M. Brivet

Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deficiency : clinical characteristics and diagnostic considerations in 30 patients

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an enzyme catalyzing the dehydrogenation of long-chain fatty acids in the first step of mitochondrial fatty acid oxidation. Using an ETF (electron transfer flavoprotein, the physiological electron acceptor of VLCAD) reduction assay, we identified VLCAD deficiency in cultured skin fibroblasts or liver tissue from 30 patients in 27 families. They clinically presented two phenotypes: a severe presentation characterized by an early onset of symptoms, with hypertrophic cardiomyopathy and a high incidence of death, and a mild form with hypoketotic hypoglycaemia, resembling MCAD (medium-chain acyl-CoA dehydrogenase) deficiency. Cells isolated from patients who develop cardiomyopathy characteristically accumulate longer-chain length acylcarnitines (hexadecanoylcarnitine and tetradecanoylcarnitine) when incubated with palmitate. However, cells from patients with the hypoglycaemic presentation produced relatively shorter-chain-length intermediates (mainly dodecanoylcarnitine). Inhibition of carnitine palmitoyl transferase I, in vitro, eliminated these intermediates with cells from both phenotypes indicating their intramitochondrial origin. Although the explanation for these distinct biochemical findings is not obvious, the correlation with the two phenotypes provides an opportunity for accurate prognosis and early implementation of appropriate treatment. Prenatal diagnosis of this life-threatening disorder was successfully performed in seven pregnancies in six of those families by assay of trophoblasts or amniocytes. In an at risk family, diagnosis of an affected fetus by measurement of VLCAD activity in noncultured chorionic villi allowed termination of the pregnancy before 13 weeks of gestation.

Defects in activation and transport of fatty acids.

The oxidation of long-chain fatty acids in mitochondria plays an important role in energy production, especially in skeletal muscle, heart and liver. Long-chain fatty acids, activated to their CoA esters in the cytosol, are shuttled across the barrier of the inner mitochondrial membrane by the carnitine cycle. This pathway includes four steps, mediated by a plasma membrane carnitine transporter, two carnitine palmitoyltransferases (CPT I and CPT II) and a carnitine-acylcarnitine translocase. Defects in activation and uptake of fatty acids affect these four steps: CPT II deficiency leads to either exercise-induced rhabdomyolysis in adults or hepatocardiomuscular symptoms in neonates and children. The three other disorders of the carnitine cycle have an early onset. Hepatic CPT I deficiency is characterized by recurrent episodes of Reye-like syndrome, whereas severe muscular and cardiac signs are associated with episodes of fasting hypoglycaemia in defects of carnitine transport and translocase. Convenient metabolic investigations for reaching the diagnosis of carnitine cycle disorders are determination of plasma free and total carnitine concentrations, determination of plasma acylcarnitine profile by tandem mass spectrometry and in vitro fatty acid oxidation studies, particularly in fresh lymphocytes. Application of the tools of molecular biology has greatly aided the understanding of the carnitine palmitoyltransferase enzyme system and confirmed the existence of different related genetic diseases. Mutation analysis of CPT II defects has given some clues for correlation of genotype and phenotype. The first molecular analyses of hepatic CPT I and translocase deficiencies were recently reported.

Diagnostic assessment and long-term follow-up of 13 patients with Very Long-Chain Acyl-Coenzyme A dehydrogenase (VLCAD) deficiency

Very Long-Chain Acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of mitochondrial long-chain fatty acid oxidation (FAO) most often occurring in childhood with cardiac or liver involvement, but rhabdomyolysis attacks have also been reported in adults. We report in this study the clinical, biochemical and molecular studies in 13 adult patients from 10 different families with VLCAD deficiency. The enzyme defect was demonstrated in cultured skin fibroblasts or lymphocytes. All patients exhibited exercise intolerance and recurrent rhabdomyolysis episodes, which were generally triggered by strenuous exercise, fasting, cold or fever (mean age at onset: 10 years). Inaugural life-threatening general manifestations also occurred before the age of 3 years in four patients. Increased levels of long-chain acylcarnitines with tetradecenoylcarnitine (C14:1) as the most prominent species were observed in all patients. Muscle biopsies showed a mild lipidosis in four patients. For all patients but two, molecular analysis showed homozygous (4 patients) or compound heterozygous genotype (7 patients). For the two remaining patients, only one mutation in a heterozygous state was detected. This study confirms that VLCAD deficiency, although being less frequent than CPT II deficiency, should be systematically considered in the differential diagnosis of exercise-induced rhabdomyolysis. Measurement of fasting blood acylcarnitines by tandem mass spectrometry allows accurate biochemical diagnosis and should therefore be performed in all patients presenting with unexplained muscle exercise intolerance or rhabdomyolysis.

Genotype/phenotype correlation in carnitine palmitoyl transferase II deficiency: lessons from a compound heterozygous patient.

Carnitine palmitoyl transferase II deficiency, an inherited disorder of long-chain fatty acid oxidation, may result in either a mild form (muscle disease in adults) or a severe form (hepatocardiomuscular syndrome in infants). The difference in severity between these two forms is related to a difference in levels of residual carnitine palmitoyl transferase II activity and long-chain fatty acid oxidation and in genotypes. Few data are, however, available regarding compound heterozygotes for a mild and a severe carnitine palmitoyl transferase II mutation. We report on such a patient carrying both the mild S113L substitution and the severe Y628S mutation. The patients clinical picture (cardiac arrest at 6 years) was markedly more serious than usually observed in S113L homozygotes, and suggested that mild/severe compound heterozygosity makes patients at risk from life-threatening events. Palmitate oxidation and carnitine palmitoyl transferase II activity were lower in lymphocytes from the S113L/Y628S patient than in those from a S113L homozygote. Thus, assessment of carnitine palmitoyl transferase II mutations, long-chain fatty acid oxidation, and carnitine palmitoyl transferase II activity, may help in predicting the potential severity of the muscular form of carnitine palmitoyl transferase II deficiency.

