mtDNA Variation Research Articles

Mitochondrial Genome Instability in W303-SK1 Yeast Cytoplasmic Hybrids.

Unlike most animals, some fungi, including baker's yeast, inherit mitochondrial DNA (mtDNA) from both parents. When haploid yeast cells fuse, they form a heteroplasmic zygote, whose offspring retain one or the other variant of mtDNA. Meanwhile, some mutant mtDNA (rho-), with large deletions in the nucleotide sequence, can displace wild-type (rho+) mtDNA. Consequently, offspring of zygotes with such rho- mtDNA predominantly carry the mutant variant. This phenomenon is called suppressivity. In this study, we investigated how the suppressivity of rho- mtDNA depends on the mitochondrial and nuclear genomes of the rho+ strain during crossing. Comparing two diverged laboratory strains, SK1 and W303, we measured suppressivity in crosses with four rho- strains. One rho- strain showed significantly higher suppressivity when crossed with SK1 than with W303. We then created cytoplasmic hybrids by swapping mtDNAs between these strains. Surprisingly, we found that the mtDNA of the rho+ strain, rather than its nuclear DNA, determines high suppressivity in crosses of SK1 rho+ with the rho- strain. Additionally, mtDNA replacement reduced respiration rate and growth rate on non-fermentable substrates while increasing the likelihood of functional mtDNA loss. Our data demonstrate that a mutant mtDNA variant's ability to displace another mitochondrial DNA variant in a heteroplasmic cell depends more on mtDNA sequences than on the biochemical and structural context created by the nuclear genome background.

A naturally occurring mitochondrial genome variant confers broad protection from infection in Drosophila.

The role of mitochondria in immunity is increasingly recognized, but it is unclear how variation in mitochondrial DNA (mtDNA) contributes to variable infection outcomes. To quantify the effect of mtDNA variation on humoral and cell-mediated innate immune responses, we utilized a panel of fruit fly Drosophila melanogaster cytoplasmic hybrids (cybrids), where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We observed substantial heterogeneity in infection outcomes within the cybrid panel upon bacterial, viral and parasitoid infections, driven by the mitotype. One of the mitotypes, mtKSA2 protected against bacterial, parasitoid, and to a lesser extent, viral infections. Enhanced survival was not a result of improved bacterial clearance, suggesting mtKSA2 confers increased disease tolerance. Transcriptome sequencing showed that the mtKSA2 mitotype had an upregulation of genes related to mitochondrial respiration and phagocytosis in uninfected flies. Upon infection, mtKSA2 flies exhibited infection type and duration specific transcriptomic changes. Furthermore, uninfected mtKSA2 larvae showed immune activation of hemocytes (immune cells), increased hemocyte numbers and ROS production, and enhanced encapsulation response against parasitoid wasp eggs and larvae. Our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes can provide broad protection against infections.

Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.

Mitochondrial DNA (mtDNA) mutations are frequent in cancer, yet their precise role in cancer progression remains debated. To functionally evaluate the impact of mtDNA variants on tumor growth and metastasis, we developed an enhanced cytoplasmic hybrid (cybrid) generation protocol and established isogenic human melanoma cybrid lines with wild-type mtDNA or pathogenic mtDNA mutations with partial or complete loss of mitochondrial oxidative function. Cybrids with homoplasmic levels of pathogenic mtDNA reliably established tumors despite dysfunctional oxidative phosphorylation. However, these mtDNA variants disrupted spontaneous metastasis from primary tumors and reduced the abundance of circulating tumor cells. Migration and invasion of tumor cells were reduced, indicating that entry into circulation is a bottleneck for metastasis amid mtDNA dysfunction. Pathogenic mtDNA did not inhibit organ colonization following intravenous injection. In heteroplasmic cybrid tumors, single-cell analyses revealed selection against pathogenic mtDNA during melanoma growth. Collectively, these findings experimentally demonstrate that functional mtDNA is favored during melanoma growth and supports metastatic entry into the blood.

