Mitochondrial myopathy and rhabdomyolysis associated with a novel nonsense mutation in the gene encoding cytochrome c oxidase subunit I

Mitochondrial DNA (mtDNA) mutations associated with rhabdomyolysis are rare but have been described in sporadic cases with mutations in the cytochrome b and cytochrome c oxidase (COX) genes and in 3 cases with tRNALeu mutation. We report a novel heteroplasmic G6708A nonsense mutation in the mtDNA COI gene encoding COX subunit I in a 30-year-old woman with muscle weakness, pain, fatigue, and one episode of rhabdomyolysis. Histochemical examination of muscle biopsy specimens revealed reduced COX activity in the majority of the muscle fibers (approximately 90%) and frequent ragged red fibers. Biochemical analysis showed a marked and isolated COX deficiency. Analysis of DNA extracted from single fibers revealed higher levels of the mutation in COX-deficient fibers (> 95%) compared with COX-positive fibers (1%-80%). The mutation was not detected in a skin biopsy, cultured myoblasts, or blood leukocytes. Nor was it identified in blood leukocytes from the asymptomatic mother, indicating a de novo mutation that arose after germ layer differentiation. Western blot analysis and immunohistochemical staining revealed that reduced levels of COX subunit I were accompanied by reduced levels of other mtDNA encoded subunits, as well as nuclear DNA encoded subunit IV, supporting the concept that COX subunit I is essential for the assembly of complex IV in the respiratory chain.     

Early-onset multisystem mitochondrial disorder caused by a nonsense mutation in the mitochondrial DNA cytochrome C oxidase II gene

We report the first nonsense mutation (G7896A) in the mtDNA gene for subunit II of cytochrome c oxidase (COX) in a patient with early-onset multisystem disease and COX deficiency in muscle. The mutation was heteroplasmic in muscle, blood, and fibroblasts from the patient and abundantly present in COX-deficient fibers, but less abundant in COX-positive fibers; it was not found in blood samples from the patient's asymptomatic maternal relatives. Immunoblot analysis showed a reduced concentration of both COX II and COX I polypeptides, suggesting impaired assembly of COX holoenzyme.     

Mitochondrial myopathy due to novel missense mutation in the cytochrome c oxidase 1 gene

We report a novel heteroplasmic mutation p.Y440C in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX) gene in a patient with late onset progressive painless weakness. Her muscle biopsy showed scattered COX-negative fibers and several small collections of inflammatory cells. The mutation was detected in the patient's muscle but not in her blood. The low mutant load in muscle could explain the patient's late onset of the myopathy and milder phenotype when compared to the previously published cases with MTCO1 mutations.     

Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene

We report a patient with severe encephalomyopathy and homoplasmic A5814G point mutation in the mitochondrial DNA tRNA gene for cysteine. This mutation had been reported in heteroplasmic condition in patients with different clinical phenotypes. Our results confirm the pathogenicity of the mutation and support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders with variable penetrance. This report also extends the clinical spectrum associated with the A5814G mutation.     

A novel nonsense variant in MT-CO3 causes MELAS syndrome

Both mitochondrial and nuclear gene mutations can cause cytochrome c oxidase (COX, complex Ⅳ) dysfunction, leading to mitochondrial diseases. Although numerous diseases caused by defects of the COX subunits or COX assembly factors have been documented, clinical cases directly related to mitochondrial cytochrome c oxidase subunit 3 gene (MT-CO3) mutations are relatively rare. Here, we report a 47-year-old female patient presented with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Muscle pathology revealed ragged-red fibres and remarkable COX-deficient muscle fibres. Muscle mitochondrial DNA sequencing analysis identified a novel MT-CO3 variant (m.9553G>A) that changed a highly conserved amino acid to a stop codon (p.Trp116*). This variant was heteroplasmic in multiple tissues, where the mutation load was 13% in oral epithelial cells, 89% in muscle samples, and not detectable in the peripheral blood lymphocytes. Single muscle fiber PCR analysis showed clear segregation of the mutation load with COX deficient fibres. Western blot analysis of the muscle samples revealed a significant decrease in the levels of COX1, COX2, COX3, COX4 and UQCRC2. COX respiration activity was remarkably reduced (58.84%) relative to the controls according to spectrophotometric assays. Taken together, our results indicated that this m.9553G>A variant may be responsible for the MELAS symdrome in the proband by affecting the stability and function of COX. The study expands the clinical and molecular spectrum of COX3-specific mitochondrial diseases.     

