Cytochrome c oxidase deficiency associated with the first stop-codon point mutation in human mtDNA.

We have identified the first stop-codon point mutation in mtDNA to be reported in association with human disease. A 36-year-old woman experienced episodes of encephalopathy accompanied by lactic acidemia and had exercise intolerance and proximal myopathy. Histochemical analysis showed that 90% of muscle fibers exhibited decreased or absent cytochrome c oxidase (COX) activity. Biochemical studies confirmed a severe isolated reduction in COX activity. Muscle immunocytochemistry revealed a pattern suggestive of a primary mtDNA defect in the COX-deficient fibers and was consistent with either reduced stability or impaired assembly of the holoenzyme. Sequence analysis of mtDNA identified a novel heteroplasmic G-->A point mutation at position 9952 in the patient's skeletal muscle, which was not detected in her leukocyte mtDNA or in that of 120 healthy controls or 60 additional patients with mitochondrial disease. This point mutation is located in the 3' end of the gene for subunit III of COX and is predicted to result in the loss of the last 13 amino acids of the highly conserved C-terminal region of this subunit. It was not detected in mtDNA extracted from leukocytes, skeletal muscle, or myoblasts of the patient's mother or her two sons, indicating that this mutation is not maternally transmitted. Single-fiber PCR studies provided direct evidence for an association between this point mutation and COX deficiency and indicated that the proportion of mutant mtDNA required to induce COX deficiency is lower than that reported for tRNA-gene point mutations. The findings reported here represent only the second case of isolated COX deficiency to be defined at the molecular genetic level and reveal a new mutational mechanism in mitochondrial disease.     

Maternally inherited cardiomyopathy and hearing loss associated with a novel mutation in the mitochondrial tRNA(Lys) gene (G8363A).

A novel G8363A mutation in the mtDNA tRNA(Lys) gene was associated, in two unrelated families, with a syndrome consisting of encephalomyopathy, sensorineural hearing loss, and hypertrophic cardiomyopathy. Muscle biopsies from the probands showed mitochondrial proliferation and partial defects of complexes I, III, and IV of the electron-transport chain. The G8363A mutation was very abundant (>95%) in muscle samples from the probands and was less copious in blood from 18 maternal relatives (mean 81.3% +/- 8.5%). Single-muscle-fiber analysis showed significantly higher levels of mutant genomes in cytochrome (c) oxidase-negative fibers than in cytochrome (c) oxidase-positive fibers. The mutation was not found in >200 individuals, including normal controls and patients with other mitochondrial encephalomyopathies, thus fulfilling accepted criteria for pathogenicity.     

An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy

A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation.     

Segregation patterns of a novel mutation in the mitochondrial tRNA glutamic acid gene associated with myopathy and diabetes mellitus.

We have identified a novel mtDNA mutation in a 29-year-old man with myopathy and diabetes mellitus. This T-->C transition at mtDNA position 14709 alters an evolutionarily conserved nucleotide in the region specifying for the anticodon loop of the mitochondrial tRNA(Glu). The nt-14709 mutation was heteroplasmic but present at very high levels in the patient's muscle, white blood cells (WBCs), and hair follicles; lower proportions of mutated mtDNA were observed in WBCs and hair follicles of all examined maternal relatives. In the patient's muscle, abnormal fibers showed mitochondrial proliferation, severe focal defects in cytochrome c oxidase activity, and absence of cross-reacting material for mitochondrially synthesized polypeptides. These fibers had higher levels of mutated mtDNA than did surrounding "normal" fibers. Although the percentage of mutated mtDNA in WBCs from family members were distributed around the percentage observed in the mothers, the pattern was different in hair follicles, where the mutated population tended to increase in subsequent generations. PCR/RFLP analysis of single hairs showed that the intercellular variations in the percentage of mutated mtDNA differed among family members, with younger generations having a more homogeneous distribution of mutated mtDNA in different hair follicles. These results suggest that the intercellular distribution of the mutated and wild-type mtDNA populations may drift toward homogeneity in subsequent generations.     

