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Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1–H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs (∼70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis. [ABSTRACT FROM AUTHOR]

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., (© 2025 The Author(s). Electrophoresis published by Wiley‐VCH GmbH.)

The identification of human fire victims is a challenging task in forensic medicine. The heat-induced alterations of biological tissues can make the conventional anthropological analyses difficult. Even if the DNA profile of the victim is achieved, it is possible that no match can be found in a forensic DNA database, thus hindering positive identification. In such cases, any information useful to nail down a possible identity should be collected, such as DNA methylation analysis which could provide useful investigative leads. In the present study, five age-related epigenetic markers (ELOVL2, FHL2, KLF14, C1orf132, and TRIM59) were initially analysed in blood samples of 72 living Italian individuals of known age, using a Single Base Extension (SBE) assay. An age prediction model was built by multiple linear regression including all the markers (Mean Absolute Error, MAE: 3.15 years). This model was tested on 29 blood samples collected during autopsies from burnt human remains, already identified through DNA analysis, providing a MAE of 6.92 years. The model allowed a correct prediction in 79.3% of the cases (95% prediction interval), while six cases were associated with inaccurate predictions (min-max prediction error: 9.8-37.3 years). Among the different sample variables considered to explain these results, only the DNA degradation index was a relevant factor affecting the reliability of the predictions. In conclusion, the SBE typing of blood from burnt remains proved to be a reliable tool to estimate chronological age of most of the samples, also in consideration of its cost-effectiveness and the availability of CE sequencers in every forensic genetics laboratory., (© 2024. The Author(s).)

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na 2 EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell ® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index ( p -values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

Asbestos-related diseases still represent a major public health problem all over the world. Among them, malignant mesothelioma (MM) is a poor-prognosis cancer, arising from the serosal lining of the pleura, pericardium and peritoneum, triggered by asbestos exposure. Literature data suggest the key role of iron metabolism in the coating process leading to the formation of asbestos bodies, considered to be both protective and harmful. Two sample sets of individuals were taken into consideration, both residing in Broni or neighboring cities (Northwestern Italy) where an asbestos cement factory was active between 1932 and 1993. The present study aims to compare the frequency of six SNPs involved in iron trafficking, previously found to be related to protection/predisposition to MM after asbestos exposure, between 48 male subjects with documented asbestos exposure who died of MM and 48 male subjects who were exposed to asbestos but did not develop MM or other neoplastic respiratory diseases (Non-Mesothelioma Asbestos Exposed - NMAE). The same analysis was performed on 76 healthy male controls. The allelic and genotypic frequencies of a sub-group of 107 healthy Italian individuals contained in the 1000 genomes database were considered for comparison. PCR-multiplex amplification followed by SNaPshot mini-sequencing reaction was used. The findings presented in this study show that the allelic and genotypic frequencies for six SNP markers involved in iron metabolism/homeostasis and the modulation of tumor microenvironment are not significantly different between the two sample sets of MM and NMAE. Therefore, the SNPs here considered do not seem to be useful markers for individual susceptibility to mesothelioma. This finding is not in agreement with previous literature., 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 © 2023 Grignani, Visonà, Fronda, Borrelli, Monti, Bertoglio, Conti, Fattorini and Previderè.)

PCR-MPS is an emerging tool for the analysis of low-quality DNA samples. In this study, we used PCR-MPS to analyse 32 challenging bone DNA samples from three Second World War victims, which previously yielded no results in conventional STR PCR-CE typing. The Identity Panel was used with 27 cycles of PCR. Despite that we only had an average of 6.8 pg of degraded DNA as template, 30 out of 32 libraries (93.8%) produced sequencing data for about 63/90 autosomal markers per sample. Out of the 30 libraries, 14 (46.7%) yielded single source genetic profiles in agreement with the biological identity of the donor, whereas 12 cases (40.0%) resulted in SNP profiles that did not match or were mixed. The misleading outcomes for those 12 cases were likely due to hidden exogenous human contamination, as shown by the higher frequencies of allelic imbalance, unusual high frequencies of allelic drop-ins, high heterozygosity levels in the consensus profiles generated from challenging samples, and traces of amplified molecular products in four out of eight extraction negative controls. Even if the source and the time of the contamination were not identified, it is likely that it occurred along the multi-step bone processing workflow. Our results suggest that only positive identification by statistical tools (e.g. likelihood ratio) should be accepted as reliable; oppositely, the results leading to exclusion should be treated as inconclusive because of potential contamination issues. Finally, strategies are discussed for monitoring the workflow of extremely challenging bone samples in PCR-MPS experiments with an increased number of PCR cycles., (© 2023. The Author(s).)

