MolecularBiologyofMuscular
Dystrophy

InternationalWorkshopbetweenJapanandFrance
heldatToranomonPastoral，Tokyo
On17−18January，1996

Sponsoredby：ScienceandTechnologyAgency（STA）：JapanInternationalScienceandTechnology
ExchangeCenter（JISTEC）：MinistryofHealthandWelfare
NationalInstituteofNeuroscience
NationalCenterofNeurologyandPsychiatry（NCNP）

MolecularBiologyofMuscular
Dystrophy
ProceedingofInternationalWorkshop
betwennJapanandFrance−−−−−−■￣−−−

昂ガ7二EDβy

均野roOza仇Ⅵ
旭日0月出血5日rureofNeurogdenCe

5

血血

ChjTb女eda

旭日0月出血5日rureof楠urogdenCe−−Preface
This book represents
betweenJapan

Dystrophy

andFranceheldinthe proceeding ofinternational workshop
entitled

Molecular

Biology

Tokyo，17「18January，1996．Theof

Muscularantecedent

of

the

WOrkshop dates back to1995，When Robert G．Whalen and Hideo Sugita
Organized a small workshopin Paris．Progressinthefield ofmuscular
dystrophy has clearly been facilitated bytheinternational cooperation，
exchangeofideas andgeneroussharingofbiologicalreagents．Assuch，We

WOuldlike to acknOwledge ourindebtednessto Drs・WhalenandSugitafbr
Settingthese conkrencesin motion．All ofuslook fbrward tothe next
meetingtentativelyscheduledforFranceintheAutumnof1997．
ProgressinthefieldofmuscularqystrophyoverthepastseveralyearS
hasbeenimpressive，andthecontributionofJapanandFrancewasbecoming
biggerandbigger．Especial1y，the cloning ofthe sarcoglycangenesandthe
Subsequentidentificationofthe mutationsinsome ofthe patients oflimb−
girdle muscular

dystrophy has been done byJapanese and French

researchers． This Bnding was high−lightedin this meeting． After
identincationofthe causativegeneofmusculardystrophy，mOStimportant
StePistoverifyunderlyingmechanismofmusclenecrosis，neVerthelessthe
CauSe Of muscle necrosis was deficiency of dystrophin，SarCOglycan，Orlamininmolecule．

Onemoreimportantsubjectinthisfieldistodevelopgenetherapyfor
musculardystrophy．Inli飴−threateneddiseasesuchasDuchennemuscular
dystrophy，thereisnomorehopefultreatmentthangenetherapy．Inthis
meeting，SOme Ofthe challenglng aPprOaCh using．cel1−mediated transfbras
Wellas uslng directgene tranSftr，hasbeen proposed．Progress nextftw
yearShouldtelltheadvantageanddisadvantageOfgenetherapy．
WegratefullyacknOwledge ScienceandTechnologyAgency（STA）and
JapanInternationalScience and Technology Exchange Center（JISTEC）fbr
theirsponsorshipofthismeeting．

EijiroOZAWA
Orgamizer

Contents
Preface
I. DYSTROPHIN

AND RELATEDDISORDERSISHIKAWA

Dystrophin:
Localizationfor expression
Teruhisa

and regulatory

regions
3

Harunori

1

The localizationof dystrophin
and its associatedproteinson the sarcolemma of mammalianstriated
muscle cells

MIIKE

Modulationof splicing
of intron by modifying
an intra-exon sequence which is deleted fromthe dystrophin
gene in dystrophin
Kobe

13

Masafumi MATSUOII.

DYSTROPHIN-ASSOCIATED

PROTEINS(DAPs)AND RELATED

Characterizationof the interaction
betweenSchwann cell dystroglycan
complex andendoneurial
lamininthe

Dystrophin-associatedprotein,(Al)

Sarcoglycanopathy

hypothesis

Kiichiro

Eijiro19

MATSUMURA

Mikiharu

YOSHIDA

OZAWA

syntrophin

DISORDERS

35

43

ƒÀ-and ƒÁ-sarcoglycan

Satoru NOGUCHI

From adhalinopathies
Marc JEANPIERRE

51

to sarcoglycanopathies

57

Identificationof muscle-specific calpain andƒÀ-sarcoglycangenes in progressive muscular
dystrophies
JacquesS. BECKMANN

Skeletalmuscle specificproperties
and function
KoichiSUZUKI

novel calpain,

63

p94:
79

III.

OTHER TYPES

OF MUSCULAR

DYSTROPHIESAND RELATED DISORDERS

Familial
hypertrophic
cardiomyopathy:
a disease of the cardiac sarcomere

87

Marc Y. FISZMAN

Identification
a determining

Suzie LEFEBVRE

and characterizationofgene in spinal muscular atrophy

Molecular genetic diagnosis
muscular dystrophy
Kiichiof Emery-Dreifuss

ARAHATA

Toward identification
congenital
muscular
Tatsushi93

101of the Fukuyama-type
dystrophy(FCMD) gene
113

TODA

Merosinopathies
FernandoIV.

M.S.TOME119

TOWARD GENE THERAPY

Muscle regeneration
dystrophy
Ikuya

INTO MUSCULAR

in progressive

DYSTROPHY

muscular

NONAKA

131

Local productionof IL-12p4O
myoblast rejection

prevents

allogenic
137

Kazunori KATO

Permanent systemic deliveryof therapeuticproteins from geneticallymodified myofibers

Jean Michel

HEARD

Effective

gene transfer

147

into skeletal

muscleusing
adenoviral
vectorShin'ichi
TAKEDA

149

Development of adenovirus vectors for genetherapy of Duchenne muscular dystrophy
Thierry

RAGOT

157

Adenoviraldiseases

gene therapyof neurodegenerative

Georg HAASE

Plasmid DNAtransfer into skeletal
Implications
for immune reactions
proteins
Robert

G. WHALEN

159

muscle:to expressed
169

Thelocalization ofdystrophin−andits
associated proteins・Onthesarcolemmaof
mammalianstriated muscle cells
HarunOriIshikawa

DepartmentofAnatomy，GurmaUniversitySchoolofMedicine，
Maebashi，Gunma371，Japan

Abstract

In confbcallaser microscopy combined withindirectimmun0−
fluorescencewithamonoclonalanti−dystrophinantibody，dystrophinwasseento

belocalized alongthe sarCOlemmain anonunifbrmandeven discontinuous
PatterninnormalgulneaPlgSkeletalandcardiacmusclecells．Sectionscut
tangentialtothesarCOlemmashowedalattice−likeimageoflongitudinaland
tranSVerSeStriations・Thetransverse striationswere oftenregularly spaced

resemblingthesarCOmerePattern・AnintenseirrlmunO−fluorescencestaining
WaS Seen at themyotendinousJunCtionsinskeletalmuscle cells and at theintercalateddiscsincardiacmusclecells・Thisstainingpatternsuggeststhat
dystrophinmayalsobelocalizedintheadherensJunCtionswheremyofibrils
attach・Whenmusclecellswerestainedwithbothanti−dystrophinandanti−β−dystroglycan，thestainingpatternwascompletelyidenticalsuggestingthatboth
PrOteinSareCOlocalized alongthesarCOlemma・In contrast，thestalnlng Of
Vinculinwasnotwellsuperimposedonthatofdystrophin・

Toclarifytheexactrelationshipbetweenthesarcolemmaanddystrophin
andotherrelated proteinS，Crude sarCOlemmalvesicles werepreparedfrom
rabbitandratskeletalmusclesaccordingtothemethodsdescribedbyOhlendiecketal・（1991）・When such crude sarCOlemmalvesicles were examined bynegativestainingandthinsection

electronmicroscopy，theywerefoundtobe

heterogeneousinpopulationvarylnglnSizeandshape・Amongthem，1arge
VeSiclesofirregularShq）eCOntainedfinefilamentOuSStruCtureSandvesicular
ffagments・SuchfilamentOuSStruCtureSfbrmednetworkswhichwereclosely
associatedwiththeinnersurfaceofthevesiclemembrane・Interestingly，the−1−filamentOuS netWOrks were restricted to the membrane domains where
Particulatestructuresprqjectedffomtheouter surface．Gold−hbeledwheat
germagglutinin（WGA）seleとtivelyboundtosuchlargeVeSicles．Withsome
Otherfindings，thelargeVeSicleswereconsideredtobeofsarCOlemmalorlgm．

WhenthecrudesarCOlemmalvesiclesweremountedonagridandimmun010−
gically stained withanti−dystrophinantibody after brief Triton treatment，
immunOgOldparticleswerefoundonlyonthefilamentousnetwo止sbeneaththe
VeSicle membraneS・Replica electron microscopy showed charaCteristic

granularStruCtureSOnmOStOflargevesicles．Theyseemedtocorrespondto
theparticulatestruCtureSPrqiectingfromtheoutersurfaceofvesiclesasseenin
thinsections．ExogenouslaminincouldbereconstitutedonthesarCOlemmal
VeSiclesincloseassociationwithsuchparticulatestruCtuI℃S，SuggeStingthatthey

mayrepresentdystroglycanCOmplexes．

−2−Dystrophin：localization andregulatory
reglOnS forexpression●

TeruhisaMiikel，Mn，MiyatakeMl，叫YoshiokaKl，M工，ZhaoJl，MD．，TamuraTl，Mn，KimuraSl，MD，OgawaMl，MnIkezawaMl，MD．，NiikawaN2，MD．，JinnoY2，Mn，UchinoM戸，MnYamamuraK！，

MD．，AbeK！，MnSuzukiMl，Mn
lDepartmentofChildDevelopment，KumamotoUniversitySchoolof

Medicine，2DepartmentofHumanGenetics，NagasakiUmiversitySchoolof
Medicine，3DepartmentofNeurology，41nstituteofMolecularEmbryology
andGenetics，KumamOtOUniJersitySchoolofMedicinC，1−1−1Honjo，KumamOtO860，Japan

Introduction
Althoughthegenethat，Whendefective，reSultsinDuchermemuscular
dystrophywasisolatedandcalleddystrophingene1−2），meChanism ofmuscle

degradationin this conditionis still obscure，because of the unkn0wn
PhysiologicalormechanicalfunctionofthegenetranSCript．Todeterminethe
localization andfunctional significanCe Of dystrophin，We Studied various
tissuesfromalmostentirebodyofcontrolandmdxmice，andcontrolrats，
uslng POlyclonalantibodies against dystrophin．We also studied humanmusclebiopsyspeCはnenS．

Itiskn0wnthatthemuscletypepromoterisinvoIvedinexpressionof
thedystrophingeneinskeletal，Cardiacandsmoothmuscleaswellasinglial
Cells・ItisconceivablethatthemuscletypedystrophinpromotercontainSthe
differentregulatoryreglOnSfor skeletal，Cardiacandsmoothmuscleinvivo，
but the detailis not wellkn0wn．Withthis background we started toinvestigatethemechanismofdevelopmentalandtissue−SPeCificregulation ofthemuscletypedystrophingene，eSPeCiallypromoter reglOninmice，uSlng
tranSgenicmice．

−3−

Materials andmethods
血皿瓜血血加血盟血山血中Allthetissues ffommiceandrats，including thecerebrum，Cerebellum，SPinalcord，eye，tOngue，nOSe，heart，lung，kidney，digestiveorganS，liver，SPleen，
muscle，SkinandbloodvesselswereobtainedimmediatelyafteraneStheticdeath．

IndirectimmunOfluorescenceandABCmethOdwereusedtostaincryostatsections
Ongelatinizedcoverslips．Ⅵkusedseveralkindsofdystrophinantibodies（Fig・1）・
nad堰く血cmfce

Wegeneratedtransgemicmicecarrylngthe900bpgenomicfragmentfrom
themuscletypedystrophinpromoterreglOn，fusedtothe codingreglOnOfthe
bacterial血cZgene（Fig．2）．InsertsfrompBluedyspromoter−hcZwereexcisedby Xhol−BamHl digestion，Purined with Gene Clean（BiolOl），and micro−
irtiectedinto400ftrtilizedmouseeggscollectedaftermating（Fig・3）・

Results
血m叩血血血血血血ゆ
WeobservedairrmunOhistoChemicaldystrophinreactioninsynq）ticreglOnS
SuCh as neuromuscularJunCtions，equatOrial reglOn Ofintrafusal fibers，myO−
tendinousJunCtions，OuterPlexiformlayer oftheretina，thetastebuds，PaPillary
layerofthedermis，taCtilenerveendipgsintheskinandneuronsinthebrain，aS
Wellas onthe surfacemembrane Ofske16tal，Cardiacand smoothmuscle fibers．

ThenYe．analyzedmRNAsfromtheretinaofmicewiththeuseofthereverse
tranSCriptionandfolymer．aSeChainreaction（RT−PCR）method・The5

sequences，

COrreSPOndingtothefirstexPn，■ofdystrophintran！Cripts（DT）intheretinawas

mainlythebraintype（Fig．q4），ふereasinth占3．regionofDTthatcorreq）Ondsto
the C−terminal domainof dystrophin，SOme additionalRT−PCR products weredetected．
Base戸qquenQeSinthreeofthemshowedhomologytoLtheseforpreYiopsly
reportedhumanbraintypedystrophin￣isofbrms（Fig．5）．

ー4−

伽卿fc∬血eNineteenindependent tranSgemic foundermicewereidentified，insix ofwhichtheexpressionofhcZwasdetectedintherightheart・Fourteenofthe
nineteentranSgemiclinesproducedoffspring・Varioustissuesoftransgenicadultmice were stainedwith X−galto detect血cZexpression・Six of the fburteen
tranSgenicmouselinesshowedJacZ鱒PreSSion onlyintherightheart（Fig・6）・
Thereportergeneexpressionwasdetectedfirstinthepresumptiverightatriaand
Ventricularmyocardiumoftheembryosat8．5dayspostcoitum・Wecomparedthepromotersequencetothatofthepreviouslyreportedthe
humansequenceof850bpreglOnuPStreamfromthecapsite3）andreportedthemousesequenceof400bpupstreamffomthecapsite4）・Thehomologybetween
thehumanandmouse140bpreglOnSuPStreamfromtheimitiationcodonreaches
the93％・Upstreamfromthispoint，themouseandhumanSequenCeSlookmore
divergent（66．8％homology）．−Non−SPeCifipregion，ATAandGCbox，andmuscle
SPeCincreglOnS，CArGbox，areCOnServedinhumanandmouse・Discussion
We observe・rather strongimmunOhistochemical

reactivityin−SynaPtic

reg10nS5−9）aswellasontheskeletal，Cardiacandsmoothmusclefibermembrane・Itiswell・knownthat．−theseLSyn叩ticreglOnS，WhichdlOWedimmunOhistochemical
dystrophinreaction，−QOntain・abundant SynaPSeS and／or non−PyPlinated・nerVe
endings，aS a COrnmOn histologiCalcharaCteristics．With this background we
COnSider thatthelocalizationofdystrophininthesynapticreglOnSSuggeStSits
Physiologicalfunctionintheconductionsystemratherthanamechanicalone・In
1993，Pillers et al・10）reported abnormalelectroretinograminDuchermeand

Beckermusculardystrophypatients，thatismarkedlyreduced叩plitudeoftheb−
WaVeinthedark−adaptedstate・Theresultssuggestedthatthedystrophinhassomefunctiontokeq，nOrmalfunctionintheretina・Recently，D−Sauzaetal・11）finallyreportedanovelisoformofdystrophin（Dp260）presentinthemouseretinaand
thatisrequiredfbrnormalfunctionintheretina・So，WeStilldonItknowthefu11
1engthbraintypedystrophinhasfunctionornot・But，anyWayitisnotlikelythat dystrophinplays acriticalroleinthe
COnduction system，because there has been no report of叩parent retinalal）nOrmalitiesinDMDpatients・Dystrophinmayplayadelicaterole，aCtifLgaSー5−

0nlyatriggerpropagatingcontractionwithinthecell・Dysfunctionlng Ofthe
COnductionsystemduetoadeftctindystrophinisstillanimportantfactorinthe

muscledegradationinDMD．AsfbrthevascularSyStem，nOSmOOthmusclecells
receivenerve endings，WhichareuSually severalmicrons awayfromthe cells・ThissuggeststhatthevascularSyStemmaybeoneofthemostvulnerabletissuesto

adeftctindystrophinalthoughitplayaminorroleintheconductionsystemdue
toanatOmiCalcharaCteristics12）・Iwouldliketoemphasizethatweshouldnot
lgnOrethelocalizationandfunction ofdystrophinonthe smoothmusclelayer，
especiallyonthevascularSyStem．AsfbrthetranSgenicmice，byusmgtheE・COli
血cZreporter geneinstead oftheCAT assay，it hasbeenpossibleto fbllow
Visuallythetemporalandspatialexpressionofdystrophinbothduringembryonic
andpostnatalmouse development．TheLacZgenehas advantageOfvisualizing
expressioninspeci丘corganS．

Wefoundthatthe900bppromoterregionofmousemuscletypedystrophin

WaSaCtiveonlyintherightheart，i．e．notinskeletalsmoothorleftheartmuscle．
Onthe other hand，MuntOmietal・13）reported family cases withonly severe
Cardiomyopathy，i．e．no climiCalmuscle symptoms，dueto possible deletion themuscletypedystrophinpromoter，butthecardiac−SPeCificreglOnOfthedystrophin
PrOmOter has not beenidentified to date．Our datainthis study may be
COnVemienttoexplainthecasesreportedbyMuntOmietal．
Theseresultssuggestthatseveraldelicatelydividedelementsfbr dystrophin
expressionexistinmuscletissues，SuChasleftheart，並eletalandsmoothmuscle
Cell．

−6−

Fig・1

Antidystrophin antibodies

AffinityーPurined，region−SPeCincdystrophinantibodiesusedinthisstudy．kD＝kDa

p20

【F

CSDys2≡芋≒三三芸三三‡妄
Dy53

Fig．2
MouseDystrophinPromoter−ねCZConstract

StruCtureOfdystrophinmuscletypepromoterlacZtransgene．
900bpPstI−HinCⅡfragmentderivedfromMousedystrophinpromoter
reglOnWaSfusedtothecodingreg10nOftheE．coli，lacZgene．

−7−

Fig．3
Scheme oftheprocedure fbrproducing
transgenic miceMicroinjection

tran＄gene

「OmO

←お×薄

ferti］izedeggs

CoHectionofEmbyoS

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頑Muscletype＜Braintype→Muscletype

一軍二等車∴＜Braintype

30cycles15cycles（a）：Ethidium bromide staining．（b）：Southem blots・
Reverse PCR productsin which the dystrophin first exon
WaS amPlified・The first exon of dystrophin transCriptin the retina wasmainly the brain type・ー9−

Fig．5

10・431＼▼／10，471
Human

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FragmentsⅡandⅢshowthesensesequenceofthemousedystrophincDNA，and
血agmentsIVandVItheantisensesequence・Arrowheadsindicatebordersof

deletedsequences・Asterisksshowmatchedsequencestctweenthehumanand
themousecDNA．

Fig．6

ExpressionofthetranSgeneisseenonlyintherightventricleoItheneart・−10−

Rererences
1．KoenigM，HofhanEP，BertelsonCJ，MonacoAP，FbenerC，KunkelLM：
CompletecloningoftheDuchennemusculardystrophy（DMD）cDNA

andpreliminarygenomicorgmizationoftheDMDgeneinnormalandafftctedindividuals．Cell1987；50：509−517．

2．HofhanEP，BrownRHJr，KunkelLM：Dystrophin：theproteinproduct
OftheDucherLnemuSCulardystrophylocus．Cell1987；51：919−928・
3．KlamutHJ，GangOPadhyaySB，WortonRG，etal．：Molecularand
functionalanalysisofmuscle−SPeCincpromoterreglOnOftheDuchenne
musculardystrophy．MoICellBiol1990；10：193−205．▼

4．GilgenkrantzH，HugnotJP，LambertM，etal．：Positiveandnegative
regulatoryDNAelementsincludingaCCArGGboxareinvoIvedinthe
Celltype−SPeCificexpressionofthehumandystrophingene．JBilChem
1992；267：10823−10830．

5．MiikeT，MiyatakeM，ZhaoJ，etal．：Irmnohistochemicaldystrophin
reactioninsynapticreglOnS．BrainDev1989；11：344−346．
6．MiyatakeM，MiikeT，ZhaoJ，etal．：Dystrophin：localizationand
PreSumedfunction．MuscleNerve1991；14：113−119．
7．ZhaoJ，UchinoM，YoshiokaK，etal．：Dystrophinincontrolandmdx
retina．BrainDev1991；13：135−137．
8．ZhaoJ，YoshiokaK，MiyatakeM，etal．：Dystrophinandadystrophin−relatedproteininintrafusalmusclefibers，andneuromuscularand
myotendinousJunCtions．ActaNeuropathol1992；84：141−146．
9・YoshiokaK，ZhaoJ，UchinoM，etal・：DystrophinisofbrmSand／orcross−reactiveproteinsonneuronsandglialcellsincontrolandmdxnervOuS
SyStem．JNeuroISci1992；108：214−220．
10．PillersDAM，BulmanDE，WeleberRG，etal．：Dystrophinexpressionin
thehumanretinarequiredfbrnormalfunctionasdennedby
electroretinography．NatureGenet1993；4：82−86．
11．D．SouzaVN，NguyenTM，MomisGE，etal．：Anoveldystrophinisoform
isrequiredfbrnormalretinalelectrophysiology．HumMoIGenet1995；
4：837−842．

ー11−12．MiyatakeM，MiikeT，ZhaoJ，etal．：Possiblesystemicsmoothmuscle
layerdysfunctionduetoadenciencyofdystrophiinDuchennemuscular
dystrophy．JNeuroISci1989；93：11−17．
13．MuntoniF，CauM，GanauA，etal．：Briefreport：Deletionofthe
dystrophinmuscle−PrOmOterreglOnaSSOCiatedwithX−linkeddilated
Cardiomyopathy．NEnglJMed

1993；329：921−925．

ー12−Modulationofsplicing ofintronby

modifyinganihtra−eXbn sequence
Whichis deletedffo甲thedystrophin
genein dystrophin Kobe
Masaf血血Matsuo
DivisionofGenetics，IntemationalCenterfbrMedicalResearch，
KobeUniversitySchoolofMedicine，Kdbe650，Japane−mail：matSuO＠iduna・kobe−u・aC．Jp

Together，DuchermeandBeckermusculardystrophies（DMDandBMD，respectively）arethemostcommoninheritedmusculardisease．Theyare

CauSedbymutationinthedystrophingene，isthelargestkn0wnlmmangene（1）．
Dystrophin，alarge（427kD）cytoskeletalproteinlocalizedtothesarcolerrma，isencodedbya14−kbRNAtranSCriptthatisprocessedfrom79exons（2）．
Resultsofarecentstudyconductedinourhboratoryhaveextendedthisgene
regionbymorethan500kbthroughtheidentificationofanewpromoter／exon
SequenCeuPStreamOfthekn0wn5−endofthegeneandexpandedthedystrophin
genetoarOund3，000kb（3）・Atleastsevenpromotersincludingourshave
beenidentifiedinthedystrophingene，andeachpromoterisexpressedina
tissue−Ordevelopment−SPeCificmamer，glVlngrisetomultipleisofbrmsof
dystrophin−（4）・Altemativesplicingofintronsfurtherincreasesthenumberof
dystrophinisoforms，eaCh ofwhichmayhaveaspecific，unlquefunction．
COnSideringthelargernumberofbothexonsandalternativesplicingsites，
SPlicingabnormalityrofdystrophinpre一血RNAissupposedtobemorecomplex．

InoneparticulardystrophingenemutationnameddystrophinKobe，Wefbund
thatexonskipplngduring甲licingwasinducedbythepresenceofintra−eXOndeletionmutationinthegenome，althoughallbftheconsensussequenceskn0wn
toberequiredforsplicingwereunafftcted・TYLedeletionwasdetectedbyPCR
analysis，Whichtrevealed that the productamPlified from the exon19
encompassngreglOnfromtheDMDcasewassmallerthannOmal．Thisresult

SuggeStedthe presenceofranovel血tationふithintheamPlified reglOn．
Sequenceanalysisconfirmedthatthiswasthetasebyshowingthat52bpoutof

一13−

88bpofexon19weredeletedfrom2to3bpupstreamfromthesplicedonorsite（5）．nis52bpdeletionwasconsideredtoresultinaffameShiftmutationthat
wouldcauseDMD・ThedystrophintranSCriptofdystrophinKobewasthen
analyzedusingreverse−tranSCriptionPCR（RT−PCR）・Surprisingly enough，theproductamPlinedfromthereglOnenCOmPaSSlngeXOn19wassmallerthan
predictedaccordingtotheresultsofthegenomicDNAanalysis・Sequence
analysisindicatedthatthewholeofexon19wasmisslngfro叩thedystrophin
cDNA，CauSlnganOut−Offramemutation・Inparticular，thisindicatedthatthe
deletionmutationwithinaneXOnSequenCeCOuldinduceasplicingerrorduring
maturationofmessengerRNA，eVenthoughtheknownconsenSuSSequenCeSat
the57and3−splicesitesofexon19weremaintained（6）・Thesedatasuggestthatthedeletedsequenceofexon19mayfunctionasa
Cis−aCtingelementfbrexactsplicingfbrtheupstreamanddownStreamintrons・
Toinvestigatethispotentialroleofexon19，an血YitTOSPlicingsystemuslng

artificialdystrophinmRNAprecursors（pre−mRNAs）was established・Pre−

mM containing exon18，trunCatedintron18，andexon19was spliced
PreCisely血Ylttv，Whereassplicingofintron18wasalmostcompletelyabolished
Whenthewild−tyPeeXOn19wasreplacedbythedystrophinKobeexon19（7）・
Splicingofintron18wasnotfullyreactivatedwhendystrophinKobeexon19
WaSreStOred tonear1ynormallengthbyinserting other sequencesinto thedeletedsite．Theseresultssuggestthatthepresenceoftheexon19SequencewhichismisslngindystrophinKobeismorecriticalforsplicingofintron18
thanthelengthoftheexon19sequence・CharaCteristically，theefficiencyof
SPlicingofthisintronseemedtocorrelatedwiththepresenceofapolypurine
trackwithinthedownstreameXOn19．Morerecently，SeVeralexonshaveben
ShowntOincludedpurine−richreglOnS，CalledexonrecognltlOnSequenCeSOr
ERSs，Whicharenecessaryforsplicingoftheupstreamintron（8）．ERSsare

likelytargetsfbrsplicingfactorsthatidentifyexonsequencesandpromotetheir
inclusioninthematuremRNA．
Thepossibilitythatoligonucleotidescouldbeusedasmodulatorsofgene
expression，andhenceaschemotherapeuticagents，iscurrentlyunderintense
investigation：The fnOdulation of splicing byantisense oligonucleotides has
recently attracted much attention（9，10）．Dominskiand Kole recently

describedanelegant eXPerimentin血ich aberrant SPlicinginduced by athalassemiamutationwascorrectedbyanantisense2．−0−methylribonucleotide

→14−

（2

−0−MeRNA）（10）・Thispromptedustotestwhethersplicingofdystrophin

Pre−mRNAcould alsobemodulatedby anantisenseRNA asthefirst step
towardsevaluatingthepotentialtherapeuticuseofantisenseRNAtocorrectaberrantSPlicingreactionsinpatientswithDMD・Anantisense31mer2一一0−

methylribonucleotidecomplementarytOthe5−halfofthedeletedsequencein

dystrophinKobeexon19inhibitedsplicingofwild−tyPePre−mRNAinadose−
andtime−dependentmannerinvitrosplicingsystem（7）．
Tosimulatedthisinhibitionin1ivingcells，theantisenseoligonucleotide

mixedwithLipofectAmine（GIBCOBRL）wasaddedtotheculturemediumof
EB−VirustranSformedlymPhoblastoidcells・ResultingdystrophintranSCript
WaSanalyzed by RT−neStedPCRand sequenclng・Theresultrevealedthat
SequenCe Of exon19disq）Peared from mRNA after a certain period of
incubationoflymphoblastoidcellswithantisenseoligonucleotide・ThisclearlyShowedthatexonskippingcanbeinducedbyantisenseoligonucleotide・Thisfirst血vitTOeVidencethatdystrophinpre−mRNAsplicingcanbe
modulatedbyanantisense oligonucleotide raised the possibility of a new

therapeuticapproachfbrDMD・DMDwouldbetransfbrmedtomildBMDby
Changlng a frame−Shift mutation causlng DMDintoanin−ffame mutation

CharacteristicofBMDbymodifyingthedystrophinmRNAsplicing（11）．For
examPle，thesameantisenseoligonucleotidewillbeusedtotreataDMDcase
Witha242nucleotidedeletionofexon20（Fig．1）．Ifweareabletoinduceexon19（88bp）skippinginvivo，thedystrophintranSCriptwilllackbothexons

19and20butthetranslationalreadingframeWillberestored．Asaresult，thismodulation of splicing should tranSfbrm DMDinto BMD・Thoughthis
StrategySeemStObeanalternativewaytoretardprogressionoftheclinicalSymptOmSOfDMD，mOreeXtenSivestudiesarerequiredbefbreclinicaltrialscan
begin；fbrexample，Weneedtoconfirmmodulationofsplicingbyantisense
Oligonucleotideinaninvivoexperiment．Weals6needtodevelopanefncientmethodtodeliverantisenseoligonucleotidestothenucleus．

−15−

Fig・1TheoreticaluseofantisenseoligonucleotidefbrtreatmentofDMD・TheDMD
phenotypeOfthepatientwithanexon20deletionwouldbetransformedintoaBMD
phenotypebyinducingtheexclusionofexon19fromthematuredtranSCriptinthe
PreSenCeOfantisense2l−0−MeRNA・ー16−

Re鈷Ⅰ℃nCel・

Roberts，R．G．，A．J．Coffty，M．Borrow，etal．1992．Determinationof
theexonstruCtureOfthedistalportionofthedystrophingeneby

VeCtOrettePCR．蝕omLcs13：942−950．
2・

Roberts，R・G・，A・J・Coffty，M・Borrow，etal．1993．ExonstruCtureOf

3．

thehumandystrophingene．GeL70mks16：536−538．
Nishio，H．，Y．Takeshima，N．Narita，etal．1994．Identincationofanovel
firstexoninthehumandystrophingeneandofanewpromoterlocatedmorethan500kbupstreamOfthenearestkn0wnPrOmOter・LCh・
血vegL94：1037−1042．
4．

Alm，A．H．andL．M．Kunkel．1993．ThestruCturalandfunctional

diversityofdystrophin．Nbtute伽et．3：283−291．
5・ Matsuo，M・，T・Masumura，T・Nakajima，etal．1990．Averysmall
frame−Shiftingdeletionwithinexon190ftheDuchennemuscular
dystrophygene．Bjochem．Blqpbys．Res．CbL7muL7．170：963−967．
6・Matsuo，M・，T・Masumura，H・Nishio，etal・1991・Exonskippingduring
SPlicingofdystrophinhRNAprecursorduetoanintraexondeletionin
thedystrophingeneofDuchermemusculardystrophyKobe・LaitZ．
血Veぶた87：2127−2131．

7．

Takeshima，Y．，H．Nishio，H．SakamOtO，etal．1995．Modulationofin
Vitrosplicingoftheupstreamintronbymodifyinganintra−eXOn
SequenCeWhichisdeletedfromthedystrophingeneindystrophinKobe・エ（コ血．血vegL95：515−520．

8・

Tanaka，K・，A・Watakatx，andY・Shimura・1994・Polypurinesequences
WithinadownStreameXOnfunctionasasplicingenhanCer・Mbl・CbD．
月foエ14：1347−1354．

9・ Barabino，S・M・L・，B・S・SproatandA・I・LamOnd．1992．Antisense
PrObestargetedtoanintemaldomaininU2snRNPspecificallyinhibit
thesecondstepofpre−mRNAsplicing・NucbLcAcidsRes．20：4457−
4464．

10・

Dominski，Z・andR・Kole・1993・Restorationofcorrectsplicingln
thalassemicpre−mRNAbyantisenseoligonucleotides・Proc．NbtL．Acad．
gc⊥uSA90：8673−8677．

11・

MatsuoM・1996・Duchenneβeckermusculardystrophy：frommoleculardiagnosistogenetherapy・Bm血DeTlinpress・−17−

Characterization oftheInteraction
betweentheSchwannCellDystroglycan
ComplexandEndoneurialLaminin

KiichiroMatsumura，fhkiYamada，HirokoFukuta−Ohi，SachikoFujitaandTeruOShimizu
DepartmentofNeurologyandNeuroscience，TeikyoUniversitySchoolof
Medicine，2−11−1Kaga，Itabashi−ku，Tokyo173，Japan

Introduction

Dystroglycanisencodedbyasinglegeneandcleavedintotwoproteins
α−andβ−dystroglycan

by posttranSlational processing（Ibraghimov−

Beskrovnaya et a1．，1992）．In skeletal・muSCle，α−dystroglycan

With amolecularmaSS Of156kDa binds the basallamina componentlaminin（Ibraghimov−Beskrovnayaeta1．，1992；ErvastiandCampbell，1993），andβ−dystroglycanbinds to cytoskeletal proteins dystrophin and utrophin（SeeOzawaeta1．，1995）．Lamininisaheterotrimermadeupofthreechainsof
Classesα，βandγ，andexistsin numerous trimericisofbrmsin different
tissues（SeeBurgesoneta1．，1994）．LamininαChainshaveglobulardomain（G）repeatsattheC−terminus，andlaminin−1，COmPrisedoftheα1，β1and†l
Chains，bindstoα−dystroglycanViatheGrepeatsintheαlchain（Geeeta1．，
1993）・In peripheralnerve，laminin−2，COmPrised oftheα2，β1and†1
Chains，isexpressedintheendoneurium（LeivoandEngvall，1988；SaneSeta1．，
1990）・AIpha−dystroglycanwithamolecularmaSSOf120kDaisexpressed
SurrOundingperipheralnervefibers血viYOandbindsboth1aminin−1and2血
Vitro（Yamadaet a1．，1994）．Recently，1amininα2chainwas showntO be

deficientincongenitalmusculardystrophyand4Ymice，Whichhavemuscular
dystrophyandperipheraldysmyelination（Arahataeta1．，1993；Sunadaeta1．，
1994；Tomeeta1．，1994；Xueta1．，1994）．Furthermore，theexpressionof
laminininthebasallaminaisassociatedwithSchwarmcelldevelopment（Leivo
andEngvall，1988；ObremskiandBunge，1995）．These findingssuggest arole fbrtheinteraction of dystroglycan

Withlamininin peripheral

myelinogenesis．Thegoalofourlaboratoryistoprovethishypothesis．As−19−

aninitialstep towardsthis goal，We have charaCterizedthe dystroglycan−
1aminininteractioninperipheralnerve（Yamadaeta1．，1996）．
Characterization ofthedystroglycan complexinperipheral
nerVe

TheresultsofcLSManalysisofrabbitsciaticnerveareShowninFigl．
ImmunOreaCtivities fbr

