出芽酵母を用いた遺伝性大腸癌の新しい遺伝子診断法の開発

出芽酵母を用いた遺伝性大腸癌の新しい遺伝子診断

法の開発

著者

石岡 千加史

出芽酵母を用いた遺伝性大腸癌の新しい遺伝子診断法の開発

(08670549)

平成8年度 平成9年度科学研究費補助金(基盤研究(C)(2))研究成果報告書

平成10年3月

研究代表者  石 岡 千 加 史

(東北大学加齢医学研究所癌化学療法研究分野)

研究組織

研究代表者:石岡千加史(東北大学加齢医学研究所痛化学療法研究分野)

研究分担者‥ 金丸龍之介(東北大学加齢医学研究所痛化学療法研究分野)

研究経費

平成S年度

平成9年度

計

1 ,200千円

1,100千円

2,300千円

研究発表

(1) Shimodaira･ H･, Filose, N･, Shibata, H･, Suzuki, T., Radice, P., Kanamaru, R., Friend,

S･H･,Kilodner, R･D･,and lshioka, C･ Funcdonalanalysis ofhMLHl gene mutadonsin

Saccharomyces cerevisiae. ｢発表予定｣

(2) Jia, L･-Q･, Osada, M･, Ishioka, C･, Gamo, M., kawa, S., Suzuki, T., Shimodaira, H.,

Niitmi, T･, Kudo, T･, Akiyama･ M･,Kimura, N･, Matsuo, M･, Mizusawa, H., Tanaka, N.,

Koyama･ H･･ Namba･ M･, Kanamaru, R･,and Kuroki, T･ Screeming･the p53 status of human cen

血es uslng a yeaStfunctionalassay･ MoJ･ CaRt血ogeJ7･ 19 : 243-253, 1997.

(3) Ishioka, C･, Suzuki, T･, FitzGerald, M., Krainer, M., Shimodaira, H., Shimada, A.,

Nomizu, T･, Isselbacher･ K･ J･, Hatx3r, D･, and Kanamaru, R･加tectiOn of heterozygous加nca血g

mutationsinthe BRCAland Ape genes by uslng a rapid screemng assay ln yeast. L}oc. Nan. Acad.

Sci., USA 94: 2449-53, 1997.

(4) Mihara･ K･･ Miyazaki･ M･･ Kondo･ T･, Fushimi, K･, TsuJ'i, T･, Inoue, Y･, Fukaya, K.,

Ishioka, C･,and Namba･ M･ Yeastfunctionalassay of the p53 gene status in humanoe止Iines

maintained in our laboratory･ AcLa Med･ Okayama. 51 : 261-265, 1997.

(5) Delia, D･, Goi, K･, Mizutani, S･, Yamada, T., Aiello, A., Fontanella, E., Lamorte, G.,

Iwata, S･, Ishioka, C･, Krajewski, S., Reed, J.C.,and Pierotti, M.A. Dissociation between all

cyck arrest aJld apoptosis can o∝urinLi-FraumBmiceus heterozygous for p53 gene mutations.

Ov]cogeL]e 14 : 213712147, 1997.

(6) Goi, K･, Takagi, M･, Iwata, S･, Delia, D.,Asada, M., Donghi, R., Tsunematsu, Y.,

Nakazawa･ S･･ Yamamoto･ T･･ Yokota･ J･, Tamura, K･, Saeki, Y･, Utsunormya, J., Takahashi, T. ,

Ishioka･ C･, Eguchi･ M･･ and Kamata･ N･･ Mizutmi･ S･ DNA damage-associated dysreguration of

the cen{ycleand apoptosis controlincenswithgerm-line p53 mutadon. CanceTRes. 57 : 1 8951

1902, 1997.

00010174426

研究目的

本研究は､遺伝性大腸癌のうち遺伝性非ポリープ性大腸癌(HNPCC)及び家族性大腸腺腫

症家系スクリーニングのための簡便な方法を開発する事を目的とする｡このため､ (1)

