Age estimation of burnt human remains through DNA methylation analysis.

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.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

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 Na2EDTA 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.

The role of single nucleotide polymorphisms related to iron homeostasis in mesothelioma susceptibility after asbestos exposure: a genetic study on autoptic samples.

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.

An mRNA Profiling Study of Vaginal Swabs from Pre- and Postmenopausal Women.

Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6-20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay's success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.

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.

Dead migrants in the Mediterranean: genetic analysis of bone samples exposed to seawater.

In April 2015, a fishing boat that departed from Libya with about 1,000 migrants on board sank in the Mediterranean Sea. Most of the migrants were packed in the hull of the boat and drowned in the shipwreck. After fifteen months, the ship was recovered from the seabed and brought to a Sicilian naval area for forensic investigations. Skeletal remains belonging to more than 700 people were retrieved. A selected sample composed of 80 victims was considered in order to evaluate the possibility of achieving genetic profiles useful for a positive identification from these challenging specimens. The molecular features of the DNA recovered from a significant number of real casework samples exposed to seawater for long periods of time were described for the first time. Three different DNA extraction protocols and three different commercial kits were employed in order to generate genetic profiles based on the characterization of 21 autosomal STR loci. The combination of multiple DNA extractions and the cross-checking of multiple PCR amplifications with different kits allowed to obtain reliable genetic profiles characterized by at least 16 STR markers in more than 70% of the samples. The factors that could have affected the different quality of the genetic profiles were investigated and the bone preservation was examined through microscopic and macroscopic analyses. The approach presented in this study could be useful in the management of the genetic analysis of bone samples collected in other similar DVI scenarios. The genetic profiles recovered from the bone samples will be compared in kinship analysis to putative relatives of the victims collected in Africa in order to obtain positive identifications.

Genetic individual identification from dried urine spots: A complementary tool to drug monitoring and anti-doping testing.

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.

Analysis of Cannabinoids and Metabolites in Dried Urine Spots (DUS).

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.

A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples.

Purpose: Whatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results. Methods: Twenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography-mass spectrometry methods. Results: We were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent. Conclusions: This study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood. Supplementary Information: The online version contains supplementary material available at 10.1007/s11419-020-00567-2.

Assessment of the Precision ID Identity Panel kit on challenging forensic samples.

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.

Highly degraded RNA can still provide molecular information: An in vitro approach.

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.

Disaster victim identification by kinship analysis: the Lampedusa October 3rd, 2013 shipwreck.

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.

Prolonged DNA hydrolysis in water: A study on DNA stability.

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.

XY (SRY-positive) Ovarian Disorder of Sex Development in Cattle.

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.

Producing standard damaged DNA samples by heating: pitfalls and suggestions.

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.

Challenges in the identification of dead migrants in the Mediterranean: The case study of the Lampedusa shipwreck of October 3rd 2013.

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.

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples.

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.

Efficacy and safety of thalidomide for the treatment of severe recurrent epistaxis in hereditary haemorrhagic telangiectasia: results of a non-randomised, single-centre, phase 2 study.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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 (p<0·0001 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. INTERPRETATION: 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. FUNDING: Telethon Foundation.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

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.

Assessment of DNA damage by micellar electrokinetic chromatography.

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.