撤i酷響謎薔盛Si鮒錯闘詳濫盟

S10Cコ EMIcAL ANDコ
ARH●
10ココヽおI●AL iESEハ
nCH(loM14コ
ヽ工CA口 ONヽ 236,151-154(1997)
LE NO RC976919
Down― Regulation Of the Defective Transcttpts
ofthe Werner's Syndrome Gene in the Celis of Patients
Y t t a k o Y a r la■
be,Masanobu Su8■
m o t O , M i s a k o S a t o h , N o■
・
■
■■
臨 S u z u 蔦, M i n o r u S u g a w a r a ,
MaltOto GotO,■and Yasuhiro Furuichi・
A C p r ご貴タ
ロこでぁr ″
sけ
力″々 2 θθ I C r″
あo コL F a れ。そ″ヵ% 拓 “″空争切“ 2 Z 7 J “P 。な o ″どヽ
D の。, か
″で
,1と
″" o とo し
Ogサ
ザ R んで
免わ o ユ危腕コo 2 カ
″″θ於滋 α″。
> 0 お″Z α ? 。●
ル, " o た4 駒ゎo ヱ 7 θJ 4 p o ″
,pj施
み2 8 ヱ ] あ″。, ″
Recttvod J_。 2 1997
Werller's sy_droI Ic(wS),all addt proge■
ia,Is a ro‐
00SSIVK gelletic dlsord●
■。allsed by tho lnIItatiolls血
the DNA tthcttc gelle(wRN)IIItht stuⅢ
,a ooIIPar_
at市。■。ItheHl blot allattsis Was ltlade for lyrAl・
p。
RNAsextactedと
nonsense cOd。 ■s FIbroblasts and B_IInIIPhoい lastoid
cell llnes ttansfo■
Iled by the EPstein_Bari予■
■usKEB"つ
狂 e h70 m奇
Or SOurces oF cells IIoII Patlel■ tS tl■
at are
avallable io■ cellular studles The HIRN nuta位
on is
。I n a b r o b l a s t s adx ■
B IPPLoblastoid
●clls of ws Paticnts,IelatNss of paticllts,aIId BIt IIllal
indviduals Tl■
e lev。ls ofllutant wI』
N IttRNAと。コ P,
ticIIt cells、マ0■e SigoJ8。 a■lt1710Hrerthallthoseofmtact
ridllals by all averヽ
mRNA iom the cells ofH。
コ匝al illd■
■
age of 70% FllrtherI Iorc,all extre‐
oⅢ loW leA● 1● f
WRN IIIRNA(s),PIesul labし
aコ Jb輸口
■o o f I H u t a ■
lt alld
's ta‐
mtact xnRN4s,,マ
as oLsewed br the patlellし
■ly
ltlellbers who caH甘
。Ile llutated allcle TLeso■
。suts
s竹 oI唱 ,suggost that a rdatwdy low lcvcl cf Lellcaso
llRNA is suflcicnt to PIovcnt tl■
e oIIset of Wo■ lloゴs
syndroIIle 。
7れな
lq●
delnic Press
ヽVerner's syndromo(ヽ VS)is an aut。国olnal recesslve
diso■dol causing srtll toIIs of Prelnature agl■ g, sucl■
as F7hlte ha■l,balaness,cataracts,and osteoPo■
。SiS(1認帯ぎ縄唯 i培論
:]モ督t忌翼胡鮮品協
some 8,v hiel■ encodes a p■ Otell■sbincmtly si・
淵
rular
to the 2 c。
力RecQ―tX pe D卜
しヽh。licases(5)At loast 19
撤
署キ薔
器
鑑 :謎
端鞘
語
韻
called variegated tians10catlon IIIosaicisコ
o(11),and ab一
noIIIal telome■ ed■ ■■amics(12)LIttle,hOwover,is
known about the nI。 leo■aI Inechal■Isms、マhereby the
WRN hdicase gene n■
utatton causes these abno■ mali‐
tles oi abo■lt the IIletabollsln of the transc五 Pts oF tlle
ltlutated gene
We and others sh。、
ved by IHT PCR o■ northem blot
analysis thatthe mRNA oFthe mlRN hellcase gene Fras
tlanscibocl in botl■ f■
broblasts antl ttBV trmsbltlned
