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The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents – electrons – into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate. Inherited bi-allelic pathogenic variants in five of the seven components of the MAS have been described hitherto and cause a wide spectrum of symptoms including early-onset epileptic encephalopathy. This review provides an overview of reported patients suffering from MAS deficiencies. In addition, we give an overview of diagnostic procedures and research performed on patient-derived cellular models and tissues. Current cellular models are briefly discussed and novel ways to achieve a better understanding of MAS deficiencies are highlighted. [ABSTRACT FROM AUTHOR]

Correct identification and quantification of different sterol biomarkers can be used as a first-line diagnostic approach for inherited metabolic disorders (IMD). The main drawbacks of current methodologies are related to lack of selectivity and sensitivity for some of these compounds. To address this, we developed and validated two sensitive and selective assays for quantification of six cholesterol biosynthesis pathway intermediates (total amount (free and esterified form) of 7-dehydrocholesterol (7-DHC), 8-dehydrocholesterol (8-DHC), desmosterol, lathosterol, lanosterol and cholestanol), two phytosterols (total amount (free and esterified form) of campesterol and sitosterol) and free form of two oxysterols (7-ketocholesterol (7-KC) and 3β,5α,6β-cholestane-triol (C-triol). For quantification of four cholesterol intermediates we based our analytical approach on sterol derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). Quantification of all analytes is performed using UPLC coupled to an Orbitrap high resolution mass spectrometry (HRMS) system, with detection of target ions through full scan acquisition using positive atmospheric pressure chemical ionization (APCI) mode. UPLC and MS parameters were optimized to achieve high sensitivity and selectivity. Analog stable isotope labeled for each compound was used for proper quantification and correction for recovery, matrix effects and process efficiency. Precision (2.4%–12.3% inter-assay variation), lower limit of quantification (0.027 nM–50.5 nM) and linearity (5.5 μM (R2 0.999) – 72.3 μM (R2 0.997)) for phyto- and oxysterols were determined. The diagnostic potential of these two assays in a cohort of patients (n = 31, 50 samples) diagnosed with IMD affecting cholesterol and lysosomal/peroxisomal homeostasis is demonstrated. [Display omitted] • Quantification of sterols in plasma is important in the field of IMD. • UPLC-Orbitrap-HRMS methods were developed and validated. • PTAD derivatization was used for quantification of desmosterol, lanosterol, 7/8-DHC. • Developed methods are highly sensitive, selective and need 100–125 μL plasma. • 190 control samples and 50 samples from patients with IMD were studied. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. Unlabelled Image [ABSTRACT FROM AUTHOR]

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. 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. 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. We show evidence for the presence of PL reductase activity in humans. Further studies are needed to identify the responsible protein. 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. • Pyridoxine strongly accumulates in CSF of PLP-treated patients, thus suggesting the existence of a PN-forming enzyme; • Our in vitro studies show that all mammalian cell lines tested reduce PL to PN in a time- and dose-dependent manner; • We expand the number of enzymes with a role in human vitamin B6 metabolism; • PL reductase(s) may play an important role in limiting the intracellular accumulation of PL and PLP. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. Unlabelled Image [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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). 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. By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome. • A hypermorphic GLS variant in combination with incremental GLS inhibition results in different glutamine/glutamate ratios. • DI-HRMS provides an in-depth metabolic fingerprint recapitulating part of the metabolic consequences of enhanced GLS activity • High GLS activity solely is sufficient to cause a truncated TCA cycle. • High GLS activity may negatively impact pyrimidine metabolism. [ABSTRACT FROM AUTHOR]

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 6. 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. Unlabelled Image • PNPO deficient zebrafish (Danio rerio) and HEK293 cell line were generated using CRISPR/Cas9 gene editing technology. • PNPO deficiency resulted in strong shortening of the survival and in development of fatal spontaneous seizures. • PLP treatment increased survival and normalized PLP, pyridoxal, 4-pyridoxic acid, GABA, glutamate and glycine levels. • PLP treatment did not prevent accumulation of pyridoxamine, pyridoxamine 5'-phosphate and several essential amino acids. • Accumulation of pyridoxamine 5'-phosphate may play a role in the residual clinical phenotype of PNPO deficiency. [ABSTRACT FROM AUTHOR]

