Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with encephalomyelopathy

Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with e n ce ph a Iom yelo pa t h y C. Marsac, MD, D. Stansbie, MD, FRCPath, G. Bonne, MSc, J. Cousin, MD, P. Jehenson, MD, PhD, C. Benelli, PhD, J.-P. Leroux, MD, and G. Lindsay, PhD From the INSERMU75 Institute, Facult6 Necker, Paris, France; the Department of Chemical Pathology, Bristol Royal Infirmary, Bristol, England; H6pital Saint-Antoine, Lille, France; the Service Hospitalier Frederic Jotiot, CEA, 9 France; INSERMU30, H6pital Necker, Paris, France; and the Department of Biochemistry, University of Glasgow, Scotland, United Kingdom A m o n g the m a n y m e t a b o l i c e n c e p h a l o m y e l o p a t h i e s c a u s e d b y d e f i c i e n c i e s in the p y r u v a t e d e h y d r o g e n a s e c o m p l e x (PDHC), n e a r l y all i n v o l v e its E1 subunit. We d e s c r i b e two new f a m i l i a l cases of PDHC d e f i c i e n c y with e n c e p h a l o m y e l o p a t h y , c h r o n i c l a c t i c a c i d e m i a , a n d a n o r m a l Et subunit of PDHC but d e f i c i e n c y in a n o t h e r c o m p o n e n t . A c t i v i t y of PDHC was m e a s u r e d in cultured skin fibroblasts a n d s k e l e t a l muscle, and i m m u n o b l o t studies were p e r f o r m e d on mitoc h o n d r i a l extracts from skin fibroblasts. Spectra of muscle tissue, o b t a i n e d in v i v o with phosphorus 31 n u c l e a r m a g n e t i c resonance, were r e c o r d e d both at rest a n d with exercise. The PDHC a c t i v i t y was m a r k e d l y r e d u c e d to 10% to 20% of n o r m a l v a l u e s in both cultured skin fibroblasts a n d skeletal muscle. Immunob l o t t i n g of skin fibroblast m i t o c h o n d r i a l extracts showed a s p e c i f i c d e f i c i e n c y in the protein X c o m p o n e n t of PDHC but normal E4, E2, a n d E3 c o m p o n e n t s . Spectra o b t a i n e d with 3tp n u c l e a r m a g n e t i c r e s o n a n c e showed a l t e r a t i o n s c o m p a t i b l e with those found in m i t o c h o n d r i a l m y o p a t h i e s . This is the s e c o n d d e s c r i p t i o n of an e n c e p h a l o m y e l o p a t h y a s s o c i a t e d with a s p e c i f i c a b s e n c e of the l i p o y l - c o n t a i n i n g protein X c o m p o n e n t , which has a structural role in the f o r m a t i o n of a f u n c t i o n a l PDHC. (J PEDIATR1993;123:9t5-20) The mammalian pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate to acetyl coenzyme A and plays an essential role in aerobic energy metabolism.l Functional defects of various subunits of the PDHC have been described and are usually manifested clinically as encephalomyelopathies. In many cases (more than 100), the defect has affected the dihydrolipoyl decarSupported by grants from the Association Franqaise Contre les Myopathies (MM/NN/864) and by the Institut Electricit6 Sant6 (92.031 CS). Submitted for publication March 30, 1993; accepted July 20, 1993. Reprint requests: C. Marsac, MD, INSERM U75, Facult6 Necker, 156 rue de Vaugirard, 75015 Paris, France. Copyright 9 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/20/50219

boxylase ( E l ) subunit of the complex. 26 Recently, Robinson et al. 7 described one defect in the dihydrolipoyl transacetylase (E2) subunit and two abnormalities of the lipoyl-bearing protein X component in patients with lactic El E2 E3 NMR PCr PDHC Pi PME

Dihydrolipoyl decarboxylase Dihydrolipoyl transacetylase Dihydrolipoyl dehydrogenase Nuclear magnetic resonance Phosphocreatine Pyruvate dehydrogenase complex Inorganic phosphate Phosphomonoesters

acidemia and PDHC deficiency. One patient appeared to have a missing X component; the other had two distinct bands for the X protein.

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Marsac et al.

