Ingrid Tein

Impaired skin fibroblast carnitine uptake in primary systemic carnitine deficiency manifested by childhood carnitine-responsive cardiomyopathy

ABSTRACT: Evidence is emerging that primary systemic carnitine deficiency, a potentially lethal but eminently treatable inborn error of fatty acid oxidation, involves a cellular defect in the uptake of carnitine. We present four unrelated children with primary carnitine-responsive cardiomyopathy, weakness (with or without hypoketotic hypoglycemic encephalopathy), low serum and/or tissue carnitine concentrations, and severe renal carnitine leak. Di-carboxylic acids were absent in the urine of three children who were tested, and all four had a rapid and dramatic improvement in cardiac function, strength, and somatic growth after carnitine therapy. We studied carnitine uptake in cultured skin fibroblasts from all four children and seven of the eight healthy nonconsanguinous parents. [3H]L-car-nitine uptake was evaluated in vitro under linear time kinetics. Substrate concentrations were varied from 0.1 to 1000 μM. Physiologic uptake was determined at carnitine concentrations between 0.1 and 50 μM. Nonspecific uptake was determined at a concentration of 10 mM. The four patients had negligible uptake throughout the physiologic range, implying a marked deficiency in the specific high-affinity, low-concentration, carrier-mediated uptake mechanism. At a concentration of 5 μmol/L, the mean velocity of uptake in the four patients was 2% of control values. Their parents showed intermediate maximal rates of carnitine uptake ranging from 13 to 44% of control Vmax values, but normal Km values, suggesting that the hetero-zygotes had a reduced number of normal functioning carnitine transporters. The observed reduction in Vmax values for the parents supports an autosomal recessive inheritance pattern and may be a more sensitive indicator of heterozygosity than serum carnitine concentrations. We conclude that carnitine uptake studies in cultured skin fibroblasts are important for diagnosis, screening of siblings and heterozygote parents, understanding pathogenesis, and investigating the molecular basis of this disease. Given the frequent history of previously unexplained sibling deaths in these familes, early identification of presymptomatic siblings and oral institution of carnitine prophylaxis may decrease immediate and long-term morbidity and mortality.

Pyridoxine dependent epilepsy and antiquitin deficiency Clinical and molecular characteristics and recommendations for diagnosis, treatment and follow-up

Antiquitin (ATQ) deficiency is the main cause of pyridoxine dependent epilepsy characterized by early onset epileptic encephalopathy responsive to large dosages of pyridoxine. Despite seizure control most patients have intellectual disability. Folinic acid responsive seizures (FARS) are genetically identical to ATQ deficiency. ATQ functions as an aldehyde dehydrogenase (ALDH7A1) in the lysine degradation pathway. Its deficiency results in accumulation of α-aminoadipic semialdehyde (AASA), piperideine-6-carboxylate (P6C) and pipecolic acid, which serve as diagnostic markers in urine, plasma, and CSF. To interrupt seizures a dose of 100 mg of pyridoxine-HCl is given intravenously, or orally/enterally with 30 mg/kg/day. First administration may result in respiratory arrest in responders, and thus treatment should be performed with support of respiratory management. To make sure that late and masked response is not missed, treatment with oral/enteral pyridoxine should be continued until ATQ deficiency is excluded by negative biochemical or genetic testing. Long-term treatment dosages vary between 15 and 30 mg/kg/day in infants or up to 200 mg/day in neonates, and 500 mg/day in adults. Oral or enteral pyridoxal phosphate (PLP), up to 30 mg/kg/day can be given alternatively. Prenatal treatment with maternal pyridoxine supplementation possibly improves outcome. PDE is an organic aciduria caused by a deficiency in the catabolic breakdown of lysine. A lysine restricted diet might address the potential toxicity of accumulating αAASA, P6C and pipecolic acid. A multicenter study on long term outcomes is needed to document potential benefits of this additional treatment. The differential diagnosis of pyridoxine or PLP responsive seizure disorders includes PLP-responsive epileptic encephalopathy due to PNPO deficiency, neonatal/infantile hypophosphatasia (TNSALP deficiency), familial hyperphosphatasia (PIGV deficiency), as well as yet unidentified conditions and nutritional vitamin B6 deficiency. Commencing treatment with PLP will not delay treatment in patients with pyridox(am)ine phosphate oxidase (PNPO) deficiency who are responsive to PLP only.

