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Mitochondrial disorders
Current Paediatrics (1997) 7, 123-127 © 1997 Pearson Professional Ltd
Mini-symposium: Metabolic disease
Mitochondrial disorders
S. Rahman, J. V. Leonard
Clinical features associated with mitochondrial disease in childhood are summarized in Box 1. Some distinct clinical syndromes are recognized (Box 2), but many patients do not fit into these defined syndromes. It is often the unusual combination of clinical features which suggests a possible diagnosis of respiratory chain disease. Pedigree analysis may be helpful, although genetic heterogeneity means that any mode of inheritance is
INTRODUCTION
The mitochondrion is the cell organelle responsible for production of energy by the process of oxidative phosphorylation. Electrons are progressively oxidized in the respiratory chain, polypeptides of which are encoded by two genomes, that of the nucleus and that within the mitochondrion itself. Disorders of the respiratory chain display marked clinical, biochemical and genetic heterogeneity and diagnosis is notoriously difficult. It is likely that the underlying diagnosis is unrecognized in a significant number of cases. Consequently, the prevalence of these disorders is not known. Mitochondrial diseases are generally classified biochemically, but with recent advances there are the beginnings of a molecular genetic classification.
Box 1 Clinical features associated with mitochondrial disease Neuromuscular
CLINICAL FEATURES
These disorders may present at any age and are clinically very heterogeneous, ranging from fatal neonatal lactic acidosis to a mild adult-onset myopathy. Historically, mainly neuromuscular diseases were recognized as being of mitochondrial origin. These are, consequently, the most well characterized. However, almost all tissues are dependent on mitochondrial energy production and so one might expect multisystem disease. Accordingly, an increasingly large number of nonneuromuscular mitochondrial diseases are being described and a clue to the diagnosis is often the involvement of several apparently unrelated systems.
Ophthalmological
Cardiac Renal Hepatic Gastrointestinal
Haematological Endocrine
Shamima Rahman MRCP, MRC Clinical Training Fellow, James
V. Leonard PhD FRCP, Professor of Paediatric Metabolic Disease, Institute of Child Health, 30 Guilford Street, London W C I N 1EH, UK. Correspondence and requests for offprints to SR.
123
Developmental delay/regression Seizures (especially myoclonic) Ataxia Encephalopathic and stroke-like episodes Dystonia Hypotonia Weakness Fatigue Peripheral neuropathy Sensorineural deafness Optic atrophy Pigmentary retinopathy Progressive external ophthalmoplegia (PEO) Cataracts Cardiomyopathy (usually hypertrophic) Conduction defects Renal tubular dysfunction particularly Fanconi syndrome Liver failure especially in infancy (may be induced by valproate) Pancreatic exocrine insufficiency Recurrent vomiting Chronic diarrhoea Enteropathy with partial villous atrophy Sideroblastic anaemia Pancytopenia Diabetes mellitus Short stature Hypoparathyroidism Hypothyroidism Hypogonadism
124
Current Paediatrics
Box 2 Recognized mitochondrial syndromes Syndrome
Summary of clinical features
Leigh (subacute necrotizing encephalopathy)
Psychomotor regression in infancy or childhood, hypotonia, lactic acidosis and brainstem dysfunction, associated with symmetrical basal ganglia/brainstem lesions on CT/MRI
MERRF (myoclonic epilepsy with ragged red fibres)
Myoclonic epilepsy or action myoclonus. Myopathy with ragged red fibres (RRF) on muscle biopsy. Clinical spectrum includes cerebellar ataxia, deafness, dementia and multiple lipomata
MELAS Stroke-like episodes with focal CT/MRI brain abnormalities, lactic acidosis, RRF myopathy, seizures (mitochondrial (focal/generalized), dementia, recurrent headache, vomiting. Clinical spectrum includes diabetes mellitus, encephalomyopathy with deafness and progressive external ophthalmoplegia (PEO) lactic acidosis and stroke-like episodes) Pearson (marrow pancreas) syndrome
Refractory sideroblastic anaemia, vacuolization of marrow precursors, exocrine pancreatic dysfunction. Survivors develop KSS in adolescence
Kearns-Sayre syndrome (KSS)
Invariant triad of onset < 20 years, PEO, pigmentary retinopathy plus at least one of heart block/cerebellar syndrome/raised CSF protein > 1 g/l
NARP (neurogenic muscle weakness, ataxia, retinitis pigmentosa)
Developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal weakness and sensory neuropathy. Clinical spectrum overlaps with Leigh syndrome
LHON (Leber's hereditary optic neuropathy)
Painless subacute bilateral loss of vision with central visual field defects, abnormal colour vision and optic atrophy. Onset usually 18-30 years but may be in childhood. Marked male predominance
MNGIE (mitochondrial neurogastrointestinal encephalomyopathy)
Gastrointestinal dysmotility, peripheral neuropathy, myopathy, ophthalmoplegia
mtDNA depletion syndrome
Hypotonia, developmental delay, lactic acidosis, liver failure, Fanconi syndrome
possible. Maternal inheritance is highly suggestive of a mitochondrial D N A (mtDNA) mutation, whilst defects of nuclear D N A may be inherited as autosomal dominant, autosomal recessive or sexlinked traits. Furthermore, clinical features may vary widely even between affected members of a single family.
