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- mRNA profiling and donor association of mock casework samples: Results of a 3rd and 4th EDNAP collaborative exercisePublication . Hänggi, Nadescha Viviane; Amorim, António; Afonso Costa, Heloísa; Andersen, Jeppe Dyrberg; Kampmann, Marie-Louise; Courts, Cornelius; Neis, Maximilian; Syndercombe-Court, Denise; Giangasparo, Federica; Fonneløp, Ane Elida; Johannessen, Helen; Hadrys, Thorsten; Fürst, Angelika; Parson, Walther; Niederstätter, Harald; Sidstedt, Maja; Aili Fagerholm, Siri; Sijen, Titia; van den Berge, Margreet; Hanson, Erin; Ballantyne, Jack; Haas, CordulaSimultaneous identification and association of body fluids to donors can serve as a powerful tool in the criminal investigation of mixed traces. Massively parallel sequencing of mRNA targets not only identifies the origin of the body fluids but may also provide additional contextual information about the body fluid donors of a (binary) mixture using coding region SNPs (cSNPs). Within the European DNA Profiling Group (EDNAP), two consecutive collaborative exercises (3rd and 4th EDNAP exercise) were organized, with the objective to evaluate the performance of two previously published high-resolution mRNA sequencing assays. In the 3rd EDNAP exercise, the BFID-cSNP-BSS assay (cSNPs for blood, saliva, and semen) was evaluated, while in the 4th EDNAP exercise, the BFID-cSNP-6F assay (cSNPs for six fluids/tissues, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin) was tested. Each RNA cSNP assay was accompanied by a genomic DNA assay for the genotyping of the cSNPs in the individual(s) or body fluid donor(s) of interest. A total of 11 laboratories participated in one or both collaborative exercises. In each exercise, the participating laboratories received a set of 16 standardized mock case stains for analysis and were encouraged to analyze additional, self-prepared stains and reference samples. Laboratories could participate using either the Ion Torrent S5™ or the Illumina MiSeq™ sequencing system. The results of the 16 mock case stains were very encouraging in both exercises, as body fluid components could be reliably identified for most of the stains. Since successful donor association depends on the number of body fluid markers covered in the sequencing results, we found that for stains consisting of blood, menstrual blood, vaginal secretion or a mixture thereof, the cSNPs provided substantial genetic discriminatory information for successful association of the respective body fluid to its donor. In mixtures, the difficulty in interpreting the cSNP genotypes might be attributed to the masking effect of the other body fluid(s) present. Body fluid identification and donor association of skin samples proved to be a significant challenge. In conclusion, body fluid identification and donor association using the BFID-cSNP-BSS and -6 F assays is a promising and effective method across laboratories and sequencing platforms.
- The Genetic Structure of Cape Verdean Population Revealed by Y-Chromosome STRsPublication . Cainé, Laura; Costa, Rita; Fadoni, Jennifer; Amorim, António; AmorimBackground/Objectives: Y-chromosomal short tandem repeats (Y-STR) are genetic markers widely used in forensic and population genetics. However, despite their importance, many populations remain under-represented in published studies and genetic databases. One such population is the Cape Verdean, which, despite its unique history of admixture between European and sub-Saharan African populations, continues to be under-represented in global Y-STR reference databases. This study aims to characterize the Y-STR haplotype diversity and paternal lineage composition of the Cape Verdean population using a high-resolution STR panel. Methods: A total of 143 unrelated Cape Verdean men were analyzed using a set of 26 Y-STR loci, including rapidly mutating markers. Allele and haplotype frequencies were calculated, along with standard forensic parameters such as gene and haplotype diversity. Paternal lineages were inferred, and genetic relationships with other populations were evaluated using distance-based and graphical methods. Results: A total of 135 haplotypes were detected, with 88.8% being unique, yielding a haplotype diversity of 0.999. The most common haplogroups reflected both West African and European ancestry. Genetic distance analysis positioned the Cape Verdean population between African and European groups, supporting its intermediate and admixed genetic background. Conclusions: This study provides the first high-resolution Y-STR dataset for Cape Verdeans, contributing valuable reference data for forensic casework and population genetic studies. The results highlight the utility of extended Y-STR panels in admixed populations and underscore the need to enhance the representation of admixed populations in international forensic reference databases
