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Heteroplasmic mtDNA mutation (T----G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high.

A female infant showing lacticacidemia, hypotonia, and neurodegenerative disease died at 7 mo of age. Autopsy revealed lesions typical of Leigh disease, both in the basal ganglia and in the brain stem. A maternal aunt and uncle died 1 year and 5 mo, respectively, after following a similar clinical course, while another uncle, presently 33 years of age, has retinitis pigmentosa and ataxia and is mentally retarded. PCR restriction-digest analysis of mtDNA isolated from the proband revealed a T-to-G change at position 8993, creating a new AvaI restriction site. The mutation present in the ATP 6 gene results in the substitution of an arginine residue for a leucine. The indexed patient had greater than 95% abnormal mtDNA in her skin fibroblasts, brain, kidney, and liver tissues, as measured by laser densitometry. The maternal aunt who died at age 1 year had greater than 95% abnormal mtDNA in her lymphoblasts. The uncle with retinitis pigmentosa had 78% and 79% abnormal mtDNA in his skin fibroblasts and lymphoblasts, respectively, while an asymptomatic maternal aunt and her son had no trace of this mutation. The mother of the index case had 71% and 39% abnormal mtDNA in her skin fibroblasts and lymphoblasts, respectively, showing that the heteroplasmy can be variable, on a tissue-specific basis, within one individual. This shows that mtDNA mutations at 8993 can produce the clinical phenotype of Leigh disease in addition to the phenotype of ataxia and retinitis pigmentosa described by Holt et al.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Mitochondrial

Segregation and manifestations of the mtDNA tRNA(Lys) A-->G(8344) mutation of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome.

We have studied the segregation and manifestations of the tRNA(Lys) A-->G(8344) mutation of mtDNA. Three unrelated patients with myoclonus epilepsy and ragged-red fibers (MERRF) syndrome were investigated, along with 30 of their maternal relatives. Mutated mtDNA was not always found in the offspring of women carrying the tRNA(Lys) mutation. Four women had 10%-33% of mutated mtDNA in lymphocytes, and no mutated mtDNA was found in 7 of their 14 investigated children. The presence of mutated mtDNA was excluded at a level of 3:1,000. Five women had a proportion of 43%-73% mutated mtDNA in lymphocytes, and mutated mtDNA was found in all their 12 investigated children. This suggests that the risk for transmission of mutated mtDNA to the offspring increases if high levels are present in the mother and that, above a threshold level of 35%-40%, it is very likely that transmission will occur to all children. The three patients with MERRF syndrome had, in muscle, both 94%-96% mutated mtDNA and biochemical and histochemical evidence of a respiratory-chain dysfunction. Four relatives had a proportion of 61%-92% mutated mtDNA in muscle, and biochemical measurements showed a normal respiratory-chain function in muscle in all cases. These findings suggest that > 92% of mtDNA with the tRNA(Lys) mutation in muscle is required to cause a respiratory-chain dysfunction that can be detected by biochemical methods. There was a positive correlation between the levels of mtDNA with the tRNA(Lys) mutation in lymphocytes and the levels in muscle, in all nine investigated cases. The levels of mutated mtDNA were higher in muscle than in lymphocytes in all cases. In two of the patients with MERRF syndrome, muscle specimens were obtained at different times. In both cases, biochemical measurements revealed a deteriorating respiratory-chain function, and in one case a progressive increase in the amount of cytochrome c oxidase-deficient muscle fibers was found.

Adult

Precision ID mtDNA Whole Genome Panel and sequencing of telogen hairs - perspectives for validation and implementation in casework.