BIOCHEMICAL CLASSIFICATION
Strictly speaking, mitochondrial diseases encompass defects of all mitochondrial proteins including enzymes involved in pyruvate metabolism, fatty acid I~-oxidation, the Krebs cycle and many others. However, in practice the term mitochondrial disease is usually taken to refer to defects of the mitochondrial respiratory chain (RC) and of oxidative phosphorylation, which comprise five complexes. Complex II is the only RC complex encoded entirely on the nuclear
genome; the others have both mitochondrial and nuclear-encoded subunits (Table 1). A defect in one complex may have several different clinical manifestations. Complex I deficiency is frequently seen with point mutations of mitochondrial transfer R N A (tRNA) genes including myoclonic epilepsy with ragged red fibres (MERRF) and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndromes. Being the largest complex, with the greatest number of mitochondrially-encoded subunits, it is one that is commonly affected by defects of the mitochondrial genome. Complex I deficiency is also described as a probable autosomal recessive trait presenting as fatal infantile myopathy with lactic acidosis, cataract and cardiomyopathy or neurodegenerative disease including Leigh syndrome. The clinical spectrum of complex IV deficiency includes lactic acidosis with isolated myopathy, or combined with Fanconi's syndrome, cardiomyopathy or Leigh syndrome.
Table 1 Respiratory chain complexes Complex
Name
Complex I Complex II Complex III Complex IV Complex V
NADH ubiquinone oxidoreductase Succinate ubiquinone oxidoreductase Ubiquinol cytochrome c oxidoreductase Cytochrome c oxidase ATP synthase
mtDNA encoded subunits
Nuclear encoded subunits
7 0 1 3 2
~34 4 10 10 11 or 12
Mitochondrial disorders Thus, defects of different complexes may have very similar clinical and pathological findings. For example, deficiencies of complex I or complex IV, as well as deficiency of the pyruvate dehydrogenase complex (PDHC), may cause Leigh syndrome. Both complex I and complex IV deficiency may produce a benign reversible infantile myopathy, clinically indistinguishable from the fatal infantile myopathies associated with defects of these enzymes. The underlying mechanism in these reversible myopathies is postulated to be a transient defect in a developmentally regulated subunit. In other patients deficiencies of multiple RC enzyme complexes may be present, in particular combined deficiencies of complexes I and IV. There may also be an associated deficiency of PDHC, implying an underlying defect of a common regulatory gene.
G E N E T I C CLASSIFICATION The genetic classification of mitochondrial disorders is complicated because of the contribution of both the mitochondrial and nuclear genomes. The mitochondrial genome is a 16 659 base pair (bp) circular D N A molecule encoding 13 polypeptide components of the respiratory chain, 22 tRNAs and two ribosomal RNAs (rRNAs). Mitochondrial genetics differs from classical Mendelian inheritance in that m t D N A is exclusively maternally inherited and, unlike nuclear genes which have two alleles, is present in multiple copy numbers. There are between two and ten m t D N A molecules per mitochondrion and hundreds or thousands of mitochondria per cell. This amplification of the mitochondrial genome allows for the phenomenon of heteroplasmy, whereby varying proportions of mutant and normal m t D N A coexist
within a cell. This in turn leads to variable tissue expression and contributes to the clinical heterogeneity. The term homoplasmy is used to indicate the presence of either 100% wild type or 100% mutant m t D N A in a tissue. However, the vast majority of mitochondrial proteins are encoded on the nuclear genome and are imported into the mitochondria from the cytoplasm using a complex translocation machinery which is also encoded on the nucleus. Several nuclear factors control m t D N A replication, transcription and translation and it is probable that defects of nuclear D N A play a major aetiological role in mitochondrial disease. Defects of m t D N A
Pathogenic m t D N A mutations include gross structural rearrangements (single deletions, multiple deletions, duplications) and point mutations (Table 2). Deletions are usually single, heteroplasmic and sporadic. Thirty percent of patients with deletions have an identical 'common' deletion of 4977 bp which includes several protein coding and t R N A genes and one of the origins of replication of the m t D N A molecule. Clinical features associated with this deletion are variable, ranging from progressive external ophthalmoplegia (PEO) to Pearson syndrome. This clinical heterogeneity probably reflects the degree of heteroplasmy and the tissue distribution of the deleted m t D N A molecules. More than 50 point mutations in m t D N A have already been described, involving tRNA, rRNA and protein coding genes. These mutations are usually heteroplasmic. As with deletions, there is no strict genotype-phenotype correlation. For example, the 8993 neurogenic muscle weakness, ataxia, retinitis pigmentosa (NARP) mutation seems to cause Leigh syndrome
Table 2 Geneticclassification
Mutation Defects of mtDNA Single deletions Duplications or duplication/deletions Point mutations tRNA genes e.g. nt 3243 nt 8344 Protein coding genes e.g. nt 8993 nt 3460, 11778, 14484 Defects of nuclear DNA Protein coding genes e.g. ComplexII Complex I ~ mutations Complex IV J not yet found Regulatory genes (none yet described) Defects of intergenomiccommunication (genes responsiblenot yet isolated) Multiple mtDNA deletions mtDNA depletion
125
Inheritance
Clinical features
Usually sporadic Sporadic or maternal
PEn, KSS, Pearson Pearson, proximalrenal tubulopathy
Maternal MELAS MERRF Maternal NARP, Leigh LHON
Autosomal recessive ?Autosomal recessive ?Autosomal recessive
Leigh Leigh, fatal infantile Mynpathy,benign Reversiblemyopathy
Autosomal dominant Autosomal recessive ?Autosomal recessive
PEn PEn, MNGIE Fatal infantile multisystemdisorder
126
Current Paediatrics
only when present at very high levels. This threshold effect is also seen with other point mutations. Conversely, the same phenotype may be associated with a number of distinct mutations, e.g. several tRNA mutations produce MELAS, and mutations more usually associated with MERRF and MELAS may also cause Leigh syndrome. More than 15 point mutations are associated with Leber's hereditary optic neuropathy (LHON). These are usually homoplasmic (mtDNA 100% mutant). Whilst some are primary mutations, others seem to act synergistically to increase the probability of blindness. There is also evidence for the presence of nuclear genes which modify the effect of mtDNA mutations, including a putative X-linked gene determining susceptibility to visual loss in LHON and potentially explaining the marked sex difference observed in this disorder. Defects of nDNA
It is thought that defects in nuclear genes are responsible for much of childhood RC disease as mutations of mtDNA are uncommon in childhood. However, in contrast to the large number of mtDNA mutations reported in the literature, only one nuclear gene mutation leading to RC disease has been described to date. This is a homozygous point mutation in the flavoprotein subunit gene of complex II and was found in two French sisters with Leigh syndrome. Defects of complex ! and IV are also inherited as autosomal recessive traits, but no mutations have been identified yet. Linkage analysis in families with PEO associated with multiple deletions has localized affected genes to chromosome 10 in a large Finnish pedigree and to chromosome 3 in some Italian families. In other families with PEO, multiple deletions appear to be inherited as an autosomal recessive trait. Defects of several nuclear genes may therefore produce the same phenotype. Children with mtDNA depletion usually present in early infancy with hypotonia, liver and renal failure and lactic acidosis. Cell fusion studies, in which the patient's nucleus is replaced with a control nucleus, have shown correction of the defect, implying involvement of nuclear gene(s) in this disorder. Thus at least two disorders of mtDNA are caused by failure of communication between the nuclear and mitochondrial genomes.
INVESTIGATION Unfortunately, no single diagnostic test is available and it is rarely possible to pinpoint the underlying molecular defect. Consequently, a range of diagnostic tests are necessary, aiming to confirm the presence of a respiratory chain defect and to try to identify the molecular defect. Initial laboratory investigations aim to demonstrate increased lactate concentrations and define the extent of organ involvement. Measurement
of lactate is fraught with difficulties as artefactual elevation may follow struggling or venous stasis during venepuncture. Ideally, the sample should be taken at rest from a previously sited catheter and immediately deproteinized in perchloric acid. Measurements should be repeated on a number of occasions, both fasting and postprandial. Persistent elevation is suggestive of a defect of the RC or of pyruvate metabolism, but other causes of lactic acidosis, such as tissue hypoxia and disorders of gluconeogenesis and long chain fat oxidation, need to be excluded. Conversely, a normal lactate does not discount the possibility of a mitochondrial disorder. Measurement of lactate in cerebrospinal fluid is less prone to artefactual variation but has not been critically evaluated. Simultaneous measurement of lactate and pyruvate may be helpful. The lactate:pyruvate ratio is often high in RC defects and low in disorders of pyruvate metabolism, but these rules are not universally upheld. Routine biochemistry may demonstrate abnormal function of other organs, such as deranged liver or renal tubular function. Neuro-imaging by computed tomography or magnetic resonance imaging may demonstrate intracranial lesions such as symmetrical lesions involving the basal ganglia and peri-aqueductal grey matter in Leigh syndrome and occipital and posterior temporal cortical lesions in MELAS. Magnetic resonance spectroscopy may be used to measure regional