Complementationn analysis of fibroblasts from peroxisomal fattyy acid oxidation deficient patients show high frequency off bifunctional protein deficiency plus intragenic complementation:: Unequivocal evidence for differential defectss in the same enzyme protein

T Inn the last few years many patients have been reported with a defect in peroxisomal fatty acid P-oxidationn of unknown origin. Using a combined approach based on direct activity measurementss of straight chain acyl-CoA oxidase and complementation analysis after somatic cell fusionn of fibroblasts, we have now classified 13 patients into 4 distinct groups representing differentt gene defects. Remarkably, we found intragenic complementation in group 2 so that groupp 2 is in fact made up of 3 distinct subgroups. The underlying basis for this peculiar phenomenonn probably has to do with the fact that bifunctional protein harbours two catalytic activitiess including enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. In group 2AA enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase are defective whereas in groupp 2B and 2C either the hydratase or 3-hydroxyacyl-CoA dehydrogenase component of thee bifunctional protein is deficient. INTRODUCTION N Peroxisomess are subcellular organelles which play an indispensable role in cellular metabolism.. The importance of peroxisomes in man is stressed by the existence of a group of inheritedd disorders in man in which there is an impairment in one or more peroxisomal functionss (1, 2). The prototype of this group of disorders is the Zellweger syndrome in which morphologicallyy distinguishable peroxisomes are absent (3) due to mutations in one of the geness involved in peroxisome biogenesis {1). Onee of the most important functions of peroxisomes, at least in higher eukaryotes, is the P-oxidationn of fatty acids and fatty acid derivatives (4, 5). Although the mechanism of P-oxidationn in peroxisomes is identical to that in mitochondria, p-oxidation in the two organelless fulfils separate purposes. Indeed, mitochondria catalyse the oxidation of the bulk off fatty acids derived from our daily diet, notably long chain fatty acids, whereas peroxrsoTTreŝ areii mvbivèu Triune"p-oxïdauon öi a'bisun'ct set of tarty a'cias," including very longg chain fatty acids (C24:0, C26:0 etc.), diand trihydroxycholestanoic acid, the precursors off the primary bile acids chenodeoxycholate and cholate, and pristanic acid (2, 6, 10, 14tetramethylpentadecanoicc acid). The latter is the a-oxidation product of phytanic acid (see (6))-Inn the last few years many patients have been reported with a defect in peroxisomal p-oxidationn of unknown origin (see (2) for references). Resolution of the underlying defect(s) inn these patients is difficult especially since the individual peroxisomal P-oxidation enzyme activitiess are very hard to measure especially in fibroblasts with the exception of acyl-CoA oxidase.. This is due to the presence of the mitochondrial P-oxidation enzymes catalysing the samee reactions, making differential analysis of the peroxisomal p-oxidation enzymes in