HNPCCの原因遺伝子であるミスマッチ修復遺伝子群を出芽酵母に発現し､酵母自身のミ

スマッチ修復系におよぼす影響を指標としたアツセイ系を開発する｡ (2) APC遺伝子の

変異は､そのアミノ酸コード領域にフレームシフトまたはストップコドンにを生じる変異

であることから､ APCcDNA断片を下流のマーカー遺伝子に結合し､酵母に発現させたと

きにマーカー遺伝子が発現するか否かを指標とするアツセイ系を開発する｡ (3)臨床検

体(該当患者由来の末梢血)について､ (1)および(2)のアツセイ系を使用､その有

用性について検討する｡

研究実施計画

(1)出芽酵母を用いたDNAミスマッチ修復遺伝子およびAPC遺伝子の診断系開発

発現ベクターを用いて､ミスマッチ修復遺伝子群のアツセイ系を開発する｡また､ APC遺

伝子のフレームシフトまたはストップコドン変異を検出できるアツセイ系を開発する｡

(2)酵母のアッセイ系を用いた臨床検体のスクリーニング

収集した臨床検体を用いて､ (1)のアツセイ系をテストする｡

研究成果概要

(1) DNAミスマッチ修復遺伝子およびAPC遺伝子の診断システムの開発

(1-a)ヒトDNAミスマッチ修復遺伝子の診断システムの開発

Amプロモーターによって発現する各種hMSIZ2, hMl, hPMS2発現ベクターを作成した｡

このうちhMZBlを野生型出芽酵母に発現した場合､酵母のミスマッチ修復系が競合阻害

されることが判明､診断用アツセイの指標になる可能性が示された｡酵母のミスマッチ修

復系の異常を検出するための新しいマーカーとして､ GT繰り返し配列をⅠ.acz遺伝子の翻

訳開始部位の下流に挿入したリポーターベクターを開発した｡ hMLHl発現ベクターを改

良し､ hMLHIPCR断片を容易に酵母に発現できるgaprepairsystemを開発した.このgap

repairsystemと新しいリポーターベクターを用いて､遺伝性非ポ1) -プ性大腸がん(HNPCC)

家系から見つかった既知のhMul変異(25種類)について､酵母ミスマッチ修復系の競合阻

害性について検札23種類の変異のうちミスセンス変異を含む8種類の変異はこの競合阻

害性を示さず､正常hA4LHlt区別できた｡研究成果は国際学術論文として公表予定であ

る｡

(llb)LVKプロモーターによってAPC遺伝子断片を発現するプラスミドを複数作成した｡各

種∬C断片の下流に各種マーカー遺伝子を挿入し､ APC断片とマーカー遺伝子から翻訳

される融合タンパク質を酵母に発現させた｡その結果､ uRA3遺伝子と融合した場合に､

URA3機能が保持されることが判明し､アツセイ用ベクターとした0 (1)同様にAPCPCR断

片を容易に酵母に発現できるgaprepairsystemを開発し､既知のAPC変異(そのほとんど全

てが､ナンセンス変異やフレームシフト変異である)についてアツセイした｡その結果､

全ての変異APC断片を挿入した場合､ uRA3融合タンパク質が発現せず(Ura-)､正常APC

断片(Ur且+)と区別できた｡その詳細は国際学術論文として公表している｡

(2)臨床検体の収集および目的遺伝子断片の増幅

遺伝性大腸癌の患者検体(末梢血液)を収集し､核酸(RNA, DNA)を抽出､ pcRにて目的遺伝

子を増幅し､上記(1-a), (llb)のアッセイ系を用いてhMLHLおよびAPC遭伝子変異について

検索した｡その結果､これまでに1家系のhA4LHlのミスセンス変異および8家系のAPC変

異を同定している｡その詳細は国際学術論文として公表している｡また､ A托変異の検出

ついては(2)のアツセイは発症前診断にも有用であった｡現在さらに家系を収集してアツ

セイ系の有用性について検討している｡

Novel method for detection of heterozygous truncating mutations in the

familial adenomatous polyposis and familial breast and ovarian cancer

genes, APC and BRCAJ, usJng a rapid screenlng assay in SacchaTOmyCeS

●       ●

● ●

Cer'e VISlae.

Chikashi Ishiokal I Thkao Suzukil I Michael FitzGerald2, Michael K血ner2,

Hideki Shimodairal, A血Shimadal, Tadashi Nomizu3, Dmiel Haber2 and

Ryunosuke Kanamarul

lmparhent of ClimiCalOncology, hstitute of Development Agingand Cancer,

Tohoku Umiversity, Sendai980, Japan

2Massachusetts GeneralHospitalCancer Center, Charlestown, MA 02 129, USA

3Deparhent of Surgery, Hoshi GeneralHospital, Koriyama 963, Japan

Correspondence should be addressed to Chkashi Ishioka,

¶le identificadon of novel gem-line mutationsintumor suppressor genes

presents a major difficultyindleir imidalcharacteri2adon, as well asinthe adaptadon of

reliableand effecdve approaches to climiCaldiagnosdcs. 刀le large size Of many of these

genesand dle factthat gem-line mutadons are heterozygous have comphcatedanalysis

based exclusively on nucleodde sequenclng. We have previously described a functional

assay to detect germ-line mutationsinp53, a genethat is affected primarily bymissense

1

mutadons, and whose funcdon as a transcripdonalactivator canbe testedinyeast.

