New insight into vitamin B6 metabolism and related diseases

Rúben José Jesus Faustino Ramos New insight into vitamin B6 metabolism and related diseases

Nieuw inzicht in het metabolisme van vitamine B6 en aanverwante ziekten

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. H.R.B.M. Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op

donderdag 10 oktober 2019 des ochtends te 10.30 uur

Cover design: Gianluca Di Vincenzo Thesis layout: Guus Gijben door Printed by: Proefschrift AIO

ISBN: 978-94-92801-99-9 Rúben José Jesus Faustino Ramos

© Rúben José Jesus Faustino Ramos, 2019 geboren op 18 april 1984 te Évora, Portugal All rights are reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the prior written permission of the author.

Promotor: TABLE OF CONTENTS Prof. dr. N.M. Verhoeven-Duif

Copromotor: Chapter 1 General introduction and outline of the thesis 7 Dr. J.J.M. Jans Chapter 2 Vitamin B6 is essential for de novo 29

Chapter 3 Biallelic GOT2 mutations cause a treatable malate- 51 aspartate shuttle related encephalopathy

Chapter 4 Metabolic consequences of GOT2 deficiency 91

Chapter 5 Serine biosynthesis flux as diagnostic tool for serine 115 biosynthesis defects

Chapter 6 Discovery of pyridoxal reductase activity as part of 129 human vitamin B6 metabolism

Chapter 7 General Discussion 153

Appendix Nederlandse samenvatting 164 Summary 168 Acknowledgements 172 List of publications 176 Curriculum Vitae 178

Dit proefschrift werd (mede) mogelijk gemaakt met financiële steun van Metakids Foundation. 1

Chapter 1

General introduction General introduction

Vitamin B6

Vitamin B6 refers to six structurally related compounds that have a 2-methyl- 1 3-hydroxypyridine structure in common but have different C4 and C5 chemical moieties: pyridoxal (PL; aldehyde group at C4; -CHO), pyridoxine (PN; alcohol group at C4; -CH2OH), pyridoxamine (PM; amine group at C4; -CH2NH2), and their respective 5’-phosphate esters pyridoxal 5’-phosphate (PLP), pyridoxine 5’-phosphate (PNP) and pyridoxamine 5’-phosphate (PMP) (Snell, 1953). 4-Pyridoxic acid (PA) is the main catabolism of vitamin B6 (Hufft and Perlzweig, 1944) (Figure 1). Figure 1

Pyridoxal Pyridoxine Pyridoxamine Catabolism Product H O HO H2 H2N H2 C C C HO HO HO OH OH OH Pyridoxic acid H C N 3 H3C N H C N 3 HO O C HO Pyridoxal 5’-phosphate Pyridoxine 5’-phosphate Pyridoxamine 5’-phosphate OH H C N H N H 3 H O HO H2 2 2 C O OH C O OH C O OH HO P HO P HO P O OH O OH O OH H C N H C N 3 3 H3C N

Figure 1. Chemical structures of vitamin B6 vitamers. Pyridoxal: aldehyde group at C4; pyridoxine: alcohol group at C4; pyridoxamine: amine group at C4 and the respective 5’-phosphate esters: pyridoxal 5’-phosphate; pyridoxine 5’-phosphate and pyridoxamine 5’-phosphate. Pyridoxic acid (vitamin B6 catabolism product): carboxylic group at C4.

All organisms depend on vitamin B6 for survival, but only microorganisms and plants can synthesize it de novo (Di Salvo, et al., 2011). Humans rely on vitamin B6 uptake from the diet to fulfil their needs. A minor part of the vitamin B6 pool is derived from the intestinal bacterial flora (Surtees, et al., 2006). Vitamin B6 is widely distributed in animal- and plant-derived foods. In animal-derived foods (such as beef, pork, poultry, fish, milk and eggs) it is mainly present as PLP and PMP and in smaller amounts as PL, PM and PN (McCormick, 1989). In plant-derived foods (such as cereals, vegetables and some fruits) vitamin B6 is mainly present as PN, PNP and pyridoxine-5’-β-D- glucoside (PN-glucoside) (McCormick, 1989; Clayton, 2006; Surtees et al., 2006). Pyridoxine (hydrochloride) is the most commonly used vitamer to fortify foods (Bender, 2005). Although humans cannot synthesize vitamin B6 de novo, all vitamers can be interconverted through the vitamin B6 salvage pathway.

9 General introduction

nialderied ood lantderied ood Vitamin B6 metabolism

lucoide Vitamin B6 absorption in the intestine is rapid and occurs after hydrolysis of the 1

Glucoide phosphorylated forms in the intestinal lumen by the membrane-bound intestinal drolee

alkaline (ALPL; EC 3.1.3.1) (Waymire et al., 1995) (Figure 2). PN-glucoside iet and aortion is hydrolyzed to PN by the cytosolic pyridoxine-β-D-glucoside in the intestinal mucosa (Mcmahon et al., 1997). Uptake of the unphosphorylated vitamers was first believed to occur via simple diffusion (Hamm et al., 1979; Mehansho et al.,

1979; Middleton III, 1977; Ink and Henderson, 1984), until studies performed on human- oidae ier derived intestinal epithelial Caco-2 cells and intestinal colonocytes of mice and men

etaoli creted showed the existence of specific carrier-mediated mechanisms for PN uptake (Said et ranainae rine al., 2003; Said et al., 2008). In Caco-2 and young adult mouse colonic epithelial (YAMC) luine ound cells, the carrier-mediated mechanism is specific, Na+ independent, and temperature eoloin ound

+ and pH dependent, suggesting a pyridoxine:H symport mechanism. In addition, PN irculation uptake seems to be regulated by an intracellular protein kinase A (PKA)-mediated e pathway in Caco-2 cells (Said et al., 2003), and by a Ca2+/CaM-mediated pathway in laa

YAMC cells (Said et al., 2008). PN uptake in human colonic apical membrane vesicles

(AMV) is, as described for the other two models, saturable (Said et al., 2008). Although etaoli ntracellular the liver is the main organ responsible for vitamin B6 metabolism, intestinal Caco-2 cells ntercellular enatic reaction possess all involved in B6 metabolism and convert small amounts of PN and PM into PL, secreting all three unphosphorylated B6 vitamers (Albersen et al., 2013). Figure 2. Vitamin B6 absorption and metabolism. The different vitamin B6 vitamers are present The portal circulation delivers PL, PN and PM to the liver and, once inside liver cells, in animal- and plant-derived food sources. Vitamin B6 absorption is rapid and occurs after hydrolysis of the phosphorylated forms (PLP, pyridoxal 5’-phosphate; PNP, pyridoxine 5’-phosphate and PMP, the B6 vitamers are converted to PLP through the salvage pathway (Figure 2). The pyridoxamine 5’-phosphate) in the intestinal lumen by the membrane-bound intestinal alkaline vitamin B6 salvage pathway recycles the different B6 vitamers through the action of phosphatases (ALPL). Inside the cells, PL kinase (PDXK) phosphorylates the hydroxymethyl group of the pyridoxal (PDXP; EC 3.1.3.74), the ATP-dependent pyridoxal kinase PL, PN and PM to their respective 5’-phosphate forms. Dephosphorylation of PLP, PNP and PMP is catalysed by PL phosphatase (PDXP). Aminotransferases use PLP during the interchange of the amino (PDXK; EC 2.7.1.35) and the flavin mononucleotide (FMN)-dependent pyridox(am)ine group between one and an α-keto acid, producing PMP as an intermediary in the first 5′-phosphate oxidase (PNPO; EC 1.4.3.5). PL, PN and PM are phosphorylated to their part of the reaction. PL can be oxidized to pyridoxic acid (PA) by PL oxidase and excreted in the urine. respective 5’-phosphate esters by PDXK, entrapping the phosphorylated B6 vitamers intracellularly (Hanna et al., 1997). PNPO oxidizes PNP and PMP to PLP (Mills et al., 2005). Dephosphorylation of PLP (but also PNP and PMP) is catalysed by pyridoxal phosphatase (PDXP; EC 3.1.3.74) (Jang et al., 2003). These three enzymes provide a mean of converting dietary B6 to circulating PL(P). The main circulating B6 vitamer in blood is PLP bound to albumin (accounting for 60% of the total circulating vitamin B6). PL, PN and PM are present in lower concentrations (Lumeng et al., 1974; Lumeng et al., 1980; Ink and Henderson, 1984; Spinneker et al., 2007).

10 11 General introduction

Vitamin B6 function The role of vitamin B6 in neurotransmitter metabolism

PLP, the metabolically active form of vitamin B6, is an essential in more than γ-Aminobutyric acid (GABA), the key inhibitory neurotransmitter in the central 1 160 -catalysed reactions (Percudani and Peracchi, 2009), representing 4% nervous system, is synthesized from glutamate (the main excitatory neurotransmitter) of all known cellular catalytic activities (Percudani and Peracchi, 2003). Most of the via the PLP-dependent enzyme decarboxylase (GAD, EC 4.1.1.15). PLP-dependent reactions are involved in synthesis, degradation and interconversion For a long time, deficient GABA levels were appointed as the main reason for the of amino acids (Ebadi, 1981; Surtees et al., 2006; Clayton, 2006; Ueland et al., 2015). clinical phenotype observed in vitamin B6 dependent epilepsy (Gospe et al., 1994). In addition, PLP is essential for biosynthesis of neurotransmitters (γ-aminobutyric In line with these observations, studies performed in zebrafish embryos showed that acid, dopamine and serotonin), sphingolipids, heme, histamine, carbohydrates and exposure to ginkgotoxin (4’-O-methylpyridoxine, a PLP antimetabolite) leads to a nucleotides. The special electrophilic characteristics of the aldehyde group of PLP seizure-like behavior. In addition, the ginkgotoxin-induced seizures were reversed at C4 derives from the existence of a protonated pyridinium hydrogen (N1) and a by GABA and/or PLP, supporting the hypothesis that the seizures were caused by phenoxide anion at C3. These stabilize the protonated state of the imine nitrogen reduced PLP, leading to imbalance between GABA and glutamate (Lee et al., 2012). during the formation of the Shiff base between PLP and substrates, while the However, data on glutamate and GABA levels in the CSF of patients suggests that phosphate group at C5 provides the anchoring point to the coenzyme (Di Salvo et GABA deficiency is not the sole cause of symptoms in pyridoxine-dependent epilepsy al., 2011). The unique environment created by the apoenzyme protein determines (Goto et al., 2001; Baumeister et al., 1994). Indeed, vitamin B6-deficient patients the catalytic potential of PLP, the specificifty of the holoenzyme and may also display biochemical features of aromatic L-amino acid decarboxylase the type of reaction (Percudani and Peracchi, 2009). Structurally, PLP-dependent (AADC, EC 4.1.1.28) deficiency. AADC is a PLP-dependent enzyme that catalyzes the enzymes belong to one of five fold types, with presumably independent evolutionary decarboxylation of levodopa and 5-hydroxytrypophan to dopamine and serotonin, lineages (Percudani and Peracchi, 2009). The mechanistic similarities between the respectively (Manegold et al., 2009). Biochemically, vitamin B6-deficient patients different PLP-dependent enzymes and their limited structural diversity, leads to may present with low CSF homovanillic acid (HVA) and 5-hydroxyindoleacetic acid particularly challenging difficulties when attempting to infer the function of these (5-HIAA) (Clayton, 2006; De Roo et al., 2014; Darin et al., 2016); raised CSF , proteins based exclusively on their sequences (Percudani and Peracchi, 2009). 3-ortho-methyldopa, L-Dopa and 5-hydroxytryptophan (Darin et al., 2016) as well as Importantly, the fold type of the enzyme does not determine the type of reactions raised urinary vanillyllactate (Clayton, 2006) (Figure 3). catalyzed. The reaction types are classified into three groups depending on the site of elimination or replacement of the substituents: 1) reactions at the α-carbon atom include , racemases of α-amino acid, amino acid α-decarboxylases, enzymes catalysing condensation and α-β cleavage of β-hydroxy amino acids (δ-aminolevulinic acid synthetase, serine hydroxymethyltransferase and sphyngosine synthetase); 2) reactions at the β-carbon atom include serine and dehydratases, synthetase, tryptophanase and kynureninase; and finally 3) reactions at the γ-carbon include homoserine dehydratase and cystathionine gamma- (Dakshinamurti and Dakshinamurti, 2007). Additionally, PLP and to a lesser extent PL, can also react non-enzymatically with primary amino groups of amines and amino acids although at lower rates (Di Salvo et al., 2011).

12 13 General introduction

Figure 3

Glutamine Tyrosine VLA enzyme methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20). 5-mTHF is used GLS TH BH TPH BH EC 3.5.1.2 EC 1.14.16.2 4 COMT EC 1.14.16.4 4 for remethylation of to via the vitamin B12-dependent EC 2.1.1.6 1 Glutamate L-Dopa 3OMD 5HT enzyme methionine synthase (MS, EC 2.1.1.13) (Figure 4). The principal physiological importance of homocysteine remethylation to methionine is the generation of GAD PLP AADC PLP EC 4.1.1.15 EC 4.1.1.28 S-adenosylmethionine (SAM; the most important methyl group donor in the human GABA AADC PLP 3MT Serotonin EC 4.1.1.28 COMT body), by methionine adenosyltransferase (MAT, EC 2.5.1.6) (Ducker and Rabinowitz, GT MAO MAO PLP EC 2.1.1.6 EC 2.6.1.96 EC 1.4.3.4 EC 1.4.3.4 2017; Lu, 2000). Figure 4 SSA Dopamine HVA 5-HIAA MAO Diet SSADH DβH EC 1.4.3.4 EC 1.2.1.24 EC 1.14.17.1 Betaine Choline Succinate Norepinephrine MHPG MAO PNMT EC 1.4.3.4 EC 2.1.1.28 Methionine Epinephrine 5-VMA DMG MAO Serine EC 1.4.3.4 BHMT MATI/II

Figure 3. Metabolism of GABA, serotonin and the catecholamines. In bold the PLP-dependent Glycine THF SAM X enzymes: GAD, glutamate decarboxylase; GT, GABA aminotransferase and AADC, aromatic PLP SHMT L-amino acid decarboxylase. GLS, glutaminase; SSADH, succinate-semialdehyde dehydrogenase; TH, tyrosine hydroxylase; DβH, dopamine β-hydroxylase; COMT, catechol-O-methyltransferase; 5,10-methyleneTHF MS Vitamin B12 PNMT, phenylethanolamine N-methyltransferase; MAO, monoamine oxidase; TPH, tryptophan reactions hydroxylase. GABA, γ-aminobutyrate; SSA, succinate-semialdehyde; L-Dopa, levodopa; 3OMD, MTHFR 3-O-mehtyldopa; VLA, vanillactate; 3MT, 3-methoxytyramine; HVA, homovanilate; MHPG, 3-methoxy- 5-methylTHF 4-hydroxyphenylglycol; 5-VMA, 5-vanillymandelate; 5-HIAA, 5-hydroxyindolacetate. SAH X-CH3

SAHH

The role of vitamin B6 in one-carbon metabolism Homocysteine and homocysteine remethylation and Serine transsulfuration pathways CBS PLP Cystathionine

One-carbon (1C) metabolism is a universal biochemical pathway that provides methyl CSE PLP groups for biological methylation reactions of proteins, phospholipids and nucleic acids (Friso et al., 2017). Folate and vitamin B6 play essential roles in 1C methyl transfer reactions. The biologically active form of folic acid, 5,6,7,8-tetrahydrofolate (THF), Figure 4. One carbon metabolism. In bold the PLP-dependent enzymes: SHMT, serine hydroxymethyltransferase; CBS, cystathionine β-synthase and CSE, cystathionine γ-lyase. MTHFR, works as a transporter of methyl (-CH ) groups. The PLP-dependent enzyme serine 3 methylenetetrahydrofolate reductase; MS, methionine synthase; BHMT, betaine-homocysteine hydroxymethyltransferase (SHMT, EC 2.1.2.1), catalyzes the transfer of the methyl methyltransferase; MATI/II, methionine adenosyltransferase I/II and SAHH, S-adenosylhomocysteine group of serine to THF, allowing the formation of 5,10-methylenetetrahydrofolate hydrolase. PLP, pyridoxal 5’-phosphate; SAM, S-adenosylmethionine and SAH, S-adenosylhomocysteine; (5,10-methyleneTHF) and glycine (de Koning et al., 2003). 5,10-methyleneTHF can THF, 5,6,7,8-tetrahydrofolate; 5,10-methyleneTHF, 5,10-methylenetetrahydrofolate; 5-methylTHF, 5-methyltetrahydrofolate. also be generated via the mitochondrial glycine cleavage system. This system is a four-enzyme complex containing the PLP-dependent glycine decarboxylase (da Silva et al., 2012). 5,10-methyleneTHF is converted to 5-methyltetrahydrofolate (5- mTHF), the main circulating form of folates in plasma, by the NADPH-dependent

14 15 General introduction

The transsulfuration pathway of homocysteine is formed by two PLP-dependent Classically, vitamin B6 deficiency is characterized by severe neonatal seizures that enzymes: cystathionine β-synthase (CBS, EC 4.2.1.22) and cystathionine γ-lyase (CSE, do not respond to common anticonvulsant therapy and are only controlled by PLP EC 4.4.1.1) (See Figure 4). CBS catalyzes the first step of the transsulfuration pathway, and/or PN (Baxter, 1999; Stockler et al., 2011). In addition to the neonatal seizures, 1 in which serine condenses with homocysteine forming cystathionine. CSE catalyzes most vitamin B6-deficient patients suffer from variable degrees of developmental the cleavage of cystathionine to cysteine, ammonia and α-ketobutyrate. Although delay and intellectual disability, despite the seizure control achieved with vitamin B6 PLP serves as coenzyme for both CBS and CSE, CSE exhibits greater loss of activity treatment (Van Karnebeek et al., 2016). than CBS when PLP is deficient.

Studies in rats show a moderate correlation between vitamin B6 insufficiency and Pyridoxine-dependent epilepsy (PDE) increasing plasma homocysteine levels (Stabler et al., 1997; Martinez et al., 2000), with a 40% decrease in total SHMT activity and 80% reduction in flux of one-carbon Pyridoxine-dependent epilepsy (PDE; MIM #266100) is a rare autosomal recessive units from serine to methionine (Martinez et al., 2000). Furthermore, the hepatic disorder. PDE was first described in 1954 in a patient with therapy-resistant seizures CBS activity is (largely) independent of both dietary PN availability and hepatic who achieved seizure control when treated with a multivitamin cocktail that contained PLP concentrations, while PLP-insufficiency mainly exerts its influence on CSE vitamin B6 (Hunt et al., 1954). Classically, PDE patients present with neonatal or early activity (~70% decrease) (Lima et al., 2006). Although the methyl groups, used in infantile seizures refractory to common anticonvulsants, but responsive to PN (Stockler the remethylation of homocysteine to methionine, can derive from several sources et al., 2011). The estimated incidence is of approximately 1:64,000 live births (Coughlin (serine, glycine, , formate, sarcosine, dimethylglycine and betaine), tracer II et al., 2018). The diagnosis of PDE was previously based on the clinical observation of studies show that serine is the primary source of one-carbon units in homocysteine cessation of seizures after pyridoxine administration and recurrence of seizures upon remethylation in humans (Davis et al., 2004). pyridoxine withdrawal (Clayton, 2006). In 2006, Mills and colleagues showed that PDE resulted from loss-of-function mutations in the ALDH7A1 , encoding the enzyme Although folate and vitamin B6 deficiencies are thought to increase circulating α-aminoadipic semialdehyde (α-AASA) dehydrogenase, also known as antiquitin (Mills homocysteine concentrations by decreasing the availability of 5-mTHF in humans, a et al., 2006). Since antiquitin deficiency was identified as the cause for PDE, more than causal relationship has not yet been confirmedin vivo (Davis et al., 2005; Davis et al., 2006). 200 patients have been genetically confirmed (Van Karnebeek et al., 2016). Antiquitin plays an important role in the pipecolic acid pathway of catabolism, catalyzing the oxidation of α-AASA to α-aminoadipic acid (Mills et al., 2006). Antiquitin deficiency Vitamin B6 deficiencies leads to accumulation of α-AASA, a compound that is in spontaneous equilibrium with L-Δ1-piperideine-6-carboxylate (P6C). The activated C5 methylene of the piperideine Nutritional vitamin B6 deficiency is very rare nowadays since most dietary sources ring of P6C reacts with the aldehyde moiety of PLP by Knoevenagel condensation, contain vitamin B6. In addition, isolated vitamin B6 deficiency is uncommon, leading to chemical inactivation of PLP (Mills et al., 2006; Clayton, 2006). Accumulation usually occurring in combination with other B-vitamin deficiencies (Spinneker of α-AASA, P6C and pipecolic acid serve as diagnostic markers in urine, plasma and et al., 2007). However, five inborn errors of metabolism affecting vitamin B6 are CSF of patients. Diagnosis is confirmed by mutational analysis of theALDH7A1 gene. known: i) pyridoxine-dependent epilepsy (PDE, α-aminoadipic semialdehyde Treatment consists on PN supplementation to compensate for PLP deficiency and lysine- dehydrogenase (antiquitin) deficiency; OMIM #266100); ii) hyperprolinemia type II restriction to lower the burden of lysine-derived intermediates that can be potentially (L-Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase deficiency; OMIM #239510); iii) neurotoxic (van Karnebeek et al., 2014). Although most PDE patients achieve adequate pyridox(am)ine 5’-phosphate oxidase deficiency (PNPO deficiency; OMIM #610090); seizure control with PN treatment, 75% of them still present with intellectual disability iv) hypophosphatasia (tissue non-specific (TNSALP) deficiency; and development delay (Coughlin II et al., 2018). OMIM #241500); and v) binding protein deficiency (PLPBP deficiency; OMIM #617290).

16 17 General introduction

Figure 5 L-Lysine PLP-dependent kynureninase reaction (Walker et al., 2000). Interestingly, and even though the XA pathway also involves other PLP-dependent enzymes, the L-Lysine Oxidase AASS 1 EC 1.4.3.14 EC 1.5.1.9 aminotransferase has been shown to be less sensitive to PLP deficiency, leading

2-keto-6-aminocaproic acid to accumulation of XA and its increased excretion in urine (Spinneker et al., 2007). P5CDH catalyses the second step of degradation, converting P5C to glutamate (Figure 6). In analogy to the pathomechanism in PDE, the activated C4 carbon of the P2C pyrroline ring in L-Δ1-pyrroline-5-carboxylate (P5C) reacts with the aldehyde group P2CR AASS of PLP through a Knoevenagel reaction, subsequently inactivating PLP (Walker EC 1.5.1.1 EC 1.5.1.9 L-Pipecolic acid et al., 2000; Farrant et al., 2001). Biochemically, HYRPRO2 patients have increased PIPOX concentrations of proline and P5C in plasma, and increased urinary excretion of EC 1.5.3.7 Equilibrium proline, hydroxyproline, and glycine (Flynn et al., 1989). Diagnosis is confirmed by P6C α-aminoadipic semialhdehyde mutational analysis of the P5CDH gene Seizures respond to oral supplementation ALDH7A1 with PN, although no recommendations exist regarding its dosage (Plecko, 2013). Inactivation EC 1.2.1.31 Figure 6 α-aminoadipic acid PLP AADAT L-Proline P5CDH EC 2.6.1.39 dehydrogenase Equilibrium α-ketoadipic acid L-Proline P5C L-GLU 5-semialdehyde L-GLU PC5R Figure 5. Antiquitin deficiency. P2C, L-Δ1-piperideine-2-carboxylate; P6C, L-Δ1-piperideine- Inactivation 6-carboxylate; AASS, alpha-aminoadipic semialdehyde synthase; ALDH7A1, α-aminoadipic semialdehyde dehydrogenase (antiquitin); AADAT, α-aminoadipate aminotransferase; P2CR, PLP 1-piperideine-2-carboxylate/1-pyrroline-2-carboxylate reductase; PIPOX, L-pipecolate oxidase. Figure 6. Hyperprolinaemia type II. P5C, L-Δ1-pyrroline-5-carboxylate, GLU, glutamate; PC5R, L-Δ1- Arrows in bold indicate situation in antiquitin deficiency. pyrroline-5-carboxylate reductase; P5CDH, L-Δ1-pyrroline-5-carboxylate dehydrogenase. Arrows in bold indicate situation in P5CDH deficiency. Hyperprolinaemia type II

Hyperprolinaemia type II (HYRPRO2; MIM #239510) is a rare autosomal recessive Pyridox(am)ine 5’-phosphate oxidase deficiency disorder. In 1989, Flynn and colleagues described for the first time a strong association between childhood febrile seizures and HYRPRO2 while studying 312 Irish travellers Pyridox(am)ine 5’-phosphate oxidase deficiency (PNPO deficiency; MIM #610090) is from 71 families related to a proband with the disease (Flynn et al., 1989). Classically, a rare autosomal recessive disorder caused by loss-of-function mutations in the PNPO HYRPRO2 patients present with convulsions in the childhood, usually triggered gene (Mills et al., 2005). Classically, PNPO-deficient patients present with severe epileptic by infections (Walker et al., 2000). HYRPRO2 is caused by deficient L-Δ1-pyrroline- encephalopathy from the first days of life, not responding to conventional anticonvulsant 5-carboxylate (P5C) dehydrogenase (P5CDH; EC 1.5.1.12), leading to increased PLP drugs (Clayton et al, 2003; Mills et al., 2005; Mills et al., 2014). Diagnosis of PNPO deficiency utilisation. The exact prevalence of this disorder is not yet known (Van De Ven et is achieved by measurements of low PLP in plasma and CSF (Footitt et al., 2013). Diagnosis al., 2014). The association between HYRPRO2 and vitamin B6 deficiency was first is confirmed by mutational analysis of the PNPO gene. PNPO is a flavin mononucleotide reported in a girl with severe seizures and reduced conscious in association with (FMN)-dependent oxidase responsible for the oxidation of PNP and PMP to PLP (Mills pneumonia (Walker et al., 2000). The patient had increased proline, hydroxyproline et al., 2014) (see Figure 7). Biochemically, PNPO-deficient patients may present a variety and , marginally low PLP and greatly reduced PA in plasma. In addition, of secondary biochemical findings, like decreased concentrations of homovanillic acid increased urinary excretion of xanthurenic acid (XA) was found, suggesting a vitamin (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), and increased 3-methoxytyrosine (3-MT) B6 deficient status, since the major pathway of tryptophan catabolism occurs via the and glycine in CSF. Additionally, increased urinary excretion of vanillactic acid (VLA) has

18 19 General introduction

been reported (Clayton et al., 2003; Clayton, 2006). The reported metabolite alterations Hypophosphatasia can be explained by reduced activities of the PLP-dependent enzymes: aromatic L-amino acid decarboxylase (VLA, HVA, 5-HIAA, and 3-MT) and glycine cleavage enzyme (glycine) Hypophosphatasia (HPP; MIM #241500) is an autosomal recessive disorder 1 (Clayton, 2006; Surtees et al., 2006). PNPO-deficient patients are mainly treated with PLP. characterized by deficiency of the tissue non-specific alkaline phosphatase (TNSALP; Nevertheless, the paradigm of exclusive PLP responsiveness shifted when 6 out of 9 EC 3.1.3.1) (Michael P Whyte et al., 1988). HPP was first described in a 9-week-old male PNPO-deficient patients were successfully treated with PN monotherapy. In addition, two with very low alkaline phosphatase activity in serum and tissues whom died with of those patients developed status epilepticus when PN was replaced by identical doses rickets and epilepsy (Rathbun, 1948). HPP is caused by loss-of-function mutations of PLP (Plecko et al., 2014). Even though the observed results are not fully understood, in the ALPL gene (Weiss et al., 1988). TNSALP is an ubiquitous plasma membrane- sufficient residual activity of the enzyme, a putative chaperone effect of PN in preserving bound enzyme essential for bone mineralization (Fedde and Whyte, 1990) (see the PNPO mutants from premature decay, age and riboflavin status at the time of the Figure 7). Defective skeletal mineralization that manifests as rickets (in children) and therapeutic trial may play a role in explaining the PN responsiveness (Mills et al., 2014). osteomalacia (in adults) are the classical clinical presentation of HPP patients. In 1985, Figure 7 Whyte and colleagues showed that TNSALP deficiency leads to strongly increased Extracellular plasma PLP concentrations (Whyte et al., 1985). Pyridoxine 5’-phosphate

HO H2 C O OH -/- HO P In 1995, Waymire and colleagues showed that TNSALP mice developed spontaneous O OH seizures usually dying within one day after the seizure onset (Waymire et al., 1995). H3C N TNSALP The development of seizures was addressed to the approximately 20-fold increase Pyridoxine Pyridoxine Pyridoxine 5’-phosphate

HO H2 HO H HO H 2 Pyridoxal kinase 2 in plasma PLP and PEA concentrations and significantly reduced intracellular PLP C C C O OH HO HO HO P concentrations in liver, brain, heart, kidney and muscle. Furthermore, GABA was OH OH Pyridoxal O OH H C N phosphatase -/- 3 H3C N H3C N approximately 50% reduced in the brain of TNSALP mice when compared to control littermates (Waymire et al., 1995). Biochemically, HPP patients present elevated serum phosphate levels, decreased serum ALP activity and increased urinary excretion of Pyridoxal 5’-phosphate PNPO H O phosphoethanolamine (PEA) (Chodirker et al., 1990). Several case reports have shown C O OH HO P that human infant HPP patients may develop convulsions in their first days of life O OH

H3C N TNSALP which may be controlled with PN or PLP treatment (Baumgartner-Sigl et al., 2007;

Pyridoxal Pyridoxal Pyridoxal 5’-phosphate Balasubramaniam et al., 2010; De Roo et al., 2014). H O H O Pyridoxal kinase H O C C C O OH HO HO HO P OH OH Pyridoxal O OH H C N H C N phosphatase H C N 3 3 3 Pyridoxal 5'-phosphate-binding protein deficiency

Pyridoxamine 5’-phosphate Pyridoxal 5'-phosphate-binding protein (PLPBP) deficiency, previously known H N H 2 2 Aminotransferases PNPO C O OH as proline synthetase co-transcribed (bacterial homolog) (PROSC; MIM #604436) HO P O OH deficiency is a rare autosomal recessive disorder caused by loss-of-function mutations H C N 3 TNSALP in the PLPBP gene. PLPBP is a ubiquitously expressed cytoplasmic and mitochondrial Pyridoxamine Pyridoxamine Pyridoxamine 5’-phosphate H N H H N H H N H protein, which is highly conserved throughout evolution (from bacteria to mammals), 2 2 2 2 Pyridoxal kinase 2 2 C C C O OH HO HO HO P suggesting an important cellular function (Ikegawa et al., 1999). In 2016, Darin OH OH Pyridoxal O OH and colleagues identified homozygous and compound heterozygous mutations phosphatase H3C N H3C N H3C N in the PLPBP gene of 7 patients from 5 unrelated families with early-onset vitamin Figure 7. The human vitamin B6 metabolism pathway. B6-dependent epilepsy (EPVB6D; MIM #617290). Biochemically, the plasma PLP

20 21 General introduction

concentration of PLPBP-deficient patients receiving pharmacological doses of PN/ REFERENCES PLP was increased (Darin et al., 2016; Plecko et al., 2017). Additionally, no information on untreated patients is available. The exact mechanism by which PLPBP works still Albersen, M., Bosma, M., Knoers, N. V. V. A. M., de Ruiter, B. H. B., Diekman, E. F., de Ruijter, J., 1 Verhoeven-Duif, N. M. (2013). The Intestine Plays a Substantial Role in Human Vitamin B6 remains uncertain, but the current hypothesis is that it is a PLP-carrier that prevents Metabolism: A Caco-2 Cell Model. PLoS ONE, 8(1). http://doi.org/10.1371/journal.pone.0054113 PLP from reacting with other reactive molecules, supplying it to the PLP-dependent Balasubramaniam, S., Bowling, F., Carpenter, K., Earl, J., Chaitow, J., Pitt, J., Ellaway, C. (2010). apo-enzymes, and protecting it from intracellular phosphatases (Darin et al., 2016). Perinatal hypophosphatasia presenting as neonatal epileptic encephalopathy with abnormal PLPBP-deficient patients respond to PN/PLP treatment with an immediate reduction neurotransmitter metabolism secondary to reduced co-factor pyridoxal-5′-phosphate availability. Journal of Inherited Metabolic Disease, 33(SUPPL. 3), 25–33. http://doi.org/10.1007/ in seizure frequency and severity (Darin et al., 2016). s10545-009-9012-y Baumeister, F. A. M., Gsell, W., Shin, Y. S., & Egger, J. (1994). Glutamate in Pyridoxine-Dependent Epilepsy: Neurotoxic Glutamate Concentration in the Cerobrospinal Fluid and Its Normalization Outline of the thesis by Pyridoxine. Pediatrics, 94(3), 318. Baumgartner-Sigl, S., Haberlandt, E., Mumm, S., Scholl-Bürgi, S., Sergi, C., Ryan, L., Högler, W. (2007). Pyridoxine-responsive seizures as the first symptom of infantile hypophosphatasia The studies presented in this thesis aimed on gaining new insight into the caused by two novel missense mutations (c.677T > C, p.M226T; c.1112C > T, p.T371I) of the pathophysiology of genetic vitamin B6 deficiencies and on characterizing new vitamin tissue-nonspecific alkaline phosphatase gene. Bone, 40(6), 1655–1661. http://doi.org/10.1016/j. bone.2007.01.020 B6-reponsive disorders. Although the discovery of vitamin B6 dates back to 1934 (Gyorgy, Baxter, P. (1999). Epidemiology of pyridoxine dependent and pyridoxine responsive seizures in the 1934) and pyridoxine-dependent epilepsy is known since the early 1950’s, the interest in UK. Arch Dis Child, 81, 431–433. vitamin B6 and its role in health and disease has reappeared in the last two decades due Bender, D. (2005). Water-soluble vitamins: Vitamin B6. In C. A. Geissler & H. J. Powers (Eds.) (pp. to the discoveries of loss-of-function mutations in the ALDH7A1 and PNPO as the 194–196). London, United Kingdom: Elsevier/Churchill Livingstone. Chodirker, B. N., Coburn, S. P., Seargeant, L. E., Whyte, M. P., & Greenberg, C. R. (1990). Increased genetic causes for PDE and PNPO deficiency. Very recently, a new inborn error of vitamin plasma pyridoxal-5’-phosphate levels before and after pyridoxine loading in carriers of B6 metabolism was identified, and although not fully understood, loss-of-function perinatal/infantile hypophosphatasia. Journal of Inherited Metabolic Disease, 13(6), 891–896. mutations in the PLPBP gene have been shown to cause vitamin B6 deficiency. Clayton, P. T. (2006). B6-responsive disorders: a model of vitamin dependency. Journal of Inherited Metabolic Disease, 29(2–3), 317–26. http://doi.org/10.1007/s10545-005-0243-2 Clayton, P. T., Surtees, R. A. H., DeVile, C., Hyland, K., & Haeles, S. J. R. (2003). Case report Neonatal With this work, we unveil the intracellular metabolic consequences of vitamin B6 epileptic encephalopathy. The Lancet, 361, 7741–7741. deficiency in a neuronal cell model chapter( 2). Strikingly lower concentrations of Coughlin II, C. R., Swanson, M. A., Spector, E., Meeks, N. J. L., Kronquist, K. E., Aslamy, M., Salomons, the amino acids serine and glycine, known key players in one-carbon metabolism, G. S. (2018). The genotypic spectrum of ALDH7A1 mutations resulting in pyridoxine dependent epilepsy : a common epileptic encephalopathy. were found in the vitamin B6-deficient cells. To understand the closely intertwined da Silva, V. R., Russell, K. A., & Gregory, J. F. 3rd. (2012). Vitamin B6. (J. W. Erdman, I. A. Macdonald, & H. relation between serine metabolism and vitamin B6 we developed a sensitive and Zeisel, Steven, Eds.), Present Knowledge in Nutrition: Tenth Edition (Tenth). Wiley-Blackwell. http:// accurate stable isotopic ultra-performance liquid chromatography tandem mass doi.org/10.1002/9781119946045.ch71 spectrometry (UPLC-MS/MS) method to study de novo serine biosynthesis and Darin, N., Reid, E., Prunetti, L., Samuelsson, L., Husain, R. A., Wilson, M., Clayton, P. T. (2016). Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent validated this method as diagnostic test for the detection of primary and secondary Epilepsy. The American Journal of Human Genetics, 99(6), 1325–1337. http://doi.org/10.1016/j. serine synthesis defects (chapter 5). In chapter 3, we characterize a new B6-responsive ajhg.2016.10.011 inborn error of metabolism: GOT2 deficiency. This novel disorder clinically responds to Davis, S. R., Quinlivan, E. P., Stacpoole, P. W., & Gregory III, J. F. (2006). Plasma and cystathionine concentrations are elevated but cysteine flux is unchanged by dietary vitamin serine and vitamin B6 treatment. In vitro studies of GOT2-deficient patients’ fibroblasts B-6 restriction in young men and women. J.Nutr., 136(0022–3166 (Print)), 373–378. http://doi. and GOT2-knockout HEK293 cells, allow us to characterize GOT2-deficiency and org/136/2/373 [pii] explain its connection to serine and PLP treatment. Further studies on the metabolic Davis, S. R., Scheer, J. B., Quinlivan, E. P., Coats, B. S., Stacpoole, P. W., & Gregory III, J. F. (2005). consequences of GOT2 deficiency, using untargeted and targeted metabolomics, Dietary vitamin B-6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men. American Journal of Clinical Nutrition, 81(3), 648–655. http:// are presented in chapter 4. Finally, chapter 6 provides evidence for the existence of doi.org/81/3/648 [pii] additional, yet undescribed, enzyme(s) with PL reductase activity in humans. This new enzyme is believed to play a role in the salvage pathway of vitamin B6 in mammals.

22 23 General introduction

Davis, S. R., Stacpoole, P. W., Williamson, J., Kick, L. S., Quinlivan, E. P., Coats, B. S., Gregory, J. F. 3rd. Hunt, A. D., Stokes Jr, J., McCrory, W. W., & Stroud, H. H. (1954). PYRIDOXINE DEPENDENCY: REPORT (2004). Tracer-derived total and folate-dependent homocysteine remethylation and synthesis OF A CASE OF INTRACTABLE CONVULSIONS IN AN INFANT CONTROLLED BY PYRIDOXINE. rates in humans indicate that serine is the main one-carbon donor. American Journal of Physiology. Pediatrics, 13(2), 140–145. Endocrinology and Metabolism, 286(2), E272-279. http://doi.org/10.1152/ajpendo.00351.2003 Ikegawa, S., Isomura, M., Koshizuka, Y., & Nakamura, Y. (1999). Cloning and characterization of 1 de Koning, T. J., Snell, K., Duran, M., Berger, R., Poll-The, B.-T., & Surtees, R. (2003). L-serine in human and mouse PROSC (proline synthetase co-transcribed) genes. Journal of Human Genetics, disease and development. 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Pyridoxal Phosphate in the Small Intestine of the Rat. The Journal of Nutrition, 109(9), 1542–1551. GYÖRGY, P. (1934). Vitamin B2 and the Pellagra-like Dermatitis in Rats. Nature, 133, 498. Retrieved Middleton III, H. M. (1977). Uptake of Pyridoxine Hydrochloride by the Rat Jejunal Mucosa in vitro. from http://dx.doi.org/10.1038/133498a0 Journal of Nutrition, 107(1), 126–131. Hamm, M. W., Mehansho, H., & Henderson, L. M. (1979). Transport and Metabolism of Pyridoxamine Mills, P. B., Camuzeaux, S. S. M., Footitt, E. J., Mills, K. A., Gissen, P., Fisher, L., Clayton, P. T. (2014). and Pyridoxamine Phosphate in the Small Intestine of the Rat. The Journal of Nutrition, 109(9), Epilepsy due to PNPO mutations: Genotype, environment and treatment affect presentation 1552–1559. and outcome. Brain, 137(5), 1350–1360. http://doi.org/10.1093/brain/awu051 Hanna, M. C., Turner, A. J., & Kirkness, E. F. (1997). Human Pyridoxal Kinase. Biochemistry, 272(16), Mills, P. B., Struys, E., Jakobs, C., Plecko, B., Baxter, P., Baumgartner, M., Clayton, P. T. (2006). Mutations 10756–10760. in antiquitin in individuals with pyridoxine-dependent seizures. Nature Medicine, 12(3), 307– Hufft, J. W., & Perlzweig, W. A. (1944). A PRODUCT OF OXIDATIVE METABOLISM OF PYRIDOXINE, 309. http://doi.org/10.1038/nm1366 2-METHYL-3-HYDROXY-4-CARBOXY-5 -HYDROXY-METHYLPYRIDINE (4-PYRIDOXIC ACID): I. ISOLATION FROM URINE, STRUCTURE, AND SYNTHESIS. J Biochem, 155, 345–355.

