2015_06 Curso Illa - present cristina MIOPATÍAS METABÓLICAS Y
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2015_06 Curso Illa - present cristina MIOPATÍAS METABÓLICAS Y
MIOPATÍAS METABÓLICAS Y MITOCONDRIALES CURSO DE NEUROMUSCULAR 3 de Junio de 2016 INDICE 1. Alteración del metabolismo energético del músculo. Vías metabólicas. 2. Defectos en la vía glucolítica. Enfermedad de McArdle. Enfermedad de Pompe del adulto. 3. Alteración del metabolismo/trasporte de los ácidos grasos. Déficit de CPT2 4. Enfermedades mitocondriales sistema OXPHOS. 1. Características generales 2. Características clínicas y genéticas 3. Aproximación diagnóstica y terapéutica 1274 S. DiMauro et al. Bashar Katirji Henry J. Kaminski Robert L. Ruff Editors Glucose CGI-58 ATGL TG droplets Pyruvate Fatty acids CPT-I 123 H+ Pyruvate Long chain ADP Pyruvate Malate ATP Carnitine-Acylcarnitine Translocase CPT-II VLCAD ADP Long chain Acyl-CoA Acetyl-CoA PDHC Oxaloacetate acyl-carnitine ATP TP H+ Carnitine Citrate TCA Cycle KT Isocitrate H+ SuccinyI-CoA Fumarate ETF-ox Succinate H+ I II CoQ b-Oxidation HAD 3-Hydroxyacyl-CoA 3Enoyl-CoA LCAD MCAD SCAD ETF-red ETF-DH FADH2 3-Ketoacyl-CoA Acyl-CoA AKetoglutarate Hydratase H+ H+ ADP ATP Inner introchondrial membrane Second Edition Outer mitochondrial membrane Neuromuscular Disorders in Clinical Practice H+ III Cyt c IV V Fig. 63.2 Schematic representation of mitochondrial metabolism. Abbreviations: PDHC pyruvate dehydrogenase complex, CPT carnitine palmitoyltransferase, VLCAD very-long-chain acyl-CoA dehydrogenase, TP trifunctional protein, LCAD long-chain acyl-CoA dehydrogenase, MCAD medium-chain acyl-CoA dehydrogenase, SCAD short-chain acyl-CoA dehydrogenase, HAD 3-hydroxyacyl-CoA dehydrogenase, KT 3-ketothiolase, ETFox oxidized form of electron transfer flavoprotein, ETFred reduced form of electron transfer flavoprotein, ETFDH ETF-coenzyme Q oxidoreductase, CG!-58 is the protein that activates ATGL, ATGL adipocyte triglyceride lipase (Reproduced from DiMauro and Haller [198], with permission) exercise, lipid oxidation becomes the major source of energy. Because the availability of FFA from adipose tissue is virtually unlimited, a normal person can perform moderate dynamic exercise for many hours. includes two enzymes (carnitine palmitoyltransferase [CPT] I and CPT II), a carrier molecule (l-carnitine), and a translocase (carnitine-acylcarnitine translocase, CACT). After oxidation of pyruvate through the pyruvate dehydrogenase 1275 (the electron transport chain) to oduction of water. The electron four multimeric complexes (I to carriers, coenzyme Q (or ubiquiThe energy generated by these protons from the mitochondrial een inner and outer mitochondrial n electrochemical proton gradient ne. A fifth multimeric complex triphosphate [ATP] synthase), a s the energy of the electrochemiTP, in a process known as oxidapling. The terminal pathway of mprising the electron transport own as the respiratory chain. Glycogen PLD III, V Glucose 1-P ATP VIII Phosphorylase b XIV Glucose Glucose 6-P ADP+Pi ATP ADP+Pi Fructose 6-P VII Fructose1,6 D-P XII Glyceraldehyde3-P 2X ATP correlation between the circumproblems and the different roles of sm in the provision of energy. Thus, ith glycogenoses are almost invarile, and usually strenuous, bout of at hurt, swell, or cramp up are those hat particular type of exercise. disorders of lipid metabolism, such -chain acyl-CoA dehydrogenase have no warning of an impending which usually follows prolonged be heralded by myalgia of exercisccompanied by painful cramps. In in and by itself may cause myony muscle group may be affected, les; a few patients with CPT II to the emergency room in respiraode of myoglobinuria [1]. The delPT II deficiency is easily explained ce of muscle on FFA oxidation, . Conversely, some patients with cy, a glycogen storage disease, note g on their exercise ability. This is ion of FFA, which facilitates the arbohydrate to lipid utilization. indeed the “business end” of mitore the energy generated by carbois released as ATP. This raises an turbances of glycogen and lipid ntolerance, cramps, and myoglobiduction, why did it take so long to Phosphorylase a 0, IV, XV UDPG 2X ADP+Pi ercise Intolerance ) AMP kinase P (+ AM (-) Phosphorylase ATP b kinase Lysosome Glycdogen II 2X ADP+Pi 3-P Glycerol-P IX 3-P Glycerate X 2-P Glycerate XIII Phosphoenolpyruvate XI 2X ATP Pyruvate XI Lactate Fig. 63.3 Scheme of glycogen metabolism and glycolysis. Roman numerals indicate enzymes whose deficiencies are associated with muscle glycogenoses: II acid maltase (GAA, Pompe disease), III debrancher (GDE, Cori-Forbes disease), IV brancher (GBE, Andersen disease), V myophosphorylase (PYGM, McArdle’s disease), VII phosphofructokinase (PFK, Tarui disease), VIII phosphorylase kinase (PHK), IX phoshoglycerate kinase (PGK), X phosphoglycerate mutase (PGAM), XI lactate dehydrogenase (LDH), XII aldolase A. Normal numerals indicate glycogenoses causing exercise intolerance, cramps, and myoglobinuria; italic numerals indicate glycogenoses causing fixed weakness. Abbreviations: UDPG uridine diphosphate glucose, PLD phosphorylase-limit dextrin, AMP adenosine monophosphate, ADP adenosine diphosphate, ATP adenosine triphosphate, Pi inorganic phosphate GLUCOGENOGÉNESIS DEGRADACIÓN DE TAG CADENA RESPIRATORIA GLUCOGENOLISIS TRASNSPORTE AG MITOCONDRIAL GLUCOLISIS BETAOXIDACIÓN DE AG 63 Alteración en el metabolismo energético del músculo lic Myopathies associate this syndrome with defects in the respiratory chain, the energetic pathway “par excellence”? The answer is that these patients often stay below the clinical radar because they contradict the rules of mitochondrial genetics (see below) [2]. Producción de ATP Glycogenoses In reviewing the glycogenoses causing exercise intolerance and myoglobinuria, we follow the metabolic “flow” in the glycogenolytic and glycolytic pathways rather than the historical numeration (Fig. 63.3). iMauro, Hasan Orhan Akman, n Paradas Glycogenoses bolism that cause ar disorders are Dynamic sympsfunctions, manr without painful in muscle breakc symptoms are ness, sometimes s (Fig. 63.1). age disorders, a and during exer- GAA GDE ALD GBE GYG1 PHK PYGM B-Enolase GYS1 PGM PFK PGK PGAM LDH Fixed weakness Disorders of lipid metabolism Carnitine transporter Myopathic and secondary carnitine deficiencies MCAD MADD SCAD GA II NLSDI NLSDM Exercise intolerance Cramps/myalgia Myoglobinuria CPT II LIPIN VLCAD MTP SCHAD Respiratory chain defects Complex I Complex III Complex IV ISCU CoQ10 63 nMyopathies several factors, ation of exercise Neuromuscular Disorders in ClinicalutiPractice rest, muscle uro, Akman, ositeHasan end of Orhan the Fig. 63.1 The two major clinical syndromes seen in metabolic myopaxercise (close to thies. Deficient enzymes are denoted by abbreviations or gene symbols radas n dynamic exer- as follows: GAA acid maltase (acid D-glucosidase) (GSD II), GDE glyn isometric exer- cogen debranching (GSD III), ALD aldolase (GSD IX), GBE glycogen branching (GSD IV), GYG1 glycogenin (GSD 0), PHK phosphorylase especially when kinase (GSD VIII), PYGM myophosphorylase (GSD V), GYS1 glycoeration to maxi- gen synthetase, PGM phosphoglucomutase (GSD IV), PFK phosphothe type of fuel fructokinase (GSD VII), PGK phosphoglycerate kinase (GSD IX), phosphoglycerate mutase (GSD X), LDH lactate dehydrogenase 63 Metabolic Myopathies 1275 ntensity of exer- PGAM (GSD XI), MTP mitochondrial trifunctional enzyme, MCAD mediummitochondrial membrane (the electron transport chain) to ) AMP kinase ), blood glucose chain P (+ Lysosome M A molecular oxygen with production of water. The electron multiple acyl-CoA dehydrogeacyl-CoA dehydrogenase, MADD (-) Phosphorylase ATP Glycdogen of lipid Glycogen chain consists of four multimeric complexes (I toDisorders Glycogenoses urces of energy. nase,transport SCAD acyl-CoA dehydrogenase, GAII glutaric b kinase II Fixed weakness IV) plus two smallshort-chain electron carriers, coenzyme Q (or ubiquimetabolism Phosphorylase a VIII 0, IV, XV with