B Iyttphobltttoid c』 l hnes(5,9)Ill this st■ldy,we show
that the anlounts of m ltantヽ ∇
Inマ dNA are reduced
in these cells having elther holnozygous。
■heしerozy‐
gOus mutahOn
MATERIALS AND METHODS
The ptttlclts were ぃdi雄
osed as previ。
■s,reF。■ビ
Ed(3)Bocanse
,:鼎
漕品品為盤難穐瑞
培
古
11号
甜鮎∬瑞総主
常よ
i描
潔艦磐督
艦塩盟選
群tよ
器警鳩皆
e翠
群笠
掛盤群掛規
宙
督」
絆諄
繋断
縄:濫
t7ons Avero iII the 。
cno,dallnngI orsetまh rhoo昭
tll the
C8si謎eと
樹監縦
認景
札掃
主
岩
鮮出督謎
盤建
mutatlons ng l抗。ws the「ositi。
こs oflLese ml■
t●
6onsiェ thecDN、
BIld tl。
1lst of Patiorts andェ
o rlnJ indl宙d■
lals ioⅢ wと。m the cell
l遮es wor。。btahoこ MItajo■ lis he naェ
T)
ge of one baso(C→
st nudcotiこ。4144(i■ tllo 33rd exo■)in tl o lo血■g regDn gPnerdtitrlg
a ■o w 載 ∞ c 。
3。
■。F コ3 A i t l e t l t t n t t。
e●ぱ
c oCこ
C A a t 延ふ o a c i d
緊鶴謎魯軽 , 豊富灘
欝
塁科論
甘艦
1336(in ho 9th elon)丁
the dD卜L生,餃ェ。ratlng a stop eoa。ェ。FTFTA
ot amlno acla rssidue 369(5 7)Tlle ttbroblasts were tttai■
ed froln
l To whom corrosPOndenco sh。
■ld be atldre。
。
。d FaI:+81467‐ 486595 E IIII:危 ruichコ3agen。。
。うp
Abbre、■■8oェs:WS Werェ ers syndrolce:ヽVIuv,巾七rnoIヽ野D3'Ome
響謎薔盛
Si鮒
撤 i酷
錯闘詳
濫盟
部縄路瑞盟環継鵠塩」
急
転温総盟品断
tI°
lll
lh算
metllod(14)and ollttteptaT30(15)駈
軸
。
。″g oF,o,ltl+RNA
袖銚と
轡選路紺鮒 Ъ
ざ
wero oloctiophoresod in 14.agaros。
騰塩
餃ls co五首ning ttrmattde ma
151
0006‐
291通97,2500
CoPriぶ 10199'bF Ac,aentc Prtss
知 1 「E h t s l r o p r o d l lnc hi●
n ary向
1 ■ r e s e r va●
/,ノ
V31 236,N。
1 1997
BIOCHEMICAL ANE BIOPHYSICAL RESEARCH COMMUNICAT10NS
PЮ L●
ths tl■ding more systematlcally SI‐
ce B lsallphoblas―
olls軒。、
toiclで
v fastol than ib■oblast cellsi they alloxved
■
ls to coIIPare the nRNA levds at all■
■
ost the salne
PDLs,as well as to obthn the cells IIom a「
eater
IItllnbO■
S Of Pahents whose mutatlon sltes wo■
e de‐
iL■ed ln aaclltloI. usillg amenable B‐ cells enabled us
to cttend tl■
e st■
■
dy te the normaI Iolati、cs of hete■ozェ
uS
Patlentsコ
vho
carrr oI■ly one allelo of the mutated
即
Sと町車lm 典
せ
ぃ
申
cells
wltl■ hoIIIoZygOus nut4tlo■
4
gene The B―
N0001
2●
′M
―
′―
NoIB甑
VS0801 and WS9801)o■
mutation 6(ヽ VS10201江 nd
(ヽ
_
N●007
AS′
M
_′
Noll 自[
ヽVSl1001)had muth less mPINA than the no■
Inal cells
一
N1000
S1/M
_′
NoBBI
(N0007 and N0008)at PDLs bettteen 48 and 87 0n
WS10001
打 ′M
'市 ctr t
1 / 1
average,the殉
■。unt of mRNA of patlellts was about
コ臼6ml
抑S ●
801
4 ●′P
4/4
30%of tl■ at of n。■mal individ■ ■
als,nealllng that the
町ヽ,801
セ ′M
4 / 4
■ntlo■
cells w■th a nutatlon in only one dllolo shodd have
巾S 1 0 8 0 1
" ′F
4′ 4
■■OtI!
