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Hiroshi Kido - One of the best experts on this subject based on the ideXlab platform.
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Endogenous genetic risk factor for serious heatstroke: the thermolabile phenotype of Carnitine Palmitoyltransferase II variant
Acute Medicine & Surgery, 2018Co-Authors: Jun Oda, Tetsuo Yukioka, Kazunari Azuma, Takao Arai, Junji Chida, Hiroshi KidoAbstract:Aim In serious heatstroke, elevated body temperature (>40°C) is considered the main cause of illness. Mitochondrial Carnitine Palmitoyltransferase II (CPT II) plays an important role in adenosine triphosphate (ATP) generation from long-chain fatty acids, and its thermolabile phenotype of CPT2 polymorphisms leads to ATP production loss under high fever. Whether by heatstroke or influenza, high fever suppresses mitochondrial ATP production in patients with the thermolabile phenotype of CPT2 polymorphisms. We investigated the relation between CPT2 polymorphism and severity of heatstroke with a body temperature of over 40°C. Methods We analyzed blood chemistry test results, Japanese Association for Acute Medicine Disseminated Intravascular Coagulation (JAAM DIC), Acute Physiologic and Chronic Health Evaluation II, and Sequential Organ Failure Assessment (SOFA) scores, and CPT2 polymorphisms in 24 consecutive patients with severe heatstroke at two university hospitals. Results Eleven patients carried thermolabile CPT II variants (rs2229291; c.1055T˃G [p.Phe352Cys]) (F352C), and the genotype frequency was greater in heatstroke patients than in healthy volunteers. There was no significant difference in body temperature or blood chemistry data at emergency room arrival between patients with and without the CPT II variants. However, hospital days were longer and initial antithrombin activity was significantly lower in the variant group, suggesting a possible link with early phase vascular endothelial cell dysfunction. The JAAM DIC diagnostic criteria and SOFA scores were also higher in the group. There were no differences in the serum albumin, serum creatine kinase, and fibrin degradation product levels, and platelet counts. Conclusions In addition to known risks (e.g., environmental temperature and old age), the CPT II polymorphism [F352C] can be a predisposing genetic risk factor for serious heatstroke with organ disfunction, and lower antithrombin activity.
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First Japanese Case of Carnitine Palmitoyltransferase II Deficiency with the Homozygous Point Mutation S113L
Internal Medicine, 2016Co-Authors: Atsushi Shima, Tetsuhiko Yasuno, Seiji Yamaguchi, Kenji Yamada, Miyoko Yamaguchi, Ryuichi Kohno, Hiroshi Kido, Hidetoshi FukudaAbstract:Carnitine Palmitoyltransferase II (CPT II) deficiency is a rare inherited disorder related to recurrent episodes of rhabdomyolysis. The adult myopathic form of CPT II deficiency is relatively benign and difficult to diagnose. The point mutation S113L in CPT2 is very common in Caucasian patients, whereas F383Y is the most common mutation among Japanese patients. We herein present a case of CPT II deficiency in a Japanese patient homozygous for the missense mutation S113L. The patient showed a decreased frequency of rhabdomyolysis recurrence after the administration of a diet containing medium-chain triglyceride oil and supplementation with Carnitine and bezafibrate.
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Abbreviated half-lives and impaired fuel utilization in Carnitine Palmitoyltransferase II variant fibroblasts.
PLOS ONE, 2015Co-Authors: Min Yao, Hiroshi Kido, Min Cai, Dengfu Yao, Rongrong Yang, Yuanyuan Zhang, Dengbing YaoAbstract:Carnitine Palmitoyltransferase II (CPT II) deficiency is one of the most common causes of fatty acid oxidation metabolism disorders. However, the molecular mechanism between CPT2 gene polymorphisms and metabolic stress has not been fully clarified. We previously reported that a number of patients show a thermal instable phenotype of compound hetero/homozygous variants of CPT II. To understand the mechanism of the metabolic disorder resulting from CPT II deficiency, the present study investigated CPT II variants in patient fibroblasts, [c.1102 G>A (p.V368I)] (heterozygous), [c.1102 G>A (p.V368I)] (homozygous), and [c.1055 T>G (p.F352C)] (heterozygous) + [c.1102 G>A (p.V368I)] (homozygous) compared with fibroblasts from healthy controls. CPT II variants exerted an effect of dominant negative on the homotetrameric proteins that showed thermal instability, reduced residual enzyme activities and a short half-life. Moreover, CPT II variant fibroblasts showed a significant decrease in fatty acid β-oxidation and adenosine triphosphate generation, combined with a reduced mitochondrial membrane potential, resulting in cellular apoptosis. Collectively, our data indicate that the CPT II deficiency induces an energy crisis of the fatty acid metabolic pathway. These findings may contribute to the elucidation of the genetic factors involved in metabolic disorder encephalopathy caused by the CPT II deficiency.