In forensic medicine, identifying novel biomarkers for use as diagnostic tools to ascertain causes of death is challenging because of sample degradation. To that aim, a cohort ( n = 26) of fatal traumatic brain injuries (TBIs) were tested for three candidate miRNAs (namely, miR-124-3p, miR-138-5p, and miR144-3p). For each case, three FFPE specimens (coup area (CA), contrecoup area (CCA), and the corpus callosum (CC)) were investigated, whereas the FFPE brain tissues of 45 subjects (deceased due to acute cardiovascular events) were used as controls. Relative quantification via the ∆∆Ct method returned significantly higher expression levels of the three candidate miRNAs ( p < 0.01) in the TBI cases. No difference was detected in the expression levels of any miRNA investigated in the study among the CA, CCA, and CC. Furthermore, the analyzed miRNAs were unrelated to the TBI samples' post-mortem intervals (PMIs). On the contrary, has-miR-124-3p ahas hsa-miR-144-3p were significantly correlated ( p < 0.01) with the agonal time in TBI deaths. Since the RNA was highly degraded in autoptic FFPE tissues, it was impossible to analyze the mRNA targets of the miRNAs investigated in the present study, highlighting the necessity of standardizing pre-analytical processes even for autopsy tissues.

In this article, we describe multiple analytical strategies that were first developed for forensic purposes, on a set of three bone samples collected in 2011. We analyzed a single bone sample (patella) collected from the artificially mummified body of the Baron Pasquale Revoltella (1795-1869), as well two femurs which allegedly belonged to the Baron's mother (Domenica Privato Revoltella, 1775-1830). Likely due to the artificial mummification procedures, the inner part of the Baron's patella allowed the extraction of high-quality DNA yields, which were successfully used for PCR-CE and PCR-MPS typing of autosomal, Y-specific, and mitochondrial markers. The samples extracted from the trabecular inner part of the two femurs yielded no typing results by using the SNP identity panel, whereas the samples extracted from the compact cortical part of the same bone samples allowed genetic typing, even by the employment of PCR-CE technology. Altogether, 10/15 STR markers, 80/90 identity SNP markers, and HVR1, HVR2, and HVR3 regions of the mtDNA were successfully typed from the Baron's mother's remains by the combined use of PCR-CE and PCR-MPS technologies. The kinship analysis showed a likelihood ratio of at least 9.1 × 10 6 (corresponding to a probability of maternity of 99.9999999%), and thus confirmed the identity of the skeletal remains as those of the Baron's mother. This casework represented a challenging trial for testing forensic protocols on aged bones samples. It highlighted the importance of accurately sampling from the long bones, and that DNA degradation is not blocked by freezing at -80 °C.

Europe is turning a blind eye on a humanitarian disaster unfolding at its doorsteps, with thousands of migrants dying unidentified in Mediterranean waters. Since 2014, Italy has been struggling in an almost indifferent international scenario to identify its dead migrants. Despite the lack of sufficient resources, of the difficulties in collecting post mortem data from the disseminated bodies, and of the problems of contacting and collecting ante mortem information from relatives, it has been proven, with a series of pilot studies, that not only can these bodies be identified but that relatives are also looking for their loved ones and need death certificates. This article focuses on the administrative limbo and lack of regulations obliging single states to engage in appropriate procedures to maximise identification., (© 2022. The Author(s).)

