Comparison of the optimal and suboptimal quantity of mitotype libraries using next-generation sequencing.

Optimizing analysis parameters and sample input is crucial in forensic genetics methods to generate reliable results, and even more so when working with muti-copy mitochondrial DNA (mtDNA) and low-quality samples. This study compared mitotypes based on next-generation sequencing (NGS) results derived from the same samples at two different sequencing library concentrations-30 pM and 0.3 pM. Thirty femur samples from the Second World War were used as a model for poorly preserved DNA. Quantitative PCR (qPCR) method targeting 113 bp long fragment was employed to assess the quantity of mitogenomes. HID Ion Chef™ Instrument with Precision ID mtDNA Control Region Panel was used for library preparation and templating. Sequencing was performed with Ion GeneStudio™ S5 System. Reference haplotypes were determined from sequencing samples at 30 pM library input. Haplotypes were compared between optimal (30 pM) and suboptimal (0.3 pM) library inputs. Often the difference in haplotypes was length heteroplasmy, which in line with other studies shows that this type of variant is not reliable for interpretation in forensics. Excluding length variants at positions 573, 309, and 16,193, 56.7% of the samples matched, and in two samples, no sequence was obtained at suboptimal library input. The rest of the samples differed between optimal and suboptimal library input. To conclude, genotyping and analyzing low-quantity libraries derived from low-quality aged skeletonized human remains therefore must be done with caution in forensic genetics casework.

Measure quantity of mitochondrial DNA in aged bones or calculate it from nuclear DNA quantitative PCR results?

Mitochondrial DNA (mtDNA) is of great value in forensics to procure information about a person when a next of kin, personal belongings, or other sources of nuclear DNA (nDNA) are unavailable, or nDNA is lacking in quality and quantity. The quality and reliability of the results depend greatly on ensuring optimal conditions for the given method, for instance, the optimal input of the copy number (CN) in next-generation sequencing (NGS) methods. The unavailability of commercial quantitative PCR (qPCR) methods to determine mtDNA CN creates the necessity to rely on recommendations to infer mtDNA CN from nDNA yield. Because nDNA yield varies between individuals, tissues, parts of the same tissue, and because mtDNA CN varies between tissues, such assumptions must be examined for a specific context, rather than be generalized. This study compares mtDNA CN calculated from nDNA yield and qPCR measured mtDNA CN. Seventy-five femurs from the Second World War victims were used as samples; they were cut below the greater trochanter, surface contaminants were removed by mechanical and chemical cleaning, samples were fully demineralized, and DNA was isolated. PowerQuant® Kit (Promega) was used to analyze DNA yield. An in-house method was used to determine mtDNA CN. Comparison of mtDNA CN from nDNA derived calculations and measured mtDNA CN highlighted vast differences. The results emphasize the need to perform qPCR to assess mtDNA CN before NGS analyses of aged bones' mitogenomes rather than estimating mtDNA CN from nDNA yield to ensure the quality and reliability of the results of NGS analysis.

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.

Isometric artifacts from polymerase chain reaction-massively parallel sequencing analysis of short tandem repeat loci: An emerging issue from a new technology?

The recent introduction of polymerase chain reaction (PCR)-massively parallel sequencing (MPS) technologies in forensics has changed the approach to allelic short tandem repeat (STR) typing because sequencing cloned PCR fragments enables alleles with identical molecular weights to be distinguished based on their nucleotide sequences. Therefore, because PCR fidelity mainly depends on template integrity, new technical issues could arise in the interpretation of the results obtained from the degraded samples. In this work, a set of DNA samples degraded in vitro was used to investigate whether PCR-MPS could generate "isometric drop-ins" (IDIs; i.e., molecular products having the same length as the original allele but with a different nucleotide sequence within the repeated units). The Precision ID GlobalFiler NGS STR panel kit was used to analyze 0.5 and 1 ng of mock samples in duplicate tests (for a total of 16 PCR-MPS analyses). As expected, several well-known PCR artifacts (such as allelic dropout, stutters above the threshold) were scored; 95 IDIs with an average occurrence of 5.9 IDIs per test (min: 1, max: 11) were scored as well. In total, IDIs represented one of the most frequent artifacts. The coverage of these IDIs reached up to 981 reads (median: 239 reads), and the ratios with the coverage of the original allele ranged from 0.069 to 7.285 (median: 0.221). In addition, approximately 5.2% of the IDIs showed coverage higher than that of the original allele. Molecular analysis of these artifacts showed that they were generated in 96.8% of cases through a single nucleotide change event, with the C > T transition being the most frequent (85.7%). Thus, in a forensic evaluation of evidence, IDIs may represent an actual issue, particularly when DNA mixtures need to be interpreted because they could mislead the operator regarding the number of contributors. Overall, the molecular features of the IDIs described in this work, as well as the performance of duplicate tests, may be useful tools for managing this new class of artifacts otherwise not detected by capillary electrophoresis technology.

