15 Defects of the Respiratory Chain

Transcription

1 15 Defects of the Respiratory Chain Arnold Munnich 15.1 Clinical Presentation Fetuses Neonates Infants Children and Adults Metabolic Derangement Genetics Mutations in Mitochondrial DNA Mutations in Nuclear DNA Genetic Analysis of Respiratory Chain Deficiencies Genetic Counseling and Prenatal Diagnosis Diagnostic Tests Screening Tests Enzyme Assays Histopathological Studies Magnetic Resonance Spectroscopy of Muscle and Brain Treatment and Prognosis 207 References 208

2 198 Chapter 15 Defects of the Respiratory Chain III The Respiratory Chain The respiratory chain is divided into five functional units or complexes embedded in the inner mitochondrial membrane. Complex I [reduced nicotinamide adenine dinucleotide (NADH)-coenzyme Q (CoQ) reductase] carries reducing equivalents from NADH to CoQ and consists of different polypeptides, seven of which are encoded by mitochondrial DNA. Complex II (succinate-coq reductase) carries reducing equivalents from reduced flavin adenine dinucleotide (FADH 2 ) to CoQ and contains five polypeptides, including the flavin adenine dinucleotide-dependent succinate dehydrogenase and a few non-heme-iron-sulfur centers. Complex III (reduced-coq-cytochrome-c reductase) carries electrons from CoQ to cytochrome c and contains 11 subunits. Complex IV (cytochrome-c oxidase) catalyzes the transfer of reducing equivalents from cytochrome c to molecular oxygen. It is composed of two cytochromes (a and a 3 ), two copper atoms, and 13 different protein subunits. The respiratory chain catalyzes the oxidation of fuel molecules by oxygen and the concomitant energy transduction into ATP. During the oxidation process, electrons are transferred to oxygen via the energy-transducing complexes: complexes I, III, and IV for succinate and complexes III and IV for FADH 2 derived from the -oxidation pathway via the electron-transfer flavoprotein (ETF) and the ETF-CoQ oxidoreductase system. CoQ (a lipoidal quinone) and cytochrome c (a lowmolecular-weight hemoprotein) act as shuttles between the complexes. The flux of electrons is coupled to the translocation of protons (H + ) into the intermembrane space at three coupling sites (complexes I, III, and IV). This creates a transmembrane gradient. Complex V (ATP synthase) allows protons to flow back into the mitochondrial matrix and uses the released energy to synthesize ATP. Three molecules of ATP are generated for each molecule of NADH oxidized. CI CII CIII CIV CV outer membr. inner membr. H + H + H + Q C H + matrix NADH Succinate O 2 electron flux Pi proton flux ATP ADP. Fig The mitochondrial respiratory chain. ADP, adenosine diphosphate; ATP, adenosine triphosphate; c, cytochrome c; CI, complex I (NADH-coenzyme-Q reductase); CII, complex II (succinate-coenzyme-q reductase); CIII, complex III (reducedcoenzyme-q-cytochrome-c reductase); CIV, complex IV (cytochrome-c oxidase); CV, complex V (ATP synthase); NADH, reduced nicotinamide adenine dinucleotide; Pi, inorganic phosphate; Q, coenzyme Q

3 15.1 Clinical Presentation Respiratory chain deficiencies have long been regarded as neuromuscular diseases only. However, oxidative phosphorylation (i.e. ATP synthesis by the respiratory chain) is not restricted to the neuromuscular system but proceeds in all cells that contain mitochondria (. Fig. 15.1). Most non-neuromuscular organs and tissues are, therefore, also dependent upon mitochondrial energy supply. Due to the twofold genetic origin of respiratory enzymes [nuclear DNA and mitochondrial (mtdna)], 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. The diagnosis of a respiratory chain deficiency is difficult to consider initially when only one abnormal symptom is present. In contrast, this diagnosis is easier to consider when two or more seemingly unrelated symptoms are observed. The treatment, mainly dietetic, does not markedly influence the usually unfavorable course of the disease Clinical Presentation Due to the ubiquitous nature of oxidative phosphorylation, a defect of the mitochondrial respiratory chain should be considered in patients presenting (1) with an unexplained association of neuromuscular and/or non-neuromuscular symptoms, (2) with a rapidly progressive course, and (3) with symptoms involving seemingly un related organs or tissues. The disease may begin at virtually any age.. Table 15.1 summarizes the most frequently observed symptoms. Whatever the age of onset and the presenting symptom, the major feature is the increasing number of tissues affected in the course of the disease. This progressive organ involvement is constant, and the central nervous system is almost consistently involved in the late stage of the disease. While the initial symptoms usually persist and gradually worsen, they may occasionally improve or even disappear as other organs become involved. This is particularly true for bone marrow and gut. Indeed, remarkable remissions of pancytopenia or watery diarrhea have been reported in infants who later developed other organ involvements. Moreover, several patients whose disease apparently started in childhood or adulthood were retrospectively shown to have experienced symptoms (transient sideroblastic anemia, neutropenia, chronic watery diarrhea, or failure to thrive) of unexplained origin in early infancy. Similarly, a benign reversible infantile myopathy with hypotonia, weakness, macroglossia, respiratory distress, and spontaneous remission within 12 years has been described. Certain clinical features or associations are more frequent at certain ages and have occasionally been identified as distinct entities, suggesting that these associations are not fortuitous. However, considerable overlap in clinical features leads to difficulties in the classification of many