Biochemical and molecular studies of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency in cultured human fibroblasts

Sammanfattning: Doctoral thesis at Karolinska Institute Nikolaos Venizelos Inborn defects in mitochondrial B-oxidation of fatty acids are a group of diseases affecting humans usually early in life. At least 15 separate defects affecting the mitochondrial fatty acid B-oxidation in man have been identified . At an early stage of the study we described 5 patients with 3-hydroxydicarboxylic aciduria, suggesting a 3-hydroxyacyl-CoA dehydrogenase deficiency. They had elevated serum concentrations of 3-hydroxy fatty acids with chain lengths of 14-18 carbon atoms. Similar accumulation could also be detected retrospectively by GC-MS analysis of stored filter paper blood samples from some of the patients and from two elder siblings who had died unexpectedly in early infancy. The disorder is due to a defect in the recently discovered mitochondrial membrane-bound trifunctional enzyme (TP) which catalyses the three last steps of the mitochondrial U-oxidation of fatty acids. The activities of 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and 3 ketoacyl-CoA thiolase were measured in fibroblasts from 17 patients with 3 hydroxydicarboxylic aciduria. 16 of them had a deficiency in long-chain 3 hydroxyacyl-CoA dehydrogenase (LCHAD) activity (23% of control levels), whereas long-chain enoyl-CoA hydratase and long-chain 3-ketoacyl-CoA thiolase activities were variably and partially deficient. Prenatal diagnosis of LCHAD deficiency was performed by analysis of the enzyme activities in a chorionic villi biopsy of a pregnancy at risk. The diagnosis was then confirrned in liver tissue and cultured fibroblasts from the aborted foetus. The defect has been further characterised at the protein and gene levels in twelve of the LCHAD deficient patients. Specific antibodies were raised against synthetic peptides corresponding to part of the a and B subunits of the trifunctional enzyme, affinity purified and used for immunoblot analysis of fibroblast extracts. The results indicated norrnal levels of both subunits in all patients. Southern blot RFLP analysis in fibroblasts showed that eleven of these patients were homozygous and one was heterozygous for a G => C point-mutation at nucleotide position 1528 in the a-subunit of the LCHAD. Four parents of homozygous children were tested and found to be heterozygous for the same G1528C mutation. The results show that this mutation is common in patients with LCHAD deficiency. Intact cultured fibroblasts have been studied using the formation of tritiated water from [9,10-3H]-palmitic acid. Palmitate oxidation was reduced in all patients with known B-oxidation defects and in 10 of 16 patients with respiratory chain defects. The results indicate that the tritium release assay has a low specificity for B-oxidation defects but is valuable as a broad screening method also detecting many respiratory chain defects. Key words: Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, trifunctional protein, cultured human fibroblasts. ISBN 91-628-2301-9 Stockholm 1997