Translation of MT-ATP6 pathogenic variants reveals distinct regulatory consequences from the co-translational quality control of mitochondrial protein synthesis.

Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogeneous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders.

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes.

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.

GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation.

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.

A variant in MRPS14 (uS14m) causes perinatal hypertrophic cardiomyopathy with neonatal lactic acidosis, growth retardation, dysmorphic features and neurological involvement.

Dysfunction of mitochondrial translation is an increasingly important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient, there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts, mitochondrial translation was impaired, and ectopic expression of a wild-type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel.Collectively, our data demonstrate pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly.

In-frame deletion in canine PITRM1 is associated with a severe early-onset epilepsy, mitochondrial dysfunction and neurodegeneration.

We investigated the clinical, genetic, and pathological characteristics of a previously unknown severe juvenile brain disorder in several litters of Parson Russel Terriers. The disease started with epileptic seizures at 6-12 weeks of age and progressed rapidly to status epilepticus and death or euthanasia. Histopathological changes at autopsy were restricted to the brain. There was severe acute neuronal degeneration and necrosis diffusely affecting the grey matter throughout the brain with extensive intraneuronal mitochondrial crowding and accumulation of amyloid-ß (Aß). Combined homozygosity mapping and genome sequencing revealed an in-frame 6-bp deletion in the nuclear-encoded pitrilysin metallopeptidase 1 (PITRM1) encoding for a mitochondrial protease involved in mitochondrial targeting sequence processing and degradation. The 6-bp deletion results in the loss of two amino acid residues in the N-terminal part of PITRM1, potentially affecting protein folding and function. Assessment of the mitochondrial function in the affected brain tissue showed a significant deficiency in respiratory chain function. The functional consequences of the mutation were modeled in yeast and showed impaired growth in permissive conditions and an impaired respiration capacity. Loss-of-function variants in human PITRM1 result in a childhood-onset progressive amyloidotic neurological syndrome characterized by spinocerebellar ataxia with behavioral, psychiatric and cognitive abnormalities. Homozygous Pitrm1-knockout mice are embryonic lethal, while heterozygotes show a progressive, neurodegenerative phenotype characterized by impairment in motor coordination and Aß deposits. Our study describes a novel early-onset PITRM1-related neurodegenerative canine brain disorder with mitochondrial dysfunction, Aß accumulation, and lethal epilepsy. The findings highlight the essential role of PITRM1 in neuronal survival and strengthen the connection between mitochondrial dysfunction and neurodegeneration.

Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome.

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson-Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).

Defective mitochondrial ATPase due to rare mtDNA m.8969G>A mutation-causing lactic acidosis, intellectual disability, and poor growth.

Mutations in mitochondrial ATP synthase 6 (MT-ATP6) are a frequent cause of NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or Leigh syndromes, especially a point mutation at nucleotide position 8993. M.8969G>A is a rare MT-ATP6 mutation, previously reported only in three individuals, causing multisystem disorders with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia or IgA nephropathy. We present two siblings with the m.8969G>A mutation and a novel, substantially milder phenotype with lactic acidosis, poor growth, and intellectual disability. Our findings expand the phenotypic spectrum and show that mtDNA mutations should be taken account also with milder, stable phenotypes.

Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.

Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.

SDHA mutation with dominant transmission results in complex II deficiency with ocular, cardiac, and neurologic involvement.

Isolated defects of the mitochondrial respiratory complex II (succinate dehydrogenase, SDH) are rare, accounting for approximately 2% of all respiratory chain deficiency diagnoses. Here, we report clinical and molecular investigations of three family members with a heterozygous mutation in the large flavoprotein subunit SDHA previously described to cause complex II deficiency. The index patient presented with bilateral optic atrophy and ocular movement disorder, a progressive polyneuropathy, psychiatric involvement, and cardiomyopathy. Two of his children presented with cardiomyopathy and methylglutaconic aciduria in early childhood. The daughter deceased at the age of 7 months due to cardiac insufficiency. The 30-year old son presents with cardiomyopathy and developed bilateral optic atrophy in adulthood. Of the four nuclear encoded proteins composing complex II (SDHA, SDHB, SDHC, SDHD) and currently known assembly factors SDHAF1 and SDHAF2 mainly recessively inherited mutations have been described in SDHA, SDHB, SDHD, and SDHAF1 to be causative for mitochondrial disease phenotypes. This is the second report presenting autosomal dominant inheritance of a SDHA mutation.© 2016 Wiley Periodicals, Inc.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial ß-barrel proteins specialized in mitochondrial genome inheritance.

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a ß-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a ß-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

Supernumerary proteins of the human mitochondrial ribosomal small subunit are integral for assembly and translation.

