SyndrOme to five markers

LETTERS TO NATURE
Genetic linkage of Werner's
SyndrOme to five markers
on chromosome 8
Makoto Goto*,Mark Rubenstein十
Kathryn WoodsS&Dennis Drayna†
,James Weberや
￨￨
,
十Departments of rMolecular Biology and s scientific Computing,
Genentech inc,460 Point San Bruno Boulevard′ South San Francisco′
California 94080,USA
米Department of Rheumatology,TOkyo Metropolitan Otsuka Hospital′
2_8-l Minami_Otsuka,Tokyo 170,」 apan
Medical Research,1000 N Oak Street,
キMarshfield Foundation for「
Marshfield,Wisconsin 54449,USA
‖TO WhOm cOrrespondence should be addressed
亀盟罪学鑑児縄協艦円溜謂船コ縦跳掛撚品 r鞘翌器
normal ageing indicates that these two conditions may have
pathogenetic コ mechanisms in conl】 “on3-5.The WS mutation has
PleiOtroPiC efFects,and Patients and their cells show many difFeren‐
ces compared with normalsS.DesPite extensive study of the clinical
and biochemical features of this disorder, the pril■
ary genetic
defect remains unknown.We have undertaken a genetic linkage
study in an efFort to identify the locus of the primary defect6.Here
we report close genetic linkage of the WS mutation to a grouP of
markers on chromosome 8.
WC Studied 21 Japanese faェ 111les7-16.Thcse Falnilies originated
in 16 difFercnt prefectures,and apart froHl an increased preva―
lence in lbaragi and its ncighbouring prefectures,were widely
distributed throughout Japan.Thirtccn of thcse faェ 11lies con―
taincd cOnsanguineous lnarriages(mostly flrst cousins),and two
others were suspected to be consanguineous. Eleven of the
卜とてく
崎０卜り的Ｑ
０ヽ的り的Ｑ
Recombination units
FIG l Results of LINKMAP analysis of Werner's syndrome gene location,
The locaHons of the marker ioci were fixed as followsi D8S87←
10 units→
‐5 units→D8S■65,with the D86871ocus set as zero The distances
Дttκ
ユく
between marker loci are those obtained from three_point iLINK analysis
(analysing the marker loci only)in our werner's syndrome two_generation
families SimHarly,the marker order was the mostllkely given by three point
iLINK`But the most ilkely order of D3S8/一
月NK■―D8S工65 was onty three
times that of the second mostlikely order D8S87-D8S165-Attκ
立.The lod
score was evaluated in 10 increments perinterval Analysis exctuded families
represented by only a single inbred affected individual
NATURE ・
VOL 355 。
20 FEBRUARY■
992
735
LETTERS TO NATURE
fanlilics consisted of a single gcncration,nine had two gener―
ations,and one had three gcncrations.Seven falnllics containcd
several afFected siblings.The sample consisted of 63 individuals,
31 of which were afeゃ
ted(for detalls of the pedigrees,see
Supplementary lnformation).Hcparinized venous blood was
obtained fron■all individuals with inforincd conscnt.Perlnanent
cell lines、
vcre established by Epstein―Barr transforIIlation of
_
lymphocytes17,。 r by sllnian virus-40 transforlnation of abr。
TABLE■
Locus
Chromosome 8 markerioci
Marker
A‖elestt
ANκ 立
ankyrin
D8S87
Mfd39
D8S165
ヽ
lfdl17
107
■■3
145
生49
15■
153
■55
142
146
148
■50
■52
154
112
114
116
118
120
122
124
■26
128
130
■32
134
165
167
171
173
175
189
19■
193
195
197
199
blasts18.
°
cr謎 :か
靴況穐:ittintttttn盟
,S瑞 ∬ 酬 3活「棚
stature;(2)premature senesccncc;(3)sclerOderma―
like skin
changes,and(4)cndocrinc abnorlmalities.Atleastthrcc ofthcsc
four mttOr signs wcrc required for thc diagnosis.Diagnosis was
紺 :紺s2地
d撒艦縦lhttp鮮
拠鮒品sttn熊
粧
blasts in 20 individuals18,21. Individuals wcre assigned as
unaFected only if they werc lnore than 30 years old.
