Biomedical subjects

Ronald J A Wanders

Publications and source records attributed to Ronald J A Wanders.

At least 55 records · Page 3Linked to original sources

PubMed2005

Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping.

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive disorder of L: -isoleucine catabolism. Little is known about the clinical presentation associated with this enzyme defect, as it has been reported in only a limited number of patients. Because the presence of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families with SBCADD, both with seizures and psychomotor delay as the main clinical features. One family illustrates the fact that affected individuals may also remain asymptomatic. In addition, the normal level of newborn blood spot C5-acylcarnitine in one patient underscores the fact that newborn screening by MS/MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease-causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5' splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A>G) induces missplicing only when in the context of non-consensus (weak) 5' splice sites. Statistical analysis of the sequences shows that the wild-type versions of 5' splice sites in which +3A>G mutations cause exon skipping and disease are weaker on average than a random set of 5' splice sites. This finding is relevant to the interpretation of the functional consequences of this type of mutation in other disease genes.

Alternative Splicing↗

PubMed2005

Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis.

D-bifunctional protein (DBP) deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. The clinical presentation of DBP deficiency is usually very severe, but a few patients with a relatively mild presentation have been identified. In this article, we report the mutational spectrum of DBP deficiency on the basis of molecular analysis in 110 patients. We identified 61 different mutations by DBP cDNA analysis, 48 of which have not been reported previously. The predicted effects of the different disease-causing amino acid changes on protein structure were determined using the crystal structures of the (3R)-hydroxyacyl-coenzyme A (CoA) dehydrogenase unit of rat DBP and the 2-enoyl-CoA hydratase 2 unit and liganded sterol carrier protein 2-like unit of human DBP. The effects ranged from the replacement of catalytic amino acid residues or residues in direct contact with the substrate or cofactor to disturbances of protein folding or dimerization of the subunits. To study whether there is a genotype-phenotype correlation for DBP deficiency, these structure-based analyses were combined with extensive biochemical analyses of patient material (cultured skin fibroblasts and plasma) and available clinical information on the patients. We found that the effect of the mutations identified in patients with a relatively mild clinical and biochemical presentation was less detrimental to the protein structure than the effect of mutations identified in those with a very severe presentation. These results suggest that the amount of residual DBP activity correlates with the severity of the phenotype. From our data, we conclude that, on the basis of the predicted effect of the mutations on protein structure, a genotype-phenotype correlation exists for DBP deficiency.

17-Hydroxysteroid Dehydrogenases↗

PubMed2005

Role of Pex19p in the targeting of PMP70 to peroxisome.

Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.

ATP-Binding Cassette Transporters↗

PubMed2005

Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmic reticulum damage.

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.

ATP Binding Cassette Transporter, Subfamily D↗

PubMed2005

Novel metabolic and molecular findings in hepatic carnitine palmitoyltransferase I deficiency.

Detection of hepatic carnitine palmitoyltransferase I (CPT IA) deficiency by metabolite screening may be problematic. The urine organic acid profile is generally said to be normal and no abnormal or increased acylcarnitine species are evident on bloodspot tandem MS examination. We diagnosed CPT IA deficiency presenting with acute encephalopathy +/- hypoglycemia and hepatomegaly in one Bukharan Jewish and two Palestinian Arab infants from consanguineous families. CPT1A mutation analysis identified two novel nonsense mutations, c.1737C>A (Y579X) and c.1600delC (L534fsX), extending the known genetic heterogeneity in this disorder. A distinctive organic aciduria was observed in all three patients, even several days after initiation of treatment and resolution of symptoms. Abnormal findings included a hypoketotic dicarboxylic aciduria with prominence of the C12 dicarboxylic (dodecanedioic) acid. This C12 dicarboxylic aciduria suggests that CPT I may play a role in uptake of long-chain dicarboxylic acids by mitochondria after their initial shortening by beta-oxidation in peroxisomes. In addition, increased excretion of 3-hydroxyglutaric acid was detected in all three patients, a finding previously observed only in glutaric aciduria type 1, ketosis, and short-chain hydroxyacyl-CoA dehydrogenase deficiency. Examination of urine organic acids with awareness of these metabolic findings may lead to improved diagnosis of this seemingly rare disorder.

Adult↗

PubMed2005

Identification of the human mitochondrial FAD transporter and its potential role in multiple acyl-CoA dehydrogenase deficiency.

