Post-mortem genetic investigation in sudden cardiac death victims: complete exon sequencing of forty genes using next-generation sequencing.

Sudden cardiac death (SCD) in young people is predominantly caused by genetic causes as cardiomyopathies. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disease and is responsible for the major proportion of SCD in the young. The purpose of this study was to identify the genetic variants present in young SCD victims with HCM characteristics. From the Portuguese records of autopsies performed at the National Institute of Legal Medicine and Forensic Sciences, North Delegation, 16 young (16-50 years) SCD victims whose death was suspected to be a manifestation of HCM were selected. Using next-generation sequencing, the coding regions of 40 genes associated with HCM, candidates, or strongly related to HCM-phenocopies were investigated. The victims included in this study were all males, with a mean age of 33.4 ± 11.7 years, left ventricle mean thickness of 21.5 ± 6.28 mm, and the majority of deaths occurred during sleep (36%). A pathogenic or likely pathogenic variant was identified in six out of 16 (37.5%) victims, in the most common HCM genes (MYBPC3 and MYH7). Our results indicate that molecular autopsy of SCD victims contributes to a more precise identification of a cause of death, and this can be used in the prevention of SCD cases through family screening of first relatives who may carry the same pathogenic variant.

Contribution to the Development of Guidelines in the Analysis of Biological Evidence in Sexual Assault Investigations.

This investigation intends to study materials and techniques used for biological evidence collection in sexual assault cases and is divided into two stages: in stage one, methods for biological evidence collection (the single swab (including three variants) and the "double swab technique") were compared; in stage two, swabs' component material was compared. The sampling was composed of 42 heterosexual couples who provided mock samples. The collection methods in which the whole swab is covered by evidence presented significantly better outcomes (p < 0.001), such as the "double swab technique." Additionally, nylon swabs proved to present significantly better features regarding the capacity of sample elution, providing significantly higher amounts of DNA (p ≤ 0.034). This study provides guidelines for better collection of biological evidence regarding the collection method using a swab and the proper swab material to utilize.

A Comparison Among Three Multiplex Y-STR Profiling Kits for Sexual Assault Cases.

Biological evidence of sexual assault is one of the most difficult sample types to analyze in forensic laboratories. Y-STR markers are thus a valuable tool for analyzing these samples. The aim of this project was to compare three Y-STR commercial kits by analyzing their amplification performance on casework samples. Overall, 247 trace samples were analyzed with a Yfiler® Plus PCR Amplification Kit (Applied Biosystems), PowerPlex® Y23 (Promega® ) System and AmpFLSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems). Comparing the amplification performance of the three kits, the first two were significantly more sensitive than the latter (p < 0.001). For samples, with a male DNA quantity less than 0.5 ng, the PowerPlex Y23® kit was the most sensitive and best performing kit, followed by the Yfiler® Plus kit (p = 0.009). In conclusion, the Yfiler® Plus and PowerPlex Y-23® kits are viable alternatives to older kits for samples with low amounts of male DNA.

The Use of Laser Microdissection in Forensic Sexual Assault Casework: Pros and Cons Compared to Standard Methods.

Sexual assault samples are among the most frequently analyzed in a forensic laboratory. These account for almost half of all samples processed routinely, and a large portion of these cases remain unsolved. These samples often pose problems to traditional analytic methods of identification because they consist most frequently of cell mixtures from at least two contributors: the victim (usually female) and the perpetrator (usually male). In this study, we propose the use of current preliminary testing for sperm detection in order to determine the chances of success when faced with samples which can be good candidates to undergo analysis with the laser microdissection technology. Also, we used laser microdissection technology to capture fluorescently stained cells of interest differentiated by gender. Collected materials were then used for DNA genotyping with commercially available amplification kits such as Minifiler, Identifiler Plus, NGM, and Y-Filer. Both the methodology and the quality of the results were evaluated to assess the pros and cons of laser microdissection compared with standard methods. Overall, the combination of fluorescent staining combined with the Minifiler amplification kit provided the best results for autosomal markers, whereas the Y-Filer kit returned the expected results regardless of the used method.

Technical Note: "Mitochondrial and nuclear DNA approaches for reliable identification of Lucilia (Diptera, Calliphoridae) species of forensic interest from Southern Europe".

