PEDIATRICS Vol. 105 No. 3 March 2000, pp. 598-603

Childhood Encephalopathies and Myopathies: A Prospective Study in a Defined Population to Assess the Frequency of Mitochondrial Disorders

Johanna Uusimaa, MD^*, , Anne M. Remes, MD^{*, §}, Heikki Rantala, MD, Leena Vainionpää, MD, Riitta Herva, MD, Katri Vuopala, MD^{, ¶}, Matti Nuutinen, MD, Kari Majamaa, MD^{*, §}, and Ilmo E. Hassinen, MD^*

From the ^* Departments of Medical Biochemistry,  Pediatrics, ^§ Neurology, and  Pathology, University of Oulu, Oulu, Finland; and the ^¶ Department of Pathology, Lapland Central Hospital, Rovaniemi, Finland.

	ABSTRACT

Top Abstract Methods Results Discussion References

Objectives.  To assess the frequency of mitochondrial abnormalities in muscle histology, defects in respiratory chain enzyme activities, and mutations in mitochondrial DNA (mtDNA) in children with unexplained psychomotor retardation in the population of Northern Finland.

Background.  The frequency of mitochondrial diseases among patients with childhood encephalopathies and myopathies is not known. Frequencies are difficult to estimate because the clinical presentation of these disorders is variable.

Methods.  A total of 116 consecutive patients with undefined encephalopathies and myopathies were enrolled during a 7-year period in a hospital serving as the only neurologic unit for a pediatric population of 97 609 and as the only tertiary level neurologic unit for a pediatric population of 48 873. Biochemical and morphologic investigations were performed on muscle biopsy material, including oximetric and spectrophotometric analyses of oxidative phosphorylation, histochemistry, electron microscopy, and molecular analysis of mtDNA.

Results.  Ultrastructural changes in the mitochondria were the most common finding in the muscle biopsies (71%). Ragged-red fibers were found in 4 cases. An oxidative phosphorylation defect was found in 26 children (28%), complex I (n = 15) and complex IV (n = 13) defects being the most common. Fifteen percent of patients (n = 17/116) with unexplained encephalomyopathy or myopathy had a probable mitochondrial disease. Common pathogenic mutations were found in the mtDNA of only 1 patient (.9%).

Conclusions.  The common known mutations in mtDNA are rarely causes of childhood encephalomyopathies, which is in contrast to the considerable frequency of the common MELAS mutation observed among adults in the same geographical area. Biochemically and morphologically verified mitochondrial disorders were nevertheless common among the children, making the analysis of a muscle biopsy very important for clinical diagnostic purposes.  Key words:  mitochondria, encephalomyopathies, children, mutation, respiratory chain, MELAS, mitochondrial DNA.

Mitochondrial encephalomyopathies comprise a group of multisystem disorders with considerable heterogeneity in clinical presentation and with onset from infancy to late adulthood.¹ Biochemically, they are characterized by defects in substrate transport, substrate utilization, the Krebs cycle, or the respiratory chain. The diagnosis of these disorders is difficult because a given biochemical dysfunction can be manifested in varying clinical presentations, and a given clinical presentation may be attributable to variable biochemical dysfunctions.² Some ancillary examinations are available for the clinical diagnosis of patients with a suspected mitochondrial disease. Many patients have increased serum or cerebrospinal fluid (CSF) lactate³ and brain imaging may reveal focal lesions, cortical atrophy, or basal ganglia calcification.¹ Muscle histology may show ragged-red fibers (RRF), representing subsarcolemmal mitochondrial proliferation, and changes in the size, shape, and structure of the mitochondria may be seen by electron microscopy.³ The morphologic abnormalities are not specific to mitochondrial myopathies, however.³

Some mitochondrial encephalomyopathies are attributable to mutations in the mitochondrial DNA (mtDNA), which codes for 13 protein subunits within complexes I, III, IV, and V and for 2 recombinant RNAs and 22 transfer RNAs. Point mutations in transfer RNA genes have been found in patients with the syndromes of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS)⁴ and myoclonus epilepsy and RRF (MERRF).⁵ A point mutation in the adenosine triphosphatase 6 gene has been found in patients with neuropathy, ataxia, and retinitis pigmentosa (NARP)⁶ and in the patients with Leigh's disease.⁷ Furthermore, the mtDNA mutations include deletions,⁸ duplications,⁹ and depletion of mtDNA.¹⁰

The frequency of mitochondrial diseases among patients with childhood encephalopathies and myopathies is not known. Therefore, we set out to identify children with an unknown encephalopathy or myopathy in a defined population in Northern Finland and investigated abnormalities in muscle histology, respiratory chain enzyme activities, and mitochondrial DNA to estimate the frequency of mitochondrial diseases.

