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Genotype; Cancer phenotypes; Genetic tumour Genotip; Fenotips del càncer; Tumor genètic Genotipo; Fenotipos del cáncer; Tumor genético Background Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype–phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing. Methods This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype–phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ2, and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test. Findings From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1–93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66–57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18–29·39], p=0·0017) and gastric cancer (7·81 [2·03–29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004). Interpretation CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria. Funding European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme. European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme.

Microsatellites (or short-tandem repeats (STRs)) are widely used in anthropology and evolutionary studies. Their extensive polymorphism and rapid evolution make them the ideal genetic marker for dating events, such as the age of a gene or a population. This usage requires the estimation of mutation rates, which are usually estimated by counting the observed Mendelian incompatibilities in one-generation familial configurations (typically parent(s)–child duos or trios). Underestimations are inevitable when using this approach, due to the occurrence of mutational events that do not lead to incompatibilities with the parental genotypes (‘hidden’ or ‘covert’ mutations). It is known that the likelihood that one mutation event leads to a Mendelian incompatibility depends on the mode of genetic transmission considered, the type of familial configuration (duos or trios) considered, and the genotype(s) of the progenitor(s). In this work, we show how the magnitude of the underestimation of autosomal microsatellite mutation rates varies with the populations’ allele frequency distribution spectrum. The Mendelian incompatibilities approach (MIA) was applied to simulated parent(s)/offspring duos and trios in different populational scenarios. The results showed that the magnitude and type of biases depend on the population allele frequency distribution, whatever the type of familial data considered, and are greater when duos, instead of trios, are used to obtain the estimates. The implications for molecular anthropology are discussed and a simple framework is presented to correct the naïf estimates, along with an informatics tool for the correction of incompatibility rates obtained through the MIA.

Background: Dementia due to Alzheimer’s disease (AD) is a complex neurodegenerative disorder, which much of heritability remains unexplained. At the clinical level, one of the most common physiological alterations is the slowing of oscillatory brain activity, measurable by electroencephalography (EEG). Relative power (RP) at the conventional frequency bands (i.e., delta, theta, alpha, beta-1, and beta-2) can be considered as AD endophenotypes. Objective: The aim of this work is to analyze the association between sixteen genes previously related with AD: APOE, PICALM, CLU, BCHE, CETP, CR1, SLC6A3, GRIN2 β, SORL1, TOMM40, GSK3 β, UNC5C, OPRD1, NAV2, HOMER2, and IL1RAP, and the slowing of the brain activity, assessed by means of RP at the aforementioned frequency bands. Methods: An Iberian cohort of 45 elderly controls, 45 individuals with mild cognitive impairment, and 109 AD patients in the three stages of the disease was considered. Genomic information and brain activity of each subject were analyzed. Results: The slowing of brain activity was observed in carriers of risk alleles in IL1RAP (rs10212109, rs9823517, rs4687150), UNC5C (rs17024131), and NAV2 (rs1425227, rs862785) genes, regardless of the disease status and situation towards the strongest risk factors: age, sex, and APOE ɛ4 presence. Conclusion: Endophenotypes reduce the complexity of the general phenotype and genetic variants with a major effect on those specific traits may be then identified. The found associations in this work are novel and may contribute to the comprehension of AD pathogenesis, each with a different biological role, and influencing multiple factors involved in brain physiology.

Migraine is a complex neurovascular disorder affecting one billion people worldwide, mainly females. It is characterized by attacks of moderate to severe headache pain, with associated symptoms. Receptor activity modifying protein (RAMP1) is part of the Calcitonin Gene-Related Peptide (CGRP) receptor, a pharmacological target for migraine. Epigenetic processes, such as DNA methylation, play a role in clinical presentation of various diseases. DNA methylation occurs mostly in the gene promoter and can control gene expression. We investigated the methylation state of the

PICALM and CLU genes have been linked to alterations in brain biochemical processes that may have an impact on Alzheimer’s disease (AD) development and neurophysiological dynamics. The aim of this study is to analyze the relationship between the electroencephalographic (EEG) activity and the PICALM and CLU alleles described as conferring risk or protective effects on AD patients and healthy controls. For this purpose, EEG activity was acquired from: 18 AD patients and 12 controls carrying risk alleles of both PICALM and CLU genes, and 35 AD patients and 12 controls carrying both protective alleles. Relative power (RP) in the conventional EEG frequency bands (delta, theta, alpha, beta, and gamma) was computed to quantify the brain activity at source level. In addition, spatial entropy (SE) was calculated in each band to characterize the regional distribution of the RP values throughout the brain. Statistically significant differences in global RP and SE at beta band (p-values < 0.05, Mann–Whitney U-test) were found between genotypes in the AD group. Furthermore, RP showed statistically significant differences in 58 cortical regions out of the 68 analyzed in AD. No statistically significant differences were found in the control group at any frequency band. Our results suggest that PICALM and CLU AD-inducing genotypes are involved in physiological processes related to disruption in beta power, which may be associated with physiological disturbances such as alterations in beta-amyloid and neurotransmitter metabolism.

Short tandem repeats are extremely abundant in the human genome and widely used in population and forensic genetics. The conventional approach to estimate mutation rates for STRs involves the detection of Mendelian incompatibilities and their frequency quantification, in family duos or trios. In this work, we aim to measure the impact this estimation methodology implies in the underestimation of mutation rates. We computed two independent experiences simulating one single-step mutation and one double step mutation in each pedigree. The pedigree with the highest number of compatibilities observed (hidden mutations) was mother-daughter duo, followed by the father-daughter duo, mother-son duo and, finally, mother-father-child trio, for the one and two-steps simulations. The estimation of X-STRs mutation rates from simple counting proved to be biased through systematic underestimation in an extent which depends on allele frequencies distribution of the marker, type of pedigree and number of repeat units involved.

