Publications by authors named "Ronald J A Wanders"

357 Publications

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Do not Improve with Carnitine Supplementation.

Front Pharmacol 2020 12;11:616834. Epub 2021 Jan 12.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Amsterdam Gastroenterology and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam, Netherlands.

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.
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http://dx.doi.org/10.3389/fphar.2020.616834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883678PMC
January 2021

Enantiomer-specific pharmacokinetics of D,L-3-hydroxybutyrate: Implications for the treatment of multiple acyl-CoA dehydrogenase deficiency.

J Inherit Metab Dis 2021 Feb 5. Epub 2021 Feb 5.

University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Section of Metabolic Diseases, Groningen, The Netherlands.

D,L-3-hydroxybutyrate (D,L-3-HB, a ketone body) treatment has been described in several inborn errors of metabolism, including multiple acyl-CoA dehydrogenase deficiency (MADD; glutaric aciduria type II). We aimed to improve the understanding of enantiomer-specific pharmacokinetics of D,L-3-HB. Using UPLC-MS/MS, we analyzed D-3-HB and L-3-HB concentrations in blood samples from three MADD patients, and blood and tissue samples from healthy rats, upon D,L-3-HB salt administration (patients: 736-1123 mg/kg/day; rats: 1579-6317 mg/kg/day of salt-free D,L-3-HB). D,L-3-HB administration caused substantially higher L-3-HB concentrations than D-3-HB. In MADD patients, both enantiomers peaked at 30 to 60 minutes, and approached baseline after 3 hours. In rats, D,L-3-HB administration significantly increased C and AUC of D-3-HB in a dose-dependent manner (controls vs ascending dose groups for C : 0.10 vs 0.30-0.35-0.50 mmol/L, and AUC: 14 vs 58-71-106 minutes*mmol/L), whereas for L-3-HB the increases were significant compared to controls, but not dose proportional (C : 0.01 vs 1.88-1.92-1.98 mmol/L, and AUC: 1 vs 380-454-479 minutes*mmol/L). L-3-HB concentrations increased extensively in brain, heart, liver, and muscle, whereas the most profound rise in D-3-HB was observed in heart and liver. Our study provides important knowledge on the absorption and distribution upon oral D,L-3-HB. The enantiomer-specific pharmacokinetics implies differential metabolic fates of D-3-HB and L-3-HB.
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http://dx.doi.org/10.1002/jimd.12365DOI Listing
February 2021

Peroxisomal Metabolite and Cofactor Transport in Humans.

Front Cell Dev Biol 2020 11;8:613892. Epub 2021 Jan 11.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC Location AMC, University of Amsterdam, Amsterdam, Netherlands.

Peroxisomes are membrane-bound organelles involved in many metabolic pathways and essential for human health. They harbor a large number of enzymes involved in the different pathways, thus requiring transport of substrates, products and cofactors involved across the peroxisomal membrane. Although much progress has been made in understanding the permeability properties of peroxisomes, there are still important gaps in our knowledge about the peroxisomal transport of metabolites and cofactors. In this review, we discuss the different modes of transport of metabolites and essential cofactors, including CoA, NAD, NADP, FAD, FMN, ATP, heme, pyridoxal phosphate, and thiamine pyrophosphate across the peroxisomal membrane. This transport can be mediated by non-selective pore-forming proteins, selective transport proteins, membrane contact sites between organelles, and co-import of cofactors with proteins. We also discuss modes of transport mediated by shuttle systems described for NAD/NADH and NADP/NADPH. We mainly focus on current knowledge on human peroxisomal metabolite and cofactor transport, but also include knowledge from studies in plants, yeast, fruit fly, zebrafish, and mice, which has been exemplary in understanding peroxisomal transport mechanisms in general.
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http://dx.doi.org/10.3389/fcell.2020.613892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829553PMC
January 2021

Fatty Acid Oxidation in Peroxisomes: Enzymology, Metabolic Crosstalk with Other Organelles and Peroxisomal Disorders.

