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Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1–5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field.

Introduction Defining extreme temperatures as the cause of death remains challenging. It is mostly based on circumstantial, macroscopic and microscopic features. Methods We retrospectively compared groups of cases of fatal hypothermia, fatal hyperthermia and non-extreme temperature-related deaths. We analysed specific histological findings, focusing on samples from the liver, pancreas and kidney. Results Between 1 January 2013 and 31 December 2016, 15 autopsies were performed for deaths related to extreme temperatures. They included 11 cases of fatal hypothermia (group A), four cases of fatal hyperthermia (group B) and eight controls (group C). Perinuclear hepatocyte vacuolisation was observed in seven cases of hypothermia, one case of hyperthermia and four controls. Pancreatic cytoarchitecture was well preserved in two cases of hypothermia, one case of hyperthermia and two controls. No particular microscopic feature was found in pancreatic samples. Renal epithelial tubular cell vacuolisation was observed in seven cases of hypothermia and one case of hyperthermia, while it was absent in all controls. Chromogranin A (CgA) was markedly positive in the pancreatic tissue of five cases of fatal hypothermia and one control, and mildly positive in one case of fatal hyperthermia. No significant p-values were observed for any comparisons ( p > 0.05), except when hypothermia cases group were compared to the control group for the Armanni–Ebstein phenomenon test ( p = 0.0078). Conclusions Although our study did not find a specific microscopic marker, hepatocyte vacuolisation, the Armanni–Ebstein phenomenon and pancreatic CgA positivity, taken together, may be useful tools to confirm hypo- and hyperthermia-related deaths, in addition to circumstantial and macroscopic findings.

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq&trade, SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq&trade, SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer&rsquo, s instructions.

The HIrisPlex-S system, targeting a total of 41 SNPs, allows the simultaneous eye, hair and skin color prediction from DNA. In the present study, we developed a massive parallel sequencing (MPS) multiplex assay in order to genotype all the HIrisPlex-S markers in degraded casework samples. PCR amplicons sizes of target regions were kept below 180 bp, in order to allow analysis of degraded DNA samples. Individuals with known phenotype, artificially degraded DNA samples and a set of 2800M control DNA dilutions were sequenced on a Ion PGM System, in order to evaluate the concordance testing results and the forensic suitability of this 41-plex MPS assay. Full and reliable profiles could be obtained with 0.1 ng of input DNA. The increment of the number of PCR cycles results in improvement of sensitivity or in typing results but an increase of artifacts were also observed.

Degraded DNA samples often fail to produce profiles suitable for individual identification with the conventional nuclear short tandem repeat markers. However, recovery of genetic profiles from degraded DNA is sometimes possible with mitochondrial DNA (mtDNA). mtDNA is present in much higher copy number in the human cell than nuclear DNA; it is maternally inherited and is not subject to the phenomenon of genetic recombination. Therefore excluding the occurrence of mutational events, the mitochondrial molecules will be passed without modification to the mother’s children and maternal relatives share an identical mtDNA genome. The main exploitation of mtDNA in forensics is related to the reconstruction of genetic relationships in case of missing person’s identification or mass disaster investigations. In addition, mtDNA analysis can provide useful results in forensic samples with damaged DNA as bone remains, teeth, hair, and hair shafts. This chapter will review the features of mtDNA, the steps necessary to obtain results in forensic casework, and issues important to interpreting results.

BACKGROUND: Microhaplotypes or microhaps (MH) were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, genotyping of degraded DNA, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. In order to make inference about a set of microhaps useful in forensic casework with low amount of degraded DNA and useful in kinship analysis, several microhaps were tested by massive parallel sequencing (MPS) assay. METHODS: We have investigated the effectiveness of 29 microhaps in a set of real forensic samples together with artificially degraded DNAs. Moreover, we explore the informativeness of 87 microhaplotypes in relationship analysis through a simulation of different kinship testing scenarios typically encountered in forensic identification. RESULTS: The MPS coverage analysis showed a good performance of the designed panel. Full profiles could be obtained with 0.1 ng of input DNA even with highly degraded samples. The increment of the number of PCR cycles does not result in an improvement in genotyping results in samples with low amounts of DNA, as the increase of drop-in and drop-out events were observed at 25 number of PCR cycles. No correlation between amplicons size and occurrence of drop-outs and drop-ins was observed. Kinship simulations showed that full siblings and half siblings relationships would be readily distinguished respect unrelated condition using the 87 microhaps panel. CONCLUSIONS: Results shown that microhaps could be a powerful tool for individual identification, relationship resolution and that they are sensitive and reliable in degraded DNA typing.

