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The activity of animals and insects at the crime scene can provide useful elements to reconstruct the dynamics of the event. Any insect that interacts with wet body fluids can produce artifacts which can be confused with human bloodstains. Considering that flies are the early colonizers of the crime scene and first players of the process of contamination, the problem is to distinguish stains produced by fly regurgitation or fecal elimination on a crime scene. Actually, fly artifacts are morphologically very similar to impact, projected, sneezed, and expirated bloodstains and cannot be reliably distinguished using presumptive or confirmatory tests for identification of human blood. Several techniques have been proposed to differentiate insect-derived artifacts based on morphological approaches and immunological assay. Recently, a DNA-based method by the analysis of the cytochrome c oxidase subunit I (COI) gene has been designed for the distinction of fly artifacts from human bloodstains on spots characterized by different morphological features and deposited by fly on a glass surface. Here, we present a study to assess the feasibility of the molecular analysis of fly artifacts deposited on different substrates as glass, paper, plexiglass and cotton and to evaluate the COI amplification success at different time intervals up to two years after fly artifacts deposition. Our results showed that the deposition time seems do not affect the detection of fly artifacts DNA, while a substrate interference was demonstrated. [ABSTRACT FROM AUTHOR]

According to the ISFG guidelines on the use of X-chromosome, the biostatistical evaluation in kinship analysis is based on a likelihood ratio approach, but since in calculation linkage and recombination events should be accounted for, an accurate estimate of mutation and recombination rates of X-markers analyzed is required. Due to the mode of genetic transmission and physical location, sometimes X-chromosome markers can be more informative than autosomal STRs and their analysis could be considered a supplementary tool in DNA testing. The increased demand to forensic laboratories for kinship investigations in complex cases explains the need not only to enlarge the Italian population database for 12 X-STRs routinely used for forensic application, but also to investigate the recombination fractions among markers. A collaborative exercise of the Italian Speaking Working Group Ge.F.I. was organized to evaluate mutation and recombination events in 12 X-STRs included in the Investigator Argus X12 kit. In order to explore the segregation stability, three-generation families (grandpa-mother-son) and two-generation families (mother-sons, father-daughters) for a total of 269 pedigrees were analyzed and calculations to estimate the recombination fractions between pairs of markers and mutation rates were performed. The statistical analysis showed evidence of inter- and intra-cluster recombination events, with almost free recombination for junction markers of linkage groups I and II and reduced recombination for linkage groups III and VI. We observed one- and two-step mutations, with an average value of 2.9 × 10−3. [ABSTRACT FROM AUTHOR]

OBJECTIVE: A number of studies have shown the role of expanded Bone Marrow-derived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were unde-tectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes. [ABSTRACT FROM AUTHOR]

BACKGROUND: Autologous chondrocyte implantation (ACI) is a cell-based treatment that can be used to regenerate chondral defects. European legislation specifically classifies such produced chondrocytes as "medicinal for advanced cell therapy" that have to be manufactured in pharmaceutical factories according to specific rules, named Good Manufacturing Practices (GMPs). One main requirement of cell manipulation in advanced therapy is to prevent the risk of any contamination. AIM: The aim of this study was to verify if chondrocyte cultures suitable for ACI were free of cross-contamination by means of DNA profiling techniques. MATERIALS AND METHODS: Cell cultures were carried on in a Hospital Cell Factory in compliance with European current Good Manufacturing Practices. DNA profiling, by means of Short Tandem Repeats and miniShort Tandem Repeats analyses, was performed on expanded chondrocytes and their related control blood samples. Mitochondrial DNA was analysed to further confirm the results and to evaluate possible mutations occurred in the samples. RESULTS: Our findings demonstrated the absence of cross-contamination between chondrocyte cultures and, thus, their identity maintenance until the end of the manipulation. CONCLUSIONS: DNA profiling technique can be a suitable test for quality control not only for chondrocyte manipulation, but for cell therapy in general. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondrial DNA (mtDNA) U/K and J/T are sister haplogroups within the superhaplogroup R. They are both common in Europe, with a combined overall frequency similar to the one reported for H, the most common European haplogroup (40–50%). In this study, we selected 159 Italian subjects, already ascribed to U/K and J/T by RFLP typing, and assigned each mtDNA to specific clades/subclades by investigating at least one diagnostic coding region SNP. For each sister haplogroup, one multiplex PCR and one SNaPshot minisequencing reaction were set up targeting 16 U/K and 7 J/T coding region SNPs. Each mtDNA sample was clearly assigned to a specific subclade, which could be further subdivided into several minor sub-branches according to peculiar HVS I/II motifs. Such a molecular dissection of haplogroups U/K and J/T could be extremely useful to reduce the overall analysis time and labor intensive sequencing procedures in high volume forensic casework, for example when it is important to rapidly exclude samples in order to restrict the number of suspects. [Copyright &y& Elsevier]

Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Although it is known that vagina undergoes characteristic age-related morphological and physiological changes over a lifetime, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups. In this collaborative study involving ten GeFI (Italian working group of ISFG) laboratories, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of vaginal swabs obtained from female volunteer donors in their reproductive years (n = 84) and postmenopausa (n = 55). Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa "observed" in the tested sample according to scoring protocol) b) average peak height ratios between vaginal-specific markers within mRNA profiling replicates. Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated. [ABSTRACT FROM AUTHOR]

Abstract: Within an Italian collaborative exercise on the extended haplotype of the Y-chromosome, 1288 subjects were typed by the AmpFlSTR YFiler Amplification Kit (AB Applied Biosystems) and other 526 were typed by the PowerPlex Y® System (Promega). The sampling scheme included either a “regional” or a “local” recruitment, the first referring to individuals born in the region of the participating lab, the second referring to individuals coming from small villages. Total sample sizes were N =954 and 860, respectively. A significant decrease of haplotype diversity was found in the local samples. The results may be of interest in forensic applications of the Y-chromosome. [Copyright &y& Elsevier]