Shed hair is a commonly encountered type of forensic evidence. Shed telogen hairs generally contain insufficient or highly degraded nuclear DNA for STR profiling; however, mtDNA analysis of telogen hair and hair shafts remains possible. We validated whole mitochondrial genome (mtGenome) sequencing using the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific) and subsequently implemented the panel for the analysis of telogen hair, buccal, and casework samples. We analysed 90 diluted DNA samples containing 3-3,600 mtDNA copies, shed telogen hairs and their corresponding mtDNA from buccal swabs from 91 individuals, and 11 archived DNA extracts from hair samples in criminal cases. Complete mtGenome sequences were consistently recovered in 99% of samples across DNA dilution series at DNA input levels as low as 47 mtDNA copies, demonstrating the assay's robustness under low-template conditions. We obtained complete and reproducible mtGenome sequences with ≥ 327 mtDNA copies/µL from telogen hair samples. After applying ISFG recommendations and excluding low-confidence discrepancies associated with high-strand bias, heteroplasmic variants and sequencing artifacts, mtGenome sequence concordance increased from 93.4% to 100%. None of the 16 negative controls produced complete mtDNA sequences. Six negative controls showed low-level mtDNA signal (2-8 variants), consisting predominantly of common polymorphisms. These samples did not yield complete mtGenome sequences and showed no correspondence to any of the analysed samples. Finally, archived telogen hair samples from criminal cases presented complete mtGenome sequences with an average read depth of 1,037x.Our findings highlight the reliability of mtDNA analysis of telogen hairs using the Precision ID mtDNA Whole Genome Panel for implementation in forensic casework.

Forensic casework

A new chick mitochondrial DNA-binding protein exhibits sequence-specific interaction near heavy-strand replication origin: cleavage activity, stimulation of mtDNA synthesis, and enhancement in transformed fibroblasts.

We have identified a new, double-strand-dependent, mtDNA-binding protein in chick embryo fibroblast (CEF) mitochondria (and inner-membrane-matrix preparations) which demonstrates both an exclusive specific affinity for the displacement loop (D-loop) control region of chick mtDNA and intramitochondrial levels that reflect corresponding changes in mtDNA replication activity both in vivo and in vitro. This approximately 36 kDa protein (designated aMDP1, avian mitochondrial DNA-binding protein 1) was identified by elution and renaturation following SDS-polyacrylamide gel electrophoresis and by direct isolation from specific mtDNA-protein complexes excised from mobility shift gels. Analysis of the entire 16.7-kb mt genome determined that a MDP1 mediates cleavage of chick mtDNA in vitro at three H- and two L-strand sequence-specific target sites located within a 90-bp A + T-rich genomic tract, theoretically capable of forming stable secondary structures, approximately 200 bases upstream from the H-strand origin (OH) of replication. Furthermore, gel-isolated aMDP1 relaxes supercoiled mtDNA, and exogenous addition of the protein, in a permeabilized in vitro system, preferentially stimulates the synthesis of H-strand sequences which hybridize to OH-containing fragments. Oncogenic transformation of CEF by Rous sarcoma viruses results in a threefold elevated level of aMDP1, directly correlating with a similarly increased level of mtDNA replication in vivo. Heterologous chick-human cross-competition experiments showed that aMDP1 also selectively interacts with human (HeLa) D-loop region mtDNA, possibly reflective of an evolutionary importance for aMDP1 interaction in the region. Functionally, we hypothesize that aMDP1 may operate in conjunction with other mtDNA-binding proteins, important in replication and transcription, by potentiating duplex unwinding either prior to or during an initial stage of H-strand synthesis. Together, these results suggest that aMDP1 is a good potential candidate for a nucleus-encoded regulatory protein which communicates with the mt genome during the replication process.

Animals

Mechanism of age-related accumulation of mtDNA mutations in human blood.