Targeted nanopore sequencing using the Flongle device to identify mitochondrial DNA variants

Variants in mitochondrial genomes (mtDNA) can cause various neurological and mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Given the 16 kb length of mtDNA, continuous sequencing is feasible using long-read sequencing (LRS). Herein, we aimed to show a simple and accessible method for comprehensive mtDNA sequencing with potential diagnostic applications for mitochondrial diseases using the compact and affordable LRS flow cell “Flongle.” Whole mtDNA amplification (WMA) was performed using genomic DNA samples derived from four patients with mitochondrial diseases, followed by LRS using Flongle. We compared these results to those obtained using Cas9 enrichment. Additionally, the accuracy of heteroplasmy rates was assessed by incorporating mtDNA variants at equimolar levels. Finally, mtDNA from 19 patients with Parkinson’s disease (PD) was sequenced using Flongle to identify disease risk-associated variants. mtDNA variants were detected in all four patients with mitochondrial diseases, with results comparable to those obtained from Cas9 enrichment. Heteroplasmy levels were accurately detected (r2 > 0.99) via WMA using Flongle. A reported variant was identified in three patients with PD. In conclusion, Flongle can simplify the traditionally cumbersome and expensive mtDNA sequencing process, offering a streamlined and accessible approach to diagnosing mitochondrial diseases.

Relation of mitochondrial DNA copy number and variants with the clinical characteristics of polycystic ovary syndrome

Mounting evidences suggests mitochondrial dysfunction as a novel contributor in the pathogenesis of PCOS. Herein, we analyzed mtDNA copy number, a biomarker of mitochondrial function in women with PCOS and non-PCOS participants and study its correlation with their clinical characteristics. In this study, we further analyzed association of 383 mtDNA variants, as reported previously by us, with characteristic traits of PCOS and perform structural analysis of mutated protein. Our results indicate relative mitochondrial DNA (mtDNA) copy number to be significantly reduced in women with PCOS compared to non-PCOS group and significantly inversely related to waist to hip ratio (WHR), triglycerides and positively related to high density lipoprotein-cholesterol (HDL-C). After adjustment of the age in the PCOS group, significantly negative correlation of mtDNA copy number with WHR was observed. Unsupervised hierarchical clustering analysis revealed rare, low heteroplasmic mtDNA variants such as 12556G, 1488T, 9200G, 9670G, 3308G, 14480G, 15914T and 5426G to be strongly associated with PCOS related traits. Among these variants, variant 12256G in ND5 gene affected both the flexibility and overall stability of the protein structure. This study is first to reveal significant correlation of mtDNA copy number with WHR in women with PCOS indicating link between mitochondrial dysfunction with central obesity in PCOS. we also first time showed association of rare mtDNA variants with characteristics traits of PCOS highlighting the clinical significance of rare mtDNA variants, which may cumulatively act as early predictors of risk of PCOS and its related comorbidities which may help in the management of PCOS.

Quantifying constraint in the human mitochondrial genome.

Mitochondrial DNA (mtDNA) has an important yet often overlooked role in health and disease. Constraint models quantify the removal of deleterious variation from the population by selection and represent powerful tools for identifying genetic variation that underlies human phenotypes1-4. However, nuclear constraint models are not applicable to mtDNA, owing to its distinct features. Here we describe the development of a mitochondrial genome constraint model and its application to the Genome Aggregation Database (gnomAD), a large-scale population dataset that reports mtDNA variation across 56,434 human participants5. Specifically, we analyse constraint by comparing the observed variation in gnomAD to that expected under neutrality, which was calculated using a mtDNA mutational model and observed maximum heteroplasmy-level data. Our results highlight strong depletion of expected variation, which suggests that many deleterious mtDNA variants remain undetected. To aid their discovery, we compute constraint metrics for every mitochondrial protein, tRNA and rRNA gene, which revealed a range of intolerance to variation. We further characterize the most constrained regions within genes through regional constraint and identify the most constrained sites within the entire mitochondrial genome through local constraint, which showed enrichment of pathogenic variation. Constraint also clustered in three-dimensional structures, which provided insight into functionally important domains and their disease relevance. Notably, we identify constraint at often overlooked sites, including in rRNA and noncoding regions. Last, we demonstrate that these metrics can improve the discovery of deleterious variation that underlies rare and common phenotypes.

