J. Inher. Metab. Dis. 19 (1996) 521-527 © SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Clinical presentation of mitochondrial disorders in childhood A. MUNNICH*, A. ROTIG, D. CHRETIEN, V. CORMIER, T. BOURGERON, J.-R BONNEFONT, J.-M. SAUDUBRAY and P. RUSTIN Ddpartement de Gdndtique et Unitd de Recherches sur les Handicaps Gdndtiques de l 'Enfant- Unitd INSERM U-393, H@ital des Enfants Malades, 149, rue de Skvres - 75743 Paris Cedex 15, France

*Correspondence

Summary: Respiratory-chain deficiencies have long been regarded as neuro- muscular diseases. In fact, oxidative phosphorytation, i.e. adenosine triphosphate (ATP) synthesis by the respiratory chain, does not occur only in the neuromuscular system. Indeed, a number of non-neuromuscular organs and tissues are dependent upon mitochondrial energy supply. For this reason, a respiratory-chain deficiency can theoretically give rise to any symptom, in any organ or tissue, at any age and with any mode of inheritance, owing to the twofold genetic origin of respiratory enzymes (nuclear DNA and mitochondrial DNA, mtDNA). In recent years, it has become increasingly clear that genetic defects of oxidative phosphorylation account for a large variety of clinical symptoms in childhood. Among 100 patients with respiratory-chain deficiencies identified in our centre, 56% presented with a non- neuromuscular symptom and 44% were referred for a neuromuscular problem. It appears that the diagnosis of a respiratory-chain deficiency is difficult initially when only one symptom is present. In contrast, this diagnosis is easier to consider when two seemingly unrelated symptoms are observed

In the last five years, more than 1000 children have been referred to us for investigation of their mitochondrial respiratory chain (1041 patients). Of these, 23.5% had resph'atory enzyme deficiency and/or mtDNA rearrangements. Among our 234 respiratory enzyme- deficient patients, 35% had complex I, 3% complex II, 11.5% complex III, 32% complex IV, 1.5% complex V, 10% complexes I+ IV and 7% generalized enzyme deficiency. As expected from the ubiquitous nature of oxidative phosphorylation, a broad spectrum of clinical features was present at the early stage of the disease. Interestingly, 44% presented with a neuromuscular disease but in 56% the presenting symptom was not neuromuscular. Organs initially involved included liver, heart, endocrines, kidney, gut, brain, skeletal muscle and skin (Munnich et al 1996).

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METABOLIC INVESTIGATIONS

How these patients with seemingly unrelated symptoms were identified as at risk for a mitochondrial disease deserves extensive explanation. Since the respiratory chain transfers NADH to oxygen, a disorder of oxidative phosphorylation should result in a markedly altered redox status in plasma. This feature is the consequence of the functional impairment of the Krebs cycle due to the excess of NADH and the lack of NAD, with a secondary elevation of blood lactate and increased lactate/pyruvate (L/P) and ketone body molar ratios in affected individuals. This is particularly true in the post-absorptive period, when more NAD is required for adequately metabolizing 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 towards ketogenesis. For this reason, our current screening for genetic defects of oxidative phosphorylation includes the determination of lactate, pyruvate, ketone bodies and their molar ratios in fasted and fed individuals (Robinson 1989; Munnich et al 1996).

Elevated plasma lactate, L/P and ketone body molar ratios are indices of respiratory enzyme deficiency, while a low ketone body molar ratio is an index of Krebs cycle disorder or pyrnvate carboxylase deficiency. High plasma lactate with low L/P ratio points towards pyruvate dehydrogenase deficiency.

NEUROMUSCULAR PRESENTATION

Muscle weakness, atrophy, hypotonia, peripheral neuropathy, cerebellar ataxia and leukodystrophy have frequently been described as presenting symptoms in mitochondrial disorders. Indeed, 44% of our patients presented with neuromuscular symptoms, Leigh syndrome, MERRF or MELAS. As far as neuromuscular presentations are concerned, we would emphasize succinate dehydrogenase (SDH) deficiency as a possible cause of Leigh disease. We have previously observed two sisters born to first-cousin parents who developed poor sucking, recurrent attacks of psychomotor regression, blindness and leukodystrophy at 12 months of age. Their redox status was markedly abnormal and widespread SDH deficiency was detected in all tissues tested. A point mutation in the SDH Fp subunit has been found in these patients and therefore represents the first nuclear gene mutation in a respiratol-y enzyme deficiency (Bourgeron et al 1995).