Mitochondrial ribosomes (mitoribosomes) have undergone substantial evolutionary structural remodeling accompanied by loss of ribosomal RNA, while acquiring unique protein subunits located on the periphery. We generated CRISPR-mediated knockouts of all 14 unique (mitochondria-specific/supernumerary) human mitoribosomal proteins (snMRPs) in the small subunit to study the effect on mitoribosome assembly and protein synthesis, each leading to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Surprisingly, the stability of mS37 was reduced in all our snMRP knockouts of the small and large ribosomal subunits and patient-derived lines with mitoribosome assembly defects. A redox-regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

Progressive mitochondrial dysfunction in cerebellar synaptosomes of cystatin B-deficient mice.

The involvement of mitochondrial dysfunction in cystatin B (CSTB) deficiency has been suggested, but its role in the onset of neurodegeneration, myoclonus, and ataxia in the CSTB-deficient mouse model (Cstb-/-) is yet unknown. CSTB is an inhibitor of lysosomal and nuclear cysteine cathepsins. In humans, partial loss-of-function mutations cause the progressive myoclonus epilepsy neurodegenerative disorder, EPM1. Here we applied proteome analysis and respirometry on cerebellar synaptosomes from early symptomatic (Cstb-/-) mice to identify the molecular mechanisms involved in the onset of CSTB-deficiency associated neural pathogenesis. Proteome analysis showed that CSTB deficiency is associated with differential expression of mitochondrial and synaptic proteins, and respirometry revealed a progressive impairment in mitochondrial function coinciding with the onset of myoclonus and neurodegeneration in (Cstb-/-) mice. This mitochondrial dysfunction was not associated with alterations in mitochondrial DNA copy number or membrane ultrastructure. Collectively, our results show that CSTB deficiency generates a defect in synaptic mitochondrial bioenergetics that coincides with the onset and progression of the clinical phenotypes, and thus is likely a contributor to the pathogenesis of EPM1.

Molecular Rapid Diagnostics Improve Time to Effective Therapy and Survival in Patients with Vancomycin-Resistant Enterococcus Bloodstream Infections.

Delays in appropriate antibiotic therapy are a key determinant for deleterious outcomes among patients with vancomycin-resistant Enterococcus (VRE) bloodstream infections (BSIs). This was a multi-center pre/post-implementation study, assessing the impact of a molecular rapid diagnostic test (Verigene® GP-BC, Luminex Corporation, Northbrook, IL, USA) on outcomes of adult patients with VRE BSIs. The primary outcome was time to optimal therapy (TOT). Multivariable logistic and Cox proportional hazard regression models were used to determine the independent associations of post-implementation, TOT, early vs. delayed therapy, and mortality. A total of 104 patients with VRE BSIs were included: 50 and 54 in the pre- and post-implementation periods, respectively. The post- vs. pre-implementation group was associated with a 1.8-fold faster rate to optimized therapy (adjusted risk ratio, 1.841 [95% CI 1.234-2.746]), 6-fold higher likelihood to receive early effective therapy (<24 h, adjusted odds ratio, 6.031 [2.526-14.401]), and a 67% lower hazards for 30-day in-hospital mortality (adjusted hazard ratio, 0.322 [0.124-1.831]), after adjusting for age, sex, and severity scores. Inversely, delayed therapy was associated with a 10-fold higher risk of in-hospital mortality (aOR 10.488, [2.497-44.050]). Reduced TOT and in-hospital mortality were also observed in subgroups of immunosuppressed patients in post-implementation. These findings demonstrate that the addition of molecular rapid diagnostic tests (mRDT) to clinical microbiology and antimicrobial stewardship practices are associated with a clinically significant reduction in TOT, which is associated with lower mortality for patients with VRE BSIs, underscoring the importance of mRDTs in the management of VRE infections.

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy.

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome.

BACKGROUND: DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. METHODS: mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. RESULTS: We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. CONCLUSIONS: mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

The roles of assembly factors in mammalian mitoribosome biogenesis.

As ancient bacterial endosymbionts of eukaryotic cells, mitochondria have retained their own circular DNA as well as protein translation system including mitochondrial ribosomes (mitoribosomes). In recent years, methodological advancements in cryoelectron microscopy and mass spectrometry have revealed the extent of the evolutionary divergence of mitoribosomes from their bacterial ancestors and their adaptation to the synthesis of 13 mitochondrial DNA encoded oxidative phosphorylation complex subunits. In addition to the structural data, the first assembly pathway maps of mitoribosomes have started to emerge and concomitantly also the assembly factors involved in this process to achieve fully translational competent particles. These transiently associated factors assist in the intricate assembly process of mitoribosomes by enhancing protein incorporation, ribosomal RNA folding and modification, and by blocking premature or non-native protein binding, for example. This review focuses on summarizing the current understanding of the known mammalian mitoribosome assembly factors and discussing their possible roles in the assembly of small or large mitoribosomal subunits.

Quantitative 1-step DNA methylation analysis with native genomic DNA as template.

BACKGROUND: DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis. METHODS: The assay is based on combining methylation-sensitive FastDigest(R) endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest. RESULTS: With our novel approach, we correctly diagnosed the imprinting disorders Prader-Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay's accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies. CONCLUSIONS: Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.

Therapeutic Manipulation of mtDNA Heteroplasmy: A Shifting Perspective.

Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease.