Southern Africa's Great Escarpment as an amphitheater of climate-driven diversification and a buffer against future climate change in bats.

Hosting 1460 plant and 126 vertebrate endemic species, the Great Escarpment (hereafter, Escarpment) forms a semi-circular "amphitheater" of mountains girdling southern Africa from arid west to temperate east. Since arid and temperate biota are usually studied separately, earlier studies overlooked the biogeographical importance of the Escarpment as a whole. Bats disperse more widely than other mammalian taxa, with related species and intraspecific lineages occupying both arid and temperate highlands of the Escarpment, providing an excellent model to address this knowledge gap. We investigated patterns of speciation and micro-endemism from modeled past, present, and future distributions in six clades of southern African bats from three families (Rhinolophidae, Cistugidae, and Vespertilionidae) having different crown ages (Pleistocene to Miocene) and biome affiliations (temperate to arid). We estimated mtDNA relaxed clock dates of key divergence events across the six clades in relation both to biogeographical features and patterns of phenotypic variation in crania, bacula and echolocation calls. In horseshoe bats (Rhinolophidae), both the western and eastern "arms" of the Escarpment have facilitated dispersals from the Afrotropics into southern Africa. Pleistocene and pre-Pleistocene "species pumps" and temperate refugia explained observed patterns of speciation, intraspecific divergence and, in two cases, mtDNA introgression. The Maloti-Drakensberg is a center of micro-endemism for bats, housing three newly described or undescribed species. Vicariance across biogeographic barriers gave rise to 29 micro-endemic species and intraspecific lineages whose distributions were congruent with those identified in other phytogeographic and zoogeographic studies. Although Köppen-Geiger climate models predict a widespread replacement of current temperate ecosystems in southern Africa by tropical or arid ecosystems by 2070-2100, future climate Maxent models for 13 bat species (all but one of those analyzed above) showed minimal range changes in temperate species from the eastern Escarpment by 2070, possibly due to the buffering effect of mountains to climate change.

Unveiling the genetic structure of pig population in a Himalayan state Uttarakhand through microsatellite and mitochondrial DNA analyses.

This study traced the maternal lineage of the domestic swine populations using mitochondrial DNA control region markers and genetic diversity using microsatellite markers in Uttarakhand, an Indian state situated at the foothills of the world's youngest (geo-dynamically sensitive) mountain system, "the Himalayas". Analysis of 68 maternally unrelated individuals revealed 20 haplotypes. The maternal signature of the Pacific, Southeast Asian, European, and ubiquitously distributed Chinese haplotypes was present in Uttarakhand's domestic pig population. The D3 haplotype reported in wild pigs from North India was also identified in 47 domestic samples. A unique gene pool, UKD (Uttarakhand Domestic), as another lineage specific to this region has been proposed. Genotypes were analyzed, using 13 sets of microsatellite markers. The observed (Ho) and expected (He) heterozygosities were 0.83 ± 0.02 and 0.84 ± 0.01, respectively. The average polymorphic information content value of 0.83 ± 0.01 indicated the high informativeness of the marker. The overall mean FIS value for all the microsatellite markers was low (F = 0.04, P < 0.01). Seven loci deviated from Hardy-Weinberg equilibrium (HWE) at a significant level (p < 0.05). Two clusters were identified, indicating overlapping populations. These results suggested that though belonging to different maternal lineages, the traditional management practices in Uttarakhand have allowed for genetic mixing and the sharing of genetic material among pig populations. It could contribute to increased genetic diversity but might also result in the loss of distinct genetic characteristics or breed purity of the local breeds if not carefully managed.

Mitochondrial DNA in bronchoalveolar lavage fluid is associated with the prognosis of idiopathic pulmonary fibrosis: a single cohort study.