It is also important to remember that respiratory enzyme deficiency is a cause of recurrent myoglobinuria. We have observed a child born to second-cousin Tunisian parents after a normal pregnancy and delivery (birth weight 2700g). At 3 weeks and at 3 months of age, she had recurrent attacks of trunk and limb hypotonia with muscle stiffness, lethargy and recurrent episodes of myogtobinuria with elevated sarcoplasmic enzymes (creatine kinase 97000U/L) but normal plasma lactate and L/P ratios. She died at 6 months of age of a terminal haemolytic uraemic syndrome. Her dystrophin gene and fatty acid oxidation were normal, but urinary Krebs cycle intermediates pointed towards a mitochondrial disorder and a complex IV deficiency was found in this patient. It is important, therefore, to consider respiratory-chain deficiency as a cause of recurrent myoglobinuria in childhood (Saunier et al 1995). Concerning diagnosis, it is worth noting that 60% of muscle enzyme deficiencies were expressed in lymphocytes in our series, making circulating leukocytes a useful tool for diagnosis of mitochondrial myopathies.

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LIVER PRESENTATION

In the period reported, 26 respiratory enzyme-deficient children were referred to us with a liver disease as the presenting symptom. This included early-onset hepatic failure (before 10 days, 45%, Cormier et al 1991), delayed onset hepatocellular dysfunction (2-48 months, 40%) and Alpers syndrome (15%). Their redox status in plasma was usually disturbed (70% of cases) and the outcome was rapidly fatal (75% of cases). Severe neurological involvement was constant and frequently triggered by valproate in both early- onset form and Alpers syndrome. By contrast, the course of the disease was milder in the delayed-onset form. Liver pathology included steatosis and micronodular cirrhosis, and enzymatic studies showed complexes I, IV and multiple enzyme deficiencies. Interestingly, almost half of the liver enzyme defects were expressed in lymphocytes and 60% in skeletal muscle. The expression of the enzyme deficiency in circulating lymphocytes is particularly helpful in patients who cannot undergo liver biopsy due to impaired coagulation subsequent to hepatic failure. Also of interest, no mtDNA deletion or mutation was found in this series (Cormier et al 1995).

Recognizing the mitochondrial origin of Alpers poliodystrophy is difficult when the enzyme deficiency is liver-specific. Indeed, we have observed several cases of Alpers syndrome with myoclonic jerks, psychomotor retardation and fatal hepatic coma (triggered by valproate) whose redox status in plasma was normal and enzyme deficiency was apparently limited to liver (Chabrol et al 1994). It is therefore important to avoid valproate in patients at risk of respiratory enzyme deficiency and to systematically include the study of liver respiratory enzyme activity in the diagnosis of Alpers syndrome and myoctonus epilepsy.

CARDIAC PRESENTATION

The cardiac presentation of mitochondrial disorders includes sudden death, heart block and, most importantly, cardiomyopathy. In the period reported, 29 patients have been referred to us for the diagnosis of cardiomyopathy. Among them, 50% had respiratory enzyme deficiency. The age of onset ranged from 1 day to 18 years. Half of our patients presented with apnoea, dyspnoea, cyanosis and bronchitis in the neonatal period. The other haft had a delayed onset with murmur or heart failure. It is important to note that the redox status was consistently disturbed in the neonatal-onset form but frequently subnormal in the delayed-onset form. In addition, almost all respiratory enzyme-deficient cardio- myopathy patients had a concentric hypertrophic hypokinetic cardiomyopathy. The outcome was rapidly fatal in 56% of cases, but slowly progressive in 44% of cases, prompting the consideration of heart transplantation in these patients. Genetic defects of oxidative phosphorylation therefore represent a major cause of cardiomyopathy in children (Rustin et al 1994; Bonnefont et al, unpublished observation).

Barth syndrome is an important subtype of cardiomyopathy as this X-linked condition may account for the excess of boys in our series (sex ratio 1.8). It is an (ante- or) neonatal onset cardiomyopathy with cyclic neutropenia and a rapidly fatal outcome, but no clinical expression in carrier females. It is therefore important to look carefully at blood counts in boys with neonatal-onset cardiomyopathy, as the recent mapping of the disease gene to chromosome Xq28 makes prenatal diagnosis theoretically feasible (Bolhuis et al 1991).

