J. Inher. Metab. Dis. 15 (1992) 448-455 © SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
Clinical Aspects of Mitochon rial Disor ers A. MUNNICH, P. RUSTIN, A. ROTIG, D. CHRETIEN, J.-P. BONNEFONT, C. NUTTIN, V. CORMIER,A. VASSAULT, P. PARVY, J. BARDET, C. CHARPENTIER, D. RABIER, J.-M. SAUDUBRAY Unitd de Recherches sur les Handicaps Gdn(tiques de l'Enfant I N S E R M U-12, D@artement de Pddiatrie and D@artment de Biochimie, H@itaI des EnfantsMalades, 149 rue de SOvres, 75743 Paris Cedex 15, France
Summary: Mitochondrial disorders have long been regarded as neuromuscular diseases only. In fact, owing to the ubiquitous nature of the oxidative phosphorytation, a broad spectrum of clinical features should be expected in mitochondrial disorders. Here, we present eight puzzling observations which give support to the view that a disorder of oxidative phosphorylation can give rise to any symptom in any organ or tissue with any apparent mode of inheritance. Consequently, we suggest giving consideration to the diagnosis of a mitochondrial disorder when dealing with an unexplained association of symptoms, with an early onset and a rapidly progressive course involving seemingly unrelated organs. Determination of lactate/pyruvate and ketone body molar ratios in plasma can help to select patients at risk for this condition.
Since the original report by Luft and colleagues (1962), mitochondrial disorders have been regarded as neuromuscular diseases. In fact, oxidative phosphorylation, which includes the oxidation of fuel molecules by oxygen and the concomitant energy transduction into ATP, is not only present in the central nervous system and muscular systems. Indeed, a number of non-neuromuscular organs and tissues are highly dependent upon mitochondrial energy supply (Wallace t989). Consequently, a disorder of oxidative phosphorylation could theoretically give rise to a wide range of symptoms in any organ or tissue. In a&tition, any mode of inheritance should be expected, owing to the particular genetic origin of mitochondrial respiratory enzymes (Wallace 1989). For this reason, the diagnosis of a mitochondrial disorder is a difficult one to consider early, particularly when the primary symptom is the only one present. By contrast, this diagnosis is easier to consider when two seemingly unrelated symptoms are observed. This paper presents evidence in support of this view and demonstrates that genetic defects of oxidative phosphorylation account for a variety of neuromuscular and non-neuromuscular symptoms in childhood. 448
Clinical Aspects of MitochondriaI Disorders
449
METHODS
Determination of the oxidoreduction status in plasma: Since the respiratory chain transfers N A D H to oxygen, a disorder of oxidative phosphorylation should result in a markedly altered oxidoreduction status in plasma. This feature is a consequence of the functional impairment of the Krebs cycle, due to the excess of N A D H and the lack of NAD, with secondary elevation of blood lactate and an increase of the ketone body and lactate/pyruvate molar ratios in affected individuals. This is particularly true in the post-absorptive period, when more N A D is required for the adequate metabolism of glycolytic substrates. Similarly, as a consequence of the Krebs cycle impairment, ketone body synthesis increases after meals, instead of decreasing, owing to the channelling of acetyl-CoA toward ketogenesis (RStig et al 1990). Consequently, current screening for genetic defects of oxidative phosphorylation must include the determination of lactate, pyruvate, ketone bodies and their molar ratios in both fasted and fed individuals (Table I). When the test is not conclusive, one must try to make it more sensitive by a glucose (or pyruvate) loading test, by an exercise test, or by looking for an abnormal oxidoreduction status in the cerebropsinal fluid (Table 1). Assessment of the mitochondrial respiratory enzymes: The metabolic investigations listed in Table 1 are routine indices of an abnormal oxidoreduction status and should prompt further enzyme investigations on fresh tissues. These investigations include the measurement of oxygen consumption by isolated mitochondria (polarography) and the assessment of enzyme activities by spectrophotometric studies (needle biopsy of the liver; open muscle biopsy under local anaesthesia, 100 200 mg of fresh tissue, usually deltoid) (Sengers et al 1984; Chr6tien et al 1990). Results are presented not only as absolute values but as ratios as well. Indeed, it has recently been observed that a constant ratio of respiratory enzyme activities is a consistent feature, regardless of the tissue tested (Rustin et al 1991). Under these conditions, patients whose absolute values were in the low normal range could be diagnosed as enzyme deficient. Although this does not hold for generalized defects, it does improve the ability to recognize localized deficiencies in the respiratory chain (Rustin et al 1991). Table 1 Metabolic investigations
Standard screening 1. Lactate/pyruvate molar ratios in plasma (fasted and fed) = redox status in the cytoplasm 2. Ketone body molar ratios in plasma (fasted and fed) = redox status in the mitochondria 3. ~Paradoxical' hyperketonaemia (]~d) 4. Urinary organic acids (GC-MS) Urinary lactate, ketone bodies Krebs cycle intermediates When inconclusive 5. Glucose (or pyruvate) loading test (2g/kg) 6. Lactate/pyruvate molar ratios in the CSF (standard or after glucose loading test) 7. Redox status after exercise test
J. Inher. Metab. Dis. 15 (1992)
450
M u n n i c h et al.
