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.

Cerebral hyperperfusion and decreased cerebrovascular reactivity correlate with neurologic disease severity in MELAS.

OBJECTIVE: To study the mechanisms underlying stroke-like episodes (SLEs) in MELAS syndrome. METHODS: We performed a case control study in 3 siblings with MELAS syndrome (m.3243A>G tRNA(Leu(UUR))) with variable % mutant mtDNA in blood (35 to 59%) to evaluate regional cerebral blood flow (CBF) and arterial cerebrovascular reactivity (CVR) compared to age- and sex-matched healthy study controls and a healthy control population. Subjects were studied at 3T MRI using arterial spin labeling (ASL) to measure CBF; CVR was measured as a change in % Blood Oxygen Level Dependent signal (as a surrogate of CBF) to repeated 10 mmHg step increase in arterial partial pressure of CO2 (PaCO2). RESULTS: MELAS siblings had decreased CVR (p ≤ 0.002) and increased CBF (p < 0.0026) compared to controls; changes correlated with disease severity and % mutant mtDNA (inversely for CVR: r = -0.82 frontal, r = -0.91 occipital cortex; directly for CBF: r = +0.85 frontal, not for occipital infarct penumbra). Mean CVR was reduced more in frontal (p < 0.001) versus occipital cortex (p = 0.002); mean CBF was increased more in occipital (p = 0.001) than frontal (p = 0.0026) cortices compared to controls. CBF correlated inversely with CVR (r = -0.99 in frontal; not in occipital infarct penumbra) suggesting that increased frontal resting flows are at the expense of flow reserve. INTERPRETATION: MELAS disease severity and mutation load were inversely correlated with Interictal CVR and directly correlated with frontal CBF. These metrics offer further insight into the cerebrovascular hemodynamics in MELAS syndrome and may serve as noninvasive prognostic markers to stratify risk for SLEs. CLASSIFICATION OF EVIDENCE: Class III.

Possible valproate teratogenicity.

The teratogenic risk of maternal valproic acid therapy and the prenatal effects on growth and morphogenesis have been difficult to determine, in part, because of the small number of epileptic women who receive valproic acid as the sole anticonvulsant therapy. An increased incidence of open neural tube defects has been suggested and other isolated case reports have noted the presence of certain dysmorphic features. We present a patient whose defects in morphogenesis appear to be associated with valproic acid exposure only, and suggest a clinical phenotype that our patient shares with other children exposed to prenatal valproic acid therapy. Features described previously include dysmorphic facies with hypertelorism, prominent forehead, low flat nasal bridge, low-set or odd-shaped ears, and micrognathia with growth deficiency of prenatal or postnatal onset. A striking and perhaps unique feature in our patient consists of hypoplasia of the lateral margins of the zygomatic arches. We discuss the pharmacokinetics of valproic acid in pregnancy and in the neonatal period.

Long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency neuropathy: response to cod liver oil.

Docosahexaenoic acid (DHA) deficiency has recently been documented in several children with long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHADD). We studied a 13-year-old boy with LCHADD who had limb girdle myopathy, recurrent myoglobinuria, and progressive sensorimotor axonopathy with demyelination. At 11 years of age, he was started on cod liver oil extract, high in DHA content. Over 12 months, he demonstrated a marked clinical recovery. Nerve conduction studies (NCS) demonstrated reappearance of previously absent posterior tibial and peroneal nerve responses and the amplitudes on motor ulnar and median NCS markedly increased from 7- to 14-fold, respectively.

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 micromol/L; free, 11.7 micromol/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.

Normal muscle CPT1 and CPT2 activities in hepatic presentation patients with CPT1 deficiency in fibroblasts. Tissue specific isoforms of CPT1?

Human carnitine palmitoyltransferase (CPT) deficiency results in 2 clinical forms: a more common "muscular form" with myoglobinuria with or without delayed or impaired ketogenesis and a rare "hepatic form" with hypoketotic hypoglycemia, encephalopathy and seizures without muscular manifestations. We present 2 patients, a male (patient 1) and a female (patient 2) with infantile "hepatic" CPT deficiency and previously documented CPT1 deficiency in fibroblasts. In patient 2, a deficiency of "total" CPT activity in liver had also been previously documented. We set up an isotope exchange assay system that effectively differentiated CPT1 and CPT2 activities in muscle. We found normal CPT1 and CPT2 activities in our patients under near saturating substrate conditions. The CPT1 and CPT2 activities were suppressed to a strikingly similar degree under different kinetic conditions as compared to control muscle and were found to have similar Km values for carnitine and PCoA. With Km concentrations of carnitine, the mean residual activities of CPT1 for patients 1 and 2 were 49 and 44%, respectively (control range 40-53%); the mean residual activities of CPT2 were 60 and 46%, respectively (control range 49-59%). With Km concentrations of PCoA, the mean residual activities of CPT1 for patients 1 and 2 were 52 and 58%, respectively (control range of 52-59%); mean residual activities of CPT2 were 54% and 56%, respectively (control range of 51-68%). When the Vmax concentration of PCoA was doubled and bovine serum albumin reduced to 0.1%, the mean residual activities of CPT1 for patients 1 and 2 were 69 and 63%, respectively (control range 60-80%). In "muscular" patients, a marked absolute deficiency of CPT2 activity (less than 12% residual) was found with an apparent increased sensitivity to suppression of enzymatic activity when the Km concentration of carnitine was used. We suggest that CPT1 and CPT2 may be separate proteins. Furthermore, CPT1 itself may exist as tissue-specific isoforms being the same protein in liver and fibroblasts and a different protein in muscle. Either could be encoded for by the same or closely related genes.

Neonatal metabolic myopathies.

The primary presentations of neuromuscular disease in the newborn period are hypotonia and weakness. Although metabolic myopathies are inherited disorders that present from birth and may present with subtle to marked neonatal hypotonia, a number of these defects are diagnosed classically in childhood, adolescence, or adulthood. Disorders of glycogen, lipid, or mitochondrial metabolism may cause three main clinical syndromes in muscle, namely, (1) progressive weakness with hypotonia (e.g., acid maltase, debrancher enzyme, and brancher enzyme deficiencies among the glycogenoses; carnitine uptake and carnitine acylcarnitine translocase defects among the fatty acid oxidation (FAO) defects; and cytochrome oxidase deficiency among the mitochondrial disorders) or (2) acute, recurrent, reversible muscle dysfunction with exercise intolerance and acute muscle breakdown or myoglobinuria (with or without cramps), e.g., phosphorylase, phosphofructokinase, and phosphoglycerate kinase among the glycogenoses and carnitine palmitoyltransferase II deficiency among the disorders of FAO or (3) both (e.g., long-chain or very long-chain acyl coenzyme A (CoA) dehydrogenase, short-chain L-3-hydroxyacyl-CoA dehydrogenase, and trifunctional protein deficiencies among the FAO defects). Episodes of exercise-induced myoglobinuria tend to present in later childhood or adolescence; however, myoglobinuria in the first year of life may occur in FAO disorders during catabolic crises precipitated by fasting or infection. The following is a survey of genetic disorders of glycogen and lipid metabolism resulting in myopathy, focusing primarily on those defects, to date, that have presented in the neonatal or early infancy period. Disorders of mitochondrial metabolism are discussed in another chapter.

Diagnostic difficulties in a case of primary systemic carnitine deficiency with idiopathic dilated cardiomyopathy.

Blood spot carnitine profiles are widely used to screen for disorders of fatty acid oxidation. This case report emphasizes that a borderline concentration of free carnitine does not exclude the diagnosis of primary carnitine deficiency. Concurrent measurement of carnitine in the plasma and urine is a more sensitive test.

Metabolic myopathies.