l‐Carnitine Supplementation in Childhood Epilepsy: Current Perspectives

Summary: In November 1996, a panel of pediatric neurologists met to update the consensus statement issued in 1989 by a panel of neurologists and metabolic experts on L‐carnitine supplementation in childhood epilepsy. The panelists agreed that intravenous L‐carnitine supplementation is clearly indicated for valproate (VPA)‐induced hepatotoxicity, overdose, and other acute metabolic crises associated with carnitine deficiency. Oral supplementation is clearly indicated for the primary plasmalemmal carnitine transporter defect. The panelists concurred that oral L‐carnitine supplementation is strongly suggested for the following groups as well: patients with certain secondary carnitine‐deficiency syndromes, symptomatic VPA‐associated hyperammonemia, multiple risk factors for VPA hepatotoxicity, or renal‐associated syndromes; infants and young children taking VPA; patients with epilepsy using the ketogenic diet who have hypocarnitinemia; patients receiving dialysis; and premature infants who are receiving total parenteral nutrition. The panel recommended an oral L‐carnitine dosage of 100 mg/kg/day, up to a maximum of 2 g/day. Intravenous supplementation for medical emergency situations usually exceeds this recommended dosage.

Carnitine transport: Pathophysiology and metabolism of known molecular defects

Summary: Early-onset dilatative and/or hypertrophic cardiomyopathy with episodic hypoglycaemic coma and very low serum and tissue concentrations of carnitine should alert the clinician to the probability of the plasmalemmal high-affinity carnitine transporter defect. The diagnosis can be established by demonstration of impaired carnitine uptake in cultured skin fibroblasts or lymphoblasts and confirmed by mutation analysis of the human OCTN2 gene in the affected child and obligate heterozygote parents. The institution of high-dose oral carnitine supplementation reverses the pathology in this otherwise lethal autosomal recessive disease of childhood, and carnitine therapy from birth in prospectively screened siblings may altogether prevent the development of the clinical phenotype. Heterozygotes may be at risk for cardiomyopathy in later adult life, particularly in the presence of additional risk factors such as hypertension and competitive pharmacological agents. OCTN2 belongs to a family of organic cation/carnitine transporters that function primarily in the elimination of cationic drugs and other xenobiotics in kidney, intestine, liver and placenta. The high- and low-affinity human carnitine transporters, OCTN2 and OCTN1, are multifunctional polyspecific organic cation transporters; therefore, defects in these transporters may have widespread implications for the absorption and/or elimination of a number of key pharmacological agents such as cephalosporins, verapamil, quinidine and valproic acid. A third organic/cation carnitine transporter with high specificity for carnitine, Octn3, has been clonedin mice. The juvenile visceral steatosis (jvs) mouse serves as an excellent clinical, biochemical and molecular model for the high-affinity carnitine transporter OCTN2 defect and is due to a spontaneous point mutation in the murine Octn2 gene on mouse chromosome 11, which is syntenic to the human locus at 5q31 that harbours the human OCTN2 gene.

An additional mitochondrial tRNAIle point mutation (A-to-G at nucleotide 4295) causing hypertrophic cardiomyopathy

A third point mutation in the mitochondrial tRNAIle gene associated with hypertrophic cardiomyopathy and respiratory chain dysfunction in heart is reported. An A‐to‐G transition at nucleotide position 4295 was shown to be highly evolutionarily conserved, never present in control individuals, and to segregate with the disease. A PCR‐based diagnostic test and endomyocardial biopsies were used to detect both the biochemical deficiency and the level of heteroplasmy in heart. The implications of this new mitochondrial DNA point mutation are discussed.

Short-chain acyl-CoA dehydrogenase deficiency: a cause of ophthalmoplegia and multicore myopathy.

Objective: To determine an underlying genetic defect within the differential diagnosis of congenital multicore myopathy. Background: A 13.5-year-old girl presented with congenital-onset facial and neck weakness, slowly progressive severe limb girdle and axial myopathy, respiratory weakness, cardiomyopathy, progressive joint contractures, lumbar lordosis, progressive external ophthalmoplegia with ptosis, and cataracts. Muscle biopsy at 3 years revealed type I fiber predominance and hypotrophy, multicores with a focal decrease in mitochondria and oxidative enzymes, and internal nuclei. Methods and Results: Serum carnitine was decreased (total, 18.2 μmol/L; free, 11.7 μmol/L). Urine organic acids intermittently revealed very large amounts of ethylmalonic and methylsuccinic acids intermittently, with elevated butyrylglycine, 2-methylbutyrylglycine, and tiglylglycine. Fibroblast acylcarnitine profiles revealed marked butyrylcarnitine elevation. Electron-transferring flavoprotein-linked reduction enzymatic assay of fibroblasts with butyryl–coenzyme A (CoA) as substrate, after immunoinactivation of medium-chain acyl–CoA dehydrogenase activity, revealed a complete absence of short-chain acyl–CoA dehydrogenase (SCAD) activity. No SCAD protein was detectable with Western blot analysis. Conclusions: This patient expands the clinical phenotype of SCAD deficiency and emphasizes the need for its consideration in the differential diagnosis of progressive external ophthalmoplegia and congenital multicore myopathy.

