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2017Glut1DeficiencyFounda5onConferenceSummaryReport

gratitudeandcredittoKrisEngelstadandallthespeakersforhelpingpreparethepresentationsummaries

GlucoseTransporterType1De;iciencySyndromeisregularlyreferencedusingavarietyofterms,andtheseindividualsummarieswerenoexception.Intheinterestofclarityanduniformity,we

haveusedthetermGlut1De)iciencythroughoutthesummary.

GlucoseTransporterType1De;iciencySyndromeisalsoknownandreferencedas:Glut1De;iciency,G1D,Glut1DS,Glut-1DS,Glut1,Glut-1,Glut1,GLUT1,Glut1D,andDeVivoDisease

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GeneralAssemblyPresentationsagendaorder,preparedbyKrisEngelstadandeditedbypresenters

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Prof.Dr.JörgKlepperGlut1De;iciencyFromPediatricstoAdulthood

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JuanPascual,MD,PhDGlut1De;iciencyin2017andBeyond

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VincentPetit,DVM,PhDNewlyDevelopedTestforGlut1De;iciency

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KarthikRajasekaran,PhDAssayingDrugsforSafetyinGlut1De;iciency

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DominicD’Agostino,PhDSignalingPropertiesandTherapeuticEffectsofKetones

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EricKossoff,MDDietTherapyforGlut1De;iciency

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MackenzieCervenka,MDKetogenicDietTreatmentsandTransitionStrategies

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UmraoMonani,PhDGeneReplacementTherapyforGlut1De;iciency

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AbrahamAl-Ahmad,PhDModelingtheBlood-BrainBarrierUsingPatientDerivedStemCells:AFocusonModelingGlucoseTransport

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2017Glut1De)iciencyFoundationConferenceSummary

TableofContents

Prof.Dr.JörgKlepperChildren’sHospitalAschaffenburgAschaffenburg,Germany

Glut1De)iciencyfromPediatricstoAdulthood

BACKGROUND:GlucoseistheessentialfuelforthebrainandentersthebrainthroughtheglucosetransporterGlut1.Withoutenoughglucosethereisanenergycrisisinthebrain:glucoseandlactatelevelsarelowindicatingGlut1De;iciency.AnovelbloodtestidentifyingthereducednumberofGlut1transportersonredbloodcells(quantitativeGlut1De;iciency)hasrecentlybeendevelopedinFrance.MutationstheSLC2A1geneoftencon;irmthediagnosisbuttheabsenceofmutationsdoesnotexcludeGlut1De;iciency.The“brainenergycrisis”resultsinthreecardinalsymptoms:epilepsy,movementdisorders,andcognitive/behavioralissues.AnycombinationandanydegreeofsymptomshavebeendescribedinGlut1De;iciency.

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Legendof)igure1:Ininfancyandearlychildhoodseizuresanddevelopmentaldelayaretheprominentsymptomsthatstabilizebeyondpuberty.Incontrast,ataxia,dystonia,andparoxysmaleventsareissuesofchildhoodandadolescence.

GLUT1DEFICIENCYTHROUGHTHEDEVELOPMENTALSTAGES:

InfantsandEarlyChildhood:TheinitialpresentationofGlut1De;iciencyisoftenaseizurewithinthe;irstsixmonthsoflife.Seizurestypesarevariableandcanincludecyanoticspells(turningblue),absence(dreaming),focal(onepartofthebody),generalized(wholebody),myoclonic(musclespasms),andastatic(dropattacks).Ininfantsparoxysmaleye-headmovementspresentasaberrantgazesaccades(jumpinglikeeyemovements)withheadandeyesmovingaround.Oftentheyarethe;irstsignofGlut1De;iciency.Ininfancyaclassical3:1ketogenicdietshouldbeused.Inearlychildhooddevelopmentalimpairmentbecomesapparent.Gaitoftenisnotnormalforage(clumsy,widebased,drunkenappearance).10%ofabsenceepilepsiesstartinginthisagehavebeenshowntobeGlut1De;iciency.

Childhood: Inchildhoodandadolescencemovementdisorderstendtoworsen.Inschoolagetherecanbeabnormalgaitsuchas:broadbasedgait,dystonia,ataxia.Cognitionshiftstowardlowercognitiverange,butnotallpatientshavecognitiveissues.Speci;icimpairmentsmaybeproblemsinvisualattention,motorskillsactivelanguage,andwholepictureprocessing(can’tseetheforestforthetrees).Incontrast,strengthsinGlut1De;iciencypatientsincludelanguagecomprehension,socialinteraction,gentleness,andslower,butconstantprogressindevelopment.Theuseofketogenicdietsisvariable,increasinglythemodi;iedAtkinsDiet(MAD)isusedinGlut1De;iciency,butdataonlong-termdevelopmentaloutcomeisnotyetavailable.

Adolescence:Inadolescence¾ofGlut1De;iciencypatientsreportparoxysmal(suddenonset)eventssuchassuddenspells,motorarrest,choreaticordystonicinvoluntarymovements,drops,orotherunspeci;ieddiscomfort.Theseepisodesoftenstartinpuberty.Triggersmaybephysicalactivity,lossofketosis,andsleepdeprivation.Oftentheseeventsoccurdespiteadequateusofketogenicdietsandthuscurrentlyaredif;iculttotreat.AdolescentsoftencomplybetterwhentheModi;iedAtkinsDietisused.Thelowglycemicindexdiet(LGIT)isnotrecommendedinGlut1De;iciency.

Adults:InadulthoodsymptomsofGlut1De;iciencyoftenstabilize.Inmostcasesepilepsyiscontrolledbymildketogenicdietsand/oranticonvulsantdrugs.Ketogenicdietsareoftenreducedtothemodi;iedAtkinsDiet(MAD)ordiscontinued–itstillremainsunclearhowlongdietarytreatment

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shouldbecontinuedinadults.Paroxysmaleventsinadultsoccurandhavebeentermed“Paroxysmalexertiondystonia(PED)”.Theyaredif;iculttotreatandmayalsopresentasmigraine,writer’scramp,andalternatinghemiplegia.

MostpatientswithGlut1De;iciencywillshowcognitiveimpairmentofvariousdegreesandwillrequireshelteredenvironments,althoughsomepatientssuccessfullyobtainedacollegedegreeandareworkingandlivingindependently.Long-termadverseeffectsofhigh-fatketogenicdiettreatmentsuchaskidneystones,growthimpairment,orcardiovascularsideeffectsremainaconcern,butpreliminaryresultsindicatethatsuchsideeffectsmaybeoverrated.Westudied10Glut1De;iciencypatientsonketogenicdiets:after10yearsonthedietlipidparametersremainedwithinthenormalrangeandultrasoundofthecarotidarteriesdidnotindicateatherosclerosis(KlepperJetal,submitted).

OUTLOOK:Glut1isexpressedinothertissuessuchasmuscle,retina,placenta,heart.PotentiallytheGlut1defectcouldalsoaffectthesetissues,butsofarnoinvolvementoftheseorganshasbeenreported-theremaybecompensatorymechanismsbyotherGlut1glucosetransportersinthesetissues.

ForpatientswithGlut1De;iciencyitisrecommendedtocontinuetheketogenicdietintoadulthood.Additionaltreatmentoptionsmaybetheuseofketoneesters,suchastriheptanoin(C7)orotherarti;icialketones.Ketoneestersserveasfueltothebrainjustasketones,buthavetheadditionaleffectofre;illingtheTCAcycle(anaplerosis)thatgeneratesenergy.C7hasbeenshowntoworkinGlut1-de;icientmiceandthe;irstclinicaltrialsinGlut1De;iciencyarecurrentlyunderway.

Dr.DeVivoandhisteamatColumbiaUniversity,NYCaresuccessfullyworkingongenetherapyforGlut1De;iciency.ResultsarepromisingusingaviralvectorcarryingtheGlut1geneintocellstocorrectthegeneticdefect,butgenetherapyinpatientswillnotbeavailableforsometime.

TogeneratemoredataaboutGlut1De;iciencyworldwideanonlinepatientregistry(www.G1DRegistry.org)hasbeengenerated.AllfamilieswithGlut1De;iciencyarerequestedtoentertheirdatatoprovidenovelinsightsintoincidence,clinicalpresentation,spectrumofmutations,dietarytreatment,andsideeffects.

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JuanPascual,MD,PhDUTSouthwesternMedicalCenter Dallas,Texas

Glut1De)iciencyin2017andBeyondHowmuchenergydoesthebrainconsumecomparedtoalightbulb?Thinkoftheenergyina60Wbulb-anadultuses500W,achilduses1500W.

Neuroscientistsdothefollowing:diagnose,comprehend,andtreat.NeuroscientistsusemanydifferentdatasourcestostudyGlut1De;iciency:mousemodels,typicalhumanbrain,Glut1De;iciencypatients.WehavealotofdataaboutpatientsintheG1DRegistry.

Forenergymetabolismthebodyhasmanywaystogettothesameplace(i.e.nourishingthebrain).Wewonderifthereisadifferentwaytonourishthebrain.Glucoseismetabolized.Neurotransmittersaremade(glutamine,glutamate,GABA):ifthereisaproblemwithglucosegettingintothebrainthenthereisaproblemwiththeconstructionofneurotransmitters(whicharethenervesignalingpartofthebrain).

Brainmetabolismisinvolvedwithtwomechanisms:1)buildingchemicals(calledanabolism)orburning/breakingdownchemicals(calledcatabolism)andthebuildingblocks(eg.glucose)mustdoboth.Anaplerosisinvolveskeepingthe;lame(energyinthebrain)going.

PETscansinGlut1De;iciencyshowlowglucoseinbrain,especiallyinthalamusandthecerebralcortex,andthisisn’tnormal.AllindividualswithGlut1De;iciencyhavethis;inding.

WhatdoestheGlut1moleculelooklike? WheredothemutationsaffecttheGlut1molecule?

Themutationstendtohappenononeareaoftheprotein.Thereseemstobenocorrelationbetweenmutationsandsymptoms.Wesaw7patientswiththeR333Wmutationandtheyareallsomewhatdifferentinphenotype.

G1DRegistry:TheG1DRegistryisusefulinobtainingdataaboutpatientsastheyentertheirowndataintotheregistry.DataenteredincludesmanyquestionsaboutpatientswithGlut1De;iciencysuchas:medicalhistory,allissues,patterns,etc.Theregistryisasecurewebsite,HIPAAcompliant,andbehinda;irewall.PascualandRonenpublishedapaperontheregistryinPediatricNeurology2015(youcan;indthisonpubmed.com)

ManytypesofGlut1De;iciencyarenotedfromthisregistryandwewantedtoknowwhatisthetypicalpatientwithGlut1De;iciencylike.

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ListedbelowiswhatapatientwithGlut1De)iciencymighthaveatvariousages:3months-intermittentinvoluntarygaze6months-fragmentaryseizures1year–absenceseizures3yearsdysarthria6years-strongsocialskillsPuberty-ameliorationandmovementdisordersAdults-obsessivecompulsivetraits

UsingmicetolearnmoreaboutGlut1De)iciency:ThemousewithG1Dcanhelpuslearnaboutthedisease.TheG1Dmousehasseizures,ataxia,lowbrainglucose.WecandoEEG’sonmiceandalsoprovideaccesstobloodveinstodeliverlabelingsubstancesatthesametime.Withthismouse,wecanstartto;indoutinformationsuchas:whereinthebraintheseseizurescomefrom?

WeknowthatthewholebrainhasEEGactivityatthesametime.WecanalsousefunctionalMRI’s(FMRI)tomeasurebrainactivity(notbrainstructure).InFMRI’sweseeregionsofthebrainthatareactivewhenyouhaveaseizures.Thesomatosensorycortexandthethalamusareespeciallyactiveduringaseizure;thisisalsowhereweseelowglucoseonPETscans.Wewillfocusourattentiononthecortexandthethalamus.

Ifyoutakeasectionofamousebrain(justasmallsection-aslongasyouhaveapartofthecortexandthethalamus)theslicecanhaveaseizure.

Weaskwhyaretheseareassohyper-excitedthattheyhaveseizures?Wecanrecordelectricalactivityfromaverysmallsliceofbraininthelaboratory.Ifthereisaproblemwithcelltocellcommunicationinthebrainaseizurecanoccur.

Twokindsofactivityinthebrainareexcitationandinhibitionandtheremustbeabalanceoryouwillhaveseizures.ExcitationisgenerallynormalinthecortexofGlut1De;iciencypatients.Whereas,inhibitionisgenerallyverylowinthepatients’cortex.Thesamethinghappensinthethalamus.

