DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay.

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1-94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28-86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Determination of "new psychoactive substances" in postmortem matrices using microwave derivatization and gas chromatography-mass spectrometry.

Despite worldwide efforts aiming to ban the marketing and subsequent abuse of psychoactive substances such as synthetic cathinones and phenethylamines, there has been an alarming growth of both in recent years. Different compounds similar to those already existing are continuously appearing in the market in order to circumvent the legislation. An analytical methodology has been validated for qualitative and quantitative determinations of D-cathine (D-norpseudoehedrine), ephedrine, methcathinone, 1-(4-methoxyphenyl)-propan-2-amine (PMA), mephedrone, methedrone, 2,5-dimethoxy-4-methylamphetamine (DOM), 4-bromo-2,5-dimethoxyamphetamine (DOB), 2,5-dimethoxyphenethylamine (2C-H), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 4-iodo-2,5-dimethoxyphenethylamine (2C-I), 2-[2,5-dimethoxy-4-(ethylthio)phenyl]ethanamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4) and 2-[2,5-dimethoxy-4-(propylthio)phenyl]ethanamine (2C-T-7), in low volumes of vitreous humor (100 µL), pericardial fluid (250 µL) and whole blood (250 µL), using deutered amphetamine, ephedrine and mephedrone as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantification, intra- and interday precision and trueness, recovery and stability. The method included mixed-mode solid phase extraction, followed by microwave fast derivatization and analysis by gas chromatography-mass spectrometry operated in selected ion monitoring mode. The procedure was linear between 5 and 600 ng/mL, with determination coefficients higher than 0.99 for all analytes. Intra- and interday precision ranged from 0.1 to 13.6%, while accuracy variability was within 80-120% interval from the nominal concentration at all studied levels. The extraction efficiencies ranged from 76.6 to 112.8%. Stability was considered acceptable for all compounds in the studied matrices. The developed assay was applied to authentic samples of the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Carbon monoxide poisoning as a cause of death and differential diagnosis in the forensic practice: a retrospective study, 2000-2010.

This study presents the epidemiology and the postmortem forensic aspects in cases with a carboxihemoglobin (COHb) analysis, from autopsies performed at the Forensic Pathology Department of the Centre Branch of the National Institute of Legal Medicine and Forensic Sciences of Portugal. Between January 2000 and December 2010, 69 COHb analyses were requested in our institution. In approximately 70% of the situations, circumstantial information included a Carbon Monoxide (CO) source at the death scene. More than half of the cases presented thermal lesions, cherry-red lividity, and cherry-red blood and viscera coloration were found in, approximately, 30% of the cases. Fourteen cases were recorded as CO poisonings. The highest number of poisonings occurred in 2000, with most of the cases in winter (53.8%), in 51-60 years-old male individuals. 69.2% of the poisonings were accidental and the remainder were suicides, being fires the most frequent sources of CO (38.5%). Cherry-red lividity was present in 61.5% of the cases, and all of them presented cherry-red blood and viscera coloration. Older individuals and those with thermal lesions presented lower COHb levels, and politrauma was the most frequent cause of death among the negative cases. It is possible to conclude that the forensic aspects of CO poisonings interact in a complex way, and differential diagnosis is not straightforward. This study also emphasizes the role played by public prevention campaigns and improvement of heating appliances in reducing the number of accidental CO poisonings, and the importance of preventing urban and forest fires, the major source of CO among us.

Suicides in the Centre of Portugal: seven years analysis.