We studied two new familial cases of encephalomyelopathy that was clinically indistinguishable from the E1P D H C deficiencies but with a defect in P D H C caused by the specific absence of the X component. W e also describe the phosphorus 31 nuclear magnetic resonance exercise-test findings. CASE REPORTS The two affected children (patients 1 and 2) were brothers; the parents, of Portuguese origin, were first cousins. Two other siblings (a boy and a girl) were healthy, but two spontaneous abortions had also occurred. The two patients were born at term by spontaneous delivery. They were well at first, but progressive neurologic symptoms subsequently developed. Patient 1 had convulsions in the neonatal period and by the age of 17 months had hypotonia, nystagmus, strabismus, and profound psychomotor retardation. Electromyographic and electroeneephalographic findings were normal, and computed tomography scans showed global cerebral atrophy, which was more pronounced on the right side. Progressively, the neurologic deterioration was consistent with Leigh disease and included failure to thrive, developmental retardation, psychomotor delay, truncal hypotonia and hypertonia of the limbs, ataxia, frequent convulsions, nystagmus, and tachypnea. The child could not speak and required complete assistance. Hyperalaninemia and hyperlactatemia followed an episode of gastroenteritis treated with antibiotics in an intravenous infusion of glucose; blood lactate levels reached a peak of 33 mmol/L (normal: <2 retool/L) and fell spontaneously to a basal level of about 7 mmol/L on its withdrawal. The lactate/pyruvate ratio was around 20. Hyperammonemia or hypoglycemia was not detected. The boy was very hypotonic at the age of 5 years, had bilateral optic atrophy, and died of respiratory insufficiency at the age of 6 years. Patient 2 was less severely affected. Between the ages of 3 and 6 years he was hospitalized because of developmental retardation. He walked at 8 years, and at this time expressive language was limited to a few words. At 11 years, he had spastic diplegia with ataxia. He was incontinent, profoundly mentally retarded, and for 16 years was confined to institutional care. He did not improve despite prolonged supplementation of vitamins B1, B2, B6, and B12.At 16 years, after a brief stay in Portugal, where he had some infectious episodes, his condition deteriorated rapidly; he went into a deep coma with irreversible severe lactic acidosis and died suddenly of cardiopulmonary arrest. Skin and deltoid muscle biopsies were performed on the two patients at the ages of 4 and 14 years, respectively. An autopsy could not be performed in either case, but the clinical features were compatible with the progressive neurologic degenerative disorder known as Leigh syndrome or subacute necrotizing encephalomyelopathy. METHODS

In vitro biochemical studies Materials. Skin fibroblast cells were grown in minimal essential medium supplemented with 10% fetal calf serum, glutamine, and antibiotics. After trypsinization, fibroblasts

The Journal of Pediatrics December 1993

were obtained for enzymatic assays and also for isolation of mitochondria. The preparation of skeletal muscle mitochondria was performed directly from a fresh muscle biopsy specimen (about 50 mg). The tissue was disrupted in potassium chloride, 150 m m o l / L , and Tris-HC1 buffer, 50 m m o l / L (pH 7.4; isotonic medium), with a loose-fitting Teflon pestle in conjunction with a Potter-Elvehjem homogenizer (Bioblock, Illkizch, France). A first centrifugation at 1000g for 5 minutes removed connective tissue and nuclei in the pellet. The supernatant of the first low-speed centrifugation (fraction S) was further centrifuged for 10 minutes at 7000g and 4 ~ C. The pellet was washed once with isotonic medium and recentrifuged at the same speed. The mitochondrial pellet (fraction P) was resuspended in a minimal volume of the same buffer. Assays o f PDHC activity. Pyruvate dehydrogenase complex activity was measured in cultured skin fibroblasts and muscle mitochondria by the fluorometric method described by Solomon and Stansbie. 8 After trypsinization, fibroblasts were suspended in a buffer containing potassium chloride, 150 m m o l / L , and Tris-HC1 buffer, 50 m m o l / L (pH 7.4). The cells were sonicated (150 watts for 5 seconds) just before the assay. The assay estimated the activity of the P D H C present in the active dephosphorylated form. W e also performed assays of pyruvate decarboxylase and transacetylase activities on a pellet of fibroblasts from our two patients, using the radioactive [ 1-14C]pyruvate method 9 with and without exogen transacetylase. Spectrophotometric assay of cytochrome c oxidase involved the determination of the rate of cyanide-sensitive oxidation of 80% reduced cytochrome c at 550 nm according to the method of Appelmans et al. 1~ Succinate cytochrome c reductase activity was measured by the rate of cytochrome c reduction. 11 Pyruvate carboxylase and phosphoenolpyruvate carboxykinase assays were performed according to the modified methods of Hsia et al. 12 and Marsac et a1.,13 and of B allard and Hanson,14 respectively. Protein content was assayed by the method of Lowry et al. is Immunochemical studies. Intact pyruvate dehydrogenase multienzyme complex from bovine heart was purified to near homogeneity as described previously, 16 with minor modifications. 17 High titers of polyclonal antibodies to the native complex were raised according to a previously published protocol, is Detailed procedures for analysis of P D H C subunit profiles in mitochondrial extracts from skin fibroblasts by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting have appeared in earlier reports.18 Immunoblotting analysis was done on mitochondrial extracts derived from skin fibroblasts of the two patients, of a control subject, and of a patient who died at 3 years of age

