The increasing interest in DNA methylation (DNAm) analysis within the forensic scientific community prompted a collaborative project by Ge.F.I. (Genetisti Forensi Italiani). The study evaluated a standardized bisulfite conversion-based Single Base Extension (SBE) protocol for the analysis of the methylation levels at five age-predictive loci (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59). The study encompassed three phases: (1) setting up and validating the protocol to ensure consistency and reproducibility; (2) comparing fresh peripheral blood with blood spots; and (3) evaluating sources of intra- and inter-laboratory variability. Samples from 22 Italian volunteers were analyzed by 6 laboratories in replicates for a total of 528 records. From phase I emerged that the choice of genetic sequencer significantly contributed to inter-laboratory data variation, resulting in separate regression analyses performed for each laboratory. In phase II, blood spots were found to be a reliable source for DNAm analysis, despite exhibiting increased experimental variation compared to fresh peripheral blood. In phase III, a strong correlation between the individual's predicted and true ages was observed across different laboratories. Analysis of variance (ANOVA) of the residuals indicated that one-third of the total variance could be attributed to laboratory-specific factors, whereas two-thirds could be attributed to inter-individual biological differences. The leave-one-out cross-validation (LOO-CV) method yielded an overall mean absolute deviation (MAD) value of 4.41 years, with an average 95% confidence interval of 5.24 years. Stepwise regression analysis proved that a restricted model (ELOVL2, C1orf132/MIR29B2C, and TRIM59) produced results virtually indistinguishable from the five-loci model. Additionally, the analysis of samples in replicates greatly improved the fit of the regression model, balancing the slight effects of intra-laboratory variability. In conclusion, the bisulfite conversion-based SBE protocol, combined with replicate analysis and in-lab calibration of a regression-prediction model, proves to be a reliable and easily implementable method for age prediction in forensic laboratories.

DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction’s nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs’ peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile’s characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.

The presence of background DNA (bgDNA) can hinder the evaluation of DNA evidence at the activity level, especially when the suspect is expected to be retrieved due to their habitual occupation of the investigated environment. Based on real-life casework circumstances, this study investigates the prevalence, composition, origin, and probable transfer routes of bgDNA found on personal items in situations where their owner and person of interest (POI) share the same workspace. Baseline values of bgDNA were evaluated on the participants’ personal items. Secondary and higher degree transfer scenarios of non-self DNA deposition were also investigated. The DNA from co-workers and co-inhabiting partners can be recovered from an individual’s personal belongings. Non-self DNA present on the hands and deposited on a sterile surface can generate uninformative profiles. The accumulation of foreign DNA on surfaces over time appears to be crucial for the recovery of comparable profiles, resulting in detectable further transfer onto other surfaces. For a thorough evaluation of touch DNA traces at the activity level, it is necessary to collect information not only about DNA transfer probabilities but also about the presence of the POI as part of the ‘baseline’ bgDNA of the substrates involved.

In a judiciary setting, questions regarding the mechanisms of transfer, persistence, and recovery of DNA are increasingly more common. The forensic expert is now asked to evaluate the strength of DNA trace evidence at activity level, thus assessing if a trace, given its qualitative and quantitative features, could be the result of an alleged activity. The present study is the reproduction of a real-life casework scenario of illicit credit card use by a co-worker (POI) of its owner (O). After assessing the shedding propensity of the participants, differences in DNA traces' qualitative and quantitative characteristics, given scenarios of primary and secondary transfer of touch DNA on a credit card, a non-porous plastic support, were investigated. A case-specific Bayesian Network to aid statistical evaluation was created and discrete observations, meaning the presence/absence of POI as a major contributor in both traces from direct and secondary transfer, were used to inform the probabilities of disputed activity events. Likelihood Ratios at activity level (LRα) were calculated for each possible outcome resulting from the DNA analysis. In instances where only POI and POI plus an unknown individual are retrieved, the values obtained show moderate to low support in favour of the prosecution proposition.