BACKGROUND: Extracellular mitochondrial DNA (mtDNA) is released from damaged cells and increases in the serum and bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) patients. While increased levels of serum mtDNA have been reported to be linked to disease progression and the future development of acute exacerbation (AE) of IPF (AE-IPF), the clinical significance of mtDNA in BALF (BALF-mtDNA) remains unclear. We investigated the relationships between BALF-mtDNA levels and other clinical variables and prognosis in IPF. METHODS: Extracellular mtDNA levels in BALF samples collected from IPF patients were determined using droplet-digital PCR. Levels of extracellular nucleolar DNA in BALF (BALF-nucDNA) were also determined as a marker for simple cell collapse. Patient characteristics and survival information were retrospectively reviewed. RESULTS: mtDNA levels in serum and BALF did not correlate with each other. In 27 patients with paired BALF samples obtained in a stable state and at the time of AE diagnosis, BALF-mtDNA levels were significantly increased at the time of AE. Elevated BALF-mtDNA levels were associated with inflammation or disordered pulmonary function in a stable state (n = 90), while being associated with age and BALF-neutrophils at the time of AE (n = 38). BALF-mtDNA ≥ 4234.3 copies/µL in a stable state (median survival time (MST): 42.4 vs. 79.6 months, p < 0.001) and ≥ 11,194.3 copies/µL at the time of AE (MST: 2.6 vs. 20.0 months, p = 0.03) were associated with shorter survival after BALF collection, even after adjusting for other known prognostic factors. On the other hand, BALF-nucDNA showed different trends in correlation with other clinical variables and did not show any significant association with survival time. CONCLUSIONS: Elevated BALF-mtDNA was associated with a poor prognosis in both IPF and AE-IPF. Of note, at the time of AE, it sharply distinguished survivors from non-survivors. Given the trends shown by analyses for BALF-nucDNA, the elevation of BALF-mtDNA might not simply reflect the impact of cell collapse. Further studies are required to explore the underlying mechanisms and clinical applications of BALF-mtDNA in IPF.

Leveraging new methods for comprehensive characterization of mitochondrial DNA in esophageal squamous cell carcinoma.

BACKGROUND: Mitochondria play essential roles in tumorigenesis; however, little is known about the contribution of mitochondrial DNA (mtDNA) to esophageal squamous cell carcinoma (ESCC). Whole-genome sequencing (WGS) is by far the most efficient technology to fully characterize the molecular features of mtDNA; however, due to the high redundancy and heterogeneity of mtDNA in regular WGS data, methods for mtDNA analysis are far from satisfactory. METHODS: Here, we developed a likelihood-based method dMTLV to identify low-heteroplasmic mtDNA variants. In addition, we described fNUMT, which can simultaneously detect non-reference nuclear sequences of mitochondrial origin (non-ref NUMTs) and their derived artifacts. Using these new methods, we explored the contribution of mtDNA to ESCC utilizing the multi-omics data of 663 paired tumor-normal samples. RESULTS: dMTLV outperformed the existing methods in sensitivity without sacrificing specificity. The verification using Nanopore long-read sequencing data showed that fNUMT has superior specificity and more accurate breakpoint identification than the current methods. Leveraging the new method, we identified a significant association between the ESCC overall survival and the ratio of mtDNA copy number of paired tumor-normal samples, which could be potentially explained by the differential expression of genes enriched in pathways related to metabolism, DNA damage repair, and cell cycle checkpoint. Additionally, we observed that the expression of CBWD1 was downregulated by the non-ref NUMTs inserted into its intron region, which might provide precursor conditions for the tumor cells to adapt to a hypoxic environment. Moreover, we identified a strong positive relationship between the number of mtDNA truncating mutations and the contribution of signatures linked to tumorigenesis and treatment response. CONCLUSIONS: Our new frameworks promote the characterization of mtDNA features, which enables the elucidation of the landscapes and roles of mtDNA in ESCC essential for extending the current understanding of ESCC etiology. dMTLV and fNUMT are freely available from https://github.com/sunnyzxh/dMTLV and https://github.com/sunnyzxh/fNUMT , respectively.