As one of the more specific and distinctive problems of human beings, suicide has been investigated with increasing attention all over the world. Several risk factors have been described as well as limitations arising from their study. The World Health Organization estimates that this scourge affects one million people annually, which corresponds to one death every 40s worldwide. According to recent studies, Portugal, despite the good rates (10 suicide deaths per 100,000 inhabitants), had shown an increasing trend among younger people. This work aims to characterize the evolution of the suicidal profile autopsied at the Forensic Pathology Department of the Centre Branch of the National Institute of Legal Medicine and Forensic Sciences of Portugal, analyzing several variables: age, gender, marital status, employment status, suicidal methodology, toxicological analysis and some conditions/behaviors regarding personal history (alcoholism, suicidal ideation, suicide attempts, physical illness, psychiatric disorder). All the autopsies from the 1 January, 2003 to 31 December, 2009 were analyzed. The suicide profile achieved corresponded to a man (77%), aged between 65 and 74 years old (20.4%), married (54.5%), employed, who committed suicide by hanging, in September, May or February. Clinical records include an organic health problem or psychiatric one, in addition to risk behaviors such as alcoholism, suicidal thoughts or suicide attempts. The number of suicides autopsied at the Centre Branch has increased, resembling the profile to the result of many other authors. However, new medical and social developments place hanging as the favorite suicide method in our study. Many barriers remain to overthrow but several prevention programs begin to be designed and implemented. Future evaluations and interventions at the social and medical level, including the death certification process, will be fundamental to a better realistic understanding of this phenomenon.

A validated procedure for detection and quantitation of salvinorin a in pericardial fluid, vitreous humor, whole blood and plasma using solid phase extraction and gas chromatography-mass spectrometry.

The use of vitreous humor and pericardial fluid as alternative matrices to blood and plasma in the field of forensic toxicology is described to quantitate low levels of Salvinorin A using ethion as internal standard. The method was optimized and fully validated using international accepted guidelines. The developed methodology utilizes a solid phase extraction procedure coupled to gas chromatography mass spectrometry operated in the selected ion monitoring mode. The method was linear in the range of 5.0-100ng/mL with determination coefficients higher than 0.99 in 100µL of vitreous humor and in 250µL of each matrix pericardial fluid, whole blood and plasma. The limits of detection and quantitation were experimentally determined as 5.0ng/mL, intra-day precision, intermediate precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The sample cleanup step presented mean efficiencies between 80 and 106% in the different biological specimens analyzed. According to the low volumes of samples used, and the low limits achieved using a single quadrupole mass spectrometer, which is available in most laboratories, we can conclude that the validated methodology is sensitive and simple and is suitable for the application in forensic toxicology laboratories for the routine analysis of Salvinorin A in both conventional and unconventional biological samples.

Prevalence of ethanol and illicit drugs in road traffic accidents in the centre of Portugal: An eighteen-year update.

This study presents the prevalence of ethanol and illicit drugs in fatal road traffic accident victims in the Centre of Portugal between January 1990 and December 2007. Among the violent deaths, road traffic accidents presented the highest percentage (around 35%; n=3095), but decreasing throughout the years. Accidents were preponderant in males (about 80%; n=2402), between 21 and 30 years-old. Accidents involving drivers (55%; n=1310) were of the most common, being the car the main vehicle (45%), followed by the motorcycle (40%). An alcohol analysis request was present in 50% of the cases (n=1687), but increasing each year. Ethanol concentrations >1.2g/L, the legal limit in Portugal, were found in 55% (n=283) of the cases. Concerning drugs of abuse requests, only 4.4% (n=137) and 17.3% (58 cases) of the cases included the analysis at the Forensic Pathology Department (FPD) and at the Medico-Legal Office (MLO), respectively. Among the road accident cases analysed, 18 were positive, mainly in men (84%), between 21 and 30 years-old; opiates (47.1%; n=8) and cannabinoids (50%; n=4) were the most found, at the FPD and at the MLO, respectively. In conclusion, ethanol was identified as a key factor to traffic accidents, which explains the definition of specific legislation and methods of enforcement to prohibit this form of impairing. Nevertheless, ethanol still remains the psychoactive substance most frequently identified in the blood of divers killed in road-traffic crashes, recommending additional actions of supervision and control.

Analysis of paternal lineages in Brazilian and African populations.