ichthyosis none) and cytochrome c. The energy generated by these type II, NLSDI neutral lipid storage diseases gy derived from aciduria UDPG PLD reactions is used to pump protons from the mitochondrial Carnitine transporter III, V m that cause (Chanarin-Dorfman neutral storageGlucose disease with Phosphorylase b matrix into the space betweendisease), inner and outer NLSDM mitochondrial 1-P Myopathic and secondary uscle glycogen GAA ATP membranes.CPT This creates an electrochemical proton gradient carnitine deficiencies XIV myopathy, II carnitine palmitoyltransferase II, VLCAD very-longsorders are GDE across the inner membrane. A fifth multimeric complex Glucose Glucose 6-P VO2max, aerobic ALD MCAD chain acyl-CoA dehydrogenase, SCHAD short-chain 3-hydroxyacyl(complex V or adenosine triphosphate [ATP] synthase), a amic sympctions, manhout painful dical Center, uscle breakmptoms are sometimes g. 63.1). disorders, a dical Center, uring exer- Bashar Katirji Henry J. Kaminski Robert L. Ruff Editors Second Edition 123 ADP+Pi rotary engine, converts the energy of the electrochemiMADD(Fe-S)Fructose GBE 6-P CoAtinydehydrogenase, ISCU nonheme iron-sulfur protein, CoQ10 proton gradient into ATP, in a process known as oxidaVII SCAD ATP GYG1 cal tion/phosphorylation coupling. intolerance The terminal pathway of ADP+Pi coenzyme Q10 Exercise Fructose1,6 D-P GA II oxidative metabolism, comprising the electron transport XII PHK chain and complex V,Cramps/myalgia is known as the respiratory chain. NLSDI Glyceraldehyde3-P Myoglobinuria PYGM NLSDM 3-P Glycerol-P ADP+Pi metabolism of glycogen is the crucial2Xsource of energy, and B-Enolase IX Disorders Causing Exercise Intolerance 2X ATP 3-P Glycerate GYS1 and Myoglobinuria fatigue appears to set in when glycogen is exhausted. The X PGM 2-P Glycerate CPT II there is a good correlation between the circumXIII typeIn general, of circulating substrate utilizedLIPIN during mild exercise PFK stances leading to clinical problems and the different roles of Phosphoenolpyruvate PGK 2X ADP+Pi VLCAD increase in the glycogen and lipid metabolism the provision of energy. varies with time, inand there isThus,a gradual Respiratory chain defects XI 2X ATP PGAMthe complaints of patients with glycogenoses are almost invariMTP ably related to an and over usually strenuous, bout of until, a fewPyruvate utilization ofidentifiable, FFA glucose hours XIintoLactate LDH SCHAD Complex I cramp up are those exertion, and the muscles that hurt, swell, or that have been engaged inComplex that particular type III of exercise. Fig. 63.3 Scheme of glycogen metabolism and glycolysis. Roman In contrast, patients with disorders of lipid metabolism, such numerals indicate enzymes whose deficiencies are associated with Complex IV as CPT II or very-long-chain acyl-CoA dehydrogenase muscle glycogenoses: II acid maltase (GAA, Pompe disease), III debrancher (GDE, Cori-Forbes disease), IV brancher (GBE, Andersen disISCU (VLCAD) deficiency, often have no warning of an impending ease), V myophosphorylase (PYGM, McArdle’s disease), VII episode of myoglobinuria, which10usually follows prolonged phosphofructokinase (PFK, Tarui disease), VIII1273 CoQ phosphorylase kinase eral factors, of exercise Clinical Practice, muscle utimoderate exercise and may be heralded by myalgia of exercis- (PHK), IX phoshoglycerate kinase (PGK), X phosphoglycerate mutase cience+Business Media New York 2014 ing muscles but is never accompanied by painful cramps. In (PGAM), XI lactate dehydrogenase (LDH), XII aldolase A. Normal numerals indicate glycogenoses causing exercise intolerance, cramps, end of the Fig. 63.1 The addition, in andsyndromes by itself may cause myotwoprolonged majorfasting clinical seen in metabolic myopaand myoglobinuria; italic numerals indicate glycogenoses causing fixed globinuria, in which case any muscle group may be affected, weakness. Abbreviations: UDPG uridine diphosphate glucose, PLD se (close to thies. Deficient enzymes denoted by abbreviations or gene symbols dextrin, AMP adenosine monophosphate, ADP including respiratoryare muscles; a few patients with CPT II phosphorylase-limit adenosine diphosphate, ATP adenosine triphosphate, P inorganic deficiency havemaltase been taken to the emergency room in respira-(GSD II), GDE glyacid (acid D-glucosidase) namic exer- as follows: GAA phosphate tory distress during an episode of myoglobinuria [1]. The delcogen debranching (GSD III), ALD aldolase (GSD IX), GBE glycogen eterious effect of fasting in CPT II deficiency is easily explained metric exerby the increased dependence of muscle on (GSD FFA oxidation, associate this syndrome with defects in the respiratory chain, branching (GSD IV), GYG1 glycogenin 0), PHK phosphorylase is partially blocked. Conversely, some patients with the energetic pathway “par excellence”? The answer is that ially when kinase (GSDwhich VIII), PYGM myophosphorylase (GSD V), GYS1 glycomyophosphorylase deficiency, a glycogen storage disease, note these patients often stay below the clinical radar because they on to maxi- gen synthetase, PGM phosphoglucomutase (GSD PFKthephosphoa beneficial effect of fasting on their exercise ability. This isIV), contradict rules of mitochondrial genetics (see below) [2]. the mobilization FFA, which facilitates the kinase (GSD IX), (GSDby VII), PGK ofphosphoglycerate ype of fuel fructokinaseexplained physiological switch from carbohydrate to lipid utilization. PGAM phosphoglycerate mutase (GSD X), LDH lactate dehydrogenase The respiratory chain is indeed the “business end” of mito- Glycogenoses ity of exerchondrial metabolism, where the energy generated enzyme, by carbo(GSD XI), MTP mitochondrial trifunctional MCAD mediumhydrate and lipid oxidation is released as ATP. This raises an In reviewing the glycogenoses causing exercise intolerance ood glucose chain acyl-CoA dehydrogenase, MADD multiple acyl-CoA dehydroge- GLUCOGENOSIS i interesting question: if disturbances of glycogen and lipid metabolism cause exercise intolerance, cramps, and myoglobi- and myoglobinuria, we follow the metabolic “flow” in the glycogenolytic and glycolytic pathways rather than the his- sport chain) to r. The electron complexes (I to me Q (or ubiquirated by these mitochondrial r mitochondrial proton gradient meric complex P] synthase), a e electrocheminown as oxidaal pathway of ctron transport atory chain. nce een the circumifferent roles of of energy. Thus, re almost invarienuous, bout of mp up are those of exercise. metabolism, such dehydrogenase of an impending lows prolonged algia of exercisnful cramps. In may cause myomay be affected, s with CPT II oom in respira- glucolisis anaeróbica FISIOLOGÍA DEL EJERCICIO 80% Glucógeno 128 Nutrición aplicada al deporte 40% VO2 Tasa de oxidación (kj/min) de ATP aportadas por los macronutrimentos. En este caso, la cantidad de ATP por molécula de glucosa sería de 28 y 30 ATP, según fuera la lanzadera usada. Para el caso de los ácidos grasos, la cantidad de ATP depende de la cantidad de átomos de carbono que componen al ácido graso. El ácido palmítico, un ácido graso de 16 carbonos, aporta 96 ATP por los ciclos de Krebs y 35 ATP por β oxidación (un total de 131 ATP). Glucosa FFA oxidación ácidos grasos Utilización de macronutrimentos durante el ejercicio físico Durante el ejercicio físico de ritmo estable, la contribución de lípidos e hidratos de carbono guarda relación con la intensidad y la duración del ejercicio. En relación con la intensidad, durante el ejercicio de baja intensidad existe un predominio del aporte de los lípidos al metabolismo energético. Sin embargo, a la medida que la intensidad aumenta, se incrementa de forma paulatina el aporte de los hidratos de carbono. Romjin et al. (1993) estudiaron la contribución de lípidos e hidratos de carbono a tres intensidades diferen· tes (25, 65 y 85% del VO2máx). Si bien la contribución porcentual de los lípidos fue mayor a la intensidad del 25% del · VO2máx, el gasto energético para este sustrato fue mayor a la · intensidad del 65% del VO2máx (fig. 6-13). Atchen et al. (2002), al valorar la oxidación de lípidos (g/min), determinaron que la zona de mayor oxidación de lípidos se encontraba a la · intensidad de 64% del VO2máx (límites, 55 a 72%). En la medida que la intensidad se incrementa, existe una mayor contribución de los hidratos de carbono, debido en parte a la mayor actividad glucolítica producto de la estimulación por el calcio intracelular a la fosforilasa y la mayor actividad adrenérgica generada conforme la intensidad del ejercicio se incrementa. Los hidratos de carbono (glucosa) son un sustrato ideal para trabajos de elevada intensidad; comparados con los ácidos grasos, la glucosa puede metabolizarse en el 40 30 20 10 0 30 60 90 120 150 180 210 240 Minutos Oxidación de hidratos de carbono Oxidación de lípidos Figura 6-14. Tasa de oxidación de hidratos de carbono y lípidos durante 4 horas de ciclismo al 57% del V̇O2máx. * Significativamente diferente de los 30 min. (Adaptada de Spriet, L. y Watt, M. 2003). citosol, requiere menos oxígeno para oxidarse (presenta una mayor relación oxígeno-carbono) y tiene una potencia energética mayor. Estas condiciones son relevantes si se considera que durante un esfuerzo de intensidad elevada se necesita una potencia energética alta y que en la medida que la tensión muscular y la velocidad de acortamiento muscular aumentan, la disponibilidad de oxígeno muscular decrece. En cuanto a la contribución de lípidos e hidratos de carbono en función de la duración del esfuerzo, cabe señalar que a cualquier intensidad de ejercicio, conforme la duración de éste aumenta, se observa una mayor contribución de los lípidos a la producción de energía (Spriet y Watt, 2003) (fig. 6-14). INTOLERANCIA AL EJERCICIO Cal/kg/min 300 Glucógeno muscular Triglicéridos musculares Ácidos grasos plasmáticos Glucosa plasmática 200 100 25 65 85 % del VO2máx Figura 6-13. Contribución de distintos combustibles al ejercicio continuo de distintas intensidades (adaptada de Romjin, J. y cols. 1993). 06_Peniche.indd 128 21/2/11 11:42:14 GLUCOGENOSIS V ENFERMEDAD DE McARDLE MUTACIÓN GEN PYGM 1275 ) AMP kinase P (+ AM (-) Phosphorylase ATP b kinase Lysosome Glycdogen II Glycogen 0, IV, XV UDPG Phosphorylase a PLD III, V Glucose 1-P ATP ✤ VIII Phosphorylase b ✤ XIV Glucose ✤ Glucose 6-P ✤ Fructose 6-P ✤ ADP+Pi ✤ ATP VII en Test de ejercicio Fructose1,6 D-P antebrazo XII CK basal elevada Glyceraldehyde3-P Estudio molecular dirigido 3-P Glycerol-P Prevalencia mínima: 1/150.000 Intolerancia al ejercicio (fatiga precoz/ mialgias/contracturas prolongadas/ debilidad inducidas por ejercicio) Second wind (100%) Mioglobinuria recurrente (50%) Debilidad fija proximal tardía (20%) ADP+Pi ✤ ✤ 2X ADP+Pi 2X ATP 2X ADP+Pi IX 3-P Glycerate X 2-P Glycerate XIII Phosphoenolpyruvate XI 2X ATP Pyruvate XI Lactate Fig. 63.3 Scheme of glycogen metabolism and glycolysis. Roman numerals indicate enzymes whose deficiencies are associated with muscle glycogenoses: II acid maltase (GAA, Pompe disease), III debrancher (GDE, Cori-Forbes disease), IV brancher (GBE, Andersen disease), V myophosphorylase (PYGM, McArdle’s disease), VII phosphofructokinase (PFK, Tarui disease), VIII phosphorylase kinase (PHK), IX phoshoglycerate kinase (PGK), X phosphoglycerate mutase (PGAM), XI lactate dehydrogenase (LDH), XII aldolase A. Normal numerals indicate glycogenoses causing exercise intolerance, cramps, and myoglobinuria; italic numerals indicate glycogenoses causing fixed weakness. Abbreviations: UDPG uridine diphosphate glucose, PLD phosphorylase-limit dextrin, AMP adenosine monophosphate, ADP adenosine diphosphate, ATP adenosine triphosphate, Pi inorganic phosphate Entrenamiento en ejercicio aeróbico moderado Vitamina B6 (piridoxina) Sacarosa antes del ejercicio sport chain) to r. The electron complexes (I to me Q (or ubiquirated by these mitochondrial r mitochondrial proton gradient meric complex P] synthase), a e electrocheminown as oxidaal pathway of ctron transport atory chain. nce GLUCOGENOSIS II ENFERMEDAD DE POMPE DEL ADULTO ) P (+ AM (-) ATP Lysosome Glycdogen II Glycogen 0, IV, XV UDPG III, V Glucose 1-P ATP 1275 AMP kinase VIII ✤ ✤ Phosphorylase b XIV Glucose ✤ Phosphorylase b kinase Phosphorylase a PLD MUTACIÓN GEN GAA Glucose 6-P ✤ ✤ Debilidad de cintura pélvica, pared abdominal, paraespinales y cuello Debilidad diafragmática Debilidad lingual CK < 1000 UI DBS ADP+Pi ATP ADP+Pi Fructose 6-P VII Fructose1,6 D-P XII Glyceraldehyde3-P 2X ADP+Pi 2X ATP EMG$ 3-P Glycerol-P IX 3-P Glycerate X 2-P Glycerate XIII Phosphoenolpyruvate een the circumifferent roles of 50% no diagnóstica 2X ADP+Pi of energy. Thus, XI 2X ATP re almost invariXI enuous, bout of Lactate Pyruvate mp up are those of exercise. Fig. 63.3 Scheme of glycogen metabolism and glycolysis. Roman metabolism, such numerals indicate enzymes whose deficiencies are associated with dehydrogenase muscle glycogenoses: II acid maltase (GAA, Pompe disease), III debrancher (GDE, Cori-Forbes disease), IV brancher (GBE, Andersen disof an impending ease), V myophosphorylase (PYGM, McArdle’s disease), VII lows prolonged phosphofructokinase (PFK, Tarui disease), VIII phosphorylase kinase algia of exercis- (PHK), IX phoshoglycerate kinase (PGK), X phosphoglycerate mutase nful cramps. In (PGAM), XI lactate dehydrogenase (LDH), XII aldolase A. Normal numerals indicate glycogenoses causing exercise intolerance, cramps, may cause myo- and myoglobinuria; italic numerals indicate glycogenoses causing fixed may be affected, weakness. Abbreviations: UDPG uridine diphosphate glucose, PLD s with CPT II phosphorylase-limit dextrin, AMP adenosine monophosphate, ADP oom in respira- adenosine diphosphate, ATP adenosine triphosphate, Pi inorganic Salvatore DiMauro, Hasan Orhan Akman, phosphate ria [1]. The delAcido graso (FA) carnitina and Carman Paradas easily explained Transportador de FA OCTN2 Membrana plasmática FFA oxidation, associate this syndrome with defects in the respiratory chain, e patients with the energetic pathway “par excellence”? The answer is that Fosfolípidos ATGL (PNPLA2) ge disease, note these patients oftenTriglicerido stay below the clinical radar because theyLC-Acil-CoA Diglicérido GCI-58 (ABHD5) ability. This is contradict the rules of mitochondrial genetics (see below) [2]. Diacilglicerol CPT1 h facilitates the LC-Acil-carnitina Membrana mitocondrial LIPIN1 d utilization. Fosfatidato MTP VLCAD CPT2 CACT ss end” of mito- Glycogenoses Glicerol-3-P rated by carbocarnitina MC-Acil-CoA Disorders of lipid LC-Acil-CoA Introduction Glycogenoses Fixed weakness the glycogenoses causing exercise intolerance P. This raises an In reviewing Dihidroxiacetona-3-P metabolism in the ogen and lipid and myoglobinuria, we follow the metabolic MCAD “flow” Β-oxidación Carnitine transporter SCAD that cause Acetil-CoA Inborn errors Glucolisis of glycogen and fatty acid metabolism GAA Myopathic and secondary , and myoglobi- glycogenolytic and glycolytic pathways rather than the hiscarnitine deficiencies ETFox or predominantly GDE torical numeration (Fig. 63.3). neuromuscular disorders are t take so long to exclusively MCAD ALD ETFred characterized by dynamic or static symptoms. Dynamic sympETDFDH MADD GBE toms are acute, recurrent,CoQ reversible muscle dysfunctions, manSCAD GYG1 Cadena Exercise intolerance GA II ifesting as exercise intolerance, Respiratoria myalgia with or without painful Cramps/myalgia PHK NLSDI cramps (contractures), and often culminating in muscle breakMyoglobinuria PYGM NLSDM B-Enolase down and myoglobinuria. In contrast, static symptoms are GYS1 manifested by fixed, often progressive weakness, sometimes PGM CPT II simulating dystrophic or neurogenic processes (Fig. 63.1). PFK LIPIN PGK To understand glycogen and lipid storage disorders, a VLCAD Respiratory chain defects PGAM MTP brief review of muscle metabolism at rest and during exerLDH SCHAD Complex I cise is helpful. Complex III The “fuel” utilized by muscle depends on several factors, Complex IV ISCU most importantly the type, intensity, and duration