Association between ambient particulate matter exposure and mitochondrial DNA copy number: A systematic review and meta-analysis.

BACKGROUND: Ambient particulate matter (PM) has been recognized as inducing oxidative stress, which could contribute to mitochondrial damage and dysfunction. However, studies investigating the association between ambient PM and mitochondria, particularly mitochondrial DNA copy number (mtDNA-CN), have yielded inconsistent results. METHODS: We conducted comprehensive literature searches to identify observational studies published before July 17, 2023, examining the association between ambient PM exposure and mtDNA-CN. Meta-analysis using random effects model was employed to calculate the pooled effect estimates for general individual exposures, as well as for prenatal exposure with specific trimester. Additionally, the quality and level of evidence for each exposure-outcome pair was evaluated. RESULTS: A total of 10 studies were included in the systematic review and meta-analysis. The results indicated that general individual exposure to PM2.5 (ß = -0.084, 95 % CI: -0.521, 0.353; I2 = 93 %) and PM10 (ß = 0.035, 95 % CI: -0.129, 0.199; I2 = 95 %) did not significantly affect mtDNA-CN. Prenatal exposure to PM2.5 (ß = 0.023, 95 % CI: -0.087, 0.133; I2 = 0 %) and PM10 (ß = 0.006, 95 % CI: -0.135; 0.147; I2 = 51 %) were also not significantly associated with mtDNA-CN in offspring. The level of evidence for each tested exposure-outcome pair was assessed as "inadequate." CONCLUSIONS: The findings of this systematic review and meta-analysis indicate that there is an "inadequate" strength of evidence for the association between general individual or prenatal exposure to ambient PM and mtDNA-CN. Future research necessitates studies with more rigorous design, enhanced control of confounding factors, and improved measures of exposure to substantiate our findings.

Lepocreadiidae (Trematoda) associated with gelatinous zooplankton (Cnidaria and Ctenophora) and fishes in Australian and Japanese waters.

We examined gelatinous zooplankton from off eastern Australia for lepocreadiid trematode metacercariae. From 221 specimens of 17 species of cnidarian medusae and 218 specimens of four species of ctenophores, infections were found in seven cnidarian and two ctenophore species. Metacercariae were distinguished using cox1 mtDNA, ITS2 rDNA and morphology. We identified three species of Prodistomum Linton, 1910 [P. keyam Bray & Cribb, 1996, P. orientale (Layman, 1930), and Prodistomum Type 3], two species of Opechona Looss, 1907 [O. kahawai Bray & Cribb, 2003 and O. cf. olssoni], and Cephalolepidapedon saba Yamaguti, 1970. Two species were found in cnidarians and ctenophores, three only in cnidarians, and one only in a ctenophore. Three Australian fishes were identified as definitive hosts; four species were collected from Scomber australasicus and one each from Arripis trutta and Monodactylus argenteus. Transmission of trematodes to these fishes by ingestion of gelatinous zooplankton is plausible given their mid-water feeding habits, although such predation is rarely reported. Combined morphological and molecular analyses of adult trematodes identified two cox1 types for C. saba, three cox1 types and species of Opechona, and six cox1 types and five species of Prodistomum of which only two are identified to species. All three genera are widely distributed geographically and have unresolved taxonomic issues. Levels of distinction between the recognised species varied dramatically for morphology, the three molecular markers, and host distribution. Phylogenetic analysis of 28S rDNA data extends previous findings that species of Opechona and Prodistomum do not form monophyletic clades.

Homo sapiens reached the higher latitudes of Europe by 45,000 years ago.

