mtDNA Mutations Research Articles

Significantly reduced, but balanced, rates of mitochondrial fission and fusion are sufficient to maintain the integrity of yeast mitochondrial DNA

Mitochondria exist as dynamic tubular networks and the morphology of these networks impacts organelle function and cell health. Mitochondrial morphology is maintained in part by the opposing activities of mitochondrial fission and fusion. Mitochondrial fission and fusion are also required to maintain mitochondrial DNA (mtDNA) integrity. In Saccharomyces cerevisiae, the simultaneous inhibition of mitochondrial fission and fusion results in increased mtDNA mutation and the consequent loss of respiratory competence. The mechanism by which fission and fusion maintain mtDNA integrity is not fully understood. Previous work demonstrates that mtDNA is spatially linked to mitochondrial fission sites. Here, we extend this finding using live-cell imaging to localize mtDNA to mitochondrial fusion sites. While mtDNA is present at sites of mitochondrial fission and fusion, mitochondrial fission and fusion rates are not altered in cells lacking mtDNA. Using alleles that alter mitochondrial fission and fusion rates, we find that mtDNA integrity can be maintained in cells with significantly reduced, but balanced, rates of fission and fusion. In addition, we find that increasing mtDNA copy number reduces the loss of respiratory competence in double mitochondrial fission-fusion mutants. Our findings add novel insights into the relationship between mitochondrial dynamics and mtDNA integrity.

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.

Comprehensive phenotypic assessment of nonsense mutations in mitochondrial ND5 in mice.

Mitochondrial dysfunction induced by mitochondrial DNA (mtDNA) mutations has been implicated in various human diseases. A comprehensive analysis of mitochondrial genetic disorders requires suitable animal models for human disease studies. While gene knockout via premature stop codons is a powerful method for investigating the unique functions of target genes, achieving knockout of mtDNA has been rare. Here, we report the genotypes and phenotypes of heteroplasmic MT-ND5 gene-knockout mice. These mutant mice presented damaged mitochondrial cristae in the cerebral cortex, hippocampal atrophy, and asymmetry, leading to learning and memory abnormalities. Moreover, mutant mice are susceptible to obesity and thermogenetic disorders. We propose that these mtDNA gene-knockdown mice could serve as valuable animal models for studying the MT-ND5 gene and developing therapies for human mitochondrial disorders in the future.

Mitochondrial DNA Alterations in Glioblastoma and Current Therapeutic Targets.

Metabolic reprogramming within tumor cells involves a shift towards either glycolysis or mitochondrial respiration, depending on the stage of tumor progression. Consequently, irreversible dysfunction of the mitochondria is considered a crucial mechanism driving the progression mechanism. While numerous mutations in mitochondrial DNA (mtDNA) have been identified across various tumor types, including glioblastoma, many studies have been limited in the scope, focusing on small segments of mtDNA or utilizing sequencing methods with restricted sensitivity. As a result, several potentially significant mtDNA mutations may have been underestimated, along with their heteroplasmic states, which play a crucial role in determining the phenotypic impact of mtDNA mutation. Although both somatic and germline mtDNA mutations have been observed in different tumor types, research on the mtDNA mutations linked to glioblastoma remains scarce. The mitochondrial genome encodes thirteen protein-coding genes that are essential for the proper functioning of respiratory complex chains. Alterations in mitochondrial function manifest at various levels, including structural and functional changes, impacting mitogenic, hemodynamic, bioenergetic, and apoptotic signaling pathways. These alterations often signify a reduced efficiency of the oxidative phosphorylation system and energy production in tumor cells. As the crucial role of mitochondrial dysfunction in glioma development grows, mitochondria have emerged as promising targets for therapy aimed at overcoming chemoresistance and eliminating cancer cells. This brief review outlines the association between mtDNA alteration and glioblastoma, as well as the current advancements in therapeutic strategies targeting mtDNA alterations.

Clinical and genetic landscape of optic atrophy in 826 families: insights from 50 nuclear genes.