of exercise CoQ10 but also diet and physical conditioning. At rest, muscle utilizes predominantly fatty acids. At the opposite end of the Fig. 63.1 The two major clinical syndromes seen in metabolic myopaspectrum, the energy for extremely intense exercise (close to thies. Deficient enzymes are denoted by abbreviations or gene symbols one’s maximal oxygen uptake, or VO2max, in dynamic exer- as follows: GAA acid maltase (acid D-glucosidase) (GSD II), GDE glycise or close to maximal force generation in isometric exer- cogen debranching (GSD III), ALD aldolase (GSD IX), GBE glycogen branching (GSD IV), GYG1 glycogenin (GSD 0), PHK phosphorylase cise) derives from anaerobic glycolysis, especially when kinase (GSD VIII), PYGM myophosphorylase (GSD V), GYS1 glycothere is a “burst” of activity with rapid acceleration to maxi- gen synthetase, PGM phosphoglucomutase (GSD IV), PFK phosphomal exercise. During submaximal exercise, the type of fuel fructokinase (GSD VII), PGK phosphoglycerate kinase (GSD IX), utilized by muscle depends on the relative intensity of exer- PGAM phosphoglycerate mutase (GSD X), LDH lactate dehydrogenase (GSD XI), MTP mitochondrial trifunctional enzyme, MCAD mediumtion. At low intensity (below 50 % VO2max), blood glucose chain acyl-CoA dehydrogenase, MADD multiple acyl-CoA dehydrogeand free fatty acids (FFA) are the primary sources of energy. nase, SCAD short-chain acyl-CoA dehydrogenase, GAII glutaric At higher intensities, the proportion of energy derived from aciduria type II, NLSDI neutral lipid storage diseases with ichthyosis carbohydrate oxidation increases, and muscle glycogen (Chanarin-Dorfman disease), NLSDM neutral storage disease with myopathy, CPT II carnitine palmitoyltransferase II, VLCAD very-longbecomes an important fuel; at 70–80 % VO2max, aerobic chain acyl-CoA dehydrogenase, SCHAD short-chain 3-hydroxyacyl- Metabolic Myopathies 63 ALTERACIÓN EN EL METABOLISMO DE LOS LÍPIDOS S. DiMauro, MD (*) Department of Neurology, Columbia University Medical Center, 4-424B College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032, USA e-mail: [email protected] CoA dehydrogenase, ISCU nonheme iron-sulfur (Fe-S) protein, CoQ10 coenzyme Q10 DÉFICIT DE CARNITINPALMITOIL MUTACIÓN CPT2 metabolism of glycogen is the crucialGEN source of energy, and TRANSFERASA II fatigue appears to set in when glycogen is exhausted. The (/!KMAN0H$s#0ARADAS-$0H$ Department of Neurology, Columbia University Medical Center, New York, NY, USA type of circulating substrate utilized during mild exercise varies with time, and there is a gradual increase in the utilization of FFA over glucose until, a few hours into B. Katirji et al. (eds.), Neuromuscular Disorders in Clinical Practice, DOI 10.1007/978-1-4614-6567-6_63, © Springer Science+Business Media New York 2014 1273 ✤ Intolerancia al ejercicio (prolongado) ✤ Rabdomiolisis de repetición (ayunas, ejercicio, fiebre, estrés, frío) Biopsia normal ✤ No calambres/contracturas ✤ CK basal normal ✤ Estudio molecular: p.S113L (75%) ✤ Tratamiento: dieta rica en HC, aceite MCT, bezafibrato Diagnóstico genético molecular Miopatías Metabólicas EXERCISE INTOLERANCE COMMON MUTATIONS (PYGM, AMPD1, CPT2) MINISEQUENCING PANEL p.Ser113Leu Heterozygous P PYGM AMPD1 WT C CPT2 METABOLIC MYOPATHIES NGS PANEL CPT2 GYS1 AGL PYGM LDHA PFKM PHKA1 PGAM2 ALDOA ENO3 PGM1 GYG1 AMPD1 PGK1 LPIN1 ACADVL PHKB SERCA1 ACE ACTN3 PGC1A carnitine palmitoyltransferase 2 glycogensynthase1(muscle) solutecarrierfamily22(organiccation/carnitinetransporter), SLC22A5 member5 ACADS acyl-CoAdehydrogenase,C-2toC-3shortchain ACADL Acyl-CoADehydrogenase,LongChain amylo-alpha-1,6-glucosidase,4-alpha-glucanotransferase ACADM acyl-CoAdehydrogenase,C-4toC-12straightchain phosphorylase,glycogen,muscle hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoAthiolase/enoyl-CoA lactatedehydrogenaseA HADHA hydratase (trifunctional protein),alpha subunit phosphofructokinase,muscle hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoAthiolase/enoyl-CoA phosphorylasekinase,alpha1(muscle) HADHB hydratase (trifunctional protein),betasubunit phosphoglyceratemutase2(muscle) CPT1B CarnitinePalmitoyltransferase1B(Muscle) aldolaseA,fructose-bisphosphate ETFA electron-transfer-flavoprotein,alphapolypeptide enolase3(beta,muscle) ETFB electron-transfer-flavoprotein,betapolypeptide phosphoglucomutase1 ETFDH electron-transferring-flavoproteindehydrogenase glycogenin1 GBE1 glucan (1,4-alpha-),branching enzyme 1 adenosinemonophosphatedeaminase1 PNPLA2 patatin-likephospholipasedomaincontaining2 phosphoglycerate kinase 1 ABHD5 abhydrolasedomaincontaining5 lipin1 solute carrier family 25(carnitine/acylcarnitine translocase),member acyl-CoAdehydrogenase,verylongchain SLC25A20 20 phosphorylasekinase,beta Sarcoplasmic/EndoplasmicReticulumCalciumATPase1 angiotensinIconvertingenzyme actinin,alpha3 Peroxisome Proliferator-Activated ReceptorGamma,Coactivator 1 ENFERMEDADES MITOCONDRIAL DEL SISTEMA OXPHOS Mutaciones en responsables de enfermedad: ALTERACIÓN DEADNmt LA FOSFORILACIÓN OXIDATIVA 1/5000-1/10000 Mutaciones en ADNmtMITOCONDRIALES en sujetos sanos: 1/200 ENFERMEDADES ADNn ADNmt Herencia mendeliana Herencia materna Esporádico CONTROL GENÉTICO DUAL ✤ Subunidades de los complejos (13 de las 90) 22 tRNAs y 2 rRNAs ✤ Mantenimiento del ADNmt ✤ ADNmt comunicación intergenómica ADNn Maquinaria de replicación Mantenimiento del pool de dNTPs ✤ ✤ ✤ ✤ ✤ Deleciones múltiples Depleción ADNmt Subunidades de los complejos (Leigh) Proteínas de ensamblaje Traducción del ADNmt Composición lipídica de la membrana mitocondrial Dinámica mitocondrial (DOA, CMT2A, CMT4A) Oxidative phosphorylation biogenesis and regulation AAA Membrane dynamics and composition Oxidative phosphorylation subunits V IV III I II dN dNMP dNDP dNTP Nucleotide transport and synthesis AAA mtDNA maintenance and expression CARACTERÍSTICAS DEL ADNmt ✤ 16569 pb-circular, doble cadena. 37 genes. 2 rRNA, 22tRNA, 13 polipéptidos (subunidades de la cadena). No tiene intrones. CARACTERÍSTICAS DEL ADNmt ✤ ✤ 16569 pb-circular, doble cadena. 37 genes. 2 rRNA, 22tRNA, 13 polipétidos (subunidades de la cadena). No tiene intrones. Herencia materna Herencia materna CARACTERÍSTICAS DEL ADNmt ✤ 16569 pb-circular, doble cadena. 37 genes. 2 rRNA, 22tRNA, 13 polipétidos (subunidades de la cadena). No tiene intrones. ✤ Herencia materna ✤ Poliplasmia Poliplasmia Homoplásmico 100% Normal Homoplásmico 100% Mutante Heteroplásmico 25% Mutante CARACTERÍSTICAS DEL ADNmt ✤ 16569 pb-circular, doble cadena. 37 genes. 2 rRNA, 22tRNA, 13 polipétidos (subunidades de la cadena). No tiene intrones. ✤ Herencia materna ✤ Poliplasmia ✤ Segregación mitótica Segregación mitótica CIGOTO ADN mt ADN mt mutado MITOSIS SEGREGACIÓN % ADN MUTADO 0 20 HOMOPLASMIA 40 60 HETEROPLASMIA 80 100 HOMOPLASMIA CARACTERÍSTICAS DEL ADNmt ✤ 16569 pb-circular, doble cadena. 37 genes. 2 rRNA, 22tRNA, 13 polipéptidos (subunidades de la cadena). No tiene intrones. ✤ Herencia materna ✤ Poliplasmia ✤ Segregación mitótica ✤ Efecto Umbral Efecto Umbral CIGOTO UMBRAL PATOLÓGICO mt DNA mt DNA mutado MITOSIS SEGREGACIÓN % DNA MUTADO 0 20 40 HOMOPLASMIA 60 80 100 HETEROPLASMIA CARACTERÍSTICAS DEL ADNmt ✤ 16569 pb-circular, doble cadena. 37 genes. 