Inthethalamusthereisonespeci;iccelltypethatisdisinhibitedandisalwaysreadyto;ireatalltimes.Thecellisthereticularcellofthethalamus.ThusifwecanblockthiscelltypefrombeingdisinhibitedmaybethiscouldhelpGlut1De;iciencypatients.Wewillbelookingintothis.Andwewillthinkaboutifthereisanythingthatwecantreatpatientswiththisinmind?

Weknowthefollowingabouttreatment:• WeneedtodevelopMRIdatatolookatbrainmetabolisminmice.• WecantreatGlut1De;iciencywithalternativebrainfuel.• GlucosegoesthroughKrebscycle(theKrebscycleispartofthemetabolicpathway).

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• Triheptanoinisamediumchaintriglyceridewith7carbonatoms(naturemakesevencarbonmolecules)andthisisimportantasitcanfueltheKrebscycleinawaythatevencarbonchainscan’tdo.

WeareengagedinclinicaltrialswithG1Dpatientsandtriheptanoin:Astandardregulardietis65%carbsandproteinand35%fat.Astandardketogenicdietof4:1is90%fatand10%carbsandprotein.Triheptanoinisprovidedat35%withcarbsandproteinat60%withanextra5%essentialfats.Wearealsotrying45%triheptanoin.

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VincentPetit,DVM,PhDMETAFORABiosystemsEvryCedex,France

NewlyDevelopedTestforGlut1De)iciency

BACKGROUND:

Nutrienttransportersareusedincellsintheprocessofmakingenergy.

Weareinthebusinessofdevelopingligandsthatbindtonutrienttransporters.Wecanusethesetotrackthefunctionandeffectofthesetransporters.Wecandetermineifthereareunderoroverconsumednutrients.Wedeveloptestsformetabolismincludingenergydemandincells.

Glut1De;iciencyinvolvesabloodbrainbarrierproteincalledGlut1protein.WeknowthatthereisalongtimetodiagnosisinpatientswithGlut1De;iciency.WewantedtodevelopatestthatcutsdownthattimebyevaluatingtheGlut1proteinfunction.

Weuseredbloodcellsfromapatientandattachaspeci;icligandtotheGlut1proteininthelaboratory.ThencalculatetheamountofGlut1proteinexpressedonredcells.

Thisisasimplebloodtestthatdoesn’trequirefastinganditisfullyautomated;thusdoesn’trequiremuchtechniciantime.Wehaveareportonthistechniqueinthejournal“annalsofneurology”(2017).Therewere30patientsandcontrolsinthepaper.WedeterminedthenormalrangeofGlut1expressionlevelinthecontrolpopulation,anddemonstratedthatinmostGlut1De;iciencypatients,thevaluesdroppedbyatleast20%.SomeofthevaluesfortheGlut1De;iciencypatientsfellinthenormalrange,butnocontrolvaluewasbelow80%.Sothesensitivityofthetestisn’t100%butitisveryspeci;ic.ThisissimilartoCSFglucoseasameasure.

Weranvaliditystudies-i.e.teststoseeifourresultswouldbethesameifwere-ranthetestdata.Thetestisveryreproducible.

Overallthetestis:rapid,non-invasive(afewdropsofbloodneeded),andspeci;ic.Itmaybeusefulin;indingpatientswithnon-commonphenotypes.

Thetestisstillinproductionmode.Currentlyitisnotreimbursablebyinsurance.

WehopetomakethetestavailabletotheUSA,Europeandothercountriesassoonaspossible.

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KarthikRajasekaran,PhDUTSouthwesternMedicalCenterDallas,Texas

AssayingDrugsforSafetyinGlut1De)iciencySyndrome

Glucoseisrequiredforbrainfunction.Glucoseistransferredfromthebloodtothebrainthroughthebloodbrainbarrier.Glucoseismetabolizedthroughglycolysis.Ifthereislessglucoseenteringthebrainthenthereislessglycolysis.GlucoseisbrokendownintoacetylCo-AandproducesATPaswellasneurotransmitters.Oneoftheneurotransmittersisglutamate;whichisanexcitatoryneurotransmitter(whenneuronisexposedtoglutamateit;ires).Glutamateisalsoconvertedintoaninhibitoryneurotransmitter,gamma-aminobutyricacid(GABA).WhenaneuronisexposedtoGABAitissilent(doesn’t;ire).Neuronsneedabalancebetweenexcitationandinhibition,andseizureshappenwhenthebalanceistitledinfavorofexcitationoverinhibition.

Therapeuticoptions-Anti-EpilepticDrugs(AED’s):TherearenogoodtherapeuticoptionsforGlut1De;iciency;seizuredrugsarenotveryuseful.Sometimesnormalanti-epilepsydrugsthatapparentlyworkbyboostinginhibitioncanparadoxicallyworsenoutcomes.

Wecantestanti-epilepticdrugs(AED’s)inthelaboratorywithanimalmodelstoseewhathappens.

ThereisamousemodelofGlut1De;iciency.ThesemicearemorepronetogeneralizedtonicclonicseizureswhichisoftenoneoftheinitialpresentationsofGlut1De;iciency.Inthemousemodel,weinjectmicewithchemicalsthatcancauseseizures.WhatistheminimumamountofaseizureinducingchemicalanormalmouserequiretohaveseizuresversusGlut1De;iciencymouse?Theminimumamountatwhich50%oftheanimalshaveseizuresiscalledtheeffectivedose(ED5).We;indthiseffectivedosebyinjectinganimalswithdifferentdosesofdrugsandthenstatisticallyanalyzingtheresponseoutcomes(doseresponserelationship).Wildtype(non-diseased)miceneedmoredrugtohaveaseizurethanaGlut1De;iciencymouse.

Whenwegivethechemicalpilocarpinetoinduceseizuresinthemice,weseethatGlut1De;iciencymicearemorepronetoGTCseizures.Weevaluatewhethertriheptanoinmightpreventseizures.WealsoevaluatewhetherdiazepamcanterminateseizuresinGlut1De;iciencymice.

Diazepam:Whenpilocarpineisinjectedandweadddiazepam30secondslater-whathappens?

