R. B. H. Schutgens

Peroxisomal disorders: a newly recognised group of genetic diseases

A new group of genetic diseases, in which peroxisomal functions are impaired, has recently been recognised. Although peroxisomes are subcellular organelles that are universally found in animal cells (except mature erythrocytes), their function in mammalian metabolism remained unclear until recently. Now, however, it is evident that peroxisomes play an essential role in several metabolic processes and that dysfunction of these organelles can have serious clinical consequences. Genetic diseases involving peroxisomes can be divided into two classes: firstly, those in which only a single peroxisomal function is impaired (acatalasaemia, X-linked adrenoleukodystrophy, the adult form of Refsum disease); secondly, those in which there is an impairment of more than one peroxisomal function (cerebro-hepato-renal (Zellweger) syndrome, the infantile form of Refsum disease, the neonatal form of adrenoleukodystrophy, the rhizomelic type of chondrodysplasia punctata and hyperpipecolic acidaemia). Whether genetic diseases like intrahepatic bile duct hypoplasia with elevation of trihydroxycoprostanoic acid and cerebrotendinous xanthomatosis can also be classified as peroxisomal disorders remains to be established. The aim of this paper is to summarise the rapid developments that have occurred during the last 3 years with regard to our knowledge of peroxisomal disorders and to describe briefly the clinical, morphological and biochemical characteristics of this new group of genetic disorders.

Deficiency of acyl-CoA: Dihydroxyacetone phosphate acyltransferase in patients with Zellweger (cerebro-hepato-renal) syndrome

We have recently reported on plasmalogen deficiency in tissues and fibroblasts from patients with Zellweger syndrome. In this paper we have analyzed the activity of the first enzyme in the pathway leading to plasmalogen biosynthesis, i.e. acyl-CoA:dihydroxyacetone phosphate acyltransferase in liver, brain and cultured skin fibroblasts from Zellweger patients and controls. The results indicate a severe deficiency of this enzyme in Zellweger patients. Thus, the Zellweger syndrome constitutes the first inborn error of metabolism with a deficiency in an enzyme involved in phospholipid biosynthesis. Cultured amniotic fluid cells contained an enzymatic activity comparable to that of control fibroblasts. These findings suggest a method for prenatal diagnosis of this disease.

Peroxisomal disorders in neurology

Although peroxisomes were initially believed to play only a minor role in mammalian metabolism, it is now clear that they catalyse essential reactions in a number of different metabolic pathways and thus play an indispensable role in intermediary metabolism. The metabolic pathways in which peroxisomes are involved include the biosynthesis of ether phospholipids and bile acids, the oxidation of very long chain fatty acids, prostaglandins and unsaturated long chain fatty acids and the catabolism of phytanate and (in man) pipecolate and glyoxylate. The importance of peroxisomes in cellular metabolism is stressed by the existence of a group of inherited diseases, the peroxisomal disorders, caused by an impairment in one or more peroxisomal functions. In the last decade our knowledge about peroxisomes and peroxisomal disorders has progressed enormously and has been the subject of several reviews. New developments include the identification of several additional peroxisomal disorders, the discovery of the primary defect in several of these peroxisomal disorders, the recognition of novel peroxisomal functions and the application of complementation analysis to obtain information on the genetic relationship between the different peroxisomal disorders. The peroxisomal disorders recognized at present comprise 12 different diseases, with neurological involvement in 10 of them. These diseases include: (1) those in which peroxisomes are virtually absent leading to a generalized impairment of peroxisomal functions (the cerebro-hepato-renal syndrome of Zellweger, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidaemia); (2) those in which peroxisomes are present and several peroxisomal functions are impaired (the rhizomelic form of chondrodysplasia punctata, combined peroxisomal beta-oxidation enzyme protein deficiency); and (3) those in which peroxisomes are present and only a single peroxisomal function is impaired (X-linked adrenoleukodystrophy, peroxisomal thiolase deficiency (pseudo-Zellweger syndrome), acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy) and probably, the classic form of Refsum disease.