2 rRNA, 22tRNA, 13 polipétidos (subunidades de la cadena). No tiene intrones. ✤ Herencia materna ✤ Poliplasmia ✤ Segregación mitótica ✤ Efecto Umbral Heterogeneidad clínica m.8993 T>G Ej. 70% NARP > 90% MILS Manifestaciones clínicas ptosis - oftalmoplegia externa retinopatía neuropatía óptica retraso mental convulsiones mioclonías ataxia leucoencefalopatía migrañas parkinsonismo sordera miocardiopatía alteración de la conducción síndrome de Fanconi glomerulopatía alteraciones endocrinas dismotilidad intestinal vómitos insuficiencia pancreática hepatopatía intolerancia al ejercicio debilidad neuropatía Mitochondrial Disorders 10/05/14 23:21 General patterns Specificity for mitochondrial disorders: Symmetric involvement of deep gray structures in absence of hypoxia Hyperintensity: T2 & FLAIR images Hypointensity: T1 Other: Delayed myelination Bilateral signal intensities in putamen, globus pallidus, & caudate: Leigh's Posterior cerebral hemisphere Stroke-like lesions: MELAS Other: Alpers Basal ganglia calcifications: Kearns-Sayre; MELAS White matter Diffuse signal change in central subcortical white matter (U-fibers): Kearns-Sayre Leukoencephalopathy with spared corpus callosum: MNGIE Diffuse white matter changes: Some electron transport chain disorders (I-IV) Macrocystic lesions: Complex I disorders Biochemistry analysis Muscle preferable to cultured fibroblasts Fresh or Frozen muscle may be used Combined partial defects of respiratory enzymes containing mtDNA-encoded subunits: Suggests mtDNA mutations Can be normal in mitochondrial disorders: Especially with multiple mtDNA deletion syndromes Molecular genetics: Mutation screening Positive result: Confirms diagnosis Screen for most common mutations associated with syndrome e.g. MELAS A3243G then T3271C Blood DNA: Adequate for Point mutations in tRNA genes: MELAS; MERRF Some mutations in structural genes: NARP; Lebers Single large mtDNA deletions with systemic disorders: Kearns-Sayre; Pearson Muscle DNA: Required for Multiple deletions Single mtDNA deletions in PEO & other localized disorders MELAS point mutation in oligo- or asymptomatic relatives Some point mutations in structural genes Treatment Aerobic training Exercise tolerance: Increased Serum lactate: Reduced MITOCHONDRIAL DISORDERS: CLINICAL SYNDROMES Adult onset Alexander disease: GFAP; NDUFV1 Alpers Alzheimer/Parkinsonism Amino Acid disorders: Nuclear mutations Anemia Ataxias Barth: Tafazzins; Xp28 Blindness Gyrate atrophy Optic atrophy ✤ LHON Wolfram: WFS1; WFS2 Visual loss: SFXN4 Cardiomyopathy ✤ Carnitine disorders Cartilage-Hair hypoplasia: RMRP; 9p21 CNS Infantile & Childhood onset ✤ Syndromes Congenital MD: CHKB; 2q13 Cramps Deafness ✤ Point mutations Maternal (mtDNA): Amino-glycoside induced: 12s rRNA Syndromic (HAM; MELAS; MERRF): tRNA Non-syndromic: 12s rRNA Nuclear mutations ✤ DIDMOAD: WFS1; 4p16 Deafness-Dystonia: DDP protein; Xq22 Diabetes Dystonia ✤ Encephalopathies Fatigue & Exercise intolerance Friedreich ataxia: Frataxin; 9q13 Functional defects Gastrointestinal ✤ HAM: mtDNA tRNA Ser Hepatic Occipital horn syndrome: ATPase 7a; Xq12 Huntington's chorea Ophthalmoplegia, External (PEO) Hypoglycemia Dominant: Multiple mtDNA deletions Infantile CNS: mtDNA & Nuclear mutations Maternal: mtDNA point mutations Inflammatory myopathies Recessive: mtDNA deplete; Mpl mtDNA del Sporadic: Single mtDNA deletion IM-VAMP Mitochondrial antibodies ? Immune (HyperThyroid) Kearns-Sayre: Single mtDNA deletion Pancreas Paraganglioma Leber's optic (LHON): mtDNA, MTND Parkinson's Leigh's syndrome: mtDNA & Nuclear muts Pearson's: mtDNA deletion Leukodystrophy Lipomatosis: mtRNA Lys & Nuclear Rhabdomyolysis: mtDNA Longevity SANDO Maple syrup urine disease Selenium deficiency MEGDEL: SERAC1; 6q25 ✤Skeletal & Hair Spastic paraparesis MELAS: mtRNA Leu + other Spinal muscular atrophy Menkes: ATPase 7a; Xq12 MERRF: mtRNA Lys & Ser SCO2: 22q13 MILS TK2: 16q22 ✤ MLASA Stuve-Wiedemann syndrome: 1p34 PUS1; 12q24 Sudden infant death: MTTL1; MTND1; HADHB YARS2; 12p11 Systemic disorders Toxic MNGIE: Thymidine phosphorylase; 22q13 ✤ Arsenic trioxide Myalgias Myoglobinuria AZT (Zidovudine) Myopathy syndromes Copper Infantile myopathies Germanium Fatal: mtDNA depletion ✤ Trichloroethylene "Later-onset": mtDNA depletion Valproate: Precipitates Inflammatory myopathy Liver failure in Alpers Seizures in MELAS Inclusion body myositis: Mpl mtDNA delete mtDNA depletion: "Later-onset" ✤Wilson's disease: ATPase 7B; 13q14 PM + COX- muscle fibers: Mpl mtDNA delete LGMD 1H NARP/MILS: mtATP6 Neoplasms Neuropathy Motor ± Episodic weakness: mtATP6 or 8 Síndromes clínicos CPEO MELAS MERFF SANDO MIRAS KEARNS SAYRE LHON NARP MNGIE LEIGH ALPERS http://neuromuscular.wustl.edu/mitosyn.html#clinicalsyndromes DIDMOAD Página 16 de 132 PEO C10orf2 (Tinkle) Dominante ✤ ✤ tRNALeu Maternal Deleción única Esporádica POLG Recesiva Aislada Como un síntoma más en un cuadro multisistémico: Afectación bulbar Miopatía Ataxia Retinitis pigmentosa Miopatía Lipomas Mutación A8344G tRNALys m.5688T>C Miopatía Epilepsia mioclónica Mutación tRNA Asn 70-74% 88-85% 59-67% Mutación TK2 Miopatía Debilidad facial Disfagia VMNI dT + ATP PEO tardía Caterina Garone et al Bypass therapy for Tk2 deficiency were observed in the treated + function in mutants 29-day-old d motor types (Tk2 ) and dCMP/dTMP +400dCMP/dTMP , and wild-type Tk2 mice ain in Tk2 (Sup- e distance traveled, horizontal and ing time (Fig 1C–E). Relative to tor function !/!200dCMP/dTMP in 29-day-old -matched Tk2 and TMP , and wild-type Tk2 mice howed decreases in gross muscle ance horizontal r that traveled, were independent of theand treatime weight (Fig 1C–E). Relative to Research Article ody (Supplementary Fig S1). !/!200dCMP/dTMP myopathy hed Tk2 or mitochondrial abnorand Biochemical studies demond1A–D). decreases in gross muscle RC activities and of protein levels were independent the treat- dTTP miopatía Bypass therapy for Tk2 deficiency k2!/!400dCMP/dTMP) appeared normal n decelerated and mild head tremor 400dCMP/dTMP Tk2!/! mice had a mean lifespan ) appeared normal of !/!200dCMP/dTMP D), whereas Tk2head elerated and mild tremor ! P= 0.0028; 7; Gehan–Breslow– mice had na =mean lifespan of 0dCMP/dTMP lived !/!200dCMP/dTMP to 44.3 " 9.1 days whereas Tk2 eslow–Wilcoxon test) (Fig 1B). The 0028; n = 7; Gehan–Breslow– in postmortem histological studies of dTMP lived to 44.3 " No 9.1 adverse days k2!/!200dCMP/dTMP mice. –Wilcoxon (Figin1B). The ncies, were test) observed the treated tmortem histological studies of us wild types (Tk2+) and mutants 00dCMP/dTMP +400dCMP/dTMP ight gain in Tk2 (Supmice. No adverse dTMP + ADP dCMP Depleción Deleciones múltiples Encefalomiopatía infantil TK2 Caterina Garone et al Histological and histochemical CNS studies confirmed dCMP/ dTMP efficacy Histological and histochemical CNS studies confirmed dCMP/ Efficacy of treatment in central nervous system (CNS) was demondTMP efficacy strated in histological studies that showed dramatic reductions in the numbers of vacuoles in neurons of the spinal cord and cerebellar Efficacy of treatment in central nervous system (CNS) was demonand brain stem nuclei of 13-day-old Tk2!/!200dCMP/dTMP mice relastrated in histological studies !/! that showed dramatic reductions in tive to untreated 13-day-old Tk2 mice (Fig 2A and B). Furtherthe numbers of vacuoles in neurons of the IV) spinal cord and cerebellar more, cytochrome c oxidase (COX, complex histochemistry of and brain stem nuclei of 13-day-old Tk2!/!200dCMP/dTMP mice relacerebellum revealed reduced overall COX activity in 13-day-old !/! tive to untreated 13-day-old mice (Fig 2A and B).and Furtheruntreated Tk2!/!mice (Fig 3A) Tk2 with normal activities in 1329-day-old Tk2!/!200dCMP/dTMP (Fig 3C complex and E) relative to Tk2+ more, cytochrome c oxidase (COX, IV) histochemistry of animals (Figrevealed 3B, D andreduced F). No cell-specific immunohistochemical cerebellum overall COX activity in 13-day-old differencesTk2 in !/! COXmice protein (Fig 3Gactivities and H) while untreated (Figwere 3A) detected with normal in 13- and severe reduction in complex I was identified by immunostaining of Tk2+ 29-day-old Tk2!/!200dCMP/dTMP (Fig 3C and E) relative to cerebellum of 29-day-old Tk2!/!200dCMP/dTMP (Fig 3I and J). animals (Fig 3B, D and F). No cell-specific immunohistochemical differences in COX protein were detected (Fig 3G and H) while Treatment crosses biological barriers severe reduction in complex I was identified by immunostaining of cerebellum 29-day-old Tk2!/!200dCMP/dTMP (Fig barriers, 3I and J).we To confirmofthat the treatment crosses biological assessed dNTP levels in isolated mitochondria. In 13-day-old !