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Sudden cardiac death (SCD) is one of the most common causes of death; most SCD are related to secondary arrhythmias, to structural heart disease, or to primary electrical abnormalities of the heart. A significant number of SCD, especially among young people, are due to genetic heart disorders, both with structural and arrhythmogenic abnormalities. However SCD occurs also in patients with negative clinical history, autopsy is not always conclusive for a diagnosis. Recent technological advances in DNA sequencing, have led to the commercialization of genetic testing now widely available in clinical practice. In particular, next generation sequencing, allows the large-scale and rapid assessment of entire genomes. Analysis of SCD with a NGS panel of 174 genes was performed in our laboratory in order to identify the genetic causes and thus to direct the clinician to an accurate clinical and genetic screening of relatives. Two SDC were studied: Case 1: female, 57, without story of syncope and no previously highlighted cardiac alteration, died post cardiac arrest; negative family history. Autopsy was apparently negative. Case 2: male, 52, who died during a football game; negative family history, neurological episodes occurred before death was reported by close relative. Autopsy was positive for ventricular hypertrophic. In both cases we made a genetic diagnosis.

In June 2006 a decapitated woman was found in a parking area of the motorway in the area of Prato (Florence). Since the body was beheaded and no victim’s documents or objects were present at the crime scene, identification at that time was impossible. However, DNA profile from woman’s bones were collected. In the same year (2003), a mother had reported her daughter's disappearance but the two events were not related at that time. About ten years later the mother’s DNA profile was finally acquired for a genetic identification of another girl’s body found in the Ferrara area. These genetic profiles were completely discordant. All these genetic comparisons were carried out on behalf of the prosecutors of the cities involved in the findings of the bodies and in the disappearance complaints, but due to the lack of a database the events remained disconnected. In January 2017, the head of the scientific police of Prato who had followed the investigation and questioned the mother of the missing girl found about ten years later, suggested to the magistrate to order the comparison of the mother's DNA with the genetic profile of the bones found in 2006. This comparison finally allowed the identification of the missing daughter. This story highlights the importance of having forensic DNA database to search for missing persons and how the investigator's intuition can play a key role in resolving criminal cases. In fact, databases of unknown bodies and relatives of missing persons were created in Italy as a part of national DNA database just at the beginning of 2018.

Hematopoietic stem cell transplantation (HSCT) is the predominant curative treatment for many malignant and non-malignant haematological diseases. Early detection of graft rejection and disease relapse following HSCT improves patient outcomes by allowing treatment to be initiated as quickly as possible. In order to evaluate the level of donor engraftment, mixed chimerism levels must be carefully monitored after transplantation. Short-tandem repeat (STR) genotyping is widely used to determine the proportions of donor and recipient cells after HSCT. In this study, Devyser Chimerism NGS kit in combination with a MiSeq System was introduced in our laboratory for monitoring HSCT. This system is a complete workflow solution for labs, combining a reliable testing process with a designed for-purpose analytical software. Up to 24 informative markers in a recipient/donor pair distributed through the human genome have strong discriminative power with low bias from ethnic parameters. These IND/DEL genetic markers with population independent discriminative power are distributed across 17 chromosomes and were further selected to allow sensitive detection combined with accurate and precise quantification of mixed chimerism. Streamlined, simple and robust NGS workflow uses just one multiplex PCR reaction per patient sample. Minimal hands-on time reduces assay complexity and risk of sample contamination and mix-up. User-friendly, designed-for-purpose software perfectly complements testing kit with an automatic detection of informative markers. Insertion/deletion (indel) polymorphisms have been used in the fields of forensic investigations owing to the advantages of their low mutation rates, widespread distributions in the human genome and small amplicon sizes. Thus, forensic efficiency evaluation of this system for forensic individual identification will be also tested.

In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory. To achieve this goal, the quantification of extracts before typing plays a fundamental role. Especially when touched objects need to be examined tens or hundreds of nanograms may be present, but also very few or no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination. Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Moreover, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also investigated.

Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes.

NGS has the potential to be a promising technology for recovering genetic information from challenging specimens in forensic genetics. In order to understand the role of DNA damage on the outcome of NGS, we investigated the performance of ForenSeq™ DNA Signature kit, Illumina (in its pre-commercial version) on a set of in vitro degraded trial DNA samples. After DNA quantification by qPCR, duplicate analyses of the samples were carried out. The resulting molecular products were then sequenced by using MiSeq ® system (Illumina) and analyzed using ForenSeq™ Universal Analysis Software (Illumina). The coverage and error rate of the NGS data obtained from the degraded samples were compared to the ones gathered from the unmodified DNA. The NGS data showed that the ability of recovering genotypes and the frequency of analytical artifacts are strongly influenced by the degree of damage of the template. NGS was able to call 46–17% of the STR loci and 68–26% of the SNPs in the degraded samples. In addition, when the genotypes from the degraded samples were compared to the ones recovered from the unmodified control DNA, correct typing was achieved from 39 to 4% of the STRs and from 55 to 13% of the SNPs. These data show that NGS is a powerful method for gathering genetic data from samples which failed the conventional approaches, even if in this experiment the risk of mistyping seems not to be negligible (up to 2%).

