Publications by authors named "Jos P N Ruiter"

40 Publications

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

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

Mol Genet Metab 2020 03 9;129(3):171-176. Epub 2020 Jan 9.

Department of Pediatrics, Division of Metabolic Disorders, Emma Children's Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Background: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes.

Methods: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [UC]Gal-1-P/ [C]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated.

Results: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001).

Conclusions: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.
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http://dx.doi.org/10.1016/j.ymgme.2020.01.002DOI Listing
March 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

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

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

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

Barth syndrome cells display widespread remodeling of mitochondrial complexes without affecting metabolic flux distribution.

Biochim Biophys Acta Mol Basis Dis 2018 11 1;1864(11):3650-3658. Epub 2018 Sep 1.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Amsterdam Gastroenterology & Metabolism, Amsterdam Cardiovascular Sciences, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Electronic address:

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.
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http://dx.doi.org/10.1016/j.bbadis.2018.08.041DOI Listing
November 2018

Biallelic loss of function variants in COASY cause prenatal onset pontocerebellar hypoplasia, microcephaly, and arthrogryposis.

Eur J Hum Genet 2018 12 8;26(12):1752-1758. Epub 2018 Aug 8.

Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Pontocerebellar hypoplasia (PCH) is a heterogeneous neurodegenerative disorder with a prenatal onset. Using whole-exome sequencing, we identified variants in the gene Coenzyme A (CoA) synthase (COASY) gene, an enzyme essential in CoA synthesis, in four individuals from two families with PCH, prenatal onset microcephaly, and arthrogryposis. In family 1, compound heterozygous variants were identified in COASY: c.[1549_1550delAG]; [1486-3 C>G]. In family 2, all three affected siblings were homozygous for the c.1486-3 C>G variant. In both families, the variants segregated with the phenotype. RNA analysis showed that the c.1486-3 C>G variant leads to skipping of exon 7 with partial retention of intron 7, disturbing the reading frame and resulting in a premature stop codon (p.(Ala496Ilefs*20)). No CoA synthase protein was detected in patient cells by immunoblot analysis and CoA synthase activity was virtually absent. Partial CoA synthase defects were previously described as a cause of COASY Protein-Associated Neurodegeneration (CoPAN), a type of Neurodegeneration and Brain Iron Accumulation (NBIA). Here we demonstrate that near complete loss of function variants in COASY are associated with lethal PCH and arthrogryposis.
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http://dx.doi.org/10.1038/s41431-018-0233-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244412PMC
December 2018

Identification of enzymes involved in oxidation of phenylbutyrate.

J Lipid Res 2017 05 9;58(5):955-961. Epub 2017 Mar 9.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.
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http://dx.doi.org/10.1194/jlr.M075317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408614PMC
May 2017

A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue.

Mol Genet Metab 2016 12 13;119(4):307-310. Epub 2016 Oct 13.

Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Background: N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method.

Methods: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates.

Results: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice.

Conclusion: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
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http://dx.doi.org/10.1016/j.ymgme.2016.10.004DOI Listing
December 2016

Clinical and biochemical characterization of four patients with mutations in ECHS1.

Orphanet J Rare Dis 2015 Jun 18;10:79. Epub 2015 Jun 18.

Departments of Clinical Chemistry and Pediatrics, Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, AZ, 1105, The Netherlands.

Background: Short-chain enoyl-CoA hydratase (SCEH, encoded by ECHS1) catalyzes hydration of 2-trans-enoyl-CoAs to 3(S)-hydroxy-acyl-CoAs. SCEH has a broad substrate specificity and is believed to play an important role in mitochondrial fatty acid oxidation and in the metabolism of branched-chain amino acids. Recently, the first patients with SCEH deficiency have been reported revealing only a defect in valine catabolism. We investigated the role of SCEH in fatty acid and branched-chain amino acid metabolism in four newly identified patients. In addition, because of the Leigh-like presentation, we studied enzymes involved in bioenergetics.