- Sexually Transmitted Infections: Usefulness of Molecular Methods for Microorganism Detection in Stored Sexual Assault SamplesPublication . Cainé, Laura; Eiras, Ana; Fadoni, Jennifer; Franco, Magda; Correia Dias, Maria Helena; Amorim, AntónioSexual assault is a global public health and human rights concern, with serious physical, psychological and reproductive consequences for survivors. Among these, sexually transmitted infections are particularly relevant due to their frequently asymptomatic nature and potential for long-term complications. The detection of sexually transmitted infections in forensic settings is crucial for clinical management of victims and for evidentiary support in forensic sexual crimes investigations. This study aimed to evaluate the applicability of real-time polymerase chain reaction for detecting Chlamydia trachomatis, Trichomonas vaginalis, Neisseria gonorrhoeae, and Treponema pallidum in biological samples collected from victims of sexual assault and stored under routine forensic conditions, in some cases, for up to 18 years. A total of 231 swabs from 116 individuals collected between 2004 and 2017 were analysed using real-time PCR with pathogen-specific primers and fluorescent probes. The analysis revealed 13 positive samples of T. vaginalis (5.6%) and 11 of C. trachomatis (4.8%). No positive results were obtained for N. gonorrhoeae or T. pallidum. These findings demonstrate the usefulness of real-time polymerase chain reaction for detecting sexually transmitted infections in long-term preserved forensic samples. Moreover, the ability to identify pathogen DNA in archived samples highlights the potential role of molecular diagnostics in the retrospective investigation of sexual crimes, including cold cases. It underscores the value of molecular methods as a complementary tool in forensic proceedings and survivor care.
- Y-STR Databases-Application in Sexual Crimes.Publication . Costa, Rita; Fadoni, Jennifer; Amorim, António; Cainé, LauraBackground/objectives: The Y chromosome is a crucial tool in forensic genetics due to its unique characteristics, such as its haploid inheritance and lack of recombination. Y-STRs (short tandem repeats on the Y chromosome) are widely used for identifying male genetic profiles in DNA mixtures, especially in sexual assault cases where high levels of female DNA hinder autosomal analysis. This study evaluates the applicability of Y-STRs in forensic investigations, addressing their limitations and the impact of advanced technologies, such as rapidly mutating Y-STRs (RM Y-STRs). Methods: A comprehensive literature review was conducted to analyze existing knowledge on the application of Y-STRs in sexual crimes. The study also examines the role of population databases, such as YHRD, in estimating haplotype frequencies and enhancing forensic reliability. Results: Y-STR analysis proves essential for male DNA identification in complex mixtures, with RM Y-STRs enhancing discriminatory power. However, limitations persist, particularly in cases involving closely related male lineages. The population database coverage remains insufficient in regions like Cape Verde, affecting forensic reliability. Case studies demonstrate Y-STR effectiveness in solving cold cases and sexual crimes, reinforcing the need for expanded databases and methodological advancements. Conclusions: Y-STRs play a fundamental role in forensic genetics, particularly in sexual assault investigations. Their integration with advanced sequencing technologies and expanded databases is critical for improving forensic accuracy. Ethical considerations regarding genetic data privacy and potential discrimination must be addressed through clear regulations and forensic best practices.
- Forensic Microbiology: challenges in detecting sexually transmitted InfectionsPublication . Eira, Ana; Fadoni, Jennifer; Amorim, António; Cainé, LauraSexual assault crimes consist of acts committed without consent and represent a major global issue with serious implications for victims. These acts have both short- and long-term consequences on the physical, mental, and sexual health of victims, with sexually transmitted infections (STIs) being one of the direct outcomes of such crimes. Sexually transmitted infections constitute a serious global public health problem and can lead to severe consequences. These infections may be caused by bacteria, viruses, or parasites and are transmitted through sexual contact. Some of the most common STIs among the general population and victims of sexual crimes include gonorrhoea, chlamydia, trichomoniasis, and syphilis. In most carriers, these infections are asymptomatic, making their detection particularly challenging. Considering the importance of further research in this field, the primary objectives of this study are to review the existing literature on the incidence of major STIs in victims of sexual crimes, to identify the various risk factors associated with these infections, and to explore their public health implications. Additionally, this study aims to assess different STI detection techniques, analyzing their advantages and disadvantages. Studies on this topic are crucial for better understanding the role of sexually transmitted infections in the context of sexual crimes. However, throughout this work, it was verified that point-of-care methods are a good option to allow the diagnosis to be faster and more accurate, when compared to other methods of detecting sexually transmitted infections.
