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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.

PurposeWhatman™ 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.MethodsTwenty 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.ResultsWe 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.ConclusionsThis 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.

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.

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.

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 (r2 > 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.

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.

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 ForenSeqTM 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 ForenSeqTM 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.

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

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

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.

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.

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.

This work provides a protocol for the in vitro production of damaged DNA samples. In particular, heat-mediated hydrolysis of the samples at 70 °C in ultrapure water was performed in 1.7 mL Eppendorf tubes sealed by Parafilm for 0–36 h. The chemical/physical features of the resulting samples are described. After normalization of the qPCR data, these were compared with those obtained from samples treated for 0–10 h in a previous study. Keywords: qPCR, Degraded samples, DNA quantification, DNA hydrolysis

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 CO2, ROS, NOx 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.

Hereditary haemorrhagic telangiectasia is a genetic disease that leads to multiregional angiodysplasia. Severe recurrent epistaxis is the most common presentation, frequently leading to severe anaemia. Several therapeutic approaches have been investigated, but they are mostly palliative and have had variable results. We aimed to assess the efficacy of thalidomide for the reduction of epistaxis in patients with hereditary haemorrhagic telangiectasia that is refractory to standard therapy.We recruited patients aged 17 years or older with hereditary haemorrhagic telangiectasia who had severe recurrent epistaxis refractory to minimally invasive surgical procedures into an open-label, phase 2, non-randomised, single-centre study at IRCCS Policlinico San Matteo Foundation (Pavia, Italy). We gave patients thalidomide at a starting dose of 50 mg/day orally. If they had no response, we increased the thalidomide dose by 50 mg/day increments every 4 weeks, until a response was seen, up to a maximum dose of 200 mg/day. After patients had achieved a response, they continued treatment for 8-16 additional weeks. The primary endpoint was the efficacy of thalidomide measured as the percentage of patients who had reductions of at least one grade in the frequency, intensity, or duration of epistaxis. We followed up patients each month to assess epistaxis severity score and transfusion need, and any adverse events were reported. We included all patients who received any study drug and who participated in at least one post-baseline assessment in the primary efficacy population. The safety population consisted of all patients who received any dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT01485224.Between Dec 1, 2011, and May 12, 2014, we enrolled 31 patients. Median follow-up was 15·9 months (IQR 10·1-22·3). Three (10%, 95% CI 2-26) patients had a complete response, with bleeding stopped, 28 (90%, 95% CI 74-98) patients had partial responses. Overall, all 31 (100%, 89-100) patients responded to therapy with a significant decrease in all epistaxis parameters (p0·0001 for frequency, intensity, and duration). A response was achieved by 25 (81%) patients at 50 mg/day of thalidomide, five (16%) patients at 100 mg/day, and one (3%) patient at 150 mg/day. Patients had only non-serious, grade 1 adverse effects, the most common of which were constipation (21 patients), drowsiness (six patients), and peripheral oedema (eight patients). One patient died a month after the end of treatment, but this was not deemed to be related to treatment.Low-dose thalidomide seems to be safe and effective for the reduction of epistaxis in patients with hereditary haemorrhagic telangiectasia. Our findings should be validated by further studies with larger patient populations, longer follow-up, and that also assess the benefit for quality of life.Telethon Foundation.

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

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.

In the last years, the migration flow across the Mediterranean Sea increased rapidly. More than 20,000 persons, included young men, women and children, died trying to reach the European coasts, 60% of which remained with no identity. In this scenario, forensic genetics can give a support to the positive identification of the victims. To this aim, the DNA extracted from the victims is analysed and compared to reference samples supplied by relatives, in order to find out familiar relationships. In collaboration with LABANOF, University of Milan, our laboratory is trying to set up a genetic and statistic approach suitable for victim identification of the migrants died during the shipwreck occurred near the Lampedusa island, in October 3rd, 2013. The genetic profiles of the 363 victims, collected by the Forensic Genetics Lab of the Scientific Police Service (Palermo, Italy), were initially compared among them and subsequently between victims and subsequently with the reference samples, using the software Familias3, setting up the DVI interface. The statistical approach identified four familiar groups among the victims, generally composed by a parent with two or more children, and five parent-child relationships. In addition, 20 matches were detected between the subjects seeking a relative and 17 victims, with a positive statistical support. Some critical points emerged during the analyses among which the most important were the number of relatives and genetic markers needed to reach a statistical support to identification, the lack of genetic information about the populations to which the victims belonged and the option to use lineage markers such as Y-chromosome and mtDNA polymorphisms. In conclusion, these results support the reliability of the approach and highlight the importance to collaborate with other forensic experts in order to achieve positive identifications of the bodies.