patients, and the nature, clinical course, and severity of symptoms vary among (and even within) affected individuals. It is more useful to bear in mind that the diagnosis of respiratory chain deficiency should be considered regardless of the age of onset and the nature of the presenting symptom when presenting with an unexplained association of signs with a progressive course involving seemingly unrelated organs or tissues. The non-exhaustive list of clinical profiles listed below illustrates the diversity of presentations (. Table 15.1) Fetuses Intrauterine growth retardation (below 3rd percentile for gestational age) either isolated or associated with otherwise unexplained antenatal anomalies is retrospectively detected in 20% of respiratory enzyme chain deficient patients. Antenatal anomalies are usually multiple and include polyhydramnios, oligoamnios, arthrogryposis, decreased fetal movements, ventricular septal defects, hypertrophic cardiomyopathy, vertebral and limb defects or other visceral anomalies (VACTERL association). At variance with a number of metabolic diseases which have a symptom-free period, respiratory chain deficiency may have an antenatal expression related to the time course of the disease gene expression in the embryofetal period [1] Neonates In the neonate (age less than 1 month), the following clinical profiles are seen: 4 Ketoacidotic coma with recurrent apneas, seizures, severe hypotonia, liver enlargement, and proximal tubulopathy, with or without a symptom-free period [2]. 4 Severe sideroblastic anemia (with or without hydrops fetalis), with neutropenia, thrombocytopenia, and exocrine pancreatic dysfunction of unexplained origin (Pearson marrow-pancreas syndrome) [3]. 4 Concentric hypertrophic cardiomyopathy and muscle weakness with an early onset and a rapidly progressive course (dilated cardiomyopathies are exceptional) [4]. 4 Concentric hypertrophic cardiomyopathy with profound central neutropenia and myopathic features in males (Barth syndrome) [5]. 4 Hepatic failure with lethargy, hypotonia, and proximal tubulopathy of unexplained origin [2].

4 200 Chapter 15 Defects of the Respiratory Chain. Table The most frequently observed symptoms in defects of the respiratory chain III Neonatal period (0 1 month) Central nervous system Iterative apnea, lethargy, drowsiness, near-miss Limb and trunk hypotonia Congenital lactic acidosis Ketoacidotic coma Muscle Myopathic presentation Muscular atrophy, hypotonia Stiffness, hypertonia Recurrent myoglobinuria Poor head control, poor spontaneous movement Liver Hepatic failure, liver enlargement Heart Hypertrophic cardiomyopathy (concentric) Kidney Proximal tubulopathy (De Toni-Debré-Fanconi syndrome) Infancy (1 month 2 years) Central nervous system Recurrent apneas, near-miss Recurrent ketoacidotic coma Poor head control, limb spasticity Psychomotor regression, mental retardation Cerebellar ataxia»stroke-like«episodes Myoclonus, generalized seizures Subacute necrotizing encephalomyopathy (Leigh syndrome) Progressive infantile poliodystrophy (Alpers syndrome) Muscle Myopathic features Muscular atrophy Limb weakness, hypotonia Myalgia, exercise intolerance Recurrent myoglobinuria Liver Progressive liver enlargement Hepatocellular dysfunction Valproate-induced hepatic failure Heart Hypertrophic cardiomyopathy (concentric) Kidney Proximal tubulopathy (De Toni-Debré-Fanconi syndrome) Tubulo-interstitial nephritis (mimicking nephronophtisis) Nephrotic syndrome Renal failure Hemolytic uremic syndrome Gut Recurrent vomiting Chronic diarrhea, villous atrophy Exocrine pancreatic dysfunction Failure to thrive Chronic interstitial pseudo-obstruction Endocrine Short stature, retarded skeletal maturation Recurrent hypoglycemia Multiple hormone deficiency Bone marrow Sideroblastic anemia Neutropenia, thrombopenia Myelodysplastic syndrome, dyserythropoiesis Ear Hearing loss Sensorineural deafness (brain stem or cochlear origin) Eye Optic atrophy Diplopia Progressive external ophthalmoplegia Limitation of eye movements (all directions, upgaze)»salt-and-pepper«retinopathy, pigmentary retinal degeneration Lid ptosis Cataract Skin Mottled pigmentation of photo-exposed areas Trichothiodystrophy Dry, thick and brittle hair Childhood ( 2 years) and adulthood Central nervous system Myoclonus Seizures (generalized, focal, drop attacks, photo - sensitivity, tonicoclonus) Cerebellar ataxia Spasticity Psychomotor regression, dementia, mental retardation»stroke-like«episodes Hemicranial headache, migraine Recurrent hemiparesis, cortical blindness or hemianopsia Leukodystrophy, cortical atrophy Peripheral neuropathy Muscle Progressive myopathy Limb weakness (proximal) Myalgia, exercise intolerance Recurrent myoglobinuria Heart Concentric hypertrophic or dilated cardiomyopathy Different types of heart block Endocrine Diabetes mellitus (insulin- and non-insulin dependent) Growth-hormone deficiency Hypoparathyroidism Hypothyroidism Adrenocorticotrophin deficiency Hyperaldosteronism Infertility (ovarian failure or hypothalamic dysfunction) Eye Lid ptosis Diplopia Progressive external ophthalmoplegia Limitation of eye movements (all directions, upgaze)»salt-and-pepper«retinopathy, pigmentary retinal degeneration Cataract, corneal opacities Leber hereditary optic neuroretinopathy Ear Sensorineural deafness Aminoglycoside-induced ototoxicity (maternally inherited)

