Publications by authors named "Judith J Jans"

44 Publications

Metabolomics in Severe Aortic Stenosis Reveals Intermediates of Nitric Oxide Synthesis as Most Distinctive Markers.

Int J Mol Sci 2021 Mar 30;22(7). Epub 2021 Mar 30.

Amsterdam UMC, Vrije Universiteit Amsterdam, Physiology, Amsterdam Cardiovascular Sciences, De Boelelaan 1117, 1081 HZ Amsterdam, The Netherlands.

Background: Calcific aortic valve disease (CAVD) is a rapidly growing global health problem with an estimated 12.6 million cases globally in 2017 and a 112% increase of deaths since 1990 due to aging and population growth. CAVD may develop into aortic stenosis (AS) by progressive narrowing of the aortic valve. AS is underdiagnosed, and if treatment by aortic valve replacement (AVR) is delayed, this leads to poor recovery of cardiac function, absence of symptomatic improvement and marked increase of mortality. Considering the current limitations to define the stage of AS-induced cardiac remodeling, there is need for a novel method to aid in the diagnosis of AS and timing of intervention, which may be found in metabolomics profiling of patients.

Methods: Serum samples of nine healthy controls and 10 AS patients before and after AVR were analyzed by untargeted mass spectrometry. Multivariate modeling was performed to determine a metabolic profile of 30 serum metabolites which distinguishes AS patients from controls. Human cardiac microvascular endothelial cells (CMECs) were incubated with serum of the AS patients and then stained for ICAM-1 with Western Blot to analyze the effect of AS patient serum on endothelial cell activation.

Results: The top 30 metabolic profile strongly distinguishes AS patients from healthy controls and includes 17 metabolites related to nitric oxide metabolism and 12 metabolites related to inflammation, in line with the known pathomechanism for calcific aortic valve disease. Nine metabolites correlate strongly with left ventricular mass, of which three show reversal back to control values after AVR. Western blot analysis of CMECs incubated with AS patient sera shows a significant reduction (14%) in ICAM-1 in AS samples taken after AVR compared to AS patient sera before AVR.

Conclusion: Our study defined a top 30 metabolic profile with biological and clinical relevance, which may be used as blood biomarker to identify AS patients in need of cardiac surgery. Future studies are warranted in patients with mild-to-moderate AS to determine if these metabolites reflect disease severity and can be used to identify AS patients in need of cardiac surgery.
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http://dx.doi.org/10.3390/ijms22073569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037707PMC
March 2021

Biallelic variants in HPDL, encoding 4-hydroxyphenylpyruvate dioxygenase-like protein, lead to an infantile neurodegenerative condition.

Genet Med 2021 Mar 14;23(3):524-533. Epub 2020 Nov 14.

Department of Neurosciences, University of California-San Diego, La Jolla, CA, USA.

Purpose: Dioxygenases are oxidoreductase enzymes with roles in metabolic pathways necessary for aerobic life. 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), encoded by HPDL, is an orphan paralogue of 4-hydroxyphenylpyruvate dioxygenase (HPD), an iron-dependent dioxygenase involved in tyrosine catabolism. The function and association of HPDL with human diseases remain unknown.

Methods: We applied exome sequencing in a cohort of over 10,000 individuals with neurodevelopmental diseases. Effects of HPDL loss were investigated in vitro and in vivo, and through mass spectrometry analysis. Evolutionary analysis was performed to investigate the potential functional separation of HPDL from HPD.

Results: We identified biallelic variants in HPDL in eight families displaying recessive inheritance. Knockout mice closely phenocopied humans and showed evidence of apoptosis in multiple cellular lineages within the cerebral cortex. HPDL is a single-exonic gene that likely arose from a retrotransposition event at the base of the tetrapod lineage, and unlike HPD, HPDL is mitochondria-localized. Metabolic profiling of HPDL mutant cells and mice showed no evidence of altered tyrosine metabolites, but rather notable accumulations in other metabolic pathways.

Conclusion: The mitochondrial localization, along with its disrupted metabolic profile, suggests HPDL loss in humans links to a unique neurometabolic mitochondrial infantile neurodegenerative condition.
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http://dx.doi.org/10.1038/s41436-020-01010-yDOI Listing
March 2021

Untargeted metabolic profiling in dried blood spots identifies disease fingerprint for pyruvate kinase deficiency.

Haematologica 2020 Sep 10;Online ahead of print. Epub 2020 Sep 10.

Central Diagnostic Laboratory-Research, University Medical Center Utrecht, Utrecht.

The diagnostic evaluation and clinical characterization of rare hereditary anemia (RHA) is to date still challenging. In particular, there is little knowledge on the broad metabolic impact of many of the molecular defects underlying RHA. In this study we explored the potential of untargeted metabolomics to diagnose a relatively common type of RHA: Pyruvate Kinase Deficiency (PKD). In total, 1903 unique metabolite features were identified in dried blood spot samples from 16 PKD patients and 32 healthy controls. A metabolic fingerprint was identified using a machine learning algorithm, and subsequently a binary classification model was designed. The model showed high performance characteristics (AUC 0.990, 95%CI 0.981-0.999) and an accurate class assignment was achieved for all newly added control (13) and patient samples (6), with the exception of one patient (accuracy 94%). Important metabolites in the metabolic fingerprint included glycolytic intermediates, polyamines and several acyl carnitines. In general, the application of untargeted metabolomics in dried blood spots is a novel functional tool that holds promise for diagnostic stratification and studies on disease pathophysiology in RHA.
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http://dx.doi.org/10.3324/haematol.2020.266957DOI Listing
September 2020

MetaboShiny: interactive analysis and metabolite annotation of mass spectrometry-based metabolomics data.