Nowadays, organ transplantation is considered an established medical practice that, every year, improves the quality of life of thousands of patients. However, the increasing demands for kidney transplantation are in contrast with the global lack of organs. The imbalance between supply and demand for organs has created the basis for a highly profitable black market, placing illicit organ trafficking in the broader context of human trafficking. Currently, thanks to the advancements of the analytical techniques used in laboratories, forensic genetics is able to discriminate the geographical origin of genetically distinct populations. The recent availability of genetic data regarding many populations of the world and the concomitant development of technologies and methodologies that are appropriate for the study of panels of STRs and SNPs are fundamental resources in this direction. This type of analyses, together with the creation of missing person DNA databases, may be used in cases of dubious origin of organs or in transplantation cases in which clear and comprehensive medical records of patients and donors are not available. It can also establish a scientific tool useful to contrast the illegal traffic of human kidneys. In this article, we will discuss biological and ethical aspects of this interesting perspective.

In forensic genetics, Short Tandem Repeat (STR) typing is the standard approach and the method of choice for identification testing due to its increased discrimination capacity, sensitivity and reliability [ [1] Butler J.M. Genetics and genomics of core short tandem repeat loci used in human identity testing. J. Forensic Sci. 2006; 51: 253-265 Crossref PubMed Scopus (473) Google Scholar , [2] Wang Z. Zhang S. Zhao S. Hu Z. Sun K. Li C. Population genetics of 30 insertion-deletion polymorphisms in two Chinese populations using Qiagen Investigator® DIPplex kit. Forensic Sci. Int. Genet. 2014; 11: e12-e14 Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar ]. However, they present some limitations in the analysis of degraded DNA samples, due to the relatively large size of the PCR amplicons [ [3] Jobling M.A. Gill P. Encoded evidence: DNA in forensic analysis. Nat. Rev. Genet. 2004; 5: 739-751 Crossref PubMed Scopus (412) Google Scholar , [4] Schneider P.M. Beyond STRs: the role of diallelic markers in forensic genetics. Transfus. Med. Hemother. 2012; 39: 176-180 Crossref PubMed Scopus (38) Google Scholar ]. Therefore, in the last decade, various strategies have been developed in an attempt to complement STR typing. One of these approaches is based on the use of Insertion-Deletion (InDel) multiplexes. These markers, which constitute about 20% of human genetic variation, are widely distributed in the genome, both on autosomal chromosomes and on sex chromosomes, apparently with the same density [ 5 Santos N.P. Ribeiro-Rodrigues E.M. Ribeiro-dos-Santos A.K. Pereira R. Gusmão L. Amorim A. Guerreiro J.F. Zago M.A. Matte C. Hutz M.H. Santos S.E. Assessing individual interethnic admixture and population substructure using a 48insertion-deletion (INDEL) ancestry-informative marker (AIM) panel. Hum. Mutat. 2010; 31: 184-190 Crossref PubMed Scopus (254) Google Scholar , 6 LaRue B.L. Ge J. King J.L. Budowle B. A validation study of the Qiagen investigator DIPplex(R) kit; an INDEL-based assay for human identification. Int. J. Legal Med. 2012; 126: 533-540 Crossref PubMed Scopus (88) Google Scholar , 7 Zaumsegel D. Rothschild M.A. Schneider P.M. A 21 marker insertion deletion polymorphism panel to study biogeographic ancestry. Forensic Sci. Int. Genet. 2013; 7: 305-312 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ]. They are biallelic length polymorphisms derived from a single mutational event, that consist in the presence or absence of short sequences, less than 200 bp. Furthermore, they have a low mutation rate, no stutter peaks, short amplicon size and high multiplexing capacity. Most of them present significant differences in the allele frequencies between geographically distant population groups, so they can also be used as ancestry informative markers [ [8] Santos C. Fondevila M. Ballard D. Banemann R. Bento A.M. Borsting C. Branicki W. Brisighelli F. Burrington M. Capal T. Chaitanya L. Daniel R. Decroyer V. England R. Gettings K.B. Gross T.E. Haas C. Harteveld J. Hoff-Olsen P. Hoffmann A. Kayser M. Kohler P. Linacre A. Mayr-Eduardoff M. McGovern C. Morling N. O’Donnell G. Parson W. Pascali V.L. Porto M.J. Roseth A. Schneider P.M. Sijen T. Stenzl V. Court D.S. Templeton J.E. Turanska M. Vallone P.M. van Oorschot R.A. Zatkalikova L. Carracedo A. Phillips C. EUROFORGEN-NoE Consortium Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: results of a collaborative EDNAP exercise. Forensic Sci. Int. Genet. 2015; 19: 56-67 Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar , [9] Pereira R. Phillips C. Alves C. Amorim A. Carracedo Á. Gusmão L. Insertion/deletion polymorphisms: a multiplex assay and forensic applications. Forensic Sci. Int. Genet. Suppl. Ser. 2009; 2: 513-515 Abstract Full Text Full Text PDF Scopus (50) Google Scholar ]. These markers can be conveniently analyzed on capillary electrophoresis equipment, already available in most forensic genetic laboratories. InDels can also be a good choice for population studies, parentage testing, missing person identification and ancient DNA analysis. InDels can be used when STR analysis is uninformative, due to poor quality of the DNA samples. On the other hand, InDel-based multiplexes must include a large number of markers to compensate for the lower statistical strength [ 10 Cardoso S. Sevillano R. Gamarra D. Santurtún A. Jarreta B.M. de Pancorbo M.M. Population genetic data of 38 insertion-deletion markers in six populations of the northern fringe of the Iberian Peninsula. Forensic Sci. Int. Genet. 2017; 27: 175-179 Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar , 11 Wang L. Lv M. Zaumsegel D. Zhang L. Liu F. Xiang J. Li J. Schneider P.M. Liang W. Zhang L. A comparative study of insertion/deletion polymorphisms applied among Southwest, South and Northwest Chinese populations using Investigator® DIPplex. Forensic Sci. Int. 2016; 21: 10-14 Abstract Full Text Full Text PDF Scopus (34) Google Scholar , 12 Shahzad M. Hussain M. Shafique M. Perveen R. Sheikh N. Population genetic data of 30 insertion–deletion markers in Punjabi population of Pakistan. Int. J. Legal Med. 2019; https://doi.org/10.1007/s00414-019-02029-w Crossref Scopus (4) Google Scholar ]. In 2009, an InDel multiplex was described by Pereira et al. [ [13] Pereira R. Phillips C. Alves C. Amorim A. Carracedo A. Gusmão L. A new multiplex for human identification using insertion/deletion polymorphisms. Electrophoresis. 2009; 30: 3682-3690 Crossref PubMed Scopus (177) Google Scholar ], combining 38 highly polymorphic loci in a single PCR. This multiplex combines all the advantages mentioned above regarding InDels. However, population data for these new markers are limited, particularly for Italy, where there is only one study that performed InDel analysis on Sicilian residents (Island in Southern Italy) [ [14] Pereira R. Alves C. Aler M. Amorim A. Arévalo C. Betancor E. Braganholi D. Bravo M.L. Brito P. Builes J.J. Burgos G. Carvalho E.F. Castillo A. Catanesi C.I. Cicarelli R.M.B. Coufalova P. Dario P. D’Amato M.E. Davison S. Ferragut J. Fondevila M. Furfuro S. García O. Gaviria A. Gomes I. González E. Gonzalez-Liñan A. Gross T.E. Hernández A. Huang Q. Jiménez S. Jobim L.F. López-Parra A.M. Marino M. Marques S. Martínez-Cortés G. Masciovecchio V. Parra D. Penacino G. Pinheiro M.F. Porto M.J. Posada Y. Restrepo C. Ribeiro T. Rubio L. Sala A. Santurtún A. Solís L.S. Souto L. Streitemberger E. Torres A. Vilela-Lamego C. Yunis J.J. Yurrebaso I. Gusmão L. A GHEP-ISFG collaborative study on the genetic variation of 38 autosomal indels for human identification in different continental populations. 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Recently microRNAs (miRNAs) have gained notoriety for having revealed useful biomarkers. The advantage of using microRNAs as biomarkers lies in their ease of use and the accuracy with which they can be measured as well as their extreme tissue specificity. Furthermore, these small molecules are exceptionally stable in laboratory treatments, unlike larger RNA molecules such as messenger RNA. In the forensic field, the use of microRNAs to confirm biological evidence has become a powerful tool of crucial importance for the reconstruction of a crime scene. Semen can provide significant evidence in some types of crime, particularly sexual assault. However, a diagnosis of sperm is often difficult for several reasons, such as trace degratheir ease of use and the accuracy with which they can be measured as well as their extreme tissue specificity. Furthermore, these small molecules are exceptionally stable in laboratory treatments, unlike larger RNA molecules such as messenger RNA. In the forensic field, the use of microRNAs to confirm biological evidence has become a powerful tool of crucial importance for the reconstruction of a crime scene. Semen can provide significant evidence in some types of crime, particularly sexual assault. However, a diagnosis of sperm is often difficult for several reasons, such as trace degradation and the presence of azoospermia, which are still present when using morphological, immunological and enzymatic methods. Recent studies show that the use of micro-RNAs overcomes both the problem of degradation (due to their high resistance to exogenous factors) and the absence of spermatozoa in azoospermic individuals. In this study, some semen-specific microRNAs (miR-10a-5p, miR-888-5p, miR891a-5p, miR135a-5p, miR135b-5p) on old semen stains were tested to assess their suitability in forensic casework. The expression of this set of markers was evaluated using a miRCURY LNA Universal RT microRNA PCR System. LNA technology showed interesting properties for molecular hybridization, including enhanced specificity in allele-specific PCR and could be used to overcome the difficulties of studying very short sequences. We concluded that the cellular specificity of the studied microRNA set makes it possible to identify the presence of semen in old stains with high specificity and consequently with high probative value in a court of justice.