5 15.2 Metabolic Derangement Infants In infancy (1 month to 2 years), the clinical profiles include the following: 4 Failure to thrive, with or without chronic watery diarrhea and villous atrophy; unresponsiveness to glutenfree and cow s milk protein-free diet [6]. 4 Recurrent episodes of acute myoglobinuria, hypertonia, muscle stiffness and elevated plasma levels of enzymes unexplained by an inborn error of glycolysis, glycogen olysis, fatty acid oxidation or muscular dystrophy [7]. 4 Proximal tubulopathy (de Toni-Debré-Fanconi syndrome) with recurrent episodes of watery diarrhea, rickets and mottled pigmentation of photo-exposed areas. 4 A tubulo-interstitial nephritis mimicking nephro nophtisis, with the subsequent development of renal failure and encephalomyopathy with leukodystrophy [8]. 4 Severe trunk and limb dwarfism unresponsive to growth-hormone administration, with subsequent hypertrophic cardiomyopathy, sensorineural deafness, and retinitis pigmentosa. 4 Early-onset insulin-dependent diabetes mellitus with diabetes insipidus, optic atrophy, and deafness (Wolfram syndrome) [9]. 4 Rapidly progressive encephalomyopathy with hypotonia, poor sucking, weak crying, poor head control, cerebellar ataxia, pyramidal syndrome, psychomotor regression, developmental delay, muscle weakness, and respiratory insufficiency; occasionally associated with proximal tubulopathy and/or hypertrophic cardiomyopathy. 4 Subacute necrotizing encephalomyopathy (Leigh s disease). This is a devastating encephalopathy characterized by recurrent attacks of psychomotor regression with pyramidal and extrapyramidal symptoms, leukodystrophy, and brainstem dysfunction (respiratory abnormalities). The pathological hallmark consists of focal, symmetrical, and necrotic lesions in the thalamus, brain stem, and the posterior columns of the spinal cord. Microscopically, these spongiform lesions show demyelination, vascular proliferation, and astrocytosis [10] Children and Adults In childhood (above 2 years) and adulthood, the neuromuscular presentation is the most frequent: 4 Muscle weakness with myalgia and exercise intolerance, with or without progressive external ophthalmoplegia [10]. 4 Ataxia, cerebellar atrophy, muscle weakness, seizures and mental retardation. 4 Progressive sclerosing poliodystrophy (Alpers disease) associated with hepatic failure [11]. 4 Encephalomyopathy with myoclonus, ataxia, hearing loss, muscle weakness, and generalized seizures (myoclonus epilepsy, ragged red fibers, MERRF) [10]. 4 Progressive external ophthalmoplegia (PEO) ranging in severity from pure ocular myopathy to Kearns-Sayre syndrome (KSS). KSS is a multisystem disorder characterized by the triad (1) onset before age 20 years, (2) PEO, and (3) pigmentary retinal degeneration, plus at least one of the following: complete heart block, cerebrospinal fluid (CSF) protein levels above 100 mg/dl, or cerebellar ataxia [10]. 4 Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). This syndrome is characterized by onset in childhood, with intermittent hemicranial headache, vomiting, proximal limb weakness, and recurrent neurological deficit resembling strokes (hemiparesis, cortical blindness, hemianopsia), lactic acidosis, and ragged red fibers (RRFs) in the muscle biopsy. Computed tomography (CT) brain scanning shows low-density areas (usually posterior), which may occur in both white and gray matter but do not always correlate with the clinical symptoms or the vascular territories. The pathogenesis of stroke-like episodes in MELAS has been ascribed to either cerebral blood-flow disruption or acute metabolic decompensation in biochemically deficient areas of the brain [10]. 4 Leber s hereditary optic neuroretinopathy (LHON). This disease is associated with rapid loss of bilateral central vision due to optic nerve death. Cardiac dysrythmia is frequently associated with the disease, but no evidence of skeletal muscle pathology or gross structural mitochondrial abnormality has been documented. The median age of vision loss is years, but it can occur at any age between adolescence and late adulthood. Expression among maternally related individuals is variable, and more males are affected [10]. Kjer s autosomal dominant optic atrophy is also caused by a mito chondrial dysfunction in the dynamin-related protein OPA1 [12]. 4 Neurogenic muscle weakness, ataxia, retinitis pigmentosa (NARP) and variable sensory neuropathy with seizures and mental retardation or dementia [13]. 4 Mitochondrial myopathy and peripheral neuropathy, encephalopathy, and gastrointestinal disease manifesting as intermittent diarrhea and intestinal pseudoobstruction (myo-neuro-gastro-intestinal encephalopathy, MNGIE) [10]. 4 Progressive multisystemic failure with encephalopathy, myopathy, peripheral neuropathy and renal failure Metabolic Derangement As the respiratory chain transfers electrons to oxygen, a disorder of oxidative phosphorylation should result in (1) an

6 202 Chapter 15 Defects of the Respiratory Chain III increase in the concentration of reducing equivalents in both mitochondria and cytoplasm and (2) the functional impairment of the citric acid cycle, due to the excess of reduced nicotinamide adenine dinucleotide (NADH) and the lack of nicotinamide adenine dinucleotide (NAD). Therefore, an increase in the ketone body (3-hydroxybutyrate/acetoacetate) and lactate/pyruvate (L/P) molar ratios with a secondary elevation of blood lactate might be expected in the plasma of affected individuals. This is parti c- ularly true in the post-absorptive period, when more NAD is required to adequately oxidize glycolytic substrates. Similarly, as a consequence of the functional impairment of the citric acid cycle, ketone body synthesis increases after meals, due to the channeling of acetyl-coenzyme A (CoA) towards ketogenesis. The elevation of the total level of ketone bodies in a fed individual is paradoxical, as it should normally decrease after meals, due to insulin release (paradoxical hyperketonemia). The position of the block might differentially alter the metabolic profile of the patient. At the level of complex I it impairs the oxidation of the 30 moles of NADH formed in the citric acid cycle. In