Metabolomics 2020 09 11;16(9):99. Epub 2020 Sep 11.

Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, STR3.217, PO Box 85060, 3508 AB, Utrecht, The Netherlands.

Direct infusion untargeted mass spectrometry-based metabolomics allows for rapid insight into a sample's metabolic activity. However, analysis is often complicated by the large array of detected m/z values and the difficulty to prioritize important m/z and simultaneously annotate their putative identities. To address this challenge, we developed MetaboShiny, a novel R/RShiny-based metabolomics package featuring data analysis, database- and formula-prediction-based annotation and visualization. To demonstrate this, we reproduce and further explore a MetaboLights metabolomics bioinformatics study on lung cancer patient urine samples. MetaboShiny enables rapid and rigorous analysis and interpretation of direct infusion untargeted mass spectrometry-based metabolomics data.
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http://dx.doi.org/10.1007/s11306-020-01717-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497297PMC
September 2020

Cross-Omics: Integrating Genomics with Metabolomics in Clinical Diagnostics.

Metabolites 2020 May 18;10(5). Epub 2020 May 18.

Section Metabolic Diagnostics, Department of Genetics, University Medical Centre Utrecht, Utrecht University, Lundlaan 6, 3584 EA Utrecht, The Netherlands.

Next-generation sequencing and next-generation metabolic screening are, independently, increasingly applied in clinical diagnostics of inborn errors of metabolism (IEM). Integrated into a single bioinformatic method, these two -omics technologies can potentially further improve the diagnostic yield for IEM. Here, we present cross-omics: a method that uses untargeted metabolomics results of patient's dried blood spots (DBSs), indicated by Z-scores and mapped onto human metabolic pathways, to prioritize potentially affected genes. We demonstrate the optimization of three parameters: (1) maximum distance to the primary reaction of the affected protein, (2) an extension stringency threshold reflecting in how many reactions a metabolite can participate, to be able to extend the metabolite set associated with a certain gene, and (3) a biochemical stringency threshold reflecting paired Z-score thresholds for untargeted metabolomics results. Patients with known IEMs were included. We performed untargeted metabolomics on 168 DBSs of 97 patients with 46 different disease-causing genes, and we simulated their whole-exome sequencing results in silico. We showed that for accurate prioritization of disease-causing genes in IEM, it is essential to take into account not only the primary reaction of the affected protein but a larger network of potentially affected metabolites, multiple steps away from the primary reaction.
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http://dx.doi.org/10.3390/metabo10050206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281020PMC
May 2020

Untargeted Metabolomics for Metabolic Diagnostic Screening with Automated Data Interpretation Using a Knowledge-Based Algorithm.

Int J Mol Sci 2020 Feb 1;21(3). Epub 2020 Feb 1.

Section Metabolic Diagnostics, Department of Genetics, University Medical Centre Utrecht, Utrecht University, Lundlaan 6, 3584 EA Utrecht, The Netherlands.

Untargeted metabolomics may become a standard approach to address diagnostic requests, but, at present, data interpretation is very labor-intensive. To facilitate its implementation in metabolic diagnostic screening, we developed a method for automated data interpretation that preselects the most likely inborn errors of metabolism (IEM). The input parameters of the knowledge-based algorithm were (1) weight scores assigned to 268 unique metabolites for 119 different IEM based on literature and expert opinion, and (2) metabolite Z-scores and ranks based on direct-infusion high resolution mass spectrometry. The output was a ranked list of differential diagnoses (DD) per sample. The algorithm was first optimized using a training set of 110 dried blood spots (DBS) comprising 23 different IEM and 86 plasma samples comprising 21 different IEM. Further optimization was performed using a set of 96 DBS consisting of 53 different IEM. The diagnostic value was validated in a set of 115 plasma samples, which included 58 different IEM and resulted in the correct diagnosis being included in the DD of 72% of the samples, comprising 44 different IEM. The median length of the DD was 10 IEM, and the correct diagnosis ranked first in 37% of the samples. Here, we demonstrate the accuracy of the diagnostic algorithm in preselecting the most likely IEM, based on the untargeted metabolomics of a single sample. We show, as a proof of principle, that automated data interpretation has the potential to facilitate the implementation of untargeted metabolomics for metabolic diagnostic screening, and we provide suggestions for further optimization of the algorithm to improve diagnostic accuracy.
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http://dx.doi.org/10.3390/ijms21030979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037085PMC
February 2020

Misdiagnosis of CTX due to propofol: The interference of total intravenous propofol anaesthesia with bile acid profiling.

J Inherit Metab Dis 2020 07 7;43(4):843-851. Epub 2020 Feb 7.

Department of Metabolic Diseases, University Medical Center Utrecht, Utrecht, Netherlands.