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Endomyocardial biopsy is a reliable diagnostic tool for morphological, enzymological and molecular studies in cardiomyopathy (Rustin et al 1994). Heart morphology was markedly abnormal in 8/13 cases of respiratory enzyme deficiency, interstitial fibrosis being the most frequent feature (5/13). Ragged-red fibres (1/13), fibroelastosis (2/13) and inflammation (t/13) were also observed. All types of enzyme deficiency were present (complex I, 7/17; complex II, 1/17; complex III, 3/17; complex IV, 3/17; complex V, 1/17; multiple, 2/17). Endomyocardial biopsy was particularly useful in the 40% of cases that were not expressed in skeletal muscle. We have failed to detect any mtDNA deletion or known mutation in our series (Bonnefont et al, unpublished observation).

RENAL PRESENTATION

In the period under consideration, 8 respiratory enzyme-deficient patients were referred to us with a kidney disease. Age of onset ranged from 1 to 8 years and presenting symptoms included proximal tubulopathy (5/8), tubutointerstitial nephritis (I/8), nephrotic syndrome (1/8) and renal failure (1/8). Interestingly, plasma lactate and L/P ratios were consistently normal in all 8 patients, but abnormal urinary lactate and Krebs cycle intermediates in all patients pointed towards respiratory enzyme deficiency. A widespread multiorgan involvement occurred in all cases and enzymatic studies showed complex I, ItI, and multienzyme deficiency. Four of our eight patients had mtDNA rearrangements (R6tig et al 1991, 1995a,b).

GASTROINTESTINAL PRESENTATION

The gastrointestinal presentation of respiratory-chain deficiency included recurrent vomiting, chronic diarrhoea, villous atrophy, failure to thrive and, in adults, chronic intestinal pseudo-obstruction. In the last few years, 4respiratory enzyme-deficient patients presented with gastrointestinal symptoms before 1 year of age. Their redox status in plasma was disturbed and a multiorgan involvement consistently occurred in the course of the disease. Two of them had a major mtDNA rearrangement (Cormier-Daire et al 1994).

ENDOCRINE PRESENTATION

Endocrine presentations included dwarfism, diabetes mellitus, hypoparathyroidism, hypothyroidism, central insipidus diabetes and ACTH deficiency. In the period of this study, 4 respiratory enzyme-deficient patients were referred for major growth retardation ( -4 to - 6 SD) before 1 year of age. They had normal basal and stimulated plasma growth hormone but markedly reduced plasma IGF1. Their redox status in plasma was disturbed and they developed a fatal multiorgan involvement. In two of them (and in several other cases from different groups), exogenous growth hormone precipitated the course of the disease.

Diabetes mellitus is known to occur frequently in the course of mitochondrial disorders. It is important to be aware that diabetes mellitus can also occur at early stages of the disease. In the last 3 years, we have observed 3 cases of neonatal-onset diabetes mellitus ascribed to respiratory-chain deficiency. The patients had major hyperglycaemia, hyper- lactataemia and ketosis. Widespread enzyme deficiency was noted and major mtDNA rearrangements were occasionally observed (details to be published).

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HAEMATOLOGICAL PRESENTATION

Since our elucidation of the mitochondrial origin of the Pearson syndrome (R6tig et al 1991), 21 patients have been referred to us for pancytopenia. All patients started their disease before 1 year of age. Half of them had anaemia and half had diarrhoea as the presenting symptom. Pancreatic dysfunction was present in 62% of cases. The disease was fatal before 3 years in 57% of cases and 43% of our patients are still alive (4-13 years). Interestingly, mtDNA rearrangements were consistent features in the Pearson syndrome (deletion, 72%; deletion+duplication, 28%; R6tig et al 1995c).

FACIAL DYSMORPHISM

Respiratory-enzyme deficiency is an unexpected cause of facial dysmorphism and malformations. We have observed respiratory-enzyme deficiencies in several patients with evolutive facial dysmorphism, microcephaly and ante- and postnatal growth retardation. Facial dysmorphism included round face, high forehead, featureless filtrum, low-set ears and short neck. The pathogenesis of this facial dysmorphism in mitochondrial disorders is debatable. Possible explanations include direct intoxication by accumulated metabolites, energy deficiency and/or inhibition of a key metabolic pathway. Facial dysmorphism in mitochondrial disorders shares clinical features with the fetal alcohol syndrome where pyruvate dehydrogenase inhibition by acetaldehyde has been considered (Cormier et al 1996).