theory at least, oxidation of reduced flavin adenine dinucleotide (FADH 2 ) derived from succinate producing substrates (methionine, threonine, valine, isoleucine, and odd-numbered fatty acids) should not be altered, because it is mediated by complex II. Similarly, oxidation of FADH 2 derived from the first reaction of the β-oxidation pathway should occur normally, because it is mediated by the electron transfer flavoprotein coenzyme- Q-reductase system. However, complex II deficiency should not markedly alter the redox status of affected individuals fed a carbohydrate-rich diet. A block at the level of complex III should impair the oxidation of both NAD-linked and FAD-linked substrates. Finally, given the crucial role of complex IV in the respiratory chain, it is not surprising that severe defects of cytochrome c oxidase (COX) activity cause severe lactic acidosis and markedly alter redox status in plasma Genetics Any mode of inheritance may be observed in mitochondrial diseases: autosomal recessive, dominant, X-linked, maternal, or sporadic. This variability is due to the high number of genes that encode the respiratory chain proteins, 13 of which are located in the mitochondria. mtdna encodes seven polypeptides of complex I, one of complex III (the apoprotein of cytochrome b), three of complex IV, and two of complex V. The mtdna molecules are small (16.5kb), double-stranded, circular, and contain no introns (. Fig.15.2). mtdna has a number of unique genetic features: 4 It is maternally inherited, and its mutations are, therefore, transmitted by the mother. 4 It has a very high mutation rate involving both nucleotide substitutions and insertions/deletions. During cell division, mitochondria are randomly partitioned into daughter cells. This means that, if normal and mutant mtdna molecules are present in the mother s cells (heteroplasmy), some lineages will have only abnormal. Fig Map of the mitochondrial genome. Regions encoding cytochrome b (cyt b), various subunits of reduced nicotinamide adenine dinucleotide-coenzyme Q reductase (ND), cytochrome oxidase (COX), and adenosine triphosphatase (A), and rrnas are indicated. Replication of the heavy strand starts in the displacement (D) loop at the heavy-strand origin (OH), and that of the light strand at OL

7 15.3 Genetics mtdna (homoplasmy), others will have only normal mtdna (wild type), and still others will have both normal and abnormal mtdna. In these last cells, the phenotype will reflect the proportion of abnormal mtdna. The nuclear genome encodes a number of proteins involved in mtdna replication and transcription, the protein components of the mitochondrial ribosome, multiple structural, chaperone and assembly proteins, and the remaining catalytic subunits of the respiratory enzyme complexes (other than the 13 mtdna-encoded subunits) Mutations in Mitochondrial DNA Pathological alterations of mtdna fall into three major classes: point mutations, rearrangements, and depletions of the number of copies. 4 Point mutations result in amino acid substitutions and modifications of mrna and trna. Most are heteroplasmic, maternally inherited, and associated with a striking variety of clinical phenotypes (LHON, MERRF, MELAS, NARP, Leigh syndrome, diabetes, and deafness) [14]. Interestingly, there are mutation hot spots on the mitochondrial genome as recurrent de novo mutations at specific nucleotide positions have been reported in unrelated families (ND1: G3946A; ND3: T10158C, T10191C; ND5: T12706C, G13094A, G13513A, A13514G; ND6: T14487C) [14]. 4 Rearrangements comprise deletions/duplications that markedly differ in size and position from patient to patient but usually encompass several coding and trna genes. They are usually sporadic, heteroplasmic, and unique and probably arise de novo during oogenesis or in early development (KSS, Pearson syndrome, PEO, diabetes, and deafness [14]). Occasionally, maternally transmitted mtdna rearrangements are found [7]. In other cases, autosomal dominant transmission of multiple mtdna deletions occurs, suggesting mutation of a nuclear gene essential for the function of the mitochondrial genome [15]. Three disease causing genes have been hitherto reported in autosomal dominant multiple mtdna deletions, namely POLG, ANT1 and Twinkle, encoding for the mitochondrial DNA polymerase, the ADP/ATP translocator and a mitochondrial helicase, respectively [16-18]. 4 Decreased mtdna copy number (mtdna depletion) is a recently identified group of respiratory chain deficiencies [19]. Depletions are genetically heterogeneous autosomal recessive conditions involving either a single organ or multiple tissues. The disease causing genes identified to date in mtdna depletion syndromes include POLG and two key enzymes in the mitochondrial nucleotide salvage pathway, deoxyguanosine kinase (DGUOK) [20] and thymidine kinase 2 (TK2) [21]. These deoxyribonucleoside kinases provide mitochondria with deoxyribonucleotides essential for mtdna synthesis. Mutations in DGUOK and POLG have been reported in earlyonset hepatic failure and encephalo pathy and TK2 mutation have been described in neo natal-onset devastating skeletal myopathies (7 also Chap. 35) Mutations in Nuclear DNA A few of the numerous disease-causing nuclear genes have been recently identified, including the gene of Barth syndrome (tafazzin) [22], nuclear genes for complex-i (NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFS6, NDUFS7) [43 48], complex-ii (Fp subunit of succinate dehydrogenase) [29-30], and complex-iv (SURF-1, SCO1, SCO2, COX10, COX15) [31 36] deficiencies, a MNGIE gene (thymidine phosphorylase) [37], and a gene encoding coenzyme Q 10 [37a] Genetic Analysis of Respiratory Chain Deficiencies An extensive family history, with documentation of minor signs in relatives, is of paramount importance in recognizing the mode of inheritance and in deciding on the molecular studies to be performed. Maternal inheritance indicates mtdna mutations, autosomal dominant inheritance indicates multiple mtdna deletions, and sporadic cases and autosomal recessive inheritance (consanguineous parents) indicate mtdna deletions/duplications and nuclear gene mutations, respectively. Investigations require a highly specialized, experienced laboratory and should take into account the following points: 4 The distribution of mutated mtdna molecules may differ widely among tissues, accounting for the variable clinical expression and requiring investigation of the tissue that actually expresses the disease. 