The Middle to Upper Palaeolithic transition in Europe is associated with the regional disappearance of Neanderthals and the spread of Homo sapiens. Late Neanderthals persisted in western Europe several millennia after the occurrence of H. sapiens in eastern Europe1. Local hybridization between the two groups occurred2, but not on all occasions3. Archaeological evidence also indicates the presence of several technocomplexes during this transition, complicating our understanding and the association of behavioural adaptations with specific hominin groups4. One such technocomplex for which the makers are unknown is the Lincombian-Ranisian-Jerzmanowician (LRJ), which has been described in northwestern and central Europe5-8. Here we present the morphological and proteomic taxonomic identification, mitochondrial DNA analysis and direct radiocarbon dating of human remains directly associated with an LRJ assemblage at the site Ilsenhöhle in Ranis (Germany). These human remains are among the earliest directly dated Upper Palaeolithic H. sapiens remains in Eurasia. We show that early H. sapiens associated with the LRJ were present in central and northwestern Europe long before the extinction of late Neanderthals in southwestern Europe. Our results strengthen the notion of a patchwork of distinct human populations and technocomplexes present in Europe during this transitional period.

Assessment of mitochondrial DNA copy number variation relative to nuclear DNA quantity between different tissues.

Mitochondrial DNA is a widely tested genetic marker in various fields of research and diagnostics. Nonetheless, there is still little understanding on its abundance and quality within different tissues. Aiming to obtain deeper knowledge about the content and quality of mtDNA, we investigated nine tissues including blood, bone, brain, hair (root and shaft), cardiac muscle, liver, lung, skeletal muscle, and buccal mucosa of 32 deceased individuals using two real-time quantitative PCR-based assays with differently sized mtDNA and nDNA targets. The results revealed that the quantity of nDNA is a weak surrogate to estimate mtDNA quantities among tissues of an individual, as well as tissues across individuals. Especially hair showed extreme variation, depicting a range of multiple magnitudes of mtDNA molecules per hair fragment. Furthermore, degradation can lead to fewer fragments being available for PCR. The results call for parallel determination of the quantity and quality of mtDNA prior to downstream genotyping assays.

Comparison of the optimal and suboptimal quantity of mitotype libraries using next-generation sequencing.

Optimizing analysis parameters and sample input is crucial in forensic genetics methods to generate reliable results, and even more so when working with muti-copy mitochondrial DNA (mtDNA) and low-quality samples. This study compared mitotypes based on next-generation sequencing (NGS) results derived from the same samples at two different sequencing library concentrations-30 pM and 0.3 pM. Thirty femur samples from the Second World War were used as a model for poorly preserved DNA. Quantitative PCR (qPCR) method targeting 113 bp long fragment was employed to assess the quantity of mitogenomes. HID Ion Chef™ Instrument with Precision ID mtDNA Control Region Panel was used for library preparation and templating. Sequencing was performed with Ion GeneStudio™ S5 System. Reference haplotypes were determined from sequencing samples at 30 pM library input. Haplotypes were compared between optimal (30 pM) and suboptimal (0.3 pM) library inputs. Often the difference in haplotypes was length heteroplasmy, which in line with other studies shows that this type of variant is not reliable for interpretation in forensics. Excluding length variants at positions 573, 309, and 16,193, 56.7% of the samples matched, and in two samples, no sequence was obtained at suboptimal library input. The rest of the samples differed between optimal and suboptimal library input. To conclude, genotyping and analyzing low-quantity libraries derived from low-quality aged skeletonized human remains therefore must be done with caution in forensic genetics casework.

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions.

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection - one per each body fluid type - demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.

Mitochondrial DNA copy number in Hepatitis C virus-related chronic liver disease: impact of direct-acting antiviral therapy.

