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Familial intermittent ataxia due to a defect of the E1 component of pyruvate dehydrogenase complex
Journal of the Neurological Sciences, 1989, 93:311-318 Elsevier
311
JNS 03238
Familial intermittent ataxia due to a defect of the component of pyruvate dehydrogenase complex
E1
L.A. Bindoff 1'2, M.A. Birch-Machin t'2, L. Farnsworth 1'2, D. Gardner-Medwin ~, J.G. Lindsay 3 and D.M. Turnbull ~ ~Division of Clinical Neuroscience, School of Neurosciences, 2Department of Pharmacological Sciences, University of Newcastle upon Tyne (U.K.), and 3DeparOnent of Biochemistry, University of Glasgow, Glasgow (U.K.) (Received 17 May, 1989) (Revised, received 22 June, 1989) (Accepted 23 June, 1989)
SUMMARY
Disturbances of pyruvate metabolism have been implicated in the aetiology of several neurological disorders including Leigh's disease and familial ataxia. We have re-investigated a patient whose initial description documented intermittent ataxia, a presumed disorder of pyruvate metabolism and an X-linked pattern of inheritance. Recent studies showed he had slow oxidation of pyruvate, low pyruvate dehydrogenase complex (PDC) activity and immunochemical evidence of E1 deficiency in skeletal muscle mitochondria. This is consistent with the recent finding that the gene for EI~ is on the X chromosome.
Key words: Intermittent ataxia; Pyruvate dehydrogenase complex; E ~ subunit; X-linked inheritance pattern
INTRODUCTION
The pyruvate dehydrogenase complex (PDC) catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. It is composed of multiple copies of 3 enzymes, pyruvate dehydrogenase (El) [EC 1.2.4.1], dihydrolipoamide transacetylase (E2) Correspondence to." Dr. D.M. Turnbull, Division of Clinical Neuroscience, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K. 022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
312 [EC2.3.1.12] and dihydrolipoamide dehydrogenase (E3) [EC 1.8.1.4] (Reed and Yeaman 1987). The E 1 component is a heterotetramer consisting of 2 ~-subunits and 2 fl-subunits. In addition to the 3 major catalytic enzymes, P D C contains 2 enzymes involved in its regulation, a specific kinase which inactivates P D C by phosphorylating 3 serme residues on EI~ and a phosphatase which removes the phosphate groups and activates the enzyme (Reed and Yeaman 1987). A sixth component, protein X, has been identified but its function is uncertain (Hodgson et al. 1988). P D C deficiency has been reported in a variety of conditions including fatal congenital lactic acidosis (StrOmme et al. 1976), subacute necrotising encephalomyelopathy (Leigh's disease) (Kretzschmar et al. 1986; Kerr et al. t987) and intermittent ataxia (Blass 1980; Evans 1981). Indeed, defects of P D C are thought to be the commonest cause of primary lactic acidosis (Robinson et al. 1980; Stansbie et al 1986). The majority of defects of P D C appear to involve the E~ component (Ho et al, 1986; Kerr et al. 1987; Birch-Machin et al. 1988; Wexler et al. 1988; Robinson 1988). Recently, Brown et al. (1989) located the gene for E l~ on the X chromosome, which would explain the X-linked pattern of inheritance of some of the diseases associated with deficiency of the E 1 component of P D C (Kerr et al. 1988). We re-investigated a young man with a long history of intermittent ataxia and metabolic acidosis (Livingstone et al. 1984). There is a family history of similar symptoms with an inheritance pattern suggesting X-linked transmission. P D C activity was low in skeletal muscle mitochondria and there were low amounts of immunochemically detectable E ~ and E ~ subunits. We suggest that this patient has a deficiency of the E~ subunit of P D C as the cause of his clinical syndrome.
