Second GHEP-ISFG exercise for DVI: “DNA-led” victims’ identification in a simulated air crash

The Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) has organized a second collaborative exercise on a simulated case of Disaster Victim Identification (DVI), with the participation of eighteen laboratories. The exercise focused on the analysis of a simulated plane crash case of medium-size resulting in 66 victims with varying degrees of fragmentation of the bodies (with commingled remains). As an additional difficulty, this second exercise included 21 related victims belonging to 6 families among the 66 missings to be identified. A total number of 228 post-mortem samples were represented with aSTR and mtDNA profiles, with a proportion of partial aSTR profiles simulating charred remains. To perform the exercise, participants were provided with aSTR and mtDNA data of 51 reference pedigrees —some of which deficient—including 128 donors for identification purposes. The exercise consisted firstly in the comparison of the post-mortem genetic profiles in order to re-associate fragmented remains to the same individual and secondly in the identification of the re-associated remains by comparing aSTR and mtDNA profiles with reference pedigrees using pre-established thresholds to report a positive identification. Regarding the results of the post-mortem samples re-associations, only a small number of discrepancies among participants were detected, all of which were from just a few labs. However, in the identification process by kinship analysis with family references, there were more discrepancies in comparison to the correct results. The identification results of single victims yielded fewer problems than the identification of multiple related victims within the same family groups. Several reasons for the discrepant results were detected: a) the identity/non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, b) some laboratories failed to use all family references to report the DNA match, c) In families with several related victims, some laboratories firstly identified some victims and then unnecessarily used their genetic information to identify the remaining victims within the family, d) some laboratories did not correctly use “prior odds” values for the Bayesian treatment of the episode for both post-mortem/post-mortem re-associations as well as the ante-mortem/post-mortem comparisons to evaluate the probability of identity. For some of the above reasons, certain laboratories failed to identify some victims. This simulated “DNA-led” identification exercise may help forensic genetic laboratories to gain experience and expertize for DVI or MPI in using genetic data and comparing their own results with the ones in this collaborative exercise.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Peer reviewe

Variabilidad genética en estudios de asociación. Estructura poblacional de 10 SNPs autosómicos relacionados con el metabolismo del hierro en mujeres españolas

En general, en los estudios de asociación sólo se obtiene un número reducido de SNPs con p significativas (p<10-7). Técnicas como la minisecuenciación pueden ser una excelente opción para confirmar o rechazar el interés de dichos SNPs y poder plantear su utilización en el campo del diagnóstico clínico y de la salud pública. Un total de 10 SNPs, fueron seleccionados según los resultados obtenidos en diferentes trabajos de genotipado masivo relacionados con el metabolismo del hierro. Se analizaron en 284 muestras de mujeres españolas no relacionadas, mediante una estrategia de SNaPshot. La muestra fue dividida en tres fenotipos relacionados con la absorción de hierro de acuerdo a sus valores de hemoglobina y ferritina: control, deficiente y anémico. Se calcularon distancias genéticas (FST) entre los tres grupos y con poblaciones europeas disponibles en el NCBI Reference Assembly. No se observaron distancias genéticas significativas entre todos los grupos comparados lo que confirma que los marcadores seleccionados no revelan ninguna estructuración poblacional. Por último, dos SNPs (rs1375515 y rs1800562) mostraron una distribución genotípica significativamente diferente entre los tres grupos fenotípicos, con lo que se discute su posible valor predictivo para la deficiencia del hierro.Peer Reviewe

Hepcidin, transferrin (exon 7), and hemochromatosis genotyping suggests that haplotype block analysis is the best strategy for predicting iron deficiency phenotype in women