- Sudden Cardiac Death: The Role of Molecular Autopsy with Next-Generation SequencingPublication . Santos, Agostinho; Amorim, António; Cainé, Laura; Fadoni, JenniferMolecular autopsy is a term employed to describe the investigation of the cause of death through the analysis of genetic information using biological samples collected post-mortem. Its utility becomes evident in situations where conventional medico-legal autopsy methods are not able to identify the cause of death, i.e., in sudden cardiac death (SCD) cases in young individuals, where deaths are commonly due to genetic cardiac conditions, such as cardiomyopathies and channelopathies. The recent advancement in high-throughput sequencing techniques, such as next-generation sequencing (NGS), has allowed the investigation of a high number of genomic regions in a more cost-effective and faster approach. Unlike traditional sequencing methods, which can only sequence one DNA fragment at a time, NGS can sequence millions of short polynucleotide fragments simultaneously. This parallel approach reduces both the time and cost required to generate large-scale genomic data, making it a useful tool for applications ranging from basic research to molecular autopsy. In the forensic context, by enabling the examination of multiple genes or entire exomes and genomes, NGS enhances the accuracy and depth of genetic investigations, contributing to a better understanding of complex inherited diseases. However, challenges remain, such as the interpretation of variants of unknown significance (VUS), the need for standardized protocols, and the high demand for specialized bioinformatics expertise. Despite these challenges, NGS continues to offer significant promise for enhancing the precision of molecular autopsies. The goal of this review is to assess the effectiveness of contemporary advancements in molecular autopsy methodologies when applied to cases of SCD in young individuals and to present an overview of the steps involved in the analysis of NGS data and the interpretation of genetic variants.
- X-chromosomal STRs: Metapopulations and mutation ratesPublication . Gusmão, L.; Antão-Sousa, S.; Faustino, M.; Abovich, M.A.; Aguirre, D.; Alghafri, R.; Alves, C.; Amorim, A.; Arévalo, C.; Baldassarri, L.; Barletta-Carrillo, C.; Berardi, G.; Bobillo, C.; Borjas, L.; Braganholi, D.F.; Brehm, A.; Builes, J.J.; Cainé, Laura; Carvalho, E.F.; Carvalho, Mónica; Catelli, L.; Cicarelli, R.M.B.; Contreras, A.; Corach, D.; Di Marco, F.G.; Diederiche, M.V.; Domingues, P.; Espinoza, M.; Fernandéz, J.M.; García, M.G.; García, O.; Gaviria, A.; Gomes, I.; Grattapaglia, D.; Henao, J.; Hernandez, A.; Ibarra, A.A.; Lima, G.; Manterola, I.M.; Marrero, C.; Martins, J.A.; Mendoza, L.; Mosquera, A.; Nascimento, E.C.; Onofri, V.; Pancorbo, M.M.; Pestano, J.J.; Plaza, G.; Porto, M.J.; Posada, Y.C.; Rebelo, M.L.; Riego, E.; Rodenbusch, R.; Rodríguez, A.; Rodríguez, A.; Sanchez-Diz, P.; Santos, S.; Simão, F.; Siza Fuentes, L.M.; Sumita, D.; Tomas, C.; Toscanini, U.; Trindade-Filho, A.; Turchi, C.; Vullo, C.; Yurrebaso, I.; Pereira, V.; Pinto, N.The analysis of STRs located on the X chromosome has been one of the strategies used to address complex kinship cases. Its usefulness is, however, limited by the low availability of population haplotype frequency data and lack of knowledge on the probability of mutations. Due to the large amount of data required to obtain reliable estimates, it is important to investigate the possibility of grouping data from populations with similar profiles when calculating these parameters. To better understand the partition of genetic diversity among human populations for the X-STRs most used in forensics, an analysis was carried out based on data available in the literature and new data (23,949 haplotypes in total; from these 10,445 new) obtained through collaborative exercises within the Spanish and Portuguese Working Group of the International Society for Forensic Genetics. Based on the available population data, a similarity in X-STR profiles was found in European populations, and in East Asian populations, except for some isolates. A greater complexity was found for African, South American, and South and Southeast Asian populations, preventing their grouping into large metapopulations. New segregation data on 2273 father/mother/daughter trios were also obtained, aiming for a more thorough analysis of X-STR mutation rates. After combining our data with published information on father/mother/daughter trios, no mutations were detected in 13 out of 37 loci analyzed. For the remaining loci, mutation rates varied between 2.68 × 10−4 (DXS7133) and 1.07x10−2 (DXS10135), being 5.2 times higher in the male (4.16 ×10−3) than in the female (8.01 ×10−4) germline.