Background: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder, characterised by chronic diarrhoea, xanthomas, cataracts, and neurological deterioration. CTX is caused by CYP27A1 deficiency, which leads to abnormal cholesterol and bile acid metabolism. Urinary bile acid profiling (increased m/z 627: glucuronide-5β-cholestane-pentol) serves as diagnostic screening for CTX. However, this led to a false positive CTX diagnosis in two patients, who had received total intravenous anaesthesia (TIVA) with propofol.

Methods: To determine the influence of propofol on bile acid profiling, 10 urinary samples and 2 blood samples were collected after TIVA with propofol Fresenius 7 to 10 mg/kg/h from 12 subjects undergoing scoliosis correction. Urinary bile acids were analysed using flow injection negative electrospray mass spectrometry. Propofol binding to recombinant CYP27A1, the effects of propofol on recombinant CYP27A1 activity, and CYP27A1 expression in liver organoids were investigated using spectral binding, enzyme activity assays, and qPCR, respectively. Accurate masses were determined with high-resolution mass spectrometry.

Results: Abnormal urinary profiles were identified in all subjects after TIVA, with a trend correlating propofol dose per kilogramme and m/z 627 peak intensity. Propofol only induced a weak CYP27A1 response in the spectral binding assay, minimally affected CYP27A1 activity and did not affect CYP27A1 expression. The accurate mass of m/z 627 induced by propofol differed >10 PPM from m/z 627 observed in CTX.

Conclusions: TIVA with propofol invariably led to a urinary profile misleadingly suggestive of CTX, but not through CYP27A1 inhibition. To avoid further misdiagnoses, propofol administration should be considered when interpreting urinary bile acid profiles.
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http://dx.doi.org/10.1002/jimd.12219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354202PMC
July 2020

Accurate discrimination of Hartnup disorder from other aminoacidurias using a diagnostic ratio.

Mol Genet Metab Rep 2020 Mar 27;22:100551. Epub 2019 Dec 27.

Department of Genetics, Section Metabolic Diagnostics, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Introduction: Hartnup disorder is caused by a deficiency of the sodium dependent B AT1 neutral amino acid transporter in the proximal kidney tubules and jejunum. Biochemically, Hartnup disorder is diagnosed via amino acid excretion patterns. However, these patterns can closely resemble amino acid excretion patterns of generalized aminoaciduria, which may induce a risk for misdiagnosis and preclusion from treatment. Here we explore whether calculating a diagnostic ratio could facilitate correct discrimination of Hartnup disorder from other aminoacidurias.

Methods: 27 amino acid excretion patterns from 11 patients with genetically confirmed Hartnup disorder were compared to 68 samples of 16 patients with other aminoacidurias. Amino acid fold changes were calculated by dividing the quantified excretion values over the upper limit of the age-adjusted reference value.

Results: Increased excretion of amino acids is not restricted to amino acids classically related to Hartnup disorder ("Hartnup amino acids", HAA), but also includes many other amino acids, not classically related to Hartnup disorder ("other amino acids", OAA). The fold change ratio of HAA over OAA was 6.1 (range: 2.4-9.6) in the Hartnup cohort, versus 0.2 (range: 0.0-1.6) in the aminoaciduria cohort ( < .0001), without any overlap observed between the cohorts.

Discussion: Excretion values of amino acids not classically related to Hartnup disorder are frequently elevated in patients with Hartnup disorder, which may cause misdiagnosis as generalized aminoaciduria and preclusion from vitamin B3 treatment. Calculation of the HAA/OAA ratio improves diagnostic differentiation of Hartnup disorder from other aminoacidurias.
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http://dx.doi.org/10.1016/j.ymgmr.2019.100551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938934PMC
March 2020

Retrospective evaluation of the Dutch pre-newborn screening cohort for propionic acidemia and isolated methylmalonic acidemia: What to aim, expect, and evaluate from newborn screening?

J Inherit Metab Dis 2020 05 22;43(3):424-437. Epub 2019 Dec 22.

Section Metabolic Diseases, Department of Child Health, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Utrecht University, Utrecht, The Netherlands.

Evidence for effectiveness of newborn screening (NBS) for propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) is scarce. Prior to implementation in the Netherlands, we aim to estimate the expected health gain of NBS for PA and MMA. In this national retrospective cohort study, the clinical course of 76/83 Dutch PA and MMA patients, diagnosed between January 1979 and July 2019, was evaluated. Five clinical outcome parameters were defined: adverse outcome of the first symptomatic phase, frequency of acute metabolic decompensations (AMD), cognitive function, mitochondrial complications, and treatment-related complications. Outcomes of patients identified by family testing were compared with the outcomes of their index siblings. An adverse outcome due to the first symptomatic phase was recorded in 46% of the clinically diagnosed patients. Outcome of the first symptomatic phase was similar in 5/9 sibling pairs and better in 4/9 pairs. Based on the day of diagnosis of the clinically diagnosed patients and sibling pair analysis, a preliminary estimated reduction of adverse outcome due to the first symptomatic phase from 46% to 36%-38% was calculated. Among the sibling pairs, AMD frequency, cognitive function, mitochondrial, and treatment-related complications were comparable. These results suggest that the health gain of NBS for PA and MMA in overall outcome may be limited, as only a modest decrease of adverse outcomes due to the first symptomatic phase is expected. With current clinical practice, no reduced AMD frequency, improved cognitive function, or reduced frequency of mitochondrial or treatment-related complications can be expected.
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http://dx.doi.org/10.1002/jimd.12193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317354PMC
May 2020

Pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations.