Mitochondrial myopathy in a child with a muscle-restricted mutation in the mitochondrial transfer RNAAsn gene

We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA(Asn), MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient's muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA(Asn) steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.     

Identification of a new mtDNA mutation (14724G>A) associated with mitochondrial leukoencephalopathy

We report a novel 14724G>A mutation in the mitochondrial tRNA glutamic acid gene in a 4-year-old boy with myopathy and leukoencephalopathy. A muscle biopsy showed cytochrome c oxidase-negative ragged-red fibers and biochemical analysis of the respiratory chain enzymes in muscle homogenate revealed partial complex I and complex IV deficiencies. The mutation, which affects the dihydrouridine arm at a conserved site, was nearly homoplasmic in muscle and heteroplasmic in blood DNA of the proband, but it was absent in peripheral leukocytes from the asymptomatic mother, sister, and two maternal aunts, suggesting that it arose de novo. This report proposes to look for variants in the mitochondrial genome when dealing with otherwise undetermined leukodystrophies of childhood.     

A new mtDNA mutation in the tRNA(Lys) gene associated with myoclonic epilepsy and ragged-red fibers (MERRF).

Myoclonic epilepsy with ragged-red fibers (MERRF) has been associated with an A--G transition at mtDNA nt 8344, within a conserved region of the tRNA(Lys) gene. Although the 8344 mutation is highly prevalent in patients with MERRF, it is not observed in 10%-20% of the cases, suggesting genetic heterogeneity. We have sequenced the tRNA(Lys) gene of five MERRF patients lacking the common 8344 mutation. One of these showed a novel T-->C transition at nucleotide position 8356, disrupting a highly conserved base pair in the T psi C stem. The mutant mtDNA population was essentially homoplasmic in muscle but was heteroplasmic in blood (47%). Neither 20 patients with other mitochondrial diseases nor 25 controls carried this mutation. These findings suggest that tRNA(Lys) alterations may play a specific role in the pathogenesis of MERRF syndrome.     

Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.     

A novel mutation in the mitochondrial 16S rRNA gene in a patient with MELAS syndrome, diabetes mellitus, hyperthyroidism and cardiomyopathy

Using RNase protection analysis, we found a novel C to G mutation at nucleotide position 3093 of mitochondrial DNA (mtDNA) in a previously reported 35-year-old woman exhibiting clinical features of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome together with diabetes mellitus, hyperthyroidism and cardiomyopathy. The patient also had an A3243G mutation in the tRNA(Leu(UUR)) gene and a 260-base pair duplication in the D-loop of mtDNA. The fibroblasts of the patient were cultured and used for the construction of cybrids using cytoplasmic transfer of the patient's mtDNA to the mtDNA-less rho(0) cells. RNA isolated from the cybrids was subjected to RNase protection analysis, and a C3093G transversion at the 16S rRNA gene and a MELAS-associated A3243G mutation of mtDNA were detected. The novel C3093G mutation together with the A3243G transition were found in muscle biopsies, hair follicles and blood cells of this patient and also in her skin fibroblasts and cybrids. The proportion of the C3093G mutant mtDNA in muscle biopsies of the patient was 51%. In contrast, the mutation was not detected in three sons of the proband. To characterize the impact of the mtDNA mutation-associated defects on mitochondrial function, we determined the respiratory enzyme activities of the primary culture of fibroblasts established from the proband, her mother and her three sons. The proportions of mtDNA with the C3093G transversion and the A3243G transition in the fibroblasts of the proband were 45 and 58%, respectively. However, the fibroblasts of the proband's mother and children harbored lower levels of mtDNA with the A3243G mutation but did not contain the C3093G mutation. The complex I activity in the proband's fibroblasts was decreased to 47% of the control but those of the fibroblasts of the mother and three sons of the proband were not significantly changed. These findings suggest that the C3093G transversion together with the A3243G transition of mtDNA impaired the respiratory function of mitochondria and caused the atypical MELAS syndrome associated with diabetes mellitus, hyperthyroidism and cardiomyopathy in this patient.     

