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Hum Genet(1997)99:191-193
C)Springer―Verlag 1997
Makaoto Goto・
Osamu lmamura・
Junro KuroHlitsu
Takehisaい
仁
atsumoto・ Yukako Yamabe
Yoshiki Tokutake・
Noriyuki Suzuki・
Brian Mason
Dennis Drayna・
単質inoru Sugawara
い在
asanobu Sugilnoto・
Yasuhiro Furuichi
Analysis of helicase gene mutations
in】
apanese Wernerrs syndrOme patients
Receivedi 25 July 1996/Revised:20 September 1996
Abstract The profile of helicase gene mutations was
sttdicd in 89 Japanese Wemer's syndrome(WRN)pa―
tients by exaHlining the prcvlously described lnutations l―
4 as well as a new mutation found during this study,des―
ignated lnutation 5,Of 178 chromosomes(89 paticnts),89
chromosomes(50%)had mutation 4,11(6。
2%)chrOmO_
somes had mutation l,and two chromosomes(1.1%)COn_
tained lnutation 5。
Mlltations 2 and 3 were not obscrved in
血is patient population.The remaining 76(42.7%)chrOmO中
somes had none of these mutatlons.A significant fractlon
of all paticnts(22 total patients,24.77))appear to be cOrrl―
pound heterozygotes,including those carrying mutations
of both types l and 4.The genotype analysis of the IIlark―
ers suttounding theヽ
ル貿ゴ
V helicase gene strongly suggests
that most ofthe chromosomes c劉
回
ゎ
れng either lnutation l
or 4 werc dcrivcd from two single founders.
introduction
Wemer's syndrome(WRN)is a rare autosomal recessive
disordeT causing premature aging (Epstein et al. 1966).
The Ntt mutation results in short stattre,juvenile
cataracts,atrophy of the skin as well as graying and loss
of hair, diabetes mellitus, arteriosclerosis, osteoporosis,
and neoplasia(ふ
江artin 1978;Goto et al. 1981, 1996)。
We
and others have prevlously assigncd theヽ
,貿
PV gene to the
short a□阻 of chrolnosolne 8(8pll-12)(Goto et al.1992;
Schellenbcrg et al.1992).Recently,Yu et al.(1996)iden―
tified the genc rcsponsible for WRN, which encodes a
protein significantly simllar to RecQ type DNA helicases,
and they described four typcs of gene mutations(muta―
tions l-4)in patients of either Japanese or Caucasian an―
cestry.We have investigated the rnutatlons in this gene in
a larger number of Japancsc paticntse Wc also deten『
lined
thc gcnotypes of the markers suⅡ
ounding the根 ミプV locus
in order to analyze the founders of these rnutations.
Material and lllethods
Genolnic DNA samples werc collected fron1 89 WRN patients and
75 nomal Japanese individuals.All the patients gave their infomed
consent prior to their inclusion in this stlldy.The patients were di―
agnosed as having WRN based on at least three of the following
four mttor signs and symptoms(Goto et al.1981);(1)CharaCteris―
tic habitus and stature;(2)premature senescence;(3)sclerOdema―
like skin changes;and(4)endoCrinOlogical abnorlnalities. E)NA
was extracted from either peipheral blood lcukocytes or B-lym―
phoblasts transforlned by Epstein―
Barr virus.Polymerase chain re―
action(PCR)was carried out using these DNAs as templates al■
d
spccific pttrners designed for each mutation site, and the nu…
cleotide sequences of the resultant amplicons were deterlnined.
The mutations were identined by COmparing these amplicon se中
quences with those of nomal individuals. The pril■
ers used are
listed in Table l.The genotypes of sho■
tandcnl repeat polymor―
Table l The list of PCR prirners for the detection of IIlutations l-5
M質uta― Scquence(5′
to 3′
)
tion
Nlt.Goto
Departlnent of Rheumatology,
Tokyo Nlletropolltan Otsuka Hospital,2-8-1,WIinalli―
Otsuka,
Toshilna―
ku Tokyo 170,Japan
O.Imalllura・
J=Kuromitsu・ T.NIatsumoto・ Y.Yamabe
Y.Tokutake・
N.Suzuki・ や質
.Sugawara・
WI.Sugilm9to
Y.Furuichi(E圏
)
AGENE Research lnstitute,200 Kttiwara,Kamakura,
Kanagawa,247 Japan
Fax:+81467-48-6595
B.MIason・
D.Drayna
単質
ercator Genetics lnc.,4040 Campbell Avenue,単
質enlo Park,
CA 94025,USA
3
4,5
TAA AGG
TCA CAC
GAT GTG
GGA AAA
AAA GGG
GCC ATA
CTT GTG
GGT AAA
ATT AAT
TGA GCA
GTG
TGG
TAT
TGT
AGA GGC
CAG TGT
GCT GTT
TTT ACT
GAA GCT
TAT CTG
TTG TTG
GAA AGT
CTA TAA
AGG AGT
AAC AGT G
ACC TG
ATG G
AAG CTC
CTT ATG
GTG C
ACT GG
CTG C
192
phic markers were detcrmined by PCR(Goddard et al.1996;Yu et
al.1996).