	METHODS

Top Abstract Methods Results Discussion References

Setting

Specialized medical care in Finland is organized at the provincial level, and in the province of Northern Ostrobothnia, Oulu University Hospital (OUH) provides this care. The inhabitants are entitled to its services and may seek publicly funded medical care at institutions in other parts of the country only with the permission of their own local health authority. Private hospitals account for 8.4% of total expenditure on hospital care in Finland.¹¹ This means that the patient populations of the institutions providing specialized medical care provide a fairly good impression of total morbidity in the provinces.

The Department of Pediatrics at the OUH serves as the only pediatric unit for the province of Northern Ostrobothnia and as a tertiary level pediatric unit for the remainder of Northern Finland. The total population of Northern Ostrobothnia on December 31, 1996 was 358 499 and the population below 18 years of age was 97 609. The total population of the remaining part of Northern Finland, the tertiary catchment area, was 294 965 and population below 18 years of age was 48 873. Eighty patients from the primary catchment area and 36 patients from the tertiary catchment area were enrolled in this study.

Patients

Consecutive patients with psychomotor retardation admitted to the Department of Pediatrics at OUH between January 1, 1990 and December 31, 1996 were evaluated in a standardized manner, including brain computed tomography (CT) or magnetic resonance imaging (MRI) examinations, an electroencephalography and electroneuromyography, karyotype analysis, mutation analysis for fragile-X, liver tests, determination of plasma and urinary levels of amino acids, and urinary excretion of organic acids and oligosaccharides. Patients were eligible for the study if no definite or probable cause diagnosis could be established in these investigations. A muscle biopsy for histologic, biochemical, and molecular analyses was taken with the informed consent of the parents. Clinical summaries of the examinations and diagnoses of the eligible patients are provided in Tables 1 and 2. The ethical committee of the University of Oulu approved this research.

The diagnosis of mitochondrial disease is usually based on the results of the following investigations: clinical examinations, biological studies, exercise tests, MRIs, histo-enzymologic studies, molecular biology, and finally a biochemical study of oxidative phosphorylation (OXPHOS) on muscle biopsies. Biochemical study is at present the primary tool in the search for an oxidative phosphorylation deficit.¹² OXPHOS defects consist of deficiencies in complexes I, I + III, II + III, or IV in mitochondrial respiratory chain. In our study, a diagnosis of mitochondrial disease was defined as possible if deficiencies in 1 or more complexes or ultrastructural changes in muscle mitochondria were found, as probable if both defects in OXPHOS enzymes and ultrastructural changes in muscle mitochondria were found, and as definite if known pathogenic mutations in mtDNA were found.

Mitochondrial Function

A skeletal muscle biopsy of .5 to 1.0 g was taken surgically from the quadriceps femoris muscle under general or local anesthesia. Parts of the sample were used for the isolation of DNA and for histologic typing and electron microscopy. The majority of the sample was immediately cooled to 0°C in .9% sodium chloride and the mitochondria were isolated.¹³ The activities of the respiratory chain enzymes were measured by oximetric and spectrophotometric methods.^14-17 Citrate synthetase activity was measured from the first 50 patients and it correlated well to OXPHOS enzyme activities. Mitochondrial respiration with nicotinamide adenine dinucleotide (NADH)-linked substrates, succinate, or tetramethyl-p-phenylenediamine and ascorbate was measured at 25°C in a 1-mL oxygraph chamber fitted with a Clark-type oxygen electrode.¹⁶ The activities of NADH-cytochrome c reductase and succinate:cytochrome c reductase were measured in mitochondria in the presence of .1 mM ferricytochrome c and potassium cyanide.¹⁵ Both activities were expressed as nmol · min¹mg¹ protein. NADH-ubiquinone oxidoreductase activity was assayed using Q1 as an electron acceptor.¹⁴ Cytochrome c oxidase was measured according to Cooperstein and Lazarow¹⁶ and the rate expressed in nmol · min¹mg¹ mitochondrial protein at 29 µM cytochrome c, which was the initial ferrocytochrome c concentration in the cuvette. Protein concentrations were assayed by a colorimetric method.¹⁸

