Search

Objective Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2KI ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. Methods We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). AAV-TK2 was administered to Tk2KI , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides. Results AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except kidney, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in liver and kidney, animal growth, and lifespan. Interpretation Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. This article is protected by copyright. All rights reserved.

Objective: To elucidate the relationship between mitochondrial DNA (mtDNA) alterations and a mitochondrial disease with a distinct combination of characteristic symptoms, namely episodic hyper-creatine kinase (CK)-emia and mild myopathy. Methods: We selected 9 patients with mtDNA np8291 alteration from 586 patients suspected to have a mitochondrial disease, and assessed them clinically, pathologically, and genetically. These 9 patients had undiagnosed mitochondrial myopathy with episodic hyper-CK-emia, all showing similar symptoms and progression. Results: Patients had mild muscle weakness and episodic hyper-CK-emia triggered by infections or drugs. Five of 9 patients were initially diagnosed with other conditions, such as myasthenia gravis, polymyositis, viral myositis, and drug-induced myopathy, because these conditions were acute or subacute, and 9 patients showed the same 16 mtDNA alterations, which have been reported to be nonpathological polymorphisms. Muscle biopsy revealed ragged-red fibers, highly expressed succinate dehydrogenase staining fibers, and cytochrome c oxidase–deficient fibers. Because their mitochondrial sequence data was almost the same, and 9 patients live in widely separated cities in Japan, the alterations may have arisen from a single source. Interpretation: These findings suggest that mild myopathy with episodic hyper-CK-emia associated with some of the 16 mtDNA alterations or at least with their mitochondria, could be a novel mitochondrial disease. Therefore, we propose that this disease be named as “mitochondrial myopathy with episodic hyper-CK-emia (MIMECK).” These alterations could work concomitantly and probably modify the impact of medications or other environmental factors. We believe these findings provide an insight into a novel aspect of mitochondrial disease pathogenesis. ANN NEUROL 2011;70: 486–492.

Contains fulltext : 70753.pdf (Publisher’s version ) (Closed access) OBJECTIVE: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate production rates, which we refer to as the "MEGS capacity." Currently, little is known about MEGS pathology in patients with mutations in the mitochondrial DNA. Because MEGS capacity is an indicator for the overall mitochondrial function related to energy production, we searched for a correlation between MEGS capacity and 3243A-->G mutation load in muscle of patients with the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome. METHODS: In muscle tissue of 24 patients with the 3243A-->G mutation, we investigated the MEGS capacity, the respiratory chain enzymatic activities, and the 3243A-->G mutation load. To exclude coinciding mutations, we sequenced all 22 mitochondrial transfer RNA genes in the patients, if possible. RESULTS: We found highly significant differences between patients and control subjects with respect to the MEGS capacity and complex I, III, and IV activities. MEGS-related measurements correlated considerably better with the mutation load than respiratory chain enzyme activities. We found no additional mutations in the mitochondrial transfer RNA genes of the patients. INTERPRETATION: The results show that MEGS capacity has a greater sensitivity than respiratory chain enzymatic activities for detection of subtle mitochondrial dysfunction. This is important in the workup of patients with rare or new mitochondrial DNA mutations, and with low mutation loads. In these cases we suggest to determine the MEGS capacity.

Using in situ hybridization, we studied muscle biopsy specimens from 4 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Three of the 4 patients with MELAS had a mutation at position 3243 of mitochondrial DNA (mtDNA) in the transfer RNALeu(UUR) gene, and the other patient had a mutation at position 3271 in the same transfer RNALeu(UUR) gene. Quantitative analysis using Southern blot hybridization and polymerase chain reaction showed 80 to 90% mutant mtDNA in muscle. In situ hybridization analysis showed that total mtDNAs (both normal and mutant) were extremely increased in blood vessels with high succinate dehydrogenase activity (strongly succinate dehydrogenase-reactive blood vessels) and ragged-red fibers. Cytochrome c oxidase activity in most of these reactive blood vessels and ragged-red fibers was positive. The similar morphological behavior in these vessels and fibers suggests that an increase in mutant mtDNA is responsible for mitochondrial proliferation and dysfunction in both tissues where cytochrome c oxidase is not a primarily defective enzyme. The pattern of expression of genes for mtDNA-encoded ribosomal RNA and the protein-coding region cytochrome c oxidase subunit II were similar in muscle specimens of patients with MELAS, patients with chronic progressive external ophthalmoplegia, and normal control subjects, and also between the two MELAS mutations. These results do not support the hypothesis that impaired transcription termination is a molecular defect in MELAS.

A cornerstone of mitochondrial genetics, strict maternal inheritance, has been challenged recently by the study of a patient with mitochondrial myopathy due to a sporadic 2bp deletion. The mitochondrial DNA (mtDNA) harboring the mutation was paternal in origin, whereas the patient's blood was identical to the maternal genotype. To determine whether this is a common phenomenon, we studied mtDNA sequence variation between muscle and blood from 35 patients with sporadic mitochondrial myopathies, but detected no evidence of paternal mtDNA transmission. Our findings suggest that paternal transmission of mtDNA is rare and should not alter our genetic advice to families.

