Population structure of modern-day Italians reveals patterns of ancient and archaic ancestries in Southern Europe.

European populations display low genetic differentiation as the result of long-term blending of their ancient founding ancestries. However, it is unclear how the combination of ancient ancestries related to early foragers, Neolithic farmers, and Bronze Age nomadic pastoralists can explain the distribution of genetic variation across Europe. Populations in natural crossroads like the Italian peninsula are expected to recapitulate the continental diversity, but have been systematically understudied. Here, we characterize the ancestry profiles of Italian populations using a genome-wide dataset representative of modern and ancient samples from across Italy, Europe, and the rest of the world. Italian genomes capture several ancient signatures, including a non-steppe contribution derived ultimately from the Caucasus. Differences in ancestry composition, as the result of migration and admixture, have generated in Italy the largest degree of population structure detected so far in the continent, as well as shaping the amount of Neanderthal DNA in modern-day populations.

Prevalence of alcohol and other drugs in injured drivers and their association with clinical outcomes.

OBJECTIVE: Driving under the influence of alcohol or drugs is a risk factor for motor vehicle accidents (MVAs). This issue has become an increasing concern for the governments of many European and North American countries, thereby encouraging the adoption of preventive policies. The aim of this study was to investigate the associations between major clinical outcomes and alcohol or drug abuse among drivers involved in MVAs who were referred to an Italian Emergency Department. PATIENTS AND METHODS: The study population consisted of consecutive injured drivers who were admitted to the Emergency Department following an MVA during a period of one year. The patients' blood alcohol concentrations (BACs) and the presence of the most common drugs of abuse [amphetamine, methamphetamine, methylenedioxymethamphetamine (MDMA), barbiturates, benzodiazepines, benzoylecgonine (cocaine main metabolite), cannabinoids, methadone, and opiates)] were determined and evaluated in association with major clinical outcomes and demographic data. RESULTS: Overall, 347 injured drivers were enrolled. Of the 347 enrolled patients, 164 (47.3%) had a positive BAC (greater than 5 mg/dL). A subgroup of 107 injured drivers was also screened for drugs of abuse. Thirty-seven of these subjects (34.5%) were positive for at least one drug. A statistically significant association was found between BAC and triage at admission (p<0.01), hospitalization (p<0.01), and lesions of internal organs (p=0.04). CONCLUSIONS: The results of this study show that a significant proportion of injured drivers had detectable levels of BAC and/or illegal drugs. Positive BACs were significantly associated with worse clinical outcomes. These findings suggest that the implementation of methods to prevent alcohol and drug abuse is of paramount importance in the effort to reduce the rates of MVAs and their dramatic consequences.

Diatoms in drowning cases in forensic veterinary context: a preliminary study.

In this preliminary study, a digestive method used in forensic context to extract diatoms has been applied in organs taken from ten wildlife animals belonging three species of mammals (a deer, a roe and five otters) and one species of birds (two magpies). Only four carcasses were recovered from aquatic environments (bath trough for animals, bathtub irrigation, river) and only in three cases out of ten that the cause of death was ruled out as drowning. In seven cases, the death was due to other causes: gunshot injuries for one otter, blunt trauma for a magpie, and traumatic injuries followed by motor vehicle collision in other four otters and a roe. Post-mortem examination was performed in all carcasses. The diatom test protocol was performed according to the Italian guidelines for analysis of benthic diatoms for ecological status assessment of inland waters. Five grams of lung, liver, and kidney was taken from all the animal carcasses. In some cases, additional tissue samples were also available among which brain, heart, spleen, and bone marrow. In all four cases found in water, the drowning medium was also available. Hydrogen peroxide (H2O2) digestion was performed, and identification of 21 genera of diatoms was obtained. The method proved to be suitable for the identification of diatoms in the organs of the animals drowned supporting the final diagnosis of drowning. Only in otters, all died for causes other than drowning, diatoms did not prove to be suitable for the diagnosis of drowning since their presence in the internal organs was mainly related to their main diet based on fishmeal. The authors believe that this first trial is very promising, and the results suggest that diatom test can be easily applied in forensic veterinary context.

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

Tuberculosis: medico-legal aspects.

