From unknown to known: Identification of the remains at the mausoleum of fosse Ardeatine.

During the Second World War, on 24th March 1944, 335 Italians were massacred near Rome by the occupying forces of Nazi Germany. Four months later forensic examination led to the identification of 323 out of 335 victims. After approximately 60 years, the identification of the remaining unidentified twelve victims began with anthropological and genetic analysis carried out by a team of Italian forensic experts. Anthropological analysis was performed in field in order to confirm the sex of each victim and verify the presence of only one individual in each grave for a correct sampling. Selected bone fragments for each individual were then collected and transferred to the laboratory for genetic analysis. Although the anthropological ante mortem information was limited, morphological and metrical data was collected for a possible future identification of the victims. Subsequently, the typing of autosomal loci, Y-STR and mtDNA D-loop region of all bone and available reference samples was conducted. LR and cumulative LRs obtained from autosomal STR and Y-STR results confirmed the alleged relationship between three victims and their relatives with values over 104 (one sample) and 106 (two samples). Therefore, the genetic analysis offered the families the possibility of replacing the number of the grave with the name of the victim.

A genetic portrait of the South-Eastern Carpathians based on autosomal short tandem repeats loci used in forensics.

OBJECTIVES: This work aimed to describe the genetic landscape of the Balkan Peninsula, as revealed by STR markers commonly used in forensics and spatial methods specifically developed for genetic data. METHODS: We generated and analyzed 16 short tandem repeats (STRs) autosomal genotypes in 287 subjects from ten administrative/geographical regions of Eastern Europe (Romania and the Republic of Moldova). We report estimates of the allele frequencies in these sub-populations, their fixation indexes, and use these results to complement previous spatial analyses of Southern Europe. RESULTS: In seven out of ten analyzed regional samples the heterozygosity, averaged across loci, was lower than expected. The average Fis was 0.011. Among the 16 loci, five returned a significant fixation index Fst. The composite Fst across the 16 loci, among the 10 regional samples, was 0.00417, a figure twice as large as that obtained with the same markers across the entire Northern Mediterranean. The first spatial principal component (sPC1) returned the picture of a Central-European pattern of frequencies for the Carpathians, which extended to the Southern boundary of the Balkan Peninsula. However, the 8 alleles extracted by sPC1 returned a picture of a strong reduction of the migration rate in the Carpathian region, mostly between the inner locations. CONCLUSIONS: Our results revealed an unexpected heterogeneity in the area. We believe that populations from some regions will require treatment as distinct entities when considered in forensic applications.

Exploring the mitochondrial DNA variability of the Amazonian Yanomami.

OBJECTIVES: The aim of this study was to explore the mitochondrial variability in the Yanomami population to reconstruct its demographic history and explore its genetic composition in relation to its cultural and linguistic features. METHODS: A total of 174 human head hair shafts -collected in 1958- belonging to individuals from a Yanomami group living in Santa Isabel, Brazil, were analyzed. Automated extraction of the hairs was performed, and several methods were applied to optimize the analysis of the degraded DNA. The mtDNA hypervariable segments I-II, along with the 9-bp COII-tRNALys deletion, were investigated. Using published data from the Yanomami and other Amazonian populations, several statistical analyses were carried out to explore the genetic variability within the study population. RESULTS: Ninety eight percent of the mitochondrial DNA (mtDNA) sequences analyzed belonged to Native American haplogroups, while 2% belonged to African haplogroups. Compared with the Yanomami groups previously studied, the Santa Isabel sample seemed more genetically similar to other Amazonian populations. CONCLUSIONS: Among the Yanomami samples studied to date, the Santa Isabel Yanomami show a higher genetic heterogeneity. This could be due to gene flow with non-Yanomami populations, as well as to the introduction of new mitochondrial haplotypes by gold miners. In both cases, the geographic location of Santa Isabel might have made this Yanomami village less isolated than the others, suggesting that the Rio Negro played a central role in increasing its genetic variability. On the whole, the Yanomami were quite genetically diversified, probably mirroring their great linguistic heterogeneity. Am. J. Hum. Biol. 28:846-856, 2016. © 2016Wiley Periodicals, Inc.

Forensic genetic value of a 27 Y-STR loci multiplex (Yfiler(®) Plus kit) in an Italian population sample.

The analysis of Y chromosome short tandem repeat (Y-STR) haplotypes provides important information that can be used for investigative purposes and in population studies. The Yfiler(®) Plus PCR Amplification kit (Yfiler(®) Plus, Thermo Fisher Scientific, Waltham, MA, USA) allows the multiplex amplification of 27 Y-STRs, including 7 rapidly mutating markers (RM Y-STRs). In this study, 203 unrelated males from Italy, which were subdivided into 4 different geographical groups (North, Center, South and Sardinia) were analyzed. Several intra-population diversity indexes were computed and compared to those obtained using only loci either from the minimal haplotype or the 17-plex (Yfiler(®), Thermo Fisher Scientific, Waltham, MA, USA). In addition, inter-population diversity analysis (RST) among the four Italian samples was performed. The same analysis was also used to compare the Italian sub-sets to other European populations where the Yfiler(®) Plus haplotype frequency data were available. The Sardinians were significantly differentiated from the other three Italian groups, thus requiring a specific sub-national Y-STR haplotype database. The Yfiler(®) Plus kit showed a high power of discrimination which is useful for criminal investigations, principally due to the inclusion of RM Y-STRs.

Traces of forgotten historical events in mountain communities in Central Italy: A genetic insight.

OBJECTIVES: Analysis of human genetic variation in mountain communities can shed light on the peopling of mountainous regions, perhaps revealing whether the remote geographic location spared them from outside invasion and preserved their gene pool from admixture. In this study, we created a model to assess genetic traces of historical events by reconstructing the paternal and maternal genetic history of seven small mountain villages in inland valleys of Central Italy. METHODS: The communities were selected for their geographic isolation, attested biodemographic stability, and documented history prior to the Roman conquest. We studied the genetic structure by analyzing two hypervariable segments (HVS-I and HVS-II) of the mtDNA D-loop and several informative single nucleotide polymorphisms (SNPs) of the mtDNA coding region in 346 individuals, in addition to 17 short tandem repeats (STRs) and Y-chromosome SNPs in 237 male individuals. RESULTS: For both uniparental markers, most of the haplogroups originated in Western Europe while some Near Eastern haplogroups were identified at low frequencies. However, there was an evident genetic similarity between the Central Italian samples and Near Eastern populations mainly in the male genetic pool. CONCLUSIONS: The samples highlight an overall European genetic pattern both for mtDNA and Y chromosome. Notwithstanding this scenario, Y chromosome haplogroup Q, a common paternal lineage in Central/Western Asia but almost Europe-wide absent, was found, suggesting that Central Italy could have hosted a settlement from Anatolia that might be supported by cultural, topographic and genetic evidence.

Molecular identification of vaginal fluid by microbial signature.

The discrimination of body fluids in forensic examinations can play an important role in crime scene reconstruction. Conventional methods rely on the detection of antigens or enzymatic activity, limiting detection sensitivity and specificity, particularly on old forensic samples. Methods based on human RNA analysis are not easily applicable to samples exposed to harsh and degrading environments. An alternative approach based on the identification of prokaryotic genomes was developed. Specific bacterial communities are characteristic typical of different human non-sterile body fluids: the molecular characterization of a microbial signature, and not the typing of single bacterial species, can effectively lead to univocal identification of these fluids. A multiplex real time PCR assay was developed using oligonucleotide mixtures targeting genomes specific for a selected group of bacteria. Microflora DNA (mfDNA) was extracted from vaginal, oral and fecal clinical swabs. In addition forensic samples were processed. Vaginal samples showed a strong specific signal for bacteria of the female genital tract. Oral samples clearly showed signal for bacteria present in saliva, and in fecal samples the main signal was from Enterococcaceae. Vaginal casework samples showed results comparable to freshly collected ones; moreover the DNA extracted was successfully used for STR typing. Also mixtures of body fluids were analyzed, providing a microbiological signature compatible with the presence of microbes of oral, fecal and vaginal origin. The presented method can be useful in identifying biological fluids, and it is based on DNA technologies already available in forensic laboratories and feasible for further high throughput automation.

The peopling of Europe and the cautionary tale of Y chromosome lineage R-M269.

Recently, the debate on the origins of the major European Y chromosome haplogroup R1b1b2-M269 has reignited, and opinion has moved away from Palaeolithic origins to the notion of a younger Neolithic spread of these chromosomes from the Near East. Here, we address this debate by investigating frequency patterns and diversity in the largest collection of R1b1b2-M269 chromosomes yet assembled. Our analysis reveals no geographical trends in diversity, in contradiction to expectation under the Neolithic hypothesis, and suggests an alternative explanation for the apparent cline in diversity recently described. We further investigate the young, STR-based time to the most recent common ancestor estimates proposed so far for R-M269-related lineages and find evidence for an appreciable effect of microsatellite choice on age estimates. As a consequence, the existing data and tools are insufficient to make credible estimates for the age of this haplogroup, and conclusions about the timing of its origin and dispersal should be viewed with a large degree of caution.

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.

Y chromosome haplotypes in Central-South Italy: implication for reference database.

One hundred and fifty individuals have been sampled across Central-South Italy and genotyped for Y chromosome STRs by PowerPlex Y system. Comparison with previous Italian databases revealed that majority of Y chromosome variation still need to be sampled. Identification of locus duplications, distribution of genetic variation and firstly identified alleles point to the necessity of more focused sampling strategies for reference databases.

Applicability of Nanotrap Sg as a semen detection kit before male-specific DNA profiling in sexual assaults.

A commercially available semen detection kit, Nanotrap Sg, which employs a one-step detection test based on immunochromatographic assay for the semenogelin protein, was evaluated for profiling male-specific DNA in sexual assault casework samples. While semen diluted with phosphate-buffered saline held and kept at 4 degrees C for 1 week showed a relatively strong signal intensity with Nanotrap Sg, the signal intensity was decreased by dilution after storage at 4 degrees C or freezing and thawing repeated more than three times. The reproducibility of Nanotrap Sg was tested on a total of 174 sexual assault casework samples from three forensic laboratories using intra- and interassay and no variation was observed in the semenogelin (Sg) signal. The positive signal ratio was 12.6% higher for prostate-specific antigen immunochromatographic membrane tests than Nanotrap Sg. Although spermatozoa were not confirmed in 61 (35%) out of 174 samples, Sg-positive signals could be detected from 41 (67%) of the 61 samples. Female genetic profiles could be observed in 95% of the samples, which tested negative for Sg on the Nanotrap Sg test, but no male genetic profiles could be observed. These results suggest that Nanotrap Sg can positively identify samples containing male DNA even in the absence of detectable intact spermatozoa. Further, Sg-positive signals identified samples for which male-specific DNA profiling could be performed, even if no sperm could be detected from the sample. The potential of Nanotrap Sg for identifying forensic samples with male-specific DNA was clearly demonstrated.

Alpha-amylase kinetic test in bodily single and mixed stains.

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.