Thc markcr ioci uscd in our prirnary survey of the human
D8S166
Mfd159
gciomc cOnsistcd predorninantly of cytosine一 adeninc_repcat
Hlicrosatellite lnarkers22,。
f which 128 wcrc from the WIarshfleld
collcction22 and a further 23 werc from thelitcrature.In addition,
the marker set also included a few trinucleotide(研 A―repeat)
and tctranucleotide (TTTA_ and TCTA―
repeat)markers.
臣生
arkers wcrc typed by polymerase chain rcaction(PCR)fol―
lowed by polyacrylanlide gcl resolution of the PCR products22.
We tested a total of 156 polymorphic lnarkers for linkage to
Werner's syndrome.These markcrs wcre distributed on a1l of
the autOsOmcs, with a median hctcrozygosity of 65°
/O in the
Japanese population.These markers had a 90°
/。probability of
dctecting linkagc(at 10 Ccntimorgans or less)tO∼
60°/。of the
autosomal component ofthe human gcnome.
our arst indication of linkage appcarcd when we noted that
two chromsome-8 markers, D8S87(Mfd39)and ANKF
(ankyrin), showed increascd homozygosity among attected
individuals from inbrcd falnllles.Such incrcases in homozygos―
ity arc predicted fOr rnarkers that lic close to a rare autosomal
locus23.Traditional maxilnum likelihood calculations using data
froln all the farnilics conarmcd linkagc for these two loci and
also revealcd linkagc to several other nearby markcrs. Thc
,EnarkCrs and thcir charactcristics in the Japanese population are
listcd in Tablc l;the genotypcs in the WS farnilics are avallable
as Supplementary lnforlnatiOn.
Signincantly pOSitive two_point lod scores、verc obtaincd for
linkage between WS and avc locithatlic on chromosome 8(refs
24,25).The two― point scores at several difFcrcnt values of θ are
listed in Table 2.Therc、 vas evidcncc for close linkage bctwccn
WS andthctwo lnarkers D8S』
7and ANKア
.Evidcnce forlinkage
、
vas also obtaincd fOr three apparently more distant inarkers,
D8SF55,D8Srび
びand D8srび 4,although the pairwise lod scorcs
for thesc linkagcs varicd considcrably.LPL(1lpoprotein lipase),
Mfd104
063
037
023
00■
0.57
006
0■ 3
028
0.57
0.01
001
009
0104
0.02
0.02
0.32
003
0.01
0.06
0.17
016
007
0.04
0.07
0103
0.29
005
009
008
012
004
022
0.02
0.02
0.01
006
0.45
060
0.57
087
米Alleles are the size in nucleotides of the predominant PCR product
十Allele frequencies and the observed heterozygOsity were determined
in 50 unrelated」
apanese individuals
anothcr markcr on 8p,showcd no linkage to WS in our data
set.Iterative two_point calculations using the障 ILINK program26
gave θ max Values for these six markers as listed in Table 2.
Although none ofthc pair、
vise lod scores excceded 5,Inulti―
撤選
:n艦
撒i樹為松譜
帽:・
縦鵠幣
堺桃群嵐
D8S87 NS一
Aハ r」
(r was 7.69. Finally, using thc data sct that
excluded farllilics represcntedbyjustasinglc aFecte
、
ve obtained a four_point lod scorc for the Order D8S87-り ИS―
MARKER
FIG 2 Genotypes of recombinant inbred WS
individuals individuals are offspring Of first_cousin
8 pter
marriages Numbers beneath each individual are
'denotes
the
とPと
the alleles of the markers iisted;“
レ
,′
allele containing the mutation at the VVerner′
s
D86∂ ア
syndrome locus For each individual, the arrows
indicate the intervalin which a recombination has
occurred at a melosis somewhere between the
affected individual and his or her parents'common
刀Nて ,
ancestor. Solid lines denote portions of the
chromosomes that are homozygOusi dashed tines
D36765
denote portions which′
as a resuit of recombina_