Disorders of glycogen, lipid or mitochondrial metabolism may cause two main clinical syndromes, namely (1) progressive weakness (eg, acid maltase, debrancher enzyme, and brancher enzyme deficiencies among the glycogenoses; long- and very-long-chain acyl-CoA dehydrogenase (LCAD, VLCAD), and trifunctional enzyme deficiencies among the fatty acid oxidation (FAO) defects; and mitochondrial enzyme deficiencies) or (2) acute, recurrent, reversible muscle dysfunction with exercise intolerance and acute muscle breakdown or myoglobinuria (with or without cramps) (eg, phosphorylase (PPL), phosphorylase b kinase (PBK), phosphofructokinase (PFK), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), and lactate dehydrogenase (LDH) among the glycogenoses and carnitine palmitoyltransferase II (CPT II) deficiency among the disorders of FAO or (3) both (eg, PPL, PBK, PFK among the glycogenoses; LCAD, VLCAD, short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), and trifunctional enzyme deficiencies among the FAO defects; and multiple mitochondrial DNA (mtDNA) deletions). Myoadenylate deaminase deficiency, a purine nucleotide cycle defect, is somewhat controversial and is characterized by exercise-related cramps leading rarely to myoglobinuria.

Addison's disease presenting with cerebral edema.

BACKGROUND: Increased intracranial pressure with encephalopathy has rarely been reported in Addison's disease. METHOD: Case Study. RESULTS: A 16-year-old female who presented with cerebral edema of unknown etiology was eventually diagnosed as having Addison's disease. She had early morning headaches, fatiguability, diarrhea and deterioration in school performance. She was hyponatremic with a serum sodium of 128 mmol/L and hyperkalemic with a serum potassium of 5.9 mmol/L. She had a low serum osmolality (264 mosm), high urine osmolality (533 mosm) and high urine sodium (87 mosm). She had a postural drop in blood pressure and diffuse hyperpigmentation. An ACTH stimulation test revealed a low baseline cortisol and no response to ACTH. Plasma renin activity was increased. Serum ACTH was elevated. She responded well to intravenous fluids and solu-cortef and was discharged on hydrocortisone and florinef. She remains well 18 months after the acute episode with no neurologic complaints or findings. CONCLUSION: Addison's Disease should be considered in the differential diagnosis of symptomatic cerebral edema and idiopathic intracranial hypertension.

Clinical and neurophysiologic response of myopathy and neuropathy in long-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency to oral prednisone.

The purpose of this study was to evaluate the clinical and neurophysiologic responses to oral prednisone therapy in a boy with enzymatically confirmed long-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency in biopsied muscle and cultured skin fibroblasts. This boy presented with progressive limb girdle myopathy, recurrent myoglobinuria, peripheral sensorimotor axonopathy, and intraventricular conduction delays. Prior to prednisone therapy, at age 8 years, he exhibited marked distal weakness greater than proximal weakness with a waddling and high-steppage gait, Gowers' maneuver (10 s to rise from the floor), fatigue after 3-20 yards of walking and the ability to climb only 2 stairs. Serum levels of creatine kinase rose from 34 to 4,124 U/L following mild exertion. Nerve conduction studies revealed progressive axonopathy with secondary demyelination. Four weeks after initiation of oral prednisone (0.75 mg/kg/day) therapy, there was approximately a 100% increase in power and endurance. He was able to walk at least 100 yards before tiring, could rise from sitting on the floor in 3-4 s, and was able to climb 20 steps in 30 s. There was concurrent improvement in nerve conduction studies. Prednisone was gradually withdrawn over the next 4 months to 0.19 mg/kg/day; lower doses of 0.08 mg/kg/day resulted in a marked deterioration in power to the prior state. Although 0.19 mg/kg/day did not maintain the peak power achieved at 0.75 mg/kg/day, it provided adequate baseline power and endurance. It is concluded that there was a significant clinical and neurophysiologic response to prednisone at a dosage > or = 0.16 mg/kg/day. Prednisone may stabilize muscle and neuronal plasma membranes, as well as the fatty acid oxidation enzyme complex in the mitochondrial membrane.