Changes in TP53 Gene, Telomere Length, and Mitochondrial DNA in Benign Prostatic Hyperplasia Patients.

Benign prostatic hyperplasia (BPH) is a growing issue due to an ageing population. Our study investigated the possible associations between BPH and ageing hallmarks, including the telomere length (TL) and mitochondrial genome copy number (mtDNA CN), along with genetic variations in the TP53 gene and mtDNA. Prostate tissue samples were obtained from 32 patients with BPH, together with 30 blood samples. As a healthy control group, age-matching blood DNA samples were used. For the comparison of mtDNA sequence data, 50 DNA samples of the general Latvian population were used. The full mtDNA genome was analyzed by using Next-Generation Sequencing (NGS), the TP53 gene by Sanger sequencing, and the mtDNA copy number (mtDNA CN) and telomere length (TL) byqPCR assay. The results showed that in BPH patients, telomeres in the prostate tissue were significantly longer than in blood cells, while the TL in blood cells of the healthy controls was the shortest. Also, the mtDNA amount in the prostate tissue of BPH patients was significantly greater in comparison with blood cells, and controls had the smallest mtDNA CN. We did not find any mutations in the TP53 gene that could be linked to BPH; however, in mtDNA, we found several unique mutations and heteroplasmic changes, as well as genetic changes that have been previously associated with prostate cancer. In conclusion, prolonged telomeres and changes in the mtDNA amount might be involved in the molecular mechanisms of BPH. Some of the heteroplasmic or homoplasmic mtDNA variants might also contribute to the development of BPH. Additional studies are needed to substantiate these findings.

Identification of sequence polymorphism in the D-loop region of mitochondrial DNA as a risk factor for breast cancer.

Mitochondrial DNA (mtDNA) variations affect the efficiency of the electron transport chain and production of reactive oxygen species, contributing to carcinogenesis. The D-loop region of mtDNA has emerged as a variation hotspot region in human neoplasia; however, the potential contribution of these variations in breast cancer risk prediction remains unknown. We investigated the relationship between germline single nucleotide polymorphisms (SNPs) in the entire D-loop region and breast cancer risk in Chinese women. Peripheral blood-isolated mtDNA from 2329 patients with breast cancer and 2328 cancer-free controls was examined for SNPs. In the combined cohort, we used traditional risk factors, susceptibility germline polymorphisms, and logistic regression analysis to evaluate the predictive value of susceptibility variants for breast cancer risk. We calculated the area under the receiver operating characteristic curve (AUC) as a measure. We also measured the content of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Individual polymorphisms SNP573 were significantly associated with breast cancer risk in both the discovery and validation cohorts. In the combined cohort, the AUC of the traditional risk factors was 64.3%; after adding susceptibility variants, the AUC was 64.9% (DeLong test, p = 0.007). 8-OHdG levels were significantly higher in patients with breast cancer than in controls and higher in individuals with SNP573 than in those negative for this variation. Overall, oxidative stress might be associated with the risk of breast cancer, and SNP573 might be associated with oxidative stress. Our results indicate the risk potential of polymorphisms in the D-loop region in breast cancer in Southern China.

Mitochondrial haplogroup R offers protection against obesity in Kuwaiti and Qatari populations.

