Molecular analysis of the muscle pathology associated with mitochondrial DNA deletions.

Large-scale deletions of mitochondrial DNA (mtDNA) are associated with a subgroup of mitochondrial encephalomyopathies. We studied seven patients with Kearns-Sayre syndrome or isolated ocular myopathy who harboured a sub-population of partially-deleted mitochondrial genomes in skeletal muscle. Variable cytochrome c oxidase (COX) deficiencies and reduction of mitochondrially-encoded polypeptides were found in affected muscle fibres, but while many COX-deficient fibres had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. Our results suggest that a specific ratio between mutant and wild-type mitochondrial genomes is the most important determinant of a focal respiratory chain deficiency, even though absolute copy numbers may vary widely.

Efficient and specific amplification of identified partial duplications of human mitochondrial DNA by long PCR.

The use of PCR to identify mtDNAs containing a partial duplication (dup-mtDNA) in the presence of a heteroplasmic population of mtDNAs harboring the corresponding deletion (delta-mtDNA) leads to ambiguous results: when the primers anneal in the duplicated portion of the dup-mtDNA (which is also the non-deleted region of the delta-mtDNA) and point towards the abnormal breakpoint junction, both templates are amplified indiscriminately. We have developed two different 'long PCR' approaches to amplify dup-mtDNA even in the presence of delta-mtDNA and wild-type mtDNA (wt-mtDNA). Long PCR with two primers annealing in the non-duplicated region in dup-mtDNA (equivalent to the region missing in delta-mtDNA) and whose 3' ends pointed towards the duplicated area amplified both dup-mtDNA and coexisting wt-mtDNA. We observed, however, a preferential amplification of the wt-mtDNA over that of the longer dup-mtDNAs. This problem was partly overcome by modifying the PCR conditions (extension time, amplicon length, amount of template). In order to overcome the problem of co-amplification, we developed a novel PCR method to amplify specifically dup-mtDNAs. A forward primer annealing across the breakpoint junction was used in conjunction with a backward primer annealing in the non-duplicated region. For those duplication breakpoints flanked by direct repeats, we designed a 'breakpoint loop-out' primer whose sequence omitted the repeated region, in order to avoid the annealing of this primer to wt-mtDNA. This second approach was able to amplify specifically and efficiently the dup-mtDNA in all samples analyzed, irrespective of the size of the duplication or its proportion in the samples.

A new mitochondrial point mutation in the transfer RNA(Leu) gene in a patient with a clinical phenotype resembling Kearns-Sayre syndrome.

OBJECTIVE: To report on the molecular identification of a novel heteroplasmic G-to-A transition at mitochondrial DNA position 3249 in transfer RNA(Leu) gene in a patient with a clinical phenotype resembling Kearns-Sayre syndrome. PATIENT AND METHODS: A 34-year-old patient had been suffering for more than 10 years from progressive visual failure, neurosensorial hearing loss, exercise intolerance, muscle weakness, paresthesia in the lower limbs, and difficulties swallowing. Clinical examination revealed generalized muscle wasting, ptosis, external ophthalmoplegia, and ataxia. Ophthalmologic examination showed dystrophic features in the cornea and retina. In skeletal muscle, morphologic and biochemical studies of the respiratory chain complexes were performed. Polymerase chain reaction, single-strand conformation polymorphism, and direct sequencing were used to screen for mutations in the 22 mitochondrial transfer RNA genes. RESULTS: In skeletal muscle, a significantly decreased catalytic activity of complex I was detected by spectrophotometric analysis and numerous cytochrome c oxidase-negative ragged-red fibers were seen on morphologic examination. A G-to-A substitution 3249 (G3249A) mutation was found in the transfer RNA(Leu) gene of the patient and mutant mitochondrial DNA represented 85% of the total in skeletal muscle but only 45% in leukocytes. The mutation was shown to be present in a small fraction in leukocytes from the unaffected mother and to be absent in leukocytes from the healthy sister. CONCLUSIONS: A causal relationship between a heteroplasmic G3249A transfer RNA(Leu) mutation in a patient suffering from progressive external ophthalmoplegia, retinal dystrophy, ataxia, neurosensorial hearing loss, and muscle wasting is postulated. To our knowledge, the G3249A mutation has never previously been described and was not detected in control subjects.