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Inwildtypemicethereiscessationofseizureswithdiazepam.However,ifwewaitlongenoughtostartdiazepamthereisaproblemwithseizures.

ForGlut1De;iciencymice,postpilocarpine,thereisnoresponsetodiazepam;andastheanimalagestheresponsetoevenhigherdoseofdiazepamisevenworse.Weknowthattheseanimalsdonotrespondtodiazepam.

Phenobarbital:Ifpilocarpineisinjectedandthenphenobarbital(1or30minuteslater);wegetdoseresponseinthewildtypeanimal.WegetED50valueinthewildtypemice.

InGlut1De;iciencymicethereisrespiratorydistresswithnormaldosesofphenobarbitalthatcausesED50inwildtypeanimalswithphenobarbital.

Trihpetanoin:Ontheotherhand,wehaveseenthattriheptanoinelevatestheseizurethreshold(i.emakesitmoredif;iculttohaveaseizure).TheED50doseforGlut1De;iciencyanimalsisgreaterthanwithouttriheptanoin.

AproposalforaHighThroughputscreening:TakentogetherwithdatafromDr.Klepper’sresearch,webelievethatdiazepamandphenobarbitalmayantagonizethefunctionoftheGlut1transporterpotentiallyworseningseizureoutcomes.

Notonlyisthissortofnegativeinteractionpossiblewithantiepilepsydrugs,butitisjustaspossiblewithotherdrugsthatGlut1De;iciencypatientsmaybeexposedtoforthemanagementofillnessesotherthanseizures.OurdesireandproposalistocreatealibraryofthecommonlyuseddrugsthatmaypotentiallycontraindicateGlut1function.Suchalibraryofpotentialcontraindicationofcommonlyusedoutpatientdrugsdoesnotcurrentlyexist.It’savailabilitycaninformandmakephysiciansandpatientfamiliesawareofpotentialnegativeinteractions–insomecasestheremaybeanopportunitytochooseanotherdrugfortheconditionthatdoesnothavethesenegativeeffects.

AhighthroughputwaytoidentifyharmfuldrugsbystudyingGlut1translocation.uptakeassays:WhenGlut1isonthesurfaceofthecellmembrane,itcanfunctionproperly.Whenitiswithinthecell,thenitisnotavailabletotransportglucose.WecoulduseahighthroughputlaboratoryassaytotestdrugsthatcanpotentiallyworsensymptomsinGlut1De;iciencybytranslocatingthemfromthecellsurfacetotheinsideofthecell.ThiscanbeaccomplishedusingculturedneuronsonwhichtheGlut1proteinis;luorescentlytagged,anddependinguponlocationinthecell(i.e.itspresenceonthecellsurfaceorwithinthecell)willemitastrongorweaksignal.Wecanadddifferentdrugsanddrugsindifferentconcentrationstodeterminewhichdrugsandatwhatdosemaypotentially

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antagonizeGlut1function.WecanalsousethissystemtounderstandhowlongitmaytakeforadrugtopotentiallyaffectGlut1location.

Asecondwayofidenti)icationfortheseharmfuldrugsisuptakeassays:Itisalsopossiblethatdrugsmayjustimpactglucoseuptakewithoutgettingtranslocated.Wehaveaglucoseuptakeassayusingastrocytes/neuronculturesthatcanbeexposedtodifferentconcentrationsofdifferentkindsofdrugs.Wecancreateadoseresponsecurveto;inddrugsthatinhibitglucoseuptake.Wehopeto;indagroupofdrugsthatareandareNOTtobeusedinpatientswithGlut1De;iciency.

Finallywecantestdrugsidenti;iedtoaffectGlut1translocationoruptakeintheanimalmodelofGlut1De;iciency.HerewecantestwhetherGlut1De;iciencyanimalstreatedwiththesedrugswouldbemorepronetoseizureandmovementdisorders.WecantestthesethingsinGlut1De;iciencymicewithEEG’sandalsolookformovementdisorders.Wecanlookforataxiainthesemiceona“catwalk”(atestforataxiainmice).

WereallyshouldlookatcommonpediatricmedicationsandseeiftheycauseaprobleminGlut1De;iciencypatients.Noonehasreallyevaluatedmedicationsforfever,asthma,allergies,ADHD,etc.TherearecommonlyusedmedicationsinpatientsbutnoonehasevaluatedthemforGlut1De;iciencypatients.ManyofthedrugscommonlyusedmaycauseseizuresinGlut1De;iciencypatients.WewilllookatdrugsthatGlut1De;iciencypatientsmaybeusing.Wearewaitingforfundingatthispoint.

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DominicD’Agostino,PhDUniversityofSouthFlorida,MorsaniCollegeofMedicineInstituteforHumanandMachineCognition(IHMC) Tampa,Florida

SignalingPropertiesandTherapeuticEffectsofKetones

BACKGROUND:TheUSOf;iceofNavalResearch(ONR)hassoughttoexploreandprovidemetaboliccountermeasurestoimprovethesafetyandperformanceinextremeenvironments(suchasdeepseadiving)forNavySEALs.

Navysealscanstayat50feetofseawaterdepthfor10minutesandafterthattheywillhavethepotentialforseizuresduetocentralnervoussystem(CNS)oxygentoxicity.TheNavyhasexperimentedwithseveraldrugstohelpreduceseizuresinNavydivers,suchasanti-epilepticdrugs,butthesedrugshaveunwantedsideeffectsandcanimpairwar;ightercognitionability.

ThereneedstobeastrategyforoxygentoxicityforNavySEALdiverswhouseoxygenrebreathers.

Thehyperbaricoxygenseizuremodelisimportanttouseinresearchastonic-clonicseizuresoccur;theseseizuresarereversibleandreproducible.

Wecanresearchthisinthelaboratorybyusinghyperbaricoxygenchamber;howeverrodentseizuremodelsinresearchareinformativebutnotalwayspredictive.

RESEARCH:Wecanevaluateseizuresinasliceofrodentbraintissue(inthelaboratory).Weareinterestedinthehippocampusareaforlearningandmemoryissues.

Weexposetherodentbrainslicetissuestohighlevelsofoxygen(likeaNavySEALmightneedfordeepseadives);weseeseizuresinthebrainsliceandcanstudythisphenomenonintheabsenceandpresenceofketones.Ketoneshelptoreduceseizuresevenwhenevokedbyvariousneurotoxins.