Hereditary optic neuropathies (HON) are a group of diseases due to genetic defects either in mitochondria or in nuclear genomes. The increasing availability of genetic testing has expanded a broader genetic and phenotypic spectrum of HON than previously recognized. To provide systematic insight into the genetic and phenotypic landscape of HON attributed to 50 nuclear genes, we conducted genetic analysis on part of 4776 index patients with clinical diagnosis of HON following our previous study on 1516 probands with Leber hereditary optic neuropathy (LHON) and mitochondrial DNA variants. Exome sequencing was performed in 473 patients diagnosed with nuclear gene-related HON (nHON) and 353 cases with unsolved LHON. Sequencing and variant interpretation in 50 causative nuclear genes indicated that the diagnostic yield of exome sequencing for nHON was 31.50% (149/473), while it was markedly lower at 1.42% (5/353) for LHON patients without primary mtDNA mutations. The top five implicated genes causing nHON in our in-house cohort, OPA1, WFS1, FDXR, ACO2, and AFG3L2, account for 82.46% of mutations. Although OPA1 was the most prevalent causative gene of nHON in both our cohort (53.25%) and literature review (37.09%), the prevalence of OPA1, WFS1, and FDXR differed significantly between our in-house cohort and the literature review (P-adjusted<0.001). Fundus changes in nHON could be stratified into three categories, the most common is optic atrophy at the examination (78.79%), the rarest is LHON-like optic atrophy (3.64%), and the intermediate is optic atrophy with concurrent retinal degeneration (17.57%), which was an independent risk factor for visual prognosis in nHON. A systematic genotype-phenotype analysis highlighted different genetic contributions for ocular, extraocular neurological, and extraocular non-neurological phenotypes. In addition, systemic variant analysis at the individual gene level suggested a revised interpretation of the pathogenicity of a WFS1 heterozygous truncation variant. This study provides a panoramic summary of both the genetic and phenotypic profiles of HON in real-world studies and literature. The category for nHON fundus phenotypes is built for future studies on molecular mechanisms underlying HON and targeted therapies. In addition to routine ophthalmic examinations, careful examination of the extraocular symptoms and meaningful genetic counseling are warranted for patients with nHON.

Mitochondrial Dysfunction Associated with mtDNA Mutation: Mitochondrial Genome Editing in Atherosclerosis Research.

Atherosclerosis is a complex cardiovascular disease often associated with mitochondrial dysfunction, which can lead to various cellular and metabolic abnormalities. Within the mitochondrial genome, specific mutations have been implicated in contributing to mitochondrial dysfunction. Atherosclerosis-associated m.15059G>A mutation has been of particular interest due to its potential role in altering mitochondrial function and cellular health. This study aims to investigate the role of the atherosclerosis-associated m.15059G>A mutation in the development of mitochondrial dysfunction in monocyte-- like cells. Monocyte-like cytoplasmic hybrid cell line TC-HSMAM1, which contains the m.15059G>A mutation in mtDNA, was used. The MitoCas9 vector was utilized to eliminate mtDNA copies carrying the m.15059G>A mutation from TC-HSMAM1 cybrids. Mitochondrial membrane potential, generation of reactive oxygen species, and lipid peroxidation levels were assessed using flow cytometry. Cellular reduced glutathione levels were assessed using the confocal microscopy. The oxygen consumption rate was measured using polarographic oxygen respirometry. The elimination of the m.15059G>A mutation resulted in a significant increase in mitochondrial membrane potential and improved mitochondrial efficiency while also causing a decrease in the generation of reactive oxygen species, lipid peroxidation, as well as cellular bioenergetic parameters, such as proton leak and non-mitochondrial oxygen consumption. At the same time, no changes were found in the intracellular antioxidant system after the mitochondrial genome editing. The presence of the m.15059G>A mutation contributes to mitochondrial dysfunction by reducing mitochondrial membrane potential, increasing the generation of reactive oxygen species and lipid peroxidation, and altering mitochondrial bioenergetics. Elimination of the mtDNA containing atherogenic mutation leads to an improvement in mitochondrial function.

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.

Mitochondrial genetic variations in leukemia: a comprehensive overview.

