Internal validation and improvement of mitochondrial genome sequencing using the Precision ID mtDNA Whole Genome Panel.

With the recent advances in next-generation sequencing (NGS), mitochondrial whole-genome sequencing has begun to be applied to the field of the forensic biology as an alternative to the traditional Sanger-type sequencing (STS). However, experimental workflows, commercial solutions, and output data analysis must be strictly validated before being implemented into the forensic laboratory. In this study, we performed an internal validation for an NGS-based typing of the entire mitochondrial genome using the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific) on the Ion S5 sequencer (Thermo Fisher Scientific). Concordance, repeatability, reproducibility, sensitivity, and heteroplasmy detection analyses were assessed using the 2800 M and 9947A standard control DNA as well as typical casework specimens, and results were compared with conventional Sanger sequencing and another NGS sequencer in a different laboratory. We discuss the strengths and limitations of this approach, highlighting some issues regarding noise thresholds and heteroplasmy detection, and suggesting solutions to mitigate these effects and improve overall data interpretation. Results confirmed that the Precision ID Whole mtDNA Genome Panel is highly reproducible and sensitive, yielding useful full mitochondrial DNA sequences also from challenging DNA specimens, thus providing further support for its use in forensic practice.

Insights into Punic genetic signatures in the southern necropolis of Tharros (Sardinia).

BACKGROUND: Phoenician and Punic expansions have been protagonists of intense trade networks and settlements in the Mediterranean Sea. AIMS: The maternal genetic variability of ancient Punic samples from the Sardinian necropolis of Tharros was analysed, with the aim to explore genetic interactions and signatures of past population events. SUBJECTS AND METHODS: The mtDNA HVS-I and coding region SNPs were analysed in 14 Punic samples and 74 modern individuals from Cabras and Belvì (for which the HVS-II region was also analysed). The results were compared with 5,590 modern Euro-Mediterranean sequences and 127 ancient samples. RESULTS: While contemporary groups fall within the genetic variability of other modern Sardinians, our Punic samples reveal proximity to present-day North-African and Iberian populations. Furthermore, Cabras and Belvì cluster mainly with pre-Phoenician groups, while samples from Tharros project with other Punic Sardinian individuals. CONCLUSION: This study provides the first preliminary insights into the population dynamics of the Punic site of Tharros. While the number of currently available samples does not allow definitive investigation of the connection with indigenous Sardinian groups, our results seem to confirm internal migratory phenomena in the central-western Mediterranean and female participation in the Punic mobility.

The genetic legacy of the Yaghnobis: A witness of an ancient Eurasian ancestry in the historically reshuffled central Asian gene pool.

OBJECTIVES: The Yaghnobis are an ethno-linguistic minority historically settled along the Yaghnob River in the Upper-Zarafshan Valley in Tajikistan. They speak a language of Old Sogdian origin, which is the only present-day witness of the Lingua Franca used along the Silk Road in Late Antiquity. The aim of this study was to reconstruct the genetic history of this community in order to shed light on its isolation and genetic ancestry within the Euro-Asiatic context. MATERIALS AND METHODS: A total of 100 DNA samples were collected in the Yaghnob and Matcha Valleys during several expeditions and their mitochondrial, Y-chromosome and autosomal genome-wide variation were compared with that from a large set of modern and ancient Euro-Asiatic samples. RESULTS: Findings from uniparental markers highlighted the long-term isolation of the Yaghnobis. Mitochondrial DNA ancestry traced an ancient link with Middle Eastern populations, whereas Y-chromosome legacy showed more tight relationships with Central Asians. Admixture, outgroup-f3, and D-statistics computed on autosomal variation corroborated Y-chromosome evidence, pointing respectively to low Anatolian Neolithic and high Steppe ancestry proportions in Yaghnobis, and to their closer affinity with Tajiks than to Iranians. DISCUSSION: Although the Yaghnobis do not show evident signs of recent admixture, they could be considered a modern proxy for the source of gene flow for many Central Asian and Middle Eastern groups. Accordingly, they seem to retain a peculiar genomic ancestry probably ascribable to an ancient gene pool originally wide spread across a vast area and subsequently reshuffled by distinct demographic events occurred in Middle East and Central Asia.

Genetic variability of CYP2D6, CYP2B6, CYP2C9 and CYP2C19 genes across the Italian Peninsula.