The present-day Brazilian population is a consequence of the admixture of various peoples of very different origins, namely, Amerindians, Europeans and Africans. The proportion of each genetic contribution is known to be very heterogeneous throughout the country. The aim of the present study was to compare the male lineages present in two distinct Brazilian populations, as well as to evaluate the African contribution to their male genetic substrate. Thus, two Brazilian population samples from Manaus (State of Amazon) and Ribeirão Preto (State of São Paulo) and three African samples from Guinea Bissau, Angola and Mozambique were typed for a set of nine Y chromosome specific STRs. The data were compared with those from African, Amerindian and European populations. By using Y-STR haplotype information, low genetic distances were found between the Manaus and Ribeirão Preto populations, as well as between these and others from Iberia. Likewise, no significant distances were observed between any of the African samples from Angola, Mozambique and Guinea Bissau. Highly significant Rst values were found between both Brazilian samples and all the African and Amerindian populations. The absence of a significant Sub-Saharan African male component resulting from the slave trade, and the low frequency in Amerindian ancestry Y-lineages in the Manaus and Ribeirão Preto population samples are in accordance with the accentuated gender asymmetry in admixture processes that has been systematically reported in colonial South American populations.

Analysis of paternal lineages in Brazilian and African populations

The present-day Brazilian population is a consequence of the admixture of various peoples of very different origins, namely, Amerindians, Europeans and Africans. The proportion of each genetic contribution is known to be very heterogeneous throughout the country. The aim of the present study was to compare the male lineages present in two distinct Brazilian populations, as well as to evaluate the African contribution to their male genetic substrate. Thus, two Brazilian population samples from Manaus (State of Amazon) and Ribeirão Preto (State of São Paulo) and three African samples from Guinea Bissau, Angola and Mozambique were typed for a set of nine Y chromosome specific STRs. The data were compared with those from African, Amerindian and European populations. By using Y-STR haplotype information, low genetic distances were found between the Manaus and Ribeirão Preto populations, as well as between these and others from Iberia. Likewise, no significant distances were observed between any of the African samples from Angola, Mozambique and Guinea Bissau. Highly significant Rst values were found between both Brazilian samples and all the African and Amerindian populations. The absence of a significant Sub-Saharan African male component resulting from the slave trade, and the low frequency in Amerindian ancestry Y-lineages in the Manaus and Ribeirão Preto population samples are in accordance with the accentuated gender asymmetry in admixture processes that has been systematically reported in colonial South American populations.

DNA typing of Diptera collected from human corpses in Portugal.

Medico-legal entomology, one area in the broad field of entomology, is routinely used in forensic applications. Insects are often collected from a corpse during criminal information related to the body, but requires the fast and accurate identification of the species attracted to the remains. The local entomofauna in most cases is important for explaining entomological evidence. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different species on corpses. A morphological and DNA-based methods for species identification were used in this study. Thirty-two cases are reported from indoors and outdoors conditions. Specimens were collected from corpses during autopsy procedures in the National Institute of Legal Medicine, Portugal, and cases were summarized by sex, death local, month of discovery, probable cause of death, species found and number of analyzed specimens. Just eight species, mainly Calliphoridae together with one Sarcophagidae were reported from corpses. The DNA sequencing was performed to study the mitochondrial encoded subunit I of the cytochrome oxidase gene. The aim of this work was the beginning of a database of the cadaveric entomofauna in Portugal.

Identification of West Eurasian mitochondrial haplogroups by mtDNA SNP screening: results of the 2006-2007 EDNAP collaborative exercise.

The European DNA Profiling (EDNAP) Group performed a collaborative exercise on a mitochondrial (mt) DNA screening assay that targeted 16 nucleotide positions in the coding region and allowed for the discrimination of major west Eurasian mtDNA haplogroups. The purpose of the exercise was to evaluate the stability and reproducibility of the self-developed multiplex-PCR and multiplex-single base extension kit by blind-testing saliva and hair shaft samples provided by the organizing laboratory. The overall success rate in obtaining useful results was high given that some of the participating laboratories had no previous experience with the technology and/or mtDNA analysis. The results of this collaborative exercise stimulate the expansion of screening methods in forensic laboratories to increase efficiency and performance of mtDNA typing, and thus demonstrates that mtDNA SNP typing is a powerful tool for forensic casework analysis.

Y-chromosome STR haplotypes in a southwest Spain population sample.

The Y-chromosome polymorphism of eight STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392; DYS393, DYS385) were studied in 111 unrelated individuals from the population of southwest Spain. The most common haplotype was shared by 3.6% of the sample, while 99 haplotypes were unique. The gene diversity was 0.9977.