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M a r s a c et al.

9 17

Table I. Enzyme activities in fibroblasts from patients 1 and 2 PC

PEPCK

COX

$CR

PDHC

Patient 1 (n) Patient 2 (n) Control subjects (n)

1.06 (1) 1.3 (1) 0.57 _+ 0.23 (20)

0.45 (1) 0.59 (1) 1.15 _+ 0.4 (13)

18.3-23.5 (2) 22.4-16.6 (2) 25.8 _+ 13.3 (28)

15.5 _+ 3.3 (4) 16.5 + 3.4 (4) 10.2 +_ 4 (34)

0.12 _+ 0.01 (5) 0.21 _+ 0.13 (5) 0.97 + 0.34 (26)

Activitiesof fiveenzymes--pyruvatecarboxylase(PC), phosphoenolpyruvateearboxykinase(PEPCK), cytochromec oxidase(COX), succinatecytochromec reductase (SCR), pyruvatedehydrogenasecomplex(PDHC)--were measuredon fibroblasthomogenates.Activitiesare expressedin nanomolesper minuteper milligram of proteinas mean _+SD. n, Numberof determinationsfor the iwo patients.

with severe lactic acidemia and unmeasurable PDHC activity. In vivo energy metabolism studied by 31p-NMR. The 31p. NMR spectra of the flexor digitorum muscles of the forearm were recorded at rest, during exercise, and during recovery. They were obtained on a 2-tesla magnet with a 3 cm diameter surface coil, as previously described. 19 The relative concentrations of the phosphate metabolites, phosphocreatine, inorganic phosphate, phosphomonoesters, phosphodiesters, and adenosine triphosphate were obtained by integration of the corresponding peaks, and the intracellular pH from the chemical shift of Pi relative to that of PCr. Unless specified otherwise, metabolite concentrations were normalized to the sum [PCr] + [Pi] + [PME]. The 71/2minute exercise consisted of repeated handgrips; the concentration ratio [PCr]/([PCr] + [Pi] + [PME])decreased to less than half its resting value at the end of the exercise. Patient 2 and his healthy sister and mother were successfully studied. At the time of the NMR study (1985) the patient was 14 years of age and his sister was 11. The patient was studied on two occasions. Useful spectra could not be obtained for another 4-year-old child because it is difficult to obtain intense voluntary exercise at that age. Statistics. Values are reported as mean + SD unless specified otherwise. Values were considered significantly different when they differed by more than 2 SD from the normal ones. RESULTS The results obtained during the two in vivo 31p-NMR studies of patient 2 were similar; average values are given here. Differences between the patient and his healthy sister and mother were present at rest and were as follows: low PCr (0.71 compared with 0,92 for normal subjects) and high Pi, with a Pi/ATP ratio of 0.72 (0.27 and 0.36 for the sister and mother, respectively). The PCr/Pi ratio was low (4.2 compared with 13 for the normal subjects), and PME and PDE values were increased. The pH at rest showed a slight tendency to alkalinity (7.1 compared with 7.03). The

pH decreased to 6.85 at the end of the exercise (6.65 for the sister and 6.35 for the mother). The rate of PCr recovery after the exercise was normal. The PDHC activity in cultured skin fibroblasts was markedly reduced to 12% and 22% of normal values for patients 1 and 2, respectively (Table I). With the radioactive method, we did not find any difference in individual E1 or E2 activities compared with those found in two control fibroblast lines stored under the same conditions for the same period (data not shown). In contrast, the pyruvate carboxylase activity was high and phosphoenolpyruvate carboxykinase, cytochrome c oxidase, and succinate cytochrome c reductase activities were all within the normal range. The decrease in PDHC activity was also found in skeletal muscle (fractions S and P) with a reduction in fraction S to 11% and 16% of normal values for patients 1 and 2, respectively (Table II). The immunoblot analysis (Figure) showed that in both the control subject and the "control" patient, the subunit profile of the E2, protein X, E 1a, and E 1/3 polypeptides appeared normal, whereas both of our patients had an identical defect, a complete absence of cross-reacting material corresponding to the protein X component of PDHC. Although the presence of the dihydrolipoyl dehydrogenase (E3) subunit could not be detected in these extracts because of the low sensitivity of the anti-PDHC serum, a separate study using a high-titer antiserum indicated that normal amounts of E3 were present in all these patients (data not shown). In some patients with E1-PDHC deficiency, the immunoreactive protein migration is not always associated with a decrease in E1 protein (Elc~ with or without El/3). Genetic studies of the E l a subunit of the complex in these two patients (karyotype in prometaphase) did not reveal any abnormalities (microdeletions) in this subunit. DISCUSSION We have described two patients with PDHC deficiency and demonstrated that the primary abnormality was in the X-lipoyl-containingcomponent. The great majority of the

9 18

Marsac et al.

The Journal of Pediatrics December 1993

Figure. Immunoblots of PDHC purified from bovine heart, mitochondrial extracts of fibroblasts from a control subject, our patients, and a control patient with severe lactacidemia associated with PDHC deficiency, with the use of antibody to the holoenzyme of PDHC purified from bovine heart. Lane M, 1 #g purified bovine heart PDHC; lanes 1 and 2, 10 and 40 ~g mitochondrial extracts of control fibroblasts; lanes 3 and 4, 10 and 40 ~g mitochondrial extracts from patient 1; lanes 5 and 6, 10 and 40 ~g mitochondrial extracts from patient 2; lanes 7 and 8, 5 and 20 ~g mitochondrial extracts from a control patient. The marker lane, M, shows the subunit cross-reactivity of the antiserum against the individual subunits of purified bovine heart PDHC. E3, the weakly cross-reacting band below subunit E2 in lane M, elicits the poorest immune response of all the subunits.

T a b l e II. E n z y m e activities in skeletal muscle (subcellular fractions S and P ) from patients 1 and 2

Patient 1 Patient 2 Control subjects (n)

PDHC-S

PDHC-P

COX-S

COX-P

SCR-S

$CR-P

0.62 0.86 5.48 _+ 2.27 (10)

12 ND 35.1 _+ 10.4 (10)

79.8 75.6 130 +_ 34 (14)

638 ND 783 +_ 196 (6)

32 ND 27.5 + 10 (14)

212 ND 189 _+ 61 (6)

Activities of three enzymes--PDHC, COX, and SCR--were studied on the two subcellular fractions, S (supernatant of the first low-speed centrifugation) and P (pellet of purified mitochondria). Activities are expressed in nanomoles per minute per milligram of protein as mean _+SD. n, Number of control subjects; ND, not determined.

reported cases of P D H C deficiency have involved defects in the E1 component. However, two main clinical forms seem to be distinguishable. 5 In one form, children die during the neonatal period with hypotonia and severe lactic acidosis; they have a residual P D H C activity t h a t is less t h a n 20% and therefore is strongly correlated with the severity of the

clinical features. 2 The second form corresponds to neurologic disorders in older children; the residual P D H C activity is variable (between 10% and 75%) a n d is not correlated with the severity of the clinical symptoms. O u r two patients h a d a progressive neurologic disorder a n d corresponded to the second group, in which the clinical features are fre-

The Journal o f Pediatrics Volume 123, Number 6

quently described as Leigh syndrome. Their clinical features were indistinguishable from those of patients with defects in the E1 component of PDHC. Many patients with necrotizing encephalomyelopathy have been described and have had different biochemical defects, all affecting cerebral energy metabolism: first, the E 1-PDHC deficiencies,3 but also cytochrome c oxidase deficiencies,2~ complex I deficiencies,21 and, recently, a point mutation in the mitochondrial D N A subunit 6 adenosinetriphosphatase 22 (unpublished observations). The predominance of cerebral nervous system dysfunction in PDHC deficiencies can be explained by the dependence of brain tissue on aerobic glucose metabolism and hence by a heavy reliance on PDHC function. Various mitochondrial myopathies have been described as showing one or more of the following alterations on 31p_ N M R muscle spectra23-25: increased Pi, decreased PCr, and/or decreased PCr/Pi ratio at rest; reduced decrease in pH during exercise; slow recovery of PCr or ATP or both. The abnormalities observed in the muscle of our patient 2 are therefore in agreement with those usually found in other mitochondrial myopathies. The reduced muscular acidification with exercise found here and in other mitochondrial myopathies is apparently paradoxical because of the increased lactic acid production by anaerobic glycolysis, as reflected by hyperlactacidemia. This may reflect an adaptation to increased production by increasing rates of acid extrusion from the cells.23 The increased levels of phosphomonoesters, which most likely represent sugar phosphates, probably result from increased glycolysis.At present, it seems that alterations found by 31p-NMR in mitochondrial myopathies are not specific for a given mitochondrial defect. However, the variety of existing mitochondrial defects and the small number of patients studied by N M R (the precise biochemical defect often being unknown) do not allow definite conclusions. The brain spectra were unfortunately of poor quality because of technical limitations of the N M R system available at the time. Our results indicate that our patients had an absence of protein X only. A similar molecular defect of PDHC was described by Robinson et al. 7 in another patient, a boy with hypotonia and psychomotor retardation; at 4 years of age he could neither speak nor walk. He had a 5-year-old sibling who was mentally retarded. As in the cases reported here, his parents were cousins. Treatment with vitamin B1 (thiamine, one of the coenzymes of PDHC) did not bring about any improvement, as in our patients. The individual activities of E l , E2, and E3 were normal, whereas the native PDHC activity was low and immunoblotting studies showed an absence of X component and normal bands corresponding to E1 and E2 in fibroblasts. The same author also described two new cases with chronic hyperlactatemia and PDHC deficiency. One

M a r s a c et al.

9 19

had an enzyme deficiency in the E2 component and very low E2 and X protein components on Western blotting of fibroblast proteins. The second had a decrease in overall PDHC activity and two distinct bands (instead of one) corresponding to the protein X component but no El, E2, or E3 deficiency or protein abnormality. We have shown that clinical manifestations with different degrees of severity in the same family can result from the same molecular defect of the PDHC, a complete absence of the protein X component. We speculate that the cerebral dysfunction reflects the degree of the PDHC deficiency in the brain. An obligatory step in the formation of the functional multienzyme complex is the correct association of E3 with the E2 core by the protein X component. This protein and subunit E2 are tightly associated structurally and functionally. Protein X has no intrinsic dihydrolipoyl transacetylase activity, but the lipoyl domain can be acetylated.26, 27 In mammalian cells the E2 and protein X subunits are also separate gene products. The isolated human E2 gene contains two lipoyl domains in tandem repeat at its N terminus; the protein X gene contains only a single lipoyl domain. A full-length clone for protein X from mammalian sources is not yet available, but analysis of a partial human complementary-DNA clone has revealed that it is a distinct gene (J. C. Neagle and J. G. Lindsay: unpublished data). The residual levels of PDHC activity (10% to 20%) observed in our two patients are similar to those observed in the in vitro studies of reconstituted mammalian PDHC when the protein X subunit has been removed by selective proteolysis (S. J. Sanderson and J. G. Lindsay: unpublished data). These data suggest that correct physical and functional interaction of E3 with the complex is mediated by the presence of protein X. In the absence of protein X, E3 binds very loosely to the E2 core assembly, resulting in greatly diminished overall complex activity. Further functional studies on protein X are necessary to determine whether it has additional catalytic or regulatory functions in addition to its role in E3 binding. REFERENCES

1. Reed LJ. Multienzyme complexes. Accounts of Chemical Research 1974;7:40-6. 2. Robinson BH, MacMillan H, Petrova-Benedict R, Sherwood WG. Variable clinical presentation in patients with defective E1 component of the pyruvate dehydrogenase complex. J PEDIATR 1987;111:525-33. 3. Ho L, Hu CW, Packman S, Patel P. Deficiency of the pyruvate dehydrogenase component in pyruvate dehydrogenase complex-deficient human fibroblasts. J Clin Invest 1986;78: 844-7. 4. De Meirleir L, Lissens W, Vamos E, Liebaers I. Pyruvate dehydrogenase (PDH) deficiency caused by a 21-base pair