Tuberculosis is a diffusive infectious disease whose typical behaviour differentiates it from other infectious diseases spread by human-to-human transmission (flu, chicken pox, cholera, etc.) that follow a classic epidemic pattern. Indeed, in the presence of a known source of Koch bacilli that is capable of spreading the bacteria by air, not all exposed individuals inhale the bacteria, not all those who inhale them absorb them, not all those who absorb the bacteria are unable to eliminate them, not all who are able to eliminate them do so using delayed hypersensitivity, not all those who react with delayed hypersensitivity suffer lasting tissue damage (among other things, minor), not all who suffer tissue damage have anatomical sequelae, and not all those who have anatomical sequelae, however minimal, become carriers of bacilli in the latent period. The vast majority (90-95%) of the latter - which are in any case a portion, not the totality of those exposed - remain asymptomatic throughout their lives and never develop active tuberculosis. Based on these biological characteristics and the legal concepts of "epidemic" and "disease," it becomes highly problematic, if not impossible, to assert both that tuberculosis can cause events of sufficient magnitude to be associated with the crime of "epidemic," and that the mere diagnosis of a latent tuberculosis infection is sufficient to assume the presence of an illness legally prosecutable in criminal proceedings or a disability prosecutable in civil proceedings. Furthermore, clinically apparent tuberculosis is a temporarily-and in some cases permanently-disabling condition, and in certain work environments, even with the difficulties caused by the lack of available effective diagnostic tools and the insidious behaviour of the disease in the early stages, targeted monitoring to identify other persons who may become ill is appropriate.

Biomarker discovery by plasma proteomics in familial Brugada Syndrome.

Brugada Syndrome (BS) is a polygenic inherited cardiac disease characterized by life-threatening arrhythmias and high incidence of sudden death. In this study, two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (LC-MS/MS) was used to investigate specific changes in the plasma proteome of BS patients and family members sharing the same gene mutation (SCN5AQ1118X), with the aim to identify novel disease biomarkers. Our data demonstrate that the levels of several proteins were significantly altered in BS patients compared with controls. In particular, apolipoprotein E, prothrombin, vitronectin, complement-factor H, vitamin-D-binding protein, voltage-dependent anion-selective channel protein 3 and clusterin were considerably increased in plasma sample of BS patients, whereas alpha-1-antitrypsin, fibrinogen and angiotensinogen were considerably decreased; moreover, post-translational modifications of antithrombin-III were detected in all affected individuals. On the light of these results, we hypothesize that these proteins might be considered as potential markers for the identification of disease status in BS.

Patterns of Y-STR variation in Italy.

The 17 Y-chromosomal short tandem repeats (STRs) included in the AmpFlSTR Yfiler Amplification Kit (AB Applied Biosystems) (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1) were typed in 292 samples from seven Italian regions. Population comparisons with other European samples were undertaken; for this purpose, two databases were collated from the literature: (a) 19 population samples including >2900 Yfiler profiles, and (b) 67 population samples including >15,000 minimum haplotype profiles. A total of 276 different Yfiler haplotypes were observed in Italy, and only one of them was shared among our seven population samples. The overall haplotype diversity (0.9996) was comparable to other European samples. AMOVA indicates that among population variance depends on the amount of Y-STRs used, being higher when using minimal haplotypes. This is probably due to the fact that Yfiler profiles are represented by singleton haplotypes in all the population samples raising the diversity values to the maximum theoretical value. AMOVA results seems to depend even more strongly on the amount of population samples used, the among population variance in Italy ranging from 2.82% to 11.03% (using 15 and 32 Italian populations samples, respectively). Variance is not as strongly stratified geographically within Italy, although it is notorious that latitude is more important than longitude in the distribution of variance. The results also indicated that Italy is less stratified than other European samples. The present study contributes to enrich the Y-chromosome databases regarding high-resolution Y-chromosome data sets and demonstrates that extended Y-STR profiles substantially increases the discriminatory capacity in individual identification for forensic purposes.

Allele frequencies of fifteen STRs in a representative sample of the Italian population.

Fifteen autosomal short tandem repeat (STR) markers (D3S1358, HUMTH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, HUMvWA, D8S1179, HUMTPOX and FGA) were analyzed in more than 400 unrelated individuals from nine different areas of Italy. After Bonferroni correction, no evidence of population structure was identified, either by considering each population as independent or by combining populations according to their geographic origin (North, Central and South of Italy). Forensic indexes were estimated considering all samples together. Combined power of discrimination (PD) and combined power of exclusion (PE) for the 15 tested STR loci were 0.9999999997 and 0.964708775, respectively. Low genetic distances were found between our data and those previously published for other neighboring European populations.

Population structure in the Mediterranean basin: a Y chromosome perspective.

The Mediterranean region has been characterised by a number of pre-historical and historical demographic events whose legacy on the current genetic landscape is still a matter of debate. In order to investigate the degree of population structure across the Mediterranean, we have investigated Y chromosome variation in a large dataset of Mediterranean populations, 11 of which are first described here. Our analyses identify four main clusters in the Mediterranean that can be labelled as North Africa, Arab, Central-East and West Mediterranean. In particular, Near Eastern samples tend to separate according to the presence of Arab Y chromosome lineages, suggesting that the Arab expansion played a major role in shaping the current genetic structuring within the Fertile Crescent.

1. Dissection of the vertebral artery during a basketball game: a case report.

Dissection of the vertebral artery represents a significant potential cause of strokes among young and middle-aged people. We present a case of dissection of the vertebral artery observed at the forensic autopsy of a professional basketball player who was suddenly taken ill during a practice game and died seven days after the event. A medico-legal autopsy was performed in order to establish an essential causal relationship between arterial dissection and basketball activity. No preceding trauma possibly contributing to the dissection was discovered. The histology of the arterial wall did not show specific alterations in the adventitia, the muscular layer, or the intima. In addition, the toxicological examination excluded any effects of drugs of abuse. We describe macroscopic and microscopic findings in the autopsy, and discuss possible mechanisms of the arterial rupture as well as the autopsy procedure in similar cases.

"Ancient" protocols for the crime scene? Similarities and differences between forensic genetics and ancient DNA analysis.

We provide a short overview on some current issues in the fields of forensic genetics and ancient DNA (aDNA) analysis. We discuss about the existence of the possible points of contact between the two disciplines, in terms of open problems and the inherent approach to their solution. We mainly focus on the problem of results authentication, its theoretical and technical aspects.

Non-fatherhood or mutation? A probabilistic approach to parental exclusion in paternity testing.

The occurrence of germline mutations at microsatellite loci poses problems in ascertaining non-fatherhood status in paternity testing. We describe the appropriate probabilistic analysis for computing the likelihood ratio in favour of paternity while allowing for mutation, for all 18 relevant combinations of seemingly incompatible parental genotypes. We allow arbitrary and possibly different mutation rates in paternal and maternal germlines. We describe a stationary mutation model for expressing the required allele-specific transition mutation rates in terms of overall mutation rates, and compare the likelihood ratios calculated from this and from other mutation models suggested in the literature. We also show how to derive an upper bound on the likelihood ratio, depending only on the overall mutation rate.

Y-chromosome haplotypes in Italy: the GEFI collaborative database.

A sample of 1176 males from 10 Italian regions have been typed for DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, and DYS385. Individual haplotype data are available on line. A low degree of variation is present among regions. Use of this database is specifically recommended for forensic applications in Italy.

Potassium concentration differences in the vitreous humour from the two eyes revisited by microanalysis with capillary electrophoresis.

This paper presents a study of the variability of potassium concentrations in the vitreous humour of the two eyes of the same body at identical postmortem interval. The study was carried out by collecting microsample amounts (50 microl) of vitreous humour and by using an original method of capillary electrophoresis with indirect detection. The electrophoretic separations were carried out in a pH 4.5 running buffer composed of 5 mmol/l imidazole, 5 mmol/l 18-crown-6 ether and 6 mmol/l alpha-hydroxybutyric acid. Detection was by indirect UVabsorption at 214 nm. Vitreous humour samples were collected from 57 medico-legal autopsies or external examinations of cases of sudden natural or violent deaths. All samples prior to analysis were diluted 1:20 with a 40 microg/ml aqueous solution of barium, the used internal standard, and finally injected by nitrogen pressure. The mean concentrations of potassium measured in the two eyes of all the cases included in the present study ranged from 4.1 to 23.5 mmol/l with the postmortem interval values varying from 7 to 144 h. A highly significant (P<0.0001) linear correlation was found between these two parameters as described by the equation: y=0.1698x+2.3587, r=0.89. The intra-eye variability of potassium concentrations was low with an average RSD of 3.89% (+/- 1.83 SD) (48 eyes, five samples per eye). No statistically significant difference was found between the potassium concentrations in the two eyes of the same subject in a group of 24 cases, excepting a single case.