Y chromosome specific short tandem repeats (Y-STRs) are widely used in population genetics and forensics. Since these markers do not recombine, mutation is the only source of diversity. The primary mutational mechanism leading to length changes in STRs is thought to be polymerase template slippage, and the most common change is the gain or the loss of one repeat motif. In this work, we aim to study 19 Y-STR alleles’ contraction and expansion. Alleles were grouped into tertiles: short (1st tertile), intermediate (2nd tertile) and long alleles (3rd tertile). Significant differences between repeat gains and losses were found at four markers - DYS19, DYS439 for intermediate alleles, and DYS570 and DYS626 for long alleles. When the average number is computed for the pooled loci, for short alleles, the number of repeat motif gains is higher than of repeat losses, and the opposite happens for long alleles. For intermediate alleles, the proportion between the number of repeat gains and losses is close to one. Generally, the rate of expansion decreases from the first tertile to the third, and conversely, the rate of contraction increases from the first tertile to the third. The pooled loci tertiles’ mutation rate increases from short to long alleles. Our results demonstrate that the mutation direction and rate depend on alleles’ length. The longer the allele the greater the mutation and contraction rates.

Population representative databases are necessary for the quantification of the genetic evidence in forensic casework. It is thus essential to perform a correct clustering of the samples, as genetic distances could substantially vary using different regional patterns. Consequently, the final clustering scheme for haplotype frequencies and estimates of forensic parameters can also change. In this study 914 unrelated haplotypes from residents of all the provinces of Argentina, typed for a set of 12 X-STRs, were considered. To compute differentiation analyses and HWE estimations we considered five alternative divisions of Argentina territory: i) Northwest, Northeast, Pampeana, Cuyo and Patagonia, ii) Northwest, Northeast, Center, and South, iii) Novo Cuyo, Norte Grande, Center, Patagonia, and Buenos Aires, iv) North, Center, and South, v) Northwest, Northeast, Middlenorth, Center and South, grouping the provinces through diverse patterns. Evidences of stratification were not found for four (i to iv) of the five clustering patterns considered. Our work shows that an inadequate clustering of the samples can lead to misleading results concerning population stratification, and thus multiple clustering alternatives of the same territory should be considered to ensure sound conclusions. This analysis is particularly pertinent in populations, such as Argentina, where clime, geography and demography deeply vary across the territory, which may result in non-coincident administrative and natural divisions.

To overcome the multifactorial complexity associated with the analysis and interpretation of the capillary electrophoresis results of forensic mixture samples, probabilistic genotyping methods have been developed and implemented as software, based on either qualitative or quantitative models. The former considers the electropherograms' qualitative information (detected alleles), whilst the latter also takes into account the associated quantitative information (height of allele peaks). Both models then quantify the genetic evidence through the computation of a likelihood ratio (LR), comparing the probabilities of the observations given two alternative and mutually exclusive hypotheses. In this study, the results obtained through the qualitative software LRmix Studio (v.2.1.3), and the quantitative ones: STRmix™ (v.2.7) and EuroForMix (v.3.4.0), were compared considering real casework samples. A set of 156 irreversibly anonymized sample pairs (GeneMapper files), obtained under the scope of former cases of the Portuguese Scientific Police Laboratory, Judiciary Police (LPC-PJ), were independently analyzed using each software. Sample pairs were composed by (i) a mixture profile with either two or three estimated contributors, and (ii) a single contributor profile associated. In most cases, information on 21 short tandem repeat (STR) autosomal markers were considered, and the majority of the single-source samples could not be a priori excluded as belonging to a contributor to the paired mixture sample. This inter-software analysis shows the differences between the probative values obtained through different qualitative and quantitative tools, for the same input samples. LR values computed in this work by quantitative tools showed to be generally higher than those obtained by the qualitative. Although the differences between the LR values computed by both quantitative software showed to be much smaller, STRmix™ generated LRs are generally higher than those from EuroForMix. As expected, mixtures with three estimated contributors showed generally lower LR values than those obtained for mixtures with two estimated contributors. Different software products are based on different approaches and mathematical or statistical models, which necessarily result in the computation of different LR values. The understanding by the forensic experts of the models and their differences among available software is therefore crucial. The better the expert understands the methodology, the better he/she will be able to support and/or explain the results in court or any other area of scrutiny.

Forensic genetics is a fast-growing field that frequently requires DNA-based taxonomy, namely, when evidence are parts of specimens, often highly processed in food, potions, or ointments. Reference DNA-sequences libraries, such as BOLD or GenBank, are imperative tools for taxonomic assignment, particularly when morphology is inadequate for classification. The auditing and curation of these datasets require reliable mechanisms, preferably with automated data preprocessing. Software tools were developed to grade these datasets considering as primary criterion the number of records, which is not compliant with forensic standards, where the priority is validation from independent sources. Moreover, 4SpecID is an efficient and freely available software tool developed to audit and annotate reference libraries, specifically designed for forensic applications. Its intuitive user-friendly interface virtually accesses any database and includes specific data mining functions tuned for the widespread BOLD repositories. The built tool was evaluated in laptop MacBook and a dual-Xeon server with a large BOLD dataset (Culicidae, 36,115 records), and the best execution time to grade the dataset on the laptop was 0.28 s. Datasets of Bovidae and Felidae families were used to evaluate the quality of the tool and the relevance of independent sources validation., Portuguese funds are supporting this work through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “Center of Mathematics of the University of Porto” (UID/MAT/00144/2019). Search-ON2: Revitalization of HPC infrastructure of UMinho” (NORTE-07-0162-FEDER-000086), cofunded by the North Portugal Regional Operational Programme (ON.2–O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Nádia Pinto work contract is established under the framework of Decreto-Lei nº 57/2016 de 29 de Agosto. Eduardo Conde-Sousa was supported by the project PPBI-POCI-01-0145-FEDER-022122, in the scope of Fundação para a Ciência e Tecnologia, Portugal (FCT) National Roadmap of Research Infrastructures.

