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.

Diffuse Axonal Injury-A Distinct Clinicopathological Entity in Closed Head Injuries.

The knowledge about the diffuse axonal injury (DAI) as a clinicopathological entity has matured in the last 30 years. It has been defined clinically (immediate and prolonged unconsciousness leading to death or severe disability) and pathologically (the triad of DAI specific changes). In terms of its biomechanics, DAI is occurring as a result of acceleration forces of longer duration and has been fully reproduced experimentally.In the process of diagnosing DAI, the performance of a complete forensic neuropathological examination is essential and the immunohistochemistry method using antibodies against ß-amyloid precursor protein (ß-APP) has been proved to be highly sensitive and specific, selectively targeting the damaged axons.In this review, we are pointing to the significant characteristics of DAI as a distinct clinicopathological entity that can cause severe impairment of the brain function, and in the forensic medicine setting, it can be found as the concrete cause of death. We are discussing not only its pathological feature, its mechanism of occurrence, and the events on a cellular level but also the dilemmas about DAI that still exist in science: (1) regarding the strict criteria for its diagnosis and (2) regarding its biomechanical significance, which can be of a big medicolegal importance.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

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.

Identical human papillomavirus (HPV) genomic variants persist in recurrent respiratory papillomatosis for up to 22 years.

Seventy initial and 125 follow-up tissue specimens of laryngeal papillomas, obtained from 70 patients who had had recurrent respiratory papillomatosis for from 1-22 years, were investigated for the presence of human papillomavirus (HPV) DNA and HPV E5a, LCR and/or full-length genomic variants. HPV-6 was found in 130/195, HPV-11 in 63/195, and HPV-6/HPV-11 in 2/195 samples. Within 67/70 (95.7%) patients, all follow-up HPV isolates genetically matched completely initial HPV isolate over the highly variable parts of the genome or over the entire genome. Frequent recurrence of laryngeal papillomas is a consequence of long-term persistence of the identical initial HPV genomic variant.

Highly efficient nuclear DNA typing of the World War II skeletal remains using three new autosomal short tandem repeat amplification kits with the extended European Standard Set of loci.

AIM: To perform an efficiency study of three new amplification kits with the extended European Standard Set (ESS) of loci for autosomal short tandem repeat (STR) typing of skeletal remains excavated from the World War II mass graves in Slovenia. METHODS: In the beginning of the 2011, we analyzed 102 bones and teeth using the PowerPlex ESX 17 System (Promega), AmpFiSTR NGM PCR Amplification Kit (Applied Biosystems), and Investigator ESSplex Kit (Qiagen). We cleaned the bones and teeth, removed surface contamination, and ground them into a powder using liquid nitrogen. Prior to DNA isolation with Biorobot EZ1 (Qiagen), 0.5 g bone or tooth powder was decalcified. Nuclear DNA of the samples was quantified using real-time polymerase chain reaction. All three kits used the same extract with the amplification conditions recommended by the manufacturers. RESULTS: We extracted up to 131 ng DNA/g of powder from the bones and teeth. All three amplification kits showed very similar efficiency, since DNA typing was successful with all amplification kits in 101 out of 102 bones and teeth, which represents a 99% success rate. CONCLUSION: The commercially available ESX 17, ESSplex, and NGM kits are highly reliable for STR typing of World War II skeletal remains with the DNA extraction method optimized in our laboratory.

An MRI study of the differences in the rate of thrombolysis between red blood cell-rich and platelet-rich components of venous thrombi ex vivo.