RESULTS AND DISCUSSION As expected from the ubiquitous nature of the oxidative phosphorylation, a broad spectrum of clinical features was present at the very early stage of the disease. As a consequence, the children were seen initially by various specialists, including paediatricians, neuropaediatrieians, intensive-care units, cardiologists, haematologists, nephrologists, endocrinologists, hepatogastro-enterologists, etc. Using the screening procedures described above (see Methods and Table 1), in the past 3 years 104 infants and children were found to have an abnormal oxidoreduction status in their plasma and underwent a muscle biopsy. Among them, 28 (27%) were found enzyme deficient, including 5 complex I, 2 complex II, 2 complex III, and 15 complex IV deficiency. Four had a generalized respiratory enzyme defect. Presenting symptoms included neurological and muscular diseases such as muscle weakness, myoclonic epilepsy and ragged red fibres (MERRF) and Leigh syndrome, but these accounted for less than 20% of all our patients. More interestingly, a variety of non-neuromuscular organs were initially involved in many patients (Table 2). In order to illustrate the diversity of the presenting symptoms in our respiratory enzyme-deficient patients we have looked at the clinical aspects of the disease at the time of initial consultation with the physician; that is, we have examined the clinical profile of our patients at the very early stage of the disease. Here, we report on eight puzzling and unexpected presentations of mitochondrial disorders. Presenting symptom: failure to thrive
A girl, born to unrelated healthy parents, had failure to thrive as the presenting symptom. She had anorexia, diarrhoea and vomiting in the first 12 months of age. Since she had total villous atrophy, she was considered to be gluten intolerant or milk protein intolerant, but her condition did not improve on appropriate dietary formula. When she was 20 months old, seemingly unrelated symptoms occurred, namely distal tubulopathy and metabolic acidosis with major loss of bicarbonate
Table 2 A broad spectrum of presenting symptoms
Metabolic: ketoacidotic coma Liver: hepatic failure Bone marrow: pancytopenia, macrocytic anaemia Heart: cardiomyopathy, 'near miss cot death' Kidney: tubutopathy, vitamin-unresponsive rickets Gut: diarrhoea, villous atrophy, failure to thrive Pancreas: exocrine pancreatic dysfunction Endocrine: diabetes, dwarfism, hypoparathyroidism Skin: mottled pigmentation, skin rashes CNS: neonatal hypotonia, Leigh syndrome, ataxia, MERRF, Kearns-Sayre, MELAS Eye: Leber's optic atrophy Muscle: muscle weakness, myopathic features Mu|tivisceral failure Malformations?
J. lnher. Metab. Dis.
15 (1992)
Clinical Aspects of Mitochondrial Disorders
45t
(urine pH < 6). These symptoms remained unexplained until a multiorgan involvement became obvious: liver enlargement, cerebellar ataxia, pyramidal syndrome, deafness, retinitis pigmentosa. A markedly altered oxidoreduction status in her plasma (lactate/pyruvate molar ratio > 25; normal < 20) prompted us to carry out a muscle biopsy, which provided histopathological evidence for a mitochondrial disorder (ragged red fibres). Interestingly, a severe complex III deficiency with heteroplasmic duplication of deleted mitochondrial genomes (deletion: 4.2 kb) was present in this patient (Cormier et al, personal communication).
Presenting symptom: proximal tubulopathy A 9-month-old girl presented with proximal tubulopathy, rickets and failure to thrive. Her mother had a lid ptosis with progressive external ophthalmoplegy and muscle weakness and her sister died at 5 years of age after an episode of dehydration related to a severe tubulopathy. The second sister also had a severe tubulopathy and a puzzling mottled pigmentation of photo-exposed areas. She subsequently developed a seemingly unrelated symptom, namely diarrhoea and total anorexia, but this association remained unexplained until a multiorgan involvement occurred (3 10 years): diabetes mellitus, cerebellar ataxia, myoclonic jerks, lid ptosis, progressive external ophtalmoplegy, deafness, blindness and liver involvement. Her oxidoreduction status in plasma was not markedly altered, probably owing to the combination of diabetes and tubulopathy, but a significant amount of lactate was detected in the urine. A combined deficiency of complexes III and IV was found in her skeletal muscle, with ragged red fibres and a puzzling patchwork ofcytochrome oxidase (Cox)-positive and Cox-negative fibres on cytochrome oxidase staining (courtesy of Norma Romero). Interestingly, the partial duplication of the mitochondrial genome was detected both in the proband and in her mother, suggesting a maternal inheritance of this duplication (R6tig et al 1992).