/! mice relative to Tk2+ littermates, isolated brain untreated Tk2 Treatment crosses biological barriers mitochondria showed decreased levels of dTTP (0.67 " 0.1 pmol/ mg-protein versus 1.0), while isolated biological liver mitochondria To confirm that 2.52 the "treatment crosses barriers, we Deoxypyrimidine monophosphate bypass therapy revealed dNTP reduced levels dCTP in levels (1.07 " 0.8 versus 2.9 1.0) assessed isolated mitochondria. In" 13-day-old eight (Supplementary Fig S1). for thymidineuntreated kinase 2 deficiency Tk2!/! mice relative to Tk2+ littermates, isolated brain pathy or mitochondrial abnor- 1,2 1,3 mitochondria showedEmmanuele decreased levels of 4dTTP (0.67 1" 0.1 pmol/ . Biochemical studies demonCaterina Garone , Beatriz Garcia-Diaz1, Valentina , Luis C Lopez , Saba Tadesse , Hasan B 1 1,* , Catarina M Quinzii & Michio Hirano Akman1, Kurenai versus 2.52 " 1.0), while isolated liver mitochondria activities and Oprotein levels Tanji5mg-protein revealed reduced dCTP levels (1.07 " 0.8 versus 2.9 " 1.0) Abstract Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2!/!) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2!/! mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2!/!200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial Eof dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. B D Keywords deoxycytidine monophosphate; deoxythymidine monophosphate; encephalomyopathy; therapy; thymidine kinase Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism Autosomal recessive TK2 mutations cause severe mtDNA depletion and devastating neuromuscular diseases in infants and children, as well as mtDNA multiple deletions and progressive external ophthalmoplegia in adults (Saada et al, 2001; Tyynismaa et al, 2012). To elucidate the molecular pathogenesis of TK2 deficiency, we generated a homozygous Tk2 H126N knock-in mutant (Tk2!/!) mouse that manifests a phenotype strikingly similar to the human infantile encephalomyopathy (Akman et al, 2008). Between postnatal day 10 and 13, Tk2!/! mice rapidly develop fatal encephalomyopathy beginning with decreased ambulation, unstable gait, coarse tremor, and growth retardation that rapidly progress to early death at age 14–16 days (Dorado et al, 2011). A similar phenotype was observed in the Tk2 knockout mouse (Zhou et al, 2008). In the Tk2!/! mice, loss of Tk2 activity caused dNTP pool imbalances with low dTTP levels in brain and decreased dTTP and dCTP in liver, which, in turn, produce mtDNA depletion and defects of mitochondrial respiratory chain (RC) complexes I, III, IV, and V containing mtDNA-encoded subunits, most prominently in the brain and spinal cord (Dorado et al, 2011). Based on the understanding of the pathogenesis of Tk2 deficiency, we have assessed a rationale therapeutic strategy to bypass the enzymatic defect with oral dCMP and dTMP supplementation. DOI 10.15252/emmm.201404092 | Received 20 March 2014 | Revised 22 May D E 2014 | Accepted 26 May 2014 Results dCMP/dTMP delays disease onset, prevents neuromuscular manifestations, and prolongs lifespan of Tk2-deficient mice Introduction Encoded by the nuclear DNA gene TK2, thymidine kinase 2 (TK2) is a mitochondrial matrix protein that phosphorylates thymidine and pyrimidine nucleosides to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP), onwhich, body inweight (A) and survival (B) of mutant triphosphates mice (n = 7 turn, are converted to deoxynucleotide <(dNTPs) 0.005; Gehan-Breslow-Wilcoxon test). (mtDNA) synthesis. required for mitochondrial DNA n clinical phenotype.deoxycytidine Oral treatment with dCMP+dTMP 200 mg/kg/day each in milk (Tk2!/!200dCMP/dTMP) beginning at postnatal day 4 delayed disease onset to 20–25 days (Supplementary Video S1), when the mutant mice developed a mild tremor and stopped gaining weight. In the !/! versus Tk2!/!200dCMP/dTMP , forfourth eachweek, group)(Tk2 they manifested weakness and reduced movements. In contrast, Tk2!/! mice treated from day 4 with dCMP+dTMP ATAXIA s of the dCMP/dTMP /!400dCMP/dTMP, P reated mice showing no difference in average distance traveled (C), ambulatory and resting times (D), and horizontal (XY axes) and ) over 10 min in 29-day-old mutant and control mice treated with 200 mg/kg/day or 400 mg/kg/day of dCMP/dTMP (n = 5) (Data 1 Department of Neurology, Columbia University Medical Center, New York, NY, USA istical analysis were performed on Tk2!/!200dCMP/dTMP versus Tk2+200dCMP/dTMP and Tk2!/!400dCMP/dTMP versus Tk2+400dCMP/dTMP). 2 Human Genetics Joint PhD Program, University of Bologna and Turin, Turin, Italy ! al phenotype. 3 Pediatric Clinic, University of Genoa, IRCCS G. Gaslini Institute, Genoa, Italy 4 Instituto de Biotecnología, Centro de Investigación Biomédica, Universidad de Granada, Parque Tecnológico de Ciencias de la Salud, Armilla, Spain 5 Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA *Corresponding author. Tel: +1 212 305 1048; Fax: +1 212 305 3986; E-mail: [email protected] ª 2014 The Authors ª 2014 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine 1 dCMP/dTMP on body weight (A) and survival (B) of mutant mice (n = 7 for each group)(Tk2!/! versus Tk2!/!200dCMP/dTMP, dCMP/dTMP, P < 0.005; Gehan-Breslow-Wilcoxon test). mice showing no difference in average distance traveled (C), ambulatory and resting times (D), and horizontal (XY axes) and 0 min in 29-day-old mutant and control mice treated with 200 mg/kg/day or 400 mg/kg/day of dCMP/dTMP (n = 5) (Data nalysis were performed on Tk2!/!200dCMP/dTMP versus Tk2+200dCMP/dTMP and Tk2!/!400dCMP/dTMP versus Tk2+400dCMP/dTMP). Ataxia sensitiva Neuropatía óptica bilateral Hipoacusia OPA1 dominante Ataxia sensitiva ª 2014 The Authors Oftalmoparesia Disartria POLG recesivo DIAGNÓSTICO - Clínico, de sospecha - Apoyado por pruebas complementarias (hallazgos morfológicos y bioquímicos de la biopsia muscular) - Molecular 01 APROXIMACIÓN DIAGNÓSTICA ✤ ✤ ✤ Sospecha clínica: ✤ Síntomas ✤ Tipo de herencia Test de Laboratorio ✤ CK ✤ Ácido láctico ✤ Nuevos biomarcadores FGF21 Caracterización del fenotipo ✤ EMG ✤ Neuroimagen Biopsia de músculo (gold standard, sobre todo para mutaciones primarias del ADNmt) La presencia de fibras rojo rotas no es ni necesaria ni patognomónica en el diagnóstico de las EM ✤ Presentes en adultos sanos a partir de los 50 años ✤ Hallazgo habitual en otros trastornos: Miositis por cuerpos de inclusión Distrofia oculofaríngea Otros Actividad enzimática de los complejos de la CR ✤ Normal ✤ Déficit único ✤ Déficit múltiple ✤ Déficit II+III Subunidad estrutural complejos de CR Proteína de ensamblaje de un complejo específico Genes ADNmt que controlan síntesis de proteínas (ARNt, ARNr, macrodeleción) ADNn que controla mantenimiento Deficit de CoQ10 Diagnóstico molecular Oxidative phosphorylation biogenesis and regulation AAA Membrane dynamics and composition Oxidative phosphorylation subunits V IV III I II dN dNMP dNDP dNTP AAA mtDNA maintenance and expression Nucleotide transport and synthesis mtDNA Blázquez et al.(2016) eLS (Wiley) Molecular genetics of OXPHOS disorders mtDNA DELETIONS NUCLEARGENE mtDNAMANTEINANCE • • ) - ) • ( , - Mul$ple mtDNAdele$ons SPORADIC mtDNALESION • • • • ( - - SinglemtDNAdele$on mtDNA 16,6Kb ΔmtDNA Southern-BlotmtDNADIGprobes Genes“Mito”-Nucleares(nDNA): Genesnuclearesjueganunimportantepapel enlasenfermedadesOXPHOS: § 75%pacientesinfantiles § 30%pacientesadultos -- OXPHOS NUCLEAR GENES OXPHOSbiogenesis/regulation Assembly factors SURF1,BCS1L,SDHAF1,ACAD9, TMEM70,TACO1 Lipid metabolism: AGK,TAZ Phospholipid remodeling: SERAC1 Disulfide relay system: GFER Mitochondrial homeostasis FBXL4,CLPB Fe-Scluster assembly/homeostasis ISCU,BOLA3,NFU1,GLRX5,LYRM4 Mitochondrial protein import TIMM8A,DNAJC19,SPG7 Apoptosis: AIF1,APOPT1,FASTKD2 CoQ10biogenesis COQ2,COQ6,PDSS2,ADCK4,COQ4, COQ9, PDSS2,CABC1 Mitochondrial chaperones HSPD1,CLPB,AFGL32,SPG7 mtDNA maintenance/stability mtDNA replication POLG,C10orf2,POLG2,RNASEH1 dNTP synthesis/salvage DGUOK,TK2,RRM2B,TYMP Mitochondrial fission/fusion OPA1,MFN2,FBXL4 dN dNMP dNDP dNTP OXPHOSstructural subunits Complex I NDUFV1,NDUFB3,NDUFS4 Complex II SDHA,SDHB Complex III UQCRB,CYC1,UQCRQ Complex IV COX6B1,COX7B,COX4L2 Complex V ATP5A1,ATP5E mRNA rRNA mtDNA tRNA OXPHOSprotein synthesis Translation andrelease factors TUFM,TSFM,C12orf65,GFM1 mt-tRNA modifying proteins MTO1,GTPBP3,PUS1,TRIT1,MTFMT mRNA processing enzymes LRPPRC,ELAC2,PNPT1,HSD17B10 Blázquez et al.