The Institute of Legal Medicine of Polytechnic University of Marche has been working in the field of forensic identification since 1980s, contributing actively to the development of the research and the application of new techniques. Before the DNA era, human identification was based on the analysis of surface polymorphic antigen systems of blood groups and HLA complex and then on the analysis of serum proteins and red cell polymorphic isozymes. The era of forensic DNA analysis began in 1985, when Alec Jeffreys described a genetic polymorphism in the human myoglobin gene. In 1987 Kary Mullis developed the polymerase chain reaction (PCR) and the revolutionary power of this technology was immediately perceived and the lab was quickly switched towards this new approach. The lab of the Institute of Legal Medicine is equipped with the most recent and high throughput instruments and technologies for DNA typing, has achieved the ISO/IEC 17025 accreditation for the participation to the National DNA Database of genetic profiles, and is a partner of the European network of forensic genetic labs. The lab is now one of the Italian leading centers in forensic genetics and it is actively involved in the most innovative research fields in forensic genetics.

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the “same donor” or from a “first degree relative” was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21–26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18–23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10−13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping.

The efficiency of MPS in forensic mtDNA analysis has been thoroughly proven, although a reliable and well established data evaluation still remains a critical point. Numerous bioinformatics tools have been developed, but most of them require specific operating systems and high costs, while free open-source programs with user-friendly interfaces are few. In this study, 43 full mtGenomes were sequenced using the Ion Personal Genome Machine™ (PGM™) System and analyzed utilizing the plug-in Variant Caller (TVC) of the Ion Torrent Software Suite and the mtDNA-Server (mDS), a free web-based mitochondrial analysis tool for MPS data. The outcomes of these two different analysis tools were compared to variants noted after manual inspection of the aligned reads performed using Integrative Genomics Viewer (IGV). The comparison highlighted the presence of thirty-nine discordant variant calls, which were resolved by Sanger sequencing that confirmed the presence of all variants, except for 7 deletions. The combined adoption of IGV and Sanger type sequencing confirmatory steps, in addition of TVC and mDS analysis, resulted in a more accurate variants assignment with the detection of 32 additional true polymorphisms, which were noted in the final dataset. Regarding the heteroplasmy issue, out of a total of thirty heteroplasmic variants, twenty-eight were detected by the TVC, while the mDS detected twenty-two. Overall, none of the used bioinformatics tools were the perfect choice and a secondary analysis with an expert's opinion in complete mtGenome MPS data evaluation is still required in forensic genetic analysis.

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

Massively parallel sequencing (MPS) has allowed to analyze a new type of forensic genetic marker, known as microhaplotypes (MHs). MHs appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Moreover, MHs are potentially suitable for the analysis of degraded DNA samples. We designed a new panel of 29 MHs for MPS assay, with amplicons sizes below 180 bp and we investigated its effectiveness with low amounts of degraded samples. We genotyped a set of real forensic samples together with a set of artificially degraded DNAs. Also, a sensitivity test was assessed by a set of 2800 M DNA dilutions. The Depth of Coverage (DoC) were uniform across all 29 loci, in spite of amplicons size. Genotyping results shown that full profiles can be obtained even in highly degraded samples when the amount of template range from 0.1 to 5.0 ng. Finally, the increase of the number of PCR cycles did not provide an improvement in typing results of low amounts of degraded samples as, in front of higher number of typed loci, higher frequencies of artefacts leading to mistyping are found at 25 cycles.

