Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.

Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.

Pathological mechanisms underlying single large-scale mitochondrial DNA deletions.

OBJECTIVE: Single, large-scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large-scale mtDNA deletions in skeletal muscle. METHODS: We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large-scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. RESULTS: We have defined 3 "classes" of single, large-scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. INTERPRETATION: Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex-specific protein-encoding genes. Furthermore, removal of mt-tRNA genes impacts specific complexes only at high deletion levels, when complex-specific protein-encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115-130.

Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations.

Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNA(Leu(UUR)) or with mutations in the mt-tRNA(Ile), both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNA(Lys), aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, ß30_31 and ß32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNA(Leu(UUR)) and mt-tRNA(Lys), and stabilize mutant mt-tRNA(Leu(UUR)). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs.

Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis.

Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age related mitochondrial dysfunction in tissues including brain and skeletal muscle. It is known that different mtDNA deletions accumulate in single cells, but the detailed nature of these rearrangements is still unknown. To evaluate this we used a complementary set of sensitive assays to explore the mtDNA rearrangements in individual cells from patients with sporadic inclusion body myositis, a late-onset inflammatory myopathy with prominent mitochondrial changes. We identified large-scale mtDNA deletions in individual muscle fibres with 20% of cytochrome c oxidase-deficient myofibres accumulating two or more mtDNA deletions. The majority of deletions removed only the major arc but ∼10% of all deletions extended into the minor arc removing the origin of light strand replication (OL) and a variable number of genes. Some mtDNA molecules contained two deletion sites. Additionally, we found evidence of mitochondrial genome duplications allowing replication and clonal expansion of these complex rearranged molecules. The extended spectrum of mtDNA rearrangements in single cells provides insight into the process of clonal expansion which is fundamental to our understanding of the role of mtDNA mutations in ageing and disease.

Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing.

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.

Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease.

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

Novel MTND1 mutations cause isolated exercise intolerance, complex I deficiency and increased assembly factor expression.

Complex I (CI) is the largest of the five multi-subunit complexes constituting the human oxidative phosphorylation (OXPHOS) system. Seven of its catalytic core subunits are encoded by mitochondrial DNA (ND (NADH dehydrogenase)1-6, ND4L (NADH dehydrogenase subunit 4L)), with mutations in all seven having been reported in association with isolated CI deficiency. We investigated two unrelated adult patients presenting with marked exercise intolerance, persistent lactic acidaemia and severe muscle-restricted isolated CI deficiency associated with sub-sarcolemmal mitochondrial accumulation. Screening of the mitochondrial genome detected novel mutations in the MTND1 (NADH dehydrogenase subunit 1) gene, encoding subunit of CI [Patient 1, m.3365T>C predicting p.(Leu20Pro); Patient 2, m.4175G>A predicting p.(Trp290*)] at high levels of mitochondrial DNA heteroplasmy in skeletal muscle. We evaluated the effect of these novel MTND1 mutations on complex assembly showing that CI assembly, although markedly reduced, was viable in the absence of detectable ND1 signal. Real-time PCR and Western blotting showed overexpression of different CI assembly factor transcripts and proteins in patient tissue. Together, our data indicate that the mechanism underlying the expression of the biochemical defect may involve a compensatory response to the novel MTND1 gene mutations, promoting assembly factor up-regulation and stabilization of respiratory chain super-complexes, resulting in partial rescue of the clinical phenotype.

Isoleucyl-tRNA synthetase levels modulate the penetrance of a homoplasmic m.4277T>C mitochondrial tRNA(Ile) mutation causing hypertrophic cardiomyopathy.

The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA(Ile) (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA(Ile). Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA(Ile) is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patient's clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA(Ile), while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.

Mitochondrial DNA disease: new options for prevention.

Very recently, two papers have presented intriguing data suggesting that prevention of transmission of human mitochondrial DNA (mtDNA) disease is possible. [Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N. et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 465, 82-85. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O. and Mitalipov, S. (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 461, 367-372.] These recent advances raise hopes for families with mtDNA disease; however, the successful translational of these techniques to clinical practice will require further research to test for safety and to maximize efficacy. Furthermore, in the UK, amendment to the current legislation will be required. Here, we discuss the clinical and scientific background, studies we believe are important to establish safety and efficacy of the techniques and some of the potential concerns about the use of these approaches.

Single Cell Analysis of Mitochondrial DNA Deletions.

Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.

Resistance Exercise Training Rescues Mitochondrial Dysfunction in Skeletal Muscle of Patients with Myotonic Dystrophy Type 1.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is a dominant autosomal neuromuscular disorder caused by the inheritance of a CTG triplet repeat expansion in the Dystrophia Myotonica Protein Kinase (DMPK) gene. At present, no cure currently exists for DM1 disease. OBJECTIVE: This study investigates the effects of 12-week resistance exercise training on mitochondrial oxidative phosphorylation in skeletal muscle in a cohort of DM1 patients (n = 11, men) in comparison to control muscle with normal oxidative phosphorylation. METHODS: Immunofluorescence was used to assess protein levels of key respiratory chain subunits of complex I (CI) and complex IV (CIV), and markers of mitochondrial mass and cell membrane in individual myofibres sampled from muscle biopsies. Using control's skeletal muscle fibers population, we classified each patient's fibers as having normal, low or high levels of CI and CIV and compared the proportions of fibers before and after exercise training. The significance of changes observed between pre- and post-exercise within patients was estimated using a permutation test. RESULTS: At baseline, DM1 patients present with significantly decreased mitochondrial mass, and isolated or combined CI and CIV deficiency. After resistance exercise training, in most patients a significant increase in mitochondrial mass was observed, and all patients showed a significant increase in CI and/or CIV protein levels. Moreover, improvements in mitochondrial mass were correlated with the one-repetition maximum strength evaluation. CONCLUSIONS: Remarkably, 12-week resistance exercise training is sufficient to partially rescue mitochondrial dysfunction in DM1 patients, suggesting that the response to exercise is in part be due to changes in mitochondria.

Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency.

Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.

Mitochondrial DNA mutations and human disease.

Mitochondrial disorders are a group of clinically heterogeneous diseases, commonly defined by a lack of cellular energy due to oxidative phosphorylation (OXPHOS) defects. Since the identification of the first human pathological mitochondrial DNA (mtDNA) mutations in 1988, significant efforts have been spent in cataloguing the vast array of causative genetic defects of these disorders. Currently, more than 250 pathogenic mtDNA mutations have been identified. An ever-increasing number of nuclear DNA mutations are also being reported as the majority of proteins involved in mitochondrial metabolism and maintenance are nuclear-encoded. Understanding the phenotypic diversity and elucidating the molecular mechanisms at the basis of these diseases has however proved challenging. Progress has been hampered by the peculiar features of mitochondrial genetics, an inability to manipulate the mitochondrial genome, and difficulties in obtaining suitable models of disease. In this review, we will first outline the unique features of mitochondrial genetics before detailing the diseases and their genetic causes, focusing specifically on primary mtDNA genetic defects. The functional consequences of mtDNA mutations that have been characterised to date will also be discussed, along with current and potential future diagnostic and therapeutic advances.

The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families.

Isolated complex I deficiency is the most frequently observed oxidative phosphorylation defect in children with mitochondrial disease, leading to a diverse range of clinical presentations, including Leigh syndrome. For most patients the genetic cause of the biochemical defect remains unknown due to incomplete understanding of the complex I assembly process. Nonetheless, a plethora of pathogenic mutations have been described to date in the seven mitochondrial-encoded subunits of complex I as well as in 12 of the nuclear-encoded subunits and in six assembly factors. Whilst several mitochondrial DNA mutations are recurrent, the majority of these mutations are reported in single families. We have sequenced core structural and functional nuclear-encoded subunits of complex I in a cohort of 34 paediatric patients with isolated complex I deficiency, identifying pathogenic mutations in 6 patients. These included a novel homozygous NDUFS1 mutation in an Asian child with Leigh syndrome, a previously identified NDUFS8 mutation (c.236C>T, p.P79L) in a second Asian child with Leigh-like syndrome and six novel, compound heterozygous NDUFS2 mutations in four white Caucasian patients with Leigh or Leigh-like syndrome. Three of these children harboured an identical NDUFS2 mutation (c.875T>C, p.M292T), which was also identified in conjunction with a novel NDUFS2 splice site mutation (c.866+4A>G) in a fourth Caucasian child who presented to a different diagnostic centre, with microsatellite and single nucleotide polymorphism analyses indicating that this was due to an ancient common founder event. Our results confirm that NDUFS2 is a mutational hotspot in Caucasian children with isolated complex I deficiency and recommend the routine diagnostic investigation of this gene in patients with Leigh or Leigh-like phenotypes.

Long-term survival of neonatal mitochondrial complex III deficiency associated with a novel BCS1L gene mutation.

Mutations of the BCS1L gene are a recognised cause of isolated respiratory chain complex III deficiency and underlie several fatal, neonatal mitochondrial diseases. Here we describe a 20-year-old Kenyan woman who initially presented as a floppy infant but whose condition progressed during childhood and adolescence with increasing muscle weakness, focal motor seizures and optic atrophy. Muscle biopsy demonstrated complex III deficiency and the pathogenicity of a novel, homozygous BCS1L mutation was confirmed by yeast complementation studies. Our data indicate that BCS1L mutations can cause a variable, neurological course which is not always fatal in childhood.

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy.

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Ultradeep mapping of neuronal mitochondrial deletions in Parkinson's disease.

Mitochondrial DNA (mtDNA) deletions accumulate with age in postmitotic cells and are associated with aging and neurodegenerative disorders such as Parkinson's disease. Although the exact mechanisms by which deletions form remain elusive, the dominant theory is that they arise spontaneously at microhomologous sites and undergo clonal expansion. We characterize mtDNA deletions at unprecedented resolution in individual substantia nigra neurons from individuals with Parkinson's disease, using ultradeep sequencing. We show that the number of deleted mtDNA species per neuron is substantially higher than previously reported. Moreover, each deleted mtDNA species shows significant differences in sequence composition compared with the remaining mtDNA population, which is highly consistent with independent segregation and clonal expansion. Deletion breakpoints occur consistently in regions of sequence homology, which may be direct or interrupted stretches of tandem repeats. While our results support a crucial role for misannealing in deletion generation, we find no overrepresentation of the 3'-repeat sequence, an observation that is difficult to reconcile with the current view of replication errors as the source of mtDNA deletions.

Triplex real-time PCR--an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells.

Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (OL) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings.