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Biomedical subjects

R J Wanders

Publications and source records attributed to R J Wanders.

At least 217 records · Page 12Linked to original sources
PubMed1997

Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor.

Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disease characterized clinically by a disproportionately short stature primarily affecting the proximal parts of the extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Although some patients have single enzyme deficiencies, the majority of RCDP patients (86%) belong to a single complementation group (CG11, also known as complementation group I, Amsterdam nomenclature). Cells from CG11 show a tetrad of biochemical abnormalities: a deficiency of i) dihydroxyacetonephosphate acyltransferase, ii) alkyldihydroxyacetonephosphate synthase, iii) phytanic acid alpha-oxidation and iv) inability to import peroxisomal thiolase. These deficiencies indicate involvement of a component required for correct targeting of these peroxisomal proteins. Deficiencies in peroxisomal targeting are also found in Saccharomyces cerevisiae pex5 and pex7 mutants, which show differential protein import deficiencies corresponding to two peroxisomal targeting sequences (PTS1 and PTS2). These mutants lack their PTS1 and PTS2 receptors, respectively. Like S. cerevisiae pex cells, RCDP cells from CG11 cannot import a PTS2 reporter protein. Here we report the cloning of PEX7 encoding the human PTS2 receptor, based on its similarity to two yeast orthologues. All RCDP patients from CG11 with detectable PEX7 mRNA were found to contain mutations in PEX7. A mutation resulting in C-terminal truncation of PEX7 cosegregates with the disease and expression of PEX7 in RCDP fibroblasts from CG11 rescues the PTS2 protein import deficiency. These findings prove that mutations in PEX7 cause RCDP, CG11.

Amino Acid Sequence↗
PubMed1997

Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.

Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

Adult↗
PubMed1997

Increased plasma malondialdehyde associated with cerebellar structural defects.

BACKGROUND: Malondialdehyde (MDA) in plasma is regarded as an indicator for increased lipid peroxidation. METHOD: Measurements of MDA concentrations in plasma were compared among healthy children (n = 31), patients with neurological disorders or epileptic syndromes (n = 15), and children with pontocerebellar structural defects (n = 31), where the cause or genetic defect remained unknown. RESULTS: In healthy children the median MDA value was 5.86 nmol/ml (mean (SD) value: 6.25 (1.97), range: 3.76-11.19). For the group with various neurological disorders or epilepsy, the values were similar with the median value at 5.66 nmol/ml (range 0.22-10.86). Compared with healthy controls and the neurological/ epileptic group, the 31 children with pontocerebellar structural defects had significantly increased MDA values with a median value at 11.29 nmol/ml (mean (SD) value: 11.62 (3.27), range 3.65-19.22). IMPLICATION: These findings of increased plasma MDA in the majority of children with pontocerebellar structural defects of unknown origin raised the question whether increased lipid peroxidation leads to prenatal and postnatal pontocerebellar maldevelopment or degeneration.

Adolescent↗
PubMed1997

X linked adrenoleukodystrophy: clinical presentation, diagnosis, and therapy.

X linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal metabolism, biochemically characterised by accumulation of saturated very long chain fatty acids. Accumulation of these fatty acids is associated with cerebral demyelination, peripheral nerve abnormalities, and adrenocortical and testicular insufficiency. The lowest estimated birth incidence is one per 100,000. At least six phenotypes can be distinguished, of which the two most frequent are childhood cerebral ALD and adrenomyeloneuropathy. The X-ALD gene has been identified, but thus far no relation between genotype and phenotype has been found. Diagnosis is relatively easy and can be confirmed reliably, and prenatal testing is possible in affected families. Several therapeutic options, some with promising perspectives, are available. Neurologists and other physicians seem not to be familiar with the many facets of X-ALD. In this review, the clinical presentation, the relative frequencies of the different phenotypes, and the diagnostic and therapeutic options are presented.

Adolescent↗
PubMed1997

Subcellular localization of dihydropyrimidine dehydrogenase.

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD. These results showed that almost all of the activity of DPD was located in the cytosolic fraction. To exclude any particulate-associated DPD, a light mitochondrial fraction was subsequently subjected to equilibrium density gradient centrifugation. The distribution profile of the activity of DPD and the various marker enzymes indicated that DPD from rat liver was exclusively located in the cytosol since no significant activity of DPD could be detected in any subcellular organelle.

Animals↗
PubMed1997

Lipoprotein[a] is not present in the plasma of patients with some peroxisomal disorders.

Peroxisomal disorders arise either from defects in the biogenesis of peroxisomes or from the defective synthesis of one or more peroxisomal enzymes. These defects result in metabolic disturbances in peroxisomal beta-oxidation of various fatty acids and derivatives and/or in the biosynthesis of ether lipids. In the current study, lipoprotein levels were determined in plasma samples from patients diagnosed with one of four different peroxisomal disorders. While low density lipoprotein (LDL) levels were found to be within the normal range, lipoprotein[a] (Lp[a]) could not be detected by enzyme-linked immunosorbent assay (ELISA) in plasma from patients with cerebro-hepato-renal (Zellweger) syndrome (ZS) and rhizomelic chondrodysplasia punctata (RCDP). Conversely, Lp[a] was clearly present in control plasma obtained from healthy newborns and from patients affected with one of two other peroxisomal disorders, X-linked adrenoleukodystrophy (X-ALD) and Refsum disease (RD) as determined by ELISA. The lack of Lp[a] in plasma of patients with ZS may result from defective secretion of apolipoprotein[a] (apo[a]) (the distinguishing protein component of Lp[a]), as apo[a] mRNA transcripts were clearly present in ZS livers as assessed by PCR, and intracellular apo[a] protein was detected in total liver homogenates from ZS patients as determined by Western blot analysis. Furthermore, LDL present in the plasma of ZS patients was able to associate with recombinant apo[a] in an in vitro Lp[a] assembly assay.

Adolescent↗
PubMed1997

Phytanic acid alpha-oxidation: decarboxylation of 2-hydroxyphytanoyl-CoA to pristanic acid in human liver.

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied. 2-Hydroxyphytanic acid was converted into 2-ketophytanic acid and pristanic acid. When ATP, Mg2+, and coenzyme A were added to the incubation medium, higher amounts of pristanic acid were formed, whereas the formation of 2-ketophytanic acid strongly decreased. When 2-hydroxyphytanoyl-CoA was used as substrate, there was virtually no 2-ketophytanic acid formation. However, pristanic acid was formed in higher amounts than with 2-hydroxyphytanic acid as substrate. This reaction was stimulated by NAD+ and NADP+. Pristanic acid, and not pristanoyl-CoA was found to be the product of the reaction. These results suggest the existence of two pathways for decarboxylation of 2-hydroxyphytanic acid. The first one, starting from 2-hydroxyphytanic acid, involves the formation of 2-ketophytanic acid with only a small amount of pristanic acid being formed. The second pathway, which starts from 2-hydroxyphytanoyl-CoA, does not involve 2-ketophytanic acid and generates higher amounts of pristanic acid. The first pathway, which is peroxisomally localized, was found to be deficient in Zellweger syndrome, whereas the second pathway, localized in microsomes, was normally active. We conclude that the second pathway is predominant under in vivo conditions.

Biomarkers↗
PubMed1997

A two-base deletion in exon 6 of the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene producing the skipping of exons 5 and 6 determines 3-hydroxy-3-methylglutaric aciduria.