Comparison of DNA preservation between ribs and vertebrae.

The choice of skeletal element types and their intra-bone parts is important because of differences in DNA preservation, and this must be considered when sampling bones for DNA testing. When incomplete skeletons are found, ribs and vertebrae have been shown to be the most suitable for genetic identification of bones from the torso. This study compares the preservation of DNA between 12th thoracic vertebrae and first ribs to determine which bone type is more suitable for genetic typing. The study analyzed 35 12th thoracic vertebrae and 29 first ribs from one mass grave from the Second World War with commingled skeletal remains excavated. Bone DNA preservation was estimated by measuring nuclear DNA concentration and its degradation and through short tandem repeat (STR) typing success. Previous studies performed on aged skeletal remains have shown that the DNA content of the first ribs and 12th thoracic vertebrae has high intra-bone variability, and this was considered when sampling the bones. After full demineralization extraction, the PowerQuant System (Promega) was used to measure the quantity and quality of DNA, and the GlobalFiler kit (Applied Biosystems) was used for STR typing. The results showed that DNA yield and degradation and STR typing success exhibited no statistically significant difference between first ribs and 12th thoracic vertebrae, and there was no intra-individual difference when comparing only paired bones from the same individuals. Consequently, with intra-bone DNA variability considered, the first ribs or the 12th thoracic vertebrae can be selected when sampling to genetically identify the skeletal remains of highly degraded torsos. HIGHLIGHTS: The first ribs and thoracic vertebrae are the most suitable bones for sampling from the torso. The proximal part of first rib and posterior vertebral column of the 12th thoracic vertebrae yielded the most DNA. The first ribs were compared with the 12th thoracic vertebrae, and the sampling process considered intra-bone DNA variability. The quality and quantity of nuclear DNA and success of STR typing were measured. The first ribs yielded the same DNA yields as well as STR typing success as the 12th thoracic vertebrae. When only the torso is present, it is not of high importance whether the first ribs or the 12th thoracic vertebrae are collected.

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.

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.

Genome-Wide DNA Methylation Patterns in Suicide Victims: Identification of New Candidate Genes.

Suicide is a major global public health problem with significant impact on society. According to the World Health Organization, every year about 800.000 people commit suicide, while at the global level suicide accounts for 50% of all violent deaths among men and for 71% among women. Suicide is a complex phenomenon which cannot be attributed to a single causal factor, but to a combination of simultaneous effects of multiple factors which are expressed in the form of psychological, biological and sociological indicators. Analysis of epigenetic mechanisms (methylation of the DNA, modifications of histone proteins and (networks of) miRNA), which link the interaction between genes and the environment, could importantly contribute to better understanding of suicidal behaviour. Recent studies on suicidal behaviour and DNA methylation show differences in DNA methylation pattern, with numerous sites among suicide victims. Using next generation sequencing, genome-wide studies helped identify novel candidate genes while studies of already known candidate genes (such as glucocorticoid receptor and BDNF) gave us better insight into the interplay of genetics and epigenetics. Epigenetic studies importantly contribute to elucidation of new biomarkers for suicidal behaviour. However, present studies are very different in design and often performed on very small samples, and these limitations could be overcome with more careful study preparation.

Rapidly mutating Y-STR analyses of compromised forensic samples.

Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were identified to improve differentiation of unrelated males and also to enable separating closely and distantly related males in human identity testing in forensic and other applications. RM-Yplex assay was developed as a single multiplex that is capable of simultaneously amplifying all currently known RM Y-STRs, and reproducibility and sensitivity testing were performed on reference samples. Additional analyses are necessary to test its suitability for analysing compromised forensic samples. For this purpose, we applied the RM-Yplex assay to approximately 70-year-old skeletons that were used as a model for poorly preserved, challenging forensic samples. We analysed 57 male skeletal remains (bones and teeth) from 55 skeletons excavated from the Second World War (WWII) mass graves in Slovenia. The RM-Yplex typing was successful in all 57 samples; there were 56% full profiles obtained, and in partial profiles, up to 7 locus drop-outs were observed and they appeared correlated with low DNA quantities and degradation of DNA obtained from WWII bone and tooth samples. The longest loci, DYS403S1b, DYS547, DYS627 and DYS526b, were the most often dropped-out RM Y-STRs. In spite of high frequency of drop-out events, the RM-Yplex typing was successful in all WWII samples, showing the possibility of successful amplification of at least half of the RM Y-STRs even from the most compromised samples analysed.