24 25 General introduction

Mills, P. B., Surtees, R. A. H., Champion, M. P., Beesley, C. E., Dalton, N., Scamber, P. J., Clayton, P. T. Van Karnebeek, C. D. M., Tiebout, S. A., Niermeijer, J., Poll-The, B. T., Ghani, A., Coughlin, C. R., (2005). Neonatal epileptic encephalopathy caused by mutations in the PNPO gene encoding Stockler-Ipsiroglu, S. (2016). Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum. pyridox(am)ine 5′-phosphate oxidase. Human Molecular Genetics, 14(8), 1077–1086. http://doi. Pediatric Neurology, 59, 6–12. http://doi.org/10.1016/j.pediatrneurol.2015.12.013 org/10.1093/hmg/ddi120 Walker, V., Mills, G. A., Peters, S. A., & Merton, W. L. (2000). Fits, pyridoxine, and hyperprolinaemia 1 Percudani, R., & Peracchi, A. (2003). A genomic overview of pyridoxal-phosphate-dependent type II. Archives of Disease in Childhood, 82(3), 236–237. http://doi.org/10.1136/adc.82.3.236 enzymes. EMBO Reports, 4(9), 850–854. http://doi.org/10.1038/sj.embor.embor914 Waymire, K. G., Mahuren, J. D., Jaje, J. M., Guilarte, T. R., Coburn, S. P., & MacGregor, G. R. (1995). Mice Percudani, R., & Peracchi, A. (2009). The B6 database: a tool for the description and classification lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC of vitamin B-6. Nature Genetics, 11, 45–51. http://doi.org/10.1038/ng0595-111 Bioinformatics, 10, 273. http://doi.org/10.1186/1471-2105-10-273 Weiss, M. J., Cole, D. E., Ray, K., Whyte, M. P., Lafferty, M. a, Mulivor, R. a, & Harris, H. (1988). A missense Plecko, B. (2013). Pyridoxine and pyridoxalphosphate-dependent epilepsies. Handbook of Clinical mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form Neurology (1st ed., Vol. 113). Elsevier B.V. http://doi.org/10.1016/B978-0-444-59565-2.00050-2 of hypophosphatasia. 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26 27 2

Chapter 2

Vitamin B6 is essential for serine de novo biosynthesis

Rúben J. Ramos, Mia L. Pras-Raves, Johan Gerrits, Maria van der Ham, Marcel Willemsen, Hubertus Prinsen, Boudewijn Burgering, Judith J. Jans, Nanda M. Verhoeven-Duif

J Inherit Metab Dis. 2017 Nov;40(6):883-891. Doi: 10.1007/s10545-017-0061-3 Vitamin B6 is essential for serine de novo biosynthesis

ABSTRACT INTRODUCTION

Pyridoxal 5’-phosphate (PLP), the metabolically active form of vitamin B6, plays 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. a pivotal role in brain metabolism and development (Surtees et al 2006). PLP is an PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant essential cofactor in more than 100 metabolic reactions in humans (Clayton, 2006). seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin Most of the PLP-dependent reactions, such as transamination, decarboxylation, 2 B6 deficiency is largely unknown. To shed more light on the metabolic consequences deamination, and desulfhydration, are involved in amino acid of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin metabolism (Ebadi 1981; Ueland et al 2015). PLP deficiency, either due to genetic or B6-deprived Neuro-2a cells. Significant alterations were observed in a range of dietary causes, disrupts the metabolism of neurotransmitters (γ-aminobutyric acid metabolites. The most surprising observation was a decrease of serine and glycine, (GABA), dopamine and serotonin), haeme, histamine, amino acids, carbohydrates and two amino acids that are known to be elevated in the plasma of vitamin B6 deficient nucleotides (da Silva et al 2013; Ueland et al 2015). patients. Five inborn errors of metabolism are known to affect vitamin B6 concentrations: To investigate the cause of the low concentrations of serine and glycine, a metabolic pyridoxine-dependent epilepsy (α-aminoadipic semialdehyde dehydrogenase flux analysis on serine biosynthesis was performed. The metabolic flux results showed (antiquitin) deficiency; OMIM #266100), hyperprolinemia type II (1-pyrroline-5- that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived carboxylate dehydrogenase deficiency; OMIM #239510), pyridox(am)ine 5’-phosphate cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. oxidase deficiency (PNPO deficiency; OMIM #610090), hypophosphatasia (tissue Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine non-specific alkaline phosphatase (TNSALP) deficiency; OMIM #241500) and proline de novo synthesis is critical to maintain intracellular serine and glycine. These findings synthetase co-transcribed bacterial homolog deficiency (PROSC deficiency; OMIM suggest that serine and glycine concentrations in brain may be deficient in patients #604436). These diseases, except for most cases of TNSALP, are characterized by with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations seizures, often beginning in the first days of life, not responsive to anticonvulsants observed in vitro may explain the favorable but so far unexplained response of some and only controlled by vitamin B6 supplementation (Baxter 1999; Stockler et al patients with pyridoxine-dependent epilepsy to folinic acid supplementation. 2011). Besides neonatal seizures, many patients suffer from developmental delay or intellectual disability, despite the seizure control (Baxter, 2001; van Karnebeek Keywords: 5-methyltetrahydrofolate; pyridoxal 5’-phosphate; serine de novo et al 2016; Darin et al 2016; Walker et al 2000; Mills et al 2005; Mills et al 2014). biosynthesis; vitamin B6 deficiency. Little is known about the specific biochemical changes that underlie the clinical symptoms of patients with vitamin B6 deficiency. Low GABA levels were thought to be the main reason for the epilepsy (Gospe et al 1994). GABA is the key inhibitory neurotransmitter in the central nervous system and is synthesized from glutamate (the main excitatory neurotransmitter) through the PLP-dependent enzyme glutamic acid decarboxylyase (GAD, EC 4.1.1.15). Animal studies performed in zebrafish larvae showed that upon exposure to ginkgotoxin (4’-O-methylpyridoxine, a pyridoxal 5’-phosphate antimetabolite) a seizure-like behavior develops. The ginkgotoxin- induced seizures were reversed by the addition of GABA and/or PLP to the fish water, supporting the hypothesis that the seizures are caused by reduced PLP availability, which leads to an imbalance between GABA and glutamate (Lee et al 2012). However, conflicting data on glutamate and GABA levels in the CSF of patients suffering from vitamin B6 deficiency suggest that GABA deficiency may not be the sole cause of symptoms in pyridoxine-dependent epilepsy (Goto et al 2001; Baumeister et al

30 31 Vitamin B6 is essential for serine de novo biosynthesis

1994). Several studies have shown that vitamin B6-deficient patients may display Vitamin B6 deficiency alters the metabolome of Neuro-2a cells biochemical features of aromatic L-amino acid decarboxylase (AADC) deficiency, with We compared the metabolomes of Neuro-2a cells cultured in 0 and 100 nM PL at low CSF homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) (Clayton, all time points. By direct-infusion high-resolution mass spectrometry (DI-HRMS) of 2006; De Roo et al 2014; Darin et al 2016), raised tyrosine, 3-ortho-methyldopa, the extracts we detected 15,765 features (a feature is defined as a mass over charge, L-Dopa and 5-hydroxytryptophan (Darin et al 2016). Other studies report increased m/z) of which 62 features in negative scan mode and 83 in positive scan mode were CSF and plasma concentrations of threonine (Mills et al 2005; Clayton, 2006), and/or significantly differentP ( <0.05 after Bonferroni correction) and identified using the 2 glycine (Darin et al 2016; Mills et al 2005) and/or branched chain amino acids (Darin Human Metabolome Database (HMDB, www.hmdb.ca). The most significantly altered et al 2016). Nevertheless, these results are not consistent in all studies (Levtova et al intracellular metabolites, in positive and negative scan modes, are shown in Tables 2015), underlining the complexity of vitamin B6 deficiency. Many additional vitamin 1 and 2, respectively. Among others, we observed decreased signals of the masses B6-dependent reactions may contribute to the clinical phenotype. corresponding to the amino acids serine, glycine and cystathionine, GABA, the Krebs cycle intermediates malate, citrate/isocitrate, fumarate, and pyruvate. Phosphoserine, To elucidate the pathogenesis of brain vitamin B6 deficiency, we performed untargeted the direct precursor of serine, was also decreased in B6-deficient cells. The masses of metabolomics on a neuronal cell model deprived of vitamin B6. Our observations the features that were increased corresponded to those of and glutamine indicate that additional factors next to GABA play a role in the pathogenesis of adducts (Tables 1 and 2). This increase was confirmed by targeted LC-MS/MS vitamin B6 deficiency. (Supplemental Figure S2a).

Table 1. The fifteen most significantly altered intracellular compounds (positive scan mode). RESULTS Direct infusion high resolution mass spectrometry (DI-HRMS) in positive scan mode was used to detect the overall intracellular metabolic consequence of vitamin B6 deprivation. A t-test was performed on the area under the curve (AUC, in a plot of intensities against time) for every compound. Compounds To study the consequences of vitamin B6 deficiency in neuronal cells, we developed a are sorted on the P value between PL absence and presence (100 nM). model system of vitamin B6-deficient Neuro-2a cells. Neuro-2a cells were cultured in the presence (100 nM) and absence of pyridoxal (PL). Cells were harvested at different # m/z Identification P value Change Fold Change a time points. 1 144.006 serine [M + K]+ 5.82E-15 Decrease 2.7 2 106.050 serine [M]+ 3.95E-13 Decrease 2.2 + + + -13 Vitamin B6 restriction results in intracellular PLP deficiency 3 223.075 L-cystathionine [M] ; cysteinyl-threonine [M] ; threoninyl-cysteine [M] 4.57E Decrease 4.0 4 128.032 serine [M+Na]+ 2.87E-12 Decrease 1.8 To establish whether the absence of PL in the medium resulted in intracellular vitamin 5 104.070 (α/β/γ)-aminobutyric acid [M]+; dimethylglycine [M]+ 5.14E-12 Decrease 1.5 B6 deficiency, we quantified the intracellular concentrations of PL, pyridoxamine 6 165.988 serine [M+NaK-H]+ 8.90E-11 Decrease 3.0 (PM), pyridoxine (PN), the 5’-phosphorylated forms (PLP, PMP and PNP, respectively) 7 150.014 serine [M+2Na-H]+ 4.85E-10 Decrease 1.9 and the breakdown product pyridoxic acid (PA) (Figure S1). PLP concentrations 8 207.014 glutamine [M+NaK-H]+ 8.50E-09 Increase 1.2 were significantly decreased in cells that were cultured in the absence of vitamin 9 157.011 malic acid [M+Na]+ 2.82E-08 Decrease 1.3 B6 (P<0.01 for all time points, with a 41-75% reduction), compared to 100 nM PL. PL 10 178.993 malic acid [M+2Na-H]+ 3.75E-08 Decrease 1.3 concentrations were relatively conserved, being significantly decreased at t=14 days 11 215.016 [M+Na]+, isocitric acid [M+Na]+ 4.21E-08 Decrease 1.2 only (P<0.05). Intracellular PMP concentrations were also decreased (P<0.01 at t=7 12 76.039 glycine [M]+ 5.51E-08 Decrease 1.4 + -08 and 18 days, with a 48% and 53% reduction, respectively; P<0.05 at t=4 and 14 days, 13 135.977 glycine [M+NaK-H] 9.44E Decrease 1.7 + -07 with a 27% and 49% reduction, respectively). PN, PNP, PM and PA were below the limit 14 191.040 glutamine [M+2Na-H] 3.42E Increase 1.4 15 113.995 glycine [M+K]+ 6.88E-07 Decrease 1.4 of quantification (LOQ), for both vitamin B6-deficient and –proficient conditions, at all time points. Thus, absence of PL in the medium leads to decreased concentrations a Fold change was calculated from the arithmetic mean values of the AUC of each group. of the active cofactor of vitamin B6, making this model suitable for the study of the consequences of vitamin B6 deficiency on the intracellular metabolome.

32 33 Vitamin B6 is essential for serine de novo biosynthesis

Table 2. The fifteen most significantly altered intracellular compounds (negative scan mode). Direct infusion high resolution mass spectrometry (DI-HRMS) in negative scan mode was used to dependent on extracellular serine. This proposition is strengthened by the decrease detect the overall intracellular metabolic consequence of vitamin B6 deprivation. A t-test was of phosphoserine, the immediate precursor of serine, observed in the untargeted performed on the area under the curve (AUC, in a plot of intensities against time) for every compound. metabolomics analysis (Table 2). Compounds are sorted on the P value between PL absence and presence (100 nM).

# m/z Identification P value Change Fold Change a Cystathionine was strongly decreased in B6-deficient conditions. Cystathionine 1 181.051 homovanilic acid [M]-; 4-hydroxyphenyllactic acid [M]- 2.39E-13 Decrease 1.6 is an intermediary metabolite in the homocysteine transsulfuration pathway. 2 2 104.035 serine [M]- 6.67E-12 Decrease 2.6 Cystathionine β-synthase (CBS, EC 4.2.1.22), the first enzyme in the homocysteine 3 221.060 L-cystathionine [M]-; cysteinyl-threonine [M]-; threoninyl-cysteine [M]- 4.48E-11 Decrease 3.8 transsulfuration pathway, catalyses cystathionine synthesis from homocysteine 4 129.056 3-methyl-2-ketovaleric acid [M]-;4-methyl-2-ketovaleric acid [M]- 5.91E-11 Decrease 2.1 and serine, using pyridoxal 5’-phosphate as cofactor. Our results suggest that the - -11 5 215.033 hexose [M+Cl] 6.98E Decrease 1.4 additive effects of vitamin B6 and serine deficiencies led to a strong decrease of - -10 6 87.009 [M] 2.31E Decrease 1.5 cystathionine production. 7 181.039 glutamine [M+Cl]- 6.09E-10 Increase 1.3 8 140.012 O-phosphoethanolamine, serine [M+Cl]-, 2.24E-09 Decrease 2.9 Figure 1 α/β/γ-aminobutyric acid [M+K-H]-, dimethylglycine [M+K-H]- 9 184.002 phosphoserine [M]- 1.08E-08 Decrease 3.5 Intracellular Extracellular 10 145.062 glutamine [M]-, ureidoisobutyric acid [M]-; alanyl-glycine [M]- 2.53E-08 Increase 1.3 Serine Serine 11 195.051 gluconic acid [M]-, galactonic acid [M]- 3.20E-08 Decrease 1.2 ∗∗ ∗∗ ∗∗ ∗∗ 80 ∗∗ 50 12 218.033 3-methyladipic acid [M]-, pimelic acid [M+NaCl]- 1.06E-07 Decrease 1.4 ∗∗ ∗∗ ∗∗ ∗∗ 60 Export 13 190.973 glyceraldehyde-3-P [M+Na-H]- 1.13E-07 Decrease 1.3 0 - -07 40 -50 14 243.038 glutamine [M+H2PO4] 2.37E Increase 1.3 - - -07

15 150.980 maleic acid [M] , [M] 7.49E Decrease 1.4 20 Import -100 -150 a Fold change was calculated from the arithmetic mean values of the AUC of each group. 3 8 11 15 18 3 8 11 15 18 The most striking observations were the decreased signals of serine and glycine, two Glycine Glycine µ mol/L amino acids that are increased in plasma of vitamin B6-deficient patients and animal 800 ∗∗ ∗∗ ∗ 240

nmol/mg protein ∗∗ ∗ ∗ models. Thus, we confirmed the amino acid findings by quantitative LC-MS/MS 600 180 (Figure 1 and S2). The intracellular amino acid fluctuations observed along the course 400 120 of the study were due to the media refreshment schedule. Media was refreshed every Export 200 60 24 hours before sampling. As a consequence, cells were exposed to the same medium for 2 or 3 days, depending on the collection time point. Nevertheless, intracellular 3 8 11 15 18 3 8 11 15 18 serine concentrations were significantly lower (P<0.01) at all time points, while Time (days) Time (days) intracellular glycine concentrations were lower at t=4, 11, 14 and 18 days (P<0.01, P<0.01, P<0.01 and P<0.05, respectively). Reasoning that the decrease of serine could 0 nM PL 100 nM PL be either due to reduced biosynthesis or reduced import from the culture medium, we analysed serine and glycine concentrations in the medium that was sampled from Figure 1. Serine and glycine levels are decreased in vitamin B6-deficient cells. the cells during the experiment (Figure 1). In the presence of 100 nM PL, cells were Neuro-2a cells were grown in medium in the presence (100 nM) and absence of PL for 18 days. found to have net export of serine into the medium at later time points. In contrast, in Extracellular serine and glycine concentrations are presented as the difference to the media basal vitamin B6-deficient cells import of serine from the medium exceeds export. Glycine amino acid levels. Intracellular concentrations are normalized to total protein content. All results are represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01. is exported, both in PL presence and absence but vitamin B6-deficient cells exported 17-21% less glycine than cells cultured in 100 nM PL. This suggests that biosynthesis of serine is hampered when cells are deficient in vitamin B6, making them more

34 35 Vitamin B6 is essential for serine de novo biosynthesis

Serine biosynthesis is decreased when vitamin B6 is deficient DISCUSSION To investigate serine de novo biosynthesis we performed a metabolic flux analysis. 13 Vitamin B6-deficient and -proficient cells were incubated with C6-. In the Vitamin B6 has an important role in development and functioning of the brain by vitamin B6 deficiency condition, serine synthesis was significantly decreased (P<0.05) catalysing essential reactions in neurotransmitter and amino acid metabolism (Surtees 13 with a 50% reduction at t=12 hours. Intracellular C2-glycine concentrations were et al 2006). To investigate the metabolic consequences of vitamin B6 deficiency in the significantly decreased at t=0.5 (P<0.01) and 12 hours (P<0.05), being 38% less in the brain, we employed a model system of Neuro-2a cells that were cultured in vitamin 2 B6-deficient cells at t=12 hours (Figure 2). B6-deficient medium. Neuro-2a cells have a neuronal origin and are easily cultured in high amounts. In previous (yet unpublished) work we have investigated the presence We analysed the intracellular concentrations of 5-mTHF, the other product of SHMT of the enzymes involved in vitamin B6 metabolism and found that all are present in activity, in addition to glycine, and established they were significantly decreased at t=1 these cells, confirming that they provide a suitable model. This model mimics vitamin and 3 days (**P<0.01, and *P<0.05, respectively) in vitamin B6-deficient cells (Figure 2). B6 deficiency as it results in strongly decreased intracellular concentrations of PLP (63% reduction at the latest time point) and PMP (50% reduction at the latest time point). The low PLP is a direct consequence of PL absence in the medium, whereas the decrease of PMP may reflect less transaminating activity secondary to the low PLP. It Figure 2 is unknown how the brain intracellular concentrations of vitamin B6 relate between B6-deficient and B6-proficient humans. However, some in vivo animal studies have 13C -glucose 6 documented vitamin B6 levels in the brain of B6-deficient animals. In Dakshinamurti ATP ADP et al., adult rats were fed PN-supplemented and PN-deficient diets and the PLP 13C -glucose-6-P 6 concentrations in the brains were documented. In whole brain of B6-deficient rats,

ATP PLP concentrations were 28% reduced, while in their hypothalamus the reduction ADP was 57% compared to PN-supplemented (control) rats (Dakshinamurti et al, 1985). Serine de novo biosynthesis These results are in close accordance with the ones presented in this study. Thus, we 13 13 13 C3-glycerate-3-P C3-hydroxypyruvate-3-P C3-serine-3-P 3-PGDH PSAT 13C -serine 4000 3 ∗ successfully created a vitamin B6 deficiency model system. PSPH 3000 5-mTHF 40 ∗∗ 2000 Methylation 30 ∗ 1000 Cycle 13C -serine Investigation of metabolism by untargeted metabolomics yielded a range of altered 20 3 0.5 4 12 10 metabolites: amino acids, Krebs cycle intermediates, GABA and homovanillic acid. 13 ∗ 8000 C2-glycine pmol/mg protein 1 3 THF SHMT 6000 Among the most significantly changed metabolites were serine and glycine, which Time (days) 5-mTHF 5,10-mTHF MTHFR Intensity/mg protein 4000 0 nM PL 100 nM PL ∗∗ were both decreased as validated by targeted LC-MS/MS analysis. Flux studies clearly 2000

13 13C -glycine illustrated reduced serine biosynthesis in vitamin B6-deficient Neuro-2a cells and thus C3-pyruvate 2 0.5 4 12 Time (hours) made evident that serine biosynthesis depends on vitamin B6. The PLP-dependent 0 nM PL 100 nM PL enzyme phosphoserine aminotransferase (PSAT, EC 2.6.1.52) cannot fully function in vitamin B6 deficient conditions, which is expected to result in less synthesis of Figure 2. Vitamin B6 deficiency hampers serinede novo biosynthesis and decreases 5-methyltetrahydrofolate levels. phosphoserine and serine, as observed. Neuro-2a cells were grown in the presence (100 nM) and absence of PL for 3 days. On day 3 cells 13 13 13 were incubated with C6-glucose, and the formation of the labelled C3-serine and C2-glycine was The low medium and intracellular serine concentrations are in contrast with reports analyzed at t=0.5, 4 and 12 hours after exposure. Results are represented as the mean of triplicates ± on the effect of vitamin B6 deprivation on human plasma, in which the concentrations SD. The insert reflects the steady state levels of5-methyltetrahydrofolate. For 5-methyltetrahydrofolate study, Neuro-2a cells were grown in medium in the presence (100 nM) and absence of PL for 3 days. of glycine showed an increase of 28% after two weeks of vitamin B6 depletion and The results are normalized to total protein content and represented as the mean of n=6 (t=1 day) and serine showed an increase of 47% after one week of depletion (Park et al 1971). This n=12 (t=3 days) ± SD; * P<0.05, ** P<0.01. behaviour was also observed in a 28-day vitamin B6 restriction diet study, where the

36 37 Vitamin B6 is essential for serine de novo biosynthesis

plasma levels of healthy men and women for glycine and serine showed an increase (5,10-methyleneTHF) and glycine (Koning et al 2003). 5,10-methyleneTHF is of 15% and 12%, respectively (da Silva et al 2013). Furthermore, cerebrospinal fluid converted by the enzyme methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) (CSF) studies of PNPO-deficient patients have reported elevated levels of glycine to 5-mTHF, the main circulating form of folate which can serve as a methyl donor prior to B6 supplementation (Mills et al 2005). For the one individual in this study in in the generation of S-adenosylmethionine (SAM). Indeed, the combination of less whom CSF analysis was repeated after supplementation this normalised. Elevation serine and PLP may lead to a lower activity of SHMT, explaining the decrease in the of CSF glycine has been reported to occur secondary to a deficiency of the activity intracellular concentration of 5-mTHF in vitamin B6-deficient cells. 2 of the glycine cleavage system, which is PLP-dependent. It should be noted however that this does not occur in all PNPO-deficient patients, with some only showing a Our findings are important in considering pathogenesis and treatment of patients with transient increase of glycine in CSF (Hoffmann et al 2007). Glycine levels have also vitamin B6-dependent epilepsy. Generally, it was thought that a reduction of GABA been reported to be slightly raised in the CSF of patients with mutations in PROSC concentrations in the brain of these patients was the main cause of the epilepsy, due prior to B6-supplementation (Darin et al 2016). In patients with mutations in to suboptimal activity of the PLP-dependent enzyme glutamic acid decarboxylase. ALDH7A1, however, CSF glycine levels have been reported to be normal (Hoffmann We demonstrate that low serine, glycine and 5-methyltetrahydrofolate may also et al 2007) or just slightly elevated (Mills et al 2010). Interestingly few studies report contribute to pathogenesis. Probably, these amino acids are low in brain cells in vivo, on serine levels leading us to assume that serine is kept within normal values in as suggested by the B6-deficient mouse studies (Tews 1969). Although theoretically the CSF of vitamin B6 deficient patients. Additionally, in a B6-deprivation study in uptake of serine and glycine from blood to brain may compensate a lower serine HepG2 cells, vitamin B6 deficiency yielded large increases in glycine concentrations biosynthetic capacity, two observations suggest clinical relevance of our findings. and no effect on serine (da Silva et al 2014). However, our findings in the Neuro-2a Patients with a defect in serine synthesis need high doses of serine to normalize model are in accordance with in vivo studies performed by Tews, in which mice fed serine in CSF (Koning et al 2003; van der Crabben et al 2013). Furthermore, some on a PN-deficient diet for 4 weeks presented a progressive decrease in brain serine patients with vitamin B6-dependent epilepsy clinically respond to supplementation concentrations. Brain glycine concentrations significantly decreased during the first of folinic acid, a 5-mTHF precursor (Nicolai et al 2006; Gallagher et al 2009; Stockler et 2 weeks of PN-deprivation and increased after 4 weeks when compared to normal- al 2011; Dill et al 2011; van Karnebeek et al 2016). Our work provides an explanation PN fed mice. Upon reinstating a complete-PN diet, serine and glycine concentrations for this hitherto puzzling observation. returned to control levels (Tews 1969). This suggests that a decrease in serine and concomitant decrease in glycine concentrations is tissue or cell type dependent. MATERIAL AND METHODS Serine in brain originates from two sources: uptake and biosynthesis (Koning et al 2003). Serine de novo biosynthesis is a side-pathway of glycolysis. The first and rate-limiting Cell culture step is the oxidation of 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate, Neuro-2a cells were purchased from ATCC Cell Biology Collection. Dulbecco’s by 3-phosphoglycerate dehydrogenase (PHGDH, EC 1.1.1.95). The conversion of modified eagle medium (DMEM) GlutaMAX™ (31966), B6 vitamer-free DMEM 3-phosphohydroxypyruvate to 3-phosphoserine is catalysed by phosphoserine GlutaMAX™ (custom made 31966-like) medium, fetal bovine serum (FBS; 10270), aminotransferase, a PLP-dependent enzyme. Serine biosynthesis seems to be particularly penicillin-streptomycin (P/S; 15140) and trypsin-ethylenediaminetetraacetic acid important in the brain, as illustrated by the severe clinical symptoms in patients affected (trypsin-EDTA, 0.5%) were purchased from Gibco (Invitrogen Life Technologies). with a defect in serine synthesis, including congenital microcephaly, severe epilepsy and Pyridoxal hydrochloride (PL-HCl) was purchased from Sigma-Aldrich (Steinheim, very little development (Jaeken et al 1996; Furuya, 2008). In CSF of these patients, serine Germany). Cells were grown in 75 cm2 flasks and maintained in DMEM GlutaMAX™ (both L- and D-serine) and glycine are decreased (van der Crabben et al 2013). supplemented with 10% heat-inactivated FBS and 1% P/S, in a humidified atmosphere

of 5% CO2 at 37ºC. When cells reached optimal confluence (>70%) they were washed Glycine production depends on serine availability and on the PLP-dependent twice with PBS and passed into 6-well plates (1.5 x 105 cells per well) by trypsinization enzyme SHMT. SHMT catalyses the transfer of the methyl group of serine to with 0.05% trypsin-EDTA. Confluent cells (>70%) were exposed to the experimental tetrahydrofolate (THF), allowing the formation of 5,10-methylenetetrahydrofolate medium conditions: 1:1, PBS:B6 vitamer-free DMEM GlutaMAX™ (with 10% FBS and

38 39 Vitamin B6 is essential for serine de novo biosynthesis

1% P/S), with 100 nM of PL-HCl (content of vitamin B6: PL 97.4 ± 5.6 nM; PN 2.6 ± 5-methyltetrahydrofolate (5-mTHF) 2.6 nM; PM 1.4 ± 1.3 nM; PLP 1.9 ± 1.5; PNP and PMP are below the LOQ) or without 5-mTHF was purchased from Sigma-Aldrich (Steinheim, Germany).The internal standard 13 vitamin B6 (residual content of vitamin B6: PL 4.7 ± 3.8 nM; PN 2.3 ± 0.4 nM; PM 1.1 ± C5-5-mTHF was purchased from Merck KGaA (Darmstadt, Germany). 5-mTHF analyses 1.1 nM; PLP, PNP and PMP are below the LOQ). were performed on a Waters Micromass Quattro Ultima triple quadrupole mass spectrometer (Manchester, U.K.), using an Acquity UPLC® BEH C18 (130 Å, 17 µm 2.1 x 50 Direct-Infusion High-Resolution Mass Spectrometry (DI-HRMS) mm column) (Waters, Manchester, UK), which was kept at 30°C, while the autosampler 2 Direct-infusion was performed using chip-based infusion (400 nozzles, nominal temperature was kept at 15°C. The dwell time was set at 0.3 s. The capillary voltage internal Ø 5 µm) on the TriVersa NanoMate (Advion, Ithaca, NY, USA). High-resolution was 3.00 kV and the cone voltage was 35 V. The source and desolvation temperatures mass spectrometry (140,000) was performed using a Q-ExactivePlus (Thermo were 120 and 450°C respectively. The cone gas flow rate was 158 L/hr. Quantitative Scientific GmbH, Bremen, Germany) using a scan range of m/z 70 to 600 in positive analysis was achieved using a negative ion multiple reaction monitoring (MRM) mode and negative modes. Besides mass calibration of the instrument, internal lock masses with the m/z transitions of 460.2 > 313.2 and 465.2 > 313.2 with a collision energy of 17 13 were used for high mass accuracy. Cells were harvested in biological triplicates. and 18 V, for 5-mTHF and C5-5-mTHF respectively. The specific MRM transitions were determined by direct infusion of both standards and internal standards. Untargeted metabolomics pipeline (DI-HRMS) RAW data files were converted to mzXML format using MSConvert (M.C. Chambers Metabolic flux analysis: serine de novo biosynthesis et al, 2012). The data were processed using an in-house developed untargeted Neuro-2a cells were grown in 6-well plates and maintained in DMEM GlutaMAX™. When cells metabolomics pipeline written in the R programming language (http://www.r- reached a confluence of >70%, they were washed twice with room temperature PBS and project.org). First, the mzXML files were converted to readable format by the XCMS incubated with B6 vitamer-free DMEM GlutaMAX-I (supplemented with 10% FBS, 1% P/S, package (Smith et al 2006). For every sample, peak finding was done and peaks and with either 100 nM PL-HCl or without PL-HCl) : PBS, 1:1. Cells were grown for 72 h in these with the same m/z (within 0.5*fwhm) were grouped over different samples. Peak media before collection. At 72 h the medium was refreshed with the exposition medium: groups that were not present in three out of three technical replicates in at least B6 vitamer-free DMEM GlutaMAX-I (supplemented with 10 % FBS, 1 % P/S, and without 13 one biological sample were discarded. The intensities of the technical replicates were or with 100 nM PL-HCl) : PBS ( C6-glucose, 25 mM), 1:1. Cells were harvested at T = 0.5, 4 13 averaged. Peak groups were identified using all entries in the HMDB, including their and 12 hours. Uniformly labelled C6-glucose (99%) was purchased from Cambridge + + + - 13 13 most likely adducts (Na , K , NH4 in positive mode and Cl and formate in negative Isotope Laboratories, Inc. (MA, USA). To quantify the intracellular C3-serine and C2- mode) and isotopes, using an accuracy of 3 ppm or better. The statistical analysis was glycine, we adapted the LC-MS/MS method described by Prinsen (Prinsen et al 2016). a t-test on the Area Under the Curve (AUC) in a plot of intensities against time for every metabolite. Raw metabolomics data can be supplied upon request. Protein analysis Protein concentrations were quantified using the 96-well microplate protocol of the Ultra Performance Liquid Chromatography Tandem Mass colorimetric bicinchoninic acid (BCA) PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific Spectrometry (UPLC-MS/MS) Incorporated), in accordance with the manufacturer’s protocol, with BSA as standard. Amino acids Amino acid concentrations were determined using the UPLC-MS/MS method described Statistical analysis by Prinsen (Prinsen et al 2016). Apart from adapting the range of the calibrators and Statistical significance was determined with unpaired two-tailed t-test, using quality control (QC) samples to resemble the concentrations in the samples, no further GraphPad Prism 6 (version 6.0.2, GraphPad Software Inc.) software. adaptations were needed for sample preparation or analysis of the amino acids. Acknowledgments Vitamin B6 vitamers We would like to thank Marjolein Bosma and Birgit Schiebergen-Bronkhorst for Vitamin B6 vitamers were quantified according to the method of van der Ham (van their technical assistance. This work was funded by Metakids foundation der Ham et al 2012). (www.metakids.nl).

40 41 Vitamin B6 is essential for serine de novo biosynthesis

Compliance with Ethics Guidelines REFERENCES

Conflict of interest Baumeister FA, Gsell W, Shin YS, Egger J (1994) Glutamate in Pyridoxine-Dependent Epilepsy: Neurotoxic Glutamate Concentration in the Cerobrospinal Fluid and Its Normalization by Rúben Ramos, Mia Pras-Raves, Johan Gerrits, Maria van der Ham, Marcel Willemsen, Pyridoxine. Pediatrics 94: 318-321 Hubertus Prinsen, Boudewijn Burgering, Judith Jans, and Nanda Verhoeven-Duif Baxter P (1999) Epidemiology of pyridoxine dependent and pyridoxine responsive seizures in the declare that they have no conflicts of interest. UK. Arch Dis Child 81: 431–433 2 Baxter P (2001) Pyridoxine-dependent and pyridoxine- responsive seizures. Dev Med Child Neurol 43: 416–420 Informed Consent Clayton PT (2006) B6-responsive disorders: a model of vitamin dependency. J Inherit Metab Dis This article does not contain any studies with human or animal subjects performed 29(2-3): 317–326 by the any of the authors. Chambers MC, Maclean B, Burke R, Amodei D, Ruderman DL, Neumann S, Gatto L, Fischer B, Pratt B, Egertson J, Hoff K, Kessner D, Tasman N, Shulman N, Frewen B, Baker TA, Brusniak M-Y, Paulse C, Creasy D, Flashner L, Kani K, Moulding C, Seymour SL, Nuwaysir LM, Lefebvre B, Kuhlmann F, Author Contributions Roark J, Rainer P, Detlev S, Hemenway T, Huhmer A, Langridge J, Connolly B, Chadick T, Holly K, Rúben Ramos, Judith Jans and Nanda Verhoeven-Duif designed the project and wrote Eckels J, Deutsch EW, Moritz RL, Katz JE, Agus DB, MacCoss M, Tabb DL, Mallick P (2012) A Cross- the manuscript. Rúben Ramos, Johan Gerrits and Maria van der Ham performed the platform Toolkit for Mass Spectrometry and Proteomics. Nat Biotechnol 30(10): 918-920 da Silva VR, Ralat MA, Quinlivan EP, DeRatt BN, Garrett TJ, Chi Y-Y, Reed MC, Gregory JF (2014) Targeted LC-MS/MS and DI-HRMS analysis. Rúben Ramos, Mia Pras-Raves and Marcel Willemsen metabolomics and mathematical modeling demonstrate that vitamin B-6 restriction alters one- performed the statistical analysis. Hubertus Prinsen, Maria van der Ham and Rúben carbon metabolism in cultured HepG2 cells. Am J Physiol Endocrinol Metab 307(1): E93–101 Ramos developed the methods used in the manuscript. Rúben Ramos, Judith da Silva VR, Rios-Avila L, Lamers Y, Ralat MA, Midttun Ø, Quinlivan EP, Garret TJ, Coats B, Shankar MN, Jans, Nanda Verhoeven-Duif, Hubertus Prinsen and Boudewijn Burgering critically Percival SS, Chi YY, Muller KE, Ueland PM, Stacpoole PW, Gregory JF (2013a) Metabolite profile analysis reveals functional effects of 28-day vitamin B-6 restriction on one-carbon metabolism discussed the results and revised the manuscript. and tryptophan catabolic pathways in healthy men and women. J Nutr 143(11): 1719–1727 Dakshinamurti K, Paulose CS, Siow YL (1985) Neurobiology of pyridoxine. In Reynolds RD, Leklem JE, eds. Vitamin B6: Its Role in Health and Disease. New York: AR Liss, 99-121. Darin N, Reid E, Prunetti L, Samuelsson L, Husain RA, Wilson M, El Yacoubi B, Footitt E, Chong WK, Wilson LC, Prunty H, Pope S, Heales S, Lascelles K, Champion M, Wassmer E, Veggiotti P, de Crécy- Lagard V, Mills PB, Clayton PT (2016) Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy. Am J of Hum Genet 99(6): 1325–1337 De Roo MG, Abeling NG, Majoie CB, Bosch AM, Koelman JH, Cobben JM, Duran M, Poll-The BT (2014) Infantile hypophosphatasia without bone deformities presenting with severe pyridoxine- resistant seizures. Mol Genet Metab 111(3): 404–407 Dill P, Schneider J, Weber P, Trachsel D, Tekin M, Jakobs C, Thöny B, Blau N (2011) Pyridoxal phosphate- responsive seizures in a patient with cerebral folate deficiency (CFD) and congenital deafness with labyrinthine aplasia, microtia and microdontia (LAMM). Mol Genet Metab 104(3): 362–368 Ebadi M (1981) Regulation and function of pyridoxal phosphate in cns. Neurochem Int 3: 181–206 Furuya S (2008) An essential role for de novo biosynthesis of L-serine in CNS development. Asia Pac J Clin Nutr 17(S1): 312–315 Gallagher RC, Van Hove JL, Scharer G, Hyland K, Plecko B, Waters PJ, Mercimek-Mahmutoglu S, Stockler-Ipsiroglu S, Salomons GS, Rosenberg EH, Struys EA, Jakobs C (2009) Folinic acid- responsive seizures are identical to pyridoxine-dependent epilepsy. Ann Neurol 65(5): 550–556 Gospe SM Jr, Olin KL, Keen CL (1994) Reduced GABA synthesis in pyridoxine-dependent seizures. Lancet 343: 1133–1134 Goto T, Matsuo N, Takahashi T (2001) CSF glutamate/GABA concentrations in pyridoxine-dependent seizures: etiology of pyridoxine-dependent seizures and the mechanisms of pyridoxine action in seizure control. Brain Dev 23: 24–29 Hoffmann GF, Schmitt B, Windfuhr M, Wagner N, Strehl H, Bagci S, Franz AR, Mills PB, Clayton PT, Baumgartner MR, Steinmann B, Bast T, Wolf NI, Zschocke J (2007) Pyridoxal 5’-phosphate may be curative in early-onset epileptic encephalopathy. J Inherit Metab Dis 30(1): 96–99

42 43 Vitamin B6 is essential for serine de novo biosynthesis

Jaeken J, Detheux M, Van Maldergem, L, Foulon M, Carchon H, Van Schaftingen E (1996) 3-Phosphoglycerate Supplementary experimental procedures dehydrogenase deficiency: inborn error of serine biosynthesis. Arch Dis Child 74: 542–545 de Koning TJ, Snell K, Duran M, Berger R, Poll-the BT, Surtees R (2003) L-serine in disease and development. Biochem J 371: 653–661 Sample collection and preparation for Direct-Infusion High- Lee GH, Sung SY, Chang WN, Kao TT, Du HC, Hsiao TH, Safo MK, Fu TF (2012) Zebrafish larvae exposed to Resolution Mass Spectrometry (DI-HRMS) ginkgotoxin exhibit seizure-like behavior that is relieved by pyridoxal-5’-phosphate, GABA and anti- Neuro-2a cells were washed twice with cold PBS (4ºC) and harvested by scraping epileptic drugs. Dis Model Mech 5(6): 785–795 of cells with 1.5 ml ice-cold methanol (-20ºC), into a 1.5 ml eppendorf tube. The Levtova A, Camuzeaux S, Laberge AM, Allard P, Brunel-Guitton C, Diadori P, Rossignol E, Hyland K, Clayton 2 PT, Mills PB, Mitchell GA (2015) Normal Cerebrospinal Fluid Pyridoxal 5’-Phosphate Level in a PNPO- samples were centrifuged (16200 g for 10 min at 4ºC), and the supernatant was Deficient Patient with Neonatal-Onset Epileptic Encephalopathy. JIMD Rep 22: 67-75 transferred to a new 1.5 ml eppendorf tube and stored at -80ºC until analysis was Mills PB, Camuzeaux SS, Footitt EJ, Mills KA, Gissen P, Fisher L, Das KB, Varadkar SM, Zuberi S, McWilliam R, performed. Before mass spectrometry analysis, samples were evaporated to dryness Stödberg T, Plecko B, Baumgartner MR, Maier O, Calvert S, Riney K, Wolf NI, Livingston JH, Bala P, Morel CF, at 40ºC, under a gentle stream of nitrogen. Samples were reconstituted with 800 µl Feillet F, Raimondi F, Del Giudice E, Chong WK, Pitt M, Clayton PT (2014) Epilepsy due to PNPO mutations: Genotype, environment and treatment affect presentation and outcome. Brain 137(5): 1350–1360 of methanol at room temperature. Cell extracts (in methanol) were diluted (1:1 v/v) Mills PB, Footitt EJ, Mills KA, Tuschl K, Aylett S, Varadkar S, Hemingway C, Marlow N, Rennie J, Baxter P, Dulac using 70 µl stable isotopes solution in methanol (NSK-A-amino acids and NSK-B-free O, Nabbout R, Craigen WJ, Schmitt B, Feillet F, Christensen E, De Lonlay P, Pike MG, Hughes MI, Struys EA, carnitine and acylcarnitine reference standards (Cambridge Isotope Laboratories, Jakobs C, Zuberi SM, Clayton PT (2010). Genotypic and phenotypic spectrum of pyridoxine-dependent epilepsy (ALDH7A1 deficiency). Brain 133(7): 2148–2159 Massachsetts, USA)). After dilution with 60 µl 0.3% formic acid, samples were filtered Mills PB, Surtees RA, Champion MP, Beesley CE, Dalton N, Scambler PJ, Heales SJ, Briddon A, Scheimberg I, using a preconditioned (with methanol) 96-well filter plate (Acro prep, 0.2 µm GHP, Hoffmann GF, Zschocke J, Clayton PT (2005). Neonatal epileptic encephalopathy caused by mutations NTRL, 1 ml well; Pall Corporation, Ann arbor, USA). The sample filtrate was collected in the PNPO gene encoding pyridox(am)ine 5’-phosphate oxidase. Hum Mol Genet 14(8): 1077–1086 in a 96-well plate (Advion, Ithaca, NY, USA). A volume of 13 µl was infused into the Nicolai J, van Kranen-Mastenbroek VH, Wevers RA, Hurkx WA, Vles JS (2006). Folinic acid-responsive seizures initially responsive to pyridoxine. Pediatr Neurol 34(2): 164–167 DI-HRMS system in triplicate (technical replicates). Prinsen HC, Schiebergen-Bronkhorst BG, Roeleveld MW, Jans JJ, de Sain-van der Velden MG, Visser G, van Hasselt PM, Verhoeven-Duif NM (2016) Rapid quantification of underivatized amino acids in plasma Sample collection and preparation for amino acid import, by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry. J Inherit Metab Dis 39(5): 651-660 metabolism and export quantification by UPLC-MS/MS Park YK, Linkswiler H (1971) Effect of vitamin B6 depletion in adult man on the plasma concentration and Neuro-2a samples (medium and intracellular content) were collected in biological the urinary excretion of free amino acids. J Nutr 101(2): 185–191 triplicates. The culture medium (1:1, PBS:B6 vitamer-free DMEM GlutaMAX™ (with Smith CA, Want EJ, O’Maille G, Abagyan R, Siuzdak G (2006) XCMS: processing mass spectrometry 10% FBS, 1% P/S), supplemented with 100 nM of PL-HCl or without a vitamin B6 data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78(3): 779–787 source addition) was refreshed every 24 h before samples collection. Medium was Stockler S, Plecko B, Gospe SM Jr, Coulter-Mackie M, Connolly M, van Karnebeek C, Mercimek-Mahmutoglu collected prior to cells harvesting, for amino acid import and export study. After S, Hartmann H, Scharer G, Struijs E, Tein I, Jakobs C, Clayton P, Van Hove JL (2011) Pyridoxine dependent media collection, the Neuro-2a cells were washed twice with cold PBS and scraped epilepsy and antiquitin deficiency: clinical and molecular characteristics and recommendations for with 1.5 ml ice-cold methanol (-20ºC). Samples were transferred into eppendorf tubes, diagnosis, treatment and follow-up. Mol Genet Metab 104(1-2): 48–60 Surtees R, Mills P, Clayton P (2006) Inborn errors affecting vitamin B 6 metabolism. Future Neurol 1(5): 615–620 centrifuged (16200 g for 10 min at 4ºC), and the supernatants were transferred to new Tews JK (1969) Pyridoxine deficiency and brain amino acids. Ann N Y Acad Sci 166(1): 74–82 eppendorf tubes. Pellets were discarded and the supernatants were evaporated to Ueland PM, Ulvik A, Rios-Avila L, Midttun Ø, Gregory JF (2015) Direct and Functional Biomarkers of Vitamin dryness at 40ºC, under a gentle stream of nitrogen, and reconstituted with 800 µl of B6 Status. Ann Rev Nutr 35: 33–70 methanol. The reconstituted samples were stored at -80ºC until amino acid analysis van der Crabben SN, Verhoeven-Duif NM, Brilstra EH, Van Maldergem L, Coskun T, Rubio-Gozalbo E, Berger R, de Koning TJ (2013) An update on serine deficiency disorders. J Inherit Metab Dis 36(4): 613–9 was performed. van der Ham M, Albersen M, de Koning TJ, Visser G, Middendorp A, Bosma M, Verhoeven-Duif NM, de Sain-van der Velden MG (2012) Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry. Anal Chim Acta 712: 108–114 van Karnebeek CDM, Tiebout SA, Niermeijer J, Poll-The BT, Ghani A, Coughlin CR, Van Hove JLK, Richter JW, Christen HJ, Gallagher R, Hartmann H, Stockler-Ipsiroglu S (2016) Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum. Pediatr Neurol 59: 1–7 Walker V, Mills GA, Peters SA, Merton WL (2000) Fits, pyridoxine, and hyperprolinaemia type II. Arch Dis Child 82(3): 236–237

44 45 Vitamin B6 is essential for serine de novo biosynthesis Figure S1

Sample collection and preparation for quantification of vitamin B6 A B C vitamers by UPLC-MS/MS Pyridoxal Pyridoxal 5’-phosphate Pyridoxamine 5’-phosphate 6 400 400 ** ** * Neuro-2a cells, in biological triplicates, were washed twice with cold (4ºC) PBS and * ** ** ** 300 ** 300 ** 4 * scraped with 600 µl of cold TCA. The samples were collected in eppendorf tubes and 200 200 2 centrifuged (16200 g, 10 min at 4ºC). The supernatants were stored at -80ºC until 100 100 analysis was performed. pmol/mg protein 2 3 8 11 15 18 3 8 11 15 18 3 8 11 15 18

Sample preparation for quantification of 5-methyltetrahydrofolate 0 nM PL 100 nM PL Time (days) by UPLC-MS/MS

Neuro-2a cells, in biological triplicates, were harvested using cold ascorbic acid Figure S1. Pyridoxal restriction results in intracellular vitamin B6 vitamers decrease. (150 µM, 4ºC). Before harvesting, cells were washed twice with 1 ml of cold PBS Neuro-2a cells were grown for 18 days. The media were replaced every 24 hours before cells were and scraped with 800 µl of cold ascorbate. The samples were collected into 1.5 ml harvested, and the intracellular vitamin B6 vitamers concentrations were determined by UPLC-MS/ MS. Data are normalized to total protein content and are represented as the mean of triplicates ± SD; eppendorf tubes, sonicated for 10 min, and centrifuged (16200 g, 10 min at 4ºC). * P<0.05, ** P<0.01. The supernatants were transferred to new 1.5 ml eppendorf tubes and the pellets discarded. Samples were stored at -80ºC until analysis was performed.

