Carnitine Palmitoyltransferase 1A Deficiency Research Articles

Molecular assay for detection of the common carnitine palmitoyltransferase 1A 1436(C>T) mutation

Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a metabolic disorder that occurs at a key checkpoint of fatty acid metabolism. A new form of CPT1A deficiency caused by a mutation at nucleotide 1436 (C>T), resulting in an amino acid substitution of leucine for proline at position 479 (P479L), has been isolated in Canadian First Nations and Inuit populations. The present study offers a molecular method for assessing CPT1A 1436 (C>T) mutation status. CPT1A-deficient fibroblasts from four patient fibroblast cell lines and ten patient peripheral blood spots were all analyzed by polymerase chain reaction (PCR) coupled to restriction endonuclease (RE) treatment. Genomic DNA was PCR-amplified and treated with an RE specific for normal DNA. CPT1A 1436 (C>T) mutations were identified by resistance to RE treatment. The RE-PCR assay identified homozygosity for the 1436 (C>T) mutation in four fibroblast cell lines and nine blood spots with CPT1A enzyme deficiency. In addition, the assay identified one blood spot that corresponded to the heterozygous genotype. RE-PCR assay for the 1436 (C>T) mutation provides a rapid assay for the diagnosis of CPT1A deficiency resulting from this mutation. The assay will have utility in screening populations with a high prevalence of this genotype.

Functional and Structural Basis of Carnitine Palmitoyltransferase 1A Deficiency

Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.