CASE REPORT The patient is a 22-year-old male whose clinical details and family history have been reported (Livingstone et al. 1984). He has continued to have episodes of ataxia lasting from a few hours to several days, similar to those described previously. These ataxic episodes are caused by excess exertion or intercurrent infection; exercise is also associated with muscle discomfort. Examination shows that even between attacks he has a mildly ataxic gait, pale optic discs, generalised hyperreflexiaand mild dementia. No new cases have been described in the family(Fig. 1), but the patient's brother (case IV.I) died recently during an episode associated with severe hyperventilation and ataxia.
MATERIALS AND METHODS Muscle was obtained by biopsy from vastus lateralis. A portion was processed for histological examination (Johnson 1983) and the remainder used to prepare a mitochondrial fraction (Watmough et al. 1988), The rates of substrate oxidation were measured polarographically (Sherratt et al. 1988) and spectrophotometrically (Turnbull et al. 1982). P D C activity was measured spectrophotometrically (Coore et al. 1971) in 5 0 - 1 0 0 # g of muscle mitochondriat extracts prepared as previously described (Birch-Machin et al. 1988). Citrate synthase activity was measured according to
313
l
i 2] , l 6rfi666 666 rhb
IV 1
2
3
4
5
6
7
8
9
14 15
• Clinically affected ~)" J~ Deceased Fig. 1. Family tree.
Shepherd and Garland (1969) and protein estimated by a modified Lowry method (Pedersen 1974). Western blots were performed using the discontinuous system described by Laemmli (1970) with antibodies to holo-PDC, E 1 and El~ (Birch-Machin et al. 1988).
RESULTS
Morphological and histochemical analysis of the patient's muscle was normal. There was poor oxidation of pyruvate by the patient's muscle mitochondrial fraction, but the rate of oxidation of succinate, oxoglutarate and glutamate plus malate TABLE 1 RATES OF O X I D A T I O N OF NAD ÷-LINKED SUBSTRATES AND SUCCINATE IN SKELETAL MUSCLE M I T O C H O N D R I A L FRACTIONS Rates are expressed as nmol ferricyanide reduced (in the presence of 10 mM A D P ) ' min ~• m g - ~ protein. The figures shown for controls are mean ± SD and represents the values for 13 subjects.
10 mM 10 mM 10 mM 10 mM
succinate glutamate + 1 mM malate pyruvate + 1 mM malate oxoglutarate
Patient
Controls
250 80 73 119
274 107 208 138
± ± ± ±
34 42 53 28
314
1
2
3
)
•
Mr E2
E3 X
57
EI~ 40
Ell3 29
12.4 Fig. 2. [mmunoblot analysis of PDC in human skeletal muscle mitochondrial fractions. Polypeptides in skeletal muscle mitochondrial fractions from control and patient were separated by SDS-polyaerylamide gel eleetrophoresis (10% gel) and immunoblotted using antibodies to ox heart PDC. Skeletal muscle mitoehondrial fractions from: lane 2, control (10 #g protein) and lane 3, patient (10 #g protein). Lane t. purified ox heart PDC (2/~g protein). The positions of the molecular weight standards are shown on the right. The extra bands seen in all lanes at approx. 48 Mr and 39Mr represent proteotytie fragments of E2 and are equivalent in the patient and control samples.
were normal (Table 1). The poor oxidation of pyruvate was confLrmed polarographically (patient, 29 ng atoms O . m g - 1 protein, min - ~; control 124). P D C activity was low (0.058 units enzyme activity, r a g - ~ protein; control range for 3 subjects (0.092-0.16 units, m g - ~ protein)). Citrate synthase activity was normal (0.97 units • m g - ~ protein; controls (6) 1.1 + 0.2 (mean + SD)). W h e n reacted against the antibody to h o l o - P D C there was a tow amount of E l , but normal amounts o f E 2, E 3 and X c o m p a r e d with control fractions (Fig. 2). This antibody does not react strongty against E 3 (De Marcucci et al. 1985). The low amounts o f El,, and E~t~were confLrmed using anti-E 1 antibody (Fig. 3). This shows some remaining e r o s s - r e a e t i ~ material at 42 k D a which was not seen when using anti-El~ antibody (Fig. 4) suggesting that this material is not EI~ but a breakdown product o f E z (De Marcucci and Lindsay 1985).