Accumulation of mutant mitochondrial DNA (mtDNA) heteroplasmy is among the strongest signatures of ageing1. Here we investigated the underlying mechanism by calling mtDNA sequence, mtDNA abundance and mtDNA heteroplasmic variants in human blood using whole-genome sequences from approximately 750,000 individuals. We observed that mtDNA single-nucleotide variants (mtSNVs) accumulate sharply at age 60 years, occur at low levels of heteroplasmy, exhibit little evidence of positive selection and are likely to be predominantly neutral. The mutational spectrum of mtSNVs does not reflect oxidative lesions, as is commonly invoked, but is more consistent with mtDNA replication errors. To understand why mtSNVs become detectable with age, we performed a genome-wide association study for heteroplasmic mtSNV burden, identifying germline variants near TERT, TCL1A and SMC4, all of which have been linked to clonal haematopoiesis (CH)2. Rare-variant analysis also showed that high mtSNV burden is associated with mutations in numerous CH driver genes. These genetic associations persisted even after exclusion of individuals with known CH driver mutations. Our results support a model in which 'cryptic' mtDNA mutations initially arise randomly as replication errors but are undetectable in bulk. They then become apparent only through age-related expansion of cellular clones in blood. We propose that the high copy number and mutation rate of mtDNA make it a sensitive blood-based marker of somatic mosaicism due to CH. Our work mechanistically unifies three prominent signatures of ageing: common germline variants in TERT, CH and observed accrual of mtDNA mutations.

Humans

Lack of transmission of deleted mtDNA from a woman with Kearns-Sayre syndrome to her child.

We have investigated the daughter of a woman with Kearns-Sayre syndrome. The woman had a high percentage of deleted mtDNA in muscle, but no deleted mtDNA was detected in fibroblasts, bone marrow, and peripheral blood cells by Southern blot analysis. With PCR, analytical sensitivity was significantly increased, and deleted mtDNA was detected in all examined tissues from this patient. The patient had healthy parents and nine healthy siblings. No deleted mtDNA was detected in blood from the mother of the patient. The patient had an uneventful pregnancy and delivered at term. Deleted mtDNA could not be detected in placenta by Southern blot analysis. With PCR, deleted mtDNA was detected in the majority of placental specimens. This finding may, however, be due to contamination with maternal DNA. The patient's daughter was healthy at age 5 mo, and morphologic examination of muscle was normal. No transmission of deleted mtDNA to the daughter could be detected by Southern blot and PCR analysis of peripheral blood cells, bone marrow, fibroblasts, and muscle. The presence of deleted mtDNA was excluded at a fractional level of less than 1:100,000 in all examined tissues from the daughter.

Adolescent

Evidence that a 1.6 kilobase region of Neurospora mtDNA was derived by insertion of part of the LaBelle mitochondrial plasmid.

The LaBelle mitochondrial plasmid hybridizes to a small region of the mtDNA of different Neurospora species. Here, we show that the region of homology encompasses 1385 bp of plasmid sequence and 1649 bp of mtDNA sequence. Several findings--that the region of homology is not found in the mtDNAs of other organisms, that it includes the C-terminus of the ORF encoding the plasmid DNA polymerase, and that the ORF sequence in the mtDNA is interrupted by insertions--suggest that the region was part of the plasmid that integrated into mtDNA prior to the divergence of Neurospora species. Since the LaBelle plasmid has been found in only one Neurospora strain, we infer that the plasmid was lost subsequently from most strains. The LaBelle plasmid is transcribed by the host Neurospora mitochondrial RNA polymerase and the major promoter is located upstream of the long ORF, within the region of homology to mtDNA. A promoter used for the transcription of the mitochondrial small rRNA is found at a corresponding position in Neurospora mtDNA and may have been acquired via integration of the plasmid sequence. Our results provide evidence that an autonomous infectious element may contribute to sequences that functionally constitute an organism's mtDNA.

Base Sequence

Multiple features of cell-free mtDNA for predicting transarterial chemoembolization response in hepatocellular carcinoma.