Detection of deleted mitochondrial DNA in Kearns-Sayre syndrome using laser capture microdissection.

A novel 4949-base pair mitochondrial DNA (mtDNA) deletion was detected in various tissues in a postmortem study of a patient with Kearns-Sayre syndrome (KSS). Deleted mtDNA levels were higher in skeletal muscle and brain and lower in kidney, working myocardium, and endocrine tissues (thyroid, parathyroids, pancreas, and adrenal glands). The distribution of the deletion in skeletal muscle and conducting myocardium was analyzed by means of laser capture microdissection (LCM). In skeletal muscle, the abundance of deleted mtDNA was slightly higher in cytochrome c oxidase (COX)-negative fibers (70%) than in COX-positive fibers (64%), whereas in the conducting myocardium it was lower in the atrioventricular node (9%) than in the sinus node and bundle of His (30% and 32%, respectively). In this study, LCM proved to be a reliable technique for a more accurate assessment of genotype/phenotype correlation when investigating mtDNA-related disorders.

Coenzyme Q in serum and muscle of 5 patients with Kearns-Sayre syndrome and 12 patients with ophthalmoplegia plus.

Coenzyme Q10 (CoQ) was measured in serum and muscle of 17 patients with ophthalmoplegia plus (including 5 patients with Kearns-Sayre syndrome), in muscle of 9 patients with neurogenic atrophies, 5 patients with myositis, and 5 patients with progressive muscular dystrophies (including 1 patient with oculopharyngeal dystrophy), and in serum and muscle of normal controls. CoQ was markedly decreased in serum and muscle of 1 patient with Kearns-Sayre syndrome and treatment with CoQ resulted in a significant clinical improvement. The other 4 patients with Kearns-Sayre syndrome and the patients with ophthalmoplegia plus exhibited normal concentrations of CoQ in serum and muscle. CoQ levels in muscle of patients with progressive muscular dystrophies, myositis or neurogenic atrophies were within the normal range. Concentrations of CoQ in serum and muscle of normal controls were independent of age and showed no sex difference. The data indicate that CoQ deficiency might be the specific cause of mitochondrial encephalomyopathy in 1 patient but it was not the underlying defect common to all cases with Kearns-Sayre syndrome and ophthalmoplegia plus, although the possibility of a focal CoQ deficiency affecting only single muscle fibres cannot be excluded.

Mitochondrial myopathies: divergences of genetic deletions, biochemical defects and the clinical syndromes.

Genomic Southern analysis of muscle mitochondrial (mt) DNA from 16 patients with mitochondrial myopathies was performed; 14 of 16 patients had chronic progressive external ophthalmoplegia (CPEO), while 2 patients had mitochondrial myopathies without CPEO. Eleven patients with CPEO, including 5 who exhibited the complete triad of symptoms characteristic of the Kearns-Sayre syndrome (i.e. CPEO, retinal degeneration and heart block) had heteroplasmic mtDNA with deletions ranging from 2.0 to 8.0 kb in length. There was no clear-cut correlation between the size and location of the deletions, on the one hand, and the histochemical and biochemical data or the severity of the disease, on the other.

Decreased activities in mitochondrial inner membrane electron transport system in muscle from patients with Kearns-Sayre syndrome.