In three paternity tests performed in our laboratories with a battery of autosomal STRs, we discovered three separate incompatibilities for the loci D3S1358, D8S1179 and D18S51. Since the other employed markers confirmed the paternity of the alleged father, the observed incompatibility for these markers was probably due to mutation events. However, taking into account the mutation value for the markers, the paternity index (PI) and posterior probability (W) results were very low. In these cases, we normally analyse an extra panel of polymorphic markers to increase the PI value. However, in these three cases, since the disputed children were males and the Y-chromosome is transmitted exclusively to the male lineage, we studied a battery of 10 Y-STR in the children and in the alleged fathers. Evaluation of Y-STR haplotype frequencies was deduced from a European available database.

In our laboratory, we performed a study of the D-loop region of mtDNA in unrelated individuals, natives of Tuscany (Central Italy), using an Infrared Automated DNA Sequencer (LI-COR 4200). Taking advantage of the long read characteristics of the IR Automated Sequencer, we adopted a new strategy based on a single fragment amplification and sequencing, including the entire HVI and HVII regions. Preliminary results of the variability in the population we studied were also presented.

The Y-chromosome polymorphisms represent an important tool in forensic genetics for different purposes: paternity testing, special problems of medical genetics, such as incest causing autosomal recessive diseases, analysis of mixed stains and other biological samples in criminal events. The number of Y-chromosome polymorphisms is huge, but only a well-defined number of them are used in official protocols of the international forensic genetics community. Recently, we presented a definitive haplotype of Y-STR in 107 unrelated individuals living in a Tuscany population (Central Italy) [Forensic Sci. Int. 120 (2001) 210]. To study this “minimal haplotype” we used the markers DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS385I/II [Forensic Sci. Int. 114 (2000) 31]. Subsequently, we introduced in our laboratory the use of an automated infrared DNA sequencer (LI-COR 4200). We applied this technology to study the hypervariable locus YCAII [Hum. Mol. Genet. 3 (1994) 115] for the “extended haplotype” and the polymorphism DXYS156 [Int. J. Legal Med. 110 (1997) 125], suggested by Kayser et al. [Hum. Biol. 70 (1998) 979]. We set up a new duplex PCR system for the simultaneous analysis of these loci with the automatic technology. This system was employed to obtain frequency data in the Tuscany population and to solve paternity dispute in which STR mutations were detected. A preliminary sensitivity study was also performed to evaluate the efficiency of this new duplex for stains analysis.

Next generation sequencing (NGS) represents the newest approach for massive DNA sequencing. Although this innovative approach is a well-established point in diagnostic genetics, the applicability in forensic field is still limited to sporadic pilot studies. This limit arises from the samples' complexity, in terms of poor quality/quantity ratio, leading to a difficult setting in analytic systems. In this study, first NGS forensic data with a new analytic protocol are presented; data were obtained with a pre-release version of the MiSeq system (Illumina). The system is able to sequence, at the same time, a wide panel including autosomal 63 STRs and 95 SNPs commonly used in forensic practice. In the first part of the study sensitivity and concordance studies were performed by using DNA controls with known genetic profile; also mixtures with different male/female concentrations were analysed, in order to verify the panel performance in comparison with traditional kits. In the second part of the experiment, 60 casework samples coming from different tissues, biological fluids, traces on supports, lysates, different aged/degraded samples were analysed. Bioinformatic analysis was performed with MiSeq FGx System BETA version (Illumina), an innovative software with an easy interface that permits a rapid data interpretation by filtering the big data quantity generated by the instrument. The comparison between new data and genetic profiles obtained with traditional typing on the same samples, the sensitivity and the potential of NGS approach represents an interesting starting point in introducing this innovative approach on forensic genetics scenario.