Hepatitis C virus (HCV) infection can regulate the number and dynamics of mitochondria, and is associated with a prominent hepatic mitochondrial injury. Mitochondrial distress conveys oxidative damage which is implicated in liver disease progression. The present study was conducted to assess the change of mitochondrial DNA (mtDNA) copy number in patients with HCV-related chronic liver disease and the impact of direct-acting antiviral (DAA) therapy. Whole blood mtDNA copy number was measured using real-time quantitative polymerase chain reaction at baseline and 12 weeks after the end of therapy in 50 treatment-naïve HCV-infected patients who achieved sustained viral response (SVR) after DAA therapy and 20 healthy controls. Whole blood mtDNA copy number appeared significantly lower in HCV-infected patients before therapy compared to healthy subjects (P < 0.001). Post-treatment, there was significant increase of mtDNA copy number in HCV-infected patients at SVR12 compared to the pre-treatment values (P < 0.001), meanwhile it didn't differ significantly between HCV-infected patients after therapy and healthy subjects (P = 0.059). Whole blood mtDNA copy number correlated inversely to the serum bilirubin in HCV-infected patients (P = 0.013), however it didn't correlate significantly to the serum aminotransferases, viral load or fibrosis-4 score (P > 0.05). In conclusion, chronic HCV infection has been associated with a prominent mitochondrial injury which could mediate a progressive liver disease. The improved mtDNA content after DAA therapy highlights a possible potential of these drugs to alleviate mitochondrial damage in HCV-related liver disease.

Significant decrease of maternal mitochondria carryover using optimized spindle-chromosomal complex transfer.

Mutations in mitochondrial DNA (mtDNA) contribute to a variety of serious multi-organ human diseases, which are strictly inherited from the maternal germline. However, there is currently no curative treatment. Attention has been focused on preventing the transmission of mitochondrial diseases through mitochondrial replacement (MR) therapy, but levels of mutant mtDNA can often unexpectedly undergo significant changes known as mitochondrial genetic drift. Here, we proposed a novel strategy to perform spindle-chromosomal complex transfer (SCCT) with maximal residue removal (MRR) in metaphase II (MII) oocytes, thus hopefully eliminated the transmission of mtDNA diseases. With the MRR procedure, we initially investigated the proportions of mtDNA copy numbers in isolated karyoplasts to those of individual oocytes. Spindle-chromosomal morphology and copy number variation (CNV) analysis also confirmed the safety of this method. Then, we reconstructed oocytes by MRR-SCCT, which well developed to blastocysts with minimal mtDNA residue and normal chromosomal copy numbers. Meanwhile, we optimized the manipulation order between intracytoplasmic sperm injection (ICSI) and SCC transfer and concluded that ICSI-then-transfer was conducive to avoid premature activation of reconstructed oocytes in favor of normal fertilization. Offspring of mice generated by embryos transplantation in vivo and embryonic stem cells derivation further presented evidences for competitive development competence and stable mtDNA carryover without genetic drift. Importantly, we also successfully accomplished SCCT in human MII oocytes resulting in tiny mtDNA residue and excellent embryo development through MRR manipulation. Taken together, our preclinical mouse and human models of the MRR-SCCT strategy not only demonstrated efficient residue removal but also high compatibility with normal embryo development, thus could potentially be served as a feasible clinical treatment to prevent the transmission of inherited mtDNA diseases.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

High Mitochondrial Haplotype Diversity Found in Three Pre-Hispanic Groups from Colombia.

The analysis of mitochondrial DNA (mtDNA) hypervariable region (HVR) sequence data from ancient human remains provides valuable insights into the genetic structure and population dynamics of ancient populations. mtDNA is particularly useful in studying ancient populations, because it is maternally inherited and has a higher mutation rate compared to nuclear DNA. To determine the genetic structure of three Colombian pre-Hispanic populations and compare them with current populations, we determined the haplotypes from human bone remains by sequencing several mitochondrial DNA segments. A wide variety of mitochondrial polymorphisms were obtained from 33 samples. Our results support a high population heterogeneity among pre-Hispanic populations in Colombia.

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome.

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former's inefficiency and the latter's inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional 'Correction' mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in 'Correction' mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.

Modeling-based prediction tools for preimplantation genetic testing of mitochondrial DNA diseases: estimating symptomatic thresholds, risk, and chance of success.

