Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-(14)C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.

Biochemical markers predicting survival in peroxisome biogenesis disorders.

OBJECTIVE: To identify prognostic markers reflecting the extent of peroxisome dysfunction in primary skin fibroblasts from patients with peroxisome biogenesis disorders (PBD). BACKGROUND: PBD are a genetically heterogeneous group of disorders due to defects in at least 11 distinct genes. Zellweger syndrome is the prototype of this group of disorders, with neonatal adrenoleukodystrophy and infantile Refsum disease as milder variants. Common to these three disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness. Because genotype-phenotype studies are complicated by the genetic heterogeneity among patients with PBD, the authors evaluated a series of biochemical markers as a measure of peroxisome dysfunction in skin fibroblasts. METHODS: Multiple peroxisomal functions including de novo plasmalogen synthesis, dihydroxyacetonephosphate acyltransferase (DHAPAT) activity, C26:0/C22:0 ratio, C26:0 and pristanic acid beta-oxidation, and phytanic acid alpha-oxidation were analyzed in fibroblasts from a series of patients with defined clinical phenotypes. RESULTS: A poor correlation with age at death was found for de novo plasmalogen synthesis, C26:0/C22:0 ratio, and phytanic acid alpha-oxidation. A fairly good correlation was found for pristanic acid beta-oxidation, but the best correlation was found for DHAPAT activity and C26:0 beta-oxidation. A mathematic combination of DHAPAT activity and C26:0 beta-oxidation showed an even better correlation. CONCLUSIONS: DHAPAT activity and C26:0 beta-oxidation are the best markers in predicting life expectancy of patients with PBD. Combination of both markers gives an even better prediction. These results contribute to the management of patients with PBD.

Identification of the peroxisomal beta-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid.

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.

Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels.

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are clinically overlapping syndromes, collectively called "peroxisome biogenesis disorders" (PBDs), with clinical features being most severe in ZS and least pronounced in IRD. Inheritance of these disorders is autosomal recessive. The peroxisome biogenesis disorders are genetically heterogeneous, having at least 12 different complementation groups (CGs). The gene affected in CG1 is PEX1. Approximately 65% of the patients with PBD harbor mutations in PEX1. In the present study, we used SSCP analysis to evaluate a series of patients belonging to CG1 for mutations in PEX1 and studied phenotype-genotype correlations. A complete lack of PEX1 protein was found to be associated with severe ZS; however, residual amounts of PEX1 protein were found in patients with the milder phenotypes, NALD and IRD. The majority of these latter patients carried at least one copy of the common G843D allele. When patient fibroblasts harboring this allele were grown at 30 degrees C, a two- to threefold increase in PEX1 protein levels was observed, associated with a recovery of peroxisomal function. This suggests that the G843D missense mutation results in a misfolded protein, which is more stable at lower temperatures. We conclude that the search for the factors and/or mechanisms that determine the stability of mutant PEX1 protein by high-throughput procedures will be a first step in the development of therapeutic strategies for patients with mild PBDs.

Enoyl-CoA hydratase deficiency: identification of a new type of D-bifunctional protein deficiency.

D-bifunctional protein is involved in the peroxisomal beta-oxidation of very long chain fatty acids, branched chain fatty acids and bile acid intermediates. In line with the central role of D-bifunctional protein in the beta-oxidation of these three types of fatty acids, all patients with D-bifunctional protein deficiency so far reported in the literature show elevated levels of very long chain fatty acids, branched chain fatty acids and bile acid inter-mediates. In contrast, we now report two novel patients with D-bifunctional protein deficiency who both have normal levels of bile acid intermediates. Complementation analysis and D-bifunctional protein activity measurements revealed that both patients had an isolated defect in the enoyl-CoA hydratase domain of D-bifunctional protein. Subsequent mutation analysis showed that both patients are homozygous for a missense mutation (N457Y), which is located in the enoyl-CoA hydratase coding part of the D-bifunctional protein gene. Expression of the mutant protein in the yeast Saccharomyces cerevisiae confirmed that the N457Y mutation is the disease-causing mutation. Immunoblot analysis of patient fibroblast homogenates showed that the protein levels of full-length D-bifunctional protein were strongly reduced while the enoyl-CoA hydratase component produced after processing within the peroxisome was undetectable, which indicates that the mutation leads to an unstable protein.

Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis.

In the past few years, many patients have been described who have a defect of unknown origin in the peroxisomal beta-oxidation pathway. Complementation analysis has been done by various groups to establish the extent of the genetic heterogeneity among the patients. These studies were based on the use of two established cell lines, one with a deficiency of acyl-CoA oxidase and one with a deficiency of l-bifunctional protein (l-BP), and they showed that most patients belong to the l-BP-deficient group. However, molecular analysis of the cDNA encoding l-BP in patients failed to show any mutations. The recent identification of a new d-specific bifunctional protein (d-BP) prompted us to reinvestigate the original patient with presumed l-BP deficiency. In a collaborative effort, we have now found that the true defect in this patient is at the level of the d-BP and not at the level of the l-BP. Our results suggest that most, if not all, patients whose condition has been diagnosed as l-BP are, in fact, d-BP deficient. We tested this hypothesis in nine patients whose condition was diagnosed as l-BP deficiency on the basis of complementation analysis and found clear-cut mutations in the d-BP cDNA from all patients.

D-hydroxyacyl-CoA dehydrogenase deficiency. Identification of a new peroxisomal disorder with implications for other disorders of beta-oxidation.

The second and third steps of peroxisomal beta-oxidation are catalysed by two multifunctional enzymes: D-bifunctional protein and L-bifunctional protein. Here we show that fibroblasts of a patient described as being deficient in the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein and fibroblasts of a patient described as being deficient in L-bifunctional protein do not complement one another. Using a newly developed method to measure the activity of D-bifunctional protein in fibroblast homogenates, we found that the activity of the D-bifunctional protein was completely deficient in the patient with presumed L-bifunctional protein deficiency.

Measurement of dihydroxyacetone-phosphate acyltransferase (DHAPAT) in chorionic villous samples, blood cells and cultured cells.

Dihydroxyacetone-phosphate acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.