Wecanalsostudythisatthelevelofthemitochondria,inEEG’s,EKG’s,physiologicaldata,andneurologicaldata.Wecanmeasurethelatencyofseizuresinresponsetoextremeenvironmentsthroughthismethod.

Inthisresearchwenoticedthatfastingketosiscausedneuroprotectiveeffectswhensubjectedtohighlevelsofoxygen.Wewonderedwhatthebrainenergychangewasinthecontextoffasting.

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TherewasapublishedstudyfromHarvardMedicalSchoolwheresubjectswerefastedfor40days.Whenfedanormaldietmostofthefuelisfromglucose.Yetwhenfastingfor40days,2/3ofthe

brainenergymetabolismisfromketonebodies.Whenlargeamountsofinsulinisprovidedtoapersononanormaldiet,severehypoglycemiawouldresultandthisisfatal.Yetwheninsulinwasprovidedtothesefastedpatientstheyallsurvivedandwereasymptomatictohypoglycemia.Thisrepresentsadramaticdemonstrationthatthebraincanuseketonesforfueleveninthefaceofwhatwouldtypicallybeseverehypoglycemia.

Metabolictherapyprotectiontohighoxygenlevelstatescanbeachievedwithnutritionalketosis.Gettingintoketosis-involvessustainedadherencetoaverylowcarbohydratedietwherethereisadepletionofliverofglycogen.Thiscanalsobeachievedwithprolongedfasting,butthisisnotsustainable..Ketonesaltsareformulatedwithsodium,potassium,calciumandmagnesiumattachedtoketones,primarilybeta-hydroxybutyrate.Exogenousketonescancircumventdietaryestablishedketonestogetintoketosis.

Ketonesmaybeimportantsignalingmetabolitessuchas:suppressionofoxidativestress(epigenetic),suppressin;lammation(NLRP3,reducedIL1B),increasetheGABAtoglutamateratio,andincreasetheconversionofglutamatetoGABAbyactivatingglutamicaciddecarboxylase(GAD)

Thetermanapleroticappliesinthatmetabolitesarefeedintometabolicpathwaystomakeneurotransmitters.KetonescanbypassGLUT1,GLUT3andpyruvatedehydrogenasede;iciency(PDH).TheyusetheusetheMCTtransportertoenterthebrain,andthistransporterisincreasedovertimewithsustainedadherencetonutritionalketosis

Theliveristhesiteofproductionofketones.Itmakesketonesbutcan’tuseketonesassourceoffuel.Ketonescancrossthebloodbrainbarrierandthemitochondrialouterwallveryeasily.Thus,ketonesproducedendogenouslyandexogenouslyarehighlyef;icientandreadilyavailablefuelsfortissuesandorgans,especiallythebrain.

WehavedemonstratedthatKetonesupplementationdelaysoxygenseizuresinrats.Ifaketoneesterisprovided;itissimilartotheratshavingbeenfastedfor1week(i.ethelevelofketosisishigh).Thisrapid(within30minutes)andsustained(over4-8hours)ketosiswasadesiredfeatureofthemilitarybecauseitcouldrapidlyinduceastateofneuroprotection.

Thehumanapplicationwouldbeashighas1gram/kg/dayofketoneesters,dividedinto2-4doses.Within30minutesweseeahighriseinketonesandreducedglucose.BothcanbemeasuredwithaPrecisionXtrabloodglucose/ketone(BHB)meter(Abbottlabs).device.Additionally,acetoacetateandbeta-hydroxybutyratecanbemeasuredinthelaborbyavarietyofotherdevicesthatarehittingthemarket(e.g.Kaomoji).

Westudytheneuroprotectiveeffectofexogenousketonesunderthe100%oxygenmodelinrats.Thisissimilarto10timestheamountofoxygenthanwenormallyhave.RatsgivenketoneestersdemonstratedremarkableresilienceagainstCNDoxygentoxicity(tonic-clonicseizures).Weused5ATAofoxygen,whichtypicallyproducedseizureswithin10minutes(controlanimals);whereasthosetreatedwithketoneesterswereableresisttheseizuresforover1hour.This

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neuroprotectionandanti-convulsanteffectishigherthanandknowantiseizurecompoundthatweknowof.

Wefeedratswithexogenousketonesmixedwithstandarddiet(10-20%ofthefoodbyweight)

Inmicefedwithketoneestersandnormaldiet;bloodglucosegoesdownwhenketonesgoupandwearenotsurewhythishappens.KetoneestersincreasesBHBandacetoacetateina1:1ratio.Ketoneesters(1,3-butanediolacetoacetatediester)signi;icantlyelevatestheacetoacetate;thisisimportanttoseizureactivity.Elevationsofacetoacetatearealsoseenwhenketonesaltsareadministered,butnottothesamelevelsaswiththisketoneester.

Glut1De)iciencymice)indingswithketoneesters:Glut1De;iciencymiceplusketoneestersshowhighvariabilityinresponseandtheytendtodisposeofketonesfast.Thisseemstobeapositiveindicationthattheirtissue(brainespecially)arestaredoffuel.

Aglucosetolerancetestshowshowfasttheglucoseistakenupbythesystem.Similarly,a“ketonetolerancetest”isagoodindicationthatGlut1De;iciencymicehaveahighcapacitytouseketonesforfuel,andthismayresultinlowerthanexpectedlevelsinthebloodandtissues(i.e.theyareburningketones)

Whengivenketoneestersmixedinwithfood;ketonelevelsdidnotrisetothelevelstypicallyseeninnon-diseaseanimals.

Ketonesalt20%upwasmucheasiertoadministerbecausethemicedidnotself-restrictandthiswaswelltolerated.

Therewerelittlechangesinbodyweightovertimeinketoneesterswhichtastebad,somicemightnotlikethem.However,micegivenketoneesterswerebetterathangingwire(strength)testandonRotarod(motorfunction)test.Overalltheyhadmorerobustphysicalcapabilitieswheninastateofnutritionalketosiscomparedtountreated.

Ketonesupplementationinpatients:Isitsafetouseketonesaltsinpatients?Theywouldneedtoconsumealargedose.

Ketonesaltshavebeenusedfordecadesinvariousmetabolicdiseasestates.Newtechnologiesaremakingitpossibletodevelopbio-identicalketonesaltsthataremadewithabalancedmineralformulathatwouldbewelltolerated,safeandpleasanttotasteinformsthatresemblefruitpunchorachocolateshake.Thiswouldalsomakelargedosesmorefeasible.