Methods: Metabolite, enzymatic, protein and genetic analyses were performed in four patients, including two siblings. Palmitate loading studies in fibroblasts were performed to study mitochondrial β-oxidation. In addition, enoyl-CoA hydratase activity was measured with crotonyl-CoA, methacrylyl-CoA, tiglyl-CoA and 3-methylcrotonyl-CoA both in fibroblasts and liver to further study the role of SCEH in different metabolic pathways. Analyses of pyruvate dehydrogenase and respiratory chain complexes were performed in multiple tissues of two patients.

Results: All patients were either homozygous or compound heterozygous for mutations in the ECHS1 gene, had markedly reduced SCEH enzymatic activity and protein level in fibroblasts. All patients presented with lactic acidosis. The first two patients presented with vacuolating leukoencephalopathy and basal ganglia abnormalities. The third patient showed a slow neurodegenerative condition with global brain atrophy and the fourth patient showed Leigh-like lesions with a single episode of metabolic acidosis. Clinical picture and metabolite analysis were not consistent with a mitochondrial fatty acid oxidation disorder, which was supported by the normal palmitate loading test in fibroblasts. Patient fibroblasts displayed deficient hydratase activity with different substrates tested. Pyruvate dehydrogenase activity was markedly reduced in particular in muscle from the most severely affected patients, which was caused by reduced expression of E2 protein, whereas E2 mRNA was increased.

Conclusions: Despite its activity towards substrates from different metabolic pathways, SCEH appears to be only crucial in valine metabolism, but not in isoleucine metabolism, and only of limited importance for mitochondrial fatty acid oxidation. In severely affected patients SCEH deficiency can cause a secondary pyruvate dehydrogenase deficiency contributing to the clinical presentation.
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http://dx.doi.org/10.1186/s13023-015-0290-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474341PMC
June 2015

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency.

Genet Med 2015 Dec 2;17(12):989-94. Epub 2015 Apr 2.

Department of Paediatric Gastroenterology and Metabolic Diseases, Wilhelmina Children's Hospital, UMC Utrecht, Utrecht, the Netherlands.

Purpose: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid β-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity.

Methods: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading.

Results: The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05).

Conclusion: Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.
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http://dx.doi.org/10.1038/gim.2015.22DOI Listing
December 2015

Monocarboxylate transporter 1 deficiency and ketone utilization.

N Engl J Med 2014 Nov;371(20):1900-7

From the Division of Pediatrics, Department of Metabolic Diseases (P.M.H., G.V.), and the Division of Pediatrics, Department of Pediatric Gastroenterology (R.H.J.H.), Wilhelmina Children's Hospital, and the Center for Molecular Medicine, Department of Medical Genetics (G.R.M., M.J.G., K.D., M.H., B.Z., J.J.S., N.M.V.-D., G.H.), University Medical Center Utrecht, Utrecht, Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Academic Medical Center, Amsterdam (S.F., J.P.N.R., M.T., R.J.A.W.), the Division of Pediatrics, Department of Metabolic Diseases, and Laboratory Genetic Metabolic Diseases, Maastricht University Medical Center, Maastricht (M.E.R.-G.), and the Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Medical Center, Nijmegen (M.C.V.) - all in the Netherlands; the National Centre for Inherited Metabolic Disorders, Children's University Hospital, Dublin, Ireland (A.A.M.); the Department of Pediatric Metabolism and Nutrition, Gazi University School of Medicine, Ankara, Turkey (I.O.); and the Department of Paediatric Metabolic Medicine, Sheffield Children's Hospital, Sheffield (M.J.S.), the Department of Metabolic Medicine, Great Ormond Street Hospital NHS Foundation Trust, London (M.C.), Chemical Pathology, Department of Laboratory Medicine, Salisbury (N.O.), and the Department of Clinical Biochemistry, Southampton General Hospital, Southampton (V.W.) - all in the United Kingdom.

Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.
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http://dx.doi.org/10.1056/NEJMoa1407778DOI Listing
November 2014

Identification and characterization of Eci3, a murine kidney-specific Δ3,Δ2-enoyl-CoA isomerase.