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insertion mutation in the E l a subunit. Hum Genet 1992; 88:649-52. Dahl HM, Maragos C, Brown RM, Hansen LL, Brown GK. Pyruvate dehydrogenase deficiency caused by deletion of a 7-bp repeat sequence in the Elc~ gene. Am J Hum Genet 1990; 47:286-93. Kerr DS, Ho L, Berlin CM, et al. Systemic deficiency of the first component of the pyruvate dehydrogenase complex. Pediatr Res 1987;22:312-8. Robinson BH, MacKay N, Petrova-Bebedict R, Ozalp I, Coskun T, Stacpoole W. Defects in the E2 lip0yl transacetylase and the X-lipoyl containing component of the pyruvate dehydrogenase complex in patients with lactic acidemia. J Clin Invest 1990;85:1821-4. Solomon M, Stansbie D. A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts. Anal Biochem 1984;141:337-43. Clot JP, Benelli C, Fouque F, Bernard R, Durand D, PostelVinay MC. Pyruvate dehydrogenase activity is stimulated by growth hormone in human mononuclear ceils: a new tool to meaSure GH responsiveness in man. J Clin Endocrinol Metab 1992;74:i258-62. Appelmans F, Wattiaux R, de Puve C. Tissue fractionation studies. Biochem J 1955;59:438-45. King TE. Preparation of suceinate cytochrome c reductase and the cytochrome b particle and the constitution of succinate cytochrome c reductase. Methods in Enzymology 1967;10:21625. Hsia YE, Scully KJ. Propionic acidaemia: diagnosis by enzyme assay in frozen leucocytes. J PEDIATR 1973;3:625-8. Marsac C, Augereau C, FelmanG, WolfB, HansonT, Berger R. Prenatal diagnosis of pyruvate carboxylase deficiency. Clin Chim Aeta 1982;119:121-7. Ballard F, Hanson RW. Ph0sphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver. Biochem J 1967;104:866-71. Lowry OH, Rosenbrough N J, Farr AL, Randall RJ. Protein measurements with the folin phenol reagent. J Biol Chem 1951;193:265-75. Stanley C, Perham RN. Purification of 2-oxoacid dehydrogenase complexes from ox heart by a new method. Biochem J 1980;191:147-54.

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17. De Marcucci O., Lindsay JG Component X: an immunologieally distinct polypeptide associated with mammalian pyruvate dehydrogenase multi-enzyme complex. Eur J Biochem 1985; 149:641-8. 18. Clarkson GH, King TE, Linsday JG. Biosynthesis and processing of the large and small subunits of succinate dehydrogenase in cultured mammalian cells. Biochem J 1987;244:1520. 19. Duboc D, Jehenson P, Tran Dinh S, Marsac C, Syrota A, Fardeau M. Phosphorus N M R spectroscopy study of muscular enzyme deficiencies involving glycogenolysis and glycolysis. Neurology 1987;7:663-71. 20. Van Coster R, Lombes A, De Vivo DC, et al. Cytochrome c oxidase-associated Leigh syndrome: phenotypic features and pathogenetic speculations. J Neurol Sci 1991;104:97-111. 21. Van Erven PMM, Gabreels FJM, Ruitenbeek W, et al. Subacute necrotizing encephalomyelopathy (Leigh syndrome) associated with disturbed oxidation of pyruvate, malate and 2-oxoglutarate in muscle and liver. Acta Neurol Scand 1985;72:36~42. 22. Tatuch Y, Christodoulou J, Feigenbaum A, et al. Heter0plasmic mtDNA mutation (T---~G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high. Am J Hum Genet 1992;50:852-8. 23. Arnold DL, Taylor D J, Radda GK. Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann Neurol 1985;18:189-96. 24. Eleff S. Kennaway NG, Buist NRM, et al. 31P-NMR study of improvement in oxidative phosphorylation by vitamins K3 and C in a patient with a defect in electron transport at complex III in skeletal muscle. Proc Natl Acad Sci USA 1984;81:352933. 25. Morvan D, Jehenson P, Duboc D, Syrota A. Discriminant factor analysis of 31p-NMR spectroscopic data in myopathies. Magn Reson Med 1990;13:216-27. 26. Jilka JM, Rahmatullah M, Kazemi M, Roche TE. Properties of a newly characterized protein of the bovine kidney pyruvate dehydrogenase complex. J Biol Chem 1986;261:1858-67. 27. Niu XD, Browning KS, Behal RH, Reed LJ. Cloning and nucieotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Proc Nat Acad Sci USA 1988;85:7546-50.