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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.

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.

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.

In a standard paternity testing, mother, child, and alleged father are analyzed with STR markers using commercially available kits. Since Italian civil legislation does not have thresholds to confirm a paternity, paternity is practically proven when likelihood ratio increases prior probability of paternity to posterior, accepted by court as sufficient. However, in some cases the number of markers included in a commercial kit may be insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. Additional genetic information can increase the values of the likelihood ratio regarding the detection of true parental relationships in a pedigree, while reducing the chances of false attributions (e.g. false paternities). In these cases the introduction of a 26Plex amplification system allows to examine 23-26 additional markers depending on the commercial kit used, thus increasing the statistical power of the kinship analysis. The PCR conditions were optimized for a multiplex amplification system and a new generation CE instrument. In order to demonstrate the utility of additional STRs markers, four complex kinship cases are presented.

Forensic genetics is extremely useful for the resolution of criminal cases, identification of missing persons and in paternity/kinship testing. Each and every laboratory that works in the forensic genetics area has developed its own working method independently, however, generally in accordance with international guidelines. More than thirty institutional, public and private forensic laboratories that deal with the identification/paternity testing through DNA in Italy have been surveyed, but to this day, only five public laboratories (four of the police and one of a university hospital) and two private ones are accredited. There are, however, many other laboratories that perform occasional forensic genetics activities that have not been surveyed. The need to achieve the ISO 17025:2005 accreditation may represent for these laboratories an excellent opportunity to improve their activities. Although the DNA analysis for forensic investigation is used in Italy since the beginning of the technique, the quality of the results has been called into question more than once, as it appears by many court cases in which the results of genetic tests have been subject to strong criticisms. Obviously, the ISO 17025:2005 is not sufficient to guarantee the quality of the results. It is essential to show the laboratory working method to the scientific community in order to obtain reliable and robust analytical results that can be used in court to accuse/exonerate individuals accused of a crime or to assign a true biological father to a child. Here, we show a part of the workflow validation process of the internal method used in the Forensic Genetic Service (FGS) of the Diagnostic Genetics Unit (DG) of the Careggi University Hospital. This article outlines some relevant aspects of the methods adopted to ensure robustness, reliability and reproducibility of genetic profiles used for forensic identification.

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%).

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

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.

Allele frequencies for the 13 CODIS markers plus D2S1358 and D19S433 loci were estimated in a sample of 148 unrelated individuals from Bolivia and parameters of forensic interest were calculated. Further, the STR data were analyzed using a distance-based method to assess the genetic relationships of this population with other ones living in Argentina, Brazil, Costa Rica, Mexico, Peru, Venezuela and three autochthonous populations living in the Beni Department of Bolivia (Quechua, Aymara and Beni population).

In a standard paternity testing, mother, child, and alleged father are analyzed with STR markers using commercially available kits. Since Italian civil legislation does not have thresholds to confirm a paternity, paternity is practically proven when likelihood ratio increases prior probability of paternity to posterior, accepted by court as sufficient. However, in some cases the number of markers included in a commercial kit may be insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. Additional genetic information can increase the values of the likelihood ratio regarding the detection of true parental relationships in a pedigree, while reducing the chances of false attributions (e.g. false paternities). In these cases the introduction of a 26Plex amplification system allows to examine 23-26 additional markers depending on the commercial kit used, thus increasing the statistical power of the kinship analysis. The PCR conditions were optimized for a multiplex amplification system and a new generation CE instrument. In order to demonstrate the utility of additional STRs markers, four complex kinship cases are presented.

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDyeTM800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.

Early menopause in the fragile X carriers has been well documented in several reports. All surveys demonstrated that 13-25% of fragile X carriers experienced premature ovarian failure (POF), defined as menopause before the age of 40 years. In 1995 we started screening two groups of subjects as a part of a Fragile X Research Program: 1) women previously diagnosed as fragile X carriers from the register of our center and 2) women with POF and without a family history of fragile X or other forms of mental retardation. In this study we report the preliminary data collected from 75 fragile X families; in 30 of them, POF was present in one or several subjects, all of whom had a fragile X premutation. None of the women with a full mutation experienced POF in our series of patients. We also identified 89 families without a family history of fragile X or mental retardation, and there were 108 subjects who experienced POF, of which 6.5% had a fragile X premutation. This is 70-fold higher than the background prevalence of fragile X premutation in the Italian population and suggests an association with POF. These data confirm the results of other surveys.

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.

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.