Presenting symptom: pancytopenia A girl, born to unrelated healthy parents, had a severe neonatal anaemia and hydrops fetalis. At 1 month of age, she had anaemia, neutropenia and thrombopenia with a peculiar vacuolization of marrow precursors. Her pancytopenia remained unexplained until a seemingly unrelated symptom, exocrine pancreatic dysfunction, occurred at 7 months of age. For this reason, the haematologists diagnosed Pearson marrowpancreas syndrome in this patient (McKusick 26056). However, the decisive clue was the discovery of a mild permanent metabolic acidosis (plasma bicarbonates concentration 17 mmol/L). This led us to the recognition of a permanent hyperlactataemia with a markedly abnormal oxidoreduction status in plasma (L/P molar ratio > 30) and prompted us to investigate the respiratory chain not in the muscle, as usually performed, but in the tissue which actually expresses the disease, namely the haemopoietic system. Oxygen consumption and complex I activity were found deficient in this patient. Then a progressive multiorgan involvement occurred and she died after a fatal liver failure, triggered by parenteral nutrition (R6tig et al 1990). Y. lnher. Metab. Dis. 15 (1992)
452
M u n n i c h et aI.
Major rearrangements of the mitochondrial genome were found in her leukocytes and in other tissues as well (Cormier et al 1991). Subsequently, R6tig and co-workers (1991) showed that rearrangements of the mitochondrial genome between directly repeated sequences are consistent features in the Pearson syndrome, including the patient originally reported by Pearson and colleagues (1979).
Presenting symptom: dwarfism A brother and sister had growth retardation and deafness at 8 months of age. Their severe dwarfism was related to growth hormone deficiency. They were given growth hormone, which apparently worsened the course of their disease. Indeed, when they were 5 years old, seemingly unrelated symptoms occurred, including cardiomyopathy, retinitis pigmentosa and dumbness. This prompted us to carry out several standard metabolic investigations and to eventually ascribe their disease to a cytochrome oxidase deficiency.
Presenting symptom: diabetes mellitus A girl, born to healthy parents, had an early-onset insulin-dependent diabetes mellitus at 1 year of age. She progressively developed an optic atrophy with retinitis pigmentosa and deafness. For this reason, she was considered to suffer from Wolfram syndrome (McKusick 22230) when she was 5 years old. Only at the point when multiorgan involvement became obvious could the diagnosis of mitochondrial disorder be considered: cerebellar ataxia, progressive external ophthalmoplegy, dry thick and brittle hairs, extrapyramidal syndrome and mental retardation. The CT scan showed low-density areas in peduncles and putamen. Mildly disturbed oxidoreduction status in plasma prompted us to carry out muscle biopsy and to eventually ascribe her disease to a complex III deficiency with heteroplasmic deletion (7.6kb) of the mitochondrial genome (R6tig et al, personal communication).
Presenting symptom: cardiomyopathy A girl, born to unrelated healthy parents, had a heart murmur at 4 years of age. When she was 8 years old, this murmur was ascribed to a cardiomyopathy, but this was well tolerated and compatible with a quasinormat activity and schooling. At 14 years of age, progressive muscle weakness and myalgia occurred and only at this point was the diagnosis of mitochondrial disorder first considered and eventually confirmed not by standard tests but by exercise tests only. Muscle biopsy provided evidence for cytochrome oxidase deficiency.
Presenting symptom: severe neonatal cardiomyopathy A girl, born after a term pregnancy and delivery (birth weight 3 kg, APGAR 10), had repeated episodes of cyanosis and apnoea at 5 hours of life. At 15 hours, severe hypertrophic and hypokinetic cardiomyopathy with major ketoacidosis was diagnosed (pH 6.42, base excess -39). Markedly abnormal oxidoreduction status in plasma led to the diagnosis ofcytochrome oxidase deficiency. P o s t m o r t e m investigations provided evidence for a widespread fatty deposit in heart, liver, and smooth and skeletal J. Inher. Metab. Dis. 15 (1992)
Clinical Aspects of Mitochondrial Disorders
453
muscle. Considering this rapidly fatal course, one can easily imagine that the cardiomyopathy might have been misdiagnosed if the seemingly unrelated symptom, ketoacidosis, had been ignored.
Presenting symptom: acute collapse A girl, born to consanguinous (first-cousin) healthy parents after a term pregnancy and delivery (birth weight 2.6 kg, APGAR 10), was found apnoeic and unconscious at 3 months of age. She retained a low but significant cardiac activity but was still referred as a 'near-miss cot death'. Retrospectively, a history of developmental delay with poor spontaneous movements, no smile and no following with the eyes at 3 months of age was obtained and major abnormalities consistent with Leigh syndrome were detected on the CT scan (low-density areas in brainstem, thalami and lenticular nuclei). Markedly altered oxidoreduction status in plasma (lactate/pyruvate molar ratio 27) led to the diagnosis of complex I deficiency. Presenting symptom: ketoacidotie coma A girl, born to consanguinous (first-cousin) parents after a term pregnancy (birth weight 3.4 kg, APGAR 9), had vomiting and anorexia after a 1-week symptom-free period. At 10 days of age, she had severe hypotonia and drowsiness and progressively fell into a deep ketoacidotic coma associated with mild proximal tubulopathy. Markedly disturbed oxidoreduction status in plasma (lactate/pyruvate molar ratio 30) led to the diagnosis of cytochrome oxidase deficiency. Presenting symptom: hepatic failure A boy, born to consanguinous (first-cousin) parents after a term pregnancy (birth weight 3 kg, APGAR 10), had a severe hepatomegaly and jaundice at 2 days of age. Poor sucking, vomiting and dehydration were noted and he progressively developed a severe hypotonia and iterative episodes of apnoea. At 2 weeks of age, lethargy, floppiness and poor spontaneous movements were noted. Major hepatocellular dysfunction (factor V 20%) with cytolysis (AST 229 IU/L) and mild tubulopathy were present. Permanent hyperlactataemia (10 mmol/L; normal < 2.,1.mmol/L) and markedly altered oxidoreduction status in plasma (lactate/pyruvate 30) prompted us to carry out a muscle biopsy and a needle biopsy of the liver. Panlobular fatty infiltration was found in the liver and cytochrome oxidase deficiency was present in both liver and skeletal muscle (Cormier et al 1991a; Parrot-Roulaud et al 1991). CONCLUSION The observations reported here show that a mitochondrial disorder can account for any presenting symptom in any organ and tissue at any age, with or without a symptom-free period. Any mode of inheritance can be observed: sporadic (Pearson syndrome); autosomal dominant (progressive external ophthalmoplegia and encephalomyopathy; Zeviani et al 1989; Cormier et al 1991b); autosomal recessive; or a
d. Inher. Metab. Dis. 15 (1992)
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Munnich et al.
Table 3 Screening for multiorgan involvement
Liver: hepatocellular dysfunction? Kidney: tubulopathy? Heart: cardiomyopathy? (ultrasound) Muscle: myopathic t~atures? (RRF, steatosis) Brain: C T scan Pancreas: exocrine pancreatic dysfunction? Gut: villous atrophy? Endocrine: hyperglycaemia, hypocalcaemia, GH deficiency? Bone marrow: vacuolizafion, pancytopenia? Eye: retinitis pigmentosa (ERG)? Ear: deafness? (auditory evoked potentials)
maternal inheritance (Shoffner et al 1990). For this reason, we clearly need clinical clues to the diagnosis of mitochondrial disorders. What are these clues? One should consider the diagnosis of mitochondrial disorder when dealing (i) with an unexplained association of symptoms, (ii) with an early onset and a rapidly progressive course (iii) involving seemingly unrelated organs, which share no common embryological origin and no common biological functions. Of course, mitochondrial disorders are not the only diagnosis to consider here, and the metabolic screening for abnormal oxidoreduction status in plasma (lactate/pyruvate and ketone body molar ratios) can help to identify patients who should be further investigated. Other organs should be systematically screened as their involvement represent a useful clinical clue to this diagnosis (Table 3). In conclusion, it is obvious that mitochondrial disorders account for a variety of neuromuscular and non-neuromuscular presentations. In the future, this group of diseases will probably account for other unexplained conditions, especially those associating seemingly unrelated symptoms. ACKNOWLEDGEMENTS We are extremely grateful to Monique Poussi~re for her constant and friendly cooperation and to Association Frangaise contre les Myopathies (AFM) for support. REFERENCES Chretien D, Bourgeron T, R6tig A, Munnich A, Rustin P (1990) The measurement of rotenonesensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem Biophys Res Commun 173: 26-33. Cormier V, R6tig A, Rasore Quartino A et al (1990) Widespread multitissue deletions of the mitochondrial genome in Pearson's marrow-pancreas syndrome. J Pediatr 117:599 602. Cormier V, Ricstin P, Brunefort JP et al (t991a) Hepatic failure in disorders of oxidative phosphorylation with neonatal onset. J Pediatr 119: 951-954. Cormier V, R6tig A, Tardieu M, Colonna M, Saudubray JM, Munnich A (199tb) Autosomal dominant deletions of the mitochondrial genome in a case of progressive encephalopathy. Am J Hum Genet 48: 643-648. Luft R, Ikkos D, Palmieri G (1962) Severe hypermetabolism of non-thyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical and morphological study. J Clin Invest 41: 1776-1804. J. Inher. Metab. Dis. 15 (1992)
Clinical Aspects o f Mitochondrial Disorders
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Parrot-Roulaud F, Carte M, Lamirau T et al (1991) Fatal neonatal hepatoceIlutar deficiency with lactic acidosis: a defect of the respiratory chain. J Inher Metab Dis 14: 289-292. Pearson HA, Lobel JS, Kocoshis SA et al (1979) A new syndrome of refractory sideroblastic anemia with vacuolization of marrow precursors and exocrine pancreatic dysfunction. J Pediatr 95: 976--984. R6tig A, Cormier V, Blanche Set al (1990) Pearson's marrow-pancreas syndrome: a multisystem mitochondrial disorder in infancy. J Clin Invest 86:1601 1608. R6tig A, Cormier V, Koll F, Mize C et al (1991) Site-specific deletions of the mitochondrial genome in Pearson's marrow-pancreas syndrome. Genomics 10:502 504. Rastin P, Chretien D, Bourgeron T et al (1991) An improved representation of enzyme activities for assessment of the mitochondrial respiratory chain. Lancet 2: 60. Sengers RCA, Stadhouders AM, Trijbels JHF (1984) Mitochondrial myopathies. Clinical, morphological and biochemical aspects. Eur J Pediatr 141: 192-207. Shoffner JM, Lott MT, Lezza AMS, Seibel P, Ballinger JW, Wallace DC (I990) Myoclonic epilepsy and ragged red fiber disease (MERRF) is associated with mitochondrial DNA tRNA lys mutation. Cell 6I: 931-937. Wallace D (1989) Mitochondrial DNA mutations and neuromuscular disease. Trends Genet 5: 9-13. Zeviani M, Servidei S, Gellera C, Bertini E, Dimauro S, Didonato S (1989) An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 339:309 311.