tion′
are heterozygOus とPL lies distal to AM立
DθSアδδ
(ref 33)、These individuals support an order of:
pter―とPL―(D8S3ろ NS)―Attκ立―D85立 65-D8Sttσ 693S7644
D8S立 64_cen
Observed
Frequency十 h e t e r o z y g O s i t y 十
5802
4601
4701
1231123
123,123
123,123
123,123
123,127
151,151
151,151
151,151
155,155
155,155
町町
明町
町〃
!・
(―
町町
既W
107,107
107,107
107,107
107,113
107,107
146,146
146,146
142,146
152,154
146,146
126,126
116,126
116,124
114,126
128,128
1651173
191,199
191,199
165,175
191,191
8 cen
736
NATURE ・
VOL 355 ・
20 FEBRUARY 1992
LETTERS TO NATURE
TABLE 2
Lod scores
Pairwise lod scores米
Marker
98S87
Дttκ
工
D8SIσ 5
D8Sユ σ6
D8Sユ σ4
とFL
5.1
29
■. 5
2.5
-4.6
-2.9
Recombination
fraction(θ)
005
00
Maximum pairwise lod scores十
Marker
レ
,6-98S87
怖6弘川κ立
婚の益立衛
NS―D8Sttσσ
Ns_Dgs立 6ィ
NS―とPL
θ
01050
0058
011
012
028
050
4.8
27
19
35
-16
-16
41
2.3
1.8
3.5
-0.37
-092
3.3
1.9
15
31
019
-0.56
015
020
0.10
2.5
■4
12
24
039
-0.34
0.25
1.8
1.0
0.81
1,9
0.39
-Ot21
030
1.2
0.64
046
13
028
-0.12
0,35
0.63
034
0.18
075
013
-007
023
012
0.02
0.31
0.01
-0.03
040
045
Lod
514
2.89
191
357
045
000
*Lod scores obtained in two_point analysis,using the MLINK program of the LINKAGE v5.l program package,Obtained from the Utah anonymous ftp sit
rcorona med.utah edu'26 run On a VAX 9000 computer′
rounded to two significant figures Values are obtained after breaking loops in the consanguineOus
families.The values for markerioci D3Sユ
6イand D8S■66 were obtained on an lBM_PC computer using LINKAGE v5.03 from」
Ott(Columbia University)run
on individual families and then added The data for these two markers were analysed using Only the alleles observed in eachplus
family′
an additional
a‖ele that represented the collective frequency of aH alleles that did not occur in that family The allele frequencies used were those
in the normal」
apanese
population,
十Maximum pairwise lod scores obtained as already des6ribed,using recombination increments of 0 001 units,
ANKr_D8sFび
5 oF9.92.We also cxalnincd thc support fOr gene
ordcrin our data set.Using three― point analysis,the lnost likely
order of the three c10sely linked loci was D8S87 NS―
ANKF.
But this Order was only 2.19 tilnes lmore likely than thc second
most likely Order of NS_D8S87-ANKF, and 7.6 tilnes morc
likely than the ordcr NS_ANKF_D8S8Z
We alsO used the program LINKMAP(ref.26)to dctcrmine
the rl10st likcly 10cation of the gcnc for WS in this reglon.This
analysis shows that the most likcly 10cation oF the 手
ルS iocus
lies bctwecn DJS87 and Ajヽ
「
Kr, rOughly ttve recombination
units from each.(Fig.1).Sevcral other points in this region givc
lod scorcs within l 10d OF the maxllnunl, and thus thc 900/。
conndence lilnits Ofthis 10catiOn are broadly based on this data.
MIorc data on the order ofthese 10ci、 vere obtaincd by exarnin―
ation of the gcnotypcs of attected individuals from con‐
sanguincous rnarriagcs.In thcse individuals,homozygosity con_
siderations allowcd us tO exarnine the genotypes of thc
)[き
碁
il匿具i,品欲試!:ξ
1貿 F監 8韓 rilド告亀 ;壇皆嵐甘岳8碍 Stts艦 33暑霊
that the order ofthese 10ciis 8ptcr― LPと一(D8S8ろ NS)一 ANKF一
D8srび j_D8sFびび―正
)8SFび4-cen.It is notcwOrthy that six inbred
aFected individuals(individuals 1701,4601,4801,6001,6701
and 6801)are hcterOzygous for either ANKF or D8S87
Ho、vcver,no inbrcd aFecteds are heterozygous for both.This
result suppOrts thc idea that thesc two marker loci flank thc
locus fOr WS.