The unequivocal tissue identification in forensic casework samples is a key step for crime scene reconstruction. Just knowing the origin of a fluid can sometimes be enough to either prove or disprove a fact in court. Despite the importance of this test, very few data are available in literature concerning human saliva identification in old forensic caseworks. In this work the stability of human α-amylase activity in aged samples is described by using three different methods integrated with DNA profiling techniques. This analytical protocol was successfully applied on 26-years old samples coming from anonymous threat letters sent to prosecutors who were working on "the Monster of Florence", a case of serial murders happened around Florence (Italy) between 1968 and 1985.

Fragile-X syndrome, which derives its name from the expression of a fragile site (FRAXA) at Xq27.3 associated with the phenotype, has achieved distinction as the most common inherited cause of mental retardation. It is the first disorder shown to be due to dynamic mutation in heritable instable DNA.In 1991 the mutation responsible for Fragile-X syndrome was delineated as an expansion of the trinucleotide (CGG) sequence within an evolutionarily conserved gene, at the position of the fragile-X site.The DNA of the promoter in the 5' UTR region of FMR-1 gene becomes abnormally methylated when the CGG sequence exceeds approximately 230 repeats, resulting in the transcriptional suppression of FMR-1. Based on the length of CGG repeat in the FMR-1 gene, the alleles are usually classified as normal, premutation or full mutation. CGG instability correlates with the length of repeats and number of AGGs within the FMR-1 CGG tract. In a minority of cases the Fragile-X syndrome may be due to deletion, or to point mutation in the FMR-1 gene.

Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are well established as models of Genomic Imprinting in humans, since completely different phenotypes are generated by the absence of paternal (PWS) or maternal (AS) contribution to the q11-13 region of chromosome 15 as a result of deletion or uniparental disomy. We report a preliminary study based on our experience of more than 20 years of research into the genetics of PWS and AS syndromes.Thirty nine subjects, referred from a number of Centers and Medical Doctors have been examined to either confirm or rule out a diagnosis of PWS or AS.Patients were evaluated through the Clinical Genetics and Dysmorphology Program at the Human Genetics Center, Dept. of Paediatrics, University of Florence.Clinical evaluation showed that 10 of these patients fulfilled diagnostic criteria for PWS and 8 for AS.All patients were isolated cases and the 18 nuclear families were unrelated.We adopted the staged diagnostic approach for all our families diagnosed PWS or AS families, moleucular using cytogenetic, genetic and molecular cytogenetic techniques.

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 .

In a case of robbery in which the criminals passed through the garden adorned with calamondin trees (Citrus madurensis), the investigators found in the grass six calamondin fruits, some undamaged, while others apparently bitten. The fruits were collected and sent to the laboratory for DNA analysis to verify the presence of saliva and robbers' DNA profile. A specific immunochromatographic strip test for saliva confirmed the presence of human salivary α-amylase, but similar positive results were also observed for intact calamondin and other citrus fruits. Further analysis with a specific automated amylase test confirmed the absence of amylase activity. DNA quantification and typing using a specific forensic kit revealed no human DNA presence in any fruits. This case report demonstrates for the first time the occurrence of false positives when human saliva is sought on citrus fruits.

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.

We report the case of four women and a man, all born in an Italian village during and immediately after WWII, that recently contacted our laboratory in order to perform kinship analysis. According to their claim, the propositi were the illegitimate offspring of a country gentleman and a peasant woman, given in adoption immediately after birth. A story that curiously reminded us of Jean Jacques Rousseau, Therese Levasseur and their five children. Problems connected with DNA analysis in cases where all stated relationship are questioned, and a wide range of different pedigrees could be used as hypotheses in LR calculations are discussed.

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).

Microsatellite instability (MSI) is present in hereditary conditions due to mismatch repair (MMR) gene mutations. Following MSI analysis, tumor samples are classified into MSS (stable), MSI-L (low instability), and MSI-H (high instability) based on the fraction of unstable loci. Another MSI-based classification takes into account the size difference between mutant alleles in tumor DNA compared to wild-type alleles; two types of MSI, A and B, are recognized using this approach, type A being characterized by smaller, more subtle allelic shifts compared to type B. Biallelic mutations of MMR genes are associated with pediatric cancers, including glial tumors, in Turcot syndrome type 1 (TS1). However, most TS1-associated gliomas so far analyzed did not display MSI. We investigated the frequency of MSI in a series of 34 pediatric gliomas of different grade using a panel of five mononucleotide quasimonomorphic markers. Subtle qualitative changes were observed for the majority of markers in two glioblastomas (5.9% of the total series and 33.3% of glioblastomas). In both cases, family histories were compatible with TS1, and mutations of the PMS2 and MLH1 genes were identified. In one family, the MSI patterns were compared between the glioblastoma and a colon cancer from an affected relative, showing a clear qualitative difference, with the former displaying type A and the latter type B instability, respectively. These results were confirmed using additional microsatellite markers, indicating that knowledge of the association between TS1-related glial tumors and subtle type A MSI is important for full ascertainment of TS1 patients and appropriate counselling.