The Kuwaiti and Qatari populations have a high prevalence of obesity, a major risk factor for various metabolic disorders. Previous studies have independently explored mitochondrial DNA (mtDNA) variations and their association with obesity in these populations. This study aims to investigate the role of mtDNA haplogroups and variants in obesity risk among these Gulf populations. Whole exome sequencing data from 1,112 participants (348 Kuwaitis and 764 Qataris) were analyzed for mtDNA variants. Participants were classified as obese or non-obese based on body mass index (BMI). Association analyses were performed to examine the relationship between mtDNA haplogroups and obesity, adjusting for covariates such as age and sex. Haplogroup R was found to be protective against obesity, with an odds ratio (OR) of 0.69 (p = 0.045). This association remained significant after adjusting for age and sex (OR = 0.694; 95% CI: 0.482-0.997; p = 0.048). Several mtDNA variants, particularly those involved in mitochondrial energy metabolism, showed nominal associations with obesity, but these did not remain significant after correcting for multiple testing. Haplogroup R consistently demonstrates a protective association against obesity in both Kuwaiti and Qatari populations, highlighting its potential as a biomarker for obesity risk in the Gulf region. However, further research with larger sample sizes is needed to validate these findings and clarify the role of mtDNA variants in obesity.

Taurine hypomodification underlies mitochondrial tRNATrp-related genetic diseases.

Escherichia coli MnmE and MnmG form a complex (EcMnmEG), generating transfer RNA (tRNA) 5-carboxymethylaminomethyluridine (cmnm5U) modification. Both cmnm5U and equivalent 5-taurinomethyluridine (τm5U, catalyzed by homologous GTPBP3 and MTO1) are found at U34 in several human mitochondrial tRNAs (hmtRNAs). Certain mitochondrial DNA (mtDNA) mutations, including m.3243A>G in tRNALeu(UUR) and m.8344A>G in tRNALys, cause genetic diseases, partially due to τm5U hypomodification. However, whether other mtDNA variants in different tRNAs cause a defect in τm5U biogenesis remains unknown. Here, we purified naturally assembled EcMnmEG from E. coli. Notably, EcMnmEG was able to incorporate both cmnm5U and τm5U into hmtRNATrp (encoded by MT-TW), providing a valuable basis for directly monitoring the effects of mtDNA mutations on U34 modification. In vitro, several clinical hmtRNATrp pathogenic mutations caused U34 hypomodification. A patient harboring an m.5541C>T mutation exhibited hmtRNATrp τm5U hypomodification. Moreover, using mtDNA base editing, we constructed two cell lines carrying m.5532G>A or m.5545C>T mutations, both of which exhibited hmtRNATrp τm5U hypomodification. Taurine supplementation improved mitochondrial translation in patient cells. Our findings describe the third hmtRNA species with mutation-related τm5U-hypomodification and provide new insights into the pathogenesis and intervention strategy for hmtRNATrp-related genetic diseases.

Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs.

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

Multiple distinct evolutionary mechanisms govern the dynamics of selfish mitochondrial genomes in Caenorhabditis elegans

Cells possess multiple mitochondrial DNA (mtDNA) copies, which undergo semi-autonomous replication and stochastic inheritance. This enables mutant mtDNA variants to arise and selfishly compete with cooperative (wildtype) mtDNA. Selfish mitochondrial genomes are subject to selection at different levels: they compete against wildtype mtDNA directly within hosts and indirectly through organism-level selection. However, determining the relative contributions of selection at different levels has proven challenging. We overcome this challenge by combining mathematical modeling with experiments designed to isolate the levels of selection. Applying this approach to many selfish mitochondrial genotypes in Caenorhabditis elegans reveals an unexpected diversity of evolutionary mechanisms. Some mutant genomes persist at high frequency for many generations, despite a host fitness cost, by aggressively outcompeting cooperative genomes within hosts. Conversely, some mutant genomes persist by evading inter-organismal selection. Strikingly, the mutant genomes vary dramatically in their susceptibility to genetic drift. Although different mechanisms can cause high frequency of selfish mtDNA, we show how they give rise to characteristically different distributions of mutant frequency among individuals. Given that heteroplasmic frequency represents a key determinant of phenotypic severity, this work outlines an evolutionary theoretic framework for predicting the distribution of phenotypic consequences among individuals carrying a selfish mitochondrial genome.

Mitochondrial Health Markers and Obesity-Related Health in Human Population Studies: A Narrative Review of Recent Literature.

This narrative review summarizes current literature on the relationship of mitochondrial biomarkers with obesity-related characteristics, including body mass index and body composition. Mitochondria, as cellular powerhouses, play a pivotal role in energy production and the regulation of metabolic process. Altered mitochondrial functions contribute to obesity, yet evidence of the intricate relationship between mitochondrial dynamics and obesity-related outcomes in human population studies is scarce and warrants further attention. We discuss emerging evidence linking obesity and related health outcomes to impaired oxidative phosphorylation pathways, oxidative stress and mtDNA variants, copy number and methylation, all hallmark of suboptimal mitochondrial function. We also explore the influence of dietary interventions and metabolic and bariatric surgery procedures on restoring mitochondrial attributes of individuals with obesity. Finally, we report on the potential knowledge gaps in the mitochondrial dynamics for human health for future study.

Untangling the colonization history of the Australo-Pacific reed warblers, one of the world's great island radiations.

Island radiations, such as those of the Australo-Pacific, offer unique insight into diversification, extinction, and early speciation processes. Yet, their speciation and colonization histories are often obscured by conflicting genomic signals from incomplete lineage sorting or hybridization. Here, we integrated mitogenomes and genome-wide SNPs to unravel the evolutionary history of one of the world's most geographically widespread island radiations. The Australo-Pacific reed warblers (Acrocephalus luscinius complex) are a speciose lineage including five species that have become extinct since the 19th century and ten additional species of conservation concern. The radiation spans over 10,000 km across Australo-Papua, Micronesia and Polynesia, including the Mariana, Hawaii and Pitcairn Island archipelagos. Earlier mtDNA studies suggested a stepping-stone colonization process, resulting in archipelago-level secondary sympatry of divergent mtDNA lineages in the Mariana Islands and Marquesas. These studies hypothesised that morphologically similar species on neighbouring islands arose from ecological convergence. Using hDNA from historical museum specimens and modern genetic samples, we show that incomplete lineage sorting and/or gene flow have shaped the radiation of Australo-Pacific reed warblers rather than secondary sympatry. The nuclear genome reconstructs a simpler biogeographic history than mtDNA, showing close relationships between species in the Mariana Islands and Marquesas despite their paraphyletic mtDNA lineages. Gene flow likely involved early and late colonizing waves of the radiation before the loss of ancestral dispersive ability. Our results highlight how collection genomics can elucidate evolutionary history and inform conservation efforts for threatened species.

Whole Mitochondrial Genome Sequencing Analysis of Canine Testicular Tumours.

Currently, the molecular background based on mitochondrial DNA (mtDNA) analysis of canine testicular tumours is underestimated. The available data mostly focus on histopathological evaluations, with a few reports of nuclear genome (nDNA) studies. Tumourigenesis represents a highly complex and diverse genetic disorder, which can also encompass defects in mtDNA. The aim of this study was to identify molecular changes in whole mitochondrial genome sequences obtained from dogs affected by testicular tumours. Samples of blood, tumour, and healthy tissue were collected from each animal, and mtDNA (ultimately 45 samples) was subsequently sequenced. Thereafter, protein analyses were performed to assess the impact of the identified molecular alterations on the amino acid level. The total number of observed changes included 722 SNPs, 12 mutations, 62 indels, 5 indel mutations, and 35 heteroplasmic sites. The highest number of mtDNA variants in protein-coding genes COX1, COX3, ATP6, ND1, ND4, and ND5 was observed. Interestingly, SNPs were found in 10 out of 22 tRNA genes. Most of the identified mtDNA defects were synonymous changes at the amino acid level. Also, polymorphisms and heteroplasmy were frequently observed in the variable number of tandem repeat (VNTR) regions, especially in its fragment spanning 16,138-16,358 bp. Based on the obtained results, it was possible to select 11 polymorphisms that occurred in all the tested samples (benign, malignant) and an additional five SNPs identified only in benign neoplasms. The comprehensive analysis of malignant testicular tumours demonstrated a significant diversity in their molecular profiles, with changes ranging from 17 to 101 per sample.