Identification of the molecular defect in a severe case of carnitine-acylcarnitine carrier deficiency.

Carnitine-acylcarnitine carrier (CAC) is involved in the transport of long-chain fatty acyl residues into mitochondria. CAC transfers acylcarnitines across the inner mitochondrial membrane in exchange for free carnitine. Ten patients with CAC deficiency (McKusick 212138) have previously been reported. Seven of them experienced life-threatening events in the neonatal period and a fatal outcome in childhood; they presented with hyperammonaemia, cardiomyopathy, heart beat disorders, very low β-oxidation flux, and undetectable CAC activity. Three patients had a mild form and are apparently doing well at ages from 2 years to 9 years. The human CAC cDNA sequence has been published and molecular defects in the CAC cDNA sequence were identified in two patients: a mild case (Huizing et al 1997) and a severe case (Huizing et al 1998). Mutation analysis from a severe case of CAC deficiency and her parents is reported here. A homozygous C558T transition in the CAC cDNA of the index case, resulting in a premature stop codon (R166X) was found. The presence of this C → T transition was directly confirmed on genomic DNA of the patient and her parents by analysing a PCR-amplified product of 211 bp encompassing the mutation. It is the first direct evidence of a molecular defect on the CAC genomic DNA.

Retrospective diagnosis of carnitine-acylcarnitine translocase deficiency by acylcarnitine analysis in the proband Guthrie card and enzymatic studies in the parents

Four steps are required for the transport of long-chain fatty acids (LCFA) into the mitochondrial matrix : a plasma membrane carnitine transporter maintains the intracellular supply of carnitine ; an outer mitochondrial membrane carnitine palmitoyltransferase I (CPT I) converts acyl-CoA compounds to their acylcarnitine analogues ; a carnitine-acylcarnitine translocase (CATR) mediates the transmembrane transfer of acylcarnitines ; an inner mitochondrial membrane carnitine palmitoyltransferase II (CPT II) allows the re-esterification of acylcarnitines to form acyl-CoA esters. Inherited metabolic diseases affecting CATR and CPT II are recognized as a contributing cause of life-threatening events in infancy. In these two disorders, long-chain acylcarnitines are not efficiently converted to their corresponding acyl-CoA esters, and prominent long-chain acylcarnitine species are seen in plasma. It has been speculated that increased concentrations of long-chain acylcarnitines may promote cardiac arrhythmia (Corr et al 1989). Tandem mass spectrometry has enabled quantitative analysis of acylcarnitines in small volumes of blood, including filter paper blood spots used for neonatal screening (Millington et al 1991). Determination of the acylcarnitine profile in the Guthrie card is particularly useful in case of sudden infant death when no other biological specimen had been preserved. The finding of abnormal accumulation of C 18 and C 16 species argues in favour of CATR or profound CPT II deficiency. However, the definitive diagnosis of these two disorders requires documentation of the specific enzymatic defect in cellular material. Both defects are transmitted as autosomal recessive traits. The enzymatic activities of obligate heterozygotes are approximately 50% of normal activity (Stanley et al 1992; Elpeleg et al 1993). Thus, enzymatic determinations in parents cells can be used to establish the diagnosis.

Diagnosis of carnitine acylcarnitine translocase deficiency by complementation analysis

Carnitine acylcarnitine translocase is one of the components necessary for the entry of long-chain fatty acids (LCFA) into the mitochondrial matrix, transferring acylcarnitines across the inner mitochondrial membrane in exchange for free carnitine. We have identified a new case of carnitine acylcarnitine translocase deficiency in a patient with impaired LCFA oxidation by complementation analysis. Restoration of release of tritiated water from [9,10(n)- 3 H]palmitate was used as the criterion for complementation in cultured fibroblasts

Mutational spectrum and DNA-based prenatal diagnosis in carnitine-acylcarnitine translocase deficiency.

Carnitine-acylcarnitine translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69-70insTGTGC, and 609-1g>a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.

Very long chain acyl-coenzyme A dehydrogenase deficiency in two siblings: evolution after prenatal diagnosis and prompt management.

A boy had neonatal seizure, lethargy, and metabolic acidosis at presentation. He recovered completely, but the recurrence of a similar episode with associated cardiomyopathy and dicarboxylic aciduria at 10 months of age led to the recognition of a fatty acid oxidation defect. A diagnosis of very long chain acyl-coenzyme A dehydrogenase deficiency was later made by enzyme assay in culture fibroblasts from this child, as well as in cultured amniotic cells from a sibling fetus. This prenatal diagnosis forestalled neonatal injury by close clinical and metabolic monitoring of the second infant. Early diagnosis and management should potentially improve the generally poor prognosis for patients with very long chain acyl-coenzyme A dehydrogenase deficiency.

A novel mutation of the ACADM gene (c.145C>G) associated with the common c.985A>G mutation on the other ACADM allele causes mild MCAD deficiency: a case report

A female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial β-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.