Fatty acid oxidation abnormalities in childhood-onset spinal muscular atrophy: primary or secondary defect(s)?

The purpose of this study was to further identify and quantify the fatty acid oxidation abnormalities in spinal muscular atrophy, correlate these with disease severity, and identify specific underlying defect(s). Fifteen children with spinal muscular atrophy (3 type I, 8 type II, 4 type III) were studied. Serum carnitine total/free ratios demonstrated a tendency toward an increased esterified fraction ranging 35-58% of total carnitine (normal: 25-30% of total) in younger children with types I and II. The remaining type II and III patients, older than 23 months of age at sampling, had normal esterified carnitine levels. Urinary organic acid analysis demonstrated mild to moderate medium-chain dicarboxylic aciduria in type I patients and normal, mild, or moderate increases in short-chain and medium-chain organic acids in type II patients. In the type III group, the organic acids were normal except for one patient with mild medium-chain dicarboxylic aciduria. Muscle intramitochondrial beta-oxidation was measured in 5 children (2 type I, 2 type II, and 1 type III) and a significant reduction in the activities of short-chain L-3-hydroxyacyl-CoA dehydrogenase, long-chain L-3-hydroxyacyl-CoA dehydrogenase, acetoacetyl-CoA thiolase, and 3-ketoacyl-CoA thiolase were found; however, normal crotonase activity was documented. Most strikingly, there was a marked increase (3- to 5-fold) in the activity ratios of crotonase to L-3-hydroxyacyl-CoA dehydrogenase and thiolase activities with both short- and long-chain substrates. The combined abnormalities suggest a defect in a mitochondrial multifunctional enzyme complex, distinct from the trifunctional enzyme. These abnormalities may be either primary or secondary and may respond to dietary measures to reduce the dependence on fatty acid oxidation.

Recurrent childhood myoglobinuria.

Recurrent heritable childhood myoglobinuria is a potentially fatal entity (mortality up to 35%) in which prompt diagnosis and treatment are critical. Sixty childhood cases have been reported between 1910 to 1988, most with undiagnosed etiologies. We have studied an additional 40 cases referred to CPMC (1980-1988), suggesting that this condition is largely underdiagnosed or unreported. We have found important differences between the childhood and adult-onset cases. Of 77 cases of adult-onset recurrent myoglobinuria, 45% have been diagnosed biochemically. In contrast, only 30% of the 60 childhood cases from the literature have been diagnosed; 11 with CPT deficiency and 7 with various glycolytic defects, and only 5 of our 40 childhood cases have been diagnosed, all with CPT deficiency. The 100 combined childhood cases can be divided into an exertional group (type I) with exertion as the leading precipitating factor (46 literature and 10 CPMC cases), a toxic group (type II) with infection and/or fever as the primary precipitant (14 literature and 23 CPMC cases), and 7 undefined cases. The type I group resembles the adult-onset group in which exercise is also the leading precipitating factor. There is a slight female predominance (male/female = 1:1.3) in the toxic group vs. a marked male predominance in the exertional and adult groups (4:1). Only 4 of 37 cases (11%) of the toxic group are diagnosed (all with CPT deficiency) vs. 19 of 56 cases (34%) of the exertional group (12 CPT, 7 glycolytic) and 45% of the adult group. The toxic group is also differentiated by a higher mortality rate and by the presence of additional clinical features, including ictal bulbar signs (8 of 18), encephalopathy (4 of 19), and seizures (2 of 7), as well as persistent cardiac abnormalities, developmental delay (4 of 17), and dysmorphic features (2 of 9). These clinical characteristics clearly differentiate the childhood from the adult cases and suggest the presence of more generalized disease processes and different biochemical etiologies. A study of the heritable causes of myoglobinuria is important because identification of the biochemical defect may elucidate the pathogenetic mechanism of the myoglobinuria and facilitate the development of rational treatment strategies aimed at circumventing or correcting the metabolic block.