about 65%(l e 50%■
30■品 )of nolmal cell rduヾ A
コFthtt i
W S J ●1 0 1
お ′F
`′
`
Tl■e talhng in botl■lhe shoit and〕 ong transuに PtiedonS
耶 11001
却 ′F
,■0卸￨
`フ
`
was more● bv■ous in the patlent cells thm lln the no■ ‐
WS0101
30′
M
1/4
PAF eEr
mal cells(ng 2B,lmes3 md5),stlg野
乱IIlg an at母
―
F
l′
FS C10・ "′
NODD山
nentocl
dettadation
oF
the
mm(A
andrOr
abnttmal
PN01は
Ⅲ
′F
“
′―
Nelmel
sPllcing III Patie・
lt eells, as shЮwn in otl■ er cases h
X・
hel■
nonserlso
codons
aFIFcted RNA netabolism ll■
FIG l Postttttoェ
or mutations l)4)and 6 in tlo cDNA ofhVRN
ve■
teb■
ate
cells(16,17)To
v。五母 thatlhe obsAIved doま
■。lic●so sIIt tl■
e llst efコ■8でコts andェ orlntt i■3ht8nals flon whom
航 e ccll linos wぃ。抗 tよned N,WS,aェ
d PN il■dlcateェ oII lds,Woト
radatlon ocarred spechcally on lheヽ
WЪnマll.RNA,wo
I■
ers syndromo F宙
jOnts snd Phコ
ot〕JCttlF norIEtt s』ヽ。乱S)rospoし
elalruned the levels of eTF2E2 nRNA(whose genoIIIc
trelF
lRN geno(18))(Fig
locatlon is dose to that oF the■
2B)Thelevds of OTF2E2い
しヽwere apparently un‐
aIIected by the mlRN mutatonヽ
Ve also exalmned tlIIoe
were付血 sFsEed to H)o■ こNぃ。mbranos(Amorstam)H"rittZa
n e I I I b e r s
o ft h a血 Ⅲp (a Ft il ge l 3■) a
p h e n yo " P i C a ■
'2P‐
tion せos Porf●llned、立tt a 2 x 10・ cP前血
radiolatelled ProSe
nomal lltother(PN0104)win heterozygous mutatlon
mtrsns―
pre,ared flom tl o C_torttnHl reュ on whtiindidestho 3′
(PN0102)
l a t e a r e。
事
ェ( ■
l l C l c o t l d e r e s i d u e s 3 1 9 9 5 0 6 5 ) o f l l l e w R N giegn ol()コ
) 4(lane 3), a PhenetvPically nllrmal siste■
with heterozyg。 ■
ls la■
utatnon l(lal■ e2)and a Pahent
乱 426C foェ 16 h Thelltl■ a me■ l13ranes were washed h 0 2 x SSCア
01%SDS at 42・
C For 30 nln aェ d antoradlographed for ive dd s
with compound heterozygoぃ
ll■
utatlons l and 4(lane
For qua■ tltatttrc analysis the nombrュェos、,α o StjPpod ttnd thoA
l)rrhe
Patlent
cells
showod
a
ve,y
low lev。
1。f the
w e r e r e h F b h d I Z e d 航血ュ・
T r a d d ぉ e l l o d ′r は ■, ■O b e 、 a c o n t r d
、v m ヾ . n P t N A ( 1 3 % o F t h e ■
nm。
allevel)aS expected flonI
to■omallze lLe de■ sttt lLe rci茄 ve ittcnsitics or hditdul wllN
the resdt、 ■th nbroblast cells sho、マn in Fig 2A Stu‐
lllRNII ba■
as were estIIEttd byュ
I As 1500 Bio血
ュ釣 g Allま ,zer
assun.hg tllo intenslけ
of工。
rmal to bo 100%
(Fttinlm〕
PrisinJ7,tl・ a cells IIom the apparcIIt homal iolablves
hatting a hete■ oz了岳ous r■■
ltatlon also showed reduced
lovels ofllnRNA(37%ar.d55%of no■ mal level),lower
RESULTS AND DiSCuSsioN
than the theOretlcallevel ofmRNA oxl ected to be pres‐
The lovels ofヽ ∇
]Rltt gene trttscipts 、マere colnIPared
entin heterozygous edls,lhat is,65%lft■ elatloned above
iIIいoth abrOblasts and B‐ I■
■nIPhoblastoid cell lines ob― in tl■
Is sectlon The■ eason(s)bebIェ d dis reducllon in
ta14 ed floコ a no■mal■ l■
dhidual(N0006)and aヽ
マS
intactヽVT破ヾlaNA accoltlpanied by the enhanced re‐
IIont car五 es the
duction of deleo成 ve ltlRNA is dlttcdt to exllaLI Il■ mo‐