4 mtdna rearrangements are unstable and gradually disappear in cultured cells unless uridine is included in the culture medium, thus precluding growth under standard conditions. 4 Negative results neither rule out an mtdna mutation nor provide a clue that a nuclear mutation is involved. 4 Although no clear-cut correlations between phenotypes and genotypes have been identified, certain clinical presentations hint at mutations in particular genes: Leigh syndrome with NARP, SURF1, SDH-Fp and ND1-5 mutations; Alpers syndrome with POLG [38] or DGUOK mutations; MINGIE with thymidine phosphorylase mutations, progressive external ophthalmoplegia with POLG, Twinkle or ANT1 mutations; cardioencephalomyopathy with SCO2 mutations; diabetes mellitus and deafness with MELAS mutations; Pearson syndrome and Kearns-Sayre syndrome with mtdna deletions.

8 204 Chapter 15 Defects of the Respiratory Chain III Genetic Counseling and Prenatal Diagnosis The identification of certain clinical phenotypes, listed above, allows some prediction with respect to their inheri t- ance. Moreover, it should be borne in mind that, in cases of maternal inheritance of a mtdna mutation, risk is absent for the progeny of an affected male but is high for that of a carrier female. In this case, determination of the proportion of mutant mtdna on chorionic villi or amniotic cells is a rational approach. Nevertheless, its predictive value remains uncertain, owing to incomplete knowledge of the tissue distribution of abnormal mtdna, its change during development, and its quantitative relationship to disease severity. In the absence of detectable mtdna mutations, the measurement of the activities of respiratory enzymes in cultured amniocytes or choriocytes provides the only possibility of prenatal diagnosis, particularly since few nuclear mutations have been identified. Unfortunately, relatively few enzyme deficiencies are expressed in cultured fibroblasts of probands, even when grown with uridine. For this reason, the ongoing identification of disease-causing nuclear genes will certainly help in delivering accurate prenatal diagnoses of respiratory chain deficiencies in the future Diagnostic Tests Screening Tests Screening tests include the determination of lactate, pyruvate, ketone bodies, and their molar ratios in plasma as indices of oxidation/reduction status in cytoplasm and mitochondria, respectively (. Table 15.2). Determinations should be made before and 1 h after meals throughout the day. Blood glucose and non-esterified fatty acids should be simultaneously monitored (7 Chap. 3). The observation of a persistent hyperlactatemia (>2.5 mm) with elevated L/P and ketone body molar ratios (particularly in the postabsorptive period) is highly suggestive of a respiratory chain deficiency. In addition, investigation of the redox status in plasma can help discriminate between the different causes of congenital lactic acidosis based on L/P and ketone body molar ratios in vivo [39]. Indeed, an impairment of oxidative phosphorylation usually results in L/P ratios above 20 and ketone body ratios above 2, whereas a defect of the pyruvate dehydrogenase (PDH) complex results in low L/P ratios (<10). Although little is known regarding tricarboxylic acid cycle disorders, it appears that these diseases also result in high L/P ratios, but ketone body molar ratios are lower in these conditions (<1) than in respiratory-chain defects (as also observed in pyruvate carboxylase deficiency; 7 Chap.12) [40 41]. However, these diagnostic tests may fail to detect any disturbance of the redox status in plasma. Pitfalls of metabolic screening are the following: 4 Hyperlactatemia may be latent in basal conditions and may only be revealed by a glucose loading test (2g/kg orally) or by determination of the redox status in the CSF. The measurement of CSF lactate and L/P ratio is useless when the redox status in plasma is altered. 4 Proximal tubulopathy may lower blood lactate and increase urinary lactate. In this case, gas chromatography mass spectrometry can detect urinary lactate and citric acid cycle intermediates. 4 Diabetes mellitus may hamper the entry of pyruvate into the citric acid cycle. 4 Tissue-specific isoforms may be selectively impaired, barely altering the redox status in plasma (this may be particularly true for hypertrophic cardiomyopathies). 4 The defect may be generalized but partial; the more those tissues with higher dependencies on oxidative metabolism suffer (such as brain and muscle), the more the oxidation/reduction status in plasma is impaired. 