315
5
Mr
i~!!i ~ ili~i~ii!i !
-57
EI~
i -40
-29
-12.4 Fig. 3. Immunoblot analysis ofthe E 1component ofPDC in human skeletalmuscle mitochondrialfractions. Skeletal muscle mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis (10% gel), and immunoblotted using antibodies raised against component El of ox heart PDC. Lanes 1 and 5, purified ox heart PDC (5 and 4/~g protein); lanes 2 and 4, controls (75 #g protein); lane 3, patient (75/~g protein). DISCUSSION We have confirmed a defect of pyruvate metabolism in this patient. The oxidation of pyruvate was slow both in intact mitochondria and by direct measurement of P D C activity. The site of the defect was established by Western blotting which showed very low concentrations o f EI~ and Ela subunits of PDC. A defect of the respiratory chain was excluded by the normal rates of oxidation of the other N A D ÷ -linked substrates and succinate. Recently it has been reported that the E ~ gene is located on the X c h r o m o s o m e (Brown et al. 1989). We believe the defect in our patient involves the E I. subunit because the family history suggests an X-linked disorder and we have shown loss of this subunit.
316 1
2
3
4
Mr
57 EI~
40
29
12.5
Fig. 4. lmmunoblot analysis of the E]~ component of PDC in human skeletal muscle mitochondrial fractions. Skeletal muscle mitochondrial proteins were separated by SDS-polyaerylamidegel electrophoresis (10% gd), and immunoblottedusing antibodies raised against componentE~ ofox heart PDC. Lane 1, purifiedox heart PDC (4/~gprotein); lanes 2 and 4, controls (50/ag protein); lane 3, patient (50/~g protein).
The low concentration of E 1~ is presumably due either to increased degradation of a subunit not incorporated normally into the enzyme complex (Wexler et aI. 1988) or. impaired transport of the Ela precursor into mitochondria due to lack o f the El~ precursor which may be necessary for its transport (DeMareucci et al. 1988). PDC deficiency is a significant cause of morbidity and mortality, especially in children. Despite the variable clinical presentations there do appear to be distinc~t groups within the broad category of PDC deficiency; one with a fulminant lactic acidosis presenting at birth, presumably with already established damage of the CNS (Aleck et al. 1988); one with significant lactic aeidosis (occasionally only detectable in C S F (Brown et al. 1988)), severe, progressive CNS damage (with or without evidence of Leigh's disease), including agenesis of the corpus callosum, cystic lesions, esl~ciatty in
317 basal ganglia, cerebral atrophy and ventricular enlargement, microcephaly and various dysmorphic features (Robinson et al. 1987); and a third with mild intermittent lactic acidosis, episodic ataxia, usually associated with intercurrent stress such as infection, and CNS damage which is often minor but which can sometimes lead to progressive disability (this patient; Robinson et al. 1987). Unfortunately, there appears to be little correlation between the site of the defect, even with defects of E 1, and the severity of the clinical features. Brown et al. (1989) found low concentrations of EI~ in 2 patients with severe symptoms present from birth. Wexler et al. (1988) studied the molecular nature of the defect in 11 patients with E l deficiency using monospecific antibodies and c D N A probes. They defined 3 groups: one in which they found cross-reacting material (CRM) for the ~- and fl-subunits plus m R N A for these proteins, one with no CRM, but with m R N A , and one with neither C R M or mRNA. There was no correlation between the clinical expression of the disease and the presence or absence of C R M or m R N A of the 2 subunits. Our results have shown a marked deficiency of the E l component of PDC. We believe this is due to the loss of the EI~ subunit and if so, it is the first time this has been shown to produce the milder syndrome associated with intermittent ataxia. It remains possible, however, that loss of either subunit could give rise to the findings we have described. Clearly, there is still much to learn about these disorders, but it is to be hoped that as molecular investigation advances better correlation between genetic defect and clinical disease will be established making possible accurate diagnosis, carrier detection and antenatal diagnosis.
