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PubMed2026

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↗

PubMed2026

Blood mitochondrial heteroplasmic variants and cognitive performance in late midlife: REGARDS study.

BACKGROUND: Studies linking mitochondrial DNA (mtDNA) variants to cognition yielded inconsistent findings, and the underlying mechanisms remain unclear. We investigated whether mtDNA heteroplasmic variants were associated with cognitive outcomes, including the Montreal Cognitive Assessment (MoCA), in 197 late midlife adults from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort with complete data. METHODS: MtDNA was sequenced from blood using targeted deep sequencing. Adjusted linear and mixed-effects models examined the associations by functional regions, genes, total variant burden, nonsynonymous variants, and control regions. RESULTS: Heteroplasmic variants in the control region (β = -0.44, 95% CI: -0.83, -0.05, p = 0.027) and transfer RNA (tRNA) genes (β = -1.34, 95% CI: -2.58, -0.11, p = 0.034) were associated with MoCA baseline scores. Individual variants in cytochrome c oxidase subunit 1 (CO1) (β = -1.51, 95% CI: -2.54, -0.47, p = 0.005), NADH dehydrogenase subunit 1 (ND1) (β = -2.63, 95% CI: -4.56, -0.70, p = 0.008), and Displacement Loop (D-LOOP2) (β = -2.25, 95% CI: -4.20, -0.30, p = 0.025) was associated with reduced baseline MoCA scores. The ND6 (β = −1.23, 95% CI: −2.09, − 0.37, p = 0.006), ND4 (β = −1.11, 95% CI: −2.02, − 0.20, p = 0.018), ATP Synthase Membrane Subunit 8 (ATP8; β = −1.38, 95% CI: −2.63, − 0.13, p = 0.031), and D-LOOP1 (β = −0.61, 95% CI: −1.20, − 0.01, p = 0.045) genes suggested a potential association with executive function. Longitudinal Animal Fluency Test (AFT) scores were inversely associated with heteroplasmic variants in coding regions (β = -0.10, 95% CI: -0.19, -0.006, p = 0.049), the total number of variants (β = -0.06, 95% CI: -0.11, -0.003, p = 0.037) and total nonsynonymous variants (β = -0.11, 95% CI: -0.21, -0.01, p = 0.040). Variants in the control region were associated with the greatest decline in verbal fluency (β = −0.20, 95% CI: −0.39 to − 0.002, p = 0.049). No associations were observed between mitochondrial variants and verbal memory performance or the MoCA composite scores. CONCLUSIONS: Our study indicates that mitochondrial variants measured in blood may provide insight into cognitive function during midlife. However, additional studies are needed to validate these associations and to address potential power limitations in our study.

Humans↗

PubMed2026

Protocol for Duplex Sequencing of Mitochondrial DNA in Single Human Oocytes.

Oocytes are densely packed with mitochondria, the energy-producing organelles that contain their own genome, mitochondrial DNA (mtDNA). Each cell contains multiple copies of mtDNA, with copy number varying among tissue types. Oocytes possess the highest mtDNA copy number, containing hundreds of thousands of mtDNA molecules per cell. Because mitochondria are inherited exclusively through the maternal lineage, accurate detection of mtDNA variants is essential for studies of inheritance, aging, and disease. The presence of multiple mtDNA copies allows wild-type and mutant molecules to coexist within the same cell, a condition known as heteroplasmy, in which low-frequency and de novo variants may occur at frequencies below 1%. Conventional next-generation sequencing (NGS) lacks sufficient accuracy to reliably distinguish these rare variants from errors introduced during library preparation and sequencing. Here, we present a protocol for enriching mtDNA from single human oocytes using Exonuclease V to remove linear DNA, followed by duplex sequencing library preparation for highly accurate mtDNA analysis. This workflow enables error-corrected sequencing of individual oocytes, facilitating reliable detection of low-frequency mtDNA variants and analysis of heteroplasmy and de novo mutagenesis. The protocol provides a reproducible approach for investigating mitochondrial genome variation in single oocytes using Illumina-compatible sequencing platforms.

Humans↗

PubMed1979

Characterization of variant Neurospora crassa mitochondrial DNAs which contain tandem reiterations.