brain lactate levels in situ and provides a valuable non-invasive method for investigating disorders of energy metabolism in vivo but it is relatively insensitive. Echocardiography is helpful to examine myocardial function. The order of subsequent investigations is guided by the individual patient. If the clinical features are highly suggestive of a known mtDNA point mutation, then mtDNA analysis is the first line of investigation. Most centres screen for the mtDNA point mutations associated with MELAS, MERRF and NARP/Leigh syndromes, and also for mtDNA deletions and depletion if clinically indicated. DNA for analysis may be extracted from whole blood, tissue biopsy material or cultured skin fibroblasts. If the clinical features and family history are especially suggestive of a mtDNA defect but no mutation is found on routine screening, then the entire mitochondrial genome may be sequenced. However, this is a costly and time-consuming exercise and currently only available as a research tool. In other cases evidence for a biochemical defect is sought. Assays of individual RC enzyme complexes are complicated, difficult technically and need to be performed in specialized laboratories. Tissue samples are required for these and the results are meaningful only if assayed in an affected tissue, e.g. skeletal muscle in myopathies or liver in hepatic dysfunction (if clotting permits). Some centres assay RC enzyme complexes in cultured skin fibroblasts, but these do not always express RC defects. In many cases the
Mitochondrial disorders
127
affected tissue is inaccessible (e.g. central nervous system, heart). In these instances muscle biopsy may be worthwhile as the defect may be expressed in muscle even in the absence of overt myopathy. Open muscle biopsy is the preferred method for assay of RC enzymes, as needle biopsy does not provide sufficient tissue for most laboratories. A number of other tests may be performed on the biopsied muscle, namely morphological analysis, histochemistry, immunohistochemistry and ultrastructural studies. Ragged red fibres, the morphological hallmark of mitochondrial disease in adults, are rare in childhood. The ragged appearance seen on Gomori trichrome stain is caused by subsarcolemmal accumulation of mitochondria. Electron microscopy may reveal abnormal mitochondria with paracrystalline inclusions, and histochemical staining of cytochrome oxidase may be reduced.
Genetic counselling is difficult because of the possible modes of inheritance. Although most mitochondrial disorders in childhood are thought to be inherited as autosomal recessive traits, a 25% recurrence risk cannot be given where there is a possibility of maternal inheritance. Maternally inherited m t D N A defects may have up to 100% recurrence and recurrence can only be reliably avoided by in vitro fertilization of a d o n o r egg. Except in a few families with cytochrome oxidase or complex I deficiency, prenatal diagnosis is not possible. There has been a single report of prenatal diagnosis for a m t D N A point mutation. However, this is an extremely unreliable procedure as there is a risk o f maternal contamination of the sample and very little is known about drift of m t D N A mutations during the intrauterine period.
M A N A G E M E N T AND P R O G N O S I S
CONCLUSION
Despite advances in diagnosis and classification of mitochondrial disease, treatment remains unsatisfactory. Symptomatic and supportive measures are the mainstay o f management. These include bicarbonate for acute exacerbations of acidosis, regular transfusions for persistent anaemia, treatment of fits and family support. N o specific treatments are available for the majority o f patients. Attempts to overcome the block in the energy production pathway, for example by supplementation of RC cofactors such as riboflavin and coenzyme Q and administration o f antioxidants (e.g. ascorbic acid and vitamin K), have been largely unsuccessful. Anecdotal reports of success have not been supported by controlled trials. Even if the lactic acidosis improves, the course of the disease is rarely altered. However, the design of such trials is severely hampered by the lack of homogeneity amongst these disorders, their unpredictable course and the small numbers of affected individuals. Gene therapy remains a distant goal and will be particularly difficult for defects of m t D N A as special carrier systems will be required to target mitochondria. The prognosis for individuals with these disorders is highly variable. Patients presenting in the neonatal period usually have a fulminating course although rarely the disorder may resolve (benign reversible myopathies). In infancy and childhood the outlook is p o o r for most patients, with progressive but unpredictable deterioration. Those who present later have a more indolent course.
The clinical spectrum of mitochondrial disorders is now very wide. It should be recognized that they may present at any age, with almost any symptoms and with any mode of inheritance, providing a major challenge for paediatricians.
