Identification of a common mutation in the carnitine palmitoyltransferase II gene in familial recurrent myoglobinuria patients.

Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. We have identified a missense mutation (Ser113Leu) in one patient with the classical muscular symptomatology. Transfection experiments in COS cells demonstrate that the mutation drastically depresses the catalytic activity of CPT II. The mutation results in normal synthesis but a markedly reduced steady-state level of the protein, indicating decreased stability of mutant CPT II. The Ser113Leu mutation is the most frequent cause of CPT II deficiency. The mutation can be detected easily by restriction analysis enabling molecular diagnosis of most patients and identification of heterozygous carriers.

A microdeletion in cytochrome c oxidase (COX) subunit III associated with COX deficiency and recurrent myoglobinuria.

We have identified a 15-bp microdeletion in a highly conserved region of the mitochondrially encoded gene for cytochrome c oxidase (COX) subunit III in a patient with severe isolated COX deficiency and recurrent myoglobinuria. The mutant mitochondrial DNA (mtDNA) comprised 92% of the mtDNA in muscle and 0.7% in leukocytes. Immunoblots and immunocytochemistry suggested a lack of assembly or instability of the complex. Microdissected muscle fibres revealed significantly higher portions of mutant mtDNA in COX-negative than in COX-positive fibres. This represents the first case of isolated COX deficiency to be defined at the molecular level.

Myopathy and parkinsonism in phosphoglycerate kinase deficiency.

A 25-year-old man with exertional myoglobinuria had no evidence of hemolytic anemia, but he had severe parkinsonism that was responsive to levodopa. Phosphoglycerate kinase (PGK) activity was markedly decreased in muscle, and molecular analysis of the PGK1 gene identified the p.T378P mutation that was recently reported in a patient with isolated myopathy. This case reinforces the concept that PGK deficiency is a clinically heterogeneous disorder and raises the question of a relationship between PGK deficiency and idiopathic juvenile Parkinson disease.

Muscle carnitine palmityltransferase deficiency and myoglobinuria.

Muscle carnitine palmityltransferase activity, measured by three different methods, was very low (0 to 20 percent of controls) in a patient with a familial syndrome of recurrent myoglobinuria. Long-chain fatty acyl CoA synthetase activity was normal; acetylcarnitine transferase activity was decreased by 40 percent, and carnitine content was 1.7 times higher than the mean control value. Utilization of palmitate by isolated mitochondria was more impaired than utilization of palmitylcarnitine, suggesting a more severe defect of carnitine palmityltransferase I than transferase II. Thus, myoglobinuria may be due to a genetic defect of lipid metabolism in skeletal muscle.

Incidence of acute exertional rhabdomyolysis. Serum myoglobin and enzyme levels as indicators of muscle injury.

This study was conducted to determine, on a prospective basis, the incidence of acute exertional rhabdomyolysis (AER) among recruits at the Marine Corps Recruit Depot, San Diego, Calif. Blood samples were taken from each of 337 volunteer recruits on each of their first six days of regularly scheduled training. Serum myoglobin, serum creatine phosphokinase, lactic dehydrogenase, and serum glutamic oxaloacetic transaminase values were used as indicators of muscle injury. Substantial elevations of serum enzyme activity were observed throughout the study population. Of the study population, 39.2% had serum myoglobin levels that ranged from 0.37 mug/ml to 21.9 mug/ml during the study interval. Six subjects had serum myoglobin levels consistent with those reported in clinical cases of AER. It is concluded that, in a recruit population, large numbers of men may have myoglobinemia but not be seen initially as clinical cases.

An unusual case of carnitine palmitoyl transferase deficiency.

A 36-year-old man presented with episodic exertional dyspnea, fluctuating exercise intolerance, and myoglobinuria. He never experienced cramps or myalgias. Subsequent evaluation revealed carnitine palmitoyl transferase deficiency. The unusual features of this case suggested that carnitine palmitoyl transferase deficiency may have a more diverse clinical picture than previously described.