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bezafibrate upregulates Carnitine Palmitoyltransferase II expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza associated encephalopathy
Molecular Genetics and Metabolism, 2011Co-Authors: Miyoko Yamaguchi, Junji Chida, Hiroshi KidoAbstract:Abstract Influenza-associated encephalopathy (IAE) is characterized by persistently high fever, febrile convulsions, severe brain edema and high mortality. We reported previously that a large proportion of patients with disabling or fatal IAE exhibit a thermolabile phenotype of compound variants for [1055T > G/F352C] and [1102G > A/V368I] of Carnitine Palmitoyltransferase II (CPT II) and mitochondrial energy crisis during high fever. In the present study, we studied the effect of bezafibrate, a hypolipidemic pan-agonist of peroxisome proliferator-activated receptor (PPAR), on CPT II expression and mitochondrial energy metabolism in fibroblasts of IAE patients and wild type (WT) fibroblasts from a healthy volunteer at 37 °C and 41 °C. Although heat stress markedly upregulated CPT II, CPT IA and PPAR-δ mRNA expression levels, CPT II activity, β-oxidation and ATP levels in WT and IAE fibroblasts at 41 °C were paradoxically downregulated probably due to the thermal instability of the corresponding enzymes. Bezafibrate significantly enhanced the expression levels of the above mRNAs and cellular functions of these enzymes in fibroblasts at 37 °C. Bezafibrate-induced increase in CPT II activity also tended to restore the downregulated ATP levels, though moderately, and improved mitochondrial membrane potential even at 41 °C to the levels at 37 °C in fibroblasts of IAE patients. L-Carnitine, a substrate of CPT II, boosted the effects of bezafibrate on cellular ATP levels in WT and IAE fibroblasts, even in severe IAE fibroblasts with thermolabile compound variations of F352C + V368I at 37 °C and 41 °C. The results suggest the potential usefulness of bezafibrate for the treatment of IAE.
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thermal instability of compound variants of Carnitine Palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza associated encephalopathy
Human Mutation, 2008Co-Authors: Hiroshi Mizuguchi, Miyoko Yamaguchi, Junji Chida, Hiroshi Yamada, Koji Shikata, Hiroshi KidoAbstract:Influenza-associated encephalopathy (IAE) is characterized by persistent high fever, febrile convulsions, severe brain edema, and high mortality in otherwise apparently healthy individuals. We have reported that a large proportion of patients suffering from disabling or fatal IAE, with transiently elevated serum acylCarnitine during high fever, exhibit a thermolabile phenotype of compound homo-/heterozygous variants of Carnitine Palmitoyltransferase II (CPT II, gene symbol CPT2). We characterized the enzymatic properties of five single and three compound CPT II variants in patients with IAE. The kinetic characteristics of WT and variant CPT IIs, expressed in COS-7 cells, indicated that the variants exert a dominant-negative effect on the homotetrameric protein of the enzyme. Among the variants, three compound variations found in patients with severe encephalopathy; [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile)], [c.1511C>T (p.Pro504Leu); c.1813G>C (p.Val605Leu)], and [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile); c.1813G>C (p.Val605Leu)], showed reduced activities, thermal instability, and short half-lives compared with the WT. Like other disease-causing mutant proteins, these variant proteins were poly-ubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome pathway. COS-7 cells transfected with the compound variants had their fatty acid β-oxidation decreased to 30–59% and intracellular ATP levels to 48–79%, and a marked reduction of mitochondrial membrane potential at 41°C, compared with control cells transfected with WT at 37°C. The unstable CPT II variants with decreased enzymatic activities may bring mitochondrial fuel utilization below the phenotypic threshold during high fever, and thus may play an important etiopathological role in the development of brain edema of IAE. Hum Mutat 29(5), 718–727, 2008. © 2008 Wiley-Liss, Inc.
Georgirene D Vladutiu - One of the best experts on this subject based on the ideXlab platform.
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novel mutations in the gene encoding very long chain acyl coa dehydrogenase identified in patients with partial Carnitine Palmitoyltransferase II deficiency
Muscle & Nerve, 2013Co-Authors: Paul J Isackson, Kristin A Sutton, Karl Y Hostetler, Georgirene D VladutiuAbstract:Introduction: Twenty-six patients with clinical symptoms of adult onset Carnitine Palmitoyltransferase II (CPTII) deficiency were examined. All patients had skeletal muscle CPTII enzyme activity levels indicative of heterozygosity for CPT2 mutations, however sequence analysis identified no pathogenic mutations within the CPT2 gene. Methods: Because the reaction product of CPTII is the substrate for very long-chain acyl-CoA dehydrogenase (VLCAD), we examined the ACADVL gene in these patients by sequence analysis. Results: Missense mutations within the ACADVL gene were identified in 3 of the patients. Conclusions: The locations of the altered amino acid residues within the crystal structure of VLCAD are on the surface of the molecule and may be involved in interactions with neighboring molecules. These findings support the importance of considering that mutations may be present in the ACADVL gene when a significant partial deficiency is found in CPTII activity, but no mutations in the CPT2 gene can be identified. Muscle Nerve, 2013
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Novel mutations in the gene encoding very long‐chain acyl‐CoA dehydrogenase identified in patients with partial Carnitine Palmitoyltransferase II deficiency
Muscle & Nerve, 2012Co-Authors: Paul J Isackson, Kristin A Sutton, Karl Y Hostetler, Georgirene D VladutiuAbstract:Introduction: Twenty-six patients with clinical symptoms of adult onset Carnitine Palmitoyltransferase II (CPTII) deficiency were examined. All patients had skeletal muscle CPTII enzyme activity levels indicative of heterozygosity for CPT2 mutations, however sequence analysis identified no pathogenic mutations within the CPT2 gene. Methods: Because the reaction product of CPTII is the substrate for very long-chain acyl-CoA dehydrogenase (VLCAD), we examined the ACADVL gene in these patients by sequence analysis. Results: Missense mutations within the ACADVL gene were identified in 3 of the patients. Conclusions: The locations of the altered amino acid residues within the crystal structure of VLCAD are on the surface of the molecule and may be involved in interactions with neighboring molecules. These findings support the importance of considering that mutations may be present in the ACADVL gene when a significant partial deficiency is found in CPTII activity, but no mutations in the CPT2 gene can be identified. Muscle Nerve, 2013
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Malignant hyperthermia-like syndrome and Carnitine Palmitoyltransferase II deficiency with heterozygous R503C mutation.
Anesthesia & Analgesia, 2009Co-Authors: Kirk Hogan, Georgirene D VladutiuAbstract:We describe a child who developed a malignant hyperthermia-like syndrome after exposure to succinylcholine and halothane. Many features of a typical malignant hyperthermia episode were present, including tachydysrhythmia, tachypnea, and fever in association with metabolic acidosis, hyperCKemia, myglobinemia, and rapid recovery without residual effects upon administration of dantrolene, sodium bicarbonate, and active cooling. Muscle rigidity, hypercarbia, and hyperkalemia were not observed. The patient was found to be heterozygous for a mutation in the Carnitine Palmitoyltransferase II gene (CPT2) encoding an arginine to cysteine substitution at amino acid 503 (R503C) with reduced activity of the enzyme.
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cpt2 gene mutations resulting in lethal neonatal or severe infantile Carnitine Palmitoyltransferase II deficiency
Molecular Genetics and Metabolism, 2008Co-Authors: Paul J Isackson, Michael J Bennett, Uta Lichterkonecki, M J H Willis, William L Nyhan, Reid V Sutton, Ingrid Tein, Georgirene D VladutiuAbstract:Three distinct clinical manifestations of Carnitine Palmitoyltransferase II (CPT II) deficiency have been defined including a mild adult onset myopathy, a severe infantile disorder and a lethal neonatal form. In this study we have examined the genomic DNA of five patients, 3 with the lethal neonatal form and 2 with the severe infantile form of the disease and identified two disease-causing mutations in the CPT2 gene for each patient, three of which are novel. In addition, based on currently available structural, biochemical and clinical data, we have classified all 64 known disease-causing mutations into groups with different predicted phenotypes depending on their CPT2 allelic counterparts.
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Lethal neonatal and severe late infantile forms of Carnitine Palmitoyltransferase II deficiency associated with compound heterozygosity for different protein truncation mutations.