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.

Purpose: Familial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases. Methods: We studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3–100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls. Results: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency. Conclusion: Attenuated polyposis patients without “conventional” APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.

A set of 16 amplifications following a D-optimal experimental design was planned using the most commonly selected kit and sequencer utilized in a collaborative exercise of the ISFG Italian Working Group GeFI on the molecular characterization of a depurinated DNA sample. The data were evaluated using STRvalidator in order to describe the occurrence of PCR artifacts such as peak imbalance and allele/locus drop outs. The aim of the study was to describe the role of the template amount, number of amplification cycles, volume of the PCR reaction in determining the assay sensitivity and the profiling accuracy as measured by peak heights and areas. The results showed that only by selecting an appropriate ratio of template to PCR reaction solution total volume together with an increase in the number of cycles it could be possible to obtain balanced peaks as well as minimal occurrence of other artifacts. These data are useful to model and understand the PCR artifacts occurrence described in the collaborative exercise.

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.

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.

A single DNA sample underwent controlled damaging treatments represented by UV radiations, free radicals, formaldehyde, reactive oxygen species (ROS), metal ions radical starters such as Fe3+ and Cu2+, HCl, and NaOH. Capillary electrophoresis showed that most of the treatments produced chemical alteration of the primary structure of the DNAs. In addition, both Alu probing and real-time PCR provided data lower than the controls. Both STR typing and Y-specific SNPs analysis were influenced by the altered composition of the templates. The results of this study indicate that more information on the molecular composition of the template can be extremely useful for the evaluation of the PCR-based evidences. © 2006.

A population sample from North-Central Italy was analysed to investigate the frequency distribution of subclades of mtDNA haplogroup H. A specific SNaPshot assay was set up to detect diagnostic mutations identifying subhaplogroups from H1 to H7. Haplogroup H subtyping can be useful to discriminate among individuals sharing common mtDNA HVS I/II sequences.

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 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.

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.

Population genetic data for five X-chromosomal STR loci (DXS7423, DXS6789, DXS6795, DXS9898 and DXS8377) were generated by analysing a population sample from Northwest Italy. Intensive stutter bands were observed for the DXS8377 locus. The analysis of the 40 family trios segregation showed no new mutation.

Capillary electrophoresis was used to analyse aged forensic DNA samples. A good correlation between DNA damage and reliability of PCR typing results was found.

The incidence of genetic instability affecting a set of STRs commonly used in forensic DNA analyses was assessed by performing a comparative study on 24 lung carcinomas with paired normal tissue samples. Out of 24 samples, 20 (83%) showed allele drop-out (ADO) in at least one STR locus. Allelic imbalance was detected at all the STR loci analysed. A small-cell carcinoma sample showed loss of heterozygosity (LOH), with complete deletion of one allele, at the D5S818 and D13S317 loci.

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 (RSD10%) 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.

Eight Y-chromosomal short tandem repeats (STRs), DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393 and DYS385, were typed in a population sample (n=101) of first-generation Albanian immigrants living in Italy.

A complete genetic profile of a mummified male body that had emerged from the melting ice of an Alpine Glacier was achieved. The body could be dated back to the late 1920s, due to the finding of newspaper cuttings in an internal pocket of the clothes. DNA was recovered from still well preserved internal organs and nail clippings. Autosomal STRs, Y chromosomal STRs and SNPs and mtDNA HV1 region were determined in order to provide a complete genetic survey of the mummified body. In a future perspective, after having collected general information on the possible identity of the body, all these genetic data, and especially the ones from the lineage markers, could be used to get a positive identification.

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.

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.

A mini-primer set to amplify the mtDNA control region was developed and tested on fourteen long compact bone samples (femur and humerus samples) recovered from a World War II mass grave. This approach gave successful PCR amplifications for all but one of the bone samples. The following sequencing analysis identified seven different mtDNA haplotypes, three of which shared by more than one bone sample. These haplotypes were then compared to living relatives of missing persons disappeared in that area at the end of the WWII (1945).

A sample of 1070 individuals was collected by 12 collaborating forensic laboratories from Central and Northern Italy, and typed by the so-called "NC01" triplex, including the three loci D10S1248, D14S1434, and D22S1045. Heterozygosities were 0.771, 0.717, and 0.720, respectively.