The purpose of this study was to investigate the correlation between the level of mutated mitochondrial DNA in muscle and oxidative capacity in 24 patients with mitochondrial myopathy (MM). Maximal oxygen uptake (VO(2max)), workload (W(max)), and venous plasma lactate levels were measured during an incremental cycle test to exhaustion in 17 patients with point mutations of mtDNA and in seven with single, large-scale deletions of mtDNA (chronic progressive external ophthalmoplegia [CPEO]). Results were compared with those in 25 healthy matched subjects. The mutation load in MM patients was 67 +/- 5% (range, 29 - 99%). VO(2max) and W(max) correlated with percentage of heteroplasmy (r0.82; p0.005) and were lower in patients versus healthy subjects (p0.000005). Exercise-induced peak increases in heart rate, ventilation, and resting plasma lactate levels correlated with muscle mutation load (r0.71; p0.005). Exercise-induced increases in plasma lactate correlated with muscle mutation load in CPEO patients (r = 0.95; p0.005). Impaired oxidative capacity and ragged red muscle fibers were found in CPEO and 3243A--G patients with mutation loads as low as 45 and 57%, respectively. The study indicates that oxidative capacity correlates directly with skeletal muscle mutation load in MM patients, and that the mutation threshold level for impaired oxidative metabolism in MM patients is lower than found in in vitro studies.

Sengers syndrome is characterized by congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy, and lactic acidosis, but no abnormalities have been found with routine mitochondrial biochemical diagnostics (the determination of pyruvate oxidation rates and enzyme measurements). In immunoblot analysis, the protein content of the mitochondrial adenine nucleotide translocator 1 (ANT1) was found to be strongly reduced in the muscle tissues of two unrelated patients with Sengers syndrome. In addition, low residual adenine nucleotide translocator activity was detected upon the reconstitution of detergent-solubilized mitochondrial extracts from the patients' skeletal or heart muscle into liposomes. Sequence analysis and linkage analysis showed that ANT1 was not the primary genetic cause of Sengers syndrome. We propose that transcriptional, translational, or posttranslational events are responsible for the ANT1 deficiency associated with the syndrome.

Barth syndrome is an X-linked cardiac and skeletal mitochondrial myopathy. Barth syndrome may be due to lipid alterations because the product of the mutated gene is homologous to phospholipid acyltransferases. Here we document that a single mitochondrial phospholipid species, tetralinoleoyl-cardiolipin, was lacking in the skeletal muscle (n = 2), right ventricle (n = 2), left ventricle (n = 2), and platelets (n = 6) of 8 children with Barth syndrome. Tetralinoleoyl-cardiolipin is specifically enriched in normal skeletal muscle and the normal heart. These findings support the notion that Barth syndrome is caused by alterations of mitochondrial lipids.

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.

Aerobic training has been shown to increase work and oxidative capacity in patients with mitochondrial myopathies, but the mechanisms underlying improvement are not known. We evaluated physiological (cycle exercise, 31P-MRS), biochemical (enzyme levels), and genetic (proportion of mutant/wild-type genomes) responses to 14 weeks of bicycle exercise training in 10 patients with heteroplasmic mitochondrial DNA (mtDNA) mutations. Training increased peak work and oxidative capacities (20-30%), systemic arteriovenous O2 difference (20%), and 31P-MRS indices of metabolic recovery (35%), consistent with enhanced muscle oxidative phosphorylation. Mitochondrial volume in vastus lateralis biopsies increased significantly (50%) and increases in deficient respiratory chain enzymes were found in patients with Complex I (36%) and Complex IV (25%) defects, whereas decreases occurred in 2 patients with Complex III defects (approximately 20%). These results suggest that the cellular basis of improved oxygen utilization is related to training-induced mitochondrial proliferation likely resulting in increased levels of functional, wild-type mtDNA. However, genetic analysis indicated the proportion of wild-type mtDNA was unchanged (3/9) or fell (6/9), suggesting a trend toward preferential proliferation of mutant genomes. The long-term implications of training-induced increases in mutant relative to wild-type mtDNA, despite positive physiological and biochemical findings, need to be assessed before aerobic training can be proposed as a general treatment option.

We studied mitochondrial function in vivo in 2 brothers harboring the mitochondrial DNA A3243G mutation by using magnetic resonance spectroscopy. One brother presented with recurrent strokes and had a mitochondrial respiratory chain complex I defect, with 85% A3243G mutation in his quadriceps. The maximum rate of mitochondrial ATP production in his calf, measured in vivo, was reduced to 21% of the normal mean value. The second brother had mild exercise intolerance, normal muscle histochemistry, and normal respiratory chain activity in vitro. Despite a level of the A3243G mutation of only 5.95% (SD, 4.45; range, 0.7-16.1%) within single muscle fibers from the gastrocnemius muscle, the maximum rate of mitochondrial ATP production in his calf, measured in vivo, was reduced to 35% of the normal mean value. These findings suggest that there may not be a clear genetic threshold level for the expression of the A3243G mutation in skeletal muscle in vivo.

MELAS is a mitochondrial encephalomyopathy characterized clinically by recurrent stroke-like episodes, seizures, sensorineural deafness, dementia, hypertrophic cardiomyopathy, and short stature. The majority of patients are heteroplasmic for a mutation (A3243G) in the tRNAleu(UUR) gene in mitochondrial DNA (mtDNA). In cells cultured in vitro, the mutation produces a severe mitochondrial translation defect only when the proportion of mutant mtDNAs exceeds 95% of total mtDNAs. However, most patients are symptomatic well below this threshold, a paradox that remains unexplained. We studied the relationship between the level of heteroplasmy for the mutant mtDNA and the clinical and biochemical abnormalities in a large pedigree that included 8 individuals carrying the A3243G mutation, 4 of whom were asymptomatic. Unexpectedly, we found that brain lactate, a sensitive indicator of oxidative phosphorylation dysfunction, was linearly related to the proportion of mutant mtDNAs in all individuals carrying the mutation, whether they were symptomatic or not. There was no evidence for threshold expression of the metabolic defect. These results suggest that marked tissue-specific differences may exist in the pathogenic expression of the A3243G mutation and explain why a neurological phenotype can be observed at relatively low levels of heteroplasmy.