J. Inher. Metab. Dis, 15 (1992)
Clinical Aspects of Mitochon rial Disor ers A. MUNNICH, P. RUSTIN, A. ROTIG, D. CHRETIEN, J.-P. BONNEFONT, C. NUTTIN, V. CORMIER,A. VASSAULT, P. PARVY, J. BARDET, C. CHARPENTIER, D. RABIER, J.-M. SAUDUBRAY Unitd de Recherches sur les Handicaps Gdn(tiques de l'Enfant I N S E R M U-12, D@artement de Pddiatrie and D@artment de Biochimie, H@itaI des EnfantsMalades, 149 rue de SOvres, 75743 Paris Cedex 15, France
Summary: Mitochondrial disorders have long been regarded as neuromuscular diseases only. In fact, owing to the ubiquitous nature of the oxidative phosphorytation, a broad spectrum of clinical features should be expected in mitochondrial disorders. Here, we present eight puzzling observations which give support to the view that a disorder of oxidative phosphorylation can give rise to any symptom in any organ or tissue with any apparent mode of inheritance. Consequently, we suggest giving consideration to the diagnosis of a mitochondrial disorder when dealing with an unexplained association of symptoms, with an early onset and a rapidly progressive course involving seemingly unrelated organs. Determination of lactate/pyruvate and ketone body molar ratios in plasma can help to select patients at risk for this condition.
Since the original report by Luft and colleagues (1962), mitochondrial disorders have been regarded as neuromuscular diseases. In fact, oxidative phosphorylation, which includes the oxidation of fuel molecules by oxygen and the concomitant energy transduction into ATP, is not only present in the central nervous system and muscular systems. Indeed, a number of non-neuromuscular organs and tissues are highly dependent upon mitochondrial energy supply (Wallace t989). Consequently, a disorder of oxidative phosphorylation could theoretically give rise to a wide range of symptoms in any organ or tissue. In a&tition, any mode of inheritance should be expected, owing to the particular genetic origin of mitochondrial respiratory enzymes (Wallace 1989). For this reason, the diagnosis of a mitochondrial disorder is a difficult one to consider early, particularly when the primary symptom is the only one present. By contrast, this diagnosis is easier to consider when two seemingly unrelated symptoms are observed. This paper presents evidence in support of this view and demonstrates that genetic defects of oxidative phosphorylation account for a variety of neuromuscular and non-neuromuscular symptoms in childhood. 448
Clinical Aspects of MitochondriaI Disorders
449
METHODS
Determination of the oxidoreduction status in plasma: Since the respiratory chain transfers N A D H to oxygen, a disorder of oxidative phosphorylation should result in a markedly altered oxidoreduction status in plasma. This feature is a consequence of the functional impairment of the Krebs cycle, due to the excess of N A D H and the lack of NAD, with secondary elevation of blood lactate and an increase of the ketone body and lactate/pyruvate molar ratios in affected individuals. This is particularly true in the post-absorptive period, when more N A D is required for the adequate metabolism of glycolytic substrates. Similarly, as a consequence of the Krebs cycle impairment, ketone body synthesis increases after meals, instead of decreasing, owing to the channelling of acetyl-CoA toward ketogenesis (RStig et al 1990). Consequently, current screening for genetic defects of oxidative phosphorylation must include the determination of lactate, pyruvate, ketone bodies and their molar ratios in both fasted and fed individuals (Table I). When the test is not conclusive, one must try to make it more sensitive by a glucose (or pyruvate) loading test, by an exercise test, or by looking for an abnormal oxidoreduction status in the cerebropsinal fluid (Table 1). Assessment of the mitochondrial respiratory enzymes: The metabolic investigations listed in Table 1 are routine indices of an abnormal oxidoreduction status and should prompt further enzyme investigations on fresh tissues. These investigations include the measurement of oxygen consumption by isolated mitochondria (polarography) and the assessment of enzyme activities by spectrophotometric studies (needle biopsy of the liver; open muscle biopsy under local anaesthesia, 100 200 mg of fresh tissue, usually deltoid) (Sengers et al 1984; Chr6tien et al 1990). Results are presented not only as absolute values but as ratios as well. Indeed, it has recently been observed that a constant ratio of respiratory enzyme activities is a consistent feature, regardless of the tissue tested (Rustin et al 1991). Under these conditions, patients whose absolute values were in the low normal range could be diagnosed as enzyme deficient. Although this does not hold for generalized defects, it does improve the ability to recognize localized deficiencies in the respiratory chain (Rustin et al 1991). Table 1 Metabolic investigations
Standard screening 1. Lactate/pyruvate molar ratios in plasma (fasted and fed) = redox status in the cytoplasm 2. Ketone body molar ratios in plasma (fasted and fed) = redox status in the mitochondria 3. ~Paradoxical' hyperketonaemia (]~d) 4. Urinary organic acids (GC-MS) Urinary lactate, ketone bodies Krebs cycle intermediates When inconclusive 5. Glucose (or pyruvate) loading test (2g/kg) 6. Lactate/pyruvate molar ratios in the CSF (standard or after glucose loading test) 7. Redox status after exercise test
J. Inher. Metab. Dis. 15 (1992)
450
M u n n i c h et al.