Complianceisoneofthemainissueswiththeketogenicdietandasnewcompaniesdeveloppre-packagedfoodsand“comfortketo-foods”thiswillbemuchlessofanissue.Also,workbyDr.EricKossoffhasshownthatissomecasesthelessrestrictivemodi;iedketogenic(Atkins)diet(MAD)maybejustasgood.AMADconsumedwithMCToilandsupplementalketonescouldbetheidealstrategyfortreatmentandcompliance

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Theketogenicdietdoessuppressseizuresinmanydifferentdisorders;suchasAngelmansyndrome.ThereisapublishedreportofusingketoneestersintheAngelmansyndromemousemodel(2016).ResearchatUSFismovingthistherapyintoclinicaltrialsatmultiplecenters.

Resultsindicatedanelevatedpresenceofenzymesthatcanconvertmoreglutamate(excitatory)intoGABA(stabilizing)withnutritionalketosis.HighGABAlevelshaveacalmingeffectandtheabilitytosuppressseizures.

WewanttodoastudyinGlut1De;iciencypatients,especiallysinceexogenousketoneshavebeenshowntoreducesomeofthesymptomsthatmaybeassociatedwiththisdiseaseincluding,enhancingmotorfunction,reducinganxietyandalsosuppressingseizures(includingabsenceseizuresThecombinationofMediumChainTriglyceridesandketonemineralsaltshavethebiggesteffectsonanxietyreduction.TherearecommercialproductsonthemarketthatcombineBHBsalts+MCTandthesewouldbetheformulaswewouldbeinterestedintesting.

WhenapatientwithGlut1De;iciencyexercises,symptomscanworsen.Maybeifwegiveketoneestersasasourceoffuelwhenexercisingitcanshiftfuelpreferencesuchasinelitelevelathletes.Ketosisshiftsenergymetabolismfromglucosetoketoneswitha50%reductioninlactate.Thisobservationhassigni;icantimplicationsforpatientsandtheirabilitytobemoremetabolicallyresilientduringexercise.Feedingpriortoexercisecouldbeimportant.

Ketoneshavebeenshowntobeinvolvedwithseveralpowerfulsignalingpathways,leadingtotherapeuticeffects.FeedingexogenousketonesincreaseGABAtoglutamateratio,increaseinneurotransmitters,antioxidantsareelevated,decreaseinglucose(suggestsincreaseinglucosedisposal).Manyoftheseeffectsaredesirabletraitsforadrugcompound,butnutritionalketosistendstostimulatealloftheseeffects(inamildway)withlittleornosideeffects.

ImplementationofnutritionalketosisthroughdietshouldbethefrontlineapproachforGlut1De;iciency,whereasexogenousketonesupplementationwillbeavailableinthefutureasaprescriptionmedicalfood.Severalcompaniesnowsellthemasnutritionalsupplements,butonlyproductsapprovedbya3rdparty(NSF,InformedChoice,etc)shouldbeconsidered.Thereisabloodketonemeteronthemarketnow(PrecisionXtra).TheDexcomPatchstickstotheskinandpicksupglucose,andtheseresultscanbesenttoasmartphone.Thesametechnologyisunderdevelopmentforketonemonitoring.Dietinitiationofketosistakesapprox.3-10dayswhereastakingexogenousketonesisafastwaytogetintoketosisandtosustaintherapeuticlevels.

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EricKossoff,MDJohnsHopkinsHospital Baltimore,Maryland

DietTherapyforGlut1De)iciency

Thecurrentstateofdietarytherapyfordisorders,includingGlut1De;iciencywasdiscussed.

Alotofpapersarebeingpublishedontheketogenicdiet(KD).

Thereismore;lexibilityinthedietnowandthishelpswithexpandingtheuseofdietarytherapy.

Thereare4majorrandomizedclinicaltrialsusingKDthathavebeenpublished.

TheCochraneCollaboration(2012)isapublishedpaperthatisusefulingettinginsurancetopayfortheKDasitclassi;iesdietarytherapyasvaluableandef;icacious.

90%ofGlut1De;iciencypatientshaveatleast50%seizurereductionwiththeKDinmajorstudies.

ThereisanicepaperbyMasinoandRho2012whichexplainswhythedietworks.

TheKDisnowmore;lexibleandaccessible.Therearealotofdifferentformulasonthemarket.Youcanusetheseasbakingmixesalsoandmanycompaniesarecreatingdifferentketofoods.

Thereare4majordiets

1) Classicketogenicdiet

2) Mediumchaintriglyceridesdiet(MCT)

3) Modi;iedAtkinsdiet(MAD)-createdatHopkins

4) Lowglycemicindextreatment–createdatMassachusettsGeneral

WeusetheModi;iedAtkinsdietatHopkinsasanalternativeprimarily.Thisincludeslessfat,abitmorecarbs,moreprotein,noadmissiontohospitalandnocalorieor;luidrestrictioncomparedtotheclassicketogenicdiet.Thereisnoweighingfoodsongramscale-patientscanhave15-20gramsofcarbsperday.

Underage2yearstheketogenicdietwasslightlybetterthantheMADdietforkidswithseizures.MostcentersusetheclassicKDforchildrenunderage2.

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WhatdoweknowaboutGlut1De)iciency?

3keyissuesthatparentscommonlyaskketocentersfortheirchildrenwithGlut1De;iciencyinclude:

1) TheKDisaseffectiveastheMAD?

2) Whataretheappropriateketonelevels?

3) CanIstopthedietever?

InGlut1De;iciencywearetalkingaboutabrainenergyfailurethusgivingmoreketonescouldbebetter.Howeverthisisanecdotal.

Maybeatayoungeragehigherketonesarebetter.

Therearesomenegativestothisconceptmainlythatketonesarehighbutsomepatientsstillhavesomeseizures,suggestingit’smorethanjustketoneshelping.

WhatabouttheclassicKDversustheMADdiet?SomepatientsswitchtotheMADdietanddidbetter.Thisisallcomplicatedandcouldvaryfrompatienttopatient.Thereisn’tasimplewaytotreatallpatients.

