chapter 45, genetic disorders of glycosylation essentials...

AssignedReading:Chapter45,GeneticDisordersofGlycosylationEssentialsofGlycobiology,3rdeditionChapter45AppendixTable45AArticle,DecipheringtheGlycosylomeofDystroglycanopathiesUsingHaploidScreensforLassaVirusEntry

CHAPTER45.GeneticDisordersofGlycosylation

HudsonH.Freeze,HarrySchachterandTarohKinoshitaThischapterdiscussesinheritedhumandiseasesthataffectglycanbiosynthesisandmetabolism.Representativeexamplesofdiseasesduetodefectsinseveralmajorglycanfamiliesaredescribed.DisordersaffectingthedegradationofglycansaredescribedinChapter44.

INHERITEDPATHOLOGICALMUTATIONSOCCURINALLMAJORGLYCAN

FAMILIES

Nearlyallinheriteddisordersinglycanbiosynthesiswerediscoveredinthelast20years.Theyarerare,biochemicallyandclinicallyheterogeneousandusuallyaffectmultipleorgansystems.Somedefectsstrikeonlyasingleglycosylationpathway,whileothersimpactseveral.Defectsoccur1)intheactivation,presentation,andtransportofsugarprecursors,2)inglycosidases,glycosyltransferases,and3)inproteinsthattrafficglycosylationmachineryormaintainGolgihomeostasis.Afewdisorderscanbetreatedbytheconsumptionofmonosacccharides.TherapidgrowthinthenumberofdiscoveredthesedisordersisshowninFigure45.1.

FIGURE45.1.Glycosylation-RelatedDisorders.Thegraphshowsthecumulativenumberofhumanglycosylationdisordersinvariousbiosyntheticpathwaysandtheyearoftheiridentification.Inmostcases,theyearindicatesthedefinitiveproofofgeneandspecificmutations.Inearlyyears,discoverywasbasedoncompellingbiochemicalevidence.NowdiscoveryisoftenbasedongenomicDNAsequencing.In2013alone,anewgenetically-provenglycosylationdisorderwasreported,onaverage,everytwoweeks.SelecteddisordersarelistedinTable45.1andallknowndisordersinOnlineAppendix45A.Diseasenomenclaturehasevolved.CongenitalDisordersofGlycosylation(CDG)wereoriginallydefinedasgeneticdefectsinN-glycosylation,butnowthetermisappliedtoanyglycosylationdefect,byindicatingthemutatedgenefollowedby“-CDG”suffix,e.g.,PMM2-CDG.CDGsarerareprimarilysinceembryoswithcompletedefectsinastepofglycosylationdonotusuallysurvivetobeborn,documentingthecriticalbiologicalrolesofglycansinhumans.CDGpatientsthatsurviveareusuallyhypomorphicretainingatleastsomeactivityofthepathwaysinvolved.

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1990 1993 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

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O-Fucose / O-Glucose

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Dystroglycanopathy Glycosaminoglycan N-Linked

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DEFECTSINN-GLYCANBIOSYNTHESIS

ClinicalandLaboratoryFeaturesandDiagnosis

ThebroadclinicalfeaturesofdisordersinwhichN-glycanbiosynthesisisdefectiveinvolvemanyorgansystems,butareespeciallycommoninthecentralandperipheralnervoussystems,hepatic,visual,andimmunesystems.ThegeneralityandvariabilityofclinicalfeaturesmakesitdifficultforphysicianstorecognizeCDGpatients.Thefirstwereidentifiedintheearly1980sbasedprimarilyondeficienciesinmultipleplasmaglycoproteins.Thepatientswerealsodelayedinreachinggrowthanddevelopmentalmilestones,hadlowmuscletone,incompletebraindevelopment,visualproblems,coagulationdefects,andendocrineabnormalities.However,manyofthesesymptomsareseeninpatientswithotherinheritedmultisystemicmetabolicdisorders,suchasmitochondria-baseddiseases.CDGpatientscanbedistinguishedbecausetheyoftenhaveabnormalglycosylationofcommonliver-derivedserumproteinscontainingdisialylated,biantennaryN-glycans.SerumtransferrinisespeciallyconvenientbecauseithastwoN-glycosylationsiteseachcontainingdisialylatedbiantennaryN-glycans.Differentglycoformscanberesolvedbyisoelectricfocusing(IEF)orion-exchangechromatography,butbetteraccuracyandsensitivityisachievedbymassspectrometryofpurifiedtransferrin.ThissimplelitmustestalertsphysicianstolikelyCDGpatientswithoutknowingthegeneormolecularbasisofthedisease.CDGdefectsmaybedividedintotwotypesbasedontransferringlycoforms.TypeI(CDG-I)patientslackoneorbothN-glycansduetodefectsinthebiosynthesisofthelipid-linkedoligosaccharide(LLO)anditstransfertoproteins.TypeII(CDG-II)patientshaveincompleteprotein-boundglycansduetoabnormalprocessing.Thesedifferenceswereusedtonamethedisorders,e.g.,CDG-IaandCDG-IIa,whichorientedthesearchforadefectivegene.ThissystemisgraduallybeingreplacedbytheaffectedgenenamewithaCDGsuffixsuchasPMM2-CDG.ThebiosyntheticpathwaysandlocationsofN-glycandefectsareshowninFigure45.2.

FIGURE45.2.CongenitalDisordersofGlycosylationintheN-glycosylationpathway.ThefigureshowsindividualstepsinLLObiosynthesis,glycantransfertoproteinandN-glycanprocessingsimilartoFigure9.3andFigure9.4.TheshuttlingoftheglycosylationmachinerybetweentheERandGolgiisorganizedandregulatedbycytoplasmiccomplexesincludingtheconservedoligomericGolgi(COG)complex.RedgenenamesindicateCDG.

TypeICongenitalDisordersofGlycosylation

AcompleteabsenceofN-glycansislethal.Therefore,knownmutationsgeneratehypomorphicalleles,notcompleteknockouts.AdeficiencyinanyofthestepsrequiredfortheassemblyofLLOintheER(e.g.,nucleotidesugarsynthesisorsugaradditioncatalyzedbyaglycosyltransferase)(Chapter9)producesastructurallyincompleteLLO.Becausetheoligosaccharyltransferaseprefersfull-sizedLLOglycans,thisresultsinhypoglycosylationofmultipleglycoproteins.ThismeansthatsomeN-glycansitesarenotmodified.Importantly,manydeficienciesinLLOsynthesisproduceincompleteLLOintermediates.MostoftheLLOassemblystepsarenoteasytoassay,butLLOassemblyisconservedfromyeasttohumans,andintermediatesthataccumulateinCDGpatientsoftencorrespondtotheintermediatesseeninmutantSaccharomycescerevisiaestrainswithknowndefectsinLLOassembly.Somemutantmammaliancells(e.g.,Chinesehamsterovarycells)havebeenshowntohavesimilardefects(Chapter49).Theclosehomologybetweenyeastandhumangenesenablesthenormalhumanorthologstorescuedefectiveglycosylationinmutantyeaststrains,whereasmutantorthologs

frompatientsdonot.Thisprovidessubstantialcluestothelikelyhumandefect,alongwithasysteminwhichtoperformfunctionalassays.PMM2-CDG(CDG-Ia)inTable45.1isthemostcommonCDGwithover800casesidentifiedworldwide.Thepatientshavemoderatetoseveredevelopmentalandmotordeficits,hypotonia,dysmorphicfeatures,failuretothrive,liverdysfunction,coagulopathy,andabnormalendocrinefunctions.Morethan100mutationsfoundinphosphomannomutase2(PMM2),impairconversionofMan-6-PtoMan-1-P,whichisaprecursorrequiredforthesynthesisofGDP-mannose(GDP-Man)anddolichol-P-mannose(Dol-P-Man).BothdonorsaresubstratesforthemannosyltransferasesinvolvedinthesynthesisofGlc3Man9GlcNAc2-P-P-DolanditslevelisdecreasedincellsfromPMM2-CDGpatients.PatientshavehypomorphicallelesbecausecompletelossofPMM2functionislethal.MouseembryoslackingPmm2die2–4daysafterfertilization,whereassomeofthosewithhypomorphicallelessurvive.TherearecurrentlynotherapeuticoptionsforPMM2-CDGpatients.MPI-CDG(CDG-Ib)inTable45.1iscausedbymutationsinMPI(mannose-6-phosphateisomerase).Thisenzymeinterconvertsfructose-6-Pandmannose-6-P(Man-6-P).IncontrasttoPMM2-CDG,thesepatientsdonothaveintellectualdisabilityordevelopmentalabnormalities.Instead,theyhaveimpairedgrowth,hypoglycemia,coagulopathy,severevomitinganddiarrhea,protein-losingenteropathy,andhepaticfibrosis.SeveralpatientsdiedofseverebleedingbeforethebasisofthisCDGwasknown.Mannosedietarysupplementseffectivelytreatthesepatients.Man-6-Pcanbegenerateddirectlybyhexokinase-catalyzedphosphorylationofmannose(Chapter5).ThispathwayisintactinMPI-CDGpatients.Humanplasmacontainsabout50μMmannoseduetoexportfollowingglycandegradationandprocessing.Mannosesupplementscorrectcoagulopathy,hypoglycemia,protein-losingenteropathy,andintermittentgastrointestinalproblems,aswellasnormalizetheglycosylationofplasmatransferrinandotherserumglycoproteins.Becauseorallyadministeredmannoseiswelltolerated,thisapproachisclearlyasatisfyinglyeffective,thoughnotcurative,therapyforthislife-threateningcondition.CompletelossoftheMpigeneinmiceislethalataboutembryonicday11.5.N-Glycosylationisnormal,butdeathresultsfromaccumulationofintracellularMan-6-P,whichdepletesATPandinhibitsseveralglycolyticenzymes.Providingdamswithextramannoseduringpregnancyonlyhastenstheembryo’sdemiseviathe“honeybeeeffect”,whichoccurswhenbeesaregivenonlymannoseinsteadofglucose.Thebeescontinueflyingforashorttimeandthenliterallydropdead.TheyhavelowMPIactivitycomparedtohexokinaseandthereforeaccumulateMan-6-P,whichtheydegrade,andperhapsrephosphorylate,furtherdepletingATP.EvenmoreseriousisthatMan-6-Pinhibitsseveralglycolyticenzymes.EntryofMan-6-Pintoglycolysisisveryslowandthusthebeesbecomeenergy-starvedanddiewithinafewminutes.MPI-CDGpatientshavesufficientresidualMPIactivityanddonotaccumulateintracellularMan-6-Pwhengivenmannose,thoughtheamountissufficienttocorrectimpairedglycosylation.However,thehoneybeeeffectmaybeatplayagaininMpi-hypomorphicmicebecausedamsgivenmodestamountsofmannoseduringpregnancyproducepupswithnolenses.TheeffectisquitespecifictolensdevelopmentwheretheMPIactivityisveryloweveninnormalmice.AhypomorphicmutationandincreasedsubstrateloadcombinessothatMan-6-Paccumulates.

OthertypesofCDG-IhaveabroadrangeofclinicalphenotypesincludinglowLDL,lowIgG,kidneyfailure,genitalhypoplasia,andcerebellarhypoplasia.Thereasonsfortheseeffectsareunknown.PatientswithmutationsinnearlyalltheremainingstepsofLLObiogenesishavebeenfound(Table45.1,Table45.2onlineandFigure45.1)includingdefectsindolicholbiosynthesis(cis-isoprenyltransferase,dolicholkinase,polyprenolreductase)andinaputativeLLOflippase.MutationsinfiveoftheoligosaccharyltransferasesubunitsalsocauseaCDG.