REFERENCES
1. Luft R. The developmentof mitochondrial medicine. Proc Natl Acad Sci USA 1994; 91: 8731-8738. 2. DiMauro S. Mitochondrial encephalomyopathies.In: Rosenberg R N, Prusiner S B, DiMauro S, Barchi R L, Kunkel L M (eds). The metabolic and genetic basis of neurological disease. Boston; Butterworth-Heinemann, 1993: 665-694. 3. Brown G K. Mitochondrial disorders. In: Clayton B E, Round J M (eds). Clinical biochemistry and the sick child 2nd ed. Oxford; BlackwellScientificPublications, 1994:159-172. 4. ShoffnerJ M, WallaceD C. Oxidativephosphorylation diseases. In: ScriverC R, Beaudet A L, Sly W S, Valle D (eds). The metabolic and molecular bases of inherited disease 7th ed. New York; McGraw-Hill, 1995: 1535-1609. 5. Munnich A, Rotig A, Chretien D, Saudubray J M, Cormier V, Rustin R Clinical presentations and laboratory investigations in respiratory chain deficiency.Eur J Pediatr 1996; 155: 262 274. 6. Robinson B H. Lacticacidaemia.Biochim BiophysActa 1993; 1182: 231-244. 7. WallaceD C. Mitochondrial diseases: genotype versus phenotype. Trends Genet 1993; 9: 128-133. 8. Rahman S, Blok R B, Dahl H H-M, et al. Leigh syndrome: clinical features and biochemicaland DNA abnormalities. Ann Neurol 1996; 39:343 351. 9. Macmillan C J, Shoubridge E A. Mitochondrial DNA depletion: prevalencein a paediatric population referred for neurologic evaluation. Pediatr Neurol 1996; 14: 203-210. 10. MorrisA A M, Leonard J V. The treatment of congenital lactic acidoses. J Inherit Metab Dis 96; 19: 573-580.
Mini-symposium: Metabolic disease
Mitochondrial disorders
S. Rahman, J. V. Leonard
Clinical features associated with mitochondrial disease in childhood are summarized in Box 1. Some distinct clinical syndromes are recognized (Box 2), but many patients do not fit into these defined syndromes. It is often the unusual combination of clinical features which suggests a possible diagnosis of respiratory chain disease. Pedigree analysis may be helpful, although genetic heterogeneity means that any mode of inheritance is
INTRODUCTION
The mitochondrion is the cell organelle responsible for production of energy by the process of oxidative phosphorylation. Electrons are progressively oxidized in the respiratory chain, polypeptides of which are encoded by two genomes, that of the nucleus and that within the mitochondrion itself. Disorders of the respiratory chain display marked clinical, biochemical and genetic heterogeneity and diagnosis is notoriously difficult. It is likely that the underlying diagnosis is unrecognized in a significant number of cases. Consequently, the prevalence of these disorders is not known. Mitochondrial diseases are generally classified biochemically, but with recent advances there are the beginnings of a molecular genetic classification.
Box 1 Clinical features associated with mitochondrial disease Neuromuscular
CLINICAL FEATURES
These disorders may present at any age and are clinically very heterogeneous, ranging from fatal neonatal lactic acidosis to a mild adult-onset myopathy. Historically, mainly neuromuscular diseases were recognized as being of mitochondrial origin. These are, consequently, the most well characterized. However, almost all tissues are dependent on mitochondrial energy production and so one might expect multisystem disease. Accordingly, an increasingly large number of nonneuromuscular mitochondrial diseases are being described and a clue to the diagnosis is often the involvement of several apparently unrelated systems.
Ophthalmological
Cardiac Renal Hepatic Gastrointestinal
Haematological Endocrine
Shamima Rahman MRCP, MRC Clinical Training Fellow, James
V. Leonard PhD FRCP, Professor of Paediatric Metabolic Disease, Institute of Child Health, 30 Guilford Street, London W C I N 1EH, UK. Correspondence and requests for offprints to SR.
123
Developmental delay/regression Seizures (especially myoclonic) Ataxia Encephalopathic and stroke-like episodes Dystonia Hypotonia Weakness Fatigue Peripheral neuropathy Sensorineural deafness Optic atrophy Pigmentary retinopathy Progressive external ophthalmoplegia (PEO) Cataracts Cardiomyopathy (usually hypertrophic) Conduction defects Renal tubular dysfunction particularly Fanconi syndrome Liver failure especially in infancy (may be induced by valproate) Pancreatic exocrine insufficiency Recurrent vomiting Chronic diarrhoea Enteropathy with partial villous atrophy Sideroblastic anaemia Pancytopenia Diabetes mellitus Short stature Hypoparathyroidism Hypothyroidism Hypogonadism
124
Current Paediatrics
Box 2 Recognized mitochondrial syndromes Syndrome
Summary of clinical features
Leigh (subacute necrotizing encephalopathy)
Psychomotor regression in infancy or childhood, hypotonia, lactic acidosis and brainstem dysfunction, associated with symmetrical basal ganglia/brainstem lesions on CT/MRI
MERRF (myoclonic epilepsy with ragged red fibres)
Myoclonic epilepsy or action myoclonus. Myopathy with ragged red fibres (RRF) on muscle biopsy. Clinical spectrum includes cerebellar ataxia, deafness, dementia and multiple lipomata
MELAS Stroke-like episodes with focal CT/MRI brain abnormalities, lactic acidosis, RRF myopathy, seizures (mitochondrial (focal/generalized), dementia, recurrent headache, vomiting. Clinical spectrum includes diabetes mellitus, encephalomyopathy with deafness and progressive external ophthalmoplegia (PEO) lactic acidosis and stroke-like episodes) Pearson (marrow pancreas) syndrome
Refractory sideroblastic anaemia, vacuolization of marrow precursors, exocrine pancreatic dysfunction. Survivors develop KSS in adolescence
Kearns-Sayre syndrome (KSS)
Invariant triad of onset < 20 years, PEO, pigmentary retinopathy plus at least one of heart block/cerebellar syndrome/raised CSF protein > 1 g/l
NARP (neurogenic muscle weakness, ataxia, retinitis pigmentosa)
Developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal weakness and sensory neuropathy. Clinical spectrum overlaps with Leigh syndrome
LHON (Leber's hereditary optic neuropathy)
Painless subacute bilateral loss of vision with central visual field defects, abnormal colour vision and optic atrophy. Onset usually 18-30 years but may be in childhood. Marked male predominance
MNGIE (mitochondrial neurogastrointestinal encephalomyopathy)
Gastrointestinal dysmotility, peripheral neuropathy, myopathy, ophthalmoplegia
mtDNA depletion syndrome
Hypotonia, developmental delay, lactic acidosis, liver failure, Fanconi syndrome
possible. Maternal inheritance is highly suggestive of a mitochondrial D N A (mtDNA) mutation, whilst defects of nuclear D N A may be inherited as autosomal dominant, autosomal recessive or sexlinked traits. Furthermore, clinical features may vary widely even between affected members of a single family.