FASEB J 2014 Mar 16;28(3):1365-74. Epub 2013 Dec 16.

1Department of Genetics and Genomic Sciences, Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mt. Sinai, 1425 Madison Ave., Box 1498, New York, NY 10029, USA.

Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.
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http://dx.doi.org/10.1096/fj.13-240416DOI Listing
March 2014

Impaired amino acid metabolism contributes to fasting-induced hypoglycemia in fatty acid oxidation defects.

Hum Mol Genet 2013 Dec 9;22(25):5249-61. Epub 2013 Aug 9.

Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry.

The importance of mitochondrial fatty acid β-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.
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http://dx.doi.org/10.1093/hmg/ddt382DOI Listing
December 2013

Role of isovaleryl-CoA dehydrogenase and short branched-chain acyl-CoA dehydrogenase in the metabolism of valproic acid: implications for the branched-chain amino acid oxidation pathway.

Drug Metab Dispos 2011 Jul 23;39(7):1155-60. Epub 2011 Mar 23.

Research Institute for Medicines and Pharmaceutical Sciences-iMED.UL, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.

Many biological systems including the oxidative catabolic pathway for branched-chain amino acids (BCAAs) are affected in vivo by valproate therapy. In this study, we investigated the potential effect of valproic acid (VPA) and some of its metabolites on the metabolism of BCAAs. In vitro studies were performed using isovaleryl-CoA dehydrogenase (IVD), isobutyryl-CoA dehydrogenase (IBD), and short branched-chain acyl-CoA dehydrogenase (SBCAD), enzymes involved in the degradation pathway of leucine, valine, and isoleucine. The enzymatic activities of the three purified human enzymes were measured using optimized high-performance liquid chromatography procedures, and the respective kinetic parameters were determined in the absence and presence of VPA and the corresponding CoA and dephosphoCoA conjugates. Valproyl-CoA and valproyl-dephosphoCoA inhibited IVD activity significantly by a purely competitive mechanism with K(i) values of 74 ± 4 and 170 ± 12 μM, respectively. IBD activity was not affected by any of the tested VPA esters. However, valproyl-CoA did inhibit SBCAD activity by a purely competitive mechanism with a K(i) of 249 ± 29 μM. In addition, valproyl-dephosphoCoA inhibited SBCAD activity via a distinct mechanism (K(i) = 511 ± 96 μM) that appeared to be of the mixed type. Furthermore, we show that both SBCAD and IVD are active, using valproyl-CoA as a substrate. The catalytic efficiency of SBCAD turned out to be much higher than that of IVD, demonstrating that SBCAD is the most probable candidate for the first dehydrogenation step of VPA β-oxidation. Our data explain some of the effects of valproate on the branched-chain amino acid metabolism and shed new light on the biotransformation pathway of valproate.
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http://dx.doi.org/10.1124/dmd.110.037606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127238PMC
July 2011

Toxic response caused by a misfolding variant of the mitochondrial protein short-chain acyl-CoA dehydrogenase.

J Inherit Metab Dis 2011 Apr 18;34(2):465-75. Epub 2010 Dec 18.

Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby, Denmark.

Background: Variations in the gene ACADS, encoding the mitochondrial protein short-chain acyl CoA-dehydrogenase (SCAD), have been observed in individuals with clinical symptoms. The phenotype of SCAD deficiency (SCADD) is very heterogeneous, ranging from asymptomatic to severe, without a clear genotype-phenotype correlation, which suggests a multifactorial disorder. The pathophysiological relevance of the genetic variations in the SCAD gene is therefore disputed, and has not yet been elucidated, which is an important step in the investigation of SCADD etiology.

Aim: To determine whether the disease-associated misfolding variant of SCAD protein, p.Arg107Cys, disturbs mitochondrial function.