ACKNOWLEDGEMENTS We are grateful to Dr. M.A. Johnson for performing the cytochemistry, Mrs. S. Lowe for help with the manuscript, and Dr. H. S.A. Sherratt and Dr. G. K. Brown for helpful discussions. This work was supported by the Muscular Dystrophy Group of Great Britain, the Medical Research Council and Newcastle University Research Committee.
REFERENCES Aleck, K.A., A.M. Kaplan, W.G. Sherwood and R H. Robinson (1988) In utero central nervous system damage in pyruvate dehydrogenase deficiency.Arch. Neurol., 45: 987-989. Birch-Machin, M.A., I.M. Shepherd, M. Solomon, S.J. Yeaman, D. Gardner-Medwin, H.S.A. Sherratt, J.G. Lindsay, A. Aynsley-Greenand D.M. Turnbull (1988) Fatal lactic acidosis due to a deficiency of E~ component of the pyruvate dehydrogenase complex.J. lnher. Metab. DIS., 11: 207-217. Biass, J. P. (1980) Pyruvate dehydrogenase.In: D. Burman,J. B. Holten and C.A. Pennock (Eds.), Disorders of Carbohydrate Metabolism, MTP Press, Lancaster, pp. 239-267. Brown, G.K., E.A. Haan, D.M. Kirby, R.D. Scholem,J.E. Wraith, J.G. Rogers and D.M. Danks (1988) "Cerebral" lactic acidosis: defects in pyruvate metabolismwith profound brain damage and minimal systemic acidosis. Eur. J. Pediatr., 147: 10-14. Brown, R. M., H-H.M. Dahl and G.K. Brown (1989) X chromosomelocalisation of the functional gene for the E~ subunit of the human pyruvate dehydrogenase complex. Genomics, 4:174-181.
318 Coore, H.G., R.M. Denton, B.R. Martin and P.J. Randle (1971) Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones. Biochem. J., 125:115-127. De Marcucci, O.G.L. and J.G. Lindsay (1985) Component X. An immunologically distinct potypeptide associated with mammalian pyruvate dehydrogenase multi-enzyme complex. Eur. J Biochem. 149: 641-648. De Marcucci, O. G. L.. A. Hunter and J.G. Lindsay (1985) Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes. Biochem, J., 226:509-517 De Marcucci, O.G.L., G.M. Gibb, J. Dick, and J.G. Lindsay (1988) Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex. Biochem. J.. 251: 817-823. Evans. O.B. (1981) Pyruvate carboxylase deficiency in subacute necrotising encephalomyeiopathy. Arch. Neurol.. 38: 515-5t9. Ho, L., C.W.C. Hu, S. Packman and M.S. Patel I1986) Deficiency of the pyruvate dehydrogenase component in pyruvate dehydrogenase complex-deficient human fibroblasts. Immunological identification. J. Clin. Invest.. 78: 844-847. Hodgson, J.A., O. G. L. De Marcucci and J.G. Lindsay (1988) Structure function studies on the lipoateacetyltransferase-component-X-core assembly of the ox heart pyruvate dehydrogenase complex. Eur. J. Biochem.. 