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

Allele and genotype frequencies for STR SE33 were obtained for a sample of 419 Italians in view of application in personal identification and paternity. D 2003 Elsevier Science B.V. All rights reserved.

A capillary electrophoresis (CE) method was used to study the DNA primary structure of artificially degraded DNA samples and aged/forensic specimens. CE investigated the ratio between the pyrimidinic and the purinic canonical moieties still bound on the DNA backbone and identified the presence of additional compound (i.e. un-canonical DNA bases). The DNA samples were then analysed by using molecular assays such as agarose gel, UV spectrophotometry and qPCR. All these data have been concurrently considered for a critical evaluation of the outcome of the STR typing.

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.

Allele frequencies for the miniSTR loci D10S1248, D14S1434, D22S1045 ( NC01 ) and D1S1677, D2S441, D4S2364 ( NC02 ) were determined in a population sample from North-Western Italy. No significant deviations from Hardy–Weinberg equilibrium expectations were detected. The forensic usefulness of the selected miniSTRs was confirmed typing different aged samples and analysing 20 family trios with paternity confirmed with CODIS autosomic STRs.

Allogeneic cord blood transplantation (CBT) is increasingly used in children with beta-thalassemia major. Donor/recipient mixed chimerism (MC) is frequently observed in patients with beta-thalassemia major given bone marrow transplantation (BMT) in the early post-transplant period, due to the persistence of haematopoietic and immune cells of host origin. Previous clinical data reported on these patients demonstrated that the majority of them exhibiting MC convert to full donor chimerism (FDC), some maintain MC stable over time, while a non negligible number of patients develop graft failure, in particular when increasing proportions of cells of host origin are observed in serial monitoring. Aims of the present study were to retrospectively investigate the proportion of children with beta-thalassemia major experiencing either transient, or long-lasting persistence of hematopoietic cells of host origin after CBT, as compared to those given BMT, and to analyze the relationship existing between the evolution of MC over time and clinical outcome. One hundred and six consecutive patients, 27 given CBT from an HLA-identical related donor (RD) and 79 given BMT (42 from a RD and 37 from an HLA-compatible unrelated volunteer, UD), were enrolled in the study. Donor/recipient chimerism status was evaluated by PCR-based assay, analyzing selected polymorphic short tandem repeat (STR) loci. CBT and BMT recipients received similar preparative regimens. Anti-thymocyte globulin (ATG 10 mg/Kg on days −4, −3 and −2; Fresenius, Graefelfing, Germany) was used in all patients given an UD-BMT. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (Cs-A) alone in CBT recipients; either CsA alone (12 RD-BMT recipients) or CsA in combination with methotrexate (MTX) was given to BMT patients. All patients given CBT engrafted and displayed MC early after transplantation, 13/27 converted to FDC (6 within 1 month and 7 within 1 year after CBT), 14/27 maintained stable MC, and all of them are alive, transfusionin-dependent and free of any immune-suppressive drug, the follow-up ranging between 15 and 89 months (median value 42 months). In the group of children given BMT, 24/79 (12 transplanted from UD and 12 from RD) exhibited FDC at all time points examined, 4/79 (1 transplanted from UD and 3 from RD) did not engraft and 51/79 (23 from UD and 28 from RD) displayed MC at time of haematological reconstitution. Forty out of these 51 BMT recipients with MC status converted to FDC (17 from UD and 23 from RD; 29 within 1 month and 11 within 1 year after BMT), 3/51 maintained stable MC (1 undergoing UD- and 2 RD-BMT), while 8/51 (5 from UD and 3 from RD) progressively lost the graft, returning to be transfusion-dependent. The median follow-up time was 51 months (range: 15–110 months) and 48 months (range: 16–81 months) in the RD-BMT and UD-BMT group, respectively. None of the patients given CBT experienced either grade II-IV acute GVHD, which was, by contrast, diagnosed in 3 and 7 patients given RD- and UD-BMT, respectively. Chronic GVHD was diagnosed in 8 children (2 from RD- and 6 from UDBMT). Five out of 106 pediatric patients enrolled in the study, 2 included in the RD-BMT and 3 in the UD-BMT group, died of transplantation-related complications. These results indicate that MC is frequently observed in children with beta-thalassemia after RD-CBT, without, however, predicting an increased risk of treatment failure. CBT from an HLA-identical related donor is a successful option in beta-thalassemia major, possibly also due to the establishment of an effective state of immune tolerance between host and donor that decreases the risk of both graft rejection and GVHD.