Peroxisomal Disorders: A Review

The peroxisomal disorders represent a group of inherited diseases in man in which there is an impairment in one or more peroxisomal functions. The disorders known up to now are usually subdivided into three groups depending upon whether there is a more generalized, multiple or single loss of peroxisomal functions. In this paper we will briefly describe the peroxisomal disorders known thus far with the biochemical abnormalities identified. Furthermore, we will describe a straightforward approach for the postnatal identification of patients suspected to suffer from a peroxisomal disorder which is of great importance since reliable prenatal diagnostic methods have become available for each of these disorders.

Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis.

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

Peroxisomal β-oxidation enzyme proteins in the Zellweger syndrome

Abstract The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very longchain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal β-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low M r were seen in the patients. With anti-(oxoacyl-CoA thiolase), high M r cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal β-oxidation enzymes.

L-2-Hydroxyglutaric acidaemia : clinical and biochemical findings in 12 patients and preliminary report on L-2-hydroxyacid dehydrogenase

Summaryl-2-Hydroxyglutaric acidaemia represents a newly defined inborn error of metabolism, with increased levels ofl-2-hydroxyglutaric acid in urine, plasma and cerebrospinal fluid. The concentration in cerebrospinal fluid is higher than in plasma. The other consistent biochemical finding is an increase of lysine in blood and cerebrospinal fluid, but lysine loading does not increasel-2-hydroxyglutaric acid concentration in plasma. This autosomal recessively inherited disease is expressed as progressive ataxia, mental deficiency with subcortical leukoencephalopathy and cerebellar atrophy on magnetic resonance imaging. Since these features were described in 8 patients by Barth and co-workers in 1992, 4 more patients with similar findings have been diagnosed and added to the present series.l-2-Hydroxyglutaric acid is found in only trace amounts on routine gas chromatographic screening in normal persons, and its origin, its fate and even its relevance to normal metabolism are unknown. Therefore its catabolism was studied in normal liver. Incubation of rat liver withl-2-hydroxyglutaric acid did not produce H2O2, which excluded (peroxisomal)l-2-hydroxyacid oxidase as the main route of catabolism. However,l-2-hydroxyglutaric acid is rapidly dehydrogenated if NAD+ is added as a co-factor to the standard reaction medium. This could also be demonstrated in human liver. The preliminary evidence for this enzyme activity in rats and humans,l-2-hydroxyglutaric acid dehydrogenase, is given. Further investigations are required to clarify the possible relevance to the metabolic defect inl-2-hydroxyglutaric acidaemia.

Measurement of peroxisomal fatty acid β-oxidation in cultured human skin fibroblasts

SummaryOne of the main functions of mammalian peroxisomes is the β-oxidation of a variety of fatty acids and fatty acid derivatives, including very long-chain fatty acids. Oxidation of these fatty acids is deficient in a number of different peroxisomal disorders, including the disorders of peroxisome biogenesis (Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease), X-linked adrenoleukodystrophy and a number of other disorders of peroxisomal β-oxidation of known and unknown aetiology. Accurate measurement of peroxisomal fatty acid oxidation is of utmost importance for correct postnatal and prenatal diagnosis of these disorders. In this paper we describe a straightforward and accurate assay method to measure the β-oxidation of palmitic acid (C16:0), hexacosanoic acid (C26:0) and pristanic acid in intact fibroblasts.

Human dihydroxyacetonephosphate acyltransferase deficiency : a new peroxisomal disorder

The peroxisomal disorders represent a genetically determined group of diseases in man in which there is an impairment in one or more peroxisomal functions. The different disorders known today are usually divided into three groups depending upon whether there is a generalized (A), multiple (B) or single (C) loss of peroxisomal functions (for review see Wanders et al 1988; Lazarow and Moser 1989). At present two distinct disorders have been recognized to belong to group B, which includes Zellweger-like syndrome and rhizomelic chondrodysplasia punctata (RCDP)