Advances in methods for characterization of hepatic urea cycle enzymatic activity in HepaRG cells using UPLC-MS/MS.

Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRG cells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRG monolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line.

Inhibition of N-acetylglutamate synthase by various monocarboxylic and dicarboxylic short-chain coenzyme A esters and the production of alternative glutamate esters.

Hyperammonemia is a frequent finding in various organic acidemias. One possible mechanism involves the inhibition of the enzyme N-acetylglutamate synthase (NAGS), by short-chain acyl-CoAs which accumulate due to defective catabolism of amino acids and/or fatty acids in the cell. The aim of this study was to investigate the effect of various acyl-CoAs on the activity of NAGS in conjunction with the formation of glutamate esters. NAGS activity was measured in vitro using a sensitive enzyme assay with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) product analysis. Propionyl-CoA and butyryl-CoA proved to be the most powerful inhibitors of N-acetylglutamate (NAG) formation. Branched-chain amino acid related CoAs (isovaleryl-CoA, 3-methylcrotonyl-CoA, isobutyryl-CoA) showed less pronounced inhibition of NAGS whereas the dicarboxylic short-chain acyl-CoAs (methylmalonyl-CoA, succinyl-CoA, glutaryl-CoA) had the least inhibitory effect. Subsequent work showed that the most powerful inhibitors also proved to be the best substrates in the formation of N-acylglutamates. Furthermore, we identified N-isovalerylglutamate, N-3-methylcrotonylglutamate and N-isobutyrylglutamate (the latter two in trace amounts), in the urines of patients with different organic acidemias. Collectively, these findings explain one of the contributing factors to secondary hyperammonemia, which lead to the reduced in vivo flux through the urea cycle in organic acidemias and result in the inadequate elimination of ammonia.

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.

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.

Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy.

Sensory motor neuropathy is associated with various inherited disorders including Charcot-Marie-Tooth disease, X-linked adrenoleukodystrophy/adrenomyeloneuropathy and Refsum disease. In the latter two, the neuropathy is thought to result from the accumulation of specific fatty acids. We describe here three patients with elevated plasma concentrations of pristanic acid (a branched-chain fatty acid) and C27-bile-acid intermediates. Two of the patients suffered from adult-onset sensory motor neuropathy. One patient also had pigmentary retinopathy, suggesting Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy. The third patient was a child without neuropathy. In all three patients we discovered a deficiency of alpha-methylacyl-CoA racemase (AMACR). This enzyme is responsible for the conversion of pristanoyl-CoA and C27-bile acyl-CoAs to their (S)-stereoisomers, which are the only stereoisomers that can be degraded via peroxisomal beta-oxidation. Sequence analysis of AMACR cDNA from the patients identified two different mutations that are likely to cause disease, based on analysis in Escherichia coli. Our findings have implications for the diagnosis of adult-onset neuropathies of unknown aetiology.

Mutations in MVK, encoding mevalonate kinase, cause hyperimmunoglobulinaemia D and periodic fever syndrome.

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal dismay and skin rash. Diagnostic hallmark of HIDS is a constitutively elevated level of serum immunoglobulin D (IgD), although patients have been reported with normal IgD levels. To determine the underlying defect in HIDS, we analysed urine of several patients and discovered increased concentrations of mevalonic acid during severe episodes of fever, but not between crises. Subsequent analysis of cells from four unrelated HIDS patients revealed reduced activities of mevalonate kinase (MK; encoded by the gene MVK), a key enzyme of isoprenoid biosynthesis. Sequence analysis of MVK cDNA from the patients identified three different mutations, one of which was common to all patients. Expression of the mutant cDNAs in Escherichia coli showed that all three mutations affect the activity of the encoded proteins. Moreover, immunoblot analysis demonstrated a deficiency of MK protein in patient fibroblasts, indicating a protein-destabilizing effect of the mutations.

Clinical variability of isovaleric acidemia in a genetically homogeneous population.

Isovaleric acidemia (IVA) is one of the most common organic acidemias found in South Africa. Since 1983, a significant number of IVA cases have been identified in approximately 20,000 Caucasian patients screened for metabolic defects. IVA is caused by an autosomal recessive deficiency of isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of isovaleryl-CoA and its metabolites. In total, 10 IVA patients and three carriers were available for phenotypic and genotypic investigation in this study. All patients were found to be homozygous for a single c.367 G > A (p.G123R) mutation. The amino acid substitution of a glycine to arginine resulted in a markedly reduced steady-state level of the IVD protein, which explains the nearly complete lack of IVD enzyme activity as assessed in fibroblast homogenates. Despite the genetic homogeneity of this South African IVA group, the clinical presentation varied widely, ranging from severe mental handicap and multiple episodes of metabolic derangement to an asymptomatic state. The variation may be due to poor dietary intervention, delayed diagnosis or even epigenetic and polygenetic factors of unknown origin.

Peroxisomes, lipid metabolism and lipotoxicity.

Peroxisomes play an essential role in cellular lipid metabolism as exemplified by the existence of a number of genetic diseases in humans caused by the impaired function of one of the peroxisomal enzymes involved in lipid metabolism. Key pathways in which peroxisomes are involved include: (1.) fatty acid beta-oxidation; (2.) etherphospholipid biosynthesis, and (3.) fatty acid alpha-oxidation. In this paper we will describe these different pathways in some detail and will provide an overview of peroxisomal disorders of metabolism and in addition discuss the toxicity of the intermediates of peroxisomal metabolism as they accumulate in the different peroxisomal deficiencies.

Preadipocytes of type 2 diabetes subjects display an intrinsic gene expression profile of decreased differentiation capacity.

OBJECTIVE: Insulin resistance and type 2 diabetes mellitus (T2DM) are associated with increased adipocyte size, altered secretory pattern and decreased differentiation of preadipocytes. In this study, we identified the underlying molecular processes in preadipocytes of T2DM patients, a characteristic for the development of T2DM. DESIGN AND PARTICIPANTS: Preadipocyte cell cultures were prepared from subcutaneous fat biopsies of seven T2DM patients (age 53 ± 12 years; body mass index (BMI) 34 ± 5 kg m(-2)) and nine control subjects (age 51 ± 12 years; BMI 30 ± 3 kg m(-2)). Microarray analysis was used to identify altered processes between the T2DM and control preadipocytes. RESULTS: Gene expression profiling showed changed expression of transcription regulators involved in adipogenesis and in extracellular matrix remodeling, actin cytoskeleton and integrin signaling genes, which indicated decreased capacity to differentiate. Additionally, genes involved in insulin signaling and lipid metabolism were downregulated, and inflammation/apoptosis was upregulated in T2DM preadipocytes. CONCLUSION: Decreased expression of genes involved in differentiation can provide a molecular basis for the reduced adipogenesis of preadipocytes of T2DM subjects, leading to reduced formation of adipocytes in subcutaneous fat depots, and ultimately leading to ectopic fat storage.

Increased mitochondrial content rescues in vivo muscle oxidative capacity in long-term high-fat-diet-fed rats.

Mitochondria are thought to play a crucial role in the etiology of muscle insulin resistance (IR). The aim of this study was to gain more insight into the timing and nature of mitochondrial adaptations during the development of high-fat-diet (HFD)-induced IR. Adult Wistar rats were fed HFD or normal chow for 2.5 and 25 wk. Intramyocellular lipids (IMCLs) were quantified in vivo using (1)H magnetic resonance spectroscopy (MRS). Muscle oxidative capacity was assessed in vivo using (31)P MRS and in vitro by measuring mitochondrial DNA copy number and oxygen consumption in isolated mitochondria. MRS in tibialis anterior muscle revealed 3.3-fold higher IMCL content and 1.2-fold increased oxidative capacity after 2.5 wk of HFD feeding. The latter result could be fully accounted for by increased mitochondrial content. After 25 wk of HFD, maximal ADP-stimulated oxygen consumption in isolated mitochondria oxidizing pyruvate plus malate remained unaffected, while IMCL and mitochondrial content had further increased compared to controls (5.1-fold and 1.4-fold, respectively). Interestingly, in vivo oxidative capacity at this time point was identical to controls. These results show that skeletal muscle in HFD-induced IR accompanied by IMCL accumulation requires a progressively larger mitochondrial pool size to maintain normal oxidative capacity in vivo.