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is most often caused by mutations in the genes encoding the alpha- or beta-subunit of electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETF-DH). Since not all patients have mutations in these genes, other as yet unidentified genes are predicted to be involved as well. Because all affected mitochondrial flavoproteins in MADD have FAD as a prosthetic group, the underlying defect in these patients may be due to a thus far undisclosed disturbance in the metabolism of FAD. Since a proper mitochondrial flavin balance is maintained by a mitochondrial FAD transporter, a defect of this transporter could also cause an MADD-like phenotype. In yeast, FAD is transported across the mitochondrial inner membrane by the FLX1 protein. An FLX1-mutated Saccharomyces cerevisiae strain exhibits a decreased activity of several mitochondrial flavoproteins. In the present study, we report the identification of the human mitochondrial FAD transporter. Based on sequence similarity to FLX1, we identified two human candidate genes (MFT and N111), which were cloned and characterized by functional expression in an FLX1-mutated yeast strain. Of the two candidate genes, only the previously described mitochondrial folate transporter (MFT) was able to functionally complement the FLX1 mutant. Candidates for mutations in the MFT gene are patients with a clinical suspicion of MADD but without any mutation in the alpha- or beta-subunit of ETF or ETF-DH.

Acyl-CoA Dehydrogenase↗

PubMed2005

Demonstration and characterization of phosphate transport in mammalian peroxisomes.

It is now well established that the peroxisomal membrane is not freely permeable to small molecules in vivo, which implies the existence of metabolite transporters in the peroxisomal membrane. A few putative peroxisomal metabolite transporters have indeed been identified, but the function of these proteins has remained largely unresolved so far. The only peroxisomal transporter characterized to a significant extent is the adenine nucleotide transporter, which is presumably required to sustain the activity of the intraperoxisomal very-long-chain-acyl-CoA synthetase. In addition to AMP, this acyl-CoA synthetase also produces pyrophosphate, which must be exported from the peroxisome. In the present study, we demonstrate that the peroxisomal membrane contains a transporter activity that facilitates the passage of phosphate and possibly pyrophosphate across the peroxisomal membrane. By reconstitution of peroxisomal membrane proteins in proteoliposomes, some kinetic parameters of the transporter could be established in vitro. The transporter can be distinguished from the mitochondrial phosphate transporter by its different sensitivity to inhibitors.

Animals↗

PubMed2005

Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder.

Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the I326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of protease susceptibility, suggesting a problem of mutant protein fold. Conformational analyses against urea denaturation using urea gradient gel electrophoresis or fluorescence emission from tryptophan residue revealed that the mutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as their secondary structure with slightly different extent. The thermal unfolding profiles measured by CD showed a marked lower melting temperature for I326T protein compared with that of wild-type protein. Analysis of the protein 3D-structure indicated that the Ile326 should be a core residue for folding kinetics and the substitution of Ile326 by threonine should directly alter the kinetic equilibrium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlations.

Amino Acid Motifs↗

PubMed2005

Intra-familial clinical heterogeneity: absence of genotype-phenotype correlation in primary hyperoxaluria type 1 in Israel.

BACKGROUND/AIMS: Primary hyperoxaluria type 1 (PH1) is caused by the deficiency of the liver enzyme alanine:glyoxylate aminotransferase which results in increased synthesis and excretion of oxalate. The clinical manifestations of PH1 are heterogeneous with respect to the age of onset and rate of progression. The aim of this study was to investigate possible relationships between a given genotype, the biochemical profile and the clinical phenotype. METHODS: We conducted a study of 56 patients from 22 families with PH1 from Israel. The clinical and biochemical data were compiled and the genotype was determined for each family. RESULTS: The prevalent phenotype was of early onset with progression to end-stage renal disease during the first decade of life. Fifteen PH1-causing mutations were detected in 21 families: 10 were first described in this patient population. Marked intra-familial clinical heterogeneity was noted, meaning that there was no correlation between a given genotype and the phenotype. CONCLUSIONS: The clinical course of patients with PH1 is not dictated primarily by its genotype. Other genetic and/or environmental factors play a role in determining the ultimate phenotype.

Child↗

PubMed2005

Characterization of the final step in the conversion of phytol into phytanic acid.

Phytol is a branched-chain fatty alcohol that is a naturally occurring precursor of phytanic acid, a fatty acid involved in the pathogenesis of Refsum disease. The conversion of phytol into phytanic acid is generally believed to take place via three enzymatic steps that involve 1) oxidation to its aldehyde, 2) further oxidation to phytenic acid, and 3) reduction of the double bond at the 2,3 position, yielding phytanic acid. Our recent investigations of this mechanism have elucidated the enzymatic steps leading to phytenic acid production, but the final step of the pathway has not been investigated so far. In this study, we describe the characterization of phytenic acid reduction in rat liver. NADPH-dependent conversion of phytenic acid into phytanic acid was detected, although at a slow rate. However, it was shown that phytenic acid can be activated to its CoA ester and that reduction of phytenoyl-CoA is much more efficient than that of phytenic acid. Furthermore, in rat hepatocytes cultured in the presence of phytol, phytenoyl-CoA could be detected, showing that it is a bona fide intermediate of phytol degradation. Subcellular fractionation experiments revealed that phytenoyl-CoA reductase activity is present in peroxisomes and mitochondria. With these findings, we have accomplished the full elucidation of the mechanism by which phytol is converted into phytanic acid.