Carnitine transport: pathophysiology and metabolism of known molecular defects.

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 cloned in 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.

Reversal of valproic acid-associated impairment of carnitine uptake in cultured human skin fibroblasts.

Much attention has focussed on the role of valproic acid induced secondary carnitine deficiency in predisposing children to life-threatening hepatotoxicity and a Reye-like syndrome. One mechanism by which valproic acid therapy induces serum and tissue depletion of carnitine is through inhibition of plasmalemmal carnitine uptake. In cultured control human skin fibroblasts, this effect is directly proportional to the duration of exposure and concentration of valproic acid; the maximal effect of valproic acid exposure time is achieved by 14 days, beyond which there appears to be no additional significant effect (1). To determine whether this effect is reversible, we preincubated control fibroblasts with varying concentrations (0-1700 mumol/L) of valproic acid for 14 days, washed the fibroblasts free of valproic acid, and then continued the fibroblast growth in valproic acid-free medium for periods of 4 hours to 14 days. The fibroblasts were subsequently incubated with fixed carnitine concentrations of 50 mumol/L (normal physiological concentration), 20 mumol/L (as seen in secondary carnitine deficiency disorders), or 5 mumol/L (as seen in the homozygous plasma membrane carnitine transporter defect) and the carnitine uptake was determined. The inhibitory effect of valproic acid on carnitine uptake was completely reversed, for all 3 carnitine concentration conditions, following > or = 5 days of growth in valproic acid-free medium.

Carnitine uptake defect: frameshift mutations in the human plasmalemmal carnitine transporter gene.

The genetic defect associated with carnitine uptake is characterized by progressive infantile-onset carnitine responsive cardiomyopathy, weakness, recurrent hypoglycemic hypoketotic encephalopathy, and failure to thrive. The cDNA encoding the sodium ion-dependent, high-affinity human carnitine transporter (557 amino acids) has been recently cloned and mapped to human chromosome 5q31. We herein report the first molecular characterization of the mutations responsible for the carnitine uptake defect in two unrelated patients. RT-PCR analysis of patient lymphoblasts and fibroblasts followed by sequencing of PCR products and their subclones revealed frameshift mutations in the plasmalemmal carnitine transporter. In both patients, the abnormal transcripts showed a partial cDNA deletion of nucleotides 255-1649 resulting in a predicted truncated protein of 92 amino acids. Both patients are compound heterozygotes; in one patient the second mutant allele revealed a 19-bp insertion between nucleotides 874 and 875 resulting in a frameshift yielding a predicted truncated protein of 284 amino acids, while in the second patient the second mutant allele had a deletion of nucleotides 875-1046 resulting in a predicted truncated protein of 237 amino acids.

GFP-Human high-affinity carnitine transporter OCTN2 protein: subcellular localization and functional restoration of carnitine uptake in mutant cell lines with the carnitine transporter defect.

Individuals with the plasmalemmal high-affinity carnitine transporter defect present with progressive infantile-onset carnitine-responsive cardiomyopathy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopathy, and failure to thrive. The carnitine uptake defect (CUD) has been documented in their cultured skin fibroblasts, lymphoblasts, and/or myoblasts. The cDNA encoding the high-affinity sodium-dependent human carnitine transporter OCTN2 has recently been cloned. We used the green fluorescent protein (GFP) as a living marker for positively transfected cells in our expression studies of the high-affinity carnitine transporter OCTN2 cDNA in cell lines with the CUD. Transfection of cell lines from 12 unrelated patients (nine fibroblast and three lymphoblastoid) with a GFP construct harboring the wild-type full-length OCTN2 cDNA was done using LipoTAXI. Transient and stable expression of the recombinant GFP-human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the fibroblast and stable transfection of the lymphoblastoid cell lines were achieved. There was functional restoration of carnitine uptake in the transfected mutant cell lines, thereby confirming the identity of the transfected cDNA. In addition, we report the first demonstration of the subcellular localization of an in-frame fusion GFP-human high-affinity carnitine transporter OCTN2 protein in the plasma membrane by confocal laser-scanning fluorescence microscopy.