Sample preparation for metabolic flux analysis Neuro-2a cells were grown in 6-well plates and maintained in DMEM GlutaMAX™. When cells reached a confluence of >70%, they were washed twice with room temperature PBS and incubated with B6 vitamer-free DMEM GlutaMAX-I (supplemented with 10% FBS, 1% P/S, and with either 100 nM PL-HCl or without vitamin B6 source) : PBS, 1:1. Cells were grown for 72 h in these media before collection. At 72 h the medium was refreshed with the exposition medium: B6 vitamer-free DMEM GlutaMAX-I (supplemented with 10 % FBS, 1 % P/S, and without or with 100 nM PL-HCl) : PBS 13 ( C6-glucose, 25 mM), 1:1. Cells were harvested at T = 0.5, 4 and 12 hours. Before collection, cells were washed twice with cold PBS (4ºC), and harvested by scraping with 1.5 ml ice-cold methanol. The samples were transferred into 1.5 ml eppendorf tubes, centrifuged (16200 g for 10 min at 4ºC), and the supernatants were transferred to new 1.5 ml eppendorf tubes. The samples were evaporated at 40ºC under a gentle stream of nitrogen until complete dryness, and reconstituted with 800 µl of methanol at room temperature. The reconstituted samples were stored at -80ºC until amino acid analysis was performed.

46 47 Vitamin B6 is essential for serine de novo biosynthesis

Figure S2b Figure S2a Methionine Ornithine Taurine Glutamic acid ∗∗ 400 ∗∗ ∗ 800 ∗∗ ∗∗ ∗∗ 12 8 ∗ ∗∗ 300 600 9 ∗∗ 6 200 400 6 4 100 200 3 2

3 8 11 15 18 3 8 11 15 18 2 3 8 11 15 18 3 8 11 15 18 Glutamine Lysine 200 260 ∗∗ 80 ∗∗ 32 ∗∗ 150 ∗ ∗ 195 60 ∗∗ 24 ∗∗ 100 130 ∗∗ ∗∗ 40 16 50 65 20 8

3 8 11 15 18 3 8 11 15 18 3 8 11 15 18 3 8 11 15 18 Histidine Hydroxyproline Proline 2.0 280 ∗ 60 ∗ ∗∗ 16 ∗∗ ∗∗ ∗ ∗∗ 1.5 210 ∗∗ ∗∗ 45 12 ∗∗ 1.0 140 30 8 0.5 70 15 4

3 8 11 15 18 3 8 11 15 18 Concentration (nmol/mg protein) 3 8 11 15 18 3 8 11 15 18 Threonine Tyrosine ∗∗ ∗ 100 800 Glycine 48 8 ∗∗ ∗∗ ∗ ∗ ∗ ∗ 75 600 26 ∗ ∗∗ 6 50 400 24 4

Concentration (nmol/mg protein) 25 200 12 2

3 8 11 15 18 3 8 11 15 18 3 8 11 15 18 3 8 11 15 18 Tryptophan Serine ∗∗ ∗∗ 80 ∗∗ 800 40 ∗∗ 12 ∗ ∗∗ 60 ∗∗ ∗∗ ∗∗ ∗∗ 600 30 ∗∗ ∗∗ 9 ∗ 40 400 20 6 20 200 10 3

3 8 11 15 18 3 8 11 15 18 3 8 11 15 18 3 8 11 15 18 Time (days) 0 nM PL 100 nM PL Asparagine 60 4 γ−Aminobutyric acid 80 ∗ ∗∗ ∗∗ 45 3 60 ∗∗ ∗ ∗ ∗∗ ∗∗ 30 40 2 15 20 1

3 8 11 15 18 3 8 11 15 18 3 8 11 15 18

0 nM PL 100 nM PL Time (days) Concentration (pmol/mg protein) Time (days) Figure S2. Effect of pyridoxal restriction in the intracellular amino acid levels. Neuro-2a cells were grown for 18 days. The media were replaced every 24 hours before cells were harvested, and the intracellular amino acid concentrations were determined by UPLC-MS/MS. Data are normalized to total protein content and are represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01.

48 49 3

Chapter 3

Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

Clara D.M. van Karnebeek†, Rúben J. Ramos†, Xiao-Yan Wen†, Maja Tarailo-Graovac†, Joseph G. Gleeson, Cristina Skrypnyk, Koroboshka Brand-Arzamendi, Farhad Karbassi, Mahmoud Y. Issa, Robin van der Lee, Britt I. Drögemöller,Janet Koster, Justine Rousseau, Philippe M. Campeau, Youdong Wang, Feng Cao, Meng Li, Jos Ruiter, Jolita Ciapaite, Leo A.J. Kluijtmans, Michel A.A.P. Willemsen, Judith J. Jans, Colin J. Ross, Liesbeth T. Wintjes, Richard J. Rodenburg, Marleen C.D.G. Huigen, Zhengping Jia, Hans R. Waterham, Wyeth W. Wasserman, Ronald J.A. Wanders, Nanda M. Verhoeven-Duif, Maha S. Zaki, Ron A. Wevers

† These authors contributed equally to this work

Am J Hum Genet. 2019 Sep 5;105(3):534-548. Doi: 10.1016/j.ajhg.2019.07.015 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

ABSTRACT INTRODUCTION

Early-infantile encephalopathies with epilepsy are devastating conditions mandating Infantile-onset encephalopathies with epilepsy often are devastating disorders an accurate diagnosis to guide proper management. Whole exome sequencing was with major consequences for the life of affected individuals and their families. used to investigate the disease etiology in four children from independent families Identification of an underlying diagnosis is paramount for personalized management. with intellectual disability and epilepsy, revealing biallelic GOT2 mutations. In depth Although inborn errors of metabolism do not represent the most common cause metabolic studies in individual 1 showed low plasma serine, hyper­citrullinemia, of these encepha­lo­pathies, their early identification is of utmost importance, since hyperlactatemia and hyperammonemia. The epilepsy was serine- and pyridoxine- many require targeted therapeutic measures beyond that of common antiepileptic responsive. Functional consequences of observed mutations were tested by drugs, either to control seizures, or to decrease the chance of neurodegeneration. measuring enzyme activity and by cell- and animal models. Zebrafish- and mouse Here we describe four affected individuals with a metabolic encephalopathy with 3 models were used to validate brain developmental and functional defects and to epilepsy due to a defect in the mitochondrial isoform of glutamate oxaloacetate test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate or aspartate aminotransferase (GOT; EC 2.6.1.1). This is a pyridoxal transaminase. GOT2 enzyme activity was deficient in fibroblasts with biallelic 5’-phosphate (PLP)-dependent enzyme that exists as cytosolic (GOT1) and intra­ mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role mitochondrial (GOT2) isoforms. Both isoforms catalyze the reversible intercon­version in the intracellular NAD(H) balance. De novo serine biosynthesis was impaired of oxaloacetate and glutamate into aspartate and α-ketoglutarate.­ These enzymes are in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the part of the malate-aspar­ta­te shuttle (MAS), a key player in intra­cellular NAD(H) redox highly-oxidized cytosolic NAD-redox state by pyruvate supplementation restored homeostasis (Figure 1).1,2 NADH produced in cytosolic NAD-linked dehydrogenase serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted reactions, mainly during glycolysis, is re-oxidized to NAD+ inside the mitochondria.3 in a brain developmental defect associated with seizure-like electroencephalography Since the inner mitochondrial membrane is relatively impermeable to NAD+ and spikes, which could be rescued by supplying pyridoxine in embryo water. Both NADH,4 NAD(H)-redox shuttles exist.3 The MAS provides a mechanism for net transfer pyridoxine and serine synergistically rescued embryonic developmental defects in of NADH reducing equivalents across the inner mitochondrial membrane.4 Defects zebrafish got2a morphants. The two treated individuals reacted favorably to their in the MAS have been described due to mutations in genes encoding mitochondrial treatment. Our data provide a mechanistic basis for the biochemical abnormalities in (MDH2, [MIM: 154100]) and both aspartate-glutamate carriers GOT2 deficiency that may also hold for other MAS-defects. (SLC25A12, [MIM: 603667], SLC25A13, [MIM: 603859]).5,6,7,8,9

GOT2 deficiency causes a metabolic encephalopathy with early-onset epilepsy, We report GOT2 deficiency, a MAS disorder, and present the clinical and biochemical progressive microcephaly and several biochemical abnormalities that seems phenotype of 3 unrelated families, computational analyses and experimental data to amenable to treatment. validate the deleterious impact of the identified GOT2 [MIM: 138150] variants, as well as biomarkers and therapeutic strategies for this inborn error of metabolism.

52 53 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

A B Whole exome sequencing (WES) analysis GLYCOLYSIS SERINE BIOSYNTHESIS GLYCOLYSIS SERINE BIOSYNTHESIS Family I GLUCOSE GLUCOSE GLUCOSE GLUCOSE Trio (proband-mother-father) whole exome sequencing (WES) was performed using the Agilent SureSelect kit and Illumina HiSeq 2000 (Perkin-Elmer, USA). The sequencing GAP GAP reads were aligned to the human reference genome version hg19 using Bowtie 210 1,3 DPG 1,3 DPG and mean coverage of 43X (proband), 32X (mother) and 48X (father) was achieved. GAP 3PG GAP 3PG The data was further analyzed using our semi-automated bioinformatics pipeline11: GAPDH 3PGDH GAPDH 3PGDH

1,3-DPG 2-OH-PYRUVATE 1,3-DPG 2-OH-PYRUVATE (i) the duplicates were marked and sorted using Picard, (ii) variants were called using NADH NAD+ NADH NAD+ 3-PG 3-PG SERINE SERINE SAMtools and BCFtools after indel realignment using GATK, (iii) transcripts were PEP PEP LDH annotated using snpEff,12 (iv) functional variants were prioritized for rare variants by 3 PYRUVATE MDH1 GOT1 PYRUVATE MDH1 GOT1 LACTATE comparison against the public databases [dbSNP, NHLBI Exome Sequencing Project OGC MAS AGC1/2 OGC MAS AGC1/2

MDH2 GOT2 MDH2 GOT2 Exome Variant Server, and Exome Aggregation Consortium (ExAC)] and (v) subse­ quent­ly screened under a series of Mendelian inheritance models: homozygous, MIM MIM 11 NADH NAD+ NADH NAD+ hemizygous, compound heterozygous and de novo as described previously. The ADP + Pi ATP ADP + Pi ATP UCSC Genome Browser was used to examine conservation of the affected amino OXPHOS OXPHOS acids, while the protein domains in which the variants occurred was visualized using O H O O H O 2 2 2 2 the Lollipops software.13 Figure 1. Schematic diagram showing the essential role of the malate-aspartate NAD(H) redox shuttle in the re-oxidation of cytosolic NADH. (A) The cytosol contains a variety of different NADH- generating dehydrogenases involved in glycolysis, serine biosynthesis and other path­ways. Since the Families II and III is the ultimate site of NADH-reoxidation, the NADH generated in the cytosol needs WES was performed on affected probands of family II and III. In family II, candidate to be shuttled across the mitochondrial membrane. This is brought about by so-called NAD(H) redox missense variants were identified in three genes MUC19 [MIM: 612170], GOT2 and shuttles with the malate aspartate shuttle as the most important one. The malate aspartate shuttle ZNF157 [MIM: 300024] consistent with recessive mode of inheritance after filtering requires the concerted action of six different components: cytosolic and mitochondrial malate dehydrogenase (MDH1 and MDH2), cytosolic and mito­chon­drial glutamate and prioritization of the variants. Similarly, missense variants in four genes PRKAG3 (GOT1 and GOT2), and the two mitochondrial solute carriers aspartate-glutamate (AGC1 and AGC2) [MIM: 604976], GOT2, ABCA10 [MIM: 612508] and DSG3 [MIM: 169615] were identified and 2-oxoglutarate (OGC). (B) Schematic diagram showing the consequences of an impairment in family III. The variant had to be homozygous in all probands with an allele frequency in the malate-aspartate NAD(H) redox shuttle and the important role of in gnomAD less than 0.01% and in the Greater Middle eastern population of less than in the re-oxidation of the NADH generated in the cytosol. GAP, glyceraldehyde 3-phosphate; 1,3- DPG, 1,3-diphosphoglycerate; 3-PG, 3-phospho­glycerate; PEP, phosphoenolpyruvate; GAPDH, 1%. For consan­guineous families, the variant was required to be present within the glyceraldehyde 3-phosphate dehydrogenase; 3-PGDH, 3-phosphoglycerate dehydrogenase; Linkage peak as defined by para­metric linkage analysis with LOD > 1.4 or in a ‘run of + LDH, lactate dehydrogenase; NAD , nicotinamide adenine dinucleotide (oxidized form); NADH, homozygosity’ of at least 1 Mb. In family II, the GOT2 variant (hg19:16:g.58750636G>C, nicotinamide adenine dinucleotide (reduced form); MIM, mitochondrial intermembrane space; ADP, Adenosine diphosphate; ATP, Adenosine­ triphosphate; Pi, inorganic phosphate; OXPHOS, oxidative c.784C>G, p.Arg262Gly, NM_002080.2:5166106) was the only variant falling within . the linkage peak with LOD > 1.4.

Sequence alignment MATERIAL AND METHODS Sequences of GOT2 orthologs were identified using BLAST with the human reference GOT2 protein sequence (NP_002071, corresponding to mRNA NM_002080); GOT2 The three families orthologs date back as far as yeast. Sequences were selected to cover a large Family I was enrolled into the TIDEX gene discovery project (UBC IRB approval H12- phylogenetic distance. Multiple sequence alignments were calculated using MAFFT 00067) and provided written consent for the investigations and for publication of global alignment (mafft-ginsi)14 and visualized using Jalview.15 this manuscript. Written informed consent was also obtained for the families II and III.

54 55 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

Protein modeling at -80°C until measurements were performed. Prior to further measurements, 0.01% The effects of theGOT2 variants on the protein structure and function were analyzed Triton X-100 was added and the samples were freeze-thawed for three cycles. Protein using three-dimensional protein structures. We modeled the variants based on a template concentrations of the extracts were determined on a KoneLab 20XTi system using a structure of the mature human GOT2 protein, solved as a homodimer complex at 3.0 U/CSF protein kit (ThermoFisher, Breda, The Netherlands). GOT activity was measured Å resolution (PDB ID: 5AX8, chains A and C).16 As the human structure lacked substrate using a commercial kit (Cat. No. 05531446) from Roche Diagnostics (Roche Diagnostics, and cofactor molecules, we also analyzed the mature mouse GOT2 protein (PDB ID: Almere, The Netherlands), for the quantitative determination of glutamate oxaloacetate­ 3PDB, chains A and B, 2.4 Å)17 crystallized with oxaloacetate and pyridoxal 5’-phosphate with pyridoxal 5’-phosphate activation on a Cobas C8000 analyzer (Roche (present in an intermediate state of as covalently-bound to Lys279). The human Diagnostics, Almere, The Netherlands). The assay is based on the following reaction, and mouse sequences and structures are highly similar at 95% sequence identity which is catalyzed by GOT in the mitochondrial protein extracts: 2-oxoglutarate + and an average distance between atoms < 1 Å (root-mean-square deviation, RMSD; L-aspartate → L-glutamate + oxaloacetate. This reaction is coupled to the following 3 superimposition of the protein structures). Therefore, ligand coordinates from the mouse reaction: oxaloacetate + NADH + H+ → malate + NAD+, catalyzed by malate dehydro­ structure were transferred to the human structure. The resulting 5AX8 template structure genase included in the assay kit. The GOT activity in the sample is directly proportional with ligands was submitted to SWISS-MODEL to model the GOT2 variants.18 Models were to the rate of NADH conversion, which is measured spectrophotometrically at 340 nm. of overall high quality (Global Model Quality Estimation, GMQE = 0.98). Structures were The experiment was performed in duplicate. visualized with YASARA (http://www.yasara.org). Generation of GOT2-knockout HEK293 clones by CRISPR/Cas9 GOT2 protein expression The CRISPR/Cas9 genome editing technology as described by Ran was used to Western blot analysis on mutant and control fibroblasts of GOT2 and succinate introduce a disruption of the GOT2 gene in HEK293 cells.19 To this end, oligonucleotides dehydrogenase complex flavoprotein subunit A (SDHA, a mitochondrial marker coding for a guide RNA upstream of a proto-spacer adjacent motif (PAM) site protein) was performed following standard molecular biological procedures. in 2 (5′-CCT, c.197_199) of the GOT2 gene were designed using the online Antibodies used are a GOT2 polyclonal antibody (Bethyl A304-356A-T; diluted CRISPR design tool (http://crispr.mit.edu/; sequences available upon request). The 1:1000) with the secondary antibody Alexa Fluor-680-labeled goat-anti-rabbit two oligos were annealed and subsequently cloned into the pX458 (-pSpCasq(BB)- antibody (Invitrogen Cat. No. A21109) and a monoclonal complex II SDHA antibody 2A-GFP) plasmid,19 followed by Sanger sequencing of the insert to confirm the (Abcam cat. ab14715; diluted 1:2000) with the secondary goat-anti-mouse IRDye800 correct sequence. HEK293 cells were transfected with 2 µg plasmid, and single antibody (Rockland Cat. NO. 610-132-121). Western blot analysis on the HEK293 green fluorescent protein (GFP)-positive cells were sorted into wells of a 96-well CRISPR/Cas9 cells was performed following standard biological procedures, using the plate using fluorescence-activated cell sorting (FACS) flow cytometry (S800H Cell β-actin polyclonal antibody from Sigma (Cat. No. ABT1487; diluted 1:10,000) and the Sorter, Sony) as described.19 After 3–4 weeks, DNA was isolated from the expanded secondary antibody donkey anti-mouse IRD 680. single colonies, and exon 2 of the GOT2 gene was PCR-amplified using Phire Hot Start II DNA Polymerase (ThermoFisher Scientific, Waltham, Massachusetts, USA) GOT2 activity in skin fibroblasts according to the manufacturer's instructions and subsequently Sanger-sequenced. GOT activity was measured in mitochondria-enriched fractions prepared from cultured Three clonal CRISPR/Cas9 GOT2-knockout HEK293 cell lines were generated: skin fibroblasts from an affected individual and three controls. For this purpose, 1x106 clone A3 was compound hetero­zygous for 205dup/202_203insA; clone A6 for cells were cultured in M199 medium supplemented with 10% FBS and antibiotics. Cells 205del/190_206del/203_206dup; and clone A7 homo­zy­gous for 205dup. There were were harvested by trypsinization and resuspended in ice-cold 10 mM Tris-Cl pH 7.6. All no significant putative off-target regions predicted by the online CRISPR design tool. subsequent steps were performed at 4°C. The cell suspension was homogenized by using a Potter-Elvehjem homogenizer after which 0.2 vol of 1.5 M sucrose was added. GOT2 activity in the HEK293 CRISPR/Cas9 cells The suspension was centrifuged for 10 min at 600 g. The supernatant was further The GOT activity, in the HEK293 cells, was measured spectrophotometrically using a centrifuged for 20 min at 20,000 g. The mitochondria-enriched pellet was washed coupled assay method based on the NADH-dependent conversion of oxaloacetate as twice with 10 mM Tris-Cl pH 7.6, was resuspended in the same buffer, and was stored

56 57 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

generated from L-aspartate by GOT, to malate mediated by malate dehydrogenase were either incubated with DMEM medium without glucose (DMEM, Cat. No.11966; added to the assay mixture. The absorbance at 340 nm was followed for 30 minutes supplemented with 10% FBS and 1% P/S), to which 25 mmol/L glucose or 25 mmol/L 13 at 37°C using a centrifugal analyzer (Hoffman COBAS-FARA, Hoffman, LaRoche). The uniformly labelled C6-glucose was added. Cells were harvested at t=0, 0.5, 4 and composition of the assay mixture was as follows: 100 mM potassium phosphate 10 hours. To this end, cells were washed twice with cold PBS (4ºC) and harvested by buffer pH 7.4; 100 mM L-aspartate; 0.2 mM NADH; 0.1% (w/v) Trition-X-100; 7.3 U/ml scraping with 1.5 ml ice-cold methanol. The samples were transferred into a 1.5 ml malate dehydrogenase and 25 µl of cellular homogenate, in a final volume of 250 µl. eppendorf tubes, centrifuged (16,200 g for 10 min at 4ºC) and the supernatants were The reactions were started by adding 10 mM α-ketoglutarate to the assay mixture. transferred to new 1.5 ml eppendorf tubes. The samples were evaporated at 40ºC under a gentle stream of nitrogen until complete dryness, and reconstituted with Lentiviral transduction of mutant fibroblasts with wild type GOT2 500 µl of UPLC-grade methanol (room temperature). The reconstituted samples were A synthetic GOT2 cDNA (NM_002080) with stop codon and attB sites was obtained stored at -80ºC until amino acid analysis was performed. 3 from Thermo Fisher (ThermoFisher Scientific, Waltham, Massachusetts, USA). The DNA fragment was cloned into pDONR201 and subsequently into pLenti6.2/V5-DEST Recovery studies on de novo serine biosynthesis by Gateway technology cloning (Invitrogen). The resulting expression construct The recovery studies on serine de novo biosynthesis were performed by incubating 13 was checked by Sanger sequencing. The construct was used for lentiviral particle cells with DMEM-no glucose medium, to which we added 25 mmol/L C6-glucose, production by transfection into HEK293-FT cells, as described before.20 The viral and 0, 2.5 or 5 mmol/L of glycerol or pyruvate. Samples were collected as described particle-containing supernatant was used to transduce sub­confluent fibroblasts before, at t = 0, 0.5 and 4 hours. with GOT2 mutations. After selection with blasticidin (Invivogen), the resulting cell culture was used for Western blot and GOT enzyme activity assays. As a control, the Amino acid analysis 13 13 mutant cells were transduced with lentiviral particles from HEK293 cells transfected To quantify intracellular C3-serine and C2-glycine, we adapted the UPLC-MS/MS with a pLENTI construct with a green fluorescent protein cDNA, as described before.20 method described by Prinsen.21 Apart from not using internal standards to avoid 13 13 interference with the signal of C3-serine and C2-glycine, adapting the range of the Cell Culture calibrators to our samples’ concentrations, and using quality control (QC) samples Dulbecco’s modified eagle medium (DMEM), high glucose, GlutaMAX™, pyruvate that resembled the concentrations of our samples, no further adaptations were (Cat. No. 31966); DMEM-no glucose (Cat. No. 11966); fetal bovine serum (FBS; Cat. needed for sample preparation or analysis of the amino acids. No. 10270); penicillin-streptomycin (P/S (10,000 U/mL); Cat. No. 15140) and trypsin- ethylenediaminetetraacetic acid (trypsin-EDTA (0.5%), no phenol red; Cat. No. 15400) Generation of GOT-/- mice by CRISPR/Cas9 TM 13 were purchased from Gibco (ThermoFisher Scientific). Uniformly labelled C6- The following mutations NM_002080.2:c.617_619delTCT: p.Leu209del and glucose (99%) was purchased from Cambridge Isotope Laboratories, Inc. (MA, USA). c.1009C>G: p.Arg337Gly were introduced into the mouse genome using the CRISPR/ Glucose was purchased from Sigma-Aldrich (Steinheim, Germany). Cas9 method (Table S3). The gRNAs were selected using the Crispr guide selection software from Feng Zhang’s laboratory (http://crispr.mit.edu/; Table S4). The gRNAs Fibroblasts and HEK293 cells (GOT2-WT; and GOT2-A3, -A6, and -A7 knockout clones) were subcloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9, a gift from Feng were grown in 75 cm2 flasks and maintained in DMEM, high glucose, GlutaMAXTM, Zhang (Addgene plasmid #42230). Repair templates (ssODN) were designed to pyruvate (with 10% heat-inactivated FBS and 1% P/S), in a humidified atmosphere insert the desired mutations and ultramer DNA were synthesized by Integrated of 5% CO2 at 37ºC. Cells were passaged upon reaching confluence and media was DNA Technologies (IDT). The Got2emhD335fs14* was generated while making refreshed every 48 hours. Got2emhR337G mice as a results of unrepair indels. Both mice were generated by the Institut de Recherches Cliniques de Montréal (IRCM). Briefly, about 2 picoliter Stable isotope analysis – de novo serine biosynthesis of ssODN (100 µl/ng) and pX330-U6-Got2-hSpCas9 plasmid (10 ng/µl) in 5 mM Tris, Cells were plated on 6-well plates (500.000 cells per well) and allowed to grow for 0.02 mM EDTA was microinjected into a pro­nucleus of C57BL/6J or B6C3F1 mouse 4 days. Media was refreshed 24 and 72 hours after plating. On the fourth day, cells zygotes that were transferred into the oviduct of CD-1 pseudopregnant surrogate

58 59 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

mothers according to the standard approved animal user protocols IRCM 2014-17. and phenol red. At 24 hpf, embryos were manually dechorionated. Total RNA was The Got2emhL209del was generated by the McGill transgenic core facility. Briefly, the extracted from embryos at 24 and 48 hpf using TRIzol (Invitrogen, Carlsbad, CA, USA). gRNA was transcribed in vitro using the MAXIscript T7 kit (ThermoFisher, AM1312). The RNA concentration of each sample was quantified using a NanoDrop ND-1000 About 2 picoliter of gRNA (20ng/µl), ssODN (100 ng/µl) and Cas9 RNA (50 ng/µl) spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity were microinjected into a pronucleus of C57BL/6N mouse zygotes and subsequently was verified in 1% agarose gel electrophoresis (Invitrogen).­ The RNA template was implanted in CD-1 pseudopregnant surrogate mothers according to the standard converted into cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, approved animal user protocols #4437. CA, USA). The primers used are as follows:

After weaning, the mice were transferred to the Centre de Recherche du Centre Forward primer: 5’- GCAATGGCCCTGTTCAAATC-3’; Hospitalier Universitaire (CR-CHU) of Sainte-Justine Hospital. Mouse husbandry Reverse Primer: 3’- CTGCATGCCTGCATCTCTAA-5’. 3 and experiments were done according to the approved animal user protocols #541 by the Coordonnatrice du Comité Institutionnel des Bonnes Pratiques Animales Compound Rescue experiments en Recherche (CIBPAR). This committee is following the guidelines of the Conseil Pyridoxine (P5669), serine (S4375), sodium pyruvate (P2256), and L-proline (P0380) Canadien de la Protection des Animaux (CCPA). were purchased from Sigma-Aldrich. Zebrafish embryos were distributed in 24 well plates (10 embryos per well per condition). At 6 hpf, the embryos were treated with Zebrafish husbandry one of the following compounds: 25 mM of pyridoxine, 2 mM of serine, 0.5 mM of Zebrafish were maintained at 28.5°C in a 10/14-h dark/light cycle. Protocols for sodium pyruvate, and 0.2 mM of L- proline. The plates were kept at 28°C. Survival experimental procedures were approved by the Ethics Board at St. Michael’s Hospital, and pheno­typic assays were performed at 1, 2 and 3 day post fertilization (dpf). The Toronto, Canada (Protocol ACC660). compound solutions were replaced with the corresponding fresh solutions at 1 and 2 dpf. For survival assay, heartbeat and embryo movement were considered at 2 dpf. For Morpholino knockdown phenotypic assay, five phenotypes (P1, P2, P3, P4 and P5) were characterized at 3 dpf To knock down , we designed and injected oligonucleotide based on the severity (Figure 5A). For phenotype severity calculation, the following morpholinos targeting the first ATG start codon as well as the splicing (sp) donor site "phenotype scores" were used: P0 (Normal) = 0, P1 = 1, P2 = 2, P3 = 3, P4 = 4, P5 (Dead) of the exon 2 for got2a. A standard control MO (Cont MO) was used as control. The MO = 5. Average phenotype severity was calculated with the following formula: sequences are as follows: Average phenotype severity = ∑ n x (Phenotype score) / N got2a atg-MO: 5’- CTTGCTGGATTTGAACAGGGCCATT -3’, got2a sp-MO: 5’- AGCTATTTAATATCACACCTTTCGA -3’, n: Number of embryos showing a specific phenotype in a well. N: Total initial number and standard control MO: 5’- CCTCTTACCTCAGTTACAATTTATA -3’. of embryos in a well. Treated samples were compared to non-treated conditions using one-way ANOVA test. MOs were designed by Gene Tools, LLC. Normally we injected individually 4 nl of got2a atg (8 ng/µl) and 4 nl of got2a sp (16 ng/µl) MOs. Each injection was repeated at least Electroencephalogram (EEG) three times. For phenotype severity rescue experiments, ATG-blocking MO solutions EEG recordings of zebrafish embryos were performed according to a previously were prepared at 0.4 mM final concentration. 2 nL of MO solution was injected in reported method.22,23 Low melting 1.2% agarose (BioShop) was placed within each embryo. For survival rescue experiments, 4 nL of 0.8 mM MO solution was used. recording media solution (1 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1.2 mM MgCl2, 10 mM Dextrose, 2.1 mM CaCl2). Embryos were anesthetized with 0.02% tricaine (Sigma Molecular validation of got2a splicing morpholino knockdown Aldrich). Embryos in the agarose block were immersed in recording media solution. Knockdown of got2a with sp-MO was confirmed by RT-PCR. Embryos were injected A microelectrode (1μm diameter, 2–7 MΩ) was mounted on a micromanipulator at 1 cell stage with 4 nl of got2a sp-MO (16 ng/µl), the MO was diluted in water and inserted into the front brain of zebrafish embryo at 2 dpf. Microelectrodes were

60 61 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

fabricated from 1.5 mm OD borosilicate glass, and pulled into two needles with a Table 1. Clinical, biochemical and genetic characteristics of the GOT2-deficient two-step Narishige micropipette puller. The microelectrode was back loaded with individuals. recording media solution using a 1 ml syringe with a Corning syringe filter (0.2 Individual 1 – Individual 2 – Individual 3 – Individual 4 – μm), and a 28-gauge MicroFil filament (World Precision Instruments) was attached. Family 1 Family II Family II Family III Clinical History Electrical activity was captured with an Axopatch 200B (Axon Instrument) patch Gender M F F M clamp amplifier in current clamp mode. Data was collected in pClamp 8 software Age (Years) 8 10 8 4 (Molecular Devices, USA) in gap-free acquisition mode, sampling at 10KHz, and gain Ethnicity Romanian Egyptian Egyptian Egyptian at 100 mV/pA. A recording chamber and electrophysiology rig were used to stabilize Parental Consanguinity - + + + embryos. EEG was analyzed as number of events, and duration of each event for a Unaffected siblings 0 1 1 0 Siblings First child was stillborn First child First child spontaneous Sister died at age of 5 3 single embryo. The average of number of events and event duration were calculated spontaneous abortion abortion months for each embryo. Delivery Caesarean section NVD NVD NVD Gestational age (weeks) 38 32 38 39 Rescue of seizures in got2a sp-MO by pyridoxine Birth length percentile 85th <1st 5th 50th Birth weight percentile 3rd <1st 5th 5th Two hours after got2a sp-MO injection, 25 mM of pyridoxine was added to the embryo Birth HC percentile 5th <1st 15th 15th water in a 12-well plate and incubated at 28°C. After 24 hours of incubation chorions Apgar scores 9 5 8 6 were removed manually. The medium and pyridoxine were refreshed after 24 hpf. At Neonatal period - Artificial ventilation - - 48 hpf, the embryos were analyzed by electroencephalogram. (first 20 days) Hypotonia (neonatal) + + + + Neonatal feeding + + + (mild) + difficulties Results Frequent infections + + + + Seizure onset 9 months 7 months 6 months 4 months Clinical history Seizure frequency 10-100 per day daily daily 20 per day Seizure semiology upward gaze, clonic seizures myoclonic, GTC, tonic myoclonic, GTC myoclonic, tonic with Four individuals from three unrelated families (P1-4; Table 1; families I, II and III; Figure at upper limbs (left +/- upward gaze 2A-C) suffered from similar clinical features, albeit of different severity: progressive right), head tilting to left and facial clonic movements microcephaly, failure to thrive and feeding difficulties, a metabolic encephalopathy with epilepsy from the first year of life and subsequent intellectual and motor disabilities. Pyridoxine + - - + Cerebral imaging showed cerebral atrophy and white matter abnormalities in all four supplementation individuals. Biochemically, high plasma lactate and hyperammonemia were found. Serine supplementation + - - + In individual 1, who was most severely affected, plasma serine was low and Progressive microcephaly + + + + MRI findings multicystic mild cerebral atrophy mild cerebral atrophy (mainly high; his seizure control and neurodevelopment improved considerably on pyridoxine encephalomalacia, with a hypoplastic with a hypoplastic frontoparietal) and L-serine supplementation to the extent that antiepileptic drugs could be stopped cerebral atrophy vermis and a thin vermis and a thin asymmetric dilated (supplementary clinical information, Table 1). Also individual 4, in whom treatment­ was corpus callosum corpus callosum lateral ventricles; hypoplastic vermis; started reacted favorably. His seizures diminished on pyridoxine only and were fully corpus callosum controlled by the combined treatment with pyridoxine and serine supplementation hypoplasia EEG findings - tempoparietal tempoparietal spikes bilateral frontoparietal (case description in the supplemental data). spikes frequent spikes

62 63 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

Metabolic Screening (14 months) (10 years) (8 years) (4 years) A B C Legend: (age) I I I Amino acids (blood) abnormal normal normal normal Male Female Stillbirth

Male proband Female proband Serine (ref. value) (µmol/L) 47 (70-294) 114 (88-172) 171 (88-178) 130 (88-178) II II II Glycine (ref. value) 280 (80-340) 184 (167-338) 295 (156-328) 273 (156-328) Spontaneous abortion P1 P2 P3 P4 (µmol/L) Citrulline (ref. value) 89 (7-55) 37 (20-46) 36 (21-43) 27 (21-43) (µmol/L) D Organic acids (urine) normal profile normal profile normal profile normal profile GOT2 1 30 Aminotransferase class I/II domain 430

Acylcarnitine (blood) normal profile normal profile normal profile normal profile p.Gly366Val p.Leu209del p.Arg262Gly p.Arg337Gly H. sapiens 202 P EQ S V L L L H A C A H 257 D A WA V R H F I EQ 332 N T P D L R K QW L Q 362 L K K E - G S T H NW P. troglodytes 202 P EQ S V L L L H A C A H 257 D A WA V R H F I EQ 332 N T P D L R K QW L Q 362 L K K E - G S T H NW M. musculus 202 P EQ S V L L L H A C A H 257 D A WA V R H F I EQ 332 T S P D L R K QW L Q 362 L K K E - G S S H NW Clinical Chemistry (age) 8 Months 9 Years 7 Years 3 Years C. lupus 202 P QQ S V L L L H A C A H 257 D A WA V R H F I EQ 332 T S P D L R K QW L Q 362 L K K E - G S S H NW B. taurus 202 P A Q S V I L L H A C A H 257 D A WA V R H F I EQ 332 T S P D L R K QW L H 362 L K K E - G S S H NW 250 325 355 3 G. gallus 195 P E K S I I L L H A C A H D A WA L R H F I EQ N T P E L R K EW L V L K K E - G S S H NW Blood lactate (ref. value) 5.7 (0.7-2.1) 4.2; 3 (0.5-2.2) 3.9 (0.5-2.2) 4.5 (0.5-2.2) X. tropicalis 199 P EQ S I I L F H A C A H 254 D A WA V R H F I Q E 329 T Q P D L R K EW L Q 359 L K K E - G S I H NW D. rerio 200 P E K S V I L L H A C A H 255 D A WA V R Y F I EQ 330 N T P E L Y K EW L Q 360 L K K E - G S T H NW (mmol/L) D. melanogaster 203 P E K S I V L L H A C A H 258 D A Q A V R T F E A D 333 N N E D L R A QW L K 363 L I K L - G S SQ NW C. elegans 191 P E G S V I L L H A C A H 246 D A F A L R H F I EQ 321 S N P E L K K SW L E 351 L K A E - G S T L NW S. pombe 207 P D G S I I L L H A C A H 262 D A Y A T R L F A S S 337 S N P A L R EQWA G 367 L E K D L K N K H SW Blood ammonia (ref. value) 143 (16-60) 110 (<80); 70 120 (<80) 120 (<80) A. thaliana 200 P E G S F F L L H A C A H 255 D A K S I R I F L E D 330 E D P E L K S L W L K 360 L E K L - G S P L SW (µmol/L) (11-32) Conservation

Last visit (age) (7 years) (10 years) (8 years) (4 years) E Length percentile <1st <1st <1st 25th Weight percentile <1st <1st 1st 25th rd st st st HC percentile <3 <1 <1 <1 Leu207 GOT2 Arg262 Intellectual disability profound severe severe profound (copy A) Speech no words < 10 single words no words no words

Motor disability Severe spastic quadriplegia, Spastic paraparesis, Spastic paraparesis, Severe spastic Arg337 wheelchair bound walks short distances wheelchair bound quadriplegia, Gly366 with support wheelchair bound 60° Gly366 GOT2 variants Compound heterozygous Homozygous Homozygous Homozygous Arg337 GOT2 Leu207 Arg262 (copy B) amino acid change p.Leu209del p.Arg337Gly p.Arg262Gly p.Arg262Gly p.Gly366Val OAA Asp253 Asp255 In silico prediction Arg262 PMP CADD 23.0 23.8 32.0 32.0 33.0 PROVEAN; cutoff: -2.5 na -4.25 -6.7 -6.7 -8.54 Leu208 (“damaging”) (“damaging”) (“damaging”) (“damaging”) Glu116 p.Leu209del His210 PolyPhen2 na 0.922 1.0 1.0 0.993 Glu111 Leu209 (“possibly (“probably (“probably damaging”) (“possibly damaging”) Arg337 Reference Leu331 Leu207 damaging”) damaging”) SIFT; cutoff: 0.05 na 0.243 0 0 0 Figure 2. The pedigrees, and the GOT2 variants. (A-C) Pedigrees of the families I-III. (D) Alignment (“tolerated”) (“damaging”) (“damaging”) (“damaging”) of GOT2 ortholog sequences. p.Leu209del represents the deletion of a leucine in a tri-leucine stretch. Figure S1 shows the full alignment. (E) Molecular modeling of the variants. Shown are rotated M, male; F, female; C-SECTION, Caesarean section; NVD, Normal Vaginal Delivery; NICU, Neonatal overview structures of the GOT2 homodimer complex, with variants found in affected individuals Intensive Care Unit; GTC, Generalised tonic-clonic seizures; EEG, electroencephalogram; MRI, shown in orange. Insets: p.Gly366Val (top-left), p.Arg337Gly and p.Arg262Gly (bottom-left; glycine Magnetic Resonance Imaging; HC, head circumference; na, not available. substitutions not visible), p.Leu209del (bottom-right; reference structure (blue) and variant model (orange) superimposed). While visualized in a single structure, variants are not simultaneously present in the same copy of the gene. Models are based on template structure PDB ‘5AX8’, with ligand coordinates taken from PDB ‘3PDB’. PMP, pyridoxamine 5′-phosphate;­ OAA, oxaloacetate. Hydrogen bonds in yellow; other charge interactions as dashed lines. p.Leu209del shortens a beta strand in the

64 65 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

protein core close to the active site, leading to a repositioning of loops involved in the geometry of A B the catalytic pocket, likely affecting binding of both the enzyme cofactor (pyridoxal 5’-phosphate) and substrates. p.Gly366Val has a predicted marginal effect on the protein structure, but is still well- Fibroblasts HEK293 cells conserved across evolution. For p.Arg337Gly and p.Arg262Gly, substitution of the positively-charged arginine residues with the neutral glycine residue results in a disruption of electrostatic interactions that in the wild type protein stabilize the alpha-helical organization of the protein. PatientPatient 1 Patient 2 Carrier 3 Control 1 Control 1 Patient 2 Patient 1 Carrier 4 2 GOT2-WTcrGOT2-A3crGOT2-A6crGOT2-A7 SDHA (70 kDa) GOT2

GOT2 (47 kDa) β-actin Exome sequencing and protein structural modeling to identify

GOT2 damaging mutations C D Nine candidate genes with rare, non-synonymous genetic variants were identified by Trio WES on family I. GOT2 was con­sidered the best candidate to explain the bioche­ GOT2 Activity in Fibroblasts Total GOT Activity in HEK293 cells 3 mical phenotype of the proband. The gene contained (1) a paternally inherited in- 400 800 frame deletion hg19:16:g.58752177delGAA (p.Leu209del, c.617_619delTTC, NM_ 300 600 002080), (2) a maternally inherited missense mutation hg19:16:g.58749928G>C 200 400

(p.Arg337Gly, c.1009C>G). Both variants are not present in dbSNP (version 142), 100 200 mU / mg protein

NHLBI ESP, ExAC, gnomAD or in our in-house genome database­ comprising more nmol / (min.mg protein) than 11,450 exome and genome sequences. Exome sequencing was also used for the

ControlControl 1 Control 2 Patient 3 Patient 1 Patient 2 Carrier 3 Patient 1 Carrier 4 2 probands of the other families. Three homozygous candidate missense variants were GOT2-WT crGOT2-A3 crGOT2-A6 crGOT2-A7 prioritized in the probands in family II and four in family III. Homozygous GOT2 mis­ E F sen­se variants for the families II and III were hg19:chr16: g.58750636G>C (c.784C>G, p.Arg262Gly, NM_002080) and g.58743394C>A (c.1097G>T, p.Gly366Val) respecti­ GOT2 activity in HEK293 cells GOT2 rescue in patient’s fibroblasts 100 ve­ly. The allele frequencies­ in gnomAD were < 0.01% and, within gnomAD, in the 4

75 Greater Middle Eastern population <1%. The presence and family segregation of all 3

GOT2 variants was confirmed with Sanger sequencing. % 50 2

25 1 All variants affect evolutionary highly conserved amino acids (Figures 2D, S1 and S2, and (mU/mg) / CS GO T supplementary information). They are predicted to be damaging by in silico methods

24,25,26,27 ControlControl 1 Control 2 Control 3 Control 4 5 (Table 1). Modeling of the variants in the GOT2 three-dimensional protein structure GOT2-WT crGOT2-A3 crGOT2-A6 crGOT2-A7 (Figure 2E) suggests that they reduce the catalytic activity of the enzyme and may impact Patient 1 + GFP Patient 1 + GOT2-WT the overall protein conformation, both of which may result in reduced levels of functional protein (Figure 2E; detailed analysis in supplementary information). Figure 3. GOT expression and activity. (A) Western blot showing GOT2 and the mitochondrial fraction marker SDHA (succinate dehydrogenase complex flavoprotein subunit A) in the GOT2- GOT2 deficiency in fibroblasts and GOT2-knockout HEK293 cells deficient individuals, GOT2-carriers, and two control fibroblast lines.B ( ) Western blot for GOT2 protein in GOT2-wild type and the three GOT2-knockout HEK293 cell lines. (C) GOT2 activity in Western blot analysis revealed that GOT2 was strongly deficient in fibroblasts of mitochondria-enriched fractions from fibroblasts from the GOT2-deficient individuals, GOT2-carriers, individual 1 and to a lesser extent in individuals 2-4 (Figure 3A). Three clonal CRISPR/Cas9 and three healthy controls. Graph bars represent mean ± SD. (D) Total GOT (GOT1 and GOT2 isoforms)­ GOT2-knockout HEK293 cell lines were successfully generated: clone A3 was compound activity in whole cell lysates; results are representative­ of two independent experiments. (E) GOT2 heterozygous for 205dup/202_203insA; clone A6 for 205del/190_206del/203_206dup; activity in the mitochondria-enriched fractions of GOT2-WT and the three GOT2-knockout HEK293 cell lines; results are representative of two independent experiments. (F) GOT2 rescue experiment and clone A7 was homozygous for 205dup. Western blot analysis performed on the three in fibroblasts from individual 1. GOT2 activity is restored to control levels when GOT2-deficient clonal cell lines showed that GOT2 was not detectable in any of the clones (Figure 3B). fibroblasts are transduced with theGOT2 -wild type gene. Five control fibroblast-lines and the GOT2- deficient fibroblasts transduced withoutGOT2 -wild type (+ GFP lane) were used for comparison.

66 67 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

and 13C -glycine fractions were determined in GOT2-WT (full line) and the GOT2-knockout HEK293 cell A 2 lines (dashed line). Cells were incubated with 13C -glucose, and the formation of the labelled 13C -serine 13 6 3 C3-serine production in fibroblasts 13 Legend: Controls and C2-glycine was analyzed at t=0, 0.5, 4 and 10 hours after exposure. The results are representative 0.06 Patient 1 of two independent experiments, and are normalized to total protein content and represented as the Patient 2 mean of n=3 (biological triplicates) ± SD. (C) To study the impact of glycerol and pyruvate supplemen­ tation in de novo serine production of the GOT2-knockout cell lines, we supplemented the cells with 0.04 Patient 3 these compounds (2.5 and 5 mmol/L) for 4 hours. The formation of labelled 13C -serine was analyzed at Patient 4 3 t=0, 0.5 and 4 hours after exposure. The results are normalized to total protein content and represented -Ser / tSer

3 Carrier 1 13 C 0.02 as the mean of n=3 ± SD. (D) The same study was performed for glycine. The formation of labelled C - 13 Carrier 2 2 glycine was analyzed at t=0, 0.5 and 4 hours after exposure. The results are normalized to total protein PSAT patient content and represented as the mean of n=3 ± SD. 3-PGDH patient 0 2 4 6 8 10 3 Time (hours) GOT2 enzymatic activity was determined in mitochondria-enriched fractions. B In individuals 1-4 it amounted to 8%, 21%, 21% and 18% of the mean of controls, 13C -serine production in GOT2-/- HEK293 13C -glycine production in GOT2-/- HEK293 3 2 respective­ly. The two GOT2-carriers (1 and 2) had a residual activity of 39% and 62%, 0.20 0.20 GOT2-WT cells respectively (Figure 3C). GOT enzy­matic activity measurements in whole cell lysates 0.15 0.15 GOT2-/- clones prepared from the three different GOT2-knockout clonal cell lines, which includes 0.10 0.10 the combined activity of both GOT1 and GOT2, was found to be markedly decreased -Gly / tGly -Ser / tSer 2 3 0.05 0.05 C C 13 13 (P<0.01) compared to GOT activity in wild type cells (Figure 3D). In addition, GOT2 0 2 4 6 8 10 0 2 4 6 8 10 activity in the mitochondrial­ fraction was less than 2% of the activity measured in Time (hours) Time (hours) control mitochondrial fractions (Figure 3E). C Glycerol Pyruvate 0.20 0.20 To investigate whether the defect could be rescued by wild type GOT2 cDNA, 0.15 0.15 fibroblasts of individual 1 were transduced using lentiviral particles with wild type 0.10 0.10 GOT2. After selection of transduced cells, GOT enzyme activity was measured in mito­ -Ser / tSer 3 0.05 0.05 C chondrial prepara­tions showing a strong increase of the mitochondrial GOT activity 13 GOT2-WT HEK293 cells to levels that fall within the range measured in control cells. The control transduction 0 2.5 5 0 2.5 5 GOT2-/--A3 HEK293 clone Concentration (mmol/L) experiment showed little or no effect on GOT2 activity of the cells (Figure 3F).

D Glycerol Pyruvate De novo serine biosynthesis in fibroblasts and GOT2-knockout 0.20 0.20 HEK293 cells 0.15 0.15 Because the most important clinical finding in individual 1 were serine- and 0.10 0.10 pyridoxine-responsive seizures, we evaluated the impact of GOT2 deficiency -Gly / tGly 2 0.05 0.05 C

13 on de novo serine biosynthesis. Formation of stable isotope labeled serine from GOT2-WT HEK293 cells 0 2.5 5 0 2.5 5 GOT2-/--A3 HEK293 clone labeled glucose was analyzed in fibroblasts of all GOT2-deficient individuals, two Concentration (mmol/L) GOT2-carriers, six controls and fibroblasts from individuals with a defect in serine Figure 4. De novo serine biosynthesis in mutant fibroblasts and GOT2-knockout HEK293 cells. biosynthesis (phosphoserine aminotransferase deficiency (PSATD; [MIM: 610992), 13 (A) C3-serine fractions were determined in fibroblasts of GOT2-deficient cases, the two GOT2-carriers, and 3-phosphoglycerate dehydrogenase deficiency (3-PGDHD; [MIM: 601815])). six healthy controls and two individuals with a de novo serine biosynthesis defect (3-PGDH and PSAT The fibroblasts of the GOT2 deficient individuals 1 and 4 formed less 13C -serine 13 13 3 deficien­cies). Fibroblasts were incubated with C6-glucose, and the formation of the labelled C3-serine was analyzed at t=0, 0.5, 4 and 10 hours after exposure. The results are normalized to total protein content amounting to 34% and 33% of controls, respectively. Individuals 2 and 3 produced and represented as the mean of n=3 ± SD for individual 4 and carrier 2, PSAT-deficiency and 3-PGDH- 55% and 52% of controls, respectively. In addition, the GOT2-carrier 1 produced 66% 13 13 deficiency; n=6 ± SD for individuals 1, 2 and 3, and carrier 1; and n=33 ± SD for controls. (B) C3-serine of C3-serine, while the GOT2-carrier 2 produced almost as much as controls (81%). In

68 69 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

fibroblasts from 3-PGDH- and PSAT-deficient individuals, de novo serine biosynthesis amounted to 15% and 0%, respectively (Figure 4A).

Analysis of de novo serine biosynthesis in GOT2-knockout cell lines revealed that the 13 three GOT2-knockouts had a severe reduction in C3-serine production (90-93%). In 13 addition, C2-glycine synthesis was reduced by 87-88% (Figure 4B).

We hypothesized that the GOT2 defect would result in decreased MAS activity resulting in an increased NADH/NAD+ ratio in the cytosol. To investigate whether restoring the cytosolic redox imbalance would correct the serine biosynthesis capacity of the cells we 3 incubated the GOT2-knockout cell line A3 with glycerol and pyruvate. Supplementation of the culture medium with 2.5 and 5 mmol/L glycerol had no effect on serine biosynthesis. However, serine and glycine synthesis was fully restored when cells were incubated with 2.5 or 5 mmol/L pyruvate, the substrate of lactate dehydrogena­ ­se (LDH, Figure 4C-D). This effect is explained by the re-oxidation of cytosolic NADH by LDH.

CRISPR/Cas9 Got2-knockout mice We generated mice heterozygous for the p.R337G and p.L209del mutations, and for a loss-of-function mutation (p.D335fs14*). These mice were viable and healthy, similarly to the parents of the four affected individuals. Unfortunately, no homozygous mice for any of these mutations were viable beyond early pregnancy (Table S5). Mouse embryonic fibroblasts (MEFs) were collected at the age of 14 days post coitum (dpc) and genotyped. No homozygous embryos were found suggesting early lethality for all three mutations in mice (Table S6).

Knockdown of got2a in zebrafish affects embryonic development and provokes seizure-like EEG spikes, which is rescued by pyridoxine and serine While CRISPR-based gene knockout is currently widely used to study gene function, however, gene knockdown technologies are still vital methods evaluating gene contribution to diseased phenotypes as the majority of human gene mutations retain Figure 5. Knockdown of got2a in zebrafish perturbs brain and embryonic development and residual gene activities at various degrees. Furthermore, there are essential genes that function, which can be rescued by pyridoxine and serine. (A) Phenotype severity and rescue scoring system for got2a knockdown embryos. Bright field images of 3 day post fertilization (dpf) WT could not be fully knocked out in the organism and GOT2 could be one of these genes. embryos injected with control morpholino (Cont MO), and or got2a ATG blocking morpholino (got2a Using the morpholino-based gene knockdown strategy, our previous studies had led to MO). The got2a morphants were scored in 5 different categories P1 to P5 based on the phenotype functional characterization of a number of genes in metabolic diseases.28,29 As the knockout severities. (B) Pyridoxine, serine and pyruvate decrease got2a morphant’s phenotype severity. of Got2 in mice is embryonic lethal, we knocked down the zebrafish mitochondrial got2a Number of larvae per condition were shown in parenthesis. 2 nL of morpholino (0.4 mM working solution) was injected. Compounds were added at 6 hours post-fertilization (hpf) in 24 well plates. gene by ATG- and splicing-blocking morpholinos (MO) (see supplementary Figure S3 for Every 24 hours dead embryos were removed and the compounds were replaced with fresh solution. details regarding specificity of knocking downgot2a gene). Phenotype was characterized at 3 dpf. For phenotype severity calculation, the following “phenotype scores” were used: P0: Normal: 0, P1: Small brain, enlarged yolk and mild cardiac edema: 1, P2: Smaller

70 71 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

brain, enlarged yolk, mild cardiac edema and shortened body: 2, P3: Smaller brain, enlarged yolk, severe cardiac edema and curved body: 3, P4: Very small brain, enlarged yolk, very severe cardiac Discussion edema, deformed tail and round body shape: 4, P5: Dead: 5. Average phenotype severity = ∑ n x (Phenotype score) / N. n: Number of embryos showing a specific phenotype in a well. N: Total initial GOT2 deficiency is a mitochondriopathy and our studies demonstrate that it is amenable number of embryos in a well. Treated samples were compared to non-treated conditions using one- way ANOVA test (* P< 0.05; ** P< 0.01; ns, not significant). C( ) Pyridoxine and serine increase the to therapeutic intervention. Further experience on more affected persons is needed survival of got2a morphants at 2 dpf. Number of larvae per condition is shown in parenthesis. 4 nL to confirm this. Clinically it presents as an early-onset metabolic encephalopathy with of morpholino (0.8 mM working solution) was injected. Compounds were added at 6 hours post- epilepsy, progressive microcephaly and several biochemical abnormalities.­ GOT2 fertilization (hpf) in 24 well plates. Every 24 hours dead embryos were removed and the compounds deficiency adds to the short list of mitochon­ ­driopathies responding to a specific were replaced with fresh solution. The ratio of alive to total number of larvae was counted and 30 survival percentage calculated at 2 dpf (a dead zebrafish embryo defined as having no heartbeat and pharmacological intervention. If confirmed on other persons GOT2 deficiency will no response to stimuli). Treated samples were compared to non-treated conditions­ using one-way expand the list of treatable metabolic epilepsies, recently­ reported 73 in number,31 as ANOVA test (* P< 0.05; ns, not significant). D( ) got2a knockdown provoked seizure-like EEG spikes well as the growing group of pyridoxine and/or pyridoxal 5’-phosphate responsive 3 in the forebrain that are rescued by treatment with pyridoxine in 48 hpf embryos. Newly fertilized epi­lepsies (ALDH7A1, PNPO, PLPBP, ALPL; [MIM: 107323, 603287, 604436, 171760 zebrafish embryos are injected with control morpholino (Cont MO) or got2a splicing morpholino (sp- MO). Embryos injected with got2a sp-MO showed EEG spike discharges, not present in traces from resp]).32 The importance of a thorough metabolic workup including a diagnostic embryos injected with Cont MO. (E) Analy­sis of EEG traces. Number of events in 5 minute recordings and therapeutic trial with pyridoxine and the active cofactor pyridoxal 5’-phosphate and the duration of each event in seconds (sec) in embryos injected with Cont MO, got2a sp-MO, and cannot be over­emphasized. In each of the three families a child died either during got2a sp-MO and treated with pyridoxine. Each event corresponds to a single spike discharge. Bars represent the mean ± SEM. N= 3 embryos per treatment. * P<0.05 and *** P<0.001. pregnancy, at birth or at very young age. All affected individuals in this study have some residual GOT2 enzymatic activity, which is probably essential for viability. While 18% of the embryos injected with got2a MO showed a mild phenotype at 48 Stillbirth and early childhood death may occur in families with more severe biallelic hours post fertilization (hpf), many other morphants showed a small head, slow GOT2 mutations, as also suggested by the homozygous Got2-knockout mice which circulation, bend body and pericardial edema. Bright field imaging confirmed brain were not viable and by the em­bryonic death which was observed in the zebrafish develop­mental defects (Figure 5A). To establish a quanti­tative measure for rescue of model. GOT2-deficient individuals share a phenotype of epilepsy, intellectual the got2a-MO phenotypes, we developed a scoring system to define the phenotype disability and several bioche­mical features with persons suffering from other MAS severity in 5 categories from P1 to P5 (very mild to embryonic death, Figure 5A). In defects (MDH2, SLC25A12, SLC25A13).9,33,34 GOT2 deficiency clini­cally also resembles exploring therapeutic methods, we dosed various concen­trations and combinations the three known inborn errors of serine metabolism (genes involved: PHGDH, of pyridoxine (PN), serine, pyruvate and proline in embryo water of the got2a PSAT1, PSPH; [MIM: 606879, 610936, 172480 resp]) with microcephaly, intellectual morphants. In non-toxic doses to embryos, 25 mM PN and 2 mM serine significantly developmental disorder and epilepsy. GOT2 deficiency should be considered in the rescued the phenotype severity (P<0.01). PN and serine had a synergistic effect (3 differential diagnosis of epileptic encephalopathy especially in the presence of high mM PN + 0.25 mM serine); noticeably 0.5 mM pyruvate also has some rescuing effect lactate, increased ammonia and citrulline and decreased serine. Theoretically other (P<0.05; Figure 5B). However, only PN and serine rescue the survival phenotype as MAS defects may also lead to secondary decreased serine synthesis due to cytosolic scored by beating hearts (Figure 5C). redox imbalance but low serine levels have not yet been reported in such cases.

As epilepsy and seizures are a predominant feature in the four affected individuals, we Western blot and enzyme activity data show that the GOT2 mutations in our affected performed EEG measurements on zebrafishgot2a morphants. This showed seizure-like individuals prevent GOT2 expression or render the GOT2-protein unstable. GOT2 spikes when the electrical probe was placed in the forebrain (Figure 5D) but not when deficiency impacts the malate-aspartate shuttle and subsequently the overall cellular placed in the midbrain (data not shown). Interestingly, the frequency of the seizure-like NADH/NAD+ ratio with consequences for NAD-dependent enzymes and pathways. spike discharge events in got2a morphants was rescued by supplying PN in the water Elevated blood lactate concentrations of the affected individuals can be explained of the developing embryos as demonstrated by 5 minutes EEG recording (Figure 5E left as direct consequence of the impaired re-oxidation of cytosolic NADH due to the panel); whereas duration of the events was rescued partially by PN (Figure 5E right panel), defective MAS. Interestingly, hyperlactatemia is also present in MDH2 deficiency, further supporting the therapeutic strategies in affected individuals. another disorder impairing the MAS (Figure 1).9

72 73 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

All four affected individuals had a mild but persistent hyper­ammo­nemia, with biosynthesis and possibly other NAD(H) dependent reactions and how the affected individual 1 also having mild hypercitrullinemia. Both can be explained by a secondary individuals benefit from serine- and/or pyridoxine supplementation. cycle defect in GOT2 deficiency. In humans, the amount of aspar­ta­te taken up from the blood is very low35 rendering cells highly dependent on their mitochondrial Individual 1 was only free of seizures when a combination of serine and pyridoxine aspartate production. In GOT2 deficiency, the intra­mitochondrial synthesis of aspartate was administered. PLP is an obligatory co-activator for all transaminases, including from oxaloacetate is decreased, leading to lower concen­trations of aspartate in the GOT2. It may stimulate residual GOT2 activity possibly by improving the proper mitochondrion and the cytosol. In one of the cytoplasmic steps of the in liver, folding of GOT2. the enzyme argini­nosuccinate synthetase requires aspartate as a substrate. Aspartate shortage leads to reduced argininosuccinate synthesis, and to citrulline accumulation, Interestingly, the only other individual with uncontrolled seizures (individual 4) was with hyperammonemia as a final consequence of diminished urea cycle activity.6 put on pyridoxine only therapy during eight months. His epilepsy was better controlled 3 and his cognitive functions and alertness improved. Once serine supplementation A further striking observation in individual 1 was a consistently reduced plasma serine was added to the treatment regimen his seizures were fully controlled with better concen­tration in the same range as observed in individuals with serine biosynthesis cognition and improved physical activity. In a period that serine was unavailable the defects. No mutations were found in genes implicated in the biosynthesis of serine. seizures returned and disappeared again when serine was reintroduced. Treatment 13 Stable isotope labeling studies showed decreased serine synthesis from C6-glucose could not be started in individuals 2 and 3. in fibroblasts of affected individuals and in GOT2-knockout HEK293 cells. Intracellular serine can originate from four sources: i) the diet, ii) de novo biosynthesis, iii) glycine, Theoretically, pyruvate supplementation might be considered as treatment strategy. or iv) protein and phospholipid degradation.36 The first reaction in de novo serine Re-oxidation of cytosolic NADH by pyruvate supplementation in vitro led to full biosynthesis is catalyzed by the NAD+-dependent enzyme 3-phosphoglycerate correction of serine biosynthesis in GOT2-deficient cells. This effect is explained by the dehydro­genase. The low serine and glycine produc­tion observed in GOT2 deficiency is pyruvate-induced correction of the high NADH/NAD+ ratio in the cytosol. Interestingly, most likely due to an increased NADH/NAD+ ratio as a consequence of a dysfunctional administration of sodium pyruvate with L-arginine proved effective in treating a person MAS. Pyridoxine is essential for serine de novo biosynthesis,37 therefore we cannot with a defect in SCL25A13.38 Increased lactate formation from pyruvate may form an exclude that the mechanism of pyridoxine responsiveness may be partly due to a unwanted side-effect of this approach. Defects in the MAS-system only affect the direct effect on boosting serine synthesis by a mechanism other than restoring the oxidation of cytosolic NADH and not of intramitochondrial NADH, which implies that NADH/NAD+ redox balance. the best therapy strategy would be to provide substrates which do not produce NADH in the cytosol, e.g. fatty acids especially medium chain triglycerides. For GOT2-deficient To investigate whether correction of the cytosolic NADH/NAD+ ratio could restore individuals, we suggest a diet low in carbohy­drates, high in fat and supplemented with serine and glycine de novo synthesis, we supplemented the culture medium of ketone bodies which circumvents the issue of acidification due to lactate production. GOT2-deficient HEK293 cells with glycerol or pyruvate. The rationale behind glycerol Our zebrafish modeling of GOT2 deficiency demonstrated that serine and pyridoxine addition was to stimulate the activity of the glycerol 3-phos­phate shuttle, a second treatment would likely deliver a synergetic effect. These could be further explored as system for the regeneration of NAD+ from NADH. No effect of glycerol on serine therapeutic strategies in disease management. and glycine synthesis was observed, suggesting that the MAS is the predominant NAD(H) redox shuttle, at least in HEK293 cells. After incubating with pyruvate, which Future research on this MAS defect should focus on the identification of more is enzymatically reduced to lactate in the cytosol while re-oxidizing NADH to NAD+, affected individuals, characterization of the full phenotypic and genotypic spectrum, complete normalization of serine and glycine syn­thesis was observed, supporting identification of biomarkers and other treatment targets, as well as implementation our hypothesis that serine biosynthesis is hampered by the impaired cytosolic redox and evaluation of therapeutic interventions in more cases. imbalance. GOT2 deficiency is a mitochondriopathy with metabolic consequences in mitochondria and in the due to redox imbalance. Our data explain the mitochondrial pathomechanism and clarify how GOT2 deficiency impacts serine

74 75 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

Acknowledgements References

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PLoS One 12, 1–14. a female fetus, was diagnosed intrauterine at 23 weeks of gestation as having cerebral 24. Kircher, M., Witten, D.M., Jain, P., O’Roak, B.J., Cooper, G.M., Shendure, J. (2014). A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 46, ano­malies (ventriculomegaly) and ended at term with a stillbirth. The index case was 310–315. diag­nosed in the first month of life with global developmental delay, generalized 25. Choi, Y., Chan, A.P. (2015). PROVEAN web server: A tool to predict the functional effect of amino hypotonia, abdominal spasms and feeding difficulties (breast fed till the age of 6 acid substitutions and indels. Bioinformatics 31, 2745–2747. 26. Adzhubei, I., Jordan, D.M., Sunyaev, S.R. (2013). Predicting Functional Effect of Human Missense months). Transfontanelar cerebral ultrasound performed at the age of 1 month showed Mutations Using PolyPhen-2. Curr. Protoc. Hum. Genet. 2. multi­cystic periventricular leukomalacia. At the age of 6 months he was unable to keep 27. Kumar, P., Henikoff, S., Ng, P.C. (2009). Predicting the effects of coding non-synonymous variants his head up, sit or fixate objects. At the age of 7 months a magnetic resonance imaging on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081. (MRI) brain scan showed multicystic encephalomalacia and wide cerebral atrophy. 28. van Karnebeek, C.D.M, Bonafé, L., Wen, X.-Y., Tarailo-Graovac, M., Balzano, S., Royer-Bertrand, B., Ashikov, A., Garavelli, L., Mammi, I., Turolla, L., et al. (2016). NANS-mediated synthesis of sialic Diagnostic evaluation at the age of 8 months, showed increased lactate (5.7; reference: acid is required for brain and skeletal development. Nat. Genet. 48, 777–84. 0.7-2.1 mmol/L) and mild hyperammonemia (143; reference: 16-60 µmol/L; Table S2 also 29. Wen, X.-Y., Tarailo-Graovac, M., Brand-Arzamendi, K., Willems, A., Rakic, B., Huijben, K., Da Silva, for clinical chemistry follow-up data). At the age of 9 months the first febrile seizures A., Pan, X., El-Rass, S., Ng, R. et al. (2018). Sialic acid catabolism by N-acetylneuraminate pyruvate appeared, with upward gaze, clonic seizures at upper limbs (left +/- right), head tilting lyase is essential for muscle function. JCI Insight 3, 1–20 . 30. Rahman, J., Rahman, S. (2018). Mitochondrial medicine in the omics era. Lancet 391, 2560–2574. to the left and facial clonic movements.­ These seizures became very frequent (10-100x/ 31. Sharma, S., Prasad, A.N. (2017). Inborn errors of metabolism and epilepsy: Current understanding, day), without fever. Antiepileptic treatment started with Valproate, Phenobarbital, diagnosis, and treatment approaches. Int. J. Mol. Sci. 18. Levetiracetam, Timonil, Lamotrigine and Topamax in different combinations without 32. Pena, I.A., MacKenzie, A., Van Karnebeek, C.D.M. (2017). Current knowledge for pyridoxine- dependent epilepsy: a 2016 update. Expert Rev. Endocrinol. Metab. 12, 5–20. sufficient effect. Furthermore, he was frequently hospitalized for infections. At the age of 33. Kobayashi, K., Sinasac, D.S., Iijima, M., Boright, A.P., Begum, L., Lee, J.R., Yasuda, T., Ikeda, S., 14 months, he was evaluated by metabolic specialists in Nijmegen. Thorough metabolic Hirano, R., Terazono, H., et al. (1999). The gene mutated in adult-onset type II citrullinaemia screening tests were performed on blood plasma (amino acid and acylcarnitine profile, encodes a putative mitochondrial carrier protein. Nat. Genet. 22, 159–163. guanidinoacetate, , homocysteine and methylmalonic acid) and urine (organic 34. Dahlin, M., Martin, D.A., Hedlund, Z., Jonsson, M., von Dobeln, U., Wedell, A. (2015). The ketogenic diet compensates for AGC1 deficiency and improves myelination. Epilepsia 56, 176–181. acid profile). Decreased concentrations of serine (47 µmol/L; reference: 70-294 µmol/L) 35. Palmieri, F. (2004). The mitochondrial transporter family (SLC25): Physiological and pathological and increased citrulline (89; reference: 7-55 µmol/L) were found in the plasma (Table S1 implications. Pflugers Arch. Eur. J. Physiol. 447, 689–709. also for follow-up data). Other amino acids were within reference ranges. He was promptly 36. de Koning, T.J., Snell, K., Duran, M., Berger, R., Poll-The, B.-T., Surtees R. (2003). L-serine in disease started on oral serine supplementation at the age of one year and 5 months (200 mg/ and development. Biochem. J. 371, 653–61. 37. Ramos, R.J., Pras-Raves, M.L., Gerrits, J., van der Ham, M., Willemsen, M., Prinsen, H., Burgering, kg/day, which also con­tained pyridoxine in unknown concentrations). Upon treatment B., Jans, J.J., Verhoeven-Duif, N.M. (2017). Vitamin B6 is essential for serine de novo biosynthesis. implementation his seizures became less frequent, and his abdominal spasms and sleep J. Inherit. Metab. Dis. 40, 883-891. improved. After 3 months on supple­ments the seizures reappeared (tonic with tongue 38. Mutoh, K., Kurokawa, K., Kobayashi, K., Saheki, T. (2008). Treatment of a citrin-deficient patient at the early stage of adult-onset type II citrullinaemia with arginine and sodium pyruvate. J. myoclonus), and the serine dose was increased to 400 mg/kg/day. During the subsequent Inherit. Metab. Dis. 31, 343–347. 12 months a clear effect was observed; the seizures stopped completely at the age of 1.7 years and antiepileptic treatment was weaned and stopped at the age of 1.9 years.

78 79 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

He became interested in the things around him, started to fixate and to follow objects.­ is no report on maternal exposure to teratogens during her or her sister’s pregnancy. The The upper respiratory tract infections decreased and were without complica­tions, and girl presented with neonatal feeding difficulties and drooling. Since her early stages of he was able to swallow better. His concentration and attention span in­creased and the life she presented with global development delay. At the age of 7 months the first tonic- head growth accelerated (1.5 cm increase in head circumference in less than one year to clonic seizures appeared with a daily recurrence. Her seizures were completely controlled 42.5 cm; below the 1st percentile). Due to treatment­ unavailability, at the age of 2.2 years, with antiepileptic drugs (Valproate and Lamo­tri­gine). The MRI revealed mild cerebral the therapy changed to L-serine monothera­py (400 mg/kg/day) without pyridoxine. atrophy and a thin corpus callosum. A metabolic work-up showed high blood lactate Although plasma serine concentrations had normalized, increased concentrations of (4.2; reference: 0.5-2.2 mmol/L), mild hyperammo­ne­mia (110; referen­ce: <80 µmol/L), and glycine and citrulline were reported. The carnitine profile showed increased levels of normal blood acylcarnitines and urinary organic acids. At the age of 10 years, her last long-chain acylcarni­ti­nes, but these results were appointed to blood cell contamination visit to the clinic, she had severe intellectual dis­ability and was myoclonic on entering to (visually detectable hemolysis) of the plasma sample. Oral serine (400 mg/kg/day) sleep and during sleep. She could sit and stand on her own and use her hands and was 3 together with advanced biomechanical rehabilitation therapy was maintained during able of walking few steps when supported. She could follow objects, speak 3 or 4 words two years (age 4-5 years). During this period, the parents reported a progress in his well­ and smile. She had increased limb tones and brisk reflexes, acrocyanosis and chilblains in being and development, although his abdominal spasms were still severe. The mother winter, and was unable of controlling her sphincters. Plasma amino acid analysis showed reported mild improvements in the child’s abdominal spasms and sleep pattern when normal serine (114; reference: 88-178 µmol/L) and citrulline (37; reference: 21-43 µmol/L), combined supplements­ of calcium, magnesium and pyridoxine were prescribed by the hyperlac­tatemia (3.0; reference: 0.5-2.2 mmol/L) and hyperammonemia (70; reference: 11- neurologist. At the age of 5 years, his weight was 12 kg (below the 1st percentile) and his 32 µmol/L). The younger sister was born after an unremarkable pregnancy but presented head circumference­ was 45 cm (below the 1st percentile). Neurological evaluation showed with mild neonatal feeding difficulties. Similarly to her sister, she was diagnosed with profound mental and motor retardation (corresponding to the age of 4 months), spastic drooling and developmental delay since her early stages of life. At the age of 6 months tetraplegia, severe axial muscular hypotonia, hypertonia of the limbs (mainly lower limbs), her first seizures appeared. The seizures were completely con­trolled with antiepileptic lumbar scoliosis and nystagmus. In addition, he was able to fixate and follow objects for drugs (Valproate, Lamotrigine and Leviteracetam). An MRI showed mild cerebral atrophy short periods of time and was able to respond to sounds. At the age of 5 years and 5 with a hypoplastic vermis and a thin corpus callosum. Lactate in blood was high (3.9; months, after GOT2 variants were identified, the serine dose was increased to 500 mg/kg/ reference: 0.5-2.2 mmol/L) with mild hyperammonemia (120; reference: <80 µmol/L). day. Pyridoxine was not available at the time. Between ages 5 to 7 years and 5 months he Plasma acylcarnitine profiling and urinary organic acids were unremar­kable. At the age was hospitalized many times for recurrent bacterial pneumonias, hematemesis and hiatal of 8 years, her last visit to the clinic, she had severe intellec­tual dis­ability, being able to hernia with gastro­esophageal reflux. At the age of 7 years and 5 months plasma serine sit on her own, use her hands, follow objects, smile and produce some sounds but no and glycine levels were normal while citrulline remained increased and cysteine was low. words. She had increased limb tones and brisk reflexes, acrocyanosis and chilblains in At this time pyridoxine supplementation (20 mg/kg/day) was initiated. At the age of 7 winter, and was also unable to control her sphincters. Plasma amino acid analysis showed years and 10 months (his last visit) his weight was 14 kg (below the 1st percen­tile), his normal serine (171; reference: 88-178 µmol/L) and citrulline (36; referen­ce: 21-43 µmol/L), head circum­ference was 46 cm (below the 2% percentile line) and his length was 108 cm hyperlactatemia (3.2; reference: 0.5-2.2 mmol/L) and hyper­ammonemia (75; reference: (below the 1st percentile). He has axial hypotonia with limb hypertonia and thoracic right 11-32 µmol/L). sided scoliosis. The boy is currently under a low protein diet (animal protein once per day, fruits and vegetables), and is kept on 7 g serine supplemen­tation with 75 mg pyridoxine, Individual 4 is a five-year-old boy, born to healthy consanguineous parents of Egyptian Espumisan and Nexium. descent after an unremarkable pregnancy (pedigree in Figure 2C). He presented with neonatal feeding diffi­culties and was diagnosed at the age of 4 months with severe Individuals 2 and 3 are sisters, born to a healthy consanguineous couple of Egyptian developmental delay and myoclonic­ seizures (15-20 times/day), uncontrolled by anti­ descent (pedigree in Figure 2B). A first pregnancy ended in a spontaneous first trimester epileptic drugs (Valproate, Leviteracetum,­ Vigabetrin and Clonazepam).­ The EEG revealed abortion. Individual 2 is a 10-year-old girl and individual 3 a 7-year-old girl. The oldest sister a bilateral frontoparietal­ (frequent spikes). The MRI showed cerebral atrophy (mainly was born at the gestational age of 32 weeks and was admitted to the neonatal intensive frontoparietal), asymmetric dilated lateral ventricles (more dilatation on the right), care unit (NICU) for 2 months, where she stayed on ventilation for the first 20 days. There T2/FLAIR white matter hyperintensities, a hypoplastic­ vermis and hypoplasia of the

80 81 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

corpus callosum. Plasma lactate was high (4.5; reference: 0.5-2.2 mmol/L) and ammonia the sequence corresponds to a loop in the protein structure that is part of the catalytic increased (120; reference: <80 µmol/L). Plasma acylcarnitine profiling and urinary organic site (see: protein structure analysis, Figure 2E). The absence of naturally-occurring acids were unremarkable. At the age of 4 years, his last visit to the clinic, the physical insertions or deletions in this part of the sequence indicate that a shift in the structure examination confirmed that weight and length are below the 25th percentile for age, caused by p.Leu209del may be detrimental to protein functioning. Positions 262 and 366 head circum­ference is below the 1st percentile for age and there is profound intellectual are strongly conserved, with all sequences examined presenting an arginine at position disability and severe motor retardation. He was unable to follow objects, has frequent 262 and all but one sequence presenting a glycine at position 366. infections and abnormal eye movements. Plasma amino acid analysis showed normal concentrations of serine (130; reference: 88-178 µmol/L) and citrulline (27; reference: 21- Consequences of the variants on the structure and function of the 43 µmol/L), hyperlactatemia (3.6; reference: 0.5-2.2 mmol/L) and hyper­ammonemia (79; GOT2 enzyme: Protein structure analysis reference: 11-32 µmol/L). Once the genetic basis of his disease was found he received GOT2 proteins form homodimer complexes (i.e. containing two identical subunits). 3 pyri­doxine (20 mg/kg/day in three divided doses per day) in addition to the antiepileptic Dimerization of two subunits results in the creation of two similar but separate catalytic drugs Valproate and Leviteracetum. Soon after starting pyridoxine supplementation, pockets with binding sites for the enzyme substrates (aspartate + α-ketoglutarate, or the mother reported that his epilepsy improved; the myoclonic fits were less frequent kynurenine + α-ketoglutarate) as well as the enzymatic cofactor pyridoxal 5’-phos­ in number, duration and severity. His alertness and cognitive function improved. Eight phate (vitamin B6 active form; pyridoxamine 5′-phosphate in the crystal structure). months later serine supplementation could be added to the treatment regimen (300 mg/ The variants found in the affected individuals (p.Leu209del, p.Arg337Gly, p.Arg262Gly, kg/day of serine in three divided doses per day). The combined pyridoxine plus serine p.Gly366Val) occur in the aspartate aminotransferase (Aminotransferase class I and II) treatment led to full seizure control and cognitive functions notably increased. Seizures domain, which covers the entire mature protein after the mitochondrial targeting returned in a period that there was no serine supplement available and dis­appeared signal is cleaved off. No post-translation modification has been reported for the again when serine treat­ment was reinstalled. This on/off effect convincingly­ illustrates mutated amino acids, nor are they part of any known short linear sequence motif. The the success of his treatment regimen. Currently he is weaned off antiepileptic drugs in a variants do not affect the inter­action interface between the homodimers (Figure 2E). timely fashion. The couple had a second child, a girl, diagnosed at the age of 3 months with severe developmental delay and focal and myoclonic seizures (around 10 times/ The p.Leu209del deletion leads to the shortening of a beta strand in the core of the day), uncontrolled by antiepileptic drugs. She had high blood lactate (4.2; reference: 0.5- protein, with two likely effects (Figure 2E). First, the affected strand is part of a beta 2.2 mmol/L), mild hyper­ammonemia (130; reference: <80 µmol/L) with a normal blood sheet structure, the connecting loops of which form the lower part of the catalytic acylcarnitine profile and normal urinary organic acids. She died at the age of 5 months. pocket of the enzyme. Modeling of the leucine deletion predicts that, in order to accommodate the shorter beta strand, there will be a shift in the positioning of the Consequences of the variants on the structure and function of the loop that follows the beta strand and which is involved in coordinating the enzyme GOT2 enzyme: Sequence analysis cofactor. Other changes include the repositioning of histidine 210, which in the wild The four positions in which GOT2 variants were found in the four affected individuals are type structure sits at only 5Å away from the cofactor. Given their strong evolutionary strongly evolutionarily­ conserved as observed in both multiple alignment of orthologous conservation (Figure S1 and S2), the beta sheet loop will be critical for the geometry protein se­quences (p.Leu209del, p.Arg337Gly, p.Arg262Gly, p.Gly366Val; Figure 2D, of the catalytic cavity, and shortening a beta strand inside the sheet (as in the case Figure S1) and in genome alignment (Figure S2). Position 337 in a few distant orthologs of p.Leu209del) will likely disrupt the precise positioning of the chemical moieties is occupied by lysine or histidine residues instead of an arginine. All these amino acids responsible for executing the catalytic reaction. A second effect is that disturbances are positively charged, indicating the importance of a positively-charged residue at this in the beta sheet likely pro­pagate to the surrounding structure and may affect protein position in the sequence and structure. p.Leu209del represents a deletion of one leucine stability. Thus, p.Leu209del is predicted to impact GOT2 catalytic activity and stability. residue in a tri-leucine repeat (Leu207, Leu208, Leu209). Positions 207, 208 and 209 are all strongly conserved for leucine, or otherwise contain residues that are all hydrophobic The p.Arg337Gly and p.Arg262Gly variants likely destabilize the protein due to a loss and accommodate a beta strand secondary structure. Strikingly, the ~12 amino acid of electrostatic interactions. Arg337 is located at the start of a long helix that forms sequence following the tri-leucine repeat shows even stronger con­ser­vation. This part of the entire surface of the protein on the far side of the homodimer complex (Figure 2E).

82 83 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

The positively-charged side chain of Arg337 sits on the inside of a 90 degree hinge Supplemental Figures between the long surface helix and a shorter helix pointing into the core of the protein. Arg337 is part of an electrostatic network that includes interactions with the backbone of Leu331 (part of the shorter helix preceding the hinge), and interactions with the negati­vely-charged side chains Glu111, Glu116 and Asp255 (though these are relatively long-distance at ~7-8Å). These interactions seem critical for the formation of the hinge and the overall helical organization of the protein. Arg262 is part of the same larger electro­static interaction network involved in the positioning of several helices, forming charge interactions with Glu116, Asp253 and Asp255. Substitution of either Arg262 or Arg337 by a neutral glycine residue, which has no side chain atoms, will result in a 3 loss of the electrostatic interactions. Taken together, p.Arg337Gly and p.Arg262Gly will likely de­stabilize the protein and reduce functional protein folding.

The p.Gly366Val variant is part of a turn at the surface of the far end of the structu­ re relative to the catalytic core and interaction interface (Figure 2E). While replace­ ­ ment of a glycine reduces flexibility, the Gly366 torsion angles are in the allowed region of the Ramachandran plot for valine, suggesting limited impact on the turn. Modeling of the variant in the structure shows negligible local structural changes. Thus, while Gly366 is well-conserved across evolution, the impact of the Gly366Val variant is unclear from a structural point of view. Various explanations are possible including reduced overall protein stability and folding capacity, altered potential for interaction with other proteins or increased proteolytic degradation.

Zebrafish got2a knockdown by splicing blocking morpholino (sp-MO) To validate the got2a ATG-blocking morpholino phenotype, we designed a splicing blocking morpholino (sp-MO) to target the splicing donor site of the 2nd exon, which will result intron-2 retention and therefore frameshift of zebrafish got2a protein. After micro­injection of sp-MO into newly fertilized embryos, we observed similar embryonic pheno­types (Figure S3A) as compared to ATG-blocking phenotypes

(Figure 5A), including various degrees of small head, slow circulation, bent body and Figure S1. Multiple sequence alignment of GOT2 orthologs. pericardial edema. These phenotypes are classified as mild (52%), medium (44%) and Sequences were identified using BLAST with the human reference GOT2 protein sequence and severe (4%). We then used RT-PCR to validate the aberrant mRNA splicing in sp-MO selected to cover a large phylogenetic distance. Residues are shaded in blue according to the percentage of residues in each column that agree with the consensus sequence (shown below the morphants. In embryos injected with control MO, it detested a 522bp wild-type (WT) alignment). Positions involved in the mutations of the affected individuals are indicated in orange. band; whereas in got2a sp-MO embryos, it detected a band of 985bp from aberrant Note that p.Leu209del represents a deletion of one leucine residue in a tri-leucine repeat. splicing, which was sequenced to validate the aberrant splicing event (Figure S3B).

84 85 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

(A) (C)

3

(D) (B)

Figure S2. Genome alignment of GOT2. (A) p.Leu209del variant. (B) p.Arg337Gly variant. (C) p.Arg262Gly variant. (D) p.Gly366Val variant. Evolutionary conservation was assessed using the Vertebrate Multiz Alignment & Conser­ ­vation (100 Species) track and visualized in the UCSC genome browser, https://www.ncbi.nlm.nih.gov/ pubmed/29106570.

86 87 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy

Mice Forward primers Reverse primers Got2emhD335fs14* 5’‑GTGAGCGTGTGGGAGCCTT-3’ 5’‑ACTTGAATTCAAGGAAGGTAGG-3’ Got2emhR337G wt: 5’‑CTGACTTCTCCAGACTTGCGG-3’ 5’‑ACTTGAATTCAAGGAAGGTAGG-3’ mut: 5’‑CTGACGTCTCCGGATTTAGG-3’ Got2emhL209del wt: 5’-CTCCTGCATGCCTGCGCTC-3’ 5’‑GAATCCACGTGTGGGTTGG-3’ mut: 5’‑GTCTTATTACACGCATGTGCAC-3’

Table S3. Mice genotyping.

Mice gRNA Repair template (ssODN) Got2emhR337G 5’‑TCCTGACTTCTCCAGACTTG-3’ 5’ CCGTCCCCTGTATTCCAACCCACCTCTCAATGGGGCCCGGATCGCAGCAACCATT 3 CTGACGTCTCCGGATTTAGGTAAGCAATGGTAACGATTACTAGCTGTTCCGTGCTA CAGCTCCATGAATGGAAAAG-3’ Got2emhL209del 5’‑GGTTGTGAGCGCAGGCATGC-3’ 5’ ACAGCCAAAAATCTCGATTGTTTCTCCTTAGAAAATCCCAGAGCAGAGTGTCTTATT Figure S3. Zebrafishgot2a gene knock-down by splicing blocking morpholino ACACGCATGTGCACACAACCCCACCGGCGTGGACCCGCGTCCCGAGCAGTGGAAGGA (A) got2a deficiency affects embryonic development. Bright field images of 48 hpf embryos injected GATAGCGTCCG-3’ with control morpholino (Cont MO) (a). Embryos injected with got2a splicing morpholino (got2a sp-MO) present three different phenotypes mild (b), medium (b’), severe (b’’). Scale bar: 25 µM. Table S4. CRISPR/Cas9 mice. (B) Validation of got2a sp-MO by RT-PCR using RNAs from embryos at 24 hpf and 48 hpf that were injected with Cont MO or got2a sp-MO. Mice +/+ +/- -/- Female Male Got2emhD335fs14* 35 48 0 53 30 Supplemental Tables Got2emhR337G 65 106 0 65 106 emhL209del Got2 68 87 0 79 76 Age (years [y], months [m]) Reference range (months) Table S5. Genotype distribution of the Got2 CRISPR/Cas9 mice. Amino acids (µmol/L) 1y, 2m 1y, 5m 2y, 2m 7y, 5m 12-24 > 24 Citrulline 89 71 83 78 7-55 21-43 Serine 47 53 81 105 70-294 88-178 MEF 14.5 dpc +/+ +/- -/- Glycine 280 318 396 271 80-340 156-328 Got2emhD335fs14* 1 3 - Got2emhR337G 2 5 - Table S1. Initial diagnostic plasma amino acid profile and follow-up data of GOT2-deficient individual Got2emhL209del 4 6 - 1. Values in bold are below or above the age-dependent reference range. Serine (with pyridoxine) treatment was started immediately after the metabolic screening at the age of 1y, 5 m. Table S6. Mouse embryonic fibroblasts (MEF) genotype distribution of the Got2 CRISPR/Cas9 mice.

Age (months) Reference range (< 18 months) 8 11 12 15 Lactate (mmol/L) 5.7 na na na 0.7-2.1 GPT (IU/L; = ALAT) 61 40 84 na < 37 GOT (IU/L; = ASAT) 75 43 85 39 < 31 Ammonia (µmol/L) 143 na na na 16-60

Table S2. Clinical chemistry findings in the plasma of the GOT2-deficient individual 1.Values in bold are above the reference range; na – not available

88 89 4 Chapter 4

Metabolic consequences of GOT2 deficiency

Rúben J Ramos, Clara D.M. van Karnebeek, Jolita Ciapaite, Mia Pras-Raves, Hans R Waterham, Ronald J Wanders, Judith JM Jans, Nanda M Verhoeven-Duif

Manuscript in preparation Metabolic consequences of GOT2 deficiency

ABSTRACT INTRODUCTION

Mitochondrial glutamate oxaloacetate transaminase (GOT2) deficiency is an autosomal Glutamate oxaloacetate transaminase 2 (GOT2, EC 2.6.1.1), also known as aspartate recessive neurometabolic disorder that has been recently described in four patients aminotransferase 2, is a pyridoxal 5’-phosphate-dependent enzyme that localizes with generalized hypotonia, epilepsy and developmental delay. GOT2 is an enzyme of to the mitochondrial matrix. GOT2, together with the cytosolic isoform (GOT1; the malate aspartate shuttle (MAS). To date, four genetic deficiencies in the MAS have glutamate oxaloacetate transaminase 1), the cytosolic and mitochondrial malate been described, caused by mutations in the genes encoding the aspartate glutamate dehydrogenases (MDH1 and MDH2, respectively), and the solute carriers aspartate- carriers (AGC1 and AGC2), the mitochondrial malate dehydrogenase (MDH2) and glutamate (AGC1 and AGC2) and 2-oxoglutarate (OGC; also known as malate-α- GOT2. An increased NADH/NAD+ ratio in the cytosol is considered to play a role in ketoglutarate carrier) constitute the malate aspartate shuttle (MAS). the pathogenesis of these defects. To further understand the pathophysiology of GOT2 deficiency we analyzed the metabolome of GOT2-deficient HEK293 cells The MAS is responsible for maintenance of the cytosolic NAD(H) homeostasis and generated by CRISPR/Cas9. Untargeted metabolomics on whole cell lysates showed the transport of reducing equivalents from the cytosol to the mitochondria (Amoedo alterations of several amino acids and intermediates of energy metabolism. Targeted et al. 2016). So far, four genetic MAS-deficiencies caused by mutations in the two 4 metabolomics confirmed decreases in alanine, aspartate, pyruvate, fumarate and aspartate-glutamate carriers (AGC1 and AGC2), MDH2 and GOT2 have been described malate. Glutamine, glutamate, methionine, ornithine, arginine, histidine and lysine in humans. and the lactate/pyruvate ratio were strongly increased in the GOT2-deficient cells. Next, we tested the hypothesis that supplementation of pyruvate could be beneficial SLC25A12 encodes the aspartate-glutamate carrier isoform 1 (AGC1 or aralar), while by normalizing the NADH/NAD+ ratio. Pyruvate supplementation resulted in the SLC25A13 encodes the aspartate-glutamate carrier isoform 2 (AGC2 or citrin). The amelioration of several of the metabolic abnormalities, suggesting a potential role in two carriers are co-expressed but have different tissue expression patterns. AGC2 is treatment of GOT2 deficiency. predominantly expressed in the liver (Palmieri 2013), while AGC1 is predominant in the adult central nervous system (Ramos et al. 2003). Both AGCs are responsible for the export of aspartate from the mitochondrial matrix to the cytosol, while glutamate and a proton are imported (Palmieri 2004). AGC1 deficiency leads to global cerebral hypomyelination, arrested psychomotor development, hypotonia and seizures (Wibom et al., 2009; Falk et al., 2014; Dahlin et al. 2015), whereas AGC2 deficiency causes neonatal intrahepatic cholestasis (NICCD, OMIM # 605814) and adult-onset type II citrullinemia (CTLN2, OMIM # 603471). CTLN2 patients suffer from disturbances of consciousness, convulsions and coma, and death may arise due to cerebral edema within a few years of onset (Kobayashi et al. 1999).

Three unrelated MDH2 deficient patients were recently reported, presenting with generalized hypotonia, psychomotor delay and refractory epilepsy (Ait-El- Mkadem et al. 2017). MDH2 catalyzes the reversible oxidation of malate and NAD+ to oxaloacetate and NADH, and is also important for the tricarboxylic acid (TCA) cycle (Ait-El-Mkadem et al. 2017). The most recently discovered MAS defect is GOT2 deficiency (van Karnebeek et al, 2019). GOT2 catalyzes the reversible interconversion of glutamate and oxaloacetate to aspartate and α-ketoglutarate in the mitochondrion (Fig. 1). The four described GOT2-deficient patients presented with early onset metabolic encephalopathy with epilepsy, progressive microcephaly and several

92 93 Metabolic consequences of GOT2 deficiency

maintain the cytosolic and mitochondrial­ NADH/NAD+ redox balance. Cytosolic NADH is mainly biochemical abnormalities (van Karnebeek et al, 2019). All patients had a mild but formed during glycolysis­ through glyceraldehyde 3-phosphate dehydroge­ ­nase (GAPDH) activity persistent hyperammonemia, with patient 1 also having mild hypercitrullinemia. and de novo serine formation by 3-phosphogly­ce­ra­te de­hydro­genase (3-PGDH). GOT2 is an essential Both can be explained by a secondary urea cycle defect in GOT2 deficiency due to member of the MAS. GOT2 catalyzes the mitochondrial­ formation of aspartate and α-ketoglutarate from oxaloacetate and glutamate. (GOT1, cytosolic glutamate oxaloacetate transaminase; GOT2, low concentrations of aspartate, the product of the GOT2-catalyzed enzyme reaction. mitochondrial glutamate oxaloacetate transami­ ­na­se; MDH1, cytosolic malate dehydrogenase; MDH2, mitochondrial malate dehydrogenase; AGC, aspartate-glutamate carrier; OGC, 2-oxoglutarate GLU αKG carrier; PDH, ; αKG, alpha-ketoglutarate; ASP, aspartate; ARG, arginine; ASS, argininosuccinate; CIT, citrulline; 1,3-DPG, 1,3-diphosphoglycerate; GAP, glyceraldehyde 3-phosphate; MDH1 + GOT1 GLU, glutamate; NAD , nico­tinamide adenine di­nucleotide (oxidized form); NADH, nicotina­mi­de ASP OAA MALATE adenine di­nucleo­tide (reduced form); ORN, ornithine; OAA, oxaloacetate; PEP, phosphoenolpyruvate;

NADH NAD+ 3-PG, 3-phosphoglycerate.

FUMARATE A remarkable observation in patient 1 was a consistently reduced concentration of serine in plasma. Stable isotope labelling studies in fibroblasts from the patients and ARG UREA 13 GOT2-knockout HEK293 cells showed decreased serine synthesis from C6-glucose 4 ASS (van Karnebeek et al, 2019). The first reaction in de novo serine biosynthesis is catalysed by the NAD+-dependent enzyme 3-phosphoglycerate dehydrogenase. The ORN UREA CYCLE low serine in GOT2 deficiency can be attributed to an increased NADH/NAD+ ratio as a consequence of a dysfunctional MAS. ASP CIT ORN OGC Complete normalization of serine and glycine synthesis was observed when the MALATE NAD+ CIT redox balance was improved by supplementing pyruvate to the medium of the cells, MDH2 NADH supporting our hypothesis that serine biosynthesis is hampered by the altered NADH/ FUMARATE OAA ACETYL-CoA AGC1/2 ASP NAD+ ratio. Two of the patients were treated with the cofactor of GOT2, pyridoxal GOT2 5’-phosphate (both patients) and serine (patient 1), with positive impact on their NADH SUCCINATE PDH seizures (van Karnebeek et al, 2019). + CITRATE NAD GLU Pyruvate supplementation might be considered as additional treatment strategy SUCCYNYL-CoA PYRUVATE ISOCITRATE for patients with GOT2 deficiency. Because GOT2 deficiency was only recently

αKG discovered, very little is known about its impact on cellular metabolism. Here, we unravel, in a detailed in vitro study, the metabolic consequences of GOT2 deficiency using a combination of untargeted and targeted metabolomics, with the main goal of identifying new biomarkers and treatment strategies. We further investigate the GLYCINE SERINE 2-OH-PYRUVATE

NADH effect of pyruvate addition aiming at correcting the metabolic abnormalities, based 3-PGDH NAD+ on our previous work in which we demonstrated complete correction of de novo GAPDH GLUCOSE GAP 1,3-DPG 3-PG PEP PYRUVATE serine synthesis, in GOT2-deficient HEK293 cells, by pyruvate supplementation (van

NAD+ NADH Karnebeek et al, 2019).

Fig. 1. The role of the malate-aspartate shuttle in the re-oxidation of cytosolic NADH and its interplay with the urea and tricarboxylic acid (TCA) cycles. Neither NAD+ nor NADH can be transported across intracellular membranes.­ The malate-aspartate shuttle (MAS) is essential to

94 95 Metabolic consequences of GOT2 deficiency

RESULTS We compared the intracellular metabolome of GOT2-wild type (WT) and GOT2- deficient HEK293 cells. The 100 most significantly altered compounds are presented To study the overall metabolic consequences of GOT2 deficiency, we performed in Table S1. The most significantly decreased features were glycolytic intermediates, direct-infusion high-resolution mass spectrometry (DI-HRMS) on whole cell lysates of TCA cycle intermediates and amino acids (Table S1). GOT2-deficient HEK293 cells. The analysis resulted in the detection of 11,760 HMDB (Human Metabolome Database; www.hmdb.ca)-identified features (defined as a Citrate α−ketoglutarate signal with a specific mass-to-charge ratio, m/z) in negative scan mode and 15,147 100 * 12 * in positive scan mode. 75 9 50 6 Concentration Aspartate Glutamate Glutamine (nmol/mg protein) 25 3 *** *** *** 24 *** 500 *** 800 *** WT A3 A6 WT A3 A6 18 375 600 12 250 400 Succinate Fumarate Malate 4 6 125 200 4 2.0 *** 16 * *** *** 3 ** 1.5 12 WT A3 A6 WT A3 A6 WT A3 A6 2 1.0 8 Alanine Methionine Ornithine 1 0.5 4 *** ** *** * 800 32 8 ** *** WT A3 A6 WT A3 A6 WT A3 A6 600 24 6 400 16 4 Lactate Pyruvate Lactate/Pyruvate *** 200 8 2 3000 * 40 *** 1000 *** *** 2250 30 750 WT A3 A6 WT A3 A6 WT A3 A6 1500 20 500 Arginine Histidine Lysine 750 10 250 * 16 40 * 120 ** * ** WT A3 A6 WT A3 A6 WT A3 A6 12 ** 30 90 8 20 60 Fig. 3. GOT2 deficiency leads to altered TCA cycle intermediates and increased redox status. The 4 10 30 intracellular TCA cycle intermediates profile of GOT2-wild type (WT) and GOT2-knockout (A3 and A6) HEK293 clones was analyzed. Cells were cultured in the presence of pyruvate (1 mmol/L). Decreases of fumarate and pyruvate were found, while succinate and the lactate/pyruvate ratio (redox status WT A3 A6 WT A3 A6 WT A3 A6 marker) were increased. The results are normalized to total protein content and represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01, *** P<0.001.

Concentration (nmol/mg protein) To validate these findings, we performed targeted liquid chromatography-tandem mass spectrometry of amino acids and intermediates of energy metabolism. In Fig. 2. GOT2 deficiency leads to altered amino acid concentrations. The intracellular amino acid GOT2-deficient cells increases in glutamate and glutamine (P<0.001), methionine, profile of GOT2-wild type (WT) and GOT2-knockout (A3 and A6) HEK293 clones was analyzed. Cells ornithine, arginine, histidine and lysine (P<0.05) were observed. In contrast, aspartate were cultured in the presence of pyruvate (1 mmol/L). Decreases of aspartate and alanine were found. On the other hand glutamate, glutamine, methionine, ornithine, arginine, histidine and lysine and alanine were strongly decreased (P<0.01) (Fig. 2). Other amino acids were not were increased. The results are normalized to total protein content and represented as the mean of significantly altered in the GOT2-deficient cells when compared to WT cells (Fig. S1). triplicates ± SD; * P<0.05, ** P<0.01, *** P<0.001.

96 97 Metabolic consequences of GOT2 deficiency

Fumarate and pyruvate were decreased (P<0.001), while the lactate/pyruvate ratio showed a tendency to normalize, even though most amino acids remained abnormal (P<0.001) and succinate were strongly increased (P<0.05) (Fig. 3). (Fig. 4 and S2). The increased concentrations of glutamate and glutamine levels were corrected by pyruvate, whereas aspartate and alanine remained strongly decreased Aspartate Glutamate Glutamine (P<0.05 and P<0.01, respectively). The lactate/pyruvate ratio normalized (Fig. 5). The *** *** ** * *** ** 24 *** *** ** *** 600 *** *** *** 800 other intermediates of the TCA cycle showed a tendency towards normalisation. *** *** *** * *** *** 18 450 600 * ** 12 300 400 Citrate α−ketoglutarate

6 150 200 100 ** * * 16 * ** * ** 75 12 ** * 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 *

Concentration 50 8 Alanine (nmol/mg protein) Methionine Ornithine Pyruvate concentration 25 4 ** *** * (mmol/L) ** 1200 *** *** ** 28 ** *** * *** 12 *** *** *** * ** ** *** 0 1 2.5 5 0 1 2.5 5 900 21 9 ** ** ** 4 600 14 6 Succinate Fumarate Malate 300 7 3 4 4 20 ** * ** * ** ** 3 ** ** * ** 3 15 ** *** *** * *** ** 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 2 2 ** *** *** 10 Arginine Histidine Lysine 1 1 5 * * * ** ** * 12 ** ** ** 32 140 * * ** * ** *** * 9 24 105 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 ** 6 16 70 Lactate ** Pyruvate Lactate/Pyruvate * ** ** 3 8 35 2800 * ** 80 ** 1000 ** *** ** ** 2100 60 750 ** 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 *** *** *** 1400 40 *** *** *** 500 700 20 250 *

0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 Concentration (nmol/mg protein) WT A3 A6

Pyruvate concentration (mmol/L) Fig. 5. Pyruvate completely normalizes the lactate/pyruvate ratio and rescues most of the Fig. 4. Pyruvate partially rescues the GOT2 deficient amino acid profile. The intracellular amino GOT2 deficient TCA cycle intermediates profile. The intracellular TCA cycle intermediates profile acid profile of GOT2-wild type (WT) and GOT2-knockout (A3 and A6) HEK293 clones under different of GOT2-wild type (WT) and GOT2-knockout (A3 and A6) HEK293 clones under different pyruvate pyruvate concentrations was analyzed and compared. Cells were cultured with four different concentrations was analyzed and compared. Cells were cultured with four different pyruvate pyruvate concentrations (0, 1, 2.5 and 5 mmol/L). The results are normalized to total protein content concentrations (0, 1, 2.5 and 5 mmol/L). The results are normalized to total protein content and and represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01, *** P<0.001. represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01, *** P<0.001.

To test the hypothesis that supplementation with pyruvate would correct the NADH/NAD+ balance and thereby normalize the metabolome, cells were exposed to pyruvate. Addition of increasing concentrations of pyruvate resulted in rising intracellular concentrations of pyruvate, however, concentrations remained still lower than in the control cells. Pyruvate in the culture medium strongly impacted the concentrations of amino acid and energy metabolites. The amino acid profile

98 99 Metabolic consequences of GOT2 deficiency

DISCUSSION knockdown Neuro2a cells (Profile et al, 2017). We here demonstrate that, like AGC1 deficiency, GOT2 deficiency may result in decreases of N-acetylaspartate (table S1). NADH, formed in the cytosol during glycolysis and serine biosynthesis, needs to be re- oxidized to NAD+ to maintain the cytosolic redox balance. As there is no direct transfer Glutamine and glutamate accumulate in GOT2 deficient cells. Glutamine is present of NAD+ and NADH over the mitochondrial membrane, cytosolic NAD+ regeneration in the medium, and glutamate is formed from glutamine by the enzyme glutaminase relies on two shuttle mechanisms: the MAS and the glycerol-3 phosphate shuttle. (GLS, EC 3.5.1.2). The accumulation of glutamine and glutamate may be attributed to Recently, we reported four patients affected with a novel defect of the MAS: GOT2 hampered glutamate metabolism directly due to GOT2 deficiency, since glutamate is deficiency (van Karnebeek et al, 2019). the substrate of GOT2.

In this study, we investigated the intracellular metabolic consequences of GOT2 Fumarate and malate were decreased in GOT2-deficient cells, while succinate deficiency to understand the pathophysiology of the disease and find targets for was increased. Increase in succinate and decrease in fumarate concentration treatment. Untargeted metabolomics of GOT2-deficient HEK293 cells revealed a (substrate and product, respectively) suggest inhibition of succinate dehydrogenase range of abnormalities in amino acid and intermediates of energy metabolism. The (SDH; EC 1.3.5.1). The increases of ornithine and arginine may be ascribed by to 4 most striking abnormality observed in the GOT2-deficient cells was the extremely hampered urea cycle due to lack of aspartate. The increased concentrations of low intracellular concentration of pyruvate. This finding can be attributed to the methionine, lysine and histidine, all essential amino acids, suggest diminished dysfunctional MAS, resulting in an elevated NADH/NAD+ ratio in the cytosol. This will utilization in GOT2-deficient cells. However, the precise underlying molecular causes subsequently result in decreased glycolysis, producing less pyruvate, and increased remain to be explained. conversion of pyruvate into lactate, an enzymatic reaction that depends on the NADH/NAD+ ratio. (Fig. 1). Intracellular lactate was not significantly increased in the We recently reported on decreased serine concentrations in blood of a GOT2-deficient GOT2-deficient cells probably due to lactate excretion into the medium, however, patient and decreased de novo synthesis of serine in the patients’ fibroblasts (van the lactate/pyruvate ratio in GOT2 deficient cells was strongly increased. As alanine Karnebeek et al, 2019). In that study, we presented complete normalization of in vitro is the direct product of pyruvate conversion in a reaction catalysed by alanine serine biosynthesis by pyruvate supplementation. Here, we demonstrate that pyruvate aminotransferase (ALT, EC 2.6.1.2), the low concentrations of alanine can be explained is taken up by the cells when supplemented in medium, resulting in normalization by pyruvate deficiency. of part of the metabolome. However, aspartate, alanine and pyruvate levels were not completely corrected and remained low, whereas glutamine, glutamate, arginine and Human cells have a very limited uptake of aspartate, and thus are highly dependent the lactate/pyruvate ratio aberrations were (almost) completely corrected. on mitochondrial aspartate production. A deficiency of GOT2 disables the intramitochondrial synthesis of aspartate from oxaloacetate, resulting in lower In conclusion, GOT2 deficiency has broad consequences for the intracellular concentrations of aspartate both in the mitochondrion and the cytosol. Indeed, metabolome. Our data shows that, at least in vitro, these consequences can be partly in our model system, aspartate was consistently and strongly decreased in GOT2- reversed by pyruvate supplementation. Care should be taken when treating patients deficient cells. This is in line with the hyperammonemia and abnormal citrulline levels with pyruvate, since it may lead to increased lactate formation. However, promising measured in our patients. results with sodium-pyruvate treatment in a CTLN2-deficient patient showed no lactate increase (Mutoh et al. 2008). Avoiding glycolysis and switching to fatty acid Aspartate is, via N-acetylaspartate, the precursor of the neurotransmitter peptide oxidation by implementing a ketogenic diet, as has been tried in patients with AGC1 N-acetylaspartylglutamate (NAAG). Insufficient levels of cytosolic aspartate have also deficiency, may also be beneficial for patients with GOT2 deficiency. Some effects been reported to lead to deficient N-acetylaspartate production (Dahlin et al. 2015). described in this paper, however, will not be overcome by switching to fatty acid In addition, brain and primary neuronal cultures of mice affected with AGC1 (the oxidation, and probably the lack of pyruvate and aspartate will still render metabolism mitochondrial aspartate transporter) deficiency presented with decreased levels of inefficient. Metabolic changes may be tissue-dependent, among others depending aspartate and N-acetylaspartate (Jalil et al. 2005). Similar results were observed in AGC1 on the relative importance of the malate aspartate and the glycerol 3-phosphate

100 101 Metabolic consequences of GOT2 deficiency

shuttles for balancing NADH/NAD+. Upregulation of the glycerol 3-phosphate days. When cells reached optimal confluence (>80%) they were exposed to DMEM shuttle activity by provision of substrates and cofactors might be beneficial, as it medium without glucose (supplemented with 10% FBS and 1% P/S), to which would ameliorate the NADH/NAD+ ratio. Furthermore, provision of aspartate may be 25 mmol/L glucose was added. Medium was supplemented with three different considered to overcome its severe deficiency. pyruvate concentrations (1, 2.5 and 5 mmol/L). Cells were harvested after 1 and 24 hours of incubation by first washing the cells with cold PBS (4ºC), and then scraping Our study clearly shows that GOT2 is essential in amino acid metabolism (especially with 1.5 ml ice-cold methanol. The samples were transferred into 1.5 ml eppendorf aspartate, glutamate and alanine metabolism) and energy metabolism. Furthermore, tubes, centrifuged (16,200 g for 10 min at 4ºC), and the supernatants were transferred (partial) correction of the NADH/NAD+ ratio through pyruvate supplementation leads to new 1.5 ml eppendorf tubes. The samples were evaporated at 40ºC under a stream to improvement of the pyruvate-related metabolic alterations. In addition, sodium of nitrogen until complete dryness, and reconstituted with 500 µl of UPLC-grade pyruvate and L-arginine treatment has already proved effective in patients with citrin methanol (room temperature). The reconstituted samples were stored at -80ºC until deficiency, another MAS defect (Mutoh et al. 2008). further analysis.

Untargeted metabolomics 4 MATERIAL AND METHODS Direct-Infusion High-Resolution Mass Spectrometry (DI-HRMS) was performed on whole cell lysates (GOT2-wild type and GOT2-knockout HEK293 clones) as described Cell culture by Ramos (Ramos et al., 2017). The bioinformatics pipeline was adapted, as will be HEK293 cells were purchased from ATCC Cell Biology Collection. Dulbecco’s modified published elsewhere (Rumping et al. submitted) eagle medium (DMEM) high glucose, GlutaMAX™, pyruvate (Cat. No. 31966), DMEM medium without glucose (Cat. No.11966), fetal bovine serum (FBS; Cat. No. Features (defined as a signal with a specific mass-to-charge ratio, m/z) were sorted on 10270), penicillin-streptomycin (P/S (10,000 U/mL); Cat. No. 15140) and trypsin- P-values, and identified using the Human Metabolome Database (HMDB, www.hmdb.ca). ethylenediaminetetraacetic acid (trypsin-EDTA, 0.5%, no phenol red; Cat. No. 15400) Features identified as pollutants, drugs, food or food additives were discarded from were purchased from Gibco (ThermoFisher Scientific). Glucose and sodium pyruvate the list. (purum, >99.0%) were purchased from Sigma-Aldrich (Steinheim, Germany). Amino acid analysis Generation of GOT2-knockout HEK293 clones by CRISPR/Cas9 Intracellular amino acids were measured using the UPLC-MS/MS method described The CRISPR/Cas9 genome editing technology was used to introduce a disruption of by Prinsen (Prinsen et al. 2016). Apart from adapting the range of the calibrators to the GOT2 gene in HEK293 cells as described by van Karnebeek (van Karnebeek, 2019). our samples’ concentrations, and using quality control (QC) samples resembling the concentrations of our samples, no further adaptations were made to the analysis. Maintenance of HEK293 cells in culture HEK293 cells (GOT2-WT; and GOT2-A3, -A6, and -A7 knockout clones) were grown Analysis of energy metabolites in 75 cm2 flasks and maintained in DMEM high glucose GlutaMAX™, pyruvate UPLC/MS-grade methanol (MeOH) and acetonitrile (ACN); the standards formate, (supplemented with 10% heat-inactivated FBS and 1% P/S), in a humidified citrate, isocitrate, fumarate, pyruvate, succinate, lactate, malate and α-ketoglutarate; 0 13 13 atmosphere of 5% CO2 at 37 C. Cells were passaged upon reaching confluency (>80%) and the internal standards C2-succinate and C3-lactate, were obtained from Sigma- 2 and media was refreshed every 48 hours. Aldrich (Zwijndrecht, the Netherlands). The internal standard H4-α-ketoglutarate was purchased from Cambridge Isotope Laboratories (Massachusetts, USA). Water was Pyruvate supplementation provided by a Millipore system. Samples were analysed on a Waters ACQUITY™ ultra- Cells were washed twice with PBS and plated onto 6-well plates (5 x 105 cells per well) performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA). by trypsinization (0.05% trypsin-EDTA) after reaching optimal confluence (>80%). Medium was refreshed 24 and 72 hours after plating and cells were cultured for 4 Detection of the analytes was carried in a Waters Xevo™ triple quadrupole tandem

102 103 Metabolic consequences of GOT2 deficiency

mass spectrometer (Waters Corp., Manchester, UK) with a Z-spray electrospray ACKNOWLEDGEMENTS ionization (ESI) source operating in both positive and negative ion modes. We would like to thank Johan Gerrits and Yuen Fung for their valuable technical The chromatographic separation was performed on an Acquity HSS C18 column assistance. This work was funded by Metakids foundation (www.metakids.nl). (2.1x100 mm, 1.7 μm particle size) equipped with an Acquity UPLC Van GuardTM Pre- Column (Waters, Milford, USA). The column was maintained at 30°C and the sample volume injected was 10 μl. AUTHOR CONTRIBUTIONS

Optimal chromatographic separation was achieved at a constant flow-rate of 0.3 R.J.R., J.J.J. and N.M.V-D. designed the study. R.J.R. conducted the studies. H.W. mL/min using a gradient with solvent A (0.1% formic acid in milliQ-water; v/v) and generated the GOT2-knockout HEK293 cells. R.J.R. and M.L.P.-R. performed the solvent B (100% ACN) as follows. Initial conditions were 1% solvent B. From 0 to 4.0 statistical analysis. R.J.R., M.L.P.-R., J.C., J.J.J., and N.M.V-D. analysed the data. R.J.R., min solvent B was set at 1%. From 4.0 to 4.5 min solvent B was increased from 1% to C.v.K., R.J.A.W., J.C., J.J.J. and N.M.V-D. prepared the manuscript. All authors edited 100%. From 4.5 to 5.0 min solvent B was set at 100% and from 5.0 to 5.1min solvent B the manuscript and approved submission for publication. 4 was decreased from 100% to 1%. The column was equilibrated for 0.9 min (5.1 to 6.0 min) in the initial conditions. Total run time was 6 min including column equilibration. Chromatographic data were collected and analyzed with Waters MassLynx v4.1 software. Quantification was achieved for each analyte using linear regression analysis of the peak area ratio analyte/IS (weighed 1/X) versus concentration.

Calibration samples were prepared in the concentration range of 0.05 to100 µM for citrate, succinate, fumarate and malate; 0.05 to 500 µM for lactate; and 0.5 to 100 µM for α-ketoglutarate and pyruvate. Internal standards were added to 500 µL of whole cell lysate extract (methanol) and evaporated under a gentle flow of nitrogen. The extracts were dissolved in 50 µL of solvent A and analyzed.

Statistical analysis Statistical significance for untargeted metabolomics was determined by comparison of the intracellular metabolome of GOT2-wild type (WT) to GOT2-deficient HEK293 cells using unpaired T-tests (with Bonferroni correction) on the area under the curve (AUC in a plot of intensities versus time).

Statistical significance for targeted metabolomics was performed using unpaired two-tailed t-test, using GraphPad Prism 6 (version 6.0.2, GraphPad Software Inc.) software.

104 105 Metabolic consequences of GOT2 deficiency

REFERENCES SUPPLEMENTARY MATERIAL

Ait-El-Mkadem S, Dayem-Quere M, Gusic M, et al (2017) Mutations in MDH2, Encoding a Krebs Asparagine Proline Valine * Cycle Enzyme, Cause Early-Onset Severe Encephalopathy. Am J Hum Genet 100:151–159. doi: 40 300 ** 140 10.1016/j.ajhg.2016.11.014 * 30 225 105 Alfarouk KO, Verduzco D, Rauch C, et al (2014) Glycolysis, tumor metabolism, cancer growth and dissemination. A new pH-based etiopathogenic perspective and therapeutic approach to an old 20 150 70 cancer question. Oncoscience 1:777. doi: 10.18632/oncoscience.109 10 75 35 Amoedo ND, Punzi G, Obre E, et al (2016) AGC1/2, the mitochondrial aspartate-glutamate carriers. Biochim Biophys Acta - Mol Cell Res 1863:2394–2412. doi: 10.1016/j.bbamcr.2016.04.011 WT A3 A6 WT A3 A6 WT A3 A6 Dahlin M, Martin DA, Hedlund Z, et al (2015) The ketogenic diet compensates for AGC1 deficiency and improves myelination. Epilepsia 56:e176–e181. doi: 10.1111/epi.13193 Leucine Isoleucine Serine Falk MJ, Li D, Gai X, et al (2014) AGC1 Deficiency Causes Infantile Epilepsy , Abnormal Myelination , * 120 ** 120 ** 60 and Reduced N -Acetylaspartate. JIMD Rep 14:77–85. doi: 10.1007/8904 90 90 45 Jalil MA, Begumi L, Contreras L, et al (2005) Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier. J Biol Chem 280:31333– 60 60 30 4 31339. doi: 10.1074/jbc.M505286200 30 30 15 Kobayashi K, Sinasac DS, Iijima M, et al (1999) The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein. Nat Genet 22:159–163. doi: 10.1038/9667 WT A3 A6 WT A3 A6 WT A3 A6 Mutoh K, Kurokawa K, Kobayashi K, Saheki T (2008) Treatment of a citrin-deficient patient at the early stage of adult-onset type II citrullinaemia with arginine and sodium pyruvate. J Inherit Glycine Citrulline Phenylalanine Metab Dis 31:343–347. doi: 10.1007/s10545-008-0914-x 800 4 80 Palmieri F (2013) The mitochondrial transporter family SLC25: Identification, properties and * 600 3 60 physiopathology. Mol Aspects Med 34:465–484. doi: 10.1016/j.mam.2012.05.005 Palmieri F (2004) The mitochondrial transporter family (SLC25): Physiological and pathological 400 2 40 implications. Pflugers Arch Eur J Physiol 447:689–709. doi: 10.1007/s00424-003-1099-7 200 1 20 Prinsen HCMT, Schiebergen-Bronkhorst BGM, Roeleveld MW, et al (2016) Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) WT A3 A6 WT A3 A6 WT A3 A6 coupled with tandem mass-spectrometry. J Inherit Metab Dis 39:651–660. doi: 10.1007/s10545- 016-9935-z Tyrosine Tryptophan Threonine * * Ramos M, Del Arco A, Pardo B, et al (2003) Developmental changes in the Ca2+-regulated 80 ** 12 ** 500 mitochondrial aspartate-glutamate carrier aralar1 in brain and prominent expression in the 60 9 375 spinal cord. Dev Brain Res 143:33–46. doi: 10.1016/S0165-3806(03)00097-X Ramos RJ, Pras-Raves ML, Gerrits J, et al (2017) Vitamin B6 is essential for serine de novo biosynthesis. 40 6 250 J Inherit Metab Dis 40:883–891. doi: 10.1007/s10545-017-0061-3 20 3 125 Roberts SJ, Lowery MS, Somero GN (1988) Regulation of binding of phosphofructokinase to myofibrils in the red and white muscle of the barred sand bass, Paralabrax nebulifer (Serranidae). WT A3 A6 WT A3 A6 WT A3 A6 J Exp Biol 137:13–27 Rumping L, Pras-Raves ML, Gerrits J, et al (2019) Metabolic fingerprinting reveals extensive Taurine Hydroxyproline ** consequences of GLS hyperactivity. Submitted 240 4 Schollmeyer P, Klingenberg M (1961) Oxaloacetate and adenosinetriphosphate levels during 180 3 inhibition and activation of succinate oxidation. Biochem Biophys Res Commun 4:43–7 Van Karnebeek CDM, Ramos RJ, Wen X-Y, et al (2019) Biallelic GOT2 mutations cause a treatable 120 2 Concentration malate-aspartate shuttle related encephalopathy. Am J Hum Genet 105(3):534-548 60 1 (nmol/mg protein) Wibom R, Lasorsa FM, Töhönen V, et al (2009) AGC1 Deficiency Associated with Global Cerebral Hypomyelination. N Engl J Med 361:489–495 WT A3 A6 WT A3 A6 Fig. S1. Effect of GOT2 deficiency in the intracellular amino acid concentrations. The intracellular amino acid profile of GOT2-wild type (WT) and GOT2-knockout (A3 and A6) HEK293 clones was determined by UPLC-MS/MS. Cells were cultured in the presence of pyruvate (1 mmol/L). The results are normalized to total protein content and represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01. 106 107 Metabolic consequences of GOT2 deficiency

Table S1. The 100 most significantly altered intracellular compounds.Direct infusion high Asparagine Proline Valine resolution mass spectrometry (DI-HRMS) in positive and negative scan mode was used to detect the * ** * * * ** overall intracellular metabolic consequence of GOT2 deficiency. A t-test was performed on the area 60 ** *** 300 ** ** 140 ** ** under the curve (AUC, in a plot of intensities against time) for every compound. Compounds are 45 *** 225 105 *** * sorted on the P value between GOT2-WT and GOT2-knockout HEK293 clones. 30 150 70 Scan Fold m/z Compound P value Change 15 75 35 mode Change 190.97282 L/D-glyceraldehyde 3-phosphate [M+Na-H]-; dihydroxyacetone 3.91E-09 - Decrease 5.7 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 phosphate [M+Na-H]-; L/D-malic acid [M+NaCl]- 205.06818 galactitol [M+Na]+; [M+Na]+; mannitol [M+Na]+; L-iditol 7.45E-08 + Decrease 12.4 Leucine Isoleucine Serine + ** [M+Na] * ** * ** ** * ** 100 100 ** 80 - -08 ** * * 87.00870 pyruvic acid; malonic semialdehyde; ascorbic acid [M-2H] ; 9.86E - Decrease 323.3 - 75 75 60 D-glucurono-6,3-lactone [M-2H] 144.96737 pyruvic acid [M+NaCl]-; malonic semialdehyde [M+NaCl]- 6.24E-07 - Decrease 5.3 50 50 40 ** 133.01423 L/D-malic acid; pyruvic acid [M+For]-; malonic semialdehyde 9.16E-07 - Decrease 272.2 25 25 20 [M+For]- 178.99255 L/D-malic acid [M+2Na-H]+ 1.92E-06 + Decrease 3.5 4 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 132.98707 pyruvic acid [M+2Na-H]+; malonic semialdehyde [M+2Na-H]+ 3.23E-06 + Decrease 4.6 - - -06 Glycine Citrulline Phenylalanine 125.00104 butyric acid [M+K-H] ; isobutyric acid [M+K-H] ; L/D-lactic acid 3.67E - Decrease 1.9 [M+Cl]-; hydroxypropionic acid [M+Cl]-; glyceraldehyde [M+Cl]-; ** * * ** - - 800 * * * ** 4 60 ** dihydroxyacetone [M+Cl] ; methoxyacetic acid [M+Cl] 600 3 * * 45 141.06574 1,3-dimethyluracil; imidazolepropionic acid; methylimidazoleacetic 4.11E-06 + Decrease 4.2 acid; Pi-methylimidazoleacetic acid; nicotinic acid [M+NH ]+; 30 4 400 2 + + picolinic acid [M+NH4] ; isonicotinic acid [M+NH4] 200 1 15 172.99858 alpha-ketoisovaleric acid [M+NaCl]-; (2)-methylacetoacetic acid 4.42E-06 - Decrease 2.4 [M+NaCl]-; levulinic acid [M+NaCl]-; 2-oxovaleric acid [M+NaCl]-; 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 glutarate semialdehyde [M+NaCl]- 242.09956 pantothenic acid [M+Na]+ 6.28E-06 + Decrease 2.4 Tyrosine Tryptophan Threonine 115.00361 fumaric acid; maleic acid; acetyl citrate [M-2H]-; propynoic acid 7.43E-06 - Decrease 3.3 ** - 80 12 * 500 * * [M+For] ** ** ** ** * ** ** -06 60 ** 9 375 129.05574 2-methyl-3-ketovaleric acid; 3-methyl-2-oxovaleric acid; 9.85E - Decrease 3.1 ketoleucine; 2-ketohexanoic acid; mevalonolactone; 3-oxohexanoic 40 6 250 acid; adipate semialdehyde 20 3 125 165.01332 (S)-3,4-dihydroxybutyric acid [M+2Na-H]+; 2,4-dihydroxybutanoic 9.89E-06 + Decrease 3.6 acid [M+2Na-H]+;4-deoxyerythronic acid [M+2Na-H]+; + + 0 1 2.5 5 0 1 2.5 5 0 1 2.5 5 4-deoxythreonic acid [M+2Na-H] ; L/D-erythrose [M+2Na-H] - -05 187.00093 L/D-lactic acid [M+H2PO4]-; hydroxypropionic acid [M+H2PO4] ; 1.46E - Decrease 1.7 Taurine Hydroxyproline - - glyceraldehyde [M+H2PO4] ; dihydroxyacetone [M+H2PO4] ; methoxyacetic acid [M+H PO ]- 240 ** ** ** ** 6.0 * 2 4 *** 207.07799 trans-4,5-epoxy-2(E)-decenal [M+K]+ 1.86E-05 + Decrease 1.5 180 4.5 ** 130.05900 homovanillic acid sulfate [M-2H]- 2.00E-05 - Decrease 3.0 120 3.0 - -05 Concentration

(nmol/mg protein) 231.99930 N-acetyl-L-aspartic acid [M+NaCl] ; N-formyl-L-glutamic acid 2.12E - Decrease 2.6 WT A3 A6 60 1.5 - - Pyruvate concentration [M+NaCl] ; 2-amino-3-oxoadipate [M+NaCl] (mmol/L) + -05 364.91582 N-acetylgalactosamine 4,6-disulfate [M+H-H2O] 2.22E + Increase 2.9 0 1 2.5 5 0 1 2.5 5 115.04001 alpha-ketoisovaleric acid; methylacetoacetic acid; levulinic 2.31E-05 - Decrease 2.9 acid; 2-oxovaleric acid; 2-methylacetoacetic acid; glutarate Fig. S2. Effect of pyruvate supplementation on GOT2 deficient amino acid profile.The semialdehyde intracellular amino acid profile of GOT2-wild type (WT) and GOT2-knockout (A3 and A6) HEK293 202.97288 oxoglutaric acid [M+NaCl]-; 3-oxoglutaric acid [M+NaCl]- 2.32E-05 - Decrease 2.3 clones under different pyruvate concentrations was determined by UPLC-MS/MS and compared. 128.03166 L/D-serine [M+Na]+ 2.42E-05 + Increase 2.6 Cells were cultured with four different pyruvate concentrations (0, 1, 2.5 and 5 mmol/L). The results 150.01366 L/D-serine [M+2Na-H]+ 2.76E-05 + Increase 4.8 are normalized to total protein content and represented as the mean of triplicates ± SD; * P<0.05, ** P<0.01, *** P<0.001.

108 109 Metabolic consequences of GOT2 deficiency

254.08011 propionylcarnitine [M+K-H]-; pantothenic acid [M+Cl]- 3.03E-05 - Decrease 2.7 130.05088 4-hydroxyproline; N-acetyl-L-alanine; propionylglycine; 1.310E-04 - Decrease 2.3 - -05 5-aminolevulinic acid; L-glutamic gamma-semialdehyde; 381.23188 stearic acid [M+HSO4] 3.45E - Increase 1.6 89.01377 4-hydroxyphenylpyruvic acid [M-2H]-; 2-hydroxy-3-(4- 4.14E-05 - Decrease 4.4 3/4-hydroxy-L-proline; 5-amino-2-oxopentanoic acid; 4-hydroxy-2- hydroxyphenyl)propenoic acid [M-2H]-; 3-hydroxyphenylpyruvic pyrrolidinecarboxylic acid; cis/trans-3-hydroxy-L-proline; nopalinic - - acid [M-2H]- acid [M-2H] ; 2-pyrrolidinone [M+For] - -04 189.98883 2-amino-3-phosphonopropionic acid [M+Na-H]-; L/D-aspartic acid 4.20E-05 - Decrease 2.6 292.98343 6-phosphonoglucono-D-lactone [M+Cl] ; 2-keto-3-deoxy-6- 1.37E - Increase 2.4 - [M+NaCl]- phosphogluconic acid [M+Cl] ; 3-hydroxy-3-carboxymethyl-adipic acid [M+KCl]- 252.97183 citric acid [M+NaK-H]+; isocitric acid [M+NaK-H]+; D-threo-isocitric 4.58E-05 + Decrease 3.0 + -04 acid [M+NaK-H]+; diketogulonic acid [M+NaK-H]+; 2,3-diketo- 144.04186 benzamide [M+Na] 1.43E + Increase 1.1 L-gulonate [M+NaK-H]+; (1R,2R)-isocitric acid [M+NaK-H]+; 204.12288 L-acetylcarnitine 1.48E-04 + Decrease 2.7 D-glucaro-1,4-lactone [M+NaK-H]+ 264.08153 pantothenic acid [M+2Na-H]+ 1.49E-04 + Decrease 2.2 133.05068 2,3-dihydroxyvaleric acid; 1-deoxy-D-xylulose; deoxyribose; 5.19E-05 - Decrease 3.7 276.06182 pantothenic acid [M+NaCl]- 1.56E-04 - Decrease 3.5 (R)-2,3-dihydroxy-isovalerate; (R)-glycerol 1-acetate; butyric acid 157.01067 L/D-malic acid [M+Na]+ 1.58E-04 + Decrease 3.3 - - [M+For] ; isobutyric acid [M+For] 176.02939 4-hydroxyproline [M+2Na-H]+; N-acetyl-L-alanine [M+2Na-H]+; 1.65E-04 + Decrease 1.7 191.04434 2-phenyl-2-butenal [M+2Na-H]+; heptanoic acid [M+NaK-H]+; 6.20E-05 + Increase 1.3 propionylglycine [M+2Na-H]+; 5-aminolevulinic acid [M+2Na-H]+; 2/5-methylhexanoic acid [M+NaK-H]+; 2-hydroxy-3- L-glutamic gamma-semialdehyde [M+2Na-H]+; 3/4-hydroxy-L- methylpentanoic acid [M+NaCl]+; (5R)-5-hydroxyhexanoic acid proline [M+2Na-H]+; 5-amino-2-oxopentanoic acid [M+2Na-H]+; + + 4 [M+NaCl] ; 5-hydroxyhexanoic acid [M+NaCl] ; L/D-leucic 4-hydroxy-2-pyrrolidinecarboxylic acid [M+2Na-H]+; cis/trans-3- acid [M+NaCl]+; (2)-hydroxyisocaproic acid [M+NaCl]+; (R)- hydroxy-L-proline [M+2Na-H]+ + 3-hydroxyhexanoic acid [M+NaCl] ; 6-hydroxyhexanoic acid 167.04678 naphthalene epoxide [M+Na]+ 1.78E-04 + Decrease 1.4 [M+NaCl]+; 2-methyl-3-hydroxyvaleric acid [M+NaCl]+ 150.97669 L/D-lactic acid [M+NaK-H]+; hydroxypropionic acid [M+NaK-H]+; 1.80E-04 + Decrease 2.7 -05 173.00905 cis/trans-aconitic acid; dehydro-ascorbic acid 6,.41E - Decrease 2.7 glyceraldehyde [M+NaK-H]+; dihydroxyacetone [M+NaK-H]+; 235.99292 L-aspartyl-4-phosphate [M+Na]+; N-acetyl-L-aspartic acid 6.43E-05 + Decrease 4.5 methoxyacetic acid [M+NaK-H]+ + + [M+NaK-H] ; N-formyl-L-glutamic acid [M+NaK-H] ; 2-amino-3- 218.10332 pantothenic acid; hexanoylglycine [M+For]-; isovalerylalanine 1.84E-04 - Decrease 3.0 + oxoadipate [M+NaK-H] [M+For]-; isovalerylsarcosine [M+For]-; N-acetylleucine [M+For]- + -05 366.04041 beta-citryl-L-glutamic acid [M+2Na-H] 7.05E + Decrease 2.0 149.99267 beta-alanine [M+NaK-H]+; L/D-alanine [M+NaK-H]+; sarcosine 1.91E-04 + Decrease 2.6 153.08846 adenine [M+NH4]+; heptanoic acid [M+Na]+; 2-methylhexanoic 7.62E-05 + Increase 1.2 [M+NaK-H]+ + + acid [M+Na] ; 5-methylhexanoic acid [M+Na] 269.98415 dopamine 4-sulfate [M+K-H]-; dopamine 3-O-sulfate [M+K-H]- 1.97E-04 - Decrease 1.3 + + -05 147.04152 4-methylcatechol [M+Na] ; 4-hydroxybenzyl alcohol [M+Na] 8.72E + Increase 1.4 481.23583 1,25-dihydroxyvitamin D3-26,23-lactone [M+K-H]- 1.98E-04 - Decrease 1.8 + -05 193.06245 4-oxo-2-nonenal [M+K] 8.75E + Decrease 1.4 342.04472 beta-citryl-L-glutamic acid [M+Na-H]- 2.01E-04 - Decrease 2.1 - - -05 190.07207 isobutyrylglycine [M+For] ; N-butyrylglycine [M+For] ; allysine 9.11E - Decrease 3.1 176.00835 L/D-proline [M+NaK-H]+; acetamidopropanal [M+NaK-H]+; 2.10E-04 + Decrease 3.0 - - [M+For] ; 4-acetamidobutanoic acid [M+For] ; (S)-5-amino- acetylglycine [M+NaCl]+; L-2-amino-3-oxobutanoic acid - - 3-oxohexanoate [M+For] ; 2-keto-6-aminocaproate [M+For] ; [M+NaCl]+; L-aspartate-semialdehyde [M+NaCl]+ (S)-2-amino-6-oxohexanoate [M+For]- 132.03019 L/D-aspartic acid 2.11E-04 - Decrease 2.6 161.01836 alpha-ketoisovaleric acid [M+2Na-H]+; methylacetoacetic acid 9.47E-05 + Decrease 1.9 199.03655 3-methyladipic acid [M+K]+; pimelic acid [M+K]+; 2.14E-04 + Decrease 1.6 [M+2Na-H]+; levulinic acid [M+2Na-H]+; 2-oxovaleric acid 3,3-dimethylglutaric acid [M+K]+; 2-methyladipic acid [M+K]+; [M+2Na-H]+; 2-methylacetoacetic acid [M+2Na-H]+; glutarate 2-ethylglutaric acid [M+K]+; ethyl methyl-succinate [M+K]+ semialdehyde [M+2Na-H]+ 89.02441 L/D-lactic acid; hydroxypropionic acid; glyceraldehyde; 2.19E-04 - Decrease 1.9 347.07491 cis-beta-D-glucosyl-2-hydroxycinnamate [M+Na-H]-; galactose- 9.49E-05 - Decrease 1.7 dihydroxyacetone; methoxyacetic acid; L/D-glucose [M-2H]-; beta-1,4-xylose [M+Cl]- L/D-galactose [M-2H]-; L/D-mannose [M-2H]-; myoinositol [M-2H]-; + + -05 148.96103 pyruvic acid [M+NaK-H] ; malonic semialdehyde [M+NaK-H] ; 9.61E + Decrease 4.1 3-deoxyarabinohexonic acid [M-2H]-; beta-D-glucose [M-2H]-; L/D- + oxalic acid [M+NaCl] fructose [M-2H]-; allose [M-2H]-; L/D-sorbose [M-2H]-; alpha-D- 171.00639 glycerol 3-phosphate; beta-glycerophosphoric acid; propionic acid 9.94E-05 - Increase 1.8 glucose [M-2H]-; L/D-tagatose [M-2H]-; beta-D-galactose [M-2H]-; - - - - [M+H2PO4] ; L/D-lactaldehyde [M+H2PO4] ; 2,3-dihydroxyvaleric scyllitol [M-2H] ; L/D-gulose [M-2H] ; dihydroxyacetone (dimer) acid [M+K-H]-; 1-deoxy-D-xylulose [M+K-H]-; deoxyribose [M-2H]-; L-galactose [M-2H]-; levoinositol [M-2H]-; acetaldehyde [M+K-H]-; (R)-2,3-dihydroxy-isovalerate [M+K-H]-; (R)-glycerol [M+For]- - - 1-acetate [M+K-H] ; phenylglyoxylic acid [M+Na-H] ; threonic 161.04546 2/3-hydroxyadipic acid; 3-hydroxymethylglutaric acid; 2-hydroxy- 2.19E-04 - Decrease 1.9 - acid [M+Cl] 2-ethylsuccinic acid; alpha-ketoisovaleric acid [M+For]-; + -04 - - 104.10680 neurine; iso-valeraldehyde [M+NH4] 1.04E + Increase 3.1 methylacetoacetic acid [M+For] ; levulinic acid [M+For] ; 152.04708 3-methylene-indolenine [M+Na]+ 1.04E-04 + Decrease 1.4 2-oxovaleric acid [M+For]-; 2-methylacetoacetic acid [M+For]-; 183.04162 4-hydroxycyclohexylcarboxylic acid [M+K]+ 1.10E-04 + Decrease 1.1 glutarate semialdehyde [M+For]-

110 111 Metabolic consequences of GOT2 deficiency

191.04016 L/D-glutamine [M+2Na-H]+; ureidoisobutyric acid [M+2Na-H]+; 2.29E-04 + Increase 2.2 362.74970 PC(14:0/18:3(6Z,9Z,12Z)) [M-2H]-; PC(14:0/18:3(9Z,12Z,15Z)) [M- 4.35E-04 - Decrease 1.9 alanylglycine [M+2Na-H]+ 2H]-; PC(14:1(9Z)/18:2(9Z,12Z)) [M-2H]-; PC(18:2(9Z,12Z)/14:1(9Z)) 360.75256 PE(18:3(6Z,9Z,12Z)/P-18:1(11Z)) [M-2H]-; 2.37E-04 - Decrease 2.0 [M-2H]-; PC(18:3(6Z,9Z,12Z)/14:0) [M-2H]-; PE(18:3(6Z,9Z,12Z)/P-18:1(9Z)) [M-2H]-; PE(18:3(9Z,12Z,15Z)/ PC(18:3(9Z,12Z,15Z)/14:0) [M-2H]-; PE(15:0/20:3(5Z,8Z,11Z)) [M- P-18:1(11Z)) [M-2H]-; PE(18:3(9Z,12Z,15Z)/P- 2H]-; PE(15:0/20:3(8Z,11Z,14Z)) [M-2H]-; PE(20:3(5Z,8Z,11Z)/15:0) 18:1(9Z)) [M-2H]-; PE(18:4(6Z,9Z,12Z,15Z)/P-18:0) [M-2H]-; PE(20:3(8Z,11Z,14Z)/15:0) [M-2H]- [M-2H]-; PE(20:4(5Z,8Z,11Z,14Z)/P-16:0) 211.07283 4-hydroperoxy-2-nonenal [M+K]+ 4.37E-04 + Decrease 1.2 [M-2H]-; PE(20:4(8Z,11Z,14Z,17Z)/P-16:0) 228.96487 glycerol 3-phosphate [M+NaCl]-; beta-glycerophosphoric acid 4.66E-04 - Increase 1.7 [M-2H]-; PE(P-16:0/20:4(5Z,8Z,11Z,14Z)) [M+NaCl]- - [M-2H] ; PE(P-16:0/20:4(8Z,11Z,14Z,17Z)) 161.99382 5-hydroxymethyl-2-furanoate [M+Na-H]- 4.79E-04 - Increase 6.9 [M-2H]-; PE(P-18:0/18:4(6Z,9Z,12Z,15Z)) [M- 253.96982 phosphocreatinine [M+NaK-H]+ 4.87E-04 + Decrease 1.7 2H]-; PE(P-18:1(11Z)/18:3(6Z,9Z,12Z)) [M-2H]-; + -04 PE(P-18:1(11Z)/18:3(9Z,12Z,15Z)) [M-2H]-; 267.15637 1,11-undecanedicarboxylic acid [M+Na] 4.91E + Increase 1.2 PE(P-18:1(9Z)/18:3(6Z,9Z,12Z)) [M-2H]-; PE(P- 303.08362 N-acetylaspartylglutamic acid; ribothymidine [M+For]-; 5.55E-04 - Decrease 2.0 18:1(9Z)/18:3(9Z,12Z,15Z)) [M-2H]- imidazoleacetic acid riboside [M+For]-; 3-methyluridine [M+For]- 153.03112 delta-hexanolactone [M+K]+; trans-hex-2-enoic acid [M+K]+ 2.39E-04 + Decrease 1.4 454.74682 kinetensin 1-7 [M-2H]- 5.86E-04 - Decrease 1.7 175.03591 ureidosuccinic acid 2.47E-04 - Decrease 2.9 240.09784 3-mercaptolactate-cysteine [M-H]- 6.85E-04 - Decrease 1.3 145.01421 2/3-oxoglutaric acid 2.49E-04 - Decrease 2.8 147.02986 citramalic acid; L/D-2/3-hydroxyglutaric acid; ribonolactone; 7.79E-04 - Decrease 1.7 4 335.04367 4-hydroxy-5-(dihydroxyphenyl)-valeric acid-O-methyl-O-sulphate; 2.73E-04 - Increase 2.0 D-xylono-1,5-lactone; 2-hydroxyglutarate; 2-ketobutyric acid - - 4-Hydroxy-5-(3'-hydroxyphenyl)-valeric acid-3'-O-sulphate [M+For] ; acetoacetic acid [M+For] ; 2-methyl-3-oxopropanoic acid - - [M+For]-; 4-hydroxy-5-(4'-hydroxyphenyl)-valeric acid-4'-O- [M+For] ; succinic acid semialdehyde [M+For] ; (S)-methylmalonic - - - - acid semialdehyde [M+For] ; 4-hydroxycrotonic acid [M+For] sulphate [M+For] ; 3,4,5-trimethoxycinnamic acid [M+HSO4] ; cis/ - 226.94930 D-glyceraldehyde 3-phosphate [M+NaCl]-; dihydroxyacetone 8.62E-04 - Decrease 1.5 trans-2,3,4-trimethoxycinnamate [M+HSO4] - 214.01113 L-aspartyl-4-phosphate; N-acetyl-L-aspartic acid [M+K]+; 2.75E-04 + Decrease 4.8 phosphate [M+NaCl] N-formyl-L-glutamic acid [M+K]+; 2-amino-3-oxoadipate [M+K]+ 187.07295 benzenebutanoic acid [M+Na]+; 3-phenylbutyric acid [M+Na]+ 4.92E-04 + Decrease 1.4 197.05727 cis/trans-4-hydroxycyclohexylacetic acid [M+K]+; 3-oxooctanoic 2.77E-04 + Decrease 1.2 139.01547 senecioic acid [M+K]+; 2-ethylacrylic acid [M+K]+; 4.96E-04 + Decrease 1.6 acid [M+K]+; alpha-ketooctanoic acid [M+K]+ 3-methylbutyrolactone [M+K]+ + -04 129.06562 dihydrothymine; pyrrole-2-carboxylic acid [M+NH ]+; 5.32E-04 + Decrease 2.4 197.11467 7-aminomethyl-7-carbaguanine [M+NH4] ; 3-hydroxynonanoic 2.78E + Increase 1.2 4 + + acid [M+Na] 2-acetyloxazole [M+NH4] 145.02603 benzoic acid [M+Na]+; 4-hydroxybenzaldehyde [M+Na]+ 2.89E-04 + Decrease 1.5 226.10469 L-acetylcarnitine [M+Na]+ 2.93E-04 + Decrease 2.1 146.04880 histamine [M+Cl]- 2.94E-04 - Decrease 3.3 174.03145 2-phenylacetamide [M+K]+ 2.99E-04 + Decrease 1.4 195.07800 4-hydroxynonenal [M+K]+ 3.02E-04 + Decrease 1.3 195.13545 capric acid [M+Na]+ 3.08E-04 + Increase 1.2 212.05271 glutarylglycine [M+Na]+; N-acetylglutamic acid [M+Na]+ 3.10E-04 + Increase 1.5 320.06266 beta-citryl-L-glutamic acid 3.40E-04 - Decrease 2.2 209.09362 cis/trans-4-decenoic acid [M+K]+; 8-methylnonenoate [M+K]+; 3.46E-04 + Decrease 1.4 5-decenoic acid [M+K]+; 2-exo-hydroxy-1,8-cineole [M+K]+; 2-hydroxycineol [M+K]+ + + -04 183.09907 7-methylguanine [M+NH4] ; 1/2/3-methylguanine [M+NH4] ; 3.58E + Increase 1.2 7-hydroxyoctanoic acid [M+Na]+; hydroxyoctanoic acid [M+Na]+; 3-hydroxyoctanoic acid [M+Na]+; (R)-2/3-hydroxycaprylic acid [M+Na]+ 190.06019 3-methylhistidine [M+Na-H]- 4.00E-04 - Decrease 1.7 190.99255 2/3-oxoglutaric acid [M+2Na-H]+ 4.25E-04 + Decrease 2.2 566.26449 LysoPE(0:0/22:5(4Z,7Z,10Z,13Z,16Z)) [M+K]+; 4.28E-04 + Decrease 1.8 LysoPE(0:0/22:5(7Z,10Z,13Z,16Z,19Z)) [M+K]+; LysoPE(22:5(4Z,7Z,10Z,13Z,16Z)/0:0) [M+K]+; LysoPE(22:5(7Z,10Z,13Z,16Z,19Z)/0:0) [M+K]+

112 113 Chapter 5 5

Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

Rúben J. Ramos, Paul J. Benke, Andrea Dieckmann, Dries Dobbelaere, Anne Fuchs, Guillaume Grolez, Peter M. Van Hasselt, Clara D.M. van Karnebeek, Karine Mention, Pasi I. Nevalainen, Cristina Skrypnyk, Judith J. Jans, Nanda M. Verhoeven-Duif

Submitted Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

ABSTRACT INTRODUCTION

De novo serine biosynthesis defects are genetic aminoacidopathies that lead to Serine is a non-essential amino acid that, in the human body, originates from four deficient levels of serine and glycine in blood, CSF and tissues. Classically, serine sources: 1) dietary intake; 2) de novo biosynthesis starting with the glycolytic intermediate deficient patients present with congenital microcephaly, severe psychomotor 3-phosphoglycerate; 3) synthesis by serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) retardation, intractable seizures and skin abnormalities. Diagnosing serine from glycine; and 4) protein and phospholipid turnover (de Koning et al 2003). De novo biosynthesis defects is challenging because subtle decreases of serine levels may be serine biosynthesis is an essential source of intracellular serine, as genetic defects in this missed during diagnostic work-up and the enzymatic activity tests are insufficiently pathway are not compensated by the other three sources (Van Der Crabben et al 2013; robust. The existence of secondary serine deficiencies contributes even further to the El-Hattab et al 2016). The de novo serine biosynthesis pathway starts with the conversion complexity of diagnosing serine biosynthesis defects. We developed a stable isotopic of 3-phosphoglycerate into 3-phosphohydroxypyruvate catalyzed by the enzyme liquid chromatography mass spectrometry-based method to analyse the formation 3-phosphoglycerate dehydrogenase (3-PGDH; EC 1.1.1.95). 3-Phosphohydroxypyruvate 13 13 of C3-serine from uniformly labelled C6-glucose in cultured fibroblasts. The results is further metabolized into 3-phosphoserine by 3-phosphoserine aminotransferase obtained show complete concordance with the genetic diagnosis of all included (PSAT; EC 2.6.1.52), after which 3-phosphoserine is dephosphorylated into serine by patients and also allowed the diagnosis of secondary serine biosynthesis defects. This 3-phosphoserine phosphatase (PSP; EC 3.1.3.3) (Figure 1). In humans, mutations have generic test assesses a complete metabolic pathway, potentially rendering separate been described in the three genes (PHGDH, PSAT1 and PSPH) encoding the de novo serine enzymatic assays unnecessary. biosynthesis enzymes. Clinical phenotypes of these defects overlap and are heterogeneous 5 (Jaeken et al 1996; Klomp et al 2000; Vincent et al 2015; Veiga-da-Cunha et al 2004; Hart et Keywords: de novo serine biosynthesis; serine biosynthesis defects; serine; glycine; al 2007; Tabatabaie et al 2009; Tabatabaie et al 2010; Shaheen et al 2014; Acuna-Hidalgo stable isotope liquid chromatography tandem mass spectrometry. et al 2014; Mattos et al 2015; Méneret et al 2012; El-Hattab et al 2016; Brassier et al 2016), ranging from the severe Neu-Laxova syndrome of multiple congenital abnormalities, intractable seizures, microcephaly and intellectual disability to milder expression of some or all of these findings, progressive polyneuropathy in adults(Acuna-Hidalgo et al 2014; El-Hattab et al 2016). The variability of phenotypes, as well as the amenability to treatment with oral L-serine and glycine, are thought to depend on the degree of residual enzyme activity (Acuna-Hidalgo et al 2014; Shaheen et al 2014; El-Hattab et al 2016).

The biochemical hallmark of serine deficiency is a decreased concentration of serine in cerebrospinal fluid (CSF) and a low to borderline concentration in plasma, with low to normal glycine concentrations (de Koning 2017). Biochemical abnormalities are more pronounced and consistent in CSF than in plasma as plasma serine and glycine concentrations are affected by meals and can be normal when patients are not fasting (Van Der Crabben et al 2013; de Koning 2017). Age-related reference values for serine in CSF and plasma are essential to detect serine deficiency (Van Der Crabben et al 2013). The diagnosis is confirmed by enzyme activity measurements and mutation analysis. While enzymatic assays for all enzymes involved in serine biosynthesis exist, they are not always conclusive (Hart et al 2007) and are complicated by problems in acquiring the substrate (3-phosphohydroxypyruvate) required for the 3-PGDH activity assay (de Koning 2017). Mutation analysis of all three genes may be warranted, but needs functional confirmation (El-Hattab et al 2016).

116 117 Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

Figure 1

13 C6-glucose treatable intellectual disability and epilepsy disorders (van Karnebeek et al. 2014; van Karnebeek et al. 2018). Therefore, a prompt and correct diagnosis is essential to optimize patient outcomes. Here we describe a method to analyse the flux through the pathway from glucose to serine and glycine in cultured skin-derived fibroblasts. This method provides a tool to detect the disorders of serine synthesis by functional 13C -glucose Cytosol 6 analysis of an entire metabolic pathway.

13C -glucose-6-P 6 RESULTS NAD+ NADH + H+ glutamate α-ketoglutarate

13C -3-P-glycerate 13C -3-P-hydroxypyruvate 13C -3-P-serine 13C -serine 13 3 3 3 3 In cells collected immediately after addition of C6-glucose-containing medium, 3-PGDH PSAT PSPH 13 13 THF C3-serine and C2-glycine were undetectable as expected. At t=0.5, 4 hours and 10 SHMT 13 13 hours, C3-serine and C2-glycine were detected in increasing amounts (Figure 2A).

13 5,10-mTHF C3-pyruvate As enrichment of serine at t=10 hours was most reliably detected (based on the 13C -glycine 2 lowest standard deviations (SD) of signal intensities between replicates) (Table 1), this time point was used to define the reference range of 0.028-0.042 (mean of 13C - 3 5 Figure 1. Schematic of the de novo serine biosynthesis pathway. De novo serine biosynthesis 13 serine/total serine +/- SD). C2-Glycine enrichment was detected at t=4 and t=10 is a side pathway of glycolysis. Firstly, 3-phosphoglycerate (3-P-glycerate) is oxidized to hours. However, enrichment was very low (low signal intensities for 13C -glycine and 3-phosphohydroxypyruvate (3-P-hydroxypyruvate), by 3-phosphoglycerate dehydrogenase (3- 2 PGDH). Next, phosphoserine aminotransferase (PSAT) transfers the amino group of glutamate to inconsistency between the biological replicates) making it hard to detect potentially 3-phosphohydroxypyruvate, generating 3-phosphoserine (3-P-serine) and α-ketoglutarate. Lastly, decreased values in the patients’ fibroblasts (Figure 2B). 3-P-serine is dephosphorylated to serine by phosphoserine phosphatase (PSPH). Additionally, serine hydroxymethyltransferase (SHMT) catalyses the transfer of serine’s amino group to tetrahydrofolate 13 Table 1. De novo C3-serine production at t=10 hours in control fibroblasts (THF) generating glycine and 5,10-methylenetetrahydrofolate (5,10-mTHF). 13 C3-serine/total serine (n=3) Average Standard deviation Coefficient of Variation (SD) (CV %) The difficulty in diagnosing serine biosynthesis defects is augmented by the existence Control 1 day 1 0,035 0,002 6,9 of secondary serine deficiencies. Low serine and glycine concentrations have been day 2 0,037 0,008 21,1 reported in CSF and plasma of patients with severe encephalopathy associated with Control 2 day 1 0,036 0,009 25,5 Control 3 day 1 0,034 0,005 15,5 viral infection, brain edema and multiple organ failure (Keularts et al 2010; Surtees Control 4 day 1 0,035 0,004 12,0 ; et al 1997 Van Der Crabben et al 2013). Additionally, inborn errors of metabolism Control 5 day 1 0,044 0,004 9,5 that relate to serine metabolism, including homocystinuria and disorders of folate day 2 0,035 0,008 22,5 metabolism, have been reported to alter serine concentrations in CSF and plasma Control 6 day 1 0,026 0,004 16,1 (Surtees et al 1997). Low concentrations of serine in CSF and plasma have also been observed in patients with severe respiratory chain defects, most likely because of abnormal redox state, leading to decreased NAD+ levels, the cofactor of 3-PGDH (van Karnebeek et al 2019; Van Der Crabben et al 2013).

In contrast to catabolic defects that ultimately lead to strong increases of specific amino acids, anabolic aminoacidopathies can be missed due to their subtle biochemical alterations. Serine defects are part of the expanding group of rare

118 119 Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

Figure 2

A B 13 In cells from the 3-PGDH-deficient patient only 16% of C3-serine was formed as 13 13 de novo C3-serine synthesis de novo C2-glycine synthesis 13 compared to the controls. C3-serine was not detectable in the two PSAT deficient 0.048 0.04 patients, in line with their severe clinical presentations (severe psychomotor retardation,

0.036 0.03 intractable seizures and spasticity). Cells from the patient with a secondary defect in serine synthesis due to mitochondrial glutamate oxaloacetate transaminase (GOT2) 0.024 0.02 13 deficiency showed a C3-serine formation of 43% of controls. In fibroblasts from the 13

-serine/total serine 3-PGDH-carrier, C -serine formation was in the control range (118%).

3 0.012 0.01 -glycine/total glycine 3 2 C C 13 13 Cells from the two patients with decreased concentrations of serine in blood and/or 0 2 4 6 8 10 0 2 4 6 8 10 13 time (hours) time (hours) CSF, but without a genetically confirmed diagnosis, formed normal amounts of C3- serine (111% and 158%, respectively) (Figure 3). Control 1 (n=6) Control 2 (n=3) Control 3 (n=3) Control 4 (n=3) Control 5 (n=6) Control 6 (n=3)

13 13 13 DISCUSSION Figure 2. De novo C3-serine and C2-glycine formation in control fibroblasts. A. C3-serine formation 13 over time shows reliable detection and low standard deviations (SD) at t=10 hours. B. C2-glycine formation over time is low, being only reliably detected at t=10 hours. Results are represented as the The complexity of de novo serine biosynthesis defects, both clinical and biochemical, 5 mean of n=3 replicates (Controls 2, 3, 4 and 6) and n=6 replicates (Controls 1 and 5) ± SD. and the unsatisfactory results obtained with the current diagnostic tools led us to Figure 3 develop a stable isotope-based LC-MS/MS method for serine synthesis in skin-derived 0.060 fibroblasts. This approach is conceptually different from currently used assays since it

0.045 examines the efficacy of a complete pathway, instead of individual enzymes.

0.030 Cells from the 3-PGDH-deficient patient with residual enzyme activity, as analysed by a specific 3-PGDH assay, had strongly decreased serine synthesis (16% of controls). It

-serine/total serine 0.015 3

C is known that most 3-PGDH-deficient patients have considerably low enzyme activity, 13 ranging between 12 and 25% of the lower range of reference values (Van Der Crabben 0 2 4 6 8 10 time (hours) et al 2013; Tabatabaie et al 2009). In this study we also included cells from a patient with low 3-PGDH activity in fibroblasts but with inconclusive mutation analysis Controls 3-PGDH-carrier (only a single variant of uncertain significance, VUS). These cells had a normal serine PSAT-deficient(i) GOT2-deficient synthesis (118%), ruling out a serine biosynthesis defect and further demonstrating PSAT-deficient(ii) Unknown(i) 3-PGDH-deficient Unknown(ii) that the flux method can be more informative than the 3-PGDH enzyme test. The two 13 13 PSAT-deficient patients had no detectable synthesis of C3-serine from C6-glucose. The first patient has a homozygous mutation c.296_297delinsTG (p.Ala99Val). The Figure 3. De novo 13C -serine biosynthesis in fibroblasts of controls and patients.13 C -serine was 3 3 p.Ala99Val variant has already been described in the literature (Acuna-Hidalgo undetectable on the fibroblasts of both patients (i and ii) with phosphoserine aminotransferase (PSAT) deficiency. 3-Phosphoglycerate dehydrogenase (3-PGDH) deficient fibroblasts formed only et al 2014; El-Hattab et al 2016) although due to a missense mutation (c.296C>T). 13 16% of C3-serine, while the patient with mitochondrial glutamate oxaloacetate transaminase The second PSAT-deficient patient had compound heterozygous variants, with a (GOT2) deficiency formed 43% of13 C -serine, when compared to controls. The fibroblasts of the two 3 c.296_297delinsTG mutation and a VUS (Table 2). Our method demonstrates that this 13 patients with unknown serine deficiency and the 3-PGDH carrier produced C3-serine in the range of the controls. Results are represented as the mean of n=24 replicates (control fibroblasts) and n=3 latter variant should be classified as pathogenic. replicates (patients fibroblasts) ± SD.

120 121 Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

Our method is more sensitive than enzymatic methods and serves as a screen for the de Table 2. Clinical, enzymatic, molecular and biochemical information of the enrolled patients novo serine biosynthesis pathway. Although enzymatic assays for all the proteins involved Diagnosis Age Sex 3-PGDH Mutation analysis Serine Serine in serine biosynthesis exist, their results may not be reliable (Hart et al 2007). Inconclusive (yrs) activity* enrichment formation enzyme activity results were reported in a PSAT-deficient patient with low PSAT activity (mean + SD) (%) Compound heterozygous variants in PHGDH: (≈50% of controls), but not sufficiently low to allow definite diagnosis of a deficiency 3-PGDH-def. uk F 10 0.000-0.012 16 c.138+2dup p.? and c.1129G>A (p.Gly377Ser). disorder (Hart et al 2007). An additional problem of the 3-PGDH assay is the difficulty in Heterozygous variant in PHGDH: c.355A>T (p.Arg119Trp) 3-PGDH-carrier 10 M 15 0.038-0.045 118 acquiring the substrate, 3-phosphohydroxypyruvate, required for the enzyme assay (de (VUS). Homozygous variants in PSAT1: c.296_297delinsTG Koning 2017). A drawback of the flux method is its inability to pinpoint the exact enzyme PSAT(i)-def. 15 M 51 0 0 (p.Ala99Val). defect in cases of low serine synthesis. When a serine biosynthesis defect is biochemically Compound heterozygous variants in PSAT1: confirmed by a reduced flux, the genetic analysis of PHGDH, PSAT1 and PSPH genes c.296_297delinsTG (p.Ala99Val) and c.698A>C PSAT(ii)-def. 8 F 57 0 0 (p.Gln233Pro) (VUS). should be considered. A second drawback is the inability of using glycine enrichment 13 No mutations in PHGDH and PSPH genes as a diagnostic biomarker since the signal intensity of newly formed C2-glycine is very Compound heterozygous variants in GOT2: low and inconsistently found in our experimental conditions. This makes it hard to detect GOT2-def. 9 M np c.617_619delTCT (p.Leu207del) and c.1009C>G 0.012-0.018 43 (p.Arg337Gly) potentially decreased glycine values in the fibroblasts of the patients. Unknown(i) 59 F 30 No mutations in PHGDH, PSAT1 and PSPH genes 0.020-0.059 111 Unknown(ii) 50 M np No mutations in PHGDH, PSAT1 and PSPH genes 0.052-0.060 158 Here we describe a robust and sensitive method for diagnosing de novo serine 5 biosynthesis defects. This method can be used to examine serine synthesis pathway VUS, variants of uncertain clinical significance; M, male; F, female; uk, unknown; np, not performed. *3-PGDH activity reference range: 20-75 nmol/min.mg protein. when amino acid test results are abnormal, or Exome test results suggestive. This approach is an example of a more generic test of a complete metabolic pathway to The two adult patients with unexplained low serine concentrations in CSF had a validate biochemical or next generation sequencing findings, circumventing the proficient de novo serine biosynthesis. The low serine levels reported in these two need to develop separate assays for the individual enzymes of the pathway. patients were likely due to other causes than genetic metabolic disturbances in the de novo serine pathway. MATERIAL AND METHODS Our method allows not only the diagnosis of primary serine defects, but also secondary defects, as observed by the reduced serine production in the GOT2- Patients deficient patient. In this case, an increased cytosolic NADH/NAD+ ratio due to a defect Seven patients with low serine concentrations in CSF and/or plasma and six controls in the malate-aspartate shuttle explains the observed serine deficiency. Because were included in this study (Table 2). The cohort included one 3-PGDH-deficient 3-PGDH is a NAD+-dependent enzyme, accumulation of NADH hampers 3-PGDH patient, one patient with a low serine in CSF and heterozygous mutation in 3-PGDH, activity and consequently reduces the production of serine. It is important to note two PSAT-deficient patients, one mitochondrial glutamate oxaloacetate transaminase that glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12), a glycolytic (GOT2)-deficient patient, and two patients with yet unexplained low concentrations enzyme upstream of 3-phosphoglycerate formation, is also NAD+-dependent. The of serine in CSF. Patients’ details are presented in table 2. All diagnoses were confirmed increased NADH/NAD+ ratio may also hamper this enzyme activity, contributing to by mutation analyses. less 3-phosphoglycerate formation and consequently less serine synthesis. These findings confirm the hypothesis that serine synthesis is influenced by the cytosolic Cell Culture NADH/NAD+ ratio, and explains the serine deficiency in a patient with severe Dulbecco’s modified eagle medium (DMEM), high glucose, GlutaMAX™, pyruvate respiratory chain defect (Van Der Crabben et al 2013) (Cat. No. 31966); DMEM-no glucose (Cat. No. 11966); fetal bovine serum (FBS; Cat. No. 10270); penicillin-streptomycin (P/S (10,000 U/mL); Cat. No. 15140) and trypsin- ethylenediaminetetraacetic acid (trypsin-EDTA (0.5%), no phenol red; Cat. No. 15400)

122 123 Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

TM 13 were purchased from Gibco (ThermoFisher Scientific). Uniformly labelled C6- Table 3. Mass spectrometry (MS/MS) parameters used for the analysis of the amino acid standards glucose (99%) was purchased from Cambridge Isotope Laboratories, Inc. (MA, USA). Amino Acid RT Concentration Cone Voltage Collision Energy MRM-transition Glucose was purchased from Sigma-Aldrich (Steinheim, Germany). (min) (µM) (V) (V) m/z Serine 8.5 1000 15 10 106.0 → 60.0 1,2,3-13C -serine 8.5 400 15 10 109.0 → 61.9 2 3 Fibroblasts were grown in 75 cm flasks and maintained in DMEM, high glucose, Glycine 7.8 2000 15 6 76.0 → 30.1 GlutaMAXTM, pyruvate (with 10% heat-inactivated FBS and 1% P/S), in a humidified 13 1,2- C2-glycine 7.8 600 15 6 78.0 → 31.1 atmosphere of 5% CO2 at 37ºC. Cells were passaged upon reaching confluence and RT, retention time; MRM, multiple reaction monitoring medium was refreshed every 48 hours.

Stable isotope analysis: de novo serine biosynthesis Statistical analysis Cells were plated on 6-well plates (400.000 cells per well), and allowed to grow for 7 Statistical significance was determined with unpaired two-tailed t-test, using days. Medium was refreshed at days 2, 4 and 6. On the 7th day, cells were incubated GraphPad Prism 6 (version 6.0.2, GraphPad Software Inc.) software. with DMEM medium without glucose (DMEM, Cat. No.11966; supplemented with 13 10% FBS and 1% P/S), to which 25 mmol/L uniformly labelled C6-glucose was added. Cells were harvested at t=0, 0.5, 4 and 10 hours. Before collection, cells were Compliance with Ethics Guidelines washed twice with cold PBS (4ºC) and harvested by scraping with 1.5 ml ice-cold 5 methanol. Samples, in biological triplicates, were collected in 1.5 ml eppendorf Conflict of interest tubes, centrifuged (16,200 g for 10 min at 4ºC), and the supernatants were transferred Rúben Ramos, Paul Benke, Andrea Dieckmann, Dries Dobbelaere, Anne Fuchs, to new 1.5 ml eppendorf tubes. Samples were concentrated by evaporation at 40ºC Guillaume Grolez, Peter van Hasselt, Clara van Karnebeek, Karine Mention, Pasi I. under a nitrogen stream until complete dryness and reconstituted with 500 µl of Nevalainen, Cristina Skrypnyk, Judith Jans and Nanda Verhoeven-Duif declare that UPLC-grade methanol (room temperature). The reconstituted samples were stored at they have no conflicts of interest. -80ºC until amino acid analysis was performed. Informed Consent Amino acid analysis All procedures followed were in accordance with the ethical standards of the Serine and glycine were purchased from Sigma-Aldrich (Zwijndrecht, the Netherlands). responsible committee of the University Medical Centre Utrecht and with the Helsinki 13 13 1,2,3,- C3-serine and 1,2- C2-glycine were purchased from Cambridge Isotope Declaration of 1975, as revised in 2000. Laboratories (Massachusetts, USA). UPLC-grade acetonitrile (ACN) and methanol were purchased from Biosolve (Valkenswaard, the Netherlands). To determine the Author Contributions intracellular synthesis of serine and glycine, we adapted the LC-MS/MS method Rúben Ramos, Judith Jans and Nanda Verhoeven-Duif designed the project and wrote described by Prinsen (Prinsen et al 2016) to whole cell lysates. Biological triplicates for the manuscript. Rúben Ramos optimized and performed the LC-MS/MS analysis. 13 each time point were analysed, and the ratios of C3-serine/total serine (total serine Rúben Ramos performed the statistical analysis. Paul Benke, Andrea Dieckmann, Dries 13 12 13 13 = C3-serine + C3-serine) and C2-glycine/total glycine (total glycine = C2-glycine + Dobbelaere, Anne Fuchs, Guillaume Grolez, Peter van Hasselt, Clara van Karnebeek, Karine 12 C2-glycine) were calculated to determine de novo biosynthesis of serine and glycine. Mention, Pasi Nevalainen and Cristina Skrypnyk observed, collected and diagnosed the 13 Internal standards were not used to avoid interference with the signal of C3-serine patients. All authors critically discussed the results and revised the manuscript. 13 and C2-glycine. The mass spectrometry (MS/MS) parameters used are described in table 3. To validate each MS/MS analysis, a set of quality control (QC) samples was used. Besides adapting the range of the calibrators to our samples’ concentrations, and using QC samples that resembled the signal intensities of our samples, no further adaptations were needed for sample preparation or analysis of the amino acids.

124 125 Serine biosynthesis flux as diagnostic tool for serine biosynthesis defects

ACKNOWLEDGMENTS Shaheen R, Rahbeeni Z, Alhashem A, et al (2014) Neu-laxova syndrome, an inborn error of serine metabolism, is caused by mutations in PHGDH. Am J Hum Genet 94:898–904. doi: 10.1016/j. ajhg.2014.04.015 We are grateful to the patients and families for their participation in this study, and to Surtees R, Bowron A, Leonard J (1997) Cerebrospinal fluid and plasma total homocysteine and the clinical and laboratory specialists for their collaborations. related metabolites in children with cystathionine beta-synthase deficiency: the effect of treatment. Pediatr Res 42:577–82. doi: 10.1203/00006450-199711000-00004 This work was funded by Metakids foundation (www.metakids.nl). Tabatabaie L, De Koning TJ, Geboers AJJM, et al (2009) Novel mutations in 3-phosphoglycerate dehydrogenase (PHGDH) are distributed throughout the protein and result in altered . Hum Mutat 30:749–756. doi: 10.1002/humu.20934 Tabatabaie L, Klomp LW, Berger R, de Koning TJ (2010) l-Serine synthesis in the central nervous REFERENCES system: A review on serine deficiency disorders. Mol Genet Metab 99:256–262. doi: 10.1016/j. ymgme.2009.10.012 Van Der Crabben SN, Verhoeven-Duif NM, Brilstra EH, et al (2013) An update on serine deficiency Acuna-Hidalgo R, Schanze D, Kariminejad A, et al (2014) Neu-laxova syndrome is a heterogeneous disorders. J Inherit Metab Dis 36:613–619. doi: 10.1007/s10545-013-9592-4 metabolic disorder caused by defects in enzymes of the l-serine biosynthesis pathway. Am J Van Karnebeek CDM, Ramos RJ, Wen X-Y, et al (2019) Biallelic GOT2 mutations cause a treatable Hum Genet 95:285–293. doi: 10.1016/j.ajhg.2014.07.012 malate-aspartate shuttle related encephalopathy. Am J Hum Genet 105(3):534-548 Brassier A, Valayannopoulos V, Bahi-Buisson N, et al (2016) Two new cases of serine deficiency van Karnebeek CDM, Sayson B, Lee JJY, et al (2018) Metabolic Evaluation of Epilepsy: A Diagnostic disorders treated with l-serine. Eur J Paediatr Neurol 20:53–60. doi: 10.1016/j.ejpn.2015.10.007 Algorithm With Focus on Treatable Conditions. Front Neurol 9:. doi: 10.3389/fneur.2018.01016 de Koning TJ De, Klomp LWJ, Oppen ACC Van, et al (2004) Prenatal and early postnatal treatment van Karnebeek CDM, Shevell M, Zschocke J, et al (2014) The metabolic evaluation of the child with an in 3-phosphoglycerate-dehydrogenase deficiency. Lancet 364:2221–2222. doi: 10.1016/S0140- intellectual developmental disorder: Diagnostic algorithm for identification of treatable causes 6736(04)17596-X and new digital resource. Mol Genet Metab 111:428–438. doi: 10.1016/j.ymgme.2014.01.011 5 de Koning TJ (2017) deficiencies. J Inherit Metab Dis 40:609–620. doi: 10.1007/ Veiga-da-Cunha M, Collet JF, Prieur B, et al (2004) Mutations responsible for 3-phosphoserine s10545-017-0063-1 phosphatase deficiency. Eur J Hum Genet 12:163–166. doi: 10.1038/sj.ejhg.5201083 de Koning TJ, Duran M, Van Maldergem L, et al (2002) Congenital microcephaly and seizures due Vincent JB, Jamil T, Rafiq MA, et al (2015) Phosphoserine phosphatase (PSPH) gene mutation in an to 3-phosphoglycerate dehydrogenase deficiency: Outcome of treatment with amino acids. J intellectual disability family from Pakistan. Clin. Genet. 87:296–298 Inherit Metab Dis 25:119–125 de Koning TJ, Snell K, Duran M, et al (2003) L-serine in disease and development. Biochem J 371:653–61. doi: 10.1042/BJ20021785 El-Hattab AW, Shaheen R, Hertecant J, et al (2016) On the phenotypic spectrum of serine biosynthesis defects. J Inherit Metab Dis 39:373–381. doi: 10.1007/s10545-016-9921-5 Hart CE, Race V, Achouri Y, et al (2007) Phosphoserine Aminotransferase Deficiency: A Novel Disorder of the Serine Biosynthesis Pathway. Am J Hum Genet 80:931–937. doi: 10.1086/517888 Jaeken J, Detheux M, Maldergem L Van, et al (1996) 3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis. Arch Dis Child 74:542–545 Keularts IMLW, Leroy PLJM, Rubio-Gozalbo EM, et al (2010) Fatal cerebral edema associated with serine deficiency in CSF. J Inherit Metab Dis 33:0–4. doi: 10.1007/s10545-010-9067-9 Klomp LWJ, De Koning TJ, Malingré HEM, et al (2000) Molecular Characterization of 3-Phosphoglycerate Dehydrogenase Deficiency—a Neurometabolic Disorder Associated with Reduced L-Serine Biosynthesis. Am J Hum Genet 67:1389–1399. doi: 10.1086/316886 Mattos EP, da Silva AA, Magalhães JAA, et al (2015) Identification of a premature stop codon mutation in the PHGDH gene in severe Neu-Laxova syndrome-evidence for phenotypic variability. Am J Med Genet Part A 167:1323–1329. doi: 10.1002/ajmg.a.36930 Méneret A, Wiame E, Marelli C, et al (2012) A serine synthesis defect presenting with a Charcot- Marie-Tooth-like polyneuropathy. Arch Neurol 69:908–911. doi: 10.1001/archneurol.2011.1526 Prinsen HCMT, Schiebergen-Bronkhorst BGM, Roeleveld MW, et al (2016) Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry. J Inherit Metab Dis 39:651–660. doi: 10.1007/s10545- 016-9935-z

126 127 Chapter 6 6 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Rúben J. Ramos, Monique Albersen, Esmee Vringer, Marjolein Bosma, Susan Zwakenberg, Fried Zwartkruis, Judith J. M. Jans, Nanda M. Verhoeven-Duif

Biochim Biophys Acta Gen Subj. 2019 Jun;1863(6):1088-1097. Doi: 10.1016/j. bbagen.2019.03.019 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

ABSTRACT INTRODUCTION

Background: Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6. Mammals Vitamin B6 is present in the human body as six interconvertible vitamers: pyridoxal (PL; cannot synthesize vitamin B6, so they rely on dietary uptake of the different B6 forms, aldehyde group at C4’), pyridoxine (PN; alcohol group at C4’), pyridoxamine (PM; amine and via the B6 salvage pathway they interconvert them into PLP. Humans possess group at C4’), and their 5’-phosphate esters pyridoxal 5’-phosphate (PLP), pyridoxine three enzymes in this pathway: pyridoxal kinase, pyridox(am)ine phosphate oxidase 5’-phosphate (PNP) and pyridoxamine 5’-phosphate (PMP) (Snell 1953) (Figure 1). and pyridoxal phosphatase. Besides these, a fourth enzyme has been described in Vitamin B6 is catabolized to 4-pyridoxic acid (PA) which is excreted in urine (Hufft and plants and yeast but not in humans: pyridoxal reductase. Perlzeig, 1944). PLP, the metabolically active form of vitamin B6, is an essential cofactor in human metabolism. A total of 56 PLP-dependent enzymes are currently known to Methods: We analysed B6 vitamers in remnant CSF samples of PLP-treated patients exist in humans according to the B6 database (Percudani and Peracchi, 2009). Most and four mammalian cell lines (HepG2, Caco2, HEK293 and Neuro-2a) supplemented PLP-dependent reactions involve the metabolism of amino acids, neurotransmitters with PL as the sole source of vitamin B6. (such as γ-aminobutyric acid, dopamine, serotonin, epinephrine and norepinephrine), nucleic acids and carbohydrates (Ueland et al., 2015). 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 Although all organisms depend on vitamin B6 to survive, only microorganisms show that all cell lines reduce PL to PN in a time- and dose-dependent manner. We and plants can synthesize it de novo (Fitzpatrick et al., 2007). Mammals acquire compared the amino acid sequences of known PL reductases to human sequences B6 vitamers from the diet and convert them to PLP, using the vitamin B6 salvage and found high homology for members of the voltage-gated potassium channel pathway (Di Salvo et al., 2011) (Figure 1). Vitamin B6 enters the cells after hydrolysis beta subunits and the human aldose reductases. Pharmacological inhibition and of the phosphorylated forms by the membrane-bound tissue non-specific alkaline knockout of these proteins show that none of the candidates is solely responsible for phosphatase (TNSALP; EC 3.1.3.1) (Waymire et al., 1995). Once inside the cells, PL kinase 6 PL reduction to PN. (PDXK; EC 2.7.1.35) phosphorylates the hydroxymethyl group of PL, PN and PM to their respective 5’-phosphate forms (Hanna et al., 1997). Pyridox(am)ine phosphate oxidase Conclusions: We show evidence for the presence of PL reductase activity in humans. (PNPO; EC 1.4.3.5) catalyses the oxidation of PNP and PMP to PLP (Mills et al., 2005). Further studies are needed to identify the responsible protein. Dephosphorylation of PLP, PNP and PMP, as catalysed by PL phosphatase (PDXP; EC 3.1.3.74), is one of the mechanisms that cells have to control the amount of intracellular General significance: This study expands the number of enzymes with a role in B6 PLP concentrations (Jang et al., 2003). An additional enzyme with a role in the B6 salvage salvage pathway. We hypothesize a protective role of PL reductase(s) by limiting the pathway has been reported in yeast and plants, but never in humans. This enzyme, PL intracellular amount of free PL and PLP. reductase (EC 1.1.1.65), is a member of the aldo-keto reductase (AKR) family (Nakano et al., 1999) and catalyses the reduction of PL to PN, simultaneously oxidizing NADPH to Keywords: Vitamin B6 vitamers, pyridoxal reductase, vitamin B6 salvage pathway, NADP+ (Guirard and Snell, 1988). PL reductase activity was first reported in the budding KCNAB2, AKR1B1 and AKR1B10. yeast Saccharomyces cerevisiae. Guirard and Snell purified the enzyme and showed that between a pH of 6.3 and 7.1 (the intracellular pH of S. cerevisiae) its equilibrium lies towards PN formation. In addition, the authors proposed that formation of PLP followed the route: PL → PN → PNP → PLP, since PN was the preferred substrate for PL kinase in this yeast (Guirard and Snell, 1988).

130 131 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Figure 1 Extracellular Cytosol In 1998, Yagi et al reported that the fission yeast Schizosaccharomyces pombe Pyridoxine 5’-phosphate accumulated PN intracellularly not only when exposed to PN, but also during

HO H2 C O OH incubation with PL (Yagi et al., 1998). The same group subsequently purified the PL HO P reductase of S. pombe and characterized its catalytic activity (Nakano et al., 1999). O OH + H3C N ALP Several years later, plr1 was identified as the PL reductase-encoding gene in S. pombe (Morita et al., 2004), but the existence of other enzyme(s) with PL reductase Pyridoxine Pyridoxine Pyridoxine 5’-phosphate HO H + 2 HO H2 Pyridoxal kinase HO H2 activity was also proposed since strains of S. pombe with deleted plr1 still produced C C C O OH HO HO HO P low amounts of PN when incubated with PL (Morita et al., 2004). OH OH Pyridoxal O OH H C N phosphatase 3 H3C N H3C N The presence of proteins with PL reductase activity has also been reported in plants. AtPLR1 was shown to catalyse the reduction of PL to PN in Arabidopsis thaliana

Pyridoxal (Herrero et al., 2011). AtPLR1 knockdown lines were shown to increase the expression Pyridoxal 5’-phosphate PNPO H O Reductase C O OH of two other salvage pathway genes (PDX3, encoding the PL (PN, PM) kinase and HO P SOS4, encoding PNP(PMP) oxidase), while little to no changes were observed in the O OH expression of the de novo pathway genes (PDX1.1, PDAX1.2, PDX1.3 and PDX2). In H3C N ALP addition, AtPLR1 was significantly up-regulated when PDX3 and SOS4 were knocked Pyridoxal Pyridoxal Pyridoxal 5’-phosphate H O H O Pyridoxal kinase H O down, leading to the conclusion that the AtPLR1-protein plays a role in the vitamin C C C O OH HO HO HO P B6 salvage pathway (Herrero et al., 2011). OH OH Pyridoxal O OH

H3C N H3C N phosphatase H3C N In this study, we provide evidence for the presence of protein(s) with PL reductase 6 activity in humans. Analysis of B6 vitamers in cerebrospinal fluid (CSF) samples of PLP- Pyridoxamine 5’-phosphate treated patients revealed surprisingly high concentrations of PN, thus suggesting the H N H 2 2 Aminotransferases PNPO C O OH existence of a PN-forming enzyme. In addition, using in vitro studies, we discovered HO P a cell-type-, time- and dose-dependent behaviour of PL reductase activity in various O OH

H3C N ALP mammalian cell lines. Further studies are needed to identify the protein responsible

Pyridoxamine Pyridoxamine Pyridoxamine 5’-phosphate for PL reductase activity in humans. Knockout models for several genes encoding H N H H N H H N H 2 2 2 2 Pyridoxal kinase 2 2 the potential human PL reductase and pharmacological inhibition studies of these C C C O OH HO HO HO P candidates unfortunately did not allow for the discovery of the responsible enzyme. OH OH Pyridoxal O OH phosphatase H3C N H3C N H3C N MATERIAL AND METHODS Figure 1. The human vitamin B6 metabolic pathway. Membrane-bound alkaline phosphatase (ALP) dephosphorylates circulating PLP, PMP and PNP to their corresponding unphosphorylated forms (PL, PM and PN), in order to cross the cellular membrane. Inside the cells, PL kinase phosphorylates Cell culture the hydroxymethyl group of PL, PN and PM into their respective 5’-phosphate forms. Pyridox(am)ine General steps phosphate oxidase (PNPO) catalyses the oxidation of PNP and PMP to PLP. Dephosphorylation of PLP, HepG2, Neuro-2a, Caco2 and HEK293 cells were purchased from the ATCC Cell Biology PNP and PMP is catalysed by PL phosphatase. Aminotransferases use PLP during the interchange of Collection. Dulbecco’s modified eagle medium (DMEM) GlutaMAX™ (31966), vitamin the amino group between one amino acid and an α-keto acid, producing PMP as an intermediary in the first part of the reaction. The dashed arrow represents the place that PL reductase may occupy in B6-free DMEM GlutaMAX™ (custom made 31966-like), fetal bovine serum (FBS; vitamin B6 metabolism. 10270), penicillin-streptomycin (P/S; 15140) and trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA, 0.5%) were purchased from Gibco (Invitrogen Life Technologies).

132 133 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Pyridoxal hydrochloride (PL-HCl) was purchased from Sigma-Aldrich (Steinheim, Colonies were picked from these plates after 1 week and expanded. To confirm Germany). Cells were grown in 75 cm2 flasks and maintained in DMEM GlutaMAX™ KCNAB2 absence, genomic DNA from candidate clones was isolated using the (supplemented with 10% heat-inactivated FBS (FBS-HI) and 1% P/S), in a humidified QIAamp DNA Micro kit (QIAGEN). The targeted genomic region was amplified by PCR atmosphere of 5% CO2 at 37ºC. When cells reached optimal confluence (>80%) they using the forward primer 5′-CTGAGCACCGACGGGATAAT-3′ and the reverse primer were washed twice with room temperature (RT) PBS and plated in 96-well plates by 5′-GCTGCATTCCCAATGACCAA-3’. PCR fragments were sequenced and analyzed by trypsinization with 0.05% trypsin-EDTA. TIDE (https://tide.nki.nl/). If TIDE predicted PCR products to only contain indels resulting in reading frame shifts, PCR fragments were cloned into pJET (Thermo Evaluation of PL reduction to PN Scientific). Multiple pJET clones were sequenced to identify the exact nucleotide Cells were grown in 96-well plates and kept in DMEM GlutaMAX™ (supplemented with sequence of targeted alleles. In most cases, sequencing results were consistent with

10% FBS-HI and 1% P/S), in a humidified atmosphere of 5% CO2 at 37ºC. Media were TIDE predictions. HepG2 clones with out of frame alleles only were considered to refreshed with DMEM GlutaMAXTM (suppl. with 10% FBS-HI and 1% P/S) 24 h before be mutant. For AKR1B10 the same approach was used but the genomic was exposure to different PL concentrations. On the day of exposure, confluent wells were amplified with the forward primer: 5′-TCCCTTGGGGTTATTTAGAG-3′, and reverse pre-incubated for 1 hour with vitamin B6-free DMEM GlutaMAX™ (with 10% FBS-HI primer: 5′-AGAGTTCTTGCTGCCAACC-3’. Absence of AKR1B1 was demonstrated by and 1% P/S). Vitamin B6-free DMEM GlutaMAX™ medium with FBS-HI was analysed Western blot analysis following standard molecular biology procedures, using a and the contribution to the B6 vitamers concentrations proved to be minimal: PL rabbit anti-AKR1B1 polyclonal antibody (Abcam cat. Ab62795; diluted 1:5000); mouse 10 nM; PN 5 nM; PM 2 nM; while no phosphorylated vitamers (PLP, PNP and PMP) monoclonal anti-GAPDH (EMD Millipore cat. MAB374; diluted 1:5000) and anti-RaIA were detectable. The pre-incubation step was introduced to remove intracellular PN (BD transduction cat. 610222; diluted 1:5000) antibodies were used as loading from the cells. After pre-incubation, cells were exposed to different experimental controls. Coding sequences of KCNAB2, AKR1B1 and AKR1B10 and protein sequences conditions: cells were either exposed to vitamin B6-free DMEM GlutaMAX™ (with 10% are provided in Supplementary figure 1. FBS-HI and 1% P/S) without PL (or any other source of vitamin B6) or to medium 6 supplemented with PL (0.1, 100 and 1000 µmol/L), in a humidified atmosphere of 5% Pharmacological inhibition of PL reductase activity

CO2 at 37ºC. Triplicates of each condition were collected at t=4, 24 and 48 hours after 3,4-Dihydroxyphenylacetic acid (DOPAC), rutin, resveratrol, zopolrestat, tolrestat exposure. Media of the four PL conditions (0, 0.1, 100 and 1000 µmol/L), kept at 37ºC and oleanolic acid were purchased from Sigma-Aldrich (Steinheim, Germany). Stock in the absence of cells, were collected at the same time points. In the absence of cells, solutions (100 mmol/L) of each inhibitor were prepared in 1% DMSO. HepG2 cells no spontaneous PN formation was observed under the experimental conditions. The were grown in 96-well plates. When optimal confluence was reached, each well was residual content of PN (derived from the 10% FBS-HI supplement and PL-supplement washed twice with RT PBS and pre-incubated for 1 hour with vitamin B6-free DMEM impurity) was subtracted to PN secreted by cells. GlutaMAX™ (with 10% FBS-HI and 1% P/S). After pre-incubation, cells were incubated for 20 minutes with vitamin B6-free medium and 100 µmol/L of each inhibitor (DOPAC, Generation of knockout clones by CRISPR-Cas9 in HepG2 cells rutin, resveratrol, zopolrestat, tolrestat and oleanolic acid). Subsequently, cells were HepG2 cells were transiently transfected with pSpCas9(BB)-2A-GFP (PX458) incubated with medium containing equimolar concentrations (100 µmol/L) of each (Ran et al., 2006) encoding sgRNAs targeting KCNAB2, AKR1B1 or AKR1B10. inhibitor and of PL. Medium, in triplicate, was collected at t=15 and 30 minutes sgRNAs were designed with http://crispor.tefor.net/ (Haeussler et al., 2016) and after exposure to the experimental conditions. The amount of PN present in these target coding sequences just upstream of a series of conserved residues, which media was subtracted from the residual content of PN (derived from the 10% FBS-HI in KCNAB2 have been defined as catalytic residues by Weng et al (Weng et al., supplement and PL-supplement impurity). 2006) (see also figure 3). For KCNAB2 the following sgRNAs were used: sgRNA2: ATCTTACCTCATCGGTGATC, sgRNA3: ATCGGTGATCTGGCCTCCGA or sgRNA4: AGACTTCTGCTGTATCGAAG; for AKR1B1 sgRNA2: GTGAAGGTGGCCATTGACGT and for AKR1B10 sgRNA1: GTGGCCATTGATGCAGGATAT. In all cases GFP-positive cells were sorted using a FACSAria II flow cytometer (BD) and plated in 10 cm dishes.

134 135 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Figure 2

Vitamin B6 vitamer analysis in CSF samples of PLP-treated patients HEK293 Neuro-2a and medium samples of the different mammalian cell lines 1.2 3

Vitamin B6 vitamers were quantified in remnant CSF samples of two PLP-treated 0.9 2.25 patients according to the UPLC-MS/MS method described by van der Ham et al (van der Ham et al., 2012). The medium samples from our in vitro studies were analysed 0.6 1.5 using the same method. Apart from adapting the range of the calibrators to the 0.3 0.75 samples’ concentrations, and dilution of the medium samples (1:10 and 1:100) with TCA, no further adaptations were needed for sample preparation or vitamer analysis. 4 24 48 4 24 48 µ mol/L) Caco 2 HepG2 Statistical analysis 6 240 Unpaired two-tailed t-tests were performed using GraphPad Prism 6 (version 6.0.2,

GraphPad Software Inc.) software. Pyridoxine ( 4.5 180

3 120

RESULTS 1.5 60

Pyridoxine accumulates in cerebrospinal fluid of children on PLP 4 24 48 4 24 48 treatment Time (hours) B6 vitamers were analysed in CSF samples of two children treated with PLP (Table 1). Legend: In addition to PL, PLP, PM and PA, metabolites commonly elevated in vitamin B6- 6 Pyridoxal: 0.1 µmol/L Pyridoxal: 100 µmol/L Pyridoxal: 1000 µmol/L treated patients, PN was strongly elevated (368 and 168 nmol/L; ref. range: <0.03 nmol/L (Albersen et al., 2012)) (Table 1). Contamination of the PLP supplement with Figure 2. Pyridoxine accumulates in the medium of four mammalian (Hek293, Neuro2a, Caco2 and PN, as a potential cause for the high PN level, was excluded: a single tablet of PLP HepG2) cell lines. Pyridoxine secretion in the culture medium is dose- and cell type-dependent, and (50 mg) was found to contain only 15 ng of PN (0.00003%, i.e. less than 0.1 nmol). observed in all of the four studied cell lines, upon PL supplementation. In addition to the patients described above, table 1 displays the plasma B6 vitamer profile of reported PLP-treated patients (Footitt et al., 2013; Mathis et al., 2016). Human homologs of PL reductase To identify the human pyridoxal reductase enzyme, the Basic Local Alignment Search Mammalian cells reduce pyridoxal to pyridoxine Tool (BLAST, NCBI) was used to identify possible homologues of PL reductase from yeast Four mammalian cell lines (mouse neuroblastoma (Neuro-2a) cells; human (plr1+ in S. pombe; accession number: CAB16409.1; GI number: 2414666) and plant embryonic kidney (HEK293) cells, human hepatocellular carcinoma (HepG2) cells, (AtPLR1 in A. thaliana; accession number: NP_200170.2; GI number: 30696358). The and human colorectal adenocarcinoma (Caco2) cells) were incubated with increasing highest homology scores for both plr1+ and AtPLR1 were for members of the auxiliary concentrations of PL to study in vitro reduction of PL to PN. Secretion of PN was beta subunits of the voltage-gated potassium channel family (KCNAB; EC 1.1.1.-) observed in all the tested cell lines in a dose- and time-dependent fashion, when and the human family (AR; EC 1.1.1.21). Based on PL was added to the culture medium (Figure 2). In contrast, no PN secretion was (Figure 3), we tested three candidate proteins as human pyridoxal reductases: the observed in any of the tested cell lines when PL was absent from the culture medium auxiliary β2 subunit of the voltage-gated potassium channel (KCNAB2), and the (data not shown). The highest secretion of PN was observed in HepG2 cells, followed aldose reductase (AKR1B1) and aldose reductase-like (AKR1B10) proteins. by Caco2, Neuro-2a and HEK293 cells (Figure 2). Therefore, subsequent studies on reduction of PL to PN were performed in HepG2 cells.

136 137 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Table 1. B6 vitamer concentrations in CSF and plasma samples of children treated with pyridoxal 5’-phosphate A Vitamin B6 vitamers (nmol/L) Diagnosis Body Therapy, Ref. and Clinical Age PLP PL PMP PM PNP PN PA fluid Dosage information Ref. <2 weeks 19-221 16-199 < 5.4d 0.3-3.3 nd < 0.03d 1.9-52 CSF range a >2 weeks 8-76 14-103 < 5.4d 0.3-1.4 < 0.03d 0.9-11 1 CSF No ATQ nor PNPO 1 d PLP, 90 mg/day 54 3940 nd 63 nd 368 548 deficiencies 2 CSF ATQ deficiency 17 d PLP, 100 mg/day 129 2078 nd 19 nd 168 136

Ref. 16.4- Plasma 4.3 y – 16 y 46-321 4.6-18.1 nd-9.3 nd nd nd-0.62 range b 139 Known IEM affecting vitamin B6 metabolism 3 b Plasma PNPO deficiency 2 y 2 m PLP, 30 mg/ 580 426.8 18 192.7 43 575 792.8 kg/day 4 b Plasma PNPO deficiency 10 y 2 m PLP, 30 mg/ 632.6 5798 101 2731 77.2 598.8 7926.3 kg/day

Seizures fully responsive to vitamin B6 (No ATQ nor PNPO deficiencies) 5 b Plasma PLP responsive 6 y 5 m Lamotrigine 709.4 7893 nd 28 nd 32 7331 and PLP, 30 mg/ kg/day

Seizures partially responsive to vitamin B6 (No ATQ nor PNPO deficiencies) 6 6 b Plasma Asperger 13 y 3 m PLP, 30 mg/ 306.4 8452.5 4.1 5.1 nd 2.7 5031.9 Syndrome, kg/day seizures, PLP responsive 7 b Plasma Partially PLP 2 y 7 m PLP, 30 mg/ 478.4 102.4 6.8 nd nd 0.31 144.6 responsive kg/day

Ref. Plasma < 18 y 10-289 4-85 na < 1 na < 1 5-564 range c

8 c Plasma PNPO deficiency 3 y PLP, 50 mg/day 1080 4180 na 1180 na 1210 3340 4 y PLP, 690 mg/day 919 6800 na 1180 na 1570 9610 9 c Plasma PNPO deficiency na PN + PLP, 100 + 412 7640 na 2050 na 11100 4940 90 mg/day a Albersen et al., 2012; b Footitt et al., 2013; c Mathis et al., 2016; d Determined limit of quantification (LOQ) of this B6 vitamer. nd, not detected; na, not available; PLP, pyridoxal 5’-phosphate; PL, pyridoxal; PMP, pyridoxamine 5’-phosphate; PM, pyridoxamine; PNP, pyridoxine 5’-phosphate; PN, pyridoxine; PA, 4-pyridoxic acid; PNPO, pyridox(am)ine 5’-phosphate oxidase; ATQ, antiquitine; CSF, cerebrospinal fluid; y, years; m, months; d, days.Reference ranges were established using samples from patients not affected by disorders of vitamin B6 metabolism and not treated with vitamin B6.Values outside the reference range are shown in bold.

138 139 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism Figure 4 A B

KCNAB2-WT

KCNAB2-/-_1.1

B

200

150

100

pyridoxine (%) 50

Figure 3. A. Amino acid sequence alignment of two known PL reductases from yeast (plr1+ in S. -/- pombe) and plant (AtPLR1 in A. thaliana) with the human KCNAB1, KCNAB2, KCNAB3, AKR1B1 and Controls KCNAB2 AKR1B10 sequences. The alignment was done with ClustalX and manually improved on the basis C of secondary structure prediction using Jpred (http://www.compbio.dundee.ac.uk/jpred). Predicted alpha-helices are marked yellow, beta-sheets are marked turquoise. Catalytic residues that 15 minutes incubation 30 minutes incubation function in aldo-keto reductase activity in KCNAB2 (Weng et al., 2006) and are conserved in AKR ns 6 ns proteins are marked by red asterisks below the sequence alignment. Cas9 sites (-3 relative 200 ns 200 ns ns to the PAM sequence) are located in (KCNAB2, AKR1B1) or immediately downstream of (AKR1B10) 150 150 ns codons that have been marked green. B. Pairwise alignment of the proteins shown in A using the NCBI Basic Local Alignment Tool (BLAST). For each combination the percentage of identity is shown 100 100 in bold with the number of residues used to calculate this percentage below. In between brackets the pyridoxine (%) 50 pyridoxine (%) 50 percentage of similarity is shown. Note that the regions of yeast PLR1 aligned to AKR-members are short. However, PLR1 from Arabidopsis, which has also been functionally characterized, does show significant similarity over extended regions to both KCNAB and AKR members. This is also reflected in Rutin Rutin DOPAC DOPAC the E-values (shown in grey cells). No Inhibitor Resveratrol No Inhibitor Resveratrol Figure 4. KCNAB2 protein as a potential human pyridoxal reductase. A. Confirmation of KCNAB2- knockout in HepG2 cells, after CRISPR/Cas9, was achieved by sequencing. Representative clone: The β2 subunit of the voltage-gated potassium (Kv) channel comparison of the sequence of KCNAB2 wild type cells and KCNAB2_2.5 clone, which contains a To verify whether KCNAB2 represents the human PL reductase, KCNAB2-/- HepG2 cells homozygous deletion of 17 nucleotides (marked with an asterisk in the wild type sequence). The were generated by CRISPR/Cas9 technology. Despite complete absence of KCNAB2 proto-spacer adjacent motif (PAM) sequence is marked in grey. B. PN secretion in KCNAB2-deficient HepG2 clones. All results are represented as the mean of triplicates ± SD; * P<0.05. C. The effect of protein (Figure 4A), the amount of PN secreted to the culture medium, after 4 hours specific KCNAB2 inhibitors on PN secretion. All results are represented as the mean of triplicates ± SD; of incubation with 100 µmol/L of PL, was not reduced when compared to control ns, not significant. cells, thus disproving the hypothesis of KCNAB2 representing the sole human PL reductase (Figure 4B). Pharmacological inhibition studies with KCNAB2-specific inhibitors (3,4-dihydroxyphenylacetic acid (DOPAC), rutin and resveratrol) (Alka et al., 2014) showed that none of these inhibitors lead to significant decreases of PN secretion (Figure 4C), thus corroborating the results obtained with the KCNAB2- knockout clones.

140 141 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Figure 5 Cas9, was achieved by sequencing. Representative clone: comparison of the sequence of AKR1B10 A B wild type cells and AKR1B10-/-_1.4 clone, which contains a homozygous deletion of 11 nucleotides 2.3 2.11 3.7 2.12 2.02 2.03 2.06 2.07 (marked with an asterisk in the wild type sequence). The PAM sequence is marked in grey. D. PN AKR1B1 AKR1B1 secretion in AKR1B1-/- AKR1B10-/- and double knockout HepG2 clones. All results are represented as the mean of triplicates ± SD; ns, not significant. E. The effect of AKR1B1- and AKR1B10-specific GAPDH RaIA inhibitors on PN secretion. All results are represented as the mean of triplicates ± SD; * P<0.05.

C The human aldose reductase (AR) The human proteins with the second highest sequence homology to plr1+ and AtPLR1 AKR1B10-WT were the aldose reductase and aldose reductase-like proteins (AKR1B1 and AKR1B10, respectively). To study the role of these proteins in reduction of PL to PN, we generated AKR1B1-/-, AKR1B10-/- and AKR1B1/10-/- knockout HepG2 cells. Absence of AKR1B1-/- and AKR1B1/10-/- was confirmed by Western Blot analysis (Figures 5A and 5B), while AKR1B10-/-_1.4 absence of AKR1B10 was confirmed by sequencing (Figure 5C). No differences in PN secretion, after 4 hours of incubation with 100 µmol/L PL, were observed between D HepG2 control cells and AKR1B1-/-, AKR1B10-/- and AKR1B1/10-/- knockout clones ns 250 (Figures 5D). Pharmacological inhibition studies with AKR1B1-specific inhibitors 200 ns ns (tolrestat and zopolrestat) and the AKR1B10-specific inhibitor (oleanolic acid) (Zhang 150 et al., 2013) were also performed. Zopolrestat strongly inhibited PN secretion (47% 100

pyridoxine (%) (P<0.05) inhibition at t=15 minutes and 61% inhibition (P<0.05) at t=30 minutes) 50 (Figure 5E), while the other two inhibitors did not significantly affect PN secretion. 6

-/- -/- -/-

Controls AKR1B1 AKR1B10 AKR1B1/10 Controls AKR1B1 Controls AKR1B10 DISCUSSION E 15 minutes incubation 30 minutes incubation Three decades have passed since PL reductase activity was discovered in yeast 200 * 200 * (Guirard and Snell 1988), followed more recently by its description in plants (Herrero

150 150 et al., 2011). No mammalian PL reductase has been described to date. However,

100 100 the presence of a reductase acting on PL is suggested by reports on increased PN concentrations in plasma of PLP-treated patients (with PNPO deficiency or with other pyridoxine (%) 50 pyridoxine (%) 50 seizure disorders responsive to vitamin B6) (Footitt et al., 2013; Mathis et al., 2016).

Tolrestat Tolrestat Presence of PN in CSF and plasma has mainly been observed in PN-treated patients, No Inhibitor Zopolrestat No Inhibitor Zopolrestat Oleanolic acid Oleanolic acid being absent in untreated subjects (Footitt et al., 2013; Mathis et al., 2016). Here, we Figure 5. AKR1B1 and AKR1B10 proteins as potential human pyridoxal reductases. A. Confirmation confirm the in vivo reduction of PL to PN by demonstrating the presence of PN in CSF of AKR1B1-knockout in the HepG2 cells, after CRISPR/Cas9, was achieved by Western Blot analysis. of two PLP-treated patients. Furthermore, experiments in cultured cells reveal that Complete absence of AKR1B1 in clones 2.3 and 2.12 at the protein level is observed, while clones PN is rapidly formed from PL in a time- and dose-dependent and cell-type specific 2.11 and 3.7 contain an intact AKR1B1 gene. GAPDH is shown as a loading control. B. Confirmation of AKR1B1/10-double knockouts, after CRISPR/Cas9: Western blot showing absence of AKR1B1 in fashion. Liver (HepG2) cells secrete the highest amount of PN, followed by intestinal AKR1B10-/- clone 1.4 (clones 2.02 and 2.06) at the protein level. Clones 2.03 and 2.07 contain an intact (Caco2), neuronal (Neuro-2a) and kidney (HEK293) cells. These data suggest that PL AKR1B1 gene. RalA is shown as a loading control. C. AKR1B10-knockout confirmation, after CRISPR/ reductase activity shows tissue dependence. Likewise, there is tissue dependency

142 143 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

of other enzymes acting on PL. PL can be phosphorylated by pyridoxal kinase, The use of zopolrestat, an AKR1B1-specific inhibitor, strongly inhibited PN-secretion converted into pyridoxic acid by and/or in our HepG2-WT cell model. Nevertheless, we were unable to confirm PL reductase and, as presented by our work, reduced to PN. The interplay between uptake of PL function of AKR1B1 in a genetic AKR1B1-knockout cell line, showing that zopolrestat and these enzymes determines the amount of PN formed. may be inhibiting other proteins, rather than AKR1B1, with a role in reducing PL to PN. The use of oleanolic acid, the AKR1B10-specific inhibitor, failed to inhibit PL Based on homology to the known PL reductase amino acid sequences of S. pombe and A. reduction. Redundancy between AKR1B1 and AKR1B10 but also with other cellular thaliana, we tested three candidate proteins as human pyridoxal reductases: the auxiliary reductases likely occurs. To study the redundancy between both aldose reductases β2 subunit of the voltage-gated potassium (Kv) channel (KCNAB2) and the aldose we generated AKR1B1/AKR1B10-/- knockouts. These double knockouts secreted the reductase and aldose reductase-like (AKR1B1 and AKR1B10, respectively) proteins. same amounts of PN as the control cell lines. Interestingly, when PL reductase was identified in S. pombe (Morita et al., 2004), the authors had already proposed the The voltage-gated potassium channels are the most complex class of the voltage-gated presence of other enzymes with PL reductase activity in those yeast strains besides ion channels both from a functional and a structural point of view (Tipparaju et al. the one encoded by the plr1+ gene. 2008). Their diverse functions include regulation of neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle Although our in vitro studies were unable to identify the enzyme responsible for contraction, and cell volume (Tipparaju et al., 2008). The β-subunits of the potassium reducing PL to PN, we present strong evidence for its existence. We clearly show that channels are cytoplasmic and associate in a 4:4 stoichiometry with the N-terminus of CSF samples of patients treated with PLP have increased PN levels, corroborating the membrane-spanning α-subunits (McCormack et al., 2002; Tipparaju et al., 2005; the existence of enzymes with PL reductase activity in humans. Similar results have Torres et al., 2007). Furthermore, the β-subunits are members of the AKR superfamily, been reported in plasma samples of PLP-treated patients, even though up to date containing an active site composed of conserved catalytic residues, a coenzyme and a no explanation was given for this puzzling finding. In our study, all mammalian cell substrate (Liu et al., 2001; Bähring et al., 2001). The Kvβ2-subunit is the most lines reduced PL to PN and secreted PN to the culture medium in a time- and dose- 6 strongly expressed Kv1-associated β-subunit protein (McCormack et al., 2002). Although dependent way. Although these findings are not entirely understood, PL reductase(s) the catalytic and kinetic mechanisms of Kvβ2 are poorly understood and its substrate may play an important role in cellular detoxification and protection, since it is a well- specificity is unknown, valuable information on its substrate preference is available in the known fact that PLP, and to a lesser extent PL, react non-enzymatically with primary literature. The β2-subunit has a strong affinity for the reduced pyridine coenzyme NADPH, amino groups of amines and amino acids through their adehyde group at C4’ (Di suggesting that the protein functions mainly as a reductase rather than as an oxidase Salvo et al., 2011). The electrophilic characteristics of the aldehyde group at C4’ derive (Liu et al., 2001). Besides oxidation of NADPH to NADP+, the β2-subunit catalyses the from the existence of a protonated pyridinium hydrogen (N1’) and a phenoxide anion reduction of both aldehydes and ketones. Interestingly, the β2-subunit reduces preferably at C3’. These stabilize the protonated state of the imine nitrogen during the formation aldehydes and aromatic substrates (Tipparaju et al., 2008), all chemical characteristics of of the Shiff base between PL(P) and substrates (Di Salvo et al., 2011). PN, on the

PL, which has an aromatic pyridine ring and an aldehyde functional group at C4’ (Figure 1). other hand, has a hydroxymethyl group (-CH2OH) at C4’, leading to a lower reactivity Deletion of KCNAB2 and the use of KCNAB2-specific inhibitors however, failed to decrease towards amino groups and therefore less cellular toxicity. PL to PN reduction, suggesting that KCNAB2 does not function as the sole human PL reductase. However, redundancy in enzyme activity between the three β-subunits (β1, β2 and β3) may occur, minimizing the deleterious effect of aβ 2-subunit deletion. CONCLUSIONS

The second family of proteins with the highest amino acid homology to the known Here, we show that mammalian cells, like plants and yeast, secrete PN in a PL (time PL reductases were the human aldose reductases. Even though the primary structure and dose) dependent fashion, suggesting the existence of PL reductase activity. of the yeast PL reductase shows low identity with the known human aldo-keto Physiologically, PL reductase(s) may serve to limit intracellular accumulation of the reductases (AKRs), the secondary structure was reported to be similar to that of the reactive aldehydes, PL and PLP, protecting the cell from unwanted reactions that human aldose reductase (Nakano et al., 1999). could lead to inactivation of important metabolites.

144 145 Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

Clause from BBA (Authorship) REFERENCES

Rúben Ramos, Judith Jans and Nanda Verhoeven-Duif designed the study. Rúben Albersen M, Groenendaal F, van der Ham M, et al (2012) Vitamin B6 Vitamer Concentrations in Cerebrospinal Fluid Differ Between Preterm and Term Newborn Infants. Pediatrics 130:e191– Ramos, Fried Zwartkruis, Susan Zwakenberg, Esmee Vringer and Marjolein Bosma e198. doi: 10.1542/peds.2011-3751 performed the study and collected the data. Rúben Ramos, Fried Zwartkruis, Monique Alka K, Dolly JO, Ryan BJ, Henehan GTM (2014) New inhibitors of the Kvβ2subunit from mammalian Albersen, Judith Jans and Nanda Verhoeven-Duif interpreted the data. Rúben Ramos, Kv1 potassium channels. Int J Biochem Cell Biol 55:35–39. doi: 10.1016/j.biocel.2014.07.013 Judith Jans and Nanda Verhoeven-Duif wrote the article. All authors revised it critically Bähring R, Milligan CJ, Vardanyan V, et al (2001) Coupling of Voltage-dependent Potassium Channel Inactivation and Active Site of Kvβ Subunits. J Biol Chem 276:22923–22929. doi: and approved the final version to be submitted. 10.1074/jbc.M100483200 Di Salvo ML, Contestabile R, Safo MK (2011) Vitamin B 6 salvage enzymes: Mechanism, structure and regulation. Biochim Biophys Acta - Proteins Proteomics 1814:1597–1608. doi: 10.1016/j. Funding bbapap.2010.12.006 Fitzpatrick TB, Amrhein N, Kappes B, et al (2007) Two independent routes of de novo vitamin B 6 biosynthesis: not that different after all. Biochem J 407:1–13. doi: 10.1042/BJ20070765 This work was supported by Metakids foundation (www.metakids.nl). Footitt EJ, Clayton PT, Mills K, et al (2013) Measurement of plasma B6 vitamer profiles in children with inborn errors of vitamin B6 metabolism using an LC-MS/MS method. J Inherit Metab Dis 36:139–145. doi: 10.1007/s10545-012-9493-y Guirard BM, Snell EE (1988) Physical and kinetic properties of a pyridoxal reductase purified from bakers’ yeast. Biofactors 1:187–192 Haeussler M, Schonig K, Eckert H, et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:1–12. doi: 10.1186/s13059-016-1012-2 Hanna MC, Turner AJ, Kirkness EF (1997) Human Pyridoxal Kinase. Biochemistry 272:10756–10760 Herrero S, González E, Gillikin JW, et al (2011) Identification and characterization of a pyridoxal 6 reductase involved in the vitamin B6 salvage pathway in Arabidopsis. Plant Mol Biol 76:157–169. doi: 10.1007/s11103-011-9777-x Hufft JW, Perlzweig WA (1944) A PRODUCT OF OXIDATIVE METABOLISM OF PYRIDOXINE, 2-METHYL- 3-HYDROXY-4-CARBOXY-5 -HYDROXY-METHYLPYRIDINE (4-PYRIDOXIC ACID): I. ISOLATION FROM URINE, STRUCTURE, AND SYNTHESIS. J Biol Chem 155:345–355 Jang YM, Kim DW, Kang T-C, et al (2003) Human Pyridoxal Phosphatase. J Biol Chem 278:50040– 50046. doi: 10.1074/jbc.M309619200 Liu SQ, Jin H, Zacarias A, et al (2001) Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels. Chem Biol Interact 130–132:955–962. doi: 10.1016/S0009- 2797(00)00248-9 Mathis D, Abela L, Albersen M, et al (2016) The value of plasma vitamin B6 profiles in early onset epileptic encephalopathies. J Inherit Metab Dis 39:733–741. doi: 10.1007/s10545-016-9955-8 McCormack K, Connor JX, Zhou L, et al (2002) Genetic Analysis of the Mammalian K + Channel β Subunit Kvβ2 ( Kcnab2 ). J Biol Chem 277:13219–13228. doi: 10.1074/jbc.M111465200 Mills PB, Surtees RAH, Champion MP, et al (2005) Neonatal epileptic encephalopathy caused by mutations in the PNPO gene encoding pyridox(am)ine 5′-phosphate oxidase. Hum Mol Genet 14:1077–1086. doi: 10.1093/hmg/ddi120 Morita T, Takegawa K, Yagi T (2004) Disruption of the plr1+ Gene Encoding Pyridoxal Reductase of Schizosaccharomyces pombe. J Biochem 135:225–230. doi: 10.1093/jb/mvh026 Nakano M, Morita T, Yamamoto T, et al (1999) Purification , Molecular Cloning , and Catalytic Activity of Schizosaccharomyces pombe Pyridoxal Reductase. 274:23185–23190 Ran FA, Hsu PD, Wright J, et al (2006) Genome engineering using the CRISPR-Cas9 system. 8:1–10. doi: 10.1038/nprot.2013.143.Genome

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Snell EE (1953) Summary of Known Metabolic Functions of Nicotinic Acid, Riboflavin and Vitamin B6. Physiol Rev 33:509–524 SUPPLEMENTARY MATERIAL Tipparaju S, Barski O, Srivastava S, Bhatnagar A (2008) Catalytic mechanisme and substrate specificity of the b-subunit of the voltage-gated potassium (Kv) channel. Biochemistry 47:8840– 8854. doi: 10.1021/bi800301b.Catalytic Tipparaju SM, Saxena N, Liu S, et al (2005) Differential regulation of voltage-gated K ϩ channels by oxidized and reduced pyridine nucleotide coenzymes. October 40202:366–376. doi: 10.1152/ ajpcell.00354.2004. Torres YP, Morera FJ, Carvacho I, Latorre R (2007) A marriage of convenience: ??-subunits and voltage-dependent K + channels. J Biol Chem 282:24485–24489. doi: 10.1074/jbc.R700022200 Ueland PM, Ulvik A, Rios-Avila L, et al (2015) Direct and Functional Biomarkers of Vitamin B6 Status. Annu Rev Nutr 35:33–70. doi: 10.1146/annurev-nutr-071714-034330 van der Ham M, Albersen M, de Koning TJ, et al (2012) Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry. Anal Chim Acta 712:108–114. doi: 10.1016/j.aca.2011.11.018 Weng J, Cao Y, Moss N, Zhou M (2006) Modulation of Voltage-dependent Shaker Family Potassium Channels by an Aldo-Keto Reductase. J Biol Chem 281:15194–15200. doi: 10.1074/jbc. M513809200 Yagi T, Tanouchi A, Hiraoka Y (1998) Growth phase-dependent active transport of pyridoxine in a fission yeast, Schizosaccharomyces pombe. FEMS Microbiol Lett 161:145–150. doi: 10.1016/ S0378-1097(98)00066-4 Zhang L, Zhang H, Zhao Y, et al (2013) Inhibitor selectivity between aldo-keto reductase superfamily members AKR1B10 and AKR1B1: Role of Trp112 (Trp111). FEBS Lett 587:3681–3686. doi: 10.1016/j.febslet.2013.09.031 6

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6

Supplementary figure .1 Coding sequences of KCNAB2, AKR1B1 and AKR1B10 and protein sequence indicated in single letter code. Catalytic residues are marked red. Pam sequences of guide RNAs used in this study are marked blue. Cas9 sites are located 3 bp downstream of the PAM sequences indicated in KCNAB2, 3 bp upstream in the case of AKR1B1 and AKR1B10.

150 151 Chapter 7

General Discussion 7 General Discussion

Vitamin B6 is essential for normal brain metabolism and development (Surtees et al. 2006). Pyridoxal 5’-phosphate (PLP; the metabolically active form of vitamin B6) acts as a cofactor in more than 160 enzyme-catalysed reactions (Percudani and Peracchi, 2009). PLP deficiency can be caused by either nutritional limitations, inborn errors of vitamin B6 metabolism or genetic defects in metabolic pathways that lead to decreased bioavailability or inactivation of intracellular PLP (Clayton 2006).

This thesis describes the discovery of a new inborn error of metabolism: mitochondrial glutamate oxaloacetate transaminase (GOT2) deficiency (Chapters 3 and 4). The four known affected patients suffer from epilepsy and severe developmental delay. As GOT2 is a PLP-dependent enzyme, some of the patients were treated with pyridoxine (PN), with positive clinical results. Probably, PLP acts by promoting the residual GOT2-enzyme activity (enzyme activity in the four affected patients ranged from 8 to 21%), stimulating the malate aspartate shuttle (MAS) in which GOT2 is an important player. A striking finding in the patient with the lowest GOT2-enzyme activity was a decreased concentration of serine in blood. Low concentrations of serine in blood and CSF have previously been described in patients with a serine biosynthesis defect, disorders with similar clinical phenotypes to GOT2 deficiency. In the GOT2-deficient patients, primary defects of serine synthesis were excluded and we demonstrated that the low serine was secondary to disturbances in the cytosolic redox balance. This is an important finding for two reasons: 1) supplementation of serine is a treatment option that has already shown beneficial effects on seizure reduction; and 2) our findings led to the hypothesis that any cause of cytosolic redox imbalance may lead 7 to a secondary serine deficiency. Thus, also for patients with other disorders of the MAS and related pathways, serine supplementation may be beneficial. Interestingly, serine has not been reported to be altered in the other three known MAS defects (mitochondrial malate dehydrogenase, MDH2; aralar, SLC25A12; and citrin, SLC25A13 deficiencies). Reasons for this may be the fact that subtle decreases of serine levels may be missed during diagnostic work-up because 1) lactate, pyruvate and other organic acids are more prominently altered; and 2) serine biochemical abnormalities are more pronounced and consistent in CSF than in plasma (the most abundant type of sample to arrive the laboratory) because plasma serine concentrations are affected by meals and can be normal when patients are not fasting (Van Der Crabben et al. 2013; de Koning 2017). Future studies in the Utrecht metabolic laboratory will investigate this hypothesis by studying the in vitro serine biosynthesis in genetically modified cell lines with the individual defects of the MAS.

155 General Discussion

GOT2 deficiency not only results in a decreased concentration of serine, but has a as a cofactor, we hypothesized that other metabolic disturbances contribute to range of other biochemical consequences. All patients have increased concentrations the phenotype. To investigate the metabolic consequences of low intracellular of lactate and ammonia in blood. The increased lactate is the direct consequence concentrations of PLP, in Chapter 2 we performed intracellular metabolomics of the impaired re-oxidation of cytosolic NADH due to defective MAS, whereas the experiments with neuronal cells. We observed that in these cells, PLP deficiency results increase in ammonia is caused by aspartate deficiency. In humans, the amount of in low concentrations of serine and glycine. This was surprising, as in B6-deficient aspartate taken up from the blood is very limited (Palmieri 2004), rendering cells patients increased concentrations of serine and glycine in plasma have been reported. highly dependent on their mitochondrial aspartate production. In GOT2 deficiency, To elucidate the cause of the low intracellular serine and glycine we performed a the intramitochondrial synthesis of aspartate from oxaloacetate is blocked, leading study of serine and glycine formation from the uniformly labelled isotopomer 13 to low aspartate both in the mitochondria and cytosol. Aspartate shortage leads to of glucose ( C6-glucose). In PLP-deficient cells, serine and glycine synthesis was reduced argininosuccinate synthase (ASS, EC 6.3.4.5) activity, citrulline accumulation, decreased. Serine synthesis is PLP-dependent, as phosphoserine aminotransferase diminished urea cycle activity, and ammonia accumulation. In the most severely (PSAT, EC 2.6.1.52) is a PLP-dependent enzyme. Thus, in PLP-deficiency, the pathway affected patient, citrulline was also consistently increased. does not fully function, resulting in low serine in this neuronal cell model. It is known that the serine synthesis pathway is particularly important in brain, and less in other Our study of the metabolome of GOT2-deficient HEK293 cells demonstrated tissues. In liver, both serine and glycine are metabolized by PLP-dependent enzymes. abnormalities in amino acid and energy metabolism (Chapter 4). Extremely low We assume that in this organ serine and glycine accumulate in case of PLP-deficiency, intracellular concentrations of pyruvate were found and may be caused by two resulting in high plasma concentrations as observed. phenomena: decreased glycolysis and increased conversion of pyruvate into lactate. In line with this finding were the low concentrations of glyceraldehyde 3-phosphate/ Taken together, both a cytosolic redox imbalance and a deficiency of PLP may result dihydroxyacetone phosphate, suggesting inhibition of glycolysis already in the first in hampered serine biosynthesis. This is important, as serine is not only important in irreversible step of glycolysis. the brain as such, but conversion of serine into glycine is of utmost importance for one-carbon metabolism, as it results in the formation of 5-methyltetrahydrofolate We demonstrated that pyruvate deficiency leads to alanine deficiency, as alanine (5-mTHF). We demonstrated that a decrease of serine synthesis results in lower is the direct product of pyruvate transamination. Accumulation of glutamine and concentrations of intracellular 5-mTHF, which may contribute to pathogenesis of 7 glutamate, as observed in the GOT2 deficient cells, may be due to hampered glutamate patients with vitamin B6-dependent epilepsy. This finding may explain the very metabolism as a direct consequence of deficient GOT2 activity, as glutamate is a puzzling observation made in 2009 that PN dependent epilepsy and folinic acid substrate of this enzyme. responsive seizures, which we long considered to be separate entities, were both caused by the same mutations in ALDH7A1 (Gallagher et al. 2009). Probably, the Our demonstration that in vitro supplementation of pyruvate to GOT2-deficient cells supplementation of folinic acid in these patients results in correction of a 5-mTHF results in normalization of part of the metabolic abnormalities may be of importance deficiency and clinical improvement. However, it remains unknown why the response to affected patients. However, in vivo supplementation of pyruvate must be done was so positive in some patients as PLP was not supplemented and thus probably with great care to avoid the occurrence of lactic acidemia. Encouraging results were was still deficient. found in patients affected with defects in SLC25A13, when administration of sodium pyruvate and L-arginine proved to be both effective and safe (Mutoh et al. 2008). We mainly gained our knowledge from in vitro experiments. Confirmation of these results in human brain cells would be ideal but not possible. Primary neuronal A second focus of this thesis were the vitamin B6 deficient epilepsies, a group of cultures from rats and mice may overcome the problem of karyotype instability genetic disorders that have an overlapping phenotype with epilepsy as the most observed in neuroblastoma cell lines but are still not human (Shipley et al. 2016). An striking symptom. The common denominator of these disorders is a cellular deficiency alternative may be the use of the human SH-SY5Y neuroblast-like cells, which have of PLP which is thought to result in decreased concentrations of the inhibitory a stable karyotype (47 ) and can efficiently be differentiated to mature neurotransmitter GABA. However, as PLP is involved in a range of enzyme reactions human neurons. Nevertheless, the use of induced pluripotent stem cell (iPSCs)-

156 157 General Discussion

derived neurons from patients with genetic vitamin B6 deficiencies may be the best in Chapter 4, instead of having to rely on several individual biochemical tests to model to understand these human neurodevelopmental diseases (Dolmetsch and achieve similar results. Furthermore, it allows the discovery of new biomarkers and Geschwind 2011) and can be used in future experiments. pathomechanisms, without having to depend on the constraint of knowing, a priori, which metabolic pathways or metabolites to focus on. In Chapter 6 we made a puzzling observation when we analysed concentrations of B6 vitamers in CSF of two patients that were treated with PLP. In these samples, we The DI-HRMS approach, a method without an up-front separation (chromatographic) observed high concentrations of PN. To date, no enzyme that catalyses the conversion step has a short analysis time (3-minutes), making it a high-throughput method that of PL(-P) into PN has been described in and this reaction is considered not enables the analysis of hundreds of samples in one day. On the other hand, the lack to occur. To study the in vitro reduction of PL into PN, we exposed mammalian cell of a separation step also has technical limitations, the main being the impossibility lines to PL and found that PN is formed in a time-dependent and cell-type specific to discriminate between molecules that share the same elemental composition (i.e. fashion. structural isomers with the same accurate mass) (Giavalisco et al. 2009; Kirwan et al. 2014; Lin et al. 2010). This limitation was overcome by validation of the results by We used the BLAST tool to identify possible human homologues of PL reductase targeted and quantitative LC-MS methods. from yeast and plants and the three proteins (KCNAB2, AKR1B1 and AKR1B10) with the highest homology scores were studied. Despite great efforts we were unable Spin-off from the scientific work described in this thesis is multiple. The knowledge is to identify the gene(s) that encoded the enzyme(s) responsible for this conversion. of direct use for diagnosis: the description of a new disease will result in identifying Thus, although we have proved the existence of protein(s) with PL reduction activity additional patients, allowing timely treatment and genetic counselling. Our work also in human cells, we have yet to identify this enzyme. gives important new leads for treatment of defects of the MAS and defects in vitamin B6 metabolism. Future studies should focus on the other two members of the KCNAB family (β1 and β3) and other protein candidates that share homology with other PL reductases. We From a methodological point of view the spin-off is also multiple: this thesis presents can also consider the use of siRNA libraries for known reductases and replicate our the first successful findings of our in house developed DI-HRMS pipeline applied to studies of PN formation from PL. It would be interesting to find out whether this assess the intracellular metabolome. We also developed a robust and miniaturized 7 enzyme activity is important for detoxification and protection. PLP and PL react non- high-throughput cell culture-based method that allows the study of the effect of enzymatically with primary amino groups of amines and amino acids through their pharmacological inhibitors or treatments on vitamin B6 metabolism. Furthermore, 13 aldehyde group at C4 (Di Salvo et al. 2011). PN, on the other hand, has a hydroxymethyl we validated the method of serine synthesis from uniformly labelled C6-glucose for group (-CH2OH) at C4, leading to a lower reactivity towards amino groups and less diagnostic use. It is an example of a test in which not a single enzyme is measured, cellular toxicity. Physiologically, PL reductase(s) may serve to limit intracellular but an entire pathway. Tests of this type will become more and more important in the accumulation of the reactive aldehydes, protecting the cell from unwanted reactions future for the functional validation of gene variants of unknown significance that are that could lead to inactivation of important metabolites. However, PN formation increasingly found in clinical whole exome and whole genome sequencing. may also occur by promiscuous enzymes and be of little importance for cellular metabolism. When the human PL reductase enzyme has been elucidated, generating knockout systems will further reveal its importance.