(2016) eLS (Wiley) Molecular genetics of OXPHOS disorders mt-aminoacyl tRNA synthetases AARS2,DARS2,RARS2,EARS2, YARS2,HARS2,LARS2, Mitoribosomal proteins MRPS16,MRPS22,MRPL3, MRPL12,MRPL44 Genes implicados en defectos de la cadena respiratoria agrupados por vías metabólicas REVIEW INSIGHT Table 1 | Products of genes that are known to be mutated in respiratory chain disorders grouped by pathway Oxidative phosphorylation subunits mtDNA maintenance and expression Oxidative phosphorylation biogenesis and Nucleotide regulation transport and synthesis Membrane dynamics and composition TWINKLE, MTFMT, GFM1, LRPPRC, MPV17, MRPS16, MRPS22, POLG, POLG2, TRMU, TSFM, TUFM, C12orf65, MTPAP, MRPL3, SARS2, YARS2, HARS2, MARS2, AARS2, RARS2, EARS2, DARS2, TACO1, MTO1, RMND1, PNPT1, PUS1 Complex I: NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, ACAD9, FOXRED1, NUBPL Complex II: SDHAF1, SDHAF2 Complex III: BCS1L, HCCS, TTC19 Complex IV: COX10, COX15, ETHE1, FASTKD2, SCO1, SCO2, SURF1, COX14, COA5 Complex V: ATPAF2, TMEM70 Fe–S: ABCB7, FXN, ISCU, NFU1, BOLA3, GLRX5 Other: DNAJC19, GFER, HSPD1, SPG7, TIMM8A, AIFM1, AFG3L2 ADCK3, AGK, COQ2, COQ6, COQ9, DRP1, MFN2, OPA1, PDSS1, PDSS2, TAZ, SERAC1 Nuclear encoded Complex I: NDUFA1, NDUFA2, NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFB3, NDUFB9, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2 Complex II: SDHA, SDHB, SDHC, SDHD Complex III: UQCRB, UQCRQ Complex IV: COX4I2, COX6B1 Complex V: ATP5E mtDNA encoded Complex I: ND1, ND2, ND3, ND4, ND4L, ND5, ND6 Complex III: CYTB Complex IV: COX1, COX2 Complex V: ATP6, ATP8 12S rRNA, tRNATyr, tRNATrp, tRNAVal, tRNAThr, tRNASer1, tRNASer2, tRNAArg, tRNAGln, tRNAPro, tRNAAsn, tRNAMet, tRNALeu1, tRNALeu2, tRNALys, tRNAIle, tRNAHis, tRNAGly, tRNAPhe, tRNAGlu, tRNAAsp, tRNACys, tRNAAla DGUOK, RRM2B, SLC25A3, ANT1, SUCLA2, SUCLG1, TK2, TYMP List of gene products was generated through synthesis of existing compilations of genes known to be mutated in respiratory-chain disease47,48, as well as review of the literature. found to be widespread folate deficiency combined with methanol however, the success rate is projected to be much lower in milder toxicity from homemade rum55. Formate, a by-product of metha- cases of disease, or those with adult onset. nol metabolism, accumulates in the setting of folate deficiency and How can we explain these unsolved cases? Most exome studies of causes inhibition of complex IV. singleton cases have been powered to identify recessive mutations in Certain medications have long been known to have toxic effects on mitochondrial proteins. It is possible that these unsolved cases are a mitochondrial function. Owing to the bacterial origins of the mito- result of dominant-acting, subtle recessive or regulatory mutations chondrial ribosome, mtDNA translation can be adversely affected by with incomplete penetrance, all of which are difficult to identify antibiotics such as aminoglycosides, which can cause sensorineural over the background of polymorphisms. Alternatively, these cases deafness when administered at high doses. Individuals with certain could be due to mutations in regions of DNA that are not targeted mtDNA mutations in the 12S ribosomal rRNA — estimated to have a for sequencing. Some cases could also have purely environmental population prevalence of 0.19% — are predisposed to deafness from causes, as with the Cuban blindness epidemic. A tantalizing posREVIEW INSIGHT aminoglycosides, and can experience hearing loss even with short sibility is that a subset of these unsolved cases is due to complex exposure to the recommended doses 56,57. The viral origins of the genetic inheritance, as a result of interactions between gene varia b mitochondrial DNA polymerase make it susceptible to nucleoside ants that each have weak or synergistic effects, known as synergistic 63 Stroke, ataxia, epilepsy, encephalopathy analogue antivirals (including fialuridine). This class of drugs is used heterozygosity . Targeted exome sequencing studies have reported and migraines commonly for HIV, and can cause side effects such as lactic acidosis that healthy controls will typically carry a burden of about 15–20 58 Optic neuropathy, Deafness through mtDNA depletion . heterozygous, loss-of-function protein alleles within their mitoretinopathy and external chondrial proteomes62. This high burden of deleterious alleles is Microorganisms are an emerging class of environmental modifi-opthalmoplegia ers, ranging from gut microbiota to viruses. Gut bacteria are drivers probably toleratedCardiomyopathy because of the robustness of mitochondrial netand conduction of disease progression in ethylmalonic encephalopathy. The causal Liverworks. However, defects it is possible that mutations that affect multiple failure CPK in themellitus same or parallel pathways, may conspire gene, ETHE1, encodes an enzyme that detoxifies sulphur compounds, genes, operating Diabetes Ácido that láctico which are released by gut microbiota, and its loss results in H2S Anaemia to yield pathology. Notably, there is suggestive evidence supRenal failure Intestinal pseudoaccumulation, with subsequent inhibition of complex IV and shortports synergistic interactions between mtDNA and nuclear DNA FGF21 obstruction chain acyl-CoA dehydrogenase. Treatment with metronidazole (anand diarrhoea variants64. Defining the genetic architecture of the large number of Muscle weakness, antibiotic that reduces gut microbial content) and N-acetylcysteine unsolved sporadicexercise casesintolerance, of mitochondrial disease represents the next cramps, atrophy, hypotonia (which promotes glutathione-mediated detoxification of H2S) results Peripheral major challenge ofandmitochondrial genetics. in clinical improvement59. Although a role for viral infections in the neuropathy Biopsia course of mitochondrial disorders has not been identified, experi- Mitochondrial ripplesmúsculo and responses 60 MELAS, MERFF, mental studies indicate that some viruses, such as HIV , can modu- Despite remarkable progress in defining the genes and environmenCadena respiratoria CPEO late complex tal triggers that underlie mitochondrial disease, their pathogenesis NARP,I activity. SANDO, Figure 1 | Phenotypic spectrum of mitochondrial disorders. a, Common seen on a modified Gomori-trichrome-stained skeletal-muscle section; clinical manifestations of mitochondrial b, Clinicalenigma. images anterior four-chamber cross-section ofoffer a heart that shows signs of hypertrophic remains almost adisorders. complete Many medical textbooks depicting pathology from patients with a variety of mitochondrial disorders. cardiomyopathy, including cardiomegaly and asymmetrical septal déficit múltiple LHON, Unsolved cases ofKSS mitochondrial disease thedéficit oversimplified explanation that manifests in tissues with Clockwise from top left, 3-Tesla único fluid-attenuated inversion-recovery brain disease hypertrophy; plain abdominal radiograph, showing massive bowel distention magnetic resonance imaging demonstrating Leigh syndrome lesions, which (arrow) in the setting of chronic intestinal pseudo-obstruction without Over the past 25 years, most of the mitochondrial disease genes have the highest ATP demand, or because of oxidative damage. Although are characterized in this image by a hyperintense signal within the caudate and evidence of mechanical obstruction; and bone-marrow aspirate sample that Análisis ADNmt en tejido SB/L-PCR putamen bilaterally seen onprobably an axial cut through the basal ganglia;to disease, has been stained for iron to demonstrate a ringed sideroblast (arrow) — been identified in familial forms of disease, in which it is possible these(arrows) factors contribute the picture is much herencia retinal image of theherencia acute phase of Leber’s