Chronic progressive external ophthalmoplegia in patients with large heteroplasmic mitochondrial DNA deletions: an immunocytochemical study

Summary Immunocytochemical studies with a holocomplex antibody battery in patients with chronic progressive external ophthalmoplegia, with and without large mitochondrial DNA deletions, revealed positive (and often increased) immunoreactivity for all complexes studied in histochemically cytochrome oxidase (COX)-negative areas, suggesting a compensatory upregulation of these components. Similar findings were observed with subunit-specific probes directed against both nuclear- and mitochondrially encoded gene products. Comparison of staining intensities between the different complexes revealed significantly more variability in COX-negative than COX-positive fibres, suggesting disordered stoichiometric control during up-regulation. These differences were confirmed using statistical models. This data challenges the view that COX-negative fibre segments have little or no mitochondrially coded protein translation.Supported by the National Health and Medical Research Council     

A novel mitochondrial DNA deletion producing progressive external ophthalmoplegia associated with multiple sclerosis

We report a previously undescribed 7676 base pair mitochondrial (mt)DNA deletion involving genes of complex I, complex IV subunits 2 and 3 (cytochrome oxidase [Cox] II, III), adenosine triphosphatase 8 and 6, cytochrome b and 8 transfer (t)RNA genes producing myopathy and progressive external ophthalmoplegia (PEO) in a 44-year-old right-handed Caucasian man with features of multiple sclerosis (MS). We performed complete mtDNA sequencing and deletion analysis, spectrophotometric analysis of muscle and platelet respiratory chain activity, measurement of platelet mitochondrial membrane potential with the potentiometric dye JC-1 and magnetic resonance spectroscopy (MRS) and MRI studies of normal-appearing and lesional cerebral tissue. The deletion resulted in significant respiratory chain deficiency in muscle and blood and abnormalities of the platelet mitochondrial membrane potential. However, cerebrospinal fluid analysis, magnetic resonance spectroscopy and MRI features suggested inflammatory central nervous system demyelination rather than a primary respiratory chain disorder. We conclude that this novel mtDNA deletion causing myopathy and PEO is associated with severe muscle and platelet cellular energetic abnormalities. Furthermore, clinical and paraclinical features of multiple sclerosis were found. The potential pathomechanistic interaction between mtDNA variation and multiple sclerosis is reviewed.     

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     

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     

Clonal expansion of mitochondrial DNA with multiple deletions in autosomal dominant progressive external ophthalmoplegia

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 singl emuscle fibers and by in sit 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 mitochondral DNA deletions or cytochorme c oxidase-deficient muscle fibers. Our result indicate that a nuclear factor predisposes to the developement 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.     

Chronic progressive external ophthalmoplegia: A correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies

Deleted mitochondrial DNA (mtDNA) has been shown to coexist with normal mtDNA (heteroplasmy) in muscles from chronic progressive external ophthalmoplegia, including Kearns-Sayre syndrome. In this study, we correlated heteroplasmic mtDNA abnormality with clinical, biochemical and histological findings with the following results: (1) large deletions ranging from 1.8 to 8.8 kb in 22 muscle specimens from 28 patients who had ophthalmoplegia clinically and focal cytochromec oxidase (CCO) deficiency by histochemistry, (2) no difference in clinical and biochemical findings between patients with and without mtDNA deletions, (3) no relationship between the size, site or populations of deleted mtDNA and respiratory chain enzyme activities in muscles, (4) positive correlation between the number of CCO-deficient fibers and the populations of deleted mtDNA, and (5) higher incidence of CCO-negative fibers in patients with deleted mtDNA than in those with no deletion of mtDNA. These results suggest that deleted mtDNA is, at least in part, responsible for focal CCO deficiency as a phenotypic expression and that the investigation on pathogenetic mechanism of focal CCO deficiency may provide a clue to understanding the underlying pathophysiology in this disorder.     

Multiple mitochondrial DNA deletions in a patient with mitochondrial myopathy and cardiomyopathy but no ophthalmoplegia

Deletions of muscle mitochondrial DNA are known in mitochondrial myopathy patients who have chronic progressive external ophthalmoplegia (CPEO). A 41-year-old patient with no apparent family history of this condition suffers from hypertrophic cardiomyopathy, slight muscle atrophy, and weakness of the extremities, but not from CPEO. A muscle biopsy showed the presence of ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. This combination of clinical features in our patient is atypical in mitochondrial myopathy with demonstrable deleted muscle mitochondrial DNA. Pleomorphic clinical expression is suggested. © John Wiley & Sons, Inc.     

