Evaluation of critical aspects in clinical and forensic management of sexual violence: A multicentre Ge.F.I. project.

Violence against women is a violation of human rights, crossing all cultures, classes, levels of education, earnings, ethnic and age groups. We conducted a retrospective study to review forensic records of sexual assault examinations carried out in different Italian health facilities and to correlate these findings with the results of the forensic DNA analyses. The goal was to determine which factors could have affected the obtained results, to identify the fundamental aspects to search for while examining a sexual assault victim in order to gather useful evidence to identify the offender and reconstruct the dynamics of the fact. We analysed 102 cases that occurred between 2006 and 2017, coming from ten participating laboratories. Despite a relatively limited number of cases, this study shows that the ability to ascertain the presence of male biological material in the samples collected is not a problem for forensic laboratories and seems to be influenced by other factors, such as how much time elapsed between the event and the sampling, the availability of the aggressor's biological material on the victim and the identification of biological fluids/stains. Therefore, the need for health structures to adopt specific protocols has been highlighted. It is necessary for health structures to define specific pathways and adopt homogeneous procedures or operational protocols, and it is essential to provide adequate training for health personnel. The results of the study could be useful in drafting and revising protocols/guidelines implemented in Italian hospital. Issues related to the limited number of analyses requested by Italian Authorities are also discussed.

The importance of forensic storage support: DNA quality from 11-year-old saliva on FTA cards.

Storage conditions influence the integrity of the recoverable DNA from forensic evidence in terms of yield and quality. FTA cards are widely used in the forensic practice as their chemically treated matrix provides protection from the moment of collection to the point of analysis with current STR typing technology. In this study, we assess the recoverability and the integrity of DNA from 11-year-old saliva on FTA cards using a forensic quantitative real-time polymerase chain reaction (qPCR) commercial assay. The quality after long-term storage was investigated in order to evaluate if the FTA device could assure enough stability over time, applying some internally validated quality criteria of the STR profile. Furthermore, we used a 3D interpolation model to combine the quantitative and qualitative data from qPCR to calculate the minimum optimal DNA input (MODI) to add to the downstream PCR reaction based on the quantitative and qualitative data of a sample. According to our results, when saliva sample is properly transferred onto FTA cards and then correctly stored according to the manufacturer's instructions, it is possible to recover sufficient amounts of DNA for human identification even after more than a decade of storage at ambient temperature. Degradation affected the quality of results especially when the Degradation Index exceeds the value of 2.12, requiring modifications of the standard internal workflow to improve the genotyping quality. Above this value, the application of a "corrective factor" to the PCR normalization process was necessary in order to adjust the recommended manufacturer's PCR DNA input taking into account the degradation level. Our results demonstrated the importance to consider in predictive terms the parameters obtained with the real-time quantification assay, both in terms of quantity (DNA concentration) and of quality (DI, inhibition). Informatics predictive tools including qPCR data together with the variables of storage duration and conditions should be developed in order to optimize the DNA analysis process.

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

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

An ancient Mediterranean melting pot: investigating the uniparental genetic structure and population history of sicily and southern Italy.

Due to their strategic geographic location between three different continents, Sicily and Southern Italy have long represented a major Mediterranean crossroad where different peoples and cultures came together over time. However, its multi-layered history of migration pathways and cultural exchanges, has made the reconstruction of its genetic history and population structure extremely controversial and widely debated. To address this debate, we surveyed the genetic variability of 326 accurately selected individuals from 8 different provinces of Sicily and Southern Italy, through a comprehensive evaluation of both Y-chromosome and mtDNA genomes. The main goal was to investigate the structuring of maternal and paternal genetic pools within Sicily and Southern Italy, and to examine their degrees of interaction with other Mediterranean populations. Our findings show high levels of within-population variability, coupled with the lack of significant genetic sub-structures both within Sicily, as well as between Sicily and Southern Italy. When Sicilian and Southern Italian populations were contextualized within the Euro-Mediterranean genetic space, we observed different historical dynamics for maternal and paternal inheritances. Y-chromosome results highlight a significant genetic differentiation between the North-Western and South-Eastern part of the Mediterranean, the Italian Peninsula occupying an intermediate position therein. In particular, Sicily and Southern Italy reveal a shared paternal genetic background with the Balkan Peninsula and the time estimates of main Y-chromosome lineages signal paternal genetic traces of Neolithic and post-Neolithic migration events. On the contrary, despite showing some correspondence with its paternal counterpart, mtDNA reveals a substantially homogeneous genetic landscape, which may reflect older population events or different demographic dynamics between males and females. Overall, both uniparental genetic structures and TMRCA estimates confirm the role of Sicily and Southern Italy as an ancient Mediterranean melting pot for genes and cultures.