The constant replenishment of tricarboxylic acid (TCA) cycle intermediates, or anaplerosis, is crucial to ensure optimal TCA cycle activity in times of high biosynthetic demand. In inborn metabolic diseases, anaplerosis is often affected, leading to impaired TCA cycle flux and ATP production. In these cases, anaplerotic compounds can be a therapy option. Triheptanoin, a triglyceride containing three heptanoate chains, is thought to be anaplerotic through production of propionyl- and acetyl-CoA. However, the precise mechanism underlying its anaplerotic action remains poorly understood. In this study, we performed a comprehensive in vitro analysis of heptanoate metabolism and compared it to that of octanoate, an even-chain fatty acid which only provides acetyl-CoA. Using stable isotope tracing, we demonstrate that both heptanoate and octanoate contribute carbon to the TCA cycle in HEK293T cells, confirming direct anaplerosis. Furthermore, by using labeled glucose and glutamine, we show that heptanoate and octanoate decrease the contribution of glucose-derived carbon and increase the influx of glutamine-derived carbon into the TCA cycle. Our findings also point towards a change in redox homeostasis, indicated by an increased NAD + /NADH ratio, accompanied by a decreased lactate/pyruvate ratio and increased de novo serine biosynthesis. Taken together, these results highlight the broad metabolic effects of heptanoate and octanoate supplementation, suggesting that therapeutic efficacy may strongly depend on specific disease pathophysiology. Furthermore, they underline the need for careful selection of fatty acid compound and concentration to optimize anaplerotic action., Competing Interests: Conflicts of Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Aminoacyl-tRNA synthetases (ARSs) couple tRNAs with their corresponding amino acids. While ARSs can bind structurally similar amino acids, extreme specificity is ensured by subsequent editing activity. Yet, we found that upon isoleucine (I) restriction, healthy fibroblasts consistently incorporated valine (V) into proteins at isoleucine codons, resulting from misacylation of tRNAIle with valine by wildtype IARS1. Using a dual-fluorescent reporter of translation, we found that valine supplementation could fully compensate for isoleucine depletion and restore translation to normal levels in healthy, but not IARS1 deficient cells. Similarly, the antiproliferative effects of isoleucine deprivation could be fully restored by valine supplementation in healthy, but not IARS1 deficient cells. This indicates I > V substitutions help prevent translational termination and maintain cellular function in human primary cells during isoleucine deprivation. We suggest that this is an example of a more general mechanism in mammalian cells to preserve translational speed at the cost of translational fidelity in response to (local) amino acid deficiencies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell-derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients' lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.