α−，β−dystroglycan

andlaminin−2werelocalized

SurrOundingthe outermostlayer ofmyelindleathofnervefibers（Fig．1）．
Ontheotherhand，immunOreaCtivitiesforutrophinandDpl16，adystrophin
geneproductspecifictoperipheralnerve（Byerseta1．，1993），reSembledthat
for Schwann Cell marker S−100 and waslocalizedin the Schwam cell
CytOPlasm（Fig．1）．
Membrane−aSSOCiated cytoskeletaland peripheralmembrane PrOteinS，butnotintegralmembrane PrOteins，areknOwn to be extracted from themembraneSby alkalinetreatment（ErvastiandCamPbe11，1991）．Based on
this，WePerfbrmedakalineextractionofthecrudebovineperipheralnerve
membraneS・Alpha−dystroglycanof120kDawasextractedatpH11，WhileP−
dystroglycanOf43kDawasnotextractedatpHllor12（Fig．2）．Together
Withthefactthatα−dystroglycanCOrreq）Onds totheN−terminalhalfofthe
dystroglycan

PreCurSOr PrOtein containing the slgnal peptide but no

tranSmembranedomainandβ−dystroglycanCOrreSPOndstotheC−terminalhalf
COntainingatranSmembranedomain（Ibraghimov−Beskrovnayaeta1．，1992），
theseresultssuggestedthatperipheralnerveα一dystroglycanWaSaPeripheral
mernl）raheprotein，Whileperipheralnerveβ一dystroglycanWaS anintegral
membranePrOtein・Theextracellularmatrixproteinlamininα2chainof320

kDawaspartiallyextractedatpH11andalmostcompletelyextractedatpH12（Fig・2），tOgether withtheβ1￣and†l chainS（not shown）．Dp116and

utrophin，Of116and395kDarespectively，WerebothextractedatpHll（Fig．
2），ingoodagreementwiththepredictionfromtheprimaryStruCturethatthey
arecytoskeletalproteinS（Bybrseta1．，1993；Tinsleyeta1．，1994）．
Weisolated，uSing

WGAandlaminin

affinitychromatogrq）hies，α一dystroglycanandits associatedproteins fromthe digitonin extracts of the
CrudebovineperipheralnertemembraneS．Asexpected，1aminin−bindingα−

dystroglycanWaSisolatedbytheseprocedures、（Fig．3）．Beta−dystroglycan，Whichdid not bindlamininin blot overlay，WFS alsoisolated（Fig・3），−20−

SuggeStingthatitwascomplexedwithα−dystroglycan．Ontheother hand，Dp116andutrophinwerenotisolatedbytheseprocedures（Fig．3）．
Thedatapresentedhere，takentogether，indicatethat（1）α−dystroglycan
isaneXtraCellular

Peripheral membrane glycoprotein whichlinksβ−dystroglycanin the Schwann Cell outer membrane Withlaminin−2inthe
endoneurialbasallamina，and（2）dystrophinhomologues，Dpl16andutrophin，
are CytOSkeletalproteins ofthe Schwarm cell cytoplasm（See Fig．7）．It
remainstobedeterminedifafractionofDp116andutrophinareCOmPlexed
WithP−dystroglycanbeneaththeSchwanncelloutermenibrane（SeeFig．7）．
CharaCterization ofcarbohydrate chains ofSchwann cellα−dystroglycan
Weisolated，uSing WGAandlaminin affinitychromatographies，α−dystroglycanffomthealkalineextractsofthecrudebovineperipheralnerve
membraneS・Wetesteditsreactivitywithmonoclonalantibodiesspecificfor
Carbohydrate chainS・

AIpha−dystroglycan was stainedintensely withmonoclonalantibodyanti−HNK−1，indicatingthatithad3−Sulfatedglucuronyl−
Substitutedoligosaccharide（Fig．4a）（Choueta1．，1986）．Ontheotherhand，
itwasnotstainedwithmonoclonalantibodiesagainstheparanSulfate，keratan
Sulfate，SialylTn，LewisX，SialylLewisX（Fig．4a）or sialylLewisA（notShown）．
Thelectin−binding−PrOPertiesofSchwanncellα−dystroglycanareShowninFig・4b・Schwann Cell（X−dystroglycanWaS Stained positivewith WGA
Whichrecognizesclusterofsialicacidsand／orbisectingGIcNAc（Fig．4切，but
WaS nOt Stainedwith PHA−E4，alectin specific fbr bisecting GIcNAc（notShown），indicating thatit contained cluster of sialic acids．It was stained

POSitivewithboth肋ackjaamuL・et7SLsagglutinin（MAA），alectinspecificfor
Sialic acidlinkedα2−3to galactose，andpeanut agglutinin（PNA），alectin
SPeCific fbr unsubstituted Ser／Thr−linked disaccharide Galβ1−3GalNAc unit
（Fig・4b）・It was also stainedpositive withJacalinwhich also recognizes
Galβ1−3GalNAc（Fig．4叫．It was not stained with Sbmbucus nlvalik
agglutinin（SNA）whichrecognizessialicacidlinkedα2−6togalactose（Fig．

4帆

TheseresultsindicatethatSchwamcellα−dystroglycanissialylatedα2−

3togalaQtOSein0−glyans・SchwmIICellα−dystroglycanWaSStaineqpositive
withVVAJB4（Fig・4b），indicatingthatlitcontainednon−reducingterminalβ−−21−

GalNAc・It was stainedpositive with both ConAand Chlantbusnjvalik
agglutinin（GNA）（Fig．4b），indicatingthatit containedN−1inked mamose
residues．Itwasnotstainedwith上btuL・aStTamOnlumagglutinin（DSA）which
recogmizesGalβ1−4GIcNAcincomplexandhybridN−glycanS，in0−glycanS
and GIcNAcin0−glycanS（Fig．4b）．The results of staining with Con−A，
GNA，PHA−E4and DSA，When combined，SuggeSt thatthe N−glycan（S）of
SchwannCellα−dystroglycanCOuldbehigh−mannOSetyPe．SchwannCellα−dystroglycanWaSnOtStainedwithlectinS・SPeCificfbrvarioustypesoffucose
residues，AAL，UEA−1（Fig．4b），LCAorLotus（notshown）．
Role ofcarbohydrate chains ofSchwann cellα−dystroglycanin

laminin binding
To seeif carbohydrate residues ofα−dystroglycanareinvoIvedin

lamininbinding，WeteSted，byblotoverlayassay，thebinding oflamininto
Schwann Cellα−dystroglycan

treated with

deglycosylation enzymes．

Treatmentofα−dystroglycanWithbothA uL・ea血cjensneuraminidase（Fig．5）
and V choleTaeneuraminidase（not shown）greatlyreducedthe binding oflaminin．Treatmentwith0−glycosidase，tOgetherwithneuraminidase，didnot
havefurther efftcts on the binding oflaminin（Fig．5）．The binding of
lamiminwasnot afftctedby treatmentwithN−glycosidaseF，Chondroitinase

ABC，heparinase，heparitinase or keratanaSe（Fig．5）．When nitrocellulose
transfersofα−dystroglycanweretreatedwithneuraminidase，thebinding oflaminintoα−dystroglycan was reduced（Fig．6a）．Thepresence ofO．l M

Sialicacid，butnotO．lMGIcNAc，intheblotoverlaymediuminhibitedthebindingoflaminintoα−dystroglycan（Fig．6b）．Inhibitionoflaminin−binding

bysialicacidwasquantifiedbyELISA．Twoformsofsialicacid，N−aCetyl
andN−glycolylneuraminicacids，andcolominicacid，POlymerOfsialicacids，
inhibitedthebindingoflaminin（Fig．6C）．
Our findings suggest a role fbr sialylation ofα−dystroglycanin thebindingoflamimin．The results thus farindicate thatα−dystroglycanis a

mucin−tyPeglycoproteininbrain（SmalheiserandKim，1995），Cardiacmuscle（BranCaCCioet a1．，1995）andperipheralnerve（this study）．A mucin−1ike
motifis detectableinthe centralreg10n Ofα−dystroglycan，and，indeed，Cardiacmuscleα−dystroglycanWaSShowntOCOnSistoftwoglobulardomains

connectedbyardd−Shapedsegment（BranCaCCioeta1．，1995），COnSistentwith−22一thepredictionthatamucindomainisextendedintoarigidrod・Thedensely
ClusteredarrayOf0−glycanSinthemucindomainmaypresentterminalsialic
acidsinapolyvalentmannerfbrtheinteractionwithlaminin（Fig．7亘）．
Besidesα−dystroglycan，1aminin binds a variety of glycolipidsand

PrOteinS（seeKennedyeta1．，1983；Geeeta1．，1993）．Atpresent，itisunClear
if sialylationaloneis re甲OnSiblefor the binding ofα−dystroglycanWith
laminin，becauselaminin was suggested to recognlZe highly charged
Oligosaccharides on proteinsratherthanmonosaccharides（Kermedyet a1．，
1983）．Inthis respect，itis ofinterest to note that，SimilartO brainα−
dystroglycan（Smalheiserand Kim，1995），Schwann Cellα−dystroglycanis

HNK−1Positive・Furthermore，theHNK−1Carbohydrateepitope，foundina
number of neuralcell adhesion molecules，Playsimportant rOlesin celladhesionandhasbeenimplicatedinthebindingtolaminin（Ktinemundeta1．，
1988；Hall et a1．，1993）．It would bethusinteresting to seeifthis
Carbohydratestructureis alsoinvoIvedintheinteractionofα−dystroglycan
Withlaminin．

ACKNO lJEDGMENTS

ThiswofkwassupportedbytheMuscularDystrophyAssociationof
America，grantSffomCiba−GeigyFoundation（Japan）fbrthePromotionof
Science，NitohFoundationandUeharaMemorialFoundation，theScience
ResearchPromotionFundffomJapanPrivateSchooIPromdtionFouhdation，ResearchGrant（5A−2）forNervOuSandMentalDisordersfromtheMinistry
OfHealthandWelfare，andGrants−in−AidfbrScientificResearchonPriorlty
Areas，fbrScientincResearch（05454262，06454280，06670670andO6770463）andfbrScientihcResearchonDevelopmentalareas（05557037）
fromtheMinistryofEducation，Science，SportsandCulture．

−23−

ComponentsofDystrogTycanCompTexinPeripheraINerve

FIG．1．⊂LSMimmunofluorescent analysis ofrabbit sciatic
nerVeShownarethe cLSMimmunofluorescentimagesofrabbitsciatic nerve．Alpha−dystroglycan（DGl），β−dystroglycan（DG2），lamininα2chain（M），lamininβlchain（Bl），lamininαlchain（A），Ⅰわ116，utrOphin（UTR）andS−1∝）proteinwere
detectedby antibodies anti−FPD，43DAG／8D5，2G9，4ElO，11D5，VIA42 DRPl and
15E2E2，reSpeCtively．Bar，10llm．

ー24−

AlkaIineExtractionofPeripherilNeNeMdmbranes

α−DG

UTR

β−DG Lamα2

晶
′
︑

曹

川
や
捌
け
．
⁚
・
⁝
州
⁚

㌻︑一−ポ一

ヽ㍉一

÷∵8㌧ご
〜 β＿ご
÷ 身
㌫打ぎざぎざffささ

▼千一▲

窪寺書法
儀軌
腰潜盛

へ

FIG．2．Alkaline extraction ofthe crudebovine peripheral
nerve membranes
120l110fthepHllextracts（firstlane），thepH12extractsofthepHllpellets（SeCOndlane）andthepH12pellets（thirdlane）ofthecrudebovineperipheral
nerve membraneswereseparatedby3−12％SDS−PAGEandtransferred tonitroCellulose membranes．Alpha−dystroglycan（α一DG），β−dystroglycan（β−DG），
lamininα2chain（Lamα2），Ppl16andutrophin（UTR）were detectedby

antibodiesIIH6，43DAG／8D5，2D9，DYS2andanti−DRP，reSpeCtively．The30kDa
banddetectedby43m妊ン8D5ispresumedtobeaproteolyticfragment orthe
unglycosylatedformofβ一dystroglycan．＿Molecular．weightstandards（Dax10−3）areShown ontheleft．

−25−

ComponentsofPeripheraINerveDystrogIycanComplex■︑．．︑︐・︐柏r．■■．■t．．；．

′

′
㌍

72−

−

格

納

46−

爾胃胃冒澤壷毀損揖観

t．一見−

ノー■

1

圏闇闇聞樹肇・⁝・．消閑讃司渕瑚

109−

一
l

FIG．3．Characterizationofperipheral nerveα−dystroglycan

andits associated proteins
Alpha−qyStrOglycan andits associatedproteinswereisolatedfromthe digitonin
extractsofthecrude bovine peripheralnerve membranesbyWGA andlaminin

affinitychromatographies．120llloftheEmeluatesofthelaminin−Sepharose
WereSeparatedby3−12％SI義一PAGEandtransferredtonitrocellulose membranes．
Alpha一句′StrOglycan（α−DG），β一郎／StrOglycan（P−DG），Ⅰや116andutrophin（UTR）

Were detectedby antibodiesIIH6，43mG／8D5，DYS2andanti−DRP，reSPeCtively．

Lam2（yLindicatestheidenticalnitrocellulosetransfer overlaidwithlaminin−2．
Heavily−glycosylatedα−dystroglycan was notidentifiedclearly onthe SIE gel

Stained with（ユ旧maSSie blue（CB）（Yamada etStandards（DaxlO13）areshownontheleft．

−26−

al．，1994）．

Molecular weightMonocIonaIAntibodyReactivityofPeripheralNerve
a−DystrogTycan

109−

72−

︑吏ざ

何て

一ぎ

古訂

古︶

ゼ匂

FIG．4．Characterization ofcarbohydrate Chains ofSChwann cellα−dystroglycan（a）AIpha−dystroglycan（3llg）wasseparatedby3−12％SI蔦−PAGE，tranSferredto
nitroCellulosemembranes，andthen reaCtedwithmonoClonalantibodies against
α−qyStrOglycan（ⅠIH6），Sialyl Th（STn），Lewis X（LeX），Sialyl Lewis X（SLeX），

heparan sulfate（HS），keratan sulfate（KS），OrmOnOClonalantiboqyanti−HNK−1．
（b）Theidenticalnitrocellulose transfers as shownin（a）were reactedwith
Variouslectins．Molecularweightstandards（DaxlO￣3）areshownontheleft．

−27−

梅

毎
里−＿＿…＿＿

28

の
N

く凸
寸

1
1

物象
卓−＿＿＿＿…‖＿＿＿＿＿＿＿＿＿＿

か旬

も匂
わ恥

％

物

上軸

JもtぷいuO亡き○霊巴d︵？OtXd邑spJ召uヨSJ鳥芯きJdt己UtO∑．S已コUU︻SnOでd＞吉富pUJUd巴巴むき︵ヱu−uき○霊Sd SJUhS已已︼USOtn∃UUOJJ召te召已Up芯 ト ︵屯︶〉ゎu巾Uh︼ぎ﹂lSh凸・古山奉読タ 野蒜丘d葛d−○のむモ鼠OJdぎ壱u石・u苛む﹂

ム旬

．宅t苫JuO已＞石膏巴三M岳X占︶sp﹂召已ヨSU忠芯きJdtnUUtO∑．︵tg邑三軍H︶NJO︵−告邑宅−︶l占召召雲
重きp3．︒岳亡U雲p已d︑S呂已q∈賀USOtntt︒BJl竃OJp巴JUhSu芸．凹等d・賃S式llMぶp32邑︒SSdき︵望M︶udUと野とS合もp雲盲︒リム的占．︒S層雲己已コ呂J08己︒Sqd芸J u＝雇主d壱u望p名声コ⊆芦意ず雲合・8S曇り筍u二〇Jl已OU．︵︼EU望︒S昌︒l已ぷJ︒ゴdUH︶US書芸−氏名．︵dUH︶US昌て已芸．︵壱喜8︶U等US昌宏〇七u〇七︑︵htud＋Jゴ芸︶
US召琶Uと甲Opud霊召竃召已n呂fn芸︶USdヨu召巴n呂云t？邑hUS召登り怠・N毒きp雲巴；窒ueとぎJJS合・月dtくゝ何てgOTu羞∈巾﹂

旬

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． −

▲ い た P

︹ ご

1m○︻

tN卜

1mN

ヨUとぎJ︺S合・ピコ︒U ddd芦月US p︒︺d︻ぉOUと的名○︺点月日d 0ぎ竃月四・S・営﹄hd︼﹂gON・u羞∈巾﹂旬． t ナ ． 〜

箸％㌔転句旬％ 箸％㌔転句旬％

こu巾呈研○烏合きざ﹂芸IP芯丘d葛dPUlq■hsO呈謬凸OINPu三・u董Eq﹂−○ぎ苛u芯一■−

BindingofLamjnintoPeripheralNerveα−Dystrog］ycanaNeuraminidase，。9●｝ 胃

が㌔

．001

．01

1

1

10

100

mM

FIG．6．（a）Efftct ofneuraminidase treatment onthebindingof
laminin toSchwann cellα−dystroglyCan

Alpha−（妙StrOglycan（3llg）wasseparatedby3−12％SⅨ−PAGEandtransferredto
nitroce11ulose membranes．The nitrocellulose transfers wereincubatedwithor
Withoutneuraminidase（＋and−，reSPeCtively），andthenoverlaidwithlaminin−1．
Molecularweightstandard（DaxlO■3）isshownontheleft．

（b）Efftct ofsialic acidonthebinding oflaminin toSchwann
Cellα−dystroglycan（blot overlay）
Alpha−（卸StrOglycan（311g）was separated by3−12％

S【万一PAGE，tranSferred tonitroCellu10Semembranes，andthenoverlaidwithlaminin−1inthe presence of
O．l M sialic acid，0．1M GIcNAc orinthe absence of these sugars（Control）．

Molecularweight standard（Daxl0−3）isshownontheleft．
（C）Efftct ofsialic acidonthebindingoflaminln toSchwann
Cellα−dystroglycan（ELISA）
InthepresenceofvariousconcentrationsofN−aCetylneuraminicacid（NAcNA），

N−glycolylneuraminicacid（NGlyNA），COlominic acid，GIcNAc orNaQ，laminin−1
WaSincubatedwithα一句／StrOglycan（1llg）coatedonto microtiter wells．Boundlaminin−1was quantifiedby ELISA．Points arethe percentage comparedtothe
Valuesofthewellsincubatedin theabsenceofinhibitors．Pointsrepresentthemean of triplicate wellsin one experiment representative of three similar
experiments．

−30−

Myelin

Pa．m
me
t
COMy
Semicompacted﹁ノーし﹁うーしJL

Semicompacted

MyelinlnnerMembrane

BasalLamina

OuterMembrane

Utro芝；＝：フ⊃／？lc3Dp116 CybpTasm

CompactMyelin

FIG．7．Hypotheticalmodelofdystroglycan−laminininteractioninperipheralnerve（a），（b）Alpha−andP一句′StrOglycanarelocalizedin the Schwann cell outermembrane，While Ppl16andutrophinarelocalizedintheSChwanncellcytoplasm．
（C）Alpha−dystroglycan（a），Whichisanextracellularperipheralmembrane
glycoprotein，linksβ−dystroglycan（b）intheSchwann celloutermembranewith
laminin−2intheendoneurialbasallamina．Themucindomainofα−dystroglycan
isextendedintoarigidrod．ClustersofsialicaCidresiduesinO−glycansofα−

dystroglycan arepresumedtobeinvolvedintheinteractionwiththeG−domainof
lamininα2chain．

−31−

Re鈷1℃nCeS

Arahata，K・，Hayashi，Y・K・，Koga，R・，Goto，K・，Lee，J・H・，Miyagoe，Y・，Ishii，H．，Tsukahara，T．，Takeda，S・，Woo，M・，Nonaka，I・，Matsuzaki，T・
andSugita，H．（1993）Laminininanimalmodelsfbrmusculardystrophy＿一一defectoflaminin−Minskeletalandcardiacmusclesandperipheral

nerveOfthehomozygousdystrophic￠伸mice・PLt，C・々n・Acad・B69，
259−264．

Brincaccio，A．，Schulthess，T．，Gesemam，M．andEngel，J．（1995）Electron
microscopICeVidencefbramucin−likereglOninchickmuscleα−

dystroglycan・FE・B・S・Lett・368，139−142・
Burgeson，R・E・，ChiquetM・，DeutzmarnR・，EkblomP・，EngelJ・，Kleinman
H．，MartinG．R・，MeneguzziG・，PaulssonM・，SaneSJ・，Timpl，R・，
Tryggvason，K．，Yamada，Y・andYurchenco，P・D・（1994）Anew

nomenclatureforthelaminins．肋tLixBioIw14，209−211・
Byers，T．J．，LidovH．G．W．andKunkel，L・M・（1993）Analtemative
dystrophintranSCriptspeci丘ctoperipheralnerve・NbtuLeGd】et・4，77−
81．

Chou，D．K．H．，Ilyas，A．A．，EvanS，J．E．，Costello，C・，Quarles，R・H・and
Jungalwala，F．B．（1986）StruCtureOfsulfatedglucuronylglycolipidsin
thenervOuSSyStemreaCtmgWithHNK−1antibodyandsomeIgM

paraproteinsinneuropathy・LBiol・αem・261，11717−11725・
Cullen，M．J．，Walsh，J．andNichoIson，L．V．B．（1994）Immunogold
localizationofthe43−kDadystroglycanattheplasmamembranein
controlanddystrophichumanmuSCle・ActaNtuLtPathol・87，349−354・
Ervasti，J．M．andCampbell，K．P．（1991）Membraneorganizationofthe
dystrophin一glycoproteincomplex・Cb〟66，1121−1131・

ErvaSti，J．M．andCamPbell，K．P．（1993）Aroleforthedystrophin−glycoproteincomplexasatranSmembranelinkerbetweenlamininandactin．L CbLtBbJ．122，809−823．

Gee，S．H．，Blacher，R．W．，Douville，P．J．，Provost，P．R．，Yurchenco，P・D・
andCatbonetto，S．（1993）Laminin−bindingprotein120frombrainis
closelyrelatedtothedystrophin−aSSOCiatedglycoprotein，dystroglycan，
andbindswithhighaffinitytothemajorheparinbindingdomainof

laminin．LBioL．Chem．268，14972−14980．

−32−

Hall，H．，Liu，L．，Schachner，M．andSchmitz，B．（1993）TheL2什INK−1
Cafbohydratemediatesadhesionofneuralcellstolaminin．EuT．L〃由mgd．5，34−42．
Hori，H・，KanamOri，T・，Mizuta，T・，YamaguChi，N・，Liu，Y・andNagai，Y・（1994）HumanlamininMchain：ePitopeanalysisofitsmonoclonal
antibodiesbyimmunOSCreenlngOfcDNAclonesandtissueexpression・L
βゐdem．116，1212−1219．
Ibraghimov−Beskrovnaya，0・，ErvaSti，J・M・，Leveille，C・J・，Slaughter，C．A．，
Semett，S・W・andCamPbell，K．P．（1992）PrimarystruCtureOf
dystrophin−aSSOCiatedglycoproteinslinkingdystrophintothe
extracellularmatrix．NbtuLe355，696−702．
Kennedy，D．W．，Robbach，D．H．，Martin，G．R．，Momoi，T．andYamada，K．
M・（1983）Theadhesiveglycoproteinlamininisanagglutinin．LCbH

印がわJ．114，257−262．
Ktinemund，V．，

Jungalwala，F．B．，Fischer，G．，Chou，D．K．H．，Keilhauer，G．

andSchaclmer，M．（1988）TheL2田NK−1Catbohydrateofneuralcell
adhesionmoleculesisinvoIvedincellinteractions．LaLtBLol．106，
213−223．
Leivo，I・andEngvall，E・（1988）Merosin，aPrOteinspeci丘Cfbrbasement
membranesofSchwamcells，Striatedmuscle，andtrophoblast，isexpressedlateinnerveandmuscledevelopment．Ptvc．NbtL．Acad．Sci．

ui485，1544−1548．
Matsumura，K・，Yamada，H．，Shimizu，T．andCamPbell，K．P．（1993）
Difftrentialexpressionofdystrophin，utrOPhinanddystrophin−aSSOCiatedPrOteinsinperipheralnerve．FEB．S．Lett．334，281−285．
Obremski，Ⅴ・J・andBunge，M．B．（1995）Additionofpurinedbasallamina

moleculesenablesSchwanncellensheathmentofsympatheticnemitesin
Culture．DeY．Bjol．168，124−137．
Ozawa，E・，Yoshida，M・，Suzuki，A・，Mizuno，Y・，Hagiwara，Y・andNoguchi，S・（1995）Dystrophin−aSSOCiatedproteinsinmusculardystrophy・HhLP・
肋血C．Ge乃eL4，171ト1716．
Sanes，J．R．，Engvall，E．，Butkowski，R．andHunter，D．D．（1990）Molecular
heterogeneityofbasallaminae．IsoformSOflamininandcollagenIVat
theneuromuscularJunCtionandelsewhere．LC〟BLol．111，1685−1699．

−33−

Smalheiser，N．R．andKim，E．（1995）Purificationofcranin，alamininbindingmembranePrOtein：identitywithdystroglycanandreassessment
Ofitscafbohydratemoieties．J．BLol．Chem．270，15425−15433・
Sunada，Y．，Bemier，S．M．，Kozak，C．A．，Yamada，Y．andCamPbell，K．P．
（1994）Deficiencyofmerosinindystrophic句′miceandgeneticlinkage
OflamininMchaingeneto㊥′locus．LBjoL．CheL7］．269，13729−13732・Tome，F．M．S．，Evangelista，T．，Leclerc，A．，Sunada，Y・，ManOle，E・，
Estoumet，B．，Barois，A．，Campbell，K．P．andFardeau，M．（1994）
Congenitalmusculardystrophywithmerosinde丘ciency・CR・Acad・ScL

JⅡロ317，351−357．
Xu，H．，Christmas，P．，Wu，X．R．，Wewer，U．M．andEngvall，E．（1994）
DefectivemusclebasementmembraneandlackofM−1aminininthe

dystrophic的mouse．PTOC．♪ねtl．AcadSbl．【店A91，5572−5576・
Yamada，H．，Shimizu，T．，Tanaka，T．，CamPbell，K．P．andMatsumura，K・（1994）Dystroglycanisabindingproteinoflamininandmerosinin
Peripheralnerve．FEB．S．Lett．352，49−53．

Yamada，H．，Chiba，A．，Endo，T．，Kobata，A．，Anderson，L．V．B．，Hori，H．，
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−34−

Dystrophin−aSSOCiated protein，SyntrOPhin（Al）

MikiharuYoshida†，AndrewH・Ahn‡，AtsushiSuzuki†，MaryDilysS．Anderson‡，ChrisA．
Feener‡，SaraSelig‡，YasukoHagiwara手，LouisM・Kunkel‡，andEgiroOzawa†（†NationalInstituteofNeuroscience，‡BostonChildren▼sHospital）

Dystrophin，theproteinresponsibleforDuchenne′Beckermusculardystrophy，isPurifiedasacomplexwithseveralproteinscalleddystrophin−aSSOCiatedproteins（DAPs）fromdigitonin−SOlubilizedrabbitskeletalmusclel1，2］．AmongDAPs，theonewithamolecularmaSS

Ofabout60，000isnamedAlor59DAP・WehavepreviOuslyshownthatthisDAPisactuallya
heterogeneousgroupofproteinsconsistingofanacidic（α−Al）andadistinctbasic（β−Al）
COmPOnent【3］・HerewereportthecloningofacDNAfbrahumanbasicisoformOfAlanddemonstratebycomparisonbetweenourresultsandothersthatthereareatleastthreeAIs（allOfwhichwillbehenceforthnamedsyntrophins），namelytwobasicones（βlandβ2）andone
Weaklyacidicone（α）・TheheterogeneousnaturesofAIwefbundarethusmostlyclarined．

dpNACわ〃玩gげβ−AJ
Alisseparatedintotripletbandsbyl−dimensionalSDS′PAGEofpurifiedrabbit
dystrophin−DAPcomplex【1，2］．Forcloningweatfirstdeterminedtheinternalaminoacid

SequenCeSOfthetripletbands，reSPeCtively（Tablel）［41・01igonucleotides fbrprobeswere

designedonthebasisofthesequenceNo・1（OrNo．4）andalibraryofhumanfttalmuscle
九gtlOcDNAwasscreenedbythem・Asinglepositiveclonewasplaque−Purified，Subcloned
intoEcoRI−CutBluescriptIISK（＋）vectorandsequenced．The2979−bpsequenceencodesa

Slnglelargeopenreadingframe，StartlngfromaninitiatorATGwithinthecontextofaKozak
COnSenSuS・Theclonehadnopoly（A）tailbutcontainedthesequencewhichseemstobethe
POlyadenylationslgnal・Thededucedproteiniscomposedof538aminoacidsandconsideredto
beacytoskeletalprotein，becausenoslgnalsequenceandnotransmembranesequencewere

hund・Thecalculatedmolecularmassof58kDaandthecalculatedpIof9・Oagreewellwiththeー35−

electrophoreticallydeterminedsizeandchargeofβ−All3］・Thededucedhumanaminoacid
sequencecontainsexactmatchestothesequencedpolypeptides1−50riginatedfromthetoptwo
bands（TableI），despitethefactthatthepartialpeptidesequenceswerederivedfromrabbit・On
thecontrary，thesequences6−1lwerenotfbundinthededucedsequence・

Tablel・PartialpolypeptidesequencesofAlSequence

Band No．
⊥

Ala

Aminoacidse

ATPYVRRGSPVS

2RDエーL工3

RQ★KP★LVVL
4Sameasl

5

S工SⅣQR★GVR
6Sameas7

7

AR★VP工．RHAYVS★★★℡PSDP
8

L★AGTGAGAGG
9

工★SADGQD
0⊥

LLLYGGLPQ℡L工Aで★L⊥⊥LAA★℡★QLVDG

Tablel．TheDAPwasresoIvedintoatripletAla，一b，and−CbySDS／PAGEanddigestedwith

V8protease・ThepolypeptidesreleasedwerethenseparatedbyHPLCandmicrosequenced・An
asteriskmeanstheresidueatthatpositionwasnotdetermined・01igonucleotidesdesignedfbrthis
studyarereversetranslations

ofsequence：5一一GCIACICCITAKGTIAARAARGG−3

・

PositionswithRweresynthesizedwithacombinationofbothdAanddGandthosewithaK
WereSynthesizedwithacombinationofdTanddG・Duringthesequencingofthishumanβ−AIcDNA，thesequencesofothercandidateAl
genesinmouse，rabbit，andTbTPedobecameavailablel5，6］andarealignedwithoursequence・

Thesequences6−11，WhichareorlglnatedfromthebottombandexceptNo・6，Wereidenticalto
thededucedaminoacidsequenceofrabbitcandidate59DAP−1［6］andareclosetothatofmouse
SyntrOPhin−1［5］．Theseproteinsareconsideredtobeα−Al，becausetheircalculatedpIsbothー36−

are6・4・Interestingly，Werealizedmousesyntrophin−2［5］tobeabasicproteinbytheisoelectric
POintcalculatedbasedonitspartialpublishedsequence（pI＝9．4），butitdidnotmatchtoourβ−
Alasdescribedbelow．
AsthecDNAwasbeingsequenced，Weidenti丘edarelativewithinthehumanESTdata

base，a210−bpESTfromahumanmusclecDNAlibrary・Itsdeducedaminoacidsequencehad
48％identityatbesttothatofβ−Al．However，WefoundthattheESTisratherclosetothe
deducedmousesyntrophin−2sequence，becausetheaminoacididentityreachedto82％
betweenthem・WethusconsidereditmostprobablethattheESTisthehumanhomologueof
mousesyntrophin−2・UsingthisESTsequencee，Wehavenowclonedthehumanhomologueof
SyntrOPhin−2，Whichisdistinctfromβ−Al［7］・WethuscopcludethatthereareatleastthreeAIs，tWObasicAIsandoneweaklyacidicAl．Sofar，SeVeralnameswereusedforthese
PrOteins・Toavoidconfusion，WeWillhereafterusesyntrophinasastandardnamefbrAIs・We
useβ1−andβ2−forthebasiconesandα−fbrtheacidiconetodiscriminatethose．β−AIwe
Clonedandsyntrophin−2arethusnamedβ1−andβ2−SyntrOPhins，reSPeCtively，Whileα−Al，SyntrOPhin−1and59DAP−larenamedα−SyntrOPhin・
Wedeteminedthegenelocationsofhumansyntrophinsincludingα−SyntrOPhinwhich
WeClonedbytheuseofpublishedsequencesofmouseandrabbit0−SyntrOPhin［5，6］．They
Were8q23−24，16q23−24and20qll．2fbrβ1−，β2−andα−SyntrOPhins，reSPeCtivelyl7］．

血m〟仰あわrAJldかね好み〃什叩あわ∫WehavenowknownthepresenceofP2−SyntrOPhinbymolecularcloning，butwehave
notdetectedityetontheproteinlevel．Usinganti−β1−SyntrOPhinantibodywhichwepreparedandtheotheranti−SyntrOPhinantibodiesprovidedbyProf・S・C・FroehnerofNorthCarolina
Universlty，WeeXaminedthedystrophin−DAPcomplexpurinedfromrabbitskeletalmuscleby
immunoblotanalysis［8］．Intheblotsseparatedbyl−dimensionalSDSIPAGE（Fig．1），the
antibodyagainst

β1．reactswiththetoptwoofthetripletsbands，Whileantibodyagainst

αreacts withthebottomband・This resultwellagrees withthefactsthatthe sequenced
POlypeptidesderivedfromthetoptwobandsandthosefromthebottombandwerefbundinthe
deducedsequencesofhumanβ−Alandrabbit59DAP−1，reSPeCtively．Ontheotherhand，We

ー37−

繭
＄yntrOPhjns

Fig・l・ImmunoblotpattemSOfdystrophin−DAPcomplexwithvariousantibodies・Dystrophin−
DAPcomplexwasseparatedbyl−dimensionalSDS／PAGE・Fromtheleftlane，6．5，4，1．3，0．1，
andO・5LlgOfsamPleswereloaded・Anti−Pl−Synistheantibodyagainsthumanβ1−SyntrOPhin［8］．
Anti−α−andami−β2−Synsaretheantibodiesagainstsyntrophins．．α

and．．β2．T，reSPeCtively，Which

WerepreParedbasedonthemousesyntrophinsequencesl101・ab1351isthemonoclonalantibody
againstTbtPedo syntrophin［101．

foundthatthemonoclonalantibodyagainstTbTPedosyntrophin（ab1351）reactedwithallthe
tripletbands，althoughthebindingabilitytoα−SyntrOPhinwasmuchstrongerthanβ1−
SyntrOPhin・Webarelydetectedβ2−SyntrOPhinwithitsspecificantibody，Whenalargequantity
Ofsampleisloadedonthegel，SuggeStingthatβ2−isoformispresentinlessminorfractionthan
Others・TherefbreitisnaturalthatwefailedtodetecttheisofomOntheproteinlevelfbrmerly・Severalbandswerestained，mOStOfwhichapparentlyoverlaptheothersyntrophinbands，but
itseemsthatthereisaslightdifftrencebetweenβ1−andβ2−SyntrOPhins．
Theblotsseparatedby2−dimensionalPAGEareshowninFig・2・Asexpectedfromthe
aboveresults，theantibodyagainst