A novel two-base deletion in the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene was found in a Spanish patient with homozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by RT-PCR of the mRNAs showed that the gene was transcribed into three different mRNAs. One showed the complete deletion of exons 5 and 6 located between nucleotides 348 and 561 of the HL cDNA. The second transcript showed deletion of exon 6 only, and the third contained a two-base deletion CT in exon 6, corresponding to nucleotides 504 and 505 of the HL cDNA. These aberrant mRNAs are predicted to encode three abnormal HMG-CoA lyase proteins; the first (from skipped exons 5 and 6) lacks 71 amino acids, which represents 24% of the mature protein; the second, (from the skipping of exon 6, producing a frameshift) contains only 192 amino acids, the last 26 of which are missense amino acids preceding a stop codon; the third contains only 175 amino acids, the last 7 of which are missense. Northern blot analysis showed that the HL mRNA levels of the patient were 4% of the control. PCR quantitative analysis indicated that the mRNA lacking exons 5 and 6 was the most abundant, representing 88% of the total. The other two mRNAs represented 8% and 4%, respectively. In the genomic DNA only one CT deletion was found at positions +7 and +8 at beginning of exon 6. No mutations were observed in the splice donor, splice acceptor, or pyrimidine-rich sequences of the intronic regions flanking exons 5 and 6. All three aberrant mRNAs resulted only from the deletion of nucleotides CT. We suggest that this deletion may affect the interaction between the small nuclear ribonucleoproteins (snRNPs) and exon 6, and that, as a result, the abnormal splicing of the pre-mRNA produces two different aberrant transcripts.

Adolescent↗
PubMed1996

Metabolic aspects of peroxisomal disorders.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in one or more peroxisomal functions. This paper discusses the current state of knowledge on these disorders with particular emphasis on the metabolic abnormalities in these diseases.

Cholesterol↗
PubMed1996

Phytanoyl-CoA hydroxylase is present in human liver, located in peroxisomes, and deficient in Zellweger syndrome: direct, unequivocal evidence for the new, revised pathway of phytanic acid alpha-oxidation in humans.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which accumulates in a number of inherited diseases in human. Because beta-oxidation is blocked by the methyl group at C-3, phytanic acid first undergoes decarboxylation via an alpha-oxidation mechanism. The structure and subcellular localization of the phytanic acid alpha-oxidation pathway have remained enigmatic through the years, although they have generally been assumed to involve phytanic acid and not its CoA-ester. This view has recently been challenged by the findings that in rat liver phytanic acid first has to be activated to its CoA-ester before alpha-oxidation and by the discovery of a new enzyme, phytanoyl-CoA hydroxylase, which converts phytanoyl-CoA to 2-hydroxyphytanoyl-CoA. We now show that this newly discovered enzyme is also present in human liver. Furthermore, we show that this enzyme is located in peroxisomes and deficient in liver from Zellweger patients who lack morphologically distinguishable peroxisomes, which provides an explanation for the long-known deficient oxidation of phytanic acid in these patients. These results suggest that phytanic acid alpha-oxidation is peroxisomal and that it utilizes the coenzyme A derivative as substrate, thus giving further support in favour of the new, revised pathway of phytanic acid alpha-oxidation.

Cell Compartmentation↗
PubMed1996

Common missense mutation G1528C in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Characterization and expression of the mutant protein, mutation analysis on genomic DNA and chromosomal localization of the mitochondrial trifunctional protein alpha subunit gene.

Mitochondrial trifunctional protein (MTP) is a recently identified enzyme involved in mitochondrial beta-oxidation, harboring long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain 3-ketothiolase activity. A deficiency of this protein is associated with impaired oxidation of long-chain fatty acids which can lead to sudden infant death. Furthermore, it is clear that this inborn error of fatty acid oxidation is very frequent, second to medium chain acyl-CoA dehydrogenase deficiency. In most patients only the LCHAD activity of MTP is deficient with near normal activity of the two other enzyme activities of the complex. We recently described the occurrence of a frequent G1528C mutation in the cDNA coding for the a subunit of MTP. Using S. cerevisiae for expression of wild type and mutant protein we show that the G1528C mutation is directly responsible for the loss of LCHAD activity. Furthermore, we describe a newly developed method allowing identification of the G1528C mutation in genomic DNA. The finding of an 87% allele frequency of the G1528C mutation in 34 LCHAD deficient patients makes this a valuable test for prenatal diagnosis. Finally, we show that the gene encoding the alpha subunit of MTP is located on chromosome 2p24.1-23.3.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae.

Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta-oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium-chain fatty acids are activated inside peroxisomes hby the acyl-CoA synthetase Faa2p. On the other hand, long-chain fatty acids are imported from the cytosolic pool of activated long-chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.

ATP-Binding Cassette Transporters↗
PubMed1996

[Smith-Lemli-Opitz syndrome; a special defect in cholesterol metabolism].

In a male neonate dysmaturity, microcephalia, a high nasal bridge, a long philtrum, broad dental ridges, schisis of the palatum molle, retrognathia, a small penis with a chorda, a small scrotum, bilateral inguinal hernia and bilateral syndactyly of the second and third toes were observed. The presence of the Smith-Lemli-Opitz (SLO) syndrome was suspected. By gas chromatography a severely decreased plasma cholesterol level (0.27 mmol/l) was found and an increased plasma 7-dehydrocholesterol level (0.24 mmol/l). The SLO syndrome is caused by a block in the cholesterol biosynthesis due to the autosomal recessive deficiency of 7-dehydrocholesterol reductase. The patient's condition improved with use of a cholesterol-enriched diet.

Abnormalities, Multiple↗
PubMed1996

Non-rhizomelic and rhizomelic chondrodysplasia punctata within a single complementation group.

Several patients have been described recently who suffer from a non-rhizomelic type of chondrodysplasia punctata (CDP), but who show all the biochemical abnormalities characteristic of the rhizomelic form of chondrodysplasia punctata (RCDP), a peroxisomal disorder. We have used protease protection experiments and microinjection of reporter-protein-encoding expression plasmids to show that peroxisomal thiolase fails to be imported into peroxisomes in cells from non-rhizomelic CDP patients, as has already been found in cells from classical RCDP patients. Furthermore, complementation analysis after somatic cell fusion indicates that the non-rhizomelic CDP patients are impaired in the same gene as classical RCDP patients. We conclude that defects in a single gene can give rise to both clinical phenotypes.

Acetyl-CoA C-Acetyltransferase↗
PubMed1996

3-Hydroxy-3-methylglutaryl CoA lyase (HL): mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients.

3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, we found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene.

Animals↗
PubMed1996

Variant rhizomelic chondrodysplasia punctata (RCDP) with normal plasma phytanic acid: clinico-biochemical delineation of a subtype and complementation studies.

Rhizomelic chondrodysplasia calcificans punctata (RCDP) is an autosomal recessive peroxisomal disorder which affects phytanic acid oxidation and de novo biosynthesis of plasmalogens in liver and fibroblasts. Peroxisomal thiolase is present in its unprocessed precursor form (44 kDa). We studied a mentally retarded 9-year-old girl with cataracts and atypical bone dysplasia. Neurological findings were mild compared to classic RCDP. Plasma phytanic acid was normal. Results of de novo plasmalogen synthesis and phytanic acid oxidation studied in cultured skin fibroblasts were intermediate between normal controls and classic RCDP. Peroxisomal thiolase was present only as the unprocessed 44 kDa protein. Taken together these results suggest that we are dealing with a variant form of RCDP with clinical and biochemical abnormalities much milder as compared to classic RCDP. In order to establish the genetic relationship between our patient and classic RCDP patients complementation studies were carried out. Earlier studies had already shown that fibroblasts from all RCDP patients studied belong to a single complementation group. Fibroblasts from our patient could also be assigned to this complementation group suggesting that the phenotypic variability results from different mutations within the same gene.

Cells, Cultured↗