4 The defect may be confined to complex II, barely altering (in principle) the redox status in plasma. When diagnostic tests are negative, the diagnosis of a respiratory chain deficiency may be missed, especially when only the presenting symptom is present. By contrast, the diagnosis is easier to consider when seemingly unrelated symptoms are observed. For this reason, the investigation of patients at risk (whatever the presenting symptom) includes the systematic screening of all target organs, as multiple organ involvement is an important clue to the diagnosis (. Table 15.2) Enzyme Assays The observation of an abnormal redox status in plasma and/or evidence of multiple organ involvement prompts one to carry out further enzyme investigations. These investigations include two entirely distinct diagnostic procedures that provide independent clues to respiratory-chain deficiencies: polarographic studies and spectrophotometric studies. Polarographic studies consist of the measurement of oxygen consumption by mitochondria-enriched fractions in a Clark electrode in the presence of various oxidative substrates (malate with pyruvate, malate with glutamate, succinate, palmitate, etc.). In the case of complex I deficiency, polarographic studies show impaired respiration with NADH-producing substrates, whilst respiration and phosphorylation are normal with FADH-producing substrates (succinate). The opposite is observed in the case of complex II deficiency, whereas a block at the level of complexes III or IV impairs oxidation of both NADH- and FADH-producing substrates. In complex V deficiency, res-

9 15.4 Diagnostic Tests Table Screening of the respiratory chain Standard screening tests (at least four determinations per day in fasted and 1-h-fed individuals) Plasma lactate Lactate/pyruvate molar ratio: redox status in cytoplasm Ketonemia (paradoxical elevation in fed individuals) -hydroxy butyrate/acetoacetate molar ratio: redox status in the mitochondria Blood glucose and free fatty acids Urinary organic acids (GC-MS): lactate, ketone bodies, citric acid cycle intermediates Provocative tests (when standard tests are inconclusive) Glucose test (2 g/kg orally) in fasted individuals, with determination of blood glucose, lactate, pyruvate, ketone bodies and their molar ratios just before glucose administration, and then every 30 min for 3 4 h (7 Chap. 3) Lactate/pyruvate molar ratios in the CSF (only when no elevation of plasma lactate is observed) Redox status in plasma following exercise Screening for multiple organ involvement Liver: hepatocellular dysfunction Kidney: proximal tubulopathy, distal tubulopathy, proteinuria, renal failure Heart: hypertrophic cardiomyopathy, heart block (ultrasound, ECG) Muscle: myopathic features (CK, ALAT, ASAT, histological anomalies, RRF) Brain: leukodystrophy, poliodystrophy, hypodensity of the cerebrum, cerebellum and the brainstem, multifocal areas of hyperintense signal (MELAS), bilateral symmetrical lesions of the basal ganglia and brain stem (Leigh) (EEG, NMR, CT scan) Peripheral nerve: distal sensory loss, hypo- or areflexia, distal muscle wasting (usually subclinical), reduced motor nerve conduction velocity (NCV) and denervation features (NCV, EMG, peripheral nerve biopsy showing axonal degeneration and myelinated-fiber loss) Pancreas: exocrine pancreatic dysfunction Gut: villous atrophy Endocrine: hypoglycemia, hypocalcemia, hypoparathyroidism, growth hormone deficiency (stimulation tests) Bone marrow: anemia, neutropenia, thrombopenia, pancytopenia, vacuolization of marrow precursors Eye: PEO, ptosis, optic atrophy, retinal degeneration (fundus, ERG, visually evoked potentials) Ear: sensorineural deafness (auditory evoked potentials, brain-stem-evoked response) Skin: trichothiodystrophy, mottled pigmentation of photo exposed areas ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; CK, creatine kinase; CT, computed tomography; ECG, electrocardiogram; EEG, electroencephalogram; EMG, electromyogram; ERG, electroretinogram; GC MS, gas chromatography mass spectrometry; MELAS, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes; NCV, nerve conduction velocity ; NMR, nuclear magnetic resonance; PEO, progressive external ophthalmoplegia; RRF, ragged red fiber piration is impaired with various substrates, but adding the uncoupling agent 2,4-dinitrophenol or calcium ions returns the respiratory rate to normal, suggesting that the limiting step involves phosphorylation rather than the respiratory chain [42]. It is worth remembering that polarographic studies detect not only disorders of oxidative phosphorylation but also PDH deficiencies, citric acid cycle enzyme deficiencies, and genetic defects of carriers, shuttles, and substrates (including cytochrome c, cations, and adenylate), as these conditions also impair the production of reducing equivalents in the mitochondrion. In these cases, however, respiratory enzyme activities are expected to be normal. While previous techniques required gram-sized amounts of muscle tissue, the scaled-down procedures available now allow the rapid recovery of mitochondria-enriched fractions from small skeletal muscle biopsies ( mg, obtained under local anesthetic), thus making polaro graphy feasible in infants and children [43]. Polarographic studies on intact circulating lymphocytes (isolated from 10 ml of blood on a Percoll cushion) or detergent-permeabilized cultured cells (lymphoblastoid cell lines, skin fibroblasts) are also feasible and represent a less invasive and easily reproducible diagnostic test [44]. The only limitation of these techniques is the absolute requirement of fresh material: no polarographic studies are possible on frozen material. Spectrophotometric studies consist of the measurement of respiratory enzyme activities separately or in groups, using specific electron acceptors and donors. They do not require the isolation of mitochondrial fractions and can be carried out on tissue homogenates. For this reason, the amount of material required for enzyme assays is very small and can easily be obtained by needle biopsies of liver and kidney, and even by endomyocardial biopsies [4]. Similarly, a 25-ml flask of cultured skin fibroblasts or a lymphocyte pellet derived from a 10-ml blood sample are sufficient for extensive spectrophotometric studies. Samples should be frozen immediately and kept dry in liquid nitrogen (or at 80 C). Particular attention should be given to apparently pa r- adoxical cases were respiratory enzyme activities are separately normal but deficient when tested in groups (I III,