Some children had had a muscle biopsy taken as part of the initial clinical evaluation. A definite diagnosis other than a mitochondriopathy was reached in 12 of these cases (verified syndromes or brain malformations [8], growth hormone deficiency [1], sequel of neonatal meningitis [1], transient muscle pain [1], benign nocturnal jerks [1]). The respiratory chain activities of these 12 patients served as control values: complex I, 42.8 ± 28.7; complex I + III, 166.9 ± 70.1; complex II + III, 49.4 ± 31.2; and complex IV, 183.8 ± 70.4 (each expressed in nmol min¹ mg¹; mean ± standard deviation). The respiratory chain activities were considered decreased when values were below 1 standard deviation of control values.

Histopathology

Consecutive cryostat sections of skeletal muscle were stained with hematoxylin-eosin, periodic acid-Schiff, Gomori trichrome, Sudan black, and Berlin blue. NADH-tetrazolium reductase, cytochrome oxidase, and adenosine triphosphatase were also routinely used as stainings for enzyme histochemistry. The specimens for electron microscopy were fixed in 4% formaldehyde/1% glutaraldehyde buffered to pH 7.4 with phosphate buffer and postfixed in OsO₄ and embedded in Epon LX-112 (Electron Microscopy Sciences, Fort-Washington, PA). Ultra-thin sections were contrasted with uranyl acetate and lead citrate and examined in a Philips LS electron microscope (Philips Export B.V., Eindhoven, The Netherlands) In the "Results" section in Table 5, "mild mitochondrial changes" means only a slightly increased number of mitochondria in electron microscopy or slight alterations in size and shape and "severe mitochondrial changes" means a markedly increased number or pronounced alterations in size and shape.

mtDNA

Total genomic DNA was isolated from frozen skeletal muscle of 115 patients and from blood of 1 patient by the standard sodium dodecyl sulfate-proteinase K method. mtDNA deletions were detected in Southern blot analysis using ³²P-labeled whole mtDNA or polymerase chain reaction (PCR)-generated mtDNA fragments as a probe. The common 5-kb mtDNA deletion (8469-13 447) was also detected by amplifying the specific region by PCR.

The common mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes point mutation in transfer RNA Leu^(UUR) (nt 3243) was detected by Apa I digestion of the appropriate mtDNA fragment in the presence of ³⁵S-deoxyadenosine triphosphate.¹⁹ The myoclonus epilepsy and RRF point mutation (at nt 8344) was detected by Bgl I digestion of a mtDNA fragment amplified in the presence of a mismatched primer and ³⁵S-deoxyadenosine triphosphate.²⁰ The digested product was electrophoresed through a 6% nondenaturing polyacrylamide gel, which was dried and autoradiographed at 72°C overnight using Kodak XAR film with an intensifying screen (Kodak, Rochester, NY). The films were analyzed with a Bioimage scanner and image processing apparatus (Millipore, Ann Arbor, MI). A T-to-G substitution mutation at nt 8993 was detected by amplifying a mtDNA fragment around the mutation using primers corresponding to nt 8278 to 8296 and nt 10 382 to 10385. The amplified 2.1-kb fragment was digested with the Ava I restriction enzyme and electrophoresed through 1% agarose gels. The 2.1-kb fragment was digested in the presence of the T-to-G mutation to give 2 fragments of 710 bp and 1390 bp.

Statistical Analysis

Fisher's exact test was used to analyze the frequencies of clinical symptoms in patients with complex I and IV deficiencies.