INTRODUCTION: Forensic genetic laboratories analyse samples included in paraffin to verify the genetic correspondence of histological samples, from living subjects or cadavers, in cases where there is a suspicion of contamination of samples with tissues of other patients. PRESENTATION OF THE CASE: A case of a man subjected to a gastrectomy as a result of a histological diagnosis of gastric adenocarcinoma after endoscopic biopsies is reported. The microscopic analysis on the gastric tissue after the gastrectomy excluded the presence of cancer. Having suspected a diagnostic error, a microscopic revision of the biopsies was performed and confirmed the presence of cancer cells but led to a hypothesis that there had been contamination with foreign intestinal tissue. The genetic analysis performed on various pieces of tissue, despite the reduced amount of biological material, succeeded in identifying the presence of two incomplete genetic profiles, one of which belonged to a subject of the opposite sex. DISCUSSION: The case raised many questions about the process of setting up histological specimens. Even though it is impossible to identify the healthcare professionals responsible for contamination, the organizational error during the management of biopsies has significantly affected the clinical case of the patient, who underwent a gastrectomy for cancer that was not present. CONCLUSION: This case is not simply an example of diagnostic error and related unnecessary surgery, but it has raised some doubts about patient management and it has led us to some medical-legal cause for reflection in the field of professional liability. (C) 2019 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd.

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).

Pulmonary ThromboEmolism (PTE) is an important disease for legal medicine. Because of their sudden lethal onset, generally medicolegal autopsies show few clinical information when PTE is the cause of death. During medicolegal autopsies, the autopsy operator must answer to important questions. For example, autopsy operator can need to assess the casual relationship between PTE and recent accident, such as trauma or long air travel. Furthermore, the autopsy operator needs to investigate the pathology of PTE as a cause of sudden cardiovascular death. It is relatively simple to confirm a fatal massive thromboembolus in the initial stage of thoracic investigations, but sometimes it might be difficult to distinguish this from postmortem clot. In such cases histopathological examination can help in the differentiation. Histological examination is also required for observation of chronological changes of the thrombi. Chronological evaluation is an important factor especially to determine whether the death coincides with the date of a specific accident/event or instead there is an earlier onset of PTE. In addition, histological sections sometimes show additional information, such as tumor fragments in cases of malignancy or small fragments of bone marrow in cases of active resuscitation, that can be useful in a medicolegal scenario. Furthermore, new diagnostic tools are arising, which they can be very helpful in the individuation of this frequently underdiagnosed disease. The goal of our work is to investigate these aspects through the review of the recent literature.

In the process of development of modern medicine, organ transplants represent one of the most important and more meaningful achievements, allowing to extend and improve the lives of patients around the world. However, increasing requests and the global lack of organs have created the basis for a highly profitable black market in organs that promotes criminal enterprise.