10 206 Chapter 15 Defects of the Respiratory Chain III II III, III V) as these are possible cases of coenzyme Q 10 (CoQ 10 ) deficiency, a potentially treatable condition due to an inborn error of quinone synthesis [37a]. CoQ 10 plays a pivotal role in the mitochondrial respiratory chain. It distributes the electrons between the various dehydrogenases and the cytochrome segments of the respiratory chain. It is in large excess compared to any other component of the respiratory chain and forms a kinetically compartmentalized pool, the redox status of which tightly regulates the activity of the dehydrogenases. Since conclusive evidence of respiratory chain deficiency is given by enzyme assays, the question of which tissue should be investigated deserves particular attention. In principle, the relevant tissue is the one that clinically expresses the disease. When the skeletal muscle expresses the disease, the appropriate working material is a microbiopsy of the deltoid. When the hematopoietic system expresses the disease (i.e., Pearson syndrome), tests should be carried out on circulating lymphocytes, polymorphonuclear cells, or bone marrow. However, when the disease is predominantly expressed in the liver or heart, gaining access to the target organ is far less simple. Yet, a needle biopsy of the liver or an endomyocardial biopsy are usually feasible. If not, or when the disease is mainly expressed in a barely accessible organ (brain, retina, endocrine system, smooth muscle), peripheral tissues (including skeletal muscle, cultured skin fibroblasts, and circulating lymphocytes) should be extensively tested. Whichever the expressing organ, it is essential to take skin biopsies from such patients (even postmortem) for subsequent investigations on cultured fibroblasts. It should be borne in mind, however, that the in vitro investigation of oxidative phosphorylation remains difficult regardless the tissue tested. Several pitfalls should be considered: 4 A normal respiratory enzyme activity does not preclude mitochondrial dysfunction even when the tissue tested clinically expresses the disease. One might be dealing with a kinetic mutant, tissue heterogeneity, or cellular mosaicism (heteroplasmy; see below). In this case, one should carry out extensive molecular genetic analyses, test other tissues, and (possibly) repeat investigations later. 4 Apart from overt misdiagnosis (i.e., confusion of Pearson syndrome with Schwachman syndrome) and false respiratory enzyme deficiencies (particularly common in non-expert centers), we are now aware of true secondary respiratory enzyme deficiency in: (1) other inborn errors of metabolism, namely propionic acidemia, TCA cycle disorders (fumarase deficiency), fatty acid oxidation disorders (long chain and 3-hydroxy long-chain acyl-coa dehydrogenase deficiency) and mevalonate kinase deficiency); (2) primary central nervous system (CNS) disorders, particularly Friedreich ataxia, where iron load causes a free radical-mediated iron sulfur cluster injury in the respiratory chain; (3) chromosomal microdeletions, unbalancing the stoechiometry of the respiratory chain (i.e., 1p36 deletion, 5q deletion of the NSD1 gene in the Sotos syndrome). 4 The scattering of control values occasionally hampers the recognition of enzyme deficiencies, as normal values frequently overlap those found in the patients. It is helpful to express results as ratios, especially as the normal functioning of the respiratory chain requires a constant ratio of enzyme activities [45]. Under these conditions, patients whose absolute activities are in the low normal range can be unambiguously diagnosed as enzyme deficient, although this expression of results may fail to recognize generalized defects of oxidative phosphorylation. 4 No reliable method for the assessment of complex I activity in circulating or cultured cells is presently available, because oxidation of NADH-generating substrates by detergent-treated or freeze-thawed control cells is variable, and the rotenone-resistant NADH cytochrome c reductase activity is very high in this tissue. 4 The phenotypic expression of respiratory enzyme deficiencies in cultured cells is unstable, and activities return to normal values when cells are grown in a standard medium [46]. The addition of uridine (200 mm) to the culture medium avoids counterselection of respiratory enzyme-deficient cells and allows them to grow normally, thereby stabilizing the mutant phenotype (uridine, which is required for nucleic acid synthesis, is probably limited by the secondary deficiency of the respiratory chain-dependent dihydro-orotate dehydrogenase activity) [47]. 4 Discrepancies between control values may indicate faulty experimental conditions. Activities dependent on a single substrate should be consistent when tested under non-rate-limiting conditions. For example, normal succinate cytochrome c reductase activity should be twice as high as normal succinate quinone dichlorophenolindophenol (DCPIP) reductase activity (because one electron is required to reduce cytochrome c, while two are required to reduce DCPIP). 4 Incorrect freezing may result in a rapid loss of quinonedependent activities, probably due to peroxidation of membrane lipids. Tissue samples fixed for morphological studies are inadequate for subsequent respiratory enzyme assays Histopathological Studies The histological hallmark of mitochondrial myopathy is the RRF, which is demonstrated using the modified Gomori trichrome stain and contains peripheral and inter-myofibrillar accumulations of abnormal mitochondria. Although the diagnostic importance of pathological studies is un-