Serum oxidative stressors levels and association of mtDNA variants with type 2 diabetes mellitus in the Central India population

BackgroundThe mitochondrial genome has a high rate of mutability which plays a major role in pathogenic mutations. These mutations are implicated with mitochondrial dysfunction increasing the vulnerability to Diabetes Mellitus (DM). AimThe study aimed to evaluate the changes in oxidative markers and analyse the specific mitochondrial DNA variants that contributed to DM in the central Indian population. MethodTo assess mitogenomic alteration, Sanger sequencing was used to identify the single nucleotide polymorphisms (SNPs) or variants, while ELISA kits were used to evaluate oxidative stress. ResultsThe levels of 8-Hydroxy-2-deoxyguanosine (8-OHdG) and Malondialdehyde (MDA) in type 2 diabetes mellitus (T2DM) were significantly higher than in healthy individual (HI). In T2DM (3371.80 ± 1110.7 pg/ml) the levels of 8-OHdG were significantly greater and were found to be nine times higher compared to the control group (P < 0.001). Additionally, MDA level which is indicative of lipid peroxidation, in the diabetic groups (39.34 ± 23.05 ng/ml) contributed to 18 times higher than the control groups (2.16 ± 2 ng/ml). Moreover, 52 variants were found in our population, among which C10400T variants were significantly prevalent and clustered with the A10398G. These variants confirmed a strong association, on analysis for linkage disequilibrium (r2), with a slightly higher r2 value in the T2DM group (0.92) compared to controls (0.85), indicating a stronger link with diabetes. According to multivariate regression analysis, variants associated with the mitogenome such as C16192T (CI: 0.004 to 1.30, p = 0.028) were found to play a protective role against T2DM. Furthermore, A3384G and G16129A may contribute to the protective role against the risk of developing diabetes. ConclusionThe study demonstrates that diabetic patients are more vulnerable to certain mtDNA variants, directly linked to increased hyperglycemia. Elevated free radical-mediated oxidative stress likely affects these mtDNA variants.

Variant load of mitochondrial DNA in single human mesenchymal stem cells

Heteroplasmic mitochondrial DNA (mtDNA) variants accumulate as humans age, particularly in the stem-cell compartments, and are an important contributor to age-related disease. Mitochondrial dysfunction has been observed in osteoporosis and somatic mtDNA pathogenic variants have been observed in animal models of osteoporosis. However, this has never been assessed in the relevant human tissue. Mesenchymal stem cells (MSCs) are the progenitors to many cells of the musculoskeletal system and are critical to skeletal tissues and bone vitality. Investigating mtDNA in MSCs could provide novel insights into the role of mitochondrial dysfunction in osteoporosis. To determine if this is possible, we investigated the landscape of somatic mtDNA variation in MSCs through a combination of fluorescence-activated cell sorting and single-cell next-generation sequencing. Our data show that somatic heteroplasmic variants are present in individual patient-derived MSCs, can reach high heteroplasmic fractions and have the potential to be pathogenic. The identification of somatic heteroplasmic variants in MSCs of patients highlights the potential for mitochondrial dysfunction to contribute to the pathogenesis of osteoporosis.

Long-term coexistence of mtDNA variations and nuclear responses of host

Long-term coexistence of mtDNA variations and nuclear responses of host

Mitochondrial DNA control-region and coding-region data highlight geographically structured diversity and post-domestication population dynamics in worldwide donkeys.

Donkeys (Equus asinus) have been used extensively in agriculture and transportations since their domestication, ca. 5000-7000 years ago, but the increased mechanization of the last century has largely spoiled their role as burden animals, particularly in developed countries. Consequently, donkey breeds and population sizes have been declining for decades, and the diversity contributed by autochthonous gene pools has been eroded. Here, we examined coding-region data extracted from 164 complete mitogenomes and 1392 donkey mitochondrial DNA (mtDNA) control-region sequences to (i) assess worldwide diversity, (ii) evaluate geographical patterns of variation, and (iii) provide a new nomenclature of mtDNA haplogroups. The topology of the Maximum Parsimony tree confirmed the two previously identified major clades, i.e. Clades 1 and 2, but also highlighted the occurrence of a deep-diverging lineage within Clade 2 that left a marginal trace in modern donkeys. Thanks to the identification of stable and highly diagnostic coding-region mutational motifs, the two lineages were renamed as haplogroup A and haplogroup B, respectively, to harmonize clade nomenclature with the standard currently adopted for other livestock species. Control-region diversity and population expansion metrics varied considerably between geographical areas but confirmed North-eastern Africa as the likely domestication center. The patterns of geographical distribution of variation analyzed through phylogenetic networks and AMOVA confirmed the co-occurrence of both haplogroups in all sampled populations, while differences at the regional level point to the joint effects of demography, past human migrations and trade following the spread of donkeys out of the domestication center. Despite the strong decline that donkey populations have undergone for decades in many areas of the world, the sizeable mtDNA variability we scored, and the possible identification of a new early radiating lineage further stress the need for an extensive and large-scale characterization of donkey nuclear genome diversity to identify hotspots of variation and aid the conservation of local breeds worldwide.

Mitochondrial related variants associated with cardiovascular traits.

Cardiovascular disease (CVD) is responsible for over 30% of mortality worldwide. CVD arises from the complex influence of molecular, clinical, social, and environmental factors. Despite the growing number of autosomal genetic variants contributing to CVD, the cause of most CVDs is still unclear. Mitochondria are crucial in the pathophysiology, development and progression of CVDs; the impact of mitochondrial DNA (mtDNA) variants and mitochondrial haplogroups in the context of CVD has recently been highlighted. We investigated the role of genetic variants in both mtDNA and nuclear-encoded mitochondrial genes (NEMG) in CVD, including coronary artery disease (CAD), hypertension, and serum lipids in the UK Biobank, with sub-group analysis for diabetes. We investigated 371,542 variants in 2,527 NEMG, along with 192 variants in 32 mitochondrial genes in 381,994 participants of the UK Biobank, stratifying by presence of diabetes. Mitochondrial variants showed associations with CVD, hypertension, and serum lipids. Mitochondrial haplogroup J was associated with CAD and serum lipids, whereas mitochondrial haplogroups T and U were associated with CVD. Among NEMG, variants within Nitric Oxide Synthase 3 (NOS3) showed associations with CVD, CAD, hypertension, as well as diastolic and systolic blood pressure. We also identified Translocase Of Outer Mitochondrial Membrane 40 (TOMM40) variants associated with CAD; Solute carrier family 22 member 2 (SLC22A2) variants associated with CAD and CVD; and HLA-DQA1 variants associated with hypertension. Variants within these three genes were also associated with serum lipids. Our study demonstrates the relevance of mitochondrial related variants in the context of CVD. We have linked mitochondrial haplogroup U to CVD, confirmed association of mitochondrial haplogroups J and T with CVD and proposed new markers of hypertension and serum lipids in the context of diabetes. We have also evidenced connections between the etiological pathways underlying CVDs, blood pressure and serum lipids, placing NOS3, SLC22A2, TOMM40 and HLA-DQA1 genes as common nexuses.