Differences in SNP genotype distributions between complex and simple suicides.

Dysregulations in serotonin neurotransmission can be a strong contributing factor in suicide and impulsive-aggressive personality traits. Victims of suicide form a heterogeneous group in terms of planning, lethality and number of used methods. In this study, we tested single nucleotide polymorphisms (SNPs) of the monoamine oxidase (MAO) A and B genes on the Slovenian population, which has one of the highest suicide rates in the world. Genotyping was performed on 77 victims of complex suicide, 406 victims of simple suicide and 289 controls. The differences in allele distribution were investigated with the two-tailed χ2 test. Haplotype analysis was performed on 740 subjects. Significant or tendency for significant differences in distribution was observed for all studied polymorphisms in the MAOA gene when comparing male victims of complex suicide, victims of simple suicide and controls. Minor allele frequencies in male victims of complex suicide were A 6.7% for rs3027407, C 13% for rs909525 and T 12.7% for rs1137070; in victims of simple suicide, A 36.3% for rs3027407, C 39.5% for rs909525 and T 36.4% for rs1137070; and in controls, A 25.2% for rs3027407, C 30.4% for rs909525 and T 25.8% for rs1137070. The distribution analysis of polymorphism rs1799836 in the MAOB gene and all studied polymorphisms in the MAOA gene in females failed to show any significant results. GTC haplotype (for rs3027407, rs909525, rs1137070) in MAOA polymorphisms was more frequent in victims of complex suicide compared to that in controls and victims of simple suicide. Compared to victims of complex suicide, male victims of simple suicide were more often carriers of MAOA alleles that are, according to literature, associated with higher levels of impulsivity and anger. These differences in SNP distribution could serve as an additional method of differentiating between victims of complex and victims of simple suicide. Further studies including psychological autopsies should be carried out in order to identify personality traits and behavioural differences among distinct groups of suicide victims.

Epigenetics and suicide: investigating altered H3K14ac unveiled differential expression in ADORA2A, B4GALT2 and MMP14.

Background: Environmental factors make an important contribution to suicide. Histone tails are prone to different modifications, leading to changes of chromatin (de)condensation and consequently gene expression. Materials & methods: Level of H3K14ac was studied with chromatin immunoprecipitation followed by high-throughput DNA sequencing. Genes were further validated with RT-qPCR; using hippocampal tissue. Results: We showed lowered H3K14ac levels in individuals who died by suicide. The genes ADORA2A, B4GALT2 and MMP14 showed differential expression in individuals who died by suicide. Identified genetic and protein interactions among genes show interactions with suicide-related genes. Conclusion: Further investigations of histone modifications in association with DNA methylation and miRNA are needed to expand our knowledge of the genes that could significantly contribute to suicide.

[Box: see text].

Reduced blood alcohol concentration in suicide victims in response to a new national alcohol policy in slovenia.

BACKGROUND: Addiction is a major social and health problem. Studies on suicide and alcohol at the individual and aggregated level have confirmed a link between alcohol and suicide. AIM: To assess the impact of the new national alcohol policy in Slovenia on the blood alcohol concentration (BAC) in BAC-positive suicide victims before, during and after the implementation of the new national alcohol policy in 2003. METHOD: Blood samples were collected by forensic pathologists during medicolegal autopsies of suicide victims in order to establish their BAC levels at the time of death. BAC was measured using two routine independent headspace gas chromatography methods (HSS-GC-FID) and expressed in grams per kilogram. RESULTS: During the period before the implementation of the act which limited the availability of alcohol in Slovenia, the BACs of BAC-positive suicide victims were higher than those tested in the period after the implementation of the act. CONCLUSION: Despite certain limitations, this study demonstrates that legislation measures restricting alcohol availability may be an effective measure of BAC reduction in BAC-positive suicide victims.

Tryptophan hydroxylase 2 (TPH 2) single nucleotide polymorphisms, suicide, and alcohol-related suicide.