Maternally inherited hypertrophic cardiomyopathy due to a novel T-to-C transition at nucleotide 9997 in the mitochondrial tRNA(glycine) gene.

We report a unique heteroplasmic T-to-C transition at nucleotide 9997 in the mitochondrial tRNA(glycine) gene in a multiplex family who manifested nonobstructive cardiomyopathy. The degree of mtDNA heteroplasmy generally correlated with the severity of the symptoms. This T-to-C transition disrupts hydrogen bonding in the region adjacent to the acceptor stem of the tRNA molecule. The thymine residue at position 9997 is highly conserved in mammals, as well as in various vertebrates and invertebrates. A PCR diagnostic test for the presence of the 9997 T-to-C transition revealed that the base change was always present in high proportion in affected family members, not present in unaffected family members, and never present in control subjects from various ethnic groups (25 groups sampled, 42 individuals), thus ruling out the possibility that this change represents a polymorphic variant in the general population. The degree of heteroplasmy in lymphoblast cultures also correlated with the level of enzyme activity present for cytochrome c oxidase (complex IV) and succinate cytochrome c oxidoreductase (complexes II and III). The absence of previously reported mtDNA mutations associated with hypertrophic cardiomyopathy was verified by both PCR diagnostic procedures and sequence analysis. All mitochondrial tRNA genes, as well as genes encoding ATPase subunits 6 and 8, were sequenced and found not to possess base changes consistent with the clinical profile. More detailed biochemical and molecular biological investigations are discussed.

Characterization of the human plasmalemmal carnitine transporter in cultured skin fibroblasts.

Carnitine is an essential cofactor for long-chain fatty acid oxidation. We characterized the human carnitine transporter in vitro in a cultured skin fibroblast model both at the previously established Km concentration of carnitine uptake in fibroblasts (5 mumol/liter) and at 0.05% Km (0.25 mumol/liter). A rapid exponential dose-dependent decrease in mean percentage of carnitine uptake was demonstrated with increasing concentrations of nigericin, but no significant decrease was found with equimolar amounts of valinomycin. This would suggest that the Na+ gradient is integral to carnitine transport function. Interference of the Na+ (out-in) gradient by nigericin may be secondary to cytoplasmic acidification by this K+ proton ionophore. The rate of uptake was fully saturated at an extracellular Na+ concentration of 150 mmol/liter. Replacement of 150 mmol/liter extracellular Na+ with Li+ resulted in an 80 and a 50% reduction, and replacement with K+ and Rb+ ions resulted in a 100 and an 85 to 90% reduction in carnitine uptake, respectively, at carnitine concentrations of 0.25 and 5 mumol/liter, underlining the specific requirement for the Na+ ion. The effects of different site-specific respiratory chain toxins, namely, rotenone (complex I), antimycin A (complex III), and potassium cyanide (KCN) (complex IV) on carnitine uptake was also examined. A rapid exponential dose-dependent decrease in mean percentage of carnitine uptake with increasing concentrations of inhibitors was demonstrated. These data suggest either a metabolic energy requirement of the carnitine transporter or interference of the Na+ (out-in) gradient by a proton gradient (in-out) secondary to the accumulation of intracellular H+ ions, due to the action of the respiratory chain toxins, further suggesting that the transporter is sensitive to and inhibited by intracellular H+ ions. The effects of several sulfhydryl-binding agents, namely 2,4-dinitrofluorobenzene, N-ethylmaleimide, and mersalyl acid, were examined, and a significant inhibition of carnitine uptake was demonstrated, suggesting that free sulfhydryl groups are also integral to the import function of the human fibroblast transporter.