Results and discussion
Of 89 WRN patients,35(39.3%)were hOmOzygous for
mutation 4,one(1.1%)Was hOmozygous for mutation lラ
six(6.7%)were positivc for both mutations l and 4,one
was homozygous for a new mutation(deSignated muta―
tion 5),13(14.6%)displaycd a single copy of lnutation 4,
three displayed a single copy of mutation l,and the re中
maining 30(33.8%)were negative for all the nve muta_
tlons(TablC 2).No pttients were obselwed with mutation
2 or 3. Thc results are simplified when expressed at the
chromosome lcvel:of the total 178 chromosomes, 89
chromosomes(50%)had mutation 4,1l chromosomcs
(6.2%)mutatiOn l,two chromosomes(1.1%)mutatiOn 5,
while the remaining 76 chromosomes(42.7%)had neither
of these mutations. Exalninatlon of the faIIlilies of the
compound hetcrozygotes with both mutatlons l and 4
confimed that these mutations have not arisen de novo
since they were identifiable in their parentsi the pedigree
of patient WS0101 ls sho、
vn as a representative result in
Fig。1.In this case,Inutation 4 was carried by the rnother's
famlly and lnutation l by the father's farl11ly.Exalninatlon
of 75 nomal Japanese individuals revealed none of these
five mutations.
We have found a new base insertion mutation(muta中
tion 5)in one patient who is homozygous for this muta―
tion.This lnutation occurs at nucleotide residue 4146 near
the prevlously described mutation l:A is inserted in the
sequencc of GCGAGC to glve rise to GCG△
AGC,result―
ing in a translational frame shift and generation of a stop
codon 38 bp downstream.Interestingly,this paticnt had a
unique type of ostcosarcoma(Goto et al.1996)。
Nonc of
the falnily members heterozygote for lnutations l,4 or5
showed any typical symptoms of WRN.
Next,we carried outthe genotypic analysis of the poly―
morphic lnarkers around the眼
＼ハ
「
gene:W4369,D8S156,
D8S2168, D8S2150, D8S2162 and D8S2186. Tight al―
lelic associations were found among the five markers ex―
cept for D8S2186.Of the total of 82 chromosomes con―
taining mutation 4,73(89%)had the genotypes of 37-46-
73-47-38(34)alleles at thc markcrs W6369,D8S156,
D8S2168, ]D8S2150, and D8S2162; only four chromo―
somes had the 34 allele at the D8S2162 markcr.One of
the two chromosomes of lhrce patients homozygous for
mutation 4 had the genotype 29-46-69-47-38.Two chro―
mosomes of one patient homozygous forrnutation 4 had a
unique genotypc,31-42-57-53-38.These findings strongly
sllggest that mutatibn 4 has arisen several tilnes in the
Japanese populatlon.For lnutatlon l,cight out of ten chro―
mosomes showed thc genotype 35-46-69-47-38,whlle the
remaining t、
vo had slightly different genotypes.These re―
sults suggest that rnost of the chromosomes canら /ing mu―
tation l or 4 arc derived from different single founders.
Table 2 List ofthe mutations in 89 Japancse WemOr's syndrome
patients.MItutations l-4 are reported by Yu et al.(1996),whilC mu_
tation 5 is described in this paper.Among the 89 patients,65 pa―
tients were unrelated to each othcr while the reェ
lalning 24 patients
、vere brothers and sisters derived fron■
1 l fanlllies,and the parents
of at least 29 patients wcre conimed to be consaguineous in their
marriage.(+Nltutation is positive;一
mutation is negative,ND not
done)
Type of rnutation
Number Percent―
of age
patients
ND /
十
/十
T/一
/ /
十
ん ―
/一
/ /
+/―
一
/一
/ 一
/―
Total
39.3
1.1
1.1
6.7
14.6
3.4
33.8
100
1/0 1/4
Fig. l The pedigree of family WS0101. Theう
どαごたs?″α
/ビwith
mutation 1/4 indicates the Wemer's syndromc patient.(4-単
質utation
4,7 mutation l,θ
neither l nor 4,れ冴not done)
The prcscnt results confim the findings by Yu et al.
(1996)that a DNA helicase gene ofthe RecQ type is mu中
tated in WRN patients.The results,howevcr,also indicate
that there are other unknown mutations which could ac―
count for the disease in about 50ワ
う
of Japanese patients,It
is also noted that therc are a significant number of com―
pound heterozygote patients(tota1 22 patients, 24.79/0);
six patients with both mutations l and 4,as well as 16 pa―
tients who presumably carry othcr unidentified mutations
together with mutation l or 4.These reslllts suggest the
following points:(1)a Significant number of cases have
arisen due to high carrier frcquencies in thc Japanese pop―
ulation,and(2)different types of rnutation cause a silnilar
defectin the WRN helicase.
Acknowledgements Wc thank the following individuals for their
contributions to the present investigation as well as valuable dis―
cussion:単
質r.A.Shimamoto,NIs S.Kitao,Dr.K.Ichikawa,NIs K.
Sugawara,Ms M.Satoh,Mr.Y Kataoka,Ms K.Fttita,and Ms C.
Itoh of AGENE Research lnstitute,as wcll as Dr.W.Thomas of
M質ercator Genetics lnc.,Calif.
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