We report on 4 male patients with clinical, radiological, and muscle biopsy findings typical of the mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) phenotype. Skeletal muscle mitochondrial DNA (mtDNA) analysis showed that all patients harbored a heteroplasmic G13513A mutation in the ND5 subunit gene. One of these cases (Patient 1) presented with symptoms characteristic of Leber's hereditary optic neuropathy (LHON) 2 years before the first stroke-like episode. Quantitative analysis in several postmortem tissue sections showed that the relative proportions of mutant mtDNA were generally lower than those reported with other pathogenic mtDNA mutations. Single-fiber polymerase chain reaction studies demonstrated significantly higher amounts of mutant mtDNA in ragged red fibers (RRFs) compared with non-RRFs. This study indicates that the G13513A transition is likely to be pathogenic, that it can cause an LHON/MELAS overlap syndrome, and that it may be a more frequent cause of MELAS than previously recognized.

Five patients with chronic progressive external ophthalmoplegia (CPEO) and 27 healthy controls were examined by near-infrared spectroscopy (NIRS) for the noninvasive and direct quantitative measurement of muscle oxygen consumption and forearm blood flow. NIRS measurements were obtained in rest and during static isometric handgrip exercise at 10% of the maximum voluntary contraction (MVC) force. A significantly decreased oxygen consumption at rest as well as during exercise was found in patients with CPEO. Our results suggest that NIRS is able to discriminate between CPEO patients and healthy controls, which makes NIRS a valuable tool in the diagnostic workup of patients suspected to have a mitochondrial myopathy.

We report the first molecular defect in an NADH-dehydrogenase gene presenting as isolated myopathy. The proband had lifelong exercise intolerance but no weakness. A muscle biopsy showed cytochrome c oxidase (COX)-positive ragged-red fibers (RRFs), and analysis of the mitochondrial enzymes revealed complex I deficiency. Sequence analysis of the mitochondrial genes encoding the seven NADH-dehydrogenase subunits showed a G-to-A transition at nucleotide 11832 in the subunit 4 (ND4) gene, which changed an encoded tryptophan to a stop codon. The mutation was heteroplasmic (54%) in muscle DNA. Defects in mitochondrially encoded complex I subunits should be added to the differential diagnosis of mitochondrial myopathies.

We report seven unrelated families with mitochondrial tRNA(Ser(UCN)) gene mutations at three different loci. A novel G7497A mutation is found in two families, both of which present with progressive myopathy, ragged-red fibers, lactic acidosis, and deficiency of respiratory chain complexes I and IV. This mutation presumably affects the tertiary tRNA(Ser(UCN)) dihydrouridine interaction. Mutations 7472 insC and T7512C, found in three and two families, respectively, are associated with myoclonus epilepsy, deafness, ataxia, cognitive impairment, and complex IV deficiency. No ragged-red fibers or ultrastructural abnormalities are seen. It is interesting that 6 of our 7 index patients are apparently homoplasmic, indicating a minor pathogenetic power of the tRNA(Ser(UCN)) mutations.

The cause and pathophysiology of dystonia remain unknown. The recent identification of mitochondrial complex I deficiency in platelets from patients with sporadic focal dystonia suggests that a defect of energy metabolism may be relevant in a proportion of patients. We have addressed the possible contribution of mitochondrial DNA (mtDNA) to the complex I deficiency in dystonia by the use of genome transfer technology. Platelets from patients deficient for complex I were fused with A549 p0 (mtDNA-less) cells to form cybrids comprising the A549 nucleus and dystonia mtDNA. Mixed cybrid cell lines were analyzed for 9 controls and 9 dystonia patients, and clonal cybrid lines were generated for 2 control and 2 dystonia patients. Subsequent biochemical analysis showed that the dystonia complex I defect was complemented in both the mixed and the clonal cybrid lines. These results contrast with similar studies in mitochondrial myopathy and Parkinson's disease patients, in which the mitochondrial defect was maintained in at least a proportion of A549 cybrids, and suggest that the complex I defect in dystonia is not caused by an mtDNA mutation.

In mitochondrial myopathy, severely impaired muscle oxidative capacity poses a dilemma for metabolic regulation in exercise. We inquired whether fuel mobilization during exercise in mitochondrial myopathy is adjusted to the reduced capacity to oxidize substrate, or if fuel is mobilized in excess of oxidative capacity. Hormonal and metabolic responses to 20 minutes of cycle exercise were studied in 4 patients with mitochondrial myopathy working at near maximal effort and in 4 healthy matched controls. On 2 separate days, controls were studied at the same absolute (A) workload (9 +/- 3 W) and the same relative (R) workload (77 +/- 9 W) as performed by the patients. During exercise, average glucose production was higher in patients (28 +/- 5 micromol min(-1) kg(-1)) than in controls at both workloads (A, 12 +/- 1; R, 18 +/- 2 micromol min(-1) kg(-1)). Exercise-induced increases in plasma glucose, growth hormone, epinephrine, norepinephrine, corticotropin, and lactate, and decreases in plasma insulin and pH were also larger in patients compared with findings in controls at both workloads. In conclusion, mitochondrial myopathies are associated with excessive neuroendocrine responses and mobilization of glucose during exercise. These responses augment ATP synthesis but result in progressive accumulation of nonoxidized substrates. Apparently, substrate mobilization and neuroendocrine responses in exercise are linked to oxidative demand rather than to oxidative capacity in working muscle.

We describe a patient with the mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) phenotype in whom initial investigations in skeletal muscle failed to show any histochemical or biochemical defect. Subsequent analysis of the mitochondrial genome identified a new heteroplasmic mutation in the valine transfer RNA gene, the first described in this region.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a maternally inherited disorder, is usually associated with a point mutation in mitochondrial DNA (mtDNA) at position 3,243 in the tRNA Leu(UUR) gene. To further study the pathogenesis of MELAS, we analyzed tissues from 8 MELAS-3,243 patients. Southern blot analysis showed an increase in the ratio of mtDNA to nuclear DNA in almost all tissues examined, implying that mitochondrial proliferation is ubiquitous and is not confined to ragged-red fibers in muscle. By northern blot analysis, we demonstrated increased steady-state levels of RNA 19, a polycistronic transcript corresponding to the 16S rRNA + tRNA Leu(UUR) + ND1 genes (which are contiguous in the mtDNA) in heart, kidney, and muscle. These results provide further evidence that altered mitochondrial nucleic acid metabolism may have pathogenic significance in MELAS.

A 35-year-old woman with features of Kearns-Sayre syndrome consisting of progressive ptosis, ophthalmoparesis, mitochondrial myopathy, and pigmentary retinopathy also had autoimmune polyglandular syndrome type 11 (Addison's disease, autoimmune insulin-dependent diabetes mellitus, Hashimoto's thyroiditis, and primary ovarian failure). There was no history of similarly affected relatives. Analysis of muscle mitochondrial DNA (mtDNA) revealed a 2,532-bp deletion of the type seen in Kearns-Sayre syndrome as well as a heteroplasmic A3243G mutation in the tRNA-Leu(UUR) gene of the type seen in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The patient's blood and her mother's blood harbored the A3243G mutation but not the deletion, and the maternal grandmother's blood had neither mutation. In muscle, the species of mtDNA harboring the deletion was exclusively associated with the species harboring the A3243G mutation, suggesting that the point mutation predisposed to the large-scale deletion. The mtDNA species with both mutations accounted for 88% of total muscle mtDNA. Other and as yet unrecognized point mutations in mtDNA might also be associated with, and possible causally related to, large-scale mtDNA deletions.

In 2002, paternal inheritance of muscle mitochondrial DNA (mtDNA) was reported in a patient with exercise intolerance and a mitochondrial DNA (mtDNA) mutation restricted to skeletal muscle. To evaluate whether paternal inheritance is a common phenomenon, we studied 10 sporadic patients with skeletal muscle-restricted mtDNA mutations: five harbored mtDNA point mutations in protein-coding genes and five had single mtDNA deletions. We performed haplotype analysis and direct sequencing of the hypervariable regions 1 and 2 of the D-loop in muscle and blood from the patients and, when available, in blood from their parents. We did not observe paternal inheritance in any of our patients.

To evaluate the possible role of cytokines in human immunodeficiency virus (HIV)-associated muscular disorders, we performed immunocytochemistry for interleukin-1 alpha, -1 beta, and -6 and tumor necrosis factor-alpha on frozen muscle biopsy specimens from HIV-infected patients with various myopathies (HIV polymyositis in 5, HIV-wasting syndrome in 5, zidovudine myopathy in 10) and from seronegative individuals (normal muscle in 2, mitochondrial cytopathies in 10). The HIV-infected patients showed positive reactivities in vessels (interleukin-1) and in inflammatory cells (mainly interleukin-1 and tumor necrosis factor-alpha), including perivascular hemosiderin-laden macrophages in 5 patients. In zidovudine myopathy, a majority of AZT fibers (i.e., ragged-red fibers with marked myofibrillar changes) showed mild to marked expression of interleukin-1. Expression of interleukin-1 in the other mitochondrial myopathies was much weaker. Interleukin-1 beta messenger RNA was demonstrated in muscle fibers by in situ hybridization, implying that interleukin-1 was produced in muscle cells. Immunoelectron microscopy showed that interleukin-1 alpha was mainly bound to mitochondrial membranes in AZT fibers. Proinflammatory and destructive effects of the studied cytokines might be responsible for several myopathological changes observed in HIV-infected patients, including inflammation and hemosiderin deposits in muscle tissue, and prominent myofibrillar breakdown in AZT fibers.

We examined muscle sections from 3 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), using single-fiber polymerase chain reaction, histochemistry, and in situ hybridization. Most type 1 ragged-red fibers showed positive cytochrome c oxidase activity at the subsarcolemmal region, while type 2 ragged-red fibers had little cytochrome c oxidase activity. However, there was no difference in the amount of total (mutant and wild-type) mitochondrial DNAs (mtDNAs) and the proportion of mutant mtDNA between type 1 and type 2 ragged-red fibers. These observations suggest that mitochondrial proliferation and nuclear factors affect muscle pathology, including cytochrome c oxidase activity, in MELAS. Total mtDNAs were greatly increased in ragged-red fibers (about 5-17 times over those in non-ragged-red fibers). The proportion of mutant mtDNA was significantly higher in ragged-red fibers (88.1 +/- 5.5%) than in non-ragged-red fibers (63.2 +/- 21.6%). Thus, the amount of wild-type mtDNA as well as mutant mtDNA was increased in ragged-red fibers in MELAS, failing to support the contention of a replicative advantage of mutant mtDNA. The proportion of mutant mtDNA was significantly higher in the strongly succinate dehydrogenase-reactive blood vessels (83.2 +/- 4.2%) than in non-succinate dehydrogenase-reactive blood vessels (38.8 +/- 16.2%). It seems likely that systemic vascular abnormalities involving cerebral vessels lead to the evolution of stroke-like episodes in MELAS.

Fifty consecutive patients infected with human immunodeficiency virus type 1 (HIV-1) were evaluated regarding the prevalence of HIV-related myopathies and the relevance of zidovudine-related mitochondrial myopathy. Disease stage, total lifetime intake of zidovudine, anthropometric and nutritional parameters, muscle strength, and histochemical and immunohistochemical findings in muscle specimens were recorded. The series was divided into two groups, patients with a total lifetime intake of zidovudine under 200 gm and those with a total lifetime intake over 200 gm. A control group included 50 healthy people matched for age and sex. HIV-related myopathy was defined by the presence of at least one of the classic pathological reactions in muscle, while zidovudine-related myopathy was defined by the presence of ragged red fibers in any percentage

Microphotometric enzyme assay was used to study cytochrome c oxidase activity in single human skeletal muscle fibers. The assay techniques combine the precise localization of enzyme activity provided by histochemical methodology with the precise quantitation of a sensitive assay system. Abnormalities of cytochrome c oxidase were investigated using microphotometric enzyme assay in 12 patients with Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia, or Leigh's syndrome. Control values were obtained using muscle biopsy specimens from 20 juvenile and 18 adult subjects with no evidence of neuromuscular disease. In the patients with Leigh's syndrome due to cytochrome c oxidase deficiency, the abnormality was found to be expressed uniformly throughout the muscle fiber population. In contrast, patients with Kearns-Sayre syndrome or chronic progressive external ophthalmoplegia showed abnormal heterogeneity of cytochrome c oxidase activity. In many cases, extreme degrees of variability were seen, with fibers containing high activity adjacent to fibers with no detectable activity. Mitochondrial DNA analysis showed that most of the patients with Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia had major rearrangements of mitochondrial DNA. It was concluded that the extreme variability of cytochrome c oxidase activity detected using microphotometric enzyme assay was an indicator of a probable abnormality of mitochondrial DNA. Conversely, cytochrome c oxidase defects in muscle which show a homogeneous distribution are more likely to be associated with defects of the nuclear genome.

Phosphorus magnetic resonance spectroscopy monitors muscle energy metabolism by recording the ratio of phosphocreatine to inorganic phosphate at rest, during exercise, and during recovery from exercise. In mitochondrial diseases, abnormalities may appear during some or all these phases. Low phosphocreatine-inorganic phosphate ratios at rest are not disease-specific, but can be increased by drug therapy in several myopathies. Phosphorus magnetic resonance spectroscopy can also record intracellular pH and thus identify disorders of glycogen metabolism in which the production of lactic acid is blocked during ischemic exercise. The measurements of accumulated sugar phosphate intermediates further delineate glycolytic muscle defects. Myophosphorylase deficiency responds to intravenous glucose administration with improved exercise bioenergetics, but no such response is seen in phosphofructokinase deficiency. The muscular dystrophies show no specific bioenergetic abnormality; however, elevation of phospholipids metabolites and phosphodiesters was detected in some cases. While phosphorus magnetic resonance spectroscopy remains primarily a research tool in metabolic myopathies, it will be clinically useful in identifying new therapies and monitoring their effects in a variety of neuromuscular disorders.

Intramuscular blood vessels were examined with succinate dehydrogenase stain in skeletal muscle biopsy specimens from 6 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Almost all arteries had large granular deposits with high succinate dehydrogenase activity in their walls. Electron microscopic examination of serial frozen sections of these biopsies showed that the smooth muscle cells of the strongly succinate dehydrogenase-reactive blood vessels contained markedly proliferated mitochondria, characteristic of patients with MELAS. The presence of strongly succinate dehydrogenase-reactive blood vessels in muscle biopsy specimens provides an important clue toward understanding the underlying pathogenetic mechanism in patients with MELAS as well as another approach to the diagnosis of this disorder.

We have defined the genetic defect in a large family first described in one of the earliest reports of suspected mitochondrial myopathy, as the mutation T14709C in the mitochondrial transfer RNA(Glu) (mt-tRNA(Glu)) gene. Extraordinarily, this mutation has attained homoplasmy (100% mutated mt-tRNA(Glu)) on at least three independent occasions in this family and has done so in one individual who remains asymptomatic with no clinical evidence of disease. Heteroplasmy (dual populations of mutated and wild-type mtDNA) usually is regarded as one of the primary diagnostic criteria for pathogenicity and previous reports of the T14709C mutation detail heteroplasmy in a variety of tissues. In contrast, homoplasmy of mt-tRNA mutations generally has been regarded as evidence of a benign nature, with rare exceptions that result in organ-specific phenotypes. Discovering that T14709C, a common and severe mt-tRNA mutation, can attain homoplasmy without symptoms or clinical signs of disease has profound implications for the identification and prevalence of other pathogenic mt-tRNA mutations. Furthermore, variation in phenotype between homoplasmic individuals implies a crucial contribution from the nuclear genetic environment in determining the clinical outcome of mt-tRNA mutations.

We identified two novel heteroplasmic mitochondrial DNA point mutations in the gene encoding the ND5 subunit of complex L a 12770A-->G transition identified in a patient with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and a 13045A-->C transversion in a patient with a MELAS/Leber's hereditary optic neuropathy/Leigh's overlap syndrome. Biochemical analysis of muscle homogenates showed normal or very mildly reduced complex I activity Histochemistry was normal. Our observations add to the evidence that mitochondrial ND5 protein coding gene mutations frequently associate with the MELAS phenotype, and it highlights the role of complex I dysfunction in MELAS.