Leukemias are a group of heterogeneous hematological malignancies driven by diverse genetic variations, and the advent of genomic sequencing technologies facilitates the investigation of genetic abnormalities in leukemia. However, these sequencing-based studies mainly focus on nuclear DNAs. Increasing evidence indicates that mitochondrial dysfunction is an important mechanism of leukemia pathogenesis, which is closely related to the mitochondrial genome variations. Here, we provide an overview of current research progress concerning mitochondrial genetic variations in leukemia, encompassing gene mutations and copy number variations. We also summarize currently accessible mitochondrial DNA (mtDNA) sequencing methods. Notably, somatic mtDNA mutations may serve as natural genetic barcodes for lineage tracing and longitudinal assessment of clonal dynamics. Collectively, these findings enhance our understanding of leukemia pathogenesis and foster the identification of novel therapeutic targets and interventions.

Maintaining mitochondrial DNA copy number mitigates ROS-induced oocyte decline and female reproductive aging

Oocytes play a crucial role in transmitting maternal mitochondrial DNA (mtDNA), essential for the continuation of species. However, the effects of mitochondrial reactive oxygen species (ROS) on mammalian oocyte maturation and mtDNA maintenance remain unclear. We investigated this by conditionally knocking out the Sod2 gene in primordial follicles, elevating mitochondrial matrix ROS levels from early oocyte stages. Our data indicates that reproductive aging in Sod2 conditional knockout females begins at 6 months, with oxidative stress impairing oocyte quality, particularly affecting OXPHOS complex II and mtDNA-encoded mRNA levels. Despite unchanged mtDNA mutation load, mtDNA copy numbers exhibited significant variations. Strikingly, reducing mtDNA copy numbers by reducing mtSSB protein, crucial for mtDNA replication, accelerated reproductive aging onset to three months, underscoring the critical role of mtDNA copy number maintenance under oxidative stress conditions. This research provides new insights into the relationship among mitochondrial ROS, mtDNA, and reproductive aging, offering potential strategies for delaying aging-related fertility decline.

Selection promotes age-dependent degeneration of the mitochondrial genome.

Somatic mutations in mitochondrial genomes (mtDNA) accumulate exponentially during aging. Using single cell sequencing, we characterize the spectrum of age-accumulated mtDNA mutations in mouse and human liver and identify directional forces that accelerate the accumulation of mutations beyond the rate predicted by a neutral model. "Driver" mutations that give genomes a replicative advantage rose to high cellular abundance and carried along "passenger" mutations, some of which are deleterious. In addition, alleles that alter mtDNA-encoded proteins selectively increased in abundance overtime, strongly supporting the idea of a "destructive" selection that favors genomes lacking function. Overall, this combination of selective forces acting in hepatocytes promotes somatic accumulation of mutations in coding regions of mtDNA that are otherwise conserved in evolution. We propose that these selective processes could contribute to the population prevalence of mtDNA mutations, accelerate the course of heteroplasmic mitochondrial diseases and promote age-associated erosion of the mitochondrial genome.

Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.

Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats

AbstractMitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

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 tRNAGlu 14693A > G Mutation, an "Enhancer" to the Phenotypic Expression of Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON), a maternally inherited ocular disease, is predominantly caused by mitochondrial DNA (mtDNA) mutations. Mitochondrial tRNA variants are hypothesized to amplify the pathogenic impact of three primary mutations. However, the exact mechanisms remained unclear. In the present study, the synergistic effect of the tRNAGlu 14693A > G and ND6 14484T > C mutations in three Chinese families affected by LHON is investigated. The m.14693A > G mutation nearly abolishes the pseudouridinylation at position 55 of tRNAGlu, leading to structural abnormalities, decreased stability, aberrant mitochondrial protein synthesis, and increased autophagy. In contrast, the ND6 14484T > C mutation predominantly impairs complex I function, resulting in heightened apoptosis and virtually no induction of mitochondrial autophagy compared to control cell lines. The presence of dual mutations in the same cell lines exhibited a coexistence of both upregulated cellular stress responses to mitochondrial damage, indicating a scenario of autophagy and mutation dysregulation within these dual-mutant cell lines. The data proposes a novel hypothesis that mitochondrial tRNA gene mutations generally lead to increased mitochondrial autophagy, while mutations in genes encoding mitochondrial proteins typically induce apoptosis, shedding light on the intricate interplay between different genetic factors in the manifestation of LHON.

