Comparative analysis of DNA preservation in permanent and deciduous teeth of adults and non-adults: Implications for archaeological and forensic research.

This study investigates the preservation of DNA in different categories of teeth, including permanent and deciduous, fully developed and not fully developed, in both adults and non-adults. Teeth were sampled from a modern-era cemetery in Ljubljana, Slovenia. DNA extraction was performed using a full demineralisation protocol. DNA quantity and quality were assessed using qPCR analyses, and autosomal STR typing was conducted to verify genetic profiles. Results revealed significant differences in DNA preservation among various tooth categories. Fully developed permanent teeth of adults exhibited the highest DNA yields, attributed to their fully developed roots and thicker cementum, which is rich in DNA. Deciduous teeth, with thinner enamel and cementum, showed lower DNA preservation regardless of developmental stage. Non-adult teeth generally yielded less DNA compared to adults, even when considering only fully developed permanent teeth, indicating factors beyond developmental stage. These findings suggest that, in archaeological and forensic contexts, researchers should prioritize fully developed permanent teeth for DNA analysis due to their superior preservation. Additionally, this study underscores the importance of considering tooth type and developmental stage when selecting samples for genetic analysis in cases where petrous bone is unavailable, expanding our understanding of DNA preservation in human remains.

SNP analysis of challenging bone DNA samples using the HID-Ion AmpliSeq™ Identity Panel: facts and artefacts.

PCR-MPS is an emerging tool for the analysis of low-quality DNA samples. In this study, we used PCR-MPS to analyse 32 challenging bone DNA samples from three Second World War victims, which previously yielded no results in conventional STR PCR-CE typing. The Identity Panel was used with 27 cycles of PCR. Despite that we only had an average of 6.8 pg of degraded DNA as template, 30 out of 32 libraries (93.8%) produced sequencing data for about 63/90 autosomal markers per sample. Out of the 30 libraries, 14 (46.7%) yielded single source genetic profiles in agreement with the biological identity of the donor, whereas 12 cases (40.0%) resulted in SNP profiles that did not match or were mixed. The misleading outcomes for those 12 cases were likely due to hidden exogenous human contamination, as shown by the higher frequencies of allelic imbalance, unusual high frequencies of allelic drop-ins, high heterozygosity levels in the consensus profiles generated from challenging samples, and traces of amplified molecular products in four out of eight extraction negative controls. Even if the source and the time of the contamination were not identified, it is likely that it occurred along the multi-step bone processing workflow. Our results suggest that only positive identification by statistical tools (e.g. likelihood ratio) should be accepted as reliable; oppositely, the results leading to exclusion should be treated as inconclusive because of potential contamination issues. Finally, strategies are discussed for monitoring the workflow of extremely challenging bone samples in PCR-MPS experiments with an increased number of PCR cycles.

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.

Nails as a primary sample type for molecular genetic identification of highly decomposed human remains.

For identification of badly preserved cadavers, only a few tissues can be used as a source of DNA, mostly bones and teeth, from which sampling and DNA extraction are difficult and time-consuming. In most highly decomposed remains, the nails are preserved. The aim of this study was to evaluate nails as an alternative source of DNA instead of bones and teeth in demanding routine identification cases. An automated extraction method was optimized on nails obtained from 33 cadavers with a post-mortem interval (PMI) up to 5 years. The commercially available EZ1 Investigator Kit (Qiagen) was used for extraction, and the G2 buffer included in the kit was replaced with TNCa buffer, and DTT was added for digestion of 5 mg of nail. The DNA was purified in a Biorobot EZ1 device (Qiagen), quantified using the PowerQuant System (Promega), and STR typing was performed with the NGM kit (TFS). From 0.3 to 270 µg DNA/g of nail was obtained from the samples analyzed, with an average yield of 36 µg DNA/g of nail. Full STR profiles were obtained from all nails except one. The optimized extraction method proved to be fast and highly efficient in the removal of PCR inhibitors, and it yields high amounts of DNA for successful STR typing. Nails were implemented as the primary sample type for obtaining DNA from highly decomposed and partially skeletonized cadavers in routine forensic identification cases in our laboratory.