A cardinal feature of impaired skeletal muscle oxidative metabolism in mitochondrial myopathies is a limited ability to increase the extraction of O(2) from blood relative to the increase in O(2) delivery by the circulation during exercise. We investigated whether aerobic forearm exercise would result in an abnormal increase in venous effluent O(2) in patients with impaired skeletal muscle oxidative phosphorylation attributable to mitochondrial disease. We monitored the partial pressure of O(2) (PO(2)) in cubital venous blood at rest, during handgrip exercise, and during recovery in 13 patients with mitochondrial myopathy and exercise intolerance and in 13 healthy control and 11 patient control subjects. Resting and recovery venous effluent PO(2) were similar in all subjects, but during exercise venous PO(2) paradoxically rose in mitochondrial myopathy patients from 27.2 +/- 4.0mmHg to 38.2 +/- 13.3mmHg, whereas PO(2) fell from 27.2 +/- 4.2mmHg to 24.2 +/- 2.7mmHg in healthy subjects and from 27.4 +/- 9.5mmHg to 22.2 +/- 5.2mmHg in patient controls. The range of elevated venous PO(2) during forearm exercise in mitochondrial myopathy patients (32 to 82mmHg) correlated closely with the severity of oxidative impairment as assessed during cycle exercise. We conclude that measurement of venous PO(2) during aerobic forearm exercise provides an easily performed screening test that sensitively detects impaired O(2) use and accurately assesses the severity of oxidative impairment in patients with mitochondrial myopathy and exercise intolerance.

In a double-blinded, placebo-controlled, crossover study in seven mitochondrial myopathy patients (MM), we investigated whether lowering of lactate with dichloroacetate (DCA) can improve exercise tolerance and oxidative capacity in MM. DCA lowered plasma lactate at rest and during exercise (from 10.5 +/- 2.0 to 5.0 +/- 1.6 mM; p = 0.005) but did not improve maximal work load or VO2 in cycle exercise or phosphorous magnetic resonance spectroscopy (31P-MRS)-assessed indices of muscle oxidative metabolism. This indicates that lactate acidosis is not the primary cause of exercise intolerance in MM.

In this study we present incidence, point prevalence, and mortality figures of mitochondrial encephalomyopathies in a population-based study of children from western Sweden. Through the screening of registers and review of medical records, we identified 32 patients under 16 years of age from the study population who were diagnosed between January 1, 1984, and December 31, 1998. The incidence of mitochondrial encephalomyopathies in preschool children (

Ragged-red fibers (RRFs) are found more frequently in highly oxidative type I fibers than in glycolytic type II fibers in the muscle of many patients with mitochondrial myopathies. Neurotrophin-4 (NT-4), a neuronal signaling molecule, is also expressed in skeletal muscle, predominantly in type I fibers. We found that NT-4 protein and mRNA were present in both type I and type II fibers but were up-regulated in RRFs of patients with mitochondrial encephalomyopathies; it is noteworthy that NT-4 was not up-regulated in muscle fibers from healthy aerobically trained athletes. Thus, NT-4 might represent a member of a new class of candidate molecules involved in the compensatory adjustments of muscle fibers to oxidative dysfunction, and may even play a role as a signaling molecule for mitochondrial proliferation.

We identified a single thymidine insertion at nucleotide position 5537 (T5537i) in the mitochondrial DNA transfer RNA gene for tryptophan in a family in which the proband had a progressive neurological disorder and his brother died in infancy of Leigh syndrome. Muscle biopsy from the proband showed subsarcolemmal proliferation of mitochondria and decreased activities of oxidative metabolism enzymes, in particular complex IV. Complex IV was also severely reduced in autopsy tissues, including heart and brain tissues, from the Leigh syndrome infant. The novel T5537i mutation was very abundant in tissues from the proband and the infant (>92%) and less abundant (range, 42-89%) in blood, hair follicles, and skin fibroblasts from 4 maternal relatives, 3 of whom showed a neuropsychiatric disturbance. The mutation was not found in more than 100 control subjects. The degree of heteroplasmy in blood correlated well with the severity of the clinical presentation, suggesting specific segregation with the disease.

Sporadic progressive external ophthalmoplegia and Kearns-Sayre syndrome are usually associated with single large-scale mitochondrial DNA deletions in muscle. In progressive external ophthalmoplegia with autosomal dominant inheritance, multiple mitochondrial DNA deletions have been reported. We studied several members of a Swedish family with autosomal dominant progressive external ophthalmoplegia and multiple mitochondrial DNA deletions by polymerase chain reaction analysis of single muscle fibers and by in situ hybridization, combined with enzyme histochemical analysis. Muscle fiber segments with deficiency of cytochrome c oxidase, which is partially encoded by mitochondrial DNA, had accumulated mitochondrial DNA with deletions and showed reduced levels of wild-type mitochondrial DNA. The deletions varied between individual muscle fibers. There was one predominant deletion in each cytochrome c oxidase-deficient muscle fiber segment. Sequencing of the deletion breakpoints showed that most but not all of the deletions were flanked by direct repeats. Young, clinically affected individuals of this family without limb muscle symptoms did not show mitochondrial DNA deletions or cytochrome c oxidase-deficient muscle fibers. Our results indicate that a nuclear factor predisposes to the development of somatic multiple mitochondrial DNA deletions. Mitochondrial DNA with multiple different deletions shows clonal expansion, which leads to mitochondrial myopathy with ragged-red fibers and muscle weakness.