Methods: We have developed a cell model system, stably expressing either the SCAD wild-type protein or the misfolding SCAD variant protein, p.Arg107Cys (c.319 C > T). The model system was used for investigation of SCAD with respect to expression, degree of misfolding, and enzymatic SCAD activity. Furthermore, cell proliferation and expression of selected stress response genes were investigated as well as proteomic analysis of mitochondria-enriched extracts in order to study the consequences of p.Arg107Cys protein expression using a global approach.

Conclusions: We found that expression of the p.Arg107Cys variant SCAD protein gives rise to inactive misfolded protein species, eliciting a mild toxic response manifested though a decreased proliferation rate and oxidative stress, as shown by an increased demand for the mitochondrial antioxidant SOD2. In addition, we found markers of apoptotic activity in the p.Arg107Cys expressing cells, which points to a possible pathophysiological role of this variant protein.
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http://dx.doi.org/10.1007/s10545-010-9255-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063561PMC
April 2011

Rosuvastatin lowers coenzyme Q10 levels, but not mitochondrial adenosine triphosphate synthesis, in children with familial hypercholesterolemia.

J Pediatr 2011 Mar;158(3):458-62

Department of Pediatrics, Academic Medical Center, Amsterdam, The Netherlands.

Objective: To investigate whether statin therapy affects coenzyme Q10 (CoQ10) status in children with heterozygous familial hypercholesterolemia (FH).

Study Design: Samples were obtained at baseline (treatment naïve) and after dose titration with rosuvastatin, aiming for a low-density lipoprotein cholesterol level of 110 mg/dL. Twenty-nine patients were treated with 5, 10, or 20 mg of rosuvastatin for a mean period of 29 weeks.

Results: We found a significant (32%) decrease in peripheral blood mononuclear cell (PBMC) CoQ10 level (P = .02), but no change in PBMC adenosine triphosphate synthesis (P = .60). Uncorrected plasma CoQ10 values were decreased significantly, by 45% (P < .01). In contrast, ratios of plasma CoQ10/total cholesterol and CoQ10/low-density lipoprotein cholesterol remained equal during treatment.

Conclusions: In children with FH, rosuvastatin causes a significant decrease in cellular PBMC CoQ10 status but does not affect mitochondrial adenosine triphosphate synthesis in children with FH. Further studies should address whether (rare) side effects of statin therapy could be explained by a deterioration in CoQ10 status.
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http://dx.doi.org/10.1016/j.jpeds.2010.08.015DOI Listing
March 2011

The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results.

J Inherit Metab Dis 2010 Oct 20;33(5):479-94. Epub 2010 May 20.

Department of Clinical Chemistry, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Oxidation of fatty acids in mitochondria is a key physiological process in higher eukaryotes including humans. The importance of the mitochondrial beta-oxidation system in humans is exemplified by the existence of a group of genetic diseases in man caused by an impairment in the mitochondrial oxidation of fatty acids. Identification of patients with a defect in mitochondrial beta-oxidation has long remained notoriously difficult, but the introduction of tandem-mass spectrometry in laboratories for genetic metabolic diseases has revolutionalized the field by allowing the rapid and sensitive analysis of acylcarnitines. Equally important is that much progress has been made with respect to the development of specific enzyme assays to identify the enzyme defect in patients subsequently followed by genetic analysis. In this review, we will describe the current state of knowledge in the field of fatty acid oxidation enzymology and its application to the follow-up analysis of positive neonatal screening results.
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http://dx.doi.org/10.1007/s10545-010-9104-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946543PMC
October 2010

Antioxidant dysfunction: potential risk for neurotoxicity in ethylmalonic aciduria.

J Inherit Metab Dis 2010 Jun 5;33(3):211-22. Epub 2010 May 5.

Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, Aarhus N, Denmark.