The recent introduction of polymerase chain reaction (PCR)-massively parallel sequencing (MPS) technologies in forensics has changed the approach to allelic short tandem repeat (STR) typing because sequencing cloned PCR fragments enables alleles with identical molecular weights to be distinguished based on their nucleotide sequences. Therefore, because PCR fidelity mainly depends on template integrity, new technical issues could arise in the interpretation of the results obtained from the degraded samples. In this work, a set of DNA samples degraded in vitro was used to investigate whether PCR-MPS could generate "isometric drop-ins" (IDIs; i.e., molecular products having the same length as the original allele but with a different nucleotide sequence within the repeated units). The Precision ID GlobalFiler NGS STR panel kit was used to analyze 0.5 and 1 ng of mock samples in duplicate tests (for a total of 16 PCR-MPS analyses). As expected, several well-known PCR artifacts (such as allelic dropout, stutters above the threshold) were scored; 95 IDIs with an average occurrence of 5.9 IDIs per test (min: 1, max: 11) were scored as well. In total, IDIs represented one of the most frequent artifacts. The coverage of these IDIs reached up to 981 reads (median: 239 reads), and the ratios with the coverage of the original allele ranged from 0.069 to 7.285 (median: 0.221). In addition, approximately 5.2% of the IDIs showed coverage higher than that of the original allele. Molecular analysis of these artifacts showed that they were generated in 96.8% of cases through a single nucleotide change event, with the C > T transition being the most frequent (85.7%). Thus, in a forensic evaluation of evidence, IDIs may represent an actual issue, particularly when DNA mixtures need to be interpreted because they could mislead the operator regarding the number of contributors. Overall, the molecular features of the IDIs described in this work, as well as the performance of duplicate tests, may be useful tools for managing this new class of artifacts otherwise not detected by capillary electrophoresis technology., (© 2022 The Authors. Electrophoresis published by Wiley-VCH GmbH.)

The collection of liquid biological matrices onto paper cards (dried matrix spots [DMS]) is becoming an alternative sampling strategy. The stability over time of molecules of interest for therapeutic, sport drug monitoring, and forensic toxicology on DMS has been recently investigated representing a reliable alternative to conventional analytical techniques. When a tampering of a urine sample in drug monitoring or doping control cases is suspected, it could be relevant to know whether genetic profiles useful for individual identification could be generated from urine samples spotted onto paper (dried urine spot [DUS]). To understand the influence of sex, storage conditions, and time on the quality and quantity of the DNA, five female and ten male urine samples were dispensed onto Whatman 903 paper and sampled after different storage conditions over time, from 1 to 12 weeks. Direct PCR was performed starting from 2-mm punches collected from each spot amplifying a panel of markers useful for individual identification. The female DUS stored in different conditions produced genetic profiles fully matching the reference samples. The same result was obtained for the male DUS but using urine 30X concentrated by centrifugation instead of the original samples. Our data show that this approach is valid for genetic individual identification of urine samples spotted onto paper cards up to 12 weeks after deposition and could be easily incorporated in anti-doping or drug screening protocols to help on the suspicion of evidence tampering or to solve questions on the reliability of samples collection., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists - International Society of Forensic Genetics (Ge.F.I. - ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of -1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes., Competing Interests: Conflict of Interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Biodurability is one of the main determinants of asbestos hazardousness for human health. Very little is known about the actual persistence of asbestos in lungs and its clearance, nor about differences in this regard between the different mineralogical types of asbestos. The aim of the present study was to evaluate the amount, the dimensional characteristics and the mineralogic kinds of asbestos in lungs (measured using SEM-EDS) of a series of 72 deceased subjects who were certainly exposed to asbestos (mainly crocidolite and chrysotile) during their life. Moreover, we investigated possible correlations between the lung burden of asbestos (in general and considering each asbestos type), as well as their dimension (length, width, and l/w ratio) and the duration of exposure, the latency- in case of malignant mesothelioma (MM), the survival and the time since the end of exposure. In 62.5% of subjects, asbestos burden in lungs was lower that the threshold considered demonstrative for occupational exposure. In 29.1% of cases no asbestos was found. Chrysotile was practically not detected. The mean length of asbestos fibers and the length to width ratio were significantly related to the duration of exposure to asbestos. No other statistically significant correlations were found between the amount and dimensional characteristics of asbestos (nor with the relative amount of each asbestos type) and the other chronological variables considered. In conclusion, it was pointed out that chrysotile can be completely removed from human lungs in <8 years and, instead, amphiboles persist much more time. The present results suggest, as well, that the finding of no asbestos in lungs cannot rule out the attribution of MM to asbestos (in particular, chrysotile) inhaled in an occupational setting. This point is of crucial importance from a legal point of view., Competing Interests: CC acted as experts for the court, the public prosecutors and the defense in asbestos-related litigations NOT related to the subjects of this paper. The remaining 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 Visonà, Capella, Bodini, Borrelli, Villani, Crespi, Colosio, Previderè and Belluso.)