Mitapivat is an investigational, oral, small-molecule allosteric activator of pyruvate kinase (PK). PK is a regulatory glycolytic enzyme that is key in providing the red blood cell (RBC) with sufficient amounts of adenosine triphosphate (ATP). In sickle cell disease (SCD), decreased 2,3-DPG levels increase the oxygen affinity of hemoglobin, thereby preventing deoxygenation and polymerization of sickle hemoglobin. The PK activator mitapivat has been shown to decrease levels of 2,3-DPG and increase levels of ATP in RBCs in patients with SCD. In this phase 2, investigator-initiated, open-label study (https://www.clinicaltrialsregister.eu/ NL8517; EudraCT 2019-003438-18), untargeted metabolomics was used to explore the overall metabolic effects of 8-week treatment with mitapivat in the dose-finding period. In total, 1773 unique metabolites were identified in dried blood spots of whole blood from ten patients with SCD and 42 healthy controls (HCs). The metabolic phenotype of patients with SCD revealed alterations in 139/1773 (7.8%) metabolites at baseline when compared to HCs (false discovery rate-adjusted p  < 0.05), including increases of (derivatives of) polyamines, purines, and acyl carnitines. Eight-week treatment with mitapivat in nine patients with SCD altered 85/1773 (4.8%) of the total metabolites and 18/139 (12.9%) of the previously identified altered metabolites in SCD (unadjusted p  < 0.05). Effects were observed on a broad spectrum of metabolites and were not limited to glycolytic intermediates. Our results show the relevance of metabolic profiling in SCD, not only to unravel potential pathophysiological pathways and biomarkers in multisystem diseases but also to determine the effect of treatment., Competing Interests: Minke A. E. Rab and Richard van Wijk received research funding from Axcella Health Inc. and Pfizer. Eduard J. van Beers and Richard van Wijk are consultants for Pfizer. Minke A. E. Rab, Eduard J. van Beers, and Richard van Wijk received research funding and are consultants for Agios Pharmaceuticals Inc. M.H.C's institution has received investigator‐initiated research and travel grants as well as speaker fees over the years from the Netherlands Organisation for Scientific Research (NWO) and Netherlands National Research Agenda (NWA), the Netherlands Organization for Health Research and Development (ZonMw), the Dutch Innovatiefonds Zorgverzekeraars, Baxter/Baxalta/Shire/Takeda, Pfizer, Bayer Schering Pharma, CSL Behring, Sobi Biogen, Novo Nordisk, Novartis, and Nordic Pharma, and for serving as a steering board member for Roche, Bayer, and Novartis for which fees go to the Erasmus MC as an institution. Erfan Nur receives research funding from Novartis and Emmaus and participates in the advisory board of Novartis. Bart J. Biemond received research funding from Sanquin, Global Blood Therapeutics/Pfizer, and Novartis and participated in advisory boards of Novartis, Bristol‐Myers Squibb/Celgene, Global Blood Therapeutics/Pfizer, Novo Nordisk, and CSL Behring. The remaining authors declare no competing financial interests., (© 2024 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13 C and/or 15 N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD ( n  = 9 SCA, n  = 5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p  < 0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p  = 0.0012), 9.6% (2.9%-15.1%; p  = 0.0013), and 13.3% (5.7%-25.5%; p  = 0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p  = 0.4324), 58% (48%-72%; p  = 0.0185), and 71% (49%-82%; p  = 0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD., Competing Interests: R.v.W. and M.R. received research funding from Axcella Therapeutics and Pfizer. E.v.B. and R.v.W. are consultants for Pfizer. E.v.B., R.v.W., and M.R. receive research funding and are consultants for Agios Pharmaceuticals Inc. M.J.K. and R.W. are employees of Axcella Therapeutics. The remaining authors declare no competing financial interests., (© 2024 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway.

Two siblings showed increased galactose and galactose-related metabolites in neonatal screening. Diagnostic workup did not reveal abnormalities in any of the known disease-causing enzymes involved in galactose metabolism. Using whole-exome sequencing, we identified a homozygous missense variant in PPA1 encoding the cytosolic pyrophosphatase 1 (PPA1), c.557C>T (p.Thr186Ile). The enzyme activity of PPA1 was determined using a colorimetric assay, and the protein content was visualized via western blotting in skin fibroblasts from one of the affected individuals. The galactolytic activity of the affected fibroblasts was determined by measuring extracellular acidification with a Seahorse XFe96 analyzer. PPA1 activity decreased to 22% of that of controls in the cytosolic fraction of homogenates from patient fibroblasts. PPA1 protein content decreased by 50% according to western blot analysis, indicating a reduced stability of the variant protein. The extracellular acidification rate was reduced in patient fibroblasts when galactose was used as a substrate. Untargeted metabolomics of blood samples revealed an elevation of other metabolites related to pyrophosphate metabolism. Besides hyperbilirubinemia in the neonatal period in one child, both children were clinically unremarkable at the ages of 3 and 14 years, respectively. We hypothesize that the observed metabolic derangement is a possible mild manifestation of PPA1 deficiency. Unresolved abnormalities in galactosemia screening might result in the identification of more individuals with PPA1 deficiency, a newly discovered inborn metabolic disorder (IMD).