In forensic entomology, rapid and unambiguous identification of blowfly species is a critical prerequisite for accurately estimating the post-mortem interval (PMI). The conventional diagnosis of cadaveric entomofauna based on external characters is hampered by the morphological similarities between species, especially in immature stages. Genetic analysis has been shown to allow precise and reliable diagnosis and delimitation of insect species. Nevertheless, the taxonomy of some species remains unresolved. This study was focused on improving the effectiveness and accuracy of analysis based on the widely used cytochrome c oxidase subunit I barcode region (COI barcode, 658 bp), complemented by other mitochondrial and nuclear regions, such as cytochrome b (Cyt-b, 307 bp) and the second internal transcribed spacer (ITS2, 310-331 bp), for the identification of Southern European blowflies. We analyzed a total of 209 specimens, collected from 38 human corpses, belonging to three Calliphoridae genera and seven species: Chrysomya (Ch. albiceps), Calliphora (C. vicina and C. vomitoria), and Lucilia (L. sericata, L. ampullacea, L. caesar and L. illustris). These species are the most common PMI indicators in Portugal. The results revealed that unambiguous separation of species of the Lucilia genus requires different loci from the barcode region. Furthermore, we conclude that the ITS2 (310-331 bp) molecular marker is a promising diagnostic tool because its inter-specific discriminatory power enables unequivocal and consistent distinctions to be made, even between closely related species (L. caesar-L. illustris). This work also contributes new genetic data that may be of interest in performing species diagnosis for Southern European blowflies. Notably, to the best of our knowledge, we provide the first records of the Cyt-b (307 bp) locus for L. illustris and the ITS2 (310-331 bp) region for Iberian Peninsula Lucilia species.

Cytochrome b as a useful tool for the identification of blowflies of forensic interest (Diptera, Calliphoridae).

In Forensic Entomology the fast and accurate identification of insects collected at the scene of events is essential if errors are to be avoided in estimating infestation times of interest and determining the post-mortem interval (PMI). Traditional identification based on morphological characteristics can be complicated due to physical similarities between different species, especially at immature stages. Genetic analysis provides a fast and reliable identification method. In this paper, molecular analysis is focused on a fragment of 307bp of the mitochondrial DNA region that codes for cytochrome b (cyt b). Six species belonging three genera of Calliphoridae of forensic interest (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia caesar, Lucilia ampullacea, Chrysomya albiceps) were collected and identified. These are the most common species of cadaveric entomofauna on the Atlantic seaboard of the Iberian Peninsula. The results show the robustness of the cyt b locus as a diagnostic tool, since its nucleotide variability enables reliable distinctions to be drawn between species. This study also contributes new consense sequences which may be of interest for correct species identification.

Genetic data of a Brazilian population sample (Santa Catarina) using an X-STR decaplex.

The study of X-chromosomal short tandem repeats (X-STRs) can complement the analysis of autosomal and Y-STRs. A decaplex system for the X-chromosome genetic markers, DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789, was used to study a population sample of Santa Catarina, Brazil. 184 individuals (72 female and 112 male samples) were typed. DNA was amplified in a multiplex reaction and the automatic detection performed using capillary electrophoresis. Allele frequencies and some forensic parameters were calculated.

Mitochondrial DNA diversity in a population from Santa Catarina (Brazil): predominance of the European input.

The state of Santa Catarina (Brazil) is known to have represented a cultural crossroads in South America due to several historic migrations mainly from Europe and Africa. We set out to scrutinize whether the genetic imprint of these migrations could be traced through analysis of the matrilineal gene pool of the Catarinenses. The entire control region of the mitochondrial DNA was studied in 80 healthy and maternally unrelated individuals. The analysis of haplogroup distribution revealed that this population is extremely heterogeneous, showing the coexistence of matrilineal lineages with three different phylogeographic origins. European lineages are the most frequent due mainly to the impact of relatively recent migratory waves from Europe. In spite of this, Native American lineages and African lineages incorporated with the slave trade are also present in noticeable proportions. The strikingly high variability generated by intense gene flow is mirrored in a high sequence diversity (0.9930) and power of discrimination (0.9806). Thus, analysis of the entire mitochondrial DNA control region emerges as a valuable tool for forensic genetic purposes in this highly admixed population, an attribute common to several present-day Latin American populations.

