MitoVisualize: A resource for analysis of variants in human mitochondrial RNAs and DNA

We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (1) the position and effect of variants in mitochondrial transfer RNA (tRNA) and ribosomal RNA (rRNA) secondary structures alongside curated variant annotations, (2) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications, and (3) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular. MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/.

Human mitochondrial DNA diversity is compatible with the multiregional continuity theory of the origin of Homo sapiens

Confidence intervals for estimates of human mtDNA sequence diversity, chimpanzee-human mtDNA sequence divergence, and the time of splitting of the pongid-hominid lineages are presented. Consistent with all the data used in estimating the coalescence time for human mitochondrial lineages to a common ancestral mitochondrion is a range of dates from less than 79,000 years ago to more than 1,139,000 years ago. Consequently, the hypothesis that a migration of modern humans (Homo sapiens) out of Africa in the range of 140,000 to 280,000 years ago resulted in the complete replacement, without genetic interchange, of earlier Eurasian hominid populations (Homo erectus) is but one of several possible interpretations of the mtDNA data. The data are also compatible with the hypothesis, suggested earlier and supported by fossil evidence, of a single, more ancient expansion of the range of Homo erectus from Africa, followed by a gradual transition to Homo sapiens in Europe, Asia, and Africa.

2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication.

Human Mitochondrial DNA: Particularities and Diseases

Mitochondria are the cell’s power site, transforming energy into a form that the cell can employ for necessary metabolic reactions. These organelles present their own DNA. Although it codes for a small number of genes, mutations in mtDNA are common. Molecular genetics diagnosis allows the analysis of DNA in several areas such as infectiology, oncology, human genetics and personalized medicine. Knowing that the mitochondrial DNA is subject to several mutations which have a direct impact on the metabolism of the mitochondrion leading to many diseases, it is therefore necessary to detect these mutations in the patients involved. To date numerous mitochondrial mutations have been described in humans, permitting confirmation of clinical diagnosis, in addition to a better management of the patients. Therefore, different techniques are employed to study the presence or absence of mitochondrial mutations. However, new mutations are discovered, and to determine if they are the cause of disease, different functional mitochondrial studies are undertaken using transmitochondrial cybrid cells that are constructed by fusion of platelets of the patient that presents the mutation, with rho osteosarcoma cell line. Moreover, the contribution of next generation sequencing allows sequencing of the entire human genome within a single day and should be considered in the diagnosis of mitochondrial mutations.

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.

Extracting Phylogenetic Information of Human Mitochondrial DNA by Linear Autoencoder

We used a linear autoencoder (LAE) and its learning dynamics to analyze the high-order structure of human mitochondrial DNA (mtDNA). A total of 360 complete human mtDNA sequences were collected from the MITOMAP database and transformed into 1024-dimensional vectors of pentanucleotide frequencies. We compressed those into a three-dimensional (3D) coordinates by an LAE at each step of training by gradient descent with respect to the quadratic error function. Along the time axis of training epochs, the compressed 3D coordinates were gradually clustered and separated in accordance with the order of the genetic distance in the phylogenetic tree of human mtDNA haplogroups. This suggests that there is an association between the learning dynamics of LAE and the high-dimensional structure of human mtDNA sequences, similar to that of phylogenetic analysis and evolutionary pathways: the five clusters eventually contained only a single haplogroup of L0, M, N, R, and U, while the L3 cluster contained a small number of M members and The packing was comparable to that realized in learning dynamics similar to genetic classification and evolutionary pathways by LAE in principal component analysis (PCA), but somewhat denser than PCA.

The population frequency of human mitochondrial DNA variants is highly dependent upon mutational bias

Genome-wide association studies (GWASs) typically seek common genetic variants that can influence disease likelihood. However, these analyses often fail to convincingly link specific genes and their variants with highly penetrant phenotypic effects. To solve the 'missing heritability problem' that characterizes GWASs, researchers have turned to rare variants revealed by next-generation sequencing when seeking genomic changes that may be pathogenic, as a reduction in variant frequency is an expected outcome of selection. While triage of rare variants has led to some success in illuminating genes linked to heritable disease, the interpretation and utilization of rare genomic changes remains very challenging. Human mitochondrial DNA (mtDNA) encodes proteins and RNAs required for the essential process of oxidative phosphorylation, and a number of metabolic diseases are linked to mitochondrial mutations. Recently, the mtDNAs of nearly 200,000 individuals were sequenced in order to produce the HelixMT database (HelixMTdb), a large catalog of human mtDNA variation. Here, we were surprised to find that many synonymous nucleotide substitutions were never detected within this quite substantial survey of human mtDNA. Subsequent study of more than 1000 mammalian mtDNAs suggested that selection on synonymous sites within mitochondrial protein-coding genes is minimal and unlikely to explain the rarity of most synonymous changes among humans. Rather, the mutational propensities of mtDNA are more likely to determine variant frequency. Our findings have general implications for the interpretation of variant frequencies when studying heritable disease.

Non-destructive extraction of DNA from preserved tissues in medical collections

Museum and medically fixed material are valuable samples for the study of historical soft tissues and represent a pathogen-specific source for retrospective molecular investigations. However, current methods for the molecular analysis are inherently destructive, posing a dilemma between performing a study with the available technology thus damaging the sample - or conserving the material for future investigations. Here we present an unprecedented non-destructive alternative that facilitates the genetic analysis of fixed wet tissues while avoiding tissue damage. We extracted DNA from the fixed tissues as well as their embedding fixative solution, to quantify the DNA that was transferred to the liquid component. Our results prove that human ancient DNA can be retrieved from the fixative material of stored medical specimens and provide new options for the sampling of valuable curated collections.Method summaryWe compared the metagenomic content of historical tissues and their embedding liquid to retrieve DNA from the host and specified pathogens based on the diagnosis of the sample. We applied ancient DNA research techniques, including in-solution hybridization capture with DNA baits for human mitochondrial DNA, Mycobacterium tuberculosis, Mycobacterium leprae, and Treponema pallidum.