Animals↗

PubMed2005

Long-chain fatty acid oxidation during early human development.

Patients with very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD)/mitochondrial trifunctional protein (MTP) deficiency, disorders of the mitochondrial long-chain fatty acid oxidation, can present with hypoketotic hypoglycemia, rhabdomyolysis, and cardiomyopathy. In addition, patients with LCHAD/MTP deficiency may suffer from retinopathy and peripheral neuropathy. Until recently, there was no indication of intrauterine morbidity in these disorders. This observation was in line with the widely accepted view that fatty acid oxidation (FAO) does not play a significant role during fetal life. However, the high incidence of the gestational complications acute fatty liver of pregnancy and hemolysis, elevated liver enzymes, and low platelets syndrome observed in mothers carrying a LCHAD/MTP-deficient child and the recent reports of fetal hydrops due to cardiomyopathy in MTP deficiency, as well as the high incidence of intrauterine growth retardation in children with LCHAD/MTP deficiency, suggest that FAO may play an important role during fetal development. In this study, using in situ hybridization of the VLCAD and the LCHAD mRNA, we report on the expression of genes involved in the mitochondrial oxidation of long-chain fatty acids during early human development. Furthermore, we measured the enzymatic activity of the VLCAD, LCHAD, and carnitine palmitoyl-CoA transferase 2 (CPT2) enzymes in different human fetal tissues. Human embryos (at d 35 and 49 of development) and separate tissues (5-20 wk of development) were used. The results show a strong expression of VLCAD and LCHAD mRNA and a high enzymatic activity of VLCAD, LCHAD, and CPT2 in a number of tissues, such as liver and heart. In addition, high expression of LCHAD mRNA was observed in the neural retina and CNS. The observed pattern of expression during early human development is well in line with the spectrum of clinical signs and symptoms reported in patients with VLCAD or LCHAD/MTP deficiency.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2005

dif-1 and colt, both implicated in early embryonic development, encode carnitine acylcarnitine translocase.

It has always been assumed that during development the embryo and fetus depend only on glycolysis for energy generation and that they do not oxidize fatty acids. Recently, however, we found abundant expression and activity of fatty acid oxidation (FAO) enzymes in the human embryo and fetus. In a search for FAO gene expression during development we came across two embryonic differentiation genes: differentiation defective (dif-1) and congested-like trachea (colt) of Caenorhabditis elegans and Drosophila melanogaster, respectively. Earlier studies showed that expression of these two genes is essential during developmental stages with high energy requirements. Both dif-1 and colt encode proteins with sequence similarity to the mitochondrial carnitine acylcarnitine carrier (CACT), which suggests that the DIF-1 and COLT proteins might be functional orthologues of CACT. To investigate this, we expressed both dif-1 and colt in Saccharomyces cerevisiae. Our results show that DIF-1 and COLT can functionally complement a yeast CACT deletion strain and thus function as carnitine acylcarnitine transporters. This finding is well in line with the recent observation that embryos are capable of oxidizing fatty acids and furthermore implies that FAO is essential during early embryonic development when the energy demand is high.

Amino Acid Sequence↗

PubMed2005

Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse.

In the present paper, we describe a novel method which enables the analysis of tissue acylcarnitines and carnitine biosynthesis intermediates in the same sample. This method was used to investigate the carnitine and fatty acid metabolism in wild-type and LCAD-/- (long-chain acyl-CoA dehydrogenase-deficient) mice. In agreement with previous results in plasma and bile, we found accumulation of the characteristic C14:1-acylcarnitine in all investigated tissues from LCAD-/- mice. Surprisingly, quantitatively relevant levels of 3-hydroxyacylcarnitines were found to be present in heart, muscle and brain in wild-type mice, suggesting that, in these tissues, long-chain 3-hydroxyacyl-CoA dehydrogenase is rate-limiting for mitochondrial beta-oxidation. The 3-hydroxyacylcarnitines were absent in LCAD-/- tissues, indicating that, in this situation, the beta-oxidation flux is limited by the LCAD deficiency. A profound deficiency of acetylcarnitine was observed in LCAD-/- hearts, which most likely corresponds with low cardiac levels of acetyl-CoA. Since there was no carnitine deficiency and only a marginal elevation of potentially cardiotoxic acylcarnitines, we conclude from these data that the cardiomyopathy in the LCAD-/- mouse is caused primarily by a severe energy deficiency in the heart, stressing the important role of LCAD in cardiac fatty acid metabolism in the mouse.

Acyl-CoA Dehydrogenase, Long-Chain↗

PubMed2005

Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis.

Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis. We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts.

Acylation↗

PubMed2005

Neonatal screening for defects of the mitochondrial trifunctional protein.

Long-chain l-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has been included in the routine neonatal screening program by the German screening commission. As tandem mass spectrometry (TMS) does not discriminate between the different defects of the mitochondrial trifunctional protein (MTP) screening for isolated LCHAD deficiency includes the detection of long-chain 3-ketoacyl-CoA thiolase and complete MTP deficiencies as well. We identified 11 patients with abnormalities of the MTP out of 1.2 million newborns screened in our laboratory during the last 6 years. Treatment was started on the day the screening result was obtained (day 3 to day 9 of life). Seven of these newborns developed satisfactorily during an observation period of up to 64 months. They had isolated LCHAD deficiency, four of them caused by the typical mutation (1528 G>C), three others had no molecular genetic analysis done or were shown to have previously unknown mutations. Four children did not survive, two of them showing complete deficiency of MTP and two showing deficiency of long-chain 3-ketoacyl-CoA thiolase. We conclude that, despite the rarity of the disease, screening for MTP deficiencies is justified based on the following criteria: improved quality of life for patients with isolated LCHAD deficiency, absence of stigmatisation of babies showing mild variants without necessity of treatment, no significant increase of the total number of false positive screening results, no false negative results to our knowledge. Finally, extension of analysis to MTP deficiencies is achieved without additional costs for screening laboratories already using TMS.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2005

Developmental changes of bile acid composition and conjugation in L- and D-bifunctional protein single and double knockout mice.

Peroxisomal beta-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this beta-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4alpha, farnesoid X receptor, and peroxisome proliferator receptor alpha did not appear to be involved in the up-regulation of the transferase.

17-Hydroxysteroid Dehydrogenases↗

PubMed2005

Evidence for two enzymatic pathways for omega-oxidation of docosanoic acid in rat liver microsomes.

We studied the omega-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (omega-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, omega-oxidation of C22:0 produced not only the hydroxylated product, omega-hydroxy-C22:0, but also the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). When rat liver microsomes were incubated with omega-hydroxy-C22:0 in the presence of either NAD+ or NADPH, C22:0-DCA was formed readily. Formation of C22:0-DCA from either C22:0 or omega-hydroxy-C22:0 with NADPH as cofactor was inhibited strongly by miconazole and disulfiram, whereas no inhibition was found with NAD+ as cofactor. Furthermore, omega-oxidation of C22:0 was reduced significantly when molecular oxygen was depleted. The high sensitivity toward the more specific cytochrome P450 inhibitors ketoconazole and 17-octadecynoic acid suggests that hydroxylation of C22:0 and omega-hydroxy-C22:0 may be catalyzed by one or more cytochrome P450 hydroxylases belonging to the CYP4A and/or CYP4F subfamily. This study demonstrates that C22:0 is a substrate for the omega-oxidation system in rat liver microsomes and that the product of the first hydroxylation step, omega-hydroxy-C22:0, may undergo further oxidation via two distinct pathways driven by NAD+ or NADPH.

Animals↗

PubMed2005

Identification of novel mutations in classical galactosemia.

Classical galactosemia is an autosomal recessive disorder of galactose metabolism due to galactose-1-phosphate uridyltransferase (GALT) deficiency. Treatment through restriction of dietary galactose intake is lifesaving, but, in spite of this diet, most patients develop abnormalities. In this paper we report the mutational spectrum of classical galactosemia in a cohort of 123 Dutch patients, all with biochemically proven classical galactosemia. In the human GALT gene, which is located on chromosome 9p13, we identified 24 different mutations, including nine mutations that have not been reported previously. The novel mutations include five missense mutations (c.152G>A/p.R51Q, c.404C>T/p.S135W, c.687G>T/p.K229N, c.756G>T/p.Q252H, and c.1140A>C/p.X380C), a frame shift mutation (c.410dupT), a splice site mutation (c.821-2A>G), a possible branch point mutation (c.508-29delT), and a large deletion encompassing at least exons 1-11. Six of these novel mutations were found in patients of Dutch descent: p.R51Q, p.S135W, p.K229N, p.Q252H, p.X380C, and c.410dupT.

Base Sequence↗