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the "same donor" or from a "first degree relative" was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21-26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18-23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10 -13 , which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Violence against women is a violation of human rights, crossing all cultures, classes, levels of education, earnings, ethnic and age groups. We conducted a retrospective study to review forensic records of sexual assault examinations carried out in different Italian health facilities and to correlate these findings with the results of the forensic DNA analyses. The goal was to determine which factors could have affected the obtained results, to identify the fundamental aspects to search for while examining a sexual assault victim in order to gather useful evidence to identify the offender and reconstruct the dynamics of the fact. We analysed 102 cases that occurred between 2006 and 2017, coming from ten participating laboratories. Despite a relatively limited number of cases, this study shows that the ability to ascertain the presence of male biological material in the samples collected is not a problem for forensic laboratories and seems to be influenced by other factors, such as how much time elapsed between the event and the sampling, the availability of the aggressor's biological material on the victim and the identification of biological fluids/stains. Therefore, the need for health structures to adopt specific protocols has been highlighted. It is necessary for health structures to define specific pathways and adopt homogeneous procedures or operational protocols, and it is essential to provide adequate training for health personnel. The results of the study could be useful in drafting and revising protocols/guidelines implemented in Italian hospital. Issues related to the limited number of analyses requested by Italian Authorities are also discussed., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical-chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r 2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