In our studies, we used direct infusion high resolution mass spectrometry (DI- HRMS) to study the intracellular metabolome. This approach offers a “helicopter view” of metabolites under specific conditions. It has the tremendous advantage of revealing, with the use of only one test, the global metabolic alterations of vitamin B6-insufficiency in Chapter 2 and the pathophysiology of GOT2 deficiency

158 159 General Discussion

REFERENCES

Clayton PT (2006) B6-responsive disorders: a model of vitamin dependency. J Inherit Metab Dis 29:317–26. doi: 10.1007/s10545-005-0243-2 de Koning TJ (2017) Amino acid synthesis deficiencies. J Inherit Metab Dis 40:609–620. doi: 10.1007/ s10545-017-0063-1 Di Salvo ML, Contestabile R, Safo MK (2011) Vitamin B 6 salvage enzymes: Mechanism, structure and regulation. Biochim Biophys Acta - Proteins Proteomics 1814:1597–1608. doi: 10.1016/j. bbapap.2010.12.006 Dolmetsch R, Geschwind DH (2011) Neurons. Cell 145:2000–2001. doi: 10.1016/j.cell.2011.05.034. The Gallagher RC, Van Hove JLK, Scharer G, et al (2009) Folinic acid-responsive seizures are identical to pyridoxine-dependent epilepsy. Ann Neurol 65:550–6. doi: 10.1002/ana.21568 Giavalisco P, Köhl K, Hummel J, et al (2009) 13C isotope-labeled metabolomes allowing for improved compound annotation and relative quantification in liquid chromatography-mass spectrometry-based metabolomic research. Anal Chem 81:6546–6551. doi: 10.1021/ac900979e Kirwan JA, Weber RJM, Broadhurst DI, Viant MR (2014) Direct infusion mass spectrometry metabolomics dataset: A benchmark for data processing and quality control. Sci Data 1:1–13. doi: 10.1038/sdata.2014.12 Lin L, Yu Q, Yan X, et al (2010) Direct infusion mass spectrometry or liquid chromatography mass spectrometry for human metabonomics? A serum metabonomic study of kidney cancer. Analyst 135:2970. doi: 10.1039/c0an00265h Mutoh K, Kurokawa K, Kobayashi K, Saheki T (2008) Treatment of a citrin-deficient patient at the early stage of adult-onset type II citrullinaemia with arginine and sodium pyruvate. J Inherit Metab Dis 31:343–347. doi: 10.1007/s10545-008-0914-x Palmieri F (2004) The mitochondrial transporter family (SLC25): Physiological and pathological implications. Pflugers Arch Eur J Physiol 447:689–709. doi: 10.1007/s00424-003-1099-7 Percudani R, Peracchi A (2009) The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC 7 Bioinformatics 10:273. doi: 10.1186/1471-2105-10-273 Shipley MM, Mangold CA, Szpara ML (2016) Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line. J Vis Exp 1–11. doi: 10.3791/53193 Surtees R, Mills P, Clayton P (2006) Inborn errors affecting vitamin B 6 metabolism. Future Neurol 1:615–620. doi: 10.2217/14796708.1.5.615 Van Der Crabben SN, Verhoeven-Duif NM, Brilstra EH, et al (2013) An update on serine deficiency disorders. J Inherit Metab Dis 36:613–619. doi: 10.1007/s10545-013-9592-4

160 Appendix

Nederlandse samenvatting Summary Acknowledgements Curriculum Vitae List of publications

163 Nederlandse samenvatting

Nederlandse samenvatting Vitamine B6-afhankelijke patiënten presenteren zich normaal gesproken met ernstige convulsies in de neonatale periode, die niet reageren op de reguliere anti- Vitamine B6 is een wateroplosbare vitamine die in het lichaam aanwezig is in zes convulsieve therapieën, maar alleen op therapeutische doses van PLP en/of PN. Naast vitameren, die qua structuur op elkaar lijken. Dit zijn pyridoxal (PL), pyridoxine (PN), deze neonatale convulsies hebben veel patiënten een achterstand in de ontwikkeling pyridoxamine (PM) en voor elk van deze vormen de 5’-fosfaatester: pyridoxal-5- en een verstandelijke beperking, beide variërend in ernst, en onafhankelijk van of de fosfaat (PLP), pyridoxine-5-fosfaat (PNP) en pyridoxamine-5-fosfaat (PMP). PLP is de convulsies onder controle te krijgen waren met behandeling met vitamine B6. actieve vorm van vitamine B6. Deze vitamine is essentieel voor de overleving van alle organismenAlleen bacteriën, gisten en planten kunnen vitamine B6 produceren. De studies die in dit proefschrift beschreven staan, hebben bijgedragen aan nieuwe Omdat het menselijk lichaam zelf geen vitamine B6 kan aanmaken, zijn mensen inzichten in de pathofysiologie van genetische vitamine B6-deficiënties. Daarnaast volledig afhankelijk van de opname van vitamine B6 uit het dieet en het hergebruiken beschrijven we een nieuwe vitamine B6-afhankelijke ziekte. van de andere B6 vitameren om zo opnieuw PLP te kunnen vormen. In hoofdstuk 1 is de huidige kennis over vitamine B6 beschreven, met als focus de Vitamine B6 komt de cel binnen na hydrolyse van de gefosforyleerde vitameren door rol van vitamine B6 in gezondheid en ziekte, de enzymen die een rol spelen in het membraangebonden alkalisch fosfatase (ALPL). Intracellulair fosforyleert PL-kinase vitamine B6-metabolisme in het menselijk lichaam en de bekende metabole ziekten (PDXK) de hydroxymethylgroep van PL, PN en PM. De opnieuw gefosforyleerde die invloed hebben op het vitamine B6 metabolisme. vitameren worden op deze manier ingesloten in de cel. De intracellulaire concentratie van PLP wordt gereguleerd door pyridox(am)ine fosfaatoxidase (PNPO), Ook al gaat de ontdekking van vitamine B6 terug tot de vroege jaren 30 van de dat de oxidatie van PNP en PMP naar PLP katalyseert, en PL-fosfatase (PDXP), dat vorige eeuw en is pyridoxineafhankelijke epilepsie bekend sinds de jaren 50 van de de defosforylatie van PLP, PNP en PMP katalyseert. Pyridoxal oxidase oxideert PL, vorige eeuw, toch begrijpen we de pathofysiologie van een vitamine B6-deficiëntie waardoor 4-pyridoxinezuur ontstaat. Dit is het voornaamste afbraakproduct van onvoldoende. In hoofdstuk 2 hebben we het begrip van de pathofysiologie vitamine B6 en dit wordt uitgescheiden in de urine. uitgebreid door de intracellulaire moleculaire consequenties van een vitamine B6- deficiëntie te bestuderen in neuronale cellen. Voorheen werd gedacht dat lage PLP is een essentiële co-factor in meer dan 160 metabole reacties. PLP is vooral concentraties gamma-aminoboterzuur (GABA) en hoge concentraties glutamaat belangrijk voor het metabolisme van het brein en voor de ontwikkeling, omdat veel van door verlaagde activiteit van glutaminezuur decarboxylase (GAD) de belangrijkste de PLP-afhankelijke enzymen onderdeel uitmaken van het aminozuurmetabolisme metabole gevolgen waren van onvoldoende PLP-beschikbaarheid. We laten zien dat, (glutamaat, aspartaat, L- en D-serine, glycine) en de biosynthese van neurotransmitters naast tekorten van GABA, neuronale cellen ook tekorten hebben aan serine, glycine (gamma-aminoboterzuur, dopamine, serotonine). Daarnaast speelt PLP een rol in het en 5-methyltetrahydrofolaat (5-mTHF). Onze studie laat zien dat een vitamine B6- metabolisme van sphingolipiden, heem, histamine, koolhydraten en nucleotiden. deficiëntie de biosynthese van serine verstoort. Dit leidt naast een tekort aan serine ook tot lage concentraties van glycine en 5-mTHF, waarschijnlijk door een verlaagde Er zijn vijf metabole ziekten bekend die invloed hebben op het vitamine B6- activiteit van serinehydroxymethyltransferase (SHMT). Vitamine B6 is dus essentieel metabolisme: pyridoxineafhankelijke epilepsie (alfa-amino-adipine semialdehyde voor de serinebiosynthese in neuronale cellen, en serinebiosynthese is noodzakelijk dehydrogenase - antiquitine - deficiëntie; OMIM #266100), hyperprolinemie type II voor het handhaven van de intracellulaire concentraties van serine en glycine. Deze (1-pyrroline-5-carboxylaat dehydrogenase deficiëntie; OMIM #239510), pyridox(am) bevindingen verklaren waarom sommige patiënten met een vitaminepyridoxine ine fosfaatoxidasedeficiëntie (PNPO-deficiëntie; OMIM #610090), hypofosfatasemie afhankelijke epilepsie klinisch lijken te reageren op suppletie met folinezuur (een (niet- weefselspecifiek alkalisch fosfatase - TNSALP - deficiëntie; OMIM #241500), precursor van 5-mTHF). Dit is van groot belang bij het beredeneren van een goede en deficiëntie van het pyridoxal-5-fosfaatbindend eiwit (PLPBP deficiëntie; OMIM behandeling voor patiënten met pyridoxine afhankelijke epilepsie. #617290). Naast deze ziekten zijn er nog andere, niet verder omschreven, vitamine B6-responsieve aandoeningen waar zowel wij (patiënt 1 in hoofdstuk 6) als anderen over hebben gerapporteerd.

164 165 Nederlandse samenvatting

In hoofdstuk 3 en 4 beschrijven we een nieuwe vitamine B6-afhankelijke genetische reduceert tot PN, in een tijd- en dosisafhankelijk patroon. Vervolgens hebben we ziekte: mitochondriële glutamaat-oxaloacetaat transaminase (GOT2) deficiëntie. Het de aminozuurvolgorde van bekende PL reductases vergeleken met menselijke bestuderen van het gehele exoom bij patiënten met een verstandelijke beperking en aminozuurvolgorden en vonden een grote overeenkomst tussen de bètadelen epilepsie zorgde ervoor dat er in vier patiënten genetische mutaties geïdentificeerd van de spanningsafhankelijke kaliumkanalen en de humane aldosereductases. zijn in het GOT2-gen, die leiden tot een volledig verlies van de activiteit van het Medicinale remming en het genetisch inactief maken van deze eiwitten liet zien dat GOT2- enzym. Functionele testen in fibroblasten van deze patiënten en hun ouders, geen een van de kandidaten uitsluitend verantwoordelijk was voor de PL-reductie in diermodellen (zebravissen en muizen) en in celmodellen (HEK293-cellen waarbij naar PN. Er zijn meer studies nodig om de eiwitten verantwoordelijk voor PL-reductie het GOT2- gen gemuteerd is) lieten zien dat de mutaties geïdentificeerd in patiënten te identificeren, maar onze bevindingen tonen wel al aan dat er meer enzymen zijn inderdaad leiden tot een tekort aan GOT2enzymactiviteit. We hebben onderzocht met een rol in het humane vitamine B6 -metabolisme dan vooralsnog gedacht werd. waarmee deze patiënten te behandelen zouden zijn en lieten zien dat GOT2- We verwachten dat de PL-reductase(s) een beschermende rol hebben voor de cel, deficiëntie een ziekte is die reageert op PLP. Twee patiënten, die het meest ernstig omdat ze de concentraties van vrij PL en PLP kunnen beperken. Deze metabolieten zijn aangedaan, worden nu behandeld met PLP en daarmee zijn de convulsies geheel kunnen namelijk niet-enzymatisch, via de aldehydegroep op C4, met de primaire onder controle. Daarnaast suggereren onze bevindingen ook dat suppletie met aminogroepen van amines en aminozuren reageren. pyruvaat een belangrijke therapeutische optie zou kunnen zijn om de biochemische veranderingen die ontstaan door GOT2- deficiëntie te kunnen normaliseren. Onze Concluderend dragen we met deze laatste studie bij aan nieuwe inzichten studies laten zien dat GOT2-deficiëntie de biosynthese van serine verstoort door in het vitamine B6-metabolisme van zoogdieren, door te laten zien dat er PL middel van een verhoogde NADH/NAD+-ratio. We verwachten daarom dat het nuttig reductaseactiviteit is in het menselijk lichaam (hoofdstuk 6). We laten daarnaast is om serinebiosynthese te bestuderen in andere defecten in de malaat-aspartaat zien dat een verstoorde serinebiosynthese het gevolg is van vitamine B6-deficiëntie shuttle en in de mitochondriële ademhalingsketen. Dit zijn allen ziektes met een (hoofdstuk 2). We hebben een nieuwe vitamine B6-afhankelijke metabole ziekte verhoogde NADH/NAD+-ratio. beschreven: GOT2- deficiëntie (hoofdstuk 3 en 4) en aangetoond dat een secundair defect in de serinebiosynthese bijdraagt aan de pathofysiologie van deze ziekte. Om In hoofdstuk 5 beschrijven we een gevoelige en accurate UPLC MS/MS methode studies naar primaire en secundaire serinedefecten te verbeteren, hebben we een met stabiele isotopen, die als screeningsmethode kan dienen voor het bestuderen methode ontwikkeld die de metabole flux kan meten (hoofdstuk 5). van de serinebiosynthese in gekweekte cellen. We hebben aangetoond dat deze methode sensitiever is dan de huidige methodes gebaseerd op enzymdiagnostiek. Deze methode geeft de mogelijkheid om de gehele serine-biosynthese in één keer te analyseren in plaats van het meten van geïsoleerde enzymen.

Tot slot laten we in hoofdstuk 6 zien dat er PL-reductase activiteit is in het menselijk lichaam. We startten onze studie toen we een sterke verhoging van PN hadden gedetecteerd in het hersenvocht van twee patiënten die behandeld werden met PLP. Deze hoge PN-concentraties in lichaamsvloeistoffen zijn normaal bij patiënten met een vitamine B6-tekort die behandeld worden met PN, maar worden normaal gesproken niet gerapporteerd of niet gezien bij patiënten die behandeld worden met PLP. Voor zover wij weten is PN=toename onder PLP- behandeling beschreven in twee studies, maar hierin wordt geen verklaring gegeven voor deze bevindingen. We verwachtten dat net als bacteriën, gisten en planten, ook mensen een PL–reductase- enzym hebben. Om het bestaan van PL-reductaseactiviteit te onderzoeken, hebben we vier cellijnen van zoogdieren met PL behandeld. We vonden dat elke cellijn PL

166 167 Summary

Summary Classically, vitamin B6-deficient patients present with severe neonatal seizures that do not respond to common anticonvulsant therapy and are only controlled Vitamin B6 is a water soluble vitamin that is present in the human body as six by pharmacological doses of PLP and/or PN. In addition to the neonatal seizures, structurally related vitamers: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), most patients suffer from variable degrees of developmental delay and intellectual and their respective 5’-phosphate esters pyridoxal 5’-phosphate (PLP), pyridoxine disability, despite the seizure control achieved with vitamin B6 treatment. 5’-phosphate (PNP) and pyridoxamine 5’-phosphate (PMP). PLP is the metabolically active form of vitamin B6 and, although it is essential for survival of all organisms, only The studies presented in this thesis resulted in new insight into the pathophysiology bacteria, yeast and plants synthesize it de novo. Because humans cannot synthesise of genetic vitamin B6 deficiencies. In addition, a new vitamin B6-reponsive disorder vitamin B6, they rely solely on its uptake from the diet and on the vitamin B6 salvage is described. pathway to recycle the different B6 vitamers to PLP. In chapter 1 we discussed the current knowledge on vitamin B6. We especially Vitamin B6 enters the cells after hydrolysis of the phosphorylated forms by the focused on the known roles of vitamin B6 in health and disease; the enzymes known membrane-bound alkaline phosphatase (ALPL). Intracellularly, PL kinase (PDXK) to play a role in the human vitamin B6 metabolism; and the known inborn errors of phosphorylates the hydroxymethyl group of PL, PN and PM to their respective vitamin B6 metabolism. 5’-phosphate forms, entrapping these vitamers inside the cell. Pyridox(am)ine phosphate oxidase (PNPO) catalyses the oxidation of PNP and PMP to PLP, while Although the discovery of vitamin B6 dates back to the early 1930’s and pyridoxine- dephosphorylation of PLP, PNP and PMP is catalysed by PL phosphatase (PDXP), dependent epilepsy is known since the 1950’s, the pathophysiology of vitamin B6 allowing the cells to control the intracellular PLP concentration. Pyridoxal oxidase deficiency is still not completely understood. Therefore, in chapter 2, we expanded oxidizes PL to 4-pyridoxic acid (PA), the main catabolic product of vitamin B6, which our understanding on the intracellular metabolic consequences of vitamin B6 is excreted in the urine. deficiency on a neuronal cell model. Previously, low γ-aminobutyrate (GABA) and high glutamate levels due to reduced glutamic acid decarboxylase activity were believed PLP is an essential cofactor in more than 160 metabolic reactions. Since most PLP- to be the most clinically relevant metabolic consequence of PLP insufficiency. We dependent reactions are involved in the metabolism of amino acids (glutamate, showed that, in addition to deficient levels of GABA, neuronal cells have decreased aspartate, L- and D-serine, glycine) and biosynthesis of neurotransmitters intracellular levels of serine, glycine and 5-methyltetrahydrofolate (5-mTHF). Our (γ-aminobutyric acid, dopamine and serotonin), PLP is especially relevant for normal study showed that vitamin B6 insufficiency strongly impairs the de novo serine brain metabolism and development. In addition, PLP is important in the metabolism synthesis and consequently leads to low glycine and 5-mTHF levels, probably due to of sphingolipids, heme, histamine, carbohydrates and nucleotides. decreased serine hydroxymethyltransferase activity. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to Five inborn errors of metabolism exist that affect vitamin B6 metabolism: pyridoxine- maintain intracellular serine and glycine. These findings explain why some patients dependent epilepsy (α-aminoadipic semialdehyde dehydrogenase (antiquitin) with vitamin B6-dependent epilepsy clinically respond to supplementation of folinic deficiency; OMIM #266100), hyperprolinemia type II (1-pyrroline-5-carboxylate acid, a 5-mTHF precursor, and are especially relevant when considering pathogenesis dehydrogenase deficiency; OMIM #239510), pyridox(am)ine 5’-phosphate oxidase and treatment of patients with vitamin B6-dependent epilepsy. deficiency (PNPO deficiency; OMIM #610090), hypophosphatasia (tissue non-specific alkaline phosphatase (TNSALP) deficiency; OMIM #241500), and pyridoxal phosphate In chapters 3 and 4 we characterized a new genetic disease that results in a vitamin binding protein deficiency (PLPBP deficiency; OMIM #617290). In addition to these, B6-responsive condition: mitochondrial glutamate oxaloacetate transaminase (GOT2) there are other, yet uncharacterized, vitamin B6-responsive conditions, as we (patient 1 deficiency. Whole exome sequencing in four children with intellectual disability and in chapter 6 of this thesis) and others have reported. epilepsy resulted in the identification of loss-of-function mutations in GOT2. Our functional studies in fibroblasts of the patients and their parents, in animal models (zebrafish and mice) and cell models (GOT2-knockout HEK293 cells) proved that

168 169 Summary

the mutations result in deficient GOT2 enzyme activity. We then explored putative In conclusion, with this study we add novel insight into mammalian metabolism treatment options and showed that GOT2 deficiency is a PLP-responsive disorder, as of vitamin B6 by presenting compelling evidence for the existence of PL reductase the two most severely affected patients are nowadays under vitamin B6 treatment activity in humans (chapter 6). In addition, we show that impaired serine synthesis and their seizures are completely under control. Furthermore, our findings suggest is an important metabolic consequence of vitamin B6 deficiency (chapter 2). We that serine and pyruvate supplementation may be important therapeutical options described and characterized a new vitamin B6-responsive inborn error of metabolism, to correct the biochemical abnormalities produced by GOT2 deficiency. Our in vitro GOT2 deficiency (chapters 3 and 4), and revealed that a (secondary) de novo serine studies showed that GOT2 deficiency impairs the de novo serine synthesis due to an biosynthesis defect may partially underlie the pathophysiology of this disease. To increased NADH/NAD+ ratio. Therefore, we hypothesize that it is relevant to assess the improve studies on primary and secondary de novo serine biosynthesis defects we de novo serine synthesis in other malate aspartate shuttle defects and mitochondrial developed a metabolic flux method (chapter 5). respiratory chain disorders (diseases with increased NADH/NAD+ ratio).

In chapter 5, we described a sensitive and accurate stable isotope-based UPLC- MS/MS (ultra-performance liquid chromatography tandem mass spectrometry) method that serves as a screening tool in the study of de novo serine biosynthesis in cultured cells. This method proved to be more sensitive than the current enzymatic methods, allowing the analysis of the complete metabolic pathway instead of testing individual enzymes. Finally, in chapter 6 we provided compelling evidence for the presence of PL reductase activity in humans. Our study was initiated when a strong accumulation of PN was detected in cerebrospinal fluid (CSF) samples of two PLP- treated patients. This PN accumulation in human biofluids is a common feature of B6-deficient patients treated with PN, but normally unreported or overlooked when PLP is the pharmacological treatment. To our knowledge, only two studies have reported PN accumulation after PLP treatment, although no explanation for the observed results was given. We hypothesized that, like bacteria, yeast and plants, humans also possess a PL reductase enzyme. To further investigate the existence of PL reductase activity, we treated four mammalian cell lines with PL and found that all cell lines reduce PL to PN in a time- and dose-dependent manner. We then 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 genetic knockout of these proteins show that none of the candidates is solely responsible for PL reduction to PN. Although further studies are needed to identify the responsible PL reductase protein(s), our findings clearly expand the number of enzymes with a role in vitamin B6 salvage pathway. We hypothesize a protective role of PL reductase(s) by limiting the intracellular amount of free PL and PLP, metabolites known to react non- enzymatically with primary amino groups of amines and amino acids through their aldehyde group at C4.

170 171 Acknowledgements

Acknowledgements came roughly at the middle of my PhD track, but you quickly became one of my dearest Dutch friends. For several times my cheeks suffered from laughing so hard “It’s the job that’s never started that takes longest to finish” – Samwise Gamgee while you were teaching me “interesting” (to say the least) Dutch words! Dank je wel! Glen, the PhD student from “the other side of the hospital”. We had a lot of fun in As we approach the end of this manuscript, I hope my creativity and tired brain still those Masterclasses and retreats. Thank you for those coffees and great (scientific and make justice to all of you that made my Dutch experience possible. non-scientific) discussions!

During this PhD I met wonderful people. People that will stay in my heart until the Monique, I still remember a time when it was just the two of us with Nanda and Judith end of my days. Not only were these “new” friends essential, but also the strength and in our meetings. Funny to see how the group grew in those four years. Thank you for support from my family and friends that stayed back home made it unforgettable. always rising my scientific reasoning. Jolita, Marjolein, Melissa and Sanne, the other Thank you all for playing the main role in this great adventure. ladies in our metabolic “oval table” discussions, thank you for all the times in the lab, for the brainstorms and your support. My first acknowledgment goes to Nanda, the supervisor of this thesis. Nanda, you opened the doors of your lab to me and allowed me to work alongside brilliant My PhD would have never been possible without the help and support of everyone people in a stimulating environment. I still remember the first e-mail I sent you in in the UMCU metabolic lab. A bit like a family, I met the mothers and fathers of the the summer of 2013. At some point I wrote: “…it is my goal to achieve experience lab, the crazy uncles and funny cousins, the pushy and the loving and caring siblings. in a renowned laboratory abroad such as yours and I would be most honoured to My big thanks to everyone! I hope to make justice to everyone and not let anyone have the opportunity of doing a PhD thesis with you as my mentor in the field of behind! Thank you Mia, Maria, Karen, Helma, Harrold, Martina, Martin, Deena, Isa, IEM”. Thank you for believing in me. I am a better scientist thanks to this invaluable Raymond, Birgid, Ans, Mirjam, Gerda, Arno, Suzana, Arda, Monique de Sain, Berthil, experience! Secondly, to Judith, my co-promotor and daily supervisor. Thank you for Astrid, Marcel Dasselar, Marcel Willemsen, Monique de Groijer, Johan, Yuen Fung, all your positive and kind words when I most needed them, and for your guidance Can and Edwin. Thank you for your help, patience and guidance during these years. and work discussions whenever we sat together. We went through quite a journey in Of all of you, some became especially important to me and my stay in Utrecht. So these four years. Thank you for believing in me. forgive me the ones I am not mentioning now, but I need to especially thank Deena, Martin and Johan. Deena, thank you for making my stay in the Netherlands better. A special thanks to Nine Knoers for the role you played in my PhD. Thank you for With you I found that Utrecht also has good coffee (my deepest thanks!) and coffee always make me feel welcome at your door. It has been an honour to sit with you and places. I found great places to eat… places I went back so often (with you for the discuss my projects. To all the members of my PhD committee a special thank you. large majority of times!). Thank you for receiving me in your house and making me Dr. Tom de Koning, prof. dr. Boudewijn Burgering, prof. dr. Peter van Tintelen, prof. feel as a family member. Martin, you were like an older pushy brother to me. You have dr. Celia Berkers and prof. dr. Ronald Wanders thank you for agreeing in being such your ways of doing stuff, but you always had my back! Thanks for everything! Johan, important pieces on my PhD. a special thank you to one of the nicest guys I have ever met. Keep on doing your amazing trips, recording them with your amazing photographic eye and spreading To my “metabolic” PhD colleagues and dear friends Lynne, Hanneke and Glen. Lynne, that enthusiasm. Keep strong my friend! you were the one that most chaperoned me and my PhD. You sat right (actually left) to me almost since the very beginning of my journey in Utrecht. We used to have Maaike Merlo, it’s funny how a failed Seahorse experiment brought such a great sunlight in our office, do you remember that? Many will never know that natural friendship. Thank you for all those cappuccino/coffee and cake breaks. With you I had light once entered that (huge) window! We did a lot of awesome stuff together: my first Dutch wedding experience! What a blast that day was!! For sure, you are one trips and congresses, singing (even in Dutch), dancing, eating and drinking (litters of the people I will forever keep in touch! of cappuccinos downstairs)! I will miss (already do) our talks and laughs. “I mean, it’s the end of an ERA”… thank GOD for FRIENDS. Dank je wel Lynne! Hanneke, you

172 173 Acknowledgements

To “my students” Maaike, Esmee and Emma a big thank you. You made me grow as Alsya, Samu and Bea, the remaining “LTI cool-gang”, you always invited me to your parties, a teacher (even though I am not one!), as a scientist and ultimately as a person. Your lunches and dinners, for all your friendship a big thank you! questions pushed me to be better at what I was doing and to constantly learn more! I wish you the greatest successes in your lives. To the beautiful people of the Biltstraat house, a huge thank you! You made my start in the Netherlands almost an easy task. Matteo, you were the first person I met in the house Aos meus paraninfos Sandra e Tiago um ENORME obrigado! Vocês dois acompanharam making you the oldest friend I have from this PhD adventure. I still remember the day I esta minha aventura quase desde o dia UM. Sandra, tu foste a primeira pessoa que met you in the other side of the hallway right across the door of my room. That same day conheci no WKZ. Lembro-me como se fosse hoje de descer as escadas no meu segundo we went to have dinner in the city centre – Kapsalon – do you remember? Thank you dia de trabalho e ouvir falar Português (com sotaque centro/norte, viste como não for always being present my friend! Silvia and Firdose, you were from FAR the two most disse nortenho?). Que sentimento bom! Parece que estava destinado a conhecer-te. important pieces in my adaptation to the Dutch life. Dottoressa Pisoni, it is very simple: Desde esse dia, posso dizer muito orgulhosamente, que és uma das minhas pessoas there are no words to express my gratitude and to tell you how important you were for my mais especiais! Perdi a conta aos “banhos” que tomamos quando voltavamos a casa mental health along these years. Your friendship and presence during that first year of my de bicicleta (engraçado tendo em conta que raramente chove nesse país). Obrigado PhD kept me focused and strong. And we will for sure be friends until the end of our lives! por sempre arranjares tempo para me ouvires, obrigado por tantos almoços (que tu Dosei, you were always a beacon of kindness and friendship in the house. Those dinners cozinhavas e partilhavas comigo) nos inúmeros fins de semana de trabalho no hospital, on the 2nd floor of the house will forever be engraved in my mind and heart! Thank you for e pelos milhares de cafés que tomamos juntos (na “tua minha” chaveninha)! Tiago, o everything! Kostas and Maria, my favourite Greek couple! Thank you for the many game segundo português que conheci na Holanda, e o meu outro paraninfo! Parece haver aqui nights and Greek goodies. I will always remember the cookie sale during King’s Day! Even um padrão! Muito obrigado por tudo meu amigo! Estiveste sempre lá, nas melhores e with all that rain we sold all of those YUMMY cookies (while eating almost as many as piores alturas. Obrigado por teres sempre uma palavra positiva! Muito obrigado a ambos we sold)! Thanks for keeping strong and being such a great example of friendship. To my por me honrarem em ter aceite ser os meus paraninfos! brilliant British friends, Aisha and Romin, it is true that sometimes stereotypes are wrong, but I do not recall ever in my life of drinking so much tea as when we were all living Friends are definitely the family we chose. Never has this sentence made more sense together. Funny enough, even after we went leaving on our own we still kept drinking or be truer than during this journey in the Netherlands. Going abroad without knowing tea (of course cocktails and beers also entered our drink-menu!) whenever we met. Ahhh anyone is a challenging deed. But I am a lucky guy, and in the Netherlands I met some good times! Thanks for everything! Pavla, thank you for all those evenings in the garden of the nicest and most beautiful people. People that I will carry with me and care for the of the house, where we chilled out and laughed while drinking a nice glass of wine! rest of my life. Aos meus brilhantes amigos Portugueses, dessas andanças FFULianas, que sempre me Ana, tu e o Tiago fizeram-me sempre sentir como família. Não houve feriado (Português apoiaram e a quem tenho obrigatoriamente de agradecer a enorme amizade: Sara, ou Holandês) que vocês me deixassem sozinho. Fui convidado assíduo da vossa casa Ruben, Paula, Nicolau, Andreia Carvalho e Andreia Luz, muito obrigado por tudo! Não em jantares, lanches, patuscadas e nos inevitáveis jogos do Glorioso. Muito obrigado seria justo tentar enumerar o quanto já passamos juntos, por isso aqui fica esta simples por tudo! Inês, a piquena, e última portuguesita a juntar-se a nós em Utrecht, mas que homenagem! OBRIGADO! aquisição!! Nunca me esquecerei desses fins de tarde a sofrer com a insanidade do sr. Shaun T em tua casa! Dias muito porreiros! O meu maior e mais sentido agradecimento vai para a minha família. Vocês mesmo longe nunca estão (nem estarão) ausentes. Obrigado mãe Maria e pai Manuel, vocês Clarinha (Chiaretta) and Joao Lucas (Gianluca), how nice it is to aportuguesar your sao os primeiros a sofrer nas minhas horas menos boas e os primeiros a celebrar as names! Grazie mille per tutto! Thank you for all your help and camaraderie. Gianluca I vitórias (por mais pequenas que sejam!). Vocês sao o meu Norte e o meu porto de would specially like to thank you for the beautiful art work of my thesis cover! You will abrigo! Muito obrigado por tudo e por me fazerem ser quem sou! Ao meu irmão forever be an essential part of this thesis! Thank you! Barbara, Daniele and Marzia the Emilio, cunhada Diana e o sobrinho mais lindo do mundo Simao um enorme obrigado rest of my Italian-LTI family! I will never forget you guys! Thank you for everything! Elena, por sempre acreditarem em mim! Esta tese é tanto minha quanto vossa!

174 175 List of publications

List of publications Coelho AI, Trabuco M, Ramos RJ, Silva MJ, Tavares de Almeida I, Leandro P, Rivera I, Vicente JB (2014) Functional and structural impact of the most prevalent missense mutations in classic galactosemia. Mol Genet Genomic Med 2(6):484-96. Van Karnebeek CDM*, Ramos RJ*, Wen XY*, Tarailo-Graovac M*, Gleeson JG, Skrypnyk C, Brand-Arzamendi K, Karbassi F, Issa MY, van der Lee R, Drögemöller BI, Koster J, Rousseau J, Campeau PM, Wang Y, Cao F, Li Santos D, Batoréu MC, Tavares de Almeida I, Davis Randall L, Mateus ML, Andrade V, Ramos RJ, Torres E, M, Ruiter J, Ciapaite J, Kluijtmans LAJ, Willemsen MAAP, Jans JJ, Ross CJ, Wintjes LT, Rodenburg RJ, Huigen Aschner M, Marreilha dos Santos AP (2013) Evaluation of neurobehavioral and neuroinflammatory end- MCDG, Jia Z, Waterham HR, Wasserman WW, Wanders RJA, Verhoeven-Duif NM, Zaki MS, Wevers RA (2019) points in the post-exposure period in rats sub-acutely exposed to manganese. Toxicology 314(1):95-9 Biallelic GOT2 mutations cause a treatable malate-aspartate shuttle related encephalopathy. Am J Hum Ventura F, Leandro P, Luz A, Rivera I, Silva M, Ramos RJ, Rocha H, Fonseca H, Gaspar A, Diogo L, Martins Genet 105(3):534-548. *These authors contributed equally to this work E, Leão-Teles E, Vilarinho L, Tavares de Almeida I (2014) Retrospective Study of the Medium-Chain Acyl- Kim SG, Becattini S, Moody TU, Shliaha PV, Littmann ER, Seok R, Gjonbalaj M, Eaton V, Fontana E, Amoretti CoA Dehydrogenase Deficiency in Portugal.Clin Genet 85(6):555-61. L, Wright R, Caballero S, Wang ZX, Jung HJ, Morjaria SM, Leiner IM, Qin W, Ramos RJ, Cross JR, Narushima Mendes MI, Colaço HG, Smith DE, Ramos RJ, Pop A, van Dooren SJ, Tavares de Almeida I, Kluijtmans S, Honda K, Peled JU, Hendrickson RC, Taur Y, van den Brink MRM, Pamer EG (2019) Microbiota-derived LA, Janssen MC, Rivera I, Salomons GS, Leandro P, Blom HJ (2014) Reduced response of Cystathionine lantibiotic restores resistance against vancomycin-resistant Enterococcus. Nature 572(7771):665-669. Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene Ramos RJ, Albersen M, Vringer E, Bosma M, Zwakenberg S, Zwartkruis F, Jans JJM, Verhoeven-Duif NM mutations in Homocystinuria patients. J Inherit Metab Dis 37(2):245-54. (2019) Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism. Biochim Coelho AI, Ramos RJ, Gaspar A, Costa C, Oliveira A, Diogo L, Garcia P, Paiva S, Martins E, Teles EL, Biophys Acta Gen Subj 1863(6):1088-1097. Rodrigues E, Cardoso MT, Ferreira E, Sequeira S, Leite M, Silva MJ, de Almeida IT, Vicente JB, Rivera I (2014) Caldeira-Araújo H, Ramos RJ, Florindo C, Rivera I, Castro R, Tavares de Almeida I (2019) Homocysteine A frequent splicing mutation and novel missense mutations color the updated mutational spectrum of Metabolism in Children and Adolescents: Influence of Age on Plasma Biomarkers and Correspondent classic galactosemia in Portugal. J Inherit Metab Dis 37(1):43-52. Genotype Interactions. Nutrients 11(3) pii:E646. Santos D, Batoreu MC, Almeida I, Ramos RJ, Sidoryk-Wegrzynowicz M, Aschner M, Marreilha Dos Santos Janeiro P, Jotta R, Ramos RJ, Florindo C, Ventura FV, Vilarinho L, Tavares de Almeida I, Gaspar A (2019) AP (2012) Manganese alters rat brain amino acids levels. Biol Trace Elem Res 150(1-3):337-41. Follow-up of fatty acid β-oxidation disorders in expanded newborn screening era. Eur J Pediatr Rivera I, Mendes D, Afonso A, Barroso M, Ramos RJ, Janeiro P, Oliveira A, Gaspar A, Tavares de Almeida 178(3):387-394. I (2011) Phenylalanine hydroxylase deficiency: Molecular epidemiology and predictable BH4- Rumping L, Tessadori F, Pouwels PJ, Vringer E, Wijnen JP, Bhogal AA, Savelberg SM, Duran KJ, Bakkers MJ, responsiveness in South Portugal PKU patients. Mol Genet Metab 104 Suppl:S86-92 Ramos RJ, Schellekens PA, Kroes HY, Klomp DW, Black GC, Taylor RL, Bakkers JP, Prinsen HC, Knaap MS, Castro R; Barroso M, Rocha M, Esse R, Ramos RJ, Ravasco P, Rivera P, Tavares de Almeida I (2010) The TCN2 Dansen TB, Rehmann H, Zwartkruis FJ, Houwen RH, Haaften G, Verhoeven-Duif NM, Jans JJ, Hasselt PM 776C>G polymorphism correlates with vitamin B12 cellular delivery in healthy adult population. Clin (2018) GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay. Biochem 43(7-8):645-9. Hum Mol Genet 28(1):96-104.

Jeanclos E, Albersen M, Ramos RJ, Raab A, Wilhelm C, Hommers L, Lesch KP, Verhoeven-Duif NM, Gohla A (2018) Improved cognition, mild anxiety-like behavior and decreased motor performance in pyridoxal phosphatase-deficient mice. Biochim Biophys Acta Mol Basis Dis 1865(1):193-205.

Ramos RJ, Pras-Raves ML, Gerrits J, van der Ham M, Willemsen M, Prinsen H, Burgering B, Jans JJ, Verhoeven-Duif NM (2017) Vitamin B6 is essential for serine de novo biosynthesis. J Inherit Metab Dis 40(6):883-891.

Massafra V, Milona A, Vos HR, Ramos RJ, Gerrits J, Willemsen ECL, Ramos Pittol JM, Ijssennagger N, Houweling M, Prinsen HCMT, Verhoeven-Duif NM, Burgering BMT, van Mil SWC (2017) Farnesoid X Receptor Activation Promotes Hepatic Amino Acid Catabolism and Ammonium Clearance in Mice. Gastroenterology 152(6):1462-1476.e10.

Wamelink MM, Ramos RJ, van den Elzen AP, Ruijter GJ, Bonte R, Diogo L, Garcia P, Neves N, Nota B, Haschemi A, Tavares de Almeida I, Salomons GS (2015) First two unrelated cases of isolated sedoheptulokinase deficiency: A benign disorder? J Inherit Metab Dis 38(5):889-94.

176 177 Curriculum Vitae

Rúben Ramos was born in Évora, Portugal, on 18 April 1984. He grew up in Avis, a picturesque medieval village in the heart of Alentejo. At the age of 15 he moved to Évora for his secondary education. After graduating secondary school, with the age of 18, he moved to Lisbon to study Pharmaceutical Sciences at the Faculdade de Farmácia da Universidade de Lisboa, Portugal, receiving is MSc degree in 2008. In the same year he started working as a senior technician at the Research Institute for Medicines and Pharmaceutical Sciences (iMed.ULisboa), in the Metabolism and Genetics group. There he received training in the field of rare metabolic diseases, being especially involved in the diagnosis and follow-up of patients with inborn errors of metabolism disorders. In 2009 he started a second MSc degree in Clinical Chemistry in the Faculdade de Farmácia da Universidade de Lisboa. He graduated in 2013, under the supervision of Prof. Isabel Tavares de Almeida. His work, in collaboration with Prof. Cornelis Jakobs and Dr. Mirjam Wamelink from the Free University of Amsterdam, the Netherlands, allowed the characterization of a new metabolic disorder in the pentose phosphate pathway. In 2014 he moved to the Netherlands to conduct his PhD studies in the department of Genetics at the University Medical Center Utrecht, under the supervision of Prof. Nanda Verhoeven-Duif and Dr. Judith Jans. His project focused on the metabolism of vitamin B6, especially aimed on understanding the intracellular metabolic consequences of vitamin B6 deficiency and how to improve diagnosis and treatment of vitamin B6 deficiencies. Rúben currently works as a senior research assistant at the department of Cell Metabolism at Memorial Sloan Kettering Cancer Center in New York, United States of America. The results of his PhD research are the subject of this thesis.

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