The primary genetic risk factor for late onset Alzheimer&rsquo, s disease (LOAD) is the APOE4 allele of Apolipoprotein E (APOE) gene. The three most common variants of APOE are determined by single nucleotide polymorphisms (SNPs) rs429358 and rs7412. Our aim was to estimate allele and genotype frequencies of APOE variants in an Iberian cohort, thus helping to understand differences in APOE-related LOAD risk observed across populations. We analyzed saliva or buccal swab samples from 229 LOAD patients and 89 healthy elderly controls (&ge, 68 years old) from Northern Portugal and Castile and Le&oacute, n region, Spain. The genotyping was performed by Sanger sequencing, optimized to overcome GC content drawbacks. Results obtained in our Iberian LOAD and control cohorts are in line with previous large meta-analyses on APOE frequencies in Caucasian populations, however, we found differences in allele frequencies between our Portuguese and Spanish subgroups of AD patients. Moreover, when comparing studies from Iberian and other Caucasian cohorts, differences in APOE2 and APOE4 frequencies and subsequent different APOE-related LOAD risks must be clarified. These results show the importance of studying genetic variation at the APOE gene in different populations (including analyses at a regional level) to increase our knowledge about its clinical significance.

Brain waves, measured by electroencephalography (EEG), are a powerful tool in the investigation of neurophysiological traits and a noninvasive and cost-effective alternative in the diagnostic of some neurological diseases. In order to identify novel Quantitative Trait Loci (QTLs) for brain wave relative power (RP), we collected resting state EEG data in five frequency bands (&delta, &theta, &alpha, &beta, 1, and &beta, 2) and genome-wide data in a cohort of 105 patients with late onset Alzheimer&rsquo, s disease (LOAD), 41 individuals with mild cognitive impairment and 45 controls from Iberia, correcting for disease status. One novel association was found with an interesting candidate for a role in brain wave biology, CLEC16A (C-type lectin domain family 16), with a variant at this locus passing the adjusted genome-wide significance threshold after Bonferroni correction. This finding reinforces the importance of immune regulation in brain function. Additionally, at a significance cutoff value of 5 &times, 10&minus, 6, 18 independent association signals were detected. These signals comprise brain expression Quantitative Loci (eQTLs) in caudate basal ganglia, spinal cord, anterior cingulate cortex and hypothalamus, as well as chromatin interactions in adult and fetal cortex, neural progenitor cells and hippocampus. Moreover, in the set of genes showing signals of association with brain wave RP in our dataset, there is an overrepresentation of loci previously associated with neurological traits and pathologies, evidencing the pleiotropy of the genetic variation modulating brain function.

Alzheimer&rsquo, s disease (AD) is the most prevalent cause of dementia, being considered a major health problem, especially in developed countries. Late-onset AD is the most common form of the disease, with symptoms appearing after 65 years old. Genetic determinants of AD risk are vastly unknown, though, &epsilon, 4 allele of the ApoE gene has been reported as the strongest genetic risk factor for AD. The objective of this study was to analyze the relationship between brain complexity and the presence of ApoE &epsilon, 4 alleles along the AD continuum. For this purpose, resting-state electroencephalography (EEG) activity was analyzed by computing Lempel-Ziv complexity (LZC) from 46 healthy control subjects, 49 mild cognitive impairment subjects, 45 mild AD patients, 44 moderate AD patients and 33 severe AD patients, subdivided by ApoE status. Subjects with one or more ApoE &epsilon, 4 alleles were included in the carriers subgroups, whereas the ApoE &epsilon, 4 non-carriers subgroups were formed by subjects without any &epsilon, 4 allele. Our results showed that AD continuum is characterized by a progressive complexity loss. No differences were observed between AD ApoE &epsilon, 4 carriers and non-carriers. However, brain activity from healthy subjects with ApoE &epsilon, 4 allele (carriers subgroup) is more complex than from non-carriers, mainly in left temporal, frontal and posterior regions (p-values &lt, 0.05, FDR-corrected Mann&ndash, Whitney U-test). These results suggest that the presence of ApoE &epsilon, 4 allele could modify the EEG complexity patterns in different brain regions, as the temporal lobes. These alterations might be related to anatomical changes associated to neurodegeneration, increasing the risk of suffering dementia due to AD before its clinical onset. This interesting finding might help to advance in the development of new tools for early AD diagnosis.

Unbiased estimation of germinal mutation rates are essential for population and evolutionary genetic analyses and in any forensic kinship evaluation. A major source of bias in diploid or haplodiploid loci was identified – ‘hidden’ or ‘covert’ mutations (i.e. those not causing Mendelian incompatibilities) and ways to overcome it proposed. Morever, the identification of (a) the parental meiosis in which mutation has occurred, as well as (b) the allele of origin and the mutated one, are problematic. A pseudo most parsimonious approach goes on being used although it was already shown that misclassification of mutations has a significant impact, overestimating one-step mutations and underestimating all others. In forensics, current publication and other publicly obtainable data formats ignore both types of bias and do not allow more than the estimation of average incompatibility rates per locus. These values are biased and, in kinship analyses, estimates of biallelic specific mutation rates considering also its parental origin, should be used for correct likelihood calculations. We urge the forensic community to undertake the task of preparing guidelines for archival, retrieval and most specially, publication of data in a format that will allow a more accurate estimation of this crucial parameter, providing a solid ground for the formulation and testing of mutation models. For this, the publication of the complete set of observed genotypes in duos and/or trios in the population sample is mandatory.