线粒体肌病和脑肌病患者骨骼肌细胞线粒体DNA缺失分析

目的为了检测线粒体肌病和脑肌病患者的骨骼肌细胞的线粒体ＤＮＡ的缺失情况。方法从６例原发性线粒体肌病和１例脑肌病患者的骨骼肌活检标本中，提取总ＤＮＡ，以线粒体ＤＮＡ全长为探针进行分子杂交。结果发现１例ＭＥＲＲＦ患者有５ｋｂ的线粒体ＤＮＡ基因缺失，另１例线粒体肌病患者有１５ｋｂ的线粒体ＤＮＡ基因缺失，剂量分析表明缺失型线粒体ＤＮＡ分别占总线粒体ＤＮＡ的１９．３％和１０．７％。结论线粒体ＤＮＡ基因缺失是线粒体疾病的重要病因之一     

Ophthalmoplegia,demyelinating neuropathy,leukoencephalopathy, myopathy,and gastrointestinal dysfunction with multiple deletions of mitochondrial DNA: A mitochondrial multisystem disorder in search of a name

This article describes a 37-year-old woman with progressive external ophthalmoplegia, peripheral neuropathy, and chronic intractable diarrhea. Laboratory studies disclosed lactic acidosis, ragged red fibers lacking cytochrome c oxidase, high-normal muscular mitochondrial enzymes, demyelinating neuropathy, leukoencephalopathy and multiple mitochondrial DNA deletions. This is the fourth patient described with this clinical syndrome, which represents a separate entity among multisystemic mitocnondrial disorders. The patient described here is the first with this syndrome to have multiple mitochondrial DNA deletions. © 1994 John Wiley & Sons, Inc.     

Widespread tissue distribution of multiple mitochondrial DNA deletions in familial mitochondrial myopathy

We used Southern blot analysis and the polymerase chain reaction to analyze the tissue distribution of multiple mitochondrial DNA (mtDNA) deletions in a 45-year-old man with familial mitochondrial myopathy. Southern blots showed two major types of abnormal mtDNA with approximately 4- and 8-kilobase deletions in the skeletal and extraocular muscles. The amount of muscle mtDNA with deletions correlated approximately with the severity of muscle involvement. The polymerase chain reaction showed multiple mtDNA deletions even in clinically asymptomatic tissues, the pattern of which differed with the type of tissue. Nucleotide sequences of several mtDNAs with deletions showed that the deletions were flanked by direct repeats consisting of 3 to 12 nucleotides. Leukocytes from the patient's affected sister and his mother exhibited the same mtDNA deletion pattern. Most of the same deletions were present in leukocytes obtained from the patient's father. © 1994 John Wiley & Sons, Inc.     

Muscle fatigue,lactate, and pyruvate in mitochondrial myopathy with progressive external ophthalmoplegia

We studied muscle fatigue and serum lactate and pyruvate levels in 20 patients with mitochondrial myopathy with progressive external ophthalmoplegia (PEO). Fatigue was assessed in the adductor pollicis muscle (AP) using a low-intensity exercise protocol (20 min). Forces (TFs) and relaxation times of ulnar nerve evoked twitches, compound muscle action potentials (CMAPs), and maximal voluntary contractions (MVCs) were monitored. Serum lactate and pyruvate levels were independently measured at rest and after exercise on a bicycle (15 min, 30 W). Most patients showed abnormal fatigue of the AP with a reduction of TFs and MVCs and normal CMAPs. The reduced TFs were significantly correlated with lactate levels at rest (r = −0.60, P < 0.05) and less so with those after exercise (r = −0.47, P < 0.05). Pyruvate levels revealed a similar correlation although they were widely scattered. We conclude that abnormal fatigue in PEO is metabolic, is localized beyond the muscle fiber membrane, and involves the electromechanical coupling and the contractile apparatus. Serum lactate levels at rest are good predictors of fatigue in PEO. © 1996 John Wiley & Sons, Inc.     