BACKGROUND: Suicide has been identified as a serious public health problem that is often accompanied by alcohol misuse and dependence. It seems that suicide is a result of an interplay between distal (e.g. genetic loading, family history of suicide) and proximal factors (e.g. existence of psychiatric disorder, events conferring acute stress), as well as their interactions. However, like suicide, alcohol dependence seems to be a multifactorial disorder caused by genetic and environmental factors. Serotonergic dysfunction has been implicated to be involved in the pathophysiology of substance abuse, and has also an important role in suicidal behaviour. Studies investigating suicide, alcohol-related suicide and the rate limiting enzyme of serotonin synthesis, tryptophan hydroxylase 2 (TPH2), remain to date rather limited. RESULTS: Recent studies of TPH2 showed a range of strong, mild or no association with suicide and alcohol-related suicide, depending on a study group and genetic variants tested. Overall, to date the clinical effects seems to be quite modest. Among suicide victims with more impulsive and verbal aggressive behaviour more alcohol misuse or dependency was present. CONCLUSIONS: Suicide and alcoholism are often comorbid disorders with a complex nature. They are both strongly linked to serotonin modulation, and therefore association studies of SNPs in genes from the serotonergic system could provide an insight into the genetic background of such disorders. However, based on current results we cannot draw any conclusions, but further research to clarify the interplay between serotonergic system dysfunction, suicide, alcohol dependence, impulsivity and the role of TPH2 enzyme is needed.

The role of brain-derived neurotrophic factor in the pathophysiology of suicidal behavior.

Suicidal behaviour is a major public health concern. It is known that the pathogenesis of suicidal behaviour involves altered neural plasticity, resulting in the aberrant stress response of the central nervous system to environmental factors. Indeed, altered brain structure and function was found in suicide victims. Neurotrophins are growth factors that are involved in the regulation of structural, synaptic, and morphological plasticity and in the modulation of the strength and number of synaptic connections and neurotransmission. Brain-derived neurotrophic factor (BDNF) the most studied and the most widely distributed among neurotrophins binds to a tropomyosin-related kinase B (TrkB) receptor and to a pan75 neurotrophins receptor. It has been reported that BDNF production is decreased in all patients with suicidal behaviour and in all suicide victims regardless of a psychiatric diagnosis. It was also found that the mRNA and protein level of BDNF was significantly lower in both the prefrontal cortex and the hippocampus of suicide subjects. Different mechanisms could be involved in the regulation of BDNF gene expression, among which epigenetic mechanisms seem to play a key role. However, also for a functional polymorphism (rs6265) Val66Metit has been shown that the Met allele is associated with the reduced BDNF activity. Further, a recent meta-analysis including 12 studies showed a trend for the Met-carrying genotypes and Met allele conferring risk for suicide. Among included studies, our study with the largest sample size, indicated that the combined Met/Met and Met/Val genotypes of the BDNF Val66Met variant could be the risk factor for violent suicide in female subjects and for suicide in victims exposed to childhood trauma. In accordance with previous reports, our findings demonstrate that aberrant regulation of BDNF synthesis is associated with suicidal behaviour.

Targeted sequencing approach: Comprehensive analysis of DNA methylation and gene expression across blood and brain regions in suicide victims.

OBJECTIVES: Epigenetic mechanisms are involved in regulation of many pathologies, including suicidal behaviour. However, the factors through which epigenetics affect suicidal behaviour are not fully understood. METHODS: We analysed DNA methylation of eight neuropsychiatric genes (NR3C1, SLC6A4, HTR1A, TPH2, SKA2, MAOA, GABRA1, and NRIP3) in brain regions (hippocampus, insula, amygdala, Brodmann area 46) and blood of 25 male suicide victims and 28 male control subjects, using bisulphite next-generation sequencing. RESULTS: Comparing mean methylation values, notable changes were observed in NR3C1 (insula p-value = 0.05), HTR1A (insula p-value < 0.001, blood p-value = 0.001), SKA2 (insula p-value = 0.03, blood p-value = 0.016), MAOA (blood p-value < 0.001), GABRA1 (insula p-value = 0.05, blood p-value = 0.024) and NRIP3 (hippocampus p-value = 0.001, insula p-value = 0.002, amygdala p-value = 0.014). Comparing methylation pattern between blood and brain, similarity was observed between blood and insula for HTR1A. Gene expression analysis in hippocampus revealed changes in expression of NR3C1 (p-value = 0.049), SLC6A4 (p-value = 0.017) and HTR1A (p-value = 0.053). CONCLUSIONS: Results provide an insight into the altered state of DNA methylation in suicidal behaviour. Epigenetic differences could therefore affect suicidal behaviour in both previously known and in novel neuropsychiatric candidate genes.

A Multisample Approach in Forensic Phenotyping of Chronological Old Skeletal Remains Using Massive Parallel Sequencing (MPS) Technology.

