mtDNA Heteroplasmy Research Articles

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former’s inefficiency and the latter’s inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional ‘Correction’ mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in ‘Correction’ mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.

Abstract P1032: Substrate Optimization For Patients With Melas And Leigh Syndrome Using Induced Pluripotent Stem Cells-derived Cardiomyocytes

Mitochondrial DNA (mtDNA) mutations increase the risk of health issues with overlapping clinical phenotypes. We propose that patient-derived induced pluripotent stem cells (iPSCs) can serve as surrogates for studying mitochondrial diseases, provided a standardized protocol is used to mitigate effects of mtDNA heteroplasmy. Accordingly, we reprogrammed peripheral blood mononuclear cells (CytoTune Kit) from mtDNA disease patients with epilepsy enrolled in clinical trials. After culturing the reprogrammed cells, six iPSC cell colonies were selected (Alkaline Phosphatase Live Stain) from each donor, pooled, and sorted by SSEA4+ marker at passage 6. Pluripotency was confirmed using OCT4 immuno-staining and RT-PCR for OCT4, NANOG, and SOX2 at passage 10. Because two of the patients (m.3243A>G MT-TL1, MELAS, and m.T8993G MT-ATP6, Leigh Syndrome) showed signs of improvement on drug therapy, we differentiated their iPSCs into cardiomyocytes (iCMs) and characterized them using immuno-staining (cTnT). We then screened the substrate utilization preferences of the iCMs using Phenotype MicroArray Mammalian assays (Biolog). The MELAS-iCMs failed to metabolize lactic acid, L-ornithine, and branched-chain amino acids, but performed better than control iCMs in the presence of arginine and glycerol phosphate. In contrast, LS-iCMs exhibited distinctly different substrate preferences and thrived in the presence of acetoacetic acid and α-keto-butyric acid with reduced oxidative stress (AmplexRed). The failure of MELAS-iCMs to survive in the presence of only lactic acid recapitulates a characteristic feature of MELAS, underscoring the potential of iPSCs for disease modeling. Our findings suggest that diverse outcomes to nutritional and pharmaceutical interventions must be tailored to each patient. To validate the pharmacological efficacy of iCMs, we will continuously evaluate their response in patients who are refractory to treatment.

Neuropathological hallmarks in autopsied cases with mitochondrial diseases caused by the mitochondrial 3243A>G mutation.

The mitochondrial (m.) 3243A>G mutation is known to be associated with various mitochondrial diseases including mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Their clinical symptoms have been estimated to occur with an increased mitochondrial DNA (mtDNA) heteroplasmy and reduced activity of oxidative phosphorylation (OXPHOS) complexes, but their trends in the central nervous system remain unknown. Six autopsied mutant cases and three disease control cases without the mutation were enrolled in this study. The mutant cases had a disease duration of 1-27 years. Five of six mutant cases were compatible with MELAS. In the mutant cases, cortical lesions including a laminar necrosis were frequently observed in the parietal, lateral temporal, and occipital lobes; less frequently in the frontal lobe including precentral gyrus; and not at all in the medial temporal lobe. The mtDNA heteroplasmy in brain tissue samples of the mutant cases was strikingly high, ranging from 53.8% to 85.2%. The medial temporal lobe was preserved despite an inhospitable environment having high levels of mtDNA heteroplasmy and lactic acid. OXPHOS complex I was widely decreased in the mutant cases. The swelling of smooth muscle cells in the vessels on the leptomeninges, with immunoreactivity (IR) against mitochondria antibody, and a decreased nuclear/cytoplasmic ratio of choroidal epithelial cells were observed in all mutant cases but in none without the mutation. Common neuropathological findings such as cortical laminar necrosis and basal ganglia calcification were not always observed in the mutant cases. A high level of mtDNA heteroplasmy was observed throughout the brain in spite of heterogeneous cortical lesions. A lack of medial temporal lesion, mitochondrial vasculopathy in vessels on the leptomeninges, and an increased cytoplasmic size of epithelial cells in the choroid plexus could be neuropathological hallmarks helpful in the diagnosis of mitochondrial diseases.