Demographic histories, isolation and social factors as determinants of the genetic structure of Alpine linguistic groups.

Great European mountain ranges have acted as barriers to gene flow for resident populations since prehistory and have offered a place for the settlement of small, and sometimes culturally diverse, communities. Therefore, the human groups that have settled in these areas are worth exploring as an important potential source of diversity in the genetic structure of European populations. In this study, we present new high resolution data concerning Y chromosomal variation in three distinct Alpine ethno-linguistic groups, Italian, Ladin and German. Combining unpublished and literature data on Y chromosome and mitochondrial variation, we were able to detect different genetic patterns. In fact, within and among population diversity values observed vary across linguistic groups, with German and Italian speakers at the two extremes, and seem to reflect their different demographic histories. Using simulations we inferred that the joint effect of continued genetic isolation and reduced founding group size may explain the apportionment of genetic diversity observed in all groups. Extending the analysis to other continental populations, we observed that the genetic differentiation of Ladins and German speakers from Europeans is comparable or even greater to that observed for well known outliers like Sardinian and Basques. Finally, we found that in south Tyroleans, the social practice of Geschlossener Hof, a hereditary norm which might have favored male dispersal, coincides with a significant intra-group diversity for mtDNA but not for Y chromosome, a genetic pattern which is opposite to those expected among patrilocal populations. Together with previous evidence regarding the possible effects of "local ethnicity" on the genetic structure of German speakers that have settled in the eastern Italian Alps, this finding suggests that taking socio-cultural factors into account together with geographical variables and linguistic diversity may help unveil some yet to be understood aspects of the genetic structure of European populations.

Detecting genetic isolation in human populations: a study of European language minorities.

The identification of isolation signatures is fundamental to better understand the genetic structure of human populations and to test the relations between cultural factors and genetic variation. However, with current approaches, it is not possible to distinguish between the consequences of long-term isolation and the effects of reduced sample size, selection and differential gene flow. To overcome these limitations, we have integrated the analysis of classical genetic diversity measures with a bayesian method to estimate gene flow and have carried out simulations based on the coalescent. Combining these approaches, we first tested whether the relatively short history of cultural and geographical isolation of four "linguistic islands" of the Eastern Alps (Lessinia, Sauris, Sappada and Timau) had left detectable signatures in their genetic structure. We then compared our findings to previous studies of European population isolates. Finally, we explored the importance of demographic and cultural factors in shaping genetic diversity among the groups under study. A combination of small initial effective size and continued genetic isolation from surrounding populations seems to provide a coherent explanation for the diversity observed among Sauris, Sappada and Timau, which was found to be substantially greater than in other groups of European isolated populations. Simulations of micro-evolutionary scenarios indicate that ethnicity might have been important in increasing genetic diversity among these culturally related and spatially close populations.

Capillary electrophoresis of multigene barcoding chloroplast markers for species identification of botanical trace evidence.

The analysis of nonhuman biological evidence both animal and botanical to find out the correct species of a sample comes as a great help to crime investigators. Particularly, forensic botany may be useful in many criminal and civil cases, e.g., for linking an individual to a crime scene or physical evidence to a geographic location, or tracking marijuana distribution patterns.Despite many molecular techniques for species identification so far applied, botanical evidences are still overlooked by forensic scientists due to the lack of reproducible and efficient protocols standardized across a wide range of different organisms and among different laboratories.Recently, the term "DNA barcoding" has been coined to describe the use of a short gene sequence from a standardized region of the genome as a molecular tool for species identification. DNA barcodes have been successfully applied to a number of animal groups and introduced in forensic science with the application of the mitochondrial gene COI. Building on this success, ongoing investigations have searched for the best barcode to apply to all land plants. Here we describe the basic protocol based on amplification and sequence analysis of barcoding markers for land plants considering the latest developments of Plant DNA barcoding Project. The aim of this chapter is to provide forensic scientists an accurate and reliable tool for assigning unidentified botanical specimens to the correct species as powerful mainstay in investigations, increasing the contributions from nonhuman DNA to forensics.

Y-STR variation in Albanian populations: implications on the match probabilities and the genetic legacy of the minority claiming an Egyptian descent.

Y chromosome variation at 12 STR (the Powerplex® Y system core set) and 18 binary markers was investigated in two major (the Ghegs and the Tosks) and two minor (the Gabels and the Jevgs) populations from Albania (Southern Balkans). The large proportion of haplotypes shared within and between groups makes the Powerplex 12-locus set inadequate to ensure a suitable power of discrimination for the forensic practice. At least 85% of Y lineages in the Jevgs, the cultural minority claiming an Egyptian descent, turned out to be of either Roma or Balkan ancestry. They also showed unequivocal signs of a common genetic history with the Gabels, the other Albanian minority practising social and cultural Roma traditions.