171: 609-614. Johnson, M.A. (1983) Skeletal muscle. In: M.L Filipe and B.D. Lake (Eds.), Histochemistry in Pathology, Churchill Livingstone, Edinburgh and London. pp, 89-113. Kerr, D.S., U Ho. C.M. Berlin, K.F. Lanoue, J. Towfighi, C.L. Hoppel, M.M. Lusk, C.M. Gondek and M.S. Patel (1987) Systemic defect of the first component of the pyruvate dehydrogenase complex. Pediatr. Res., 22: 312-318. Kerr. D. S., S.A. Berry, M.M. Lusk, L. Ho and M. S. Patel (1988) A deficiency of both subunits of pyruvate dehydrogenase which is not expressed in fibrobtasts. Pediatr. Res., 24: 95-100. Kretzschmar, H. A., S. J. DeArmond, T. K. Koch, M. S. Patel, C. J, L. Newth, K. A. Schmidt and S. Packman (1987) Pyruvate dehydrogenase complex deficiency as a cause of subacute neerotisingencephalopathy (Leigh disease). Pediatrics, 79:370-373 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T~ Nature, 227: 680-685. Livingstone, I.R.. D. Gardner-Medwin and R,J.T. Pennington (1984) Familial intermittent ataxia with possible X-linked recessive inheritance. J. Neurol. Sci.. 64: 89-97. Pedersen, G.L. (I 977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem., 83: 346-356. Reed, L.J. and S.J. Yeaman (1987) Pyruvate dehydrogenase. In: P.D. Boyer and E.G. Krebs (Eds. ~, The Enzymes, Vol. 18, Academic Press, New York and London, pp. 77-95. Robinson, B.H. (1988) Cell culture studies on patients with mitochondrial diseases: molecular defects in pyruvate dehydrogenase. J. Bioenerg. Biomemb., 20: 3t3-323. Robinson. B. H., J. Taylor and W.G. Sherwood (1980) The genetic heterogeneity of lactic acidosis: occurrence of recognisable inborn errors of metabolism in a pediatric population with lactic acidosis. Pediatr. Res.. 14: 956-962. Robinson, B. H., H. MacMillan. R. Petrova-Benedict and W.G. Sherwood (1987) Variable clinical presentation in patients with defective E~ component of pyruvate dehydrogenase complex~ J. Pediatr.. 11 t: 525-533. Shepherd, D. and P.B. Garland (19731 Citrate synthase from rat liver. Methods Enzymol.. 13:11-16. Sherratt, H. S. A., N.J. Watmough, M.A. Johnson and D.M. Turnbull (1988) Methods for study of normal and abnormal skeletal muscle mitochondria. Methods Biochem. Anal., 33: 243-335. Stansbie, D., S.J. Wallace and C. Marsac (1986) Disorders of the pyruvate dehydrogenase complex. J. Inherit. Metab. Dis., 9: 105-119. Strtimme, J. H., O. Borud and P.J. Moe (1976) Fatal lactic acidosis in a newborn attributable to a congenital defect in pyruvate dehydrogenase. Pediatr. Res., 10: 60-66. TurnbuI1, D. M., H. S. A. Sherratt. D.M. Davies and A. G. Sykes (1982) Tetracyano-2,2-bipyridineiron (!It), an improved electron acceptor for the speetrophotometric assay offloxidation and succinate dehydrogenase m intact mitochondria. Biochern. J.. 206: 511-516. Watmough, N.J., A. K.M.J. Bhuiyan, K. Bartlett. H. S.A. Sherratt and D.M. Turnbull (1988) Skeletal muscle mitochondrial fl-oxidation. A study of the products of [U-14C]hexadecanoate oxida~tion by h.p.l.c, using continuous on-line radiochemical detection. Biochem. J., 253: 541-547. Wexler, I.D., D.S. Kerr, L. Ho, M.M. Lusk, R.A. Pepin, A.A. Javed, J.E. Mote. B.W. Jesse, T.J. Thekkumkara, G. Pons and M. S. Patel (1988) Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency. Proc. Natl. Acad. Sci. USA, 85: 7336-7340.