Two variant mtDNA types ((types IIa and HI-10) have been identified in individual subcultures of the extra-nuclear [poky] mutant of Neurospora crassa. Eco RI digests of type IIa mtDNA are characterized by an extra band, alpha, Mr = 1.4 Mdal, which arises from tandemly inserted reiterations of a 1.4 Mdal sequence. Restriction enzyme analysis and Southern hybridization experiments show: that the 1.4 Mdal repeats are located at the junction of Eco RI-4 and -6, that the repeats contain sequences ordinarily present in Eco RI-4 and -6, that the repeats are oriented head-to-tail and that the number of repeats per molecule (n) varies from n = 0 to n = 8, with about half of the molecules containing no repeats. The 1.4 Mdal repeats appear to be actively mained in type IIa mtDNA populations as a result of a specific alteration in mtDNA. Data are presented which suggest that this alteration may be located near small deletions and/or sequence changes in Eco RI-3 and -10, fragments almost exactly opposite the site of the repeats on the genome. The second variant, HI-10 mtDNA, arose in a heteroplasmic strain in which type IIa mtDNA was one component. The most striking feature of HI-10 mtDNA is the up to 5-fold amplification of an 18 Mdal segment extending from Eco RI-4 (the site of the 1.4 Mdal repeats) through the rRNA genes. Eco RI digests show that HI-10 possesses characteristic features of type IIa mtDNA, including the 1.4 Mdal repeats and the alteration in Eco RI-10. HI-10 mtDNA also shows a novel Eco RI fragment, beta, Mr = 2.9 Mdal. The variant Neurospora mtDNAs may be generated by mechanisms analogous to those which give rise to defective mtDNAs of yeast petite mutants. The possible consequences of defective mtDNAs in obligately aerobic organisms are discussed.

Base Sequence↗

PubMed2026

"Tissue-specific mitochondrial dysfunction in keratoconus: An integrated structural, genomic, and functional analysis".

PURPOSE: Keratoconus (KC) is a progressive corneal ectasia characterized by stromal thinning, conical protrusion, and irregular astigmatism, leading to visual impairment. Although oxidative stress is implicated in KC, the role of mitochondrial dysfunction remains unclear. We evaluated mitochondrial structural, genomic, and functional abnormalities in corneal tissues and blood from KC patients. METHODS: This prospective study enrolled 110 KC patients and 55 controls. Transmission electron microscopy (TEM) and immunohistochemistry (IHC) were performed on epithelial and stromal tissues from 10 KC to 5 control corneas assessing mitochondrial morphology, oxidative phosphorylation (OXPHOS) complexes and pro-apoptotic protein NOXA. Whole mitochondrial DNA (mtDNA) sequencing and relative mtDNA copy number analysis were performed on paired blood and corneal tissues from 50 KC patients and 35 controls including both epithelial and stromal samples. Gene expression of mitochondrial biogenesis and oxidative stress-related genes was analysed by qRT-PCR in corneal epithelium from independent 50 KC patients and 15 controls. RESULTS: TEM revealed cristolysis, membrane disruption, and reduced mitochondrial density in KC corneas. IHC showed reduced expression of OXPHOS complexes and increased NOXA expression (p < 0.05). Sequencing identified 1107 mtDNA variants, with more variants in corneal tissues than matched blood (929 vs. 576; p = 0.0002). Recurrent likely pathogenic variants were enriched in complex I-encoding genes (ND4, ND5). KC corneas showed reduced mtDNA copy number, downregulated POLRMT, upregulated NOX4, and significant downregulation of multiple antioxidant genes (p < 0.0001). CONCLUSION: KC patients exhibit tissue-specific mitochondrial abnormalities and impaired oxidative stress regulation, supporting a role for mitochondrial dysfunction in disease pathogenesis and highlighting potential therapeutic targets.

Corneal pathology↗

PubMed2026

Transmitochondrial pigs reveal causal effects of mitochondrial DNA on backfat thickness via nuclear epigenetic reprogramming.