Exercise-induced recurrent myoglobinuria: defective activity of inner carnitine palmitoyltransferase in muscle mitochondria of two patients.

In carnitine palmitoyltransferase (CPT) deficiency, it is not known whether the outer (CPT-I) and the inner (CPT-II) mitochondrial activities are equally altered. By two different assays, we found that CPT activity in fresh intact mitochondria of two patients was normal or increased, indicating an active outer CPT. In controls and in one of the two patients, the isotope-exchange assay was also evaluated after disruption of mitochondria by sonication: in controls the activity almost doubled because of the contribution of inner CPT to the assay, but in the patients it did not increase, indicating absence of the CPT-II activity. After further disruption of mitochondria by freezing and all-glass homogenization, CPT activity in patients decreased to 36% and 10% of control. These data suggest CPT deficiency was limited to the inner mitochondrial activity. The alteration could be explained by mutation of the membrane factor that determines in situ differences between CPT-I and II.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence.

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

Combined defects of muscle phosphofructokinase and AMP deaminase in a child with myoglobinuria.

A 14-year-old boy with exercise-related myalgia and cramps had several episodes of myoglobinuria since early childhood. An episode at 2 years of age caused acute renal failure. Histochemical and biochemical analysis of muscle showed a combined defect of phosphofructokinase (PFK) and adenosine monophosphate (AMP) deaminase. DNA analysis showed that the patient was homozygous for a G-to-C substitution at codon 39 of the PFK gene (previously described in an Italian patient) and for the common mutation found in AMP deaminase deficiency.

Myoglobinuria and carnitine palmityltransferase (CPT) deficiency: studies with malonyl-CoA suggest absence of only CPT-II.

A 23-year-old man suffered since adolescence from recurrent myoglobinuria. His ketone body production during fasting was normal. Muscle, liver, and platelet carnitine palmityltransferase (CPT) ranged from 4 to 27% of control by isotope exchange and backward assays. Forward CPT activity was 34% of control in liver, whereas in muscle and platelets it was either normal or absent depending on the experimental conditions. CPT residual activity was studied with malonyl-CoA, a physiologic inhibitor of CPT-I (sensitive fraction) in rat liver mitochondria. In our patient, the insensitive fraction was missing in muscle, liver, and platelets, while the sensitive fraction was increased considerably in the same tissues. Similar results were obtained in platelets of two other patients with CPT deficiency. Increased malonyl-CoA sensitive CPT and decreased malonyl-CoA insensitive CPT suggest absence of only the CPT-II isoenzyme in these patients.

Carnitine palmityl transferase deficiency: clinical variability, carrier detection, and autosomal-recessive inheritance.

A 21-year-old man had recurrent myoglobinuria; his 28-year-old sister had symptoms of fatigability. During prolonged fasting, serum free fatty acid rose in both siblings, but only the sister produced ketone bodies and had elevated creatine phosphokinase activity. Carnitine palmityl transferase (CPT) activity was less than 30% of normal in muscle and platelets. Liver biopsy disclosed a low level of the enzyme in the brother. The parents had intermediate levels of the enzyme in platelets. CPT deficiency seems to have an autosomal-recessive pattern of inheritance and a variable phenotypic expression.

Carnitine palmityl transferase deficiency: myoglobinuria and respiratory failure.

A 51-year-old man presented with acute respiratory failure and myoglobinuria precipitated by an infection. Carnitine palmityltransferase (CPT) deficiency was documented in muscle, leukocytes, and liver. The enzyme defect in liver, previously suspected and now documented, explained the decreased production of ketone bodies during fasting observed in this patient as well as others with muscle CPT deficiency. Decreased utilization of long-chain fatty acids and decreased availability of ketone bodies can deprive the muscle of crucial sources of energy and, in certain conditions, may precipitate myoglobinuria.

Cytochrome c oxidase deficiency presenting as recurrent neonatal myoglobinuria.