Adv Exp Med Biol 2020 ;1299:55-70

Departments of Clinical Chemistry and Pediatrics, Amsterdam Gastroenterology Endocrinology Metabolism, Laboratory Genetic Metabolic Diseases and Emma Children's hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Peroxisomes play a central role in metabolism as exemplified by the fact that many genetic disorders in humans have been identified through the years in which there is an impairment in one or more of these peroxisomal functions, in most cases associated with severe clinical signs and symptoms. One of the key functions of peroxisomes is the β-oxidation of fatty acids which differs from the oxidation of fatty acids in mitochondria in many respects which includes the different substrate specificities of the two organelles. Whereas mitochondria are the main site of oxidation of medium-and long-chain fatty acids, peroxisomes catalyse the β-oxidation of a distinct set of fatty acids, including very-long-chain fatty acids, pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid. Peroxisomes require the functional alliance with multiple subcellular organelles to fulfil their role in metabolism. Indeed, peroxisomes require the functional interaction with lysosomes, lipid droplets and the endoplasmic reticulum, since these organelles provide the substrates oxidized in peroxisomes. On the other hand, since peroxisomes lack a citric acid cycle as well as respiratory chain, oxidation of the end-products of peroxisomal fatty acid oxidation notably acetyl-CoA, and different medium-chain acyl-CoAs, to CO and HO can only occur in mitochondria. The same is true for the reoxidation of NADH back to NAD. There is increasing evidence that these interactions between organelles are mediated by tethering proteins which bring organelles together in order to allow effective exchange of metabolites. It is the purpose of this review to describe the current state of knowledge about the role of peroxisomes in fatty acid oxidation, the transport of metabolites across the peroxisomal membrane, its functional interaction with other subcellular organelles and the disorders of peroxisomal fatty acid β-oxidation identified so far in humans.
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http://dx.doi.org/10.1007/978-3-030-60204-8_5DOI Listing
February 2021

Disorders of flavin adenine dinucleotide metabolism: MADD and related deficiencies.

Int J Biochem Cell Biol 2021 Mar 3;132:105899. Epub 2020 Dec 3.

Human Metabolomics, North-West University, Potchefstroom, South Africa. Electronic address:

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD), or glutaric aciduria type II (GAII), is a group of clinically heterogeneous disorders caused by mutations in electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) - the two enzymes responsible for the re-oxidation of enzyme-bound flavin adenine dinucleotide (FADH) via electron transfer to the respiratory chain at the level of coenzyme Q10. Over the past decade, an increasing body of evidence has further coupled mutations in FAD metabolism (including intercellular riboflavin transport, FAD biosynthesis and FAD transport) to MADD-like phenotypes. In this review we provide a detailed description of the overarching and specific metabolic pathways involved in MADD. We examine the eight associated genes (ETFA, ETFB, ETFDH, FLAD1, SLC25A32 and SLC52A1-3) and clinical phenotypes, and report ∼436 causative mutations following a systematic literature review. Finally, we focus attention on the value and shortcomings of current diagnostic approaches, as well as current and future therapeutic options for MADD and its phenotypic disorders.
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http://dx.doi.org/10.1016/j.biocel.2020.105899DOI Listing
March 2021

An autosomal dominant neurological disorder caused by de novo variants in FAR1 resulting in uncontrolled synthesis of ether lipids.

Genet Med 2020 Nov 26. Epub 2020 Nov 26.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Department of Clinical Chemistry, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands.

Purpose: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu).

Methods: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics.

Results: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production.

Conclusion: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.
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http://dx.doi.org/10.1038/s41436-020-01027-3DOI Listing
November 2020

Neonatal carnitine concentrations in relation to gestational age and weight.

JIMD Rep 2020 Nov 8;56(1):95-104. Epub 2020 Sep 8.

Reference Laboratory for Neonatal Screening, Centre for Health Protection Dutch National Institute for Public Health and the Environment Bilthoven The Netherlands.

Background: Free carnitine has been measured in the Dutch newborn screening (NBS) program since 2007 with a referral threshold of ≤5 μmol/L, regardless of gestational age or birthweight. However, several studies suggest that carnitine concentrations may depend on gestational age and birthweight. We evaluated differences in postnatal day-to-day carnitine concentrations in newborns based on gestational age (GA) and/or weight for GA (WfGA).

Methods: A retrospective study was performed using data from the Dutch NBS. Dried blood spot (DBS) carnitine concentrations, collected between the 3rd and 10th day of life, of nearly 2 million newborns were included. Individuals were grouped based on GA and WfGA. Median carnitine concentrations were calculated for each group. Mann-Whitney tests, and chi-square tests were applied to test for significant differences between groups.

Results: Preterm, postterm, and small for GA (SGA) newborns have higher carnitine concentrations at the third day of life compared to term newborns. The median carnitine concentration of preterm newborns declines from day 3 onwards, and approximates that of term newborns at the sixth day of life, while median concentrations of postterm and SGA newborns remain elevated at least throughout the first 10 days of life. Carnitine concentrations ≤5 μmol/L were found less frequently in SGA newborns and newborns born between 32 and 37 weeks of gestation, compared to term newborns.

Conclusions: Median carnitine concentrations in NBS DBS vary with day of sampling, GA, and WfGA. It is important to take these variables into account when interpreting NBS results..
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http://dx.doi.org/10.1002/jmd2.12162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653253PMC
November 2020

The Saccharomyces cerevisiae ABC subfamily D transporter Pxa1/Pxa2p co-imports CoASH into the peroxisome.