PURPOSE: To test whether T(1)-weighted MRI can detect the differences in the rate of thrombolysis induced by recombinant tissue plasminogen activator (rt-PA) between platelet-rich regions and red blood cell (RBC)-rich regions of venous thrombi ex vivo. MATERIALS AND METHODS: Each of 21 venous thrombi ex vivo (8 pulmonary emboli and 13 in situ thrombi) was dissected along the longitudinal axis. Half of it was analyzed for the presence of platelet, fibrin, and RBC components by immunohistochemistry and the other half was imaged serially by high-resolution T(1)-weighted three-dimensional MRI to assess the progression of thrombolysis. The MR images were analyzed for proportions of the remaining platelet-rich and RBC-rich regions. RESULTS: Laminated platelet-rich regions, corresponding to Zahn lines, were confirmed immunohistochemically and by MRI in 18/21 venous thrombi. In T(1)-weighted MR images (TE/TR = 10/105 ms) the mean signal intensity of platelet-rich regions was on average 2.3 higher than that of RBC-rich regions. The rate of thrombolysis in platelet-rich regions was on average 30% lower than in RBC-rich regions. After 120 min of thrombolysis the proportion of lysed platelet-rich regions was 0.27 ± 0.04 versus 0.40 ± 0.08 in RBC regions, which resulted in 1.4% decrease of lysed thrombus volume per 1% increase of platelet-rich content. CONCLUSION: Venous thrombi are most often composed of interspersed platelet-rich and RBC-rich regions. T(1) -weighted MRI is capable of noninvasive discrimination between those two components of venous thrombi ex vivo which have a different susceptibility to thrombolysis by rt-PA.

Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia.

This paper describes molecular genetic identification of one third of the skeletal remains of 88 victims of postwar (June 1945) killings found in the Konfin I mass grave in Slovenia. Living relatives were traced for 36 victims. We analyzed 84 right femurs and compared their genetic profiles to the genetic material of living relatives. We cleaned the bones, removed surface contamination, and ground the bones into powder. Prior to DNA isolation using Biorobot EZ1 (Qiagen), the powder was decalcified. The nuclear DNA of the samples was quantified using the real-time polymerase chain reaction method. We extracted 0.8 to 100 ng DNA/g of bone powder from 82 bones. Autosomal genetic profiles and Y-chromosome haplotypes were obtained from 98% of the bones, and mitochondrial DNA (mtDNA) haplotypes from 95% of the bones for the HVI region and from 98% of the bones for the HVII region. Genetic profiles of the nuclear and mtDNA were determined for reference persons. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and mtDNA of all persons that had been in contact with the skeletal remains. When comparing genetic profiles, we matched 28 of the 84 bones analyzed with living relatives (brothers, sisters, sons, daughters, nephews, or cousins). The statistical analyses showed a high confidence of correct identification for all 28 victims in the Konfin I mass grave (posterior probability ranged from 99.9% to more than 99.999999%).

Suicide, stress and serotonin receptor 1A promoter polymorphism -1019C>G in Slovenian suicide victims.

Implication of serotonergic system in suicide and suicide attempts has been discussed for several years. One of the most abundant serotonin receptors in the mammalian brain is the receptor 1A (5-HT1A); studies of its polymorphisms and suicide have provided very inconsistent results so far. The suggestion that the G allele depresses HTR1A autoreceptor expression, and therefore reduces serotonergic neurotransmission that might predispose to depression and suicide, made the promoter polymorphism -1019C>G a very promising candidate gene. In our study we analyzed promoter polymorphism -1019C>G on 323 suicide victims and 190 controls (all of Slovenian origin), taking into account sex, suicide method, and in case of suicide victims also stressful life events. Differences in the distributions of genotype and allele frequencies were not statistically significant between suicide victims and control group, and the same was found for distributions according to sex and suicide method. For 62 suicide victims information about stressful life events in the month prior to the suicide and in childhood was provided. For analysis we combined CG/GG genotypes and compared them to the CC genotype. More stressful life events in the month prior to the suicide were reported for the subgroup with CC genotype (mean number of events = 2.53; SD = 1.50) in comparison to subgroup with CG/GG genotypes (mean number of events = 1.58; SD = 1.32; P < 0.05). However, subgroups of suicide victims with CC or CG/GG genotypes did not differ regarding numbers of reported stressful life events in childhood (P > 0.05). Our study provides no evidence for the implication of HTR1A promoter polymorphism in suicide in general, but it suggests further studies that would take into account the interconnected network of suicide completion, genetic background and stress, beside other risk factors.

Expression of cyclooxygenase-1 and cyclooxygenase-2 in the normal human heart and in myocardial infarction.