DERMATOLOGICAL SYMPTOMS

Hair and skin anomalies are frequent in mitochondrial disorders. Yet they are inconstant, delayed and associated with other symptoms. They include mottled pigmentation of photoexposed areas (R6tig et al 1991), hypertrichosis, trichothiodystrophy and dry, thick and brittle hair (R6tig et al 1993). Clinically, the hair is dry, short and sparse, with alopecia. The hair shafts break easily. Electron microscopy shows irregular hair shafts with prominences, depressions, twists, grooves, transverse fractures and severe alteration of the cuticle (Bodemer et al, in preparation).

DIAGNOSIS AND CLINICAL OUTCOME OF MITOCHONDRIAL DISEASES

Genetic defects of oxidative phosphorylation can give rise to any symptom in any organ

or tissue at any age with any mode o f inheritance. This diagnosis should be considered in a patient with an unexplained association of symptoms with a rapidly progressive course involving seemingly unrelated organs. Neonatal (36% before 1 month) and infantile onset was frequently observed in our series (1-24 months, 44%). Onset after 2 years was less frequent (20%). Whatever the age at onset, thisdiagnosis is difficult to consider early when only one symptom is present. It is an easier diagnosis to make when two or more seemingly unrelated symptoms are observed. In this case, one should carry out metabolic investigations (see above). Nevertheless, one should be aware of the pitfalls in the metabolic screening (Munnich et al 1996).

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(1) Normal plasma lactate in basal conditions does not rule out this diagnosis. Instead, one should carry out a glucose loading test (2 g/kg orally) and test the redox status in the cerebrospinal fluid (CSF). CSF lactate is useless when the redox status in plasma is altered.

(2) Proximal tubulopathy may lower blood lactate. For this reason, the search for urinary lactate and Krebs cycle intermediates using gas chromatography mass spectrometry is mandatory in suspected cases of respiratory-chain deficiency.

(3) Diabetes mellitus may hamper entry of pyruvate in the Krebs cycle. (4) Organ-specific enzyme deficiency may barely alter the redox status (cardio-

myopathy). (5) The deficiency may be generalized but partial: the higher the tissue dependency on

oxidative phosphorylation (brain, muscle), the greater the redox impairment in plasma.

Which tissues should be investigated? The relevant tissue is the one which expresses the disease: blood leukocytes in the Pearson syndrome, muscle biopsy in myopathy, needle biopsy of the liver in hepatic failure, endomyocardial biopsy in cardiomyopathy. If the relevant tissue is not accessible, one should test leukocytes and muscle, as the diagnostic yield in these tissues is relatively high (40-60% in our series). Whichever the expressing organ, one should take skin biopsy for fibroblast culture and prenatal diagnosis. In our series, 40-60% of respiratory-enzyme deficiencies were expressed in cultured skin fibroblasts, making prenatal diagnosis feasible in a significant proportion of cases.

Although the initial symptom usually persists and gradually worsens, it may occasionally improve or even disappear. Spontaneous remission of infantile myopathy, pancytopenia, cardiomyopathy and villous atrophy are exceptional but possible. In addition, the number of affected tissues usually increases in the course of the disease, whatever the age of onset and the presenting symptom. The central nervous system is almost consistently involved in the late stage of the disease. Some cases remained organ- specific as we have observed in cardiomyopathy and hepatic failure. When evidence of organ-specific enzyme deficiency is present (heart, liver), it is reasonable to consider organ transplantation.

CONCLUSION

Mitochondrial disorders have long been regarded as neuromuscular diseases only. It appears now that the clinical spectrum of respiratory-chain deficiency is remarkably large, especially as oxidative phosphorylation is present not only in the central nervous system but in all organs and tissues as well. For this reason, genetic defects of oxidative phos- phorylation might account for other syndromes, especially those associating seemingly unrelated symptoms. Determination of lactate, pyruvate, ketone bodies and their molar ratios in plasma can help in selecting patients at risk for mitochondriaI disorders.

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