Forensic botany: species identification of botanical trace evidence using a multigene barcoding approach.

Forensic botany can provide significant supporting evidence during criminal investigations. However, it is still an underutilized field of investigation with its most common application limited to identifying specific as well as suspected illegal plants. The ubiquitous presence of plant species can be useful in forensics, but the absence of an accurate identification system remains the major obstacle to the present inability to routinely and correctly identify trace botanical evidence. Many plant materials cannot be identified and differentiated to the species level by traditional morphological characteristics when botanical specimens are degraded and lack physical features. By taking advantage of a universal barcode system, DNA sequencing, and other biomolecular techniques used routinely in forensic investigations, two chloroplast DNA regions were evaluated for their use as "barcoding" markers for plant identification in the field of forensics. We therefore investigated the forensic use of two non-coding plastid regions, psbA-trnH and trnL-trnF, to create a multimarker system for species identification that could be useful throughout the plant kingdom. The sequences from 63 plants belonging to our local flora were submitted and registered on the GenBank database. Sequence comparison to set up the level of identification (species, genus, or family) through Blast algorithms allowed us to assess the suitability of this method. The results confirmed the effectiveness of our botanic universal multimarker assay in forensic investigations.

Species identification through DNA "barcodes".

Conventional methods for forensic species identification are mainly based on immunological procedures, which have limited applications for old and degraded specimens. The mitochondrial cytochrome b gene sequence has emerged in forensics among molecular methods. Recent investigations in the taxonomic field have suggested that a DNA-based identification system may aid the resolution of animal diversity and classification using sequence analysis and phylogenetic links. Selected gene sequences can be viewed as a genetic "barcode," which is enclosed in every cell, and barcoding is a standardized approach for characterizing species using short DNA sequences as a diagnostic biomarker for organisms. The aim of this study was to evaluate the potential of barcode mitochondrial genes, such as the cytochrome c oxidase sub 1 (COI) and the 16S rRNA gene, as a forensic tool. We developed a new approach for species testing and identification with a singleplex PCR amplification that will be useful not only in criminal casework but also in biosecurity, food authentication, investigation against poaching or illegal trade of endangered species, and wildlife enforcement. Seven fragments ranging from 157 to 541 bp (base pairs) in humans were selected from COI and 16S rRNA genes by different redesigned sets of primers suitable for forensic purposes. The specificity of each primer pair was evaluated with a single PCR reaction on different substrates, and the diversity values were calculated by statistical tests to select a set of markers that could be useful in different caseworks. A case example of forensic species identification is also presented.

J1-M267 Y lineage marks climate-driven pre-historical human displacements.

The present day distribution of Y chromosomes bearing the haplogroup J1 M267(*)G variant has been associated with different episodes of human demographic history, the main one being the diffusion of Islam since the Early Middle Ages. To better understand the modes and timing of J1 dispersals, we reconstructed the genealogical relationships among 282 M267(*)G chromosomes from 29 populations typed at 20 YSTRs and 6 SNPs. Phylogenetic analyses depicted a new genetic background consistent with climate-driven demographic dynamics occurring during two key phases of human pre-history: (1) the spatial expansion of hunter gatherers in response to the end of the late Pleistocene cooling phases and (2) the displacement of groups of foragers/herders following the mid-Holocene rainfall retreats across the Sahara and Arabia. Furthermore, J1 STR motifs previously used to trace Arab or Jewish ancestries were shown unsuitable as diagnostic markers for ethnicity.

Slow and fast evolving markers typing in Modena males (North Italy).

One hundred thirty male individuals, strictly selected for their geographical origin and for typical regional surnames were submitted to the analysis. 17 STRs (short tandem repeats) loci and 19 SNPs binary markers (single nucleotide polymorphisms) of male-specific region of the Y chromosome (MSY) were typed to well characterize the selected population of Modena province. The availability of joint distribution of MSY haplotypes and haplogroup frequencies is becoming an important tool for both human evolutionary studies and forensic investigation, but large databases of complete Y-lineages are needed for a better understanding of the power of the combined use of Y-specific polymorphisms. A total of 129 haplotypes and 9 haplogroups were found and R1b haplogroup with a frequency of 67.7% was the most frequent, as expected because of the geographical location of the sample (Northwestern Italy). The Modena Y-lineages (STRs and SNPs independently) were also compared with published data of other neighbouring populations' samples.