INTRODUCTION: Cyclooxygenase is a key enzyme in prostanoid synthesis. It exists in two isoforms: cyclooxygenase-1 (COX-1), which is constitutively expressed in cells and tissues maintaining normal homeostasis, and cyclooxygenase-2 (COX-2), which is normally not present in most cells, but can be induced by various stimuli. Little is known about the significance of COX isoforms in the normal human heart and in myocardial infarction (MI). Thus, we aimed to investigate the immunohistochemical expression of COX-1 and COX-2 in the normal human heart and in MI. METHODS: Our study included autopsy samples of heart tissue from 15 healthy individuals who died in accidents, and from 40 patients with MI who died few hours to a month after the onset of symptoms. Immunohistochemistry was performed by a sensitive peroxidase-streptavidin method on formalin fixed, paraffin-embedded tissue, using monoclonal antibodies against COX-1 and COX-2. RESULTS: In normal hearts, COX-1 was found in endothelial and smooth muscle cells of blood vessels and in endothelial cells of the endocardium. In MI, it was expressed in inflammatory cells, as well as in myofibroblasts and capillaries of granulation and fibrous tissue. COX-2 was either not present or it was present in occasional myocytes in the normal hearts. In MI, its expression was induced in cardiomyocytes as well as in interstitial inflammatory cells, and in capillaries and myofibroblasts in granulation tissue. CONCLUSIONS: Our results suggest that COX-1 is associated with normal homeostasis in the heart, whereas COX-2 probably mediates inflammatory reaction in MI. It appears that both COX-1 and COX-2 are associated with the healing processes and scar formation after MI.

Characterization of pulmonary emboli ex vivo by magnetic resonance imaging and ultrasound.

INTRODUCTION: Magnetic resonance imaging (MRI) and transesophageal ultrasound (US) are promising methods for detection and characterization of central pulmonary emboli. Both methods employ different physical principles. We tested how US and MRI characterized pulmonary emboli ex vivo. METHODS: Thirty six ex vivo pulmonary emboli, obtained during routine autopsies of patients who died of massive pulmonary embolism, were subjected to US imaging (linear vascular probe, 5.7-10 MHz) and to high resolution three-dimensional T1-weighted spin-echo MRI. In another 3 pulmonary thromboemboli and 2 tumor emboli, we compared MRI with immunohistochemistry to platelets, red blood cells and renal carcinoma cells. We also studied model clots in vitro (retracted and non-retracted red whole-blood clots, platelet aggregates and compacted and non compacted fibrin-rich plasma clots) with MRI and US. RESULTS: T1-weighted MR images of pulmonary thromboemboli consistently showed dark regions that corresponded to red cell-rich regions and bright layers that corresponded to platelet aggregates, but bright signal was obtained also from viable carcinoma cells and necrotic regions in tumor emboli. US images provided less structural detail than MRI, but clot retraction or compaction increased image brightness. The correlation between US and MRI characteristics of pulmonary emboli was poor. CONCLUSIONS: T1-weighted MRI of pulmonary emboli is capable of non-invasive assessment of the red cell-rich and platelet-rich components of pulmonary thromboemboli. US imaging shows increased brightness with clot retraction or compaction. Thus, both methods detect clot characteristics that influence susceptibility to thrombolytic treatment.

Genetic changes in Slovenian patients with gastric adenocarcinoma evaluated in terms of microsatellite DNA.