Objective The aim of this study was to evaluate the effect of electroencephalographic (EEG) volume conduction in different measures of functional connectivity and to characterize the EEG coupling alterations at the different stages of dementia due to Alzheimer's disease (AD). Approach Magnitude squared coherence (MSCOH), imaginary part of coherence (iCOH), lagged coherence (lagCOH), amplitude envelope correlation (AEC), synchronization likelihood (SL), phase lag index (PLI), phase locking value (PLV), and corrected imaginary PLV (ciPLV) were applied to: (i) synthetic signals generated with a Kuramoto-based model of several coupled oscillators; and (ii) a resting-state EEG database of real recordings from 51 cognitively healthy controls, 51 mild cognitive impairment (MCI) subjects, 51 mild AD (AD mil ) patients, 50 moderate AD (AD mod ) patients, and 50 severe AD (AD sev ) patients. Main results Our results using synthetic signals showed that PLI was the least affected parameter by spurious influences in a simulated volume conduction environment. Results using real EEG recordings showed that spontaneous activity of MCI patients is characterized by a significant coupling increase in the [Formula: see text] band. As dementia progresses, this increase in the [Formula: see text] band became more pronounced, and a significant widespread decrease in [Formula: see text] band appeared at the last stage of dementia. Significance Our results revealed that the estimation of functional EEG connectivity using PLI could reduce the bias introduced by the spurious influence of volume conduction, and it could increase the insight into the underlying brain dynamics at different stages of the AD continuum.

Made available in DSpace on 2020-12-12T01:14:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 The GHEP-ISFG organized a collaborative study to estimate mutation rates for the markers included in the Investigator Argus X-12 QS kit Qiagen. A total of 16 laboratories gathered data from 1,612 father/mother/daughter trios, which were used to estimate both maternal and paternal mutation rates, when pooled together with other already published data. Data on fathers and mothers’ age at the time of birth of the daughter were also available for ∼93 % of the cases. Population analyses were computed considering the genetic information of a subset of 1,327 unrelated daughters, corresponding to 2,654 haplotypes from residents in several regions of five countries: Argentina, Brazil, Ecuador, Portugal and Spain. Genetic differentiation analyses between the population samples from the same country did not reveal signs of significant stratification, although results from Hardy-Weinberg and linkage disequilibrium tests indicated the need of larger studies for Ecuador and Brazilian populations. The high genetic diversity of the markers resulted in a large number of haplotype combinations, showing the need of huge databases for reliable estimates of their frequencies. It should also be noted the high number of new alleles found, many of them not included in the allelic ladders provided with the kit, as very diverse populations were analyzed. The overall estimates for locus specific average mutation rates varied between 7.5E-04 (for DXS7423) and 1.1E-02 (for DXS10135), the latter being a troublesome figure for kinship analyses. Most of the found mutations (∼92 %) are compatible with the gain or loss of a single repeat. Paternal mutation rates showed to be 5.2 times higher than maternal ones. We also found that older fathers were more prone to transmit mutated alleles, having this trend not been observed in the case of the mothers. Institute of Pathology and Molecular Immunology from University of Porto (IPATIMUP) Instituto de Investigação e Inovação em Saúde I3S Universidade do Porto CMUP Centro de Matemática da Universidade do Porto Section of Forensic Genetics Department of Forensic Medicine Faculty of Health and Medical Sciences University of Copenhagen Laboratório de Diagnóstico por DNA (LDD) Universidade do Estado do Rio de Janeiro Centro de Genética Forense Poder Judicial de Córdoba Laboratorio Regional de Investigación Forense Tribunal Superior de Justicia de Santa Cruz UNESP-Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Laboratório de Investigação de Paternidade-NAC Instituto Nacional de Medicina Legal e Ciências Forenses I.P. Serviço de Genética e Biologia Forenses Delegação do Centro Escuela de Medicina Facultad de Ciencias de la Salud Universidad de Las Américas (UDLA) Laboratorio de ADN de la Fiscalía General del Estado Departamento de Biología Servicio de Criminalística de la Guardia Civil Laboratorio Regional de Genética Forense Poder Judicial de Río Negro Instituto Nacional de Medicina Legal e Ciências Forenses I.P. Serviço de Genética e Biologia Forenses Delegação do Norte PRICAI-Fundación Favaloro Laboratorio de Análisis de ADN Facultad de Ciencias Médicas Universidad Nacional de Cuyo LabGenetics: Laboratorio de Genética Clínica S.L. Genomic Engenharia Molecular Molecular Laboratorio de Genética Forense Poder Judicial de la Provincia de La Pampa Laboratorio MANLAB Área de Filiaciones UNESP-Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Laboratório de Investigação de Paternidade-NAC

The analysis of X-chromosome STRs is useful in certain kinship cases for which autosomal markers provide insufficient statistical power. Particularly, powerful results are achieved in paternity cases with a daughter, when the alleged father is not accessible for analysis, contrarily to his unquestioned mother or daughter. However, representative haplotype frequencies for this type of markers are not available for some populations, as is the case of Argentina, which prevents the quantification of the proof in routine forensic analyses. In this work we present haplotype frequencies for the 12 X-chromosome STRs included in the Investigator Argus X-12 kit, as well as segregation data, obtained from the analysis of the genetic profiles of 457 father-daughter duos, which gave us information on 914 (unrelated) haplotypes from residents of all Argentinian provinces. Fil: García, M.G.. Laboratorio Manlab; Argentina Fil: Catanesi, Cecilia Ines. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Penacino, G. A.. Fundación Ingen; Argentina. Colegio Oficial de Farmacéuticos y Bioquímicos; Argentina Fil: Gusmão, L.. Universidade do Estado de Rio do Janeiro; Brasil. Universidad de Porto; Portugal Fil: Pinto, N.. Universidad de Porto; Portugal

Alzheimer&rsquo, s disease (AD) is a neurodegenerative disorder with high prevalence, known for its highly disabling symptoms. The aim of this study was to characterize the alterations in the irregularity and the complexity of the brain activity along the AD continuum. Both irregularity and complexity can be studied applying entropy-based measures throughout multiple temporal scales. In this regard, multiscale sample entropy (MSE) and refined multiscale spectral entropy (rMSSE) were calculated from electroencephalographic (EEG) data. Five minutes of resting-state EEG activity were recorded from 51 healthy controls, 51 mild cognitive impaired (MCI) subjects, 51 mild AD patients (ADMIL), 50 moderate AD patients (ADMOD), and 50 severe AD patients (ADSEV). Our results show statistically significant differences (p-values &lt, 0.05, FDR-corrected Kruskal&ndash, Wallis test) between the five groups at each temporal scale. Additionally, average slope values and areas under MSE and rMSSE curves revealed significant changes in complexity mainly for controls vs. MCI, MCI vs. ADMIL and ADMOD vs. ADSEV comparisons (p-values &lt, 0.05, FDR-corrected Mann&ndash, Whitney U-test). These findings indicate that MSE and rMSSE reflect the neuronal disturbances associated with the development of dementia, and may contribute to the development of new tools to track the AD progression.