BACKGROUND: Transarterial chemoembolization (TACE) is the primary treatment modality for advanced HCC, yet its efficacy assessment and prognosis prediction largely depend on imaging and serological markers that possess inherent limitations in terms of real-time capability, sensitivity, and specificity. Here, we explored whether multiple features of cell-free mitochondrial DNA (cf-mtDNA), including copy number, mutations, and fragmentomics, could be used to predict the response and prognosis of patients with HCC undergoing TACE treatment. METHODS: A total of 60 plasma cell-free DNA samples were collected from 30 patients with HCC before and after the first TACE treatment and then subjected to capture-based mtDNA sequencing and whole-genome sequencing. RESULTS: Comprehensive analyses revealed a clear association between cf-mtDNA multiple features and tumor characteristics. Based on cf-mtDNA multiple features, we also developed HCC death and progression risk prediction models. Kaplan-Meier curve analyses revealed that the high-death risk or high-progression-risk group had significantly shorter median overall survival (OS) and progression-free survival than the low-death risk or low-progression-risk group (all p<0.05). Moreover, the change in cf-mtDNA multiple features before and after TACE treatment exhibited an exceptional ability to predict the risk of death and progression in patients with HCC (log-rank test, all p<0.01; HRs: 0.36 and 0.33, respectively). Furthermore, we observed the consistency of change between the cf-mtDNA multiple features and copy number variant burden before and after TACE treatment in 40.00% (12/30) patients with HCC. CONCLUSIONS: Altogether, we developed a novel strategy based on profiling of cf-mtDNA multiple features for prognosis prediction and efficacy evaluation in patients with HCC undergoing TACE treatment.

Humans

Tissue segregation of a heteroplasmic mtDNA mutation in MERRF (myoclonic epilepsy with ragged red fibers) encephalomyopathy.

The distribution of the causal 8344A-->G mtDNA mutation has been examined in six tissues of a patient with myoclonic epilepsy with ragged red fibers (MERRF), to study the developmental genetics of this type of mitochondrial disorder, and to determine the pathophysiological importance of the mtDNA heteroplasmy generally observed in such patients. Heteroplasmy of the mtDNA was observed in all six tissues (cerebellum, cerebrum, pancreas, liver, muscle, and heart) suggesting that, whereas the mtDNA mutation is relatively new, the mutated population must have existed before the formation of the three primary embryonic layers. The tissue distribution reveals significant variations in the ratio between the mutated and the normal mtDNA species, indicating the randomness of mtDNA segregation during developmental cell division and differentiation events. The result suggests the existence of tissue-specific nuclear factor(s) that determines the expression of the 8344A-->G mutation in various tissues; in MERRF syndrome, expression is mainly in the central nervous system.

Adult

New approaches to dating suggest a recent age for the human mtDNA ancestor.

The most critical and controversial feature of the African origin hypothesis of human mitochondrial DNA (mtDNA) evolution is the relatively recent age of about 200 ka inferred for the human mtDNA ancestor. If this age is wrong, and the actual age instead approaches 1 million years ago, then the controversy abates. Reliable estimates of the age of the human mtDNA ancestor and the associated standard error are therefore crucial. However, more recent estimates of the age of the human ancestor rely on comparisons between human and chimpanzee mtDNAs that may not be reliable and for which standard errors are difficult to calculate. We present here two approaches for deriving an intraspecific calibration of the rate of human mtDNA sequence evolution that allow standard errors to be readily calculated. The estimates resulting from these two approaches for the age of the human mtDNA ancestor (and approximate 95% confidence intervals) are 133 (63-356) and 137 (63-416) ka ago. These results provide the strongest evidence yet for a relatively recent origin of the human mtDNA ancestor.

Animals

Trans-Mitochondrial Cybrid Generation from mtDNA Patient Platelets: An Efficient Protocol Optimizing Colony Selection and Functional Validation.