Biochim Biophys Acta Mol Basis Dis 2020 03 21;1866(3):165607. Epub 2019 Nov 21.

Department of Genetics, University Medical Center Utrecht, 3584 EA Utrecht, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, 3584 EA Utrecht, the Netherlands.

Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo zebrafish develop seizures resulting in only 38% of pnpo zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.
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http://dx.doi.org/10.1016/j.bbadis.2019.165607DOI Listing
March 2020

Metabolic fingerprinting reveals extensive consequences of GLS hyperactivity.

Biochim Biophys Acta Gen Subj 2020 03 14;1864(3):129484. Epub 2019 Nov 14.

Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands. Electronic address:

Background: High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways.

Methods: We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS).

Results And Conclusions: Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected.

General Significance: By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome.
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http://dx.doi.org/10.1016/j.bbagen.2019.129484DOI Listing
March 2020

Assessing the Pre-Analytical Stability of Small-Molecule Metabolites in Cerebrospinal Fluid Using Direct-Infusion Metabolomics.

Metabolites 2019 Oct 18;9(10). Epub 2019 Oct 18.

Section Metabolic Diagnostics, Department of Genetics, University Medical Center Utrecht, Utrecht University, Lundlaan 6, 3584 EA Utrecht, The Netherlands.

Metabolomics studies aiming to find biomarkers frequently make use of historical or multicenter cohorts. These samples often have different pre-analytical conditions that potentially affect metabolite concentrations. We studied the effect of different storage conditions on the stability of small-molecule metabolites in cerebrospinal fluid to aid a reliable interpretation of metabolomics data. Three cerebrospinal fluid pools were prepared from surplus samples from the Amsterdam Dementia Cohort biobank. Aliquoted pools were exposed to different storage conditions to assess the temperature and freeze/thaw stability before final storage at -80 °C: storage up to four months at -20 °C and up to one week at either 5-8 °C or 18-22 °C and exposure to up to seven freeze/thaw cycles. Direct-infusion high-resolution mass spectrometry was performed, resulting in the identification of 1852 m/z peaks. To test the storage stability, principal component analyses, repeated measures analysis of variance, Kruskal‒Wallis tests, and fold change analyses were performed, all demonstrating that small-molecule metabolites in the cerebrospinal fluid (CSF) are relatively unaffected by 1‒3 freeze/thaw cycles, by storage at -20 °C up to two months, by storage at 5-8 °C for up to 72 h, or by storage at 18-22 °C for up to 8 h. This suggests that these differences do not affect the interpretation of potential small-molecule biomarkers in multicenter or historical cohorts and implies that these cohorts are suitable for biomarker studies.
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http://dx.doi.org/10.3390/metabo9100236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835587PMC
October 2019

Inborn errors of enzymes in glutamate metabolism.

J Inherit Metab Dis 2020 03 11;43(2):200-215. Epub 2019 Oct 11.

Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Glutamate is involved in a variety of metabolic pathways. We reviewed the literature on genetic defects of enzymes that directly metabolise glutamate, leading to inborn errors of glutamate metabolism. Seventeen genetic defects of glutamate metabolising enzymes have been reported, of which three were only recently identified. These 17 defects affect the inter-conversion of glutamine and glutamate, amino acid metabolism, ammonia detoxification, and glutathione metabolism. We provide an overview of the clinical and biochemical phenotypes of these rare defects in an effort to ease their recognition. By categorising these by biochemical pathway, we aim to create insight into the contributing role of deviant glutamate and glutamine levels to the pathophysiology. For those disorders involving the inter-conversion of glutamine and glutamate, these deviant levels are postulated to play a pivotal pathophysiologic role. For the other IEM however-with the exception of urea cycle defects-abnormal glutamate and glutamine concentrations were rarely reported. To create insight into the clinical consequences of disturbed glutamate metabolism-rather than individual glutamate and glutamine levels-the prevalence of phenotypic abnormalities within the 17 IEM was compared to their prevalence within all Mendelian disorders and subsequently all disorders with metabolic abnormalities notated in the Human Phenotype Ontology (HPO) database. For this, a hierarchical database of all phenotypic abnormalities of the 17 defects in glutamate metabolism based on HPO was created. A neurologic phenotypic spectrum of developmental delay, ataxia, seizures, and hypotonia are common in the inborn errors of enzymes in glutamate metabolism. Additionally, ophthalmologic and skin abnormalities are often present, suggesting that disturbed glutamate homeostasis affects tissues of ectodermal origin: brain, eye, and skin. Reporting glutamate and glutamine concentrations in patients with inborn errors of glutamate metabolism would provide additional insight into the pathophysiology.
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http://dx.doi.org/10.1002/jimd.12180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078983PMC
March 2020

MDH1 deficiency is a metabolic disorder of the malate-aspartate shuttle associated with early onset severe encephalopathy.

