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.
10
20
30
40
50
60
70
80 N
umber of D
isorders
1990 1993 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
90
100
1981
O-Fucose / O-Glucose
O-GalNAc / O-GlcNAc
Glycolipid
Dystroglycanopathy Glycosaminoglycan N-Linked
GPI-Anchor
2014 2015
110
2016
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
AutosomalrecessiveGPI-anchordeficiency
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
AutosomalrecessiveGPI-anchordeficiency
PIGM Firstα-MannosyltransferaseinGPIbiosynthesis
610293 610273 Sz,portalveinthrombosis,portalhypertension
2006 PMID:16767100
Hyperphosphatasiamentalretardationsyndrome
PIGV Secondα-MannosyltransferaseinGPIbiosynthesis
239300 610274 Hyperphosphatasiawithmentalretardationsyndrome1(HPMRS)
2010 PMID:20802478
AutosomalrecessiveGPI-anchordeficiency
PIGN GPIEthanolaminePhosphatetransferase1 614080 606097 Severeneurologicimpairment,Sz,lackofdevelopment,multiplecongenitalanomalies,earlydeath
2011 PMID:21493957
Hyperphosphatasiamentalretardationsyndrome
PIGO GPIEthanolaminePhosphatetransferase3 614749 614730 Hyperphosphatasiawithmentalretardationsyndrome2(HPMRS)
2012 PMID:22683086
AutosomalrecessiveGPI-anchordeficiency
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
AutosomalrecessiveGPI-anchordeficiency
PGAP1 LipidremodelingstepsofGPI-anchormaturation
615802 611655 IDwithencephalopathy 2014 PMID:24784135
Hyperphosphatasiamentalretardationsyndrome
PGAP2 LipidremodelingstepsofGPI-anchormaturation
614207 615187 Hyperphosphatasiawithmentalretardationsyndrome3(HPMRS)
2013 PMID:23561846PMID:23561847
Hyperphosphatasiamentalretardationsyndrome
PGAP3 LipidremodelingstepsofGPI-anchormaturation
615716 611801 Hyperphosphatasiawithmentalretardationsyndrome4(HPMRS)
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
Walker-Warburgsyndrome(MDDGA8)
POMGNT2 β1,4GlcNAc-transferase 614830 614828 Brainmalformations,variouseyemalformations
2012 PMID:22958903
Walker-Warburgsyndrome(MDDGA10)
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
Walker-Warburgsyndrome(MDDGA12)
POMK O-Mankinase 615249 615247 Walker-Warburgsyndrome,brainandeyemalformations,elevatedserumCK
2013 PMID:23929950PMID:23519211
Walker-Warburgsyndrome(MDDGA13)
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.
www.sciencemag.org SCIENCE VOL 340 26 APRIL 2013 479
REPORTS
on
May
22,
201
6ht
tp://
scie
nce.
scie
ncem
ag.o
rg/
Dow
nloa
ded
from
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).
26 APRIL 2013 VOL 340 SCIENCE www.sciencemag.org480
REPORTS
on
May
22,
201
6ht
tp://
scie
nce.
scie
ncem
ag.o
rg/
Dow
nloa
ded
from
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)].
www.sciencemag.org SCIENCE VOL 340 26 APRIL 2013 481
REPORTS
on
May
22,
201
6ht
tp://
scie
nce.
scie
ncem
ag.o
rg/
Dow
nloa
ded
from
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.
26 APRIL 2013 VOL 340 SCIENCE www.sciencemag.org482
REPORTS
on
May
22,
201
6ht
tp://
scie
nce.
scie
ncem
ag.o
rg/
Dow
nloa
ded
from
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.
www.sciencemag.org SCIENCE VOL 340 26 APRIL 2013 483
REPORTS
on
May
22,
201
6ht
tp://
scie
nce.
scie
ncem
ag.o
rg/
Dow
nloa
ded
from
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
Editor's Summary
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
Viruses and Congenital Disorders
This copy is for your personal, non-commercial use only.
Article Tools
http://science.sciencemag.org/content/340/6131/479article tools: Visit the online version of this article to access the personalization and
Permissionshttp://www.sciencemag.org/about/permissions.dtlObtain information about reproducing this article:
is a registered trademark of AAAS. ScienceAdvancement of Science; all rights reserved. The title Avenue NW, Washington, DC 20005. Copyright 2016 by the American Association for thein December, by the American Association for the Advancement of Science, 1200 New York
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last weekScience
on
May
22,
201
6ht
tp://
scie
nce.
scie
ncem
ag.o
rg/
Dow
nloa
ded
from