mtDNA Mutations Research Articles

Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.

Mitochondrial DNA (mtDNA) encodes a variety of rRNAs, tRNAs, and respiratory chain complex proteins. The integrity of mtDNA supports the mitochondrial functions and plays an essential role in numerous physiological and pathological processes. Mutations in mtDNA cause metabolic diseases and aging. The mtDNA within the human cells are packaged into hundreds of nucleoids within the mitochondrial matrix. Knowledge of how the nucleoids are dynamically distributed and organized within mitochondria is key to understanding mtDNA structure and functions. Therefore, visualizing the distribution and dynamics of mtDNA within mitochondria is a powerful approach to gain insights into the regulation of mtDNA replication and transcription. In this chapter, we describe the methods of observing mtDNA and its replication with fluorescence microscopy in both fixed and live cells using different labeling strategies.

Detection of UV-Induced Deletions in Mitochondrial DNA.

Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.

Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.

Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.

Mechanisms regulating mitochondrial DNA quality control

Maintenance of genome integrity is essential for cellular survival. There are mechanisms utilized by the cells to sense and respond to assaults on genomic DNA. These mechanisms are conserved across all domains of life and are collectively called the DNA damage response pathways. However, eukaryotic cells also have extrachromosomal DNA in mitochondria (mtDNA), which is indispensable for mitochondrial function, and hence cell survival. Indeed, impaired mitochondrial activity arising due to mutations in mtDNA has been found to be associated with many human pathologies. Despite its importance, our understanding of how cells ensure mtDNA genome integrity is limited. Since mitochondria do not encode for machinery required for the maintenance of their own genomes, they depend on the nucleus for replication, transcription, and repair processes. This adds a layer of complexity with the requirement for organelle crosstalk and coordination in response to mtDNA damage. This review summarizes recent findings that provide new insights into mechanisms involved in mtDNA quality control, acting at the level of mtDNA or organelle and also discusses a few new avenues of research towards a comprehensive understanding of the “mtDNA damage response”.

Multidrug resistance transporter-1 dysfunction perturbs meiosis and Ca2+ homeostasis in oocytes.

Oocyte quality remains the most important and unsolved issue in reproduction. Our data show that multidrug resistance transporters and oocyte mitochondria are involved in determining oocyte quality in a mouse model. Multidrug resistance transporter-1 (MDR-1) is a transmembrane ATP-dependent effluxer present in organs that transport a variety of xenobiotics and by-products. Previous findings by our group demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Considering the importance of these organelles in the female gamete, we assessed the impact of MDR-1 dysfunction on mouse oocyte quality, with a particular focus on the meiotic spindle organization, aneuploidies, Ca2+ homeostasis, ATP production and mtDNA mutations. Our results demonstrate that young Mdr1a mutant mice produce oocytes characterized by lower quality, with a significant delay in the germinal vesicle to germinal vesicle breakdown transition, an increased percentage of symmetric divisions, chromosome misalignments and a severely altered meiotic spindle shape compared to the wild types. Mutant oocytes exhibit 7000 more SNPs in the exomic DNA and twice the amount of mitochondrial DNA (mtDNA) SNPs compared to the wild-type ones. Ca2+ analysis revealed the inability of MDR-1 mutant oocytes to manage Ca2+ storage content and oscillations in response to several stimuli, and ATP quantification shows that mutant oocytes trend toward lower ATP levels compared to wild types. Finally, 1-year-old mutant ovaries express a lower amount of SIRT1, SIRT3, SIRT5, SIRT6 and SIRT7 compared to wild-type levels. These results together emphasize the importance of MDR-1 in mitochondrial physiology and highlight the influence of MDR-1 on oocyte quality and ovarian aging.

Potential Roles of mtDNA Mutations in PCOS-IR: A Review

Polycystic ovary syndrome (PCOS) is the most common heterogeneous endocrine disease that affecting females in reproductive age. Insulin resistance (IR), an important molecular basis for PCOS, accounts for at least 75% of women carrying this syndrome. Although there have been many studies on PCOS-IR, the detailed mechanisms are not fully understood. As essential hub for energy generation, mitochondria are critical to insulin secretion and normal function, whereas mutations in mitochondrial DNA (mtDNA) result in mitochondrial dysfunctions contributing to the pathophysiology of PCOS-IR via the regulation of balance of oxidative stress (OS), energy deficiency, or hormone metabolism. In the current review, we summarize the clinical and molecular features of PCOS-IR and discuss molecular mechanisms related to mtDNA mutations.