Y-chromosomal STR haplotype diversity in males from Santa Catarina, Brazil.

One-hundred and nine unrelated and healthy males from Santa Catarina, Brazil were included in this study. Allele frequencies and gene diversities for the loci DYS456, DYS458 and DYS448 were calculated. A comparison between our population and others was performed.

Analysis of 10 X-linked tetranucleotide markers in mixed and isolated populations.

We have applied a recently described X-STRs decaplex to characterize four population groups of the Iberian Peninsula, including two well mixed populations and two relatively isolated ones from Northern Spain, in order to get a better insight about the characteristics of X-STRs in those population types between-population differences in allelic frequencies were relatively small. Nevertheless, Fst values were between 0.2 and 2.7%, figures higher than usually reported for autosomic STRs. This result suggests that when forensic cases originate from relatively isolated groups in western Europe, and a specific reference database is not available, it is probably safe to include a Fst-based correction in the calculations of matching or kinship probabilities.

A GEP-ISFG collaborative study on the optimization of an X-STR decaplex: data on 15 Iberian and Latin American populations.

In a collaborative work carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG), a polymerase chain reaction multiplex was optimized in order to type ten X-chromosome short tandem repeats (STRs) in a single reaction, including: DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08, and DXS7423. Using this X-decaplex, each 17 of the participating laboratories typed a population sample of approximately 200 unrelated individuals (100 males and 100 females). In this work, we report the allele frequencies for the ten X-STRs in 15 samples from Argentina (Buenos Aires, Córdoba, Río Negro, Entre Ríos, and Misiones), Brazil (São Paulo, Rio de Janeiro, Paraná, and Mato Grosso do Sul), Colombia (Antioquia), Costa Rica, Portugal (Northern and Central regions), and Spain (Galicia and Cantabria). Gene diversities were calculated for the ten markers in each population and all values were above 56%. The average diversity per locus varied between 66%, for DXS7133, and 82%, for DXS6809. For this set of STRs, a high discrimination power was obtained in all populations, both in males (> or =1 in 5 x 10(5)) and females (> or =1 in 3 x 10(9)), as well as high mean exclusion chance in father/daughter duos (> or =99.953%) and in father/mother/daughter trios (> or =99.999%). Genetic distance analysis showed no significant differences between northern and central Portugal or between the two Spanish samples from Galicia and Cantabria. Inside Brazil, significant differences were found between Rio de Janeiro and the other three populations, as well as between São Paulo and Paraná. For the five Argentinean samples, significant distances were only observed when comparing Misiones with Entre Ríos and with Río Negro, the only two samples that do not differ significantly from Costa Rica. Antioquia differed from all other samples, except the one from Río Negro.

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.

Genetic data of 4 X-chromosomal short tandem repeats in a North of Portugal population.

POPULATION: One hundred unrelated females and 100 unrelated males, autochthonous, healthy, from the North of Portugal.

Genetic data of four X-chromosomal STRs in a population sample of Santa Catarina, Brazil.

POPULATION: A total of 184 healthy unrelated individuals (70 females and 114 males), autochthonous from Santa Catarina, Brazil.

Allele frequencies of sixteen STRs in the population of Northern Portugal.

Allele frequencies of sixteen autossomal short tandem repeats (STRs), D3S1358, VWA, D16S539, D8S1179, D21S11, D18S51, TH01, FGA, D5S818, D13S317, D7S820, TPOX, CSF1PO, Penta D, Penta E (included in the PowerPlex 16 kit), and the SE33 (PowerPlex ES Monoplex System SE33) were determined in a sample of 200 healthy unrelated individuals from the north of Portugal.

Allele frequencies and haplotypes of 8 Y-chromosomal STRs in the Santa Catarina population of southern Brazil.

Allele frequencies and haplotypes of eight Y-chromosomal short tandem repeats (STRs), DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392 and DYS393 were determined in a sample of 109 males from Santa Catarina. The origin of this southern Brazilian population is mainly from Portuguese people, namely from Azores archipelago.