In the majority of patients with mitochondrial encephalomyopathies, signs and symptoms appear in the first three decades of life. Here we report on a group of 9 older patients (> 69 years old) with late-onset skeletal myopathy characterized by focal accumulations of deleted mitochondrial DNAs (mtDNAs) and altered muscle energy status, suggestive of a primary mitochondrial disease. The clinical phenotype was somewhat variable. However, all patients shared a common feature of insidious moderate proximal muscle weakness; some also showed fatigability and axial muscle weakness. In situ hybridization analysis demonstrated accumulations of messenger RNAs transcribed from deleted mtDNAs in a relatively large number of muscle fibers in the patient group. These fiber segments appeared as ragged red with the modified Gomori trichrome stain and hyperreactive with a modified succinate dehydrogenase stain. Most were negative for cytochrome c oxidase activity. On transverse sections their mean frequency was 0.69% (trichrome) and 1.97% (succinate dehydrogenase) significantly above control levels. Multiple mtDNA deletions were demonstrated by the polymerase chain reaction in both the patients and an age-matched control group, but not in younger control subjects. Phosphorus 13 magnetic resonance spectroscopy of resting muscle showed a decreased phosphocreatine-inorganic phosphate ratio in the patient group. The myopathy in this group of patients appears to result from mitochondrial dysfunction related to the clonal expansion of different mtDNA deletions in individual fiber segments. While the origin of the mtDNA mutations is not clear, the phenotype seems to represent an exaggerated form of what is observed in the normal aging process.

The use of zidovudine (AZT) for the treatment of acquired immunodeficiency syndrome (AIDS) induces a DNA-depleting mitochondrial myopathy, which is histologically characterized by the presence of muscle fibers with "ragged-red"-like features, red-rimmed or empty cracks, granular degeneration, and rods (AZT fibers). Because dysfunctioning muscle mitochondria may lead to defects of beta-oxidation of fatty acids, we examined the degree of neutral fat accumulation and muscle carnitine levels in the muscle biopsy specimens from 21 patients with AZT-induced myopathic symptoms of varying severity. Six patients with no AZT fibers had normal endomyofibrillar lipid deposits and muscle carnitine levels; 7 patients with fewer than 5 AZT fibers per field had a mild (+) to moderate (++) increase in lipid droplets, and reduced muscle carnitine levels (3 patients); and 8 patients with more than 5 AZT fibers had severe muscle changes, a ++ to marked ( ) increase in lipid droplets, and reduced muscle carnitine levels (6 patients). Serial sections showed lipid globules often within "cracks" or vacuoles of the abnormal muscle fibers. We conclude that the muscle mitochondrial impairment caused by AZT results in (1) accumulation of lipid within the muscle fibers owing to poor utilization of long-chain fatty acids, (2) reduction of muscle carnitine levels probably due to decreased carnitine uptake by the muscle, and (3) depletion of energy stores within the muscle fibers. The findings may have potential therapeutic implications in the treatment of AZT-induced myopathic symptoms using oral carnitine supplementation.

A female patient with mitochondrial myopathy had a mitochondrial DNA mutation at nucleotide pair 3243, commonly seen in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), but unlike MELAS patients, she had no central nervous system symptoms. Muscle weakness, which was most severe when she was 7 years old, improved gradually with age. Comparison of two muscle biopsies obtained at an interval of 12.5 years (7 and 20 years of age, respectively), revealed that the number of ragged-red fibers was markedly decreased and histochemical cytochrome c oxidase activity increased in parallel with the decrease in population of mutant genomes.

Zidovudine can induce a mitochondrial myopathy with ragged-red fibers and partial cytochrome c oxidase deficiency. In an attempt to improve histological assessment of zidovudine myopathy, we evaluated cytochrome c oxidase histochemical reaction in the muscle of 10 patients with biopsy-proven zidovudine myopathy (Group 1), 10 myopathic zidovudine receivers without typical histopathological features of zidovudine myopathy (Group 2), and 10 human immunodeficiency virus (HIV)-infected patients not treated by zidovudine who had an immunohistological profile of HIV-associated myopathy or other neuromuscular disorders (Group 3). Among zidovudine receivers, cytochrome c oxidase deficiency was found in 10 of 10 patients from Group 1 and 7 of 10 from Group 2. No cytochrome c oxidase deficiency was observed in patients not treated by zidovudine. When present, cytochrome c oxidase-negative fibers accounted for 2 to 28% of fibers, and there was no difference for the number of cytochrome c oxidase-negative fibers between Group 1 and Group 2. Most patients with cytochrome c oxidase deficiency that could be evaluated clinically after muscle biopsy improved after withdrawal of zidovudine (5 of 7 in Group 1, 5 of 5 in Group 2). Patients who did not improve had an HIV-associated myopathy concurrently with zidovudine myopathy. We conclude that cytochrome c oxidase reaction may be used as a reliable marker of zidovudine mitochondrial toxicity in HIV-infected patients with muscular symptoms.

We reviewed 10 patients (5 males, 5 females) with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The age of symptom onset ranged from 3 months to 12 years. All had lactic acidosis, multiple stroke-like events with secondary neurological deficits, radiological changes of progressive brain infarction, and muscle biopsy showing ragged-red fibers. In patients with earlier onset of symptoms (< 2 yr), involvement tended to be more diffuse, with failure to thrive and early onset of delayed development. Patients whose symptoms appeared later tended to have focal neurological deficits with migraine-like headache, and a rate of cognitive regression reflecting the rapidity of disease progression. Radiological changes included multiple areas of infarction with initial predilection for parietal occipital areas, progressing to generalized atrophy. Pathological findings in muscle biopsies included type 1 fiber predominance, ragged-red fibers, increased intermyofibrillar lipid deposition, and abnormal mitochondria. Four patients showed mitochondrial DNA tRNA mutation at position 3,243. No difference was noted in clinical, radiological, or pathological findings in patients with and without this mutation, suggesting that multiple sites of point mutation may give rise to mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes.