On October 3rd, 2013 a boat carrying more than 500 migrants coming mostly from the Horn of Africa (Eritrea, Somalia, Ethiopia) sank near Lampedusa, a small Italian Island in the middle of the Mediterranean Sea. The recovered bodies were examined by a forensic team, and post mortem data (anthropological and odontological records, and DNA) were collected for identification. Genetic profiles based on 16 autosomal STRs were acquired from both victims and putative relatives recruited following an international call. The final genetic database included 363 victims and 43 reference persons from 36 independent families recruited until mid-2017, who were missing 35 first-degree and 6 second-degree relatives. A pairwise blind search approach was used to identify familial relationships within the victims and between victims and putative relatives. Two statistics were calculated, the Identity by State (IBS) and the Identity by Descent (IBD), the latter by using the DVI module of the FAMILIAS3 software to compute LRs and posterior probabilities. The putative identifications were confirmed in pedigree analysis using the information provided by the relatives. In selected cases, additional autosomal and lineage (Y-chromosome and mtDNA) markers were typed. Some critical points were highlighted: the lack for accurate allele and haplotype frequencies in African populations, especially for the lineage markers, and the need for a shared approach to the biostatistical interpretation of the results in DVI. In the end, 29 first-degree (parent-child and full sibs) out of 35 missing (83%), and 3 out of 6 of second-degree relatives (50%) showed a high statistical confidence for a positive identification. This study represents the first attempt to systematically deal with the genetic identification of African migrants who died in the Mediterranean Sea. The methodological and statistical approach used in this study was proved to be reliable and appropriate for future genetic identifications in other similar mass disasters., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A rectrospective study was conducted on the effect of the long term storage of 122 DNA samples resuspended in water, one of the elution media still suggested by well established protocols. These DNA samples come from four different kinds of forensically relevant samples (saliva swabs, FTA card bloodstains, nails and II° World War bones) extracted in 2008-2018 and stored at - 20°C (n=113 of groups #1-#5) and at +4°C (n=9 of the group #6), respectively. At the time of the present study (2019), quantitative PCR (qPCR) was employed as tool for assessing the degradation of the samples. The employment of the Human Quantifiler Kit showed that the median loss of DNA ranged from 17.8% to 66.6% in groups #1-#5 while it was 85.0% in group #6. However, it is likely that these values represent an underestimation due to the shortness of the qPCR probe (62 bp). Noteworthy, the DNA loss was statistically significant in each of the six groups (p values ≤ 0.0167). Thus, in agreement with the data on spontaneous DNA decay, no forensic DNA sample should be stored in water for long term periods. In conclusion, the results of this technical note warn against the use of water for this purpose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro. Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 °C for 0-18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes ("Protocol P") and in Eppendorf tubes provided with screwcaps ("Protocol S"). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the "Protocol S" affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2-3 orders of magnitude. The chemical-physical reasons of these findings are discussed together with the role of potential factors such as enhanced reactivity of CO 2 , ROS, NO x and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of degradation is well documented could be helpful in ISO/IEC 17025 validation procedures and in proficiency testing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Every year thousands of migrants die during the endeavour to reach the Italian coasts, making the Mediterranean the theatre of one of the greatest tragedies of mankind. Over 60% of these victims is buried unidentified: one of the reasons behind this is related to the specific difficulties and lack of strategies concerning AM and PM data collection. The present article describes how Italy is trying to face the problem of migrant identification, thanks to the collaboration between government, the Italian national police and universities. In particular, this is the first pilot study carried out to identify the victims of the second greatest tragedy of its kind off the Italian coast, near Lampedusa, on October 3rd 2013, which caused 366 victims. The present article shows the strategies conceived to collect postmortem and especially antemortem data and to compare them to identify matches, using medicolegal, anthropological, odontological and genetic approaches. Thirty-one victims out of 53 missing sought by relatives were identified (58.5%). The type and the quality of antemortem data available, generally photos and videos, pinpoints the importance of the face and the body for identification when the bodies are well preserved and how DNA analyses may at times present difficulties. In fact, critical points emerged concerning especially the lack of genetic information of the populations to which the victims belonged, the number of genetic markers needed to reach a statistical support for the identification and the need to adopt lineage markers such as mitochondrial DNA and Y-chromosome polymorphisms to identify parental relationships. This pilot study however has proven that families continue to seek their relatives and that it is possible, as well as mandatory, to identify migrant victims in spite of the difficulties in the collection of antemortem and postmortem data. In addition, considering the peculiar scenario, novel strategies for positive identification have to be defined in each field (anthropological, odontological and genetic) as well as in combination., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In mammals, the sex of the embryo depends on the SRY gene. In the presence of at least one intact and functional copy of this genetic factor (XY embryo) undifferentiated gonads will develop as testicles that subsequently determine the male phenotype. When this factor is not present, i.e., in subjects with 2 X chromosomes, an alternative pathway induces the development of ovaries, hence a female phenotype. In this case study, we describe a female cattle affected by a disorder of sex development (DSD). The subject, despite having a chromosomal XY constitution, did not develop testicles but ovaries, although they were underdeveloped. Moreover, genetic analysis highlighted the presence of the SRY gene with a normal coding region in both blood- and tissue-derived DNA. A chimeric condition was excluded in blood by sexing more than 350 cells and by allele profile investigation of 18 microsatellite markers. Array CGH analysis showed the presence of a not yet described 99-kb duplication (BTA18), but its relationship with the phenotype remains to be demonstrated. Gonadal histology demonstrated paired ovaries: the left one containing a large corpus luteum and the right one showing an underdeveloped aspect and very few early follicles. To our knowledge, we describe the first case of XY (SRY+) DSD in cattle with a normal SRY gene coding sequence., (© 2018 S. Karger AG, Basel.)

In most mammals, the sex of an individual is genetically determined by the Y chromosome-specific SRY gene. The presence of at least one functional copy of this gene determines the development of the primordial gonads into testes. However, testicular tissue does develop in the absence of SRY, albeit rarely, which is the case in testicular XX (SRY-negative) disorder of sex development (DSD). This condition is very important for studying the process of sexual determination because it allows the identification of genetic factors that are able to promote the male developmental pathway in the absence of SRY and thereby enables a better understanding of this process. Until now, this condition has been identified in various animal species but has never been reported in cat. In this study, we describe the first case of an XX (SRY-negative) DSD cat. The cat possesses a tortoiseshell coat associated with male-like external genitalia, including normal scrotum with 2 palpably normal testicles. Histological analysis confirmed the presence of the testes, and cytogenetic and genetic analyses showed a female karyotype associated with the absence of the SRY gene. Finally, sequencing of the RSPO1 gene revealed no mutation, and FISH analysis of the SOX9 locus did not reveal any large abnormalities., (© 2017 S. Karger AG, Basel.)

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by fetal macrosomia, macroglossia, and abdominal wall defects. BWS patients are at risk to develop Wilms tumor, neuroblastoma, hepatoblastoma, and adrenal tumors. A young woman with BWS features, but with inconclusive genetic evidence for the disease, came to clinical observation for signs of virilization at the age of 16 years. An adrenocortical tumor was diagnosed and surgically resected. The tumor underwent 2 local relapses that were also surgically treated. The patient was also operated to remove a breast fibroadenoma. SNP arrays were used to analyze chromosome abnormalities in normal and tumor samples from the patient and her parents. The patient presented genome-wide mosaic paternal uniparental disomy (patUPD) both in the adrenocortical and the breast tumors, with different degrees of loss of heterozygosity (LOH). The more recent relapses of the adrenocortical tumor showed a loss of part of chromosome 17p that was absent in the first tumor. Analysis of a skin biopsy sample also showed mosaic patUPD with partial LOH, while no LOH was detected in leukocyte DNA. This case shows that virilizing adrenocortical tumors may be a clinical feature of patients with BWS. The SNP array technology is useful to diagnose genome-wide patUPD mosaicism in BWS patients with an inconclusive molecular diagnosis and underlines the tumorigenic potential of the absence of the maternal genome combined with an excess of the paternal genome., (© Georg Thieme Verlag KG Stuttgart · New York.)

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural" and "urban" samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 © 2012 Elsevier Ireland Ltd. All rights reserved.)

A DNA sample was partially degraded by scalar heat-acid treatments to study the extent of apurinic-apyrimidinic (A-P) lesions produced along the molecule. A CE-UV method allowed us to measure the rate of depurination at pH 5.0 and 70°C which was calculated to be 5.41×10(-6)  s(-1) for adenine and 6.27×10(-6)  s(-1) for guanine. CE identified depurination on treated samples when it occurred with a loss of >4% of the basic moieties. The molecular features of the A-P enriched samples were investigated by using molecular assays (agarose gel electrophoresis, UV spectrophotometry and quantitative PCR) and the consistency of the results of the STR typing were compared with the degree of depurination of the PCR template. The treated DNA samples showed molecular features such as fragmentation, altered OD(260) /OD(280) ratios and decreased ability of the quantitative PCR to synthesise the human target, related to the severity of depurination. A satisfactory correlation between the degree of damage and the amount of residual PCR-sensitive target sequences was also demonstrated (r(2) =0.9717). The conventional and mini-STR typing of the samples showed that the genetic outcome was influenced by a depurination damage that exceeded 4% when locus drop-outs and artefactual PCR results were evident. As the success of STR typing depends on the integrity of the DNA recovered from the samples, the CE-UV, physical and molecular assays described here are proposed as a set of useful methods in the analysis of certain forensic and clinical samples, for a critical evaluation of the outcome of the genetic testing., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The relative contributions to modern European populations of Paleolithic hunter-gatherers and Neolithic farmers from the Near East have been intensely debated. Haplogroup R1b1b2 (R-M269) is the commonest European Y-chromosomal lineage, increasing in frequency from east to west, and carried by 110 million European men. Previous studies suggested a Paleolithic origin, but here we show that the geographical distribution of its microsatellite diversity is best explained by spread from a single source in the Near East via Anatolia during the Neolithic. Taken with evidence on the origins of other haplogroups, this indicates that most European Y chromosomes originate in the Neolithic expansion. This reinterpretation makes Europe a prime example of how technological and cultural change is linked with the expansion of a Y-chromosomal lineage, and the contrast of this pattern with that shown by maternally inherited mitochondrial DNA suggests a unique role for males in the transition., Competing Interests: The authors have declared that no competing interests exist.

