The Baron Pasquale Revoltella's Will in the Forensic Genetics Era

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 × 106 (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

Italian mitochondrial DNA database: results of a collaborative exercise and proficiency testing

Abstract 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/¬7025 AluI-was shared by 20 subjects. The majority of mtDNAs detected in the Italian population fell into the most common wes

T lymphocytes of recipient origin may contribute to the recovery of specific immune response toward viruses and fungi in children undergoing cord blood transplantation

AbstractPatients 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. (Blood. 2004;103:4322-4329

Donor/recipient mixed chimerism does not predict graft failure in children with β-thalassemia given an allogeneic cord blood transplant from an HLA-identical sibling

Background Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with β-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 β-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 β-thalassemia given a cord blood transplant from a relative

GENETIC INDIVIDUAL IDENTIFICATION FROM DRIED URINE SPOTS (DUS): A COMPLEMENTARY TOOL TO DRUG MONITORING AND ANTI DOPING TESTING

8: 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.nonemixedGrignani, Pierangela; Manfredi, Alessandro; Monti, Maria Cristina; Moretti, Matteo; Morini, Luca; Visonà, Silvia Damiana; Fattorini, Paolo; Previderè, CarloGrignani, Pierangela; Manfredi, Alessandro; Monti, Maria Cristina; Moretti, Matteo; Morini, Luca; Visonà, Silvia Damiana; Fattorini, Paolo; Previderè, Carl

SNP analysis of challenging bone DNA samples using the HID‑Ion AmpliSeq™ Identity Panel

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 reliableoppositely, 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

Isometric artifacts from polymerase chain reaction-massively parallel sequencing analysis of short tandem repeat loci: an emerging issue from a new technology?

5siThe 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 GlobalFilerTM 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, etc.) 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 a 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 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. This article is protected by copyright. All rights reserved.noneopenPajnič, Irena Zupanič; Previderè, Carlo; Zupanc, Tomaž; Zanon, Martina; Fattorini, PaoloPajnič, Irena Zupanič; Previderè, Carlo; Zupanc, Tomaž; Zanon, Martina; Fattorini, Paol