BIOCHEMICAL CLASSIFICATION
Strictly speaking, mitochondrial diseases encompass defects of all mitochondrial proteins including enzymes involved in pyruvate metabolism, fatty acid I~-oxidation, the Krebs cycle and many others. However, in practice the term mitochondrial disease is usually taken to refer to defects of the mitochondrial respiratory chain (RC) and of oxidative phosphorylation, which comprise five complexes. Complex II is the only RC complex encoded entirely on the nuclear
genome; the others have both mitochondrial and nuclear-encoded subunits (Table 1). A defect in one complex may have several different clinical manifestations. Complex I deficiency is frequently seen with point mutations of mitochondrial transfer R N A (tRNA) genes including myoclonic epilepsy with ragged red fibres (MERRF) and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndromes. Being the largest complex, with the greatest number of mitochondrially-encoded subunits, it is one that is commonly affected by defects of the mitochondrial genome. Complex I deficiency is also described as a probable autosomal recessive trait presenting as fatal infantile myopathy with lactic acidosis, cataract and cardiomyopathy or neurodegenerative disease including Leigh syndrome. The clinical spectrum of complex IV deficiency includes lactic acidosis with isolated myopathy, or combined with Fanconi's syndrome, cardiomyopathy or Leigh syndrome.
Table 1 Respiratory chain complexes Complex
Name
Complex I Complex II Complex III Complex IV Complex V
NADH ubiquinone oxidoreductase Succinate ubiquinone oxidoreductase Ubiquinol cytochrome c oxidoreductase Cytochrome c oxidase ATP synthase
mtDNA encoded subunits
Nuclear encoded subunits
7 0 1 3 2
~34 4 10 10 11 or 12
Mitochondrial disorders Thus, defects of different complexes may have very similar clinical and pathological findings. For example, deficiencies of complex I or complex IV, as well as deficiency of the pyruvate dehydrogenase complex (PDHC), may cause Leigh syndrome. Both complex I and complex IV deficiency may produce a benign reversible infantile myopathy, clinically indistinguishable from the fatal infantile myopathies associated with defects of these enzymes. The underlying mechanism in these reversible myopathies is postulated to be a transient defect in a developmentally regulated subunit. In other patients deficiencies of multiple RC enzyme complexes may be present, in particular combined deficiencies of complexes I and IV. There may also be an associated deficiency of PDHC, implying an underlying defect of a common regulatory gene.
