Therapeutic potential of somatic cell nuclear transfer for degenerative disease caused by mitochondrial DNA mutations

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal

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

The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells.

Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present. We show that three human mt aminoacyl-tRNA syntethases, namely leucyl-, valyl-, and isoleucyl-tRNA synthetase are able to improve both viability and bioenergetic proficiency of human transmitochondrial cybrid cells carrying pathogenic mutations in the mt-tRNA(Ile) gene. Importantly, we further demonstrate that the carboxy-terminal domain of human mt leucyl-tRNA synthetase is both necessary and sufficient to improve the pathologic phenotype associated either with these "mild" mutations or with the "severe" m.3243A>G mutation in the mt-tRNA(L)(eu(UUR)) gene. Furthermore, we provide evidence that this small, non-catalytic domain is able to directly and specifically interact in vitro with human mt-tRNA(Leu(UUR)) with high affinity and stability and, with lower affinity, with mt-tRNA(Ile). Taken together, our results sustain the hypothesis that the carboxy-terminal domain of human mt leucyl-tRNA synthetase can be used to correct mt dysfunctions caused by mt-tRNA mutations

A novel mitochondrial DNA m.7507A > G mutation is only pathogenic at high levels of heteroplasmy

We present a Dutch family with a novel disease-causing mutation in the mitochondrial tRNA(Ser(UCN)) gene, m.7507A>G. The index patient died during the neonatal period due to cardio-respiratory failure and fatal lactic acidosis. A second patient, his cousin, has severe hearing loss necessitating cochlear implants and progressive exercise intolerance. Laboratory investigations of both patients revealed combined deficiencies of the enzyme complexes of the mitochondrial respiratory chain in several tissues. Reduced levels of fully assembled complexes I and IV in fibroblasts by BN-PAGE associated with (near) homoplasmic levels of the m.7507A>G mutation in several tissues and a severe reduction in the steady-state level of mt-tRNA(Ser(UCN)) in fibroblasts were observed. The novel mitochondrial DNA mutation was shown to segregate with disease; several healthy maternal family members showed high heteroplasmy levels (up to 76 +/- 4% in blood and 68 +/- 4% in fibroblasts) which did not lead to any alterations in the activities of the enzyme complexes of the respiratory chain in fibroblasts or clinical signs and symptoms. We hereby conclude that the m.7507A>G mutation causes a heterogeneous clinical phenotype and is only pathogenic at very high levels of mtDNA heteroplasmy

Human mitochondrial leucyl tRNA synthetase can suppress non cognate pathogenic mt-tRNA mutations

Disorders of the mitochondrial genome cause a wide spectrum of disease, these present mainly as neurological and/or muscle related pathologies. Due to the intractability of the human mitochondrial genome there are currently no effective treatments for these disorders. The majority of the pathogenic mutations lie in the genes encoding mitochondrial tRNAs. Consequently, the biochemical deficiency is due to mitochondrial protein synthesis defects, which manifest as aberrant cellular respiration and ATP synthesis. It has previously been reported that overexpression of mitochondrial aminoacyl tRNA synthetases has been effective, in cell lines, at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs. We now show that leucyl tRNA synthetase is able to partially rescue defects caused by mutations in non-cognate mt-tRNAs. Further, a C terminal peptide alone can enter mitochondria and interact with the same spectrum of mt-tRNAs as the entire synthetase, in intact cells. These data support the possibility that a small peptide could correct at least the biochemical defect associated with many mt-tRNA mutations, inferring a novel therapy for these disorders

Accurate Measurement of Mitochondrial DNA Deletion Level and Copy Number Differences in Human Skeletal Muscle

<div><p>Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied <i>MT-ND1</i>/<i>MT-ND4</i> multiplex mtDNA deletion assay and a recently developed <i>MT-ND1</i>/<i>B2M</i> singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.</p></div

Cardiomyopathies due to homoplasmic mitochondrial tRNA mutations: morphologic and molecular features

Isolated hypertrophic cardiomyopathy may represent the sole clinical feature of a mitochondrial disorder in adult patients. The clinical outcome is characterized by a rapid progression to dilation and failure. A mitochondrial etiology in these cases is not obvious at clinical investigation and may represent an unexpected finding at autopsy or after cardiac transplant. We describe the morphologic, biochemical, and molecular features of hearts from 3 transplanted patients with isolated mitochondrial cardiomyopathy caused by homoplasmic mutations in the MTTI gene, coding for mitochondrial isoleucine tRNA (mt-tRNA(Ile)). On gross examination, the 3 hearts showed a symmetric pattern of hypertrophy. At histology, cardiomyocytes were hypertrophic and showed sarcoplasmic vacuoles filled with granules that stain with antimitochondrial antibodies. On frozen sections, the combined cytochrome c oxidase (COX)/succinate dehydrogenase stain showed a large prevalence of COX-deficient cardiomyocytes. Mitochondrially encoded COX subunit I was almost absent on immunohistochemistry, whereas the nuclear-encoded COX subunit IV was normally expressed. Ultrastructural analysis confirmed the marked mitochondrial proliferation. Biochemical studies of cardiac homogenates revealed a combined respiratory chain defect. Quantitative restriction fragment length polymorphism analysis of DNA from cardiac homogenate confirmed that the mt-tRNA mutations were also detected in the patient's blood. High-resolution Northern blot analysis showed a marked decrease in the steady-state level of mt-tRNA(Ile), confirming pathogenicity. In conclusion, pathologists play a major role in unraveling the mitochondrial etiology of isolated hypertrophic cardiomyopathies, provided that a detailed diagnostic flowchart is followed. Once the mitochondrial etiology is clearly defined, molecular analyses on the heart are an invaluable tool to assign mutation pathogenicity. (C) 2013 Elsevier Inc. All rights reserved