Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction

We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1374–1381, 1998     

Toxic myopathy with multiple deletions in mitochondrial DNA associated with long‐term use of oral anti‐viral drugs for hepatitis B: A case study

Oral nucleoside analogs (NAs) reduce hepatitis B virus (HBV) replication by inhibiting HBV DNA polymerase. However, NAs can also affect human mitochondrial DNA (mtDNA) polymerase, which can lead to mtDNA depletion (quantitative abnormality). Indeed, several mitochondrial myopathy cases have been reported in which a reduced mtDNA copy number was induced by oral NAs for hepatitis B. Herein, we report a case of toxic myopathy with multiple mtDNA deletions (qualitative abnormality) associated with long‐term use of NAs for hepatitis B. A 68‐year‐old woman, who underwent long‐term treatment with lamivudine and adefovir for chronic hepatitis B, developed proximal muscle weakness in the four extremities. Neurological examination showed mild proximal muscle weakness and atrophy in the four extremities. Upon admission to our hospital, her blood lactate/pyruvate ratio during an aerobic exercise test was elevated. Myogenic patterns were observed in lower limb muscles on electromyographic examination. Muscle magnetic resonance imaging revealed diffuse atrophy of proximal muscles in the four extremities with no signal changes. A biopsy from the biceps brachii muscle showed an abnormally large variation in fiber size, scattered muscle fibers with decreased cytochrome c oxidase activity, and ragged‐red fibers. Analysis of mtDNA from skeletal muscle revealed no decrease in copy number but increased incidence of multiple deletions, including a deletion of 4977 base pairs (known as the common deletion) reflecting oxidative stress‐induced mtDNA damage. This case study indicates that long‐term oral antiviral therapy for hepatitis B can induce chronic oxidative damage to mtDNA resulting in qualitative mtDNA abnormalities and toxic myopathy.     

Sensory ataxic neuropathy with ophthalmoparesis caused by POLG mutations

Mutations in POLG gene are responsible for a wide spectrum of clinical disorders with altered mitochondrial DNA (mtDNA) integrity, including mtDNA multiple deletions and depletion. Sensory ataxic neuropathy with ophthalmoparesis (SANDO) caused by mutations in POLG gene, fulfilling the clinical triad of sensory ataxic neuropathy, dysarthria and/or dysphagia and ophthalmoparesis, has described in a few reports. Here we described five cases of adult onset autosomal recessive sensory ataxic neuropathy with ophthalmoplegia. All patients had ataxia, neuropathy, myopathy, and progressive external ophthalmoplegia (PEO). The muscle pathology revealed ragged-red and cytochrome c oxidase (COX) negative fibers in three patients. However, deficiencies in the activities of mitochondrial respiratory chain enzyme complexes were not detected in any of the patients' muscle samples. Multiple deletions of mtDNA were detected in blood and muscle specimens but mtDNA depletion was not found. Due to these diagnostic difficulties, POLG-related syndromes are definitively diagnosed based on the presence of deleterious mutations in the POLG gene.     

Defects of the mitochondrial respiratory chain complexes in three pediatric cases with hypotonia and cardiac involvement.

Three children displaying hypotonia, cardiac involvement and defects of the mitochondrial respiratory chain complexes are reported. The first case showed severe neonatal hypotonia, failure to thrive, hepatomegaly, dilation of the right cardiac cavities, profound lactic acidosis and amino aciduria. The boy died at the age of 7 weeks. In the second case hypotonia, severe cardiomyopathy, cyclic neutropenia, lactic acidosis and 3-methylglutaconic aciduria occurred. The boy died at the age of 27 months. The third case presented at the age of 16 months as an acute hypokinetic hypertrophic cardiomyopathy with transient hypotonia and mild lactic acidosis. Spontaneous clinical remission occurred. In all cases muscle biopsy was performed. Morphological studies failed to show ragged-red fibers but there was lipid storage myopathy and decreased cytochrome c oxidase activity. Biochemical studies confirmed the cytochrome c oxidase deficiency in muscle in all cases. It was associated with complex I III deficiency in case 1 and with severe deficits of all respiratory chain complexes in case 2. Post-mortem studies in case 1 indicated that complex IV was reduced in the liver but not in the heart and quantitative analysis of mtDNA revealed a depletion in muscle. Cases 1 and 2 shared some clinical features with fatal infantile myopathy associated with cytochrome c oxidase deficiency, while case 3 displayed a very unusual clinical presentation. The histochemical enzyme reaction of cytochrome c oxidase is useful for the diagnosis of mitochondrial myopathy because ragged-red fibers may be lacking. Finally, biochemical measurement of the different mitochondrial respiratory chain complexes is required because multiple defects are frequent and occasionally related to mtDNA depletion.     