Unusual concurrent detection by polymerase chain reaction of Bartonella henselae and parvovirus b19 in an immunocompetent child with erythema nodosum and hepatic granulomatous disease.

We report an unusual case of documented Bartonella henselae genotype I from hepatic tissue in an Italian immunocompetent girl presenting with erythema nodosum and hepatic granulomata. Polymerase chain reaction (PCR) was performed on biopsied liver sample to confirm the etiologic role of B. henselae and to identify the genetic variant of this organism. A PCR on the same liver biopsy for parvovirus B19 was also positive, but the clinical meaning of this was not clear.

Cancerous tissues in forensic genetic analysis.

Microsatellites or short tandem repeats (STRs) markers are important tools for mapping disease-causing genes by linkage, for performing investigations in forensic medicine, for population genetic studies and for studying genetic modifications in tumors. In forensic applications neoplastic tissues can be used as a source of genetic information for personal identification or paternity testing when no other specimen is available. Cancer tissues can show microsatellite instability (MSI) and loss of heterozygosity (LOH) also for the STRs used in the forensic field. In this study, we screened 56 sporadic gastrointestinal carcinomas in order to provide further data for the evaluation of the incidence of allelic alterations for 15 STR loci and the suitability of using cancerous tissues in forensic applications. Sixty-six percent of the cancerous tissues were found to possess allelic alterations of the microsatellites analyzed with a high incidence of MSI-L (microsatellite instability low) when compared to the corresponding normal tissue. The most frequently altered loci were D18S51, VWA, and FGA. From a forensic perspective, great care must be taken in evaluating the DNA typing results obtained from cancerous tissue samples.

Validation of a large Italian Database of 15 STR loci.

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.

Post-mortem diagnosis of encephalitis in a 75-year-old man associated with human herpesvirus-6 variant A.

An HHV-6 variant A infection is described in a 75 year-old man in association with meningoencephalitis identified at autopsy. The patient presented with fever and anorexia, then he developed altered consciousness, motor weakness, progressive lethargy, and coma, and died 21 days after hospital admission. Histopathological examination showed perivascular lymphocytic infiltrates in the central nervous system (CNS). Serum and cerebral spinal fluid (CSF) samples drawn from the patient were tested for viruses by a nested polymerase chain reaction (nPCR). HHV-6 primers A and C [Aubin et al., 1991: J Clin Microb 29: 367-372] and HS6AE and HS6AF from [Dewhurst et al. (1993): J Clin Microb 31: 416-418] disclosed a 750 bp genomic product of HHV-6 in both types of biological samples. Restricted site analysis showed that the HHV-6 DNA amplified belonged to the variant A of the virus. Short sequences of HHV-6 DNA could also be detected in the DNA extracted from formalin-fixed, paraffin-embedded sections of CNS tissues by use of one (GM5 and GM6) of three pairs of HHV-6 primers that were selected. Immunohistochemical examination of brain sections, employing a specific monoclonal antibody directed against the HHV-6 gp 102 protein, detected the viral antigen in neurons and glial cells.

Development of a heptaplex PCR system to analyse X-chromosome STR loci from five Italian population samples. A collaborative study.

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.

Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors.

Kaposi sarcoma (KS) is a vascular tumor that can develop in recipients of solid tissue transplants as a result of either primary infection or reactivation of a gammaherpesvirus, the KS- associated herpesvirus, also known as human herpesvirus-8 (HHV-8). We studied whether HHV-8 and the elusive KS progenitor cells could be transmitted from the donor through the grafts. We used a variety of molecular, cytogenetic, immunohistochemical and immunofluorescence methods to show that the HHV-8-infected neoplastic cells in post-transplant KS from five of eight renal transplant patients harbored either genetic or antigenic markers of their matched donors. These data suggest the use of donor-derived HHV-8-specific T cells for the control of post-transplant KS.

Allele sharing in first-degree and unrelated pairs of individuals in the Ge F I AmpFlSTR Profiler Plus database.

Eleven Italian forensic laboratories participated in a population study based on the AB Profiler Plus loci with proficiency testing. The validated database, including 1340 individuals, is available on-line. Tests for Hardy-Weinberg equilibrium, gametic unbalance, and heterogeneity of gene frequency were generally not significant. Gene frequencies at each locus were consistent with those of two previously published Italian studies, but different from a third. Individuals of each subsample were paired, and the total number of alleles shared across the nine loci was determined in each pair. The analysis was replicated over the total sample. In addition, two samples of mother-child pairs (N=315) and full-sib pairs (N=91) were subjected to allele sharing analysis. The resulting distributions were sufficiently distinct from the sample of unrelated pairs as to be of practical usefulness.