The Journal of Pediatrics, 2002Co-Authors: Georgirene D Vladutiu, David Smail, Elizabeth J. Quackenbush, Bryan E. Hainline, Simone Albers, Michael J BennettAbstract:Abstract We describe a lethal neonatal form of Carnitine Palmitoyltransferase II (CPT II) deficiency with compound heterozygosity for 2 truncation mutations (Q413fs and 109AGC → GCAGC). A new phenotype for a severe late infantile form of CPT II deficiency with hypoglycemia is associated with compound heterozygosity for the severe Q413fs mutation and a mild point mutation (P50H). (J Pediatr 2002;141:734-6)
F Demaugre - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of Carnitine Palmitoyltransferase II deficiency with hepatocardiomuscular expression
American Journal of Human Genetics, 1996Co-Authors: Jeanpaul Bonnefont, Jean-marie Saudubray, Franco Taroni, Claude Cepanec, J P Leroux, Patrizia Cavadini, Michèle Brivet, F DemaugreAbstract:Carnitine Palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the SER-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, with out consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency.
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infantile form of Carnitine Palmitoyltransferase II deficiency with hepatomuscular symptoms and sudden death physiopathological approach to Carnitine Palmitoyltransferase II deficiencies
Journal of Clinical Investigation, 1991Co-Authors: F Demaugre, Jeanpaul Bonnefont, M Colonna, Claude Cepanec, J P Leroux, Jean-marie SaudubrayAbstract:Reported cases of Carnitine Palmitoyltransferase II (CPT II) deficiency are characterized only by a muscular symptomatology in young adults although the defect is expressed in extra-muscular tissues as well as in skeletal muscle. We describe here a CPT II deficiency associating hypoketotic hypoglycemia, high plasma creatine kinase level, heart beat disorders, and sudden death in a 3-mo-old boy. CPT II defect (-90%) diagnosed in fibroblasts is qualitatively similar to that (-75%) of two "classical" CPT II-deficient patients previously studied: It resulted from a decreased amount of CPT II probably arising from its reduced biosynthesis. Consequences of CPT II deficiency studied in fibroblasts differed in both sets of patients. An impaired oxidation of long-chain fatty acids was found in the proband but not in patients with the "classical" form of the deficiency. The metabolic and clinical consequences of CPT II deficiency might depend, in part, on the magnitude of residual CPT II activity. With 25% residual activity CPT II would become rate limiting in skeletal muscle but not in liver, heart, and fibroblasts. As observed in the patient described herein, CPT II activity ought to be more reduced to induce an impaired oxidation of long-chain fatty acids in these tissues.
Jean-marie Saudubray - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of Carnitine Palmitoyltransferase II deficiency with hepatocardiomuscular expression
American Journal of Human Genetics, 1996Co-Authors: Jeanpaul Bonnefont, Jean-marie Saudubray, Franco Taroni, Claude Cepanec, J P Leroux, Patrizia Cavadini, Michèle Brivet, F DemaugreAbstract:Carnitine Palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the SER-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, with out consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency.
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Recurrent metabolic decompensation in profound Carnitine Palmitoyltransferase II deficiency.
The Journal of Pediatrics, 1993Co-Authors: Orly Elpeleg, Jean-marie Saudubray, Adina Joseph, David Branski, Elisabeth Holme, Ernst Christensen, Alisa GutmanAbstract:A 3-year-old boy had recurrenl episodes of lethargy, encephalopathy, and hepatomegaly accompanied by hypoglycemia, elevated liver aminotransferase and creatine kinase values, and nonketotic dicarboxylic aciduria; the serum Carnitine level was moderately reduced. Carnitine Palmitoyltransferase II activity was decreased in lymphocytes and fibroblasts. Therapy with L -Carnitine and a diet low in long-chain triglycerides did not prevent recurrent episodes.
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infantile form of Carnitine Palmitoyltransferase II deficiency with hepatomuscular symptoms and sudden death physiopathological approach to Carnitine Palmitoyltransferase II deficiencies
Journal of Clinical Investigation, 1991Co-Authors: F Demaugre, Jeanpaul Bonnefont, M Colonna, Claude Cepanec, J P Leroux, Jean-marie SaudubrayAbstract:Reported cases of Carnitine Palmitoyltransferase II (CPT II) deficiency are characterized only by a muscular symptomatology in young adults although the defect is expressed in extra-muscular tissues as well as in skeletal muscle. We describe here a CPT II deficiency associating hypoketotic hypoglycemia, high plasma creatine kinase level, heart beat disorders, and sudden death in a 3-mo-old boy. CPT II defect (-90%) diagnosed in fibroblasts is qualitatively similar to that (-75%) of two "classical" CPT II-deficient patients previously studied: It resulted from a decreased amount of CPT II probably arising from its reduced biosynthesis. Consequences of CPT II deficiency studied in fibroblasts differed in both sets of patients. An impaired oxidation of long-chain fatty acids was found in the proband but not in patients with the "classical" form of the deficiency. The metabolic and clinical consequences of CPT II deficiency might depend, in part, on the magnitude of residual CPT II activity. With 25% residual activity CPT II would become rate limiting in skeletal muscle but not in liver, heart, and fibroblasts. As observed in the patient described herein, CPT II activity ought to be more reduced to induce an impaired oxidation of long-chain fatty acids in these tissues.
