to our standard workflow. The 15 samples that yielded no or a limited number (2–17/21) of autosomal STR markers as well four bone control samples that provided a full profile with the conventional PCR/CE test were typed in duplicate by the GlobalFiler NGS kit. Despite the degradation of the samples, which resulted in lower coverage and a lower % of on-target reads, reliable sequencing data were obtained from 16/19 samples. The use of a threshold of 30× for the locus call led to a consensus profile (cp) of 20–31/31 STR autosomal loci in 10 samples and to a cp of 8–10/31 loci in two samples, whereas the four control samples yielded a cp of 26–31/31 loci. Finally, the data of the NGS typing were combined with those of the CE typing. This last task allowed us to recover (on average) three alleles per sample and to increase the number of the heterozygous patterns in 37 cases. In total, the combined approach proposed here made possible the genetic typing of 65–100% of the autosomal STR markers in 10/15 (66.6 %) skeletal remains that yielded no or very poor results with the conventional PCR/CE approach. However, because several artefacts (such as allelic drop-out and allelic drop-in) were scored, the risk of mistyping cannot be neglected.

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

AimTo present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina.MethodsSamples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach.ResultsA total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications.ConclusionDNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them.

A Comparative Analysis of the AmpFlSTR Identifiler and PowerPlex 16 Autosomal Short Tandem Repeat (STR) Amplification Kits on the Skeletal Remains Excavated from Second World War Mass Graves in Slovenia

Aim. This paper describes a comparative study of two different amplification kits for autosomal STR typing of skeletal remains excavated from Second World War mass graves in Slovenia. We analyzed 92 bones and teeth and compared the genetic profiles obtained using the AmpFlSTR IdentifilerTM Amplification Kit (Applied Biosystems) and the PowerPlex 16 System (Promega). Methods. We cleaned the bones and teeth, removed surface contamination and ground them into a powder using liquid nitrogen. Prior to deoksiribo nucleic acid (DNA) isolation with Biorobot EZ1 (Qiagen), 0.5g bone or tooth powder was decalcified. The nuclear DNA of the samples was quantified using the real-time polymerase chain reaction (PCR) method. The amplification protocols for both kits were optimised for old skeletal remains. Results. We extracted 0.4 to 100 ng DNA/g of powder from the bones and teeth. Both amplification kits showed very similar efficiency on the 70 year old bones and teeth, since DNA typing was successful in 81 out of the 92 bones and teeth with both amplification kits, which represent an 88% success rate. Conclusions. The comparative study indicated that the commercially available Identifiler and PowerPlex 16 PCR amplification kits are reliable for short tandem repeat (STR) typing of World War II skeletal remains with the DNA extraction method and PCR amplification conditions optimised in our laboratory, since very often complete STR profiles of autosomal DNA were obtained with both kits.

Performance of the Human Quantifiler, the Investigator Quantiplex and the Investigator ESSplex Plus kit for quantification and nuclear DNA typing of old skeletal remains

Aim. We tested the performance of two real-time polymerase chain reaction (PCR) human quantification kits (Human Quantifiler (Applied Biosystems) and Investigator Quantiplex kit (Qiagen)) and forensic identification short tandem repeat (STR) Investigator ESSplex Plus (Qiagen) kit on approximately 70 years old skeletal remains. Methods. We analysed 54 bones and teeth excavated from Second World War mass graves in Slovenia. Genomic DNA was obtained from 0.5 g of bone or tooth powder after total demineralization. The DNA was purified in a Biorobot EZ1 (Qiagen) device. The same extract was used for quantification and STR typing with all three kits using the amplification conditions recommended by the manufacturers. Results. In almost two thirds of the samples the results of quantification were up to 6 times higher using the Human Quantifiler than using the Investigator Quantiplex kit, because of the degradation of DNA in old skeletal remains and amplification of shorter DNA fragment with the Human Quantifiler kit. The autosomal STR typing with the Investigator ESSplex Plus kit was successful in 52 out of the 54 samples, which represent a 96% success rate. Conclusion. The commercially available Investigator ESSplex Plus kit can be used for STR typing of old skeletal remains with the DNA extraction method optimised in our laboratory and without any changes to the manufacturers' PCR amplification protocols. The Human Quantifiler kit and Investigator Quantiplex kit together can be used for estimation of the degree of DNA degradation in compromised old bone samples.