Deletions of muscle mitochondrial DNA in mitochondrial myopathies: sequence analysis and possible mechanisms.

Forty per cent of patients with mitochondrial myopathies, a diverse group of multisystem diseases predominantly affecting skeletal muscle and the brain, have large deletions of a proportion of muscle mitochondrial DNA (mt DNA). These appeared to be identical in 13 of 28 cases, contained within the region 8286-13595 bp. Analysis of the deletion junction in two cases showed a 13 nucleotide sequence which occurred in the normal genome as a direct repeat flanking the region deleted in the mutant mt DNAs. Mt DNA deletions may arise from recombination or slippage between short sequence repeats during replication.     

Experimentally induced defects of mitochondrial metabolism in rat skeletal muscle. Biological effects of the NADH: coenzyme Q reductase inhibitor diphenyleneiodonium.

An animal model for the human condition of mitochondrial myopathy has been established and characterized physiologically and biochemically. The NADH: coenzyme Q reductase inhibitor diphenyleneiodonium [Bloxham (1979) Biochem. Soc. Trans. 7, 103-106] was either infused acutely in vivo into rat hind limb or injected chronically into rats. Both modes of delivery resulted in a reduced muscle oxidative capacity and increased fatigue. Analysis of muscle metabolites by h.p.l.c. and 31P-n.m.r. indicated that ATP concentrations were similar to control values during periods of stimulation and these were maintained by the phosphocreatine pool. During the recovery period after muscle stimulation in the experimental animals the muscle pH remained depressed and the rate of phosphocreatine synthesis was markedly delayed as compared with controls. Factors thought to be involved in the fatigue response are discussed in relation to this model.     

A new mitochondrial disease associated with mitochondrial DNA heteroplasmy.

A variable combination of developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy occurred in four members of a family and was maternally transmitted. There was no histochemical evidence of mitochondrial myopathy. Blood and muscle from the patients contained two populations of mitochondrial DNA, one of which had a previously unreported restriction site for AvaI. Sequence analysis showed that this was due to a point mutation at nucleotide 8993, resulting in an amino acid change from a highly conserved leucine to arginine in subunit 6 of mitochondrial H(+)-ATPase. There was some correlation between clinical severity and the amount of mutant mitochondrial DNA in the patients; this was present in only small quantities in the blood of healthy elderly relatives in the same maternal line.     

Mitochondrial DNA polymorphism in mitochondrial myopathy

Summary In order to test the hypothesis that mitochondrial myopathy may be caused by mutation of the mitochondrial (mt) genome, restriction fragment length polymorphism in leucocyte mt DNA has been studied in 38 patients with mitochondrial myopathy, 44 of their unaffected matrilineal relatives, and 35 normal control subjects. Previously unreported mt DNA polymorphisms were identified in both patients and controls. No differences in restriction fragment patterns were observed between affected and unaffected individuals in the same maternal line, and there was no evidence of major deletion of mt DNA in patients. This study provides no positive evidence of mitochondrial inheritance in mitochondrial myopathy, but this has not been excluded.     

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.     

Mitochondrial Disease: A Historical,Biochemical, and London Perspective

Roland Luft is credited with describing the first truly mitochondrial disorder in the late 1950s and early 1960s. Cases such as his have proven to be exceptionally rare. Some years later, methods of mitochondrial analysis--enzymatic, polarographic, and spectroscopic, which had been developed primarily by groups in Philadelphia--were applied to the study of mitochondria isolated from skeletal muscle biopsies of patients thought to have defects of oxidative phosphorylation. In the vanguard of these investigations were groups in New York and London. John Clark led the latter group. Application of biochemical studies, more recently supplemented by molecular mtDNA and nuclear DNA studies, have revealed that mitochondrial disorders are among the most common of all metabolic disorders.     

Interaction of di-iodinated 125I-labelled alpha-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat.

1. Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibres were characterized by their interaction with di-iodinated 125I-labelled alpha-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22 deggrees C). 2. The time course and concentration dependence of 125I-labelled alpha-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labelled alpha-bungarotoxin (1.4--200 nM) the bimolecular-association rate constant, k + 1, was (2.27 +/- 0.49) x 10(4)M-1.S-1 (mean +/- S.D., N = 10). In concentration-dependence experiments, k + 1 was 2.10 x 10(4)M-1.S-1 and 1.74 x 10(4) M-1.S-1 with 10 and 135 min incubations respectively. In association experiments the first-order rate constant was proportional to the 125I-labelled alpha-bungarotoxin concentration. 125I-Labelled alpha-bungarotoxin dissociation was first order with a dissociation constant, k-1, less than or equal to 3 x 10(-6)S(-1) (half-life greater than or equal to 60 h.) The results indicated a single class of high-affinity toxin-binding sites at the end-plate with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 +/- 0.46) x 10(7) (mean +/- S.D., n = 22) per rat end-plate. 3. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labelled alpha-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. In contrast agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labelled alpha-bungarotoxin binding. 4. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (delta So = +0.46 kJ.mol-1.K-1) than the antagonist tubocurarine (delta So = +0.26 kJ.mol-1.K-1). 5. The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. 6. The affinity and specificity of 125I-labelled alpha-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibres were the same. 7. The study of binding only requires milligram amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.