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R J Wanders

Publications and source records attributed to R J Wanders.

At least 55 records · Page 3Linked to original sources
PubMed2000

Biochemical and genetic aspects of mevalonate kinase and its deficiency.

Mevalonate kinase (MK) is an essential enzyme in the mevalonate pathway which produces numerous cellular isoprenoids. The enzyme has been characterized both at the biochemical and the molecular level in a variety of organisms. Despite the fact that mevalonate kinase is not the rate-limiting enzyme in isoprenoid biosynthesis, its activity is subject to feedback regulation by the branch-point intermediates geranyldiphosphate, farnesyldiphosphate and geranylgeranyldiphosphate. Recently, the importance of mevalonate kinase was demonstrated by the identification of its deficiency as the biochemical and molecular cause of the inherited human disorders mevalonic aciduria and hyperimmunoglobulinemia D and periodic fever syndrome. The pathophysiology of these disorders is not yet understood, but eventually will give insight into the in vivo role of mevalonate kinase and isoprenoid biosynthesis with respect to the acute phase response and fever. The subcellular localization of mevalonate kinase is still a matter of debate. The enzyme could be localized predominantly in the cytosol, or in peroxisomes, or it is associated differentially with peroxisomes. Here we review the biochemical and molecular properties of MK, and discuss its biological significance, the regulation of its enzyme activity and finally its subcellular localization.

Amino Acid Sequence↗
PubMed2000

Biochemical and genetic aspects of 7-dehydrocholesterol reductase and Smith-Lemli-Opitz syndrome.

In recent years, several inherited human disorders caused by defects in cholesterol biosynthesis have been identified. These are characterized by malformations, multiple congenital anomalies, mental and growth retardation and/or skeletal and skin abnormalities indicating a pivotal role of cholesterol in morphogenesis and embryonic development. The first recognized and most common of these developmental disorders is Smith-Lemli-Opitz syndrome, an autosomal recessive trait caused by mutations in the DHCR7 gene resulting in a deficiency of the encoded sterol Delta(7)-reductase, alternatively called 7-dehydrocholesterol reductase (EC 1.3.1.21). This enzyme catalyzes the final step in cholesterol biosynthesis, which is the reduction of the Delta(7) double bond of 7-dehydrocholesterol to produce cholesterol.

Animals↗
PubMed2000

Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenases in isoleucine and valine metabolism.

Acyl-CoA dehydrogenase (ACAD) defects in isoleucine and valine catabolism have been proposed in clinically diverse patients with an abnormal pattern of metabolites in their urine, but they have not been proved enzymatically or genetically, and it is unknown whether one or two ACADs are involved. We investigated a patient with isolated 2-methylbutyrylglycinuria, suggestive of a defect in isoleucine catabolism. Enzyme assay of the patient's fibroblasts, using 2-methylbutyryl-CoA as substrate, confirmed the defect. Sequence analysis of candidate ACADs revealed heterozygosity for the common short-chain ACAD A625 variant allele and no mutations in ACAD-8 but a 100-bp deletion in short/branched-chain ACAD (SBCAD) cDNA from the patient. Our identification of the SBCAD gene structure (11 exons; >20 kb) enabled analysis of genomic DNA. This showed that the deletion was caused by skipping of exon 10, because of homozygosity for a 1228G-->A mutation in the patient. This mutation was not present in 118 control chromosomes. In vitro transcription/translation experiments and overexpression in COS cells confirmed the disease-causing nature of the mutant SBCAD protein and showed that ACAD-8 is an isobutyryl-CoA dehydrogenase and that both wild-type proteins are imported into mitochondria and form tetramers. In conclusion, we report the first mutation in the SBCAD gene, show that it results in an isolated defect in isoleucine catabolism, and indicate that ACAD-8 is a mitochondrial enzyme that functions in valine catabolism.

Alternative Splicing↗
PubMed2000

Succinyl-CoA:3-ketoacid CoA transferase (SCOT): cloning of the human SCOT gene, tertiary structural modeling of the human SCOT monomer, and characterization of three pathogenic mutations.

The activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT; locus symbol OXCT; EC 2.8.3.5) is the main determinant of the ketolytic capacity of tissues. Hereditary SCOT deficiency causes episodic ketoacidosis. Here we describe the human SCOT gene, which spans more than 100 kb and contains 17 exons, on chromosome 5p13. We report pathogenic missense mutations in three SCOT-deficient patients designated GS04, 05, and 06. GS04 is a G219E/G324E compound; GS05 is a V221M homozygote, and GS06 is a G324E homozygote. We constructed a tertiary structural model of human SCOT by homology modeling based on the known structure of Acidaminococcus fermentans glutaconate CoA transferase. The model predicts that V221 and G219 are on the dimerizing surface, whereas G324 is near the active site. SCOT activity was reduced to a comparable degree in all three patients, but in a transient expression assay in SCOT-deficient fibroblasts, cDNAs containing G219E and G324E produced no detectable activity, whereas V221M constructs yielded approximately 10% of the control peptide level and detectable specific activity. Interestingly, GS05 had the mildest clinical course reported to date and detectable levels of SCOT protein in fibroblasts.

Acidosis↗
PubMed2000

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.

Acyl-CoA Oxidase↗
PubMed2000

Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum's disease.

Refsum's disease (RD) is an inherited neurological syndrome biochemically characterized by the accumulation of phytanic acid in plasma and tissues. Patients with RD are unable to degrade phytanic acid due to a deficient activity of phytanoyl-CoA hydroxyl-ase (PhyH), a peroxisomal enzyme catalysing the first step of phytanic acid alpha-oxidation. To enable mutation analysis of RD at the genome level, we have elucidated the genomic organization of the PHYH gene. The gene is approximately 21 kb and contains nine exons and eight introns. Mutation analysis of PHYH cDNA from 22 patients with RD revealed 14 different missense mutations, a 3 bp insertion, and a 1 bp deletion, which were all confirmed at the genome level. A 111 bp deletion identified in the PHYH cDNA of several patients with RD was due to either one of two different mutations in the same splice acceptor site, which result in skipping of exon 3. Six mutations, including a large in-frame deletion and five missense mutations, were expressed in the yeast Saccharomyces cerevisiae to study their effect on PhyH activity. The results showed that all these mutations lead to an enzymatically inactive PhyH protein.

Amino Acid Substitution↗
PubMed2000

[Identification of the gene for hyper-IgD syndrome: a model of modern genetics].

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is a rare autosomal recessive disorder. Patients suffer from recurrent attacks (3-6 days) with fever, abdominal distress, lymphadenopathy, skin lesions and arthralgias. Patients display a constantly elevated serum IgD which serves as a biological marker of the disease. Recently, the gene for HIDS was discovered by two independent groups using positional and functional cloning methods. One group used linkage analysis (positional cloning) and was able to locate the gene for HIDS on the long arm of chromosome 12 (12q24). Mevalonate kinase was an interesting candidate gene because patients with a near complete absence of this enzyme (mevalonic aciduria) do exhibit attacks of fever. Indeed subsequent data showed that there was a decreased enzyme activity due to missense mutations in the mevalonate kinase gene. The other group detected slightly elevated urinary excretion of mevalonic acid during attacks in a HIDS patient (functional cloning). The enzyme activity of mevalonate kinase was lower in cultured cells and sequence analysis identified several missense mutations in cDNA encoding for mevalonate kinase. Mevalonate kinase is a key enzyme in the cholesterol synthesis pathway and it is rather surprising that a defect in the cholesterol metabolism can cause a periodic inflammatory disease such as HIDS.

Biomarkers↗
PubMed2000

Molecular cloning and expression of human L-pipecolate oxidase.

In higher eukaryotes L-lysine can be degraded via two distinct routes including the saccharopine pathway and the L-pipecolate pathway. The saccharopine pathway is the primary route of degradation of lysine in most tissues except the brain in which the L-pipecolate pathway is most active. L-pipecolate is formed from L-lysine via two enzymatic reactions and then undergoes dehydrogenation to Delta(1)-piperideine-6-carboxylate. At least in humans and monkeys, this is brought about by the enzyme L-pipecolate oxidase (PIPOX) localized in peroxisomes. In literature, several patients have been described with hyperpipecolic acidaemia. The underlying mechanism responsible for the impaired degradation of pipecolate has remained unclear through the years. In order to resolve this question, we have now cloned the human L-pipecolate oxidase cDNA which codes for a protein of 390 amino acids and contains an ADP-betaalphabeta-binding fold compatible with its identity as a flavoprotein. Furthermore, the deduced protein ends in -KAHL at its carboxy terminus which constitutes a typical Type I peroxisomal-targeting signal (PTS I).

Amino Acid Sequence↗
PubMed2000

Molecular and biochemical characterization of rat gamma-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis.

