Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise.

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.

Study of InDel genetic markers with forensic and ancestry informative interest in PALOP's immigrant populations in Lisboa.

The migratory phenomenon in Portugal has become one of the main factors for the genetic variability. In the last few years, a new class of autosomal insertion/deletion markers-InDel-has attracted interest in forensic genetics. Since there is no data for InDel markers of Portuguese-speaking African countries (PALOP) immigrants living in Lisboa, our aim is the characterization of those groups of individuals by typing them with at least 30 InDel markers and to compare different groups of individuals/populations. We studied 454 bloodstain samples belonging to immigrant individuals from Angola, Guinea-Bissau, and Mozambique. DNA extraction was performed with the Chelex® 100 method. After extraction, all samples were typed with the Investigator® DIPplex method. Through the obtained results, allelic frequencies show that all markers are at Hardy-Weinberg equilibrium, and we can confirm that those populations show significant genetic distances between themselves, between them, and the host Lisboa population. Because of this, they introduce genetic variability in Lisboa population.

Sequencing CYP2D6 for the detection of poor-metabolizers in post-mortem blood samples with tramadol.

Tramadol concentrations and analgesic effect are dependent on the CYP2D6 enzymatic activity. It is well known that some genetic polymorphisms are responsible for the variability in the expression of this enzyme and in the individual drug response. The detection of allelic variants described as non-functional can be useful to explain some circumstances of death in the study of post-mortem cases with tramadol. A Sanger sequencing methodology was developed for the detection of genetic variants that cause absent or reduced CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles. This methodology, as well as the GC/MS method for the detection and quantification of tramadol and its main metabolites in blood samples was fully validated in accordance with international guidelines. Both methodologies were successfully applied to 100 post-mortem blood samples and the relation between toxicological and genetic results evaluated. Tramadol metabolism, expressed as its metabolites concentration ratio (N-desmethyltramadol/O-desmethyltramadol), has been shown to be correlated with the poor-metabolizer phenotype based on genetic characterization. It was also demonstrated the importance of enzyme inhibitors identification in toxicological analysis. According to our knowledge, this is the first study where a CYP2D6 sequencing methodology is validated and applied to post-mortem samples, in Portugal. The developed methodology allows the data collection of post-mortem cases, which is of primordial importance to enhance the application of these genetic tools to forensic toxicology and pathology.

Genetic portrait of Lisboa immigrant population from Angola with mitochondrial DNA.

Portugal has been considered a country of emigrants, nevertheless in the past decades the number of immigrants has grown throughout all the country. This migratory flux has contributed to a raise of heterogeneity at multiple levels. According to statistical data, at the end of 2012 the total number of Angolan immigrants in Portugal equalled about 20,000 individuals. A territorial predominance has been found for the metropolitan region of Lisboa. Angola is a country located in the Atlantic coast of Africa. The presence of Bantu people and the colonisation by Portuguese people on Angolan territory are considered to be the major modulators of the genetic patterns in Angola. Mitochondrial DNA is known for its features that enable an approach to the study of human origin and evolution, as well to the different migration pathways of populations. This genetic marker can also contribute to ascertaining the identity of individuals in forensic cases. The main aim of this study was to determine the genetic structure of the Angolan immigrant population living in Lisboa. Therefore, a total of 173 individuals, inhabitants in Lisboa, nonrelated and with Angolan ancestry were studied. Total control region of mitochondrial DNA was amplified from position 16,024 to position 576 using two pairs of primers - L15997/H016 and L16555/H639. The majority of the identified haplotypes belong to mtDNA lineages known to be specific of the sub-Saharan region. Our results show that this immigrant population inhabitant in Lisboa presents a genetic profile that is characteristic of African populations. This study also demonstrates the genetic diversity that this immigrant population introduces in Lisboa. This does not contradict the historical data concerning colonization of Angola, since this was made mainly by male European individuals, who did not contribute with their maternal information of mtDNA. Lisboa immigrant population from Angola can be accessed via EMPOP dataset with accession number EMPOP662.

Distribution of Y-chromosomal haplotypes in the Central Portuguese population using 17-STRs.

17 Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS456, DYS391, DYS392, DYS393, DYS385 a/b, DYS458, DYS439, DYS635, GATA H4.1, DYS437, DYS438 and DYS448) were determined for 303 unrelated males, living in Central Portugal, using the AmpFlSTR YFiler PCR Amplification kit (Applied Biosystems). A total of 287 different haplotypes were found, 272 being unique. The overall haplotype diversity (HD) was determined as 0.9996, a value similar to other YFiler data sets. Y-STR polymorphisms in Central Portugal population, using YFiler, provide a powerful discrimination tool for routine forensic applications.