311
JNS 03238
Familial intermittent ataxia due to a defect of the component of pyruvate dehydrogenase complex
E1
L.A. Bindoff 1'2, M.A. Birch-Machin t'2, L. Farnsworth 1'2, D. Gardner-Medwin ~, J.G. Lindsay 3 and D.M. Turnbull ~ ~Division of Clinical Neuroscience, School of Neurosciences, 2Department of Pharmacological Sciences, University of Newcastle upon Tyne (U.K.), and 3DeparOnent of Biochemistry, University of Glasgow, Glasgow (U.K.) (Received 17 May, 1989) (Revised, received 22 June, 1989) (Accepted 23 June, 1989)
SUMMARY
Disturbances of pyruvate metabolism have been implicated in the aetiology of several neurological disorders including Leigh's disease and familial ataxia. We have re-investigated a patient whose initial description documented intermittent ataxia, a presumed disorder of pyruvate metabolism and an X-linked pattern of inheritance. Recent studies showed he had slow oxidation of pyruvate, low pyruvate dehydrogenase complex (PDC) activity and immunochemical evidence of E1 deficiency in skeletal muscle mitochondria. This is consistent with the recent finding that the gene for EI~ is on the X chromosome.
Key words: Intermittent ataxia; Pyruvate dehydrogenase complex; E ~ subunit; X-linked inheritance pattern
INTRODUCTION
The pyruvate dehydrogenase complex (PDC) catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. It is composed of multiple copies of 3 enzymes, pyruvate dehydrogenase (El) [EC 1.2.4.1], dihydrolipoamide transacetylase (E2) Correspondence to." Dr. D.M. Turnbull, Division of Clinical Neuroscience, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K. 022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
312 [EC2.3.1.12] and dihydrolipoamide dehydrogenase (E3) [EC 1.8.1.4] (Reed and Yeaman 1987). The E 1 component is a heterotetramer consisting of 2 ~-subunits and 2 fl-subunits. In addition to the 3 major catalytic enzymes, P D C contains 2 enzymes involved in its regulation, a specific kinase which inactivates P D C by phosphorylating 3 serme residues on EI~ and a phosphatase which removes the phosphate groups and activates the enzyme (Reed and Yeaman 1987). A sixth component, protein X, has been identified but its function is uncertain (Hodgson et al. 1988). P D C deficiency has been reported in a variety of conditions including fatal congenital lactic acidosis (StrOmme et al. 1976), subacute necrotising encephalomyelopathy (Leigh's disease) (Kretzschmar et al. 1986; Kerr et al. t987) and intermittent ataxia (Blass 1980; Evans 1981). Indeed, defects of P D C are thought to be the commonest cause of primary lactic acidosis (Robinson et al. 1980; Stansbie et al 1986). The majority of defects of P D C appear to involve the E~ component (Ho et al, 1986; Kerr et al. 1987; Birch-Machin et al. 1988; Wexler et al. 1988; Robinson 1988). Recently, Brown et al. (1989) located the gene for E l~ on the X chromosome, which would explain the X-linked pattern of inheritance of some of the diseases associated with deficiency of the E 1 component of P D C (Kerr et al. 1988). We re-investigated a young man with a long history of intermittent ataxia and metabolic acidosis (Livingstone et al. 1984). There is a family history of similar symptoms with an inheritance pattern suggesting X-linked transmission. P D C activity was low in skeletal muscle mitochondria and there were low amounts of immunochemically detectable E ~ and E ~ subunits. We suggest that this patient has a deficiency of the E~ subunit of P D C as the cause of his clinical syndrome.
CASE REPORT The patient is a 22-year-old male whose clinical details and family history have been reported (Livingstone et al. 1984). He has continued to have episodes of ataxia lasting from a few hours to several days, similar to those described previously. These ataxic episodes are caused by excess exertion or intercurrent infection; exercise is also associated with muscle discomfort. Examination shows that even between attacks he has a mildly ataxic gait, pale optic discs, generalised hyperreflexiaand mild dementia. No new cases have been described in the family(Fig. 1), but the patient's brother (case IV.I) died recently during an episode associated with severe hyperventilation and ataxia.