This orderis supported by results fronl othcrs who have found
the order 8pter―D8S』み ANKF-8cen(ref.27)and 8pter_D8S87D8SFび 5-D8SFびび_D8Srび 4_8cen(J.W.,unpublished rcsult).Al―
though cvidence forthc order oftheヽ
ルS10cus is notlargc,these
results suggest the ordcr of loci in this reglon is 8pter―
LpL_
D8S87 NS_ANKF_D8SFび
5_D8Srびび_D8sFび 4-8ccn.
Werner's syndrome is a clinically complcx disease, in that
virtually all tissucs ofthc body seem to be aFcctcd.The genetics
of this disOrder,howevcr,argue for a relativcly sllnple,single
gene mutation as the cause of thcsc diverse symptoms. For
cxample, thc diseasc is rare and displays a clear autosomal
rccessivc modc Ofinhcritancc3,7.In addition,Werner'ssyndrome
is highly penetrant,and the WS phenotype is quite consistcnt
among the affectcd individuals. Our linkages clustered on
chromOsOme 8 are in agreemcnt with thc idea of a mutation at
a single locus as the cause of this disease.
NATURE ・
VOL 355 ・
20 FEBRUARY 1992
The closest marker,D8SJz is knOWn to bc tightly linked to
the genc for tissue plasllinogen activator,which lies On chromo―
somc 8,band p12(ref.28)。 Another apparcntly close marker,
ANKF,has been indcpendcntly localized to thc same chromo―
somal region,8pll.1_21.1(rcfs 24,29).Although the other
markers havc been physically localized only to chromosome 8
(J.W.,unpublished results),these data strongly suggest that the
WS rnutation lies close to or within the region 8p12
Wcrncr's syndrome has bcen reported worldwidel'3,30-32,and
although the clinical phcnotype ofヽVerner's syndrome is siinllar
in all populatlons,itis conceivablc thatthe WS phenotypc could
be conferrcd by diFcrent mutations in other, non_Japancsc
populations.This possibllity can now bc quickly evaluatcd using
these c10sely linked markers. These markers can be used for
presymptOmatic,prenatal and carricr diagnosis in WS falnilies
of Japanese and perhaps Other ancestries. They may also be
useful to evaluatc the hypothcsis that other progeroid syn―
dromes, such as HutchinsOn_Gulllford progeria and Roth―
mund― ThompsOn syndrome,may be due to diFerent rnutations
at this samc locus2,5.The Closest markcrs also provide a starting
point for eFOrts to clone the gene that is defective in WS.It is
possible that IIlore detailed analysis of thc Werner's syndrome
mutation may lead to a greatcr understanding of thc factor or
factors that dcter■
line the characteristic lifespan in man.
□
R e c e i v e d
5
A u g u s t
a c c 9e 9p 1t e d
6
D e c e m b e r ■
■ W e r n e r , o t h eK si e sl9 ,(0 U■
4 n) 巾
2 Thannhauser,S」 Ann lr74 A4ed 23,559(■945)
,
3 Epstein,C」 ,Marttn,GM,Schuitz,A L&Motuisky′
AGんイ
θJrc力
θ45,177-22■ (■
966)
4 Salk,D用口印 Cettθ
と62,■-15(1982)
tter's SypJrο
5 И/θ
tte an」打り
仰a,力巨ing(eds Sa kァD,Fujiwara,Y&Marun,GM)(剛
enum,New
York,1985)
6 Botstein,D,Wh te,R,Skoin ck,M&Davis,RA何
立わり
何 Cenet 32,3■ 4-33■ (■
989)
7 Goto,M,Tanimoto,K Hor uchi,Y&Sasazuki,T Clrr7 6θ ,θ4■9,8-■5(■98■)
8 Matsunagaァ
￨,wata,S&Yoshida,T ttβ立Opわ紛,169,743(■975)
9 Chi′K θをつた,4/esiヵβ立つθ″"ai 50,824-827(1988)
■O Aok,R &Gon,H 力
, 立筋ed 77,125(■ 988)
■■ Mo高,S eralヵ ,ュ 6e的,425,486490(■ 988)
■2 Fujisawa,A,Shoji,」 ,Terada,H&Khano,Sヵ
幻サOp力的a13■,6785-689(■ 989)
■3 Mashiyama,S,Mizoi,K,TakahashL A,Suzuki′ J&Sako,T ttβ サNeurOsυrg■6,75-78(1988)
■4 Hayash,N,K ura,Y,Aoiki,M &Tamak,T フ
ο
リ,ユ θrrん
ρ 3■,■87■一生873(■985)
15 0kuno,N er al Draberes 33,179(■ 990)
16 Shndo′ Y,Akiyama,」ァ
Matsumoto,K,Takaser Y&Hashimoto,Tユ
De拘竹at■3,396-398(■ 986)
17 Thottey_Lawsoni D A,Chess,L &Strorninger,」 L ノexp ル修d■ 46.495-508(1977)
18 Matsumura,T,Nagataァ M,KOn sh,R &Goto,M nス
びvancesわ gxperlmenta′んイ
θ」lcわ
θanJ
B′
0′
og/!‖をrr7θ
/sS/p」 rome a,び Hり用θtt Aき
嘔 V0 190(eds Salk,D,Fujiwara,Y &Martn,
G M)313-330(Plenurn,New York,1985)
737
LETTERS TO NATURE
Bostonl■987)
,θ
ουS DISeases(ed Gomez,MR)242-246(ButterwOrths′
19 Goto,M in Nθ uroo2:動
20 Goto,M&Murata,K Clrr7て力加ムθra 85,■0■―■06(1978)
′
,e/sS/ndrome
anび
Og/iИゎ
竹Jrcゎ
θanびaro′
ya,cθs lr7 EXperrmentarん
21 Hanaokar F θr arin tt」
FuJwara,Y&Marun,GM)439-457(Plenum,New York,1985)
出uma,スきngvOI■ 90(eds Salk,D′
わutt Cenei 44,388-396(1989)
22 Weberi」 L&May,PE Amサ
■570(1987)
23 Lander,ES&Botstein,D Scrence 236,1567…
,e9,4S7&8M,i9w4a-′
9 5 ( 1 9 8 8 )
仰
2 4 K k a t a n , M " C h i y o , H , O z a k , MS "H Sり
h i6 kθ
8,4038(1990)
25 Weber,」 L′Kwitek,AE,May,PE′ Patterson,D&Drabkin,H rVuclercスcrJs F7es■
―
向 6θ,θi 36,460-465(■ 984)
M 湾何ユかυ
26 Lathrop,GM&Lalouel,」
27 Blanton,S H θ :a16enθ ttrcs■■,857-869(■ 99■)
i cel1 69,o440,784(■985)
28 Yang,Fengr T に,Opdenakke「,G"Voickaert,G&Francke,U Cy:οger7θ
s■ 9,969(199■ )
l′
C R /Vuclerc Acrds R●
29 Po ymeropOulos,M H,Rath,DS,Ziao,H&Mer高
30 Aram′ H&Fatourechi,V θ υrls■4,2■5…218(1974)
3■ Cettmeler D er al ttwm cenθt 62,25-30(1982)
筋9 a 7 , 3 0 9 - 3 1 ■
3 2 S a m a n t r a y , S K , S a m a n t r aSy,′」
o h n s o n , S C & B h a k t a v i z i a mA′A w s t t t Z サ
(1977)
cs■,■38-■44(1987)t
33 Sparkes,R S eral ce,θ 何′
SUPPLEMENTARY INFORMAT10N Requests should be addressed to the LondOn edttoHal ottice of
ACKNOWLEDGEMENTS This work was supported by Genentechinc,by the Human Science FoundaHon,
Japan,and by a grant from the NIH(JW)We thank K Nishセ awa and」 Tomfohrde for techttcal
assistancei the many ndividuals from families attected with Werners SyndrOmei the Japanese一
Amettcan biood donorsi the oligonucieoddes synthesis grOup
T Tada,K Okumurar K Nishioka,Y Mizushima′ D Botstein and R Whte for encouragement and the
es famiい
physicians and sc enttsts in Japan who assisted in the collecHon of samples from affected
738
at
Genentechi
S
Aotsuka,V
NATURE
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