Novel CSF biomarkers of GLUT1 deficiency syndrome: implications beyond the brain’s energy deficit

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic CSF profiles from 11 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from O- glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O -glucosylation. Since many proteins are O -glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The study was funded by Stofwisselkracht under the project name of "Innovative diagnostics in cerebrospinal fluid of patients with neurometabolic disorders" (KLM Coene and MM Verbeek). MAAP Willemsen received an unrestricted grant from the Glut1 Deficiency Foundation (Owingsville, USA). The authors gratefully acknowledge the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) for the support of the FELIX Laboratory. This work was supported by the NWO division Exact and Natural Sciences (grant numbers TTW 15769, TKI-LIFT 731.017.419) and Radboud University through an interfaculty collaboration grant. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Commissie Mensgebonden Onderzoek Radboudumc of the Radboud University Medical Center gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data that support the findings of this study are available from the corresponding author, upon reasonable request. * 2DOG : 2-deoxy-D-glucose 3OMG : 3-O-methyl-D-glucose DHA : dehydroascorbic acid DKG : diketogulonic acid EGF : epidermal growth factor-like GLUT1DS : glucose transporter type 1 deficiency syndrome HILIC-IRIS : hydrophilic interaction liquid chromatography infrared ion spectroscopy HMDB : human metabolome database IEM : inborn error of metabolism IP-LC-MS/MS : ion-pair liquid chromatography tandem mass spectrometry KDT : ketogenic diet therapy m/z : mass-to-charge ratio NGMS : next-generation metabolic screening PPP : pentose-phosphate pathway QC : quality control RPLC-MS : reversed-phase liquid chromatography mass spectrometry RT : retention time Xyl-Glc : xylose-α1-3-glucose Xyl-Xyl-Glc : xylose-α1-3-xylose-α1-3-glucose