The present study shows biochemical data on skeletal muscle from 5 patients with Kearns-Sayre syndrome (KSS). Enzyme activities per muscle wet weight in the electron transport system of inner mitochondrial membrane were not significantly different in KSS from those in normal subjects except one patient with long duration of symptoms. On the other hand, mitochondrial contents were increased and enzyme activities per mitochondrial protein in the electron transport system were markedly decreased in the muscle of all cases. These results suggest that the decreased enzyme activity of the mitochondrial electron transport system in each mitochondrion may result in a compensatory increase in mitochondrial contents in the muscle of KSS.

Pyruvate dehydrogenase complex deficiency and altered respiratory chain function in a patient with Kearns-Sayre/MELAS overlap syndrome and A3243G mtDNA mutation.

Combined alteration of the pyruvate dehydrogenase complex and respiratory chain function is described in a 21 year-old male patient with overlapping MELAS (mitochondrial encephalomyopathy, lactic acidosis, and 'stroke-like' episodes) and Kearns-Sayre syndrome. Progressive external ophthalmoplegia, pigmentary retinopathy and right bundle branch block were present when he experienced the first 'stroke-like' episode at 18 years old. The A>G tRNALeu(UUR) point mutation at nucleotide 3243 of the mitochondrial DNA was predominant in muscle tissue (79%) and present, but at lower levels in fibroblasts (49%) and blood cells (37%). Biochemical analysis revealed diminished activities of pyruvate dehydrogenase (23%) and respiratory chain complexes I and IV (57%, respectively) in muscle, but normal activities in the fibroblasts. Immunochemical studies of the muscular pyruvate dehydrogenase components showed normal content of E1alpha, E1beta and E2 protein. Molecular screening of the E1alpha gene did not indicate a nuclear mutation. These observations suggest that mitochondrial DNA defects may be associated with altered nuclear encoded enzymes which are actively imported into mitochondria and constitute components of the mitochondrial matrix. Biochemical workup of mitochondrial disorders should not be restricted to the respiratory chain even if mitochondrial DNA mutations are present.

Mitochondrial DNA deletions and cytochrome c oxidase deficiency in muscle fibres.

We have studied cytochrome c oxidase (COX) deficient muscle fibre segments in 6 patients with mitochondrial myopathy and deletions of mitochondrial DNA (mtDNA). The distribution of transcripts of normal and mutated mtDNA in skeletal muscle sections was studied by in situ hybridization. The results were compared with the enzyme histochemical activity of COX and the immunohistochemical distribution of mtDNA encoded and nuclear DNA encoded subunits of COX. In all cases a proportion of the muscle fibres (less than 1-30% of the fibres in cross-sections) had low COX activity and high activity of succinate dehydrogenase (COX deficient muscle fibres). Transcripts of normal and deleted mtDNA showed the same distribution within the tissue as the corresponding mtDNA, indicating that the deleted mtDNA is transcribed. The COX deficient muscle fibres showed accumulation of transcripts of deleted mtDNA, which had a similar distribution as the accumulated mitochondria within these fibres. With few exceptions, there was a low level of transcripts of normal mtDNA in these COX deficient fibres. Immunohistochemical analysis revealed low levels of immunoreactive material using antiserum to the mtDNA encoded subunits II/III as well as the nuclear DNA encoded subunit IV of COX in all COX deficient muscle fibres. The fraction of deleted mtDNA in muscle ranged from 43 to 87%. There was no correlation between the proportion of COX deficient muscle fibres and the fraction of deleted mtDNA. In 2 cases the deletion did not involve any COX gene. One of these cases had 87% deleted mtDNA but less than 1% COX deficient muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic progressive external ophthalmoplegia: a correlative study of quantitative molecular data and histochemical and biochemical profile.