Apoptosis and necrosis mediate skeletal muscle fiber loss in age‐induced mitochondrial enzymatic abnormalities

Sarcopenia, the age‐induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age‐dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age‐dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC‐abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Congenital encephalomyopathy and adult-onset myopathy and diabetes mellitus: different phenotypic associations of a new heteroplasmic mtDNA tRNA glutamic acid mutation.

We report the clinical, biochemical, and molecular genetic findings in a family with an unusual mitochondrial disease phenotype harboring a novel mtDNA tRNA glutamic acid mutation at position 14709. The proband and his sister presented with congenital myopathy and mental retardation and subsequently developed cerebellar ataxia. Other family members had either adult-onset diabetes mellitus with muscle weakness or adult-onset diabetes mellitus alone. Ragged-red and cytochrome c oxidase (COX)-negative fibers were present in muscle biopsies. Biochemical studies of muscle mitochondria showed reduced complex I and IV activities. The mtDNA mutation was heteroplasmic in blood and muscle in all matrilineal relatives analyzed. Primary myoblast, but not fibroblast, cultures containing high proportions of mutant mtDNA exhibited impaired mitochondrial translation. These observations indicate that mtDNA tRNA point mutations should be considered in the differential diagnosis of congenital myopathy. In addition they illustrate the diversity of phenotypes associated with this mutation in the same family and further highlight the association between mtDNA mutations and diabetes mellitus.     

The age-related accumulation of a mitochondrial DNA control region mutation in muscle, but not brain, detected by a sensitive PNA-directed PCR clamping based method

The peptide nucleic acid (PNA)-directed PCR clamping technique was modified and applied to the detection of mitochondrial DNA mutations with low heteroplasmy. This method is extremely specific, eliminating false positives in the absence of mutant molecules, and highly sensitive, being capable of detecting mutations at the level of 0.1% of total molecules. Moreover, the reaction can be multiplexed to identify more than one mutation per reaction. Using this technique, the levels of three point mutations, the tRNA^Leu(UUA) 3243 mutation causing mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS); the tRNA^Lys 8344 mutation causing myoclonic epilepsy and ragged red fibers (MERRF); and the nucleotide position 414 mutation adjacent to the control region promoters, were evaluated in human brain and muscle from individuals of various ages. While none of the mutations were detected in brain samples from individuals ranging in age from 23 to 93, the 414 mutation could be detected in muscle from individuals 30 years and older. These data demonstrate that the 3243 and 8344 mutations do not accumulate with age to levels greater than 0.1% in brain and muscle. By contrast, the 414 mutation accumulates with age in normal human muscle, though not in brain. The reason for the striking absence of the 414 mutation in aging brain is unknown.     

Impairment of mitochondrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia

We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNA(Ile). The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient's healthy mother. Functional characterization of the mutation revealed a six-fold reduced rate of tRNA(Ile) precursor 3' end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNA(Ile) displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNA(Ile) precursor 3' end processing and contribute to the molecular pathomechanism of this mutation.     

Mitochondrial myopathy in a child with a muscle-restricted mutation in the mitochondrial transfer RNA gene

We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA^Asn, MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient’s muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA^Asn steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.     

Oxidative phosphorylation in mitochondria from different fiber types of chicken muscles

Isolated mitochondria from different types of muscle fibers from chickens 3 to 5 weeks were studied to evaluate the comparative oxidation of various substrates. Pectoralis (alphaW fibers), lateral adductor (betaR fibers), and medial adductor (alphaR fibers) were the muscles used. Oxygen consumption rates, RCR, and ADP/O ratios were measured to study mitochondrial function. Mitochondria from pectoralis muscle utilized pyruvate, succinate, L-glutamate, alpha-glycerophosphate, and beta-hydroxybutyrate. Mitochondria from the other two muscle types utilized all of those substrates except alpha-glycerophosphate. In each muscle type utilization of NADH was minimum and was not coupled with phosphorylation of ADP. Thus, in alphaW muscles oxidation of alpha-glycerophosphate may play an important role in transport of cytoplasmic NADH to the mitochondrial respiratory chain. In alphaR and betaR muscles "shuttle" systems other than alpha-glycerophosphate oxidation, e.g., beta-hydroxybutyrate, may perform that important role.