Tracking Rare Single Donor and Recipient Immune and Leukemia Cells after Allogeneic Hematopoietic Cell Transplantation Using Mitochondrial DNA Mutations.

Combined tracking of clonal evolution and chimeric cell phenotypes could enable detection of the key cellular populations associated with response following therapy, including after allogeneic hematopoietic stem cell transplantation (HSCT). We demonstrate that mitochondrial DNA (mtDNA) mutations coevolve with somatic nuclear DNA mutations at relapse post-HSCT and provide a sensitive means to monitor these cellular populations. Furthermore, detection of mtDNA mutations via single-cell assay for transposase-accessible chromatin with select antigen profiling by sequencing (ASAP-seq) simultaneously determines not only donor and recipient cells but also their phenotype at frequencies of 0.1% to 1%. Finally, integration of mtDNA mutations, surface markers, and chromatin accessibility profiles enables the phenotypic resolution of leukemic populations from normal immune cells, thereby providing fresh insights into residual donor-derived engraftment and short-term clonal evolution following therapy for post-transplant leukemia relapse. As throughput evolves, we envision future development of single-cell sequencing-based post-transplant monitoring as a powerful approach for guiding clinical decision-making. Significance: mtDNA mutations enable single-cell tracking of leukemic clonal evolution and donor-recipient origin following allogeneic HSCT. This provides unprecedented insight into chimeric cellular phenotypes of early immune reconstitution, incipient relapse, and quality of donor engraftment with immediate translational potential for future clinical post-transplant monitoring and decision-making.

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the PolgD257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed invivo and exvivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration.

Defective post-transcriptional modification of tRNA disrupts mitochondrial homeostasis in Leber’s hereditary optic neuropathy

Leber's Hereditary Optic Neuropathy (LHON) is a rare, maternally inherited eye disease, predominantly due to the degeneration of retinal ganglion cells (RGCs). It is associated with a mitochondrial DNA (mtDNA) point mutation. Our previous study identified that the m.15927G>A homoplasmic mutation damaged the highly conserved basepairing (28C-42G) in anticodon stem of tRNAThr, caused deficient t6A modification and significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr, and led to mitochondrial dysfunction. Meanwhile, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we manifested that defective nucleotide modification induced by the m.15927G>A mutation interfered with the expression of nuclear genes involved in cytoplasmic proteins essential for oxidative phosphorylation system (OXPHOS), thereby impacting the assemble and integrity of OXPHOS complexes. As a result of these mitochondrial dysfunctions, there was an imbalance in mitochondrial dynamics, particularly distinguished by an increased occurrence of mitochondrial fission. Excessive fission compromised the autophagy process, including initiation phase, formation and maturation of autophagosome. Both Parkin-mediated mitophagy and receptor-dependent mitophagy were significantly impaired in cybrids haboring the m.15927G>A mutation. These changes facilitated intrinsic apoptosis, as indicated by increased cytochrome c release and elevated levels of apoptosis-associated proteins (e.g., BAK, BAX, cleaved caspase 9, cleaved caspase 3, and cleaved PARP) in the mutant cybrids. This study demonstrates that the m.15927G>A mutation contributes to LHON by dysregulating OXPHOS biogenesis, aberrant quality control, increased autophagy, inhibited mitophagy, and abnormal apoptosis.

Accumulation of mitochondrial DNA with a point mutation in tRNALeu(UUR) gene induces brain dysfunction in mice