Hereditary optic neuropathy associated with demyelinating diseases of the central nervous system

Demyelinating optic neuritis and hereditary optic neuropathy (HON) take a leading place among the diseases, the leading clinical syndrome of which is bilateral optic neuropathy with a simultaneous or sequential significant decrease in visual acuity. Optic neuritis can occur at the onset or be one of the syndromes within multiple sclerosis (MS), neuromyelitis optica spectrum disorders (NMOSD), and myelin oligodendrocyte glycoprotein (MOG) antibody disease (MOGAD). HON are a group of neurodegenerative diseases, among which the most common variants are Leber's hereditary optic neuropathy (LHON), associated with mitochondrial DNA (mtDNA) mutations, and autosomal recessive optic neuropathy (ARON), caused by nuclear DNA (nDNA) mutations in DNAJC30. There are phenotypes of LHON «plus», one of which is the association of HON and CNS demyelination in the same patient. In such cases, the diagnosis of each of these diseases causes significant difficulties, due to the fact that in some cases there are clinical and radiological coincidences between demyelinating and hereditary mitochondrial diseases.

Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.

Over the last 10years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.

Prevalence and Clinical Characteristics of Mitochondrial DNA Mutations in Korean Patients With Sensorineural Hearing Loss.

Mutations in mitochondrial DNA (mtDNA) are associated with several genetic disorders, including sensorineural hearing loss. However, the prevalence of mtDNA mutations in a large cohort of Korean patients with hearing loss has not yet been investigated. Thus, this study aimed to investigate the frequency of mtDNA mutations in a cohort of with pre- or post-lingual hearing loss of varying severity. A total of 711 Korean families involving 1,099 individuals were evaluated. Six mitochondrial variants associated with deafness (MTRNR1 m.1555A>G, MTTL1 m.3243A>G, MTCO1 m.7444G>A and m.7445A>G, and MTTS1 m.7471dupC and m.7511T>C) were screened using restriction fragment length polymorphism. The prevalence of the six variants was also analyzed in a large control dataset using whole-genome sequencing data from 4,534 Korean individuals with unknown hearing phenotype. Overall, 12 of the 711 (1.7%) patients with hearing loss had mtDNA variants, with 10 patients from independent families positive for the MTRNR1 m.1555A>G mutation and 2 patients positive for the MTCO1 m.7444G>A mutation. The clinical characteristics of patients with the mtDNA variants were characterized by post-lingual progressive hearing loss due to the m.1555A>G variant (9 of 472; 1.9%). In addition, 18/4,534 (0.4%) of the Korean population have mitochondrial variants associated with hearing loss, predominantly the m.1555A>G variant. A significant proportion of Korean patients with hearing loss is affected by the mtDNA variants, with the m.1555A>G variant being the most prevalent. These results clarify the genetic basis of hearing loss in the Korean population and emphasize the need for genetic testing for mtDNA variants.

Germline TFAM levels regulate mitochondrial DNA copy number and mutant heteroplasmy in C. elegans.

The mitochondrial genome (mtDNA) is packaged into discrete protein-DNA complexes called nucleoids. mtDNA packaging factor TFAM (mitochondrial transcription factor-A) promotes nucleoid compaction and is required for mtDNA replication. Here, we investigate how changing TFAM levels affects mtDNA in the Caenorhabditis elegans germ line. We show that increasing germline TFAM activity boosts mtDNA number and significantly increases the relative proportion of a selfish mtDNA mutant, uaDf5 . We conclude that TFAM levels must be tightly controlled to ensure appropriate mtDNA composition in the germ line.