Novel economical, accurate, sensitive, single-cell analytical method for mitochondrial DNA quantification in mtDNA mutation carriers.

Although a variety of analytical methods have been developed to detect mitochondrial DNA (mtDNA) heteroplasmy, there are special requirements of mtDNA heteroplasmy quantification for women carrying mtDNA mutations receiving the preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PD) in clinic. These special requirements include various sample types, large samplenumber, long-term follow-up, and the need for detection of single-cell from biopsied embryos. Therefore, developing an economical, accurate, high-sensitive, and single-cell analytical method for mtDNA heteroplasmy is necessary. In this study, we developed the Sanger sequencing combined droplet digital polymerase chain reaction (ddPCR) method for mtDNA quantification and compared the results to next-generation sequencing (NGS). A total of seventeen families with twelve mtDNA mutations were recruited in this study. The results showed that both Sanger sequencing and ddPCR could be used to analyze the mtDNA heteroplasmy in single-cell samples. There was no statistically significant difference in heteroplasmy levels in common samples with high heteroplasmy (≥ 5%), low heteroplasmy (< 5%), and single-cell samples, either between Sanger sequencing and NGS methods, or between ddPCR and NGS methods (P > 0.05). However, Sanger sequencing was unable to detect extremely low heteroplasmy accurately. But even in samples with extremely low heteroplasmy (0.40% and 0.92%), ddPCR was always able to quantify them. Compared to NGS, Sanger sequencing combined ddPCR analytical methods greatly reduced the cost of sequencing. In conclusion, this study successfully established an economical, accurate, sensitive, single-cell analytical method based on the Sanger sequencing combined ddPCR methods for mtDNA heteroplasmy quantification in aclinical setting.

Capability of the iSeq 100 sequencing system from Illumina to detect low-level substitutions in the human mitochondrial genome

The significance of mtDNA heteroplasmy in forensic and medical genetics has increased recently because massively parallel sequencing (MPS) technologies enable more accurate and precise detection of minority nucleotide variants. Recent reports have shown that detection of low-level substitutions may depend on library preparation or sequencing protocol, and can vary for different MPS platforms. The MiSeq (Illumina) and Ion S5 (Thermo Fisher Scientific) are mainly used for heteroplasmy detection, but no data are available regarding the iSeq 100, an Illumina platform of the smallest throughput. Notably, unlike the other systems, the machine utilizes sequencing by synthesis one-channel chemistry to determine DNA sequences. Thus, it is important to verify the capability of the iSeq 100 system to determine mitochondrial haplotypes and detect heteroplasmic substitutions. In this study, previously determined entire mitochondrial genomes were sequenced with the iSeq 100 system. Each mitogenome was sequenced twice, giving approximately 2000x and 10,000x coverage. All homoplasmic mutations and minority variants above the 19 % level detected with the iSeq 100 system were also observed after dideoxy sequencing. Moreover, all heteroplasmic substitutions above the 2 % level were consistently detected with SBS one-channel chemistry. However, detection of low-level mtDNA variants may require additional, confirmatory experiments. In summary, the iSeq 100 system enables reproducible and accurate sequencing of human mitochondrial genomes. Detection of mtDNA minority variants depends on the laboratory protocol and sequencing platform used, but homoplasmic mutations and heteroplasmy above the 2 % level can be correctly detected with the iSeq 100 system.

Nanopore long-read next-generation sequencing for detection of mitochondrial DNA large-scale deletions.

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both short-read NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA.

Single-cell multi-omics of mitochondrial DNA disorders reveals dynamics of purifying selection across human immune cells.

Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.