A set of eighty-two forensic samples with different levels of degradation, as well five in vitro damaged samples were analyzed by the Precision ID Identity Panel. PCR amplifications were performed with scalar amount of DNA (from 1 ng to 12 pg) and through different number of cycles. A minimum coverage of 50 x was adopted for “locus call”. Very informative profiles (based on about 65–70% of the loci) were obtained even in highly degraded samples when the amount of template range from 0.1 to 1.0 ng. When dealing with low amount of degraded DNAs, no more than half of the loci were typed, and the risk of mistyping (due to drop out phenomena) increased dramatically. The employment of a high number of PCR cycles is discussed.

Osteogenesis imperfecta (OI) is a rare disease of collagen synthesis causing bone fragility. Also called “glass bone disease” since it manifests as spontaneous fractures, it is classified into nine types, both with dominant and recessive transmission. In 95% of cases OI is caused by mutations in COL1A1 and COL1A2 genes encoding the alpha1 and alpha2 chains of type 1 collagen, mainly null variants caused by frame-shift/nonsense mutations or splicing defects. In infants the differential diagnosis include not-accidental trauma, so child abuse. Families suspected of abuse often provide an unverified history of frequent fractures; conversely, the family history of individuals with OI often does not reveal any other affected individuals because of a de novo pathogenic variant in the proband or the presence of a mild phenotype in relatives. Therefore, legal medicine unit with DNA lab is crucial in these cases since it could early collect living or autopsy samples when a child abuse is suspected and then test DNA. We set up a MPS (massively parallel sequencing) panel including the coding regions of COL1A1 and COL1A2 and other 11 genes known to cause OI. We presented a case of suspected abuses in 2-month-old baby. MPS libraries were sequenced by Ion Torrent PGM platform; pathogenic variants and VUS (variants of uncertain significance) were confirmed by Sanger sequencing and familial segregation study was performed to better characterize the clinical significance of the mutation. This study remarks that MPS could help not only for identification, ancestry/phenotyping or molecular autopsy applications but also for forensic investigation over child abuse. The usefulness of this assay for diagnostic projects on victims of abuse together with post-mortem cases is discussed.

This study investigated the involvement of ADH4 gene polymorphisms in the susceptibility to alcohol use disorders.Thirty-eight single-nucleotide polymorphisms (SNPs) in and around the ADH4 gene were investigated in 136 Italian alcoholics and 276 healthy controls. A new approach based on a bioinformatic method selected 26 SNPs that may affect the splicing sites, destroying or creating binding sites of splicing regulatory proteins.Case-control comparisons for allele and genotype frequencies showed that ADH4 SNPs were associated with alcohol dependence but not with alcohol abuse. The association signal was strongest for rs1009145, rs13148577 (both P=0.0008) and rs7689753 (P=0.0007), whose minor alleles were predicted to alter the target protein sequences involved in mRNA splicing. A pairwise linkage disequilibrium analysis showed that all SNPs except five were located in a single haplotype block. Six haplotype tag SNPs were selected to infer haplotypes and to estimate their frequency distributions. A logistic regression analysis confirmed the association between ADH4 variants and alcohol dependence when sex, age, years of education, marital status and the allele genotype, haplotype and diplotype data of the six haplotype tag SNP were considered. Haplotype ATAAAT, which contained the minor allele of rs10009145 and the major allele of rs7689753, increased the risk of alcohol dependence, whereas haplotype GGGGAT, bearing the major allele of rs10009145 and the minor allele of rs7689753, protected against it. Again, there was no evidence of an association with alcohol abuse.These data suggest that ADH4 intronic variants play a role in alcohol dependence susceptibility in Italian populations. Functional studies are needed to establish the role of the genetic variations that seem to affect the splicing mechanism.

Microhaplotypes (microhaps) are defined as loci of two or more SNP within the span of a single sequencing run with three or more common allelic combination (haplotypes) of the SNPs. Microhaps appear to be useful in forensics for individual identification, ancestry inference, estimating relationships, and deconvoluting mixtures. The most important issue is identifying and characterizing a set of microhaps with the optimum characteristics for specific purposes and developing a suitable genotyping technology. The MPS technologies are now making microhaplotypes a new type of forensic marker: a single sequence read can cover the expanse of the microhaplotypes and these loci become phase-known. In the present study we selected a panel of 89 microhaps, from The ALlele FREquency Database and we evaluated these loci on 73 Italian samples. The results make the panel very informative for resolving DNA mixture, for identification and for recostruction of biological relationships. Microhaps will become a very promising new type of forensic marker when MPS is the technology being used for genotyping.