Cytochrome c oxidase deficiency in the muscle of patients with zidovudine myopathy is segmental and affects both mitochondrial DNA- and nuclear DNA-encoded subunits

Zidovudine (AZT) can induce a mitochondrial disorder associated with mitochondrial (mt) DNA depletion affecting skeletal muscle, heart, and liver. Zidovudine myopathy is characterized by ragged-red fibers and partial cytochrome c oxidase (COX) deficiency. We evaluated at a single fiber level the expression of COX II (mtDNA-encoded) and COX IV (nuclear DNA-encoded) subunits in 12 HIV-infected patients with zidovudine myopathy. We also evaluated COX activity on longitudinal muscle sections in one patient. In all patients, evaluation of the expression of COX II and COX IV subunits showed focal deficiency. All fibers negative for COX II or COX IV were negative by COX histochemistry; 32–92% (median 61%) of COX-negative fibers were negative for COX II antigens, and 7–58% (median 28%) were negative for COX IV antigens. One hundred and thirty-nine of 317 COX-negative fibers 139 (43.8%) were selectively negative for COX II; 28 of 317 (8.8%) COX-negative fibers were selectively negative for COX IV. A study of longitudinal distribution of COX activity demonstrated that COX deficiency was segmental with blurred borders, as previously observed in patients with myoclonus epilepsy with ragged-red fibers. We conclude that proteins encoded by mtDNA are predominantly, but not exclusively, involved in zidovudine myopathy. Our results confirm the value of single muscle fiber evaluation in the assessment of mitochondrial abnormalities related to zidovudine. Received: 8 July 1999 / Revised: 6 October 1999 / Accepted: 12 October 1999     

Mitochondrial DNA copy number threshold in mtDNA depletion myopathy

The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome.     

Progressive depletion of mtDNA in mitochondrial myopathy

The authors studied seven patients with mitochondrial DNA (mtDNA) myopathy. Over time, there was a progressive depletion of mtDNA, which preferentially affected wild-type mitochondrial genomes. This suggests that loss of wild-type mtDNA is a major feature of mtDNA myopathy, and preventing wild-type mtDNA depletion has treatment implications.     

Selective muscle fiber loss and molecular compensation in mitochondrial myopathy due to TK2 deficiency

A 12-year-old patient with mitochondrial DNA (mtDNA) depletion syndrome due to TK2 gene mutations has been evaluated serially over the last 10 years. We observed progressive muscle atrophy with selective loss of type 2 muscle fibers and, despite severe depletion of mtDNA, normal activities of respiratory chain (RC) complexes and levels of COX II mitochondrial protein in the remaining muscle fibers. These results indicate that compensatory mechanisms account for the slow progression of the disease. Identification of factors that ameliorate mtDNA depletion may reveal new therapeutic targets for these devastating disorders.     

Association of myopathy with large-scale mitochondrial dna duplications and deletions: Which is pathogenic?

We identified large-scale heteroplasmic mitochondrial DNA (mtDNA) rearrangements in a 50–year-old woman with an adult-onset progressive myopathy. The predominant mtDNA abnormality was a 21.2–kb duplicated molecule. In addition, a small population of the corresponding partially deleted 4.6–kb molecule was detected. Skeletal muscle histology revealed fibers that were negative for cytochrome c oxidase (COX) activity and had reduced mtDNA-encoded COX subunits. By single-fiber polymerase chain reaction analysis, COX-negative fibers contained a low number of wild-type or duplicated mtDNA molecules (ie, nondeleted). In situ hybridization demonstrated that the abnormal fibers contained increased amounts of mtDNA compared with normal fibers and that most of the genomes were deleted. We concluded that deleted mtDNA molecules were primarily responsible for the phenotype in this patient.     

Single muscle fiber analysis of mitochondrial myopathy,encephalopathy, lactic acidosis,and stroke-like episodes (MELAS)

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.     