Mitochondrial DNA (mtDNA) polymorphisms have been associated with production traits in farm animals, including backfat thickness in pigs, yet direct in vivo evidence establishing a causal link between specific mtDNA haplotypes and fat deposition remains limited. In this study, we generated transmitochondrial pigs (mitopigs) by combining the Dapulian nuclear genome with Wuzhishan mtDNA via somatic cell nuclear transfer, introducing 23 mtDNA mutations relative to controls. Mitopigs exhibited significantly increased backfat thickness at 5 months, a difference that persisted in their offspring, without significant differences in body weight, body size, or litter size. Fibroblasts derived from mitopigs exhibited reduced mtDNA copy numbers, decreased expression of mitochondrial biogenesis genes (PPARA, PPARGC1A, RRM2B, and LRPPRC), impaired mitochondrial respiration, elevated reactive oxygen species (ROS), and upregulated adipogenic transcription factors (CEBPA, CEBPB, and PPARG). Consistent with these fibroblast findings, backfat tissue of mitopigs showed corresponding upregulation of adipogenic transcription factors and downregulation of mitochondrial biogenesis genes. Integrated transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses revealed nuclear transcriptional reprogramming that was closely associated with differential DNA methylation, predominantly affecting mitochondrial function and lipid metabolism pathways. Mitopig fibroblasts also showed a pro-inflammatory response to lipopolysaccharide stimulation, with elevated expression of IL-12, NOS2, RELA, and TNF-α. Our findings provide direct in vivo evidence that mtDNA variants regulate adiposity in pigs through mitochondrial dysfunction, oxidative stress, and nuclear epigenetic modulation, highlighting the potential for incorporating mtDNA haplotype information into pig breeding programs as a complementary strategy to nuclear genomic selection.

Adipogenesis↗

PubMed2026

URMD-Seq: A high-throughput method for scalable detection of ultra-rare mutations in the human mitochondrial genome.

The study of mitochondrial genetics has long been limited to polymorphisms and high frequency mutations owing in part to technical and technological limitations in reliably detecting and quantifying rare somatic mutations. Over the past decade or so, the study of rare somatic mitochondrial DNA (mtDNA) variants has expanded and continues to garner increasing interest in a wide range of research fields. Here, we describe Ultra-Rare Mutation Detection-Sequencing (URMD-Seq), a high-throughput method that combines unique molecular identifier (UMI)-based library preparation and Next Generation Sequencing (NGS) for the accurate and scalable detection of ultra-rare mutations in the mtDNA control region. Our method exploits degenerate primers to label individual mtDNA molecules. This is followed by several purification, quantification and amplification steps, to obtain high quality amplicons for sequencing on the Illumina MiSeq platform. Our approach enables the use of total genomic DNA extract as starting point for the assay, overcoming the need for organelle isolation and/or mtDNA enrichment, hence broadening the type of specimen that can be studied, while offering cost and time benefits. The assay described herein has been demonstrated to reliably measure variants present at on average 0.09%, but as low as 0.03%, variant allele frequency in a variety of tissues, including fresh and frozen biobanked specimens. Using this protocol, library preparation of 300 specimens can be completed by a single individual with general nucleic acid handling experience in approximately 20 days. Given its flexibility and scalability, URMD-Seq is particularly well suited for epidemiological studies using a large number of specimens.

Humans↗

PubMed2026

DNM1L depletion leads to accelerated heteroplasmy shifting of m.10191C allele through ATG7-dependent pathways.

Nucleotide composition bias in mitochondrial DNA (mtDNA) makes the heavy strand prone to form a DNA secondary structure called a guanine quadruplex (G4). This secondary structure has been shown to inhibit polymerase processivity in vitro. We previously identified pathogenic mtDNA variants that lead to increased G4-forming propensity, including a T to C mutation at m.10191 (m.10191 T > C) that causes Leigh syndrome. Cells treated with G4 binding agent (G4BA) berberine show a reduction in m.10191C pathogenic heteroplasmy levels. To help better understand the underlying mechanism behind berberine-induced heteroplasmy shift, we examined the relationship between mitochondrial fission and berberine-mediated shift. Here we show that knockdown of the fission factor DNM1L leads to an accelerated heteroplasmy shift towards the healthy mtDNA allele, lowering m.10191C by 10% in 3 weeks, compared to the 5 weeks required for berberine alone. The specific mechanism involves ATG7, as knockdown of ATG7 is able to partially delay this accelerated heteroplasmy shift. Taken together, we show that DNM1L knockdown is able to accelerate berberine-induced m.10191C heteroplasmy shifting through an autophagy-related mechanism.

Humans↗

PubMed2026

Pathogenic POLRMT variants in mice impair mtDNA transcription and affect perinatal survival.