- Phytocanabinoids and synthetic cannabinoids: from recreational consumption to potential therapeutic use – a reviewPublication . Teixeira, Helena M.Cannabinoids are part of the most popular group of illicit substances in the Western world. The word “cannabinoid” refers to any chemical substance, regardless of structure or origin, that binds to the body’s cannabinoid receptors and that has effects similar to those produced by the Cannabis plant. Regarding their origin, cannabinoids can be classified into endocannabinoids, phytocannabinoids and synthetic cannabinoids. The behavioral and physiological effects of cannabinoids have received particular attention over the last few decades, including sensations of euphoria, relaxation and loss of concentration, with their repeated use being associated with short and long-term side effects, including respiratory and cardiovascular disorders, cognitive changes, psychoses, schizophrenia and mood disorders. On the other hand, recent investigations have proposed a promising therapeutic potential of cannabinoid-based drugs for a wide range of medical situations, including neurological and psychiatric disorders, among other indications. The growing popularity in the use of cannabinoid-based compounds, both for recreational and therapeutic purposes, has been accompanied by an equally continuous and growing evolution of knowledge regarding their potential harmful and beneficial effects. However, there are several open questions and challenges to be answered, which require more and better investigations. This article’s main objectives are: i) to understand the importance of the action of cannabinoids in humans; ii) identify the different types of cannabinoids that exist and understand the differences in their action; iii) distinguish the legislative framework for cannabinoid consumption; iv) identify the possible adverse effects of cannabinoid consumption, as well as their potential benefits; v) know the existing medical-scientific evidence in terms of therapeutic potential, particularly in relation to aspects of safety and efficacy; vi) encourage critical thinking about the recreational consumption and therapeutic use of cannabinoids, based both on currently available evidence and gaps in knowledge.
- Determination of Arylcyclohexylamines in Biological Specimens: Sensors and Sample Pre-Treatment ApproachesPublication . Pelixo, Rodrigo; Barroso, Mário; Gallardo, Eugenia; Rosado, TiagoArylcyclohexylamine (ACH) compounds represent a predominant faction within new psychoactive substances. Due to their powerful dissociative effects, they are used in recreational contexts but also in situations of drug-facilitated sexual assault, and therefore, they are a constant target of analysis by forensic experts. In recent years, their consumption has been notably high, especially the use of ketamine, presenting daily challenges for laboratories in the determination of this and other ACH analogues. This review comprises the recent strategies that forensic specialists use to identify and quantify ACH compounds in the laboratory with more traditional analytical techniques and technology, and on the point-of-care testing via sensor technology. The study focuses on analogues of phencyclidine (PCP), ketamine, and eticyclidine, highlighting the consistent need for higher sensitivity in the analysis of various samples collected from real cases and simulations of possible matrices. The review also emphasises the ongoing research to develop more sensitive, quicker, and more capable sensors.
- Volumetric Absorptive Microsampling in ToxicologyPublication . Pires, Bruno; Catarro, Gonçalo; Soares, Sofia; Gonçalves, Joana; Rosado, Tiago; Barroso, Mário; Araujo, André R. T. S.; Gallardo, EugeniaVolumetric absorptive microsampling (VAMS) is an emerging technique in clinical and forensic toxicology. It is recognized as a promising alternative to traditional sampling methods, offering an accurate and minimally invasive means of collecting small volumes of biological samples, such as blood, urine, and saliva. Unlike conventional methods, VAMS provides advantages in terms of sample stability, storage, and transportation, as it enables samples to be collected outside laboratory environments without requiring refrigeration. This review explores several VAMS methodologies, with a particular focus on its application for the quantification of drugs and other substances in clinical and forensic toxicology. It compares VAMS to other microsampling techniques, such as dried blood spots (DBSs), highlighting VAMS's superiority in addressing issues related to sample volume consistency and environmental impact. Despite its advantages, VAMS also presents certain limitations, including higher costs and difficulties in detecting underfilled samples. Overall, VAMS stands out as a microsampling technique with the potential to enhance patient compliance and operational efficiency, positioning itself as a viable tool for toxicological analysis in both clinical and forensic contexts.