Hum Genet 2019 Dec 19;138(11-12):1247-1257. Epub 2019 Sep 19.

Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, 12713, Saudi Arabia.

The reversible oxidation of L-malate to oxaloacetate is catalyzed by NAD(H)-dependent malate dehydrogenase (MDH). MDH plays essential roles in the malate-aspartate shuttle and the tricarboxylic acid cycle. These metabolic processes are important in mitochondrial NADH supply for oxidative phosphorylation. Recently, bi-allelic mutations in mitochondrial MDH2 were identified in patients with global developmental delay, epilepsy and lactic acidosis. We now report two patients from an extended consanguineous family with a deleterious variant in the cytosolic isoenzyme of MDH (MDH1). The homozygous missense variant in the NAD-binding domain of MDH1 led to severely diminished MDH protein expression. The patients presented with global developmental delay, epilepsy and progressive microcephaly. Both patients had normal concentrations of plasma amino acids, acylcarnitines, lactate, and urine organic acids. To identify the metabolic consequences of MDH1 deficiency, untargeted metabolomics was performed on dried blood spots (DBS) from the patients and in MDH1 knockout HEK293 cells that were generated by Crispr/Cas9. Increased levels of glutamate and glycerol-3-phosphate were found in DBS of both patients. In MDH1 KO HEK293 cells, increased levels of glycerol-3-phosphate were also observed, as well as increased levels of aspartate and decreased levels of fumarate. The consistent finding of increased concentrations of glycerol-3-phosphate may represent a compensatory mechanism to enhance cytosolic oxidation of NADH by the glycerol-P-shuttle. In conclusion, MDH1 deficiency is a new metabolic defect in the malate-aspartate shuttle characterized by a severe neurodevelopmental phenotype with elevated concentrations of glycerol-3-phosphate as a potential biomarker.
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http://dx.doi.org/10.1007/s00439-019-02063-zDOI Listing
December 2019

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

N Engl J Med 2019 09;381(12):1185

University Medical Center Utrecht, Utrecht, the Netherlands

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http://dx.doi.org/10.1056/NEJMc1907427DOI Listing
September 2019

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

Aspartylglycosamine is a biomarker for NGLY1-CDDG, a congenital disorder of deglycosylation.

Mol Genet Metab 2019 08 9;127(4):368-372. Epub 2019 Jul 9.

Section Metabolic Diagnostics, Department of Genetics, Utrecht University, University Medical Centre Utrecht, Lundlaan 6, 3584 EA Utrecht, the Netherlands. Electronic address:

Background: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently.

Methods: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases.

Results: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8-8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of -0.1 (range: -2.1-4.0) in DBS of healthy controls and patients with other diseases.

Discussion: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible.
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http://dx.doi.org/10.1016/j.ymgme.2019.07.001DOI Listing
August 2019

Pathophysiology of propionic and methylmalonic acidemias. Part 1: Complications.

J Inherit Metab Dis 2019 09 7;42(5):730-744. Epub 2019 Aug 7.

Section Metabolic Diseases, Department of Child Health, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Utrecht University, Utrecht, The Netherlands.

Over the last decades, advances in clinical care for patients suffering from propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) have resulted in improved survival. These advances were possible thanks to new pathophysiological insights. However, patients may still suffer from devastating complications which largely determine the unsatisfying overall outcome. To optimize our treatment strategies, better insight in the pathophysiology of complications is needed. Here, we perform a systematic data-analysis of cohort studies and case-reports on PA and MMA. For each of the prevalent and rare complications, we summarize the current hypotheses and evidence for the underlying pathophysiology of that complication. A common hypothesis on pathophysiology of many of these complications is that mitochondrial impairment plays a major role. Assuming that complications in which mitochondrial impairment may play a role are overrepresented in monogenic mitochondrial diseases and, conversely, that complications in which mitochondrial impairment does not play a role are underrepresented in mitochondrial disease, we studied the occurrence of the complications in PA and MMA in mitochondrial and other monogenic diseases, using data provided by the Human Phenotype Ontology. Lastly, we combined this with evidence from literature to draw conclusions on the possible role of mitochondrial impairment in each complication. Altogether, this review provides a comprehensive overview on what we, to date, do and do not understand about pathophysiology of complications occurring in PA and MMA and about the role of mitochondrial impairment herein.
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http://dx.doi.org/10.1002/jimd.12129DOI Listing
September 2019

Pathophysiology of propionic and methylmalonic acidemias. Part 2: Treatment strategies.

J Inherit Metab Dis 2019 09 9;42(5):745-761. Epub 2019 Jul 9.

Section Metabolic Diagnostics, Department of Biomedical Genetics, Centre for Molecular Medicine, University Medical Centre Utrecht, Utrecht University, Utrecht, The Netherlands.