Mitochondrial dysfunction and oxidative stress are central to the molecular basis of several human diseases associated with neuromuscular disabilities. We hypothesize that mitochondrial dysfunction also contributes to the neuromuscular symptoms observed in patients with ethylmalonic aciduria and homozygosity for ACADS c.625G>A-a common variant of the short-chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) enzyme in the mitochondrial fatty acid oxidation pathway. This study sought to identify the specific factors that initiate cell dysfunction in these patients. We investigated fibroblast cultures from 10 patients with neuromuscular disabilities, elevated levels of ethylmalonic acid (EMA) (>50 mmol/mol creatinine), and ACADS c.625G>A homozygosity. Functional analyses, i.e., ACADS gene and protein expression as well as SCAD enzyme activity measurements, were performed together with a global nano liquid chromatography tandem mass spectroscopy (nano-LC-MS/MS)-based screening of the mitochondrial proteome in patient fibroblasts. Moreover, cell viability of patient fibroblasts exposed to menadione-induced oxidative stress was evaluated. Loss of SCAD function was detected in the patient group, most likely due to decreased ACADS gene expression and/or elimination of misfolded SCAD protein. Analysis of the mitochondrial proteome in patient fibroblasts identified a number of differentially expressed protein candidates, including reduced expression of the antioxidant superoxide dismutase 2 (SOD2). Additionally, patient fibroblasts demonstrated significantly higher sensitivity to oxidative stress than control fibroblasts. We propose that reduced mitochondrial antioxidant capacity is a potential risk factor for ACADS c.625G>A-associated ethylmalonic aciduria and that mitochondrial dysfunction contributes to the neurotoxicity observed in patients.
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http://dx.doi.org/10.1007/s10545-010-9086-6DOI Listing
June 2010

Mitochondrial long chain fatty acid beta-oxidation in man and mouse.

Biochim Biophys Acta 2009 Aug 22;1791(8):806-15. Epub 2009 May 22.

Department of Clinical Chemistry, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, The Netherlands.

Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.
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http://dx.doi.org/10.1016/j.bbalip.2009.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763615PMC
August 2009

Carnitine-palmitoyltransferase 2 deficiency: novel mutations and relevance of newborn screening.

Am J Med Genet A 2008 Nov;146A(22):2925-8

Department of Pediatric Kidney-, Liver- and Metabolic Diseases, Children's Hospital Hannover Medical School, Hannover, Germany.

We report on a newborn male, born at term after an uneventful pregnancy presenting with a pathological acylcarnitine profile in routine newborn screening on the third day of life. The profile showed characteristic elevations of C14:0-, C16:0-, C16:1- and C18:1-acylcarnitines, while the ratio of (C16 + C18:1)/C2 was increased, suggesting CPT2- or carnitine-acylcarnitine-translocase- deficiency. The acylcarnitine profile in blood taken on day 9 was normal with breast milk feeding. No dicarboxylic aciduria was found. In fibroblasts, the activity of CPT2 was decreased to 25%, overall oxidation of the long-chain fatty acids was reduced to 10% of control values. Sequence analysis of the CPT2 gene showed heterozygosity for two previously undescribed mutations in exon 4: c.748-749delAA (truncating), and c.1436A > G (p.Tyr479Cys; missense) mutations. The asymptomatic parents were found to be heterozygous, the mother carries the c.748-749delAA and the father the c.1436A > G mutation. The boy is now 2.5 years old; no clinical symptoms associated with the marked impairment of long-chain fatty acid oxidation have occurred. Confirmation of mitochondrial fatty acid oxidation defects from an initial abnormal newborn-screening by tandem mass spectrometry should include enzyme and, if possible, molecular genetic analysis despite a normal 2nd screening. Biochemical testing of urine (organic acids) may be unrevealing.
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http://dx.doi.org/10.1002/ajmg.a.32545DOI Listing
November 2008

Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation.

Biochim Biophys Acta 2007 Sep 10;1767(9):1126-33. Epub 2007 Jul 10.

Centro de Patogénese Molecular, Unidade de Biologia Molecular e Biopatologia Experimental, (UBMBE) Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.

The effect of the antiepileptic drug valproic acid (VPA) on mitochondrial oxidative phosphorylation (OXPHOS) was investigated in vitro. Two experimental approaches were used, in the presence of selected respiratory-chain substrates: (1) formation of ATP in digitonin permeabilized rat hepatocytes and (2) measurement of the rate of oxygen consumption by polarography in rat liver mitochondria. VPA (0.1-1.0 mM) was found to inhibit oxygen consumption and ATP synthesis under state 3 conditions with glutamate and 2-oxoglutarate as respiratory substrates. No inhibitory effect on OXPHOS was observed when succinate (plus rotenone) was used as substrate. We tested the hypothesis that dihydrolipoyl dehydrogenase (DLDH) might be a direct target of VPA, especially its acyl-CoA intermediates. Valproyl-CoA (0.5-1.0 mM) and valproyl-dephosphoCoA (0.5-1.0 mM) both inhibited the DLDH activity, acting apparently by different mechanisms. The decreased activity of DLDH induced by VPA metabolites may, at least in part, account for the impaired rate of oxygen consumption and ATP synthesis in mitochondria if 2-oxoglutarate or glutamate were used as respiratory substrates, thus limiting the flux of these substrates through the citric acid cycle.
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http://dx.doi.org/10.1016/j.bbabio.2007.06.007DOI Listing
September 2007

Studies on the extra-mitochondrial CoA -ester formation of valproic and Delta4 -valproic acids.

Biochim Biophys Acta 2007 Apr 23;1771(4):533-43. Epub 2007 Jan 23.

UBMBE, Centro de Patogénese Molecular, Faculdade de Farmácia da Universidade de Lisboa, Av Prof Gama Pinto, Lisboa, Portugal.

The hypothesis whether valproic acid (VPA) and its main microsomal metabolite, Delta(4)-valproic acid, can be activated to the respective CoA esters in the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differential centrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA and cofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix as believed until now. Furthermore, the activation of Delta(4)-VPA is demonstrated in vitro. Valproyl-CoA and Delta(4)-valproyl-CoA were detected after in vitro incubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoA was characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants (K(m)(app)) were estimated for the reaction substrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extra-mitochondrial VPA activating enzymes. The present findings suggest additional mechanisms of mitochondrial dysfunction associated with VPA, and they may contribute to the further understanding of the toxic effects associated with this drug.
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http://dx.doi.org/10.1016/j.bbalip.2007.01.010DOI Listing
April 2007

Single-base substitution at the last nucleotide of exon 6 (c.671G>A), resulting in the skipping of exon 6, and exons 6 and 7 in human succinyl-CoA:3-ketoacid CoA transferase (SCOT) gene.

Mol Genet Metab 2007 Mar 13;90(3):291-7. Epub 2006 Dec 13.

Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1194, Japan.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT, EC 2.8.3.5) is the key enzyme for ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We identified a homozygous point mutation (c.671G>A) , which is a single-base substitution at the last nucleotide of exon 6, in a Turkish patient (GS12) with SCOT deficiency. This point mutation resulted in the skipping of exon 6, and exons 6 and 7 in human SCOT genes. To understand why the c.671G>A causes exons 6 and 7 skipping, nuclear RNA was separated from cytoplasmic RNA and both were analyzed by RT-PCR. In nuclear RNA, SCOT mRNA with exon 6 skipping was predominant and mRNA with exons 6 and 7 skipping was hardly detected, whereas the latter became one of major mRNA species in cytoplasmic RNA. This discrepancy was interpreted as follows: exon 6 skipping causes a frameshift and nonsense-mediated RNA decay in the cytosol, so mRNA with exon 6 skipping was unstable. On the other hand, SCOT mRNA with exons 6 and 7 is a minor transcript but it retains the reading-frame and is stable in cytosol. As a result, the latter mRNA is more abundant under steady-state conditions as compared to the former mRNA.
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http://dx.doi.org/10.1016/j.ymgme.2006.10.010DOI Listing
March 2007

Mutations in the gene encoding 3-hydroxyisobutyryl-CoA hydrolase results in progressive infantile neurodegeneration.

Am J Hum Genet 2007 Jan 30;80(1):195-9. Epub 2006 Nov 30.

Department of Clinical Chemistry and Pediatrics, Emma Children's Hospital, Amsterdam, The Netherlands.

Only a single patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency has been described in the literature, and the molecular basis of this inborn error of valine catabolism has remained unknown until now. Here, we present a second patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency, who was identified through blood spot acylcarnitine analysis showing persistently increased levels of hydroxy-C(4)-carnitine. Both patients manifested hypotonia, poor feeding, motor delay, and subsequent neurological regression in infancy. Additional features in the newly identified patient included episodes of ketoacidosis and Leigh-like changes in the basal ganglia on a magnetic resonance imaging scan. In cultured skin fibroblasts from both patients, the 3-hydroxyisobutyryl-CoA hydrolase activity was deficient, and virtually no 3-hydroxyisobutyryl-CoA hydrolase protein could be detected by western blotting. Molecular analysis in both patients uncovered mutations in the HIBCH gene, including one missense mutation in a conserved part of the protein and two mutations affecting splicing. A carefully interpreted acylcarnitine profile will allow more patients with 3-hydroxyisobutyryl-CoA hydrolase deficiency to be diagnosed.
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http://dx.doi.org/10.1086/510725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1785315PMC
January 2007

An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry.

Mol Genet Metab 2007 Jan 28;90(1):24-9. Epub 2006 Aug 28.

Department of Clinical Chemistry and Pediatrics, Laboratory Genetic Metabolic Diseases (F0-224), Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands.

Carnitine palmitoyl transferase I (CPTI), which converts acyl-CoA and carnitine into acyl-carnitine and free CoASH, is the rate limiting enzyme of hepatic mitochondrial beta-oxidation. CPTI-deficiency is a severe disorder characterized by Reye-like attacks with hypoketotic hypoglycemia, hepatomegaly, elevated liver enzymes and hyperammonemia. We developed a simple tandem-MS-based assay to measure CPTI activity in human fibroblasts. Surprisingly, a large part of the palmitoyl-carnitine formed in our assay by CPTI was degraded into C14- to C2-acyl-carnitines. Degradation of the product of CPTI leads to under estimation of the CPTI activity. When we used potassium cyanide to inhibit enzymes downstream of CPTI and thereby degradation of the product, we measured four times more CPTI activity than the previous methods. This inhibition is essential for correct calculation of CPTI activity. In fibroblasts of CPTI-deficient patients, CPTI activity was not detectable and this assay can be used for the diagnosis of CPTI-deficiency.
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http://dx.doi.org/10.1016/j.ymgme.2006.07.006DOI Listing
January 2007

Variations in IBD (ACAD8) in children with elevated C4-carnitine detected by tandem mass spectrometry newborn screening.

Pediatr Res 2006 Sep 20;60(3):315-20. Epub 2006 Jul 20.

Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby Sygehus, 8200 Aarhus N, Denmark.

The isobutyryl-CoA dehydrogenase (IBD) enzyme is involved in the degradation of valine. IBD deficiency was first reported in 1998 and subsequent genetic investigations identified acyl-CoA dehydrogenase (ACAD) 8, now IBD, as the gene responsible for IBD deficiency. Only three individuals homozygous or compound heterozygous for variations in the IBD gene have been reported. We present IBD deficiency in an additional four newborns with elevated C(4)-carnitine identified by tandem mass spectrometry (MS/MS) screening in Denmark and the United States. Three showed urinary excretions of isobutyryl-glycine, and in vitro probe analysis of fibroblasts from two newborns indicated enzymatic IBD defect. Molecular genetic analysis revealed seven new rare variations in the IBD gene (c.348C>A, c.400G>T, c.409G>A, c.455T>C, c.958G>A, c.1000C>T and c.1154G>A). Furthermore, sequence analysis of the short-chain acyl-CoA dehydrogenase (SCAD) gene revealed heterozygosity for the prevalent c.625G>A susceptibility variation in all newborns and in the first reported IBD patient. Functional studies in isolated mitochondria demonstrated that the IBD variations present in the Danish newborn (c.409G>A and c.958G>A) together with a previously published IBD variation (c.905G>A) disturbed protein folding and reduced the levels of correctly folded IBD tetramers. Accordingly, low/no IBD residual enzyme activity was detectable when the variant IBD proteins were overexpressed in Chang cells.
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http://dx.doi.org/10.1203/01.pdr.0000233085.72522.04DOI Listing
September 2006

Fatty acid oxidation in the human fetus: implications for fetal and adult disease.