βlreactswiththebasicseriesofspots，Whileantibody−38−

basic urea−lEF acidic

α−SyntrOPhin＋β1−SyntrOPhin（ab1351）α−SyntrOPhin（antj−α−Syn）

β1−SyntrOPhin（anti−β1−Syn）

β2−SyntrOPhin（anti−β2−Syn）

Fig・2・Immunoblotpatternsofdystrophin−DAPcomplexseparatedby2−dimensionalPAGE
Withvariousantibodies・Fromthetoppanel，1，10，4and12LLgOfsampleswereloadedonthe
gels・Atthelstelectrofocuslng，Sampleswereloadedfromthebasicsideexceptthebottompanel，
inwhichβ2−SyntrOphinwasfocusedonlywhenthesamplewasloadedfromtheacidicside．
AntibodiesusedarethesameaSthoseinFig．1．

ー39−

agalnStαreactswiththeacidicseriesofspots．Ontheotherhand，ab1351reactedwithbothacidicandbasicseriesofspotsinagreementwiththerpsultshowninFig．1．Wecould．detect
β2−SyntrOPhinasabよsicseriesofspotssimilartothatofβ1−SyntrOPhin．Bytheseblots，
however，WeCOuldnotclearlyshowthedifftrencebetweenbothbasicsyntrophins．Thenwe
attemptedtoanalyzethemonasheetofblotbythedoublestalnlngmethod，andwefoundthat
theaveragepositionofP2−SyntrOPhinspotswasslightlymorebasicthanthatofP1−SyntrOPhin（datanotshown）［8］．

Here，WeShowedbymolecularclonlngthatthereareatleastthreekindsofsyntrophins

andidentifiedthemontheproteinlevelinrabbitskeletalmuscle．Wedeteminedthegene
locations ofhumanβ1−，β2−andα−SyntrOPhins as8q23−24，16q23−24and20qll．2，
respectivelyl7］．Theselocationsexcludesyntrophinsffomthehumanneuromusculardiseases

Withaknownposition．
Curiously，eaChsyntrophinwasshowntobestillcomposedofmanyspotswhichwere
SeParatedby2−dimensionalPAGE・Apartofthespotscanbeexplainedbyphosphorylation，
becausetheydisappearedbythealkiliphosphatasetreatmentofdystrophin−DAPcomplex【3］．

0therspotsmaysuggestthepresenceofisofbrmsderivedfromthealternativelyspliced

mRNAs，becauseseveralsizesofmRNAsareobservedbyNorthemanalysesofsyntrophinsl4，7］andbecaus？thegenestruCtureSOfmousesyntrophinscomposedofseveralexonswere
recentlyreportedl9］

Wehaveshownelsewherethatα−andβ−isofbrmsofsyntrophinstandemlyassociatedin
thisorderwithdystrophinattheverylimitedareaintheneighborhoodoftheC−teminal［7，11，

12］・Wehavealsoshownthata90−kDaDAP，AO，alsoassociateswithdystrophininthesite

Whereβ−isoformsassociate（β−Site）［11］．Sinceitmightbedifficultfortheseproteinsto
associatewithdystrophininthissiteatthesametime，WeatPreSentSPeCulatethatβ−SyntrOPhins（andAO）mayalternatelyassociatewithdystrophinintheP−Site．Recently，itis
reportedbyimmunohistochemistrythatα−SyntrOPhinisdetectedonthecellsurfacemembrane

includingtheneuromuscularjunction（NMJ）ofratskeletalmuscle，butβ2−SyntrOPhinisonly−40−detectedatNMJl10］．Thisresultmaysuggestthatβ−SiteofdystrophinatNMJisoccupied
Withβ2−SyntrOPhin．WeattemptedtoexaminethesubcellularlocalizationofP1−SyntrOPhin，but
Ourantibodywasnotusefu1forhistochemistry．Sincethesubcellularlocalizationofeach
isofbrmofsyntrophinoverdevelopmentmayleadtoabetterappreciationofthefunctionof
SyntrOPhins，gOOdantibodiesagainstsyntrophinsincluding

β1

aredesired．

Rereren（：eSll］Yoshida，M．andOzawa，E．（1990）J．Biochem．108，748−752［2］ErvaSti，J・M・，Ohlendieck，K．，Kahl，S．D．，Gaver，M．G．，andCampbell，K．P．（1990）∧b〃re345，315−319［3］Yamamoto，H．，Hagiwara，Y．，Mizuno，Y．，Yoshida，M．，andOzawa，E．（1993）J．

βわCゐem．114，132−139［4】Ahn，H．A．，Yoshida，M．，Anderson，M．S．，Feener，C．A．，Selig，S．，Hagiwara，Y．，

Ozawa，E．，andKunkel，L．M．（1994）Proc．Natl．AcadSci．USA．91，4446−4450．
［5］Adams，M．E．，Butler，M．H．，Dwyer，T．M．，Peters，M．F．，Mumane，A．A．，and

Froehner，S．C．（1993）Neuron．11，531−540．
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（1995）J．Biol．Chem．inpressl8］Yoshida，M．，Yamamoto，H．，Noguchi，S．，Mizuno，Y．，Hagiwara，Y．，andOzawa，E・（1995）Fe如上e比367，31ト314［9］Adams，M．E．，Dwyer，T．M．，Dowler，L．L．，White，R．A．，andFroehner，S．C．（1995）J・

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SARCOGLYCANOPATHY HYPOTHESIS
EijiroOzawa，YujiMizun0，SatoruNoguchiandMikiharu
YoshidaNationalInstituteofNeuroscience，NCNP，Kodaira，Tokyo187Japan

Recently，a

neW

WOrd

SarCOglycanopathyhasbeenintroducedtorepresentagroupoflimb−girdlemusculardystrophyintheplaceof severe
childhoodautosomalrecessivemusculardystrophy，namelySCARMD（l）・PresentthemesarehowthesarCOglycanOPathyconcq）thasbeenfbrmed
andwhatproblemsarelefttobesoIved．

1．Dystrophin−aSSOCiatedproteins
Sincewddiscoveredthatdystrophinispresentinassociationwithmuscle
membranein1988（2），Wehavestudiedtheproblemhowdystrophinmoleculeis
PreSentinsubsarCOlemmalundercoat of cytoskeleton．Duringthe study ofthissubiect，neWPrOblemswereraisedconcernlngthemoleculararChitectureofdystrophinanddystrophin−aSSOCiatedproteinsorbrieflyDAPs（3）・DAPswereassortedalmostsimultaneOuSlybyCappbell

sgroupandour

groupin1990andeachgroupgavethedifftrentnameStODAPs（4・5）・Onthis
OCCaSion，itisnecessarytOemPhasizethedif托renceinthenameSOfthe43kD
PrOteinwhichlooked doubletsin1−dimensional gel electrophoresis．

CamPbellIsgroupreportedthatthedoubletsareimmunOlogicallythesame（6）・Andtheynamedit43DAG．
Ontheotherhand，WeCOnSideredthatthedoubletsrepresentdifferent
twoproteins，andthennamedA3aandA3b，reSPeCtively．
Later，WefbundthatthesetwoareCOmPletelydifferentproteinS．They

differinimmunOlogicalreactivities，isoelectricpoints，PePtidemaps，COmPlex
formationpatternsand tissue distribution patterns（7）・Recently，they areShowntObedifferentatthecDNAlevel．Andnow，A3aandA3bareCalled
β−dystroglycanandβ−SarCOglycan，reSPeCtively．

WhendystrophinandDAPcomplexextractedftommusclemembrane
fractionby digitonin−treatment，arefurther treatedwithocty1−glucosideand−43−fractionatedwithgelfiltrationcolurrm，Wefbundthatthecomplexisseparated
into roughly three fractions（7）・The void fraction contained dystrophinbinding with syntrophin group．

The firstfraction was composed ofdystroglycanCOmPlex，namely，α−andβ−dystroglycanS．Thesecondfraction

COntained50DAG（adhalin），A3band35DAG．L Sincetheyare foundto be
PreSent aS a COmPlex，We named this complex sarCOglycan

COmPlex（7）・

Iもwever，thereareStillsomeotherproteinstobeclassifiedsuchas25DAPas
membersofsarCOglycanCOmPlex．25DAPisconsideredasatranSmembrane
PrOteinandpresentveryclosetoeitherdystroglycancomplexor sarCOglycan
COmPlex．

Takingtheexperimentalresultsmainlyofourgroupandalsoafewof
Othergroupsintoconsideration，WeCOnStruCtedamodelhowdystrophinand
DAPsareassociatedwithmusclemembrane・Fig・1showsourupdatedmodel・
DystrophinbindsactinfilamentSatitsN−terminus（9），andbindsatitscarboxyl
Side ofthemolecule withβ−dystroglycan（10），Whichinturnbinds withα−dystroglycan（7）・α−dystroglycanisaneXtraCellularPrOteinwhichbindswith

laminin（6），One Of the maincomponents of basallamina．Thus，these
COmPOnentS COnStruCt

anaXis connectlng the extracellularmatrixandSubsarcolemmalcytoskeletonnetwork（1）・
SarCOglycancomplex，Ontheotherhand，doesnotseemtoserveasthe
memberofthecornectingaxis（1）・Presumably，SarCOglycanCOmplexplaysa
roleotherthantheconnectingaxis．
Concernlng SyntrOPhin group，theprevious q）eaker，Dr・Yoshida has
reviewedthepresentstatusoftheresearch．
ThenomenclatureofthesarCOglycancomplexsofarkn0wniscompared・
Thiscomplexiscomposedofatleast3proteins・Theyareadhalin，A3band
35DAG（7）・Recently，allthe cDNAsare Clonedandthese proteinSare
renamedtobeα一，β−andY−SarCOglycanS（S・11・12）．Theorderofα，βandγisthedecreaslngOrderofmolecularmaSSOftheproteinsandatthesametime
theorderofclonlngOfcDNA．
2・SCARMDanditsresponsiblegenes
SCARMDisagroup ofthemuscle dystrophies dueto the deftct of
SarCOglycancomplex（13）・AlthoughthisnamebearSSeVereandchildhood，thisdiseaseisnow considerednotnecessarilytobe severeand restrictedto theー44−Patientsin childhood，andis sometimes called Duchenne−like autosomal
recessivemusculardystrophy．

In1992，Matsumuraetal．（14）ShowedthatinSCARMDmuscle，50DAG
Or adhalinismisslng．Thiswasthefirstindicationofthelossofacertain
PrOteininthe SCARMD muscle・This gave strongimpression to theresearchersthatSCARMDisadiseaseduetothedefectofadhalin．
Atthesameyear，namelyin1992，BenOthmaneetal．（15）dlOWedthat

TunisianSCARMDislinkedto13q12．Butinthenextyear，Passos−Buenoet
al・（16）inSanPauloshowedthatBrazilianSCARMDisnotlinkedto13q・And
theyclaimedthatSCARMDisnotasinglediseasebutisagroupofdiseases
Whicharegeneticallyheterogeneous・

In1994，WefbunduslnglmmunOlogicalstainingofSCARMDmuscles
WhatislackinginSCARMDmuscleisnotconfinedtoadhalin，butA3band
35DAGarealsolacking・Likeothergroups，WeCOnfirmedothercomponents
SuChasdystrophin，dystroglycanCOmplexetc．arenOrmalinSCARMDmuscle．
This showed that thewhole members of sarcoglycanCOmPlexarelostin

SCARMDmuscle，althoughother componentsareintact（17）．BasedonthesefindingsaswellasthefindingthatSCARMDisgeneticallyheterogeneous，We
PrOPOSed ahypothesisthat SCARMDis causedby theloss of sarCOglycan
complex（17）・

Thehypothesisisas fbllows：First，Whenany OneOfthe sarCOglycan
geneisdefective，thismayresultinthelossoftheproteinproductofthegene・Second，WhentheproteinismisslngWhichisthegeneproduct，this causes
failurFinformlngthe．complexandtheincompletecomplexisnotsufficientenoughtobefixedtothesarCOlemma・Andlossofthewholecomplexensues・
Third，thelossofthecomplexcausestheSCARMDphenotype．Therefore，
genedefectofanyOneCOmPOnentOfsarCOglycanCOmPlex causesthesame
disease・ThismaybethereasonofgeneticheterogeneityofSCARMD．

Whenweproposedthishypothesis，nOSarCOglycangenehadbeenkn0wn
toberesponsibletoSCARMD．
Assoonasourpaperpresentingthishypothesiswasaccepted，Roberds
etal・（11）reportedclonlngOfhumanadhalincDNA・Itismqppedat17q21andthegeneisresponsiblefbrSCARMD・Theyshowedasinglebasereplacement

inthe each allele of adhalin gene of SCjuMD patientlinked to17q・
Thereafter，Onthesebasis，thewordadhalinopathywaspopularlyused・Some

ー45−

PeOPleextendingadhalinopathy

the

usage

of

this

word

by

uslngtheword

secondary

fbrSCARMDwhichwerenotlinkedtotheadhalingene・

Our next problem was whether the other sarCOglycan

geneS

are

responsible fbr SCARMD or not．It wasinFebruary Of1995，When weStartedtheexperimentstoclonethecDNAsofA3band35DAG．Atthattime，adhalinhadbeenshowntOberesponsiblefor SCARMD，andclassifiedtobe
LGMD2D（11・18）・SCARMDlinkedto13q12wasclassifiedtobeLGMD2C
butitsresponsiblegenewasnotknown・
Bythe beginnlng OfJune1995，We SuCCeededto clone A3b cDNA
togethermainlywith

Kunkel−sgroupin

BostonandHoffmansgroupinPittsburgh・UsingourdataonpartialpolypeptidesequencesofA3b，Kunkel−sgroup9loned thecDNAand determinedgenelocusto be at4q12・And
bffman一sgroupfoundmutations

on

A3b

gene

ofSCARMD

patient・We

furtherexaminedA3bin4q12−1inkedgenedefectmuscleatproteinleveleither

biochemicallyandimmunOhistochemically．Theseresultswerepublishedin
theNovemberissueofNature￣Geneticsin1995（8）．

Since Campbell−s group also obtained the same reSults，their paper
appearedintandemwithours（19）・Onthepublication，thenameOftheprotein
A3bwasdeterminedtobeβ−SarCOglycanbytheproposalofLouKunkeland
Withtheagreement ofrelatedresearChers．Atthesametime，thename Of
adhalinwaschangedtoα−SarCOglycan．

Untilthe end ofJune，Noguchisucceeded to clone rabbit cDNA of
35DAGuslng OurmOnOClonalantibodyasaprobe．Thenourgroup cloned
humangeneandfoundthatthemRNAisexpressedexclusivelyintheskeletal
andcardiacmuscles，namelystriatedmuscles，aSWehadpreviouslyshownby
immunoblotanalysis．KunkelTsgroupmappedthegeneat13q12andfbund

themutations onthe gene ofthe SCjuMD patientslinked to13q12．We
renamed35DAGtobeY−SarCOglycan．ThiswaspublishedinNovember3rd
issueofSciencelastyeaf（12）・
Inbothβ−andY−SarCOglycangenemutations，themuscleswerefoundto
beal，SentfromwholesarCOglycan complexasour hypothesis（17）predicted・

Therefbre，WeCOnSiderthatSCAMcanbecalledsarcoglycanOPathy．
3．0nnewclassificationofLGMD
Ithasbeendescribedthatlimb一girdlemusculardystrophiesareClassified

ー46−

On

the genetical point of view．

According to this classification，Y−

SarCOglycanopathyisLGMD2C，α−SarCOglycanOPathyisLGMD2Dandβ−
SarCOglycanopathyshouldbe2E．
Thereis，however，aPrOblem whether thereare Stillsome types oflimb−girdle autosomal recessive muscular

dystrophy to be classified as

SarCOglycanOpathy．HoffmannTsgroupfoundthatthereisatleastonemore
COmPOnentOfthecomplex、tObecalled8−SarCOglycanandthedystrophywhose
responsiblegeneis8−SarCOglycangene，namely，8−SarCOglycanOPathy．
In the above−mentioned classification oflimb−girdle muscular
dystrophy（18），itistheruleto glVethe succeeding alphabetto the newly
discovered gene mutation．Therefore，the next autosomal recessivelimb−
girdle should be

2F．On the other hand，there

are Still some other

SarCOglycanOPathyandtheremaybesomeadditionalautosomalrecessivelimb−
girdlemusculardystrophieswhichdonotbelongtosarCOglycanopathy．Ifwe

COntinueto use theruleofthepresent classification，itispossiblethatthe
Phenotypically different muscular

dystrophies will bearrangedintheintermingledmanner・WecannOtPredictthatthenextmemberoflimb−girdle
belongstosarcoglycanOPathyornot．
Therefbre，thefbllowlngPrOblemshouldbeseriouslyconsideredthat
thesarCOglycanOPathyisclassifiedseparatelyfromtheotherautosomal
recessivelimb−girdlemusculardystrophiesandbeglVenthenumber3．In
thiscontext，α−，β−andY−SarCOglycanOPathyshouldbecalledLGMD3A，3B

and3C，reSpeCtively・Inthiswaytheclassincationmaybecomeclearerandmoreusefu1．

−47−

−48−

Referencesl・OzawaE・et

al・‥Dystrophin−aSSOCiatedproteinsinmusculardystrophy・

Hum．Mol．Genet．4：1711−1716（1995）
2・ArahataK・etal・：Immunostainingofskeletalandcardiacmusclesurface
membraneWithantibodyagainstDuchermemusculardystrophypeptide・
Nahre333：861−863（1988）
3・TinsleyJ・M・etal・：IncreaslngCOmPlexity ofthedystrophin−aSSOCiatedPrOteincomplex．Proc．Natl．Acad．Sci．USA91：8307−8313（1994）
4・ErvastiJ・M・et al・：Deficiency of a glycoprotein component of thedystrophincomplexindystrophicmuscle．Nature345：315−319（1990）
5・YoshidaM・andOzawaE・‥GlycoproteincomplexanChoringdystrophinto

SarCOlemma・J・Biochem．1P8：748−752（1990）
6・Ibraghimov−Beskrovnaya

O・etal・‥Prlmary

StruCtureOf

dystrophin−associated glycoproteinslinking dystrophin tothe extracellularmatrix．
Nature355：696−702（1992）
7・Yoshida M・et al・：Dissociation of the complex of dystrophinandits
associatedproteinsintoseveraluniquegroupsbyn−OCtylβ−D−glycoside．
Eur．J．Biochem．222：1055−1061（1994）
8・B6nnemannC・G・etal・：β−SarCOglycan（A39）mutationscauseautosomal
recessivemusculardystrophywithlossofthesarCOglycanCOmPlex・Nature
Genet．11：266−272（1995）
9・WinderS・J・etal・‥Dystrophinandutrophin：themisslnglinks！FEBSLett・

369：27−33（1995）

10・SuzukiA・et al・：MolecularOrganization at the glycoprotein−COmPlex−bindingsiteofdystrophin．Eur．J．Biochem．220：283−292（1994）
11・RoberdsS・L・et al・：Missense mutationsin theadhalin genelinked toautosomalrecessivemusculardystrophy．Cel178：625−633（1994）

12・Noguchi S・et al・：Mutationsinthe dystrophin−aSSOCiated protein−Y−
SarCOglycaninchromosome13musculardystrophy・Science270‥819−822（1995）
13・H狐lidaM・B・etal・‥Severechildhoodmusculardystrophyaffectingboth

SeXeSandfrequentinTunisia．MuscleNerve6：469−480（1983）
14・Matsumura

K・et

al・‥

Deficiency

of

the

50K

dystrophin−aSSOCiatedglycoproteininseverechildhoodautosomalrecessivemusculardystrophy・Nahre359：320−322（1992）ー49−

15．OthmaneK．B・etal・：LinkageofTunisianautOSOmalrecessiveDucherLne−
likemusculardystrophytothepericentromericreglOnOfchromosome13q・
NatureGenet．2：315−317（1992）
16．Passos−Bueno M．R．et al．：Genetic heterogeneity

for Duchenne−likemusculardystrophy（DLMD）based onlinkageand50DAG analysis・
Hum．Mol．Genet．2：1945−1947（1993）
17．MizunO Y．et al．：Selective defect of complex

inSevere childhood

autosomalrecessivemusculardystrophymuscle・Biochem・Biophys・Res・

Comn．203：979−983（1994）
18・BushbyK・M・D・etal・‥Thelimb−girdlemusculardystrophies−PrOPOSalfor

anewnomenclature．Neuromusc．Disord．5：337−343（1995）
19．LimL．E．etal．：β−SarCOglycan：CharaCterizationandroleinlimb−girdle
musculardystrophylinkedto4q12．NatureGenet・11：257−265（1995）−50−

β−andY・SarCOgIycan

SatoruNoguchi＊，CarstenG．B6nnemann‡，ElizabethM．McNally‡，RqjuModiS，KamelBenOthmaneⅧ，YltjiMizuno＊，YasukoHagiwara＊，MikiharuYoshida＊，
HidekoYamamoto＊，EmanuelaGussoni‡，DavidDuggan§，CorradoAngelini＃，PeterH．DentonⅧ，TheodorosKyriakides†，LefkosMiddleton†，FaycalHentatiO，MorjiBenHamida◇，IkuyaNonaka＊，JefftryM．VanceⅧ，EricP．Hoffman§，LouisM．Kunkel‡，EiiiroOzawa＊（＊NationalInstituteofNeuroscience，‡BostonChildren・sHospital，§UniversltyOfPittsburghSchoolofMedicine，ⅦDuke
University MedicalCenter，＃Universita diPadova，†CyprusInstitute of
NeurologyandGenetics，◇InstituteofNeurology，Tunisia）

Dystrophin−aSSOCiated glycoproteins are clarifiedinto two groups，
dystroglycancomplexandsarcoglycancomplex，Onthebasis ofbiochemical
PrOPertiesandthetissuedistributionoftheseproteins．SarCOglycancomplexis
COmPOSedof50−kDaα一SarCOglycan（adhalin），43−kDaβ−SarCOglycan（A3b）and

35−kDaY−SarCOglycan（35DAG）．Sarcoglycancomponentswereshowntobe
Selectivelydeficientinthemusclesofpatientsofseverechildhoodautosomal

recessive muscular dystrophy（SCARMD）．Based on these findings，We
POStulatedthesarcoglycanopathyhypothesisthat SCARMDisadiseasecaused
bytheprlmarydeftct ofoneofthesarcoglycangenesl・Adeftctofanyoneof
the sarcoglycan component causes the secondary destabilization of the
SarCOglycancomplexresultinginthecommonhistologicalphenotypeoftheloss
Ofawholesarcoglycancomplex．Recentlyα−SarCOglycanwas showntobe

prlmarily responsible to SCARMD2・Therefore the othercomponents of
SarCOglycancomplex，β−andY−SarCOglycan，WereCandidatesfortheresponsible
genesfbrSCARMD．
NowweisolatedrabbitandhumancDNAsencodingβ−SarCOglycanwith
an oligonucleotide derivedfrom a part of a determinedinternalpeptide
ー51−sequencefromβ−SarCOglycan3．β−Sarcoglycanwas318aminoacidresiduesandthismolecularmassiswell−COnSistentwithapparentmolecularmassfbrthe
glycopeptidase−Fdigestedβ−SarCOglycanontheSDS−PAGE・Rabbitandhuman
γSarCOglycancDNAswereisolatedwithaspecificmonoclonalantibody4・Y−

Sarcoglycanwas291residues・Thesesequenceswerewell−COnServedbetween
humanandrabbitwith97％identityforβ−SarCOglycanand87％identityfbrY−
SarCOglycan，reSPeCtively・BothproteinsarenoveltypeIIsingletransmembrane
PrOteinsthatcontainlargeextracellulardomainatC−terminalsideandsmall
intracellular domain at N−terminal side．There are three and one conserved
potentialasparagine−linkedglycosylationsitesinC−teminalregionofβ−andY−
sarcoglycan，reSPeCtively．The sequence homology betweenβ−and†−
SarCOglycanwasnotdetectedbuttheseproteinshadsomecharacteristiccommon
struCtureS，Whichareapotentialphosphorylationsiteinintracellulardomainanda cluster offour conserved cysteine residuesin the distalportions ofthe
extracellularC−termini．Furthermoretheputativesecondarystructuresofthese
PrOteins were similar to each other as showninFigure・The extracellulardomainsofallthreecomponentsarerichintheβ−turnStruCture・Acoupleof
α−helicesiscommonlypresentinthecenteroftheextracellulardomainsinthree
PrOteins．
TheNorthemanalysisshowedthatmRNAofβ一SarCOglycanisexpressedatthehighestlevelinskeletalmuscleandheartandatthesubstantiallylower
levelinbrain，kidney，Placenta，PanCreaS andlung・Ontheotherhand，theexpressionofmRNAofY−SarCOglycanwasconnnedinskeletalmuscleandheart
WhichisconsistentwithourpreviousdataoftheimmunochemicalsurveyOfthe
Y−SarCOglycanproteininprlmatetissues．
Thehumangenesforβ−andY−SarCOglycanweremappedtochromosome
4q12and13q12，reSPeCtively．Thissamereg10n，13q12wasimplicatedinNorth
AfricanSCARMDfamiliesbylinkageanalysis．Anotherreg10n，4q12didn

−52−

t丘t

thelocusreportedtorelatetomusculardystrophiesfbrwhichachromosomal
asslgnmentisknownwithoutadefinedprimarygeneticdeftct．

α・SarCOglycan

β−SarCOglycan

γSarCOglycan

V 崇：ニCOSidesugars「慧㌘？霊SPho ration ≡≡言霊：ta。州。
Figure・ThepredictedstruCtureSOfthesarcoglycancomponents・
ThesecondarystruCtureSWerePredictedbythemethodof P．Y．ChouandG．D．Fasman（1978）・ThepredictedstruCtureSOfthethreecomponentsaresimilartoeachother．They
haveslngletransmembranedomains，andlargerextracellularandsmallerintracellulardomains・Fiveconservedcysteineresiduesarepresentintheextracellulardomainsofall
thecomPOnentS・Themutationpositionsidentifiedintheβ−andY−SarCOglycangenesin
SCARMDpatientsareshownbyarrows．

・Thereforewetriedtonndoutthemusculardystrophypatientswiththemutationofβ−SarCOglycangene．Weselected62musclebiopsiesinwhichto
analyzeβqsarcoglycanmRNAfbrmutations．Thesebiopsieswerechosenfrom
amuscle biopsybankofover2，400patients，andmetthe criteria：1）H＆E

Stalnlng Ofcryosections showlng histopathologicalftatures consistent withmuscular dystrophy；2）normaldystrophin molecular mass and amount；3）
VariableimmunostalnlngPatternSOftheeitherdystrophin，adhalinofmerosin

ー53−oncryosection；and4）serumCPKvaluesinexcessof1，000IU／1・Forinitial
screenlng，WePerformedSSCPanalysisontheRT−PCRproductamplifiedfrom

RNAisolatedfromeachofthe62biopsleS・Wefbundasinglepatientwithgives
fouraberrantconformers．Inoneconformer−Palr，aTtoGconversionwas
identified which resultedin a nonsense codon（Y184X）．In the second
confbrmer−Palr，an8basepairduplicationafteraminoacidposition125wasfoundwhichcausedaframeshiftandanimmediatestop−COdonatnucleotide
129．Themutationswereconnrmedby PCRanalysisofgenomicDNAfrom
the patient andbothparents・Analysis ofgenomic DNAfrom theparents
showedthepatient■smothertobeheterozygousfbrtheTtoGconversionand

fathertobeheterozygousfortheduplication，COnfirmlngautOSOmalrecessive
inheritance ofthismusculardystrophy・Theseprematurestopmutationsare
predictedto severelytruncatetheprotein，ablating mostofitsextracellulardomain，includingthetwoorthreeasparaglneglycosylationsitesandthecluster
OfcysteineresiduesinC−terminal（SeeFigure）・

Theminimalcandidatereg10nforSCARMDwasrecentlyrefinedtoa
smallreg10nSurrOundingD13S232andlinkagedisequilibriumwiththemarker
D13S232suggests thatboth alleles oftheresponsiblegeneshouldcarryan
identicalmutationin North african families．To determine the homozygous
mutationresponsiblefbrSCARMD，WePreParedRNAfromamusclebiopsy
taken from an affected Tunisian SCARMD patient whose familyis
consangulneOuSanddemonstrateslinkagedisequilibriumwithD13S232and
perfbrmedPCRforY−SarCOglycangeneR）llowedbydirectsequenclng・Oneof
thethymineresiduesfromnucleotides645to649washomozygouslydeletedin
thepatient・ThisdeletionwasconnrmedinthegenomicDNAfromthispatient・Thismutationwasalsofoundinanaffectedsiblinglnthisfamilyandintwo
additionalTunisianSCARMDfamilies．Thedeletionofonethyminechanged
thereadingframe afteramino−aCid175，CreatlngaPremature StOPCOdon atamino acid193．This aberrant Y−SarCOglycan would retain theits−54−

transmembraneanchoranditsasparaglne−linkedglycosylationsitebutlosethe
Clusterofcysteineresiduespresentinthedistalportionoftheproteinasshown
inFigure・ToascertainwhetherY−SarCOglycanmutationscontributetosporadic

CaSeSOfSCARMD，WeanalyzedY−SarCOglycancDNAfrOmmusclebiopsleSOf
fburJapanesepatientswhosemuscledisplayeddystrophicarchitectureonH＆E
Stalnlng，deficiencyofsarcoglycancomponents，andpreservationofnormal
dystrophinstalnlng・SSCPanalysisofthereglOn丘omnucleotide516to8470f
theY−SarCOglycangeneshowedaslnglepatientgivestwoaberrantconfbrmerS
andaconfbrmerbandwassequenced・Itcorrespondedtoadeletionof73base
PalrS，PrOducingaprematurestopcodonataminoacid1700ftheaberrantY−
SarCOglycan・Inthiscase，theC−terminalonethirdoftheY−SarCOglycanprotein
WaSlost，includingthedistalcysteinecluster（seeFigure）．
Theimmunostainingpatterns showedthe common phenotype that
SarCOglycan complexis selectivelylost from muscle membrane．
Immunostainingofthemusclebiopsyofthepatientswithafftctedβ−SarCOglycan
gene demonstratedaprimary deficiency ofP−SarCOglycan and secondary
deficiencies ofα−andY−SarCOglycanin contrast to the preservation ofdystrophin，β−dystroglycanandmerosinstaining・Immunostainingof13q12−
1inkedpatientsmuscleshowedα−andβ−SarCOglycandeficienciesbesidethe
denciencyofY−SarCOglycan・Thissuggeststhatonecomponentofsarcoglycan
COmPlexis required for the stabilization and／Orlocalization of two other
COmPOnentS，therebymaintalnlngtheintegntyofthesarcoglycancomplexinthe
membrane．

Theseresultsshowthatβ−andY−SarCOglycanareresponsibletoSCARMD
aswellasα−SarCOglycan・Themutationsinβ−andγ−SarCOglycans notonly
affectedtheprlmarily−mutatedproteinsbutalsodisrupttheintegrltyOfthe

entiresarcoglycancomplex・Thesefactsfurthersuppoftsourhypothesis・We
PrOPOSedthenameHsarcoglycanopathyMasacollectivenameforthesetypesof
thedisease calledasSCARMD−55−

【Re鮎rences］

1．E．Ozawa，M．Yoshida，A．Suzuki，Y．Mizuno，Y．Hagiwara，andS．Noguchi：
Dystrophin−aSSOCiatedproteinsinmusculardystrophy・HumanMolecular
Genetics，4，1711−1716，1995

2．S．L．Roberds，F．Leturcq，V．Allamand，F・Piccolo，M・Jeanplerre，R・D・Anderson，L．E．Lim，J．C．Lee，F．M．S．Tome，N．B．Romero，M．Fardeau，
J．S．Beckmann，J．−C．Kaplan，andK．P．Campbell：Missensemutaationsin
theadhalingenelinkedtoautosomalrecessivemusculardystrophy・Cel178，625−633，1994

3．C．G．B6nnemann，R．Modi，S．Noguchi，Y．Mizuno，M．Yoshida，E・Gussoni，

E．M．McNally，D．J．Duggan，C．Angelini，E．P．Hofhan，E・Ozawa，andL・
M．Kunkel：Dystrophin−aSSOCiatedproteinβ−SarCOglycan（A3b）mutations
CauSe autOSOmalrecessive muscular dystrophy with disintegration ofthe

SarCOglycancomplex．NatureGeneticsll，266−273，1995

4．S．Noguchi，E．M．McNally，K．BenOthmane，Y．Hagiwara，Y・Mizuno，M・
Yoshida，H．Yamamoto，C．G．B6nnemann，E．Gussoni，P．H．Denton，T．
Kyriakides，L．Middleton，F．Hentati，M．Ben Hamida，I・，Nonaka，J・M・Vance，L．M．Kunkel，andE．Ozawa：Mutationinthedystrophin−aSSOCiatedPrOteinY−SarCOglycaninchromosome13musculardystrophy・Science270，819−822，1995−56−

Fromadhalinopathiesto
SarCOglycanopathies
M・Jeanpierrel，A．Carriel，F．Piccolol，F．Leturcql，C．deTomal，
K・AzibiZ，C．Beldjordl，L．Merlini3，T．Voit4，N．Romero5，Y．Sunada7，
F．M．S．Tom66，M．Fardeau6，K．P．CamPbe117，J．｛．Kaplan1
1I・aboratoiredeBiochimieetGen6tiqueMo16culaires，PavillonCassini
123，130ulevarddePort−Royal，Paris，France，2cHUBologhine，Algiers，Algeria，3Instit・OrtopedicoRizzoli，130logna，Italy，4UmiV・−Knder妃imik，

Essen，Gemany，5Hop・R・Debre，Paris，France，6INSEM153，Paris，France，
7Univ．ofIowaColl．Med，IowaCity，USA

Dys仕OPhin，thedefectivegeneproductinDuchenneandBeckermuscular
dystrophy，is associated with a complex of sarcolemmal glycoproteins
believedtolinkthecytoskeletonofmusclefiberstolamlnln，amaJOrCOmPOnent
oftheextracellularmatriⅩ1．2．

It has been suggested that deficienciesin any component of thetransmembranelinker may disrupt thelink between the cytoskeleton andextracellularmatrixandleadtoassociatedexerciseinducedmusclenecrosis34．
Thelargeoligomericcomplexwithdystrophiniscomprisedofthreesub−
FOmPlexes5・

1）Dystroglycancomplex
α−dystroglycan（156DAGorAO）

β−dystroglycan（43DAGorA3a）2）Sarcoglycancomplex
α−SarCOglycan（Adhalinor50DAG＿OrA2）β−SarCOglycan（43DAGorA3b）
Y−SarCOglycan（35DAGorA4）
3）Syntrophincomplex
α−SyntrOPhin（59DAPorAl）

β1−SyntrOPhin（59DAPorAl）
β2−SyntrOPhin（59DAPorAl）−57−

Anadditionalsarcolemmalprotein（25DAPorA5）has beenshownto
COPurifywiththesecomplexesbuthasnotbeenextensivelystudied・Thethreecomponents ofthe sarcogbcan complex are muscle−SPeCi五C

glycoproteinsunliketheubiquitousdystroglycancomplex6B．
A dramatic reduction of cL−SarCOglycan（Adhalin or

50DAG）wasObservedin patients affected with autosomal recessive muscular dystrophy（SCARMD）9￣10，Withsomereductionoftheothermembersofthesarcoglycan

COmPlex．Actuallythediseaseisphenotypicallyandgenotypicallyheterogenous，Sincein some cases the（α一SarCOglycan（adhalin）geneitselfis mutated（LGMD2D at17q21．2）11，12，Whereasin other cases the adhalinopathyisSeCOndary，themorbidlocusbeingeithertheβ−SarCOglycangene（I．GMD2Eat
4q12）13−140r

theY−SarCOglycan（LGMD2C

at13q12）15￣17．The

respective

incidence of the three categoriesis unknown yet．SCARMD wasinitiallydescribedinTumisia18andfurtherreportedinpatientsfromotherNorthAfricanand Middle−Eastern countries．SCARMD standsfor the most severe muscular
dystrophies，andisoftenconsideredastheworstendofacontinuumratherthan
adiscreteentity，distinct丘omthemilderforms19・

Wepresentaseriesof32unrelatedfamilieswithmutationsinthealpha−
SarCOglycan

gene，therefore

qualifyingas〟prlmary

adhalinopathies〃・They

WerefoundafterprlmaryDGGEscreenlngOfgenomicDNAfromlOOunrelated
Patients with proximal muscle dystrophy・In our sampleα−SarCOglycano−Pathies accountfor about50％ofadhalin deficient patients・We fbund22
difkrentmutations（2nonsens，3frarrKShiftinsertionsordeletions，4splicemutatio．ns，13missense）．Allthemissensemutationsareintheextracellulardomain，andwerefoundin2nofthemutatedchromosomes．

Fivemissensemutations（40fwhichinvoIvingaq】Gdinucleotide）were
foundmorethanoncewithastrikingprevalenceoftheArgC77）Cysmutation（30％ofthemutatedchromosomes），andoftheArg（284）Cysmutation（13％of
the mutated chromosomes）．The recurrent mutations are carried byunrelatedsubjectsondifferenthaplotypebackgrounds・ThedinucleotideCpG
isa

hotspot

formutationinthehumangenomeasaresultofthemodi丘cation

Ofthe5．cytosinebycellularDNAmethyltransferasesandtheconsequenthighfrequency of spontaneous deamination of

5−methyl cytosine（5mC）tothymidine20．TheArg（77）CysmutationhasalsobeendescribedinJapan21andBrazi122．TheArg（98）Hishasbeen alsoidentifiedinanAfro−American girl−58−

with childhood−OnSet muSCular dystrophy23・This girlwas a compound
heterozygote，andtheothermutationwasArg（98）Cysthatcouldbeexplaihedbyadistinctmutationofthesameq）Gdinucleotide．
The severity of the phenotype varies greatly．Patients with nullmutationsaremoreseverelyaffected．Abroadrangeofseverityisobservedin
missense mutations・Homozygous patients fbr the Arg（284）Cys mutations
exhibitamilderdiseasethanthosewiththeArg（77）Cysmutation．Verymildlimb−girdlemusculardystrophiescouldbeunderdiagnosedsincesomepatients
COuldonlycomplainofbeingeasilytired，muSCleweaknessbeingnotreadily
evidentuponphysicalexamination・Inthesemildcases，muSClebiopsleSare
not routinely performed and sarcog吋Can deficiencies not diagnosed．The
actualfrequencyof（ユーandY−SarCOglycanmutationscouldthusbehigherthan

estimatedfrominvestigadonofSevereChildhood−OnSetAutosomalRecessive
MuscularDystrophycases23−25
Thewidespread geographic orlgln Ofthe32patientsofthisseries（22from Western Europe，6from North Africa，4from NorthAmerica）
SuggeStSthatα−SarCOglycanopathiesare producedbyworld−Wideindependent

mutations．InNorthrAfticawhereSCARMDhadbeenprlmarilydescribedthis
SeVere Phenotypeis due either toα−

Or tO Y−SarCOglycanopathies・The

PrOPOrtionofsarcoglycandeficiencylnmuSCulardystrophiesvariesfrom2％to12％24−25・Thisvariationcouldbeexplainedby samplebiasorcouldmirror
actualpopulationdifftrencesinmusculardystrophies・Itwillbeinteresting tounderstandtheimpactofthedifferentmissense
mutations on theinteractions of adhalin with the other member of the
SarCOglycancomplex．

Acknowled ements．

ThisworkwasfundedinpartbyAFM（AssociationFraneaisecontreles
Myopathies）．Dr K・P・Campbellis anInvestigator ofthe Howard Hughes
MedicalInstitute．

−59−

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ComplexasaTransmembraneLinkerBetweenLamininandActin．JCbLl

助，1993，722，809−823．
2・Suzuki〉A・）Yoshida，M・，Hayashi，K・〉Mizuno）Y・，Hagiwara，Y・＆Ozawa，
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Dystrophin−ThreeDystrophin−AssociatedProteinsBindDirectlytothe

Carboxy−TerminalPortionofDystrophin・血rJBibcbem，1994，220，283−292．

3・Mizuno，Y・，Yoshida）M・〉Nonaka，I・）Hirai，S・＆Ozawa）E・Expressionof
utrophin（dystrophin−relatedprotein）anddystrophin−aSSOCiatedglycoproteinsinmusclesfrompatientswithDuchennemuscular

dystrophy．血cIeNttve，1994，1ろ206−16．
4・Campbell，K・P・Threemusculardystrophies：lossofcytoskeleton−
extracellularmatrixlinkage．CbLT，1995，8q675−9．
5・Ozawa，E・，Yoshida，M・，Suzuki，A・，Mizuno，Y・，Hagiwara，Y・＆Noguchi，
S・Dystrophin−aSSOCiatedproteinsinmusculardystrophy・HumMblGeLZeち

1995，4，11711−11716．
6・Roberds，S・L・，Anderson，R・D・，IbraghimovbeskrovnayaI0・＆Campbell，
K・P・PrimaryStruCtureandMuscle−Spec漬cExpressionofthe50−kDa
・Dystrophin−AssociatedGlycoprotein（Adhalin）．J点MCnem，1993，268，
23739−23742．

7・Ahn，A・H・〉Yoshida，M・〉Anderson，M・S・，Feener）C・A・，Selig，S・，Hagiwara，Y・，Ozawa，E・＆Kunkel，LM．ClonlngOfhumanbasicAl，adistinct59−
kDadystrophin−aSSOCiatedproteinencodedonchromosome8q23−24・
伽cJ由〟Acad艮ゴUgA，1994，夕J，4446−50．
8・Yamamoto，H・，Mizuno，Y．，Hayashi，K．，Nonaka，Ⅰ．，Yoshida，M．＆Ozawa，

E・Expressionofdystrophin−aSSOCiatedprotein35DAG（A4）and50DAG（A2）iscon貢nedtostriatedmuscles．J月わchem7bkvo，1994，115J62−7．
9・Matsumura，K・，Tomi）F・M・S・，Collin〉H・，Azibi，K・〉Chaouch，M・，Kaplan，
J・−C・，Fardeau，M・＆Campbell，K・P・De頁ciencyofthe50Kdystrophin−associatedglycoproteininseverechildhoodautosomalrecessivemuscular

dystrophy．ぬtzLtt，，1992，359，320−322．

−60−

10・Fardeau，M・，Matsumura，K・，Tome，F・M・S・，Collin，H・，Leturcq，F・，Kaplan，
J・C・＆CamPbell，K・P・De丘ciencyofthe50kDaDystrophinAssociatedGlycoprotein（Adhalin）inSevereAutosomalRecessiveMuscular
DystrophiesinChildrenNativefromEuropeanCountries．CRAcadScl

J丁年7，1993，3柏799−804．
11・Roberds，S・L・，Leturcq，F・，Allamand，V・，Piccolo，F・，Jeanplerre，M・，Anderson，R．D．，Lim，L．E．，Lee，J．C．，Tome，F．M．，Romero，N．B．etal．
Missensemutationsintheadhalingenelinkedtoautosomalrecessive
musculardystrophy．CblLl994，78，625−33．

12・Piccolo，F・，etal・Primaryadhalinopathy：aCOmmOnCauSeOfautosomal
recessivemusculardystrophyofvariableseverity・NbtzLLeGenetics，1995，
Jq243−245．
13・Lim，L・E・，etal・β−SarCOglycan：Characterizationandroleinlimb一girdle

musculardystrophylinkedto4q12・NbtuteGenetics，1995，11，257−285．
14・B6nnemamm，C．G．，etal．β−SarCOglycan（A3b）mutationscauseautosomal
recessivemusculardystrophywithlossofthesarcoglycanCOmPlex・♪加U躇Ge刀e〟cg，1995，〃，266−273．
15・BenOthmane，K・，etal・LinkageofTunisianautOSOmalrecessiveDucheme−
likemusculardystrophytothepericentromericreglOnOfchromosome
13q．♪加日代Gene。cg，1992，2，315−317．
16・Azibi，K・，etal・Severechildhoodautosomalrecessivemusculardystrophy
Withthedeficiencyofthe50kDadystrophin−aSSOCiatedglycoprotein

mapstochromosome−13q12．HumMblGenet1993，2，1423−1428．
17・Noguchi，S・，etal・Mutationsinthedystrophin−aSSOCiatedproteinY−

SarCOglycaninchromosome13musculardystrophy・Sc血ce，1995，270，
819−822．
18．BenHamida，M．，Fardeau，M．＆Attia，N．Severechildhoodmuscular
dystrophyafftctingbothsexesandfrequentinTunisia・MuscleNtTYe，1983，氏469−480．
19・Bushby，K・M・D・＆Becknam，J・S・Thelimb一girdlemusculardystrophies・

Proposalfbranewnomenclature．NtummuscDjAotdi1995，5；337−343．
20・Cooper，D・N・＆Krawczak，M・CytosinmethylationandthefateofCpG
dinucleotidesinvertebretegenome．HumanGenetics，1989，83，181−188．
21・Kawai，H・，Akaike，M・，Endo，T・etal・Homozygousadhalingenemutations
inpatientswithmalignantlimb−girdletype（autosomalrecessiveー61−childhoodpelvic−girdle）muscularduytrophywithadhalindeficiency・J（コ血血Ve銘1995，血pne55．
22．PassosBueno，M．R．，Moreira，E・，Vainzof，M・，maI血edain，J・，Marie，SK・，Pereira，L，Akiyama，J・，Roberds，S・L・，CamPbell，K・P・＆Zatz，M・Acommonmissensemutationintheadhalingeneinthreeunrelated
BrazilianfamilieswitharelativelymildfbrmOfautosomalrecessivelimb−girdlemusculardystrophy・HumatlMblGenet1995，4，1163−1167・
23．り皿ggren，A．，Duggan，D・，McNally，E・，Boylan，K・B・，Gama，C・H・，Kunkel，
L．M．＆Hofhan，E・P・Primaryadhalindenciencyasacauseof

musculardystrophyinpatientswithnormaldystrophin・AmnutDl，1995，3g，367−372．

24・Hayashi，Y・K・，Mizuno，Y・，Yoshida，M・，Nonaka，I・，Ozawa，E・＆Arahata，
K．Thefrequencyofpatientswith50−kddystrophin−aSSOCiatedglycoprotein（50DAGoradhalin）deficiencyinamusculardystrophy

patientpopulationinJapan：irrmnocytochemicalanalysisof50DAG，
43DAG，dystrophin，andutrophin・抽uLDlqgy，1995，
25．Stec，Ⅰ．，Kress，W・，Meng，G・，Mdller，B・，Mdller，C・R・＆Grimm，T・
Estimateofsevereautosomalrecessivelimb−girdlemusculardystrophy
払GMD2C，LGMD2D）amongsporadicmusculardystrophymales‥astudyof415families・Jbumalofmedicalgenetics，1995，32，930−933・ー62−

Identification ofmuscle−SPeCific calpaln
andβ−SarCOglycan genesinprogressive
muscular dystrophies
J・S・Beckmannl，Ⅰ．Richardl，QBrouxl，F．FougerousSel，V．

Allamandl，NChianmilkulchai1，・LE・Lim2，F・Dudos2，N・Bourgl，LBrenguierl，C．Roudautl，Y．Sunada2，J．Meyer2，C．Moomaw3，CSlaughter3，F．M．S．恥1164，nCohenS，CEJackson6，KP．Campbe112
andM．Fardeau4

1G6n6thon／CNRSURA1922，Evry，2HowardHughesMedicalInstitute，
IowaCity，3UniversityofTexas，HowardHuguesMedicalInstitute，Dal1as，
4INSERMU153，Paris，5cEPH−FondationJeanDau＄Set，PariS，6HenryFord

Hospital，Detroit．

Introduction
Limb−girdlemusculardystrophies（LGMD）areagrOuP Ofinherited
neuromusculardiseases presenting clinical heterogeneity．

LGMDs

are

Characterizedbyprogressiveweaknessofthepelvicandshouldergirdlemuscles，
andtheirgeneticetiologyhasyettobeelucidated・Thesedisordersmaybe
inheritedeither asanautOSOmaldominantorrecessivetrait，thelatterbeing
morecommonwithaneStimatedprevalbnceoflO．5（Emery，1991）．Invie示ofthelackofaconsensusonthespecificnosologicaldefinitionofeachentity，itwas
PrOPOSed at the30th−31th ENMC workshop to temporarilylump theseautosomal recessive entities under the common denomination of LGMD2（BushbyandBeckmann，1995）・Thefactthatthisdecisionisfarfromhaving
generatedaunanimoussupportamOngClinicianSinvoIvedinthisarea，illustrates
thecomplexityofthismedicalandscientificproblem・
ThefirstdemonstrationofageneticbasisforanyformofLGMDwas

Obtainedin1991usingbiallelicRFLPmarkers（Becknanneta1．，1991）．This
StudywasperfbrmedinageneticisolatefromtheIsleofLaReunion，localisingbylinkagearecessiveform（LGMD2A，MIMnumber253600）tochromosome
15q・Confirmationofthislocalizationwassubsequentlyreportedinnorthern

IndianaAmish（Youngeta1．，1992）andBrazilian（Passos−Buenoeta1．，1993）

ー63−

Pedigrees・Thelatterandsubsequentworkdemonstratedthattheautosomal
recessive forms（LGMD2）constitute a genetically heterogeneous group
invoIvingfivegenes・Thesewereshowntomaptoeitherchromosomes2p13−
P16（LGMD2B，Badliretal・，1994），13q12（LGMD2C，BenOthmaneetal・，

1992；Azibieta1．，1993；Noguchietal．，1995），15q15．1（LGMD2A，Becknam
eta1．，1991），17q12−q21．33（LGMD2D，Roberdsetal・，1994；McNallyetal・，
1994）and4q12（LinVDuclos／Brouxeta1．，1995；B6mmemannetal・，1995）・The
LGMD2D gene was recentlyidentified：it encodesfor the50kDa adhalin
glycoprotein（Roberdseta1．，1994；McNallyetal・，1994；Picolloetal・，1995），
nowknownasα−SarCOglycan．TheLGMD2Clocuswasalsojustidentified：it

encodesfbrthe35kDaY−SarCOglycan（Noguchieta1．，1995）．BothsarcoglycanS
aremembersofthedystrophinglycoproteincomplex．
OureffbrtsfocussedmainlyontheLGMD2AandLGMD2Eloci・These
genes wereidentified following either a classical positional cloning（fbrLGMD2A）or acombinedpositionalandfunctionalcloning（fbr LGMD2E）
Strategies・ThevariousstepsinvoIvedinthisquestarereviewedhere・

MappingofaChromosome15RegionInvoIvedin
LGMD2A
FouryearSagO，ftwhighlyinfbrmativegeneticmarkerswereavailable．
Fortunately，theGenethonprogramhad startedandhadalreadyproduced a

SCOreOfnew markers（Weissenbachet a1．，1992；Gyapayet a1．，1994）．In
Parallel，We also set to developln a targetted fashion markers from theLGMD2Aregion（Fougerousseeta1．，1994；PereiradeSouzaeta1．，1994）・
Aftergenotyplngthenewlyavailablechromosome15microsatellitemarkers，
twowerefoundtoflankthediseaselocuswithinanintervalthatwasassessed，
basedonsegregationstudiesontheCEPHreftrencefamilies，aSSPannlng7
CentiMorganS．

The screenlng Of the CEPH YAClibraries with theCOrreSPOnding probes allowed theisolation of YACs which were usedin
fluorescence血sltuhybridizationtode丘netheLGMD2Acytogeneticintervalas
15q15．1−15q21．1（Fougerousse et a1．，1994）．Difftrent approaches were

PurSuedfortheestablislmentofthephysicalmapofthisarea．Thisallowed
eventuallytheassemblyofanuninterruptedYACcontigspannlnganeStimatedl0−12megabases（Figurel），WithacurrentaverageSTSresolutionof50kb，Orfbrthe36polymorphicmicrosatellitesonthismap，Of300kb．Twelvegenesand−64一25geneticmarkerswereinitiallypositionedinthiscontig，Whichisconstituted
OfaminimumoflOclones．

PrefdrentialLocalizationofI．GMD2AintheProximalPart

OfalcM15q15．1−q15．31nterval
NewmicrosatellitemafkersdevelopedusingtheYACsftomthe10−12Mb
LGMD2AYACcontigweregenotypedonlarge，COnSangulneOuSLGMD2A
Pedigreesfromdifferentorigins（Allamandeta1．，1995a）．Theidentificationof
recombinationeventsinthesefamiliesallowedtherestrictionoftheLGMD2A
reglOntOaneStimatedl cMinterval，equivalentto q）PrOXimately3−4Mb（Figurel），bracketedbyD15S514andD15S222．Linkagedisequilibriumdata

OngeneticisolatesfromtheIsleofLaReunionandfromtheAmishcommunity
SuggeStedapreferentiallocationoftheLGMD2AgeneintheproximalpartOf
thisreglOn，anareaCOVeredbyYAC774G4．
Furthermore，analysisoftheinterrelatedpedigreesffomtheIsleofLa
Reunionrevealedtheexistenceofatleastsixdifftrentcarrierhaplotypes・This
allelicheterogeneityisincompatiblewiththeapL・LOnPreSumedexistenceofa−
fbunderefftct，andsuggeststhatmultipleLGMD2Amutationsmaysegregatein
thispopulation．

An STSMapoftheI・GMD2ARegion
As YAC clone774G4spanS a reglOn Showlng Slgnificantlinkage
disequilibriumwithLGMD2A，thisl・6MblongYACwastentativelyorganizedintoacontigofoverlapplngCOSmidclones，mainlybySTSsscreenlngand
inter−AluPCRhybridization・Atotalof70STSs，including13transcription
unitsand7microsatellitescoversthis−1・6MbreglOnWithanaVerageOf3
COSmidsperSTSandoneSTSevery25kb（Richardeta1．，1995a）．

Aprimary expressionmapofchromosome15q15
regioncontainingtherecessiveI・GMD2Agene
InordertoprogresstowardtheidentificationofthegeneinvoIvedin
LGMD2A，aPrlmarytranSCriptionmapoftheLGMD2AgenomicreglOnWaS
generated（Chiannilkulchaietal・，1995）・ThedirectcDNAselectionstrategy
WaSuSedwith3YACscoverlngthecandidatereg10nandtwodifferentmuscle
CDNAlibraries・SeventeendifferenttranscriptionunitswereidentifiedamOng

ー65−171cDNAfragmentsanalysed・Fivesequencescorrespondedtokn0wngeneS，
andtwelvetonewones・TheywerecharaCterizedfbrtheirsequences，PhysicalpositionswithintheYACandcosmidcontigs，andexpressionpatterns・Among
those specifically tranSCribedinmuscle，the calpaingene−Whose cDNA
sequencewaskn0wnSince1989（Sorimachietal・，1989）−aPPearedtobeagood
positionalcandidateforLGMD2A，althoughitwasnotconsidered，aPnOLi，aSafunctionalcandidate．

Mutationsintheproteolyticenzyme，Calpain3，CauSe
LGMD2AThe CANP3gene，enCOdingthelarge Subunit ofthe muscle−SPeCific
calcium−aCtivatedneutralprotease3・Thiscysteineproteasebelongsto thefamilyofintracellularCalpainS，requlrlngCalciumfortheircatalyticactivities（fbrareviewseeSaidoetal．1994）．It亭genOmicorganisationwasdetermined，

bysequenclngCOSmidscontainingthisgene，Whichiscomposedof24exons
SPreadovermorethan40kb（Richardetal・1995b）・
ValidationofacandidategenerequlreStheidentificationofthepathogenic
mutations．Atotalof16nonsense，SPlicesite，frameShiftormissensemutations
inthismuscle−SPeCificgenewereinitiallyfoundtocosegregatewiththedisease
inLGMD2Afamilies，SixofwhichwerefbundwithinReunionislandLGMD2A
patients（Richardetal．，1995b）．AdigenicinheritanCemOdelwasproposedto
accountfbrtheunexpectedpresenceofmultipleindependentmutationsinthis

sma11inbredpopulation・OneofthemutationsencounteredintheisleofLa
Reunion，WaSfoundtobeasynonymousmissensemutationleadingtoapredicted

Gly−tO−Glysubstitution・Asamatteroffact，tOreVealthismutation，Wehadto
screenlymphocyteillegitimatetrahscriptionproducts（Chellyeta1．，1989），Since
theCANP3locusshowsatissue−SPeCificpatternofexpressionandfbrlackof
muscletissuesfromthislikefrommostpatients・Inthismanner，WeCOuld
demonstratethatthisapparentlyneutralmutationcreatesanewsplicesite，andis
thusefftctivelyequivalenttoanullmutant（RichardandBeckmarm，1995）・Last
butnotleast，the findingthatcalpainmutations causeLGMD2Aisthe first
demonstrationofanenZymaticratherthanaStruCturalproteindefectcauslnga
musculardystrophy，adeftctthatmayhaveregulatoryconsequences，PedlaPSin
Signaltransduction（Richardeta1．，1995b）．

−66−

馳vingidentifiedthe role ofthecalpain geneinLGMD2A，WeneXt
analysedthesegregationofmarkersflankingtheLGMD2Alocusandcamiedout
asearChforCANP3mutationsinLGMD2pedigreesffomvariousgeographic
OriginS．Altogether，42distinctmutationswereunCOVered（23missense，11
frameShift，2nonsense，4splicesite，and2smallin−framedeletions），dispersed
throughouttheentirelengthoftheCANP3gene（unpublisheddata）．Thesedata
indicatethatmusculardystrophycausedbymutationsinthemusclecalpaingene
arefbundinpatientsfromallcountriessofareXamined，Withno・Predominant
mutation，andfurthersupportthewideheterogeneityofmoleculardefbctsinthis
rare disease．

Characterisation ofthemurine canp3gene
CDNAclonesencodingmousemuscle−SPeCificcalpainwereselectedby
hybridizationandsequenced（RichardandBecknann，●1996）．Themousegene

encodesfbranmRNAofsimilarsizetothehumanCANP3mRNA，directingthe
Symthesisofan821aminoacidslongprotein・ApartialcharaCterizationofthegenomicorganisationofthecanp3genesuggeststhatitfollowsthesameOVerall
StruCtureaSitshumanhomologue・Theresultsobtainedonsomaticcellhybrid
Panelindicateseitheralocalizationonmousechromosome20rOnChromosome
4・InformationofsyntenyconservationwithhumanSuPPOrtthechromosome2
localization・Interspeciescomparisonofthecanp3DNAsequencesshowsahighlevelofidentitybothattheDNAandaminoacidlevels．Thededucedaminoacid
SequenCeShareS98・7％，93．5％and77．3％identitywiththerat，humanand

Chickencodingsequences，reSPeCtively（RichardandBecknarm，1996）．The
degreeofsimilarityisindicativeofthefundamentalbiologicalrole ofthis
PrOtein・Togetherwiththeotherkn0wnanimalsequences，themousecanP3
SequenCeWOuldbeusefultoassessmissensemutationsinLGMD2Apatients・Thissequenceinfbrmationcanbeusedtostudythespacio−temPOralandtissue−
SPeCificexpressionaswellastodevelopknock−OutmOuSemOdels・Itrepresents
thusausefultoolfbrgene−regulation，Physiopathologicalstudiesofthediseaseas
Wellasfbrtherapeuticinvestigations．

一67−

Geneticheterogeneity ofautosomalrecessive LGMDina
geneticisolate（Amish）andevidenceforanewlocus
Cases of autosomal recessivelimb−girdle musculardystrophyamOng

membersof the old order ofAmidl Ofnorthernand southernIndiana WerealreadydescribedbyJacksonandCarey（1961）andJacksonandStrehler（1968）・

ThefamiliesofthesecommunitiesareinterrelatedbymultipleconsangulneOuS
linksandcommonancestrywhichcanbetracedbacktothe19thcenturyinthe
CantOnOfBem，Switzerland．ItisuponstudyingthesefamiliesthatYoungetal・（1992）confirmedthechromosome1510calisationoftheLGMD2Alocus．All

AmishLGMDpatientsfromnorthernIndianaWereSubsequentlyshowntObehomozygousforthesameArg769GlumissenseCANP3mutation（Richardetal・，
1995b）．

Allamandetal．（1995b）reportedtheexclusionoftheLGMD2Alocusin

SixAmish kindreds from southernIndianathatare related by multiple
COnsangulneOuSlinks tothe same nOrthernIndiana familiesin which the
invoIvementofthechromosome15locuswaspreviouslydemonstrated．ThesefindingsdisclosedgeneticheterogeneityofLGMDinthisIndianaAmishisolate．
Inview ofthehighconsanguinityandthe similarClinicalpresentationofall

AmishLGMDpatients，thisresultwastotallyunexpected．FurthermOre，genetic
analysesalsoruledoutthepossibleinvoIvementoftheotherknownLGMD2loci，thusdemonstratingthatamutationwithinatleastoneadditionallocusleads
tothiscondition．SeveralcandidategenesputativelyinvoIvedinneuromusculardisorders were also excluded．A systematic genome−Wide genetic study
undertakenwithsixofthesefamilieseventuallyuncoveredgeneticlinkagetothe

Pericentromericregion of chromosome4（LimIDuclosβroux et a1．，1995），
therebyprovidinglegitimacytotheLGMD2Elocus．

Identification ofthe LGMDElocus
MeanWhile，thehumanCDNAofβ−SarCOglycan，a43kDa dystrophin−associatedglycoprotein（alsoknownasA3b），WaSClonedandmappedtothesame
Chromosome4region（Lim／Duclos／Brouxeta1．，1995）．Severalchromosome4
Pericentromeric markersand aβ−SarCOglycanintragenic polymorphic CA

repeatcosegregatedperftctlywiththediseaseinAmishfamiliesfromSouthem
IndianaWithautosomalrecessivelimb−girdlemusculardystrophy・Inaddition，reconstruCtionofhaplotypesallowedidentificationofchromosomesdescending

ー68−

β

from acommOnanCeStOr・Furthermore，aThr−tO−Arg missense mutation，●βSarCOglycangene thatleads to a dramatically reduced expression of

一

PreSentinahomozygOuS Statein allpatients，WaSidentified withinthe

SarCOglycaninthesarCOlemmaandaconcomitantlossofα−andY−SarCOglycan，

thereby disruptingtheintegrity of the dystrophin glycoproteincomplex（LinVDuclosβrouxet al・，1995）・SimilareVidencewere also obtained by
B6nnemanetal・（1995）・TTleSedatademonstratedthatadeftctinβ−SarCOglycan

isresponsiblefortherecessivemusculardystrophyinsouthernIndianaAmishfamilies．Thustheβ−SarCOglycangeneisthefifthlocus（LGMD2E）inVoIvedin
autosomalrecessivelimb−girdlemusculardystrophy，andthethirdsarCOglycan
involvedinamyopathy（TableI）．

Conclusion
Theyearthatjustendedseemsto havebeenagoodvintageyearfbr
geneticistsandclinicianSinterestedinautosomalrecesiveprogressivemuscular

dystrophies・Indeedinalittle overayear，fbur LGMD2geneshavebeen
identified，threeofwhichbelongtothedystrophin−aSSOCiatedproteincomplex，
thefourthonebeinganenZyme（TableI）．Thesediscoveriesraisemore
questionsthananSWerS・Butthisisnotamatterofcomplaint・Theyarealsoa

newsourceofhope・Thisknowledgecan，Ontheonehand，alreadybeusedto
leammoreaboutthephysio−Path010gicalprocessesinvoIved．Ontheotherhand，Carefulexaminationofthephenotype−genOtyPerelationships（e．g．Fardeaueta1．，
1996）willenableaclearerd6finitionofthespecificnosologicalboundariesof

eachoneofthesesimilar，yetdifferent，Clinicalentities，aknowledgethatcould
beofgreatimmediatebenefittothepatientsthemself・Andlastbutnotleast，it
OPenSPrOSPeCtSfbrnewtherapeuticavenues．

Acknowledgments
WeareParticularlyindebtedtoallpatients，theirfamiliesandclinicianS．
WethankDrs．M．Conneally，J．Tischfield，M．R．Passos−BuenoandM．Zatzfor
theirparticipationinpartsofthiswofk・WealsoacknowledgemanyCOlleagues
forproviding clones，Chromosome4specificcosmidlibrary；microsatellite
repeatprobes；forperformlngmuSClebiopsleS；andfinally，membersfromour
respectivelabfbrtheirhelp・ThisresearChwassupportedbygrantSfromthe

AmeriCanMuscularDystrophyAssociationandAssociationFran9alSeCOntreles

ー69−

Myopathies（AFM），andthe Groupement de Recherche Europeen surle
Genome．V．A・isfundedbyagrantfromAFM・K・P・Cisaninvestigatorofthe

HowardHughesMedicalInstitute・−70−

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With■mOrethanOneCharacterizedSTSaredrawn）・ThenameSOftheSTSsaregivenabovethe

linerepresentadon ofthe contig・Tbe position ofpolymorphicmicrosatellite markersis
indicatedbythepresenceofareddotabovethem訂kername・TbeSTSsaredrawnequidistant
exceptwhentheyareissuedfromthesamegene（andhencearearbitrar皿yordered），butforthe
5，endofthe丘briuingene・A可acentmarkerswhichcmnOtbeorientedwithrespecttothismap

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theCEPHlibraries・Uprighttriangles：STSswhichfailedtoyieldYACsinthisscreenlng．
Barsonfu111inesindicatepositiveSTSs．Dottedlinesmeanthataclonewasnotfoundtobe

POSitiveforaglVenintemalSTS・Anows attheendofthecloneindicatethataclonewas
foundtobenegativeforthea可acentSTSs・Theredlinesrepresenttheminimaltilingpath
COnSistingoflOYACclonesspanningthecontig・Thechromosome15YACend−STSs（RorOareshowninfu11squares，andYACend00nStitutedofrepeatedsequen∝SaSdottedsquares．
Tbeendsare afbitrarilydrawnwheneverboth ends failed tomqp withinthe condg・One
extremityOfclone911B6，adoubleinsertandmitoticallyunStableYAC，isshownasahatched
Square，inthemiddleofthecontig，aSthisclonewasshowntobepositiveforextemalSTSs．
A drawlng Showing approxlmate G−banding pattern of the corresponding reglOn Of
Chromosome15isplacedabovethecontigpresentation．Tnesmallestrecombinantinterval
de貴ningtheLGMD2AreglOnisshownbythebluever（icalarrowsontheregionalchromosome
map，andthedistancespanmingtherecombinantintervalsisglVenabove．

−72−

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MammalianGenome（inpress）．
Richard，I・，0・Broux，V・Allamand，F・Fougerousse，N．Chiamilkuchar，N．
Bourg，LBrenguler，C・Devaud，P・Pasturaud，C．Roudaut，D．Hillaire，
M・−R・Passos−Bueno，M・Zatz，J・A・Tischfield，M・Fardeau，C．E．Jackson，
D・CohenandJ・S・Becknam（1995b）．Anovelmechmismleadingto
musculardystrophy：mutationsincalpain3Causelimbgirdlemuscular
dystrophytype2A．Cel181：27−40．
Roberds，S・L・，Leturcq，F・，Allamand，V・，Piccolo，F・，JeanPlerre，M．，

Anderson，R・D・，Lim，L・E・，Lee，J・C・，Tome，F．M．S．，Romero，N．B．，
Fardeau，M・，BecknannJ・S・，Kaplan，J．−C．andCamPbell，K．P．（1994）．
Missensemutationsintheadhalingenelinkedtoautosomalrecessive
musculardystrophy．Cel178，625−633．
Saido，T・，Sorimachi，H・andSuzuki，K．（1994）．Calpain：neWPerSPeCtivesin
moleculardiversityandphysiological−PathologicalinvoIvement．FASEB
J．8，814−822．

Sorimachi，H・，Imqioh−Ohmi，S．，Emori，Y．，Kawasaki，H．，0lm0，S．，Minami，Y・andSuzukiK・（1989）．MolecularcloningofanovelmammalianCalcium−dependantPrOteaSedistinctfrombothm−andmu−tyPe・Specinc
expressionofthemRNAinskeletalmuscle．J．Biol．Chem．264，201（）6−
20111．

ー77−

Weissenbach，J．，G・Gyapay，C・Dib，A・Vignal，J・Morissette，P・Millasseau，G・
VaysseixandG．M．Lathrop（1992）・Asecond−generationlinkagemapof
thehumangenOme・Nature359，794−801・

Young，K・，T・Foroud，P・WilliamS，C・E・Jackson，J・S・Beckmarm，D・Cohen，
P．M．Conneally，J．TischfieldandM・E・Hodes（1992）・Confirmationof
LinkageofLimb−GirdleMuscularDystrophytoChromosome15・
Genomics13：1370−71．

−78−SkeletalMuscleSpecificNovelCalpaln，p94：
Properties andFunction
HiroyukiSorimachil，Kayokomnbaral，SumikoKimura2，Shoichi
Ishiural，andKoichiSuzukil
lInstituteofMolecularandCellularBiosdences，UmiversityofTokyo，Bunkyo−ku，Tokyo113and2DepartmentofBiology，FacultyofSdence，
ChibaUniversity，Inage−ku，Chiba263，Japan

TheactivityofintracellularPrOteaSeSmuStberegulatedstrictlyin
Ordertopreventco−eXistingcytosolicproteinsfromrandomdegradation．If
thisactivityregulationsystemdoesnotworkproperly，thelevelofproteaseactivitylnCreaSeS Or decreases than the normallevelresultinginrapid
degradation or accumulation of proteinsleading to various disorders．