Functional respiratory chain studies in subjects with chronic progressive external ophthalmoplegia and large heteroplasmic mitochondrial DNA deletions.

The functional consequences of large heteroplasmic mtDNA deletions were investigated in a group of 6 patients with chronic progressive external ophthalmoplegia (CPEO) syndromes. State III respiration rates corrected for age were low with site I and II substrates in all cases and cytochrome oxidase activity was depressed. The severity of impairment varied and is consistent with inclusion of a variable percentage of non-functioning mitochondria (with deleted mtDNA) in the pellet. Western blot studies with a holocomplex antibody battery revealed no abnormalities in subunit content of complexes III and IV. A deficiency of several complex I subunits in 3 cases suggests that abnormal nuclear-mitochondrial regulation of complex I assembly may follow large mtDNA deletions.     

TWINKLE gene mutation: report of a French family with an autosomal dominant progressive external ophthalmoplegia and literature review

Background: Multiple mitochondrial DNA (mtDNA) deletions usually have a mendelian inheritance secondary to mutation in nuclear genes. One of these is the Twinkle gene whose mutation is responsible for autosomal dominant progressive external ophthalmoplegia (PEO). The number of reported cases with mainly myopathic symptoms and possible nervous system involvement related to Twinkle gene mutation is limited. We present a new French family of whom two members displayed myopathy and neuropathy associated with PEO, and we perform a clinical review in light of other observations reported in the literature. Methods: The proband, one son and the daughter have been investigated. Southern blot analysis and long‐range PCR assay have been performed from muscle biopsy specimens. Coding exons and flanking intron regions of polymerase gamma (POLG) and DNA helicase (Twinkle) genes were sequenced. Results: Multiple mitochondrial DNA deletions have been found and sequencing of the Twinkle gene showed the change p.R374Q. Conclusion: Two other families from the literature also had the R374Q mutation. Symptoms reported in association with this mutation were myopathy, peripheral neuropathy, dysarthria and/or dysphagia, respiratory insufficiency and parkinsonism. Respiratory insufficiency caused by chest wall weakness was reported in other families with different Twinkle gene mutations, and one might provide exercise intolerance, dysarthria and/or dysphagia as symptoms in favor of the diagnosis. Occurrence of impressive emaciation was a peculiarity in our family.     

POLG1, C10ORF2, and ANT1 mutations are uncommon in sporadic progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

The authors sequenced POLG1, C10ORF2, and ANT1 in 38 sporadic progressive external ophthalmoplegia patients with multiple mitochondrial DNA (mtDNA) deletions. Causative mutations were identified in approximately 10% of cases, with two unrelated individuals harboring a novel premature stop codon mutation (1356T>G). None had a mutation in C10ORF2 or ANT1. In the majority of patients, the primary nuclear genetic defect is likely to affect other unknown genes important for mtDNA maintenance.     

Progressive external ophthalmoplegia characterized by multiple deletions of mitochondrial DNA: unraveling the pathogenesis of human mitochondrial DNA instability and the initiation of a genetic classification

Over the last decade, many sporadic and familial cases have been reported with multiple deletions of mitochondrial DNA (mtDNA) in postmitotic tissues. Most patients suffer from progressive external ophthalmoplegia (PEO) and may have a nuclear gene defect that predisposes to the accumulation of mtDNA deletions. Recently, positional cloning has led to the discovery of mutations in four such nuclear genes. Some mutations are dominant and others recessive. In all autosomal mutations, defective mtDNA replication and/or repair are probably responsible for the generation of secondary mtDNA deletions. There are also data suggestive of a prominent pathogenic role for disturbed nucleotide metabolism. We here present a tentative genotype-phenotype correlation. Since clinical presentations are heterogeneous and overlap with different previously described clinical syndromes, we advocate the use of a genetic, instead of a clinical, classification of disorders with multiple mtDNA deletions.     

Mitochondrial myopathy and ophthalmoplegia in a sporadic patient with the 5698G-->A mitochondrial DNA mutation

We describe a second patient carrying the 5698G→A transition in the mitochondrial DNA gene encoding tRNA^Asn, who has an apparently isolated mitochondrial myopathy with chronic progressive external ophthalmoplegia. A muscle biopsy showed the presence of ragged-red and COX-negative fibres. Analysis of the mutation load on single muscle fibres showed significant segregation of the 5698G→A with COX-depleted fibres. These results indicate that the 5698G→A is pathogenic.     