The penultimate step in carnitine biosynthesis is mediated by gamma-trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), a cytosolic NAD(+)-dependent aldehyde dehydrogenase that converts gamma-trimethylaminobutyraldehyde into gamma-butyrobetaine. This enzyme was purified from rat liver, and two internal peptide fragments were sequenced by Edman degradation. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA containing an open reading frame of 1485 base pairs encoding a polypeptide of 494 amino acids with a calculated molecular mass of 55 kDa. Expression of the coding sequence in Escherichia coli confirmed that the cDNA encodes gamma-trimethylaminobutyraldehyde dehydrogenase. The previously identified human aldehyde dehydrogenase 9 (EC 1.2.1.19) has 92% identity with rat trimethylaminobutyraldehyde dehydrogenase and has been reported to convert substrates that resemble gamma-trimethylaminobutyraldehyde. When aldehyde dehydrogenase 9 was expressed in E. coli, it exhibited high trimethylaminobutyraldehyde dehydrogenase activity. Furthermore, comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities. In addition, the highest V(max)/K(m) values were obtained with gamma-trimethylaminobutyraldehyde as substrate. This indicates that human aldehyde dehydrogenase 9 is the gamma-trimethylaminobutyraldehyde dehydrogenase, which functions in carnitine biosynthesis.

ATP-Binding Cassette Transporters↗
PubMed2000

Molecular mechanism of detectable catalase-containing particles, peroxisomes, in fibroblasts from a PEX2-defective patient.

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.

Amino Acid Sequence↗
PubMed2000

Molecular basis of very long chain acyl-CoA dehydrogenase deficiency in three Israeli patients: identification of a complex mutant allele with P65L and K247Q mutations, the former being an exonic mutation causing exon 3 skipping.

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is a life-threatening disorder of mitochondrial fatty acid beta-oxidation. We identified four novel mutations in three unrelated patients. All patients had the severe childhood form of VLCAD deficiency with early onset and high mortality. Immunoblot analysis revealed that VLCAD protein was undetectable in patients 2 and 3, whereas normal-size VLCAD protein and an aberrant form of VLCAD (4kDa smaller) were detected in patient 1. As expected, null mutations were found in patients 2 and 3: patient 2 is homozygous for a frameshift mutation, del 4 bp at 798-801, and patient 3 is homozygous for a nonsense mutation 65C>A(S22X). Patient 1 was homozygous for a complex mutant allele containing two alterations, including a 194C>T transition (P65L) and 739A>C transversion (K247Q); in the case of P65L, the amino acid change does not reduce enzyme activity. However, the nucleotide change resulted in exon 3 skipping, whereas the latter K247Q mutation had a drastic effect on enzyme activity. We verified these events by in vivo splicing experiments and transient expression analysis of mutant cDNAs. The P65L mutation locates 11 bases upstream of a splice donor site of intron 3. This is an example of an exonic mutation which affects exon-splicing.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed2000

A case of methemoglobinemia type II due to NADH-cytochrome b5 reductase deficiency: determination of the molecular basis.

Clinical, biochemical and molecular findings in a patient with methemoglobinemia type II are described. Furthermore, a comparison between methemoglobinemia type I and type II, both caused by a deficiency of NADH-cytochrome b5 reductase (b5R), is made. Although the clinical pictures of type I and II are strikingly different, mutations in the diaphorase (DIA1) gene located on chromosome 22 have been described in both types. In the present patient, two newly identified mutations, both leading to a stop codon in exon 4 (Gln77Ter) and in exon 6 (Arg160Ter), were found. Identification of different mutations at different positions in the DIA1 gene might shed light on the clinical and biochemical differences between methemoglobinemia type I and type II.

Child↗
PubMed2000

Hepatic peroxisomes in isolated hyperpipecolic acidaemia: evidence supporting its classification as a single peroxisomal enzyme deficiency.

Hyperpipecolic acidaemia is still regarded as a peroxisomal assembly deficiency. The enzyme responsible for the accumulation of pipecolic acid is located in the peroxisomes in man. We studied the appearance and alterations of peroxisomes in liver biopsy material from three unrelated children suffering from isolated hyperpipecolic acidaemia, in which only the metabolism of pipecolic acid is disturbed, using light and electron microscopy after cytochemical staining for visualisation of peroxisomes. Morphometric results showed the presence of normal-sized to small peroxisomes, an increase in number and abnormally shaped organelles, suggesting enhancement of metabolic efficiency. In one case enlarged organelles were observed. Skin fibroblasts were studied in all patients: their peroxisomes appeared to be normal. The obvious presence of peroxisomes in isolated HPA indicates that this disorder should be classified as a single peroxisomal enzyme deficiency.