Aims Alcoholism is a multifactorial, genetically influenced disorder. It is a major health and social issue, a highly frequent disease and a cause of premature death. It is also the most expensive addictive disorder due to morbidity, mortality, societal and legal problems. Besides their involvement in alcohol-related fatalities, forensic scientists are also required to assess driving and working ability as well as permanent invalidity due to alcohol-related conditions. Greater knowledge of the genetic basis of alcoholism could improve prevention by identifying specific risk factors and mechanisms, leading to effective therapeutic strategies and eventually to personalized treatments. Methods This overview of the recent scientific literature on the genetic basis of alcoholism summarizes the analytical strategies currently applied to the identification of candidate genes involved in alcohol-use disorders (AUDs) and discusses some genes and related phenotypes that have been shown to influence the risk of alcoholism. Results Alcoholism is a complex heterogeneous genetic disease. It is a quantitative disorder, in which the combined incidence of multiple genetic factors and environmental factors varies from one subject to another. Family, twin and adoption studies indicate that 50-60% of the risk of alcoholism is due to genetic factors. Risk loci for AUDs include both genes involved in alcohol pharmacokinetics and pharmacodynamics as well as genes moderating neurophysiological responses such as impulsivity, disinhibition, sensation-seeking and externalizing behaviours. Alcoholism also co-exists with other addictions and psychiatric disorders. Such co-morbidity suggests the existence of shared aetiological factors. Conclusions Despite several genes that influence the risk for AUDs having been identified, the genetic bases of alcoholisn remain largely unknown. Particularly the mechanism of action or the understanding of the physiology of some genes, as well as the gene-environment interactions, is still unknown. Technological progress and advances in transcriptomics, epigenomics and proteomics are expected to enhance our knowledge of the genetic susceptibility to alcoholism.

Introduction Reciprocity with primary caregivers drives subjects’ adaptive abilities towards the construction of a Personal Meaning Organization (PMO) that is useful with respect to their developmental environment. Those with inward personalities read environmental signals through their internal activations; those with outward personalities read internal activations through the environment. It has recently been shown that PMO development may also have physiological and genetic bases. Because the serotonin receptor 2A gene (HTR2A) is one of the serotoninergic genes associated with personality and behaviour, it is expected to exert an influence on PMO development. The aim of this work was to explore possible correlations between HTR2A gene single nucleotide polymorphisms (SNPs) and the inward/outward PMO. Materials and methods We enrolled 149 healthy, unrelated members of our hospital staff (65 males, 84 females; mean ages 42 ± 10.3 and 37.6 ± 8.7, respectively). Each was classified as having an inward or outward PMO by two trained psychotherapists using a Post-Rationalist Clinical Interview and two self-rating questionnaires for PMO. A mini-sequencing assay was used to assess 20 SNPs at the HTR2A locus in genomic DNA samples from each participant. Results Three intronic HTR2A SNPs (rs2770304, rs55948462, and rs61948318) were found to be associated with the development of an inward rather than an outward PMO. Conclusions These preliminary results indicate that gene polymorphisms can influence the outcome of PMO development. The gene associations identified in this study support the view that attachment behaviour is also genetically based. This evidence is clinically relevant for a better understanding of the efficacy of pharmacological and psychotherapeutic strategies, and it suggests that further investigation is warranted to discover new associations between PMO development and serotonin gene variants.