The analysis of X-chromosomal markers can be important in different situations where the application of autosomal and Y- chromosomal STR markers are not sufficient to solve the cases. Currently, the Argentine population lacks a representative database on X-chromosomal markers, regarding allele and/or haplotype frequencies, and mutation rates. The absence of this information represents an important limitation for their routine use in laboratories, preventing the achievement of a quantitative, statistically supported evaluation. In order to estimate mutation rates for the twelve X-chromosomal markers included in the Argus X-12 kit, 345 father-daughter duos were genotyped. The samples were selected from all provinces of Argentina and the biological relationship of paternity was previously confirmed by the analysis of autosomal STR markers for all duos, for which likelihood ratios higher than 106 were achieved. A total of 21 mutations over 4140 allelic transmissions were observed at DXS7132, DXS10134, DXS10079, DXS10146, DXS10101, DXS10103, DXS10074, DXS10148 and DXS10135 loci. The overall X-STRmutation rate observed was 5.1×10−3 (95% CI, 3.1×10−3–7.7 ×10−3) and all the genotypic configurations were explainable by the gain or loss of a single repeat. Finally, it should be noted that the overall mutation rate observed in this work resulted higher in comparison with some other reports, likely due to only father-daughter duos had been considered. Indeed, these findings are in agreement with previous works suggesting higher mutation rates for males, due to the higher number of germline divisions they experience., Instituto Multidisciplinario de Biología Celular

In kinship testing powerful statistical results are usually obtained when genetic information is required to be shared between a set of individuals, under the assumption of one of the hypotheses. This is the case when the hypotheses parenthood or identity are compared with the hypothesis of no relatedness, even when only a pair of individuals is analyzed. In this work we will consider kinship problems where the sharing of genetic information is not required when a pair of individuals is analyzed, such as is the case of the hypotheses full-siblings or avuncular. Statistical evaluation was computed through the quantification of likelihood ratios, assuming the genotypic configuration of 100,000 simulated families, for each of the kinship problems analyzed: full-siblings vs. unrelated, full-siblings vs. half-siblings, half-siblings vs. unrelated, avuncular vs. unrelated, first cousins vs. unrelated, and half-first cousins vs. unrelated. For each of the six studied cases we have obtained results allowing us to weight the informative power impact of increasing the number of markers and of the addition of an extra individual, considering (a.) the use of different sets of STRs (from 8 to 35), and (b.) the introduction of a third undoubted relative. Based on these results we were able to provide recommendations for each case both on the minimal number of STRs to be used and on the third relative whose genetic analysis should be privileged.

The use of biostatistical software programs to assist in data interpretation and calculate likelihood ratios is essential to forensic geneticists and part of the daily case work flow for both kinship and DNA identification laboratories. Previous recommendations issued by the DNA Commission of the International Society for Forensic Genetics (ISFG) covered the application of bio-statistical evaluations for STR typing results in identification and kinship cases, and this is now being expanded to provide best practices regarding validation and verification of the software required for these calculations. With larger multiplexes, more complex mixtures, and increasing requests for extended family testing, laboratories are relying more than ever on specific software solutions and sufficient validation, training and extensive documentation are of upmost importance. Here, we present recommendations for the minimum requirements to validate bio-statistical software to be used in forensic genetics. We distinguish between developmental validation and the responsibilities of the software developer or provider, and the internal validation studies to be performed by the end user. Recommendations for the software provider address, for example, the documentation of the underlying models used by the software, validation data expectations, version control, implementation and training support, as well as continuity and user notifications. For the internal validations the recommendations include: creating a validation plan, requirements for the range of samples to be tested, Standard Operating Procedure development, and internal laboratory training and education. To ensure that all laboratories have access to a wide range of samples for validation and training purposes the ISFG DNA commission encourages collaborative studies and public repositories of STR typing results. Published by Elsevier Ireland Ltd.

Mild cognitive impairment (MCI) is a pathology characterized by an abnormal cognitive state. MCI patients are considered to be at high risk for developing dementia. The aim of this study is to characterize the changes that MCI causes in the patterns of brain information flow. For this purpose, spontaneous EEG activity from 41 MCI patients and 37 healthy controls was analyzed by means of an effective connectivity measure: the phase slope index (PSl). Our results showed statistically significant decreases in PSI values mainly at delta and alpha frequency bands for MCI patients, compared to the control group. These abnormal patterns may be due to the structural changes in the brain suffered by patients: decreased hippocampal volume, atrophy of the medial temporal lobe, or loss of gray matter volume. This study suggests the usefulness of PSI to provide further insights into the underlying brain dynamics associated with MCI.

The aim of this study was to analyze electroencephalographic (EEG) background activity in Alzheimer’s disease (AD) and mild cognitive impairment (MCI) by means of multiscale spectral entropy (MSSE). To achieve this goal, five minutes of EEG activity were acquired from 18 cognitive healthy controls, 10 MCI subjects and 32 AD patients. Our results showed statistically significant differences (p-values < 0.05, Kruskal-Wallis test) in MSSE values for all scale factors. Additionally, a 3D receiver operating characteristic (ROC) curve was used to assess the discrimination ability of MSSE among the 3 groups, showing a good three-way discrimination power (volume under the surface of 0.6). These results suggest that MSSE can be a useful measure to characterize neural alterations in AD, even at early stages.

