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

Abstract

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.

Introduction

The oxidation of fatty acids (FA) in mitochondria requires a series of enzymes which are located either in the membranes and active with long-chain FA, or soluble enzymes in the mitochondrial matrix which are most active with short- and medium-chain substrates. Mitochondrial FA oxidation represents the major energy-producing pathway under conditions of fasting. FA are the preferred fuel for heart and are also an essential source of energy for skeletal muscle during prolonged exercise. In liver, FA are used to synthesize ketone bodies which serve as fuel for the brain and muscle. Equally important, FA oxidation by the liver provides energy for gluconeogenesis and ureagenesis. This explains why the 12 inherited defects of FA oxidation show a rather similar range of clinical features with hepatic, cardiac and muscle involvement 1, 2.

The pathway for oxidizing FA proceeds by several steps [3]: after activation to a coenzyme A (CoA) thioester, the long-chain FA (e.g., ≥16 carbons) are shuttled across the inner mitochondrial membrane as acylcarnitine esters. Within the mitochondrial matrix, the β-oxidation cycle sequentially shortens the FA by two carbons in a series of four reactions (acyl-CoA dehydrogenase, enoyl-CoA hydratase, l-3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase) until it is completely converted to acetyl-CoA. Until a few years ago, it was believed that these oxidative steps were all catalysed by monofunctional matrix soluble enzymes. In 1992, two membrane-bound enzymes of β-oxidation, active on long-chain FA, were identified: the very-long-chain acyl-CoA dehydrogenase (VLCAD) [4], and the trifunctional protein [5]bearing all three of the enzyme activities (enoyl-CoA hydratase, l-3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase). VLCAD presents some homology with the other acyl-CoA dehydrogenases but it is a homodimer instead of a homotetramer as are the others 4, 6. The specific activity of VLCAD toward palmitoyl-CoA is ten times higher than that of LCAD [4], and VLCAD is a rate-limiting enzyme in the long-chain FA oxidation system [7]. VLCAD clearly plays a major role in dehydrogenation of long-chain FA. In 1993, we characterized the first VLCAD-deficient patient by determining the ETF-linked dehydrogenation of palmitoyl-CoA in both membrane and soluble fractions from homogenates of fibroblasts and demonstrated that membrane-bound VLCAD activity was missing [8]. Subsequently, five other patients with VLCAD deficiency were diagnosed using immunoblot analysis or measuring palmitoyl-CoA dehydrogenation in the presence and absence of antibodies versus acyl-CoA dehydrogenases 9, 10. VLCAD deficiency is inherited as an autosomal recessive trait.

We report here our experience with VLCAD deficiency in 30 patients from 27 unrelated families. Seven prenatal diagnoses of this life-threatening disorder of long-chain fatty acid oxidation have been performed, either on trophoblastic cells or on amniocytes, in six families.