The role of complex I genes in MELAS: a novel heteroplasmic mutation 3380G>A in ND1 of mtDNA

While Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) is typically associated with mutations in the mitochondrial tRNA(Leu) gene, mutations in complex I subunit genes of the mtDNA have emerged as a second significant cause. Here we report a novel mutation in the mitochondrial complex I subunit gene ND1 in a patient with late-onset MELAS. The 3380G>A mutation shows very good evidence of pathogenicity as it is heteroplasmic, undetectable in controls, alters a highly conserved amino acid, and is more abundant in ragged-red than in normal muscle fibers. These findings support the significant role of complex I mutations in MELAS.     

Recurrent myoglobinuria due to a nonsense mutation in the COX I gene of mitochondrial DNA

OBJECTIVE: To elucidate the molecular basis of a mitochondrial myopathy associated with recurrent myoglobinuria and cytochrome c oxidase (COX) deficiency in muscle. BACKGROUND: Recurrent myoglobinuria is typically seen in patients with inborn errors of carbohydrate or lipid metabolism, the main sources of energy for muscle contraction. Relatively little attention has been directed to defects of the mitochondrial respiratory chain in patients with otherwise unexplained recurrent myoglobinuria. METHODS: Having documented COX deficiency histochemically and biochemically in the muscle biopsy from a patient with exercise-induced recurrent myoglobinuria, the authors sequenced the three mitochondrial DNA (mtDNA)-encoded COX genes, and performed restriction fragment length polymorphism analysis and single-fiber PCR. RESULTS: The authors identified a nonsense mutation (G5920A) in the COX I gene in muscle mtDNA. The mutation was heteroplasmic and abundantly present in COX-negative fibers, but less abundant or absent in COX-positive fibers; it was not found in blood or fibroblasts from the patient or in blood samples from the patient's asymptomatic mother and sister. CONCLUSIONS: The G5920A mutation caused COX deficiency in muscle, explaining the exercise intolerance and the low muscle capacity for oxidative phosphorylation documented by cycle ergometry. The sporadic occurrence of this mutation in muscle alone suggests that it arose de novo in myogenic stem cells after germ-layer differentiation. Mutations in mtDNA-encoded COX genes should be considered in patients with recurrent myoglobinuria.     

The mutations m.5628T>C and m.8348A>G in single muscle fibers of a patient with chronic progressive external ophthalmoplegia

We identified a double mutation in a patient with chronic progressive external ophthalmoplegia, located in the tRNA(Ala) (m.5628T>C) and tRNA(Lys) (m.8348A>G) genes. Both mutations were previously described separately and considered pathogenic, however the same mutations were also reported as polymorphisms or phenotype modulator. We analyzed the proportion of each mutation in isolated muscle fibers by single fiber-polymerase chain reaction to investigate the contribution of each mutation to mitochondrial deficiency. Our findings demonstrated that the mutations were heteroplasmic in skeletal muscle and both mutations were present in all single muscle fibers. The proportions of the m.5628T>C mutation were not significantly different between normal and cytochrome-c-oxidase (COX) deficient fibers. However, a significant higher proportion of the m.8348A>G mutation was observed in COX deficient fibers. Homoplasmic m.8348A>G was only observed in COX negative fibers. In conclusion, we provide a piece of evidence toward the pathogenicity of the m.8348A>G mutation and suggest that m.5628T>C is probably a neutral polymorphism.     

Progressive encephalopathy and complex I deficiency associated with mutations in MTND1

Complex I of the oxidative phosphorylation system is composed of at least 45 subunits, seven of which are encoded by mitochondrial DNA (mtDNA). In this study we have investigated two children with complex I deficiency in muscle mitochondria. Patient 1 had cerebellar ataxia from early infancy and an abnormal MRI of the brain compatible with Leigh syndrome (LS). The course was rapidly progressive with frequent exacerbations and death at 2 years and 10 months of age. Patient 2 had a lactic acidosis in the newborn period and had a severe psychomotor developmental retardation. In her teens she developed hypertrophic cardiomyopathy and died at 26 years of age because of cardiac insufficiency. Sequencing analysis of mitochondrial encoded ND genes (MTND) showed two DE NOVO mutations in MTND1 in both patients. Patient 1 had a novel heteroplasmic G3890A mutation, R195Q. Patient 2 had a heteroplasmic G3481A mutation, E59K. The G3890A mutation in patient 1 is the first identified mutation in MTND1 in association with LS and complex I deficiency. The findings in this patient as well as in patient 2 demonstrate new clinical expressions of mutations in MTND1. The findings in patient 2 also illustrates that MTND mutations may be pathogenic even at a low percentage.     