Effects of short-term zidovudine exposure on mitochondrial DNA content and succinate dehydrogenase activity of rat skeletal muscle cells

Long-term use of zidovudine (AZT) may cause mitochondrial abnormalities in various tissues, including a toxic myopathy in AIDS patients associated with mitochondrial DNA (mtDNA) depletion. In the present study, we examine the short-term (48 h) effect of AZT (10, 30 and 100 microg/ml) on the mitochondrial succinate dehydrogenase (SDH) and mtDNA content of rat cultured skeletal muscle. The effect of AZT on cytochrome c oxidase (COX) enzyme was also analyzed. The histochemical quantitative analysis of SDH showed that AZT 10, 30 and 100 microg/ml increased by 7%, 9% and 13% the mitochondrial content. Conversely, treatment of rat cultures with 10 to 100 microg/ml AZT reduced the mtDNA content by 23% to 66%, when compared to control values. The spontaneous contraction and the COX activity were not modified by up to 100 microg/ml AZT. Taken together, these results show that short-term treatment with AZT can induce severe myotoxicity that involves mitochondrial proliferation and mtDNA depletion in the rat cultured myotubes. Our results also indicate that rat cultured skeletal muscle might be a valuable in vitro assay to evaluate the effect of drugs on mitochondria to predict their potential to induce mitochondrial toxicity.     

Oculopharyngeal somatic myopathy in a patient with a novel large-scale 3,399 bp deletion and a homoplasmic T5814C transition of the mitochondrial DNA

We report a 65-year-old woman with a sporadic form of progressive oculopharyngeal somatic myopathy due to a novel large-scale 3,399 base pair (bp) deletion of the mitochondrial DNA (mtDNA) and co-occurrence of a homoplasmic T5814C transition. The onset of myopathy began from chronic progressive external ophthalmoplegia (CPEO) at age of 20 years. Bulbar weakness, neck and proximal limb paralysis, slowly progressed to eventual respiratory failure. The plasma levels of pyruvate (1.5 mg/dL) and lactate (20.2 mg/dL) were elevated. Muscle biopsy showed decreased enzymatic activity of cytochrome c oxidase, but no ragged-red fibers. Electron microscopy showed "parking-lot" paracrystalline inclusions in the enlarged mitochondria suggestive for mitochondrial myopathy. Sequencing of the whole mitochondrial genome of the patient's muscle and leukocytes showed 3,399 bp deletion of the mtDNA from nucleotide position 8,024 to 11,423 and a homoplasmic thymidine to cytosine transition at nucleotide position 5,814 of the tRNA(Cys) gene of mtDNA (T5814C). T5814C was absent in the white blood cells of the patient's daughter and in 205 normal controls. We conclude that a large-scale deletion may coexist with T5814C transition in patients with sporadic form of mitochondrial cytopathy manifested by slowly progressive oculopharyngeal somatic myopathy.     

Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations

The mitochondrial DNA (mtDNA) depletion syndrome is a genetically heterogeneous group of diseases caused by nuclear gene mutations and secondary reduction in mtDNA copy number. We describe a patient with progressive muscle weakness and increased creatine kinase and lactate levels. Muscle weakness was first noted at age 1.5 years and he died of respiratory failure and bronchopneumonia at age 3.5 years. The muscle biopsy showed dystrophic features with ragged red fibers and numerous cytochrome c oxidase (COX)-negative fibers. qPCR analysis demonstrated depletion of mtDNA and sequence analysis of the mitochondrial thymidine kinase 2 (TK2) gene revealed two novel heterozygous variants, c.332C > T, p.(T111I) and c.156 + 5G > C. Quantitative analysis of mtDNA in single muscle fibers demonstrated that COX-deficient fibers showed more pronounced depletion of mtDNA when compared with fibers with residual COX activity (P < 0.01, n = 25). There was no evidence of manifestations from other organs than skeletal muscle although there was an apparent reduction of mtDNA copy number also in liver. The patient showed a pronounced, albeit transient, improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake, suggesting that nutritional modulation may be a therapeutic option in myopathic mtDNA depletion syndrome.     

Multiple mitochondrial DNA deletions in hereditary inclusion body myopathy

We have recently described an autosomal dominant hereditary inclusion body myopathy (h-IBM). Clinically it is is characterized by congenital joint contractures and slowly progressive, proximal muscle weakness and ophthalmoplegia. There is deterioration of muscle function between 30 and 50 years of age. While young patients show minor pathological changes in muscle, the middle-aged and old patients show rimmed vacuoles and inclusions of filaments measuring 15–18 nm in diameter. Except for the absence of significant inflammation the histopathology is similar to that found in sporadic inclusion body myositis (s-IBM). In s-IBM mitochondrial alterations including cytochrome c oxidase (COX) -deficient muscle fibers are common. These are due to multiple mitochondrial DNA (mtDNA) deletions. In this study we investigated the occurrence of mitochondrial alterations in autosomal dominant h-IBM. Young affected individuals showed no mitochondrial changes but three patients aged 38, 51 and 59 years, respectively, showed ragged red fibers and COX-deficient muscle fibers. Polymerase chain reaction analysis showed multiple mtDNA deletions. By in situ hybridization clonal expansions of mtDNA with deletions were demonstrated in COX-deficient muscle fibers. Most of the analyzed deletion breakpoints showed nucleotide repeats flanking the deletions. The results show that COX-deficient muscle fibers and somatic mtDNA deletions are present in this family with h-IBM. The same factors may be involved in the development of mtDNA deletions in s-IBM and this family with h-IBM. Received: 13 July 1999 / Revised: 6 October 1999 · Accepted: 12 October 1999     