OBJECTIVES: Adenocarcinoma of the stomach is a relatively frequent malignant disease in Slovenia. We investigated the frequency of microsatellite instability (MSI) and loss of heterozygosity (LOH) in gastric carcinomas from the Slovenian population to determine their prognostic significance. METHODS: We evaluated MSI of mismatch repair associated loci and LOH on loci associated with following tumour suppressors: APC, nm23, Rb and p53. Results of the multiplex-PCR amplifications were correlated with clinicopathological factors for 73 patients. RESULTS: LOH was found in 52% of informative samples (20.5% LOH-H; 31.5% LOH-L). We found correlation of MSI with low-frequency LOH (LOH-L) in 11% of cases and with high-frequency LOH (LOH-H) tumours in 4% of cases. LOH-H and high-frequency MSI (MSI-H) were not associated. LOH was found in APC 36%, p53 33%, Rb 24% and nm23 33% of informative samples, whereas MSI was found in 30% of samples (12% MSI-H; 18% MSI-L). LOH-H status was associated with ulceration (P=0.029). LOH-N status was associated with diagnosis at higher TNM status (0.074) and infiltrative growth (P=0.006). Interestingly, in 6% of samples we found MSI on LOH loci as well. MSI-H was associated with higher age at diagnosis (r=0.24; P=0.04), antral location (r=0.252; P=0.04), intestinal type (P=0.044), expansive growth (P=0.001), tubular type (0.014), better differentiation (P=0.01), less nodal involvement (0.006) and better survival (P=0.022). The poorest prognosis was found in patients with both low-frequency MSI (MSI-L) and low-frequency LOH (LOH-L) tumours. CONCLUSION: The experimental design presented in the study may be of potential value for clinicians: at least five relevant markers for both MSI and LOH analysis may be needed to evaluate a gastric cancer (GC) patient's clinical status.

Fingerprint recovery from human skin surfaces.

A study was conducted to investigate whether certain dactyloscopic powders and reagents can recover latent fingerprints on human skin surfaces. Four fingerprint powders, Magnetic Jet Black, Magnetic Silver, Silver Special, Swedish Black, and two other methods, cyanoacrylate fuming (CA) and Ruthenium tetroxide (RTX), were used. Having examined skin surfaces with a forensic light source, we observed that the fingerprint impressions remained visible up to 15 min after intentionally placing them on the skin surface of living subjects and dead bodies. Finger marks were recovered and positive results were achieved with Magnetic Black and Swedish Black powder on living subjects. On dead bodies finger marks treated with cyanoacrylate were visible but those treated with RTX, Swedish Black and Magnetic Jet Black powder were useful for potential comparison. On dead bodies best results were obtained with RTX method.

Prediction of autosomal STR typing success in ancient and Second World War bone samples.

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.

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.

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31-46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.

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.

Computer simulation of the accident with nine victims.

In the paper we wish to emphasise the significance of vehicle driving dynamics analysis in the collision phase and occupant load analysis by means of using a software environment. Thereby we also wish to present the results of the simulation of the course of a traffic accident with nine victims that arose from a collision between an Audi A6 passenger car and the VW Caravelle van. In treating the traffic accident the forensic expert was faced with the questions about what caused the injuries to the front passenger in the Audi A6 passenger car, about the way the two vehicles had collided, about their collision velocities, about the way the two vehicles were handled and about the causes that originated the traffic accident. The critical situation on the road was a consequence of the tiredness of the van driver, the inadequate use of the passive safety systems and overloading the van.

Searching for the mother missed since the Second World War.

The aim of the study was to perform the genetic identification of a human cranium from a Second World War gravesite in Slovenia and find out if it belonged to the mother of a woman used as a family reference. Both genetic and anthropological examinations were carried out. The genetic examination was performed on 2 molars and petrous bone. Prior to DNA isolation 0.5 g of tooth and bone powder was decalcified. The DNA was purified in a Biorobot EZ1 (Qiagen) device. The nuclear DNA of the samples was quantified and short tandem repeat (STR) typing performed using two different autosomal and Y-STR kits. Up to 22.4 ng DNA/g of powder was obtained from samples analyzed. We managed to obtain nuclear DNA for successful STR typing from the left second molar and from the petrous bone. Full autosomal genetic profile including amelogenin locus revealed the male origin of the cranium that was further confirmed by the analyses of Y-STRs. The same conclusions were adopted after the anthropological analysis which identified the cranium as that of a very young Caucasoid male. The male origin of the cranium rejected the possibility of motherhood for the compared daughter. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and Y-STRs of all persons that had been in contact with the analyzed cranium and no match was found.

Highly efficient automated extraction of DNA from old and contemporary skeletal remains.

We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation - ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories.