Molecular characterization of L-CPT I deficiency in six patients: Insights into function of the native enzyme

Carnitine palmitoyltransferase I (CPT I) catalyzes the formation of acylcarnitine, the first step in the oxidation of long-chain fatty acids in mitochondria. The enzyme exists as liver (L-CPT I) and muscle (M-CPT I) isoforms that are encoded by separate genes. Genetic deficiency of L-CPT I, which has been reported in 16 patients from 13 families, is characterized by episodes of hypoketotic hypoglycemia beginning in early childhood and is usually associated with fasting or illness. To date, only two mutations associated with L-CPT I deficiency have been reported. In the present study we have identified and characterized the mutations underlying L-CPT I deficiency in six patients: five with classic symptoms of L-CPT I deficiency and one with symptoms that have not previously been associated with this disorder (muscle cramps and pain). Transfection of the mutant L-CPT I cDNAs in COS cells resulted in L-CPT I mRNA levels that were comparable to those expressed from the wild-type construct. Western blotting revealed lower levels of each of the mutant proteins, indicating that the low enzyme activity associated with these mutations was due, at least in part, to protein instability. The patient with atypical symptoms had ∼20% of normal L-CPT I activity and was homozygous for a mutation (c.1436C→T) that substituted leucine for proline at codon 479. Assays performed with his cultured skin fibroblasts indicated that this mutation confers partial resistance to the inhibitory effects of malonyl-CoA. The demonstration of L-CPT I deficiency in this patient suggests that the spectrum of clinical sequelae associated with loss or alteration of L-CPT I function may be broader than was previously recognized.