Diseases caused by mitochondrial DNA mutations

This article highlights data on diseases caused by mitochondrial DNA mutations. The purpose of the review was to reveal existing diseases that arise as a result of mtDNA mutations. Mitochondrial diseases are diseases that are most often caused by genetically determined structural and functional disorders of mitochondria, and as a result, the energy supply of cells is disrupted. All mitochondrial diseases are transmitted through the maternal line, so if mutations are detected in time, they can be blocked and the further inheritance will be stopped. It is suggested that the role of mitochondrial DNA in certain diseases began to develop rapidly in 1988 when the first mutations in mitochondrial DNA were discovered. To understand the course and development of mitochondrial DNA, it is necessary to understand the structure and functional properties of the mitochondrial cell. MtDNA is a circular DNA molecule and is localized in mitochondria. Such organelles can replicate, transcribe, and translate their own DNA independently of nuclear DNA. Mitochondrial DNA can mutate more than 10 times more often than nuclear DNA. MtDNA has no protective functions against the phenomena of mutations. A mitochondrial cell can contain both mutant DNA and normal DNA. In genetics, such a condition is called heteroplasmy, which allows the survival of a lethal mutation. Single deletions, large deletions, and multiple deletions that are transmitted autosomally and have different phenotypic manifestations are the primary cause of the development of mitochondrial diseases. Scientists also identify systemic manifestations of mitochondrial DNA mutations. they include endocrine manifestations (diabetes), neurological diseases, gastrointestinal manifestations (acid-alkaline imbalance), and pulmonary manifestations (myoclonic epilepsy, hypoventilation abnormalities). Several main principles of treatment of mitochondropathies are distinguished: following a diet; additional introduction of cofactors involved in enzymatic reactions of energy metabolism (thiamine, riboflavin, nicotinamide, lipoic acid, biotin, carnitine); prescription of drugs, capable of carrying out the function of transferring electrons in the respiratory chain (vitamins K1 and K3, ascorbic acid).

Detecting mitochondrial mutations associated with aminoglycoside ototoxicity by noninvasive prenatal testing.

Numerous diseases and disorders are associated with mitochondrial DNA (mtDNA) mutations, among which m.1555A > G and m.1494C > T mutations in the 12 S ribosomal RNA gene contribute to aminoglycoside-induced and nonsyndromic hearing loss worldwide. A total of 76,842 qualified non-invasive prenatal (NIPT) samples were subjected to mtDNA mutation and haplogroup analysis. We detected 181 m.1555A > G and m.1494C > T mutations, 151 of which were subsequently sequenced for full-length mitochondrial genome verification. The positive predictive values for the m.1555A > G and m.1494C > T mutations were 90.78% and 90.00%, respectively, a performance comparable to that attained with newborn hearing screening. Furthermore, mitochondrial haplogroup analysis revealed that the 12 S rRNA 1555A > G mutation was enriched in sub-haplotype D5[p=0, OR=4.6706(2.81-7.78)]. Our findings indicate that the non-invasive prenatal testing of cell-free DNA obtained from maternal plasma can successfully detect m.1555A > G and m.1494C > T mutations.

High-frequency and functional mitochondrial DNA mutations at the single-cell level

Decline in mitochondrial function underlies aging and age-related diseases, but the role of mitochondrial DNA (mtDNA) mutations in these processes remains elusive. To investigate patterns of mtDNA mutations, it is particularly important to quantify mtDNA mutations and their associated pathogenic effects at the single-cell level. However, existing single-cell mtDNA sequencing approaches remain inefficient due to high cost and low mtDNA on-target rates. In this study, we developed a cost-effective mtDNA targeted-sequencing protocol called single-cell sequencing by targeted amplification of multiplex probes (scSTAMP) and experimentally validated its reliability. We then applied our method to assess single-cell mtDNA mutations in 768 B lymphocytes and 768 monocytes from a 76-y-old female. Across 632 B lymphocyte and 617 monocytes with medium mtDNA coverage over >100×, our results indicated that over 50% of cells carried at least one mtDNA mutation with variant allele frequencies (VAFs) over 20%, and that cells carried an average of 0.658 and 0.712 such mutation for B lymphocytes and monocytes, respectively. Surprisingly, more than 20% of the observed mutations had VAFs of over 90% in either cell population. In addition, over 60% of the mutations were in protein-coding genes, of which over 70% were nonsynonymous, and more than 50% of the nonsynonymous mutations were predicted to be highly pathogenic. Interestingly, about 80% of the observed mutations were singletons in the respective cell populations. Our results revealed mtDNA mutations with functional significance might be prevalent at advanced age, calling further investigation on age-related mtDNA mutation dynamics at the single-cell level.

Mitochondrial Dysfunction and Therapeutic Perspectives in Cardiovascular Diseases.