It is very important to generate phenotypic results that are reliable when processing chronological old skeletal remains for cases involving the identification of missing persons. To improve the success of pigmentation prediction in Second World War victims, three bones from each of the eight skeletons analyzed were included in the study, which makes it possible to generate a consensus profile. The PowerQuant System was used for quantification, the ESI 17 Fast System was used for STR typing, and a customized version of the HIrisPlex panel was used for PCR-MPS. The HID Ion Chef Instrument was used for library preparation and templating. Sequencing was performed with the Ion GeneStudio S5 System. Identical full profiles and identical hair and eye color predictions were achieved from three bones analyzed per skeleton. Blue eye color was predicted in five skeletons and brown in three skeletons. Blond hair color was predicted in one skeleton, blond to dark blond in three skeletons, brown to dark brown in two skeletons, and dark brown to black in two skeletons. The reproducibility and reliability of the results proved the multisample analysis method to be beneficial for phenotyping chronological old skeletons because differences in DNA yields in different bone types provide a greater possibility of obtaining a better-quality consensus profile.

A fast and highly efficient automated DNA extraction method from small quantities of bone powder from aged bone samples.

An efficient extraction method is important for obtaining high-quality DNA from degraded aged bone samples. An automated full-demineralization method using the EDTA and DNA Investigator Kit (Qiagen) combined with Qiagen's biorobots was optimized in our laboratory in the past to extract the DNA from 500 mg of aged bone samples. The purpose of this research was to further improve the method with the aim of reducing the required sample material, shortening the extraction time, and achieving higher throughput. To process extremely small samples, the amount of bone powder was reduced to 75 mg, EDTA was replaced with reagents from the Bone DNA Extraction Kit (Promega), and decalcification was shortened from overnight to 2.5 h. Instead of 50 ml tubes, 2 ml tubes were used, which allows higher throughput. The DNA Investigator Kit (Qiagen) and EZ1 Advanced XL biorobot (Qiagen) was used for DNA purification. A comparison between both extraction methods was made on 29 Second World War bones and 22 archaeological bone samples. The differences between both methods were explored by measuring nuclear DNA yield and STR typing success. After cleaning the samples, 500 mg of bone powder was processed using EDTA, and 75 mg of powder from the same bone was processed using the Bone DNA Extraction Kit (Promega). DNA content and DNA degradation were determined using PowerQuant (Promega), and the PowerPlex ESI 17 Fast System (Promega) was used for STR typing. The results showed that the full-demineralization protocol using 500 mg of bone was efficient for Second World War and archaeological samples, and the partial-demineralization protocol using 75 mg of bone powder was only efficient for the Second World War bones. The improved extraction method-for which significantly lower amounts of bone powder can be used, the extraction process is faster, and higher throughput of bone samples is possible-is applicable for genetic identification of relatively well-preserved aged bone samples in routine forensic analyses.

Extracellular vesicles from cerebrospinal fluid revealed changes in miR-19a-3p and miR-4516 expression in Slovene male suicides.

Suicide is an important public-health concern, with more than 700,000 people dying by suicide yearly. It is a multifactorial phenomenon, shaped by the effects of sociodemographic, environmental and biological factors. The latter two factors can be linked through epigenetic studies, which examine differences in gene expression that are not due to changes in the DNA sequence itself. Epigenetic mechanisms include micro RNAs (miRNAs), which have a direct effect on already translated mRNA, leading to either decay or translational repression of the target mRNA. MiRNA molecules have been identified as cargo of extracellular vesicles (EVs) used by cells for long-distance communication, and pathophysiological changes in miRNA in brain cells may be reflected in cerebrospinal fluid (CSF) vesicles. In this study we investigated the presence and differential expression of selected miRNAs in EVs from the CSF of male suicide completers and controls. Western blot and nanoparticle tracking analyses confirmed the presence of small and medium sized EVs. Of the miRNA analyzed (miR-16-5p, miR-19a-3p, miR-34c-5p, miR-17-5p, miR-4286, miR-26b-5p, miR-381-3p, and miR-4516) miR-19a-3p and miR-4516 reached statistical significance with p-values of 0.0408 and 0.0168, respectively. Mir-4516 and miRNA-19a-3p have been previously studied in suicide, and target SLC6A4 and TNF-α expression, correspondingly. Approximately 70% of known miRNAs are expressed in the central nervous system, and therefore represent an important biomarker potential. Investigating the cargo of CFS and blood EVs would further support the identification of miRNAs with clinical use potential.