Juvenile Leigh syndrome, optic atrophy, ataxia, dystonia, and epilepsy due to T14487C mutation in the mtDNA-ND6 gene: a mitochondrial syndrome presenting from birth to adolescence

An increasing number of reports describe mutations in mitochondrial DNA coding regions, especially in mitochondrial DNA- encoded nicotinamide adenine dinucleotide dehydrogenase subunit genes of the respiratory chain complex I, as causing early-onset Leigh syndrome. The authors report the molecular findings in a 24-year-old patient with juvenile-onset Leigh syndrome presenting with optic atrophy, ataxia dystonia, and epilepsy. A brain magnetic resonance imaging revealed bilateral basal ganglia and thalamic hypointensities, and a magnetic resonance spectroscopy revealed an increased lactate peak. The authors identified a T14487C change causing M63V substitution in the mitochondrial ND6 gene. The mutation was heteroplasmic in muscle and blood samples, with different mutation loads, and was absent in the patient's mother's urine and blood samples. They suggest that the T14487C mtDNA mutation should be analyzed in Leigh syndrome, presenting with optic atrophy, ataxia, dystonia, and epilepsy, regardless of age.     

A novel mitochondrial tRNAPhe mutation causes MERRF syndrome

A woman with typical features of myoclonic epilepsy with ragged red fibers (MERRF) had a novel heteroplasmic mutation (G611A) in the mitochondrial DNA tRNA phenylalanine gene. The mutation was heteroplasmic (91%) in muscle but undetectable in accessible tissues from the patient and her maternal relatives. Single-fiber PCR analysis showed that the proportion of mutant genomes was higher in cytochrome c oxidase (COX)-negative ragged red fibers (RRFs) than in COX-positive non-RRFs. This report shows that typical MERRF syndrome is not always associated with tRNA lysine mutations.     

A novel mitochondrial ND5 (MTND5) gene mutation giving isolated exercise intolerance

We describe a patient with isolated exercise intolerance caused by a new, maternally inherited mutation in mitochondrial DNA. The heteroplasmic T>C transition at position 13271 in MTND5 affects a highly conserved base and segregates with the disease, being present at highest levels in skeletal muscle fibres showing abnormal mitochondrial accumulation. This is the 15th mutation affecting the MTND5 subunit of respiratory chain complex I and confirms this protein as an important site for disease with phenotypes ranging from MELAS and infantile encephalopathies to isolated syndromes affecting a single tissue such as Leber hereditary optic neuropathy and now skeletal muscle.     

Identification and characterization of the novel m.8305C>T MTTK and m.4440G>A MTTM gene mutations causing mitochondrial myopathies

We report on two novel mtDNA mutations in patients affected with mitochondrial myopathy. The first patient, a 44-year-old woman, had bilateral eyelid ptosis and the m.8305C>T mutation in the MTTK gene. The second patient, a 56-year-old man, had four-limb muscle weakness and the MTTM gene m.4440G>A mutation. Muscle biopsies in both patients showed ragged red fibers and numerous COX-negative fibers as well as a combined defect of complex I, III and IV activities. The two mutations were heteroplasmic and detected only in muscle tissue, with a higher mutation load in COX-negative fibers. Additionally, both mutations occurred in highly conserved mt-tRNA sites, and were not found by an in silico search in 30,589 human mtDNA sequences. Our report further expands the mutational and phenotypic spectrum of diseases associated with mutations in mitochondrial tRNA genes and reinforces the notion that mutations in mitochondrial tRNAs represent hot spots for mitochondrial myopathies in adults.     

De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency

Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leigh's disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.     

Atypical MELAS associated with mitochondrial tRNA gene A8296G mutation

We report on a unique patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) presenting optic atrophy, cardiomyopathy, and bilateral striatal necrosis before stoke-like episodes became apparent. Skeletal muscle total mitochondrial DNA analysis identified a heteroplasmic A to G point mutation in the tRNA^Lys gene at position 8296. Skeletal muscle pathology revealed typical MELAS findings, including ragged-red fibers cytochrome c oxidase positive strongly succinate dehydrogenase-reactive blood vessels. Recent reports describe the 8296 mutation identified in patients with diabetes mellitus or myoclonus epilepsy with ragged-red fibers, not MELAS. We conclude that the 8296 mutation is likely to be pathogenic and that it may be not only a mutation responsible for diabetes mellitus or myoclonus epilepsy with ragged-red fibers but also for MELAS.     