Cystic fibrosis transmembrane conductance regulator in human muscle: Dysfunction causes abnormal metabolic recovery in exercise.

Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using 31P‐magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high‐intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end‐exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle.

Mild trifunctional protein deficiency is associated with progressive neuropathy and myopathy and suggests a novel genotype-phenotype correlation.

Human mitochondrial trifunctional protein (TFP) is a heterooctamer of four alpha- and four beta-subunits that catalyzes three steps in the beta-oxidation spiral of long-chain fatty acids. TFP deficiency causes a Reye-like syndrome, cardiomyopathy, or sudden, unexpected death. We delineated the molecular basis for TFP deficiency in two patients with a unique phenotype characterized by chronic progressive polyneuropathy and myopathy without hepatic or cardiac involvement. Single-stranded conformation variance and nucleotide sequencing identified all patient mutations in exon 9 of the alpha-subunit. One patient is homozygous for the T845A mutation that substitutes aspartic acid for valine at residue 246. The second patient is a compound heterozygote for the T914A that substitutes asparagine for isoleucine at residue 269 and a C871T that creates a premature termination at residue 255. Allele-specific oligonucleotide hybridization studies revealed undetectable levels of the mRNA corresponding to the mutant allele carrying the termination codon. This study suggests a novel genotype-phenotype correlation in TFP deficiency; that is, mutations in exon 9 of the alpha-subunit, which encodes a linker domain between the NH2-terminal hydratase and the COOH-terminal 3-hydroxyacyl-CoA dehydrogenase, result in a unique neuromuscular phenotype.

A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn's disease locus (IBD5).

Organic cation transporters function primarily in the elimination of cationic drugs in kidney, intestine, and liver. The murine organic cation/carnitine (Octn) transporter family, Octn1, Octn2, and Octn3 is clustered on mouse chromosome 11 (NCBI Accession No. NW_000039). The human OCTN1 and OCTN2 orthologs map to the syntenic IBD5 locus at 5q31, which has been shown to confer susceptibility to Crohns disease. We show that the human OCTN3 protein, whose corresponding gene is not yet cloned or annotated in the human reference DNA sequence, does indeed exist and is uniquely involved in carnitine-dependent transport in peroxisomes. Its functional properties and inferred chromosomal location implicate it for involvement in Crohns disease.

Skeletal muscle metabolic dysfunction in obesity and metabolic syndrome.

Obesity and the related metabolic syndrome have become a worldwide epidemic. Inactivity appears to be a primary causative factor in the pathogenesis of this obesity and metabolic syndrome. There are two possible, perhaps not mutually exclusive, events that may lead to intramyocellular lipid accumulation and mitochondrial dysfunction in patients with obesity. First, obesity, with high intake-associated lipid accumulation in muscle may interfere with cellular mitochondrial function through generation of reactive oxygen species leading to lipid membrane peroxidative injury and disruption of mitochondrial membrane-dependent enzymes. This in turn leads to impaired oxidative metabolism. Secondly, a primary defect in mitochondrial oxidative metabolism may be responsible for a reduction in fatty acid oxidation leading to intramyocellular lipid accumulation as a secondary event. Non-invasive techniques such as proton (1H) and phosphorus (31P) magnetic resonance spectroscopy, coupled with specific magnetic resonance imaging techniques, may facilitate the investigation of the effects of various ergometric interventions on the pathophysiology of obesity and the metabolic syndrome. Exercise has positive effects on glucose metabolism, aerobic metabolism, mitochondrial density, and respiratory chain proteins in patients with metabolic syndrome, and we propose that this may be due to the exercise effects on AMP kinase, and a prospective physiological mechanism for this benefit is presented. A physiological model of the effect of intramyocellular lipid accumulation on oxidative metabolism and insulin mediated glucose uptake is proposed.