Acyl-CoA Oxidase↗
PubMed2000

Secondary respiratory chain defect in a boy with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: possible diagnostic pitfalls.

UNLABELLED: We report on a boy who suffered from microcephaly, growth retardation, cardiomyopathy and hepatic dysfunction. When he had his first febrile infection at the age of 3 months he showed metabolic decompensation. Laboratory parameters and clinical features were compatible with a beta-oxidation defect or a respiratory chain disorder. Measurement of beta-oxidation enzymes showed long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency; determination of respiratory chain complex activities revealed complete absence of complex I, II, III and IV activities in skeletal muscle and reduced activities of complexes II and IV in cultured fibroblasts, with secondary dysregulation of ATP synthase. The patient was found to be homozygous for the MTP:G1528 C mutation (LCHAD-deficiency). CONCLUSION: This patient had LCHAD deficiency as his primary metabolic disorder, leading to secondary inhibition of respiratory chain enzymes by 'toxic' metabolites.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

Carnitine-acylcarnitine translocase deficiency: metabolic consequences of an impaired mitochondrial carnitine cycle.

We describe a patient with carnitine-acylcarnitine translocase deficiency (MIM 212138), who presented with neonatal generalized seizures, heart failure, and coma. Laboratory evaluation revealed hypoglycemia, hyperammonemia, lactic acidemia, hyperuricemia, and mild dicarboxylic aciduria. The fact that total plasma carnitine (7.1 micromol/l [20-30]) and free carnitine (1.9 micromol/l [12-18]) were low together with a high acylcarnitine/free carnitine ratio of 2.7 [0.4-1.0] prompted acylcarnitine analysis. This revealed the presence of large amounts of long-chain derivatives including C(16:0), C(16:1), C(18:1), C(18:2). Based on these findings carnitine-acylcarnitine translocase deficiency was suspected which was confirmed by enzyme studies in fibroblasts. The underlying complex metabolic consequences of this defect are reviewed. Prenatal diagnosis was performed in a subsequent pregnancy and a defect ruled out by measurement of carnitine-acylcarnitine translocase activity in cultured chorionic villi cells. As the clinical recognition of a life-threatening fatty acid oxidation disorder may be difficult, defects in this pathway should be considered in any child with coma, an episode of a Reye-like syndrome, and cardiomyopathy. Since routine laboratory tests often do not provide clues about potential disorders and profiles of urinary organic acids may not be characteristic, we recommend to measure free carnitine and acylcarnitines in plasma in any child with hyperammonemia, hypo/hyperketotic hypoglycemia or lactic acidemia for prompt treatment, proper genetic counseling, and potential prenatal diagnosis.

Ammonia↗
PubMed2000

Defective inactivation of leukotriene B4 in patients with Sjögren-Larsson syndrome.

In 6 patients with Sjögren-Larsson syndrome (SLS), the urinary excretion of leukotriene B(4) (LTB(4)) and omega-hydroxy-LTB(4) was found to be highly elevated, whereas omega-carboxy-LTB(4) was absent. This abnormal pattern of urinary excretion of LTB(4) and its metabolites appears to be unique to patients with SLS and offers a new approach to the diagnosis of this disorder. Moreover, defective inactivation of LTB(4) might be of pathophysiologic significance in the disease.

Case-Control Studies↗
PubMed2000

Muscular carnitine palmitoyltransferase II deficiency in infancy.

An 8-month-old female presented with febrile myoglobinuria. The activity of carnitine palmitoyltransferase (CPT) II was decreased to 16% of the control mean, and the oxidation of the long-chain fatty acids was reduced to 25% of the mean in the fibroblasts. Homozygosity for the common mutation, S113L, was identified in the CPT II gene. Residual CPT II activity of more than 10% of the mean and homozygosity for the common mutation S113L are usually associated with a milder reduction of long-chain fatty acid oxidation to about 80% of the control and with a later age of clinical onset. The early clinical presentation in the present patient is unique and was associated with a marked impairment of long-chain fatty acid oxidation, possibly because of other genetic factors. CPT II deficiency should be included in the differential diagnosis of isolated myoglobinuria in infancy.

Carnitine O-Palmitoyltransferase↗