The potential and difficulties of the application of genome wide data in forensics are analyzed. We argue that, besides statistical, computational, ethical, economic and technical validation problems, the state of the art of population genetics theory is insufficient to deal with the forensic use of this type of data. In order to keep the current standards of quantifying and reporting genetic evidence, namely in kinship analyses and identification, substantial improvement in the theoretical framework should be reached, since to obtain genome-wide results is to provide the experts with data that they cannot quantify the corresponding evidentiary value. Therefore, while a satisfactory, generalized theoretical and biostatistical modelling is not achieved, it may well be wiser to improve the already established approaches to a limited, pre-defined number of validated genetic markers, amenable to a consensual handling and reporting. Whole genome population analyses will prove extremely useful in selecting the best suited and most efficient of those markers.

While traditional forensic genetics has been oriented towards using human DNA in criminal investigation and civil court cases, it currently presents a much wider application range, including not only legal situations sensu stricto but also and, increasingly often, to preemptively avoid judicial processes. Despite some difficulties, current forensic genetics is progressively incorporating the analysis of nonhuman genetic material to a greater extent. The analysis of this material—including other animal species, plants, or microorganisms—is now broadly used, providing ancillary evidence in criminalistics in cases such as animal attacks, trafficking of species, bioterrorism and biocrimes, and identification of fraudulent food composition, among many others. Here, we explore how nonhuman forensic genetics is being revolutionized by the increasing variety of genetic markers, the establishment of faster, less error-burdened and cheaper sequencing technologies, and the emergence and improvement of models, methods, and bioinformatics facilities. MA, FP, MO, NP, AML and VG are supported by the Portuguese Foundation for Science and Technology (FCT), European RegionalDevelopment Fund (ERDF) and Programa Operacional Potencial Humano,through the grants IF/00955/2014, IF/01356/2012, SFRH/BPD/66071/2009, SFRH/BPD/97414/2013, IF/01262/2014 and SFRH/BPD/76207/2011, respectively. MA was also supported by the “Ramón y Cajal” grant RYC-2015-18241 from the Spanish Government. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. The funders had no role in the preparation of the article.

Different mutation models have been developed considering the genotypic observations of parent(s)/offspring duos or trios, even though, for autosomal transmission, only Mendelian incompatibilities, not mutations, are able to be identified. The most commonly considered mutation models are the so-called "Equal", "Proportional", "Stepwise" and "Extended Stepwise", all implemented in the software Familias. In this work we simulated 100,000 profiles (duos and trios) of parent-child, full-siblings, and half-siblings, assuming a specific database for 17 autosomal STRs and probabilities of incompatibility inferred from the American Association of Blood Banks (AABB) report, 2008. Using the R version of the software Familias, we calculated the likelihood ratios where the probability of the genotypic configuration of the individuals assuming each of the pedigrees was compared with the probability of the same observations assuming unrelatedness. In the case of full-siblings, the comparison assuming half-sibship as the alternative pedigree was also considered. The results show that for profiles generated assuming the above mentioned pedigrees, except for unrelated, the use of different mutation models with parameters inferred from the proportion of observed mendelian incompatibilities does not result in major differences, which also indicates that the consideration of hidden mutations does not have a major influence in the final result. Future work should be developed to measure the impact for cases where a close relative of the father, such as a brother, is analyzed as the putative father in a standard paternity test.

The increasing relevance of human Y-STRs in forensic science demands reliable estimates of their mutation rates. Therefore, a collaborative study was carried out by the Spanish and Portuguese working group of the International Society for Forensic Genetics (GHEP-ISFG) in the interest of extending the data on Y-chromosomal short tandem repeat (Y-STR) mutation rates. Sixteen Y-STRs were considered in the analyses: DYS456, DYS389I, DYS389II, DYS390, DYS458, DYS19, DYS385, DYS391, DYS392, DYS393, DYS439, DYS635, DYS437, DYS438, DYS448, GATA H4. Among the sample of 1598 father-son duos analyzed, 46 mutations were observed, 45 of which were a single-step change and 1 was a double-step change. A total of 28 repeat losses were observed against 18 gains, with a ratio of 1:1.5. Eleven duos showing double alleles at the Y-STR loci DYS19, DYS391, DYS439 and DYS448 without allelic discrepancy between the father-son duo were also observed. This new data was added to the previous studies from the GHEP-ISFG working group, totalizing 63 496 allele transmissions (varying between 2298 and 7347 per locus). The average mutation rate across all 16 Y-STRs loci was 0.00187 (95% CI 0.00155–0.00224). The average mutation rates per marker varied between 0.00057 (95% CI 0.00007–0.00206) at DYS438 and 0.00606 (95% CI 0.00375–0.00925) at DYS439.

In paternity testing the genetic profiles of the individuals are used to compare the relative likelihoods of the alleged father and the child being related as father/offspring against, usually, being unrelated. In the great majority of the cases, analyses with the widely used sets of short tandem repeat markers (STRs) provide powerful statistical evidence favouring one of the alternative hypotheses. Nevertheless, there are situations where the final statistical result is ambiguous, mostly because the alleged father shows incompatible genotypes at a few loci along with a very high paternity index in the remaining systems. In these cases, the possibility that the alleged father is actually a close relative of the real one (son, father or brother) can reasonably be raised. In such cases, when the statistical evidence obtained is considered as insufficient, the common practice is to extend the set of analysed markers. In this context, many authors have suggested that bi-allelic markers, such as single nucleotide (SNP) or insertion/deletion (Indel) polymorphisms, are markers of choice, as they are incomparably less prone to mutation than STRs. In this work we address the soundness of this claim and the consequences of this strategy, analyzing the a priori odds both for (a) expected number of Mendelian incompatibilities, and (b) expected values for the final likelihood ratios. Moreover, one hundred real pairs of second degree relatives, typed for two sets of markers: 15 STRs plus 38 Indels, were used to simulate paternity testing. Our data show that, for the number of markers commonly considered, the results from an extended battery of SNPs or Indels should be interpreted with caution when relatives are possibly involved.

Some practical and theoretical aspects of evaluation of evidence based on the likelihood ratio (LR) in kinship cases are discussed. If relationships are complex or if complicating factors like mutation, correction for population structure or silent alleles need to be accounted for, available software may fail. We present an explicit general formula for non-inbred pairwise cases. Equipped with this formula it is possible to evaluate, say, how strongly a shared rare allele, points towards a specific relationship. Moreover, a general expression as the one presented, adds to the understanding of models and the underlying biological mechanisms. It is also useful for checking software and defining the limitations of programs. Some ideas for improving software may also be generated by the derivation of exact expressions. We argue that a proportional mutation model is well suited from a pragmatic point of view and derive some theoretical properties of this model. Several examples based on the general pairwise formula and its implementation in the freely available R package mut are presented.

The existence of pedigrees belonging to the same kinship class (i.e. indistinguishable through independent markers) is well known in the forensic community, and theoretical frameworks for autosomal and X-chromosomal markers were already developed for a pair of individuals. Nevertheless, studies for the cases where a greater number of individuals is available for testing are still lacking. With this work, we intend to pave the way for a theoretical and general framework, identifying the individuals/relatives that should be chosen to distinguish autosomal analyses between such pedigrees. In this work we identify the individuals/relatives that are non-informative for calculations (the pedigrees under discussion remaining indistinguishable independently of their genetic profile), as well as those that will likely be very informative, influencing the statistical outcome. For example, given the respective genotypes, to compare the likelihoods of the father of the individual B to be: (a.) the father, or (b.) a full—brother, of the individual A, the hypotheses H 1 : "The individual A is paternal half-sibling of the individual B", and H 0 : "The individual A is paternal uncle/aunt of the individual B" are considered. It is proved that considering just individuals A and B the hypotheses are equally likely. In this work we show that the same is also true for the case where the mother of B is available for testing, notwithstanding the hypotheses being differently weighted if the mother of A is considered. Similar considerations are done for other kinship hypotheses and/or individuals/relatives. Irrelevance of the genetic profile of some specific relatives are mathematically demonstrated, and data obtained from 20,000 simulated families are presented for the other cases.

In studies involving pedigree reconstruction and kinship estimation, it is acknowledged that some pedigrees have the same algebraic expressions for the joint genotypic probabilities and are, therefore, indistinguishable when considering only genetic information, no matter what the mode of transmission considered. Indeed, although standard forensic practice considers solely unlinked autosomal markers, the existence of pedigrees with the referred theoretical property (that are then said to belong to the same kinship class) is possible when considering any kind of genetic transmission. The research on genetic relatedness has always been linked to the root concept of identity-by-descent (IBD). However, although the basic theoretical core for autosomal transmission has been long formalised, a general method allowing the decision if two pedigrees linking two non-inbred individuals are distinguishable using unlinked autosomal markers along with the respective IBD partitions (and consequently the algebraic expressions for the joint genotypic probabilities) was only recently published. In this work X-chromosomal transmission will be at stake, considering that the analytical framework for X-chromosomal markers has been recently established and the importance of X-chromosome markers for these questions has been steadily growing, particularly in forensics, as a tool both to complement the information given by autosomes in complex kinship testing cases and to differentiate pedigrees belonging to the same autosomal kinship class. Therefore, here it will be presented a formal and mathematically well supported framework where a general counting rule is given, allowing a secure and expeditious decision on the usefulness of typing (unlinked) X-chromosomal markers on pairwise kinship testing involving two non-inbred individuals. Moreover the counting rule now presented allows the derivation of algebraic expressions for the joint genotypic probabilities associated with any pedigree.

Quantification of kinships between two individuals using unlinked autosomal markers rests upon the identity-by-descent (IBD) probabilities among their four alleles at a locus because they determine the algebraic expressions of the joint genotypic probabilities. Nevertheless, some pedigrees share the same IBD probabilities and are therefore indistinguishable using those markers. Examples of these pedigrees were previously described, such as the case of half-siblings, grandparent-grandchild and avuncular, but a general analysis has not been attempted. The aim of this study is to present a systematic and mathematically supported framework where considering unlinked autosomal markers complete sets of indistinguishable pedigrees linking two non-inbred individuals are generally derived. In our work, complete sets of pedigrees with the same IBD partitions are formally established and mathematically treated, considering kinships linking any pair of non-inbred individuals, whether they are related just maternally or paternally, or both. Moreover, general expressions for IBD partitions, and consequently for joint genotypic probabilities, are derived considering a simple counting rule based on two ‘atom’ pedigrees: parent-child and full-siblings. Besides the theoretical formalization of the problem, the developed framework has potential applications in forensics as well as in breeding strategies design and in conservation studies.

Mutation is a fundamental topic in forensic genetics since the calculation of kinship likelihoods depends on estimates of germinal mutation rates. Germinal mutation rates have been computed by simply proportioning the number of true parent(s)-child genotypic configurations inconsistent with Mendelian segregation. For STRs, current technology is based on PCR fragment size determination and germinal mutations are detected when parent-child do not share alleles' length. For technical reasons, it is uncommon to obtain the sequence composition of STR alleles, and the identification of the allele that mutated is based on assumptions. It has been assumed that one-step are much more common than multi-step mutations. Whenever a genotypic parent-child configuration is compatible with Mendelian rules by a single-step mutation, this is the one assumed, despite other possible mutational events. Multi-step events are therefore evoked exclusively when a single-step cannot reconcile the observation with the true kinship, leading to an overestimation of single step mutation rates. For any mode of transmission other than purely haploid it is theoretically impossible to identify both the allele at "origin" (in parents) and at "destination" (in offspring). Therefore, a more sophisticated statistical framework than the simple proportioning is then required to properly evaluate mutation rates. Moreover, the possibility of the occurrence of silent alleles should be simultaneously modeled since it has to be considered whenever an apparently homozygous individual is involved.