TypeIICongenitalDisordersofGlycosylation

CDG-IIdisorders(Figure45.2)affectN-glycanprocessingandincludedefectsinglycosyltransferases,nucleotidesugartransporters,vacuolarpHregulators,andmultiplecytoplasmicproteinsthattrafficglycosylationmachinerywithinthecellandmaintainGolgihomeostasis.InB4GALT1-CDG,glycansshowedthelossofbothgalactose(Gal)andsialicacid(Sia)fromtransferrinbecauseoflossofβ1-4galactosyltransferaseIactivity.AsimilarglycanpatternoccursintheX-linkedSLC35A2-CDG,duetolossofUDP-Galtransporteractivity.Surprisingly,withinafewyearsafterbirth,abnormalglycosylationbecomesnormal.ThisisprobablyduetosomaticmosaicismofcellscarryingthemutatedSLC35A2geneandunaffectedcells,andselectionagainsttheaffectedcells.PatientswithleukocyteadhesiondeficiencytypeII(LAD-IIorSLC35C1-CDG)werefoundtohavemutationsinSLC35C1,encodingaGDP-Fucose(Fuc)transporter.Heretransferrinsialylationwasnormal,sothisdefectwasnotdetectedbytheusualtest,butsomeserumproteinsandO-linkedglycansonleukocytesurfaceproteinsweredeficientinFuc.Oneleukocyteproteincarriesaselectinligandglycan,sialylLewisx,thatmediatesleukocyterollingpriortoextravasationofleukocytesfromcapillariesintotissues(Chapter34).Thisdefectgreatlyelevatescirculatingleukocytesanddecreasesleukocyteextravasationsopatientshavefrequentinfections.AfewpatientsrespondedtodietaryFuctherapy.SialylLewisxreappearedontheirleukocytes,andcirculatingneutrophilspromptlyreturnedtonormallevels.FucisconvertedintoFuc-1-PbyfucosekinaseandthentoGDP-FucbyGDP-Fucpyrophosphorylase(Chapter5).FucsupplementsmustincreasetheamountofGDP-Fucenoughtocorrectthedefect.AmousemodelofFucdeficiencylacksdenovobiosynthesisofGDP-FucfromGDP-Man(Chapter5).ThemicediewithoutFucsupplements,butprovidingFucinthedrinkingwaterrapidlynormalizestheirelevatedneutrophils.ThetreatmentalsocorrectsabnormalhematopoeisisresultingfromdisruptedO-Fuc-dependentNotchsignaling.CDG-IIdefectsarealsocausedbymutationsintheeight-subunitconservedoligomericGolgi(COG)complex,whichhasmultiplerolesintraffickingwithintheGolgi.COG7-CDG(Table45.1)wasdiscoveredfirst.TraffickingofmultipleglycosyltransferasesandnucleotidesugartransportersweredisruptedinCOG7-CDG.ThemutationaffectsthesynthesisofbothN-andO-glycansandglycosaminoglycan(GAG)chains.MutationshavenowbeenfoundinallCOG-subunitsexceptCOG3.MammaliancellsdeficientinCOG1,COG2,COG3,andCOG5alsoshow

variousdegreesofalteredglycosylation.VariousmutationsinavacuolarH+-ATPasesubunitalsodisruptmultipleglycosylationpathways,presumablyduetoanincreaseinGolgipHandconcomitantdecreaseinglycosyltransferaseactivities.OthergeneticdefectsthatimpairN-glycansynthesisincludeI-celldisease,whichresultsfromthelackofMan-6-PonlysosomalenzymeN-glycans(Chapter33).AnunusualdisordercalledHEMPASthatleadstoabnormalredcellshapeandinstability(hemolysis)duetomutationsinSEC23B,anotherintracellulartraffickingproteinthatproducesabnormalredbloodcellglycansinseveralpathways.Inaninterestingtwistitispossibletohavediseasescausedby“excessive”glycosylation.Forexample,Marfansyndromeresultsfrommutationsinfibrillin1(FBN1)andoneofthesecreatesanN-glycosylationsitethatdisruptsmultimericassemblyofFBN1.Thismaynotbeanisolatedcase.Asurveyofnearly600knownpathologicalmutationsinproteinstravelingtheER-Golgipathwayshowedthat13%ofthemcreatenovelglycosylationsites.ThisisfargreaterthanpredictedbyrandommissensemutationsandmaymeanthathyperglycosylationleadstoanewclassofCDGs.

GALACTOSEMIA

GalactosemiareferstomutationsinthreegenesinvolvedinGalmetabolism.In“Classicalgalactosemia”,Gal-1-Puridyltransferase(GALT;Figure45.3)isdeficient.ThisresultsinexcessGal-1-PanddecreasedsynthesisandavailabilityofUDP-Gal.DefectsinUDP-Gal-4’-epimerase(GALE;Figure45.3)orgalactokinase(GALK;Figure45.3)alsocausethedisease,buttheyaremorerare.

FIGURE45.3.UDP-Galsynthesisandgalactosemia.Themostcommonformofgalactosemiaisduetoadeficiencyofgalactose-1-phosphateuridyltransferase(GALT).ThisenzymenormallyutilizesGal-1-PderivedfromdietaryGal.IntheabsenceofGALT,Gal-1-Paccumulates,alongwithexcessiveGalanditsoxidativeandreductiveproductsgalactitolandgalactonate(notshown).UDP-GalsynthesismayalsobeimpairedintheabsenceofGALT,butnotcompletelybecauseUDP-Gal-4’-epimerase(GALE)canformUDP-GalfromUDP-Glcandcansupplythedonortogalactosyltransferasesrequiredfornormalglycoconjugatebiosynthesis.

GALT-deficientinfantsfailtothriveandhaveenlargedliver,jaundice,andcataracts.Alactose-freedietamelioratesmostoftheacutesymptomsbyreducingtheamountofGalenteringthepathwayandtheaccumulationofGalandGal-1-P.ReducingGaldecreasesgalactitolandgalactonate,whichareproducedviareductiveoroxidativemetabolismofGal,respectively.Galactitolisnotmetabolizedfurtherandhasosmoticpropertiesthatcontributetocataractformation.Unfortunately,aGal-freedietapparentlydoesnotpreventtheappearanceofcognitivedisability,ataxia,growthretardation,andovariandysfunctionthatarecharacteristicofthisdisease.Thelong-termcomplicationsintreatedGALT-deficientindividualsmaybeduetosmallamountsoftoxicmetabolitesthataccumulate.Alternatively,thecomplicationsmayreflectdysfunctionsthatoriginatedduringfetallife.GALTdeficiencymaydecreaseUDP-Galandgalactosylatedglycans.HypogalactosylationofglycoproteinsandglycolipidshasbeenobservedinsomeGALT-deficientindividuals.ButinadditiontolossofGalonglycans,somepatientswhomistakenlyreceiveGalalsosynthesizetransferrinthatismissingbothN-glycans.ThebasisofthecombinedabsenceofGal/SiaandentireN-glycansisnotunderstood,butthepatternreturnstonormalwhenthepatientsareplacedonaGal-freediet.

MUSCULARDYSTROPHIES

FIGURE45.4.O-Manglycanbiosyntheticpathway.ThebiosyntheticpathwaysofrepresentativeO-Manglycansareshown.Genesthatcauseadisorderareindicatedinred.Threemaingroupsareidentified:coreM1-3.AllO-Manglycansareinitiatedoneitheraserine(S)orathreonine(T)intheacceptorproteinusingtwoenzymesprotein-O-mannosyltransferase1and2(POMT1/2)andDol-P-Mandonor.SeveralgenesinthebiosynthesisofDol-P-Man(GMPPB,DPM1-3)aredeficientinsomepatientswithdystroglycanopathy,whereasothers(DOLK,DPM1,PMM2)causeamoregeneralizedCDG.MannosereceiveseitherGlcNAcβ1,2-toformcoreM1orβ1,4GlcNActoformcoreM3.Mutationsinbothgenes

(POMGNT1andPOMGNT2)cancauseadystroglycanopathy.CoreM2canbeformedifabranchingβ1-6GlcNAcisadded.CoreM1andM2areelongatedbyGalandmayterminatewithFuc,Sia,GlcAwithoptionalsulfation.Noneofthegenesresponsibleforaddingtheterminalsugarshavebeenassociatedwithdystroglycanopathies.Aftertheadditionoftheβ1-4GlcNAc,coreM3iselongatedwithGalNAc,andthentheManis6-O-phosphorylatedviaaspecifickinase(POMK,alsoknownasSGK196).FKTNandFKRPcanacttoadd2ribitol-5-phosphateunitstotheGalNAcresiduethatisthenextendedwithasingleXylandGlcAbyTMEM5andB4GAT1respectively.Ribitol-5-phosphateusedbyFKTNandFKRPissynthesizedfromCDP-ribitolbyISPD.Mutationsinthesegenes(B3GALNT2,POMK,FKTN,FKRP,ISPD,TMEM5andB4Gat1)cancauseadystroglycanopathy.ThelastdefinedstepincoreM3biosynthesisisusingthisXyl-GlcAprimerforthestepwiseadditionofXylandGlcAtoformaGAG-likerepeatingdisaccharidetermedmatriglycan.ThisiscatalyzedbytheenzymeencodedbyLARGE(oritshomologueGYLTL1B)withdualglycosyltransferaseactivities.Matriglycanbindslaminintoα-DGanditsreductionorlossisbelievedtobethecauseofmostofthedystroglycanopathies(POMGNT1-mutationsbeingtheexception).

CongenitalMuscularDystrophies

MutationsalteringO-Manglycans(Chapter13),primarilyonα-dystroglycan(α-DG)causeatleastsixteentypesofcongenitalmusculardystrophy(CMD)termedDystroglycanopathies(Figure45.4)α-DGatneuromuscularjunctionslinksskeletalmusclecellcytoskeletontolamininintheextracellularmatrix.Theclinicalspectrumofdystroglycanopathiesisbroad,rangingfromverysevere,andoftenlethal,musculo-oculo-encephalopathies,suchasWalkerWarburgsyndrome(WWS),muscle-eye-braindisease(MEB),andFukuyamacongenitalmusculardystrophy(FCMD)tomilderformsoflimb-girdlemusculardystrophy.Geneticanalysisofthesedisordershasbeenindispensablefordiscoveringfunctionalglycansandtheirbiosynthesis.ThecomplexpathwayispresentedinFigure45.4andChapter13.ThepathwayisinitiatedintheERbythetransferofMantoSer/ThrviatheproteinO-mannosyltransferasecomplexcontainingPOMT1andPOMT2(Table45.1).IntheGolgi,thispathwaygeneratesover20O-Manglycansinmammals.AnunusualfeatureononesubsetofO-ManglycansistheexistenceofaMan-6-PgeneratedbyPOMK.TheMan-6-PcontainingCoreM3glycanconsistsofMan-6-P,GlcNAc(transferredbyPOMGNT2)andGalNAc(transferredbyB3GALNT2).Thetrisaccharidecoreisextendedbytwounitsofribitol-5-phosphateandasinglerepeatofxylose(Xyl)andglucuronicacid(GlcA).Thisstructureiselongatedbyanalternatingdisaccharide(β1-3Xylα1-3GlcA).Tworibitol-5-phosphatesaresequentiallytransferredbyFKTN(fukutin)andFKRP(fukutin-relatedprotein)fromCDP-ribitol,whichisgeneratedbyISPD.XylandGlcAinthesinglerepeataretransferredbyTMEM5andB4GAT1,respectively.TheelongationbythealternatingdisaccharideiscatalyzedbyLARGE.Thepolymericglycan(matriglycan)isrecognizedbyseveraldiagnosticmonoclonalantibodiesandisnecessaryforthebindingoflamininandothermoleculestoα-DG.O-Manglycanscanalsoplayatraitorousrole,becausetheyarereceptormoleculesforLassaVirusentryintocells.Thisfeaturewascleverlyexploitedtoscreenlibrariesandidentifygenesrequiredforvirusentry.ThemethodcorrectlyidentifiedallpreviouslyknownWWS-causinggenesandpredictednewculprits.Sofar,sixteengeneshavebeenproventocauseaDystroglycanopathy(POMT1,POMT2,POMGNT1,FKTN,FKRP,ISPD,LARGE,POMGNT2,TMEM5,

B3GALNT2,POMK,B4GAT1,GMPPB,DPM1,DPM2,DPM3)ofwhichthree(DPM1–3)arerequiredforN-glycanandGPI-anchorbiosynthesis.