	RESULTS

Top Abstract Methods Results Discussion References

Clinical Examination of Patients

A total of 116 children (median age of onset: .8 years; range: 1 day to 11.6 years) with an encephalopathy or myopathy of unknown cause were prospectively enrolled for the study (Table 1). Their clinical features are shown in Table 2. The most common abnormalities in electroencephalography were slowing of the background activity (18%), focal irritation (13%), generalized spike-and-wave discharges (7%), and hypsarrhythmia (5%). Electroneuromyography showed myopathy (13%), neurogenic degeneration (7%), and lower motor neuron disease (2%). Brain CT or MRI revealed cortical atrophy in 18% of cases and intracranial calcifications in 6%. In addition, there were 42 patients with MRI and CT changes suggestive of mitochondrial disease and 27 of these patients had abnormal mitochondrial ultrastructure, and 9 patients of 42 had an OXPHOS defect. There was 1 patient with high T2 signal intensity in basal ganglia in MRI. However, her OXPHOS functions were normal and the muscle biopsy material was not sufficient for electron microscopic examination.

Blood lactate was intermittently or constantly elevated (>1.8 mmol/L) in 58% of cases (mean: 3.12 mmol/L; range: 1.08-7.86 mmol/L), whereas CSF lactate was elevated in 32% (mean: 1.94; range: .93-4.28 mmol/L). There was 1 patient who had high CSF lactate with normal blood lactate and all other patients with high CSF lactates also had increased blood lactate values.

Patients With Mitochondrial Aberration

Electron microscopy of the skeletal muscle revealed ultrastructural changes in the mitochondria of 68 patients (Table 3). Increased amounts of mitochondria were found in 50% of the patients, and variation in the size and shape of the mitochondria was found in 33%. There were 4 patients with severe mitochondrial changes in the possible group, 1 in the probable group, and none in the definite group. The most common abnormal findings in light microscopy were type 2-fiber atrophy (6%), fat accumulation (6%), degenerative changes (4%), and myopathic changes (3%). RRF were found in 4 patients (3%).

Decreased activities of the respiratory chain enzymes were found in 26 of the 94 examined patients (28%) by oximetric and spectrophotometric measurement of isolated muscle mitochondria (Table 3). The ratios of different complexes detected OXPHOS defects in a similar manner as the absolute respiratory control enzyme activities. Respiratory control ratios with NADH substrates for controls and for patients with OXPHOS defects were 10.8 and 10.4, respectively, meaning that mitochondria were intact. Decreased activity of complex I was found in 15 patients, making this the most common defect. It was found in combination with a defect of complex III in 9 patients. Diminished activity of complex IV was found in 13 patients, 4 of them having a combined defect. Four patients had combined defects of complexes I to IV, and 1 patient had decreased activity of complexes II + III only. The criteria and the number of patients with suspected mitochondrial disorders have been shown in Table 4. In some cases, there were difficulties to establish a mitochondrial disorder because of insufficient muscle biopsy material for adequate biochemical and/or morphologic analysis.

No large scale deletions in mtDNA were found by Southern blot analysis nor was the common 5-kb deletion observed in PCR analysis. The transfer RNA Leu^(UUR) mutation at nt 3243 was found in 1 patient (Table 5; patient 3). The degree of heteroplasmy of the mutation was 83% in muscle and 64% in blood. No mutations at nt 8344 or 8993 were found in these 116 patients.

Clinical Features of Patients With a Biochemically and Ultrastructurally Defined Mitochondrial Disorder

There were 17 patients (Table 5) with an OXPHOS defect and ultrastructural changes in mitochondria and 19 patients with a normal OXPHOS and normal mitochondria in electron microscopy (Table 3). The 2 groups did not differ markedly in their clinical features. In contrast, hypotonia and developmental retardation was found in 5 of the 9 patients with an isolated complex IV defect but in only 1 of the 9 with combined complex I and III defects (P = .07). In addition, 3 of the 9 patients with an isolated complex IV defect had psychomotor retardation with hypotonia and epilepsy; whereas, none of the 9 patients with complex I and III deficiencies had this combination of symptoms. The blood lactate concentrations of these patients did not differ from those of the group with normal OXPHOS and normal electron microscopy. There were no differences in clinical features of patients between possible and probable groups.

	DISCUSSION

Top Abstract Methods Results Discussion References

This cohort of children with unexplained encephalomyopathy or myopathy represents fairly well the level of morbidity in the province of Northern Ostrobothnia, where the Department of Pediatrics at the Oulu University Hospital serves as the only pediatric unit. The frequency of ultrastructural mitochondrial aberration was 71%, that of a mitochondrial biochemical decrease was 28%, and that of a mtDNA mutation was <1% in 116 consecutive patients. The high frequency of ultrastructural changes suggests that the investigation of muscle ultrastructure may not be specific for the diagnosis of mitochondrial disorders. Other diseases that may lead to secondary changes in the mitochondria include other metabolic diseases, for instance defects in fatty acid -oxidation and inflammatory myopathies, ie, inclusion body myositis²¹ and polymyositis.²² Indeed, we found 37 patients with abnormal mitochondria or increased fat in their muscle tissue in whom no OXPHOS defect was detected (Table 3). In contrast, morphologic abnormalities were not observed in the muscle biopsies of all the patients with an OXPHOS defect. This makes diagnosis difficult, especially in sporadic cases.

Decreased activities of OXPHOS enzyme complexes were found in 26 of 94 children (28%), and a similar frequency of respiratory chain defects has been found in 2 previous series of cases.^23,24 In another study, a biochemically defined mitochondrial disorder was found in 20 of 50 children with central nervous system (CNS) symptoms or neuromuscular disease combined with hyperlactataemia.² These frequencies suggest that an OXPHOS defect is fairly common among children with encephalomyopathy. The activities of complexes I and IV were most commonly affected in our patients. It has been shown that complex I and I + III activities do not vary in children of different ages, but complex IV activity seems to decrease with age.²⁵ Complex I deficiency has been observed in a variety of mitochondrial myopathies in children^26,27 and has recently been shown to be a common cause of Leigh disease.^28,29 Numerous cases of complex IV deficiencies have been reported presenting either as a myopathy or as a multisystem disorder with CNS symptoms as the dominating clinical feature.

The most common mtDNA mutations were rare among our patients, as we found only 1 boy with the A3243G mutation, suggesting a frequency of .9%. Mutations in the mtDNA have been found at a frequency of 8% among children with CNS or neuromuscular disease and with hyperlactatemia,² whereas the corresponding frequency among similarly defined patients in our cohort would be 1.7%. The A3243G mutation is found more commonly among various adult patient populations with a frequency of ~1% among unselected patients with diabetes mellitus,³⁰ 6% among young adults with occipital stroke,³¹ and 7% among young adults with sensorineural hearing loss.³² Indeed, its frequency has been found to be as high as 16.3/100 000 in the adult population in Northern Finland.³²

Of the 79 patients in whom both mitochondrial ultrastructure and the OXPHOS activities had been determined, 36 had either both tests normal or both abnormal, whereas there were 37 children in whom mitochondrial ultrastructure was abnormal but OXPHOS was normal, suggesting that either the sensitivity of OXPHOS measurement is low or the specificity of electron microscopy is low. We defined the patients with both mitochondrial ultrastructure and OXPHOS abnormality as having a clinical probability of mitochondrial disease. The clinical features of these 17 patients (Table 5) were very variable, with encephalopathy, muscular hypotonia, and epilepsy occurring as common features, whereas ataxia, spasticity, and short stature were found occasionally. Serum lactate was increased in half of the children, and 3 of the 4 patients with RRF found in the cohort belonged to this group. The patient with the A3243G mutation (Table 5; patient 3) had muscle RRF together with ultrastructural abnormalities in the mitochondria and a complex IV deficiency in the respiratory chain. Multiple defects in OXPHOS enzymes have been found previously in patients with the A3243G mutation.^33,34 Studies on cell cybrids harboring a mixture of 3243A:T and 3243G:C genomes have shown that there is a marked decrease in complex I activity when the proportion of 3243G:C is 60% to 90%, whereas cybrid cell lines containing a very high level of 3243G:C have been shown to be defective in complexes III and IV as well.³⁵

We conclude that the commonly known mtDNA mutations are a rare cause of childhood encephalomyopathies, whereas a decrease in OXPHOS activity and morphologic changes in mitochondrial structure are more common. Either ultrastructural changes or OXPHOS defect in muscle samples may raise the possibility of a mitochondrial disorder. However, these disorders are not clinically distinct; therefore, we suggest that the diagnosis of probable mitochondrial disease requires the demonstration of both ultrastructural changes in mitochondria and decreased activity of 1 or more respiratory chain enzymes in a muscle biopsy. Mitochondrial dysfunction may be one of the most common causes of disease among the children with unexplained encephalomyopathy or myopathy as we found that 15% of such patients could be diagnosed with a probable mitochondrial disease. However, there are undoubtedly patients with normal polarographic and oxidative phosphorylation studies who have undetected mitochondrial DNA disease. Thus, the overall incidence of mitochondrial DNA disease is likely even higher that we have found.