Two families with a point mutation in mtDNA associated with myoclonic epilepsy and ragged-red fiber disease showed pronounced clinical heterogeneity. The mothers of the two families had adult-onset myopathy with ragged-red fibers, partial deficiency of cytochrome c oxidase, and sensory neuropathy. Members of the first family had variable clinical features of progressive ataxic-myoclonic encephalomyopathy and of the other family, primarily adult-onset myopathy. There was a point mutation from A to G at nucleotide pair 8344 located in the tRNALys gene of the mtDNA of all patients tested, three in Family 1, and the mother of Family 2. This clinical heterogeneity may reflect the effects of varying proportions of mutant and wild-type mtDNA in the different organ systems in each individual.

The Golgi apparatus plays a key role in the posttranslational processing of polypeptides destined for secretion, incorporation into plasma membranes, and fast axoplasmic transport. Dispersion or fragmentation of the Golgi apparatus, experimentally induced by microtubule-disrupting agents, is associated with decreased secretion of immunoglobulins and insulin. The Golgi apparatus is also involved in targeting of lysosomal enzymes and in the endocytosis of certain hormones, receptors, and toxins. There is a paucity of information on this important organelle in human neuropathological conditions. Using an organelle-specific antiserum we have examined by immunocytochemistry the Golgi apparatus of motor neurons in the spinal cord in 4 patients with amyotrophic lateral sclerosis and 1 patient with Werdnig Hoffmann's disease, 1 with infantile neuronal degeneration, 1 with adult-type familial bulbospinal atrophy, 1 with mitochondrial myopathy with cytochrome c oxidase deficiency, 1 with centronuclear myopathy, and 1 with Duchenne's muscular dystrophy, and in 9 age-matched control subjects. In all motor neuronopathies examined and in the patient with mitochondrial myopathy, 20 to 85% of neurons counted had "fragmented" Golgi apparatus. In age-matched control subjects and the other 2 patients with myopathies, 0 to 1.65% of motor neurons had fragmented Golgi apparatus. These findings suggest that the Golgi apparatus of motor neurons is involved in patients with amyotrophic lateral sclerosis and related motor neuron diseases, and perhaps in patients with certain fatal primary myopathies.

A T-to-C transition mutation at nucleotide position 3,250 in the mitochondrial tRNA(Leu)(UUR) gene was present in a family with mitochondrial myopathy. Two of three muscle biopsies examined had complex I (NADH-ubiquinone oxidoreductase) deficiency. Heteroplasmy of wild and mutant mitochondrial DNA was detected by Nae I digestion of the polymerase chain reaction products with a modified primer. This was found in blood or muscle samples or both from all seven members examined. Similar to the 3,243 mutation in most patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), the new mutation site was located in the dihydrouridine loop and embedded in the binding region of mitochondrial transcription termination factor. Elucidation of the effects of this mutation may help clarify the role of mitochondrial tRNAs and transcription termination.

A 9-year-old girl and an 11-year-old boy had ptosis, progressive external ophthalmoplegia, pigmentary retinopathy, and sensorineural hearing loss. The girl had diabetes mellitus and the boy had hypoparathyroidism. Both children also developed recurrent vomiting and cerebral infarcts with lactic acidosis. Muscle biopsy specimens showed ragged-red fibers and Southern analysis demonstrated a distinct heteroplasmic deletion of muscle mitochondrial DNA in each patient but no evidence of the point mutation in the transfer RNALeu(UUR) gene recently identified in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). These 2 children had combined features of Kearns-Sayre syndrome and MELAS, suggesting that mitochondrial DNA deletions occasionally can have pleomorphic clinical expression.

Phosphorus magnetic resonance spectroscopy allows noninvasive measurement of the intracellular phosphate-containing metabolites and intracellular pH in localized volumes of human muscle and brain in vivo. This technique was used to study 8 patients with a mitochondrial cytopathy (myoclonus epilepsy with ragged red fibers). Phosphorus magnetic resonance spectroscopy of resting gastrocnemius muscle demonstrated significantly increased relative intracellular inorganic phosphate concentrations (p less than 0.0005) and decreased phosphocreatine to inorganic phosphate concentration ratios (p less than 0.01) in the patients, although only 3 had myopathic signs or symptoms. We propose, therefore, that phosphorus magnetic resonance spectroscopy of resting skeletal muscle is a useful clinical test in evaluation of progressive myoclonus epilepsy. In contrast to results from muscle, however, the relative phosphate metabolite concentrations and intracellular pH in central volumes of the brains of these patients were normal, despite evidence from our previous positron emission tomography studies suggesting that there is diffuse impairment of cerebral oxidative metabolism.

By using a combination of Southern blot hybridization analysis, polymerase-chain reaction amplification, and direct nucleotide sequencing, we studied deletions of mitochondrial DNA (mtDNA) in several nonfamilial patients with progressive external ophthalmoplegia and Kearns-Sayre syndrome, and in some of their direct relatives. Results suggest that the heteroplasmic mtDNA populations are already present at a very early stage of development, and that there is no direct transmission of mtDNA heteroplasmy by maternal inheritance.

The hypothesis that brain mitochondria are directly affected in several phenotypes associated with disordered oxidative phosphorylation was tested using phosphorus 31 (31P) magnetic resonance spectroscopy. Abnormal phosphorylation potentials in skeletal muscle have been demonstrated by 31P magnetic resonance spectroscopy in patients with mitochondrial cytopathies (heritable disorders of oxidative phosphorylation), but abnormalities of phosphorylation potentials in other organs have not been documented. Several lines of evidence suggest that these mutations may affect mitochondria in nonmuscle tissue. In this study we found that phosphocreatine-to-ATP ratios in brain were significantly reduced and that calculated brain ADP concentrations, phosphorylation potentials, and percentage of maximal rate of ATP synthesis were significantly altered in the 5 patients examined. This study indicates a primary abnormality of mitochondrial function in the brain, even in the absence of clinically evident cerebral dysfunction.