Whileα-DGisthemajorcarrierofLARGE-modifiedO-Manglycans,cadherinscarryO-Manglycansimportantfortheirrolesincell-celladhesion.ClusteredprotocadherinsthatcontainO-Manglycansareregulatedduringbraindevelopmentandformlargeroligomers.TheO-Manglycansseemtoorientcadherindomainsforcriticalinteractions.O-ManinclusteredprotocadherinsmayhelpexplainocularandbrainmalformationsindisorderssuchasWWS.ClinicalcriteriapreviouslydefinedtheDystroglycanopathies,butnowtheyaredefinedbythemutatedgeneasmutationsingenesofthepathwayfittheclinicalcriteria.WWSisthemostsevereCMD.Patientsliveabout1yearandhavemultiplebrainabnormalities,andseveremusculardystrophy.About20%ofpatientshavemutationsinPOMT1andafewhavemutationsinPOMT2.OthershavedefectsinFKTNandFKRP,butmutationsinbothalsocausemilderformsofmusculardystrophy.POMGNT1ismutatedinmuscle–eye–braindisease(MEB),whichischaracterizedbysymptomssimilarto,butmilderthan,WWS.ThemostseverelyaffectedMEBpatientsdieduringthefirstyearsoflife,butthemajorityofmildcasessurvivetoadulthood.Fukuyamamusculardystrophy(FCMD)iscausedbyasingle3-kb3’-retrotransposoninsertionaleventintotheFKTNgene,whichoccurred2000–2500yearsago.ThispartiallyreducesthestabilityofthemRNA,makingitarelativelymildmutation.FCMDisoneofthemostcommontypesofCMDinJapanwithacarrierfrequencyof1/188.Fktn-nullmicediebyE9.5inembryogenesisandappeartohavebasementmembranedefects.Congenitalmusculardystrophytype1C(MDC1C)isarelativelymilddisorderthatiscausedbymutationsinFKRP.PatientswithMDC1D,alimb-girdlemusculardystrophy,containmutationsinLARGE,originallydescribedinmyodystrophicmice(myd,nowcalledLargemyd).Theproteinhastwoglycosyltransferasesignatures(DXD)indifferentdomainsthataccountforxylosyl-andglucuronosyl-transferaseactitivitiesrespectively(Chapter13).GNEMyopathyRecessivemutationsinGNEcauseadult-onsetGNEmyopathy(previouslynamedhereditaryinclusionbodymyopathytype2(HIBM2)orNonakamyopathy)(Table45.1).Itoccursworldwide,butonemutation(p.Met745Thr)isespeciallycommonamongPersianJews(1:1500)andoccursinthekinasedomain(Chapter5).GNEmutationsoccurinvariouscombinationsinbothGNEenzymaticdomains,andvariablyaffectenzymeactivity.Themutationsmoderatelyreduceenzymaticactivityandreducesialylationinmousemodels.Siaisefficientlysalvagedfromdegradedglycoproteins,butthereislittleinformationonthecell-typepreferenceorage-dependentcontributionsofthedenovoversussalvagepathways.Gne-nullmicedieduringembryogenesisandmostmicehomozygousfortheknock-inp.Met743Thrmutationdieafewdaysafterbirthbecauseofseverehematuriaandproteinuria,notmyopathy.Glomerularabnormalitiesinthepodocytebasementmembraneresultfromundersialylatedinfootpodocytessuchaspodocalyxinandnephrin.ProvidingN-acetylmannosamine(ManNAc)tothepupsintheneonatalperiodrescuessomeofthemand

increasessialylationofpodocalyxinandnephrin.Mutantp.Met743ThrsurvivorswhodidnotreceiveManNAcaftertheneonatalperioddevelopadult-onsethyposialylationofmuscletissue,whichcanberescuedbyoralManNActherapyatadultage.OralManNAcisbeingtestedasatherapyforGNEmyopathypatients,aswellasforpatientswithprimaryglomerulardiseases(focalsegmentalglomerulosclerosis,minimalchangedisease,membranousnephropathy).Anothermousemodel,carryingatransgenicGnemutation(p.Asp207Val)commonintheJapanesepopulation,developsapathologicaladult-onsetmusclephenotypeinvolvingβ-amyloiddepositionthatprecedestheaccumulationofinclusionbodies.ProvidingmodestamountsofSia,N-acetylmannosamineorsialyllactosetothesemicepreventsandevenreversesmuscledeterioration.Surprisingly,sialylactosesupplementsaremosteffective.Westillknowrelativelylittleabouthoweachofthesesugarsareimportedintovariouscellsorpreferentiallyusedinglycansynthesis(Chapter15).

DEFECTSINO-GalNAcGLYCANS

AdefectinO-GalNAcsynthesisbyaparticularpolypeptideGalNActransferase(GALNT3)causesfamilialtumoralcalcinosis.Thissevereautosomalrecessivemetabolicdisordershowsphosphatemiaandmassivecalciumdepositsintheskinandsubcutaneoustissues.MutationsintheO-glycosylatedfibroblastgrowthfactor23(FGF23)alsocausephosphatemia,suggestingthatGALNT3modifiesFGF23.Therareautoimmunedisease,Tnsyndrome,iscausedbysomaticmutationsintheX-linkedgeneC1GALT1C1,whichencodesahighlyspecificchaperoneCOSMCrequiredfortheproperfoldingandnormalactivityoftheβ1-3galactosyltransferaseC1GALT1neededforsynthesisofcore1and2O-glycans(Chapters10and46).

DEFECTSINPROTEOGLYCANSYNTHESIS

ProteoglycansandtheirGAGchainsarecriticalcomponentsinextracellularmatrices.Foradiscussionoftheirbiosynthesis,coreproteins,andfunction,seeChapter17.

Ehlers–DanlosSyndrome(ProgeroidType)

Ehlers–Danlossyndrome(progeroidtype)isaconnectivetissuedisordercharacterizedbyfailuretothrive,looseskin,skeletalabnormalities,hypotonia,andhypermobilejoints,togetherwithdelayedmotordevelopmentanddelayedspeech.ThemolecularbasisofthedisorderisreducedsynthesisofthecoreregionofGAGsinitiatedbyXyl.GalactosyltransferaseI(B4GALT7)theenzymethataddsGaltoXyl-Serismutatedinthisdisease.TheactivityofgalactosyltransferaseII(B3GALT6),theenzymeresponsibleforaddingthesecondGaltothecoreofaGAG,mayalsobereduced.OnepossibleexplanationforthedualeffectisthattheprimarymutationaffectstheformationorstabilityofabiosyntheticcomplexinvolvingseveralGAGbiosyntheticenzymes.

CongenitalExostosis

Defectsintheformationofheparansulfate(HS)causehereditarymultipleexostosis(HME),anautosomaldominantdiseasewithaprevalenceofabout1:50,000(Table45.1).ItiscausedbymutationsintwogenesEXT1andEXT2,whichareinvolvedinHSsynthesis.HMEpatientshavebonyoutgrowths,usuallyatthegrowthplatesofthelongbones.Normally,thegrowthplatecontainschondrocytesinvariousstagesofdevelopment,whichareenmeshedinanorderedmatrixcomposedofcollagenandchondroitinsulfate(CS).InHME,however,theoutgrowthsareoftencappedbydisorganizedcartilagenousmasseswithchrondrocytesindifferentstagesofdevelopment.About1–2%ofpatientsalsodeveloposteosarcoma.HMEmutationsoccurinEXT1(60–70%)andEXT2(30–40%).TheencodedproteinsmayformacomplexintheGolgiandbotharerequiredforpolymerizingN-acetylglucosamine(GlcNAc)α1–4andGlcAβ1–3intoHS.However,thepartiallossofonealleleofeithergeneappearssufficienttocauseHME.ThismeansthathaploinsufficiencydecreasestheamountofHSandthatEXTactivityisratelimitingforHSbiosynthesis.Thisisunusualbecausemostglycanbiosyntheticenzymesareinsubstantialexcess.ThemechanismofHMEpathologyislikelyrootedinadisruptionofthenormaldistributionofHS-bindinggrowthfactors,whichincludeFGFandmorphogenssuchashedgehog,Wnt,andmembersoftheTGF-βfamily.ThelossofHSdisruptsthesepathwaysinDrosophila.MicethatarenullforeitherExtgeneareembryoniclethalandfailtogastrulate;however,Extheterozygousanimalsareviable,anddonotdevelopexostosesonthelongbones,incontrasttopatientswithHME.However,micewithchondrocyte-specificsomaticmutationsinExt1causealossofheterzygosityanddevelopexostosesandgrowthabnormalitiesinthegrowthplatesofthosebones.HSisrequiredtoestablishandmaintaintheperichondriumphenotype,anditalsorestrainspro-chondrogenicsignalingproteinsincludingBMPsthatnormallyrestrictchondrogenesis.WithoutHS,chondrogenesisincreasesinlocalizedareasofactivelygrowingcells.

Achondrogenesis,DiastrophicDystrophy,andAtelosteogenesis

Threeautosomalrecessivedisorders,diastrophicdystrophy(DTD),atelosteogenesistypeII(AOII),andachondrogenesistypeIB(ACG-IB),allresultfromdefectivecartilageproteoglycansulfation.Theseformsofosteochondrodysplasiahavevariousoutcomes.AOIIandACG-IBareperinatallethalbecauseofrespiratoryinsufficiency,whereasDTDpatientsdevelopsymptomsonlyincartilageandbone,includingcleftpalate,clubfeet,andotherskeletalabnormalities.ThoseDTDpatientssurvivinginfancyoftenliveanearlynormallifespan.AllofthesedisordersresultfromdifferentmutationsintheDTDgene(SLC26A2)thatencodesaplasmamembranesulfatetransporter.Unlikemonosaccharides,sulfatereleasedfromdegradedmacromoleculesinthelysosomeisnotsalvagedwell.Theheavydemandforsulfateinboneandcartilageproteoglycansynthesisprobablyexplainswhythesymptomsaremostevidentintheselocations.DefectsintheUDP-GlcA/UDP-N-acetylgalactosamine(GalNAc)Golgi

transporter(SLC35D1)causeSchneckenbeckendysplasia.Patientshaveboneabnormalitiessimilartothoseseeninotherchrondrodysplasias,andamousemodelofthediseaseshowssimilarfeatures.

MacularCornealDystrophy

KeratansulfateI(KS-I)inthecorneaisanN-linkedoligosaccharidewithpoly-N-acetyllactosaminerepeats(Galβ1-4GlcNAcβ1-3)variablysulfatedatthe6-positions.Macularcornealdystrophy(MCD),anautosomalrecessivedisease,causesthecorneatobecomeopaqueandcorneallesionstodevelop.TwotypesofMCDhavebeendescribed.MCDIappearstobeduetoadeficiencyinsulfatingtherepeatingunits.BothGalandGlcNAcaresulfatedinKS;sulfationofGalandGalNAcinCSarealsoaffectedinMCDpatients.