Mutation is a topic of intense research and raises important problems in forensics. Since the markers of choice in current forensic genetics analyses are microsatellites or Short Tandem Repeat Polymorphisms (STRs), mutation is sufficiently common to cause difficulties in evaluating DNA evidence in a significant proportion of cases but at the same time rare enough to turn the estimation of the corresponding probability of occurrence into a hard task. We address these issues using the simplest model of transmission: the Y chromosome specific STRs. Within this model, and under an explicit set of definitions and involved assumptions, we developed the theoretical framework required for the study of allelic transitions in gametogenesis, identifying the required parameters and associated probabilities and finally we discuss the estimation of these parameters and their application in forensics. We conclude that (i) for forensic casework the relevant parameter for incorporation in a likelihood ratio is biallelic specific (i.e. the mutation rate estimate corresponds to the probability of the specific allelic transition observed) and (ii) for these estimates as well as in order to provide data for testing mutation models the absolute frequency of mutated and non-mutated transmissions per allele, along with the description of the observed mutations should be reported.

The study of blood relatedness among individuals is rooted in the concept of identity-by-descent (IBD). Two alleles are called IBD if they descend from the same ancestral allele. The probability of two individuals exhibiting a certain genotype at a specific locus, assuming that they are related by a defined pedigree, depends only on IBD probabilities and on the frequency of the alleles at that locus in the population.

This paper is motivated by power considerations in connection with relationship testing. Given the true relationship between a set of individuals, a claimed relationship between the same individuals, and a set of genetic markers, we compute the power of exclusion, i.e., the probability that the genotypes will be incompatible with the claimed relationship. If exclusion is impossible, as will be the case if it is required for instance to distinguish between sibs and half sibs, we rather obtain the distribution of the likelihood ratio. The problem we are addressing can also be seen as a standard way of measuring the ability of a battery of tests to resolve claimed family relationships. In particular, simple exclusion probabilities are regularly calculated worldwide as a part of designing forensic marker sets. Our approach to these problems is guided by a natural way of calculating exclusion probabilities on a computer. We present a user friendly implementation for this as part of the R package paramlink, originally designed by one of the authors (MDV) for pedigree manipulations and likelihood computations. By doing so we are able to handle problems more challenging than we have seen in the literature. Specifically, we deal with complex pedigrees with arbitrary inbreeding and conditioning. We present examples for autosomal as well as X-linked markers and some formulae to validate the results. The examples indicate a wide range of applications. Details are presented for an immigration case where previously reported calculations are extended to account for possible inbreeding and known genotypes. The supplementary material includes a tutorial on how to perform these calculations in paramlink.

In paternity testing the informativeness of genetic markers is traditionally measured through the probability of finding, in randomly chosen individuals, inconsistencies with parent to child Mendelian rules of transmission. This statistic, called power of exclusion (PE), paternal exclusion chance or probability, can be defined for duos (mother not typed) or trios (random false fathers are matched against mother/child pairs) and performed both for autosomal and X-chromosomal markers (restricted to paternity testing involving daughters). PE is an a priori statistic, in the sense of not depending on the individual's genetic data of a case, being dependent however on the estimates of genetic markers allele (or haplotype) frequencies. We have studied the behaviour of this statistic in situations where the randomness assumption is not met, because either (a) the alleged – and false – father is related to the true one, or (b) there is a non-negligible level of background relatedness in the population. For the first case, we derived general (autosomal and X-chromosomal) PE formulas for duos and trios for any genealogy linking alleged father and child, highlighting that the PE of each marker only depends on a single kinship parameter associated with their pedigree. In this case we also estimate a lower bound for the number of extra markers needed to be analysed to achieve the same global power as for unrelated individuals. In the second situation, we demonstrate that for realistic values of the coancestry coefficient the decrease in PE due to population inbreeding is very moderate even when duos are analysed. In this work, beyond the aforementioned issues, we also discuss the suitability of assuming the pedigree father–daughter for calculating the X-PE, since X-markers are not the tool of choice in laboratorial routine when the alleged father is available for testing. Indeed, X-markers are particularly useful in situations where the alleged father is not available for testing but experts are able to type the mother or a daughter of his. Such increase of power is due to the paternal genealogies: half- and full-sisters, and grandmother–granddaughter, having a non-null X-PE even when only duos are analysed in contrast to what happens for autosomes. Algebraic expressions for these cases are also presented.

Ancestry-informative markers (AIMs) show high allele frequency divergence between different ancestral or geographicallydistant populations. These genetic markers are especially useful in inferring the likely ancestral origin of an individual orestimating the apportionment of ancestry components in admixed individuals or populations. The study of AIMs is of greatinterest in clinical genetics research, particularly to detect and correct for population substructure effects in case-controlassociation studies, but also in population and forensic genetics studies. This work presents a set of 46 ancestry-informativeinsertion deletion polymorphisms selected to efficiently measure population admixture proportions of four different origins(African, European, East Asian and Native American). All markers are analyzed in short fragments (under 230 basepairs)through a single PCR followed by capillary electrophoresis (CE) allowing a very simple one tube PCR-to-CE approach. HGDP-CEPH diversity panel samples from the four groups, together with Oceanians, were genotyped to evaluate the efficiency ofthe assay in clustering populations from different continental origins and to establish reference databases. In addition, otherpopulations from diverse geographic origins were tested using the HGDP-CEPH samples as reference data. The resultsrevealed that the AIM-INDEL set developed is highly efficient at inferring the ancestry of individuals and provides goodestimates of ancestry proportions at the population level. In conclusion, we have optimized the multiplexed genotyping of46 AIM-INDELs in a simple and informative assay, enabling a more straightforward alternative to the commonly availableAIM-SNP typing methods dependent on complex, multi-step protocols or implementation of large-scale genotypingtechnologies. This work was partially supported through PhD grants to RP (SFRH/BD/30039/2006), NP (SFRH/BD/37261/2007), and CS (SFRH/BD/75627/2010)awarded by the Portuguese Foundation for Science and Technology (FCT) and co-financed by the European Social Fund (Human Potential Thematic OperationalProgramme). This work was also partially supported by the project Xunta de Galicia INCITER09 208 163 PR (PI: Victoria Lareu) SI