Current forensic mitochondrial (mt)DNA databases are limited in representative population data of African origin. We investigated HVS-I/HVS-II sequences of 120 Tunisian and Moroccan healthy male donors applying stringent quality criteria to assure high quality of the data and phylogenetic alignment and notation of the sequences. Among 64 Tunisians, 56 different haplotypes were observed and the most common haplotype (16187T 16189C 16223T 16264T 16270T 16278T 16293G 16311C 73G 152C 182T 185T 195C 247A 263G 309.1C 315.1C; haplogroup (hg) L1b) was shared by four individuals. 56 Moroccans could be assigned to 52 different haplotypes where the most common haplotype was of West Eurasian origin with the hg H sequence motif 263G 315.1C and variations in the HVS-II polyC-stretch (309.1C 309.2C) shared by six samples. The majority of the observed haplotypes belong to the west Eurasian phylogeny (50% in Tunisians and 62.5% in Moroccans). Our data are consistent with the current phylogeographic knowledge displaying the occurrence of sub-Saharan haplogroup L sequences, found in 48.4% of Tunisians and 25% of Moroccans as well as the presence of the two re-migrated haplogroups U6 (7.8% and 1.8% in Tunisians and Moroccans, respectively) and M1 (1.6% in Tunisians and 8.9% in Moroccans).

To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%). The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries. High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population.

To define the Y-chromosome genetic structure in Apennine populations, 17 Y-chromosome short tandem repeats (Y-STRs) and 37 Y-single nucleotide polymorphisms (Y-SNPs) were typed in 162 subjects living in the upland area of the Marches (Central Italy). A total number of 155 haplotypes (haplotype diversity was 0.9994) and 14 SNP haplogroups were observed. Testing high-resolution Y-chromosome data sets, e.g. using Yfiler and SNPs, increases the discriminatory capacity in individual identification for forensic purposes. It is also useful in autochthonous population and micro-population studies to highlight the most informative loci for evolutionary aims.

Introduction Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inherited disease predisposing to the development of colorectal cancers and several other malignancies (endometrium, ovaries, stomach, small bowel, hepatobiliary and urinary tract). HNPCC is caused by germline mutations in any of the MisMatch Repair (MMR) genes. Mutations in MLH1 and MSH2 account for almost 90% of all identified ones. About 15% of mutations identified in MSH2 are missense ones. Patients and methods We studied one family, fulfilling Amsterdam II criteria, referred to our Center for genetic counselling. The proband, and some of her relatives, have been investigated for microsatellite instability (MSI), immunohistochemical MMR protein staining and by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Results All patients carried the same novel MSH2 germline missense mutation (R359S) in exon 7, which determines the substitution of an Arginine, which is a basic amino acid, with a polar Serine residue (R359S). The mutation was associated with lack of expression of MSH2 protein and high microsatellite instability in tumour tissues. The same mutation has been detected in one healthy relative. Conclusions The mutation here reported shows a high correlation with phenotype. The mutation is located in an evolutionary conserved domain. Taken together, our findings suggest evidence that the amino acid substitution can be interpreted as pathogenetic.

This work describes an efficient and rapid test for typing 37 single nucleotide polymorphisms (SNPs) of the non-recombining region of Y chromosome (NRY) from a minimal amount of DNA using six PCR multiplexes. Markers were drawn following a hierarchical strategy based on the phylogenetic tree of Y chromosome proposed by the Y Chromosome Consortium [The Y Chromosome Consortium, A nomenclature system for the tree of human Y-chromosomal binary haplogroups, Genome Res. 12 (2002) 339-348]. Two multiplexes--arbitrarily named MY1 and MY2--were developed to explore the basal branches of the tree encompassing all the major clades A-R: MY1 for markers M35, M89, M172, M170, M9, M173, M45 and MY2 for markers M52, M216, M174, M181, M201, M91, M96, M214. Four multiplexes able of typing the more superficial branches typical of most frequent European haplogroups E3b, J2, R1 and I, were also developed and named MY-E3b (M78, M107, M224, M165, M148, M81), MY-J2 (M158, M68, M47, M102, M137, M67), MY-R1 (M17, M269, M18, P25, SRY10831.2) and MY-I (M72, M223, M26, M21, M161). SNP genotyping was carried out by hot-start PCR amplification with primers yielding fragments between 63 and 210 nucleotides, followed by minisequencing reaction based on dideoxy single-base extension and capillary electrophoresis of extension products. The sequential application of these multiplexes is a robust and effective resource for typing the most frequent European Y-SNP haplogroups, and appears to be suitable for forensic purposes and evolutionary studies.