MusculardystrophyisaneXamPleofthefbrmercaseanddepositionofamyloid
PrOteinsinAIzheimer．sdiseaseisthelattercase・Understandingoftheactivity

regulationsystemofintracellularPrOteaSeSisthereforeessentialtoclarifythemolecularmeChanismsofvariousdiseasesarlSlngfromtheal）nOrmallevelof
PrOteaSeaCtivity．
Calpainisamosttypicalcytosoliccysteineproteinaseinanimalcells
andtheactivityisregulatedbycalciumionsandaproteinaceousinhibitor，Calpastatin（forreviewsandreferencessee（1−3））．Itplaysapivotalrolenot
Onlyasamodulatorincalcium−dependentcellulareventsbutalsoasaninitiator
Ofdegradationofproteins，eSPeCiallyshortlivedproteinS・Calpainisbelieved
tobere甲OnSibleformuscleatrophyinDuchermemusculardystrophy・We
havebeenstudyingphysiologicalfunctionandregulationofactivityofcalpain・
Quiterecently，SeVeralnewcalpainspecieshavebeenfound．Someofthemare
expressedexclusivelyincertaintissuesincontrasttoubiquitousexpressionof

Calpainthusfarkn0wn・ThesetissuespecificnovelcalpalnSPeCiesaremOre
SuitablethanubiquitouscalpainSinanalyzingphysiologicalfunctionandactivity

regulationsystem・臨rewesummarizeourrecentstudiesperformedmainly
uslngSkeletalmusclespecificcalpain，P94・ー79−1）NewFamilyMembersofCalpain

Foralongtime，tWOisozymeS，LLandm−CalpalnSWithdistinctcalcium
sensitivitiesareknown．Theseare COmPOSed ofa distinct catalytic80kDa
subunit（80K）and a commOn30kDa regulatory subunit（30K）・Recently
severalnovelcalpainspeciesincludingathirdubiquitouscalpain，〟mtype（4），havebeenidentified，indicatlngthatcalpainconstitutesalargefamily・These

arehomologsof80Kofpandm−CalpainsassummarizedinFig・1・Skeletal
musclespecificcalpain，P940rnCL−1（novelcalpainlargeSubunit），COntains
three阜nsertionsequences，ISl，IS2andNS・Itmustbementionedthatan

alternativesplicingshortproductwithoutthecalciumbindingdomainisfbund
fbrstomachspeci丘ccalpain（nCL−2／nCL−2）・anddrosophilacalpain（calpA／calp

Al）．Thesenovelcalpainspeciesincludingaltemativesplicingproductsarenot
minorspeciesatleastasfarasthemRNAlevelisconcemed・ForinstanCe，themRNAlevelsofLl，m，nCL−2，andnCL−2−instomachareeSSentiallythesame，
indicatingthatthesespeciesmusthavetheirownimportantfunction・Further，
theamOuntOfp94mRNAinskeletalmuscleisatleastoneorderofmagnitude

higherthanthosefbrpandm−CalpainS・Nevertheless，nOneOfthemexceptfor
Phn−Calpainhasbeenisolatedasaproteinandcharacterizationofthesecalpains
remainstobeanimportantissue．

2）Thelarge80kDaSubunitisanActiveCalpainSpeCies
Previousdissociationandreassociationstudiesofcalpainhaveshown
that80Khasintrinsicproteaseactivity，but30Kisessentialfbrexpressionoffull
enzymeactlVlty，SuggeStlngthatbothsubunitsarerequiredforfullactivity・
Sincenovelcalpainseemstoexistasamonomer ofan80Khomologue，Wereexaminedrenaturationof80Kafterdissociationfrom30Kinureaandfbund
that80KalonecouldregainfullenzymeactlVltyafterprolongedincubation（ca．30days at40C）（5）．Addition of30K to80K accelerated the rate ofrenaturationbutdidnotafftctthefinallevelofactivityrecovery．AnE．coli
ChaperoninGroEhadsimilareffbctbutonlyinthepresenceofATP，SuggeStlng
that30KfunctionsasachaperonratherthanaSanaCtivatorof80K・Inaddition，dissociated80Kshowedenhanced calciumsensitivityidenticaltothethat ofactivatedN−terminallymodifiedcalpainpreparedbypreincubationincalcium匝）・

ThecalciumsensitivityofLlandm−Calpainsisnothighenoughtobeactivein
Vivoandmusttherefbrebeincreasedbyacertainmechanism・Autolysiswhich−80−

modifiestheN−terminalreg10nOfbothsubunitshaslongbeenproposedasan
activationmechamism・TheN−terminallymodified80Kpreparedbyautolysis
Showedthesameenzymaticpropertiesasthoseofintact80Kincludingcalcium
SenSitivity・Moreover，ithasbeenshownthatcalpaincompriSingN−terminallymodinedsubunitstendstodissociatemoreeasily・Theseresultsclearlyindicate
thatautolysisisnotthecauseofactivationbutdissociationof80Kftom30K
COrreSPOndstoactivation・Inconclusion，30KcannOWbeinterpretedas aregulatorysubunitessentialfbrregulationof80KactivityinresponsetochangeS
incalciumconcentrations．

3）PropertiesofMuscleSpecincCalpainp94
AlthoughthemRNAlevelofp94isslgnificantlyhigherthatpand
m−Calpains，identificationofp94asanenzymeis verydifficult・In vitro
tranSCriptionandtranSlationexperimentofp94cDNAindicatesthatnewlytranslated p94degrades very rapidly with a half−life of ca．30min．This
degradationisautocatalytic，becausevariouspointmutantSOftheactivesiteCys，

HisandAsnresiduesarestable・ExperimentswithtrunCationmutantSreVealed
thatIS2isresponsibleforthisrq）idauto−degradation・IS2mightcontaina
Slgnalfbrrapidproteindegradation・Althoughisolationofintactp94ishardly
POSSible，P94Canbedetectedhistochemicallyincellsaftertransfectionofits
CDNA・P94existsinbothcytosolandnucleuswhichiscontroledbyanuclear

translocationslgnallocatedinIS2．Whenratskeletalmuscleisstainedwith
antibody，aSimilarStainingpatterisobserved・Sincethep94messagecannot
bedetectedinculturedmusclecellsbutexistsinskeletalmuscle，themessage
mustappearatacertainstageofmusclecelldifferentiation・P94inthenucleus
might regulate difftrentiation by regulating thelevelof muscle specifictranscnptlOnfactorssuchasMyoD，myOgenin，etC．
4）p94BindstoCormectin／ritin

Toidentifyaregulatorysubunitofp94，ifany，ahumanSkeletalcDNA
libraryWaSSCreenedbytheyeasttwo−hybridsystemuslnganinactivepoint

mutantofp94wheretheactivesiteCys−129isreplacedwithserine．Weused
theLlandm−CalpainlargeSubunit（LICLandmCL）andcalpastatinasacontrol

fbrthisscreenlng・TheresultsshowedthaりlCLandmCLinteractwith30K
butcalpastatinwhichisknowntoboundcalpainonlyinthepresenceofcalcium−81−doesnotbindtocalpain・Theresultssuggestthattheyeasttwo−hybridsystem
worksinlowcalciumconditionsinyeastsothatonlytheinteractionwhich
occursintheabsenceofcalciumCanbedetected．Under theconditionsused，
p94didnotbindto30Kbut32positivecloneswereobtained（乃・Comparlngthe

sequencesofthe cloneswiththecompleteamino acid sequenceofhumanconnectin（8），itwasidentifiedthat31clonesencodesequencesidenticaltothe
C−terminalsequenceofcornectinandthattheremalnlngOneClonecorresponds
tothecormectinsequenceoftheN21ineposition・Connectinisagiantelastic
muscleproteinofca．3000kDaspanningtheZlinetoMlineinmyofibrils（Fig・2）．The N−and C−termini of connectinlocate at the Z−and M−1ines，
respectively・CormectinissusceptibletosplitattheN21inewhichis1200kDa

downstreamffomtheN−terminusofcormectinortheZ−line．吋drolysisof
cormectinatthispositionisoftenobservedduringpreparationofconnectinand
oneoftypicalfeaturesobservedwithmusclesofDuchennemusculardystrophy・AlthoughthephysiologicalmeanlngOfthissplittinglSnOtClear，Calpainor
calciumisreportedtobeinvoIved・Thepresentresultsthatp94bindstotheNilinesuggestthatp94mightberesponsibleforthe甲litting・Sincebindingof
p94totheN2−1ineismuchstrongerthanthattotheC−terminalreglOn，further
preciseexperiments were perfbrmswith the former case・Using various
deletionmutantsthebindingsiteofp94for connectinwasidentifiedasIS2・ThisisconsistentwiththefactthatP・andm−CalpainswithoutIS2donotbind
connectin．Itshouldbenotedthatp94specificsequenceIS2isresponsiblefor
nuclearlocalization，raPidautolysisandbindingtoconnectin・IS2thusplaysa
CruCialroleinthephysiologicalfunctionofp94・Toexaminep94−COnneCtinbindinglnVivo，Weisolatedmyofibrilsand
examinedcolocalizationofp94andcormectin．SDS−gelelecetrophoresisandWesternblottingsuslngP94antibodiesindicatedthatintactp94existedinthe
myofibri1fraction・However，OnCeCOnneCtWaSeXtraCtedffommyofibrilsbya
buffercontaininghighsalt，P94degradedveryrapidlyintofragments・Since

isolatedp94isunstable，thepresenceofintactp94suggeststhatitsactivityis
regulatedwhenboundtoconnectin・Further，histochemicaldetectionofp94by
itsspecincantibodies，gaVeSlgnalsattheZ−bandandatpositionscorresponding
totheN2−1ine（7），being consistentwiththeresultsofthetwohybridsystem，althoughwehavenotyetidentifiedclonescorrespondingtotheZ−1inestaining・
Sincecormectinis one ofthebiggestproteins，tO Obtainclones forthe N−

一82−

tertminalreglOnisverydimcult．
5）Correlationbetweenp94andMuscularDystrophy
Quiterecentlymutationsinthep94geneareShowntOCauSelimb
girdletype2Amusculardystrophy，LGMD2AP）・Variousmutationsincluding
POintmutationsandnonsensemutantswhichyieldshorterp94withoutthe
PrOteaSedomainarefbund・Thusaloss−Of−functionofp94mustbethecauseof
LGMD2A・Inthe caseofDuchennetypemusculardystrophy，deftctsin
dystrophinchangeSthemembranepermeabilityofmusclecellsresultingin
increaseintheintracellularcalciumconcentrationwhichtriggersactivationof
Calpainanddegradationofmuscleproteins・InthecaseofLGMD2A，Similar
degradationofmuscleproteinsisobserved，SuggeStingparticipationofcalpainin
thiscaseaswell・Itistherefbreessentialtoelucidateaslgnalingpathwayfrom
inactivationofp94toactivationofcalpainanddegradationofskeletalmuscle
PrOteinS・ThisslgnaltranSductionsystemwillinvoIvep94bindingproteinS，neighboringproteins andsubstrate proteinS，SuCh asconnectin，Calpastatin，
myotoninproteinkinase，etC・Itishopedthatpreciseanalysisofthisslgnal
tranSductionsystemwilleventuallyclarifythemechanismofmuscledegradation
fbrvariousmusculardystrophies・ー83−

Large S u bu n it（
80K ）S m aIIS u b un it（
30K I

Ca2＋−bindingDomain

ⅣL＞

Protease
Domain

PCL
mCL
H／mCL
P94（nCL・1）
lskeletalmuscLe］

nCL−2
lStOmaCh］

esplicingnCL−2■nCL−3
ldigestivetissues］

Sm−CL（∫Cあおわ∫〃椚α肌α〃∫β〃O

Dm−CaIpain（CaIpA）
alternativesplicing

prO∫呼，力〟α∽gh〃qgα∫／どう

Ca小A■lsingleoptic］obe］

CerCL2（Cbg〃〃祓α∂戯由g晦α〝∫）Ce−CL3An−CL
U甲g瑠㌦〟∫〃肋血〃∫）Fig・1・SchematicdomainstruCtureOfcalpainfhmilymembers．

ー84−

．白き〇月SOS一dS芯hn︶U己lS︼Tq篭hEU⁝ldE08SPu巾山上．占0呂已80︼寸乳葛ぎ⁝p月由・N・的に．召00日8葛巴nIU貞SO焉EjUSO召叫宕−d已き〇月SO岳SOCOでNMPOljOSこOSRO芸SOdO声

Pu帽山上q85

REFERENCES（1）H．Sorimachi，T．C．Saido，K．Suzuki：FEBSLett．，343，1−5，1994．
（2）T．C．Saido，H．Sorimachi，K．Suzuki：FASEBJ．，8，814−822，1994．
（3）K．Suzuki，H．Sorimachi，T．Yoshizawa，K．KinbaraandS．Ishiura：Biol．
Chem．Hoppe−Seyler，376，523−529，1995．
（4）H．Sorimachi，T．Tsukahara，M．Okada−Ban，H．Sugita，S．Ishiura，K．
Suzuki：Biochim．Biophys．Acta，1261，381−393，1995．
（5）T．Yoshizawa，H．Sorimachi，S．Tomioka，S．Ishiura，K．Suzuki：FEBSLett．，
358，101−103，1995．
（6）T．Yoshizawa，H．Sorimathi，S．Tomioka，S．Ishiura，K．Suzuki：Biochem．
Biophys．Res．Commun．，208，376−383，1995．
（7）H．Sorimachi，K．Kinbara，S．Kimura，M．Takahashi，S．Ishiura，N．
Sasagawa，N．SoriamChi，H．Shimada，K．Tagawa，K．MaruyamaandK．
Suzuki：J．Biol．Chem．，270，31158−31162，1995．
（8）S．LabeitandB．Kolmerer：Science，270，293−296，1995．
（9）I．Richard，0．Broux，V．Allamand，etal．：Cell，81，27−40，1995．

−86−

Familial Hypertrophic Cardiomyopathy：
A disease ofthe cardiac sarcomere
Man：Y．Fiszman
INSERMU153，HopitalPiti6−Salp6triere，Paris，France

Familial hypertrophic cardiomyopathy（FHC）isinherited as anautosomaldominantdisorderthatischaraCterizedbyhypertrophy，Oftenofthe
leftventricle，WithpredominantinvoIvementoftheinterventricularSePtumin
theabsenceofothercauseofhypertrophysuchashypertensionorvalvularheartdisease・Thepredominant CardiacpathologylSmyOCyte hypertrophy andSarCOmeredisarray，thefbrmerisfbundinmostcardiatdiseaseswhilethelatter

isahallmarkofFI肥・Theventriculardiastolicfunctionisusuallyimpaired
andtheclinicalmanifestationsrangefromabenignasymPtOmaticcourseto
SeVereheartfailureandsuddendeath．

FI肥isnotonlyheterogeneousphenotypically，butitisalsogeneticallyheterogeneous．

In1989，thefirstlocus（CMHl）wasidentifiedonchromosome14q11−
q12（5）・Sincetheoこandβmyosinheavychaingenesarelocalizedinthesame

reg10n，theybecameObviouscandidatesasdiseasegenes．Indeed，amissense
mutationwasthereafterdetectedatcodonposition403inthegenewhichcodes
fortheβmyosinheavychainβ）．sincethisfinding，mOrethan29different
POintmutationshavebeendescribed（fbrreviewsee（9））．
Besidesthisallelicheterogeneity，itbecameVeryraPidelyobviousthat
FHCwasalsoheterogeneousintermoflociand4lociweresubsequently
identified on chromosomelq3（CMHZ）（13），15q2（CMIB）（10），11p13−q13（CMH4）Q）and7q3（CMH5）（7）．
ThediseasegenescorrespondingtoCMIBandCMIBwereidentified
andshowntocodefbrcardiactroponin−T（cTnT）andα−trOPOmyOSin（α一TM）

respectively（11）・Lastly，thegenecorrespondingtoCMI剃wasalsoidentified
amdshowntocodefbrthecardiacMyosin−BindingProteinC（MyBP−C）（l・12）−87−Ⅰ）−β−Myosin HeavyChain
Myosinisthemolecularmotorwhichdrivesshorteningofthesarcomere，
hencemusclecontraction・MyosinconsistsoftwoheavychainSandtwopalrS
oflightchainsallofwhichexistasvariousisofbrmsthatareSPeCificforthe
varioustypeofmuscles・Intheadulthumanheart，themqormyosinheavy
chainistheβisofbrmWhichisalsoexpressedinslowfibersofskeletalmuscle・
From a structuralpoint ofview，myOSinheavy chainis a complex
molecule．TheN−terminusformsaglobularheadwhichcontainsthefixation
Sitesforthemyosinlightchainsaswellasallthenecessaryelementstogenerate
movementofactinrelativetomyosinduringATPhydrolysis・TheC−terminus
partofthemoleculehasarodshapeandisinvoIvedinthepolymerisationofthemoleculetoformthethickfilaments．
AllmutationsassociatedwithFIICaremissensepointmutationsandare
localizedintheglobularheadorinthehead−rOdjunctionofthemolecule・The
three−qimensionalstruCtureOftheβmyosinheavychainisunkn0wnbuth3CauSe

theprlmarySequenCeOfmyosinishighlyconserved，itcanbeexpectedthatthe
three−dimensionalstruCtureSOfallmyosinmoleculeswillbeverysimilarhence
makingitpossibletousethethree−dimensionalstructureofchicken skeletal
myosintointerprettheeffectsinducedbythevariousmutations・Allthe
mutationsinthecardiacaminoacidsequencemaptowell−definedpartSOfthe
three−dimensionalstructureofchicken奴eletalmyosin（8）・TheyareClusteredar0undspecificlocationsintheheadandinparticular，OnegrOuPISaSSOCiatedwiththeactinbindinginterface，aSeCOndisclosetotheactivesiteandathirdgroupISClosetotheinterfaceoftheheavychainwiththeessentiallightchain・
Recently，takingadvantageOfthefactthatβmyosinheavychainisalsoexpressed
inslowtwitchmusclefibers，aStudywasconductedonskeletalmusclebiopsleS
obtainedfrompatientswiththreedistinctβmyosinheavychainmutations（6）．
Twoofthesemutations，Onenearthebindingofessentiallightchainandoneat
theactininterface，Showdepressedvelocityofshorteningwhilethethird，atthe
endoftheATP−bindingpocket，Showsnomodificationofthemyosinproperties・
Although thereis variabilityinthe natureand the extend offunctional
impairmentsin skeletalfibers containing differentβmyosin heavy chain
mutation，itisverylikelythatthesefunctionalalterationsconstitutetheprlmary
Stimulusfbrthecardiachypertrophy．

ー88−ⅠⅠ）−α−Tropo？yOSinandCardiacTroponinT
Tropomyoslnand Troponin Tinteract with oneanOther

andareCOmpOnentSOfthethinfilamentinthesarcomere．Asfbrthemyosin，multiple
isofbrmSeXistwhichshowmuscletypespecificity・TwomutationshavebeendescribedfortheαtrOPOmyOSinwhichboth

alterthenetchargeofthemoleculeandafftctareglOnClosetooneofthebinding
SitesofTroponinTtotropomyosin．
AsfbrthecardiactroponinT，fiveoutofsevenmutationschangethenet
ChargeofthemoleculeandarelocalizedinapartofthemoleculeinvoIvedintheinteractionwithtropomyosin・AsixthmutationinducesaberantSPlicingwhich
resultsinaprematureterminationofthemoleculewhich，agaln，Willnotinteract
Withtropomyosin．
Asfbrtheβmyosinheavychain，itisverylikelythatallthesemutations
alterthekineticoftheinteractionbetweenthinandthickfilamentSWhichresults
inamodincationinthespeedofcontractionofthecardiacsarcomereleadingto
COmPenSatOryhypertrophy．
ⅠⅠⅠ）−CardiacMyosinBhdingProtein C

Themyosin−bindingproteinCisalsofoundinthesarcomerewhereitis
arrayedtranSVerSelyinthesarCOmereA−bandsandbindmyosinheavychainin
thethickfilament，POSSiblyactininthethinnlamentandtitininelastic丘1amentS．

hparticular，thedomainofmyosininteractionislocalizedattheCterminusend
Ofthemolecule．

ThefunctionoftheMyBP−Cisnotwellunderstood，however，reCent
StudieshaveshownthatpartialextractionofhlyBP−Cfromratskinnedcardiac
andrabbitskeletalmusclefibersleadstoanincreasedrateofcontractionand
increasestheCa＋＋sensitivityofthefbrce−Velocitycurve㈹・Theseresultstend

toindicatethatMyBP−CplaysanimportantrOleinregulatingmusclecontraction
Three distinct mutations have beenidentified，tWO Of which afftct
SPlicingofthetranscriptandresultinprematureterminaisonoftheproteinthus
leadingtothefbrmationofavariantlackingthemyosinbindingdomain・TileSe
mutations couldinduce cardiachypertrophythrough aperturbation of the
SarCOmerefunction．

ー89−Inconclusion，allthegeneswhichhavebeenidentifiedsofar asbeing
responsiblefbrFIICcodeforacomponentofthesarCOmericmachinerywhich
reinfbrcethehypothesis thatFHCis apathologyof thesarCOmereandthat
hypertrophyisacompensatorymechanisminresponseofanalterationofthe

musclecontraction．Itremaintobeseenifthemidentifiedgenesalsobelongto
血es訂ne血nily．

−90−

REFERENCES

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Weissenbach，H・P・Vosberg，M・Fiszman，M．Kom可daandK．
Schwartz・1995・Cardiacmyosinbindingprotein−Cgeneacceptorsite
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NatureGenet．11：438−440．
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Epstein・1995・StruCturalinterpretationofthemutationsintheβ−Cardiacmyosinthathavebeenimplicatedinfamilialhypertrophic

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−91−

9．Schwartz，K．，LCarrier，P．GuicheneyandM．Komajda．1995．The
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A．Bowcock，H．P．Vosberg，J．G．SeidmanandC．E．Seidman．
1993．Afamilialhypertrophiccardiomyopathylocusmapsto
Chromosome15q2．Proc・Natl・Acad・Sci・USA・90：6270−6274・

11．Thierfelder，L．，H．Watkins，C．MacRae，R．Lamas，W．McKenna，
H．−P．Vosberg，J．G．SeidmanandC・E・Seidman・1994・0ト

tropomyosinandcardiactroponinTmutationscausefamilial
hypertrophiccardiomybPathy：adiseaseofthesarcomere・Cell・
77：701−712．

12．Watkins，H．，D．Conner，L．Thierfelder，J．A．Jarcho，C・MacRay，
W．J．McKenna，B．J．Maron，J．G．SeidmanandC．E．Seidman．
1995．Mutationsinthecardiacmyosinbindingprotein−Cgeneon
Chromosomellcausefamilialhypertrophiccardiomyopathy・Nature
Genet．11：434−437．

13．Watkins，H．，C．MacRae，L Thierfelder，Y．H．Chou，M．
Frenneaux，W．J．McKeena，J．G．SeidmanandC．E．Seidman．
1993．Adiseaselocusforfamilialhypertrophiccardiomyopathymapsto
Chromosomelq3．NatureGenet・3：333−337・−92t

Identification andcharacterization ofa
determining geneinspinalmuscularatrophy
SuzieLefebvre，LydieBurglen，LouisViollet，PhilippeBurlet，

01ivierClermOnt，Am01dMumichandJudithMdb
Umit6deRecherchessurlesHandicapsG6n6tiquesdelTnfant，INSERM
U393，IFREM，HopitaldesEnfants−Malades，PariS，France

Spinal muscular

atrOPhies（SMA）are a grOuP Of neuromusculardisorderscharacterizedbydegenerationofanteriorhorncellsofthespinalcord
leadingtolimbandtrunkparalysiswithmuscularatrophy・Theyrepresentthe

SeCOndmostcommOnfatalrecessiveautosomaldisorderaftercysticfibrosiswith
anincidence oflin60001ivebirths．Based on clinicalcriteriafrom theInternational SMA Consortiumthatinclude age of onset，milestones ofdevelopmentandageofsurvival，theSMAareClassifiedinto3forms．The
mostacutefbrmorWerdnig−tbfharmdisease（typeI）ischaraCterizedbya
SeVerehypotoniawithanonsetearlyafterbirthorwithinthenext3monthS．
Thisdiseasemaybedistinguishedfromtheintermediateform（typeII），and
juvenile disease（typeIIIor Kugelberg−Welander）．TypeIIchildren will
acqulretheabilitytositunaidedbutwillneverwalk・TypeIIISMAhasalater
OnSetand will usually progress to a chronic course・The underlying
biochemicaldeftct（S）remainsunknown．Itleadsustodevelopapositional
ClonlngStrategythatallowstheidentincationofadiseasegenebyitspositiononthegenome．
Bymeansoflinkageanalysis，WeandothershaveshownthatallthreefbrmSOfSMAmaptochromosome5qll．2−q13．3（1−4），SuggeStingthattheyare
allelicdisorders・Theseresultswerethestartingpointfbrahighresolution
genetic mqp・The geneticinterval between the flanking markers was
SuCCeSSively reducedfrom9centimorganS CM）to2CMbetween markers
AFM265wf5andAFM281yh9（5）・Thisintervalwasclonedintoa4−Megabaseyeastartificial chromosome（YAC）contig spanning the diseaselocus by
COmbining chromosomal walking with theidentification of new highly−93−POlymOrPhicmarkers・The5q13chromosomalreglOnischaraCterizedbythe
PreSenCe Of specificlow−COPy rePeatS（6）．Indeed，POlymorphic markers

detectedeither2（C212andC272）or3（C161）lociwithinthe5q13region．In
addition，nO reCOmbination event was detected betweenlociidentified bymarkersC212−C272andtheSMAlocus，indicatingthatthesemarkersareclose
tothediseasegene．
Totestthehypothesisthatthelowcopyrepeatsofthe5q13reglOnmight
triggerlarge−SCalerearrangementS，Weanalyzedtheallelesegregationatloci

detectedbyC212andC272in201non−COnSangulneOuSSMAfamilies・Absence
andincompleteparentalcontributionswere observedin9affected children・Southernblotanalysisconfirmedthepresedceofeitherinheritedordenovodeletionsinthesepatients．Inaddition，anheterozygOSltydeficiencyatloci
detectedbymarkersC212andC272wasobservedin18％oftypeIpatients．
Theseresultsindicatedthat deletionevents ofthe5q13reglOninSMAare
Statistipally associated withthe severe form ofthe disease（typeI）・Interestingly，（おnovodeletionsmightaccountforthelowsegregationratioin
SMA（7）．Theymightalsoaccountfbrthe叩Parentgeneticheterogeneityof
the disease．The presence oflow−COPy rePeatS On Chromosome5q13may
accountfbrtheinstabilityofthereg10nandtriggerfrequentdeletionsbymeans
Of unequal crosslng−OVer eVentS

in

SMA．

A genetic map allowed toCharaCterizetheextentofthe5q13reglOninvoIvedintheserearrangementS．
Studying allpolymorphicDNA markersderivedfromthe YACcontig，WeObservedthatthesmallestdeletionsoccurredwithinareglOnborderedbyloci
detectedbymarkerC161andtheclusterC212−C272．Theseresultssuggested
thattheSMAlocuslieswithinanintervalof1200KbentirelycontainedinYAC
Clone903Dl．
The two strategiesundertaken consistedin charaCterization of the

SmallestdeletioninSMA patients withtheidentificationof candidate genes
Withinthecriticalregion．PFGE（PulseFieldGelElectrophoresis）analysisof
thecriticalreglOnreVealed ahigh degreeoflengthVariation ofrestriction
fragmentsin bothcontroIsand SMAindividuals usmg different restriction
enzymes．Clearly，thisvariabilityhamPeredourabilitytorecognlZeabnormal
restrictionfragmentsinSMApatients（8）．Thisobservationemphasizesthe
genomiccomplexityofthecriticalreg10n．Inregardofthiscomplexity，afine
Physical map ofthe YAC contlg COVerlng the SMA critical reglOn WaS−94−

established・ThepositionoftherestrictionsitesandDNAmarkersprovided
evidencefbralargeinvertedduplicationofanelementthatwasdividedintoEtel
andEcenaccordingtolocation・SouthemblotanalysisusmgprobeHe3enables
to distinguish Etel ffom Ecen・The analysis of SMA patients born to
COnSangulneOuSParentSPrOVidedevidencefbrgenomicrearrangementSinEtel
OnbothmutantChromosomesandsupportedthelocationoftheSMAgeneinEtel
betweenprobeHe3andma止erC272・ThiscriticalreglOnisentirelycontained
ina140Kb丘agment．
WeisolatedacandidategenenamedSMN（SurvivalMotorNeuron）gene
bythedetectionofinterspeciesconservationuslngaSubclonefromaphage
COntainingmarkerC272（8）．ScreeningofhumanfttalbiaincDNAlibraryallowedtheisolationof70VerlappngcDNAclones・Northernblotanalysis
revealedthepresenceofawidelyexpressedl・7Kbtranscript・Thisgeneisalso
expressedinthespinalcord・SequenceanalysisrevealedanOPenreadingffame
encodingaputativeproteinof32Dawithnohomologywith sequencesin
databases・Specifichybridizationwithnon−OVerlapplngYACscontainingonly
EcenorEtelprovidedevidencefbrtheduplicationofSMNgene・But，Onlythe
telomericversionwashighlycandidatebyitsposition・The organization and structure of bothSMN geneandits highly
homologouscounterpart（CentrOmericcopy）weredetemined（9）．Thegeneis
about20Kbinlengthandiscomposedofnineexons・Thesequencedataofthe
5

endofthegeneshowthatmarkerC272iswithinaputativereglOnOfgene

regulation・ThesequenceanalysisofeachexonanditsflankingreglOnSallowed
thedetectionof5discrepanCiesbetweenthecentromericandSMNgenesin
introns6，7andexons7and8．
TheanalysisofSMNexons7and8revealedthatallcontroIscarriedthe
SMNgeneand95％ofthemalsocamiedthecopygene（8）．Ontheotherhand，
SMNexon7isabsentin98，6％ofpatientsindependentlyoftheclinicalsubtype・
Inaddition，thethreeSMApatientsretainingSMNexon7camiedeitherapoint
mutation（Y272C）orshortdeletioムSintheconsensussplicesitesofSMNintrons
6and7・Furthermore，inarecentcolhborationwiththegroupofMontserrat
BaigetinBarCelona54unrelatedSpanishSMAfamilieswereanalyzed（10）．
NinetyonepercentofpatientslackedSMNexon70nbothmutantChromosomes．
FourpatientsretainingSMNexon7Carrieda4bpdeletionresultinglna
frameshiftinexon3withaprematurestopcodon・Thelastpatientretainingー95−

SMNexon7hadalimitedgeneconversionchanglngSMNexonandintron7into
itshighlyhomologouscopy・TheseobservationsglVeStrOngSuPPOrttOthe

viewthatmutationsoftheSMNgenearereSPOnSiblefbrtheSMAphenotype・
ThesefindingshavedirectclinicalapplicationsintheSMAdiagnosis・The
absenceofSMNexon7inpatientsmakesmoleculardiagnosiseasyintypicalandvariant SMA（11，12）．Furthermore，direct prenatal diagnosis becomes飴asible．
TheseresultsstronglysuggestthattheSMNgeneanditscopymustbe
difftrentinsomerespects・NorthernblotandRT−PCRanalysesrevealedthat
bothgenesaretranSCribedandthatadifferentialsplicingofexon7enablesthe
SMNgeneanditscopytobedistinguished（8）・Indeed，RT−PCRanalysisfrom
exon6to8revealedthatthecentromericcopygenebutnotSMNgeneundergoes
alternativesplicingofexon7toproducetranSCriptslackingthisexonanda
putativeproteinwithadifferentC−terminalend・Investigationsattheprotein
levelshouldhelpaddresslngthisquestion・
Weinvestigatedthecorrelationbetweentheclinicalphenotypeandthe
genotypeattheseloci（13）．AtotaloflO6patientswereclassifiedintotypeI（44），tyPeII（31）andtypeIII（31）andanalyzedusingSMN，markersC212and
C272，andNAIPmq）PlnguPStreamanddownStreamfromSMNrespectively・
NAIPistheneuronalapoptosisinhibitorproteingenecharaCterizedbyAlex
Mackenziels team（14）．NAlP exon5is q）eCific to Etel．The combined
analysisofallmarkersshowedthatalargeprOPOrtionoftypeI（43％）CarriedlargeSCaledeletionsinvoIvingbothSMNanditsflankingmarkers（C212／C272
andNAIPexon5），aSCOmParedwithnoneofthepatientswithtypeIIorIIISMA・
The presence oflarge SCale deletionsinvoIving theselociis specific to
Werdnig−Hoだmann（typeI）andallowsonetopredicttheseverityofthedisease
inour series．Iiowever，Smaller rearrangementSCanStillresultinasevere
Phenotypeas27％ofSMAtypeIpatientslackedtheSMNgenebutnotthe
CZ12−C2720rNAIPloci．Thesedatasuggestthatothergeneticmechanisms
mightbeinvoIvedinthevariableclinicalexpressionofthedisease・Elucidating
thefunctionofthegeneproductswillbeimportantfbrtheunderstandingofthe
PathogenesisofSMA．
Recently，Weandothers（15，16，17）analyzedunusualSMApedigrees・Inthese families，boththeaffectedandthe apparentlynon−affected siblings
Carriedthesame・haplotype．Theseobservationssuggestedeitheramisdiagnosis−96−Orgeneticheterogeneityofthedisease．However，theabsenceofSMNgene
COnfirmedthediagnosisofSMApatientswithadiseaselocuslinkedtotheSMN．

ThefactthattheSMNgenewaslackinginthenon−SymPtOmaticsiblingmaybe
duetoeitheranincompletepenetranCeOfthegeneoranadditionalgeneticevent
takingplaceinthenon−affectedbutnotintheaffbctedsiblings・Thissituationis
Observedonlyinthemildestformsofthedisease．Thissituationisrareand
representslessthan1％outof302SMAfamilies．
Carefulanalysis ofthe SMN geneandits copy atthe genomic，thetramscriptionalandtheproteinlevelshouldcontributetotheunderstandingof

thisextremeclinicalvariability・ThecreationofananimalmodelbySMNgene
targetingwillbeveryhelpfultounderstandthephysiopath010gyofthedisorder．

ー97−References
1． Brzustowicz，L．M．，Lelmer，T．，Castilla，L・H・，etal・GeneticmapplngOf
chronicchildhoodonsetspinalmuscularatrophytochromosome
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2． MelkiJ．，Abdelhak，S．，Sheth，P．，etal．Genefbrchronicproximal
spinalmuscularatrophiesmapstochromosome5q・Nature344，
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3．

MelkiJ．，Sheth，P．，Abdelhak，S．，etal．Mappingofacute（typeI）spinal
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4．

Gilliam，T．C．，Brzustowic，L．M．，Castilla，etal．Genetichomogeneity
betweenacuteandchronicfbrmSOfspinalmuscularatrophy・Nature（1990）345，823−825．

5． ClermOnt，0．，Burlet，P・，Burglen，L・，Leftbvre，S・etal・Useofgenetic
andphysicalmapplngtOlocatethespinalmuscularatrophylocus
betweentwonewhighlypolymorphicDNAmafkers・TheAmerican
JoumalofHumanGenetics（1994）54，687−694．
6． MelkiJ．，Lefbbvre，S．，Burglen，L．，etal．Denovoandinherited
deletionsofthe5q13reglOninspinalmuscularatrophies・Science（1994）264，1774−1777．
7． Zerres，K．HabilitatiOnsschrift．UniversitatBonn，1988．
8． Leftbvre，S．，Burglen，L．，Reboullet，S．，ClermOnt，0・，Burlet，P・，Viollet，L．，Benichou，B．，Cruaud，C．，Millasseau，P．，Zeviani，M．，Le
Paslier，D．，Frezal，J．，Cohen，D．，Weissenbach，J．，Murmich，A．and
Melki，J．Identificationandcharacterizationofaspinalmuscularatrophydetemininggene・Cell（1995）80，155−165・9・ Burglen，L・，Lefebvre，S・，ClemOnt，0・，Burlet，P・，Viollet，L・，Cruaud，C・，
Munnich，A．andMelki，J．StruCtureandOrganizationoftheHunan
SurvivalMotorNeuron（SMN）gene．Genomics．Inpress．
10． Bussaglia，E，ClermOnt，0．，Tizzan0，E．Lefebvre，S・，Burglen，L・，
Cruaud，C．，Urtizberea，J．A．，Colomer，J．，Munnich，A．，Baiget，M．and
Melki，J．Aframe−Shiftdeletioninthesurvivalmotorneurongene
inSpamishspinalmuscularatrophypatients・Natureqenetics（1995）
11，335−337．

−98−11． Burglen，L．，Spiegel，R．，Ignatius，J．，CobbenJ．M．，Landrieu，P．，
Lefebvre，S．，Munmich，A．andMelkiJ．SMNgenedeletioninvariant
Ofinfantilespinalmuscularatrophy．TheLanCet（1995）346，316−
317．

12・ Clermont，0．，Burlet，P．，Lefebvre，S．，Burglen，L．，Munnich，A．and
Melki，J・SMNgenedeletionsinadult−OnSetSPinalmuscularatrophy・
TheLanCet（1995）346，1712−1713．

13． Burlet，P．，Burglen，L．，ClemOnt，0．，Lefebvre，S．，Viollet，L．，Munnich，
A・andMelki，J・Largescaledeletionsofthe5q13reglOnareSPeCific
toWerdnig−Hofhamdisease．J．Med．Genet．Inpress．
14・ Roy，N・，Mahadevan，M・S・，McLeanetal・ThegenefbrNAIP，anOVel
PrOteinwithhomologytobaculoviruSinhibitorsofapoptosis，isPartiallydeletedinindividualswithspinalmuscularatrophy．Cell（1995）80，167−78．
15・ Brahe，C．，Servidei，S．，Zappata，S．，Ricci，E．，Tonali，P．andNeri，G．
Genetichomogeneitybetweenchildhood−OnSetandadult−OnSet
autosomalrecessivespinalmuscularatrophy．LanCet（1995）346，
74ト742．
16．

Zerres，K．，Rudnik−Schonebom，S．，Forkert，R．andWirth，B．Genetic
basisofadult−OnSetSPinalmuscularatrophy．LanCet（1995）346，
1162．

・17・ ClemOnt，0．，Burlet，P．，Lefebvre，S．，Burglen，L．，Munnich，A．and
Melki，J．LanCet（1995）346，1712−1713．

一99−

MoleculargeneticdiagnosisofEmeW一Drt，ifuss

musculardystmphy

KichiArahatal，MegumuOgawalタYoshihirt）Kuran02，AtsushiUmetani3，

ⅨanakoGotol，YukikoKHayashil）AtsushiNaganol，4，SmiyaKawada3，
GtQjirt｝nmSe，SachioKawai6，RhyozoOkada6，SllinichimKubol・4，粘m

Bong−Yoonland ToshifumiTsukaharal

1・DepartmentofNeuromhscularResearch，NationalInstituteofNeuroscience，NCNP，Tokyo
2・FttiirebioCentralResearchInstitute，Tokyo．
3・FujirebioObihiroResearchLaboratory，Obihiro．
4・The3rdDepartmentofInternalMedicine，TokyoMedicalCollege，Tokyo・
5・DepartmentofNeurology，KanazawaMedicalCollege，Ishikawa．
6・DepartmentofCardiology，JuntendoUniversityMedicalSchool，Tokyo・

Con℃SPOndenceshouldbeaddrttSSedto；DI：KiichiArahata，M．D．lK．A．】
DepartmentofNeummuSCtdarReseat℃h，NationalInstituteofNeumscience，NCNP，
4−1−10gawa−higashi，Kodaira，Tokyo187，JAPAN
TEL：＋81（0）423412711
FAX：＋81（0）423461742

E−mai1：arahata＠ncnaxp・nCnp・gOJP−101−

X−linkedEmery−Dreifussmusculardystrophy（EDMD：MM310300）isnrst
describedbyDrs．DreifussandHoganinalargeVirginianfamilyih1961・
Soonafter，in1966，Dr．Emery，tOgetherwithDr．Dreifuss，rePOrtedthis
diseaseasanewfbrmOfX−1inkedmusculardystrophyafterdetailedclimical，biochemical，andclinicalgeneticobservation（1，2）．EDMDisadiseasecharaCterizedbythetriadof；（i）Slowlyprogressivemusclewastingand
Weaknesswithahumero−PerOnealdistribution，（ii）contracturesoftheelbows，Achillestendonsandposteriorneck，and（iii）cardiomyopathywithconduction
blockwhichisoflenlift−threaterung．In1994，mutationsintheSrA geneat
theXq2810cushavebeenfbundinpatientswithEDMD（3）・ThenameS7u
isonglnated丘omDr．StefanoRivellaencodedanewproteinnamed

emerinsclone−A．Importantly，thisgene（3）．Weraisedantibodiesagainst

Syntheticpeptidefragmentspredicted丘omemerincDNA，tOelucidatethe

Subcellularlocalizationoftheprotein（4）．Bytheseamibodies，WefoundpositivenuclearmembranestalIllnglnSkeletal，Cardiacandsmoothmusclesin
thenormalcontroIsandinpatientswithawiderangeofneuromusculardiseases otherthan EDMD who hada novelnonsense mutationinthe SrAgene（4）．InthenomalcontroIsbutnotinEDMD，aPrOteinimmunoreactive
WiththeantibodieswhichhadarelativemolecularWeightof34kDaexist・
Thesefindingssuggestthespecincdeficiencyofemerininthenuclear

一102−

membraneofmusclecellsinEDMDandthepotentialinvoIvementofemerin
inthepath010gicalprocessofthedisease．

EmerinThehumanSrA geneintheteromericreglOnOfXq28locusis2147bplong，
andhasanopenreadingframeof762ntswhichencodesaserine−richprotein
Of254aminoacidsnamed

emerin（3，5）・EmerinmRNAshowsubiquitous

tissuedistributionwiththehighestexpressioninskeletalandcardiacmuscles（3）・ThecDNAsequenceofemerinpredictsanewtai1−anChoredmembrane
PrOteinthathassimilaritiesinaminoacidsequencetothymopoietins（TMPO）
intheN−andC−teminalregions（−41％）．

Developmentofanti−emerinantibodies
TwopeptidefragmentsSRSSLDLSYYPTSSST（peptideEDl：173−188）andFMQAEEGNPF（peptideED2：245−254）withanextraCystotheN−teminus
WereSynthesized，andcorjugatedwithKLH（4）．ThepeptideEDlhasno
homologytootherknOwnprotein，ThepeptideED2haslittlehomologywith
thymopoietinsβandγ．PolyclonalantiserawereraiSedinNewZealandWhitemalerabbits（anti−EDlandED2）．Monoclonalanti−EDlandED−2
antibodieswerealsoestablisheduslngaCOnVentionaltechnique・Specincityoftheantibodieswasexaminedbyimmunoblot，indirectELISA−103−

andimmunocytochemiCalanalyses．Thepolyclonalanti−ED2antiserumWaS
passedthroughana抗mityCOluⅡm（ProtOnTMKit）Coupledwithasynthetic
peptideCNTFtoavoidthecross−reaCtivitywiththymopoietinsβandγ・

hlIIlunOCytOChemistry
Fro，ZenSkeletalandcardiacmusclebiopsy（Orautopsy）specimenswerepro−

CeSSedfbrimmunOCytOChemistry．Thespecificityoftheimmunostainingwas
examinedbyreplacementofthepnmaryamibodieswithpreimmunerabbit
SerumOrimmunOglobulin，andpre−absorptlOnOfthepnmaryamibodieswith
theantlgenPePtidesEDlandED2・ForthespeciBcidentificadonofnuclear

membrane，eaChtissuesectionwasdoublestainedforimmunOfluorescencewithanti−EDl（OrED2）antibodyandanti−humannuclearmembraneantibody
MAB1274．

Cellularlocalizationofemerin（Figs．1−3）
Wefbundde負ciencyoftheimmunOStalnlngfornuclearmembranesinthe

EDMDskeletalandcardiacmuscles．Theemerin−POSitiveneurologicaldiseasesincluded；Paikinsomism，mOtOrneurOndiseases，metabohcmyopa−

thies，inflammatOrymyOPathies，andmusculardystrophiesotherthanEDMD・
ThenegativestainlngOfmuscle缶bernuclearmembraneagalnSttheantibodies

inpatientswithEDMDwasqultedistinctfromotherdiseasesexamined・ー104−Tissues otherthanSkeletalandcardiacmuscles
ThepositivenuclearmembranestainingofthecontroIsectionswasnot
restrictedtotheskeletalandcardiacmuscles．Itwas alsofoundinothercell
types，i・e・，VaSCularandvisceralsmoothmuscles（Fig・ld），PrimaryspermatO−
Cyteandfollicularcellofthethyroidgland（datanotshown）．Ontheother
hand，neurOnS，Spleencells，renaltubularcellsandhepatocyteshadnoclearly
dehneatedstaimingarOundthenuclearmembrane（datanotshown），indicating
thatthesecellshaveundetectableamountoftheproteinatthenuclear

membraneorhavedifftrentsubcellulardistributionffommyogeniccells‥

hm肌ulOblotanalysis（Fig．4）

Subcellular丘actionationstudiesrevealedthatthe34kDaproteinwas
associatedwiththenuclear丘actionofcontroIsample，butwasabsentin
EDMD・Theanti−ED2antibodiesalsodetectedthe34kDaprotein
inthecontrolmuscle，butwasagalnabsentinEDMD．

AmutationoftheS7A gene
OurEDMDpatientshadanonsensemutationatposition735（GtoA）ofthe
emerincDNAthatchangedatryptophancodon（TGG）toastopcodPn（TAG）・TheGtoAmutationat血cleotide735wasconflrmedbythe
restrictionenzyme（BfaI）digestionoftheamplifiedgenomicDNA．The

ー105−

motherofthebrotherpatientswasconsideredtobeaheterozygousasymp−
tomaticcamierofthedisease．SSCPanalysisofthePCRamph丘edproducts

ofgenomicDNAoftheSrA genealsorevealedthatthebrotherpatientsand
theirmotherhadmobilityshiftsoftheconfbrmerSbands・Noneofthetwenty
丘vecontroIshadanynuCleotidechangesatcDNAposition735，indicatlng
thatthismutation−isnotapolymorphism．

SummaryandConclusions
First，PairedimmunofluorescencestalnlngOftissuesectionswiththeami−EDl（OrED2）amibodyandtheMAB1274markerrevealed畠SpeCincde缶ciencyof
emerininthenuclearmembraneofskeletal，Cardiacmuscles，andvascularsmoothmuscleinEDMD・Theslgni頁canceofthisobservationwasfurther

deteminedbytheimmunOblotanalysis・Thesefindingsindicatethatemerinis
loca】izedin thenuclearmembranes ofnormalskeletal，Cardiacmuscle，andsmoothmusclecells，andsuggestastruCturaland／orfunctionalroleforemerin
inthe nuclear membranes．
Second，nOtallkindsofcelltypesshownuclearmembranestainlng，equally・Amongthecontrolhumannon−muSCletissues，neurOnS，SPleencells，renaltubularcellsandhepatocytesexhibitedmarkedlydiminishedor
undetectableimmunostainlngOfthenuclearmembranes・Theseresultsimply
the（i）presenceoftissue−SPeCificalternativeemerintranscript（S），Or（ii）othertissuerestrictedprotein（S）thatlinksemerintothenuclearmembranewhchis−106−absentin＿nOn−muSCletissues．UbiqutousexpressionofemerinmRNAand

PrOteinwillbeattributed，atleastinpart，tOthepresenceofemerininthe
nuclearmembrane ofvascular smooth muscle cells．

Acknowledgements
WewishtothanktoDr．HideoSugita（PresidentoftheNCNP，Japan）forhis
Sincereencouragementandadviceduringthisstudy．WealsothankDrs．
TadaytlkiIshihara，KyozoYonemoto，KoSahashi，ReikoNamba，Masae
Ono，KoichiSuda，EriArikawa−Hirasawa，Yu−ichiGotoandIhlyaNonakafbr
PrOVidinguscontroltissuesamples．Thissttldyhasbeensupportedby

ResearchGrantsfrohtheMinistryofHealthandWelfhre，Japan，andgrants
fromtheMimistryofEducation，ScienceandCulture．

ー107−

隅g．1

−108−

mg・4

−109−

12

隅g．5

だ還U U双岩讐蒜﹁ ▲↓ト■

uOコLUS三一一q
uO手心一心凸 DuO石和︸⊃∈︸u石d □S︸uむコdn︵ヒ≧凸山 u傭Pむ︸Uむ︺むP SuO；d︸⊃≡ー110−FigureLegends
ng．lIndirectimmunofluorescencestalmngOfskeletalmusclesectionswithmonoclonalanti−EDl
antibody・（a，C−d）Controlnormalmusclesectionsfromapatientwithnon−neurOmuSCular
diseases（takenatthetimeoforthopedicsurgerywithinformedconsent）．In（a）EDl＋nuclear

membraneOfskeletalmusclefibresisvisualized，WhilemusclesectiOnsftomapatientwith

Emery−DreifussmusculardystrophywhohadamutationintheSmgene，theEDlmarker
Showednodetectableimmunostainidgofthemyonucleai（b）．Atahighermagnincation，nuclear membraneindentation canbe visualized as spike−like prqjections（C）．Nuclear
membraneOfvascularsmoothmusclecellsshowpositiveimmunostainingfortheantibody（d）．
Bar＝20microns．

ng．2
Frozensectionsofhumancardiacmusclesstainedwithmonocronalanti−EDlantibodyby
indirectimmunofluorescence・NotethecleatnuclearmembraneStainingofcontrol（a）cardiac
muscle・Bycontrast，therewasnoimmunostainingofthenuclearmembraneofEDMD（b）．
Bar＝20microns．

mg．3
Pairedimmunofluorescence stalnlng Ofmuscle sections with polyclonalanti−EDl rabbit
antiserum（visualizedbygreenfluorescence）andmonoclonalanti−humannuclearmembrane
antibody MAB1274（visualized by red rhodaminefluorescence）．On doubleexposure，in
COntrOlmusclesectionfromapatientwithpolymyositis，EDl＋nuclearmembraneisvisualized

inyellowcolorwhilesarcolemmalmembraneremainsgreen（a）．Ontheotherhand，muSCle
SeCtionfromapatientwithEmery−Dreifussmusculardystrophy，themyonucleairemainsred
COlorduetonegativeimmunostainingofthegreenEDlmarker（b）．Bar＝20microns．

ng．4
Immunoblotanalysisofemerin・Theimmunoreactivecomponentofamolecularweightof34
kDaproteininacontrolmuscle（lanel；arrOW）wasnotdetectedinEmery−Dreifussmuscular
dystrophy（1ane2）．

ー111−lReferences］

1．Emery，A．H．E．andDreifuss，F．E．UnusualtypeofbenignX−linkedmusculardystrophy・
J．Neurolo．Neurosurg．Psychiatr．29，338−342，1966．

2．Emery，A・H・E・Emery−Dreifussmusculardystrophyandotherrelateddisorders・British

Med．Buue．45，772−787，1989．

3．Bione，S．，etal・IdemificationofanovelX−linkedgeneresponsibleforEmery−Dreifuss
musculardystrophy．NatureGenet．8，323−327，1994．

4．Sabine，M．，etal．IdentificationofnovelmutationsinthreefamilieswithEmery−Dreifuss
musculardystrophy．Hum・Mol・Genet・4：1853−1857，1995

5．Bione，S．，etal．IdentificationofnewmutationsintheEmery−Qreifussmusculardystrophy
geneandevidenceofgeneticheterogeneltyOfthedisease・Hum・MoLGenet・4：1859−1863，
1995．

6．Nigro，V．，etal．SSCP detection ofnovelmutationsin patients with Emery−Dreifuss
musculardystrophy：definitionofasmallC−terminalreglOnrequiredforemerinfunction・Hum・

Mol．Genet．4：2003−2004，1995．
7．Nagan0，A．，etal．EmerindeficiencyatthenuclearmembraneinpatientswithEmery−
Dreifussmusculardystrophy．NatureGenet．12：254−259，1996．

8．Manilal，S．，etal．TheEmery−Dreifussmusculardystrophyprotein，emerin，isanuclear
membraneprotein．Hum．Mol．Genet．5：801−808，1996．
9．Yamada，T・andKobayashiT．AnovelemerinmutationinaJapanesepatientwithEmery−
Dreifussmusculardystrophy．Hum．Genet．97：693−694，1996．

−112−

Towardidentification oftheFukuyama−
type congenitalmusculardystrophy（FCMD）gene
TatsushiTm，MDりPhD．

DepartmentofHumanGenetics，GraduateSchoolofIntemationalHealth，
UmiversityofTokyo，Tokyo，Japan

Fukuyamatype congenitalmusculardystrophy（FCMD），the second
mostcommonfbrmofmusculardystrophyinねpan，isanautOSOmalrecessive
SeVeremuSCulardystrophy，aSSOCiatedwithbrainanOmaliesduetoadefectin
themlgrationofneuronsl）．

AsafirststeptoelucidatethegeneticdeftctofFCMD，WePerfbrmed
geneticlinkageanalysesuslng COnSangulneOuSFCMDfamiliesmainly，and
localizedtheFCMDlocustoarOundD9S580nChromosome9q31−332）（fig．1）．
Ibwever，thisreglOnrangedover20cMandwaspresumedtoincludemore
也an400genes．
cM betweenloci D9S127

and CA246，

V

b

ノ

homozygOSitym叩Plngln Patientsborn to consangulneOuS ParentS

y

5

d
m

reglOn Of approximatelyb

FollowlngOurinitialmapplng，Wefurther definedthelocuswithina

recombination analysesin0ther families3・4）．we also fbundevidence forlinkagedisequilibriumbetweenFCMDandmfd220（D9S306）inthiscandidate
reglOn・We suspectedthat the FCMD genecouldlie within1Mb of the
mm220locuslocatedon9q313）（fig．1）．
SinceFCMDis prevalentamOngtheJapanese whichis geneticallyisolatedpopulation，linkagedisequilibriummapplngisfeasibleasinFinland5−6）．

fk｝WeVer，nO markers have been discovered very close to mfd220．We
COnStruCtedayeastartificialchromosome（YAC）contig7）containingmfd220，
developed new microsatellite markers fromthe contig，determined their
POSitiononYACs，andallele−tyPedFCMDandnormalindividualswiththesenewmarkers8）・HP excessM values9），rePreSenting thestrength oflinkage

disequilibrium，WereO・32，0・81，0・95，0・60，andO・45atmfd220，M8，J12，−113−

GATA，and A4，reSPeCtively・DistanCeSbetweenFCMDandeachmarker
were

presumedto＿belMb，160kb，30kb，450kb，and740kb，reSPeCtively・TheseputativedistanCeSWereCOnSistentwitheachmarker−spositiononYACs・Thus，theFCMDgenecouldliewithinareglOnOflessthanlOOkbcontaining
J12andweareabletoperformhighly−focusedpositionalcloning8）（fig・2）・
WefurtherexaminedhaplotypesofFCMDchromosomesatafewloci
ar0undJ12，WhichshowedmaximumvalueofPexcess．Theresultsindicatedthat77％ofFCMD−bearmgChromosomescarriedananCeStralhq）lotypeand
that88％ofFCMDpatientscarriedtwoanCeStralhaplotypeshomozygously，
Or OneanCeStral haplotypeand one putative newly mutated／Chromosome

heterozygOuSly・So far，We have not seen this haplotypeinthe normal
chromosomes，yet．These data verifyour hypothesis of a slngle disease
founderand suggest thatthese markers canbe used forthe diagnosis ofsporadicFCMD，tOagreateXtent，althoughtheyarepolymorphicmarkers8）・
Duringtheafbrementionedprocess，neWfindingsareasfollows：1）Therearefewknownproteinslocatedon9q31．TheFCMDgeneproductmaybea）anunknOwnadhesionmoleculerelatedtoneuronalmigrationorb）anunknownprotein1inkedwiththedystrophin−dystroglycan−1aminin

COmPlex，becauseimmunOStalnlngOftheconstituentsofthiscomplexis
reducedinFCMDmuscles．
2）Asdescribedabove，mOStFCMDpatientsatpresentarederivedfroma
singleanCeStralfbunderS）・
3）Usingtheselinkedmarkers，Prenatalandcarrierdiagnosesarefeasiblein
FCMDfamilies10−12）
4）Theprogressoflinkagedisequilibriummappingallowedustodiagnose
WhetherasporadiccasehasFCMDornot，byhaplotypeanalysisuslngVery
closemarkers8）．

5）AtypicalFCMD（whocouldwalk）familieshavebeenlinkedtothe9q31
markers．¶leSeCaSeSCanbelongtoaseriesofclinicalspectrumOfFCMD．
6）IthasbeendiscussedwhetherFCMDandWalker−Warburgsyndrome（WWS）belongtothesamediseaseentityornot．Weanalyzedafamilyin
Which3siblingswereafftctedwitheitherFCMDorWWS．Theresults
SuggeStedthatbothFCMDandWWSsiblingssharedtheidentical
COmbinationofmutationsoneitheralleleoftheFCMDlocus．Although−114−

WWScouldbegeneticallyheterogeneous，SOmeWWScasescouldbe
CauSedbymutationsintheFCMDgene13）・
7）AneuropathologicalstudyofanfbtuswithprenataldiagnosisofFCMD
revealedthatthebreachesoftheglialimitanSmaybetheprlmaryCauSeOf
micropolygynaseeninthisdiseaselO）・

Insummary，followlngOurinitiallinkageanalysisoftheFCMDgene
to chromosome

9q31−33，We defined thelocus within

a

5

cM，by

homozygOSitymapplngandrecombinationmapplng，andthenfoundlinkage
disequilibrium．Furthermore，bylinkage disequilibrium mapplng，Weare

abouttocomedownontheFCMDgeneitself．

ー115−

2
1
1フL3
1フL3

q

3111 つLZつL﹁−フL 3
1フしつこフLフLフJフー33 3︵−■﹃蒜蓮；■﹄■±

P

4 321 321 1
2 222 1 11 1

D9S1763cM

33…129

轄品≧雪CAZ46

Candidate：ごM］

regl0n

5．5cH
D9S58

Z．8cM
D9SlOS

4．9cM
D9S59

Figl・PhysicalandgeneticlocatiOnsoflinkedmafkersonchromosome9q・1
CM＝aboutlMb．neFCMDgenecouldliewithinaregiOnbetweenD9S127

andCA246and within1Mb ofthe mfd220locus on9q31showing血kage
disequilibrium・−116−

VFICsspannlngtheFCMDcandidateregion
N：Notl，M：Mlul，Nr：Nrul
Nr
M

588kb

MMNr

r

−．−J■′

M

NM

N………………llこノMmtd228

Nr

M

M

I、＼、

M

l﹁．；；；．

N

R
T
R
rU

NM

l■﹁●：：■．−．

M

1−．−⁝．﹂．M／M

L l T ． ： ⁚ ： ：l＼⁝．．⁝．．

LTl1−11−11111一⁝⁝■1⁝■−一−一⁝−粥川47珊ほ98︐
q一774q一2
日コ日日コ日日コ‖

D9S127

M

J8・
J12

1．88−

日．75−

8．58−

P eHCeSS

8．32

distanceotF［MD（kb l198

8．75

238

8．818．95

168

旦8

日．810．68

8．45

168

748

458

Fig2・AYAC contigoftheFCMD genereglOpaJldunkage disequilibrium
mqpplng・ Newly developed miCrosatellite markers are positioned・ ne
maximumPexcess valueis obtainedatJ12．Putativedistancesbetween FCMD
andeachmafkerarealsorepresented・neFCMDgenecouldliewithinareg10n
OflessthanlOOkbcontainingJ12・ー117−

REFERENCE

1）FukuyamaY，OsawaM，SuzukiH：BrainDev3：1−30，1981
2）TodaT，SegawaM，NomuraYetal．：NatureGenet5：283−286，1993
3）TodaT，kegawaS，OkuiKetal．：AmJHumGenet55：946−950，1994
4）TodaT，IkegawaS，MiyakeMetal・：JpnJHumGenet（inpress）
5）HatbackaJ，delaChapelleA，MahtaniMMetal・：Cel178：1073−1087，
19946）MitchinsonHM，0−RaweAM，TaschnerPEMetal・：AmJHumGenet56：654−662，1995

7）MiyakeM，TodaT，MizunoKetal・：（submitted）
8）TodaT，MiyakeM，KobayashiKetal・：（Submitted）
9）Lehe亘iokiAE，KoskiniemiM，NorioRetal・：HumMoIGenet2：1229−
1234，1993

10）NakanoI，FbnahashiM，TakadaKetal・：ActaNeuropath01（inpress）

11）kondoE，SaitoK，TodaTetal．：AmJMedGenet（inpress）
12）YamamOtOT，KomoriT，ShibataNetal・‥（submitted）

13）TodaT，YoshiokaM，NakahoriYetal．：ArmNeuro137：99−101，1995

ー118−

MEROSINOPATHIES

femandoM・S・1址，AnneHelbling−I・ederc，PascaleGuicheney
andMichelFardeau

INSERMU・153，Ⅸveloppement，PathologiC，R6g6n6rationduSyst主me
Neuromusculaire，17rueduFer−a−Moulin，75005Paris

Merosinis thelamininisofbrm specific of muscle fiber basement
membranelLeivoandEngvall・1988］・Lamininisaheterotrimermadeupofone

heavychainandtwolightchainS，Whicharearrangedinvariouscombinationsin
diffbrentbasementmembraneslEngva111993，Tryggvason1993］．Themerosin（orlaminin−2）isfbrmedbyaheavyM（orα2）chainandtwolightchainS：Bl（Orβ1）andB2（orγ1）Purgesonetal・1994］．Apartialdeficiencyofmerosinwas

firstdemonstratedinFukuyamaCOngenitalmusculardystrophy（FCMD），uSing
antibodiesagainstlamininα2−Chain［馳yashietal．1993］．FCMDisaformof

COngenitalmusculardystrophy（CMD）charaCterizedbyitsassociationwith
SeVereCentralnervoussystemdisturbances『ukuyamaetal．1960and1981，
Nonakaand Chou1979，Osawaet al，1991］．Severalhundreds ofaffectedChildrenweredetectedinJq）an，andonlyveryfewoutside．Thediseaseis
generallyverysevere，thehighestmotoflevelreachedbythechildrenbeing

generallycrawlingonthebuttocksortheknees・Thechildrenhaveaverylow
IQ，Oftenassociatedwithepilepsy・TheCTscanShowsdecreaseindensityoftheWhitematter，Ventriculardilatationandcorticalatrophy；theneuropathologicalexaminationconsistentlyrevealsmarkedbrainchanges，WithpachygyrlaOr
micropolygyriaofthecerebralandcerebellarCOrteX『ukuyamaetal，1981；
Osawa et al，1991］．Anautosomal recessiveinheritance of FCMD was
COnSideredashighlyprobablelOsawa，1978］andthisdiseasewasmappedon
Chromosome9q31−33［Todaetal．1993and1994］．

InWesterncountriestheCMDisafrequentcauseofsevereneonatal
hypotoniaofneuromuscularOrlgln，andisoftenreferred toas classicalor
OCCidentalformofCMD『ardeau1992，Banker1994，Dub6witz1995］．Its

ClinicalmanifestationsarenOticedatbirthorinthefirstmonthsoflifeand

COnSistofmusclehypotoniaandweakness，markedlydelayedmotormilestones，ー119−Severeandearlycontractures，Oflenassociatedwithjointdeformities・Serum
creatinekinaseisusuallyraised，uPlOtimesnormal，intheear1ystagesofthe

disease，andthenrapidlydecreases．TheoccidentalCMDhasbeenconsideredas
beingusuallyclinicalcharacterizedby一一pureHmuscularinvoIvement・However，

Whennon−invasiveteclmiquesofbrainimagmgwereused，WhitematterchangeS
WeredetectedinasignificantPrOPOrtionofCMDcases［Bernier etal・1979；
Egger et al．1983；Echenne et al．1986；Trevisanet al・1991］・The

histopathological changeS rePOrtedin muscle biopsleS COnSistedinlarge
Variationinmusclefibersize，aftwnecroticandregeneratingfibers，marked
increaseinendomysialcollagentissue，VariableamOuntOffattissue，andno
CharaCteristicultrastructuralfeatures［Afifielal．1969；Zellwegeretal．1967a，
1967b］．ThesehistologicalchangeSlackofspecificity，aSOthermuscledisorders

maypresentwithsimilarfeatures・ThefrontiersofCMDwereill−definedandthediagnosisofCMDwasoftenmadeafterexclusionofthedifferenttypesof
StruCturalcongenitalmyopathies．Itwasclear，fromthedifftrentseriesofCMD
describedinWesterncountries，thatamarkedclinicalheterogeneityexistedin
thisgroup．Anautosomalrecessiveinheritancewasgenerallypostulated．
ThemarkedincreaseincomectivetissueobservedinmusclebiopsleSOf
PatientswithCMDhassuggestedthatanextracellularmatrixabnormalitycould
accountfbrthepathogenesisofthisdiseaselDuanCeetal，1980，FidzianSkaetal，
1982］butearlystudiesfailedtorevealsignincantChangeSOfcollagenandsome
OtherextracellularcomponentslStephensetal．1982；Hantaretal．1985］．

We haveinvestigated whether one component of the complex of
SarCOlemmalglycoproteins associatedwith dystrophin（dystrophinassociated
COmPlex−DGC）or oneof subunitsofthelaminin，Whichislinkedto thisCOmPlex【seeCamPbell1995andOzawa1995］couldbeinvoIvedintheclassical（OCCidental）fbrmofCMD．WedidnotobserveanySignificantchangesofthe
PrOteinsoftheDGCbutwehavefound，bybothimmunOCytOChemical（Fig．1）
andinmnoblottechniques，COmPletemerosin（1amininα2−Chain）deficiencyin
13casesbutnotinother7cases，alsodiagnosedasCMDslTomeetal．1994］．In

allthosemerosin−deficient cases，aS Wellasin otherssubsequently reported［Hayashietal．1995，Sewryetal．1995，Fardeauetal．1996，Fard3auandTome，
1996，Topalogluetal．1996，Trevisanetal．1996］，1amininα2−Chainwasabsent

not only from the basementmembrane Ofmuscle fibersbut also from thebasement membrane Of Schwann cells of intramuSCular−120−

nerVeSandneuromuscularjunctions・Incontrast，thelamininA（orα1）chain（heavychain

Oflaminin−1）wasoverexpressed，tOVaryingdegrees，arOundmusclefibersinall
merosin−deficientCMDcases−Fig・lrrom6etal．1994］．Inmusclebiopsy
SPeCimensfromnormalcontroIs，Withtheantibodiesagainstlamininα1−Chain，

thelabeling was almost exclusively confined to the bloodvesselbasement
membrane（Fig・1）・Inmostmerosin−nOn−deficientcasestheexpressionthis
PrOteinwasidenticaltothatofcontroIs．However，inseveralcases，therewas
OVereXpreSSionofthisproteinarOundaftwmusclefibers『ard3auandTome
1996］・Theanti−1amininβ1−Chainand†1−Chainantibodies showed some

Variationintheintensityofthelabelinginbothgroupsofpatients，butsimilar
VariationswereseeninotherpathologleS・Theanti−dystrophinantibodiesdidnot
ShowslgnificantChangeSinthedystrophinexpressioneitherinthemerosin−
deficientorinthemerosin−nOn−deficientcases．
Thisstudysuggestedthatthelamininα2−Chaingene（LAMA2）couldcause
themerosin−de負cientCMD，andasthisgenehadbeenlocalizedtochromosome
6q22−q23Ⅳuolteenahoetal・1994］，ageneticstudyinvoIvinghomozygOZity
mapplngandlinkageanalysiswasundertakenonapanelofconsangulneOuS
familieslivinginFranCeandTurkey・This allowedthelocalizationofthe
merosin−deficientCMDlocustoa16cMregionofchromosome6q2lHillaireet
al・1994］・Recentlywehavereaqustedthislocalizationtoa3cMregion（Fig．2），
morecentromericthanPreViouslythought［Helbling−Leclercetal．1995a］．This

localizationwasalsofoundinthreepatientswithtypicalCMDofoccidentaltype
havingtraceofmerosin：afaintstainingofvarylnglntenSltyWaSObservedby
immunOCytOChemistryarOund most muscle fibers【馳lbling−Leclerc et al．
1995a］・Mappingto6q2locuswasexcludedinmerosinnon−deficientCMD
Patients［Hillaireetal．1994］．Themerosin−nOn−deficientCMDfamiliesstudied

byusdidnotmapeithertochromosome6q20rtOChromosome9q31−33，reglOn

WheretheFCMDgenewaslocalizedbyTodaetal．（1993）．
ThesefindingsledtoclinicalreevaluationofthetwogroupsofCMD
Patients，definedaccordingthenormalpresenceorthedeficiencyofmerosin，
and whofulfilledthe criteria agreed bytheInternationalConsortium on
Congenital MuscularDystrophy［Dubowitz1994】．Theinitial evaluation

SuggeStingthatthesetwogroupscouldbedistinguishedbytheirclinicalpattems
andtheirseveritylseethereftrencetoFardeaupresentationatthe22ndENMC
1994workshop on CMD，in Dubowitz and Fardeau1995］，WaSlargely−121−confirmedby our subsequentstudies［Dubowitz1995，Fardeauet al・1996，
Fard3au and Tomg1996］．They showed that the clinicalphenotype washomogeneous and typicalin merosin−deficient CMD group butit washeterogeneousand often atypicalin merosin−nOn−deficient CMD・This
demonstratedthatthese two groups were distinctand suggested that themerosin−deficient CMD was a clinically well−individualized entity，uSually
CharaCterized by severe congenitalhypotonia，COntraCtureS，delayed motor

developmentandwhitematter hypodensitybyRMIor scanbrainimaglng『arde？uetal・1996，FardBauandTom61996］・Theseverityofthediseasewas

reflectedbythefactthat4（OutOfaseriesof150fourcases）diedbefbretheage
Of6yearS『artbauandTome，1996］．Incontrast，aSeriesof15merosin−nOn−

deficientCMDcasesformedamoreheterogeneousgroup，generallywithless
SeVerity『ard3auandTom61996］．Neonatalhypotomiawasrarelymarked，

COntraCtureSWereCOnStant，butrarelysevere．Motordevelopmentwasrarely
SeVerelydelayed．Noneofthechildreninthisserieswerereportedtohavedied・It dlOuld be noted that similar difftrences between merosin−deficientand
merosin−nOn−deficientpatientswerefbundinotherseriesofcaseslPhilpotetal・
1995，Topalogluetal．1996］．