RESULTS AND DISCUSSION As expected from the ubiquitous nature of the oxidative phosphorylation, a broad spectrum of clinical features was present at the very early stage of the disease. As a consequence, the children were seen initially by various specialists, including paediatricians, neuropaediatrieians, intensive-care units, cardiologists, haematologists, nephrologists, endocrinologists, hepatogastro-enterologists, etc. Using the screening procedures described above (see Methods and Table 1), in the past 3 years 104 infants and children were found to have an abnormal oxidoreduction status in their plasma and underwent a muscle biopsy. Among them, 28 (27%) were found enzyme deficient, including 5 complex I, 2 complex II, 2 complex III, and 15 complex IV deficiency. Four had a generalized respiratory enzyme defect. Presenting symptoms included neurological and muscular diseases such as muscle weakness, myoclonic epilepsy and ragged red fibres (MERRF) and Leigh syndrome, but these accounted for less than 20% of all our patients. More interestingly, a variety of non-neuromuscular organs were initially involved in many patients (Table 2). In order to illustrate the diversity of the presenting symptoms in our respiratory enzyme-deficient patients we have looked at the clinical aspects of the disease at the time of initial consultation with the physician; that is, we have examined the clinical profile of our patients at the very early stage of the disease. Here, we report on eight puzzling and unexpected presentations of mitochondrial disorders. Presenting symptom: failure to thrive
A girl, born to unrelated healthy parents, had failure to thrive as the presenting symptom. She had anorexia, diarrhoea and vomiting in the first 12 months of age. Since she had total villous atrophy, she was considered to be gluten intolerant or milk protein intolerant, but her condition did not improve on appropriate dietary formula. When she was 20 months old, seemingly unrelated symptoms occurred, namely distal tubulopathy and metabolic acidosis with major loss of bicarbonate
Table 2 A broad spectrum of presenting symptoms
Metabolic: ketoacidotic coma Liver: hepatic failure Bone marrow: pancytopenia, macrocytic anaemia Heart: cardiomyopathy, 'near miss cot death' Kidney: tubutopathy, vitamin-unresponsive rickets Gut: diarrhoea, villous atrophy, failure to thrive Pancreas: exocrine pancreatic dysfunction Endocrine: diabetes, dwarfism, hypoparathyroidism Skin: mottled pigmentation, skin rashes CNS: neonatal hypotonia, Leigh syndrome, ataxia, MERRF, Kearns-Sayre, MELAS Eye: Leber's optic atrophy Muscle: muscle weakness, myopathic features Mu|tivisceral failure Malformations?
J. lnher. Metab. Dis.
15 (1992)
Clinical Aspects of Mitochondrial Disorders
45t
(urine pH < 6). These symptoms remained unexplained until a multiorgan involvement became obvious: liver enlargement, cerebellar ataxia, pyramidal syndrome, deafness, retinitis pigmentosa. A markedly altered oxidoreduction status in her plasma (lactate/pyruvate molar ratio > 25; normal < 20) prompted us to carry out a muscle biopsy, which provided histopathological evidence for a mitochondrial disorder (ragged red fibres). Interestingly, a severe complex III deficiency with heteroplasmic duplication of deleted mitochondrial genomes (deletion: 4.2 kb) was present in this patient (Cormier et al, personal communication).
Presenting symptom: proximal tubulopathy A 9-month-old girl presented with proximal tubulopathy, rickets and failure to thrive. Her mother had a lid ptosis with progressive external ophthalmoplegy and muscle weakness and her sister died at 5 years of age after an episode of dehydration related to a severe tubulopathy. The second sister also had a severe tubulopathy and a puzzling mottled pigmentation of photo-exposed areas. She subsequently developed a seemingly unrelated symptom, namely diarrhoea and total anorexia, but this association remained unexplained until a multiorgan involvement occurred (3 10 years): diabetes mellitus, cerebellar ataxia, myoclonic jerks, lid ptosis, progressive external ophtalmoplegy, deafness, blindness and liver involvement. Her oxidoreduction status in plasma was not markedly altered, probably owing to the combination of diabetes and tubulopathy, but a significant amount of lactate was detected in the urine. A combined deficiency of complexes III and IV was found in her skeletal muscle, with ragged red fibres and a puzzling patchwork ofcytochrome oxidase (Cox)-positive and Cox-negative fibres on cytochrome oxidase staining (courtesy of Norma Romero). Interestingly, the partial duplication of the mitochondrial genome was detected both in the proband and in her mother, suggesting a maternal inheritance of this duplication (R6tig et al 1992).