Many X-chromosome short tandem repeats (X-STRs) have been validated for forensic use even if further studies are needed on allele frequencies and mutation rates to evaluate the extent of polymorphism in different populations and to establish reference databases useful for forensic applications and for anthropological studies. A single multiplex reaction of seven X-STRs, which includes the DXS6789, HUMARA, DXS10011, DXS7423, HPRTB, DXS6807, DXS101 loci, is presented and their allele frequency distribution in a large population sample including 556 subjects (268 females and 288 males) analysed by five forensic laboratories of Central and Northern Italy is shown. Our results demonstrate the feasibility of a single amplification/detection reaction involving seven markers of the X chromosome, which can be fruitfully used in complex kinship analysis.

Nardi B, Piva F, Turchi C, Giulietti M, Castellucci G, Arimatea E, Rocchetti D, Rocchetti G, Principato G, Tagliabracci A, Bellantuono C. HTR2A gene polymorphisms and Inward and Outward Personal Meaning Organisations.Objective:Caregiver behaviours and emotional expressions may induce development of two basic categories of constructing identity and of regulating cognitive and emotional processes: an Inward or an Outward Personal Meaning Organisation (PMO). Inwards read environmental signals through their internal activations. Their emotions are more distinct, and reciprocity is more based on physical distance (protection, loneliness). Outwards read internal activations through the environment. Their emotions are more blurred, and reciprocity is more based on a semantic sight of relations (approval, rules).It has recently been shown that PMO development may also have physiological and genetic bases. In a functional magnetic resonance imaging (fMRI) study, Inward and Outward subjects showed different amygdala activation patterns and an association with the SLC6A4 serotonin transporter gene 5-HTTLPR polymorphism.Methods:In this work, 149 healthy subjects were examined with respect to Inward and Outward PMOs. We explored the association with 10 serotonin receptor 2A (HTR2A) gene single nucleotide polymorphisms (SNPs) selected by bioinformatics methods.Results:An intronic SNP (rs55948462) was found to be significantly associated with an Inward and an Outward PMOs development. However, after statistical adjustments, these results did not remain significant.Conclusion:We did not find associations between considered SNPs and Inward/Outward PMOs. However, the role of HTR2A polymorphisms was not considered in this study and that of the other serotonin-related genes should be valued.

This study reports the selection of 22 autosomal SNPs and the setting of PCR and minisequencing multiplexes suitable for forensic purposes.

In order to define the Y-chromosome genetic structure in Apennine populations, 17 Y-chromosome microsatellites and 37 Y-single nucleotide polymorphisms were typed in 81 subjects living in Fabriano and Urbino, two small towns in the upland area of the Marches (central Italy), speaking different dialects and submitted to a limited genetic flow.

DNA recovered at a crime scene often results as damaged; this represents enormous difficulty for the correct typing because of fragmentation or the lack of DNA region of interest. In this work a set of biological samples was prepared and stored under different conditions; STRs and SNPs typing was performed at regular interval of time in order to study the effects of natural DNA degradation. Allelic/locus drop-out phenomenon for the higher molecular weight loci or no results were obtained for microsatellite analysis after 1 week. SNPs typing gave positive results depending on storage conditions and type of substrate; a nucleotide alteration (C to T) was observed for M269 locus in a sample after 3 months.

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 aim of this study was to set up a multiplex PCR of eight autosomal single nucleotide polymorphisms (SNPs) suitable for forensic purposes.

