Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia e Pediatría

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Chemical Straightening as a Source of Analytical Variability in Hair Testing: A Dual-Analyte Case Report
(Wiley, 2026-04-08) Blanco Ces, Miriam; Castro Ríos, Ana de; Cobo Golpe, María; López Rabuñal, Ángela; Cruz Landeira, Angelines; Lendoiro Belío, Elena; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Hair analysis is increasingly used in forensic toxicology; however, result interpretation may be influenced by cosmetic hair treatments. Although the effects of bleaching, dyeing, and thermal straightening have already been evaluated, data on permanent chemical hair straightening are scarce, particularly under in vivo conditions. This case report evaluates the impact of an alkaline-based permanent hair straightening procedure on the determination of an endogenous compound, gamma-hydroxybutyrate (GHB), and an exogenous drug, alprazolam, in hair. Hair samples were collected from a 39-year-old woman before and 1 month after undergoing chemical hair straightening based on ammonium thioglycolate. Adjacent hair strands corresponding to identical temporal windows were analyzed. GHB was quantified in 0.5-cm hair segments using a validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method, whereas alprazolam was analyzed in 2-cm segments using a validated LC-MS/MS method for drugs of abuse and psychoactive pharmaceuticals. All analyzed segments were positive for GHB in both samples. After the straightening procedure, endogenous GHB concentrations showed a pronounced increase compared with pretreatment levels, with fold changes ranging from approximately 6 to 43 in corresponding segments. In contrast, alprazolam concentrations in segments corresponding to the reported period of drug intake decreased by approximately 1.7- to 2-fold following chemical straightening. This case report demonstrates that permanent chemical hair straightening can produce marked and analyte-dependent effects on hair drug concentrations. These findings emphasize the importance of systematic documentation of cosmetic hair treatments and careful interpretation of hair analysis results in forensic casework, particularly when analyte concentrations are low.
Avulsión del velo posterior durante reparación valvular mitral percutánea borde-a-borde
(Sociedad Espanola de Cirugia Cardiovascular y Endovascular (SECCE), 2025-04-14) Yebra, Carlos; Bastos-Fernández, María; Martínez Monzonís, Amparo; El-Diasty, Mohammad M.; Suárez Peñaranda, José Manuel; Fernández González, Ángel Luis; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Departamento de Cirurxía e Especialidades Médico-Cirúrxicas
Relationship Between Vertigo and Consumption of Psychotropic Drugs: A Prospective Case–Control Study
(MDPI, 2025-04-08) Sánchez Sellero, Inés; Soto Varela, Andrés; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Departamento de Cirurxía e Especialidades Médico-Cirúrxicas
Background/Objectives: The association between vestibular symptoms and psychological distress has been previously studied, mainly with the use of questionnaires. The purpose of this study is to compare the consumption of psychotropic drugs between a group of patients with vertigo and a control group. Methods: A prospective cross-sectional, observational, case–control study was carried out, including 506 patients (232 with Ménière’s disease, 79 with vestibular migraine, 34 with vestibular neuritis, and 161 with benign paroxysmal positional vertigo). In total, 253 participants were included in the control group. Both groups were comparable regarding age, sex, and history of previous psychiatric diseases. Results: The percentage of patients with vertigo who consumed psychotropic drugs (41.3%) was higher than the percentage of the control group who did so (26.9%) (Fisher’s exact test, p < 0.0001; OR = 1.914, CI95% (1.377; 2.662)). The mean number of psychotropic drugs consumed was also higher (Mann–Whitney test, p = 0.0003) in cases (0.68 ± 0.959) than in controls (0.47 ± 0.889). This higher consumption in the group of patients with vertigo was found for all pharmacological groups studied, being especially relevant regarding “anxiolytics and hypnotics and sedatives” and “antidepressants”. No statistically significant differences in the consumption of psychotropic drugs between types of vestibular disorders were observed. The longer the symptoms were present, the higher the prevalence of psychotropic drug use was observed. Conclusions: A relationship between vertigo and consumption of psychotropic drugs was found. Recording the consumption of these drugs is proposed as an objective method to better understand the psychological distress that patients with vertigo may suffer from.
OralImmunoAnalyser: a software tool for immunohistochemical assessment of oral leukoplakia using image segmentation and classification models
(Frontiers Media, 2024-02-26) Abdullah AL Tarawneh, Zakarya; Pena Cristóbal, Maite; Cernadas García, Eva; Suárez Peñaranda, José Manuel; Fernández Delgado, Manuel; Mbaidin, Almoutaz; Gallas Torreira, María Mercedes; Gándara Vila, Pilar; Universidade de Santiago de Compostela. Departamento de Cirurxía e Especialidades Médico-Cirúrxicas; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Centro de Investigación en Tecnoloxías Intelixentes da USC (CiTIUS)
Oral cancer ranks sixteenth amongst types of cancer by number of deaths. Many oral cancers are developed from potentially malignant disorders such as oral leukoplakia, whose most frequent predictor is the presence of epithelial dysplasia. Immunohistochemical staining using cell proliferation biomarkers such as ki67 is a complementary technique to improve the diagnosis and prognosis of oral leukoplakia. The cell counting of these images was traditionally done manually, which is time-consuming and not very reproducible due to intra- and inter-observer variability. The software presently available is not suitable for this task. This article presents the OralImmunoAnalyser software (registered by the University of Santiago de Compostela–USC), which combines automatic image processing with a friendly graphical user interface that allows investigators to oversee and easily correct the automatically recognized cells before quantification. OralImmunoAnalyser is able to count the number of cells in three staining levels and each epithelial layer. Operating in the daily work of the Odontology Faculty, it registered a sensitivity of 64.4% and specificity of 93% for automatic cell detection, with an accuracy of 79.8% for cell classification. Although expert supervision is needed before quantification, OIA reduces the expert analysis time by 56.5% compared to manual counting, avoiding mistakes because the user can check the cells counted. Hence, the SUS questionnaire reported a mean score of 80.9, which means that the system was perceived from good to excellent. OralImmunoAnalyser is accurate, trustworthy, and easy to use in daily practice in biomedical labs. The software, for Windows and Linux, with the images used in this study, can be downloaded from https://citius.usc.es/transferencia/software/oralimmunoanalyser for research purposes upon acceptance.
Epigenome-wide analysis reveals potential biomarkers for radiation-induced toxicity risk in prostate cancer
(BioMed Central, 2025-12-01) López-Pleguezuelos, Carlos; Aguado Barrera, Miguel Elías; Carballo-Castro, Ana; Peleteiro, Paula; Calvo-Crespo, Patricia; Taboada Valladares, Begoña; Lobato-Busto, Rubén; Fuentes Ríos, Olivia; Galego Carro, Javier; Coedo-Costa, Carla; Gómez-Caamaño, Antonio; Vega Gliemmo, Ana Paula; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría
Background: Prostate cancer is the second most common cancer globally, with radiation therapy (RT) being a key treatment for clinically localized and locally advanced cases. Given high survival rates, addressing long-term side effects of RT is crucial for preserving quality-of-life. Radiogenomics, the study of genetic variations affecting response to radiation, has primarily focussed on genomic biomarkers, while DNA methylation studies offer insights into RT responses. Although most research has centred on tumours, no epigenome-wide association studies have explored peripheral blood biomarkers of RT-induced toxicities in prostate cancer patients. Identifying such biomarkers could reveal molecular mechanisms underlying RT response and enable personalized treatment. Methods: We analysed 105 prostate cancer patients (52 cases and 53 controls). Cases developed grade ≥ 2 genitourinary and/or gastrointestinal late toxicity after 12 months of starting RT, whereas controls did not. An epigenome-wide association study of post-RT toxicities was performed using the Illumina MethylationEPIC BeadChip, adjusting for age and cell type composition. We constructed two methylation risk scores—one using differentially methylated positions (MRSsites) and another using differentially methylated regions (MRSregions)—as well as a Support Vector Machine-based methylation signature (SVMsites). We evaluated RT effects on biological age and stochastic epigenetic mutations within established radiation response pathways. Gene Ontology and pathway enrichment analyses were also performed. Results: Pre-RT methylation analysis identified 56 differentially methylated positions (adjusted p-value ≤ 0.05), and 6 differentially methylated regions (p-value ≤ 0.05) associated with the genes NTM, ACAP1, IL1RL2, VOOP1, AKR1E2, and an intergenic region on chromosome 13 related to Short/Long Interspersed Nuclear Elements. Both Methylation Risk Scores (MRSsites AUC = 0.87; MRSregions AUC = 0.89) and the 8-CpG Support Vector Machine signature (SVMsites AUC = 0.98) exhibited strong discriminatory accuracy in classifying patients in the discovery cohort. Gene ontology analysis revealed significant enrichment (adjusted p-value ≤ 0.05) of genes involved in DNA repair, inflammatory response, tissue repair, and oxidative stress response pathways. Conclusions: Epigenetic biomarkers show potential for predicting severe long-term adverse effects of RT in prostate cancer patients. The identified methylation patterns provide valuable insights into toxicity mechanisms and may aid personalized treatment strategies. However, validation in independent cohorts is essential to confirm their predictive value and clinical applicability.
Evaluating the trends and impact of COVID-19 on illicit drug and benzodiazepine use in drivers: A retrospective large-scale study based on oral fluid testing
(Elsevier, 2026-02-26) Blanco Ces, Miriam; Lendoiro Belío, Elena; Cruz Landeira, Angelines; Cobo Golpe, María; López Rabuñal, Ángela; Castro Ríos, Ana de; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Background: The COVID-19 pandemic disrupted global drug markets, but consumption soon returned to pre- pandemic levels. Continuous monitoring of drug use trends is essential for effective public health responses. Methods: A total of 29,397 oral fluid specimens from roadside drug tests across Spain (January 2019–July 2024) were sent to the Toxicology Laboratory of the Institute of Forensic Sciences, University of Santiago de Compostela, for LC-MS/MS confirmation of on-site positives. Results were stratified into five periods to assess drug use trends and the impact of COVID-19. Results: Over 90% of drivers were male, and 85% were under 45 years old. Overall, 69.9% of samples were positive for cannabis, 64.9% for cocaine, 13.7% for amphetamines, 10.6% for opiates, 6.5% for ketamine, 5.4% for methadone and 6.6% for benzodiazepines/zolpidem; 56.3% showed poly-drug use. Cannabis use was higher in men, while amphetamines and benzodiazepines were more frequent in women (p < 0.001). Due to regional variability in drug use patterns and sample distribution, trends were analyzed across five geographic regions. Globally, during the strict lockdown, cocaine, opiates, methadone and benzodiazepines peaked, while cannabis and amphetamines declined, and ketamine remained stable. In the final period, cannabis reached its highest levels, and ketamine showed a marked increase. Cocaine and amphetamines returned to pre-COVID levels, while opiates, methadone and benzodiazepines declined. Statistically significant differences across the studied periods were observed in the different regions. Specifically, in the Northwest for opiates, methadone, and benzodiazepines (p < 0.001), as well as for cannabis (p < 0.05); in the East for opiates, amphetamines, cocaine, and ketamine (p < 0.001); in the Center for cocaine (p < 0.001) and ketamine (p < 0.05); and in the South and Islands for cocaine (p < 0.05). Conclusions: Although a slight impact on drug use was observed during the strict lockdown, consumption increased again for all substances (particularly ketamine), except for opiates, methadone and benzodiazepines.
Possible adverse effects of mining activity on the neurocognitive development of children in the area of Cerro de Pasco (Perú)
(Elsevier, 2026-05) Carreiro-DaCunha, Elton; Ordóñez Mayán, Lucía; Bianchini, Flaviano; Muñoz Barús, José Ignacio; Universidade de Santiago de Compostela. Departamento de Psiquiatría, Radioloxía, Saúde Pública, Enfermaría e Medicina; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
Over the last 10 years there have been a number of studies examining the effects of exposure to environmental metal pollution on the population of the area of Cerro de Pasco (Peru). These have documented the prolonged pollution of the area caused by mining activity and recorded its pathological effects on the exposed population. The present work reports associations between the concentrations of metals in the hair of the area's children and their cognitive development, investigates the neurocognitive effects of exposure, and examines the change in environmental metal concentrations over time. Significant differences in hair metal concentrations were detected between exposed (case) and non-exposed (control) populations; in the former, the mean arsenic concentration was three times that of the latter, the cadmium concentration was double, and that of lead six times that of the latter. The mean total IQ of the exposed children was 12.3 points lower than those who were not exposed. Significant correlations were detected between the lead, cadmium, arsenic, manganese and antimony concentrations of the children's (combined exposed and non-exposed) hair and TIQ. In the exposed population, marked increases in hair metal concentrations were recorded between 2016 and 2018 (200 %), later falling by 2021 (though still exceeding the 2016 concentrations). Multivariate analyses involving big data are required to determine the covariables that influence the development of TIQ in exposed children, and to determine whether high toxic metal concentrations are an independent risk factor for cognitive deficit.
Whole-Genome Sequencing in Galicia Reveals Male-Biased Pre-Islamic North African Ancestry, Subtle Population Structure, and Microgeographic Patterns of Disease Risk
(Wiley, 2025-12-13) Pardo Seco, Jacobo; Camino Mera, Alba; Bello Paderne, Xabier; Gómez Carballa, Alberto; Castelo Martínez, Lúa; Martínez Cadenas, Conrado; Martinón Torres, Federico; Salas Ellacuriaga, Antonio; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría
Galicia, at the westernmost edge of Europe, exhibits distinctive genetic traits compared to other Iberian populations. We present the first whole-genome sequencing (WGS) study of a Galician population (GALOMICS; n = 91; 17.2 M variants; https://galomics.genpob.eu), analyzed alongside WGS data from other Spanish and continental populations (n = 1078). Contrary to recent claims of extreme genetic stratification, Galicia's structure reflects broader Iberian patterns, characterized by one major genetic cluster and four minor, localized ones. Analyses of the Spanish National DNA Bank (NDNAB; n = 453) confirm this pattern, with three Galician clusters, one clearly predominant. Phylogenetic analysis places Galician clusters on terminal, recently diverged branches, challenging earlier models suggesting ancient separation. Slightly elevated homozygosity, driven by the Porto do Son cluster, suggests mild regional inbreeding. A notable North African/Middle Eastern ancestry component (13.5%–16.5%) appears, likely introduced via trans-Mediterranean contact ca. 620–670 ce, predating the Islamic conquest of 711 ce, with a subtle south-to-north gradient and a male-biased signal (Y-DNA: 21.2%; mtDNA: 1.1%). This calls for reexamining assumptions about Islamic-era ancestry. Finally, Polygenic Risk Scores for common diseases (e.g., cancer, Alzheimer's disease, diabetes, autism) show geographic variability aligned with genetic substructure, highlighting the relevance of regional genomics to public health policy.
Interferon gene expression declines over time post-COVID infection and in long COVID patients
(Taylor and Francis, 2024-08-20) Gómez Carballa, Alberto; Pischedda, Sara; Pardo Seco, Jacobo José; Gómez Rial, José; Martinón Torres, Federico; Salas Ellacuriaga, Antonio; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría
Background: Interferons (IFNs) represent a first-line defense against viruses and other pathogens. It has been shown that an impaired and uncontrolled release of these glycoproteins can result in tissue damage and explain severe progression of coronavirus disease 2019 (COVID-19). However, their potential role in Long-COVID syndrome (LC) remains debateable. Objectives: The objective of the present study is to shed further light on the possible role of IFNs (and related genes) gene expression patterns in the progression of COVID-19 and LC patients. Methods: We carried out a multi-cohort study by analyzing the IFN gene expression patterns (using different IFN gene signatures) in five cohorts of acute COVID-19 (n = 541 samples) and LC patients (n = 188), and compared them to patterns observed in three autoimmune diseases (systemic lupus erythematous [n = 242], systemic sclerosis [n = 91], and Sjögren’s syndrome [n = 282]). Results: The data show that, while the interferon signatures are strongly upregulated in severe COVID-19 patients and autoimmune diseases, it decays with the time from symptoms onset and in LC patients. Differential pathway analysis of IFN-related terms indicates an over activation in autoimmune diseases (IFN-I/II) and severe COVID-19 (IFN-I/II/III), while these pathways are mostly inactivated or downregulated in LC (IFN-I/III). By analyzing six proteomic LC datasets, we did not find evidence of a role of IFNs in this condition. Conclusion: Our findings suggest a potential role of cytokine exhaustion mediated by IFN gene expression inactivation as a possible driver of LC.
Is There a Bias Towards Males in the Diagnosis of Autism? A Systematic Review and Meta-Analysis
(Springer, 2024-01-29) Cruz, Sara; Conde-Pumpido Zubizarreta, Sabela; Costa, Ana Daniela; Araújo, Rita; Martinho, Júlia; Tubío Fungueiriño, María; Sampaio, Adriana; Cruz Guerrero, Raquel; Carracedo Álvarez, Ángel; Fernández Prieto, Montserrat; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
Autism is more frequently diagnosed in males, with evidence suggesting that females are more likely to be misdiagnosed or underdiagnosed. Possibly, the male/female ratio imbalance relates to phenotypic and camouflaging differences between genders. Here, we performed a comprehensive approach to phenotypic and camouflaging research in autism addressed in two studies. First (Study 1 – Phenotypic Differences in Autism), we conducted a systematic review and meta-analysis of gender differences in autism phenotype. The electronic datasets Pubmed, Scopus, Web of Science, and PsychInfo were searched. We included 67 articles that compared females and males in autism core symptoms, and in cognitive, socioemotional, and behavioural phenotypes. Autistic males exhibited more severe symptoms and social interaction difficulties on standard clinical measures than females, who, in turn, exhibited more cognitive and behavioural difficulties. Considering the hypothesis of camouflaging possibly underlying these differences, we then conducted a meta-analysis of gender differences in camouflaging (Study 2 – Camouflaging Differences in Autism). The same datasets as the first study were searched. Ten studies were included. Females used more compensation and masking camouflage strategies than males. The results support the argument of a bias in clinical procedures towards males and the importance of considering a ‘female autism phenotype’—potentially involving camouflaging—in the diagnostic process.
Insights Into Hereditary Alpha-Tryptasemia From a Genome-Wide Association Study in Hymenoptera Venom Anaphylaxis
(Wiley, 2026) Blanco Ramos, María Teresa; Cruz Guerrero, Raquel; Fernández-Franco, Irene; Vargas, Mª Ángeles; Carracedo Álvarez, Ángel; González Quintela, Arturo; Vidal Pan, Carmen; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Departamento de Psiquiatría, Radioloxía, Saúde Pública, Enfermaría e Medicina; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
Forensic SNP Genotyping with SNaPshot: Technical Considerations for the Development and Optimization of Multiplexed SNP Assays
(Forensic Science Review, 2017-01-01) Fondevila, M.; Børsting, C.; Phillips, C; Puente Vila, María del Carmen de la; Santos, C.; EUROFORGEN-NoE Consortium; Carracedo Álvarez, Ángel; Morling, N.; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot single-base extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides an advantage. In order to provide the basis for developing such expertise, we cover in this paper the most challenging aspects of the SNaPshot technology, focusing on the steps taken to design primer sets, optimize the PCR and single-base extension chemistries, and the important features of the peak patterns observed in typical forensic SNP profiles using SNaPshot. With that purpose in mind, we provide guidelines and troubleshooting for multiplex-SNaPshot-oriented primer design and the resulting capillary electrophoresis (CE) profile interpretation (covering the most commonly observed artifacts and expected departures from the ideal conditions).
Cerebellar Cognitive Affective Syndrome in Costa da Morte Ataxia (SCA36)
(Springer Nature Link, 2020-04-08) Martínez Regueiro, Rocío; Pombo Arias, Manuel Arturo; Cruz Guerrero, Raquel; Quintáns Castro, Beatriz; Labella Caballero, T.; Pardo, M.; Pardo, J.; García Murias, M.; Carracedo Álvarez, Ángel; Sobrido, María Jesús; Fernández Prieto, M.; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
SCA36 is an autosomal dominant spinocerebellar ataxia (SCA) affecting many families from Costa da Morte, a northwestern region of Spain. It is caused by an intronic GGCCTG repeat expansion in NOP56. In order to characterize the cognitive and affective manifestations of this cerebellar disease, a group of 30 SCA36 mutation carriers (11 preataxic and 19 ataxic patients) were assessed with a comprehensive battery of standardized tests. Phonological verbal fluency – but not semantic fluency – was already mildly impaired in preataxic subjects. In ataxic patients, both phonological and semantic fluencies were significantly below normal. Depression, while more frequent and prominent in ataxic patients, was also often present in the preataxic stage. This is the first systematic study supporting the presence of a mild cerebellar cognitive and affective syndrome in SCA36. Routine evaluation of cognitive and emotional spheres in SCA36 patients as well as asymptomatic mutation carriers should allow early detection and timely therapeutic intervention.
Development of the VISAGE enhanced tool and statistical models for epigenetic age estimation in blood, buccal cells and bones
(Aging, 2021-03-11) Woznial, Anna; Heidegger, Antonia; Piniewska-Róg, Danuta; Pośpiech, Ewelina; Xavier, Catarina; Pisarek, Aleksandra; Kartasińska, Ewa; Boroń, Michal; Freire Aradas, Ana María; Wojtas, Marta; Puente Vila, María del Carmen de la; Niederstätter, Harald; Płoski, Rafal; Spólnicka, Magdalena; Kayser, Manfred; Phillips, Christopher P.; Parson, Walther; Branicki, Wojciech; VISAGE Consortium; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium’s enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2, MIR29B2CHG, KLF14, FHL2, TRIM59 and PDE4C, and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C, MIR29B2CHG, ELOVL2, KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue
Development and Evaluation of the Ancestry Informative Marker Panel of the VISAGE Basic Tool
(MDPI, 2021-08-22) Puente Vila, María del Carmen de la; Ruiz Ramírez, Jorge; Ambroa Conde, Adrián; Álvarez Dios, José Antonio; Freire Aradas, Ana María; Mosquera Miguel, Ana; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Phillips, Christopher; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
We detail the development of the ancestry informative single nucleotide polymorphisms (SNPs) panel forming part of the VISAGE Basic Tool (BT), which combines 41 appearance predictive SNPs and 112 ancestry predictive SNPs (three SNPs shared between sets) in one massively parallel sequencing (MPS) multiplex, whereas blood-based age analysis using methylation markers is run in a parallel MPS analysis pipeline. The selection of SNPs for the BT ancestry panel focused on established forensic markers that already have a proven track record of good sequencing performance in MPS, and the overall SNP multiplex scale closely matched that of existing forensic MPS assays. SNPs were chosen to differentiate individuals from the five main continental population groups of Africa, Europe, East Asia, America, and Oceania, extended to include differentiation of individuals from South Asia. From analysis of 1000 Genomes and HGDP-CEPH samples from these six population groups, the BT ancestry panel was shown to have no classification error using the Bayes likelihood calculators of the Snipper online analysis portal. The differentiation power of the component ancestry SNPs of BT was balanced as far as possible to avoid bias in the estimation of co-ancestry proportions in individuals with admixed backgrounds. The balancing process led to very similar cumulative population-specific divergence values for Africa, Europe, America, and Oceania, with East Asia being slightly below average, and South Asia an outlier from the other groups. Comparisons were made of the African, European, and Native American estimated co-ancestry proportions in the six admixed 1000 Genomes populations, using the BT ancestry panel SNPs and 572,000 Affymetrix Human Origins array SNPs. Very similar co-ancestry proportions were observed down to a minimum value of 10%, below which, low-level co-ancestry was not always reliably detected by BT SNPs. The Snipper analysis portal provides a comprehensive population dataset for the BT ancestry panel SNPs, comprising a 520-sample standardised reference dataset; 3445 additional samples from 1000 Genomes, HGDP-CEPH, Simons Foundation and Estonian Biocentre genome diversity projects; and 167 samples of six populations from in-house genotyping of individuals from Middle East, North and East African regions complementing those of the sampling regimes of the other diversity projects.
Evaluation of the Qiagen 140-SNP forensic identification multiplex for massively parallel sequencing
(Elsevier, 2017-01-27) Puente Vila, María del Carmen de la; Phillips, Christopher P.; Santos, C.; Fondevila, Manuel; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
A new forensic 140-SNP genotyping system from Qiagen, designed for massively parallel sequencing (MPS) analysis, was evaluated using the Ion PGM™ MPS system. Assessments consisted of the sequencing of: established control DNAs that had been previously genotyped with alternative PCR and library preparation kits supplied by Thermo Fisher Scientific for the Ion PGM™ system; a simple set of artificial DNA mixtures; DNA extracted from a degraded femur; and a dilution series to gauge forensic sensitivity. In addition to the reagents for the DNA target capture PCR and library preparation, Qiagen offer an alternative sequence analysis software system (Workbench), which was assessed alongside the Ion PGM™ Genotyper software for forensic MPS analysis. The Qiagen SNP genotyping system produced full genotyping concordance with previous data obtained with a similar SNP panel on the Ion PGM™ and in comparison to genotypes listed for 139 of the 140 SNPs in 1000 Genomes. The workbench software was as reliable as Genotyper in calling genotypes, although scrutiny of sequence data with IGV revealed the problem of sequence misalignment plagues a small proportion of the 140 SNPs in the Qiagen panel, a problem already recognized in multiple MPS studies of the same markers in alternative kits. The potential for genotype miscalls from sequence misalignment in certain SNPs will require manual inspection in cases where low-level or degraded DNA reduces the sequence coverage to a point where misalignment influences individual SNP genotype quality
The global AIMs nano set: A 31-plex SNaPshot assay of ancestry-informative SNPs
(Elsevier, 2016-01-25) Puente Vila, María del Carmen de la; Santos, C.; Fondevila, Manuel; Manzo, L; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Phillips, Christopher P.; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
A 31-plex SNaPshot assay, named 'Global AIMs Nano', has been developed by reassembling the most differentiated markers of the EUROFORGEN Global AIM-SNP set. The SNPs include three tri-allelic loci and were selected with the goal of maintaining a balanced differentiation of: Africans, Europeans, East Asians, Oceanians and Native Americans. The Global AIMs Nano SNP set provides higher divergence between each of the five continental population groups than previous small-scale AIM sets developed for forensic ancestry analysis with SNaPshot. Both of these characteristics minimise potential bias when estimating co-ancestry proportions in individuals with admixed ancestry; more likely to be observed when using markers disproportionately informative for only certain population group comparisons. The optimised multiplex is designed to be easily implemented using standard capillary electrophoresis regimes and has been used to successfully genotype challenging forensic samples from highly degraded material with low level DNA. The ancestry predictive performance of the Global AIMs Nano set has been evaluated by the analysis of samples previously characterised with larger AIM sets
Inter-laboratory evaluation of the EUROFORGEN global ancestry-informative SNP panel by massively parallel sequencing using the Ion PGM™
(Elsevier, 2016-04-21) Eduardoff, M.; Gross, T. E.; Santos, C.; Puente Vila, María del Carmen de la; Ballard, David J.; Strobl, C.; Børsting, Claus; Morling, Niels; Fusco, L.; Hussing, C.; Egyed, B.; Souto, L.; Uacyisrael, J.; Syndercombe Court, Y. Denise; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Schneider, Peter M.; Parson, Walther; Phillips, Christopher P.; EUROFORGEN-NoE Consortium; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
The EUROFORGEN Global ancestry-informative SNP (AIM-SNPs) panel is a forensic multiplex of 128 markers designed to differentiate an individual's ancestry from amongst the five continental population groups of Africa, Europe, East Asia, Native America, and Oceania. A custom multiplex of AmpliSeq™ PCR primers was designed for the Global AIM-SNPs to perform massively parallel sequencing using the Ion PGM™ system. This study assessed individual SNP genotyping precision using the Ion PGM™, the forensic sensitivity of the multiplex using dilution series, degraded DNA plus simple mixtures, and the ancestry differentiation power of the final panel design, which required substitution of three original ancestry-informative SNPs with alternatives. Fourteen populations that had not been previously analyzed were genotyped using the custom multiplex and these studies allowed assessment of genotyping performance by comparison of data across five laboratories. Results indicate a low level of genotyping error can still occur from sequence misalignment caused by homopolymeric tracts close to the target SNP, despite careful scrutiny of candidate SNPs at the design stage. Such sequence misalignment required the exclusion of component SNP rs2080161 from the Global AIM-SNPs panel. However, the overall genotyping precision and sensitivity of this custom multiplex indicates the Ion PGM™ assay for the Global AIM-SNPs is highly suitable for forensic ancestry analysis with massively parallel sequencing
Global patterns of STR sequence variation: Sequencing the CEPH human genome diversity panel for 58 forensic STRs using the Illumina ForenSeq DNA Signature Prep Kit
(Wiley, 2018-07-16) Phillips, Christopher P.; Devesse, Laurence; Ballard, David; Weert, Leanne van; Puente Vila, María del Carmen de la; Melis, Stefania; Álvarez Iglesias, Vanessa; Freire Aradas, Ana María; Oldroyd, Nicola; Holt, Cydne; Syndercombre Court, Denise; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR)
The 944 individuals of the CEPH human genome diversity panel (HGDP-CEPH), a standard sample set of 51 globally distributed populations, were sequenced using the Illumina ForenSeq™ DNA Signature Prep Kit. The ForenSeq™ system is a single multiplex for the MiSeq/FGx™ massively parallel sequencing instrument, comprising: amelogenin, 27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 SNPforID+Kiddlab autosomal ID-SNPs (plus optionally detected ancestry and phenotyping SNP sets). We report in detail the patterns of sequence variation observed in the repeat regions of the 58 forensic STR loci typed by the ForenSeq™ system. Sequence alleles were characterized and repeat region structures annotated by aligning the ForenSeq™ sequence output to the latest GRCh38 human reference sequence, necessitating the reversal and re-alignment of STR allele sequences reported by the Forenseq™ system in 20 of 58 STRs (plus the reverse alleles in two Y-STRs with duplicated-inverted repeat regions). Individual population sample sizes of the HGDP-CEPH panel do not allow reliable inferences to be made about levels of genetic variability in low frequency STR alleles-where particular sequence variants are found in only a few individuals; but we assessed the occurrence of both population-specific sequence variants and singleton observations; finding each of these in a sizeable proportion of HGDP-CEPH samples, with consequences for planning the co-ordinated compilation of sequence variation on a much larger scale than was required before by forensic laboratories now adopting massively parallel sequencing
A forensic multiplex of nine novel pentameric-repeat STRs
(Elsevier, 2017-04-15) Puente Vila, María del Carmen de la; Phillips, Christopher P.; Fondevila, Manuel; Gelabert Besada, Miguel; Carracedo Álvarez, Ángel; Lareu Huidobro, María Victoria; Universidade de Santiago de Compostela. Departamento de Ciencias Forenses, Anatomía Patolóxica, Xinecoloxía e Obstetricia, e Pediatría; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS)
Pentameric-repeat short tandem repeats (STRs), consisting of loci with repeat units of five base-pairs, have the advantage of reduced stutter products compared to their tetrameric-repeat STR counterparts. This characteristic potentially helps the interpretation of mixed DNA profiles when minor component alleles may coincide with stutter peaks from the major components. To develop a simple but informative forensic multiplex with the capability to aid mixture interpretation, we designed an 11-plex assay of nine pentameric STRs new to forensic analysis plus two male- specific markers: DYS391 and the Y-Indel rs2032678 used in GlobalFiler™ (Life Technologies). East Asian-specific variation in the recently adopted Y-Indel rs2032678 is reported in this study for the first time in its forensic use as a sex marker. We estimated the levels of variation observed in the nine pentameric STRs in three of the major population groups sampled in the HGDP-CEPH human genome diversity panel: African, European and East Asian (combining individual populations as their sample sizes were too small for STR allele frequency estimations); and we include genotype data from a population sample of Northwest Spain. From this data, forensic informativeness metrics were estimated when applying the nine novel STRs in identification or kinship analyses. The assay was assessed for forensic sensitivity and ability to successfully genotype highly degraded DNA. In the profiles from the 11-plex assay we observed an average 2.15% stutter ratio in all the pentameric loci compared to 7.32% across equivalently-sized tetrameric STRs in the Promega Powerplex® ESX-17 kit

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