DEFECTSINGLYCOSYLPHOSPHATIDYLINOSITOL(GPI)–ANCHORED

PROTEINS

CompletedeletionoftheGPIpathwayinmicecausesembryoniclethality.Notsurprising,since>150membraneproteinsrequireaGPI-anchorforcellsurfaceexpression(Chapter12).HypomorphicmutationsinmultiplegenesinthepathwayleadtoapartialreductioninGPI-anchoredproteins.TheseincludePIGA,PIGQ,PIGY,PIGL,PIGW,PIGM,PIGVandPIGOinanchorassembly(Table45.1),andPIGTinthetransferoftheglycantoprotiens.Defectsinsidechainmodifications(PIGNandPIGG)andmaturationofGPIfollowingattachmenttoproteins(PGAP1,PGAP2andPGAP3),alsocauseinheritedGPIdeficiency,butnotembryonicdeath.GPIdeficiencyhasimmenseandvariableconsequencesincludingneurologicsymptoms,particularlydevelopmentaldelay/intellectualdisabilityandseizures,epilepticencephalopathy,progressivecerebraland/orcerebellaratrophy,hypotonia,corticalvisualimpairment,sensorineuraldeafnessandHirschsprungdisease.Non-neurologicphenotypesincludebrachytelephalangy,anorectalanomaly,renalabnormality,cleftpalate,heartdefect,andcharacteristicfacialfeaturessuchashypertelorism,broadnasalbridgeandtentedmouth.Othersymptomssuchasichthyosis,irondeposition,hepatosplenomegaly,diaphragmaticherniaandhepaticand/orportalveinthrombosiswerereportedinsmallfractionsoftheaffectedindividuals.ItisnoteasytocausallyrelatespecificsymptomstodeficiencyofparticularGPI-anchoredproteinexceptforfewinstances.Deficiencyoftissuenon-specificalkalinephosphatase(TNALP)accountsforseizuresinsomeofthepatients.DeathwithinayearafterbirthduetoaspirationorstatusepilepticusisnotrareamongseverelyaffectedindividualswithGPIdeficiencywhilemildlyaffectedindividualslivewithGPIdeficiency.

DEFECTSINGLYCOSPHINGOLIPID(GSL)SYNTHESIS

OnlythreedisordersinGSLsynthesisareknowninhumans.MutationsinST3GAL5causeofautosomalrecessiveAmishinfantileepilepsysyndromeandalso“SaltandPeppersyndrome”.ThisgeneencodesasialyltransferaserequiredforthesynthesisofthegangliosideGM3(Siaα2-3Galβ1-4Glc-ceramide)fromlactosylceramide(Galβ1-4Glc-ceramide).GM3isalsoaprecursorforsomemorecomplexgangliosides.Thepatients’plasmaglycosphingolipidsarenonsialylated.Incontrasttothehumanformofthedisease,micethatlackGM3donothaveseizuresorashortenedlifespan.However,mousestrainsthatarenullforthesialyltransferaseandanN-acetylgalactosaminyltransferasethatisrequiredformakingothercomplexgangliosides,dodevelopseizures,suggestingthatitistheabsenceofthesemorecomplexgangliosidesthatmaybetheunderlyingproblem(Chapter11).MutationsinB4GALNT1(alsoknownasGM2/GD2synthase)causehereditaryspasticparaplegiasubtype26.Thesepatientshavedevelopmentaldelaysandvaryingcognitiveimpairmentswithearly-onsetprogressivespasticityowingtoaxonaldegeneration.Cerebellarataxia,peripheralneuropathy,corticalatrophy,andwhite-matterhyperintensitieswerealsoconsistentacrossthedisorder.AB4galnt1−/−mouserecapitulatesseveraloftheneurologicalcharacteristicsofSPG26,mostprominentlytheprogressivegaitdisorderST3GAL3makesmorecomplexgangliosidesaswellasN-andO-glycans.ItisrequiredforthedevelopmentofhighcognitivefunctionsandismutatedinsomeindividualswithWestsyndrome.AnSt3gal3−/−mousemodelalsoexists,butthesemiceappeartohavenoovertneurologicalphenotype.GSLdisordersaredifficulttoidentifybiochemicallybecausenoconvenientbiomarkersexist.Nextgenerationsequencingwillrevealnewcandidates.ADeglycosylationdisorderEarlyon,itwasassumedthatglycosylationdisorderswouldresultfromdefectsinglycanbiosyntheticenzymes,butthatperspectivehaschanged.DiscoveryofdefectsinGolgiorganizationandhomeostasis,ERchaperonessuchasCOSMCorEDEMandinERqualitycontrolhavebroadenedtheperspective.AnewdefectintheER-associateddegradation(ERAD)continuum(Chapter39)iscausedbydefectsinNGLY1,anenzymethatcleavesN-glycansfrommisfoldedglycoproteinstransportedintothecytoplasm,priortotheirproteasomaldegradation(Table45.1).ThedefectdoesnotappeartoinducetheERADpathway,accumlateundegradedglycoproteinsinvesicles,ortriggerautophagy.Itisunclearhowthedefectcausessymptomssuchasdevelopmentaldelay,movementdisorder,seizures,andacuriouslackoftearproduction,buttheirclinicalsimilaritytootherCDGsemphasizesthatglycosylationdefectscannotsimplybedividedinto“synthesis”or“degradation”.

PHENOTYPES,MULTIPLEALLELES,ANDGENETICBACKGROUND

Phenotypicexpressionofthesamemutationcanhavehighlyvariableimpact,evenamongaffectedsiblings.Explanationsbasedonresidualactivityforthese“simpleMendeliandisorders”areneithersimplenorgenerallysatisfying.Itisoftenattributedto“genetic

background.”Aknockoutmutationmaybelethalinonehighlyinbredmousestrain,butnotinanotherbecausecompensatorypathwaysmayexist.DietaryandenvironmentalimpactsaresubstantialasseeninMPI-CDGpatientswithandwithoutoralmannosetherapy.Multiplesimultaneousorsequentialenvironmentalinsultsmayimpingeonborderlinegeneticinsufficienciestoproduceovertdisease.ACKNOWLEDGMENTSTheauthorsacknowledgecontributionofBobbyG.NgandappreciatehelpfulcommentsandsuggestionsfromAimeLopezAguilar,KekoaTaparra,KrithikaVaidyanathanandShwetaVarshney.FURTHERREADINGRosnobletC,PeanneR,LegrandD,FoulquierF.2013.Glycosylationdisordersofmembrane

trafficking.GlycoconjJ30:23-31.DobsonCM,HempelSJ,StalnakerSH,StuartR,WellsL.2013.O-Mannosylationandhuman

disease.CellMolLifeSci70:2849-2857.HuegelJ,SgarigliaF,Enomoto-IwamotoM,KoyamaE,DormansJP,PacificiM.2013.Heparan

sulfateinskeletaldevelopment,growth,andpathology:thecaseofhereditarymultipleexostoses.DevDyn242:1021-1032.

JaekenJ.2013.Congenitaldisordersofglycosylation.HandbClinNeurol113:1737-1743.KinoshitaT.2014.Biosynthesisanddeficienciesofglycosylphosphatidylinositol.ProcJpnAcad

SerBPhysBiolSci90:130-143.MaedaN.2015.Proteoglycansandneuronalmigrationinthecerebralcortexduring

developmentanddisease.FrontNeurosci9:98.NishinoI,Carrillo-CarrascoN,ArgovZ.2015.GNEmyopathy:currentupdateandfuture

therapy.JNeurolNeurosurgPsychiatry86:385-392.HennetT,CabalzarJ.2015.Congenitaldisordersofglycosylation:aconcisechartofglycocalyx

dysfunction.TrendsBiochemSci40:377-384.FreezeHH,EklundEA,NgBG,PattersonMC.2015.Neurologicalaspectsofhuman

glycosylationdisorders.AnnuRevNeurosci38:105-125.

Appendix45A.OnlineTABLE45.2Tableofknownhumanglycosylationdisorders

Disorder Gene FunctionDisorderOMIM

GeneOMIM MainClinicalFeatures Year Reference

N-LinkedPathwayDPAGT1–CDG

DPAGT1 GlcNAc-1-Ptransferase 608093 191350 ID,Hy,Sz,M,infections,earlydeath&CMS

2003 PMID:12872255

ALG1–CDG

ALG1 β1,4Mannosyltransferase 608540 605907 ID,Hy,Sz,M,infections,earlydeath 2004 PMID:14709599PMID:14973778PMID:14973782

ALG2–CDGALG2–CMS

ALG2 α1,3Mannosyltransferase 607906 607905 ID,Hy,Sz,infections,hypomyelination,hepatomegaly,earlydeathCongenitalMyasthenicSyndrome

20032013

PMID:12684507PMID:23404334

ALG3–CDG

ALG3 α1,3Mannosyltransferase 601110 608750 ID,Hy,Sz,M,opticnerveatrophy 1999 PMID:10581255

ALG6–CDG

ALG6 α1,3Glucosyltransferase 603147 604566 ID,Hy,Sz,M,ataxia 1999 PMID:10359825

ALG8–CDG

ALG8 α1,3Glucosyltransferase 608104 608103 DD,hepatomegaly,protein-losingenteropathy,coagulopathy,ascites,renalfailure,earlydeath

2003 PMID:12480927

ALG9–CDG

ALG9 α1,2Mannosyltransferase 608776 606941 ID,Hy,Sz,hepatomegaly 2004 PMID:15148656

ALG11–CDG

ALG11 α1,2Mannosyltransferase 613661 613666 ID,Hy,Sz,deafness,dysmorphism 2010 PMID:20080937

ALG12–CDG

ALG12 α1,6Mannosyltransferase 607143 607144 ID,Hy,Sz,M,recurrentinfections 2002 PMID:11983712PMID:12217961

ALG13–CDG

ALG13 UDP-GlcNActransferase 300884 300776 M,Sz,hepatomegaly,horizontalnystagmus,opticnerveatrophy,infections

2012 PMID:22492991

ALG14–CMS

ALG14 UDP-GlcNActransferase 616227 612866 CongenitalMyasthenicSyndrome 2013 PMID:23404334

RFT1–CDG

RFT1 Man5GlcNAc2flippase 612015 611908 ID,Hy,Sz,M,hepatomegaly,coagulopathy,deafness

2008 PMID:18313027

TUSC3–CDG

TUSC3 SubunitoftheOSTcomplex 611093 601385 NSID(Non-syndromicintellectualdisability)

2008 PMID:18452889PMID:18455129

MAGT1–CDG

MAGT1 SubunitoftheOSTcomplex 300716 300715 XLNSID(X-LinkedNon-syndromicintellectualdisability)

2008 PMID:18455129

DDOST–CDG DDOST SubunitoftheOSTcomplex 614507 602202 ID,DD,failuretothrive,gastroesophagealreflux,earinfections,oromotordysfunction

2012 PMID:22305527

STT3A–CDG

STT3A SubunitoftheOSTcomplex 615596 601134 ID,DD,H,M,Sz,failuretothrive 2013 PMID:23842455

STT3B–CDG STT3B SubunitoftheOSTcomplex 615597 608605 ID,DD,H,M,Sz,failuretothrive,thrombocytopenia,genitalabnormalities

2013 PMID:23842455

NGLY1–CDG

NGLY1 N-Glycanase-1 615273 610661 ID,DD,Sz,abnormalliverfunction 2012 PMID:22581936

SSR4–CDG

SSR4 Signalsequencereceptor,delta 300934 300090 M,ID,Sz,gastroesophagealreflux 2013 PMID:24218363

SSR3–CDG

SSR3 Signalsequencereceptor,gamma 606213 Sz,ID,DD,M,abnormalbrainstructure

MGAT2–CDG

MGAT2 GlcNAc-transferaseII 212066 602616 ID,feedingproblemsseverediarrhea,growthretardation,dysmorphism

1996 PMID:8808595

MOGS–CDG MOGS α1,2Glucosidase 606056 601336 Hy,Sz,hepatomegaly,hypoventilation,feedingproblems,dysmorphism,fatal,uniquetetrasaccharideinurine.