Forensic genetics is extremely useful for the resolution of criminal cases, identification of missing persons and paternity/kinship testing. The Italian town of Prato, near Florence, has been a centre of Italian textile industry since the 12th century and now it hosts a number of Chinese-run textile factories producing garments for export across Europe. In December 2013 a fire burned a factory where eleven Chinese workers were employed. The fire was due to a short circuit and it surprises the workers in the makeshift dormitory. After the fire was extinguished, remains of seven people were found by rescuers. The identification was carried out in accordance with Interpol protocols (AM and PM form) and in every case one of the three primer identifiers was satisfied. Relatives of the victims were available for information and biological samples' collection. Here, we show the protocol adopted in the forensic genetics laboratory that included the use of a wide battery of markers STRs, Y-STR and biostatistics evaluation of different assumptions about the identification of these persons.

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.

We extensively employed a real-time quantitative PCR system, together with a commercially available kit (Quantifilerk Human DNA Quantification Kit, Applied Biosystems), for the quantification of human DNA in a large variety of samples. The results we obtained were reliable, with a low deviation standard for the same sample; however, when the inhibitors in a sample were in high concentration and/or DNA degradation was present (like in postmortem matrices), we observed variable results of quantification in the same extract. Here, we report our experience using this real-time based method for quantification and typing of samples recovered in forensic casework. D 2006 Elsevier B.V. All rights reserved.

Laboratory DNA contamination is a frequent problem in forensic genetics, especially when extra PCR cycles are used to obtain acceptable amplified products from low copy number DNA samples. We have evaluated the use of the DNase I for forensic genetics studies. Initially we introduced known genomic DNA in the PCR reaction mix in order to simulate DNA contamination. Different conditions of action an inactivation of the DNase I were tested. Finally, we performed amplifications of low copy number samples employing homemade and commercially available STR amplification systems; in both cases decontamination was successful. We demonstrate that DNase I may be useful to reduce laboratory contamination in forensic genetics labs. Our protocol may be easily introduced in standard PCR protocols of commercial kits in order to be used routinely in low copy number DNA amplification.

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.

Two different automated procedures (capillary electrophoresis and infrared automated sequencer) for STR analysis on DNA obtained from highly degraded teeth were compared. Even with these two automatic protocols, negative or not much informative results were obtained. Our results confirm that in extreme conditions, it is necessary to perform more sensitive analysis (i.e. SNPs, mt-DNA) to obtain informative genetic profiles.

Here we show two new amplification systems, which were realised for the amplification of 80 highly polymorphic loci, in combination with an Infrared Automated DNA Sequencer LI-COR 4200 (LI-COR, Nebraska, USA). The two amplification systems were indicated as MU5 and MU6. The amplification system MU5 contained D12S391, F13A01, SE33 and Penta D loci, the amplification system MU6 the loci D2S1338, D19S433, F13B and Penta E. Both systems were performed using forward IRDye™800 labelled and reverse unlabelled primers, in combination with the QIAGEN Multiplex PCR kit (Qiagen, Germany). The extension of the set of STRs in our laboratory is useful to further improve our ability to resolve complex kinship cases (e.g. deficiency cases, incest, paternity tests with mutations). Moreover, these new multiplex systems can also be used for manual typing, because the STRs do not overlap in size.

The principle purpose of this study was to investigate the frequency distribution of the short tandem repeat system FXIIIB in a sample of individuals from Tuscany (Central Italy). Samples (204), taken from donors residing in the area of Florence, Prato and Pistoia, were analysed by PCR and vertical denaturing polyacrylamide gel electrophoresis. A sensitivity study was also undertaken and the method was optimised for the study of forensic samples. The research resulted in the identification of 5 alleles and 11 genotypes. The system was tested for Hardy–Weinberg equilibrium and the Central Italian population was compared with populations from Northern Italy and Austria. Moreover, we introduced the use of this microsatellite in an automatic platform, in combination with other STRs in a new multiplex amplification system. An infrared automatic DNA sequencer was used to analyse this marker. The introduction of this marker in our protocol could be useful to resolve particular intriguing cases of forensic interest, as for example incest or deficitary paternity/maternity tests, in which additional loci must be investigated.

Haplotype data of 1176 Italian males from 10 regions were obtained as a part of a collaborative validation exercise. Individual data are available at http://www.gefi-forensicDNA.it .

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.

Here we show the Y-haplotype database consisting in the loci DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, YCAII and DXYS156Y of 107 males living in Toscany (central Italy).