Background: Alcoholism is a major health and social issue, a highly frequent disease and a cause of premature death. It is also the most expensive addictive disorder being related to high morbidity and mortality, violence, accidents, and social and legal problems. It is a quantitative disorder, where the combined incidence of environmental and multiple genetic factors varies from 1 subject to another. Recent association studies have identified several genes as candidates for alcoholism, including GABA(A) receptor genes, due to their role in mediating several behavioral effects of alcohol, such as motor incoordination, anxiolysis, sedation, and withdrawal. The proposed association between the 3' half of the gene encoding the alpha-2 subunit of GABA receptor (3'-GABRA2) and alcohol use disorders (AUDs) has received several independent confirmations. Methods: In this study, 10 single nucleotide polymorphisms (SNPs) of the 3'-GABRA2 gene, previously reported to be implicated in alcohol dependence, were used to evaluate the linkage between selected SNPs and AUDs in an Italian sample and to compare findings with those of previous studies. Results: No evidence of an association was found at the allele, genotype, haplotype, or diplotype levels between the 3'-GABRA2 polymorphisms investigated and alcoholism in 149 Italian alcoholics (98 alcohol dependents and 51 alcohol abusers) and 278 controls. Conclusions: Despite previous reports, we did not find an association between AUDs and 3'-GABRA2 polymorphisms. This is probably due to the minimal comorbidity of our Italian sample suggesting that this gene is implicated in polysubstance dependence rather than in alcoholism alone. Language: en

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.

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.

The opportunities to analyse the genetic variations related to the risk of addiction are of interest to forensics, who beside their involvement in drugs-related fatalities may also be required to assess driving and working ability as well as permanent invalidity due to drugs-related conditions. Several genetic variants have been shown to be associated with heroin addiction. The most investigated gene is OPMR1 that encode the μ-opioid receptor. The purpose of this study was to examine the contribution of genetics variants in OPRM1 to the susceptibility to addiction.

Material recovered from 374 fingerprints left by eleven laboratory workers on three different substrates (glass, wood, metal) at a standard pressure time of 30 s, with and without preliminary handwashing, was submitted to morphological, quantitative, and type analysis. Morphological and agarose-gel electrophoresis analysis showed that a non-negligible amount of epidermal corneal cells presented apoptotic alterations. The quantity of DNA recovered from fingerprints ranged between 0.04 to 0.2 ng, and in a significant number of experiments no DNA was detected. Handwashing reduced the amount of DNA recovered from fingerprints. The "shedder status" of the donor was a very important factor, causing inter-individual variations in the amount of DNA left by fingerprints. Spurious alleles from laboratory-based and secondary transfer contamination, stutters, and other artifacts described when analyzing low-copy-number DNA and capable of affecting correct profiles were observed.

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

Mitochondrial DNA sequences of the hypervariable regions HVI and HVII were analysed in 83 Caucasians living in central Italy to expand the database for forensic identification purposes, and 75 different haplotypes resulting from 62 polymorphic positions in HVI and 44 in HVII were observed. The most frequent haplotype (263G, 309.1C, 315.1C) was shared by 7 individuals, 2 haplotypes were shared by 2 individuals, and 72 were unique. The genetic diversity was found to be 0.99 and the random match probability 1.9%. A condition of sequence heteroplasmy was found in only one case at nt 16311, whereas a length heteroplasmy was found in the homopolymeric stretch of cytosines 303–315. Our results indicate that in direct sequencing beyond the poly-cytosine stretch, the overlap is due to length heteroplasmy, whereas the blurred signal occurs when the stretch is composed of more than 10 cytosines.

Addiction to drugs of abuse is the major health and social issue, for its morbidity and mortality, individual and society cost, violence and legal problems involvement. The susceptibility to addiction has a complex genetic basis characterized by phenotypic and genetic heterogeneity as well as polygenicity. Both linkage mapping and association mapping have identified susceptibility loci for addiction-related phenotypes. In this study we limit our focus to gamma-amino butyric acid A receptor alpha 2 (GABRA2), one of the candidate gene in addiction, in order to investigate the genetic susceptibility of alcoholism. 10 SNPs across the 3′-GABRA2 were analyzed in an Italian sample of 149 cases and 278 healthy controls. No SNP and haplotype association of 3′-GABRA2 with AUDs was found, in contrast to previous reports in other Caucasian populations.