2000 PMID:10788335

MAN1B1–CDG

MAN1B1 α1,2Mannosidase 614202 604346 NSID(Non-syndromicintellectualdisability),delayedmotorandspeechdevelopment,variabledysmorphicfeatures,truncalobesityandmacrocephaly

2011 PMID:21763484

I-celldisease GNPTAB GlcNAc-1-Ptransferase 252500252600

607840 ID,congenitaldislocationofthehip,thoracicdeformities,hernia,hyperplasticgums,coarsefacialfeatures,restrictedjointmovement

1981 PMID:6461005

AutosomalDominantPolycysticLiverDisease

PRKCSH GlucosidaseIISubunitBeta 174050 177060 AutosomalDominantpolycysticliverdisease

2003 PMID:12529853PMID:12577059

CongenitalSevereNeutropenia

JAGN1 EndoplasmicReticulumorganization 616022 616012 CongenitalSevereNeutropenia,recurrentinfections

2014 PMID:25129144PMID:25129145

PotentialtoEffectMultiplePathwaysPMM2–CDG

PMM2 ConversionofMan-6-PtoMan-1-P 212065 601785 ID,Hy,Sz,strabismus,cerebellarhypoplasia,failuretothrive,cardiomyopathy.20%lethalityinthefirst5years

1997 PMID:9140401

MPI–CDG

MPI ConversionofFruct-6-PandMan-6-P 602579 154550 Hepaticfibrosis,coagulopathy,hypoglycemia,protein-losingenteropathy,vomiting,Noneurologicalsymptoms

1998 PMID:9525984

DHDDS–CDG

DHDDS DehydrodolicholDiphosphateSynthase 613861 608172 RetinitisPigmentosainAshkenaziJews 2011 PMID:21295282PMID:21295283

DOLK–CDG

DOLK DolKinase 610768 610746 ID,Hy,Sz,hypoglycemia,ichthyosis,dilatedcardiomyopathy,cardiacfailure

2007 PMID:17273964

SRD5A3–CDG SRD5A3 PolyprenolReductase 612379 611715 ID,Hy,eyeandbrainmalformations,nystagmus,hepaticdysfunction,coagulopathy,ichthyosis

2010 PMID:20637498

DPM1–CDG

DPM1 Dol-P-Mansynthasecomplex 608799 603503 ID,Hy,Sz,M,dysmorphism,coagulopathy

2000 PMID:10642597PMID:10642602

DPM2–CDG DPM2 Dol-P-Mansynthasecomplex 615042 603564 Dystroglycanopathy,Sz,Hy,M,dysmorphism,cerebellarhypoplasia,earlydeath

2012 PMID:23109149

DPM3–CDG

DPM3 Dol-P-Mansynthasecomplex 612937 605951 Dystroglycanopathy,dilatedcardiomyopathy,stroke-likeepisode

2009 PMID:19576565

MPDU1–CDG

MPDU1 Man-P-Dolutilization 609180 604041 ID,Sz,failuretothrive,ichthyosis-likeskindisorder,severefeedingdifficulties

2001 PMID:11733556PMID:11733564

GMPPA–CDG

GMPPA GDP-ManpyrophosphorylaseA 615510

615495 Achalasia,alacrima,andneurologicaldeficits

2013 PMID:24035193

SLC35C1–CDG SLC35C1 GDP-Fuctransporter 266265 605881 ID,Hy,Sz,M,unusualfacialappearance,dwarfism,infectionswithneutrophilia

2001 PMID:11326279

B4GALT1–CDG B4GALT1 β1,4Galactosyltransferase 607091 137060 ID,DD,Hy,macrocephaly,Dandy-Walkermalformation,coagulopathy,myopathy

2002 PMID:11901181

SLC35A1–CDG SLC35A1 CMP-Sialicacidtransporter 603585

605634

I.D,Sz,Ataxia,Bleeding,thrombocytopenia,neutropenia,RenalandCardiacinvolvement

20052013

PMID:15576474PMID:23873973

SLC35A2–CDG

SLC35A2 UDP-Galtransporter 300896 314375 ID,Sz,skeletalanomalies 2013 PMID:23561849

SLC35A3-CDG SLC35A3 UDP-GlcNActransporter 615553 605632 Autismspectrumdisorder,Hy,epilepsyandarthrogryposis

2013 PMID:24031089

SLC39A8-CDG SLC39A8 Manganesetransporter 616721 608732 Cranialasymmetry,severeinfantilespasmswithhypsarrhythmia,anddysproportionatedwarfism

2015 PMID:26637979PMID:26637978

COG1–CDGCOG1–CCMS

COG1 Golgi-to-ERretrogradetransport 611209117650

606973 ID,shortenedlongbones,facialdysmorphismandcerebrocostomandibular(CCMS-likesyndrome)

2009 PMID:16537452PMID:19008299

COG2–CDG COG2 Golgi-to-ERretrogradetransport N/A 606974 M,psychomotorretardation,Sz,liver 2014 PMID:24784932

dysfunction,hypocupremia,hypoceruloplasminemia

COG4–CDG COG4 Golgi-to-ERretrogradetransport 613489 606976 DD,Hy,Sz,nystagmus,hepatosplenomegaly,failuretothriveininfancywithrecurrentdiarrhea,earlydeath

2009 PMID:19494034

COG5–CDG

COG5 Golgi-to-ERretrogradetransport 613612 606821 ID,Hy,delayedspeech,ataxia 2009 PMID:19690088

COG6–CDG

COG6 Golgi-to-ERretrogradetransport 614576 606977 Severeneurologicdisorder,Sz,vomiting

2010 PMID:20605848

COG7–CDG

COG7 Golgi-to-ERretrogradetransport 608779 606978 Hy,M,growthretardation,adductedthumbs,failuretothrive,cardiacanomalies,wrinkledskin,earlydeath

2004 PMID:15107842

COG8–CDG

COG8 Golgi-to-ERretrogradetransport 611182 606979 ID,Hy,Sz 2007 PMID:17331980PMID:17220172

ATP6V0A2–CDGWrinklyskinsyndrome

ATP6V0A2 GolgipHRegulator 219200278250

611716 Cutislaxa,congenitalhipdislocation,jointhyperlaxity,dysmorphism,feedingproblems,lateclosurethefontanelles,varyingCNSinvolvement

2008 PMID:18157129

TMEM165–CDG TMEM165 GolgiRegulatorpHandCalciumHomeostasis 614727 614726 ID,Hy,M,shortstature,dysmorphism,eyeabnormalities,hepatomegaly,skeletaldysplasia

2012 PMID:22683087

TMEM199–CDG TMEM199 Golgitrafficking 616829 616815 Mildphenotypeofhepaticsteatosis,elevatedaminotransferases,alkalinephosphatase,andhypercholesterolemia,lowserumceruloplasmin

2016 PMID:26833330

CCDC115–CDG CCDC115 Golgihomeostasis 616828 613734 Storage-disease-likephenotypeinvolvinghepatosplenomegaly,whichregressedwithage,highlyelevatedbone-derivedalkalinephosphatase,elevatedaminotransferases,andelevatedcholesterol,incombinationwithabnormalcoppermetabolismandneurologicalsymptoms

2016 PMID:26833332

Congenitalmyasthenicsyndrome

GFPT1 Glutamine-fruct-6-Ptransaminase1 610542 138292 Congenitalmyasthenicsyndromewithtubularaggregates

2011 PMID:21310273

Achondrogenesistype1A TRIP11 Golgistructure 200600 604505 Lethalachondrogenesis,deficientossification

2010 PMID:20089971

PGM1–CDG PGM1 ReversibleconversionofGlc-1-PandGlc-6-P 614921 171900 Neurologicallynormal,splituvula, 2012 PMID:22492991

Glycogenstoragedisease14

612934 hepatopathy,hypoglycemia,rhabdomyolysis,dilatedcardiomyopathy,cardiacarrest,malignanthyperthermia

Hyper-IgEsyndrome(HIES)

PGM3 ReversibleconversionofGlcNAc-1-PandGlcNAc-6-P

615816 172100 Severeatopy,increasedserumIgElevels,immunedeficiency,autoimmunity,andmotorandneurocognitiveimpairment

2014 PMID:24589341PMID:24698316

Neutropenia,severecongenital4

G6PC3 Glc-6Phosphatase,catalytic,3 612541 611045 Severecongenitalneutropenia,recurrentinfections,prominentsuperficialveins,cardiacabnormalities

2011 PMID:21385794

GlycogenstoragediseaseIbandIc

G6PT1 Glc-6-Ptransporter 232220232240

602671 Neutrophildysfunction 2011 PMID:21385794

Non-syndromicI.DWestsyndrome

ST3GAL3 N-Acetyllactosaminideα-2,3Sialyltransferase 611090615006

606494 NSID(Non-syndromicintellectualdisability),Infantilespasms,hypsarrhythmia

20112013

PMID:21907012PMID:23252400

Cranio-lenticulo-suturaldysplasia(CLSD)

SEC23A Golgitrafficking 607812 610511 Late-closingfontanels,suturalcataracts,facialdysmorphism,skeletaldefects

2006 PMID:16980979

CongenitalDyserythropoieticAnemia(CDA-II)

SEC23B Golgitrafficking 224100 610512 Disruptederythropoiesiswithmultinucleatederythroblastsinbonemarrow

2009 PMID:19561605

AutosomalDominantPolycysticLiverDisease

SEC63 Golgitrafficking 174050 608648 AutosomalDominantpolycysticliverdisease.

2004 PMID:15133510

GPIAnchorPathwayX-LinkedGPI-anchordeficiencyParoxysmalNocturnalHemoglobinuria

PIGA GlcNAc-PIsynthesisprotein 300868300818

311770 Dysmorphism,Hy,Sz,variableCNS,cardiac,urinarysystems,earlydeathComplement-mediatedhemolysis

19932012

PMID:8500164PMID:22305531

AutosomalrecessiveGPI-anchordeficiency

PIGQ GlcNAc-PIsynthesisprotein N/A 605754 SevereDD,SZ,earlydeath 2014 PMID:24463883


PIGY GlcNAc-PIsynthesisprotein 616809 610662 SevereDD,SZ,earlydeath 2015 PMID:26293662

CHIMESyndromeHyperphosphatasiamentalretardationsyndrome

PIGL GlcNAc-PIde-N-Acetylase 280000 605947 ID,colobomas,heartdefect,early-onsetichthyosiformdermatosis,earanomalies(conductivehearingloss)Hyperphosphatasiamentalretardationsyndrome

2012 PMID:22444671

Westsyndromeand PIGW Acylatestheinositolringof 616025 610275 Westsyndrome,hyperphosphatasia 2013 PMID:24367057

hyperphosphatasiawithmentalretardationsyndrome

phosphatidylinositolinGPI-anchorbiosynthesis

withmentalretardationsyndrome


PIGM Firstα-MannosyltransferaseinGPIbiosynthesis

610293 610273 Sz,portalveinthrombosis,portalhypertension

2006 PMID:16767100

Hyperphosphatasiamentalretardationsyndrome

PIGV Secondα-MannosyltransferaseinGPIbiosynthesis

239300 610274 Hyperphosphatasiawithmentalretardationsyndrome1(HPMRS)

2010 PMID:20802478


PIGN GPIEthanolaminePhosphatetransferase1 614080 606097 Severeneurologicimpairment,Sz,lackofdevelopment,multiplecongenitalanomalies,earlydeath

2011 PMID:21493957


PIGO GPIEthanolaminePhosphatetransferase3 614749 614730 Hyperphosphatasiawithmentalretardationsyndrome2(HPMRS)

2012 PMID:22683086


PIGG GPIEthanolaminePhosphatetransferase2 N/A N/A DD/ID,Hy,Sz 2016 PMID:26996948

AutosomalrecessiveGPI-anchordeficiencyParoxysmalNocturnalHemoglobinuria

PIGT GPITransamidasecomplex 615398615399

610272 ID,Hy,Sz,abnormalskeletal,endocrine,ophthalmologicabnormalitiesandhypophosphatasiaComplement-mediatedhemolysis

20132013

PMID:23636107PMID:23733340


PGAP1 LipidremodelingstepsofGPI-anchormaturation

615802 611655 IDwithencephalopathy 2014 PMID:24784135




2013 PMID:23561846PMID:23561847




2014 PMID:24439110

DystroglycanopathyWalker-Warburgsyndrome(MDDGA1,B1,C1)

POMT1 O-Mannosyltransferase 236670613155609308

607423 Walker-Warburgsyndrome,brainmalformations,variouseyemalformations,elevatedserumCK

2002 PMID:12369018

Walker-Warburgsyndrome(MDDGA2,B2,C2)

POMT2 O-Mannosyltransferase 613150613156613158

607439 Walker-Warburgsyndrome,brainmalformations,variouseyemalformations,elevatedserumCK

2005 PMID:15894594

Muscle-eye-braindisease(MDDGA3,B3,C3)

POMGNT1 O-MannosylGlycanGlcNAc-transferase 253280613151613157

606822 ID,severeearly-onsetmuscleweakness,brainmalformations,variouseyemalformations,elevatedserumCK

2001 PMID:11709191

Fukuyama-typecongenitalmusculardystrophy(MDDGA4,B4,C4)

FKTN Ribitol-5-phosphatetransferase 253800613152611588

607440 Hy,ID,Sz,generalizedmuscleweakness,elevatedserumCK

1998 PMID:9690476

Congenitalmuscular FKRP Fukutin-RelatedProtein,ribitol-5-phosphate 613153 606596 Hy,feedingdifficulties,hypertrophy 2001 PMID:11592034

dystrophytype1C(MDDGA5,B5,C5)

transferase 606612607155

oflowerlimbmuscles,wastingofshouldergirdle,variableneurologicalinvolvement,elevatedserumCK

Congenitalmusculardystrophytype1D(MDDGA6,B6)

LARGE XylandGlcAtransferase 613154608840

603590 ID,whitematterchanges,elevatedserumCK

2003 PMID:12966029

Walker-Warburgsyndrome(MDDGA7)

ISPD CDP-ribitolsynthetase 614643 614631 Brainmalformations,variouseyemalformations,elevatedserumCK

2012 PMID:22522420PMID:22522421


POMGNT2 β1,4GlcNAc-transferase 614830 614828 Brainmalformations,variouseyemalformations

2012 PMID:22958903


TMEM5 Xyl-transferase 615041 605862 Brainmalformations,facialclefts,retinaldysplasia,gonadaldysgenesis.

2012 PMID:23217329

Congenitalmusculardystrophy(MDDGA11)

B3GALNT2 β1,3GalNAc-transferase2 615181 610194 I.D,Hy,Sz,brainmalformations,variouseyemalformations,elevatedserumCK

2013 PMID:23453667


POMK O-Mankinase 615249 615247 Walker-Warburgsyndrome,brainandeyemalformations,elevatedserumCK

2013 PMID:23929950PMID:23519211


B4GAT1 β1,4Glucuronyltransferase 615287 605517 Hy,Sz,brainmalformations,retinaldysplasia,elevatedserumCK

2013 PMID:23359570

Congenitalmusculardystrophy(MDDGA14,B14,C14)

GMPPB GDP-ManPyrophosphorylaseB 615350615351615352

615320 I.D,M,brainandeyemalformations,elevatedserumCK

2013 PMID:23768512

HereditaryInclusionbodymyopathy

GNE UDP-GlcNAc-2-epimerase/ManAckinase 600737605820269921

603824

Proximalanddistalmuscleweakness,wastingoftheupperandlowerlimbs,sparingofthequadriceps

2001 PMID:11528398

GlycosaminoglycanEhlers–Danlossyndrome B4GALT7 β1,4Galactosyltransferase7 130070 604327 ProgeroidformwithDD,short

stature,osteopenia,defectivewoundhealing,hypermobilejoints,hypotonicmuscles,loosebutelasticskin

1990 PMID:2106134

HereditaryMultipleExostoses

EXT1/EXT2 GlcA/GlcNAc-transferase 133700 608177608210

Multipleexostosesofthebone 1995 PMID:7550340

Schneckenbeckendysplasia

SLC35D1 UDP-GlcA/UDP-GalNAcGolgitransporter 269250 610804 Neonatallethalchondrodysplasia,short-limbedskeletaldysplasia

2007 PMID:17952091

Spondylo-epimetaphysealdysplasia

PAPSS2 3′-phosphoadenosine-5′-phosphosulphatesynthase

612847 603005 Short-trunkstature,skeletaldysplasia,normalintelligence,variableepiphysealandmetaphysealchanges

1998 PMID:9771708

Achondrogenesistype1B SLC26A2 SulphateAnionTransporter 222600600972256050

606718 Earlydeathinseverecases,adultsreported.AchondrogenesisIb:usuallystillbornorearlydeathofrespiratoryfailure.AtelosteogenesisII:pulmonaryhypoplasia,fatalininfants

1996 PMID:8528239

Spondylo-epimetaphysealdysplasia(SED-Omanitype)

CHST3 Chondroitin6-O-Sulfotransferase

143095 603799 Skeletaldysplasia,normalintelligence 2004 PMID:15215498

MacularcornealdystrophytypesI/II

CHST6 KeratanSulphate6-0-Sulfotransferase 217800 605294 Cornealcloudinganderosions,painfulphotophobia

2000 PMID:11017086

PeelingSkinSyndrome

CHST8 GalNAc4-OSulfotransferase1 270300 610190 Generalizedsuperficialskinpeelingfrombirth

2012 PMID:22289416

Ehlers–DanlossyndromeAdductedthumb-clubfootsyndrome

CHST14 DermatansulfateGalNAc4-OSulfotransferase1

601776 608429 Adductedthumb,clubfoot,progressivejoint,skinlaxitysyndrome

20092010

PMID:20004762PMID:20533528

Ehlers-DanloslikesyndromeorSEDwithjointhyperlaxity

B3GALT6 β1,3Galactosyltransferase6 271640615349

615291 Abnormalskeletalandconnectivetissueslaxskin,musclehypotonia,jointdislocation,andspinaldeformity

2013 PMID:23664117

Larsen-likesyndrome B3GAT3 β1,3Glucuronyltransferase3 245600 606374 Multiplejointdislocations,shortstature,craniofacialdysmorphismandcongenitalheartdefects

2011 PMID:21763480

Autosomalrecessiveshortstaturesyndrome

XYLT1 Xyl-transferase1 615777 608124 ModerateI.D,shortstature,distinctfacialfeatures,alteredfatdistribution

2014 PMID:23982343

Spondylo-OcularSyndromewithBoneFragility,Cataracts,andHearingDefects

XYLT2 Xyl-transferase2 605822 608125 Osteoporosis,cataracts,sensorineuralhearingloss,andmildlearningdefects

2015 PMID:26027496

MusculocontracturaltypeofEhlers–Danlossyndrome

DSE Dermatansulfateepimerase 615539 605942 Characteristicfacialfeatures,congenitalcontracturesofthethumbsandfeet,hypermobilityoffinger,elbow,andkneejoints,muscleweakness

2013 PMID:23704329

OtherAmishinfantileepilepsy ST3GAL5 Sia2,3Galβ1,4Glc-CerSynthase(GM3) 609056 604402 Infantile-onsetepilepsy, 2004 PMID:15502825

developmentalstagnation,blindnessSaltandPepperSyndrome ST3GAL5 Sia2,3Galβ1,4Glc-CerSynthase(GM3) 609056 604402 SevereI.D,epilepsy,scoliosis,altered

dermalpigmentation,choreoathetosis,dysmorphicfacialfeatures

2014 PMID:24026681

ComplexHereditarySpasticParaplegia

B4GALNT1 β1,4GalNAc-transferase1 609195 601873 Early-onsetspasticparaplegia,I.D,cerebellarataxia,andperipheralneuropathy,corticalatrophyandwhitematterhyperintensity

2013 PMID:23746551

Adams-OliverSyndrome4

EOGT EGF-domain-specificO-linkedO-GlcNActransferase

615297 614789 Aplasiacutiscongenita,terminaltransverselimbdefects

2013 PMID:23522784

FamilialTumoralCalcinosis

GALNT3 PolypeptideGalNAc-transferase 211900 601756 Massivecalciumdepositsinskinandtissue

2004 PMID:15133511

Tnsyndrome

C1GALT1C1 Chaperoneofβ1,3GalT 300622 300611 Hemolyticanemiawiththrombocytopenia,leukopenia

2005 PMID:16251947

Petersplussyndrome B3GLCT β1,3GlucosyltransferasespecificforO-FucoseonThrombospondintype1repeats

261540 610308 Peterseyeanomalyoftheanteriorchamber,IDandDD,prenatalgrowthdelay,postnatal,typicallydisproportionatelyshort,cleftlipwithorwithoutcleftpalate

2006 PMID:16909395

Dowling-DegosDisease2 POFUT1 ProteinO-Fucosyltransferase1specificforparticularEGFrepeats

615327 607491 Skindisordershowingreticulatehyper-andhypo-pigmentationatflexureregionssuchastheneck,axilla,andareasbelowthebreastsandgroin

2013 PMID:23684010

Dowling-DegosDisease4 POGLUT1 ProteinO-glucosyltransferase1specificforparticularEGFrepeats

615696 615618 Skindisordershowingreticulatehyper-andhypo-pigmentationatflexureregionssuchastheneck,axilla,andareasbelowthebreastsandgroin

2014 PMID:24387993

AutosomalRecessiveSpondylocostaldysostoses3

LFNG LunaticFringespecificforO-FucoseonparticularEGFrepeats

609813 602576 Spondylocostaldysostosiswithseverevertebralanomalies.

2006 PMID:16385447

CDG-CongenitaldisordersofglycosylationCMS-CongenitalMyasthenicSyndromeDol-DolicholID-IntellectualDisabilitySz-SeizuresHy-HypotoniaM-Microcephaly

DD-DevelopmentaldelayNSID-Non-syndromicintellectualdisabilityCK-Creatinekinase

ArticleDecipheringtheGlycosylomeofDystroglycanopathiesUsingHaploidScreensforLassaVirusEntry

Deciphering the Glycosylome ofDystroglycanopathies Using HaploidScreens for Lassa Virus EntryLucas T. Jae,1 Matthijs Raaben,2 Moniek Riemersma,3,4,5 Ellen van Beusekom,5Vincent A. Blomen,1 Arno Velds,1 Ron. M. Kerkhoven,1 Jan E. Carette,6 Haluk Topaloglu,7Peter Meinecke,8 Marja W. Wessels,9 Dirk J. Lefeber,3,4 Sean P. Whelan,2*Hans van Bokhoven,5* Thijn R. Brummelkamp1,10*

Glycosylated a-dystroglycan (a-DG) serves as cellular entry receptor for multiple pathogens,and defects in its glycosylation cause hereditary Walker-Warburg syndrome (WWS). At least eightproteins are critical to glycosylate a-DG, but many genes mutated in WWS remain unknown. Toidentify modifiers of a-DG, we performed a haploid screen for Lassa virus entry, a hemorrhagicfever virus causing thousands of deaths annually that hijacks glycosylated a-DG to enter cells.In complementary screens, we profiled cells for absence of a-DG carbohydrate chains orbiochemically related glycans. This revealed virus host factors and a suite of glycosylationunits, including all known Walker-Warburg genes and five additional factors critical for themodification of a-DG. Our findings accentuate the complexity of this posttranslational featureand point out genes defective in dystroglycanopathies.