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.

Within an Italian collaborative exercise on the extended haplotype of the Y-chromosome, 1288 subjects were typed by the AmpFlSTR YFiler Amplification Kit (AB Applied Biosystems) and other 526 were typed by the PowerPlex Y ® System (Promega). The sampling scheme included either a "regional" or a "local" recruitment, the first referring to individuals born in the region of the participating lab, the second referring to individuals coming from small villages. Total sample sizes were N =954 and 860, respectively. A significant decrease of haplotype diversity was found in the local samples. The results may be of interest in forensic applications of the Y-chromosome.

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.

We report an unusual paternity test case showing multiple peculiarities. Using AmpFlSTR® Profiler Plus and AmpFlSTR® Identifiler PCR Amplification kits, the alleged father and the two children were apparently homozygous at the FGA locus, but using the PowerPlex® 16 kit the three individuals were found to be heterozygous. Drop-out was caused by a single mutation event in the presumptive binding site of the reverse primer. In addition, three inconsistencies were detected between the daughter and the alleged father among 18 STR markers. The occurrence of the rare null allele at the FGA locus and case history suggested that the true father was the brother of the alleged father. Furthermore, a single-step repeat maternal mutation was also detected at D16S539. This puzzling case was solved by using multiple analytical approaches, including the use of different primer pairs, the use of a high number of STR markers, and the characterization of the mutation causing the “null allele.”

Results from a collaborative exercise with proficiency testing conducted by 20 Italian laboratories on the 15 loci included in the Identifiler ® kit were analyzed by allele sharing methods and by standard population genetics tests. The validated database, including about 1500 subjects, was merged with that of a previous exercise conducted on nine loci, and the resulting allele frequencies, subdivided by Italian region, were published on-line.

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.

Allele frequencies for 26 short tandem repeats (STRs) were obtained from a sample of 203 unrelated individuals living in the area of Florence, Prato and Pistoia (Tuscany, Central Italy). These 26 loci are in addition to the existing 13 U.S. core loci and can help provide assistance in complex kinship testing when, for example, the alleged father is not available for testing. The results were compared with U.S. Caucasian, African American and Hispanic populations.

Germline mutations of human short tandem repeat (STR) loci are expansions or contractions of repeat arrays which are not well understood in terms of the mechanism(s) underlying such mutations. Although polymerase slippage is generally accepted as a mechanism capable to explain most features of such mutations, it is still possible that unequal crossing over plays some role in those events, as most studies in humans could not exclude unequal crossing over (UCO). Crossing over can be studied by analyzing haplotypes using flanking markers. To check for UCO in mutations, we have analyzed 150 paternity cases for which more than the usual trio (mother, child, and father) were available for testing by analyzing 16 STR loci. In a total of 4900 parent-child allele transfers four mutations were observed at different loci (D8S1179, D18S51, D21S11, and SE33/ACTBP2). To identify the mutated allele and to check for UCO, we typed at least four informative loci flanking the mutated locus and used the pedigree data to establish haplotypes. By doing so we were able to exclude UCO in each case. Moreover, we were able to identify the mutations as one-repeat contractions/expansions. Our data thus support slippage as the mechanism of germline mutations in STRs.

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 present a paternity testing case in which a double incompatibility was found for two short tandem repeat (STR) markers, human fibrinogen alpha (FGA) and Penta E. Analysis of the trio (mother, father, and daughter) included the amplification with a battery of 15 autosomal short tandem repeats (STR) by using a commercially available PowerPlex 16 System kit, and the detection with an ultraviolet-automatic sequencer. The biological paternity was confirmed with 12 additional markers. Reanalysis of the trio for the same markers with different primers was carried out by using an infrared automated sequencer and infrared-fluorescent primers. High paternity index confirmed that the observed inconsistencies were due to a double mutation, which was confirmed by sequence analysis at FGA and Penta E loci. Amplification and detection results obtained by the infrared-protocol showed consistent results with those obtained by ultraviolet-protocol and a commercially available kit. This has been our first case of double mutation at FGA and Penta E in a paternity testing. The use of our approach, based on two amplification and detection formats and on the sequence analysis, confirmed the observed meiotic paternal mutations.