Abstract: We recently found a number of intermediate DYS458 alleles, indicated as .2. This allelic variant is distributed in several populations, but currently no information is available regarding the molecular structure and the genealogical correlation of chromosomes with this variant. The molecular characterisation of such allele, its worldwide distribution and the correlated evolutionary history are the subject of the present paper. Molecular and genealogical data are suggestive of a single origin for the .2 variant. Phylogeographic analysis points to either a Middle East or East African origin, but additional data is necessary to clarify this point. Our results suggest that the .2 variants is a stable polymorphism and that it could be used for population studies. [Copyright &y& Elsevier]

A collaborative exercise on DNA/RNA co-analysis and RNA cell typing involving 15 GEFI (Italian working group of ISFG) laboratories was organized in collaboration with the Netherlands Forensic Institute. Participants received: 1) PCR primers for a 19-plex mRNA profiling assay, with reference purified PCR products for each cell type targeted in the multiplex; 2) detailed protocols for DNA/RNA co-extraction, mRNA profiling, and interpretation of results; 3) a set of 8 mock forensic stains (7 single source, one a mixture of two body fluids). All but one laboratory generated correct DNA typing results. As expected, stochastic effects were seen for low template DNA extracted from a skin stain. As for mRNA profiling, the percentage of laboratories that correctly identified body fluids was ≥60% for blood, saliva, vaginal mucosa, semen, and skin. Success rates were <50% for menstrual secretions, nasal mucosa, and the mixed stain (menstrual secretions and saliva). [ABSTRACT FROM AUTHOR]

Abstract: Substance addiction is a complex chronic brain disorder, characterized by neurobiological changes leading to compulsive drug seeking and taking. Although environmental factors contribute to drug addiction, evidence showed that genetic factors with multiple genes also play a significant role. Cannabis and tobacco result as the most common widely abused substances. Epidemiological studies have strongly implicated genetics in nicotine and marijuana consuming and vulnerability to subsequent dependence, estimating the range of heritability from 34% to 78% for cannabis and approximately from 50% to 70% for nicotine. Furthermore, varying aspects of impulsive personality and principal psychiatric disorders co-occur with tobacco and cannabis dependence status. We evaluate the possibility of identifying an individual''s risk probability to become an addict, based on a genotype analysis and the different possible applications in legal medicine and forensic genetics. [Copyright &y& Elsevier]

Abstract: A sample of 1070 individuals was collected by 12 collaborating forensic laboratories from Central and Northern Italy, and typed by the so-called “NC01” triplex, including the three loci D10S1248, D14S1434, and D22S1045. Heterozygosities were 0.771, 0.717, and 0.720, respectively. [Copyright &y& Elsevier]

‘Cold’ cases involving sexual assault can be solved decades after investigations analysing DNA from stored evidences, collected when current forensic method were not developed. To evaluate the influence of time and storage conditions on presumptive test and DNA analysis, three aged seminal stains date back to the first half of the ‘900, recovered during restoration work of the Institute of Legal Medicine of the University of Bologna, were analysed by the semenogelin test, microscopic identification of spermatozoa, autosomal, Y-chromosome and X chromosome STRs, and mitochondrial DNA analysis. All samples displayed a weak positive reaction for semenogelin test and the sperm heads were clearly identified. The number of detectable autosomal and Y-STRs was strongly correlated to the DNA degradation index, except for one specimen showing a high degradation index but enough DNA to obtain a composite profile. STRs included in the Minifiler kit and smaller STRs in ESX System were reproducible in all analysed specimens. Aged seminal stains may be successfully analysed by current forensic methods, even if the selection of appropriate amplicons size based on DNA amount and its degradation index is mandatory to predict the success probability in casework. [ABSTRACT FROM AUTHOR]

Abstract: The 2011 collaborative exercise of the ISFG Italian Working Group GeFI was aimed at validating the five ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. The protocol required to type at least 50 multilocus profiles from locally resident individuals and two blind bloodstains in duplicate (i.e., using at least two different commercial kits), and to send the electropherograms to the Organizing Committee. Nineteen laboratories distributed across Italy participated, collecting a total of 960 samples. Full concordance was found for the five new miniSTRs as observed from the comparison of 13,150 alleles. The inspection of the electropherograms allowed the identification of a very limited number of mistypings in the miniSTR genotypes thus contributing to the establishment of an high quality Italian database of frequencies. [Copyright &y& Elsevier]

Abstract: Alleles frequencies for the 17 short tandem repeats (STRs) loci included in the PowerPlex® ESI 17 kit were investigated in a sample of 80 unrelated individuals from a Tibetan ethnic group living in Dharamsala in the state of Himachal Pradesh (Northern India). Due to the relative novelty of this kit, including the 5 new ENFSI recommended loci (D22S1045, D2S441, D10S1248, D1S1656, D12S391) together with SE33 locus, no information regarding all these new loci is available for the Tibetan population. The aims of the present study were to determine the genetic polymorphism of forensic validated markers and to increase the ethnical population database. [Copyright &y& Elsevier]

Abstract: We collected published and unpublished data from 17 contributing groups participating in the GeFI (Italian Forensic Geneticists). The total number of typed subjects was 1114 males and 777 females, coming from 11 regions of North, Centre, and South Italy, and Sardinia. Individual''s multilocus genotypes included 4–12 loci. The total number of typed markers was 29, scattered along the X-chromosome genetic map in several clusters; the most used marker was DXS7423 (2429 gene copies); the mean number of subjects typed per marker was 336 for males and 208 for females. Data are available online. [Copyright &y& Elsevier]