FEBS Lett 2020 Oct 28. Epub 2020 Oct 28.

Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology & Metabolism, Amsterdam UMC, University of Amsterdam, the Netherlands.

ATP-binding cassette (ABC) subfamily D transporters are important for the uptake of fatty acids and other beta-oxidation substrates into peroxisomes. Genetic and biochemical evidence indicates that the transporters accept fatty acyl-coenzyme A that is cleaved during the transport cycle and then re-esterified in the peroxisomal lumen. However, it is not known whether free coenzyme A (CoA) is released inside or outside the peroxisome. Here we have used Saccharomyces cerevisiae and isolated peroxisomes to demonstrate that free CoA is released in the peroxisomal lumen. Thus, ABC subfamily D transporter provide an import pathway for free CoA that controls peroxisomal CoA homeostasis and tunes metabolism according to the cell's demands.
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http://dx.doi.org/10.1002/1873-3468.13974DOI Listing
October 2020

Mitochondrial Fatty Acid Oxidation Disorders: Laboratory Diagnosis, Pathogenesis, and the Complicated Route to Treatment.

J Lipid Atheroscler 2020 Sep 22;9(3):313-333. Epub 2020 Sep 22.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Amsterdam Gastroenterology and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands.

Mitochondrial fatty acid (FA) oxidation deficiencies represent a genetically heterogeneous group of diseases in humans caused by defects in mitochondrial FA beta-oxidation (mFAO). A general characteristic of all mFAO disorders is hypoketotic hypoglycemia resulting from the enhanced reliance on glucose oxidation and the inability to synthesize ketone bodies from FAs. Patients with a defect in the oxidation of long-chain FAs are at risk to develop cardiac and skeletal muscle abnormalities including cardiomyopathy and arrhythmias, which may progress into early death, as well as rhabdomyolysis and exercise intolerance. The diagnosis of mFAO-deficient patients has greatly been helped by revolutionary developments in the field of tandem mass spectrometry (MS) for the analysis of acylcarnitines in blood and/or urine of candidate patients. Indeed, acylcarnitines have turned out to be excellent biomarkers; not only do they provide information whether a certain patient is affected by a mFAO deficiency, but the acylcarnitine profile itself usually immediately points to which enzyme is likely deficient. Another important aspect of acylcarnitine analysis by tandem MS is that this technique allows high-throughput analysis, which explains why screening for mFAO deficiencies has now been introduced in many newborn screening programs worldwide. In this review, we will describe the current state of knowledge about mFAO deficiencies, with particular emphasis on recent developments in the area of pathophysiology and treatment.
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http://dx.doi.org/10.12997/jla.2020.9.3.313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521971PMC
September 2020

Exploring the metabolic fate of medium-chain triglycerides in healthy individuals using a stable isotope tracer.

Clin Nutr 2020 Sep 4. Epub 2020 Sep 4.

Amsterdam UMC, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Amsterdam Cardiovascular Sciences, Amsterdam Gastroenterology Endocrinology and Metabolism, Meibergdreef 9, 1105, AZ, Amsterdam, the Netherlands; Section Metabolic Diseases, Wilhelmina Children's Hospital, University Medical Center Utrecht, Lundlaan 6, 3584, EA, Utrecht, the Netherlands. Electronic address:

Background & Aims: Medium chain triglyceride (MCT) supplementation is often recommended as treatment for patients with long-chain fatty acid β-oxidation (lcFAO) disorders, since they can be utilized as an energy source without the use of the defective enzyme. However, studies in mice and preterm infants suggest that not all medium-chain fatty acids (MCFA) are oxidized and may undergo elongation to long-chain fatty acids (LCFA). In this single blinded study, we explored the metabolic fates of MCT in healthy individuals using a C-labeled MCT tracer.

Method: Three healthy males in rest received on two test days a primed continuous infusion of glyceryl tri[1,2,3,4-C]-octanoate with either an isocaloric supplementation of 1) exclusively MCT (MCT-only) or 2) a mixture of MCT, proteins and carbohydrates (MCT-mix). Gas chromatography - combustion - isotope ratio mass spectrometry (GC-C-IRMS) was used to determine C-enrichment of long-chain fatty acids in plasma and of CO in exhaled air.

Results: When provided as single energy source, an estimated 42% of administered MCT was converted to CO. In combination with carbohydrates and proteins in the diet, oxidation of MCT was higher (62%). In both diets <1% of C-label was incorporated in LCFA in plasma, indicating that administered MCT underwent chain-elongation to LCT.

Conclusions: Although the relative MCT oxidation rate was higher when combined with carbohydrates and protein, quantitatively more MCT was oxidized when given an isocaloric meal with solely MCT. As these results were obtained in the resting state opposed to during exercise, it is too early to give a recommendation concerning the use of MCT in lcFAO disorders. The data show that in resting healthy individuals only a very small part of the MCT is traced back as LCFA in plasma, suggesting that MCT treatment does not result in a large LCFA burden, however further research on storage of MCT in tissues is warranted.

Registration: The study was registered in the Nederlands Trialregister. Protocol ID: Trial NL7417 (NTR7650).
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http://dx.doi.org/10.1016/j.clnu.2020.08.032DOI Listing
September 2020

Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes.

J Inherit Metab Dis 2020 Aug 2. Epub 2020 Aug 2.

Human and Animal Physiology, Wageningen University and Research, Wageningen, Netherlands.

Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.
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http://dx.doi.org/10.1002/jimd.12296DOI Listing
August 2020

A newborn screening approach to diagnose 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

JIMD Rep 2020 Jul 14;54(1):79-86. Epub 2020 Apr 14.

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc Olomouc Czech Republic.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed-phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age-matched controls, we found 3-methylglutaconic acid and 3-hydroxy-3-methylglutaric acid, together with 3-hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first-tier NBS analysis to screen for elevated 3-hydroxyisovalerylcarnitine. In a positive case, a second-tier analysis of 3-hydroxy-3-methylglutaric acid and 3-methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second-tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.
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http://dx.doi.org/10.1002/jmd2.12118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358667PMC
July 2020

Subclinical effects of long-chain fatty acid β-oxidation deficiency on the adult heart: A case-control magnetic resonance study.

J Inherit Metab Dis 2020 09 5;43(5):969-980. Epub 2020 Jun 5.

Department of Radiology and Nuclear Medicine, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.

Cardiomyopathy can be a severe complication in patients with long-chain fatty acid β-oxidation disorders (LCFAOD), particularly during episodes of metabolic derangement. It is unknown whether latent cardiac abnormalities exist in adult patients. To investigate cardiac involvement in LCFAOD, we used proton magnetic resonance imaging (MRI) and spectroscopy ( H-MRS) to quantify heart function, myocardial tissue characteristics, and myocardial lipid content in 14 adult patients (two with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD); four with carnitine palmitoyltransferase II deficiency (CPT2D); and eight with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD)) and 14 gender-, age-, and BMI-matched control subjects. Examinations included cine MRI, MR tagging, native myocardial T and T mapping, and localized H-MRS at 3 Tesla. Left ventricular (LV) myocardial mass (P = .011) and the LV myocardial mass-to-volume ratio (P = .008) were higher in patients, while ejection fraction (EF) was normal (P = .397). LV torsion was higher in patients (P = .026), whereas circumferential shortening was similar compared with controls (P = .875). LV hypertrophy was accompanied by high myocardial T values (indicative of diffuse fibrosis) in two patients, and additionally a low EF in one case. Myocardial lipid content was similar in patients and controls. We identified subclinical morphological and functional differences between the hearts of LCFAOD patients and matched control subjects using state-of-the-art MR methods. Our results suggest a chronic cardiac disease phenotype and hypertrophic LV remodeling of the heart in LCFAOD, potentially triggered by a mild, but chronic, energy deficiency, rather than by lipotoxic effects of accumulating lipid metabolites.
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http://dx.doi.org/10.1002/jimd.12266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539973PMC
September 2020

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Can Be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates.

Int J Mol Sci 2020 Apr 8;21(7). Epub 2020 Apr 8.

Department of Clinical and Experimental Cardiology, Heart Center, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca concentration ([Ca]) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca] changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca] in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.
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http://dx.doi.org/10.3390/ijms21072589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177397PMC
April 2020

Fibroblast-specific genome-scale modelling predicts an imbalance in amino acid metabolism in Refsum disease.

FEBS J 2020 Dec 31;287(23):5096-5113. Epub 2020 Mar 31.

Systems Medicine of Metabolism and Signalling, Laboratory of Paediatrics, University of Groningen, University Medical Centre Groningen, The Netherlands.

Refsum disease (RD) is an inborn error of metabolism that is characterised by a defect in peroxisomal α-oxidation of the branched-chain fatty acid phytanic acid. The disorder presents with late-onset progressive retinitis pigmentosa and polyneuropathy and can be diagnosed biochemically by elevated levels of phytanate in plasma and tissues of patients. To date, no cure exists for RD, but phytanate levels in patients can be reduced by plasmapheresis and a strict diet. In this study, we reconstructed a fibroblast-specific genome-scale model based on the recently published, FAD-curated model, based on Recon3D reconstruction. We used transcriptomics (available via GEO database with identifier GSE138379), metabolomics and proteomics (available via ProteomeXchange with identifier PXD015518) data, which we obtained from healthy controls and RD patient fibroblasts incubated with phytol, a precursor of phytanic acid. Our model correctly represents the metabolism of phytanate and displays fibroblast-specific metabolic functions. Using this model, we investigated the metabolic phenotype of RD at the genome scale, and we studied the effect of phytanate on cell metabolism. We identified 53 metabolites that were predicted to discriminate between healthy and RD patients, several of which with a link to amino acid metabolism. Ultimately, these insights in metabolic changes may provide leads for pathophysiology and therapy. DATABASES: Transcriptomics data are available via GEO database with identifier GSE138379, and proteomics data are available via ProteomeXchange with identifier PXD015518.
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http://dx.doi.org/10.1111/febs.15292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754141PMC
December 2020

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism.

Biochim Biophys Acta Mol Basis Dis 2020 05 10;1866(5):165720. Epub 2020 Feb 10.

Department of Genetics and Genomic Sciences, Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, Box 1498, New York, NY 10029, USA.

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.
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http://dx.doi.org/10.1016/j.bbadis.2020.165720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146961PMC
May 2020

Nutritional ketosis improves exercise metabolism in patients with very long-chain acyl-CoA dehydrogenase deficiency.

J Inherit Metab Dis 2020 07 5;43(4):787-799. Epub 2020 Feb 5.

Neuroimaging Center, Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, Groningen, The Netherlands.

A maladaptive shift from fat to carbohydrate (CHO) oxidation during exercise is thought to underlie myopathy and exercise-induced rhabdomyolysis in patients with fatty acid oxidation (FAO) disorders. We hypothesised that ingestion of a ketone ester (KE) drink prior to exercise could serve as an alternative oxidative substrate supply to boost muscular ATP homeostasis. To establish a rational basis for therapeutic use of KE supplementation in FAO, we tested this hypothesis in patients deficient in Very Long-Chain acyl-CoA Dehydrogenase (VLCAD). Five patients (range 17-45 y; 4 M/1F) patients were included in an investigator-initiated, randomised, blinded, placebo-controlled, 2-way cross-over study. Patients drank either a KE + CHO mix or an isocaloric CHO equivalent and performed 35 minutes upright cycling followed by 10 minutes supine cycling inside a Magnetic Resonance scanner at individual maximal FAO work rate (fatmax; approximately 40% VO max). The protocol was repeated after a 1-week interval with the alternate drink. Primary outcome measures were quadriceps phosphocreatine (PCr), Pi and pH dynamics during exercise and recovery assayed by in vivo P-MR spectroscopy. Secondary outcomes included plasma and muscle metabolites and respiratory gas exchange recordings. Ingestion of KE rapidly induced mild ketosis and increased muscle BHB content. During exercise at FATMAX, VLCADD-specific plasma acylcarnitine levels, quadriceps glycolytic intermediate levels and in vivo Pi/PCr ratio were all lower in KE + CHO than CHO. These results provide a rational basis for future clinical trials of synthetic ketone ester supplementation therapy in patients with FAO disorders. Trial registration: ClinicalTrials.gov. Protocol ID: NCT03531554; METC2014.492; ABR51222.042.14.
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http://dx.doi.org/10.1002/jimd.12217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384182PMC
July 2020

Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia.

Brain 2019 11;142(11):3382-3397

Manchester Centre for Genomics Medicine, St Mary's Hospital, Manchester University Hospital Foundation Trust, Health Innovation Manchester, Oxford Road, Manchester, UK.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.
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http://dx.doi.org/10.1093/brain/awz291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821184PMC
November 2019

Mild Zellweger syndrome due to functionally confirmed novel PEX1 variants.

J Appl Genet 2020 Feb 18;61(1):87-91. Epub 2019 Oct 18.

Department of Pediatrics, Nutrition and Metabolic Disease, The Children's Memorial Health Institute, Al. Dzieci Polskich 20, 04-730, Warsaw, Poland.

Zellweger spectrum disorders (ZSD) constitute a group of rare autosomal recessive disorders characterized by a defect in peroxisome biogenesis due to mutations in one of 13 PEX genes. The broad clinical heterogeneity especially in late-onset presenting patients and a mild phenotype complicates and delays the diagnostic process. Here, we report a case of mild ZSD, due to novel PEX1 variants. The patient presented with an early hearing loss, bilateral cataracts, and leukodystrophy on magnetic resonance (MR) images. Normal results of serum very-long-chain fatty acids (VLCFA) and phytanic acid were found. Molecular diagnostics were performed to uncover the etiology of the clinical phenotype. Using whole exome sequencing, there have been found two variants in the PEX1 gene-c.3450T>A (p.Cys1150*) and c.1769T>C (p.Leu590Pro). VLCFA measurement in skin fibroblasts and C26:0-lysoPC in dried blood spot therefore was performed. Both results were in line with the diagnosis of ZSD. To conclude, normal results of routine serum VLCFA and branched-chain fatty acid measurement do not exclude mild forms of ZSD. The investigation of C26:0-lysoPC should be included in the diagnostic work-up in patients with cataract, hearing loss, and leukodystrophy on MR images suspected to suffer from ZSD.
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http://dx.doi.org/10.1007/s13353-019-00523-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968987PMC
February 2020