Prolonged DNA hydrolysis in water: A study on DNA stability.

This work provides a protocol for the in vitro production of damaged DNA samples. In particular, heat-mediated hydrolysis of the samples at 70 °C in ultrapure water was performed in 1.7 mL Eppendorf tubes sealed by Parafilm for 0-36 h. The chemical/physical features of the resulting samples are described. After normalization of the qPCR data, these were compared with those obtained from samples treated for 0-10 h in a previous study.

Producing standard damaged DNA samples by heating: pitfalls and suggestions.

Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro. Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 °C for 0-18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes ("Protocol P") and in Eppendorf tubes provided with screwcaps ("Protocol S"). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the "Protocol S" affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2-3 orders of magnitude. The chemical-physical reasons of these findings are discussed together with the role of potential factors such as enhanced reactivity of CO2, ROS, NOx and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of degradation is well documented could be helpful in ISO/IEC 17025 validation procedures and in proficiency testing.

Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system.

Retrieving information about externally visible characteristics from DNA can provide investigative leads to find unknown perpetrators, and can also help in disaster victim and other missing person identification cases. Aiming for the application to both types of forensic casework, we previously developed and forensically validated the HIrisPlex test system enabling parallel DNA prediction of eye and hair colour. Although a recent proof-of-principle study demonstrated the general suitability of the HIrisPlex system for successfully analysing DNA from bones and teeth of various storage times and conditions, practical case applications to human remains are scarce. In this study, we applied the HIrisPlex system to 49 DNA samples obtained from bones or teeth of World War II victims excavated at six sites, mostly mass graves, in Slovenia. PCR-based DNA quantification ranged from 4pg/µl to 313pg/µl and on an average was 41pg/µl across all samples. All 49 samples generated complete HIrisPlex profiles with the exception of one MC1R DNA marker (N29insA) missing in 83.7% of the samples. In 44 of the 49 samples (89.8%) complete 15-loci autosomal STR (plus amelogenin) profiles were obtained. Of 5 pairs of skeletal remains for which STR profiling suggested an origin in the same individuals, respectively, 4 showed the same HIrisPlex profiles and predicted eye and hair colours, respectively, while discrepancies in one pair (sample 26 and 43) are likely to be explained by DNA quantity and quality issues observed in sample 43. Sample 43 had the lowest DNA concentration of only 4pg/µl, producing least reliable STR results and could be misleading in concluding that samples 43 and 26 originate from the same individual. The HIrisPlex-predicted eye and hair colours from two skeletal samples, suggested to derive from two brothers via STR profiling together with a living sister, were confirmed by the living sister's report. Overall, we demonstrate that after more than 70 years, HIrisPlex-based eye and hair colour prediction from skeletal remains is feasible with high success rate. Our results further encourage the use of the HIrisPlex system in missing person/disaster victim identification to aid the identification process in cases where ante-mortem samples or putative relatives are not directly available, and DNA predicted eye and hair colour information provides leads for locating them, allowing STRbased individual identification.

Extraction of DNA from Human Skeletal Material.

In recent years the recovery and analysis of DNA from skeletal remains has been applied to several contexts ranging from disaster victim identification to the identification of the victims of conflict. Here are described procedures for processing the bone and tooth samples including mechanical and chemical cleaning, cutting and powdering in the presence of liquid nitrogen, complete demineralization of bone and tooth powder, DNA extraction, DNA purification using magnetic beads, and the precautions and strategies implemented to avoid and detect contamination. It has proven highly successful in the analysis of bones and teeth from Second World War victims' skeletal remains that have been excavated from mass graves in Slovenia and is also suitable for genetic identification of relatively fresh human remains.

Slovenian population data for five new European Standard Set short tandem repeat loci and SE33 locus.

