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- Insights into the anatomical expressions of anencephaly in three infants from 17th to 19th- century Lisbon, Portugal.Publication . Lourenço, Marina; Curate, Francisco; Cunha, EugéniaObjective: This study aims to analyse and compare the cranial morphological variations in three individuals, each exhibiting different severity levels of malformations. Materials: Three nearly complete and well-preserved skeletons of infants from the S˜ao Domingos children’s necropolis in Lisbon, Portugal, dating from the 17th to early 19th centuries. Methods: Macroscopic and metric assessments were performed aimed at creating a detailed description of the skeletons. Results: The three infants exhibit an absence of the upper cranial vault, accompanied by several cranial bone alterations such as abnormal development and morphology of the occipital, sphenoid, temporal, and frontal bones. Additionally, two of the infants present maxillary and dental anomalies. Conclusions: The three infants from the S˜ao Domingos necropolis provide crucial insights into the presence of anencephaly in an historical population, representing an exceptionally rare archaeological find. The distinct cranial abnormalities strongly support the diagnosis and highlight varying severity levels of the condition. Significance: These examples enhance the recognition of anencephaly in archaeological contexts and deepen the understanding of its varied bone expressions. Examining skeletal variations within the same condition also complements the broader palaeopathological discussion of rare diseases. Limitations: The lack of soft tissue preservation reduces a comprehensive assessment of anencephaly in skeletal remains. The archaeological context presents challenges such as fragmentation. Additionally, determining postnatal survival is difficult due to the subtle or absent skeletal indicators that might suggest survival beyond birth. Suggestions for further research: Biomolecular genetics analysis could be a valuable approach for future research.
- The off-season of dental cementum investigations. A critical appraisal of season-of-death prediction in medico-legal investigationsPublication . Benoit Bertrand; Kaddoura, Ahmad; Cunha, Eugénia; Bécart, Anne; Gosset, Didier; Hédouin, Valery; Marques, CarinaIt has been suggested that death seasonality could be estimated based on the dark/ bright appearance of the outermost dental cementum deposit. The few existing studies show variable accuracy rates. The question whether estimating season-of-death based on dental cementum represents a realistic and achievable goal can arise. This work, based on a softwareassisted procedure and direct human observation, aims to critically evaluate the applicability of dental cementum to estimate the season-of-death in forensic anthropology. The sample consists of 20 canines from individuals belonging to the 21st century Identified Skeletal Collection, University of Coimbra, Portugal, and with documented biodemographic data. Fifty percent of the individuals died during the summer and 50% during the winter. Five non-decalcified 100 mm cross-sections per tooth were prepared, using a standardized protocol. Images of the region of interest, with the acellular cementum, were captured, and imported into FIJI/ImageJ software. Performance of the season-of-death predictions was very low, with 50% accuracy, score of 0.0000 for Matthews Correlation Coefficient, and F1-score of 61.5% for Summer and 28.6% for Winter, were obtained for the observer assessment. For the software model, performance was equally poor, with 52.6% accuracy, score of 0.0272 for Matthews Correlation Coefficient, and F1-score of 30.8% for Summer and 64.0% for Winter. This work is of relevance to the forensic science community by demonstrating the prospects and limitations of dental cementum as a method for estimating seasonality of death.
- The genetic history of Portugal over the past 5,000 yearsPublication . Roca-Rada, Xavier; Davidson, Roberta; Williams, Matthew P; Villalba-Mouco, Vanessa; Carvalho, António Faustino; Ravishankar, Shyamsundar; Collen, Evelyn; Haarkötter, Christian; Taufik, Leonard; Cuesta-Aguirre, Daniel R; Tente, Catarina; Calleja, Álvaro M Monge; MacRoberts, Rebecca Anne; Melo, Linda; Purnomo, Gludhug A; Souilmi, Yassine; Tobler, Raymond; Cunha, Eugénia; Tereso, Sofia; Matos, Vítor M J; Fernandes, Teresa Matos; Maurer, Anne-France; Silva, Ana Maria; Carvalho, Pedro C; Llamas, Bastien; Teixeira, João CBackground: Recent ancient DNA studies uncovering large-scale demographic events in Iberia have presented very limited data for Portugal, a country located at the westernmost edge of continental Eurasia. Here, we present the most comprehensive collection of Portuguese ancient genome-wide data, from 67 individuals spanning 5000 years of human history, from the Neolithic to the nineteenth century. Results: We identify early admixture between local hunter-gatherers and Anatolian-related farmers in Neolithic Portugal, with a northeastern-southwestern gradient of increasing Magdalenian-associated ancestry persistence in Iberia. This profile continues into the Chalcolithic, though Bell Beaker-associated sites reveal Portugal's first evidence of Steppe-related ancestry. Such ancestry has a broader demographic impact during the Bronze Age, despite continuity of local Chalcolithic genetic ancestry and limited Mediterranean connections. The village of Idanha-a-Velha emerges in the Roman period as a site of significant migration and interaction, presenting a notably diverse genetic profile that includes North African and Eastern Mediterranean ancestries. The Early Medieval period is marked by the arrival of Central European genetic diversity, likely linked to migrations of Germanic tribes, adding to coeval local, African, and Mediterranean influences. The Islamic and Christian Conquest periods show strong genetic continuity in northern Portugal and significant additional African admixture in the south. The latter remains stable during the post-Islamic period, suggesting enduring African influences. Conclusions: We reveal dynamic patterns of migration in line with cultural exchange across millennia, but also the persistence of local ancestries. Our findings integrate genetic information with historical and archeological data, enhancing our understanding of Iberia's biological and cultural heritage.
- 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, L.; Carvalho, E.F.; Carvalho, M.; Catelli, L.; Cicarelli, R.M.B.; Contreras, A.; Corach, D.; Di Marco, F.G.; Diederiche, M.V.; Domingues, P.; Espinosa, 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.; Cainé, Laura; Carvalho, Mónica; ElsevierThe 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.
- Human identification with combined anthropologic and genetic tools: two case reports in forensic medicine practicePublication . Amorim, António; Afonso Costa, Heloísa; Espinheira, Rosa; Costa, Rosa; Cunha, Eugénia; Costa Santos, JorgeThe identification of skeletonized corpses or skeletal remains, in forensic medicine practice, as well as being an imperative to human rights respect, has particular relevance and significance in psycho-social terms. If, on one hand, the Universal Declaration of Human Rights advises identification before all body inhumations, on the other hand, returning the identity to an unidentified corpse and subsequent return of the corpse to his family is, itself, the instrument which will allow for the possibility of beginning the family grieving. Forensic anthropology is the first approach towards the identification of unknown skeletonized corpses, which may lead to positive identification of the body or, sometimes, cannot go further than establish some pointers that will lead to further attempt to genetic identification. Here we report two cases of two dead bodies, unidentified, that were presented to the National Institute of Legal Medicine (INML), with instructions, from the prosecutors, to be determined possible cause and circumstances of death and achieve to individual identification. Forensic anthropology, allowed, in both cases, some indications that pointed to alleged missing individuals. Genetic study of skeletonized bodies and family members of the alleged missing individuals has turned possible, in one case, the positive identification. For one of the corpses, most likely due to the weather conditions to which he was exposed for a very considerable period, it was not possible to achieve to positive identification. However, in the latter case, we believe that the use of mitochondrial DNA study may help to settle the case with positive identification of the corps.
- 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.