Brain functions are mediated via the complex interplay between several complex factors, and hence, identifying the underlying cause of an abnormality within a certain brain region can be challenging. In mitochondrial disease, abnormalities in brain function are thought to be attributed to accumulation of mitochondrial DNA (mtDNA) with pathogenic mutations; however, only few previous studies have directly demonstrated that accumulation of mutant mtDNA induced abnormalities in brain function. Herein, we examined the effects of mtDNA mutations on brain function via behavioral analyses using a mouse model with an A2748G point mutation in mtDNA tRNALeu(UUR). Our results revealed that mice with a high percentage of mutant mtDNA showed a characteristic trend toward reduced prepulse inhibition and memory-dependent test performance, similar to that observed in psychiatric disorders, such as schizophrenia; however, muscle strength and motor coordination were not markedly affected. Upon examining the hippocampus and frontal lobes of the brain, mitochondrial morphology was abnormal, and the brain weight was slightly reduced. These results indicate that the predominant accumulation of a point mutation in the tRNALeu(UUR) gene may affect brain functions, particularly the coordination of sensory and motor functions and memory processes. These abnormalities probably caused by both direct effects of accumulation of the mutant mtDNA in neuronal cells and indirect effects via changes of systemic extracellular environments. Overall, these findings will lead to a better understanding of the pathogenic mechanism underlying this complex disease and facilitate the development of optimal treatment methods.

Robustness and reliability of single-cell regulatory multi-omics with deep mitochondrial mutation profiling.

The detection of mitochondrial DNA (mtDNA) mutations in single cells holds considerable potential to define clonal relationships coupled with information on cell state in humans. Previous methods focused on higher heteroplasmy mutations that are limited in number and can be influenced by functional selection, introducing biases for lineage tracing. Although more challenging to detect, intermediate to low heteroplasmy mtDNA mutations are valuable due to their high diversity, abundance, and lower propensity to selection. To enhance mtDNA mutation detection and facilitate fine-scale lineage tracing, we developed the single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM) approach, an integrated experimental and computational framework. Recently, some concerns have been raised about the analytical workflow in the ReDeeM framework. Specifically, it was noted that the mutations detected in a single molecule per cell are enriched on edges of mtDNA molecules, suggesting they resemble artifacts reported in other sequencing approaches. It was then proposed that all mutations found in one molecule per cell should be removed. We detail our error correction method, demonstrating that the observed edge mutations are distinct from previously reported sequencing artifacts. We further show that the proposed removal leads to massive elimination of bona fide and informative mutations. Indeed, mutations accumulating on edges impact a minority of all mutation calls (for example, in hematopoietic stem cells, the excess mutations on the edge account for only 4.3%-7.6% of the total). Recognizing the value of addressing edge mutations even after applying consensus correction, we provide an additional filtering option in the ReDeeM-R package. This approach effectively eliminates the position biases, leads to a mutational signature indistinguishable from bona fide mitochondrial mutations, and removes excess low molecule high connectedness mutations. Importantly, this option preserves the large majority of unique mutations identified by ReDeeM, maintaining the ability of ReDeeM to provide a more than 10-fold increase in variant detection compared to previous methods. Additionally, the cells remain well-connected. While there is room for further refinement in mutation calling strategies, the significant advances and biological insights provided by the ReDeeM framework are unique and remain intact. We hope that this detailed discussion and analysis enables the community to employ this approach and contribute to its further development.

Analysis of Mutational Burden of Mitochondrial Genome in Cells of Different Human Organs and Tissues.

Cells of different human organs and tissues contain different numbers of mitochondria. In these organelles, there are different copies of the mitochondrial genome, which is characteristic of a certain organ or tissue. The aim of the investigation was to analyze the results of scientific works dedicated to the analysis of heteroplasmy levels of mitochondrial genome mutations in a number of organs and tissues. Based on literature data, the level of heteroplasmy of mitochondrial genome mutations was analyzed in organs such as the liver, lungs, muscles, small intestine, large intestine, spleen, kidney, brain, heart, and hair. In addition, this parameter was studied in such tissues as leukocytes, buccal epithelium, and epithelial cells from urine. Significant differences in the mutational burden of the mitochondrial genome were found in various samples of organs and tissues. The highest heteroplasmy level for mtDNA mutations was in muscles; it was lower in buccal epithelium; and in human blood cells, the heteroplasmy level of mitochondrial mutations turned out to be significantly lower compared to other tissues. During the comparison of samples of patients with different diseases and healthy people, significant differences were found in the heteroplasmy level between some organs and tissues. The heteroplasmy level of mitochondrial genome mutations can significantly differ in the organs and tissues of individuals. In addition, in a number of literature sources, it is noted that there is a dependence on the mutational burden of the mitochondrial genome from the type of disease, sex, and age of a person.