	ACKNOWLEDGMENTS

This work was supported under a research contract with the Medical Research Council of the Academy of Finland and through grants from the Arvo and Lea Ylppö Foundation and the Sigrid Juselius Foundation.

The expert technical assistance of Anja Heikkinen and Irma Vuoti is gratefully acknowledged.

	FOOTNOTES

Received for publication Oct 20, 1998; accepted Jun 22, 1999.

Reprint requests to (K.M.) University of Oulu, Department of Neurology, Kajaanintie 52 A, FIN-90220 Oulu, Finland. E-mail: kari.majamaa{at}oulu.fi

	ABBREVIATIONS

CSF, cerebrospinal fluid; RRF, ragged-red fibers; mtDNA, mitochondrial DNA; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERFF, myoclonus epilepsy and ragged red fibers; NARP, neuropathy, ataxia, and retinitis pigmentosa; OUH, Oulu University Hospital; CT, computed tomography; MRI, magnetic resonance imaging; OXPHOS, oxidative phosphorylation; NADH, nicotinamide adenine dinucleotide; PCR, polymerase chain reaction; CNS, central nervous system.

	REFERENCES

Top Abstract Methods Results Discussion References

1. Johns DR Mitochondrial DNA and disease. N Engl J Med 1995; 333:638-644 [Free Full Text]
2. Tulinius M, Holme E, Kristiansson B, Mitochondrial encephalomyopathies in childhood. I. Biochemical and morphological investigations. J Pediatr 1991; 119:242-250 [CrossRef][Medline]
3. Shoffner JM Maternal inheritance and the evaluation of oxidative phosphorylation diseases. Lancet 1996; 348:1283-1288 [CrossRef][Medline]
4. Goto Y, Nonaka I, Horai S A mutation in the tRNA Leu^(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 1990; 348:651-653 [CrossRef][Medline]
5. Shoffner JM, Lott MT, Lezza AMS, Sabel S, Ballinger SW, Wallace DC Myoclonic epilepsy and ragged red fiber disease (MERFF) is associated with a mitochondrial DNA tRNALys mutation. Cell 1990; 61:931-937 [CrossRef][Medline]
6. Holt IJ, Harding AE, Petty RKH, Morgan-Hughes JA A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 1990; 46:428-433 [Medline]
7. Tatuch Y, Christodoulou J, Feigenbaum A, Heteroplasmic mtDNA mutation (T-G) at 8993 can cause Leigh disease where the percentage of abnormal mtDNA is high. Am J Hum Genet 1992; 50:852-858 [Medline]
8. Holt IJ, Harding AE, Morgan-Hughes JA Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331:717-719 [CrossRef][Medline]
9. Poulton J, Deadman ME, Gardiner RM Duplications of mitochondrial DNA in mitochondrial myopathy. Lancet 1989; 1:1236-239 [Medline]
10. Tritscher H-J, Andreetta F, Moraes CT, Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA. Neurology 1992; 42:209-217 [Abstract/Free Full Text]
11. NAWH National Research and Development Centre for Welfare and Health. Overview on the population's health situation: use of health services and resources. In: Health, II. Gummerus Kirjapaino Oy, Jyväskylä; 1994
12. Durrieu G, Letellier T, Antoch J, Identification of mitochondrial deficiency using principal component analysis. Mol Cell Biochem 1997; 174:149-156 [CrossRef][Medline]
13. Makinen M, Lee C-P Biochemical studies of skeletal muscle mitochondria. I. Microanalysis of cytochrome content, oxidative and phosphorylative activities of mammalian skeletal muscle mitochondria. Arch Biochem Biophys 1968; 126:75-82 [CrossRef][Medline]
14. Vuokila PT, Hassinen IE N'N-Dicyclohexylcarbodi-imide-sensitivity of bovine heart mitochondrial NADH:ubiquinone oxidoreductase. Biochem J 1988; 249:339-344 [Medline]