Strategies to draw associations between disease, genes and nutrients include the investigation of mutations and/or haplotypes. We studied the relationship between certain mutations in hepcidin (HAMP, exon 1, 2 and 3), transferrin (Tf, exon 7), and hemochromatosis (HFE, H63D and C282Y) genes, as well as their associations in haplotypes, and iron deficiency, with or without anemia, in young women. One hundred and sixty-two young, menstruating women were recruited. Hemoglobin (Hb [g/dL]), ferritin (Ft [μg/L]), total iron binding capacity, Tf saturation, hematocrit, and mean corpuscular volume were determined, and subjects were divided in 3 iron status groups: sufficient (Hb ≥12, Ft ≥20; n = 63), deficient (Hb ≥12, Ft <20 or Hb <12, Ft ≥20; n = 77), and deficient-anemic (Hb <12, Ft <20; n = 22). Mutations in the HAMP gene were detected by means of single strand conformational polymorphism analysis, in exon 7 of the Tf gene using DNA sequencing, whereas C282Y and H63D mutations in the HFE gene were detected using restriction enzymes. Among the iron-deficient women, 8 were G277S/L247L-heterozygous (except 1 L247L homozygote), and 1 was I7V-heterozygous for the mutation in the HAMP gene. Three iron-deficient, anemic women were G277S/L247L/H63D-heterozygous (except 1 H63D homozygote), and 1 was I7V-heterozygous. Two iron-sufficient females were G277S/L247L/H63D-heterozygous, 1 was G277S/L247L/H268H-heterozygous, and 2 were G277S/L247L-heterozygous. In conclusion, (1) the iron-deficient anemic group presented the highest proportion of subjects carrying mutations; (2) there are 3 possible haplotypes: G277S /L247L, G277S/L247L/H63D, and G277S/L247L/H268H, nonrestricted to any iron status; and (3) analysis of haplotype blocks is the best strategy to associate genes and iron deficiency. © 2007 Elsevier Inc. All rights reserved.Peer Reviewe

Nondestructive extraction DNA method from bones or teeth, true or false?

One of the most challenging points for ancient DNA studies on human remains is that analysing procedures involve partial or total destruction of the samples, which usually are unique and irreplaceable. This is what prevents museums, anthropologists or archaeologists from giving samples for genetic investigation. So that it is interesting to find an analysing method without destroying them. In this study, it was carried out the evaluation of a non-destructive extraction DNA protocol on 4 Neolithic individuals, from Can Gambús (Sabadell, Spain) and 4 pre Bell Beaker period individuals, from Los Cercados (Valladolid, Spain). Along this work, it was discussed the efficiency of the non-destructive protocol, valuing the obtained result, and the evidences state of preservation after the whole procedure.This work has been possible by the project HAR2009-10105 funded MINECO of the Spanish Government and the BES2010-035322FPI grant, for P-D.S., by G/6401400/8000 research project (Santander-Universidad Complutense de Madrid, Spain) for C.G, and by the HAR2011-23149 research project funded by the MICINN of the Spanish Government for J.G.Peer Reviewe

El origen de los individuos del yacimiento de Can Gambús (Sabadell, Barcelona). Resultados preliminares

El origen de las comunidades neolíticas en el noreste de la Península Ibérica ha sido estudiado básicamente a partir de la presencia de aquello conocido como “el paquete neolítico” (restos de cereales y animales domésticos, cerámica, industria pulimentada, etc.), de las dataciones por radiocarbono y más recientemente por los análisis genéticos. Aunque el foco de atención se ha dirigido hacia las primeras evidencias referentes a ese Neolítico Inicial (mediados del VI milenio cal BC), son muy pocos los estudios de periodos posteriores. En el caso del noreste de la Península Ibérica, hemos iniciado un primer trabajo sobre algunas de las sepulturas de la necrópolis de Can Gambús 1, Sabadell, Barcelona perteneciente a lo que se conoce como “Neolítico Medio o Cultura de los Sepulcros de Fosa (finales del V e inicios del IV milenio cal BC). El objetivo del presente estudio consiste en la determinación del origen biogeográfico por vía materna de cinco individuos del referido yacimiento, a través del análisis del ADN mitocondrial (mtDNA). Posteriormente a la extracción de ADN, se amplificaran dos fragmentos de las regiones hipervariables I y II del mtDNA, procediéndose a continuación a la purificación y secuenciación de las muestras. Los resultados obtenidos indican que el ADN está degradado, dado que han sido necesarios por lo menos 8 ensayos para obtener resultados fiables y reproducibles. Se ha podido obtener secuencias consenso y determinar que cuatro de los cinco individuos pertenecían al haplogrupo H6, actualmente uno de los haplogrupos presentes en Europa. Sin embargo, el análisis del quinto individuo ha revelado un haplogrupo distinto, J1c. Este resultado es particularmente importante ya que es coherente con la información previa del estudio de la paleodieta, indicando un origen biogeográfico distinto para este individuo.Peer reviewe