In humans, a-dystroglycan (a-DG) links theextracellular matrix with the cytoskeleton andis extensivelymodified by sugar chains, includ-

ing an unusual O-linked glycan (1). Mutations ingenes required for a-DG glycosylation lead tocongenital disorders, termed dystroglycanopathies.Notable is Walker-Warburg syndrome (WWS)(2), a severe muscular dystrophy with malforma-

tions of the eyes and brain, associated with de-fective binding of a-DG to its ligands, such aslaminin (3). The O-linked carbohydrate unit isalso used by pathogens to enter their host, in-cludingMycobacterium leprae (leprosy) (4), Lassavirus (LASV), and other Old World arenaviruses(5, 6). At least eight potential glycosyltransferasesare required to install the laminin-binding epitope

ona-DG (7–9), but only ~50%of theWWS casesare explained by mutations in these genes (8).

We undertook a haploid genetic approach(10) to identify host factors essential for LASVentry. For this purpose, we replaced the glyco-protein of replication-competent vesicular stoma-titis virus (VSV) with the Lassa virus glycoprotein(rVSV-GP-LASV) (fig. S1A). This virus infectsnormal human fibroblasts, whereas patient fi-broblasts carrying mutations in the WWS geneISPD (isoprenoid synthase domain containing)(8, 9) resist infection (fig. S1B). Likewise, haploid

1Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsterdam, Netherlands. 2Department of Microbiology andImmunobiology, 77 Avenue Louis Pasteur, Harvard MedicalSchool, Boston, MA 02115, USA. 3Department of Neurology,Institute for Genetic and Metabolic Disease, Radboud Uni-versity Medical Centre, 6525 GA Nijmegen, Netherlands. 4Lab-oratory of Genetic, Endocrine and Metabolic Disease, Institutefor Genetic and Metabolic Disease, Radboud University MedicalCentre, 6525 GA Nijmegen, Netherlands. 5Department of Hu-man Genetics, Nijmegen Centre for Molecular Life Sciences,Radboud University Medical Centre, Post Office Box 9101,6500HBNijmegen, Netherlands. 6Department ofMicrobiologyand Immunology, Stanford University School of Medicine, 299Campus Drive, Stanford, CA 94305, USA. 7Hacettepe UniversityChildren’s Hospital, 06100 Ankara, Turkey. 8Institut für Human-genetik, Universitätsklinikum Hamburg-Eppendorf, 20246Hamburg, Germany. 9Department of Clinical Genetics, ErasmusMedical Center, 3015 GE Rotterdam, Netherlands. 10CeMMResearch Center for Molecular Medicine of the Austrian Acad-emy of Sciences, 1090 Vienna, Austria.

*Corresponding author. E-mail: [email protected] (S.P.W.); [email protected] (H.V.B.);[email protected] (T.R.B.)

Fig. 1. Haploid genet-ic screen for cellularhost factors requiredfor rVSV-GP-LASV in-fection. Significance ofenrichment of gene-trapinsertions in the virus-selected population com-pared with nonselectedcontrol cells is indicatedon the y axis. Bubbles rep-resent genes, and bubblesize corresponds to thenumber of independentgene-trap events observedin the virus-selected pop-ulation. Significant hitsare grouped by functionhorizontally (other genesin random order). Genescarrying the majority ofgene-trap insertions inintrons were colored ifthey passed two statisti-cal tests: enrichment ofdisruptivemutations com-pared with control cells(one-sided Fisher’s exacttest, P ≤ 10−5) and biasfor gene trap–insertionevents in the transcriptional orientation of the affected gene (binomial test, P ≤ 0.05). Intron-poor genes were colored if they passed the former criterionusing a stricter cut-off (one-sided Fisher’s exact test P ≤ 10−30) (13). Data are displayed until –log(P value) = 0.001.

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human HAP1 cells (11) are also infected andkilled in an a-DG–dependent manner (fig. S2,A and B).

Mutagenized HAP1 cells were exposed torVSV-GP-LASV, and gene trap–insertion siteswere analyzed in virus-resistant cells (12). Genessignificantly enriched for mutagenic gene trap–insertion events include DAG1, encoding a-DG,with 316 independent disruptive gene-trap inser-tions and 25 other genes that are predicted orknown to be involved in glycosylation (Fig. 1 andfigs. S3 and S4) (13). Among these are LARGE,ISPD, FKTN, FKRP, POMT1, POMT2, DPM3,and C3orf39, all of which cause dystroglycano-pathies (2, 7, 14), and B3GNT1, which was un-covered as a new WWS gene during preparationof this manuscript (15). Other hits include genesinvolved in sialic acid biosynthesis, the generationof uridine diphosphate (UDP)–glucuronic acidand UDP-xylose, N-glycosylation, mannose sup-ply, and localization of glycosylating enzymes in

the Golgi apparatus. Last, we found a numberof potential enzymes that have not been linked toa-DG modification before (SGK196, TMEM5,PTAR1, ST3GAL4, and B3GALNT2) and hits thatdid not readily connect to glycosylation. None ofthe enriched genes were identified whenmutagen-ized HAP1 cells were selected with a recombinantVSV carrying the Ebola virus glycoprotein (11),which suggested that they are not required forbiology related to the VSV vector. Thus, the hap-loid screen identifies host factors required forvirus entry mediated by the Lassa glycoprotein,including the known entry receptor, known re-ceptor modifiers, and a substantial number ofadditional genes.

As virus entry is a complex succession ofevents, we teased apart the roles of the identifiedgenes through a series of comparative geneticscreens. Principally, hits could have a specificrole in a-DG glycosylation, they could affect gly-cosylation in general, or they could act in virus

entry steps unrelated to receptor binding. Weenriched mutagenized HAP1 cells for defectivepresentation of glycosylated a-DG at the cellsurface (fig. S5, A to C). This population showeda significant increase for haploid cells carryinggene-trap insertions in all known WWS genes,indicating that this mutagenesis screen was carriedout at high coverage (Fig. 2A). Genes requiredfor N-glycosylation and sialic acid biosynthesiswere not enriched, in line with the notion thatthe laminin-binding epitope on a-DG is createdthrough O- rather than N-glycosylation (1) anddoes not require the presence of sialic acid (16).An unexpected exception to this is SLC35A1,which encodes a transporter for cytidine mono-phosphate (CMP)–sialic acid (17, 18). This mayindicate that this gene is involved in the transportof other sugars needed for a-DGO-glycosylationor that it indirectly affects generation of the laminin-binding epitope. Together, this screen identifiesgenes required for a-DG modification and dis-

Fig. 2. Cell surface profiling of mutagenizedhaploid HAP1 cells. (A) Genes enriched for muta-tions in a cell population depleted for glycosylateda-DG at the cell surface. The cell population enrichedfor mutants lacking glycosylated a-DG at the cellsurface was analyzed and depicted as described inFig. 1. (B) A mutant cell population selected for di-minished cell surface heparan sulfate was obtainedas described above. Data were analyzed as previously,except that, for intron-rich genes, the cut-off for dis-ruptive mutations compared with control cells was ad-justed (one-sided Fisher’s exact test, P ≤ 10−21) (13).

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tinguishes them fromhost factorsmediatingLASVentry unrelated to a-DG binding (e.g., LAMP1and genes required for sialic acid biosynthesis).

To distinguish general glycosylation genes fromthose required specifically for the generation ofthe laminin-binding epitope on a-DG, we probedmutagenized HAP1 cells for defects in the gen-eration of heparan sulfate in a separate geneticscreen (Fig. 2B and fig. S6). The carbohydratechains present on a-DG or in heparan sulfate areboth thought to contain xylose and glucuronicacid moieties, and indeed, genes required for theirbiogenesis (UGDH and UXS1) also stood out inthis screen (19, 20). Other overlapping hits affectglycosylation globally, such as the COG complexand TMEM165 (21). PTAR1 constitutes a poten-tial prenyltransferase that has not been implicatedin glycosylation before but also appears to affectglycosylation globally (fig. S4). Finally, cells de-pleted for heparan sulfate on their surface wereenriched for mutations in heparan sulfate bio-synthesis genes (Fig. 2B and fig. S4) (19). Thisfinding suggests that although there are biochem-ical similarities between heparan sulfate and theO-carbohydrate chains on a-DG, these are, byand large, installed by separate enzymes.

Using transcription activator–like effector nu-cleases (TALENs), we generated null alleles for apanel of selected genes in HAP1 cells (fig. S7)(22), and independent clones were isolatedcarrying frameshift mutations and/or prematurestop codons (Fig. 3A and fig. S8). TALEN-inducedmutations in all genes except for ST3GAL4and LAMP1 affected a-DG glycosylation or itsability to interact with laminin (fig. S9, A to C,and fig. S10, A and B). This is in agreement withthe absence of ST3GAL4 and LAMP1 as hits inthe a-DG antibody screen (see Fig. 2A and fig.

S4). Mutant cell lines also showed increased re-sistance to viral infection, although this pheno-type was less pronounced in the SGK196mutants(Fig. 3B and fig. S10C). TALEN-induced pheno-types were reverted by complementation with therespective cDNAs (fig. S11, A and B). In sum-mary, we conclude that TMEM5, B3GALNT2,B3GNT1, SLC35A1, and SGK196 constitutegenes required for the presentation of the laminin-binding carbohydrate feature present on a-DG,whereas ST3GAL4 and LAMP1 are likely in-volved in virus infection by means other thanmodification of a-DG.

TMEM5 encodes a transmembrane proteinthat has not been assigned any function but thatcontains an exostosin family domain (E value0.0002) (fig. S12) that is also present in theheparan sulfate biosynthesis enzymes EXT1,EXT2, and EXTL3. SGK196 contains a kinase-like domain, and knockout mice develop hydro-cephalus (23), reminiscent of the brain abnormalitiesobserved in WWS patients. We sequenced thecoding exons of TMEM5 and SGK196 in a panelof 28 patients with severe dystroglycanopathy,diagnosed with WWS or muscle-eye-brain dis-ease (MEB), not carryingmutations in any knownWalker-Warburg gene. Two families with patientsthat carried homozygous mutations in TMEM5were identified. One mutant allele results in astop codon at position Arg340 [1018(C→T)]; theother family transmits an early frameshift muta-tion A47Rfs*42 [139(delG)] (Fig. 4A). The malepatient with the Arg340* mutation died at the ageof 22 months and had clinical manifestations sug-gestive of WWS (13). The female siblings car-rying the frameshift mutation had a slightly milderphenotype suggestive of MEB. A cranial mag-netic resonance image (MRI) of one of the af-

fected girls recorded at the age of 1 year showedbrainstem atrophy, dilated ventricles, widespreadpachygyria, and substantial white matter involve-ment (Fig. 4B).During revision of thismanuscript,mutations in TMEM5 have also been found infetuses displaying cobblestone lissencephaly (24).A patient with compound heterozygous muta-tions L137R and Q258R in SGK196 and typicalWWS phenotype was identified in another family(Fig. 4C). To test causality of the identified mu-tations for the disease, we supplied HAP1 cellsdeficient for either SGK196orTMEM5with cDNAsencoding the patient-derived variants. Unlike theirwild-type counterparts, these neither restoreda-DG glycosylation (Fig. 4D) nor enhanced sus-ceptibility to infectionwith rVSV-GP-LASV(fig. S13).Together, the detection and functional validationof TMEM5 and SGK196 loss-of-function muta-tions in families with WWS-MEB–type dystro-glycanopathy underlines the relevance of theidentified a-DG modifiers for human disease.