Mitochondrial gene expression is essential for oxidative phosphorylation that generates the bulk of the cellular ATP, and mitochondrial dysfunction is a common cause of human metabolic diseases. Recently, the first pathogenic variants in the only known mitochondrial RNA polymerase (POLRMT) were described in patients presenting with a wide variety of clinical manifestations, including hypotonia, short stature, and developmental delay. Here, we modeled two human pathogenic POLRMT variants by creating the corresponding substitutions in mice: the dominant S582F and the recessive R984C variant. Mice homozygous for the R984C variant showed perinatal lethality without apparent embryonic developmental defects, a finding consistent with a failure to adapt to the metabolic transition to oxidative metabolism at birth. Mice carrying the S582F variant were viable and exhibited decreased mitochondrial transcript levels due to impaired de novo transcription. However, mtDNA levels and in organello mtDNA replication remained normal, which recapitulates the molecular phenotypes observed in patients. Altogether, our findings indicate that the conserved arginine near the active site is essential for POLRMT function, while the serine in the intercalating hairpin of the N-terminal domain is required for near-genome length transcription but not primase activity. This study highlights genotype-phenotype differences and provides new insights into POLRMT function.

Animals↗

PubMed2026

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↗

PubMed1992

Mitochondrial DNA mutation and the ageing process: bioenergy and pharmacological intervention.

A comprehensive hypothesis concerning the contribution of mitochondrial DNA (mtDNA) mutations to the human ageing process is reviewed and the implications for cellular bioenergy loss and pharmacological therapy are considered. The central idea is that random mutations in the population of mtDNA molecules of each cell occur throughout life, and that this is a major contributor to the gradual loss of cellular bioenergy capacity within tissues and organs, associated with general senescence and diseases of ageing. An elaboration of four major aspects of the general proposition, together with relevant supporting data, is presented. (1) An extensive array of deletions in mtDNA of many tissues of humans and other mammals has been observed to occur in an age-related manner. (2) The preservation and selection of fully functional mtDNA molecules in the female germ line cells is proposed to occur via a human mtDNA cycle, in which selective amplification of a limited number of mtDNA templates occurs during oocyte development. This proposal explains the endowment of normal neonates with a mtDNA complement minimally contaminated by damaged mtDNA molecules. The phenomena of maternal inheritance and rapid fixation of sequence variants of mtDNA in mammals, as well as selection of cells based on mitochondrial function, are taken into account. (3) Tissue bioenergy mosaics result from accumulated mtDNA damage during ageing, representing different rates of cellular bioenergy loss within individual cells of a tissue. The random segregation of mtDNA during cell division will also further contribute to the tissue energy mosaic. Cells unable to meet their particular bioenergy demand will become non-functional, leading to cell death; the bioenergy threshold is different for the various cell types in the tissues of the body. (4) In order to bioenergetically resuscitate cells and tissues suffering from impaired mitochondrial functions as a result of the ageing process, we propose that redox compounds may be used therapeutically in the pharmacological configurations of a by-pass strategy or as a redox sink therapy. The role of these compounds is to maintain at least part of the mitochondrial respiratory chain function (by-pass) as well as to maintain adequate levels of cellular NAD+ (redox sink) for ATP synthesis, predominantly by the cytosolic glycolytic pathway, with some contribution from mitochondrial oxidative phosphorylation.

Adult↗

PubMed1992

Heteroplasmy of short tandem repeats in mitochondrial DNA of Atlantic cod, Gadus morhua.

The mitochondrial DNA of the Atlantic cod (Gadus morhua) contains a tandem array of 40-bp repeats in the D-loop region of the molecule. Variation among molecules in the copy number of these repeats results in mtDNA length variation and heteroplasmy (the presence of more than one form of mtDNA in an individual). In a sample of fish collected from different localities around Iceland and off George's Bank, each individual was heteroplasmic for two or more mtDNAs ranging in repeat copy number from two (common) to six (rare). An earlier report on mtDNA heteroplasmy in sturgeon (Acipenser transmontanus) presented a competitive displacement model for length mutations in mtDNAs containing tandem arrays and the cod data deviate from this model. Depending on the nature of putative secondary structures and the location of D-loop strand termination, additional mechanisms of length mutation may be needed to explain the range of mtDNA length variants maintained in these populations. The balance between genetic drift and mutation in maintaining this length polymorphism is estimated through a hierarchical analysis of diversity of mtDNA length variation in the Iceland samples. Eighty percent of the diversity lies within individuals, 8% among individuals and 12% among localities. An estimate of theta = 2N(eo) mu greater than 1 indicates that this system is characterized by a high mutation rate and is governed primarily by deterministic dynamics. The sequences of repeat arrays from fish collected in Norway, Iceland and George's Bank show no nucleotide variation suggesting that there is very little substructuring to the North Atlantic cod population.

