G C A T T A C G G C A T genes Article The Alkaline Phosphatase (ALPL) Locus Is Associated with B6 Vitamer Levels in CSF and Plasma Loes M. Olde Loohuis 1,† , Monique Albersen 2,†,‡, Simone de Jong 1, Timothy Wu 1 , Jurjen J. Luykx 3,4 , Judith J. M. Jans 2, Nanda M. Verhoeven-Duif 2 and Roel A. Ophoff 1,* 1 Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles (UCLA), Los Angeles, CA 90095, USA; [email protected] (L.M.O.L.); [email protected] (S.d.J.); [email protected] (T.W.) 2 Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC), 3584 EA Utrecht, The Netherlands; [email protected] (M.A.); [email protected] (J.J.M.J.); [email protected] (N.M.V.-D.) 3 Department of Psychiatry, Rudolf Magnus Institute of Neuroscience, University Medical Center (UMC), 3584 CG Utrecht, The Netherlands; [email protected] 4 Department of Translational Neuroscience, Human Neurogenetics Unit, Brain Center Rudolf Magnus, University Medical Center Utrecht (UMC), 3584 CG Utrecht, The Netherlands * Correspondence: [email protected] † These authors contributed equally to this work. ‡ Present address: Amsterdam UMC, VU Medical Center, Department of Clinical Chemistry, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Received: 3 December 2018; Accepted: 14 December 2018; Published: 22 December 2018 Abstract: The active form of vitamin B6, pyridoxal phosphate (PLP), is essential for human metabolism. The brain is dependent on vitamin B6 for its neurotransmitter balance. To obtain insight into the genetic determinants of vitamin B6 homeostasis, we conducted a genome-wide association study (GWAS) of the B6 vitamers pyridoxal (PL), PLP and the degradation product of vitamin B6, pyridoxic acid (PA). We collected a unique sample set of cerebrospinal fluid (CSF) and plasma from the same healthy human subjects of Dutch ancestry (n = 493) and included concentrations and ratios in and between these body fluids in our analysis. Based on a multivariate joint analysis of all B6 vitamers and their ratios, we identified a genome-wide significant association at a locus on chromosome 1 containing the ALPL (alkaline phosphatase) gene (minimal p = 7.89 × 10−10, rs1106357, minor allele frequency (MAF) = 0.46), previously associated with vitamin B6 levels in blood. Subjects homozygous for the minor allele showed a 1.4-times-higher ratio between PLP and PL in plasma, and even a 1.6-times-higher ratio between PLP and PL in CSF than subjects homozygous for the major allele. In addition, we observed a suggestive association with the CSF:plasma ratio of PLP on chromosome 15 (minimal p = 7.93 × 10−7, and MAF = 0.06 for rs28789220). Even though this finding is not reaching genome-wide significance, it highlights the potential of our experimental setup for studying transport and metabolism across the blood–CSF barrier. This GWAS of B6 vitamers identifies alkaline phosphatase as a key regulator in human vitamin B6 metabolism in CSF as well as plasma. Furthermore, our results demonstrate the potential of genetic studies of metabolites in plasma and CSF to elucidate biological aspects underlying metabolite generation, transport and degradation. Keywords: genome wide association study; vitamin B6; cerebrospinal fluid; plasma 1. Introduction The active form of vitamin B6, pyridoxal phosphate (PLP), functions as a co-factor in >200 enzymatic reactions in human metabolism [1,2]. Inverse relationships have been found between Genes 2019, 10, 8; doi:10.3390/genes10010008 www.mdpi.com/journal/genes Genes 2018, 9 FOR PEER REVIEW 2 of 14 1. Introduction The active form of vitamin B6, pyridoxal phosphate (PLP), functions as a co-factor in >200 Genes 2019, 10, 8 2 of 14 enzymatic reactions in human metabolism [1,2]. Inverse relationships have been found between vitamin B6 and conditions such as diabetes, oxidative stress, cardiovascular disease, inflammation vitaminand cancer B6 and [3–7]. conditions Its role such in amino as diabetes, acid oxidativeand neurotransmitter stress, cardiovascular metabolism disease, renders inflammation vitamin and B6 cancerspecifically [3–7]. essential Its role infor amino brain aciddevelopment and neurotransmitter and functioning. metabolism Lower concentrations renders vitamin of PLP B6 specifically in plasma essentialhave not foronly brain been development associated with and functioning.symptoms of Lower depression concentrations (according of to PLP the in Major plasma Depression have not onlyInventory been associated[8]), but also with with symptoms poor cognition of depression [9] and (according Alzheimer’s to thedisease Major [10,11]. Depression Inventory [8]), but alsoThe with dependence poor cognition of the [neurotransmitter9] and Alzheimer’s balance disease in [ 10the,11 brain]. on