MATERIALS AND METHODS Muscle was obtained by biopsy from vastus lateralis. A portion was processed for histological examination (Johnson 1983) and the remainder used to prepare a mitochondrial fraction (Watmough et al. 1988), The rates of substrate oxidation were measured polarographically (Sherratt et al. 1988) and spectrophotometrically (Turnbull et al. 1982). P D C activity was measured spectrophotometrically (Coore et al. 1971) in 5 0 - 1 0 0 # g of muscle mitochondriat extracts prepared as previously described (Birch-Machin et al. 1988). Citrate synthase activity was measured according to
313
l
i 2] , l 6rfi666 666 rhb
IV 1
2
3
4
5
6
7
8
9
14 15
• Clinically affected ~)" J~ Deceased Fig. 1. Family tree.
Shepherd and Garland (1969) and protein estimated by a modified Lowry method (Pedersen 1974). Western blots were performed using the discontinuous system described by Laemmli (1970) with antibodies to holo-PDC, E 1 and El~ (Birch-Machin et al. 1988).
RESULTS
Morphological and histochemical analysis of the patient's muscle was normal. There was poor oxidation of pyruvate by the patient's muscle mitochondrial fraction, but the rate of oxidation of succinate, oxoglutarate and glutamate plus malate TABLE 1 RATES OF O X I D A T I O N OF NAD ÷-LINKED SUBSTRATES AND SUCCINATE IN SKELETAL MUSCLE M I T O C H O N D R I A L FRACTIONS Rates are expressed as nmol ferricyanide reduced (in the presence of 10 mM A D P ) ' min ~• m g - ~ protein. The figures shown for controls are mean ± SD and represents the values for 13 subjects.
10 mM 10 mM 10 mM 10 mM
succinate glutamate + 1 mM malate pyruvate + 1 mM malate oxoglutarate
Patient
Controls
250 80 73 119
274 107 208 138
± ± ± ±
34 42 53 28
314
1
2
3
)
•
Mr E2
E3 X
57
EI~ 40
Ell3 29
12.4 Fig. 2. [mmunoblot analysis of PDC in human skeletal muscle mitochondrial fractions. Polypeptides in skeletal muscle mitochondrial fractions from control and patient were separated by SDS-polyaerylamide gel eleetrophoresis (10% gel) and immunoblotted using antibodies to ox heart PDC. Skeletal muscle mitoehondrial fractions from: lane 2, control (10 #g protein) and lane 3, patient (10 #g protein). Lane t. purified ox heart PDC (2/~g protein). The positions of the molecular weight standards are shown on the right. The extra bands seen in all lanes at approx. 48 Mr and 39Mr represent proteotytie fragments of E2 and are equivalent in the patient and control samples.
were normal (Table 1). The poor oxidation of pyruvate was confLrmed polarographically (patient, 29 ng atoms O . m g - 1 protein, min - ~; control 124). P D C activity was low (0.058 units enzyme activity, r a g - ~ protein; control range for 3 subjects (0.092-0.16 units, m g - ~ protein)). Citrate synthase activity was normal (0.97 units • m g - ~ protein; controls (6) 1.1 + 0.2 (mean + SD)). W h e n reacted against the antibody to h o l o - P D C there was a tow amount of E l , but normal amounts o f E 2, E 3 and X c o m p a r e d with control fractions (Fig. 2). This antibody does not react strongty against E 3 (De Marcucci et al. 1985). The low amounts o f El,, and E~t~were confLrmed using anti-E 1 antibody (Fig. 3). This shows some remaining e r o s s - r e a e t i ~ material at 42 k D a which was not seen when using anti-El~ antibody (Fig. 4) suggesting that this material is not EI~ but a breakdown product o f E z (De Marcucci and Lindsay 1985).
315
5
Mr
i~!!i ~ ili~i~ii!i !