Despite realizing increased survival rates for propionic acidemia (PA) and methylmalonic acidemia (MMA) patients, the current therapeutic regimen is inadequate for preventing or treating the devastating complications that still can occur. The elucidation of pathophysiology of these complications allows us to evaluate and rethink treatment strategies. In this review we display and discuss potential therapy targets and we give a systematic overview on current, experimental and unexplored treatment strategies in order to provide insight in what we have to offer PA and MMA patients, now and in the future. Evidence on the effectiveness of treatment strategies is often scarce, since none were tested in randomized clinical trials. This raises concerns, since even the current consensus on best practice treatment for PA and MMA is not without controversy. To attain substantial improvements in overall outcome, gene, mRNA or enzyme replacement therapy is most promising since permanent reduction of toxic metabolites allows for a less strict therapeutic regime. Hereby, both mitochondrial-associated and therapy induced complications can theoretically be prevented. However, the road from bench to bedside is long, as it is challenging to design a drug that is delivered to the mitochondria of all tissues that require enzymatic activity, including the brain, without inducing any off-target effects. To improve survival rate and quality of life of PA and MMA patients, there is a need for systematic (re-)evaluation of accepted and potential treatment strategies, so that we can better determine who will benefit when and how from which treatment strategy.
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http://dx.doi.org/10.1002/jimd.12128DOI Listing
September 2019

Identification of human D lactate dehydrogenase deficiency.

Nat Commun 2019 04 1;10(1):1477. Epub 2019 Apr 1.

Department of Genetics, University Medical Center Utrecht, Utrecht, 3584, CX, The Netherlands.

Phenotypic and biochemical categorization of humans with detrimental variants can provide valuable information on gene function. We illustrate this with the identification of two different homozygous variants resulting in enzymatic loss-of-function in LDHD, encoding lactate dehydrogenase D, in two unrelated patients with elevated D-lactate urinary excretion and plasma concentrations. We establish the role of LDHD by demonstrating that LDHD loss-of-function in zebrafish results in increased concentrations of D-lactate. D-lactate levels are rescued by wildtype LDHD but not by patients' variant LDHD, confirming these variants' loss-of-function effect. This work provides the first in vivo evidence that LDHD is responsible for human D-lactate metabolism. This broadens the differential diagnosis of D-lactic acidosis, an increasingly recognized complication of short bowel syndrome with unpredictable onset and severity. With the expanding incidence of intestinal resection for disease or obesity, the elucidation of this metabolic pathway may have relevance for those patients with D-lactic acidosis.
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http://dx.doi.org/10.1038/s41467-019-09458-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443703PMC
April 2019

Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism.

Biochim Biophys Acta Gen Subj 2019 06 27;1863(6):1088-1097. Epub 2019 Mar 27.

Department of Genetics, University Medical Center Utrecht, Utrecht, the Netherlands. Electronic address:

Background: Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6. Mammals cannot synthesize vitamin B6, so they rely on dietary uptake of the different B6 forms, and via the B6 salvage pathway they interconvert them into PLP. Humans possess three enzymes in this pathway: pyridoxal kinase, pyridox(am)ine phosphate oxidase and pyridoxal phosphatase. Besides these, a fourth enzyme has been described in plants and yeast but not in humans: pyridoxal reductase.

Methods: We analysed B6 vitamers in remnant CSF samples of PLP-treated patients and four mammalian cell lines (HepG2, Caco2, HEK293 and Neuro-2a) supplemented with PL as the sole source of vitamin B6.

Results: Strong accumulation of pyridoxine (PN) in CSF of PLP-treated patients was observed, suggesting the existence of a PN-forming enzyme. Our in vitro studies show that all cell lines reduce PL to PN in a time- and dose-dependent manner. We compared the amino acid sequences of known PL reductases to human sequences and found high homology for members of the voltage-gated potassium channel beta subunits and the human aldose reductases. Pharmacological inhibition and knockout of these proteins show that none of the candidates is solely responsible for PL reduction to PN.

Conclusions: We show evidence for the presence of PL reductase activity in humans. Further studies are needed to identify the responsible protein.

General Significance: This study expands the number of enzymes with a role in B6 salvage pathway. We hypothesize a protective role of PL reductase(s) by limiting the intracellular amount of free PL and PLP.
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http://dx.doi.org/10.1016/j.bbagen.2019.03.019DOI Listing
June 2019

Direct-infusion based metabolomics unveils biochemical profiles of inborn errors of metabolism in cerebrospinal fluid.

Mol Genet Metab 2019 05 15;127(1):51-57. Epub 2019 Mar 15.

Section Metabolic Diagnostics, Department of Biomedical Genetics, Centre for Molecular Medicine, University Medical Centre Utrecht, Utrecht University, Lundlaan 6, 3584 EA Utrecht, The Netherlands. Electronic address:

Background: For inborn errors of metabolism (IEM), metabolomics is performed for three main purposes: 1) development of next generation metabolic screening platforms, 2) identification of new biomarkers in predefined patient cohorts and 3) for identification of new IEM. To date, plasma, urine and dried blood spots are used. We anticipate that cerebrospinal fluid (CSF) holds additional - valuable - information, especially for IEM with neurological involvement. To expand metabolomics to CSF, we here tested whether direct-infusion high-resolution mass spectrometry (DI-HRMS) based non-quantitative metabolomics could correctly capture the biochemical profile of patients with an IEM in CSF.

Methods: Eleven patient samples, harboring eight different IEM, and thirty control samples were analyzed using DI-HRMS. First we assessed whether the biochemical profile of the control samples represented the expected profile in CSF. Next, each patient sample was assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients.