J Inherit Metab Dis 2006 Feb;29(1):71-5

Department of Pediatrics, G8-205, Emma Children's Hospital AMC, Academic Medical Centre, PO Box 22660, NL-1100 DD, Amsterdam, The Netherlands.

Studies in the last few years have shown a remarkably high activity of fatty acid oxidation (FAO) enzymes in human placenta. We have recently shown mRNA expression as well as enzymatic activity of long-chain FAO enzymes in the human embryo and fetus. In this study we show activity of the FAO enzymes carnitine palmitoyltranferase 1, medium-chain acyl-CoA dehydrogenase and short-chain hydroxyacyl-CoA dehydrogenase in embryonic and fetal tissues. In addition, we show the presence of different acylcarnitines in fetal liver and kidney, which substantiates the notion that the mitochondrial FAO enzymes are not only present in human fetal tissues but also metabolically active. In a glucose-rich environment FAO might be necessary for additional ATP production from fatty acids, but also for the breakdown of fatty acids that are products of the turnover of membranes in the growing fetus. The importance of FAO in the human embryo and fetus is further stressed by the fact that a higher frequency of prematurity, intrauterine growth retardation, fetal morbidity and intrauterine death is noted in long-chain FAO defects. Furthermore, in animal studies, gestational loss during early embryonic development has been observed as a consequence of disturbed FAO. Finally, there are indications that regulation of activity of FAO during fetal development might not only be important for fetal life but may also have implications for health and disease in adulthood.
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http://dx.doi.org/10.1007/s10545-006-0199-xDOI Listing
February 2006

Real-time nucleic acid sequence-based amplification assay to quantify changes in mitochondrial DNA concentrations in cell cultures and blood cells from HIV-infected patients receiving antiviral therapy.

Clin Chem 2006 Jun 6;52(6):979-87. Epub 2006 Apr 6.

Primagen, Amsterdam, The Netherlands.

Background: To study the clinical relevance of changes in mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) attributable to HIV infection and/or combination antiretroviral therapy (cART), a high-throughput molecular assay to quantify mtDNA is required.

Methods: We developed a quantitative real-time duplex nucleic acid sequence-based amplification assay in which both mtDNA and nuclear DNA are simultaneously amplified in 1 tube. The assay could accurately quantify mtDNA in a range of 15-1500 copies of mtDNA per 2 genomic copies with an intrarun variation of 11% and an interrun variation of 16%. We compared this real-time assay with the lactate/pyruvate ratios in fibroblasts incubated with glucose and exposed to zalcitabine. Additionally, we studied the effects of platelet contamination and the in vivo effects of cART on mtDNA in PBMCs from a small group of patients.

Results: Decreases in mtDNA preceded the increase in lactate/pyruvate ratios and vice versa when zalcitabine was eliminated from the culture. Platelets affected the mtDNA in PBMCs if >5 platelets per PBMC were present. Within 12 weeks, mtDNA increased and remained increased in PBMCs from patients on continuous treatment with zidovudine/lamivudine/indinavir therapy (P = 0.03), but increased if patients were switched to stavudine/didanosine therapy (P = 0.008).

Conclusion: After drug exposure, the mtDNA assay can detect changes in mtDNA concentrations in cell lines and PBMCs, when properly controlled for platelet effects, earlier than traditional assays.
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http://dx.doi.org/10.1373/clinchem.2005.062901DOI Listing
June 2006