Hereby we show a paternity test in which a deceased alleged father was suspected to be the biological father of a child affected by Down syndrome. The alleged father was died but the mother and two half-brothers of the child were available for DNA testing. A panel of 37 autosomal STRs markers and 16 Y-STRs including commercial and manual systems were used for this test. STR typing and biostatistical evaluation with Familias ® program confirmed the paternity; at D21S11 locus the child showed three different alleles but only one was identical with the mother. In order to further investigate the parental origin and the meiotic/mitotic origin of the extra chromosome 21, a panel of seven STR markers mapped on the chromosome 21 normally used for prenatal diagnostics were also performed on the mother, half-brothers and on the child. The used STR markers mapped along the whole chromosome from position 21q21.3 to 21q21.1. The results showed that the observed inconsistency was due to a maternal mutation. This study clearly shows that there was a single step mutation at D21S11 locus in the maternal germ cell, compatible with the widely reported in the literature for the origin of trisomy 21.

Although in Italy DNA analysis is used since the beginnings, the introduction of a specific legislation had a very important impact for the quality of the results. Only authorized laboratories can now implement the database with genetic profiles; these labs have to follow ISO/IEC standard and according to the Council Framework Decision 2009/905/JHA of 30 November 2009 and the Recommendation 7 of ENFSI this standard is the ISO17025/2005 accreditation. Since December 22th 2009, ACCREDIA is the only appointed Italian Accreditation authority can issue the accreditation certificate. There is no rule to define the ISO test method for the study of DNA polymorphisms in forensics, thus each laboratory should build their own analytical system, reliable and reproducible, through its own internal validation. Here, we focus on some points of the accreditation process developed in our laboratory concerning internal validation protocol.

The use of semi-automated extraction from forensic samples is useful both for contamination control and method reproducibility. However, when an accredited ISO17025 forensic laboratory starts to use a new analytical system, an internal validation procedure is needed before processing forensic samples. Here we show validation results obtained by introducing QIAcube automated station (Qiagen) for DNA extraction from teeth that enables seamless integration of automated low-throughput sample preparation into the laboratory workflow. Purification steps, in association with QIAamp DNA Investigator Kit, are thus fully automated, in order to prevent cross contamination. A total of 18 teeth stored for 4–5 years after removal and 8 teeth stored for one-two months at room temperature underwent to DNA extraction by using this system. Teeth were previously decontaminated and a conservative endodontic access to pulp chamber was performed. A blood reference of the teeth donors was also collected. After DNA extraction all samples were quantified by using Quantifiler ® Duo DNA Quantification Kit. Total DNA ranging between 0.2 and 23,900ng in 50μl of TE buffer with no inhibitors presence in samples. The obtained results confirm the robustness and reproducibility of the extraction method with QIAcube technology.

We report a judicial paternity testing with two exclusions at D2S1338 and vWA loci. Since these results suggested that the true father should be a close male relative of the tested man, mother was included into analysis. Subjects were also typed for 24 validated STRs, 11 STRs for linkage analysis, 8 X-STRs and 30 DIPs, for a total of 87 markers. No further exclusions were found. Paternity index, taking into account mutation rates for D2S1338 and vWA, was 1.45×10 13 ( W =0.99999999999993). The final odds that the true father should be the untyped brother of the alleged father, that refused DNA profiling, was 1:192.

Y-STR analysis is widely used in many fields, such as paternity testing, genealogy studies and in male/female mixtures. In many rape cases, Y-STRs are also useful for the determination of contributors' number. Here we described a father/son pair with double peaks at DYS439 and DYS635 loci. This case should focus the attention on forensic interpretation of Y-haplotype profiles, because multiple alleles at various loci do not forcibly indicate that the sample originates from a mixture. We also report a case of two half-brothers with null allele at DYS448. Since DYS439 and DYS635 loci are located in the AZFa region and DYS448 locus in the AZFc region, we performed a molecular genetics study of these regions to evaluate a possible correlation between Y-STR profiles and Y chromosome deletions involved in infertility.

Allele frequencies for 17 STRs, together with some parameters of forensic interest, were estimated in a sample of 835 unrelated individuals born in Tuscany, an Italian region. These data were compared with Italian, Chinese, Kosovo Albanian, Romanian and Tunisian populations, strongly represented in this area. No significant differences in single loci were detected, except for Chinese in comparison with all the other populations.

Human Monozygotic (MZ) Twins estimated to occur once in 250 live births, result from an abnormal behavior by embryonic cell(s) to develop as separate embryos. The MZ twins are considered genetically identical and the reduced phenotypic, genetic or chromosomal concordance represents a very important challenge for geneticists. Determination of zygosity by molecular studies usefully provides a superior replacement for the conventional studies of placenta and chorionic membranes. We utilized a protocol based on the CODIS polymorphic markers battery, by using an infrared LI-COR 4200 instrument (LICOR, Nebraska, USA), and we associated the statistical analysis method with formulae derived by the DNA-View's Kinship Module to better evaluate the genetic identity in a group of MZ twins.