A novel D104G mutation in the adenine nucleotide translocator 1 gene in autosomal dominant progressive external ophthalmoplegia patients with mitochondrial DNA with multiple deletions

Autosomal dominant progressive external ophthalmoplegia is a mitochondrial disorder characterized by multiple large deletions of mitochondrial DNA. A recent study showed pathogenic heterozygous missense mutations in the heart/skeletal muscle isoform of the adenine nucleotide translocator 1 gene in autosomal dominant progressive external ophthalmoplegia patients. In one Japanese autosomal dominant progressive external ophthalmoplegia family, we found a novel A-to-G heterozygous mutation at nucleotide 311 of the adenine nucleotide translocator 1 gene, which segregated with affected individuals and could not be detected in the genomic DNA sequence of 120 normal controls. This mutation converted a highly conserved aspartic acid into a glycine at codon 104. Polymerase chain reaction analysis of single muscle fibers showed the presence of one type of deletion in each fiber, suggesting clonal expansion of mitochondrial DNA with deletions. These findings support the pathogenesis of the adenine nucleotide translocator 1 gene mutation in human disease.     

Increase of mitochondria in vasa nervorum of cases with mitochondrial myopathy,Kearns-Sayre syndrome,progressive externaI ophthalmoplegia and MEWS*

Previous studies on patients with mitochondrial myopathy, encephalopathy. lactic acidosis, and stroke-like episodes (MELAS) revealed accumulations of mitochondria in endothelial cells. smooth muscle cells of pial arterioles, and small intracerebral arteries up to 250μm in diameter; in pericytes of capillaries, endothelial cells, and smooth muscle cells of small blood vessels in skeletal muscle; and according to preliminary results also in endothelial and smooth muscle cells of capillaries and arterioles in sural nerves. These mitochondria do not show the prominent paracrystalline inclusions which are seen in striated muscle fibres and led to the identification of this group of disorders. To corroborate our preliminary findings in peripheral nerves, additional cases have been evaluated morphometrically by electron microscopy including cases in which rnitochondrial DNA (mtDNA) mutations have been identified. In fact, an increase of the mean number and an enlargement of the mean cross-sectional area of mitochondria was noted in endothelial and smooth muscle cells of endoneurial and epineurial arterioles, and in endothelial cells and in pericytes of capillaries in sural nerves of the 20 cases with mitochondrial disorders studied. This increase was statistically significant compared to the control group. However, due to heteroplasmia, which is a common feature in mitochondrial disorders, and because of the limited number of measurable blood vessels and cases, no significant differences could be detected between the various types of mitochondrial diseases which were characterized by dflerent point mutations or deletions of mtDNA. Our findings suggest that the mitochondria play a significant role in the pathogenesis not only of myopathic and encephalopathic symptoms, but also in the pathogenesis of peripheral neuropathy which appears to be regularly associated with mitochondrial disorders.     

The 8,344 mutation in mitochondrial DNA: A comparison between the proportion of mutant DNA and clinico-pathologic findings

Ten patients, two men and eight women with mitochondrial encephalomyopathy, had an A-G mutation at nucleotide pair 8,344 in the mitochondrial DNA, the most common genetic defect in myoclonus epilepsy with ragged-red fibers (MERRF). Eight patients had the clinical and pathologic characteristics of MERRF including myoclonus, seizures, cerebellar ataxia and myopathy with ragged-red fibers. Two patients had atypical symptoms such as early onset of fatal cardiac failure and late onset of rapid mental deterioration, respectively. The striking feature in our patients with the 8,344 mutation was that four of 10 patients had cardiac involvement and two developed progressive heart failure. In the typical MERRF patients, the proportion of mutant mitochondrial DNA in their skeletal muscles, quantified by a single strand conformation polymorphism analysis, was above 85%. However, there was no significant correlation between clinical severity, histopathological findings and the proportion of mutant mtDNA in muscle biopsy samples, suggesting that non-ragged-red fibers play an important role in the phenotypic expression of the mutants.