AIM: To establish the allele distribution and statistical parameters of forensic interest for the D10S1248, D22S1045, D2S441, D1S1656, D12S391, and SE33 loci in Slovenian population and to compare allele frequencies with those from other populations. METHODS: We analyzed blood and buccal swab samples from 333 unrelated, healthy Slovenian individuals. All samples were genotyped using the AmpFlSTR NGM Kit to obtain the allele frequency data for the loci D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Samples from 113 individuals were also analyzed using the PowerPlex ESX 17 system to obtain the allele frequency data for the SE33 locus. Allele frequencies and statistical parameters of forensic interest were determined and frequency profiles compared between Slovenian and other European Caucasian populations using the Arlequin software, version 3.5.1.3. RESULTS: The investigated short tandem repeat (STR) loci in Slovenian population had a great discriminating potential with a combined discrimination power of 0.99999998. The highest discrimination power and polymorphism information content were observed for the SE33 locus, followed by loci D1S1656, D12S391, D10S1248, D2S441, and D22S1045. When Slovenian allele frequency distribution was compared with other European populations, deviations were found only for Spanish and Italian population for D2S441 and D12S391. CONCLUSION: Slovenian population does not differ significantly from other European populations in terms of allele frequency distributions for the six analyzed STR loci. Based on forensic efficiency values, SE33 may be considered the most informative locus, which makes it especially useful in forensic investigations.

Serotonin transporter gene promoter (5-HTTLPR) and intron 2 (VNTR) polymorphisms: a study on Slovenian population of suicide victims.

Several studies have been carried out to investigate how genetic variants of gene encoding for the serotonin transporter (5-HTT) may confer susceptibility to suicide. It was demonstrated that polymorphisms in the promoter region (5-HTTLPR) and in the second intron (VNTR) have functional consequences and are for this reason of particular interest in relation to various psychiatric disorders. In our study, we analyzed 5-HTTLPR and VNTR polymorphisms in 235 suicide victims and 233 controls in a Slovenian population to find a possible association of the polymorphisms and suicidal behavior. No statistically important differences between genotypes of controls and suicide group (5-HTTLPR: Pearson's chi2=1.597, df=2, P=0.455; VNTR: Pearson's chi=1.961, df=4, P=0.744), as well as no differences in allele distribution (5-HTTLPR: Pearson's chi2=0.598, df=1, P=0.467; VNTR: Pearson's chi2=0.837, df=2, P=0.654) were found, although a slightly higher frequency of LL genotype and of L allele was observed in the suicide group. Haplotype frequency analysis showed no excess of particular haplotypes between the two groups. Our study showed no association of serotonin transporter polymorphisms and suicide. The study, however, was performed on a population with a very high suicide rate (27.1 victims/100,000 citizens) and the role of 5-HTTLPR polymorphisms may be different in other populations.

Association study of seven polymorphisms in four serotonin receptor genes on suicide victims.

A number of molecular genetic studies have investigated if serotonin (5-HT) receptor subtypes are involved in the pathogenesis of depression, suicidal behavior, aggression, and impulsive behavior. Existence of many receptor subtypes for a single transmitter permits a great diversity of signaling raising the possibility that they may serve as genetic markers for suicidal behavior. Most previous studies of suicide have analyzed polymorphisms of the receptors 5-HT1A, 5-HT1B, 5-HT2A, fewer have examined 5-HT1F. We report a study of possible association between the polymorphisms in the 5-HT receptor genes (1A, 1B, 1F, and 2A) and suicidal behavior on a sample of 226 suicide victims and 225 healthy control subjects. No significant differences in genotype frequency distributions between the suicide victims and healthy control subjects were observed for four polymorphisms; three were not polymorphic. A single polymorphism, C-1420T in gene 5-HT2A, showed a slight association with suicide (chi2= 4.94, df = 2, P = 0.067), but the correlation was not statistically significant. None of the tested genetic variants of serotonin receptors appears to be associated with suicidal behavior in the Slovenian population which has a relatively high suicide rate.