Whnethefunctionalproperdes of other cancer suscepdbility genes are not wen

understood, many of these aJTe disrupted primarily by truncahg mutations. Vhuallyall

mutadonsinthe colon cancer gene APC 2-5 and 80% of mutadons inthe breast cancer

predisposi血n gene BRCA1 6 consist of nonsense or frameshift mutadons, ぬading to

the development of screcnmg assays based onthe in vL'hlD Production of truncated

pepddes, so-caned PTr assays7-1 1･ These methodsare reliableand effective, but requlre

significant levels of techmiCalexpertiseandinterpretation of results. Here we describe a

simple yeast-based method, which makes use of homologous recombinadon in

Sacchmvtnyces celleV血hze lD SeParatealklesandinvolvesthe production of a marker

fusion protein to test for trunc弧ing mutations.

The stop codon scan(SC assay) benefits from two advantages of yeast systems.

First die ability to synthesize fusion proteins withorotidine-5'-phosphate (OMP)

decarboxylase encoded bythe URA3 geneand second,the use of gap repalrand

homologous recombinadon to efficien止yinsert a PCR-generah:d sequenceintothis fusion

constructand separatethe products of different aneles･ The oudine of the assay lS

summarizedinFig･ 1･ We constructed a centromeric yeast expression vector,

PCI-HA(URA3)-2, withtwo selectable markers: LEU2and URA3 (codons 51267), which

complementthe genedc defects of the YPH499 strain, anowing growthinthe absence of

leucineand uracn. 'me URA3 gene is driven bythe sb'ong PGK promoter, tagged atthe

N-terminus by hemagglu血in(HA),and interrupted by a BanHI site to allow

inb･oducdon ofexogenous DNA fragments. To test whedler fusion of proteins tothe

N-teminus of URA3 preservesthe Ura'phenotype, 15 different coding sequen∝s of

O･8-3.4kbinsize, dedvedfrom 7 different genes wereinsertedin-frame.Allinsertions

demonstratedthe Ura'phenotype, conf+irmingthat dlis sekctable marker is not disrupted

by N-teminalfusion to a variety of proteindomains(data not shown). The Ura'

phenotype was dependent on use of the correct upstreim PrOmOterand translational

imidadon codon, as demonstrated by its loss fomowing plaαment of the URA3 fragment

inthe reverse orientadon, byinserdon of 4bpintothe BLmHI site, or byinserdon of

exogenous DNAfragments containingan out10f frameinserdon orinthe reverse

orien血on･ Synthesis of the expected fun lengthURA3 fusion protein from Ura'colonies

was co血med by irmunoblo血ganalysis usnganti-HAantibody.As predicted,

inserdon of out-of-frame sequencesinto PCI-HA(URA3)12 led to die expression of

HA-positive truncated fusion proteinsinUra- colomies (data not shown).

To test the SC assay in detecting truncahg mutationsinBRCAl,the

coding sequence was dividedinto duee overlapping fragments, which wereamplified by

RT-PCRandinsertedin-frameintothe BLmHI site of the PCI-HA(URA3)-2 vector. 1me

resul血g constructs, pCIIBRla, band c, showed preservadon of the Ura'phenotype

following introduc丘oninto yeasL The plasmids were convertedintothe corrsponding

"gap vectors.., PCI-BRlag, pCIIBRlbg and pCIIBRIcg, by removlng most Ofthe

BRG41insert, leavlng 1(氾bp offlanking BRG41 sequence toallow for homologous recombinadon (see Fig. lb and Methods). The three corresponding BRCAl

fragments werethenamplified by RT-PCRfrom peripheralblood mononuclearceus of

padents withknown BRCAl truncahg mutadonsand controls. Cotransfomadon of

unpurified PCR productsand corresponding linearized gap vectorsinto yeast anowed

homologous recombinadon and growth of leucine auxotrophs containing the

recircularized plasmid. Twenty fiveindependent transformants were assayed for growth

inthe absen従Of uracil. Fouowing gap repair WithPCR products derived from control

lymphocytes, 88-100% of transformants were Ura', demonstra血g efficient homologous

recombination of the BRG4J fragmentand reconstitution of the URA3 fusion protein

Cral)le 1). The sman fracdon of Ura- b,ansformants is pnsunably due to infidelity of

the Taq DNA polymeraseand to recombinadon error, as described previouslyl･ In

comb.ast, PCR products derived from the lymphocytes of padents with known

heterozygous BRCAl mutadonsl 1 kd to 44-64% Ura+ transformants. These included

specimens from two padents (#231and #253) witha heterozygous 2bp deledon at codon

23 (the so-Called 185delAG mutation) (gap vector PCI-BRlag), one padent (寸秒9) witha

2bp dele血n at codon 327 (gap vector pCIIBRlbg), one pa也nt (#364) witha

nonsense mutadon at codon 563 (gap vector PCI-BRlbg),and one padent (#250) witha I

bpinsertion at codon 1756 (the so-cabd 5382insC muta血n) (gap vector pCLBRIcg).