PURPOSE: Preimplantation genetic testing (PGT) has become a reliable tool for preventing the germline transmission of mitochondrial DNA (mtDNA) variants. However, procedures are not standardized across mtDNA variants. In this study, we aim to estimate symptomatic thresholds, risk, and chance of success for PGT for mtDNA pathogenic variant carriers. METHODS: We performed a systematic analysis of heteroplasmy data including 455 individuals from 187 familial pedigrees with the common m.3243A>G, m.8344A>G, or m.8993T>G pathogenic variants. We applied binary logistic regression for estimating symptomatic thresholds of heteroplasmy, simplified Sewell-Wright formula and Kimura equations for predicting the risk of disease transmission, and binomial distribution for predicting minimum oocyte numbers. RESULTS: We estimated the symptomatic thresholds of m.8993T>G and m.8344A>G as 29.86% and 16.15%, respectively. We could not determine a threshold for m.3243A>G. We established models for mothers harboring common and rare mtDNA pathogenic variants to predict the risk of disease transmission and the number of oocytes required to produce an embryo with sufficiently low variant load. In addition, we provide a table allowing the prediction of transmission risk and the minimum required oocytes for PGT patients with different variant levels. CONCLUSION: We have established models that can determine the symptomatic thresholds of common mtDNA pathogenic variants. We also constructed universal models applicable to nearly all mtDNA pathogenic variants which can predict risk and minimum numbers for PGT patients. These models have advanced our understanding of mtDNA disease pathogenesis and will enable more effective prevention of disease transmission using PGT.

Mitochondrial DNA Analysis.

Mitochondrial DNA (mtDNA) is a 16,569 base pair (bp) circular genome that is passed from generation to generation through the maternal line. mtDNA analysis in the context of the forensic science field usually involves unidentified human remains or missing persons. These cases tend to have more challenging sample types (e.g., rootless hairs, bone, blood, and saliva), and mtDNA analysis can be an additional method to assist in identification efforts. Due to the multifaceted protection of mtDNA within cells, mtDNA is able to be extracted even in cases of extreme degradation. mtDNA analysis for forensic science has been both peer-reviewed in academic journals and has been testified to in criminal court procedures since the late 1990s, allowing for consistent and reliable usage in casework. This chapter describes the general methodology of extracting, amplifying, quantifying, and analyzing an mtDNA sequence for use in forensic casework, specifically for these common items of evidence.

Diagnostic models to predict nuclear DNA and mitochondrial DNA recovery from incinerated teeth.

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).

Ancient human DNA recovered from a Palaeolithic pendant.

Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals1 who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited2-5. Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000-25,000 years. Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology.

Black Devils in Normandy-Identification of an Unknown Soldier Found in the Polish War Cemetery of Urville-Langannerie (France).

A paper dedicated to the identification of a Polish soldier from the 1st Armoured Division under the command of General Stanislaw Maczek, who fell in 1944 in Normandy, during World War II. The remains were found at the Urville-Langannerie Polish War Cemetery. A team from the Department of Forensic Genetics at the Pomeranian Medical University in Szczecin, commissioned by the Ministry of Culture Heritage and Sport, exhumed the remains in order to carry out genetic identification tests. A comprehensive anthropological analysis of the heavily degraded remains was carried out, and biological samples were secured for genetic testing. The identification of Jan Dusza is the first case of restoring the identity of an active combatant from the First Armoured Division. In the case analysis, the analysis of mitochondrial DNA in highly degraded biological material proved crucial. Genetic studies decided to reject the original historical hypothesis No. I at their preliminary stage. Regarding hypothesis No. II, a comprehensive genetic analysis of mitochondrial and autosomal DNA was carried out. Comparative material was obtained from the alleged victim's sister. Thanks to the analysis of kinship in the maternal line based on the mtDNA haplotype, it was possible to establish that the remains belong to Jan Dusza, who served in the Podhale Rifle Battalion, part of the Polish 1st Armoured Division. The research was co-financed by the Polish Ministry of Heritage and National Culture.