Two new mutations in the MTATP6 gene associated with Leigh syndrome

In this study we have analyzed the mtDNA encoded ATPase 6 and 8 genes ( MTATP6 and MTATP8) in two children with Leigh syndrome (LS) and reduced Mg (2+) ATPase activity in muscle mitochondria. In patient 1, with a mild and reversible phenotype, mutational analysis revealed a heteroplasmic T --> C mutation at nt position 9185 (T9185C) in the MTATP6. The mutation resulted in substitution of a highly conserved leucine to proline at codon 220. The proportion of the mutation was > 97 % in the patient's blood and muscle and 85 % in blood of his asymptomatic mother. Patient 2, with severe clinical phenotype and death at 2 years of age, exhibited a novel heteroplasmic T9191C missense mutation in the MTATP6, which converted a highly conserved leucine to a proline at position 222 of the polypeptide. The proportion of the mutation was 90 % in fibroblasts and 94 % muscle tissue. This mutation was absent in the patient's parents and sister suggesting that the mutation was de novo. Our findings expand the spectrum of mutations causing LS and emphasize the role of MTATP6 gene mutations in pathogenesis of LS.     

Cytochrome c Oxidase subunit I microdeletion in a patient with motor neuron disease

An out-of-frame mutation of the mitochondrial DNA-encoded subunit I of cytochrome c oxidase (COX) was discovered during investigation of a severe isolated muscle COX deficiency in a patient with motor neuron-like degeneration. The mutation is a heteroplasmic 5-bp microdeletion located in the 5′ end of the COI gene, leading to premature termination of the corresponding translation product. Western blot analysis, immunohistochemistry, and single-fiber polymerase chain reaction demonstrated a tight correlation between COX defect, COX I expression, and percentage of mutation. COX subunits II, III, and IV were decreased as well, suggesting a defective assembly of COX holoenzyme. The mutation was associated with a clinical phenotype unusual for a mitochondrial disorder, that is, an isolated motor neuron disease(MND) with some atypical findings, including early onset, preferential involvement of the upper motor neuron, and increased cerebrospinal fluid protein content. MND may arise from impaired scavenging and overproduction of free oxygen radicals, a by-product of oxidative phosphorylation (OXPHOS). Our observation suggests that OXPHOS impairment could play a role in the pathogenesis of some MND cases.     

Exercise intolerance due to cytochrome b mutation

Cytochrome b mutations are rare causes of exercise intolerance. We report an 18‐year‐old man with exercise intolerance since childhood, resting lactic acidosis, cytochrome c oxidase (COX)‐positive ragged‐red fibers, and isolated muscle complex III deficiency due to a heteroplasmic m.14849T>C mutation in cytochrome b. We review previously described patients carrying mutations in the same gene. COX‐positive ragged‐red fibers together with exercise intolerance and lactic acidemia provide a clue for the diagnosis of this rare mitochondrial disorder. Muscle Nerve, 2010     

Pyruvate dehydrogenase deficiency: The relation of the E1α mutation to the E1β subunit deficiency

We report 7 patients with pyruvate dehydrogenase (PDH) deficiency caused by mutations of the PDH-E1α subunit. Each patient had a different mutation; 4 with duplicate insertions, 1 with a deletion of tandem repeat, and 2 with point mutations. Five of the mutations were novel, thus confirming allelic heterogeneity. Immunoblot analysis revealed decreased immunoreactivity for the E1α and E1β subunits in every patient. Pulse-labeling and chase study of the E1α and E1β subunits revealed that initial synthesis of the mutant E1α subunit was normal and posttranslational degradation was complete by 48 hours. However, the post-translational degradation rate of the E1β subunit varied from one patient to another. Factors other than instability of the E1β monomer must contribute to the degradation rate of this subunit in the presence of an E1α subunit mutation. Including this series, 3 patients with thhe S312 deletion and 5 with the R302C point mutation have been reported, and all of these patients are female. These findings suggest that these two loci are hot spots for gene mutations, and may be lethal in the male fetus.     

Molecular analysis of LGMD-2B and MM patients: identification of novel DYSF mutations and possible founder effect in the Italian population

Dysferlin, the protein product of the dysferlin gene (DYSF), has been shown to have a role in calcium-induced membrane fusion and repair.

Dysferlin is absent or drastically reduced in patients with the following autosomal recessive disorders: limb-girdle muscular dystrophy type 2B (LGMD-2B), Miyoshi myopathy (MM) and distal anterior compartment myopathy.

To date, less than 45 mutations have been described in DYSF and a wide inter- and intra-familial variation in clinical phenotype has been associated with the same mutation. This observation underlines the relevance of any new report describing genotype/phenotype correlations in dysferlinopathic patient and families.

Here we present the results of clinical, biochemical and genetic analysis performed on one MM and three LGMD Italian families. By screening the entire coding region of DYSF, we identified three novel mutations (two missense substitutions and one frame shift microdeletion). The possible existence of a founder effect for the Arg959Trp mutation in the Italian population is discussed.