Background: Early diagnosis of inherited metabolic diseases (IMDs) is important because treatment may lead to reduced mortality and improved prognosis. Due to their diversity, it is a challenge to diagnose IMDs in time, effecting an emerging need for a comprehensive test to acquire an overview of metabolite status. Untargeted metabolomics has proven its clinical potential in diagnosing IMDs, but is not yet widely used in genetic metabolic laboratories. Methods: We assessed the potential role of plasma untargeted metabolomics in a clinical diagnostic setting by using direct infusion high resolution mass spectrometry (DI-HRMS) in parallel with traditional targeted metabolite assays. We compared quantitative data and qualitative performance of targeted versus untargeted metabolomics in patients suspected of an IMD ( n = 793 samples) referred to our laboratory for 1 year. To compare results of both approaches, the untargeted data was limited to polar metabolites that were analyzed in targeted plasma assays. These include amino acid, (acyl)carnitine and creatine metabolites and are suitable for diagnosing IMDs across many of the disease groups described in the international classification of inherited metabolic disorders (ICIMD). Results: For the majority of metabolites, the concentrations as measured in targeted assays correlated strongly with the semi quantitative Z-scores determined with DI-HRMS. For 64/793 patients, targeted assays showed an abnormal metabolite profile possibly indicative of an IMD. In 55 of these patients, similar aberrations were found with DI-HRMS. The remaining 9 patients showed only marginally increased or decreased metabolite concentrations that, in retrospect, were most likely to be clinically irrelevant. Illustrating its potential, DI-HRMS detected additional patients with aberrant metabolites that were indicative of an IMD not detected by targeted plasma analysis, such as purine and pyrimidine disorders and a carnitine synthesis disorder. Conclusion: This one-year pilot study showed that DI-HRMS untargeted metabolomics can be used as a first-tier approach replacing targeted assays of amino acid, acylcarnitine and creatine metabolites with ample opportunities to expand. Using DI-HRMS untargeted metabolomics as a first-tier will open up possibilities to look for new biomarkers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Willems, van der Ham, Schiebergen-Bronkhorst, van Aalderen, de Barse, De Gruyter, van Hoek, Pras-Raves, de Sain-van der Velden, Prinsen, Verhoeven-Duif and Jans.)

Background and Aim: Phenotypic expression of hypertrophic cardiomyopathy (HCM) and disease course are associated with unfavorable metabolic health. We investigated if Western diet (WD) feeding is sufficient to trigger cardiac hypertrophy and dysfunction in heterozygous (HET) Mybpc3 c.772G>A knock-in mice., Methods and Results: Wild-type (WT) and HET mice (3-months-old) were fed a WD or normal chow (NC) for 8 weeks. Metabolomic analyses on serum revealed systemic metabolic derailment in WD-fed WT and HET mice. Strikingly, only WD-fed HET mice developed cardiac hypertrophy and dysfunction, which was not driven by aggravated cardiac myosin binding protein-C haploinsufficiency. WD reduced oxidative phosphorylation and increased toxic lipids in the heart irrespective of genotype. Cardiac proteomic analyses revealed higher abundance of proteins involved in fatty acid oxidation in WD-fed mice, however this increase was blunted in HET compared to WT mice. Accordingly, cardiac metabolomic and lipidomic analyses showed accumulation of acylcarnitines in WD-fed HET vs WT mice., Conclusion: WD feeding triggered cardiac dysfunction and hypertrophy in otherwise phenotype-negative HET Mybpc3 c.772G>A mice. We propose that the presence of a HCM mutation predisposes the heart to metabolic inflexibility when subjected to systemic metabolic stress. Our study represents a novel approach to study the interplay between unfavorable metabolic health and mutation-induced defects in HCM disease development., Competing Interests: All authors report no conflict of interest., (© 2023 The Authors.)

In physiological glucose homeostasis, the liver plays a crucial role in the extraction of glucose from the portal circulation and storage as glycogen to enable release through glycogenolysis upon fasting. In addition, insulin secreted by the pancreas is partly eliminated from the systemic circulation by hepatic first-pass. Therefore, patients with a congenital porto-systemic shunt present a unique combination of (a) postabsorptive hyperinsulinemic hypoglycaemia (HH) because of decreased insulin elimination and (b) fasting (ketotic) hypoglycaemia because of decreased glycogenolysis. Patients with porto-systemic shunts therefore provide important insight into the role of the portal circulation and hepatic function in different phases of glucose homeostasis., Competing Interests: VM consults for Mirum Pharmaceuticals, Albireo, and AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 van Albada, Shah, Derks, Fuchs, Jans, McLin and van der Doef.)