High mortality rates due to cardiovascular diseases (CVDs) have attracted worldwide attention. It has been reported that mitochondrial dysfunction is one of the most important mechanisms affecting the pathogenesis of CVDs. Mitochondrial DNA (mtDNA) mutations may result in impaired oxidative phosphorylation (OXPHOS), abnormal respiratory chains, and ATP production. In dysfunctional mitochondria, the electron transport chain (ETC) is uncoupled and the energy supply is reduced, while reactive oxygen species (ROS) production is increased. Here, we discussed and analyzed the relationship between mtDNA mutations, impaired mitophagy, decreased OXPHOS, elevated ROS, and CVDs from the perspective of mitochondrial dysfunction. Furthermore, we explored current potential therapeutic strategies for CVDs by eliminating mtDNA mutations (e.g., mtDNA editing and mitochondrial replacement), enhancing mitophagy, improving OXPHOS capacity (e.g., supplement with NAD+, nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and nano-drug delivery), and reducing ROS (e.g., supplement with Coenzyme Q10 and other antioxidants), and dissected their respective advantages and limitations. In fact, some therapeutic strategies are still a long way from achieving safe and effective clinical treatment. Although establishing effective and safe therapeutic strategies for CVDs remains challenging, starting from a mitochondrial perspective holds bright prospects.

MitoSomatic: a tool for accurate identification of mitochondrial DNA somatic mutations without paired controls.

Mitochondrial DNA (mtDNA) somatic mutations play important roles in the initiation and progression of cancer. Although next-generation sequencing (NGS) of paired tumor and control samples has become a common practice to identify tumor-specific mtDNA mutations, the unique nature of mtDNA and NGS-associated sequencing bias could cause false-positive/-negative somatic mutation calling. Additionally, there are clinical scenarios where matched control tissues are unavailable for comparison. Therefore, a novel approach for accurately identifying somatic mtDNA variants is greatly needed, particularly in the absence of matched controls. In this study, the ground truth mtDNA variants orthogonally validated by triple-paired tumor, adjacent nontumor, and blood samples were used to develop mitoSomatic, a random forest-based machine learning tool. We demonstrated that mitoSomatic achieved area under the curve (AUC) values over 0.99 for identifying somatic mtDNA variants without paired control in three tumor types. In addition, mitoSomatic was also applicable in nontumor tissues such as adjacent nontumor and blood samples, suggesting the flexibility of mitoSomatic's classification capability. Furthermore, analysis of triple-paired samples identified a small group of variants with uncertain somatic/germline origin, whereas application of mitoSomatic significantly facilitated the prediction of their possible source. Finally, a control-free evaluation of the public pan-cancer NGS dataset with mitoSomatic revealed a substantial number of variants that were probably misclassified by conventional tumor-control comparison, further emphasizing the usefulness of mitoSomatic in application. Taken together, our study demonstrates that mitoSomatic is valuable for accurately identifying somatic mtDNA variants in mtDNA NGS data without paired controls, applicable for both tumor and nontumor tissues.

In Utero Exposure to Air Pollutants and Mitochondrial Heteroplasmy in Neonates.

Mitochondria are sensitive to oxidative stress, which can be caused by traffic-related air pollution. Placental mitochondrial DNA (mtDNA) mutations have been previously linked with air pollution. However, the relationship between prenatal air pollution and cord-blood mtDNA mutations has been poorly understood. Therefore, we hypothesized that prenatal particulate matter (PM2.5) and NO2 exposures are associated with cord-blood mtDNA heteroplasmy. As part of the ENVIRONAGE cohort, 200 mother-newborn pairs were recruited. Cord-blood mitochondrial single-nucleotide polymorphisms were identified by whole mitochondrial genome sequencing, and heteroplasmy levels were evaluated based on the variant allele frequency (VAF). Outdoor PM2.5 and NO2 concentrations were determined by a high-resolution spatial-temporal interpolation method based on the maternal residential address. Distributed lag linear models were used to determine sensitive time windows for the association between NO2 exposure and cord-blood mtDNA heteroplasmy. A 5 μg/m3 increment in NO2 was linked with MT-D-Loop16311T>C heteroplasmy from gestational weeks 17-25. MT-CYTB14766C>T was negatively associated with NO2 exposure in mid pregnancy, from weeks 14-17, and positively associated in late pregnancy, from weeks 31-36. No significant associations were observed with prenatal PM2.5 exposure. This is the first study to show that prenatal NO2 exposure is associated with cord-blood mitochondrial mutations and suggests two critical windows of exposure in mid-to-late pregnancy.

