Challenges and (Un)Certainties for DNAm Age Estimation in Future

Age estimation is a paramount issue in criminal, anthropological, and forensic research. Because of this, several areas of research have focused on the establishment of new approaches for age prediction, including bimolecular and anthropological methods. In recent years, DNA methylation (DNAm) has arisen as one of the hottest topics in the field. Many studies have developed age- prediction models (APMs) based on evaluation of DNAm levels of many genes in different tissue types and using different methodological approaches. However, several challenges and confounder factors should be considered before using methylation levels for age estimation in forensic contexts. To provide in-depth knowledge about DNAm age estimation (DNAm age) and to understand why it is not yet a current tool in forensic laboratories, this review encompasses the literature for the most relevant scientific works published from 2015 to 2021 to address the challenges and future directions in the field. More than 60 papers were considered focusing essentially on studies that developed models for age prediction in several sample typesinfo:eu-repo/semantics/publishedVersio

A Blood–bone–tooth model for age prediction in forensic contexts

The development of age prediction models (APMs) focusing on DNA methylation (DNAm) levels has revolutionized the forensic age estimation field. Meanwhile, the predictive ability of multi-tissue models with similar high accuracy needs to be explored. This study aimed to build multi-tissue APMs combining blood, bones and tooth samples, herein named blood–bone–tooth-APM (BBT-APM), using two different methodologies. A total of 185 and 168 bisulfite-converted DNA samples previously addressed by Sanger sequencing and SNaPshot methodologies, respectively, were considered for this study. The relationship between DNAm and age was assessed using simple and multiple linear regression models. Through the Sanger sequencing methodology, we built a BBT-APM with seven CpGs in genes ELOVL2, EDARADD, PDE4C, FHL2 and C1orf132, allowing us to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 6.06 years, explaining 87.8% of the variation in age. Using the SNaPshot assay, we developed a BBT-APM with three CpGs at ELOVL2, KLF14 and C1orf132 genes with a MAD of 6.49 years, explaining 84.7% of the variation in age. Our results showed the usefulness of DNAm age in forensic contexts and brought new insights into the development of multi-tissue APMs applied to blood, bone and teethinfo:eu-repo/semantics/publishedVersio

Chorangioma and John Clarke

Perinatal mortality is a relevant indicator of a country health status. Through the centuries, measures have been promoted to reduce modifiable risk factors or to treat installed diseases. That was the example of the English medical doctor John Clarke (1758/1760?-1851), who dedicated his life to mother-child health care [1]. Among his contributions is the report of a placental tumour in 1798, named Chorangioma placenta (CP) [2]. It may occur in primiparas or multiparas, apparently increasing with the mother’s age, with association to the mother’s hypertension or diabetes mellitus [3]. Chorangioma may appear in single or multiple pregnancies and may lead to foetal heart failure, hydrops, or sudden intra-uterine death [3]. The authors report the case of a 2 cm diameter chorangioma (Fig. 1A), which ended in premature death of the male foetus in utero at 35 weeks and 5 days, in a multiparous mother. Histopathological examination confirmed the macroscopic suspicion by disclosing a benign vascular capillary proliferation (Fig. 1B) positive for endothelial markers CD34/CD31 (Fig. 1C). Its current incidence ranges from 0.5% to 1% of analysed placentas [4] and may represent a primitive angioblastic tissue malformation, aggravated with hypoxia and/or haemodynamic changes during pregnancy. To conclude, we highlight the relevance of chorangioma as a cause of perinatal death, which is around 30% [4]info:eu-repo/semantics/publishedVersio

The role of DNA concentrations in forensic casework results : regression models application

Póster apresentado no 28th Congress of International Society For Forensic Genetics (ISFG 2019), Praga, República Checa, 9-13 de Setembro de 2019In forensic DNA typing, short tandem repeats (STRs) are the most frequently genotyped markers in order to distinguish between individuals and to relate them to a crime or to exonerate the innocent. In recent years, new controversies have arisen with the advent of more sensitive techniques, allowing profiles to be recovered from minimum amounts of DNA, hence, bringing challenges to weight of evidence evaluation for forensic DNA profiles obtained from low template DNA samples. Introduction of interpretation models, or even new weight of evidence software should be accompanied by a measure of uncertainty that is part of any biological analysis. Specially, due to stochastic effects, the reliability of the obtained profiles might differ between machinery, workflow and also PCR settings in use in different laboratories. In this work we try to understand the relation between Peak Area, DNA concentration and also size marker, using adequate regression models. Buccal swabs from 180 individuals, with unknown identity, were selected for this study. DNA was extracted with prep-n-go™ buffer and quantified using Quantifiler® Trio DNA Quantification kit in a 7500 Real-Time PCR System (Applied Biosystems). STR amplification was performed with Powerplex Fusion 6C amplification kit (Promega). Amplified PCR products were separated and detected in an Applied Biosystems® 3500 Genetic Analyzer using manufacturer’s conditions. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Statistical analysis was performed with R Studio. Our results allow having an important overview about the relation between DNA concentrations, peak area, and size of the studied genetic markers.N/