Trans-mitochondrial cybrid cell line generation represents the gold-standard method for determining pathogenicity by enabling biochemical analyses of a specific mitochondrial DNA (mtDNA) variant of interest at high and low percentages (heteroplasmy levels) within an otherwise identical mtDNA and nuclear genome background. Historically, the cybrid generation process has been tedious and poorly efficient. Here, we describe a highly efficient and effective protocol for generating trans-mitochondrial cybrid cell lines by fusing human platelets with a standard osteosarcoma 143B cell line to provide an isogenic nuclear background depleted of mtDNA (Rho0 cells). Cell isolates capture a given mtDNA genome of interest to establish stable cell lines harboring different degrees of heteroplasmy, or to compare divergent effects of distinct mitochondrial haplogroups. Because cybrids from mitochondrial patients may be more difficult to establish with standard protocols, this current methodology focuses on isolating mtDNA variants where the electron transport chain activity is affected. We here demonstrate that colony selection techniques reduce time and improve the yield of generating high-level heteroplasmy mtDNA mutant cybrid lines. A case study is provided of cybrid generation for a variant of unknown significance in MT-ND1, m.3985G>A (p.E227K). We analyze the efficiency of the cybrid generation process using this protocol and run functional studies performed by high-resolution respirometry. High-level heteroplasmy MT-ND1 m.3985G>A cybrid mutants generated by this protocol are shown to have impaired complex I-dependent mitochondrial respiration relative to wild-type control, demonstrating m.3985G>A is likely pathogenic.

Humans

Single-cell profiling of mitochondrial phenotyping-coupled mtDNA genotyping.

Simultaneously profiling mitochondrial DNA (mtDNA) heteroplasmy and phenotypic variability at the single-cell level remains a challenge due to the absence of integrated methods that map mitochondrial genotypes alongside their functional states. We introduce human single-cell mitochondrial phenotype-coupled mtDNA sequencing (scMPCDS), a platform that quantifies mtDNA mutations and heteroplasmy together with mitochondrial membrane potential and reactive oxygen species within individual cells. Unlike bulk sequencing or separate single-omics techniques, scMPCDS directly correlates mitochondrial genomic instability with functional outcomes. Using this approach, we demonstrate that DdCBE-mediated mtDNA editing induces cell-specific off-target mutations in the mitochondrial genome, which coincide with diverse phenotypic changes. Applying scMPCDS to HeLa cells and clear cell renal cell carcinoma tissues, we identify single-cell subpopulations exhibiting distinct mtDNA mutation burdens and altered bioenergetic profiles, implicating potential mitochondrial heterogeneity-driven tumor evolution. Overall, scMPCDS serves as a versatile tool to unravel mitochondrial genotype-phenotype relationships at the single-cell level in both normal and disease states, thereby advancing precise mitochondrial diagnostics and therapeutics.

Humans

Metabolic regulation of mitochondrial DNA (mtDNA) homeostasis.

Mitochondria are central hubs of cellular metabolism that harbor their own genome (mtDNA), whose maintenance is essential for both cellular and organismal homeostasis. Unlike nuclear DNA, mtDNA replicates continuously throughout the cell cycle, rendering it particularly sensitive to changes in metabolic state. Emerging evidence indicates that mtDNA homeostasis is not governed solely by dedicated replication factors but is tightly coupled to cellular metabolism. In this review, we discuss how metabolic networks shape mtDNA maintenance through three interconnected layers: mitochondrial nucleotide pools, metabolic control of the replication machinery, and stress-response pathways. This conceptual framework underscores the direct role of metabolic state in governing mtDNA replication, stability, and quality control, with significant implications for mitochondrial disease and therapeutic strategies.

Integrated stress response (ISR)

Multiple short direct repeats associated with single mtDNA deletions.