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Am J Hum Genet 2019 09 15;105(3):534-548. Epub 2019 Aug 15.

On behalf of "United for Metabolic Diseases," 1105AZ Amsterdam, the Netherlands; Department of Laboratory Medicine, Translational Metabolic Laboratory, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands. Electronic address:

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.
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http://dx.doi.org/10.1016/j.ajhg.2019.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732527PMC
September 2019

Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose.

Sci Rep 2019 07 19;9(1):10502. Epub 2019 Jul 19.

School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of β-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed.
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http://dx.doi.org/10.1038/s41598-019-46685-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642094PMC
July 2019

Prediction of disease severity in multiple acyl-CoA dehydrogenase deficiency: A retrospective and laboratory cohort study.

J Inherit Metab Dis 2019 09 17;42(5):878-889. Epub 2019 Jul 17.

Division of Metabolic Diseases, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an ultra-rare inborn error of mitochondrial fatty acid oxidation (FAO) and amino acid metabolism. Individual phenotypes and treatment response can vary markedly. We aimed to identify markers that predict MADD phenotypes. We performed a retrospective nationwide cohort study; then developed an MADD-disease severity scoring system (MADD-DS3) based on signs and symptoms with weighed expert opinions; and finally correlated phenotypes and MADD-DS3 scores to FAO flux (oleate and myristate oxidation rates) and acylcarnitine profiles after palmitate loading in fibroblasts. Eighteen patients, diagnosed between 1989 and 2014, were identified. The MADD-DS3 entails enumeration of eight domain scores, which are calculated by averaging the relevant symptom scores. Lifetime MADD-DS3 scores of patients in our cohort ranged from 0 to 29. FAO flux and [U- C]C2-, C5-, and [U- C]C16-acylcarnitines were identified as key variables that discriminated neonatal from later onset patients (all P < .05) and strongly correlated to MADD-DS3 scores (oleate: r = -.86; myristate: r = -.91; [U- C]C2-acylcarnitine: r = -.96; C5-acylcarnitine: r = .97; [U- C]C16-acylcarnitine: r = .98, all P < .01). Functional studies in fibroblasts were found to differentiate between neonatal and later onset MADD-patients and were correlated to MADD-DS3 scores. Our data may improve early prediction of disease severity in order to start (preventive) and follow-up treatment appropriately. This is especially relevant in view of the inclusion of MADD in population newborn screening programs.
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http://dx.doi.org/10.1002/jimd.12147DOI Listing
September 2019

Liver disease predominates in a mouse model for mild human Zellweger spectrum disorder.

Biochim Biophys Acta Mol Basis Dis 2019 10 15;1865(10):2774-2787. Epub 2019 Jun 15.

Amsterdam UMC, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology & Metabolism, the Netherlands. Electronic address:

Zellweger spectrum disorders (ZSDs) are autosomal recessive diseases caused by defective peroxisome assembly. They constitute a clinical continuum from severe early lethal to relatively milder presentations in adulthood. Liver disease is a prevalent symptom in ZSD patients. The underlying pathogenesis for the liver disease, however, is not fully understood. We report a hypomorphic ZSD mouse model, which is homozygous for Pex1-c.2531G>A (p.G844D), the equivalent of the most common pathogenic variant found in ZSD, and which predominantly presents with liver disease. After introducing the Pex1-G844D allele by knock-in, we characterized homozygous Pex1-G844D mice for survival, biochemical parameters, including peroxisomal and mitochondrial functions, organ histology, and developmental parameters. The first 20 post-natal days (P20) were critical for survival of homozygous Pex1-G844D mice (~20% survival rate). Lethality was likely due to a combination of cholestatic liver problems, liver dysfunction and caloric deficit, probably as a consequence of defective bile acid biosynthesis. Survival beyond P20 was nearly 100%, but surviving mice showed a marked delay in growth. Surviving mice showed similar hepatic problems as described for mild ZSD patients, including hepatomegaly, bile duct proliferation, liver fibrosis and mitochondrial alterations. Biochemical analyses of various tissues showed the absence of functional peroxisomes accompanied with aberrant levels of peroxisomal metabolites predominantly in the liver, while other tissues were relatively spared. ur findings show that homozygous Pex1-G844D mice have a predominant liver disease phenotype, mimicking the hepatic pathology of ZSD patients, and thus constitute a good model to study pathogenesis and treatment of liver disease in ZSD patients.
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http://dx.doi.org/10.1016/j.bbadis.2019.06.013DOI Listing
October 2019

A mutation creating an upstream translation initiation codon in SLC22A5 5'UTR is a frequent cause of primary carnitine deficiency.

Hum Mutat 2019 10 3;40(10):1899-1904. Epub 2019 Jul 3.

Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology and Metabolism Research Institute, University of Amsterdam, Amsterdam, The Netherlands.

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.
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http://dx.doi.org/10.1002/humu.23839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790604PMC
October 2019

Corrigendum to "Cardiolipin-deficient cells depend on anaplerotic pathways to ameliorate defective TCA cycle function" [Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1864/5(2019) 654-661].

Biochim Biophys Acta Mol Cell Biol Lipids 2019 Aug 4;1864(8):1183. Epub 2019 May 4.

Department of Biological Sciences, Wayne State University, Detroit, MI 48202, United States of America. Electronic address:

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http://dx.doi.org/10.1016/j.bbalip.2019.04.014DOI Listing
August 2019

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in .

N Engl J Med 2019 04;380(15):1433-1441

From Amsterdam University Medical Centers, University of Amsterdam, Departments of Clinical Chemistry, Pediatrics, and Clinical Genetics, Emma Children's Hospital, Amsterdam Gastroenterology and Metabolism (A.B.P.K., R.L., J.K., J. Meijer, L.A.T., M.T., M.W., R.J.A.W., H.R.W., C.D.M.K.), and United for Metabolic Diseases (A.B.P.K., R.J.A.W., H.R.W., C.D.M.K.), Amsterdam, and the Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht (J.J.F.A.V., J.H.V.), and the Project MinE ALS Sequencing Consortium (J.J.F.A.V., J.H.V.), Utrecht - all in the Netherlands; the Departments of Biochemistry and Molecular Biology and Medical Genetics, Cumming School of Medicine, and Alberta Children's Hospital Research Institute, University of Calgary, Calgary (M.T.-G.), Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute (P.A.R., M.J.J., M.S.K., J. MacIsaac, W.W.W., C.D.M.K.), the Faculty of Pharmaceutical Sciences (B.I.D., G.E.B.W., C.J.R.), and the Departments of Medical Genetics (C.M., I.-S.R.-B., W.W.W.) and Pediatrics (C.D.M.K.), University of British Columbia, Vancouver, the Zebrafish Centre for Advanced Drug Discovery, St. Michael's Hospital and University of Toronto (K.B.-A., F.K., M.L., Y.W., X.-Y.W.), the Centre for Applied Genomics, Genetics and Genome Biology, the Hospital for Sick Children (C.N., S.W.S., B.T., R.K.C.Y.), and the Department of Molecular Genetics (C.N., S.W.S., R.K.C.Y.), the McLaughlin Centre (S.W.S.), and the Departments of Medicine, Physiology, and Laboratory Medicine and Pathobiology, Institute of Medical Science (X.-Y.W.), University of Toronto, Toronto, and the Division of Medical Genetics, Department of Pediatrics, Children's Hospital Eastern Ontario, University of Ottawa, Ottawa (J.S.W., M.T.G.) - all in Canada; the Departments of Medicine and Physiology, National University of Singapore (M.A.P.), and the Translational Laboratory in Genetic Medicine, Agency for Science, Technology, and Research (M.A.P., B.S., X.X., J.Z.) - both in Singapore; Uppsala University, Department of Chemistry-Biomedical Center, Uppsala, Sweden (D.D.); Illumina, San Diego, CA (E.D., M.A.E.); Gene Structure and Disease Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD (B.H., D.K., K.U.); and the Department of Clinical Inherited Metabolic Disorders, Birmingham Children's Hospital, Birmingham, United Kingdom (S.S.).

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase () that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.
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http://dx.doi.org/10.1056/NEJMoa1806627DOI Listing
April 2019

An UPLC-MS/MS Assay to Measure Glutathione as Marker for Oxidative Stress in Cultured Cells.

Metabolites 2019 Mar 5;9(3). Epub 2019 Mar 5.

Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry, Amsterdam UMC, Location AMC, University of Amsterdam, 1105 Amsterdam, The Netherlands.