Results: the biochemical profile identified using DI-HRMS in CSF samples resembled the known profile, with - among others - the highest median intensities for mass peaks annotated with glucose, lactic acid, citric acid and glutamine. Subsequent analysis of patient CSF profiles resulted in correct 'most probable diagnoses' for all eleven patients, including non-ketotic hyperglycinaemia, propionic aciduria, purine nucleoside phosphorylase deficiency, argininosuccinic aciduria, tyrosinaemia type I, hyperphenylalaninemia and hypermethioninaemia.

Conclusion: We here demonstrate that DI-HRMS based non-quantitative metabolomics accurately captures the biochemical profile of this set of patients in CSF, opening new ways for using metabolomics in CSF in the metabolic diagnostic laboratory.
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http://dx.doi.org/10.1016/j.ymgme.2019.03.005DOI Listing
May 2019

Direct Infusion Based Metabolomics Identifies Metabolic Disease in Patients' Dried Blood Spots and Plasma.

Metabolites 2019 Jan 11;9(1). Epub 2019 Jan 11.

Section Metabolic Diagnostics, Department of Genetics, University Medical Centre Utrecht, Utrecht University, Lundlaan 6, 3584 EA Utrecht, The Netherlands.

In metabolic diagnostics, there is an emerging need for a comprehensive test to acquire a complete view of metabolite status. Here, we describe a non-quantitative direct-infusion high-resolution mass spectrometry (DI-HRMS) based metabolomics method and evaluate the method for both dried blood spots (DBS) and plasma. 110 DBS of 42 patients harboring 23 different inborn errors of metabolism (IEM) and 86 plasma samples of 38 patients harboring 21 different IEM were analyzed using DI-HRMS. A peak calling pipeline developed in R programming language provided Z-scores for ~1875 mass peaks corresponding to ~3835 metabolite annotations (including isomers) per sample. Based on metabolite Z-scores, patients were assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients. Based on DBS sample analysis, 37/42 of the patients, corresponding to 22/23 IEM, could be correctly assigned a 'most probable diagnosis'. Plasma sample analysis, resulted in a correct 'most probable diagnosis' in 32/38 of the patients, corresponding to 19/21 IEM. The added clinical value of the method was illustrated by a case wherein DI-HRMS metabolomics aided interpretation of a variant of unknown significance (VUS) identified by whole-exome sequencing. In summary, non-quantitative DI-HRMS metabolomics in DBS and plasma is a very consistent, high-throughput and nonselective method for investigating the metabolome in genetic disease.
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http://dx.doi.org/10.3390/metabo9010012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359237PMC
January 2019

The Alkaline Phosphatase (ALPL) Locus Is Associated with B6 Vitamer Levels in CSF and Plasma.

Genes (Basel) 2018 Dec 22;10(1). Epub 2018 Dec 22.

Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles (UCLA), Los Angeles, CA 90095, USA.

The active form of vitamin B6, pyridoxal phosphate (PLP), is essential for human metabolism. The brain is dependent on vitamin B6 for its neurotransmitter balance. To obtain insight into the genetic determinants of vitamin B6 homeostasis, we conducted a genome-wide association study (GWAS) of the B6 vitamers pyridoxal (PL), PLP and the degradation product of vitamin B6, pyridoxic acid (PA). We collected a unique sample set of cerebrospinal fluid (CSF) and plasma from the same healthy human subjects of Dutch ancestry ( = 493) and included concentrations and ratios in and between these body fluids in our analysis. Based on a multivariate joint analysis of all B6 vitamers and their ratios, we identified a genome-wide significant association at a locus on chromosome 1 containing the (alkaline phosphatase) gene (minimal = 7.89 × 10, rs1106357, minor allele frequency (MAF) = 0.46), previously associated with vitamin B6 levels in blood. Subjects homozygous for the minor allele showed a 1.4-times-higher ratio between PLP and PL in plasma, and even a 1.6-times-higher ratio between PLP and PL in CSF than subjects homozygous for the major allele. In addition, we observed a suggestive association with the CSF:plasma ratio of PLP on chromosome 15 (minimal = 7.93 × 10, and MAF = 0.06 for rs28789220). Even though this finding is not reaching genome-wide significance, it highlights the potential of our experimental setup for studying transport and metabolism across the blood⁻CSF barrier. This GWAS of B6 vitamers identifies alkaline phosphatase as a key regulator in human vitamin B6 metabolism in CSF as well as plasma. Furthermore, our results demonstrate the potential of genetic studies of metabolites in plasma and CSF to elucidate biological aspects underlying metabolite generation, transport and degradation.
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http://dx.doi.org/10.3390/genes10010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357176PMC
December 2018

Identification of a Loss-of-Function Mutation in the Context of Glutaminase Deficiency and Neonatal Epileptic Encephalopathy.

JAMA Neurol 2019 03;76(3):342-350

Institute of Human Genetics, University Medical Center Leipzig, Leipzig, Germany.

Importance: The identification and understanding of the monogenic causes of neurodevelopmental disorders are of high importance for personalized treatment and genetic counseling.