15. Sottocasa GL, Kuylenstierna BO, Ernster L, Bergstrand A An electron transport system associated with outer membrane of liver mitochondria: a biochemical and morphological study. J Cell Biol 1967; 32:415-438 [Abstract/Free Full Text]
16. Cooperstein SJ, Lazarow A A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem 1951; 89:665-670
17. Schapira AH, Cooper JM, Morgan-Hughes JA, Molecular basis of mitochondrial myopathies: polypeptide analysis in complex I deficiency. Lancet 1988; 1:500-503 [Medline]
18. Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Ann Biochem 1976; 72:248-254
19. Kobayashi Y, Momoi MY, Tominaga K, A point mutation in the mitochondrial tRNA Leu^(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Biochem Biophys Res Commun 1990; 173:816-882 [CrossRef][Medline]
20. Zeviani M, Gellera C, Antozzi C, Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA, tRNA Leu. Lancet 1991; 338:143-147 [CrossRef][Medline]
21. Oldfors A, Larsson NG, Lindbery C, Mitochondrial DNA deletions in inclusion body myositis. Brain 1993; 116:325-336 [Abstract/Free Full Text]
22. Molnar M, Schröder JM Pleomorphic mitochondrial and different filamentous inclusions in inflammatory myopathies associated with mtDNA deletions. Acta Neuropathol (Berl) 1998; 96:41-51 [CrossRef][Medline]
23. Munnich A, Rötig A, Chretien D, Clinical presentation of mitochondrial disorders in childhood. J Inherit Metab Dis 1996; 19:521-527 [CrossRef][Medline]
24. Zeviani M, Bertagnolio B, Uziel G Neurological presentations of mitochondrial diseases. J Inherit Metab Dis 1996; 19:504-520 [CrossRef][Medline]
25. Lefai E, Terrier-Cayre A, Vincent A, Enzymatic activities of mitochondrial respiratory complexes from children muscular biopsies: age related evolutions. Biochem Biophys Acta 1995; 1228:43-50 [Medline]
26. Morgan-Hughes JA, Schapira AHV, Cooper JM, Clark JB Molecular defects of NADH-ubiquinone oxidoreductase (complex I) in mitochondrial diseases. J Bioenerg Biomembr 1988; 20:365-380 [CrossRef][Medline]
27. Koga Y, Nonaka I, Kobayashi M, Tojyo M, Nihei K Findings in muscle in complex I (NADH coenzyme Q reductase) deficiency. Ann Neurol 1988; 24:749-756 [CrossRef][Medline]
28. Morris AAM, Leonard JV, Brown GK, Deficiency of respiratory chain complex I is a common cause of Leigh disease. Ann Neurol 1996; 40:25-30 [CrossRef][Medline]
29. Rahman S, Blok RB, Dahl H-HM, Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 1996; 39:343-351 [CrossRef][Medline]
30. Kadowaki T, Kadowaki H, Mori Y, A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. N Engl J Med 1994; 330:962-968 [Abstract/Free Full Text]
31. Majamaa K, Turkka J, Kärppä M, Winqvist S, Hassinen IE The common MELAS mutation A3243G in mitochondrial DNA among young patients with an occipital infarct. Neurology 1997; 49:1331-1334 [Abstract/Free Full Text]
32. Majamaa K, Moilanen JS, Uimonen S, Epidemiology of A3243G, the mutation for mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes: prevalence of the mutation in an adult population. Am J Hum Genet 1998; 63:447-454 [CrossRef][Medline]
33. Goto Y, Horai S, Matsuoka T, Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS): a correlative study of the clinical features and mitochondrial DNA mutation. Neurology 1992; 42:545-550 [Abstract/Free Full Text]
34. Ciafaloni E, Ricci E, Shanske S, MELAS: clinical features, biochemistry, and molecular genetics. Ann Neurol 1992; 31:391-398 [CrossRef][Medline]
35. Dunbar DR, Moonie PA, Zeviani M, Holt IJ Complex I deficiency is associated with 3243G:C mitochondrial DNA in osteosarcoma cell cybrids. Hum Mol Genet 1996; 5:123-129 [Abstract/Free Full Text]