The searchfor mutationsinLAMA2geneinmerosin−deficientCMD
Patientsallowedtofindsplicesiteandnonsensemutationsinthisgeneintwo
families［ⅠもIbling−Leclerc etal．1995b］．Thesemutationsleadto truncatedlaminin−α2proteinSandsuggestedthatmutationsintheLAMA2genecausethis

SeVerefomOfmusculardystrophy．
Itcanbeconcludedfromourstudiesthatmerosin−deficientCMD should
beconsideredasisawel1−dennedclinical，mOrPhologicalandgeneticentity．

Acknowledgments．Thiswofkwassupportedinpartbythe一一Association

Fran9aisecontrelesMyopathies（AFM）T

．

ー122−

lamimina2chain

lamininαlchain

Control

CMD

Fig．1．IrrmunocytochemiCalanalysisoflamininSα20rM（rightmicrographs）andαl

OrA（leftmicrographs）chainsinserialsectionsfromthemusclebiopsiesofanormi
COntrOl（uppermicrographs）and ach址Iwithcongenitalmusculardys仕OPhy−CMD（lowermicrographs）・Shownis absence oflamininSα2chain andoverexpression oflamininSαlchainaroundmuscleBbersintheCMDpatient．MagnificationX265．

−123−

つ一2
一

︻∋43

Chromosomell

11 1 1 1 1

つ一︵J

／

4■さ

／．

1

′0

／4cM

D6S1715

D6S407

／
／

／
／
つ 一
つ一

LAMA2

3
つ−
4
つ一

＼
＼

D6S1620

■
3

つ一

＼

＼

′
0
7

へェ一2

D6S1705

＼

＼lcM
＼

＼
＼

D6S1572

Fig・2・ScheI血crepresentationofchromosoIn36，Showingthelocalizationofdle
merosin−dencientCMDina3cMinterval，betweenD6S407and郎S1620markers・The
D6S1620washomozygousforallthepatientsstudied（Helbling−Leclercetal・1995a）・

ー124−

Reftrences

AfifiA．，ZellwegerIl，McCormickW．F．，MergnerW．Congenitalmuscular
dystrophy：lightand electron microscopIC Observations．L NtuTOl．

飴UmgU掛物止血L32：273−280，1969．
Banker BQ．Congenitalmyopathies．In：Ab，01qgy，2nd ed．A．G・Engel，C・FranZini−Armstrong，eds．，VOl．2．NewYork：McGraw−fLllBookCompany，
1275−1289，1994．

BernierJ．P．，BrookeM．，NaidichR．，CarOllJ．Myoencephalopathy：Cerebral
hypomyelinationrevealedbyCT Scamer oftheheadinamuscledisease．
Tねng．Am．∧buroJ．ASg．104：244−246，1979．
BurgesonRE，ChiquetM，DeutzmannR，EkblomP，EngelJ，KleinmanIt
MartinGR，MeneguzziG，PaulssonM，SanesJ，TimpIR，TryggvasonK，

YamadaY Yurchenco PD．Anew nomenclature for thelaminins．肋trix
月foJ．14：209−211，1994．
CamPbellK．P．Threemusculardystrophies：lossofcytoskeleton−eXtraCellularmatrixlinkage．CblL80：675−679，1995．

DuanceV．C．，StephensIIR．，DunnM．，BaileyA．J．DubowitzV．Arolefor
COllageninthepathogenesisofmusculardystrophy？♪ねtuTe284：470−472，
1980．
DubowitzV．肋scIemsoLdtt

S血ChiLdbood，2nded．London，Philadelphia：

Saunders，93−105，1995．
Dubowitz

V．一

22nd

ENMC

sponsored

worshoponcongenital

musculardystrophyheldinBaarn，TheNetherlands，14−16May1993日．臓uTOmuSC．
β血ord4：75−81，1994．
DubowitzV．，FardeauM．Workshop report：PrOCeedingsonthe27thENMC
SPOnSOredworshoponcongenitalmusculardystrophy．抽uL

OmuSC．DLsoTd．

5：253−258，1995．

EchenneB．，ArthuisM．，Bi11ardC．，CamPOS−CastelloJ．，CastelY．，DulacO．，
FontanD．，Gauthier A．，Kulakowski S．，De Meuron G．，MooreJ．R．，
Nieto−BarreraM．，PagesM．，ParainD．，PavoneL．，PonsotG．Congenital
musculardystrophy and cerebral Cr scan

anOmalies．Results of a

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7−22，1986．

ー125−

EggerJ．，Kendall B．E．，Erdohazi M．，Lake B．D．，WilsonJ．，Brett E．M．
InvoIvement of

the central nervous systemin congenital muscular

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−129−

Muscle RegenerationinProgressive
Muscular Dystrophy，NdbuyukiMurakami，YokoSaito，Yuichi（btoDepartmentofUltrastruCturalResearch，NationalInstituteof
Neuroscience，NationalCenterofNeurologyandPsychiatry（NCNP），
Kodaira，Tokyo187，Japan

Ⅰntroduction

Itiswell−kn0wnthatthemusclefiberiscq）ableofregenerationafter
necrosisnotonlyinnormalbutindystrophicmuscles1−2）・Despitethepresence

Ofnumerousregenerating fibers，the muscleweaknessis progressiveIIin
PrOgreSSive muscular

dystrophies．To understand the reason why theregenerativeprPCeSSCannOtCOmpenSateformuscledegenerationinprogressive

musculardystrophies，WePerformedacomparativestudybetween抑拘り
andmdxmice．Theformerhasprogressivemuscleweaknessleadingtodeath
atapproximately3−4monthsofage，WhilethelatterhasnoapparentClinical
SymPtOm，eVenthoughthereisobviousmusclefibernecrosisinbothanimals．

1）Muscle fiber growth and fiber type differentiation do notdifferin dy；皿dxandnormalmuscles3）
Inboth句′andmdxmice，muSClefibersizeandfibertypedifftrentiation
WereSimilaruPtO10−15daysofagewhennecroticfibersbegantOaPPear．
ApartfromthenecroticareaS，themusclefiber morphology appearedtobe
normal，i．e．therewasno distinctdifferenceinmusclefiber size，fiber type

differentiationandthenumber ofmyosatellitecells．Musclefibersprobably
undergonecrosisonlywhentheyreachacertainstageofmaturation．ThesefindingssuggestthatthereisnorelationshipbetweenmusclefibergrowthandSubsequentdiseaseprogression．

2）Thecellularresponsetomusclefibernecrosisismorestriking
inzzzdxthan4ymouse
Thenecroticprocessappearsafter15daysofageinbothanimals．The
necroticfiberstendedtobeaccompaniedbyverymarkedcellularreSPOnSein−131−mdxmuscle，WhiletherewereonlyscatteredreactivecellswhichchanCedlittle
Phagocyticactivityin句′muSCle（Figl）．Afterlmonthofageinmdxmuscles，
mostofthemuscleapartfromtheclustersofnecroticfiberswerereplacedby
CentrOnuCleated regenerating fibers，Whereasin㊥′muSCles necrotic

andregeneratingfiberswerescatteredthroughoutthemusclewithmatkedvariation
infiber sizeandinterstitial fibrosis．The earlierand moreintense cellularresponse to muscle fiber necrosisis probably the crucial eventleading to
Subsequentmoreactiveregenerativeprocessinthemdxmouse．Itis well−knownthat factors originatingfrom themacrophage stimulate satellite cell
PrOliferation，eSPeCiallyPDGF（plateletderivedgrowthfactor），bFGF（basic
fibroblastgrowthfactor）andTGF−β（transfbrminggrowthfactor−β）4）．

3）MyoD and myogenin are expressedin the early stages ofmuscle regeneration

Tounderstandhow叫わDandnv，qgen血，geneSreSPOnSiblefbrinitiation
Of myoblast proliftrationand myotube fbrmation

are eXPreSSed

in

the

regenerativeprocess，WeeXaminedthesegeneproductsimmunohistochemicallyinaneXPerimentallyinducedmyonecrosismodel．Bupivacainehydrochloride（MarCaine⑧），alocalaneStheticwhichinducesmassivefibernecrosisandrapid

regeneration，WaSirjectedintotheadultratsoleusmuscle5）．Almostallthe
fibers underwent necrosisimmediately after theinjection．Macrophages
becameeVidentat24hoursandthenecroticmusclewas丘11edwithmononuclear
Cells（mostlyacidphosphatase−POSitivemacrophages）by48hoursafterirtiection，Whensatellitecellswereactivelydividing．At72hoursafterfiber necrosis，
SmallbasophilicregeneratingfiberswithcentralnucleiwiththemorphologlC
CharacteristicsofmyotubesbegantOaPPearamOngthemononuclearcells．The
myotulxsincreasedinnumberandsizeandtheregeneratlngfibershadrapidly
almost totally replaced the necrotic fibers5−7days afterthe bupivacainehydrochlorideirtiection6）．

MyoDwasmaximallyexpressed48hoursaftermusclefiber necrosis
Whensatellitecellswereactivatedandhadstartedtoproliferate・Thesatellite
Cell，rerrmantOfthemyoblastduringmyogenesis，PlaysanimportantrOlein
musclefibersregenerationtoformthemyotube・Myogeninwasmaximally
expressedwhenthebasophilicmyotubesappearedamOngthenecroticfibers，theexpressionofbothgeneproductswassimilartOthatseenintheregenerativeー132−

PrOCeSSduringmyogenesis，butthisoccurredoveraveryshortperiod（Fig2）．

4）MyoDandmyogenin are expressedin4yandRZdxmuscles
ToclarifywhethertheAb，ODandL77yqgeL］血areeXPreSSedduringthe
regenerativeprocessinprogressivemuscular dystrophies，andwhether gene
expressionis different between mdx and句′

muSCle，We aPPlied

immunOhistochemicalstainingtobothanimalmusclesatage6weekswhenfiber
necrosisandregenerationareactive．
MyoDandmyogeninwereexpressedinbothanimals：theMyoD was
POSitiveinmononuclearCellsinthenecroticareaS，andmyogeninintheearly
regeneratingfiberswiththemorphologiccharacteristicsofthemyotube（Fig3）．
Sincethecellularresponseandregenerativeprocessweremoreactiveinthemdk
muscles，thenumbers ofMyoD andmyogeninpositivecells werefarmOre
numerousinthemdxmusclesthan鴎′muSCles．Inconclusion，therewasno
qualitative differenceinMyoDand myogenin expressions，but there were
quantitativedifftrencesbetweenmdxand句′muSCleswhichweresimplythe

resultsofthedeferrlngeXtentOfthecellularreSPOnSe，bothnecroticaswellas
regenerativeprocessesbetweenthetwoanimals．Althoughwehavenotapplied
theimmunOhistochemicalstalnlngtOhumandystrophicmuscles，Webelievethat
itishighlylikelythatboth叫印かandmyqgenLr］Playanimportantroleinmuscle
regenerationinbothirtjuredanddiseasedhumanmuSCles．

−133−

Figl．nereismassivefitx：rneCrOSiswithextensivecellularreaCtion（asterisk）inthe
mdxmousemuscle（A）．NoteanaggregateOfregeneratingfiberswithcentrany
Placednuclei（arrows）．In4ymouse（B），thereareonlyscatterednecroticBtx：rS（arrOW）withlidecellularreaCtion．Tnereismi1dendomysialfibrosis．
A，B：hemotoxylinandeosin，×450．

MyoD

Myogenin

Bupivacaine

l
0

1

2

3

4

5

days

Necrosis」

Phagocytosis SateHitecell Earlymyotube myotube myotube
Satellitecell proliferation
activation

Fig2：MyoDandmyogeninexpressioninexperimentallyinducedmusclenbrneCrOSis
followedbyregeneration．MyoDismostintensivelyexpressed2daysafter
myonecrosiswhenthesatellitecellsstarttOPrOliftrate，andmyogeninat3−4days
Whenearlymyotubefbrmationoccurs．

−134−

Fig3：MyoDisstronglyexpressedinboth4y（A，B）andmdx（C，D）mousemusclesin
necrotiC且bersfilledwithmacrophagesandproliftrabngsatellitecells．Thereisno

qualitativebutthereisquantitativedi飴rencetx：tWeenthetwoanimals．
A，C：hematoxylinandeosin，B，D：immunOStalnlngWithanti−MyoDantibody．
A−D：×600．

−135−References

1・NonakaI，TakagiA，SugitaH：Thesignificanceoftype2cmusclefibersin
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4・RobertsonTA，MaleyMAL，GroundsMD，PapadimitriouJM：Theroleof
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5・NonakaI，TakagiA，IshiuraS，NakaseH，SugitaH：Pathophysiologyof
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ActaNeuropatho160：167−174，1983
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LocalProduction ofIL−12p40Prevents

Allogeneic MyoblastRejection
KazunoriKatol，OsamuShimozatol，Ken−ichiHoshi2，Hideo

Yagital，KoOkunural，andTakeshiSato2
1DepartmentOfImmunplogy，JuntendoUniversitySchoolofMedicine，Tokyo113；and2DepartmentofNeurology，NationalCenterofNeurology
andPsychiatry，Chiba272，Japan

ABSTRACT

Thep40subunitofinterleukin12（IL−12p40）hasbeenknowntoactasan
IL−12antagOnistiL7VitTO・We

here

describeit］Vivoimmunosuppressive

effect ofIL−12p40in allogeneic myoblast transplantation・A murine
myoblastcellline，C2C12，WaStranSducedwithretrovirusvectorscarrylngthe
lacZgeneasamarkerandtheIL−12p40gene・IL−12p40secretedfromthetransfectantinhibitedtheIL−12−inducedinterferonY（IFNl）production by
SPlenocytesinvitTO・SurvivalofC2C12tranSPlantedintoallogeneicrecipients
WaSSubstantiallyprolongedwhentranSducedwithIL−12p40．Cytokine（IL−2
andIFNl）productionandcytotoxicTlymphocyte（CTL）induction against
allogeneic C2C12Wereimpairedintherecipientstransplanted withtheIL−
12p40transfectant・Delayed−tyPehypersensitivity（DTfT）req）OnSeWaS also
Slgnificantly diminishedintheIL−12p40recipient・Furthermore，Serum
antibodiesagainstβ−galactosidaseoftheT−helperl（Thl）−dependentisotypes（IgG2andIgG3）weredecreasedintheIL−12p40recipients．Theseresults
indicatethatlocallyproducedIL−12p40exertsapotentimmunosuppressive
efftct on Th1−mediatedimmunereSPOnSeS thatleadto allograft rqection・
Therefore，IL−12p40genetransductionwouldbeusefulfor preventing therqiectionofallograftssuchasmyoblastandgeneticallymodifiedowncellsthat
aretranSducedwithpotentiallyantigenicmoleculesingenetherapyincluding
dystrophintransduction．

⊥137−

INTRODUCTION
ThlcellsthatproduceIFN†andmediatecellularimmuneresponseshave
beenimplicatedinthepathogenesisofallograftr亘iection（1−2）．Recentstudies
revealed a crucialrole ofIL−12inthe Thl development（3）・IL−12is aheterodimericcytokinecomposedof35kD（P35）and40kD（P40）subunits，
thelatter of whichis responsible fbr receptor bindingP−5）It has been
demonstratedthattheexpressionofp35andp40isdifftrentiallyregulatedand
thatp40Canbeproducedasmonomerorhomodimerintheabsenceofp35，
WhichactsasanIL−12antagOnist血vILro（6−8）・Iもwever，thephysiological
functionofIL−12p401tlVIYOremainsunclear・Inthisstudy，WeeXaminedtheil］YiYOeffbctofIL−12p40by

tranSducingtheIL−12p40geneinto

myoblast

allograftandfoundapotentimmunosuppressiveeffect・Apossibleutilityof
thisstrategyforpreventingr再ectioninallogeneicmyoblasttranSPlantationfor
muscle dystrophy andin gene therapyintroducing potentially Lantlgenic
moleculesisdiscussed．

RESULTS戯ocムemfcaJ

胡d戯oJ呼元aJα∬aCk汀血。m Of

血cZ

胡d

几一J勿40肋娩C加わ．
In order to easeidentification of the tranSPlanted myoblastsin therecipientskeletalmuscle，WefirstintroducedthelacZgeneencodingE．coliP−
galactosidase（β−gal）as amarkerintothe myoblast cellline，C2C12（CZ），
OriginatedfromaC3H（Ii2k）mouse・AstabletransfectanteXPreSSingahighlevelofβ−gal（C2Z）wasestablishedbyFACSsortingafterFDGstaining（Fig．
1A）・Subsequently，C2ZwasfurtherintroducedwiththeIL−12p40geneandastabletranSfectantproducingahighlevelofIL−12p40（C2Zp40）wasselected（Fig・lB）・Moreover，IL−12p40proteinswithapparentmOlecularWeightof
40kD wereimmunOPreCipitated byanti−mOuSeIL12p40mAb from the
SuPernatantOfC2Zp40cellsunderbothnonreducingandreducingconditions（Fig・lC）・Thetripletmostlikelyrepresentsdifferentlyglycosylatedforms

OfIL12p40．
Wenextverifiedtheit7VitroeffectofIL−12p40secretedbyC2Zp40・
Culture supernatant Of C2Zp40，Which containedIL−12p40atlOO nghnl，ー138−

exhibitedanantagOnistic efftct on rIL−12−inducedIFNI production by

SPlenocytes（datanotshown）．

凸egerya。0月OfC2年仰血AJ呼eⅢe元入亜ce．

Toestlmatethein vivoeffect ofIL−12p40，We tranSPlantedC2Z and
C2Zp40into the quadriceps offu11y allogeneic C57BL／6（Ii2b）mice．
Survival of

the tranSplanted myocytes was periodically estimated byhistochemicalstainingofthetissuesectionswithaβ−galsubstrate（X−gal）and

IIE．AsrepresentedinFig．2AatdaylOafterthetranSPlantation，intactC2Z
Cellsexpressingβ−galdisappearedwithin8daysandamassiveinfiltrationof
mononuclearCellswasobserved，indicatingtheoccurrenceofatypicalcellularr再ection．In contrast，intact C2Zp40cells expressingβ一gal were well
PreSerVedin spite of amassive mononuclearinfiltration（Fig．2B）．Some
C2Zp40cellsappearedtobeincorporatedintothemuscleandwere■preserved

even after4weeks after the transplantation．These resultsindicate that
locallyproducedIL−12p40iseffbctiveinpreventlngtheallograftr亘iection．

q′わ肋e伽duc血刀andCn血血C。仰血喝40−餌軋口甲an妃d几ガce．
To analyzethe mechanisms for the prolonged allograft survival，VeestimatedcytokineproductionandCrLinductionfromtheC2Zor C2Zp40

recipients．Whenre−StimulatedwithirradiatedC2Zinvitt・0，IL−2andIFNY
PrOductionbysplenocyteswasimpairedintheC2Zp40recIPlentCOmParedto
theC2Z recipient（Fig．3A）．Furthermore，nOCTLactivitywasinducedin
thesplenocytesfromtheC2Zp40recipient（Fig．3B）．Theseresultsindicate
thatlocallyproducedIL−12p40preventedthedevelopmentofIFNY−PrOducing
ThlcellsandalloreactiveCTLprecursors血vIYO．

血路揖0月Ofj）mRe甲0月ge血C2年40Rec亘）血f．
To prove thatlocally producedIL−12p40suppresses Th1−mediated
CellularimmunereSPOnSeS血vLvo，WeneXtinvestigatedwhetheritalsoaffects
DTII DTHresponseswereelicitedintheC2ZorC2Zp40−tranSPlantedmice
bys．C．irtiectionofC2ZintoearOrfootpad，andwereslgnificantlydiminishedintheC2Zp40recipient（datanotshown），indicatingthatlocallyproducedIL−
12p40inhibited theinduction of cellularimmune reSPOnSeS rePreSented by
DTH．

−139−

鬼0りpeか血出血0月Of良肌皿血痛やっp血co血ねSeA血わ0deg．

InordertofurtherverifytheeffectoflocallyproducedIL−12p400nthe
Thldevelopment，Weanalyzedtheisotypeprofilesofserumantibodiesraised

againsttheb−galintroducedinto theallografL It hasbeenkn0wnthat the
PrOduction ofIgGl

andIgG2／3is mainly helped by Th2

and Thl，

respectively（9・10）・AscomparedwiththeC2Zrecipients，Serumantibodiesof
IgG2andIgG3isotypeswereslgnificantlydecreasedintheC2Zp40recIPlentS（Fig．4）．ThisfurtherindicatesthattheThldevelopmentinresponseto theallograftswassuppressedbythelocallyproducedIL−12p40．
DISCUSSION
Inthepresentstudy，WedemonstratedthatthetranSductionofIL−12p40
geneisanefftctive way for preventing allogeneic myoblast rqiection．

TranSPlantationofallogeneicmyocyteshasbeentriedtoimprovethemuscle
functionsinpatientswithseveremusculardystrophybutsofaraChievedlittle
SuCCeSS due to rapid r亘iection（11−12）

our present strategy would be

immediatelyapplicabletosuchacase．
Duchennemusculardystrophy（DMD）iscausedbydefectiveexpression
Ofdystrophin（13）・Sinceithasbeendemonstratedthatintroductionofnormal
dystrophintransgeneintothemdxmice，ananimalmodelfbrDMD，COrreCted
dystrophic symptoms，a Similargene therapy for DMDis expected（14−15）

However，theintroduction ofdystrophin geneintoimmunOCOmPetenthosts
innatelylackingdystrophinmightleadtor再ectionofthemodifiedcellsdueto
immune reSPOnSeS against the dystrophin・Consistent with this notion，We
recentlyfoundthatC57BL／10myoblaststranSPlantedintothemdxmiceare
acutelyrqectedinsplteOfthesamegeneticbackgroundexceptfordystrophin
andthat dystrophinitselfserves as the CTL targetantigen（manuSCriptin

Preparation）・SuchacompromisinghostimmunereSPOnSetOtheintroduced
geneproductsisalsoexpectedingenetherapiesforothergeneticdeficiencies，
SuChashemophilias，WheremyoblastsmayserveasvehiclesfbrgenetranSfer（16−17）・Therefore，COtranSductionoftheIL−12p40genealongwiththetarget
genewouldbeaneffectivestrategyfbrpreventingtherqjectionofgeneticallymodi丘edcells．

−140−

A
（⊃
（⊃

打．

○

0

Oや

0
0

ONL

十二＿

（⊃
N

Oの︻

O
tO

0▼N

⊂）
¶

N

○○︻ ONr

LUq∈コZニ00

やつ

0
▼
N

006

⊂I
く〉
（1

Fluorescencelntensity

C
kD

「

lO6−
80−

C2Zp40500

1000

Productiono目し・12p40（ng／106／24h）

Figure l．

Characterization of C2C12transfectants．（A）Expression of P−galactosidase・

Parental C2C12（C2）anditslacZ transfectants（C2Z and C2Zp40）were stained
fluorescentwith aβ一D−galac10Sidase substrate，FDG，aS describedin Materials and Me（hods，andanalyzed on a FACScan．（B）Production ofIL−12p40・Supernatants were collected after

culturing the cells（l x106／ml）for24h，and concentration ofIL12p40was measured by
ELISA．Data represent mean＋SD oflriplica（e wells．（C）Immunoprecipitation ofIL−12p40

fiomc2Zp40．C2Z（lanesland3）andC2Zp40（lanes2and4）werelabeledwilhl35S】Met andl35S］Cys，andthelabeled

proleil−S

SeCretedinto

culturesupernatants

wereimmunopreclpltated with anti−mOuSeIL−12p40 mAb−COnJugated Sepharose・ The precIPltateS
were subjected to SDS−PAGE（7．5％）under nonreducing（lalleSl and2）or ruducing（lanes 3
alld4）conditions and then to fluorography．The positions of molecular weight Standards are
illdicated at theleftill kD．

−141一Figure 2・Preservation of C2Zp40allograft．C2Z（l￠）or C2Zp40（right）cells（lxlO6）
Werelnjectedinto quadriceps of C57BL／6 mice． Ten days after the transplantation，tissue
SeCtions were prepared and stained with X−gal and HE as describedin Materials and Methods・00￠￠dd−巴−0品が∞∞︵一∈∋︶uO葛コPOaトN﹄

0

へ●∈盲d︶uO笥nPOaN・﹂−

C2Z C2Zp40

C2Z C2Zp40

−20

10

5

Figure 3・ CeHularimmune responses of C2Zp40 recIPlentS．（A）Cytokine production・
Splenocy【es from the mice trallSplanled with C2Z ropen column）or C2Zp40rclosed coLumn）
Were CO−Cultured withirradiated C2Z for

2

days．

Concentrations ofIL−2

andIFNγin the

SupernatantS Were meaSured by ELISA．（B）CTLillduclioll． SplellOCyles from the micetransplanted with C2Z（OPen Circle）or C2Zp40（Closed circLe）were co−Cultured withirradiatedC2Z for5days・Cytoly【ic activity against C2Z was measured by6h5lcr release assay at theindicated E／T ratios・Data represent mean±SD of trlplicate wells．

−142−

︵0等＜︶OUU月LOSqV
IgG2alsotypeFigure 4・Isotype profile of serum anti−β−galactosidase al叫ibody．SerumIgM，IgGl，IgG2a，
IgG2b andIgG3 anti−β−galactosidase Abs from C2Z（Shaded bars）or C2Zp40（Closed bars）
trallSplanted mice were determined byisotype−SPeCific ELISA as describedin Materials andMelhods．

Data represenl mean＋SD of triplicale samples．

lhreeindependent experimentS．

一143−

Similar results were obtainedill

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Strom，T．B．（1994）CunlqPin．血munoL．6，757−764．
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E．＆Fleischer，B．（1994）EuLILL71muL701．24，2435−2440．
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Rtide，E．＆Germann，T．（1993．）EuLIL血munol．23，2202−2208．
8・Gillessen，S．，Carvajal，D．，Ling，P．，Podlaski，F．J．，Stremlo，D．L．，
Familletti，P．C．，Gubler，U．，Presky，D．H．，Stem，A．S．＆Gately，M．K．
（1995）且丑J」血mm正25，200−206．
9．Finkelman，F．D．，Ⅰもlmes，J．，Katona，I．M．，Urban，J．F．，Beckmann，M．

P．，Park，L．S．，Schooley，K．A．，Cofhan，R．L．，Mosmann，T．R．＆Paul，W．E．（1990）Annu．ReY．血munol．8，303−333．
10．Germann，T．，Bongartz，M．，Dlugonska，H．，Hess，H．，Schmitt，E．，Kolbe，L・，KOlsch，E．，Podlaski，F．J．，Gately，M．K．＆Rtide，E．（1995）EuLIL
五mm日月0⊥25，823−829．

11．Gussoni，E．，Pavlath，G．K．，Lanctot，A．M．，Sharma，K．R．，Miller，R．
G．，Steinman，L．＆Blau，H．M．（1992）♪ねtuLe仏ond．）．356，435−438．
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Cb〟．54，447−452．

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CamPbell，K．P．，Faulkner，J．A．＆Chamberlain，J．S．（1993）NiztuLe（LoJ】dJ．364，725−729．

15．Karpati，G．，＆Acsadi，G．（1993）肋scleNtTV．16，1141−1153．
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Clift，S．，Bems，A．＆Cohen，L．K．（1995）Ptt）C．NbtI．Acad．Sci．USA．
92，1023−1027．

17．Dai，Y．，Schwarz，E．M．，Gu，D．，Zhang，W．，Sarvetnick，N．＆Vema，I．
M．（1995）伽C．♪由比Acad．gci．uSA．92，140ト1405．

−145−

Permanentsystemicdelivery oftherapeutic
PrOteins ffomgenetically modified
myofibers
N．NafEakhl，D．Bohll，C．Pinset2，D．MontarraS2，OJ〕anosl，and
J．M．Heardl
1LaboratoireR6troviruSetTransftrtG至n6tique，2LaboratoiredeBiochimie，InstitutPasteur，Paris，France

Abstract
InanattemPttO uSe Skeletalmuscles for thepermanent delivery oftherapeuticproteinsintheserum，Wehavetransducedmousepnmarymyoblast
Cultureswithretrovirusvectorsandengraftedthegenetically−mOdifiedcellsin
recipientanimals．Repeated exposures to retrovirus vectors allowed gene
tranSferintonearlOO％ofculturedcells．Irtiectionofculturedmyoblastsinto
regeneratingmusclesgaverisetoupto60％ofgenetically−mOdifiedmyofibers．
Various enhanCer−deleted retrovirus have been used，in which theexpressionofthecDNAofinterestwascontrolledbyaninternalpromoters，
includingvariouscombinationsofskeletalmuscle一甲eCificenhanCer−PrOmOter

elements．Twopotentiallytherapeuticproteinswereconsidered：alysosomal
enzyme（theβ−glucuronidase）for whichadeficientmousemodelisavailable（theMPSⅥImouse）andEpo，WhichstimulatesHbFsymthesisandhasbeen
PrOPOSedfbrtreatinghumanhemoglobinopathies．
Invitroexperimentsinmyoblastsandmyotubesindicatedthataspeci丘cexpressionin differentiated myotubes canbe achieved by uslng retrOViral

VeCtOrSCOntainingmusclespecificcontrolelementssuchastheMLCenhanCer
associated with the o−aCtin promoter or thelmmandesminenhanCerand
PrOmOter．托ghlevels ofthe reporter proteins were detectedin culture
SuPernatantS．Afterinvivoimplantation，β−glucronidase−SeCretingmyofibers
allowedacompletecorrectionofthediseasedphenotypeindeficientmicewhich
WaSStablefbratleast8months．Epo−SeCretingmyo丘bersinducedapermanent−147−

inCreaseofthehematocritwhichroseover75％．Thesedataindicatedthata
Permanent delivery of a therq）eutic protein canbe obtainedinmice by
implantinggenetically−mOdifiedmyoblasts・
However，inmanydisordersincludingEpo−reSPOnSiveanemias，genetherapymightbeproposedonlyiftranSgeneeXPreSSionlevelscanberequested・
The tetracycline resistance operon of E・COLL provides a tranSCriptional

regulatorysystemseffectiveinmammaliancells・AchimerictranSaCtivator（tTA）consistingofthetetracyclinerepressorfuSedtothetranSaCtivationdomain
OfHSV−VP16stimulatestranSCriptlOnbybindingaminimalCMVpromoter
COntaining repeats ofthe tetracycline operator（op7CMV）・Bindingis
abolishedbytetracycline，thusimpalrlngPrOmOteraCtivation・
Wehaveconstructedtwoenl1anCer−deletedretroviralvectors carrylng
eithertheEporeportergeneunderthecontroloftheop7CMVpromoterorthetTAgeneunder thecontrolofthemuscle−SPeCifichumandesminenhanCer−
PrOmOter，C2・7myoblastsweretranSducedwiththesevectorsandthelevelof
Eposecretionwasexaminedinvitroandafterengraftementinrecipientmice，
eitherinthepresenceorintheabsenceoftetracycline・Weobservedthatthe
tranSaCtivationbytTAcouldbeabolishedinthepresenceoftetracycline，bothin
CellculturesandinanimalsrecelVlngtetraCyCline．Theseexperimentsshowed
thefeasibilityofusingtetracyclinetomodulatetheinvivosecretionlevelsofa
therapeuticproteininapolyclonalcellpopulationtranSducedwithretrovirus
VeCtOrS．

一148−

Effective genetransferinto skeletal
muscle using adenoviralvector

NationalInstituteofNeuroscience，NationalCenterofNeurologyand
Psychiatry，Kodaira，Tokyo187，JAPANEffectivegenetranSftrintoskeletalmuscleisoneoftheimportant
StePS fbr genetherapy upon hereditary muscle diseases such as muscular
dystrophy・A widevariety ofmethOds havebeen developedtointroduce
DNAinto skeletalmuscle cells，including calcium phosphate precipitation，
liposome−mediatedtransfer，electropolation，retrOVira1−mediatedtranSferandadenovira1−mediatedtranSfer・Amongthem，rePlication−incompetentadeno−
VirusgenetransftrhasseveraladvantageS；thatcanintroducetranSgeneinto
non−dividingcells，and that alsoallows the promoter dependent tranSgene
expressioninthetargetedcells・

Whenrecombinantadenovirus wastranSferredintoneonatalmouse
Skeletalmuscle，tranSfectedgenecanperSistinthenucleusfbr alongtime，
howeverwhentranSferredintoadultskeletalmuscle，reCOmbinantadenovirus
COuldnotbeincorporatedintoskeletalmusclel）．
Inthispaper，Idescribeeffectiveadenovira1−mediatedgenetranSfer
intoyoungadultC57BIJlOmouseskeletalmuscle．
We have gotten three recombinant adenoviruses，AxCALacZ2），
AxSRαLacZ2）and AxMIK−IIBLacZ・hAxCALacZ，thelacZgene wasdrivenbyβ−aCtinpromoterwithCMVenhanCerandinAxSRαLacZ，thelacZ

gene wasintroducedby SV−40andITrLV−l dualpromoter・Whereasin
AxMI忙−IIBLacZ，WemadeuslngthemethodTTCOS−TPCH3），thehcZgene

WaSdrivenbythemousemuscle−SPeCinc胴genepromoter4）・

WhenAxCALacZwastransferredintomousemusclecellline，C2
undertheconditionofm．0．i．10（multiplicityofinfection），almostlOO％ofthe
Cellsrevealedβ一galactosidaseactivities．Ontheotherhand，AxMHC−IIBLacZ

didnotproduceapparentaCtivitiesinthegrowlngC2cells，butweobserved
CertainnumbersofP−galactosidasepbsitivewel1−difftrentiatedmyotubes，Whenー149−

Weevokeddifferentiationaftertheinftction．Tocomparetheeffectivenessof
thepromoters，Weinfectedthe same amOuntS Ofrecombinant adenoviruses（m．0．i．10）intoC2cellsandexaminedβ−galactosidaseactivities（Tablel）．In
thisexperiment，aftertheinftctionwithrecombinantadenoviruses，WeeVOked

difftrentiation of the C2cells by changlng the medium．Among threerecombinant adenoviruses，AxCALacZ revealedmuch higher activities than
Othertwo，AxSRαLacZandAxMHC−IIBLacZ．TheactivitiesbyAxCALacZ

atthree days aftertheinfection was290times morethanthat of vacantadenoYirusAxIW，thathasnoinsertioninSteadofEIA，EIBandE3gene
deletion．Atsevendaysaftertheirtjection，the activitybyAxCALacZ was
167timesmorethanthatbyAxIW．AxMHC−IIBLacZpresentedratherlow
β一galactosidaseactivity，buttheactivitieswereincreasedattendaysafter the

infection．Thispatternreflectedtheexpressionprofileofthe丸田C−HBgene
PrOmOter，that was only expressedin highly difftrentiated fast glycolytic
nbers4）．
In order to determine whether adenoviral vector couldinfectinto
POSt−dividing differentiated cells，C2cells wereinftcted with AxCALacZ
under m．0．i．10at various times after weevoked differentiation．Cells were
harvestedthreedaysaftertheinfectionandtheβ一galactosidaseactivityamOhg

Sameam0untSOfproteinhavebeenmeasured．Cellsontheplateswerealso
Stained with X−gal to examine whether myotubes really expressed