High prevalence of short-chain acyl-CoA dehydrogenase deficiency in the Netherlands, but no association with epilepsy of unknown origin in childhood.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of metabolism, most frequently associated with developmental delay and/or epilepsy. Most SCADD patients carry common SCAD-encoding gene ( ACADS) variants or these variants in combination with a rare ACADS mutation, in the Netherlands predominantly the c.1058C>T. Epilepsy in childhood often remains unexplained and patients with epilepsy related to SCADD may remain undiagnosed because studies for SCADD are often not performed. To test this hypothesis and to further estimate the extent of the Dutch SCADD population, we performed a study on blood spot samples in 131 paediatric patients with epilepsy and 909 anonymous newborns and investigated the presence of the 2 common ACADS variants and the rare c.1058C>T mutation. Overall, the 2 common ACADS variants and the rare c.1058C>T mutation were detected in either homozygous or compound heterozygous forms in 9.2% of the epilepsy and 7.5% of the reference group. A birth prevalence of SCADD with a mutation/variant genotype in the Netherlands as high as >1:1,000 was calculated. This is in contrast with the low number of patients diagnosed clinically and supports the hypothesis that SCADD is clinically irrelevant. Furthermore our study does not support an association between SCADD and epilepsy.

Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.

Equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in 14 horses associated with ingestion of Maple leaves (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum).

This case-series describes fourteen horses suspected of equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) also known as atypical myopathy of which seven cases were confirmed biochemically with all horses having had access to leaves of the Maple tree (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum). Assessment of organic acids, glycine conjugates, and acylcarnitines in urine was regarded as gold standard in the biochemical diagnosis of equine acquired multiple acyl-CoA dehydrogenase deficiency.

Pyruvate dehydrogenase kinase 4 expression is synergistically induced by AMP-activated protein kinase and fatty acids.

Organs are flexible as to which substrates they will use to maintain energy homeostasis. Under well-fed conditions, glucose is a preferred substrate for oxidation. During fasting, fatty acid oxidation will become a more important energy source. Glucose oxidation is decreased by fatty acids, a process in which the pyruvate dehydrogenase complex (PDH) and its regulator pyruvate dehydrogenase kinase 4 (PDK4) play important roles. It is currently unknown how energy status influences PDH activity. We show that AMP-activated protein kinase (AMPK) activation by hypoxia and AICAR treatment combined with fatty acid administration synergistically induce PDK4 expression. We provide evidence that AMPK activation modulates ligand-dependent activation of peroxisome proliferator-activated receptor. Finally, we show that this synergistic induction of PDK4 decreases cellular glucose oxidation. In conclusion, AMPK and fatty acids play a direct role in fuel selection in response to cellular energy status in order to spare glucose.

Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-(14)C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.

Organization and integration of biomedical knowledge with concept maps for key peroxisomal pathways.

MOTIVATION: One important area of clinical genomics research involves the elucidation of molecular mechanisms underlying (complex) disorders which eventually may lead to new diagnostic or drug targets. To further advance this area of clinical genomics one of the main challenges is the acquisition and integration of data, information and expert knowledge for specific biomedical domains and diseases. Currently the required information is not very well organized but scattered over biological and biomedical databases, basic text books, scientific literature and experts' minds and may be highly specific, heterogeneous, complex and voluminous. RESULTS: We present a new framework to construct knowledge bases with concept maps for presentation of information and the web ontology language OWL for the representation of information. We demonstrate this framework through the construction of a peroxisomal knowledge base, which focuses on four key peroxisomal pathways and several related genetic disorders. All 155 concept maps in our knowledge base are linked to at least one other concept map, which allows the visualization of one big network of related pieces of information. AVAILABILITY: The peroxisome knowledge base is available from www.bioinformaticslaboratory.nl (Support-->Web applications). SUPPLEMENTARY INFORMATION: Supplementary data is available from www.bioinformaticslaboratory.nl (Research-->Output--> Publications--> KB_SuppInfo)

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.

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1.

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-->Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg-mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.

Zellweger syndrome with unusual findings: non-immune hydrops fetalis, dermal erythropoiesis and hypoplastic toe nails.

The peroxisomal biogenesis disorders (PBDs) comprise the Zellweger spectrum disorders (i.e., Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease) and rhizomelic chondrodysplasia punctata. Peroxisomal biogenesis disorders can be caused by mutations in any of 13 currently known PEX genes, which encode peroxins involved in peroxisomal protein import and/or assembly of the organelle. We report here on a Turkish patient who presented with unusual clinical findings, that included non-immune hydrops, dermal erythropoiesis and hypoplastic toenails, as well as common dysmorphic features of Zellweger syndrome. The patient has also pulmonary hypoplasia, which has been reported in only a few patients with Zellweger syndrome. A peroxisomal biogenesis disorder was confirmed by enzyme analysis and abnormal very long-chain fatty acid (VLCFA) profiles in plasma and fibroblast and immunofluorescence microscopy studies. Subsequent molecular genetic analysis revealed a homozygous c.856C>T mutation (R268X) in the PEX3 gene, which made this patient the third to have a defect in this gene. In contrast to those of the two previously reported patients, the cells of this patient still contained peroxisomal membrane structures (ghosts), seen by immunofluorescence microscopy analysis. The case presented here and the two previously reported cases point out that a PEX3 gene defect may present with fairly heterogeneous clinical findings. This case also raises a possibility that hydrops fetalis may be associated with a PEX3 gene defect and that peroxisomal disorders can be considered in the etiology of hydrops fetalis as well as other cell organelle disorders when one is considering yet undiscovered complementation groups in peroxisomal disorders.

Catecholamine excretion profiles identify clinical subgroups of neuroblastoma patients.

INTRODUCTION: Analysis of urinary catecholamine metabolites is one of the primary modalities to diagnose patients with neuroblastoma. Although catecholamine excretion patterns have been recognised in the past, their biological rationale and clinical relevance remain largely unknown. Therefore, this study was designed to identify unique catecholamine excretion patterns and elucidate their underlying biology and clinical relevance. PATIENTS AND METHODS: A panel of 25 neuroblastoma cell lines was screened for catecholamine excretion. Detection of the catecholamine enzymes was performed using Western blot. Based on catecholamine enzymes presence and excreted catecholamine metabolites, excretion profiles were defined. The prevalence of these profiles was investigated in vivo using diagnostic urines from 301 patients with neuroblastoma and immunohistochemistry on primary tumours. The clinical relevance of the profiles was determined by linking the profiles to clinical characteristics and outcome of patients with neuroblastoma. RESULTS: Four excretion profiles (A-D) were identified in vitro, which correlated with the relative protein expression of the catecholamine enzymes. These profiles were also identified in urine samples from patients with neuroblastoma and correlated with the presence of the catecholamine enzymes in the tumour. Strikingly, in 66% of the patients, homovanillic acid and vanillylmandelic acid excretions were discordant with the catecholamine profiles. Clinical characteristics and outcome gradually improved from patients with profile A (predominantly high risk) towards profile D (predominantly observation), with 5-years overall survival of 35% and 93%, respectively. CONCLUSIONS: Catecholamine profiles in vitro and in vivo reflect, to a large extent, the presence of the individual catecholamine enzymes and represent distinct subgroups of patients with neuroblastoma.