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes.

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called "minimal") haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any data. In view of its collaborative character, and in order to put YHRD to its intended use, viz. the support of forensic caseworkers in their routine decision-making process, the database has been made publicly available via the Internet in February 2000. Online searches for complete or partial Y-STR haplotypes from evidentiary or non-probative material can be performed on a non-commercial basis, and yield observed haplotype counts as well as extrapolated population frequency estimates. In addition, the YHRD website provides information about the quality control test, genotyping protocols, haplotype formats and informativity, population genetic analysis, literature references, and a list of contact addresses of the contributing laboratories.

A predominantly indigenous paternal heritage for the Austronesian-speaking peoples of insular Southeast Asia and Oceania.

Modern humans reached Southeast Asia and Oceania in one of the first dispersals out of Africa. The resulting temporal overlap of modern and archaic humans-and the apparent morphological continuity between them-has led to claims of gene flow between Homo sapiens and H. erectus. Much more recently, an agricultural technology from mainland Asia spread into the region, possibly in association with Austronesian languages. Using detailed genealogical study of Y chromosome variation, we show that the majority of current Austronesian speakers trace their paternal heritage to Pleistocene settlers in the region, as opposed to more-recent agricultural immigrants. A fraction of the paternal heritage, however, appears to be associated with more-recent immigrants from northern populations. We also show that the northern Neolithic component is very unevenly dispersed through the region, with a higher contribution in Southeast Asia and a nearly complete absence in Melanesia. Contrary to claims of gene flow (under regional continuity) between H. erectus and H. sapiens, we found no ancestral Y chromosome lineages in a set of 1,209 samples. The finding excludes the possibility that early hominids contributed significantly to the paternal heritage of the region.

A repository of 14 PCR-loci Italian gene frequencies in the World Wide Web.

A collection of 6830 typing results produced by the Immunohematology Laboratory at the UCSC, pertaining to 11 STRs (FES/FPS, vWA31, HUMTH01, F13A1, MBP, D21S11, D7S460, D18S51, CD4, TPOX, CSF1PO) and 3 AmpFLPs (D1S80, APO-B, COL2A1), is publicly available as an electronic archive at a website.

Field-amplified sample stacking capillary zone electrophoresis applied to the analysis of opiate drugs in hair.

The present paper describes the methodological optimization and validation of capillary zone electrophoresis (CZE) for the determination of major opiates (morphine, codeine, 6-monoacetylmorphine, acetylcodeine, heroin) in hair samples by using a field-amplified sample stacking injection before the separation in a binary running buffer (0.1 M sodium phosphate, pH 2.5, with 40% ethylene glycol). The applied potential was 20 kV, at 25 degrees C. Detection was by UV absorption at the fixed wavelength of 214 nm or by recording the full spectrum between 190-400 nm, thus improving the analytical selectivity and identification power of CZE. Hair samples were liquid/liquid extracted; dried extracts, reconstituted with a low-conductivity solvent (0.1 mM phosphoric acid, with 80% 1-propanol), were injected by electromigration at 10 kV for 99 s, after a 0.5 mm plug of water. Under the described conditions, the limit of detection (with a signal-to-noise ratio of 3) in hair extracts was 100 pg/mL for codeine, 75 pg/mL for morphine and 6-monoacetylmorphine (6-MAM), 150 pg/mL for ethylmorphine, and 0.75 ng/mL for acetylcodeine and heroin. The precision of the method was validated for standards in pure solution by using internal standardization, providing for intraday and day-to-day assays, in terms of migration times, relative standard deviation (RSD) values < or = 0.2%, and in terms of peak areas, RSD values <5.71%.