Forensic genetic analysis of South Portuguese population with the six dye Powerplex® Fusion 6C

Poster apresentado no 28th Congress of International Society For Forensic Genetics (ISFG 2019), Praga, República Checa, 9-13 de Setembro de 2019As an improvement in efficiency and in Human Discrimination Power, the new six dye multiplex kit PowerPlex® Fusion 6C System, by Promega, available for human identification can co-amplify 27 loci, in a single reaction, have been introduced in the last years with great success. This kit allows the amplification and detection of autosomal loci included in the expanded Combined DNA Index System CODIS, plus the loci Penta D, PENTA E and SE33 as well as Amelogenin for gender determination. Furthermore, this kit includes three Y –STRs (DYS391, DYS576 and DYS570), allowing allelic attribution in a total of 27 loci. This genetic markers extension satisfies not only CODIS but also European Standard Set recommendations. Thinking about continuous human migration movements, especially in a very cosmopolitan region like Lisbon and south of Portugal, and also, in keeping population studies and actualized STR databases we decided to update our previous studies. Our sample is composed of 600 unrelated individuals, from paternity testing with laboratory identity anonymised. DNA was extracted by Prep-n-go BufferTM(Thermo-Fisher Scientific). PCR amplification was performed with PowerPlex® Fusion 6C System, according to manufacturer’s guidelines. Fragment analysis was carried out in an Applied Biosystems® 3500 Genetic Analyser. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Allele frequencies and population statistics, including Hardy-Weinberg equilibrium p-values from exact test probabilities and forensic parameters were calculated with adequate software. In conclusion, our population information was updated in order to apply most recent data in our casework weight of evidence.N/

Dental abscess and “unexpected death”...

Even though we are living in an era of major technical-scientific advances and effective antimicrobial and antiviral therapy,dental infections are still the most important predisposing factors for head and neck infections. Odontogenic infections can cause severe complications, e.g. compromised airways, tissue necrosis, deep neck infections, mediastinitis, endocarditis and sepsis. These severe odontogenic infections can be potentially life-threatening. Usually odontogenic infections respond well to a combination of surgical treatment (incision, rainage) and antibiotic therapy. However, especially when the medico-surgical therapy is installed late, cases may evolve unfavourably and be fatal. The authors report a case of a 30-year-old man who was observed on three consecutive occasions by the General Practitioner in a District Hospital, for a decayed tooth with abscess and was, then, referred to a Central Hospital. There, he was examined for the fourth time, this one by a Stomatologist at the Emergency Department, where he died. The post mortem examination revealed bacterial (Gram +) acute neutrophilic (purulent) infection of soft tissues of the mandibular region and neck with para-tracheal extension, as well as thrombosis ofthe left jugular vein. Circumstantial clinical information, post mortem findings, pathophysiology (including complications andprogression of the disease to death) are discussed, highlighting the relevance of accurate and timely diagnosis and treatmentto avoid malpractice and mortality.info:eu-repo/semantics/publishedVersio

Avaliação da eficácia da lise diferencial em amostras biológicas de processos sexuais

Poster apresentado no 18º Congresso Nacional de Medicina Legal e Ciências Forenses. Coimbra, Portugal, 21-23 de Novembro de 2019Em todos os processos de genética forense, o objetivo consiste na obtenção de um perfil genético identificativo de um ou vários indivíduos intervenientes num determinado processo. Nos casos de abuso sexual, em particular, são recolhidos vestígios biológicos constituídos por sangue, sémen, saliva, células epiteliais entre outros. Acresce ainda nestes casos, que, muitas amostras biológicas, são frequentemente constituídas por misturas muito desproporcionais de material celular das vítimas e dos supostos agressores. A maior concentração de material genético feminino relativamente à concentração de material genético masculino, pode impedir a obtenção de um perfil genético autossómico masculino ou mesmo de mistura. Nestes casos, a identificação do agressor só poderá ser efetuada recorrendo aos marcadores do cromossoma Y, que apenas identifica a linhagem paterna. Outra abordagem para estas situações seria a utilização da extração diferencial, que em algumas condições permite a obtenção de um perfil genético masculino autossómico. Com o objetivo de avaliar uma metodologia recente de extração diferencial em amostras forenses, foram selecionadas, com base nos resultados de quantificação de ADN, 10 amostras recolhidas em processos de agressão sexual. Estas amostras, foram previamente estudadas com os métodos de extração PrepFiler Express™ (Applied Biosystems) e quantificação de ADN com o método Quantifiler™ Trio DNA Quantification Kit (Applied Biosystems). Estes métodos revelaram a presença de uma mistura de material masculino e feminino com proporções de 1:10, respetivamente. O ADN de uma segunda porção destas amostras foi extraído pelo método Sampletype-i-sep® DL (Biotype), que permite a separação de ADN em misturas de material biológico de uma forma simples e com pouca manipulação das amostras, minimizando o erro e a contaminação das mesmas. A avaliação da eficácia do método de extração diferencial foi efetuada recorrendo à determinação da concentração de ADN e à amplificação de marcadores autossómicos e do cromossoma Y. Os dois métodos de extração foram comparados entre si de forma a verificar qual o método que permitiu a obtenção do perfil genético mais discriminativo. A análise dos resultados revelou que este método constituir uma mais valia importante na resolução dos casos de agressão sexual. Assim, um passo importante para a abordagem dos processos de agressão sexual, pode consistir na aplicação de métodos de separação das células espermáticas masculinas das células epiteliais femininas designado por lise diferencial, nomeadamente o método Sampletype-i-sep® DLN/