G E N E T I C CLASSIFICATION The genetic classification of mitochondrial disorders is complicated because of the contribution of both the mitochondrial and nuclear genomes. The mitochondrial genome is a 16 659 base pair (bp) circular D N A molecule encoding 13 polypeptide components of the respiratory chain, 22 tRNAs and two ribosomal RNAs (rRNAs). Mitochondrial genetics differs from classical Mendelian inheritance in that m t D N A is exclusively maternally inherited and, unlike nuclear genes which have two alleles, is present in multiple copy numbers. There are between two and ten m t D N A molecules per mitochondrion and hundreds or thousands of mitochondria per cell. This amplification of the mitochondrial genome allows for the phenomenon of heteroplasmy, whereby varying proportions of mutant and normal m t D N A coexist
within a cell. This in turn leads to variable tissue expression and contributes to the clinical heterogeneity. The term homoplasmy is used to indicate the presence of either 100% wild type or 100% mutant m t D N A in a tissue. However, the vast majority of mitochondrial proteins are encoded on the nuclear genome and are imported into the mitochondria from the cytoplasm using a complex translocation machinery which is also encoded on the nucleus. Several nuclear factors control m t D N A replication, transcription and translation and it is probable that defects of nuclear D N A play a major aetiological role in mitochondrial disease. Defects of m t D N A
Pathogenic m t D N A mutations include gross structural rearrangements (single deletions, multiple deletions, duplications) and point mutations (Table 2). Deletions are usually single, heteroplasmic and sporadic. Thirty percent of patients with deletions have an identical 'common' deletion of 4977 bp which includes several protein coding and t R N A genes and one of the origins of replication of the m t D N A molecule. Clinical features associated with this deletion are variable, ranging from progressive external ophthalmoplegia (PEO) to Pearson syndrome. This clinical heterogeneity probably reflects the degree of heteroplasmy and the tissue distribution of the deleted m t D N A molecules. More than 50 point mutations in m t D N A have already been described, involving tRNA, rRNA and protein coding genes. These mutations are usually heteroplasmic. As with deletions, there is no strict genotype-phenotype correlation. For example, the 8993 neurogenic muscle weakness, ataxia, retinitis pigmentosa (NARP) mutation seems to cause Leigh syndrome
Table 2 Geneticclassification
Mutation Defects of mtDNA Single deletions Duplications or duplication/deletions Point mutations tRNA genes e.g. nt 3243 nt 8344 Protein coding genes e.g. nt 8993 nt 3460, 11778, 14484 Defects of nuclear DNA Protein coding genes e.g. ComplexII Complex I ~ mutations Complex IV J not yet found Regulatory genes (none yet described) Defects of intergenomiccommunication (genes responsiblenot yet isolated) Multiple mtDNA deletions mtDNA depletion
125
Inheritance
Clinical features
Usually sporadic Sporadic or maternal
PEn, KSS, Pearson Pearson, proximalrenal tubulopathy
Maternal MELAS MERRF Maternal NARP, Leigh LHON
Autosomal recessive ?Autosomal recessive ?Autosomal recessive
Leigh Leigh, fatal infantile Mynpathy,benign Reversiblemyopathy
Autosomal dominant Autosomal recessive ?Autosomal recessive
PEn PEn, MNGIE Fatal infantile multisystemdisorder
126
Current Paediatrics
only when present at very high levels. This threshold effect is also seen with other point mutations. Conversely, the same phenotype may be associated with a number of distinct mutations, e.g. several tRNA mutations produce MELAS, and mutations more usually associated with MERRF and MELAS may also cause Leigh syndrome. More than 15 point mutations are associated with Leber's hereditary optic neuropathy (LHON). These are usually homoplasmic (mtDNA 100% mutant). Whilst some are primary mutations, others seem to act synergistically to increase the probability of blindness. There is also evidence for the presence of nuclear genes which modify the effect of mtDNA mutations, including a putative X-linked gene determining susceptibility to visual loss in LHON and potentially explaining the marked sex difference observed in this disorder. Defects of nDNA
It is thought that defects in nuclear genes are responsible for much of childhood RC disease as mutations of mtDNA are uncommon in childhood. However, in contrast to the large number of mtDNA mutations reported in the literature, only one nuclear gene mutation leading to RC disease has been described to date. This is a homozygous point mutation in the flavoprotein subunit gene of complex II and was found in two French sisters with Leigh syndrome. Defects of complex ! and IV are also inherited as autosomal recessive traits, but no mutations have been identified yet. Linkage analysis in families with PEO associated with multiple deletions has localized affected genes to chromosome 10 in a large Finnish pedigree and to chromosome 3 in some Italian families. In other families with PEO, multiple deletions appear to be inherited as an autosomal recessive trait. Defects of several nuclear genes may therefore produce the same phenotype. Children with mtDNA depletion usually present in early infancy with hypotonia, liver and renal failure and lactic acidosis. Cell fusion studies, in which the patient's nucleus is replaced with a control nucleus, have shown correction of the defect, implying involvement of nuclear gene(s) in this disorder. Thus at least two disorders of mtDNA are caused by failure of communication between the nuclear and mitochondrial genomes.