Functional analysis of a novel POLγA mutation associated with a severe perinatal mitochondrial encephalomyopathy

Mutations in the mitochondrial DNA polymerase gamma catalytic subunit (POLγA) compromise the stability of mitochondrial DNA (mtDNA) by leading to mutations, deletions and depletions in mtDNA. Patients with mutations in POLγA often differ remarkably in disease severity and age of onset. In this work we have studied the functional consequence of POLγA mutations in a patient with an uncommon and a very severe disease phenotype characterized by prenatal onset with intrauterine growth restriction, lactic acidosis from birth, encephalopathy, hepatopathy, myopathy, and early death. Muscle biopsy identified scattered COX-deficient muscle fibers, respiratory chain dysfunction and mtDNA depletion. We identified a novel POLγA mutation (p.His1134Tyr) in trans with the previously identified p.Thr251Ile/Pro587Leu double mutant. Biochemical characterization of the purified recombinant POLγA variants showed that the p.His1134Tyr mutation caused severe polymerase dysfunction. The p.Thr251Ile/Pro587Leu mutation caused reduced polymerase function in conditions of low dNTP concentration that mimic postmitotic tissues. Critically, when p.His1134Tyr and p.Thr251Ile/Pro587Leu were combined under these conditions, mtDNA replication was severely diminished and featured prominent stalling. Our data provide a molecular explanation for the patient´s mtDNA depletion and clinical features, particularly in tissues such as brain and muscle that have low dNTP concentration.     

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.     

Mitochondrial DNA depletion in sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is a late onset disorder of unkown aetiology. Mitochondrial changes such as cytochrome oxidase deficient fibres are a well recognised feature and mitochondrial DNA (mtDNA) deletions have also been reported, but not consistently. Since mtDNA deletions are not present in all cases, we investigated whether other types of mtDNA abnormality were responsible for the mitochondrial changes. We studied 9 patients with sIBM. To control for fibre loss or replacement with inflammatory cells, we compared sIBM patients with necrotising myopathy (n?=?4) as well as with healthy controls. Qualitative anlysis for mtDNA deletions and quantitative measurement of mtDNA copy number showed that muscle from patients with sIBM contained on average 67% less mtDNA than healthy controls (P?=?0.001). The level of mtDNA was also significantly depleted in sIBM when compared to necrotising myopathy. No significant difference in copy number was seen in patients with necrotising myopathy compared to controls. Deletions of mtDNA were present in 4 patients with sIBM, but not all. Our findings suggest that mtDNA depletion is a more consistent finding in sIBM, and one that may be implicated in the pathogenesis of the disease.     

A large-scale deletion of mitochondrial DNA in a case with pure mitochondrial myopathy and neuropathy

Here we report the findings from a male patient with myopathy and neuropathy, who has a large-scale deletion of the mitochondrial genome at nucleotides 6570–14150. In the patient’s history, muscle cramps with intermittent weakness and polyneuropathy with disturbed micturition were the predominant symptoms. Morphological examination of a muscle biopsy sample revealed numerous ragged red fibers and prominent paracrystalline intramitochondrial inclusions. The sural nerve biopsy sample disclosed a chronically progressive neuropathy, predominantly axonal in type with a minor demyelinating component. In previous studies the clinical symptoms mentioned above have been related to point mutations at various positions in the mitochondrial DNA (mtDNA). The present study is the first to describe a large (8 kb) deletion of the mtDNA which had apparently caused myopathy and polyneuropathy without encephalopathy. Received: 27 July 1995 / Revised, accepted: 4 December 1995     

Localization of mitochondrial DNA in normal and pathological muscle using immunological probes: a new approach to the study of mitochondrial myopathies.

We have studied the usefulness of anti-DNA antibodies to detect ragged-red fibers (RRF) in muscle biopsies from patients with mitochondrial myopathies. We have found that these antibodies are excellent probes for the localization of mitochondrial DNA (mtDNA) in RRF, and for the diagnosis of depletion of mtDNA in a newly described group of fatal myopathies of infancy.