Investigación de parentescos biológicos en el Neolítico del NE de la Península Ibérica: el caso de la necrópolis de Can Gambús 1, Barcelona. Resultados preliminares

Ha sido habitual en la arqueología hacer suposiciones sobre el parentesco de las personas inhumadas en un mismo enterramiento o en tumbas diferentes de una misma necrópolis. No es extraño leer que aquellas sepulturas dobles donde hay un individuo del sexo femenino y niño sean “madre e hijo/a”. Sin embargo no existe criterio objetivo para tal afirmación. En el contexto arqueológico en el que estamos trabajando, el Neolítico del noreste peninsular, hemos iniciado los primeros trabajos para reconocer qué grado de parentesco existía, no sólo en las tumbas con dos individuos sino también entre distintos individuos de la misma necrópolis De esta forma, el presente estudio tiene como objetivos la investigación de posibles relaciones familiares entre 5 individuos encontrados en tumbas individuales, así como la determinación del sexo molecular, para una posterior comparación con la información arqueológica disponible. Para la determinación genética de los parentescos, se han considerado marcadores de ADN nuclear de pequeña longitud (“mini-STRs”), así como el análisis de fragmentos cortos solapantes de las regiones hipervariables I y II del ADN mitocondrial. Este último análisis pretende analizar específicamente relaciones por vía materna y posible determinación de un mismo linaje materno. Teniendo en cuenta la antigüedad y el estado crítico de las muestras, han sido necesarios, como mínimo, 8 ensayos para obtener resultados fiables y reproducibles. Con respecto a la investigación de polimorfismos de ADN nuclear, no ha sido posible obtener resultados concluyentes. Por consiguiente, no ha sido posible la determinación del sexo molecular de los individuos, ni de parentescos cercanos, como paternidad o hermandad.Peer reviewe

Presumptive tests: A substitute for Benzidine in blood samples recognition

The nature of the sample in a forensic case is one of the most important factors, since it determines the posterior analysis, helping to define or discard its identity (like blood versus semen). A presumptive test is a qualitative analysis that allows to identify, or confirm, the presence of a substance in a sample. These determinations usually occur, after a chemical reaction, and a specific colour is produced. A false positive is another substance reacting the same way, producing the expected result. The aim of this work was to evaluate the most effective presumptive test (with fewer false-positives) when analysing products that could look and behave like blood during a forensic screening assay. Eight different products were tested, like Betadine®, and four reagents were considered: Tetramethylbenzidine, O-toluidine, Leuchomalachite green and BlueStar® Forensic (BlueStar). Each product was tested with the reagents five times − mixed with human blood (3:1), with three different animal blood (3:1), and then unmixed. Our results indicated that Leuchomalachite green is the most suitable presumptive test, since it was the reagent with less false positives.This work was supported by G/6401400/8000 research project (Santander-Universidad Complutense de Madrid, Spain) for C.Gomes.Peer reviewe

Biological kinship analysis in extremely critical samples: The case of a Spanish Neolithic necropolis

It is common in archeology and some forensic cases to make assumptions about the relationship between persons buried in the same grave, or in distinct burials but in the same cemetery. However, there is no objective criterion to make such suppositions. This study aims to investigate the biological relationship among six Neolithic individuals (4200–3400 cal BC) from the Can Gambús-1 necropolis (Barcelona, Spain) as well as, their possible mitochondrial ancestry. From at least 47 tombs, six skeletons were selected, from six individual graves, selecting from each one three integral teeth, without cavities or caries. To carry out this study, the samples were analyzed by two distinct extraction methods, one destructive and other non-destructive, and to determine a presumptive (biological) kinship between the buried individuals, small length nuclear DNA markers were considered, as well as a mitochondrial DNA analysis, in order to determine their maternal lineage. The samples were extremely degraded, requiring several attempts to achieve sequences with a standard quality, without double peaks or other artefacts. Despite the degraded state of the samples, it has been possible to analyze the mitochondrial information and to determine that the studied individuals had a European ancestry, and were not maternally related, since they did not share the same mitochondrial haplotype.This work was supported by G/6401400/8000 research project (Santander-Universidad Complutense de Madrid, Spain) for C. Gomes. This work has been partially funded by HAR2015-67323-C2-2-P.Peer reviewe