Mitochondrial DNA Mutations as Natural Barcodes for Lineage Tracing of Murine Tumor Models.

mtDNA mutations can serve as natural barcodes to enable lineage tracing in murine cancer models, which can be used to provide new insights into disease biology and to identify therapeutic vulnerabilities.

Mitochondrial DNA involvement in patients with autism spectrum disorders and intellectual disability

BackgroundAutism spectrum disorders (ASD) are neurodevelopmental disorders, with heterogeneous etiology, characterized by deficiencies in social interaction, communication, and by repetitive and stereotyped behaviors.Mitochondrial encephalomyopathies are inherited disorders of oxidative metabolism, with a wide clinical, biochemical and genetic heterogeneity, requiring a complex diagnostic flow-chart. Infantile forms often occur with defects in development, intellectual disabilities and dysmorphisms.ASD has been associated with mitochondrial respiratory chain deficiency and/or mitochondrial DNA (mtDNA) mutations. However, few studies carried out detailed clinical, instrumental, morphological, biochemical investigations, and mtDNA sequencing in ASD patients. MethodThe purpose of this study was the identification of mitochondrial dysfunction due to mtDNA variants in 19 selected subjects presenting ASD and clinical features of mitochondrial disease. For these patients we adopted a multidisciplinary approach combining clinical and laboratory investigations on muscle biopsy, with biochemical, histological and genetic techniques. ResultsThe histological examination showed myogenic or neurogenic changes in 79 % of patients; furthermore, 58 % of the patients had lipid accumulation, mitochondrial proliferation and COX deficient fibers. The biochemical investigations reported in three patients impairments involving one or more of the respiratory chain complexes. In addition, genetic studies revealed in one patient with normal histology and biochemistry multiple mtDNA deletions, and in four patients different mtDNA point mutations. ConclusionsThe present study confirms the hypothesis of an association between ASD and mitochondrial dysfunction. However, further studies in a larger group of subjects are needed.

Roles of Mitochondrial DNA Damage in Kidney Diseases: A New Biomarker

The kidney is a mitochondria-rich organ, and kidney diseases are recognized as mitochondria-related pathologies. Intact mitochondrial DNA (mtDNA) maintains normal mitochondrial function. Mitochondrial dysfunction caused by mtDNA damage, including impaired mtDNA replication, mtDNA mutation, mtDNA leakage, and mtDNA methylation, is involved in the progression of kidney diseases. Herein, we review the roles of mtDNA damage in different setting of kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD). In a variety of kidney diseases, mtDNA damage is closely associated with loss of kidney function. The level of mtDNA in peripheral serum and urine also reflects the status of kidney injury. Alleviating mtDNA damage can promote the recovery of mitochondrial function by exogenous drug treatment and thus reduce kidney injury. In short, we conclude that mtDNA damage may serve as a novel biomarker for assessing kidney injury in different causes of renal dysfunction, which provides a new theoretical basis for mtDNA-targeted intervention as a therapeutic option for kidney diseases.

Retinal involvement in mitochondrial disease

AbstractRetinal involvement in mitochondrial disease is a variable feature of mitochondrial dysfunction. The retinal pigment epithelium and photoreceptor layers can both be affected. It manifests with signs ranging from asymptomatic peripheral salt‐and‐pepper retinopathy to classic bone‐spicule pigmentary changes. The consequences can be progressive mild or severe visual field constriction and central visual acuity loss. The pathophysiological mechanisms involved in retinal dysfunction in mitochondrial disease are still being elucidated and may include a range of metabolic and bioenergetic defects.Mitochondrial conditions affecting the eye that display prominent outer retinal involvement include.(a) mitochondrial encephalomyopathy, lactic acidosis and stroke‐like episodes (MELAS) secondary to the m.3243A > G mtDNA mutation in MT‐TL1,(b) neuropathy, ataxia and retinitis pigmentosa (NARP) secondary to the m.8993 T > G mtDNA mutation in MT‐ATP6,(c) chronic progressive external ophthalmoplegia (CPEO), in particular the Kearns‐Sayre syndrome (KSS) secondary to single large‐scale mtDNA deletions, and.(d) some newer entities, such as optic neuropathy and pigmentary retinopathy seen in patients with mutations in the gene SSBP1.The features and diagnostic pathway for these conditions will be illustrated and discussed.