Comparação da extração em fase sólida com a precipitação proteica na determinação de canabinóides em amostras de sangue total

Poster apresentado no 20º Congresso de Medicina Legal e Ciências Forenses, Coimbra, 24-26 de novembro de 2022.Intodução: Em toxicologia forense, é essencial ter um método rápido e eficiente que permita usar uma pequena quantidade de amostra biológica para determinação e quantificação de baixas concentrações de canabinóides. A extração em fase sólida (SPE) tem sido uma das técnicas extrativas mais utilizadas no laboratório do Serviço de Química e Toxicologia Forenses (SQTF) na confirmação e quantificação de canabinóides, estando este procedimento acreditado. No entanto, importa sempre procurar metodologias analíticas que possam apresentar mais vantagens e suprimir aspetos menos satisfatórios, como por exemplo, custos, processos laboriosos, entre outros. Neste sentido, a introdução da precipitação proteica como técnica extrativa na rotina laboratorial pode demonstrar-se uma mais valia para o SQTF. Material e Métodos: No procedimento extrativo em amostras de sangue foi utilizada a técnica de SPE com 0,5 mL de amostra e a de precipitação proteica de 0,1 mL de sangue, para a determinação de ∆9-tetrahidrocanabinol (THC), 11-hidroxi-∆9-THC (THC-OH) e 11-nor-9-carboxi-∆9-THC (THC-COOH). A análise cromatográfica foi realizada por cromatografia líquida de alta resolução acoplada a espetrometria de massa (LC-MS/MS) nos dois equipamentos existentes no SQTF da Delegação do Centro. Na separação cromatográfica foi utilizada uma fase móvel constituída por formato de amónia aquoso 2mM (ácido fórmico 0,1 %) e metanol, em modo gradiente, com um fluxo de 0,4 mL/min e uma coluna Acquity UPLC HSS® T3 quer no equipamento da marca Waters quer no equipamento da marca Sciex. Resultados e Discussão: No equipamento da Waters, a precipitação proteica demonstra não influenciar de forma significativa a intensidade e área dos picos, bem como o ruído de fundo quando comparado com os resultados obtidos por extração em fase sólida. No equipamento da Sciex, as amostras preparadas por precipitação proteica apresentam maior intensidade, maior área e menor ruído do que as amostras preparadas por extração em fase sólida, dada a otimização dos parâmetros cromatográficos. Os autores apresentam também os resultados da validação do procedimento extrativo por precipitação proteica e análise por LC-MS/MS, no equipamento da marca Sciex. Conclusões: A substituição da técnica de extração em fase sólida de 0,5 mL de amostra pela de precipitação proteica de 0,1 mL de sangue no procedimento laboratorial de confirmação e quantificação de canabinóides, torna-o mais simples, rápido e com um menor custo associado. O método validado demonstrou ser seletivo, específico, exato, preciso, robusto e linear na gama de trabalho, cumprindo todos os critérios de aceitação estabelecidos para a análise de canabinóides em amostras de sangue por LC-MS/MS.N/

Got her mummy´s eyes: Eye color investigation using IrisPlex – preliminary study

Poster apresentado no 9th ISABS Conference on Forensic and Anthropologic Genetics,22 a 26 de junho de 2015, ilha de Brač, CroáciaSNP phenotypic markers are being studied by several groups worldwide. As a result, phenotypic loci such as the ones responsible for human eye, hair and skin color are starting to get known. These may provide information for identification of unknown sample donors in criminal casework when there are no suspects. This kind of information may be even more relevant when the sample donor possesses phenotypic characteristics which distinguish him from the population in which he is inserted. Nevertheless, this approach may be used in similar scientific areas. In this work, a DNA sample from a mummified corpse with historical and scientific interest was studied in order to discover more information about who this corps belonged to. One of the tools used in this investigation was IrisPlex, designed for eye color prediction. This study presents one example on how this methodologies may be useful, not only in forensic investigation but also in areas such as physical anthropology.N/

Implementação do laboratório de virologia e análises clínico forenses na Delegação do Centro do INMLCF

Poster apresentado no 20º Congresso Nacional de Medicina Legal e Ciências Forenses, Coimbra, 2022N/