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In April 2015, a fishing boat that departed from Libya with about 1,000 migrants on board sank in the Mediterranean Sea. Most of the migrants were packed in the hull of the boat and drowned in the shipwreck. After fifteen months, the ship was recovered from the seabed and brought to a Sicilian naval area for forensic investigations. Skeletal remains belonging to more than 700 people were retrieved. A selected sample composed of 80 victims was considered in order to evaluate the possibility of achieving genetic profiles useful for a positive identification from these challenging specimens. The molecular features of the DNA recovered from a significant number of real casework samples exposed to seawater for long periods of time were described for the first time. Three different DNA extraction protocols and three different commercial kits were employed in order to generate genetic profiles based on the characterization of 21 autosomal STR loci. The combination of multiple DNA extractions and the cross-checking of multiple PCR amplifications with different kits allowed to obtain reliable genetic profiles characterized by at least 16 STR markers in more than 70% of the samples. The factors that could have affected the different quality of the genetic profiles were investigated and the bone preservation was examined through microscopic and macroscopic analyses. The approach presented in this study could be useful in the management of the genetic analysis of bone samples collected in other similar DVI scenarios. The genetic profiles recovered from the bone samples will be compared in kinship analysis to putative relatives of the victims collected in Africa in order to obtain positive identifications., Competing Interests: Conflict of interest The authors have declared no conflict of interest and that the study was performed with the authorization of the Italian Government’s Office of the Commissioner for Missing Persons (UCPS-Ufficio Commissario Straordinario per le Persone Scomparse) and of the judicial authority., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Early-Onset Schizophrenia (EOS) is a very rare mental disorder that is a form of schizophrenia occurring before the age of 18. EOS is a brain disease marked by an early onset of positive and negative symptoms of psychosis that impact development and cognitive functioning. Clinical manifestations commonly include premorbid features of Autism Spectrum Disorder (ASD), attention deficits, Intellectual Disability (ID), neurodevelopmental delay, and behavioral disturbances. After the onset of psychotic symptoms, other neuropsychiatric comorbidities are also common, including obsessive-compulsive disorder, major depressive disorder, expressive and receptive language disorders, auditory processing, and executive functioning deficits. With the purpose to better gain insight into the genetic bases of this disorder, we developed a pilot project performing whole exome sequencing of nine trios affected by EOS, ASD, and mild ID. We carried out gene prioritization by combining multiple bioinformatic tools allowing us to identify the main pathways that could underpin the neurodevelopmental phenotypes of these patients. We identified the presence of variants in genes belonging to the Wnt, cadherin and cholecystokinin receptor signaling pathways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Drozd, Capovilla, Previderé, Grossi, Askenazy, Bardoni and Fernandez.)

Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxicological analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabinoids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites.

Purpose: Whatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results., Methods: Twenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography-mass spectrometry methods., Results: We were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent., Conclusions: This study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood., Supplementary Information: The online version contains supplementary material available at 10.1007/s11419-020-00567-2., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2021.)

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeq TM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9-10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as "low coverage" (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeq TM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1-44.8% and 10.9-58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and inexpensive MEKC method, which is able to assess base damage within DNA samples, is illustrated. After heat-acid hydrolysis of the DNA samples, both the percentage of each canonical DNA base and the relative amount of uncanonical DNA bases can be measured. This method is useful for an evaluation of the integrity of PCR templates used in several fields of investigation.

The assessment of the integrity of the DNA primary structure and the identification of canonical and modified bases are useful tools in medical, pharmaceutical, and forensic applications. In this article we report on the first microwave-assisted hydrolyses of deoxyribonucleoside-triphosphates (dNTPs) and human DNA using "Design of Experiments" methodology. We use hydrophilic interaction chromatography (HILIC) and UV detection at 260 nm for the determination of purinic and pyrimidinic bases at levels of 0.5 μM. We use a ZIC-HILIC 150mm × 2.1 mm i.d., 5 μm particle size column and ammonium formate buffers in acetonitrile for gradient separation of the analytes. We then compare the final concentrations of Thymine, Adenine, Cytosine, and Guanine with the nominal amounts of such bases in the dNTPs and DNA submitted to hydrolysis. After optimization of the hydrolysis (11.5 min, 0.15 M aqueous HCl, 150 °C), the method turns out to be suitable for the determination of products released from quantities of human DNA as low as 500 ng with precision (RSD<10%) and accuracy (REC 97-104%). These results confirm that the kinetics of the release of the bases depends on their molecular structure and show that the concentration of the substrate plays a relevant role. We conclude with a discussion of the method and a comparison to the methods described in previous studies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A CE/UV method was developed to separate by a micellar system the four DNA bases and other five purinic-pyrimidinic compounds (5-methyl-cytosine, uracil, xanthyne, hypoxanthyne and 5-bromo-uracil). Selectivity, precision, accuracy and sensitivity were assessed and proved to be suitable for the analysis of the primary structure of DNA. This method was adopted to study 16 aged samples including two Egyptian mummies, formaldehyde-fixed paraffin-embedded tissues and other forensic specimens. Lower relative values of the four canonical unmodified DNA bases (uDNAb) and more complex pherograms were found in the aged samples when compared with the modern controls. The results of the CE analysis, together with those obtained by classical molecular methods (agarose gel electrophoresis, DNase I and RNase A assays, and UV spectrophotometry), were finally evaluated for assessing the reliability of STR typing. Since samples with low uDNAb showed no amplification or unreliable STR profiles, the uDNAb value is discussed as a further quality criterion in the evaluation of the genetic data obtained from aged samples.

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.

Y-chromosomal DNA haplotypes were determined in 74 infertile and 216 control Italian males using eight biallelic markers. A significant difference in haplotype frequency was found, but could be explained by the geographical origins of the samples. The Y chromosome is thus a sensitive marker for population substructuring and may be useful for determining whether two population samples come from a single population, for example in association studies.