Animals↗

PubMed1992

Mitochondrial DNA length variation and heteroplasmy in populations of white sturgeon (Acipenser transmontanus).

Southern blot analysis was used to quantify the extent of mtDNA D-loop length variation in two populations of white sturgeon, Acipenser transmontanus. Over 42% of individuals were heteroplasmic for up to six different mtDNA length variants attributable to varying copy numbers of an 82-bp repeat sequence. Chi-square analyses revealed that the frequencies of length genotypes and the incidence of heteroplasmy were significantly different between Fraser and Columbia River sturgeon populations but not between restriction site haplotypes. Heteroplasmic fish have, on average, higher copy number than homoplasmic fish. Forty-five of 101 homoplasmic individuals carry only a single copy of the repeat, while none of the 73 heteroplasmic fish has the single repeat as the predominant variant. On the basis of differences in frequency distributions of copy number within and between fish, we suggest that (1) heteroplasmy is maintained by high recurrent mutation of multiple copy genomes, favoring increased copy number and (2) the mutation pressure toward higher copy number heteroplasmy is partially offset by selection to reduced genome size and segregation to the homoplasmic condition.

Animals↗

PubMed2026

Twelve Japanese patients with POLG-related disorders: Population-specific genetic differences of POLG variants in Japan and Europe.

BACKGROUND: POLG encodes mitochondrial DNA (mtDNA) polymerase γ. Pathogenic POLG variants cause mitochondrial diseases, including progressive external ophthalmoplegia. POLG-related disorders are relatively common in Europe, possibly because of the high prevalence of carriers in the general population, but remain rare in Japan for unclear reasons. METHODS: We performed long-range PCR on mtDNA from skeletal muscle and/or peripheral blood from 3146 patients with suspected mitochondrial disease between 1993 and 2021. We selected 167 individuals with clinical features suggestive of POLG-related disorders for POLG gene analysis; all lacked pathogenic mtDNA point mutations, and most had multiple mtDNA deletions and/or a family history of mitochondrial disease. RESULTS: Among the 167 patients (median age: 52 years, range: 0-83 years, 11% pediatric cases), we identified 12 Japanese patients with POLG-related disorders and six POLG variants, including one novel variant. The six variants were p.Y955C, p.R943H, p.T599I, p.M299L, p.Y1210* (c.3626_3629dupGATA), and the novel variant p.F377S (c.1130T>C). Neither these six variants nor the 10 previously reported cases from Japan included the POLG variants that are more frequent in Europe. We also analyzed three population databases: two whole-genome sequencing databases covering 61,000 and 9850 Japanese individuals, respectively, and one global population database (gnomAD) covering 730,000 individuals worldwide. POLG variants that are more frequent in Europe were not detected in the Japanese databases or among East Asian individuals in gnomAD. CONCLUSIONS: Our findings suggest population-specific genetic differences in POLG between Japanese and European populations, explaining the lower frequency of POLG-related disorders in Japan.

CPEO↗

PubMed2024

Increased risk of hearing loss associated with MT-RNR1 gene mutations: a real-world investigation among Han Taiwanese Population.

BACKGROUND: Previous studies have implicated inherited mutations in mitochondrial DNA (mtDNA) in sensorineural hearing loss (SNHL). However, the definitive association between mitochondrial 12S rRNA (MT-RNR1) variants and hearing loss in the population has not been well established, particularly in Asia. The objective of this retrospective cohort study was to assess the association between MT-RNR1 variants and the risk of SNHL in patients in Taiwan. METHODS: The cohort included 306,068 participants from Taiwan between January 2003 and December 2020. Participants were classified based on genetic variants, particularly mitochondrial mutations (rs267606618, rs267606619, rs267606617). MT-RNR1 variant cases were matched 1:10 with non-mutant patients by age, gender, and visit year, excluding those with pre-existing hearing loss. The primary endpoint was SNHL, identified using specific ICD-TM codes with a 90% positive predictive value. Medication exposure history was determined via self-report or electronic medical records in the hospital. Cox proportional hazard regression models were used to assess the association between MT-RNR1 variants and hearing loss, adjusting for various covariates. Kaplan-Meier survival curves and log-rank tests compared hearing loss incidence between groups. RESULTS: The mean age of the mtDNA variants group is 32.4 years, with a standard deviation of 19.2 years. The incidence density of hearing loss for the mutation group was 36.42 per 10,000 person-years (95% Confidence Interval [CI], 27.21-47.73), which was higher than the 23.77per 10,000 person-years (95% CI, 21.32-26.42) in the wild-type group (p = 0.0036). Additionally, diabetes mellitus was associated with an increased risk of developing SNHL in individuals with MT-RNR1 variants (adjusted hazard ratio = 1.76 [95% CI, 1.00-3.09], p < 0.05). CONCLUSION: This study highlights the increased risk of hearing loss in patients carrying MT-RNR1 variants, particularly those with diabetes mellitus. Future research that integrates genetic and clinical data is crucial for developing more precise interventions to monitor and treat hearing loss in this vulnerable population.

Adolescent↗

PubMed2026

Visual Detection and Stratification of Pathogenic mtDNA SNV Heteroplasmy by Balancing FnCas12a Signal Output and Allelic Discrimination.

Assessment of pathogenic mitochondrial DNA (mtDNA) single-nucleotide variant (SNV) heteroplasmy is important for molecular diagnostics, yet rapid visual profiling remains analytically challenging because an assay must combine single-nucleotide allelic discrimination, mutant-fraction-associated readout, and suitable target access. Herein, we report VISTA (visual identification and stratification of targeted mtDNA alleles), a broad-PAM FnCas12a assay that rebalances trans-cleavage signal output and mutant-wild-type discrimination for visual mtDNA SNV heteroplasmy analysis. VISTA uses unmodified FnCas12a with relaxed TTN PAM recognition and integrates crRNA spacer-length engineering with PEG8000/acBSA reaction tuning to improve the practical signal-discrimination balance without nuclease engineering. At the m.3243A>G model locus, spacer truncation enhanced mutant-wild-type discrimination, while molecular-dynamics simulations identified spacer-dependent differences between matched and mismatched complexes at the crRNA-DNA interface. The optimized assay resolved defined synthetic m.3243A>G heteroplasmy gradients by fluorescence imaging and was further adapted to lateral-flow detection. In locus-specific analyses of a deidentified collection of 74 peripheral-blood samples, fluorescence and lateral-flow readouts achieved ROC AUC values above 0.9 for mutant-allele classification after target-region amplification. Fluorescence supported heteroplasmy-associated profiling, whereas lateral flow provided a visual, semiquantitative readout for relative ranking based on the T/C ratio rather than absolute heteroplasmy measurement. VISTA therefore provides an accessible dual-readout analytical strategy for visual detection and heteroplasmy-associated profiling by tuning the FnCas12a signal output and allelic discrimination.

DNA, Mitochondrial↗

PubMed2025

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↗

PubMed2026

Mitochondrial DNA diversity in Ecuadorian populations: Recurrence of variant 16136 within haplogroup B2.

The identification of lineage-defining variants, frequently found in the coding region of mitochondrial DNA (mtDNA), is essential for refining haplogroup classification. Most mtDNA studies in South American populations have focused on the control region (CR), which has provided important insights into population structure and maternal lineage origins, although information needed for more robust phylogenetic resolution has been neglected. This study investigates the maternal genetic structure of Ecuadorian populations by combining CR and whole mitogenome analyses. Sequences from the mtDNA CR were obtained from 461 individuals (253 Mestizos and 208 Native Americans), while complete mitogenomes were sequenced for 127 individuals to improve phylogenetic resolution by identifying lineage-defining variants present in coding region. Most mtDNA haplogroups in the two population groups analyzed were of Native American origin (A2, B2, B4, C1, D1, D4), with significant differences in the distribution of specific lineages between them. Among Mestizos, African haplogroups (all within the L branches) and Eurasian haplogroups (H, K, R, U) were detected at low frequencies, whereas no African lineages were observed among Native Americans. The results obtained highlighted a heterogeneity within Ecuadorian populations that must be considered when developing mtDNA haplotype databases for forensic purposes. Whole mitogenome sequences enabled the identification of variants that refined haplogroup classifications, provided a more accurate reconstruction of the maternal genetic diversity, and improve the discrimination between Native American and Asian maternal lineages within haplogroup B4b.

Humans↗