A novel 4 bp deletion mutation in the FALDH gene segregating in a Turkish family with Sjögren-Larsson syndrome.
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Aldehyde Oxidoreductases↗
Biomedical subjects
R J Wanders
Publications and source records attributed to R J Wanders.
Stability of alkyl-dihydroxyacetonephosphate synthase in human control and peroxisomal disorder fibroblasts.
Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme that plays a key role in ether phospholipid biosynthesis. To determine the turnover of alkyl-DHAP synthase in several peroxisomal disorders, pulse-chase experiments were performed. In control fibroblasts, mature alkyl-DHAP synthase displayed a half-life of 23 +/- 12 h. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, in which alkyl-DHAP synthase cannot be imported into peroxisomes, the enzyme was mainly detected in its precursor form. This precursor form showed a much shorter half-life, 5 +/- 2 h. In contrast, when the precursor protein accumulated inside the peroxisome of a particular neonatal adrenoleukodystrophy cell line in which processing does not take place, a half-life of 18 +/- 8 h, resembling that of the mature protein in controls, was observed. In a cell line from a patient with a single deficiency in the activity of alkyl-DHAP synthase, the mature form was detected and its radioactivity decreased with a half-life of 16 +/- 7 h. Collectively, these results provide an explanation for the instability of alkyl-DHAP synthase outside its target organelle. Additionally, they indicate that both the precursor and mature form of alkyl-DHAP synthase exhibit considerable intraperoxisomal turnover.
Alkyl and Aryl Transferases↗
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.
17-Hydroxysteroid Dehydrogenases↗
PEX13 is mutated in complementation group 13 of the peroxisome-biogenesis disorders.
The peroxisome-biogenesis disorders (PBDs) are a genetically and phenotypically diverse group of diseases caused by defects in peroxisome assembly. One of the milder clinical variants within the PBDs is neonatal adrenoleukodystrophy (NALD), a disease that is usually associated with partial defects in the import of peroxisomal matrix proteins that carry the type 1 or type 2 peroxisomal targeting signals. Here, we characterize the sole representative of complementation group 13 of the PBDs, a patient with NALD (patient PBD222). Skin fibroblasts from patient PBD222 display defects in the import of multiple peroxisomal matrix proteins. However, residual matrix-protein import can be detected in cells from patient PBD222, consistent with the relatively mild phenotypes of the patient. PEX13 encodes a peroxisomal membrane protein with a cytoplasmically exposed SH3 domain, and we find that expression of human PEX13 restores peroxisomal matrix-protein import in cells from patient PBD222. Furthermore, these cells are homozygous for a missense mutation at a conserved position in the PEX13 SH3 domain. This mutation attenuated the activity of human PEX13, and an analogous mutation in yeast PEX13 also reduced its activity. The mutation was absent in >100 control alleles, indicating that it is not a common polymorphism. Previous studies have demonstrated extragenic suppression in the PBDs, but the phenotypes of patient PBD222 cells could not be rescued by expression of any other human PEX genes. Taken together, these results provide strong evidence that mutations in PEX13 are responsible for disease in patient PBD222 and, by extension, in complementation group 13 of the PBDs.
Amino Acid Substitution↗
Mutations of OCTN2, an organic cation/carnitine transporter, lead to deficient cellular carnitine uptake in primary carnitine deficiency.
Systemic primary carnitine deficiency (CDSP, OMIM 212140) is an autosomal recessive disease characterized by low serum and intracellular concentrations of carnitine. CDSP may present with acute metabolic derangement simulating Reye's syndrome within the first 2 years of life. After 3 years of age, patients with CDSP may present with cardiomyopathy and muscle weakness. A linkage with D5S436 in 5q was reported in a family. A recently cloned homologue of the organic cation transporter, OCTN2, which has sodium-dependent carnitine uptake properties, was also mapped to the same locus. We screened for mutation in OCTN2 in a confirmed CDSP family. One truncating mutation (Trp132Stop) and one missense mutation (Pro478Leu) of OCTN2 were identified together with two silent polymorphisms. Expression of the mutant cDNAs revealed virtually no uptake activity for both mutations. Our data indicate that mutations in OCTN2 are responsible for CDSP. Identification of the underlying gene in this disease will allow rapid detection of carriers and postnatal diagnosis of affected patients.
Amino Acid Sequence↗
Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.
Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.
Amino Acid Sequence↗
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.
17-Hydroxysteroid Dehydrogenases↗
Identification and characterization of three novel missense mutations in mevalonate kinase cDNA causing mevalonic aciduria, a disorder of isoprene biosynthesis.
Mevalonic aciduria is a rare autosomal recessive metabolic disorder, characterized by psychomotor retardation, failure to thrive, hepatosplenomegaly, anemia and recurrent febrile crises. The disorder is caused by a deficient activity of mevalonate kinase due to mutations in the encoding gene. Thus far, only two disease-causing mutations have been identified. We now report four different missense mutations including three novel ones, which were identified by sequence analysis of mevalonate kinase cDNA from three mevalonic aciduria patients. All mutations affect conserved amino acids. Heterologous expression of the corresponding mutant mevalonate kinases as fusion proteins with glutathione S -transferase in Escherichia coli showed a profound effect of each of the mutations on enzyme activity. In addition, immunoblot analysis of fibroblast lysates from patients using specific antibodies against mevalonate kinase identified virtually no protein. These results demonstrate that the mutations affect not only the activity but also the stability of the mutant proteins.
Adolescent↗
Isolation, characterization and mutation analysis of PEX13-defective Chinese hamster ovary cell mutants.
We isolated peroxisome biogenesis mutants ZP128 and ZP150 from rat PEX2 -transformed Chinese hamster ovary (CHO) cells, by the 9-(1'-pyrene)nonanol/ultraviolet method. The mutants lacked morphologically recognizable peroxisomes and showed a typical peroxisome assembly-defective phenotype such as a high sensitivity to 12-(1'-pyrene)dodecanoic acid/UV treatment. By means of PEX cDNA transfection and cell fusion, ZP128 and ZP150 were found to belong to a recently identified complementation group H. Expression of human PEX13 cDNA restored peroxisome assembly in ZP128 and ZP150. CHO cell PEX13 was isolated; its deduced sequence comprises 405 amino acids with 93% identity to human Pex13p. Mutation in PEX13 of mutant ZP150 was determined by RT-PCR: G to A transition resulted in one amino acid substitution, Ser319Asn, in one allele and truncation of a 42 amino acid sequence from Asp265 to Lys306 in another allele. Therefore, ZP128 and ZP150 are CHO cell lines with a phenotype of impaired PEX13.
Amino Acid Sequence↗
Defective PEX gene products correlate with the protein import, biochemical abnormalities, and phenotypic heterogeneity in peroxisome biogenesis disorders.
Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including dihydroxyacetone phosphate acyltransferase and beta oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD.
Catalase↗
Progression of abnormalities in adrenomyeloneuropathy and neurologically asymptomatic X-linked adrenoleukodystrophy despite treatment with "Lorenzo's oil".
OBJECTIVES: X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal fatty acid oxidation, biochemically characterised by the accumulation of saturated very long chain fatty acids (VLCFAs), particularly hexacosanoic acid (C26:0). Dietary treatment with a 4:1 mixture of glyceroltrioleate and glyceroltrierucate ("Lorenzo's oil") normalises plasma VLCFA concentrations, but neither ameliorates nor arrests the rapid progression of neurological symptoms in the cerebral variants of X-ALD. The efficacy of "Lorenzo's oil" in the milder phenotypes of X-ALD was assessed, as this has been much less investigated. METHODS: Twenty two patients who were treated with "Lorenzo's oil" for at least 12 months for a median period of 2.5 (range 1.0-6.0) years were studied. Two had asymptomatic ALD, four the "Addison only" variant, 13 adrenomyeloneuropathy (AMN), and three were symptomatic female carriers. RESULTS: The plasma C26:0 concentration normalised or near normalised in 19 patients (86%), in the three others it decreased significantly. Nevertheless, disability as measured with the extended disability status scale score increased mildly (0.5 (95% confidence interval (95% CI) 0.25-1.0)) in the 16 patients with neurological symptoms. Furthermore, one "Addison only" patient and one patient with AMN developed cerebral demyelination, and another "Addison only" patient developed AMN. Adrenocortical insufficiency evolved in one patient with AMN, and hypogonadism in one patient with asymptomatic ALD and two patients with AMN. Nerve conduction, evoked potential studies (SEP, BAEP, VEP), and abnormalities on cerebral MRI did not improve. On the other hand, side effects were often noted-namely, mild increases in liver enzymes (55%), thrombocytopenia (55%), gastrointestinal complaints (14%), and gingivitis (14%). We also found a mild decrease in haemoglobin concentration and leucocyte count. CONCLUSIONS: The data suggest that treatment with "Lorenzo's oil" neither improved neurological or endocrine function nor arrested progression of the disease. Furthermore, the oil often induced adverse effects. Therefore, it is advocated that "Lorenzo's oil" should not be prescribed routinely to patients with X-ALD who already have neurological deficits.
Adolescent↗
Incomplete Sjögren-Larsson syndrome in two Japanese siblings?
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Aldehyde Oxidoreductases↗
Diagnosis and follow-up of a case of peroxisomal disorder with peroxisomal mosaicism.
Peroxisomal disorder phenotypes are the result of mutations that cause defective peroxisomal assembly or alterations in the import mechanism of peroxisomal proteins that lead to multiple peroxisomal dysfunctions, or the result of a peroxisomal enzymatic deficiency with a single peroxisomal dysfunction. With complementation analysis, 16 groups have been found. Assignment of the genetic defect has been described for some of the complementation groups. We describe the clinical evolution and follow-up over 10 years of a patient who belongs to complementation group 4, although he showed a milder clinical course. It has been found in fibroblasts different peroxisome populations, normal processing and expression of beta-oxidation PTS1 and PTS2 proteins, abnormal ALD protein distribution and normal plasmalogen biosynthesis; abnormal beta-oxidation metabolites have also been detected in serum. Ultrastructural studies in liver showed peroxisomal mosaicism as in fibroblasts. It has been taken into account that peroxisomal mosaicism may lead to variability in peroxisomal diagnostic parameters, making difficult the final diagnosis in these patients.
Adolescent↗
Primary hyperoxaluria type I: a model for multiple mutations in a monogenic disease within a distinct ethnic group.
Primary hyperoxaluria type 1 is an autosomal recessive inherited metabolic disease in which excessive oxalates are formed by the liver and excreted by the kidneys, causing a wide spectrum of phenotypes ranging from renal failure in infancy to mere renal stones in late adulthood. Mutations in the AGXT gene, encoding the liver-specific enzyme alanine:glyoxylate aminotransferase, are responsible for the disease. Seven mutations were detected in eight families in Israel. Four of these mutations are novel and three occur in children living in single-clan villages. The mutations are scattered along various exons (1, 4, 5, 7, 9, 10), and on different alleles comprising at least five different haplotypes. All but one of the mutations are in a homozygous pattern, reflecting the high rate of consanguinity in our patient population. Two affected brothers are homozygous for two different mutations expressed on the same allele. The patients comprise a distinct ethnic group (Israeli Arabs) residing in a confined geographic area. These results, which are supported by previous data, suggest for the first time that the phenomenon of multiple mutations in a relatively closed isolate is common and almost exclusive to the Israeli-Arab population. Potential mechanisms including selective advantage to heterozygotes, digenic inheritance, and the recent emergence of multiple mutations are discussed.
Arabs↗
Ether lipid biosynthesis: alkyl-dihydroxyacetonephosphate synthase protein deficiency leads to reduced dihydroxyacetonephosphate acyltransferase activities.
Recent studies have indicated that two peroxisomal enzymes involved in ether lipid synthesis, i.e., dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, are directed to peroxisomes by different targeting signals, i.e., peroxisomal targeting signal type 1 and type 2, respectively. In this study, we describe a new human fibroblast cell line in which alkyl-dihydroxyacetonephosphate synthase was found to be deficient both at the level of enzyme activity and enzyme protein. At the cDNA level, a 128 base pair deletion was found leading to a premature stop. Remarkably, dihydroxyacetonephosphate acyltransferase activity was strongly reduced to a level comparable to the activities measured in fibroblasts from patients affected by the classical form of rhizomelic chondrodysplasia punctata (caused by a defect in peroxisomal targeting signal type 2 import). Dihydroxyacetonephosphate acyltransferase activity was completely normal in another alkyl-dihydroxyacetonephosphate synthase activity-deficient patient. Fibroblasts from this patient showed normal levels of the synthase protein and inactivity results from a point mutation leading to an amino acid substitution. These results strongly suggest that the activity of dihydroxyacetonephosphate acyltransferase is dependent on the presence of alkyl-dihydroxyacetonephosphate synthase protein. This interpretation implies that the deficiency of dihydroxyacetonephosphate acyltransferase (targeted by a peroxisomal targeting signal type 1) in the classic form of rhizomelic chondrodysplasia punctata is a consequence of the absence of the alkyl-dihydroxyacetonephosphate synthase protein (targeted by a peroxisomal targeting signal type 2).
Acyltransferases↗
Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation.
Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.
Amino Acid Sequence↗
Carnitine biosynthesis. Purification of gamma-butyrobetaine hydroxylase from rat liver.
gamma-Butyrobetaine hydroxylase catalyse the last step in carnitine biosynthesis, the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on Fe2+, alpha-ketoglutarate, ascorbate and oxygen. Initial attempts to purify the protein from rat liver showed that gamma-butyrobetaine hydroxylase is unstable. We, therefore, determined the influence of various compounds on the stability of gamma-butyrobetaine hydroxylase at different storage temperatures. The enzyme activity was best conserved by storing the protein at 4 degrees C in the presence of 200 g/l glycerol and 10 mM DTT. We subsequently purified the enzyme from rat liver to apparent homogeneity by liquid chromatography.
Animals↗
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.
3-Hydroxyacyl CoA Dehydrogenases↗