We studied muscle biopsies of 5 patients with Kearns-Sayre syndrome and 3 patients with chronic progressive external ophthalmoplegia all with the common deletion. Steady state levels of normal and deleted mitochondrial DNA (mtDNA) measured in each patient by quantitative PCR were correlated with histochemical and biochemical features. We found that (1) normal mtDNA levels were higher in many patients than in controls; (2) as levels of deleted mtDNA increased, so did levels of normal mtDNA; (3) cytochrome c oxidase (COX) activity and the percentage of COX negative fibers were both related to the levels of deleted mtDNA; and (4) as percentage of ragged red fibers increased, so did levels of total, deleted and normal mtDNA. The quantity of deleted mtDNA plays a key role in determining the severity of COX deficiency, which is responsible for the overaccumulation of mitochondria in muscle.

Oxidative damage to skeletal muscle DNA from patients with mitochondrial encephalomyopathies.

To estimate the oxidative damage to skeletal muscle DNA in mitochondrial encephalomyopathies, we studied the amount of 8-hydroxy-deoxyguanosine (8-OH-dG) and the localization of superoxide dismutase (SOD) in the skeletal muscles of patients with progressive external ophthalmoplegia (PEO) or Kearns-Sayre syndrome (KSS). The molar ratio of 8-OH-dG/deoxyguanosine in skeletal muscle from PEO or KSS patients was significantly higher than the control value. The ratio from patients with polymyositis or Duchenne's muscular dystrophy was not significantly elevated. Immunohistochemical staining for both Mn-SOD and Cu,Zn-SOD showed pronounced staining in the subsarcolemmal and intermyofibrillar regions of cytochrome-oxidase-negative ragged red fibers of KSS or PEO muscles. Our findings suggest that overproduction of 8-OH-dG and mitochondrial dysfunction with gene deletions are associated with each other in muscle cells of patients with PEO or KSS, and that free radicals may play an important role in the pathophysiology of mitochondrial encephalomyopathies.

Kearns-Sayre syndrome associated with de Toni-Debré-Fanconi syndrome due to cytochrome-c-oxidase (COX) deficiency.

We report a 43-year-old female with complete Kearns-Sayre syndrome, focal deficiency of cytochrome-c-oxidase (COX) and extensive deletion of the mtDNA in muscle fibers, which showed progressive insufficiency of the renal tubule: first hyperphosphaturia and hyperaminoaciduria and, later, also glucosuria (de Toni-Debré-Fanconi syndrome), a syndrome to date rarely diagnosed in association with complete Kearns-Sayre syndrome. In our opinion, this case, in view of the relationships between retinal and kidney disorders, suggests a search for de Toni-Debré-Fanconi syndrome in all patients with Kearns-Sayre syndrome also by quantitative and chromatographic methods for the assessment of aminoacids, phosphates and sugars in the urine.

[Kearns-Sayre syndrome: mitochondrial encephalomyopathy caused by deficiency of the respiratory chain]. / Le syndrome de Kearns et Sayre: encéphalomyopathie mitochondriale par déficit de la chaine respiratoire.

We report the cases of a 46 year old woman and of a 18 year-old boy who met the criteria for Kearns-Sayre syndrome. Additional atypic features were present in one case: family history, psychosis and acute respiratory failure. In both cases histoenzymatic analysis of the muscle biopsy and biochemical studies of mitochondria isolated from the muscle sample demonstrated mitochondrial myopathy associated with combined partial deficiency of complexes I and IV of the electron transfer chain. Although there is no correlation between clinical and biological data in the mitochondrial myopathies our cases confirm that such defects are involved in Kearns-Sayre syndrome. Improvement with coenzyme Q10 therapy in these patients is reported.

[Mitochondrial encephalomyopathy]. / Le encefalomiopatie mitocondriali.

Modern concepts regarding mitochondrial encephalomyopathies (ME) are summarized. Utilizing recent techniques of molecular biology we studied some cases of ME referred to the Institute of Clinical Neurology of Milan University. With these techniques we demonstrated different mitochondrial DNA deletions either in patients' muscle or in culture.

Segmental cytochrome c-oxidase deficiency in CPEO: teased muscle fiber analysis.