-57
EI~
i -40
-29
-12.4 Fig. 3. Immunoblot analysis ofthe E 1component ofPDC in human skeletalmuscle mitochondrialfractions. Skeletal muscle mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis (10% gel), and immunoblotted using antibodies raised against component El of ox heart PDC. Lanes 1 and 5, purified ox heart PDC (5 and 4/~g protein); lanes 2 and 4, controls (75 #g protein); lane 3, patient (75/~g protein). DISCUSSION We have confirmed a defect of pyruvate metabolism in this patient. The oxidation of pyruvate was slow both in intact mitochondria and by direct measurement of P D C activity. The site of the defect was established by Western blotting which showed very low concentrations o f EI~ and Ela subunits of PDC. A defect of the respiratory chain was excluded by the normal rates of oxidation of the other N A D ÷ -linked substrates and succinate. Recently it has been reported that the E ~ gene is located on the X c h r o m o s o m e (Brown et al. 1989). We believe the defect in our patient involves the E I. subunit because the family history suggests an X-linked disorder and we have shown loss of this subunit.
316 1
2
3
4
Mr
57 EI~
40
29
12.5
Fig. 4. lmmunoblot analysis of the E]~ component of PDC in human skeletal muscle mitochondrial fractions. Skeletal muscle mitochondrial proteins were separated by SDS-polyaerylamidegel electrophoresis (10% gd), and immunoblottedusing antibodies raised against componentE~ ofox heart PDC. Lane 1, purifiedox heart PDC (4/~gprotein); lanes 2 and 4, controls (50/ag protein); lane 3, patient (50/~g protein).
The low concentration of E 1~ is presumably due either to increased degradation of a subunit not incorporated normally into the enzyme complex (Wexler et aI. 1988) or. impaired transport of the Ela precursor into mitochondria due to lack o f the El~ precursor which may be necessary for its transport (DeMareucci et al. 1988). PDC deficiency is a significant cause of morbidity and mortality, especially in children. Despite the variable clinical presentations there do appear to be distinc~t groups within the broad category of PDC deficiency; one with a fulminant lactic acidosis presenting at birth, presumably with already established damage of the CNS (Aleck et al. 1988); one with significant lactic aeidosis (occasionally only detectable in C S F (Brown et al. 1988)), severe, progressive CNS damage (with or without evidence of Leigh's disease), including agenesis of the corpus callosum, cystic lesions, esl~ciatty in
317 basal ganglia, cerebral atrophy and ventricular enlargement, microcephaly and various dysmorphic features (Robinson et al. 1987); and a third with mild intermittent lactic acidosis, episodic ataxia, usually associated with intercurrent stress such as infection, and CNS damage which is often minor but which can sometimes lead to progressive disability (this patient; Robinson et al. 1987). Unfortunately, there appears to be little correlation between the site of the defect, even with defects of E 1, and the severity of the clinical features. Brown et al. (1989) found low concentrations of EI~ in 2 patients with severe symptoms present from birth. Wexler et al. (1988) studied the molecular nature of the defect in 11 patients with E l deficiency using monospecific antibodies and c D N A probes. They defined 3 groups: one in which they found cross-reacting material (CRM) for the ~- and fl-subunits plus m R N A for these proteins, one with no CRM, but with m R N A , and one with neither C R M or mRNA. There was no correlation between the clinical expression of the disease and the presence or absence of C R M or m R N A of the 2 subunits. Our results have shown a marked deficiency of the E l component of PDC. We believe this is due to the loss of the EI~ subunit and if so, it is the first time this has been shown to produce the milder syndrome associated with intermittent ataxia. It remains possible, however, that loss of either subunit could give rise to the findings we have described. Clearly, there is still much to learn about these disorders, but it is to be hoped that as molecular investigation advances better correlation between genetic defect and clinical disease will be established making possible accurate diagnosis, carrier detection and antenatal diagnosis.
ACKNOWLEDGEMENTS We are grateful to Dr. M.A. Johnson for performing the cytochemistry, Mrs. S. Lowe for help with the manuscript, and Dr. H. S.A. Sherratt and Dr. G. K. Brown for helpful discussions. This work was supported by the Muscular Dystrophy Group of Great Britain, the Medical Research Council and Newcastle University Research Committee.
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