Presenting symptom: pancytopenia A girl, born to unrelated healthy parents, had a severe neonatal anaemia and hydrops fetalis. At 1 month of age, she had anaemia, neutropenia and thrombopenia with a peculiar vacuolization of marrow precursors. Her pancytopenia remained unexplained until a seemingly unrelated symptom, exocrine pancreatic dysfunction, occurred at 7 months of age. For this reason, the haematologists diagnosed Pearson marrowpancreas syndrome in this patient (McKusick 26056). However, the decisive clue was the discovery of a mild permanent metabolic acidosis (plasma bicarbonates concentration 17 mmol/L). This led us to the recognition of a permanent hyperlactataemia with a markedly abnormal oxidoreduction status in plasma (L/P molar ratio > 30) and prompted us to investigate the respiratory chain not in the muscle, as usually performed, but in the tissue which actually expresses the disease, namely the haemopoietic system. Oxygen consumption and complex I activity were found deficient in this patient. Then a progressive multiorgan involvement occurred and she died after a fatal liver failure, triggered by parenteral nutrition (R6tig et al 1990). Y. lnher. Metab. Dis. 15 (1992)
452
M u n n i c h et aI.
Major rearrangements of the mitochondrial genome were found in her leukocytes and in other tissues as well (Cormier et al 1991). Subsequently, R6tig and co-workers (1991) showed that rearrangements of the mitochondrial genome between directly repeated sequences are consistent features in the Pearson syndrome, including the patient originally reported by Pearson and colleagues (1979).
Presenting symptom: dwarfism A brother and sister had growth retardation and deafness at 8 months of age. Their severe dwarfism was related to growth hormone deficiency. They were given growth hormone, which apparently worsened the course of their disease. Indeed, when they were 5 years old, seemingly unrelated symptoms occurred, including cardiomyopathy, retinitis pigmentosa and dumbness. This prompted us to carry out several standard metabolic investigations and to eventually ascribe their disease to a cytochrome oxidase deficiency.
Presenting symptom: diabetes mellitus A girl, born to healthy parents, had an early-onset insulin-dependent diabetes mellitus at 1 year of age. She progressively developed an optic atrophy with retinitis pigmentosa and deafness. For this reason, she was considered to suffer from Wolfram syndrome (McKusick 22230) when she was 5 years old. Only at the point when multiorgan involvement became obvious could the diagnosis of mitochondrial disorder be considered: cerebellar ataxia, progressive external ophthalmoplegy, dry thick and brittle hairs, extrapyramidal syndrome and mental retardation. The CT scan showed low-density areas in peduncles and putamen. Mildly disturbed oxidoreduction status in plasma prompted us to carry out muscle biopsy and to eventually ascribe her disease to a complex III deficiency with heteroplasmic deletion (7.6kb) of the mitochondrial genome (R6tig et al, personal communication).
Presenting symptom: cardiomyopathy A girl, born to unrelated healthy parents, had a heart murmur at 4 years of age. When she was 8 years old, this murmur was ascribed to a cardiomyopathy, but this was well tolerated and compatible with a quasinormat activity and schooling. At 14 years of age, progressive muscle weakness and myalgia occurred and only at this point was the diagnosis of mitochondrial disorder first considered and eventually confirmed not by standard tests but by exercise tests only. Muscle biopsy provided evidence for cytochrome oxidase deficiency.
Presenting symptom: severe neonatal cardiomyopathy A girl, born after a term pregnancy and delivery (birth weight 3 kg, APGAR 10), had repeated episodes of cyanosis and apnoea at 5 hours of life. At 15 hours, severe hypertrophic and hypokinetic cardiomyopathy with major ketoacidosis was diagnosed (pH 6.42, base excess -39). Markedly abnormal oxidoreduction status in plasma led to the diagnosis ofcytochrome oxidase deficiency. P o s t m o r t e m investigations provided evidence for a widespread fatty deposit in heart, liver, and smooth and skeletal J. Inher. Metab. Dis. 15 (1992)
Clinical Aspects of Mitochondrial Disorders
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muscle. Considering this rapidly fatal course, one can easily imagine that the cardiomyopathy might have been misdiagnosed if the seemingly unrelated symptom, ketoacidosis, had been ignored.
