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.

Morphologic evidence of diffuse vascular damage in human and in the experimental model of ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is a rare autosomal recessive disorder characterized by early onset encephalopathy, chronic diarrhoea, petechiae, orthostatic acrocyanosis and defective cytochrome c oxidase (COX) in muscle and brain. High levels of lactic, ethylmalonic and methylsuccinic acids are detected in body fluids. EE is caused by mutations in ETHE1, a mitochondrial sulphur dioxygenase. By studying a suitable mouse model, we found that loss of ETHE1 leads to accumulation of sulphide, which is a poison for COX and other enzymatic activities thus accounting for the main features of EE. We report here the first autopsy case of a child with a genetically confirmed diagnosis of EE, and compare the histological, histochemical and immunohistochemical findings with those of the constitutive Ethe1 (-/-) mice. In addition to COX depleted cells, widespread endothelial lesions of arterioles and capillaries of the brain and gastrointestinal tract were the pathologic hallmarks in both organisms. Our findings of diffuse vascular damage of target critical organs are in keeping with the hypothesis that the pathologic effects of ETHE1 deficiency may stem from high levels of circulating hydrogen sulphide rather than the inability of specific organs to detoxify its endogenous production.

Oestrogens ameliorate mitochondrial dysfunction in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Leber's hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Leber's hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17ß-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Leber's hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17ß-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17ß-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor ß localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Leber's hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules.

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.

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.

Isolated distal myopathy of the upper limbs associated with mitochondrial DNA depletion and polymerase gamma mutations.

OBJECTIVE: To describe an unusual clinical phenotype in an adult harboring 2 compound heterozygous polymerase γ (POLG) mutations. DESIGN: Case report. SETTING: University-based outpatient neurology clinic and pathology and genetics laboratory. PATIENT: A 27-year-old man presenting with isolated distal myopathy of the upper extremities in the absence of sensory disturbances. RESULTS: Histochemical analysis of a muscle biopsy specimen showed numerous cytochrome c oxidase-deficient fibers. Molecular analysis revealed marked depletion of muscle mitochondrial DNA in the absence of multiple mitochondrial DNA deletions. Sequence analysis of the POLG gene revealed heterozygous sequence variants in compound c.1156C>T (p.R386C) and c.2794C>T (p.H932Y) segregating with clinical disease in the family. The p.R386C change appears to be a novel mutation. CONCLUSION: Our case broadens the phenotypic spectrum of disorders associated with POLG mutations and highlights the complex relationship between genotype and phenotype in POLG-related disease.