BACKGROUND: Environmental conditions and past migratory events may have shaped genetic heterogeneity of clinically relevant enzymes involved in the phase I metabolism of the most common therapeutic drugs. AIM: To investigate the genetic variability of CYP2D6, CYP2B6, CYP2C19 and CYP2C9 across the Italian Peninsula, by sampling only ancestrally and geographically homogeneous individuals from northern, central and southern Italy. SUBJECTS AND METHODS: A total of 25 SNPs were genotyped in 174 unrelated Italian individuals by means of multiplex PCR and minisequencing reactions. CYP2D6 genotypic data were used to predict phenotypes and the phylogenetic relationships among reconstructed haplotypes were represented by means of a Median Joining Network. RESULTS: Pairwise Fisher Exact tests revealed significant differences between northern and southern Italy in the distribution of CYP2C19 genotypes, with the CYP2C19*2 allele appearing over-represented in northern Italy. Likewise, significant differences in the distribution of CYP2D6 genotypes (*4/*3, *4/*4 and *6/*4) responsible for the poor metabolizer phenotype were observed in northern with respect to both central and southern Italy. CONCLUSIONS: The north-south structuring pattern showed by CYP2D6 and CYP2C19 underline how a deeper knowledge of the geographic distribution of alleles may improve clinical practice and help to avoid hypothetical bias in drug trials.

Iron Age Italic population genetics: the Piceni from Novilara (8th-7th century BC).

BACKGROUND: Archaeological data provide evidence that Italy, during the Iron Age, witnessed the appearance of the first communities with well defined cultural identities. To date, only a few studies report genetic data about these populations and, in particular, the Piceni have never been analysed. AIMS: To provide new data about mitochondrial DNA (mtDNA) variability of an Iron Age Italic population, to understand the contribution of the Piceni in shaping the modern Italian gene pool and to ascertain the kinship between some individuals buried in the same grave within the Novilara necropolis. SUBJECTS AND METHODS: In a first set of 10 individuals from Novilara, we performed deep sequencing of the HVS-I region of the mtDNA, combined with the genotyping of 22 SNPs in the coding region and the analysis of several autosomal markers. RESULTS: The results show a low nucleotide diversity for the inhabitants of Novilara and highlight a genetic affinity of this ancient population with the current inhabitants of central Italy. No family relationship was observed between the individuals analysed here. CONCLUSIONS: This study provides a preliminary characterisation of the mtDNA variability of the Piceni of Novilara, as well as a kinship assessment of two peculiar burials.

Pope Leo XII's death: the twist to a longstanding dispute by novel historical documents and paleopathographic analysis.

Although the practice of autopsy on the Pope's corpse was performed from the 16th century, autopsy reports are only rarely analysed, and never with the aim of investigating the real causes of the death from a concomitant medical and historical point of view. Here, for the first time, we report on the discovery of new unpublished documents from the Vatican Secret Archives and their investigation by a scientific and inter-disciplinary approach. This analysis allows us to draw new conclusions on the true cause of Leo XII's mysterious death. His sudden death, that occurred on February 10th, 1829 after a short illness, particularly struck the public. Suspicions of poisoning or surgeon's guilt or inexperience and even the shadow of a venereal disease, contributed to create a "black legend" on his pontificate and death. On the contrary, the present paleopathographic analysis points toward a new conclusion. The regular use of catheterization with a silver syringe provided an easy access for bacterial superinfection, confirmed by the observed early emphysematous stage of the corpse. So, the most substantiated hypothesis concerning the cause of Leo XII's death indicates a severe form of sepsis, exacerbated by a weakened state due to chronic hemorrhoids.

Old wild wolves: ancient DNA survey unveils population dynamics in Late Pleistocene and Holocene Italian remains.

BACKGROUND: The contemporary Italian wolf (Canis lupus italicus) represents a case of morphological and genetic uniqueness. Today, Italian wolves are also the only documented population to fall exclusively within the mitochondrial haplogroup 2, which was the most diffused across Eurasian and North American wolves during the Late Pleistocene. However, the dynamics leading to such distinctiveness are still debated. METHODS: In order to shed light on the ancient genetic variability of this wolf population and on the origin of its current diversity, we collected 19 Late Pleistocene-Holocene samples from northern Italy, which we analyzed at a short portion of the hypervariable region 1 of the mitochondrial DNA, highly informative for wolf and dog phylogenetic analyses. RESULTS: Four out of the six detected haplotypes matched the ones found in ancient wolves from northern Europe and Beringia, or in modern European and Chinese wolves, and appeared closely related to the two haplotypes currently found in Italian wolves. The haplotype of two Late Pleistocene samples matched with primitive and contemporary dog sequences from the canine mitochondrial clade A. All these haplotypes belonged to haplogroup 2. The only exception was a Holocene sample dated 3,250 years ago, affiliated to haplogroup 1. DISCUSSION: In this study we describe the genetic variability of the most ancient wolf specimens from Italy analyzed so far, providing a preliminary overview of the genetic make-up of the population that inhabited this area from the last glacial maximum to the Middle Age period. Our results endorsed that the genetic diversity carried by the Pleistocene wolves here analyzed showed a strong continuity with other northern Eurasian wolf specimens from the same chronological period. Contrarily, the Holocene samples showed a greater similarity only with modern sequences from Europe and Asia, and the occurrence of an haplogroup 1 haplotype allowed to date back previous finding about its presence in this area. Moreover, the unexpected discovery of a 24,700-year-old sample carrying a haplotype that, from the fragment here obtained, falls within the canine clade A, could represent the oldest evidence in Europe of such dog-rich clade. All these findings suggest complex population dynamics that deserve to be further investigated based on mitochondrial or whole genome sequencing.

Absence of mutations in the human interferon alpha-2b gene in workers chronically exposed to ionising radiation.

Individuals chronically exposed to low-level ionising radiation (IR) run the risk of harmful and long-term adverse health effects, including gene mutations and cancer development. The search for reliable biomarkers of IR exposure in human population is still of great interest, as they may have a great implementation potential for the surveillance of occupationally exposed individuals. In this context, and considering previous literature, this study aimed to identify mutations in the human interferon alpha-2b (hIFNα-2b) as a potential biomarker of occupational chronic low-dose IR exposure linking low-IR exposure to the effects on haematopoiesis and reduced immunity. The analysis was performed in the genomic DNA of 51 uranium miners and 38 controls from Kazakhstan, and in 21 medical radiology workers and 21 controls from Italy. hIFNα-2b gene mutations were analysed with the real-time polymerase chain reaction (PCR) or Sanger sequencing. However, none of the investigated workers had the hIFNα-2b mutation. This finding highlights the need for further research to identify biomarkers for early detection of health effects associated with chronic low-dose IR exposure.

Poisoning histories in the Italian renaissance: The case of Pico Della Mirandola and Angelo Poliziano.

Giovanni Pico della Mirandola and Angelo Poliziano were two of the most important humanists of the Italian Renaissance. They died suddenly in 1494 and their deaths have been for centuries a subject of debate. The exhumation of their remains offered the opportunity to study the cause of their death through a multidisciplinary research project. Anthropological analyses, together with documentary evidences, radiocarbon dating and ancient DNA analysis supported the identification of the remains attributed to Pico. Macroscopic examination did not reveal paleopathological lesions or signs related to syphilis. Heavy metals analysis, carried out on bones and mummified tissues, showed that in Pico's remains there were potentially lethal levels of arsenic, supporting the philosopher's poisoning theory reported by documentary sources. The arsenic concentrations obtained from analysis of Poliziano's remains, are probably more related to an As chronic exposure or diagenetic processes rather than poisoning.

Multiple selective events at the PRDM16 functional pathway shaped adaptation of western European populations to different climate conditions.

Several studies highlighted the role of climate in shaping many human evolutionary processes. This occurred even in relatively recent times, having affected various human phenotypic traits, among which metabolic processes that orchestrate absorption and accumulation of substances to maintain energy homeostasis, that is critical for the survival of individuals in high energy-expenditure environments. To date, most researches have focalized on detection of climatic influence on SNPs' frequency in populations exposed to extreme environmental conditions or by comparing variation patterns between populations from different continents. In this study, we instead explored the genetic background of distinct western European human groups at loci involved in nutritional and thermoregulation processes, to test whether patterns of differential local adaptation to environmental conditions could be appreciated also at a lower geographical scale. Taking advantage from the 1000 Genomes Project data, genetic information for 21 genes involved in nutritional and thermoregulation processes was analysed for three western European populations. The applied Anthropological Genetics methods pointed to appreciable differentiation between the examined groups especially for the PRDM16 gene. Moreover, several neutrality tests suggested that balancing selection has acted on different regions of the gene in people from Great Britain, as well as that more recent positive selection could have also targeted some PRDM16 SNPs in Finn and Italian populations. These series of adaptive footprints are plausibly related to climate variability in both ancient and relatively recent times. Since this locus is involved in thermoregulation mechanisms and adipogenesis, local adaptations mediated by a pathway related to the brown adipose tissue activity could have evolved in response to changing cold temperature exposures of such populations.