Kossoff(2016)hasareportregardingdietarytherapyandseizuresinGlut1De)iciency:

92familiescompletedasurveyregardingtheiruseofdietarytherapyfortheirchildrenwithGlut1De;iciency.Itwascompletedatthe2015Glut1De;iciencyFoundationmeetinginOrlando.

Theagerangeofpatientswas1-24yearswithameanof9.9yearsand90patientshadbeentreatedwithdietarytherapy2patientsdidnothavedietarytherapy.

ThebreakdownofdietsutilizedwereKDn=59,MADn=29,MCTn=4,andLGITn=2.

Switchingwascommon;manyswitchedfromKDtoMAD,oftenontheirown.

SomewentfromMADtoKD.FortheKDtherewasawidevarietyforratios;manywerenoton4:1(about2/3’sofpatients),choosinglowerratios

46%saidtheirchildwasseizurefreeonthedietand80%hadagreaterthan90%reductionofseizures.Thiscon;irmsjusthoweffectivedietarytherapycanbeforchildrenwithGlut1De;iciency.

Whymightsomepatientsstillhaveseizures?Notclearfromthesurvey

Other;indings:

1) Ageatdiagnosis-theyoungerthebetteratstartingthediet.

2) Currentagemakesadifferenceinhavingseizures(youngerdidbetter).

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3.)TheMADequalstheKDforseizureactivityinthissurveyaccordingtoparents.

4).A4:1fattocarb/proteinratiodidnotmakeadifferenceinseizures

(lowerratioimprovedseizuresequally).

5) Checkingbloodketonesversusurineketonesmakesnodifferenceinseizureoutcome.

Extrasupplementstakeninpatientsinsurvey:

Carnitine(n=62),oralcitrates(n=25),MCToil(n=20),noonewasonC736%wereonAED’s

76/76withmovementdisordersimprovedthemovementdisordersandcognitiononadiet.

Ketones:Checkingketonelevelsvariesalotamongpatients.34%checkingblood,34%saidcheckurine,21%both,and11%checkednotatall.Didn’tseemtomatterforseizurecontrol.Worthfuturestudy.

Puberty:22patientswereator;inishedpubertyand64%saidtheyhadachangeinseizureactivity.

Sideeffectsofdietarytherapyincludesomepatientswhohadgastrointestinalissuesand1personwithacholesterolissue.

5.5yearswastheaveragetimeondiet(1familyhadbeenonfor20years).

67%wereunsurewhetherthepatientshouldcomeoffthedietinthefuture.Alsoworthfuturestudy.

Sinceour2016study,therehavebeenmorelookingatthedietandGlut1:

NewstudybyAmalou(2016)

10childrenwerestartedonMADdiet(2infants). DatashowedtherewassimilarimprovementwithMADcomparedtoKD.

Japanstudy:DatashowedthattheMADdietismorepalatableforGlut1De;iciencyandwascomparableorbetterinsomepatientstoclassicKD.

G1DRegistry:Dataintheregistryconsistsof181patients.Againalotofvaryingdiets.54%wereontheKD.

Summary:

Itisoktomakeachangeas4:1KDdoesnot;itallpatients

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OutcomesondietsarespectacularforGlut1De;iciencyandtheyareexcellentforseizures,cognitionandmovementdisorders.

Managementofdietisalsovariable,includingketonesandsupplements.

Weneedfurtherstudyforpuberty,discontinuation,correlationwithketosis,andsupplements.

InthefuturewewillevaluatecognitionandalsoadultswithGlut1De;iciency.

FuturePossibleNoveldietaryapproachestoGlut1De)iciency:

Triheptanoin

C10

Ketoneesters

Modi;iedcornstarch

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MackenzieC.Cervenka,MDJohnsHopkinsHospital Baltimore,Maryland

KetogenicDietTreatmentsandTransitionStrategies

Glut1De;iciencywasdiscoveredinthe1990’sandthe;irstpatientsdiagnosedareallnowadults.

AdultswithGlut1De;iciencycanhaveatypicalsymptomssuchasmigraine,writer’scramp,andalternatinghemiplegiawhichmaynotberecognizedassymptomsofGlut1De;iciency.

AdultpatientswithGlut1De;iciencycanalsohavethefollowingsymptoms:chronicmildencephalopathy,infrequentseizures,varyingspasticity,ataxiaandparoxysmalexertionaldystonia(PED).

Geneticmutationsaretypicallyautosomaldominantandaremostoftendenovo(nottransmittedfromaparent).

SomepatientshaveadecreaseinseizuresduringchildhoodandwhenwemeetthemasanadulttheyareonlyhavingothertypesofeventssuchasPEDs.However,theadultclinicalpictureisoftennotsodifferentthaninchildren.Butwewanttoknowjusthowtheyaredifferentthanchildren?

Dietaryconsiderations:

ThestandardAmericandietformostadultsinoursocietyincludeseatingalotofcarbs.Thedevelopingbrainneedsmoreenergy.However,theadultneedforenergyislessthanthatofayoungchild.

PatientswithGlut1De;iciencyareontheketogenicdietbutwedon’treallyknowthequantityofketonesthesepatientsneed.

Theclassicketogenicdietis90%caloriesfromfat.Theratiooffatstocarbohydratesandproteincombinedis4:1or3:1.Themodi;iedketogenicdietandtheModi;iedAtkinsDiet(MAD)arelessrestrictivethantheclassicdietandusedbysomepatientsbuttheystillmeasureketones.Theratiooffatstocarbohydratesandproteincombinedistypically2:1or1:1.

Manyadultpatientsstartthesedietsontheirown;asmanyoftheketogenicdietcentersdon’tofferdietstoadultsandmostadultepilepsycentersdonotuseketogenicdietsfortreatment.Thisisn’talwaysthebestwaytodothings.

ItwasreportedbyPong,2012that62%ofpatientswithGlut1De;iciencybecameseizurefreeontheketogenicdiet.Compliancewas84%.

Thereislittleinliteratureregardingeffectivenessofthemodi;iedketogenicdietinadults.

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TheModi;iedAtkinsDietisusedatJohnHopkinsHospitalandseveralotheradultdietcentersnationwide.FortheMAD,adultstake20gramsnetcarbsperday(;iberdoesn’tcount!).Patientsaren’trequiredtotakeacertainamountoffat,justenoughtogetintoketosis.TheMADdietequatestoabouta1:1to2:1ratiooffatstocarbohydratesandproteincombined.Whenadultsreducecarbs,manywillgointoketosiswithjustthis.Ifthepatientsdonothaveimprovementinseizuresandothersymptoms,weaskthemtostartthinkingaboutusingaclassicketogenicdiet.