Oxidative stress plays a role in the onset and progression of a number of diseases, such as Alzheimer's disease, diabetes and cancer, as well as ageing. Oxidative stress is caused by an increased production of reactive oxygen species and reduced antioxidant activity, resulting in the oxidation of glutathione. The ratio of reduced to oxidised glutathione is often used as a marker of the redox state in the cell. Whereas a variety of methods have been developed to measure glutathione in blood samples, methods to measure glutathione in cultured cells are scarce. Here we present a protocol to measure glutathione levels in cultured human and yeast cells using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC⁻MS/MS).
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http://dx.doi.org/10.3390/metabo9030045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468779PMC
March 2019

Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Mitochondrion 2019 07 22;47:298-308. Epub 2019 Feb 22.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands; Amsterdam UMC, University of Amsterdam, Surgical Laboratory, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Electronic address:

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.
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http://dx.doi.org/10.1016/j.mito.2019.02.005DOI Listing
July 2019

The cholic acid extension study in Zellweger spectrum disorders: Results and implications for therapy.

J Inherit Metab Dis 2019 03 21;42(2):303-312. Epub 2019 Feb 21.

Department of Pediatric Neurology, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.

Introduction: Currently, no therapies are available for Zellweger spectrum disorders (ZSDs), a group of genetic metabolic disorders characterised by a deficiency of functional peroxisomes. In a previous study, we showed that oral cholic acid (CA) treatment can suppress bile acid synthesis in ZSD patients and, thereby, decrease plasma levels of toxic C -bile acid intermediates, one of the biochemical abnormalities in these patients. However, no effect on clinically relevant outcome measures could be observed after 9 months of CA treatment. It was noted that, in patients with advanced liver disease, caution is needed because of possible hepatotoxicity.

Methods: An extension study of the previously conducted pretest-posttest design study was conducted including 17 patients with a ZSD. All patients received oral CA for an additional period of 12 months, encompassing a total of 21 months of treatment. Multiple clinically relevant parameters and markers for bile acid synthesis were assessed after 15 and 21 months of treatment.

Results: Bile acid synthesis was still suppressed after 21 months of CA treatment, accompanied with reduced levels of C -bile acid intermediates in plasma. These levels significantly increased again after discontinuation of CA. No significant changes were found in liver tests, liver elasticity, coagulation parameters, fat-soluble vitamin levels or body weight.

Conclusions: Although CA treatment did lead to reduced levels of toxic C -bile acid intermediates in ZSD patients without severe liver fibrosis or cirrhosis, no improvement of clinically relevant parameters was observed after 21 months of treatment. We discuss the implications for CA therapy in ZSD based on these results.
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http://dx.doi.org/10.1002/jimd.12042DOI Listing
March 2019

Impact of newborn screening for very-long-chain acyl-CoA dehydrogenase deficiency on genetic, enzymatic, and clinical outcomes.

J Inherit Metab Dis 2019 05 8;42(3):414-423. Epub 2019 Apr 8.

Department of Metabolic Diseases, Dutch Fatty Acid Oxidation Expertise Center, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

Most infants with very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) identified by newborn screening (NBS) are asymptomatic at the time of diagnosis and remain asymptomatic. If this outcome is due to prompt diagnosis and initiation of therapy, or because of identification of individuals with biochemical abnormalities who will never develop symptoms, is unclear. Therefore, a 10-year longitudinal national cohort study of genetically confirmed VLCADD patients born before and after introduction of NBS was conducted. Main outcome measures were clinical outcome parameters, acyl-CoA dehydrogenase very long chain gene analysis, VLCAD activity, and overall capacity of long-chain fatty acid oxidation (LC-FAO flux) in lymphocytes and cultured skin fibroblasts. Median VLCAD activity in lymphocytes of 54 patients, 21 diagnosed pre-NBS and 33 by NBS was, respectively, 5.4% (95% confidence interval [CI]: 4.0-8.3) and 12.6% (95% CI: 10.7-17.7; P < 0.001) of the reference mean. The median LC-FAO flux was 33.2% (95% CI: 22.8-48.3) and 41% (95% CI: 40.8-68; P < 0.05) of the control mean, respectively. Clinical characteristics in 23 pre-NBS and 37 NBS patients revealed hypoglycemic events in 12 vs 2 patients, cardiomyopathy in 5 vs 4 patients and myopathy in 14 vs 3 patients. All patients with LC-FAO flux <10% developed symptoms. Of the patients with LC-FAO flux >10% 7 out of 12 diagnosed pre-NBS vs none by NBS experienced hypoglycemic events. NBS has a clear beneficial effect on the prevention of hypoglycemic events in patients with some residual enzyme activity, but does not prevent hypoglycemia nor cardiac complications in patients with very low residual enzyme activity. The effect of NBS on prevalence and prevention of myopathy-related complications remains unclear.
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http://dx.doi.org/10.1002/jimd.12075DOI Listing
May 2019