Patient(WS10801)The WS10801 pε
・
homozygOus ll■ ■
■
tat10n 4 SmnIItt alnounts of p。
loculJ
terms,d■ le to a llェ
ェted data and knぃ vledge in
い(均
RNA● xtracted ioltlcellIInes at a siIIIIlaI PDL cdt■
lred the沌喪 arch ield deallng with tte sぃ che degladation
under lhe same condltlo■ s wα e analA7zed by no■ ■でm
of nonsense mRNA NotRIthstandng ths enign■
a,Ive
bl抗血江ysis ushg aヨ 2P Lbelled DNA ng 2A shows
boIIeve tllat our nIIdlng P・。v■des data new to mFdヾ
A
tl■
at the HIRN g。 ■e tl anscript levelオ om theヽ VS pa―
dynamics witll a Hコ xed Populatlo■ ofintact and deieC―
tlent was much less than lhat iom lhe no‐
tlal indln伊
tlve lnRl(A
ual, for eitter nbroblasts o■
B lyltlPhoblastoid cells
In gene■ al,the reduced levels ofコ =R]ヾA ll■
alr be at‐
―a c t l n I I P t N A a s s e s s e d a s a
The exPressiOn levels of β
伍 buted to a reduction in ttanscttption efIIciency or
colltrol were alnost the salne fol no■
Itlal and WS cells
nRNA dectt Tlle results ofthls st■ lけ do not necessar
The larg● ■■anschpts of about 8 kb o■
oatei
could
ily exでlude the posslbIIよ丁 of a■eductlo■ II■trarlscIIP―
軒
be the stable intermediate Prod■
lcts oftl■
e sPhhng reac‐
tlon efflcioIIcy,but a lnutatlon agectlng he transc五
Ij―
■2
→
４
→ｉ
ｍ
如
∞
tlon eftciency of theヽ
Vmヾ gene is u述
血 Ⅲ because
tlon
ヽ
Ve■ lsed B切 yttphoblastoid cell IInes to investlgate
the levels of CTTF2E2 1nRNA wore m■
152
ノ'ク
cl■
anged between
〔o1 236 No l 1997
BlocnEMIcAL■
ND BIOP口
YSICAL RESE I CH COMⅣ
81
15
RNICAT10NS
B
A
ハ
じ
4
Si々
Sizo
(kb)
(k切
0575-
0575-
44-
44-
24-
24-
i4-
■4 _
伊o●4in
6「F2E2
,や。1●
bl or Nerma
Ⅲ Ⅲ0 28
19
7
HG 2 Norlhem blot analysis oF lhc ccllsは
。In normalindi、 idiols aェせヽ
VS,前 icl■
ts 141ミヽェ。
blasへ fttom aェ oHnal ttditltltt N0006
(lane l)and flomェ p●5。■￨ヽvS10801(Iane 2)and B l沖
IPh品 1■stoid ccIIs市。D a normalindl■ d■
lal N6006(lan。 3)aェ t rlolll'VS10801(laェ o
4)(B)BⅢ
ェp上oもlastnd cons Fttomェ ormal iIIdividuals N0007(lane l)血
d Nooo8 0ano 2)血
ご付onl 13atlel■
tsヽTSOS01(lltDo 3)WS9811
―
d WSl1001(lano 6)TTo β
(lanc 4)NSlo201(lane 5),並
BCtlェana CTF2E21nRNAs on tlo s“
me ttters were hrbittZじ d bi sFccinc Probes
and were usod す o ●
。mpare andェ ormttisc tlle diュ
■o■mal and Patients(Fig 2B)Rather,the difi■
lsion Of
mutated mRNA into smallo■
sizes in the no■thern b16t
andslsis(Iヽ g 2B,lanes 3 and 5)is consistent wltl■
tl■
e
second Possibiltty thaし the dettaddtlon of mutated
then be he biologicalい。nef■t tti the down‐ ■o印ェlaton
of mutated transc■ lpts?The acで urnulaton oF deFective
mR卜しヽInay have the lollowing hマ o disadvantages l)
廿anslatiOn Of do■ ctive protelrls is not econon■ lcttl to
m R N A i s e n h a n c e d , a s w i t h l hg e1 0 nb ti un t aathe
tn edcells,and
d β 2),vith heterozsrgotes,thOse protenls are
廿iose PhosPhate isoltlerase genes(16,17)ヽ ,■aしwo■』d
harlrr■ll aue to the co血 petitI∞ with noIInal Products
Recently, we Jound that most of the mutatlons in
theヽVRN gene generate lhe PreIIatule tertta■ inatloェ of