INVESTIGATION Unfortunately, no single diagnostic test is available and it is rarely possible to pinpoint the underlying molecular defect. Consequently, a range of diagnostic tests are necessary, aiming to confirm the presence of a respiratory chain defect and to try to identify the molecular defect. Initial laboratory investigations aim to demonstrate increased lactate concentrations and define the extent of organ involvement. Measurement
of lactate is fraught with difficulties as artefactual elevation may follow struggling or venous stasis during venepuncture. Ideally, the sample should be taken at rest from a previously sited catheter and immediately deproteinized in perchloric acid. Measurements should be repeated on a number of occasions, both fasting and postprandial. Persistent elevation is suggestive of a defect of the RC or of pyruvate metabolism, but other causes of lactic acidosis, such as tissue hypoxia and disorders of gluconeogenesis and long chain fat oxidation, need to be excluded. Conversely, a normal lactate does not discount the possibility of a mitochondrial disorder. Measurement of lactate in cerebrospinal fluid is less prone to artefactual variation but has not been critically evaluated. Simultaneous measurement of lactate and pyruvate may be helpful. The lactate:pyruvate ratio is often high in RC defects and low in disorders of pyruvate metabolism, but these rules are not universally upheld. Routine biochemistry may demonstrate abnormal function of other organs, such as deranged liver or renal tubular function. Neuro-imaging by computed tomography or magnetic resonance imaging may demonstrate intracranial lesions such as symmetrical lesions involving the basal ganglia and peri-aqueductal grey matter in Leigh syndrome and occipital and posterior temporal cortical lesions in MELAS. Magnetic resonance spectroscopy may be used to measure regional brain lactate levels in situ and provides a valuable non-invasive method for investigating disorders of energy metabolism in vivo but it is relatively insensitive. Echocardiography is helpful to examine myocardial function. The order of subsequent investigations is guided by the individual patient. If the clinical features are highly suggestive of a known mtDNA point mutation, then mtDNA analysis is the first line of investigation. Most centres screen for the mtDNA point mutations associated with MELAS, MERRF and NARP/Leigh syndromes, and also for mtDNA deletions and depletion if clinically indicated. DNA for analysis may be extracted from whole blood, tissue biopsy material or cultured skin fibroblasts. If the clinical features and family history are especially suggestive of a mtDNA defect but no mutation is found on routine screening, then the entire mitochondrial genome may be sequenced. However, this is a costly and time-consuming exercise and currently only available as a research tool. In other cases evidence for a biochemical defect is sought. Assays of individual RC enzyme complexes are complicated, difficult technically and need to be performed in specialized laboratories. Tissue samples are required for these and the results are meaningful only if assayed in an affected tissue, e.g. skeletal muscle in myopathies or liver in hepatic dysfunction (if clotting permits). Some centres assay RC enzyme complexes in cultured skin fibroblasts, but these do not always express RC defects. In many cases the
Mitochondrial disorders
127
affected tissue is inaccessible (e.g. central nervous system, heart). In these instances muscle biopsy may be worthwhile as the defect may be expressed in muscle even in the absence of overt myopathy. Open muscle biopsy is the preferred method for assay of RC enzymes, as needle biopsy does not provide sufficient tissue for most laboratories. A number of other tests may be performed on the biopsied muscle, namely morphological analysis, histochemistry, immunohistochemistry and ultrastructural studies. Ragged red fibres, the morphological hallmark of mitochondrial disease in adults, are rare in childhood. The ragged appearance seen on Gomori trichrome stain is caused by subsarcolemmal accumulation of mitochondria. Electron microscopy may reveal abnormal mitochondria with paracrystalline inclusions, and histochemical staining of cytochrome oxidase may be reduced.
Genetic counselling is difficult because of the possible modes of inheritance. Although most mitochondrial disorders in childhood are thought to be inherited as autosomal recessive traits, a 25% recurrence risk cannot be given where there is a possibility of maternal inheritance. Maternally inherited m t D N A defects may have up to 100% recurrence and recurrence can only be reliably avoided by in vitro fertilization of a d o n o r egg. Except in a few families with cytochrome oxidase or complex I deficiency, prenatal diagnosis is not possible. There has been a single report of prenatal diagnosis for a m t D N A point mutation. However, this is an extremely unreliable procedure as there is a risk o f maternal contamination of the sample and very little is known about drift of m t D N A mutations during the intrauterine period.
M A N A G E M E N T AND P R O G N O S I S
CONCLUSION
Despite advances in diagnosis and classification of mitochondrial disease, treatment remains unsatisfactory. Symptomatic and supportive measures are the mainstay o f management. These include bicarbonate for acute exacerbations of acidosis, regular transfusions for persistent anaemia, treatment of fits and family support. N o specific treatments are available for the majority o f patients. Attempts to overcome the block in the energy production pathway, for example by supplementation of RC cofactors such as riboflavin and coenzyme Q and administration o f antioxidants (e.g. ascorbic acid and vitamin K), have been largely unsuccessful. Anecdotal reports of success have not been supported by controlled trials. Even if the lactic acidosis improves, the course of the disease is rarely altered. However, the design of such trials is severely hampered by the lack of homogeneity amongst these disorders, their unpredictable course and the small numbers of affected individuals. Gene therapy remains a distant goal and will be particularly difficult for defects of m t D N A as special carrier systems will be required to target mitochondria. The prognosis for individuals with these disorders is highly variable. Patients presenting in the neonatal period usually have a fulminating course although rarely the disorder may resolve (benign reversible myopathies). In infancy and childhood the outlook is p o o r for most patients, with progressive but unpredictable deterioration. Those who present later have a more indolent course.
The clinical spectrum of mitochondrial disorders is now very wide. It should be recognized that they may present at any age, with almost any symptoms and with any mode of inheritance, providing a major challenge for paediatricians.
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