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.

Background: Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with beta-thalassemia given a bone marrow transplant. We investigated the relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for beta-thalassemia., Design and Methods: Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens., Results: All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again., Conclusions: Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with beta-thalassemia given a cord blood transplant from a relative.

This work is a review of a collaborative exercise on mtDNA analysis undertaken by the Italian working group (Ge.F.I.). A total of 593 samples from 11 forensic genetic laboratories were subjected to hypervariable region (HVS-I/HVS-II) sequence analysis. The raw lane data were sent to MtDNA Population Database (EMPOP) for an independent evaluation. For the inclusion of data for the Italian database, quality assurance procedures were applied to the control region profiles. Only eight laboratories with a final population sample of 395 subjects passed the quality conformance test. Control region haplogroup (hg) assignments were confirmed by restriction fragment length polymorphism (RFLP) typing of the most common European hg-diagnostic sites. A total of 306 unique haplotypes derived from the combined analysis of control and coding region polymorphisms were found; the most common haplotype--CRS, 263, 309.1C, 315.1C/ not7025 AluI--was shared by 20 subjects. The majority of mtDNAs detected in the Italian population fell into the most common west Eurasian hgs: R0a (0.76%), HV (4.81%), H (38.99%), HV0 (3.55%), J (7.85%), T (13.42%), U (11.65%), K (10.13%), I (1.52%), X (2.78%), and W (1.01%).

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1-H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs ( approximately 70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis.

Patients undergoing allogeneic cord blood transplantation (CBT) benefit from a low risk of graft-versus-host disease (GVHD), but there are still concerns that they be able to recover an effective immune capacity early after transplantation. We investigated the ability to develop in vitro T-lymphocyte-mediated immune response toward human cytomegalovirus and Candida albicans antigens, early and late after transplantation, in children given cord blood transplants from either an HLA-identical sibling or an unrelated donor. Proliferative capacity and frequency of antigen-specific T cells were evaluated; antigen-specific CD4(+) T-cell clones were also generated and characterized for T-cell receptor repertoire diversity, cytokine phenotype, and their origin (either from donor or patient). We found that the majority of recipients can develop a specific response to viral or fungal antigens already early after transplantation. Antigen-specific T-cell clones of both donor and recipient origin contributed to the reconstitution of immune response. Antigen-specific T lymphocytes of recipient origin were detected in patients receiving a transplant from a relative, after a chemotherapy-based conditioning regimen, and who did not have GVHD. Our results document, at a clonal level, that after CBT recovery of either polyclonal or pauciclonal T-cell response toward widespread pathogens is prompt, with some patients benefiting from a contribution of recipient-derived cells.

The molecular composition of the genetic substrate recovered from seven aged forensic samples has been extensively investigated. A simple enzymatic test based on DNAseI incubation of the extracts showed that the UV-fluorescent material from the forensic specimens is composed of nucleic acids, with the DNA fraction representing at least 90% of the total amount. Since spectrophotometric determinations of the extracts showed unreliable results due to anomalous OD(260)/OD(280) ratios, quantification of the nuclease-sensitive genetic material was performed by a slightly modified agarose plate method. The first quantitative data on exogenous contamination in aged forensic samples are provided by slot-blot hybridisation of the extracts to human, bacterial and fungal probes. Only limited amounts of human and contaminant DNA were detected in the samples. The molecular integrity of the primary structure of these aged DNA samples was analysed by reversed-phase HPLC/MS. The data show a good correlation between the degree of chemical damage and the ability to hybridise to molecular probes. The ability to achieve specific genetic profiles was assessed by multiplex PCR amplification of STR loci. Our data show that accurate determination of the molecular composition of the DNA recovered from forensic samples can be extremely useful for a reliable evaluation of the PCR typing results.

Five polymerase chain reaction (PCR) products which could not be reliably typed by allele-specific oligonucleotide (ASO) probing at the human leukocyte antigen (HLA) DQA1 locus were analyzed by polyacrylamide gel electrophoresis and direct sequencing. The first method revealed the preferential amplification of only one of the two alleles in two cases. Direct sequencing of PCR products allowed unambiguous genetic typing but a high number of artifacts was observed. Several of these artifacts occurred in the sequences recognized by the ASOs. This finding provides an explanation for the mistyping in the ASO probing procedure because Taq polymerase errors both created new genetic specificities and eliminated site-specific polymorphisms. Reversed-phase HPLC-MS of the five forensic templates showed a high degree of DNA damage. These data together indicate that the risk of mistyping when using the ASO probing procedure cannot be neglected in the forensic analysis of damaged DNA samples.

The Y chromosome STRs DYS437, DYS438 and DYS439 were selected from publicly available genome databases and used to analyse an Italian population sample. A tetraplex PCR reaction including the highly informative DYS385 locus, was set up and used for the analysis of 131 male samples to determine allele frequencies and STR diversity values. The number of different haplotypes and the haplotype diversity value found from the analysis of the STRs included in the tetraplex reaction were very similar to those found from the analysis of the basic set of 7 Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392 and DYS393) previously carried out on the same population sample. By combining the allelic states of the 11 Y-chromosomal STRs we could construct highly informative haplotypes that allowed the discrimination of 93.8% (120 out of 128) of the samples tested. This approach represents a very powerful tool for individual identification and paternity testing in forensic medicine.

Polymerase chain reaction (PCR) direct sequence analysis was performed on aged forensic samples, six or thirteen years old. This method allowed unambiguous genetic typing, but PCR products from such samples showed several artifacts. Control samples generated sequence ambiguities at a frequency of 1 in 567 bases, but the aged samples had an error frequency about 30-fold higher. In order to study the molecular composition of these aged DNA samples, reversed-phase high performance liquid chromatography (HPLC) was performed. Reduced amounts of the four DNA bases were observed and anomalous peaks were found. These peaks were analyzed by ionization mass spectrometry and identified as molecular products of DNA oxidation. The frequency of sequencing artifacts was found to be proportional to the decay of the PCR templates. Although PCR fidelity is a relevant concern in the forensic analysis of damaged samples, our data indicate that the risk of mistyping is circumventable by sequencing both strands and by performing replicate amplifications from the same PCR template.

In order to evaluate the relevance of protooncogene alterations in gastric cancer and to specifically relate these alterations to types and stages of the neoplasia, we studied oncogenes of possible interest in gastric tumors with different clinical parameters. Fifty DNAs from primary gastric adenocarcinoma were analyzed, by the Southern blotting technique, for the presence of amplification or rearrangements of seven different protooncogenes: c-myc, c-erbB2, c-Ki-ras, c-Ha-ras, c-N-ras, hst, and c-mos. All the tumors analyzed were histologically classified and staged. Amplification of the following genes was found: c-myc (2 of 50), hst (3 of 50), c-erbB2 (3 of 50), and c-Ki-ras (5 of 50). The simultaneous amplification of hst (3 cases), c-myc (1 of 3), or c-Ki-ras (2 of 3) was observed. Analysis of DNAs from atrophic and metaplastic gastric mucosa (which can be regarded as preneoplastic lesions) of the 10 patients showing gene amplification demonstrated that this was limited to neoplastic cells. Considering protooncogene amplification in general (i.e., involving different genes and occurring to different degrees) and clinical parameters of tumors, we found a statistically significant association between amplification and both tumor progression and presence of metastases. Therefore, at least for the genes analyzed, amplification is a relatively infrequent phenomenon and represents a late event in the temporal development of gastric cancer.

A sample of about 300 subjects from the Italian population of the Oltrepò Pavese, in Lombardy, was studied for 6 polymorphic genetic markers: ACP1, ADA, ESD, GLO1, PGM1 subtyping and HP. The observed gene frequencies were: ACP1*A = .267, ACP1*B = .697, ACP1*C = .036; ADA*2 = .060; ESD*2 = .119; GLO1*1 = .375; PGM1*1S = .688, PGM1*1F = .095, PGM1*2S = .175, PGM1*2F = .042; HP*1 = .362.