vitamin B6 is illustrated by inbornThe errors dependence of metabolism of the neurotransmitter resulting in hampered balance in thevitamin brain onB6 vitaminmetabolism. B6 is illustratedSuch patients by inborn may errorspresent of with metabolism epilepsy resulting and intellectual in hampered disability vitamin [12]. B6 metabolism. Examples Suchof these patients genetic may disorders present with are epilepsyantiquitin and deficiency intellectual (OMIM disability #266100) [12]. [13], Examples pyridox(am)ine-5 of these genetic′-phosphate disorders oxidase are antiquitin (PNPO) deficiencydeficiency (OMIM(OMIM #266100)#610090) [[14],13], pyridox(am)ine-5hyperprolinaemia0-phosphate type II (pyrroline-5-carboxylate oxidase (PNPO) deficiency dehydrogenase (OMIM #610090) deficiency; [14], hyperprolinaemiaOMIM #239510) [15] type and II (pyrroline-5-carboxylate hypophosphatasia (alk dehydrogenasealine phosphatase deficiency; (ALPL) OMIM deficiency; #239510) OMIM [15] and#241500) hypophosphatasia [16,17]. Although (alkaline in phosphataseantiquitin and (ALPL) PNPO deficiency; deficiencies, OMIM treatment #241500) with [16,17 vitamin]. Although B6 inis antiquitinsuccessful andin antagonizing PNPO deficiencies, the epilepsy, treatment patients with with vitamin these B6 inborn is successful metabolic in antagonizing disorders will the still epilepsy, suffer patientsfrom intellectual with these disability inborn metabolic [12,18]. In disorders addition will to stillthese suffer known from causes intellectual of functional disability vitamin [12,18]. B6 In additiondeficiency, to there these knownare patients causes in ofwhom functional the caus vitamine of their B6 deficiency, vitamin B6-responsive there are patients epilepsy in whom remains the causeunelucidated. of their vitamin B6-responsive epilepsy remains unelucidated. Humans depend depend on on dietary dietary sources sources of ofvitamin vitamin B6 since B6 since they theyare unable are unable to synthesize to synthesize it. The it.different The differentB6 vitamers—pyridoxine B6 vitamers—pyridoxine (PN), pyridoxami (PN), pyridoxaminene (PM), pyridoxal (PM), pyridoxal(PL) and their (PL) respective and their respectivephosphate-esters phosphate-esters (Figure 1)—are (Figure interconvertible1)—are interconvertible through the through action theof several action ofenzymes. several enzymes.Transport Transportacross the acrosscell membrane the cell membrane is preceded is by preceded hydrolysis by hydrolysisof the phosphorylated of the phosphorylated B6 vitamers B6by vitamersmembrane- by membrane-boundbound alkaline phosphatase alkaline phosphatase (ALPL) [19]. (ALPL) The ALPL [19]. locus The ALPLhas been locus associated has been with associated PLP and with vitamin PLP andB6 in vitamin plasma B6 [20–23]. in plasma Intracellularly, [20–23]. Intracellularly, phosphorylat phosphorylationion by pyridoxal by kinase pyridoxal (PDXK) kinase [24] (PDXK)yields PNP, [24] yieldsPMP and PNP, PLP PMP (Figure and PLP1). Pyridox(am)ine (Figure1). Pyridox(am)ine phosphate oxidase phosphate converts oxidase PNP converts and PMP PNP into andthe active PMP intoco-factor, the active PLP co-factor,[25]. Release PLP of [25 vitamin]. Release B6 offrom vitamin the cell B6 fromis dependent the cell is on dependent a vitamin on B6-specific a vitamin B6-specificphosphatase phosphatase (pyridoxal phosphatase (pyridoxal phosphatase(PDXP)) [26]. (PDXP))Oxidation [26 of]. the Oxidation resulting PL of by the aldehyde resulting oxidase PL by aldehyde(AOX) [27] oxidase constitutes (AOX) the [ 27degradation] constitutes pathway the degradation of vitamin pathway B6, of which of vitamin the major B6, ofproduct, which thepyridoxic major product,acid (PA), pyridoxic is excreted acid into (PA), urine is excreted (Figure into 1) urine [28]. (FigureAlthough1)[ 28the]. Althoughenzymes theinvolved enzymes in involvedvitamin B6 in vitaminmetabolism B6 metabolism have been elucidated have been at elucidated genetic and at genetic protein and levels, protein knowledge levels, knowledgeabout human about vitamin human B6 vitamintransport B6 is transport very limited. is very At limited.the biochemical At the biochemical level, there level, is evidence there is for evidence carrier-mediated for carrier-mediated transport transport[29–31], but [29 not–31 ],a single but not human a single vitamin human B6 vitamin transporter B6 transporter has been identified has been identified to date. to date. Figure 1.1. Schematic display of vitamin B6 metabolism.metabolism. The different B6 vitamers pyridoxine (PN), pyridoxaminepyridoxamine (PM), pyridoxal (PL) and their respective phosphate-estersphosphate-esters (PNP, PMP and PLP) are interconvertibleinterconvertible