The ubiquitous presence of plant species makes forensic botany useful for many criminal cases. Particularly, bryophytes are useful for forensic investigations because many of them are clonal and largely distributed. Bryophyte shoots can easily become attached to shoes and clothes and it is possible to be found on footwear, providing links between crime scene and individuals. We report a case of suicide of a young girl happened in Siena, Tuscany, Italia. The cause of traumatic injuries could be ascribed to suicide, to homicide, or to accident. In absence of eyewitnesses who could testify the dynamics of the event, the crime scene investigation was fundamental to clarify the accident. During the scene analysis, some fragments of Tortula muralis Hedw. and Bryum capillare Hedw were found. The fragments were analyzed by a bryologists in order to compare them with the moss present on the stairs that the victim used immediately before the death. The analysis of these bryophytes found at the crime scene allowed to reconstruct the accident. Even if this evidence, of course, is circumstantial, it can be useful in forensic cases, together with the other evidences, to reconstruct the dynamics of events.

The X-chromosomal markers are increasingly used in forensic genetics, particularly for relationship testing. Their use has become a valuable tool in complex cases of kinship but rather in criminal caseworks is still quite rare. In this paper the authors present a case of sexual assault in which the use of X chromosome polymorphisms has been crucial. The victim was a young immigrant woman found dead in her home. The main suspect was her husband. However, the couple lived in a community in which the particular cultural context suggested the involvement of the other males of the husband family. The suspect lived together with his brother, his father and his uncle. Generally, a useful tool to solve cases of sexual violence is undoubtedly the use of Y chromosome, but in this case this device could not discriminate between the four males involved. An additional factor has further complicated the situation: the most important biological evidence (typed with AmpFlSTR Identifiler and AmpFlSTR NGM) showed a mixed profile in which was very difficult to discriminate the suspects profiles. To solve the casework, the authors typed the victim, the suspects and the biological traces with 6 X-STRs in an homemade esaplex. The results showed the presence of victim and her husband profiles in the biological trace excluding his brother, father and uncle's profiles.

In recent years, European working groups (ENFSI-EDNAP) have strongly encouraged the development of new amplification kits that allow to obtain DNA profiles even on difficult samples (degraded samples, presence of inhibitory substances, LCN, mixtures, etc.). To enable the amplification of difficult biological samples the new loci were designed to achieve greater resistance to inhibitors and most robust and uniform amplification. With the aim of evaluating the performance of latest generation kits in real forensic caseworks the authors retested some bone samples previously analyzed in their laboratory over the past five years with the old commercial kits. The authors compared the performance of AmpFℓSTR® NGM™ PCR Amplification kit (Applied Biosystems) with PowerPlex® ESX 17 systems (Promega). The twelve analyzed samples came from eight exhumed corpses and four bodies found outdoor in the advanced stage of putrefaction. For the genetic investigation the authors used a piece of bone taken from the femur. In conclusion, the authors can claim that the latest generation kits have proved to be decisive in all cases, including those where the previous use of traditional kits did not produce reliable and uniquely interpretable results.

A case of a woman killed in Perugia is reported. The woman was beaten to death and the body showed evidence of bites, kicks and punches. The request of the Court was to verify the presence of bites and if they belonged to humans. Morphological examination and genetic analysis with human Y-specific markers were performed in order to verify the origin of the bites. The DNA profile from the surrounded area of the traces was compared with the profile of the victim's husband (the suspect).The results showed a match between the profile of the suspect and that of the traces for all loci examined. Due to the fact that also other relatives of the husband's male lineage lived in the same house, it was not possible to identify the man who really contributed to the traces. Therefore, the analysis was implemented with autosomal STR markers, which showed a mixed genetic profile. In order to verify the number and the identity of the contributors, statistical analysis based upon peak area information was performed with Probabilistic Expert Systems.

MiniSTR loci have demonstrated to be an effective tool to recover genetic information from degraded sample due to the small PCR products. The use of these non-CODIS miniSTRs can increase the probability that a degraded sample can be typed and in addition these systems can be used in complex paternity cases where more markers are needed.Six autosomal loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441, D1S1677) were investigated in 200 individuals from Italian regions, Umbria and Sardinia.Statistical analysis and comparison between two groups were performed. (C) 2009 Elsevier Ireland Ltd. All rights reserved.