Fatty acid oxidation disorders as primary cause of sudden and unexpected death in infants and young children: an investigation performed on cultured fibroblasts from 79 children who died aged between 0-4 years.

BACKGROUND: Disorders of fatty acid metabolism are known to be responsible for cases of sudden and unexpected death in infancy. At least 14 disorders are known at present. 120 cases of sudden infant death syndrome (SIDS) had been examined for a prevalent mutation (G985) causing medium chain acyl CoA dehydrogenase deficiency, which is inherited in an autosomal recessive mode. No over-representation of either homozygous or heterozygous cases was found. AIMS: To investigate a broader spectrum of fatty acid oxidation disorders in a wider range of sudden deaths in infants and young children. METHODS: Seventy nine cases of unexpected death in infants and young children younger than 4 years old were examined for a minimum of nine fatty acid oxidation disorders, using the global [9, 10-3H] myristic acid oxidation assay in cultured fibroblasts from achilles tendon biopsies taken at postmortem examination. RESULTS: Three cases with fatty acid oxidation disorders and two carriers of the G985 mutation were found, all categorized as non-SIDS or borderline SIDS. The global assay used has the advantage of simplicity. CONCLUSIONS: These results indicate that disorders of fatty acid oxidation play a small but significant role in the cause of unexpected death in infants and young children, and that infants and children dying in this way should be regarded as high risk candidates for metabolic diseases

Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis:evidence for post-translational modification of the enzyme

Two-dimensional gel electrophoresis was used to study and compare wild type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and missense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wildtype protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation. Detection of both wild-type and K304E mutant MCAD was achieved in lymphoblastoid cells from patients and carriers of the mutation. Both spots for the wild-type but only the nonmodified spot for the K304E mutant could be detected. In lymphoblastoid cells from carriers, the intensity of the spot representing the mutant protein is much weaker than the two spots representing wild-type MCAD, emphasizing that the K304E mutant protein is more susceptible to degradation than wild-type MCAD. The absence of detectable amounts of modified K304E mutant MCAD protein in these cells suggest that the conclusion drawn from COS-7 cell expression is also valid in patient cells