11 15.5 Treatment and Prognosis disputed, the absence of RRFs does not rule out the diagnosis of mitochondrial disorder [10]. Different histochemical stains for oxidative enzymes are used to analyze the distribution of mitochondria in the individual fibers and to evaluate the presence or absence of the enzymatic activities. Histochemical staining permits an estimation of the severity and heterogeneity of enzyme deficiency in the same muscle section. Myofibrillar integrity and the predominant fiber type and distribution can be evaluated with the myofibrillar adenosine triphosphatase stain. Studies using polyclonal and monoclonal antibodies directed against COX subunits are carried out in specialized centers. For analysis, the muscle specimen taken under local anesthetic must be frozen immediately in liquid-nitrogen-cooled isopentane Magnetic Resonance Spectroscopy of Muscle and Brain Magnetic resonance spectroscopy (MRS) allows the study of muscle and brain energy metabolism in vivo. Lactate, inorganic phosphate (Pi), phosphocreatine (PCr) and intracellular ph may be measured. The Pi/PCr ratio is the most useful parameter and may be monitored at rest, during exercise, and during recovery. An increased ratio is found in most patients, and MRS is becoming a useful tool in the diagnosis of mitochondrial diseases and in the monitoring of therapeutic trials. However, the observed anomalies are not specific to respiratory enzyme deficiencies, and no correlation between MRS findings and the site of the respiratory enzyme defect can be made [10] Treatment and Prognosis No satisfactory therapy is presently available for respiratory chain deficiency. Treatment remains largely symptomatic and does not significantly alter the course of the disease. It includes symptomatic treatments, supplementation with cofactors, prevention of oxygen-radical damage to mitochondrial membranes, dietary recommendations, and avoid ance of drugs and procedures known to have a detrimental effect. It is advisable to avoid sodium valproate and barbiturates, which inhibit the respiratory chain and have occasionally been shown to precipitate hepatic failure in respiratory enzyme-deficient children [11]. Tetracyclines and chloramphenicol should also be avoided, as they inhibit mitochondrial protein synthesis. Due to the increasing number of tissues affected in the course of the disease, organ transplantations are exceptional (bone marrow, liver, heart). Symptomatic treatments include: slow infusion of sodium bicarbonate during acute exacerbation of lactic acidosis, pancreatic extract administration in cases of exocrine pancreatic dysfunction, and repeated transfusions in cases of anemia or thrombocytopenia. Recently, administration of L-arginine, a nitric oxide precursor, has been shown to significantly decrease the frequency and severity of strokelike episodes in MELAS [48]. One recently identified condition, inborn errors of coenzyme Q 10 (CoQ 10 ) synthesis [37a] deserves particular atten tion as, when recognized, this condition should be treatable by large doses of oral quinone (Ubidecarenone). In the three hitherto recognized clinical presentations (the myopathic form [49-52], the ataxic form [53] and the multisys temic form [54-56]), the respiratory enzyme activities are individually normal but they are deficient when tested in group, as CoQ 10 acts as electron shuttle between complexes in the respiratory chain. Giving oral quinones to CoQ 10 de ficient patients restores the electron flow (5 mg/ kg/day). Yet, apart from this rare situation, neither CoQ 10 nor its analogues (Idebenone) can restore electron flow in case of respiratory chain deficiency. Oral quinones are not only useless but even possibly harmful in respiratory chain deficiency. Indeed, because quinones can divert electrons from the respiratory chain, they may become pro-oxidant and possibly deleterious if reduced quinones are not reoxidized by a normally functioning respiratory chain. The low uptake of oral quinones by CoQ 10 sufficient cells probably limits their deleterious effect when given to respiratory enzyme deficient patients. By contrast, in Friedreich ataxia, where iron overload causes a free-radical induced iron sulfur cluster injury to an otherwise normal respiratory chain, idebenone (10 mg/kg/day) reoxidized on the respiratory chain has been shown to efficiently counteract the life threatening hypertrophic cardiomyopathy [57]. Treatment with riboflavin (100 mg/day) has been associated with improvement in a few patients with complex I deficiency myopathy. Carnitine is suggested in patients with secondary carnitine deficiency. Dichloroacetate or 2-chloropropionate administration has been proposed to stimulate pyruvate dehydrogenase (PDH) activity and has occasionally reduced the level of lactic acid [58], but detrimental effects of dichloroacetate have recently been reported. The dietary recommendation are a high-lipid, lowcarbohydrate diet in patients with complex I deficiency. Indeed, a high-glucose diet is a metabolic challenge for patients with impaired oxidative phosphorylation, especially as glucose oxidation is largely aerobic in the liver. Based on our experience, we suggest avoiding a hyper caloric diet and parenteral nutrition and recommend a low-carbohydrate diet in addition to the symptomatic treatment. Succinate (6 g/day), succinate-producing amino acids (isoleucine, methionine, threonine, and valine) or propionyl carni tine have occasionally been given to patients with complex I deficiency, as these substrates enter the respiratory chain via complex II.

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