Markedly reduced cytochrome c oxidase (COX) activity was found in cultured skin fibroblasts of an infant with recurrent episodes of acute myoglobinuria, hypertonia, muscle stiffness and elevated plasma levels of sarcoplasmic enzymes (creatine kinase 96950 U/l, normal below 150) since the age of 3 weeks (COX activity: 36 nmol/min/mg protein; normal 65-440; COX/succinate cytochrome c reductase ratio: 1.4, normal 3.0 +/- 0.4). The expression of the disease in cultured fibroblasts allowed us to carry out a prenatal diagnosis during the next pregnancy. Hitherto, mitochondrial respiratory chain deficiency has not been established as a cause of recurrent myoglobinuria in childhood. Since most cases of myoglobinurias remain poorly understood, we suggest giving consideration to respiratory chain deficiency in elucidating the origin of unexplained recurrent myoglobinuria in childhood, especially when seemingly unrelated symptoms are present.

Association of genetically proven deficiencies of myophosphorylase and AMP deaminase: a second case of 'double trouble'.

We studied a 25-year-old man with paresis of the limbs and neck, scapular atrophy, facial weakness, exercise intolerance and frequent episodes of myoglobinuria. Muscle histochemistry and biochemistry revealed a combined defect of myophosphorylase and AMP deaminase. Molecular genetic analysis showed that the patient was homozygous for the two most common mutations associated with myophosphorylase and AMP deaminase deficiencies. This is the second documented case of genetic 'double trouble', which should be looked for in patients with unusual severe phenotypes.

Recurrent myoglobinuria due to carnitine palmitoyltransferase II deficiency: expression of the molecular phenotype in cultured muscle cells.

Carnitine palmitoyltransferase II deficiency (CPT II) is an autosomal recessive disorder and the most frequent cause of hereditary myoglobinuria. We report the case of a young man who presented a severe fever-induced episode of rhabdomyolysis and myoglobinuria resulting in acute renal failure. Cultured skeletal muscle cells have been used for the biochemical and molecular characterization of the defect in this patient. Immunoblot analysis revealed reduced steady-state level of CPT II protein. A PCR-based method detected the common Ser113Leu substitution only in one allele, suggesting that the patient is a compound heterozygote for this common mutation and a different as yet unidentified mutation.

Heterogeneity of carnitine-palmitoyltransferase deficiency.

Episodes with muscle ache, rhabdomyolysis and myoglobinuria with or without associated renal insufficiency are characteristic of muscle carnitinepalmitoyltransferase (CPT) deficiency. However, patients differ from each other in many aspects, such as the kind of stimulus that triggers rhabdomyolysis, the ability to produce ketone bodies when fasting, whether the enzyme defect is localized in skeletal muscle or is general, and the nature of the enzyme defect, which may be in CPT I or CPT II or both. Studies of muscle, liver and fibroblasts from a patient with recurrent rhabdomyolysis spontaneously occurring or triggered by exercise or fever, revealed a CPT deficiency in the muscle and liver biopsy samples but normal CPT activity in cultured cells, differing from previously reported patients. The enzyme defect in muscle was evidenced by two different methods, but not when determined with a method that measures the formation of palmitoylcarnitine. The enzyme abnormality in the patient's liver was associated with a delayed ketone body production and with a dramatic increase in long-chain acylcarnitines in the serum when fasting. Moreover the patient was unable to build up ketones when fed long-chain triglycerides (LCT) but showed prompt ketogenic response when fed medium-chain triglycerides (MCT). The heterogeneity of clinical presentations and of the biochemical findings in patients with CPT deficiency are discussed.

Lactate dehydrogenase M-subunit deficiency: a new type of hereditary exertional myopathy.