hereditary optic neuropathy, characterized by a halo of iron-laden mitochondria around the nucleus of an herencia to follow the segregation of highly penetrant causal alleles. In demonstrating our more complicated. that there ADNmt apropiado (RFLP, SB) an optic disc with swollen nerveCellular fibre layer thatmodels is associated of disease erythrocyteindicate precursor — from a patient withis myopathy, lactic acidosis and autosómico with engorged and obscured blood vessels (arrows); ragged red fibre (arrow) anaemia syndrome. experience Massachusetts capacity for preservation ofsideroblastic ATP production through materna autosómica Estudio at dirigido ADNnGeneral Hospital, we have found that a remarkable 65 less than 25% of patients with clinical and biochemical evidence of enhanced glycolysis , and, in animal models of respiratory chain innovations . The mosaic composition of human mito- only 13 of which are mtDNA-encoded. Although typically depicted mitochondrial of an affected first-eukaryotic or dysfunction, pathology can develop without a major increase in oxi-the respiratory chain is truly chondria is evident in the organelle’s replication and translation as a linear chain operating in isolation, negdisease have strong evidence SB/L-PCR deleción machinery, with the ribosome66,67 closely resembling its bacterial hub in inhibition the network of cellular metabolism that is characterized by neg recentdeleciones second-degree relative. Several studies have applied exome dative damage . Furthermore, the facta that of respirasecuenciación NGS counterpartPanel the DNA polymerase resembling that of a viral convergence and divergence of pathways, supercomplex formation 61,62 múltiples ADNmt sequencing to establish molecular diagnoses in singleton cases (bacteriophage) . tionand can be. tolerated, andtheeven beneficial in reversibility. certain settings (Box 1), única ancestor As discussed later, molecular basis of and ADNmt certain mitochondrial pathologies clear when theof ancestry Almostchain all of the cell’s redox However, the success rate is lower than anticipated. Our experiindicates that thebecomes consequences respiratory lesions arereactions not ultimately feed into the OXPHOS of the organelle is taken into account. respiratory chain (Fig. 2b). Complexes I and II mediate two-electron ence has shown that in biochemically proven, severe cases in infants uniformly bad,the and suggests the involvement nonlinear of secuenciación During the course of evolution, organelle ceded ownership of cer- transferof from NADH and modes FADH to the mobile elecADNn: depleción / , respectively, Panel NGS ADNn defectos tain62pathways to the rest of the cell, but retained and even acquired oth- sitron carrier coenzyme Q, providing links to the tricarboxylic acid exome sequencing ADNmtshould achieve a diagnosis in about half of cases ; pathogenesis and threshold effects. Sospecha clínica Descripción del fenotipo ¿Síndrome clínico definido? SI NO 7 10 11 2 ers. For example, ribonucleotide reductase — which is used for de novo (TCA) cycle. Coenzyme Q can also receive electrons from de novo CI, CIII, CIV, CV del. multip. mantenimiento synthesis of deoxyribonucleotides — is found only in the cytosol, and pyrimidine biosynthesis, fatty-acid and amino-acid oxidation, deficiency of this enzyme causes mtDNA depletion . In0other choline 15 N O V E syndrome MBER 2 12 | V O L 4oxidation 9 1 | N(ultimately A T no U R E affecting | 3 7 9 one-carbon metabolism), factores ensamblaje POLG1,POLG2,OPA1,ANT1, cases, theLimited. mitochondria have retained a duplicated copy of the cytosolic and glycolysis. Complex III, through its ‘Q-cycle’, is an adaptor that © 2012 Macmillan Publishers All rights reserved two electrons reduced coenzyme Q and funnels indipathway, such as for tetrahydrofolate-dependent one-carbon metabo- receives ADNn Paneles NGS,from genes subunidades lism . These paralogous one-carbon pathways seem to have adopted vidual electrons to cytochrome c. Complex IV ends the respiratory C10ORF2,TK2,TYMP, implicados síntesis a different functional importance, and may be particularly relevant in chain by en accepting electronsde from cytochrome c and using them disease states such as cancer . Understanding the logic of compartmen- to fully reduce oxygen to water. Reactive oxygen species (ROS) are RRM2B, DGUOK, proteínas mt talization and paralogous pathways is an ongoing challenge. potentially toxic by-products of these reactions — especially at comSUCLG1,SUCLA2, MFN2 plexes I and III — but are buffered by dedicated superoxide disPUS1,EFG1,EFTu, Respiratory chain and its connections mutase and catalase,mtARS2, as well as glutathione, thioredoxin and protein 12 13 14 At the heart of mitochondria is the respiratory chain, the core machinery for oxidative phosphorylation (Fig. 2b). Classically, the respiratory chain is defined as four macromolecular complexes that catalyse electron transfer from reducing equivalents, which are derived from intermediary metabolism, to molecular oxygen. Free energy is conserved by coupling electron transport to the formation of a proton gradient, or proton motive force (PMF), by three of these complexes (I, III and IV), which is then dissipated by F1F0-ATPase (complex V) for ATP synthesis. These complexes are associated with the inner membrane and consist of about 90 protein components, thiol systems. Interruptions to the respiratory chain can therefore affect nucleotide pools, TCA-cycle flux, one-carbon metabolism and ROS signalling to unleash numerous ripples (discussed later). The PMF is best known for driving ATP synthesis through oxidative phosphorylation, but it is linked to many other processes (Fig. 2b). The nicotinamide nucleotide transhydrogenase relies on the PMF to regenerate mitochondrial NADPH, which is required for ROS homeostasis. Furthermore, the PMF is coupled to solute and ion transport across the inner membrane, and collapse of the PMF can halt essential biosynthetic reactions, such as Fe–S cluster 1 5 NOV E M B E R 2 0 1 2 | VO L 4 9 1 | NAT U R E | 3 7 5 © 2012 Macmillan Publishers Limited. All rights reserved Consideraciones terapéuticas ✤ Cofactores y vitaminas (decorenone, carnicor, riboflavina, ácido fólico, etc) ✤ Tratamientos específicos: ✤ ✤ MELAS: L-Arginina ✤ MNGIE: Trasplante de médula ósea ✤ Síndromes de depleción: nucleósidos Transferencia nuclear para evitar la transmisión de mutaciones en ADNmt Mensajes finales 1. Las miopatías metabólicas pueden manifestarse como intolerancia al ejercicio o como debilidad muscular fija +/- progresiva. 2. La intolerancia al ejercicio se caracteriza por aparición de mialgias/contracturas/calambres o debilidad inducida por la actividad física y se acompaña de elevación variable de la CK (diferenciar de astenia). La enfermedad de McArdle es frecuente con un prevalencia 1:15000 3. La causa más frecuente de mioglobinuria en el adulto es el déficit de CPT2 (75% p.S113L) 4. Las enfermedades mitocondriales OXPHOS pueden ser esporádicas, de herencia autosómica (dominante, recesiva o ligada X), o de herencia materna, dado que pueden se debidas a mutaciones en genes localizados en dos genomas (el ADNmt y ADNn), >150 genes implicados 5. La variabilidad clínica es elevada, con cuadros normalmente multisistémicos con afectación de SN y músculo principalmente, aunque hay fenotipos tejido-específicos (LHON, sordera neurosensorial) 6. En las EM siempre se debe tratar de alcanzar un diagnóstico molecular tanto para asegurar un consejo genético adecuado como para ofrecer un tratamiento específico en algunos casos. Unidad de Neuromuscular Neurólogos Laboratorio Anatomía Patológica Cristina Domínguez González Jesús Esteban Pérez Juan Francisco Gonzalo Ana Alonso Ortíz Miguel Ángel Martín Casanueva Alberto Blázquez Encinar Alberto García Redondo Aurelio Hernández Laín Oscar Toldos Neuropediatría Enfermería Rogelio Simón Ana Camacho Salas Noemí Núñez Enamorado Begoña Lucas Gordillo Pilar Cordero Vázquez Neumología Pedro Benavides Mañas Javier Sayas Díaz