1
2
廿ansi atlon,resultlng in theimpttred nuclear tlansPor―
Sizo
l
tation of gene p■oducts in the c。1ls Of Patients(19)Ihe
(kり
ln。1。
cular events associated w■ th the NVRN mutatlons
lllay lhus be a down■ egIIatioェof tl ansttipts(shown
0575in this Paper),the generatio■ oftruncated P■ otein and
44their impaired nuclear tl aコ
sPort
Tllis study showed the clinically II■ ■
poltant condu
24sions tl dt l)WRN IllRム Lヽlevels were sigコ incantly re
Ⅲ4 _
duced in Patienし cells,2)theヽ Vttni nttL、 oxPreSSed
by a nO■ nal ユ lelo of a heterozygous individual was
do、、 regHaled by an u血
o、■ mechanisコ (s))but 3)
J
IVS01 01
a relatlveⅢ low level of helicase mRNA was suttdent
2 PN0102
to PTevent the onset ofヽ ▽einerjs synd■ onle
・.。iN。「
ma[
13
37
55
16
，ｍ
aCtin
F‐
3
PN0104
ACKNOVVLEDGMENTS
H C t 3 N o ァ t h e r a b l o t a n a l l s i s o f t 付。
l lme tchdo] 、b m ‖y o f a
slュェd4 B‐
PatientヽTS0101 will comPound tteterozェ印 Is IItltiioェ
lymPhoblastol■
PN0102、
cells iom p●
たth a lletcrozJgouS Ⅲ
はじnt WS0101(laェ
ol))his■
■tatlo■ 1(lttlle 2Ⅲ
ヽ
7c aro gntorlllo Dr Aaroェ
J Shaddn attl o Centeribr Advttncod
Biotechェ
olo骨
y and｀た031hno(PlscatawaF,NJ)応 r critctt readlnF of
the mェ
nuscnl t and encouragomont Tlis、
vort was sIPPorted bF th0
Drug Orgmizab。
oF JaPaェ
■sIPer宙 sed bF l■e Nillistri nf H。
llh and Wolirc
.■
oIII tt StSter
and LS工
。IInal
REFERENCES
mother PN0104 with a heterozFg●
[ts m utiion 4(lttno 3)Tho protles
or,■ icnt、vS10001(lane 4)and a nor■
ld individud N0006(lttno 5)
are shown os roにァ●nce
i EPsteiniC J Marhn)CM Sh,ltz A L,and Motuls、
,AG
″α45,177_221
(1966)コ″αど'c↓
153
/デよ
B10CHEM CAL AlrEI BIOPHYRIC地
Vd 236,No l,1997
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]たosoA,Bj
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ch Y,2nd Coto M (1997)II ′
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cto,K)H611uch■ i Y j BIld Sasaz洞
は,T (1981)
C,, Co jセ 19,8-15
4 Goto,Mク
取I I I α R W I s h 底
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Caz硬ェFβケ
彦α
れわとBめ"o清 ″
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Л
々ゼ B力 ′190,541_546
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Aish RⅢ
s,J,m,T,o.alg,S,MalhII
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ll●
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255-262
H,mtt Mれ
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12 Sthdz,VP Z』
と
ここn,VA,0=bm,OE,1[cKaF,JⅢ
、acz)AAクロ租and,SD)●
nd Mal伍 ■ GM (1996)″
97,750-754
ab
,あ ?
Jarzebo‐
″ル &″ ￠F
6 0shllnEa,J,Y■l,C‐E,IIIssar C iK anづ G」 4bkOWSlu J j Salcij 13 取回ler,G(1990)を
,Vよ o崎前 (n。ldo B N and Kttpo lt M
S.Miti,T,Ntturこ
,J,0車 haa,T,Ells,JⅢ S血批 ,1を deA C,
Eds)P, 1921-1958,Ravm hess,No「
■o止
Molaragno,M I FracrarA,M,SoEI Patlod,S,Mttttllews J
14 ChoIItczttS虹
sFrv 162,
, P , 適 d S a c c h N ( 1 9 8 7 ) あ o 7 B わあ o ルテ
Outts, S,Ja■ zあ o■■cz,A Sい
。1lonbors G D and Maltl■
,
156-159
GM (1996)ど
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