Presenting symptom: acute collapse A girl, born to consanguinous (first-cousin) healthy parents after a term pregnancy and delivery (birth weight 2.6 kg, APGAR 10), was found apnoeic and unconscious at 3 months of age. She retained a low but significant cardiac activity but was still referred as a 'near-miss cot death'. Retrospectively, a history of developmental delay with poor spontaneous movements, no smile and no following with the eyes at 3 months of age was obtained and major abnormalities consistent with Leigh syndrome were detected on the CT scan (low-density areas in brainstem, thalami and lenticular nuclei). Markedly altered oxidoreduction status in plasma (lactate/pyruvate molar ratio 27) led to the diagnosis of complex I deficiency. Presenting symptom: ketoacidotie coma A girl, born to consanguinous (first-cousin) parents after a term pregnancy (birth weight 3.4 kg, APGAR 9), had vomiting and anorexia after a 1-week symptom-free period. At 10 days of age, she had severe hypotonia and drowsiness and progressively fell into a deep ketoacidotic coma associated with mild proximal tubulopathy. Markedly disturbed oxidoreduction status in plasma (lactate/pyruvate molar ratio 30) led to the diagnosis of cytochrome oxidase deficiency. Presenting symptom: hepatic failure A boy, born to consanguinous (first-cousin) parents after a term pregnancy (birth weight 3 kg, APGAR 10), had a severe hepatomegaly and jaundice at 2 days of age. Poor sucking, vomiting and dehydration were noted and he progressively developed a severe hypotonia and iterative episodes of apnoea. At 2 weeks of age, lethargy, floppiness and poor spontaneous movements were noted. Major hepatocellular dysfunction (factor V 20%) with cytolysis (AST 229 IU/L) and mild tubulopathy were present. Permanent hyperlactataemia (10 mmol/L; normal < 2.,1.mmol/L) and markedly altered oxidoreduction status in plasma (lactate/pyruvate 30) prompted us to carry out a muscle biopsy and a needle biopsy of the liver. Panlobular fatty infiltration was found in the liver and cytochrome oxidase deficiency was present in both liver and skeletal muscle (Cormier et al 1991a; Parrot-Roulaud et al 1991). CONCLUSION The observations reported here show that a mitochondrial disorder can account for any presenting symptom in any organ and tissue at any age, with or without a symptom-free period. Any mode of inheritance can be observed: sporadic (Pearson syndrome); autosomal dominant (progressive external ophthalmoplegia and encephalomyopathy; Zeviani et al 1989; Cormier et al 1991b); autosomal recessive; or a
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Table 3 Screening for multiorgan involvement
Liver: hepatocellular dysfunction? Kidney: tubulopathy? Heart: cardiomyopathy? (ultrasound) Muscle: myopathic t~atures? (RRF, steatosis) Brain: C T scan Pancreas: exocrine pancreatic dysfunction? Gut: villous atrophy? Endocrine: hyperglycaemia, hypocalcaemia, GH deficiency? Bone marrow: vacuolizafion, pancytopenia? Eye: retinitis pigmentosa (ERG)? Ear: deafness? (auditory evoked potentials)
maternal inheritance (Shoffner et al 1990). For this reason, we clearly need clinical clues to the diagnosis of mitochondrial disorders. What are these clues? One should consider the diagnosis of mitochondrial disorder when dealing (i) with an unexplained association of symptoms, (ii) with an early onset and a rapidly progressive course (iii) involving seemingly unrelated organs, which share no common embryological origin and no common biological functions. Of course, mitochondrial disorders are not the only diagnosis to consider here, and the metabolic screening for abnormal oxidoreduction status in plasma (lactate/pyruvate and ketone body molar ratios) can help to identify patients who should be further investigated. Other organs should be systematically screened as their involvement represent a useful clinical clue to this diagnosis (Table 3). In conclusion, it is obvious that mitochondrial disorders account for a variety of neuromuscular and non-neuromuscular presentations. In the future, this group of diseases will probably account for other unexplained conditions, especially those associating seemingly unrelated symptoms. ACKNOWLEDGEMENTS We are extremely grateful to Monique Poussi~re for her constant and friendly cooperation and to Association Frangaise contre les Myopathies (AFM) for support. REFERENCES Chretien D, Bourgeron T, R6tig A, Munnich A, Rustin P (1990) The measurement of rotenonesensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem Biophys Res Commun 173: 26-33. Cormier V, R6tig A, Rasore Quartino A et al (1990) Widespread multitissue deletions of the mitochondrial genome in Pearson's marrow-pancreas syndrome. J Pediatr 117:599 602. Cormier V, Ricstin P, Brunefort JP et al (t991a) Hepatic failure in disorders of oxidative phosphorylation with neonatal onset. J Pediatr 119: 951-954. Cormier V, R6tig A, Tardieu M, Colonna M, Saudubray JM, Munnich A (199tb) Autosomal dominant deletions of the mitochondrial genome in a case of progressive encephalopathy. Am J Hum Genet 48: 643-648. Luft R, Ikkos D, Palmieri G (1962) Severe hypermetabolism of non-thyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical and morphological study. J Clin Invest 41: 1776-1804. J. Inher. Metab. Dis. 15 (1992)
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