P-228 Reliability of blastomere versus trophectoderm biopsy in preimplantation genetic testing for mitochondrial DNA disorders

Abstract Study question Can a single blastomere or trophectoderm (TE) biopsy accurately reflect the heteroplasmy levels of the whole embryo for mitochondrial DNA (mtDNA) disorders? Summary answer Heteroplasmy levels of single blastomere and TE are comparable to those of the rest of the embryo, including the inner cell mass (ICM). What is known already Oocytes of women carrying mtDNA mutations can display varying mutation loads – percentage of mutated mtDNA copies. Preimplantation genetic testing (PGT) aims at reducing the risk of having affected children by screening for embryos with minimal mtDNA heteroplasmy. The technique involves either a blastomere biopsy on cleavage stage embryos or TE biopsy from blastocysts, followed by genetic analysis to select embryos suitable for transfer. However, it remains unclear whether there is a uniform segregation of heteroplasmic mtDNA during the early stages of embryogenesis, and therefore whether the mutation load from blastomere or TE biopsies are representative of the whole embryo. Study design, size, duration We investigated the suitability of PGT for mtDNA disorders by comparing the mutation load between a single blastomere recovered at the eight-cell stage, TE cells and rest of the same blastocyst (n = 15) in a mouse model heteroplasmic for the m.5024C&amp;gt;T mutation. We also compared the levels of heteroplasmy between TE and ICM of heteroplasmic human blastocysts (n = 5) and between blastomeres of arrested cleavage stage embryos (n = 4) donated by two mtDNA disease patients. Participants/materials, setting, methods To obtain heteroplasmic mouse embryos, we mated female mice carrying a mitochondrial tRNAAla mutation (m.5024C&amp;gt;T) with wild-type males. Whereas in human experiments, we analysed preimplantation embryos from two different carriers of the m.3243A&amp;gt;G mutation, associated with MELAS syndrome, who showed 16% and 32% mutation load in their peripheral blood. After embryo biopsy, NGS was performed in three independent technical replicates and profiles were compared between samples from the same embryo to assess heteroplasmy levels. Main results and the role of chance Analysis of heteroplasmic mouse embryos showed a strong correlation in mtDNA heteroplasmy between blastomere and the corresponding embryo (r2 = 0.76), and between TE and the rest of the blastocyst (r2 = 0.94) (n = 15). Moreover, a strong correlation was observed between the biopsied blastomere and TE from the same embryo (r2 = 0.90). Heteroplasmy levels in the embryos analysed ranged from 55.3% to 88.3%, and the mean overall mutation load was 73.7 ± 7.9%. Development of biopsied embryos was comparable to those of non-manipulated mutant (n = 10) and wild-type embryos (n = 10) (blastocyst rate = 85.7%). For human blastocysts (n = 5), statistical analysis showed a strong correlation in heteroplasmy levels between ICM and TE portions (r2 = 0.98). Moreover, a modest correlation was observed among blastomeres of cleavage stage human embryos (r2 = 0.60) (n = 24 from 4 embryos). The mutation load in the embryos analysed ranged from 10.3% to 57.4%, and the mean overall heteroplasmy was 42.8 ± 12.2% in cleavage stage embryos and 38.6 ± 17.3% in blastocysts. Overall, concordance in heteroplasmy levels between single blastomeres, TE portions and corresponding blastocyst was established in human and mouse embryos. Limitations, reasons for caution These results should be further validated by increasing the sample size and by the inclusion of different mtDNA mutations. Furthermore, current data are suggestive, but not definitive, to guarantee that the mtDNA mutation load in the embryo will remain constant throughout life. Wider implications of the findings Taken together, results suggest that TE biopsy may be better than blastomere biopsy for mtDNA disorders. Moreover, biopsy at the blastocyst stage is likely less harmful for embryo development. Following, we will also investigate how heteroplasmic mtDNA segregates at the peri-implantation stages after the resumption of mtDNA replication. Trial registration number Not applicable

Discrimination of monozygotic twins using mtDNA heteroplasmy through probe capture enrichment and massively parallel sequencing.

Differentiating between monozygotic (MZ) twins remains difficult because they have the same genetic makeup. Applying the traditional STR genotyping approach cannot differentiate one from the other. Heteroplasmy refers to the presence of two or more different mtDNA copies within a single cell and this phenomenon is common in humans. The levels of heteroplasmy cannot change dramatically during transmission in the female germ line but increase or decrease during germ-line transmission and in somatic tissues during life. As massively parallel sequencing (MPS) technology has advanced, it has shown the extraordinary quantity of mtDNA heteroplasmy in humans. In this study, a probe hybridization technique was used to obtain mtDNA and then MPS was performed with an average sequencing depth of above 4000. The results showed us that all ten pairs of MZ twins were clearly differentiated with the minor heteroplasmy threshold at 1.0%, 0.5%, and 0.1%, respectively. Finally, we used a probe that targeted mtDNA to boost sequencing depth without interfering with nuclear DNA and this technique can be used in forensic genetics to differentiate the MZ twins.

Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells.

Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.

Probing the pathogenicity of patient-derived variants of MT-ATP6 in yeast

The list of mitochondrial DNA (mtDNA) variants detected in individuals with neurodegenerative diseases is constantly growing. Evaluating their functional consequences and pathogenicity is not easy, especially when they are found in only a limited number of patients together with wild-type mtDNA (heteroplasmy). Owing to its amenability to mitochondrial genetic transformation and incapacity to stably maintain heteroplasmy, and the strong evolutionary conservation of the proteins encoded in mitochondria, Saccharomyces cerevisiae provides a convenient model to investigate the functional consequences of human mtDNA variants. We herein report the construction and energy-transducing properties of yeast models of eight MT-ATP6 gene variants identified in patients with various disorders: m.8843T>C, m.8950G>A, m.9016A>G, m.9025G>A, m.9029A>G, m.9058A>G, m.9139G>A and m.9160T>C. Significant defect in growth dependent on respiration and deficits in ATP production were observed in yeast models of m.8950G>A, m.9025G>A and m.9029A>G, providing evidence of pathogenicity for these variants. Yeast models of the five other variants showed very mild, if any, effect on mitochondrial function, suggesting that the variants do not have, at least alone, the potential to compromise human health.

Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing.

The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies.

Mitochondrial Heteroplasmy as a Marker for Premature Coronary Artery Disease: Analysis of the Poly-C Tract of the Control Region Sequence

Mitochondrial DNA (mtDNA) differs from the nuclear genome in many aspects: a maternal inheritance pattern; being more prone to acquire somatic de novo mutations, accumulative with age; and the possible coexistence of different mtDNA alleles (heteroplasmy). Mitochondria are key cellular organelles responsible for energy production and involved in complex mechanisms, including atherosclerosis. In this scenario, we aimed to evaluate mtDNA variants that could be associated with premature cardiovascular disease. We evaluated 188 consecutive patients presenting with premature myocardial infarction with ST elevation (STEMI) confirmed by coronary angiogram. mtDNA polymorphisms and clinical data were evaluated and compared with 271 individuals from the same population (control group). Tobacco consumption (80.85% vs. 21.21%, p < 0.01) and dyslipidemia (38.83% vs. 28.41%, p = 0.02) were significantly more frequent among STEMI patients. Moreover, C16223T mtDNA mutation and poly-C heteroplasmy were significantly more frequent among premature STEMI male patients than in controls. The OR associated C16223T mtDNA with the increased presence of cardiovascular risk factors. Our data suggest that mtDNA 16223T and heteroplasmy may be associated with unstable premature atherosclerosis disease in men. Moreover, the presence of cardiovascular risk factors (CVRFs) was associated with C16223T mtDNA, with a cumulative effect. Protective mitochondrial pathways are potential therapeutic targets. Preventing exposure to the damaging mechanisms associated with CVRFs is of utmost importance.

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.

Hybridization drives mitochondrial DNA degeneration and metabolic shift in a species with biparental mitochondrial inheritance.

Mitochondrial DNA (mtDNA) is a cytoplasmic genome that is essential for respiratory metabolism. Although uniparental mtDNA inheritance is most common in animals and plants, distinct mtDNA haplotypes can coexist in a state of heteroplasmy, either because of paternal leakage or de novo mutations. mtDNA integrity and the resolution of heteroplasmy have important implications, notably for mitochondrial genetic disorders, speciation, and genome evolution in hybrids. However, the impact of genetic variation on the transition to homoplasmy from initially heteroplasmic backgrounds remains largely unknown. Here, we use Saccharomyces yeasts, fungi with constitutive biparental mtDNA inheritance, to investigate the resolution of mtDNA heteroplasmy in a variety of hybrid genotypes. We previously designed 11 crosses along a gradient of parental evolutionary divergence using undomesticated isolates of Saccharomyces paradoxus and Saccharomyces cerevisiae Each cross was independently replicated 48 to 96 times, and the resulting 864 hybrids were evolved under relaxed selection for mitochondrial function. Genome sequencing of 446 MA lines revealed extensive mtDNA recombination, but the recombination rate was not predicted by parental divergence level. We found a strong positive relationship between parental divergence and the rate of large-scale mtDNA deletions, which led to the loss of respiratory metabolism. We also uncovered associations between mtDNA recombination, mtDNA deletion, and genome instability that were genotype specific. Our results show that hybridization in yeast induces mtDNA degeneration through large-scale deletion and loss of function, with deep consequences for mtDNA evolution, metabolism, and the emergence of reproductive isolation.

Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases. However, current methods can be imprecise, and quantifying small changes in either heteroplasmy or copy number is challenging. We developed a new approach to measure mtDNA heteroplasmy using a single digital PCR (dPCR) probe. This method is based on the observation that fluorescent-labeled probes in dPCR exhibit different intensities depending on the presence of a single nucleotide change in the sequence bound by the probe. This finding allowed us to precisely and simultaneously determine mtDNA copy number and heteroplasmy levels using duplex dPCR. We tested this approach in two different models (human and mouse), which proved faster and more internally controlled when compared to other published methods routinely used in the mitochondrial genetics field. We believe this approach could be broadly applicable to the detection and quantification of other mixed genetic variations.

UNCOVERING THE MISSING PIECE: COMPREHENSIVE REVIEW OF THE TRANSMISSION OF MITOCHONDRIAL DNA HETEROPLASMY TO THE PREIMPLANTATION EMBRYO

The objectives for this study were to: 1) Summarize the methods and key findings from published literature regarding mitochondrial DNA (mtDNA) heteroplasmy transmission and rate of de novo mutations seen in offspring; 2) Evaluate the utility of genome-wide mtDNA heteroplasmy analysis in preimplantation embryos; and 3) Discuss technical and ethical challenges of whole-genome mtDNA analysis in preimplantation embryos.

A Bioinformatics pipeline for variant discovery from Targeted Next Generation Sequencing of the human mitochondrial genome

Sequence variants of human mitochondrial DNA (mt DNA) have been implicated in a variety of disorders and conditions. Massive parallel sequencing is becoming increasingly popular due to its efficiency and cost-effectiveness. In relation to acquiring significant sequence information like levels of heteroplasmy in mt DNA, it offers a marked improvement compared to previous methods used. Here we describe a variant calling pipeline for human mitochondrial DNA using Next Generation Sequencing (NGS) data obtained by enriching the sample only for mitochondria prior to sequencing.

Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

Genomic analysis of a novel Neanderthal from Mezmaiskaya Cave provides insights into the genetic relationships of Middle Palaeolithic populations

The Mezmaiskaya cave is located on the North Caucasus near the border that divides Europe and Asia. Previously, fossil remains for two Neanderthals were reported from Mezmaiskaya Cave. A tooth from the third archaic hominin specimen (Mezmaiskaya 3) was retrieved from layer 3 in Mezmaiskaya Cave. We performed genome sequencing of Mezmaiskaya 3. Analysis of partial nuclear genome sequence revealed that it belongs to a Homo sapiens neanderthalensis female. Based on a high-coverage mitochondrial genome sequence, we demonstrated that the relationships of Mezmaiskaya 3 to Mezmaiskaya 1 and Stajnia S5000 individuals were closer than those to other Neanderthals. Our data demonstrate the close genetic connections between the early Middle Palaeolithic Neanderthals that were replaced by genetically distant later group in the same geographic areas. Based on mitochondrial DNA (mtDNA) data, we suggest that Mezmaiskaya 3 was the latest Neanderthal individual from the early Neanderthal’s branches. We proposed a hierarchical nomenclature for the mtDNA haplogroups of Neanderthals. In addition, we retrieved ancestral mtDNA mutations in presumably functional sites fixed in the Neanderthal clades, and also provided the first data showing mtDNA heteroplasmy in Neanderthal specimen.