Objective: To identify and characterize novel genes for a specific neurodevelopmental disorder characterized by refractory seizures, respiratory failure, brain abnormalities, and death in the neonatal period; describe the outcome of glutaminase deficiency in humans; and understand the underlying pathological mechanisms.

Design, Setting, And Participants: We performed exome sequencing of cases of neurodevelopmental disorders without a clear genetic diagnosis, followed by genetic and bioinformatic evaluation of candidate variants and genes. Establishing pathogenicity of the variants was achieved by measuring metabolites in dried blood spots by a hydrophilic interaction liquid chromatography method coupled with tandem mass spectrometry. The participants are 2 families with a total of 4 children who each had lethal, therapy-refractory early neonatal seizures with status epilepticus and suppression bursts, respiratory insufficiency, simplified gyral structures, diffuse volume loss of the brain, and cerebral edema. Data analysis occurred from October 2017 to June 2018.

Main Outcomes And Measures: Early neonatal epileptic encephalopathy with glutaminase deficiency and lethal outcome.

Results: A total of 4 infants from 2 unrelated families, each of whom died less than 40 days after birth, were included. We identified a homozygous frameshift variant p.(Asp232Glufs*2) in GLS in the first family, as well as compound heterozygous variants p.(Gln81*) and p.(Arg272Lys) in GLS in the second family. The GLS gene encodes glutaminase (Enzyme Commission 3.5.1.2), which plays a major role in the conversion of glutamine into glutamate, the main excitatory neurotransmitter of the central nervous system. All 3 variants probably lead to a loss of function and thus glutaminase deficiency. Indeed, glutamine was increased in affected children (available z scores, 3.2 and 11.7). We theorize that the potential reduction of glutamate and the excess of glutamine were a probable cause of the described physiological and structural abnormalities of the central nervous system.

Conclusions And Relevance: We identified a novel autosomal recessive neurometabolic disorder of loss of function of glutaminase that leads to lethal early neonatal encephalopathy. This inborn error of metabolism underlines the importance of GLS for appropriate glutamine homeostasis and respiratory regulation, signal transduction, and survival.
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http://dx.doi.org/10.1001/jamaneurol.2018.2941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439720PMC
March 2019

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Hum Mol Genet 2019 01;28(1):96-104

Department of Pediatrics, University Medical Center Utrecht, Utrecht University, Utrecht CX, The Netherlands.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.
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http://dx.doi.org/10.1093/hmg/ddy330DOI Listing
January 2019

Vitamin B6 is essential for serine de novo biosynthesis.

J Inherit Metab Dis 2017 11 11;40(6):883-891. Epub 2017 Aug 11.

Section Metabolic Diagnostics, Department of Genetics, University Medical Center Utrecht, KC02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands.

Pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.
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http://dx.doi.org/10.1007/s10545-017-0061-3DOI Listing
November 2017

Quantification of metabolites in dried blood spots by direct infusion high resolution mass spectrometry.

Anal Chim Acta 2017 Aug 26;979:45-50. Epub 2017 Apr 26.

Department of Genetics and Center for Molecular Medicine, UMC Utrecht, Utrecht, The Netherlands. Electronic address:

Diagnosis and treatment of inborn errors of metabolism (IEM) require the analysis of a variety of metabolites. These compounds are usually quantified by targeted platforms. High resolution mass spectrometry (HRMS) has the potential to detect hundreds to thousands of metabolites simultaneously. A chip-based nanoelectrospray source (chip-based nanoESI) enables the direct infusion of biological samples. Major advantages of this system include high sample throughput, no sample carryover, and low sample consumption. The combination, chip-based nanoESI-HRMS enables untargeted metabolomics of biological samples but its potential for quantification of metabolites has not been reported. We investigated whether chip-based nanoESI-HRMS is suitable for quantification of metabolites in dried blood spots (DBS). After addition of internal standards, metabolites were extracted with methanol. Aliquots of each extract were analysed by chip-based nanoESI-HRMS operating in both positive and negative mode with an m/z window of 70-600 and a resolution of 140,000. Total run time was 4.5 min per sample and a full report could be generated within 40 min. Concentrations of all 21 investigated diagnostic metabolites in DBS as quantified by chip-based nanoESI-HRMS correlated well with those obtained by targeted liquid chromatography-tandem mass spectrometry. We conclude that chip-based nanoESI-HRMS is suitable for quantification.
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http://dx.doi.org/10.1016/j.aca.2017.04.038DOI Listing
August 2017

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder.

J Clin Invest 2017 Mar 13;127(3):1005-1018. Epub 2017 Feb 13.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.
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http://dx.doi.org/10.1172/JCI90259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330740PMC
March 2017

Mutations in MDH2, Encoding a Krebs Cycle Enzyme, Cause Early-Onset Severe Encephalopathy.

Am J Hum Genet 2017 Jan 15;100(1):151-159. Epub 2016 Dec 15.

Department of Medical Genetics, National Centre for Mitochondrial Diseases, Nice Teaching Hospital, 06202 Nice, France; Nice Sophia-Antipolis University, CNRS UMR 7284, INSERM U1081, Institute for Research on Cancer and Aging, Nice, 06107 Nice, France. Electronic address:

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children.
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http://dx.doi.org/10.1016/j.ajhg.2016.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223029PMC
January 2017