In an attempt to elucidate the pathogenesis of focal cytochrome c-oxidase (COX) deficiency in skeletal muscle from patients with chronic progressive external ophthalmoplegia (CPEO), we examined the longitudinal distribution of COX activity in single muscle fibers from 6 CPEO patients with muscle mitochondrial DNA (mtDNA) deletions. A new method for teasing single muscle fibers, recently developed in our laboratory, revealed fibers with COX-positive and -negative segments in all 6 patients. The borders between the enzyme-positive and -negative segments in these fibers were sharply delineated, so that the length of each COX-negative segments could be accurately measured. The proportion of the sum of the lengths of the enzyme-negative segments to the total length of the muscle fibers correlated well with the proportion of deleted mtDNA, suggesting that abnormal mitochondria harboring mutant mtDNA may be responsible for the focal loss of COX activity.

Progressive cytochrome c oxidase deficiency in a case of Kearns-Sayre syndrome: morphological, immunological, and biochemical studies in muscle biopsies and autopsy tissues.

We report biochemical, immunological, and morphological findings in a patient with fatal Kearns-Sayre syndrome. Histochemical and biochemical findings from muscle biopsy specimens obtained 7 years apart documented the disease's evolution from a mild mitochondrial disorder affecting a small proportion of muscle fibers to a severe disorder affecting a large proportion of muscle fibers. Cytochrome c oxidase activity in muscle declined profoundly as the disease progressed, although the level of enzyme protein was normal, as shown by immunochemical techniques. Other organs were severely affected by the disease. Examination of postmortem tissue showed spongiosis in the frontal cortex, diffuse loss of Purkinje cells in the cerebellum, liver steatosis, and heart fibrosis with mitochondrial abnormalities. Cytochrome c oxidase activity was only slightly reduced in these organs.

Biochemical and molecular investigation of mitochondrial disease: an illustrative case showing the value of a multifaceted approach.

Detailed biochemical and molecular investigations in a patient with Kearns-Sayre syndrome are presented. Polarographic studies in isolated mitochondria revealed a global impairment in respiratory capacity consistent with an admixture of functional and non-functional mitochondria. Cytochrome difference spectra revealed a selective deficiency in cytochrome aa3. Western immunoblot studies revealed normal subunit content of Complexes I, III and IV. Southern blot studies of mtDNA showed a deletion of approximately 5 Kb coexisting with wild type DNA. PCR analysis confirmed that this deletion lies between the ATPase8 and NAD coenzyme Q oxidoreductase subunit 5 (ND5) genes. Breakpoint sequencing revealed a 13 nucleotide direct repeat flanking sequence (ACCTCCCTCACCA) consistent with slippage in mtDNA during replication as the mechanism of deletion. Histochemical studies of skeletal muscle revealed many cytochrome oxidase negative fibres and immunocytochemical studies showed cytochrome oxidase negative areas with abundant respiratory complex protein suggesting upregulation. The value of a multifaceted approach in unravelling the pathophysiology of mitochondrial diseases is emphasised.

A case of Kearns-Sayre syndrome with the 4,977-bp common deletion associated with a novel 7,704-bp deletion.

Mitochondria from a patient diagnosed with Kearns-Sayre syndrome (KSS) exhibited severely diminished cytochrome c oxidase activity and at least four mitochondrial DNA (mtDNA) species: 9%-11% of the fulllength mtDNA (16.6 kb), 70%-75% of a 11.7-kb population (harboring the 4,977-bp common deletion), 2%-3% of a 10.5-kb population, and 12%-17% of a 8.9-kb population. The 8.9-kb mtDNA exhibited a secondary deletion that extended 7,704 bp from nucleotide 7,979 in the cox2 gene to nucleotide 15,683 in the cytb gene. To our knowledge, this is the first description of the presence of at least two large-scale deletions of mtDNA in KSS.

Cytochrome c oxidase activity in single muscle fibers: assay techniques and diagnostic applications.

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.