For decades, genes associated withMendeliandisorders have been discovered by studyingpedigrees of affected individuals. Although ex-pedited by robust sequencing strategies, the iden-tification of causative mutations in geneticallyheterogeneous conditions remains problematic.Here, we apply a haploid genetic approach tocapture the complexity of a severe hereditarydisease in vitro. The resulting “glycosylome” ofa-DG highlights the intricate nature of this post-translational modification and identifies addition-al genes mutated in Walker-Warburg syndrome.Because polymorphisms associated with the hu-man LARGE gene are under selective pressure inareas where LASVis endemic (25), it becomes ofinterest to examine the glycosylome genes invirus-exposed populations.

Fig. 3. TALEN-induced mutations in identified genes affect sus-ceptibility to rVSV-GP-LASV. (A) HAP1 cells transfected with TALENsdisplay frameshift mutations and/or introduce premature stop codons intargeted genes. Sequences recognized by the TALENs are displayed in red

and blue. (B) The HAP1 cell lines with TALEN-induced mutations in thecorresponding genes and wild-type control cells were infected withrVSV-GP-LASV [infected cells express enhanced green fluorescent protein(eGFP)].


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References and Notes1. T. Yoshida-Moriguchi et al., Science 327, 88

(2010).2. C. Godfrey, A. R. Foley, E. Clement, F. Muntoni, Curr.

Opin. Genet. Dev. 21, 278 (2011).3. D. E. Michele et al., Nature 418, 417 (2002).4. A. Rambukkana et al., Science 282, 2076 (1998).5. W. Cao et al., Science 282, 2079 (1998).6. S. Kunz et al., J. Virol. 79, 14282 (2005).7. M. C. Manzini et al., Am. J. Hum. Genet. 91, 541

(2012).8. T. Roscioli et al., Nat. Genet. 44, 581 (2012).9. T. Willer et al., Nat. Genet. 44, 575 (2012).

10. J. E. Carette et al., Science 326, 1231 (2009).11. J. E. Carette et al., Nature 477, 340 (2011).12. J. E. Carette et al., Nat. Biotechnol. 29, 542

(2011).13. Materials and methods are available as supplementary

materials on Science Online.14. D. J. Lefeber et al., Am. J. Hum. Genet. 85, 76

(2009).15. K. Buysse et al., Hum. Mol. Genet. (2013).16. A. C. Combs, J. M. Ervasti, Biochem. J. 390, 303

(2005).17. S. K. Patnaik, P. Stanley, Methods Enzymol. 416, 159

(2006).

18. M. Eckhardt, M. Mühlenhoff, A. Bethe, R. Gerardy-Schahn,Proc. Natl. Acad. Sci. U.S.A. 93, 7572 (1996).

19. R. J. L. Hari, G. Garg, Charles A. Hales, Chemistry andBiology of Heparin and Heparan Sulfate (Elsevier,Kidlington, Oxford, UK, 2005).

20. K. Inamori et al., Science 335, 93 (2012).21. F. Foulquier et al., Am. J. Hum. Genet. 91, 15 (2012).22. N. E. Sanjana et al., Nat. Protoc. 7, 171 (2012).23. P. Vogel et al., Vet. Pathol. 49, 166 (2012).24. S. Vuillaumier-Barrot et al., Am. J. Hum. Genet. 91, 1135

(2012).25. P. C. Sabeti et al.; International HapMap Consortium,

Nature 449, 913 (2007).

Fig. 4. TMEM5 and SGK196 mutations found in patients with WWS and MEB,lacking mutations in known WWS genes. (A) Pedigree structure of consanguineous,respectively first and second cousins, families 43 and 56 segregating a TMEM5 mutation.Family 43 has an affected male with features of WWS and a stillbirth, without availableclinical records. Family 56 has two affected females with clinical features reminiscent ofMEB (13). A nonsense mutation in exon 6 was identified in family 43. Family 56 harbors aframeshift mutation in exon 1. Both mutations were homozygously present in the patient(s) and heterozygously in the parents. The unaffected boy in family 43 isheterozygous for the mutation. IC, intracellular domain; TM, transmembrane domain; EC, extracellular domain; EF, exostosin family domain. (B) Cranial MRI ofthe oldest affected female of family 56 at the age of 1 year; sagittal cut (T1-weighted image): atrophy of pons and cerebellum; axial cut (flair image): fronto-parietal pachygyria, enlarged ventricles, and abnormal white matter. (C) Compound heterozygosity of mutant SGK196 in an affected patient. Both non-consanguineous parents are heterozygous carriers of either mutation. KL, kinaselike domain (D) HAP1 cells with TALEN-induced disruption of endogenousTMEM5 or SGK196 were complemented with cDNAs encoding the mutant variants observed in patients and analyzed for presence of the a-DG laminin-bindingepitope using flow cytometry.

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Acknowledgments: We thank T. Sixma, J. Roix, S. Mukherjee,S. Hill, and S. Nijman for discussions and S. Radoshitzkyand M. Farzan for providing the plasmid encoding the LASVglycoprotein. Supported by The Netherlands GenomicsInitiative (NGI), Netherlands Organization for ScientificResearch (Vidi-91711316), and the European ResearchCouncil (ERC) starting grant (ERC- 2012-StG 309634) to T.R.B.,Prinses Beatrix Fonds (W.OR09-15) to D.J.L. and H.V.B.,European Union Framework Programme 7 Health Programme(241995 GENCODYS) to H.V.B., and NIH grants AI081842and AI057159 to S.P.W. T.R.B. is a cofounder of HaplogenGmbH, S.P.W. is inventor on a patent describing the

reverse-genetics system for VSV (International Patent no:5,789,229), J.E.C. and T.R.B. are inventors on a patent onmutagenesis in haploid or near-haploid cells (U.S. PatentApplication no: 2012/0190,011), and materials will bemade available to the academic community under aMaterials Transfer Agreement. The study was approved by theethical board of the Radboud University Nijmegen MedicalCentre, Commissie Mensgebonden Onderzoek RegioArnhem-Nijmegen Approval 2011/155 (9612-1812). Deepsequencing data have been deposited in the NCBI SequenceRead Archive (www.ncbi.nlm.nih.gov/sra) under accessionnumber SRP018361.

Supplementary Materialswww.sciencemag.org/cgi/content/full/science.1233675/DC1Materials and MethodsSupplementary TextFigs. S1 to S13Tables S1 to S4References (26–30)

5 December 2012; accepted 7 March 2013Published online 21 March 2013;10.1126/science.1233675

Potent Social Learning andConformity Shape a Wild Primate’sForaging DecisionsErica van de Waal,1,2 Christèle Borgeaud,2,3 Andrew Whiten1,2*

Conformity to local behavioral norms reflects the pervading role of culture in human life.Laboratory experiments have begun to suggest a role for conformity in animal social learning, butevidence from the wild remains circumstantial. Here, we show experimentally that wild vervetmonkeys will abandon personal foraging preferences in favor of group norms new to them. Groupsfirst learned to avoid the bitter-tasting alternative of two foods. Presentations of these optionsuntreated months later revealed that all new infants naïve to the foods adopted maternalpreferences. Males who migrated between groups where the alternative food was eaten switched tothe new local norm. Such powerful effects of social learning represent a more potent force thanhitherto recognized in shaping group differences among wild animals.

Ever since pioneering studies on the diffu-sion of a new sweet-potato washing habitin Japanese macaques (1) and milk-bottle

opening in great tits (2), accumulating field studieshave suggested that the cultural transmission ofbehavior through social learning provides manyanimals with a “second inheritance system” (3).This system complements genetic inheritance andindividual learning in shaping behavioral reper-toires (4, 5). The scope and impact of this secondsystem are important to delineate because exploit-ing the existing knowledge of others can potentiallysupport efficient adaptation to local circumstances(6). It can also generate locally differentiated be-havioral traditions, and indeed, much of the evi-dence for a role for animal culture in the wildderives from identifying local variations consist-ent with the existence of such traditions (7–9).However, owing to their observational nature,these studies lack the experimental rigor to con-firm whether putative cultural variations aresocially learned. Experiments with captive pop-ulations, by contrast, have seeded different groupswith models trained to act in different ways, suchas opening an “artificial fruit” using either of twoalternative techniques, then documenting the dif-

ferential diffusion of these variants across groups(10) and even between them (11).

These paradigms have now produced a sub-stantial corpus of laboratory studies document-ing cultural transmission in taxa as diverse asinsects (12), fish (13), and apes (11, 14). Such ex-periments in the wild remain scarce (10, 15–19),however, because in natural populations, it istypically impractical to isolate individuals fordifferential training as models. The few fieldstudies that have attempted to approximate thisapproach have generally produced evidence forweaker transmission of the seeded alternatives(15–18) than counterparts in captive populations(10, 11, 20, 21).

Here we introduce a different methodolog-ical approach, which has demonstrated two po-tent effects of social learning in the wild. Insteadof seeding behavioral variants in single models,we seeded variants in four whole groups of wildvervet monkeys, Chlorocebus aethiops, totaling109 individuals (22) (table S1 and fig. S1). Wethen investigated how two classes of individualsnaïve to the local group norm—infants and im-migrant males—responded to the particular lo-cal preferences they were exposed to. To createinitial preferences, we provisioned groups withtwo adjacent trays of maize corn, one with corndyed blue, the other pink (Fig. 1). One of these(pink in two groups, blue in two others) was madehighly distasteful so that after three monthly train-ing sessions, the distasteful alternative was rare-ly eaten or even tried (table S2 and figs. S3 andS4). After a period of more than 4 months inwhich a new cohort of identifiable infants ma-tured sufficiently to take solid foods, we againoffered the two colored foods with no distastefultreatments and tested (i) whether the naïve in-fants would copy their mother’s preference forthe locally favored color over the now equallypalatable alternative, and (ii) whether males mi-grating from a group trained to prefer one colorto a second group where the alternative colorwas preferred would be influenced by the latter.

When the corn provisions were offered againafter 4 to 6 months, a preference for the earlierpalatable alternative was maintained across fivetest trials spanning 2 months, despite both colorsnow being palatable. Some of the previously dis-tasteful food was tried and consumed (Fig. 2 and

1Centre for Social Learning and Cognitive Evolution, and ScottishPrimate Research Group, School of Psychology and Neuroscience,University of St Andrews, St Andrews KY16 9JP, UK. 2InkawuVervet Project, Mawana Game Reserve, Swart Mfolozi, KwaZuluNatal 3115, South Africa. 3Institute of Biology, University ofNeuchâtel, 2000 Neuchâtel, Switzerland.

*Corresponding author. E-mail: [email protected]

Fig. 1. Experimental set-up illustrating preferen-tial foraging. Maize corndyed either pink or bluewas provided intermittentlyin two adjacent containers.Photograph shows infantsitting on the color earliermade distasteful to itsmother, as it eats the colorcurrently preferred by itsmother and the rest ofthe group.


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originally published online March 21, 2013 (6131), 479-483. [doi: 10.1126/science.1233675]340Science

Thijn R. Brummelkamp (March 21, 2013) Wessels, Dirk J. Lefeber, Sean P. Whelan, Hans van Bokhoven and Jan E. Carette, Haluk Topaloglu, Peter Meinecke, Marja W.Beusekom, Vincent A. Blomen, Arno Velds, Ron. M. Kerkhoven, Lucas T. Jae, Matthijs Raaben, Moniek Riemersma, Ellen vanHaploid Screens for Lassa Virus EntryDeciphering the Glycosylome of Dystroglycanopathies Using

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genetic screens can be used to define the genetic architecture of a complex disease.had unique mutations among genes identified in the genetic screen. Thus, comprehensive forwardidentified candidates involved in glycosylation. Individuals from different pedigrees exhibiting WWS

andonline 21 March) screened for genes involved in O-glycosylation that affected Lassa virus infection (p. 479, publishedet al.Jae modification is also required for efficient Lassa virus infection of cells.

modifications associated with the congenital disease Walker-Warburg syndrome (WWS). This cellular -dystroglycan O-linked glycosylation result in posttranslationαMutations in genes involved in

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