Three families with a complete deficiency of the lactate dehydrogenase M subunit show exertional myoglobinuria. The response to ischemic forearm work is characteristic in these three families: an increase of venous lactate concentration after ischemic work was not observed and a marked increase of venous pyruvate was found. Glycolysis was markedly retarded in the patient's muscle in the glyceraldehyde 3-phosphate dehydrogenase (GA3PD) step. A significant increases in glyceraldehyde 3-phosphate, dihydroxyacetone phosphate and fructose 1,6-diphosphate were observed. The glycolysis retardation may be attributed to the impaired reoxidation of NADH produced by GA3PD action. The cytosolic fraction of skeletal muscle is rich in alpha-glycerophosphate dehydrogenase. This enzyme reoxidizes the excess NADH and drains triose phosphates from the glycolytic pathway under anaerobic conditions. For this reason, ATP production was significantly impaired and muscle cells were damaged in these patients. Consequently, the cytosolic enzymes and proteins such as creatine kinase and myoglobin were released into the blood stream. Otherwise, patients with a lactate dehydrogenase M-subunit deficiency do not show muscle stiffness and myoglobinuria under ordinary circumstances. They complain of muscle rigidity and sudden myoglobinuria after strenous exercise under anaerobic conditions. Thus, the lactate dehydrogenase M-subunit deficiency does not show any symptoms under ordinary circumstances, but is a latent hereditary disorder, now recognized as a new type of hereditary exertional myoglobinuria.

Hereditary deficiency of lactate dehydrogenase M-subunit.

A family with a complete deficiency of lactate dehydrogenase M-subunit was investigated. The propositus was an 18-year-old male who complained of exertional pigmenturia and easy fatigue. Marked discrepancy was observed in the ratio between creatine kinase and lactate dehydrogenase (CK/LDH). Electrophoretic analysis of serum LDH isoenzymes of the propositus demonstrated only one activity band of LDH H4. A complete lack of the LDH M-subunit was similarly demonstrated in erythrocytes, leukocytes and in the intermediate vastus muscle. LDH levels in the muscle specimen were markedly decreased in the patient, whereas CK and aspartate aminotransferase were almost the same as in a control subject. LDH isoenzymes of erythrocytes were analyzed in 5 siblings and in the parents. This demonstrated a complete lack of LDH M-subunit in 3 siblings. The ratio between H-subunit and M-subunit (H/M) in erythrocyte LDH suggested a partial absence of the M-subunit in two siblings and in the parents. An abortive increase of blood lactate and a marked increase in blood pyruvate were observed immediately after ischemic work of the forearm, accompanied by an increase in serum creatine kinase and myoglobinuria. The present case represents a newly described form of genetically determined myopathy.

Recurrent myoglobinuria in a child with mental retardation: phosphoglycerate kinase deficiency.

We report the case of an 11-year-old mentally retarded boy with recurrent myoglobinuria precipitated after a generalized tonic-clonic convulsion. No hemolysis was noted. Ischemic forearm test revealed no rise of venous lactate, suggesting a metabolic defect in an anaerobic glycolytic pathway. Histochemistry studies of the quadriceps muscle showed a normal appearance, but electron microscopy confirmed a moderate increase of the glycogen content in muscle. Direct measurement of glycolytic enzymes demonstrated a marked decrease of phosphoglycerate kinase (PGK) activity in muscle (4.4% of control mean) and hemolysate (8% of control mean). Enzyme characteristics of PGK from our patient (PGK Hamamatsu) using hemolysate demonstrated that it had normal Michaelis constants (Km), normal thermal stability, and a normal pH curve. The reason that hemolytic anemia was absent is uncertain. We concluded that a systematic enzyme analysis of the glycolytic pathway, especially of PGK, should be performed on myoglobinuric patients who are males, or who have an X-linked inheritance as suggested by the family history.

Novel mutation in the CPT II gene in a child with periodic febrile myalgia and myoglobinuria.

We have identified a novel missense mutation in the carnitine palmitoyltransferase II (CPT II) gene in a child with CPT II deficiency characterized clinically by episodes of myalgia and myoglobinuria induced by intercurrent febrile illnesses. The patient was heterozygous for a G-to-A substitution at codon 487, changing an encoded glutamic acid to a lysine (E489K), while the other allele carried the common S113L mutation. This case enlarges the spectrum of mutations in patients with CPT II deficiency, and confirms the association of the S113L mutation with the muscular form.