β一galactosidaseor not．Thehighestβ−galactosidaseactivities，Wehave gotten
WaSfromthecells，Whichweinfecttherecombinantadenovirus6days after
WeeVOked differentiation．Indeed，highlydifferentiated myotubes revealed
StrOngβ−galactosidaseactivityintheplateunderthesameintervalbetweenthe
initiation of differentiationand theinfection．

This dataimplies

th．at

adenoviralvector couldinftctintopost−dividing，eVen highly differentiatedmyotubes．WhenAxMHC−IIBLacZwasinfected，β一galactosidaseactivitywas

generallylow，buttheactivitiestendedtoincreasewhentheperiodbetween
Changlngthemediumandinfectionwasbecomlnglonger．Thisdataindicates
thatthe transgene expressionincultured muscle cellis regulated by the
PrOmOterintherecombinantadenoviruS．
Then，Weintroducedtheserecombinantadnovirusesintoyoungadultmouseskeletalmuscleinvivo．Weirtiected withAxCALacZinto anterior
tibialmuscle of5−6weeks oldC57BL／10mice（Table2）．After the various−150−

Period，WetOOkoutthemuscleandfixedthemusclespecimentostainwithβ−galactosidaseandhematoxylin−eOSin．Sevendaysafter theirjection，66％ofmusclefibersintheanterior tibialmusclewerestainedwithβ一galactosidase

andfburteendaysafter，arOund50％ofmusclecellsshowedpositiveactivities．
Ⅰもwever，β一galactosidasepositivecellsbecamegraduallyreducedandlO％of

thefiberswereremainedtobepositiveattwenty−eightdaysafterthe・irjection．
Wenoticedthat，however，eVenin180daysafter theinftction，10％of thefibersstillexpressedenzymeaCtivities，althoughactivitiesthemselveslooked
Weak．β一galactosidaseactivitieswerealsoobservednotonlyatmusclefibers

butalso atintrafusalfibersandcapsuleinmuscle甲indle，andatendothelial
Cellsinvein．
We also examinedirtiected muscle uslngimmunohistochemical
StainingagainstCD4andCD8．Sevendaysaftertheirtiection，CD4positive
Cellshave been foundintheskeletalmuscle specimen．Theinmtration oflymphocyteswereprominentfburteen daysafter theirtiectionandCD4and
CD8positive cells were often detected surroundingβ一galactosidasepositive

Cells，amOngthemCD8positivecellsgraduallydominatedandwasevidentat
twenty−eightdaysaftertheirtjection．
Wealsocomparedtheefficiencyofeachrecombinantadenovirusin
Vivo transferinto skeletal muscle（Table2）．Inthe occasion of AxMHC−
IIBLacZ，the same amOuntS Of recombinant adenovirus waslqeCtedinto
anteriortibialmuscleofthesameage，−howevertheefficiencywasdrasticallydiminishedfromgalactosidasethat

activityofat

AxCALacZ．No

SeVen

days

after

musclecells

expressed

P一

theirtiection．Infiltrationoflymphocyteswererecognizedregardlessofnoβ一galactosidasepositive cells．

Moreover，When a vacant adenoviral vector，AxIW wasintroduced，
lymphocytesinfiltrationhas been also noticedin skeletalmuscle specimen．
These dataindicate thatnot only theproduct ofinserted gene such as P−
galactosidase，but alsoleaky expressed viralproteininduceimmunologicalreactionininftctedskeletalmusclewithrecombinantadenoviruS．
Theresults，highefficienciesofgenetransferuslngadenoviralvector
hasneverbeenobtainedinprevioustransferstudiesintoadultrodentskeletal
muscles・Theefftctivegenetransferintoyoungadultrodentskeletalmuscle
COuldbeLexplainedbyl）the charaCterOfbackbone replication−incompetentadenovirus2）highlyeffectiveCAGexpressionunit，Chickβ−aCtinpromoter−151−

With CMVenhanCerandβ−globinpolyadenylation signa13）suitablemouse
Strainfbradenoviralgenetransfer．
Therecombinant adenovirus，We uSedin thisstudy，areEl／E3deleted
typeofvectorsandhave beenmade usingthe COSrrPCmethod3），Whichenables homologous recombinationmuch easierthanthe previous method．
Furthermore，the tranSCriptional orientation ofthe tranSgeneisleftward，
OPPOSitethedeleted El geneandthesame directionas theE4gene．This
OrientationmighthelptheeffectivetranSCriptionofthetransgene，becausethe
expression unit withleftward orientation revealed the higher yield oftranSgene PrOduct thanthose with rightward orientation（PerSOnal

COmmunication）．

We showed the CAG expression unit revealed high expression of
tranSgeneinskeletalmuscle comparedwith SRαPrOmOter（SV−40，ITLV−1

dualpromoter）andthe加圧肛−ILB gene promoter（myosinheavy chainIIB
PrOmOter）．Sincethese expressionunits hasthe samebackbone adenovirus
andthesameE．ColiJacZgene，thedifferenceoftheβ一galactosidaseexpression

Canbeexplainedbytheeffectivenessofthepromoter．The CAGpromoter
itselfhas been proved as strong promoterinVarious tissuesin tranSgenic
mouse studies，howeveramOng SeVeral tissues this promoter revealed the
StrOngeStaCtivitiesinskeletalmuscle5）・
The mouse strain alsolargely contributes the efficiency of the

tranSgeneeXPreSSion．Weused C57BL／10mice，becausethis strainhas theSame background as dystrophin deficient mdx mice． Whereas，Other
researchers usually preferred BALB／c micqinidection study to skeletal
muscle with recombinant adenovirusl）・There has been no comparison
betweenmousestrainininfectionstudyintoskeletalmuscle，howeverinthe
liver，Barret．al6）clearlydemonstratedprolongedpersistenceinC57BL／6and
C57BL／10micerelativeto BALB／Cwhenthe commonadenoviralvector was
infected．
Thecurrentproblemthatwehave，ishowtopreventeliminationprocess
Of the tranSgene−eXPreSSed muscle fibers to prolong the expression oftransferred gene・We presented the CD4and CD8positive cells were
ParticipatedintheeliminationprocessandnotonlythetranSgenePrOductbut
also viral proteinevoke theinfiltration of thelymphocytes・Toinduce
immunOlogicaltoleranCeagainsttranSgenePrOductssuchasβ−galactosidaseandー152−

agalnStViralproteinssuchasE2aisoneofthenextstepsforthegenetranSftr
intoskeletalmuscleuslngadenoviralvector．

−153−

．層雲OJd望︑Slでnまぶ白き〇月SG0月Sj

S蔓等竃dOS召芯010d−亀・改む卓JOhOJJOp︼雇員ISpS払巴ぎ灯心占ト・苫OpuO月S遥hdSSdOS名唱○︸Udt品・npSJUJdt

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貞きpOJUdu霊ttUUNUu−SO芸人でdOSdp電0−Udt品・nJOuOS焉d∈OU IUtq責ー154−

．sl已む眉J乱雲已Oで爪音 巴ぎUS百〇七pUーd−nOtd呂0月

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References

1）AcsadiG，JaniA，MassieBetal．
Adifferentialefficiencyofadenovirus−mediatedinvivogenetranSferinto
Skeletalmusclecellsofdiffbrentmaturity．HumMoIGenet3：579−584，
1994．

2）KanegaeY，LeeGwang，SatoYetal．
EfncientgeneactivationinmammalianCellsbyuslngreCOmbinantadenoviruSeXPreSSlngSite−SPeCi丘ccrerecombinase．NucIAcidsRes
23：3816−3821，1995．

3）MiyakeS，MakimuraM，KanegaeYetal．
EfncientgenerationofrecombinantadenoviruSeSuSlngadenoviruSDNA−terminalproteincomplexandacosmidbearingthefu11−1engthvirus
genome．ProcNatlAcadSciUSA93：1320−1324，1996・
4）TakedaS，NorthDL，DiaganaTetal．
MyogenicregulatoryfactorscanaCtivateTATA−COntamlngPrOmOter
elementsviaanE−boxindependentmechanism．JBioIChem270：15664−
15670，1995．

5）NiwaH，YamamuraK，andMiyazakiJ．
Efncientselectionfbrhigh−eXPreSSiontranSftctantswithanovel
eukaryoticvector．GenelO8：193−200，1991．
6）BarrD，TubbJ，FergusonDetal．
StrainrelatedvariationsinadenovirallymediatedtranSgeneeXPreSSion

frommousehepetocytesinvivo：ComparisonsbetweenimmunOCOmPetentandimmunodeficientinbredstrainS．GeneTherapy2：151−155，1995．

−156−

Developmentofadenovirusvectors fbrgene
therapyofDuchennemuscular dystrophy，NicolasDECON馴CK2，PauleOKM8Nl，Jean−MarieGILLIS2andMidlelPERRICAUDETl
1URA1301CNRS，InstitutGustaveRoussy，39rueCamilleDesmoulins，94805Villejuif，FRANCE．2LaboratoiredePhysiologieGきneraledes

Muscles，Universit6Cath01iquedeLouvain，55AvenueHippocrate，1200
Bruxelles，BELGIQUE．

We haveinsertedintoanadenovirus vector a dystrophincDNA

copy（6．3kb）clonedfroma65year−01dpatient，PreSentingaverymildformof
Beckermusculardystrophy．ThiscDNAcopycodesforatruncatedformof
dystrophin，COntainingalargedeletioninthecentralroddomainofthemolecule
and

named

minidystrophinT．．The

recombinantadenovirus（AdRSVmDys）

WaSintramuSCularlyirtiectedonceintodifftrentmusclesofnewbommdxmice，
takingadvantageOftherelativetoleranceoftheimmunesystemandveryhigh
SuSCePtibility ofimmature myOfibres to adenovirus．

The number ofminidystrophin−POSitivefibres could reach two−thirdoftotalmuscle fibres．
Mhidystrophinexpressionwas stableatleastfor oneyear・AmaJOrity of
nucleiinthese fibres was at the normal submembranous position while
dystrophin−deficient一mdx

minidystrophin can

myofibres

werecentronucleated・Expression

PrOteCt efficiently the fibres againstofthe necrosis−

regenerationprocessthataffectsthedystrophicmousemuscles．Theseresults
PrOmPtedustodeviseexperimentsmeanttOSeeifminidystrophin−eXPreSSlng
fibreshavealsorecoverednormalmechanicalproperties・
We have submittedisolated rapid muscles from adult mdxmice torepeated contractions with stretches，Sinceitiskn0wnthatthese eccentric
COntraCtionsproduceahighlevelofstress．Theforcedrophasbeenfoundto
bearOund60％Onaverageinthemdxmousefastmuscle，COmParedto20％on
averageinthenormalmousemuscles・Thiscorrelateswiththeimportantfibre
damagethatcanbeobservedinmdxmousemusclesections．htemediatefbrce
drops has been foundiniItiected muscles，inversely correlated with the−157−

PerCentageOffibresexpressmgdystrophinineachanimalandcorrelatedwith
thenumber ofdamagedfibres・Theseresultsleadustotheconclusionthat
muscleswithmorethan40％offibresexpresslngdystrophinwouldhaveaforce
dropalmostsimilartOthenormalmuscle・Theseexperimentsshow，for the
firsttime，afunctionalcorrectionofamuscleafteranuneVenSOmaticdystrophin
genetranSfbrinmyopathicmdxmicemodel・Toknowwhethertheefficientandlong−lastingresultsobtainedinthe
newbommicecouldbeextendedtoadults，WeeXPerimentedon：
1）theimpactofageatirtiectioninnormalandmdxmutantmiceand2）thedurationofexpressioninadultnormalandgenetically一血nosuppressed
scid mice．

Adenovirus seems to tranSduceimmature fibres much more

efficientlythanmatureones．▼Irjectionintoimmnocompetentmiceresultsina

largedestruCtionofinfbctedcellsby3weeks−time・Incontrast，WeObserveda
longtermexpressionofthetranSgeneinscidmousemyofibressincetheyare
PrOteCtedagainstthespecificimmunereq）OnSe・StrategleSbasedongenetic
mamPulationoftheadenovirusvectorandimmunOSuPPreSSivetreatmentswere
PrOPOSedtopreventthehostimmunereSPOnSe・−158−

AdenoviralGeneTherapyof
Neurodegenerative Diseases

GHaasel，B．Pettmann2，E．Vigne3，S．A虹RDraghial，
ccainaudl，LPoenarul，比SchmalbruCh4，A．Kahnl
lINSERMU・129，ICGM，24，ruedufbSt．Jacques，75014PariS；2INSERM

U・382，IBDM，CaSe907−Luminy，13288MarSeule；3cNRSURA1301，IGR，
39，rueCami11eDesmoulins，94805V山ejuif；4Instituteof
NeurOphysiology，UmiversityofCopenhagen，Blegdamsvej3，DK2200
Copenhagen，DenmarkN

Introduction

Genether叩y，thatisthetherapeuticuseofgenesasdrugs，COuldhave
two mainfields of applicationin neurodegenerative diseases，hereditarydiseasesandanydiseaseinwhichatherapeuticprotein，Can，inprlnCiple，bereplacedbyitsstruCturalgeneorcDNA．
Severalneurodegenerativediseasescouldbeaccessibletogenetherapyln

thefuture（for review：1）・Recently，tranSftr ofthetyrosinehydroxylase
gene has been reportedinananimalmodel for Parkinson disease．In
AIzheimer disease，tranSftr of genesfbr neurotrophic factors（i．e．NGF），
acetylcholinesymthesisenzymesorscavengersofamyloidβproteinhavetobe
COnSidered・Ourgenetherapyq）PrOaChesfortwoother neurodegenerative
diseases，mOtOrneurOnaldegenerationlikeamyOtrOPhiclateralsclerosis，ALS，
WhichisinmostcasesacquiredandTay−Sachsdisease，aninheritedlysosomal
StOragedisorder，Willbepresentedbelow．

Genescanbe transferredto theCNS by2mainStrategies，throughtran甲1antation of eズ

YiYO genetically modified cells or by direct

administration血vivo・uSlng Viralornon−Viralvectors・Our group wasamOngthefirsttodemonstratetheftasibilityofadenoviralgenetransferinto
theCNSusingareplicationde丘cientadenoviralvector．

−159−

Adenovirusesaremadeupofalinear36kbDNAgenomeflankedby2
invertedterminalrepeats・Their genome canbedividedinto early genes
expressedbeforeonsetofviralreplicationandlategenescodingforstructuralproteinS・Adenoviralvectorsarederivedftomserotypes20r5・Theyare
deletedinreglOnSElandE3toaccomodatefbrelgnDNA・Secondgeneration
vectorsarefurthercrlPPledbyatemperature−SenSitivemutationintheE2A
gene・Therecombinantadenovirusesareinfectiousbutreplicationdefective・
Adenoviralvectors（DEl／E3）Canbepropagatedinthe293cellline which
tranSCOmPlementsfortheEIAandEIBgenefunctions・WildtypeadenovirusesonlycausebenignsymPtOmSmOStlyintheupper
respiratorytractandtheeye・Theyhavebeenusedinattenuatedformas
vaccinesin thousands of US−SOldiers without reported side−efftcts・
Adenoviralvectors have a broad hostand celltype rangeinCluding non−dividingcells・TheycanbeproducedathightitersuptolO12pfu／ml，are
relativelystableanddrivehighleveltranSgeneeXPreSSionwithefficientpost−
tranSlationalprocesslng・InthehostcelltheyremaininanePISOmalposition・
Ontheotherhand，theircapactiyforfbrelgnDNAiscurrentlylimited
to8．5kb，thereisa riskofrecombinationevents withnaturally occurrlng
adenoviruses，andthe hostimmune response canreSultinpartialloss ofinftctedcells．TheseinflammatOryPhenomenaare Substantiallyreducedin
SeCOndgenerationadenoviralvectors・

PotentialandlimitationsofadenoviralenetransftrintotheCNS

TheadenoviralvectorusedinourinitialexpelmentSenCOdestheE・COli
enzymebeta−galactosidaseundertheLtranSCriptionalcontroloftheRSVLTR
PrOmOterandisdeletedingenereglOnSElandE3・Thisvectorwasusedto
inftctembryonicratneuralcellsinprimaryCulture（2）・Neurons，labeledby
anantiY−enOlaseantiserum，andastroglia，labeledbyananti−GFAP−antiserum，
COuldbeinfectedveryefficiently・Theexpressionlevelwasstableforatleast
OnemOnth．
Administrationinto various reglOnS Of the CNS was performed by

StereOtaCticirdection（3）．Whenthevector wasirdectedintothenucleusof
thehypoglossus，Virtuallyallneuronswithinthisstructurewereinfectedandー160−

X−Gal−Stainingextendedtoaxons・Interestingly，theadenovirusitselfdiffuses
POOrlyfromtheiItiectionsiterespectingtheanatOmicallimitsofthenucleus．
Theadenoviruswasalsol叫eCtedintotheventralhornofthespinalcord・Thisprovokessuchahighexpressionofthebeta−galactosidasethattheenzyme
diffusesintothecytoplasm，dendritesandaxonsofneuronsdespiteitsnuclear
localisationslgnal・Thisprovidesa〟GolgilikeIIcellstaining・In these experiments no cytopathic efftct has been observed foradenoviraldosesinftriorto5xlO7pfuandnogrossadverseeffectsonthe
animalshealthandbehaviorwerenoted．
Administrationinto theventricularSyStemleadstoexpressionofthe

tranSgeneinnumerousependymalcellsasvisualizedbyimmunOfluorescence．
The virus does not pass the glialimitans membrane．

Quantitative

measurementsshowedthatthenumber ofinftctedependymalcellswasstablefrom8to30daysaflerirtjectionwithameanoflinfectedcellperhundred
叫eCtedpfu．
ThespreadoftheadenovirusthroughthewholeventricularSyStemis
illustratedasependymalcellsinthecentralchannelofthelumbarSPlnalcord
areinfectedatdistance oftheirtiection site whichwasinthe suboccipitalreglOn・

Tosummarize：adenoviralgenetranSferinto alimited CNS structure
through stereotacticirjectionis feasibleand efficient．Ibwever，mOSt
neurodegenerativediseasesarecharaCterizedbyamultifocalor disseminatedPathology・Inorder toreachmorewidespreadneuronor gliapopulations
Otherstrategiesarethereforerequired：Osmoticdisruptionoftheblood−brain
barrierfollowedbyintraarterialirjectionrepresentsonepromlSlngmethod．
Alternatively，ePendymal cells，infected afler

intracerebroventricularadministration of adenovirus，might serve as cellularPlatforms fbr the
SeCretionoftherapeuticproteinS・Aprerequisitetothisq）PrOaChwouldbe
the efficient diffusion of such proteins ffom the LCRinto the brain
ParenChymeandtherein．

ThereisanOtherpossibilitytotargetPreCisereglOnSintheCNSwith

adenoviralvectors：aXOnaltransport・WhentheadenoviralvectorislqeCted
intothestriatum，labelingcannotbeobservedonlyinthestriatumbutalsoin
neuronsofthe▼Substantianlgra，indicatingthat

tranq）Orted retrogradely

theviralvectorshavebeen

from axon terminals（3，4）．In contrast，nO

ー161−histochemicalstainingwasobservedinneuronsthathaveaxonscrossmg−butnotending−intheiItjection site，SuggeStingthatviralparticlescannOt be
endocytosedbyaxonsofpassage・

An0ther examPle of viral tranSPOrt at distanCeisillustratedin theolfactorysystem（5）．Infectionofmitralcellsisshown4days after nasal
instillation of adeno P−galactosidase vector・In fact，the tranSPOrt WaSanterogradeinthiscase，andassociatedwithpassageofthevectorfromaxonal
teminalsofolfactoryneuronstomitralcellsoftheolfactorybulb・
Fromtheolfactorybulbthevectorisabletoreachlocuscoeruleuscells

血ichprqiecttotheolfactorybulb．SuchvirustranSPOrtisalsoseeninthe
neuromuscularSyStem．IntramuSCularirtiection of adenoviral vectors canleadtotheirretrogradeaxonaltranSPOrtinmotorneuronsofspinalcordand
brainstem．

−Sachsdiseaseasacandidatedisorderfbr

enethera

WeareinterestedindevelopingastrategyofgenetherapyfbrTaySachs
disease，alysosomalstoragedisorderduetodeficiencyofhexosaminidaseA，
anenZyme COmPOSedof2subunits，aandβ．The diseaseis causedby
mutationsinthegenecodingfbrtheα−Subunit・ThelossofhexosaminidaseA

activityleadstoaccumulationofundegradedGM−2ganglioside，Particularlyin
neurons，and resultsin progressive motorand mental retardation．A

quantitativecorrelationexistsbeweenresidualenzymeaCtivityandseverltyOf
thedisease（6）．
AnadenoviralvectorcarrylngtheIもxαCDNAunderdirectionofthe
RSV−LTR was used toinfect cultured fibroblastsfrom Tay−Sachs patients
devoidofHexαmRNA asshownbyNorthern−blotanalysis・Infectedcells
accumulate more specific mRNA than controI cells・Ⅰもxosaminidase A
actlVltyOfTay−Sachsfibroblastsassayedwithspecificsubstratesisrestoredby
the vector，tOtally on theartificial substrateand partially on the natural
Substrate．Furthermoreaveryhigh secretedHbxAactivityisfoundin theculturemediumofinftctedcells．
Thepartialrestorationobservedwiththenaturalsubstratecontrastswith

thehighleveloftranSgeneeXPreSSion．TheWesternblotindicatesthatthis
Phenomenonis probably due to overexpression ofα−Chain precursorsin−162−

infbctedfibroblasts，leadingtoabnormalprecursormaturationandabnormal
accumulationoftheseprecursors．
InfectionofTaySachsfibroblastswiththehexosaminidasealphavector
allowedtonormalizegangliosidemetabolismuslngthefollowlngeXPerimental

PrOtOCOl・InftctedcellswereloadedwithradiAnbelledGMlgangliosideand
Subiectedtoa7daychase・ExtractedGangliosideswerethenseparatedby
thinlayerchromatography・Innormalcells，GMlisconvertedintoGM2bylysosomalbeta−galactosidase，thenintoGM3byhexosaminidaseA・InTay−
Sachsfibroblasts，GM2camot be hydrolyzedinto GM3and accumulates．
Infectionofthese deficient fibroblastswithinCreaslng titers ofthe vector
PrOgreSSivelynormalizesthegangliosidepronle（7）．

WearenOWWOrkingon aknockoutmodelof臨xAdeficientmice
Which，although not suffering the Tay Sachs neurological symptoms，accumulateGM2inVariouscellsassoonasthesecondmonthoflift，andwe
areteStingtheefficiencyofourvectoronbothenzymedeficiencylnVarious
OrganSandabnormalgangliosideaccumulation．

Administration‡OfneurotroDhicfactorsviaEenetranSftr

NeurotrophicfactorsplayanimportantrOleinthenormaldevelopment
and maintenanCe Ofneuronal subpopulationsand may be required under

Pathologicalconditions（8，9）．Neurotrophic factorslike NGF，BDNF andNT−3，CNTF，GDNFandIGF−1are nOWinvestigatedina variety ofneurodegenerative diseases，eitherin preclinicalor clinical studies．The
Clinicalpotentialof the recombinant factors howeverislimited bytheir
PharmaCOlogicalproperties・
CiliaryneurOtrOPhicfactor，CNTF，forexamPle，hasaveryshorthalf
lifeinserumoflessthan3minutes，SeVereCytOkine−likesideeffetssuchas
fever，CaChexia，hepaticdysfunctionandisnotabletocrosstheblood−brain
barrier・ForinstanCeafterivinjectiononlyO．04％ofCNTFarefoundinthe
SPinalcord（10）・Thatis why gene tranSfer might beanadvantageOuS
alternativetodeliverCNTFandotherneurotrophicfactorsinamorestablefashionandtoapreciselocalisation．

WeconstructedanadenoviralvectorcodingfbrCNTF．OnNorthern
blotoftotalRNAsffominftctedfibroblaststwotranSCrlPtSOfexpectedsizeー163−thatusedifferentpolyAsignalsoftranSCriptionterminationarevisualized・

SecretionandbiologicalactivityoftheadenoviralCmFweredemonstratedin
achicken ciliary ganglioncellsurvivalassayuponaddition ofconditioned
mediafrom adenovirusinfectedfibroblasts．The conditioned media show arise of CNTF activity withincreaslng adenoviral dose comparable to thecontroIwithrecombinantCNTFprotein・ThereisnoCNTFsecretionafter
inftctionwithacontroladenoviruS．
血viYOadministrationoftheCNTFvectorbyintracerebroventricular

injectionleads tothe accumulation ofCmFmessenger RNAinnumerous
ependymalcells，aSdetectedbyinsituhybridizationinthethirdventricleor
byimmunOhistochemistryinthelateralventricles・
An0therwelldocumentedactionofCNTFisitsdifftrentiatingeffecton
glialcells，02Aprecursorcellsandastrocytes・Stereotacticadministrationof
theadenoCNTFvectorintothestriatumresultsinamqorincreaseinGFAP
immunostalnlng，diffuSingoutsidetheareaOftranSgeneeXPreSSiondetectedby

CmFimmunOStaining・Thisisprobablyduetosecretionofbothexogenous
andendogenousCNTFbyinftctedcells，leadingtoactivationofsurrounding
astrocytes（11）．

Neurotro hic factorsandmotorneurondiseases

Various neurotrophic factorshavebeen shovm to exert neurotrophic

efftcts on motor neurons，in vitTO（12）and血vivo（13，14）．In ourhboratory we are mostinterestedin CNTF，Ciliary neurOtrOPhic factor，
BDNF，brain−derivedneurotrophicfactor，NT−3，neurOtrOPhin−3andGDNF，
glialcell−derivedneurotrophicfactor・
The neuroprotective effect of our CNTF adenovirus has been
demonstratedusingmotorneurons（11）．Embryonicchickenmotorneurons，

PurifiedbyimmunOPannlng，donotsurviveinculturefbrmorethan4days・
Adeno−CNTFinfectionincreasesmaximalsurvivalto8dayscomparableto a
muscle extract．Thereisanadditive effect of both treatments suggesting
COOPerationofCNTFwithothertrophicfactors．
Wenext tested neurotrophic factor adenovirusesin the mouse model

PL71n（15）． 卿−mice are Suffering a progressive motor neuronaldegenerationbegirnlnginthehindlimbs．ApnlnmOuSeaged4weeks does

−164−

notspreadthetoesoftheir hindlimbsandshows amarked atrophy ofthe
Pelvicgirdle．Lateronthediseaseextendstotheforelimbsandothermuscles
andleadstodeathbeforeday52・DeathisprobablycausedbyresplratOry
failureduetodegenerationofmotoraxonsirmervatingtherespiratorymuscles・
丹qgTeSdvemotot

neuL

OnqPa叫′iswellcharaCterizedbyhistologicaland electrophysiological studies・Itis caused byanautOSOmal recessive
mutationwhichhasbeenmappedtochromosome13byJ・L・GuenetIsgroup

Whoalsointroducedthea句acentmarkerExtratoe（16）．鵜homozygOteS
dieinutero，XypL71nheterozygotes，tWOthirdsoftheprogeny，haveaclearly
Visibleextradigitandcanbeeasilyidentifiedatbirth．Thisallowsfor thetreatmentofhomozygeOuSPnOmice beforethe onset offirst clinical
SlgnSOfneuronaldegeneration．
The RT−PCR experiment demonstrateslong−term eXPreSSion of the
CNTF adenovirus after

intramuSCular

VeCtOr administrationinto newborn

mice・Vh detect the adenoviral CNTF RT−PCR productinallinjected
gastrocnemiusmusclesbutinnoneofthecontrolmuscles・▼Analogousresults

WereObtainedwiththeadenoviralvectorthatencodeBDNFandNT＿3．

MeanSurVivalofnon−treatedpLnL7miceisabout40days．Adenoβ一galactosidaseadministered byintramuscularlnJeCtion does not modifythe
SurVival・

A combination of adenoviral vectors coding for difftrent

neurotrophicfactorsimprovedsurvivaltoameanof64daysandamaximum
OflO5days・Thisrepresentsanincreaseinsurvivalofmorethan50％（G．

Haaseetal・，inpreparation）・Atday50anon−treatedsurvivingpIL7mOuSe
WaSCharaCterizedbyseverehindlimbatrophyandparalysiswhilethetreatedanimalhadmuchlesspronouncedatrophyandabettermotorperformanCe．

Wecountedthenumberofmyelinatedaxonsinthephrenicnerve．At
day25，untreatedpmnmicealreadyshowedmassiveaxonallossascomparedtocontrolmiceofthesamelitters・Thisaxonaldegeneration wasinpart
PreVentedbyintramuSCularvectoradministration．
Weare CurrentlylnVeStigating4difftrent hypothesesto explain thebenefitofneurotrophicfactorsadministeredbyadenoviralgenetranSftrinto

muscle・Adenoviralinftctionofmuscleisknowntobeveryefficientinthe
neonatalperiodandcanreSultinsustainedsecretionofrecombinantPrOteinS．
Accordingtoalstmechanismtheneurotrophicfactorscouldbetakenupby
motorneuronsattheirnerveendingsandtransportedtothecellbodiesashas−165−been shownfbr CNTF，BDNFand NT−3．Otherwisethey could have a
trophiceffectonmuscleaswassuggestedinthecaseofCNTF・Nextthey
could galn aCCeSS tO the circulationand act systemically・Finally the
adenoviralvectoritselfcouldbetran甲Ortedtothespinalcordviaretrograde
axonaltranSPOrtaShasbeenrecentlydemonstratedinmotorneurons（17）・
Combinations of neurotrophic factor adenoviruses qppear

Very

PrOmlSlng becausethe factors act on difftrent membrane reCq）tOrS，Via
diffbrentintracellularSlgnallingmecanismsandmightbenecessaryatdifftrent
momentsofneurodegenerationorfbr distinctmotorneuronsubpopulations・
Adeno CNTF，NT−3，BDNF

and GDNF

are administered−alone orin

COmbination−byintramuSCular，intravenous，intraperitonealorintracerebro−
Ventriculariqjections．

Wealsostudytheimpactoftheantiadenoviralimmuneresponsewhichmightbeattenuatedbytheuseoffuturegenerationadenoviralvectorsorafter
tolerizationbyintrathymicirtiectionintheneonate（18）・
Ofcourse，muChmoreinvestlgationswillbeneededtodetermineifsuch
Strategiesare alsousefulinhumanmotorneurondiseases，SpinalMuscular

AtrophiesandAmyotrophicLateralSclerosis・一166−

Acknowled ements
WethankProftssor S．Nicolaidisforhelpandadvicewithstereotactic
Surgery，Drs．Lisovoskiand Dreyfus from Dr．Peschanskislaboratory for
motorneuronculturesandCNTF−immunOhistochemistry．

Reftrences

1．Karpati，G．，etal．77endsNtumscl．19，49−54（1996）．
2．Caillaud，C．，etaLEuL・JNtuLVSC15，1287−91（1993）．

3．Akli，S．，efal♪加蝕ef3，224−8（1993）．
4．Ridoux，V．，efaJ．月ra血Reg．648，171−75（1994）．

5．Draghia，R．，etaLGene77zeLW2，418−423（1995）．
6．Poenanl，L．〟∫丁場dぶd12，35−46（1996）．
7．Akli，S．，etal．G即e77zeLW，Submitted，（1996）．
8．Thoenen，H．77endsNtutDSCL14，165−70（1991）．
9．Lindsay，R．M．，eta1．7h由NtutDSCL．17，182−190（1994）．
10．Sendtner，M．，etal．J．oftbeNbuLDlqgicalSbihαS124，77−83（1994）．
11．Lisovoski，F．，etal．NbtGenet，Submitted，（1996）．
12．Henderson，C．E．，etal．∧btun？363，266−70（1993）．

13．Sendtner，M．，etaLNbtuLe358，502−4（1992）．
14．Sagot，Y．，eraJ．gurJ∧ku∫Dgd7，1313−1322（1995）．

15・SchmalbruCh，H．，etal．LofNbuLtPatholqgyandEqe血entaL
摘W血訂50，192−204（1991）．

16．Brunialti，A．L．B．，etal．伽0mics29，131−135（1995）．
17．Ghadge，G．D．，etal．Gene77zeLW2，132−137（1995）．
18・GilgenkrantZ，H．，etaL馳mat7Gene77zeLWlO，（1995）．

−167−

Plasmid DNAtransferinto skeletal muscle：
Implications forimmunereactions to
expressed proteins
RobertGVVHAIBN
BiochimieCellulaire，CNRS，Con昌gedeFrance，75231Paris，France．

Abstract
PurerecombinantplasmidDNAcanbeusedfordirectgenetransferof
Variouscelltypesbutitisqpparentlymoreefficient．fbrtransducingskeletal
musclefibers・ComparisonofdirectgenetransftrinnormalandregeneratingmusclebyintramuSCularlqeCtion ofrecombinant plasmids，adenovirus or
retroviruS ShowsnakedDNAtobeequaltoorbetterthantheviralvectors．In
addition，Plasmid DNAis notintrinsicallylmmunOgenic orinflammatOry，
Wheretheviralvectorsinduceimmuneresponses，PrObablyagalnSttheviralcoat
PrOteinsaswellasagalnSteXPreSSedviralproteinS・
Wehaveattemptedtoassessthenatureoftheproblemsassociatedwith
VeCtOrtranSftrandalsoduetoexpressionofafbrelgnPrOteininmusclefibers．
Thissituationisanalogoustotheexpressionofanormalcellularproteininan
Organisminwhichamutationhasextinguishedtheexpressionoftheprotein・Forthesestudieswehaveusedvectorsexpresslngthebacterialenzymebeta−
galactosidaseandtheenvelopeproteinofthehq）atitisBvirus・Theadvantage
Ofthe viralprotein，Whichcarriesthesurfaceantigen，isthattheimmune
responsetothisimmunOgenafternormalimmunizationiswellcharacterizedand
Canthuscanbe comparedtotheimmuneresponseafterDNAtranSferinto
Skeletalmuscle．
Onestriking feature ofthese experimentalmodelsis that a single

iItiectionofDNAleadstoastrong，SuStainedandbroad−basedimmunereSPOnSe
to the proteinS・Antibodies，T cell proliftration as well as cytotoxic
lymphocytesareStrOnglyinducedafteraslngleDNAinjection．Themuscle
fibersthatbecome tranSfectedandexpress theforelgnPrOtein degenerate，ー169−

apparentlyduetoanimmunologicallymediatedmechanism．
These issues will be discussed within the context of current

immunOlogicalconcepts．Theimmun010gicalproblemsassociatedwithgene
tranSfer，andwhichhavebeenlargelyignoreduntilnow，mayCOmPrOmisegene
therapyprotocoIs．

一170−

Correspondence：

InternationalWorkshopon
MolecularBiologyofMuscularDystrophy

鱒がroOZAWAOfga刀jzer

5肋刀ワchj TAズ王DA
gecrefaly

NationalInstituteofNeuroscience
NationalCenterofNeurologyandPsychiatry4−1−10gawa−higashi
Kodaira，Tokyo187，JAPAN
Tel．and FAX 81p423−46−3864−171−