We have sequenced the breakpoints of deleted mtDNA in muscle from four children with mitochondrial myopathy and multisystem mitochondrial disorders. The deletions were 4884, 6067, 7663 and 7150 base pairs (bp) in size and affected several protein and transfer RNA genes. The sequences needed for transcription and replication of mtDNA were not affected in any case. The deletions were flanked by direct short repeats in all cases. Multiple repeats were found in case 1 and 4. Imperfect repeats were found in case 3 and 4 and this made it possible to distinguish the repeats 5' and 3' to the deletion. In both cases the 3' repeat was retained. The deletion of 7663 bp in case 3 has been reported in two other cases and may represent a second hotspot for mtDNA deletions in addition to the common deletion of 4977 bp found in one third of cases. A comparison of the breakpoint sequence of case 3 with the two other reported cases revealed that when a deletion is formed between the same repeats in different patients either the 5' or 3' repeat can be retained. This study shows that both single and multiple repeats can be associated with single mtDNA deletions and that both 5' and 3' repeated sequences can be retained. These findings are consistent with the slip-replication model for the generation of mtDNA deletions.

Base Sequence

MELAS mutation in mtDNA binding site for transcription termination factor causes defects in protein synthesis and in respiration but no change in levels of upstream and downstream mature transcripts.

The pathogenetic mechanism of the mitochondrial tRNA(LeuUUR) gene mutation responsible for the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) syndrome was investigated in transformants obtained by transfer of mitochondria from three genetically unrelated MELAS patients into human mitochondrial DNA (mtDNA)-less (rho 0) cells. Marked defects in mitochondrial protein synthesis and respiratory activity were observed in transformants containing virtually pure mutant mtDNA, as compared to the parent of the rho 0 cells (the 143B cell line) or to transformants containing exclusively wild-type mtDNA, derived from one of the patients or a maternally related asymptomatic individual. A striking protective effect against the mutation was exerted in the transformants by levels of residual wild-type mtDNA above 6%. The MELAS mutation occurs within the mtDNA binding site for a protein factor (mTERF) that promotes termination of transcription at the 16S rRNA/tRNA(LeuUUR) gene boundary. A marked decrease in affinity of purified mTERF for the mutant target sequence was observed in in vitro assays. By contrast, RNA transfer hybridization experiments failed to show any significant change in the steady-state amounts of the two rRNA species, encoded upstream of the termination site, and of the mRNAs encoded downstream, in the transformants carrying the MELAS mutation.

Acidosis, Lactic

Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy.

The segregation of mutant and wild-type mtDNA was investigated in transformants constructed by transferring human mitochondria from individuals belonging to four pedigrees with the MELAS encephalomyopathy-associated mtDNA mutation (MELAS is mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) into human mtDNA-less (rho 0) cells. Five of 13 clonal cell lines containing mixtures of wild-type and mutant mtDNAs were found to undergo a rapid shift of their genotype toward the pure mutant type. The other 8 cell lines, which included 6 exhibiting nearly homoplasmic mutant mtDNA, on the contrary, maintained a stable genotype. Subcloning experiments and growth rate measurements clearly indicated that an intracellular replicative advantage of mutant mtDNA was mainly responsible for the dramatic shift toward the mutant genotype observed in the unstable cell lines.

Cell Division

Dynamics of cytoplasmic incompatibility and mtDNA variation in natural Drosophila simulans populations.

In Drosophila simulans a cytoplasmically transmitted microorganism causes reduced egg hatch when infected males mate with uninfected females. The infection is rapidly spreading northward in California. Data on a specific mtDNA restriction site length polymorphism show that changes in the frequency of mtDNA variants are associated with this spread. All infected flies possess the same mtDNA allele, whereas the uninfected flies are polymorphic. Given that both paternal inheritance of the infection and imperfect maternal transmission have been demonstrated, one might expect instead that both infected and uninfected flies would possess both mtDNA variants. Our data suggest that imperfect female transmission of the infection (and/or the loss of the infection among progeny) is more common in nature than paternal transmission. A simple model of intrapopulation dynamics, with empirically supported parameter values, adequately describes the joint frequencies of the mtDNA variants and incompatibility types.

Animals