Background and purpose Unequivocal identification of insect specimens is a crucial step in forensic entomology and could be difficult using only morphological features, especially for immature stages. The majority of published studies consider the cytochrome oxidase subunits one (COI) analysis and the obtained sequences are representative for insect population of specific geographic regions. This work describes a different approach for genetic identification of insect specimens. Method The immature specimens found in human corpses recovered in Italy were bred to obtain adult flies. DNA was extracted and analyzed by a ∼900bp fragment of COI and COII genes, using four degenerate primers. The sequences were aligned and compared with the Diptera sequences in GenBank for species-specific identification. Results The sequenced fragment allowed an insect species-specific identification; intra-specific and geographic variations were found. COI and COII sequences were compared with GenBank sequences independently and sometimes species-specific identification was ambiguous. Conclusion The combined analysis of COI and COII fragments is a more accurate approach for Diptera species identification than single COI fragment analysis. The geographic and intra-specific variations are important for insect identification and local database set up are strongly recommended.

Alcoholic abuse is of great interest in forensic medicine, for the evaluation of driving and working ability, for permanent invalidity to alcohol-related pathologies and for the high incidence of alcohol-related deaths. It is a quantitative disorder, with a combined incidence of environmental and genetic factors. Since it was observed that variations in ADH4 gene are associated with alcoholism, SNPs mapping in different regions of ADH4 gene were selected. A new approach was applied in this study: mutations that could modify the splicing sites were selected using "Splice site prediction" available at http://www.fruitfly.orgseq_tools/sp/lice.html; in addition, since the correct splicing is finely regulated by RNA binding proteins, SNPs that could affect splicing, destroying or creating protein target sequences and changing RNA secondary structure, were predicted by a tool available at www.introni.it. 206 unrelated volunteers, diagnosed for alcohol dependence (DSM-IV), were included in this study. SNPs typing was performed setting up 2 multiplex PCRs and minisequencing reactions. 12 SNPs were selected from literature data and 26 SNPs, among 233 polymorphisms screened, were chosen by computational analyses.

At the beginning of 2006 more than 301,000 immigrants resident in Italy resulted to come from Tunisia and Morocco, 66% of which are male subjects; in addition, it is estimated that some other thousand are clandestine. Our data show that there is an increasing involvement of Tunisian and Moroccan individuals in paternity testing and in individual identification cases. For these reasons, the aim of this work was to enrich forensic Y-chromosome databases with Northern Africa data to better know markers frequency and their distribution across these populations (in YHRD there are 246 Tunisian samples and 0 Moroccans, access date to www.yhrd.org: August 2007). 103 Tunisian and Moroccan healthy male donors were typed by 17 microsatellites extended haplotype and 41 Y-SNPs. A high-resolution level database was created, including both haplotype and haplogroup for each sample. This study confirmed that precious informations might come both from Y-SNPs haplogroup distribution besides Y-STRs data.

All forensic laboratories routinely use commercial kits and softwares for automated typing; in rare cases genotyping misinterpretations or mislabellings occur. This study refers to the investigation on a D2S1338 off-ladder allele mislabelling observed in DNA profile of murdered woman. The Identifiler ® revealed heterozygosity in the range of D16S539, with a presumptive microvariant allele "14.2", based on assigned size, while PowerPlex ® 16 resulted in a homozygosity of allele "11". Singleplex amplification of D16S539 locus confirmed homozigosity. D2S1338 locus, the closest to D16S1338 in Identifiler ® , genotyped as homozigote "19", was singleplex amplified. The off-ladder peak was gel-isolated, sequenced and designed as a rare "11" allele variant [(TGCC)6(TTCC)5]. Genotype was finally designed as D16S539 "11,11" and D2S1338 "11,19". To avoid genotyping misinterpretations or mislabelling, ambiguous genotypes should be established by two commercial kits at least. Furthermore, off ladder alleles as well as allele microvariants should be assigned by direct sequencing. This issue should be considered in Criminal DNA database requirements, that is still under debate in Italy.

This study reports a paternity case analyzed by the AmpFlSTR Identifiler Kit (AB) in which father and daughter shared three rare alleles for D19S433, D18S51 and TH01 microsatellites. The case also showed an apparent exclusion, due to a mutation at the D3S 1358 microsatellite. Sequencing analysis was performed to assess the size of the rare alleles and to establish their structure, which revealed some molecular variations in regions flanking the motif repeats.