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including Onchocerca volvulus , antileiomodin-1 antibodies, vitamin B 6 deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis, and a hypothetical causal model was constructed using structural equation modelling. Of 607 children with nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio 7.04, 95% confidence interval 2.28-21.7), Necator americanus infection (odds ratio 2.33, 95% confidence interval 1.02-5.3), higher antimalarial seroreactivity (odds ratio 1.75, 95% confidence interval 1.20-2.57), higher vitamin E concentration (odds ratio 1.53 per standard deviation increase, 95% confidence interval 1.07-2.19) and lower vitamin B 12 concentration (odds ratio 0.56 per standard deviation increase, 95% confidence interval 0.36-0.87) were associated with higher odds of nodding syndrome. In a structural equation model, we hypothesized that Mansonella perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of nodding syndrome while lower vitamin B 12 concentration, Necator americanus and malaria infections resulted from having nodding syndrome. We found no evidence that Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B 6 and other factors were associated with nodding syndrome. Our results argue against several previous causal hypotheses including Onchocerca volvulus . Instead, nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect., Competing Interests: The authors report no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Glutaminase (GLS) hyperactivity was first described in 2019 in a patient with profound developmental delay and infantile cataract. Here, we describe a 4-year-old boy with GLS hyperactivity due to a de novo heterozygous missense variant in GLS , detected by trio whole exome sequencing. This boy also exhibits developmental delay without dysmorphic features, but does not have cataract. Additionally, he suffers from epilepsy with tonic clonic seizures. In line with the findings in the previously described patient with GLS hyperactivity, in vivo 3 T magnetic resonance spectroscopy (MRS) of the brain revealed an increased glutamate/glutamine ratio. This increased ratio was also found in urine with UPLC-MS/MS, however, inconsistently. This case indicates that the phenotypic spectrum evoked by GLS hyperactivity may include epilepsy. Clarifying this phenotypic spectrum is of importance for the prognosis and identification of these patients. The combination of phenotyping, genetic testing, and metabolic diagnostics with brain MRS and in urine is essential to identify new patients with GLS hyperactivity and to further extend the phenotypic spectrum of this disease., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

The 22q11.2 deletion syndrome (22q11.2DS) is characterized by a well-defined microdeletion and is associated with increased risk of neurodevelopmental phenotypes including autism spectrum disorders (ASD) and intellectual impairment. The typically deleted region in 22q11.2DS contains multiple genes with the potential of altering metabolism. Deficits in metabolic processes during early brain development may help explain the increased prevalence of neurodevelopmental phenotypes seen in 22q11.2DS. However, relatively little is known about the metabolic impact of the 22q11.2 deletion, while such insight may lead to increased understanding of the etiology. We performed untargeted metabolic analysis in a large sample of dried blood spots derived from 49 22q11.2DS patients and 87 controls, to identify a metabolic signature for 22q11.2DS. We also examined trait-specific metabolomic patterns within 22q11.2DS patients, focusing on intelligence (intelligence quotient, IQ) and ASD. We used the Boruta algorithm to select metabolites distinguishing patients from controls, patients with ASD from patients without, and patients with an IQ score in the lowest range from patients with an IQ score in the highest range. The relevance of the selected metabolites was visualized with principal component score plots, after which random forest analysis and logistic regression were used to measure predictive performance of the selected metabolites. Analysis yielded a distinct metabolic signature for 22q11.2DS as compared to controls, and trait-specific (IQ and ASD) metabolomic patterns within 22q11.2DS patients. The metabolic characteristics of 22q11.2DS provide insights in biological mechanisms underlying the neurodevelopmental phenotype and may ultimately aid in identifying novel therapeutic targets for patients with developmental disorders., (© 2022. The Author(s).)

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

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.

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.

Introduction: Hartnup disorder is caused by a deficiency of the sodium dependent B 0 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 ( p  < .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., Competing Interests: All authors state that they have no competing financial interests to declare. None of the authors accepted any reimbursements, fees or funds from any organization that may in any way gain or lose financially from the results of this review. The authors have not been employed by such an organization. The authors have not act as an expert witness on the subject of the review. The authors do not have any other competing financial interest., (© 2019 The Authors.)

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., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

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.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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.