Franco Taroni - One of the best experts on this subject based on the ideXlab platform.
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Two CPT2 mutations in three Japanese patients with Carnitine Palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes.
Human Mutation, 1998Co-Authors: Kaoru Wataya, Franco Taroni, Jun-ichi Kira, Patrizia Cavadini, Jun Akanuma, Yoko Aoki, Shigeo Kure, Federica Invernizzi, Ichiro Yoshida, Yoichi MatsubaraAbstract:Carnitine Palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.
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molecular analysis of Carnitine Palmitoyltransferase II deficiency with hepatocardiomuscular expression
American Journal of Human Genetics, 1996Co-Authors: Jeanpaul Bonnefont, Jean-marie Saudubray, Franco Taroni, Claude Cepanec, J P Leroux, Patrizia Cavadini, Michèle Brivet, F DemaugreAbstract:Carnitine Palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the SER-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, with out consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency.
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recurrent myoglobinuria due to Carnitine Palmitoyltransferase II deficiency expression of the molecular phenotype in cultured muscle cells
Journal of the Neurological Sciences, 1996Co-Authors: Jean Villard, Jean Marc Collombet, Franco Taroni, Ginette Mandon, Andreas Fischer, B MoussonAbstract: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.
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Identification of 5' regulatory regions of the human Carnitine Palmitoyltransferase II gene.
Biochimica et Biophysica Acta, 1994Co-Authors: Laura Montermini, Franco Taroni, Stefano Didonato, Elisabetta Verderio, Haowei Wang, Gaetano FinocchiaroAbstract:Abstract We have identified two partially overlapping genomic clones that contain part of the 5′ regulatory region of the human Carnitine Palmitoyltransferase II gene. The 1.2 kb region upstream the transcription start site, as defined by primer extension experiments, shows promoter activity when inserted upstream of a reporter gene and contains a putative insulin responsive element.
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molecular characterization of inherited Carnitine Palmitoyltransferase II deficiency
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Franco Taroni, Gaetano Finocchiaro, Elisabetta Verderio, Stefania Fiorucci, Patrizia Cavadini, G Uziel, Eleonora Lamantea, Cinzia Gellera, Stefano DidonatoAbstract:Abstract Deficiency of Carnitine Palmitoyltransferase II (CPTase II; palmitoyl-CoA:L-Carnitine O-Palmitoyltransferase, EC 2.3.1.21) is a clinically heterogeneous autosomal recessive disorder of energy metabolism. We studied the molecular basis of CPTase II deficiency in an early-onset patient presenting with hypoketotic hypoglycemia and cardiomyopathy. cDNA and genomic DNA analysis demonstrated that the patient was homozygous for a mutant CPTase II allele (termed ICV), which carried three missense mutations: a G-1203----A transition, predicting a Val-368----Ile substitution (V368I); a C-1992----T transition, predicting an Arg-631----Cys substitution (R631C); and an A-2040----G transition, predicting a Met-647----Val substitution (M647V). Genomic DNA analysis of family members showed that the mutations cosegregated with the disease in the family. However, screening of 59 healthy controls demonstrated that both the V368I and M647V mutations are sequence polymorphisms with allele frequencies of 0.5 and 0.25, respectively. By contrast, the R631C substitution was not detected in 22 normal individuals or in 12 of 14 CPTase II-deficient patients with the adult muscular form. Notably, 2 adult CPTase II-deficient patients were heterozygous for the ICV allele, thus suggesting compound heterozygosity for this and a different mutant allele. The consequences of the three mutations on enzyme activity were investigated by expressing normal and mutated CPTase II cDNAs in COS cells. The R631C substitution drastically depressed the catalytic activity of CPTase II, thus confirming that this is the crucial mutation. Interestingly, the V368I and M647V substitutions, which did not affect enzyme activity alone, exacerbated the effects of the R631C substitution. Biochemical characterization of mutant CPTase II in patient's cells showed that the mutations are associated with (i) severe reduction of Vmax (approximately 90%), (II) normal apparent Km values, and (IIi) decreased protein stability.