HowtheMADisusedinGlut1De)iciencypatients:

IntheG1DregistryofpatientswithGlut1De;iciency,2/3ofpatientsusedmodi;ieddiets.WeknowthattheMADcanreduceoxidativestress.

TheMADhasalsobeenusedformovementdisorders.AdultswithGlut1De;iciencyoftenhaveissueswithmovementdisordersandlessproblemswithseizures.Patientswithabetahydroxybutyratelevelof0.2-2mmol/Lhadareductioninmovementdisordersinonestudy(Leenetal.,2013).

Transitioningtoanadultdietcenterfromapediatricdietcenter:

ThetransitionmeansthatmanypatientswithGlut1De;iciencywillbemoreindependentasadults.Theymaybeaskedtochooseappropriatefoods,checkketones,andmonitorsymptoms.

Adultpatientsoftenwanttodrive.However,tobeallowedtodrive,thepatientmustbeseizurefree.Thereisarequirementforlengthoftimebeingseizurefreetogainpermissiontodriveandthesevaryfromstatetostate.Thisisalsotrueformovementdisordersthatmayimpactdrivingability.

ParentsorotheradultscanobtainguardianshipofGlut1De;iciencypatientsoncetheyturn18yearsofageiftheyarenotabletomakedecisionsforthemselves.

DisabilityinGlut1De;iciencyadultscanbesigni;icant.Adultpatientsmaybetakenoffoftheirparents’healthinsurance(agevariesbyinsurancecarrierandstate).Makesurehe/shehasMedicareandMedicaidinplace.

Transitionplanningpriortotheonsetofadultageisimportantforseveralreasons.Graduationfromthepublicschoolsystemhappensfrom18-21yearsandpatientsmayplantoparticipateindayprogramsorrequirecare/supervisionathome.Somecollegeswillhelpmakeketofoodsavailableindininghalls.

TransitioningtoAdulthood:

Atage10-13yearsstarttakingaboutatransitionplanwiththepediatricianandpediatricneurologyteam.

Atages14-15yearscontinuetoplan.

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Atages16-17yearsstartworkingforindependenceorguardianshipand;indjobprograms.Consideradvancemeetingofthechildwiththeadultneurologist.

At18yearsinitiatethetransition.

TransitionTips:

• Planaheadtopreventgapsincare.

• Avoidreinventingthewheel(takingthesametestsortryingoldtreatmentsagainthatdidn’tworkthe;irsttime).

• Planningimprovespatientandhealthcareprovidersatisfaction.

• Haveatransitiondietclinic.Thereareseveraldietclinicsthroughthenation.

Drs.KossoffandCervenkawroteapaperon“Transitioningpediatricpatientsreceivingketogenicdietsforepilepsy”describingthisexperiencein10patientswithepilepsyonketogenicdiets(2013).

Tipstoimprovedietarycompliance:

• Considermodifyingthediettomakeitlessrestrictiveandeasiertofollow.

• Ketogenicdietresourcessuchascookingclasses,cookbooksandwebsiteswithrecipes,anddietmonitoringmobileapplicationsareavailable.

• Manyketogenicfoodsarecommerciallyavailableorasmedicalfoods(requireaprescription).

• Potentialsideeffectsofthesedietscanbeconstipationandkidneystonescanbeavoidedwithgoodhydration.

Whathappensifawomanontheketogenicdietgetspregnant?

Isthereteratogenicity(harmtothefetus)?Thereisverylittleinformationintheliteratureaboutthis(vanderLouw,2017).

Therearesomepotentiallongtermsideeffectstoavoid:

Vitaminde;iciency(knownrisk)

Carnitinede;iciency(knownrisk)

Kidneystones(knownrisk)�23

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Osteopenia/Osteoporosis(knownrisk)

Cardiovasculardisease(riskunknown)

Cerebrovascularissues(riskunknown)

Preventativemeasurestohelppreventsideeffects:

Replaceheavycreamwitholiveoilormediumchaintriglycerideoilinpatientswithelevatedlipids

Reducecaloriesinobesepatients

Multivitaminsupplements

Oralcitrates(forkidneystones)

Checkforhyperlipidemiaas(inMAD)bloodLDLandtotalcholesteroloftengoupin;irst6months,thentrendbacktonormalin1-2yearsondiettherapy.Studiesinchildrenhaveshownthatin12monthstherewassomedecreaseinvascularelasticitybutthisimprovesandwasnotsigni;icantat24months(Kapetanakis,2014;Coppola,2014).

WestillneedtolearnmoreaboutadultsusingketogenicdietsforepilepsyandGlut1De)iciency:

Howlongshouldadultsstayonthesediets?

Whataboutsupplements?

Whichdietisthebest?

Whatketonelevelsarebest?

Arethereothersideeffectsthatwedon’talreadyknowabout?

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BothDr.UmraoMonaniandDr.AbrahamAl-AhmadarerecipientsofresearchgrantawardsfromtheGlut1De;iciencyFoundationandtheypresentedupdatesontheirworkattheNashvilleconference.DuetoKrisEngelstad’stravelschedule,shewasunabletoattendtheirpresentations.Wehavesomeresourcesrelatedtotheirtalkstosharebelow.

Dr.UmraoMonani,PhDColumbiaUniversityMedicalCenterNewYork,NewYork

Dr.MonanipresentedonGeneReplacementTherapyforGlut1De)iciency.

Dr.Monani’steamhaspublishedapaperrelatedtotheirresearchongenetherapy.Youcanaccessthefulltextarticlehere:

AbrahamAl-Ahmad,PhDTexasTechUniversityHealthSciencesCenterLubbock,Texas

Dr.Al-AhmadpresentedonModelingtheBlood-BrainBarrierusingPatient-DerivedStemCells:AFocusonModelingGlucoseTransport.

Dr.Al-Ahmadprovidedthepowerpointslidesforhispresentation,whichyoucanaccesshere.

OtherConferenceResources:Youmayalso;indadditionalconferenceresourcesatourwebsite,includingtheagenda,presentationslides,photos,andalistofexhibitorsandsponsors.

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