Another 6 samplesthat only contained BRCAL polymorphismsll scored as wad-type

(Table 1). The dis廿ibution of Ura'and Ura- colomies dedved from specimens withor

without BRG41 tnmca血g mutadons was reproducibleand non-overlapping (Fig. 2),

demonstratingthe reliability of this assay for diagnostic purposes.

To testthe ability of the SC assay to detectunknown mutations, we chosethe

fammalpolyposis gene APC, which is inactivated exclusively (∼93%) by truncating

mutadons located withinthe N-terminal60% of the coding sequence12･ This pordon of

theAPC CDNA was dividedinto two fragments, APCaand APCb, which containSthe

so-caned mutation cluster region (MCR)13, wasfu,ther dividedinto two ove,lapping

fragments, APCcand APCd.Asexpected, in-frame inserdon of these fragmentsinto

the BLmHI site of PCI-HA(URA3)-2 preservedthe Ura'phenotype. Wethenanalyzed

24individuals derived from sixunrelated famnies widl familialpolyposis (FA㌢) Crable 2).

Analysis of the MCR region for 6 padents (individuals I-I from Families B, D, Eand F;

individualⅠ-7 from FamLy C; individualII-2 from Family A) who were clinically

diagnosed as affected, uslng gap V∝tOr PCI-APCbg, yielded yeast b.ansformants, half of

which retainedthe Ura'phenotype (m弧1 49%, range 38-60%), consistent withthe

presence of a heterozygous truncating mutaBon (Tabk 2). To idemifythe precise

locadon of each mutadon, gap repau assays wereperformed using dleintEmalgap vectors,

PCI-APCcg and pCIIAPCd (NdeIINsll digest):individualⅡ-2 from Family Åand individualⅠ-I from Fam止y B, scored posi丘vefor mutadons widlinfragment APCc, but

not APCd, whereasthe converse was true for individualIl7from Farnily Cand

individuals Ill from Farnilies D-F. Thisanalysis was extended tothe remaJnlng 18

members of these families, idemifying 5individuals as having heterozygous trunc姐ng mutadons withinthe same fragment asthe proband (Table 2). Sixindependent

Ura-colonies were pooled and subjected to direct nucleotide sequenclng. ne SeParadon of

APCalleles resulting fromthe gap repair assay made it possible to specificdlyanaly2X:the

mutaJltallele, avoidingthe difficul血s inherentinsequenclng heterozygous mut加ions.

All specimens scored as positive by SC assay were found to have truncabng APC

mutadons: a 4bp dekBdon at codon 929 (3765de14)inFamily A, a lbpinsertion at codon

938 (2831insT) in Fandy B, a 2bp deldon atcodon 1249 (3765de12)inFamily C, a 5bp

deledon atcodon 1309 (3945de15)inbothFamilies Dand E,and a lbp deletion at codon

1322 (3983delA)inFamily F gable 2).Analysis of odler members from each fanny

demonstrated a complete concordance betweenthe results of the SC assayand direct

sequencinganalysis Crable 2).

The mutadonalanalysisthat we describe here is comparable in its efficacy to

previously described PTr teclmiquesthatinvolve PCR-amplification of gene fragments,

fonowed by in vi細tranSCription-tranSladon and resolution of encoded pepddes by

SDS-PAGE. However,the yeast-based SC assay provides a number of important techmiCal

advantages compared to dleSe加viqD, gel-based assays.Amongtheseare (i)the

ability to analyze larger (-3.5kb) DNAかagments, which托duccsthe number of PCR

reiK:也ons required to scananemire gene for trunca血g mutadons, (ii)the abnity to

detect mutationsthat arise adjacent tothe PCR-primers, which minimiZeSthe overlap

required between fragments, (ih)the separation of aneles, which gready simplifies

conflrmadon of hetero野gOuS mutadons by nucleo丘de sequencing, (iv) avoidance of

the requirement for radioisotopes and for protein gel-electrophoresis. From a technical

standpoint,the SC assay requires few mmipuladonsand dle血Ie required toperformthe

assay (four days) reflectsthat required 也 clearly visuali2XB yeaLSt COlomies after sequemial

plahg on leucineand uracn derlCient plates. ne use of a mutadonalscreenmg test

based onthe detection of trunc血ng mutationsalso has important advantagesin血e

adaptadon of such approaches to chicaldiagnostics. Proteintruncabng mutations

comprisethe majority ofinactiV血g mutadons for a number of important cancer

predisposition genes,including BRC41, BRCA2, APC,mismatch repair genes,and

potentiany AIM. FurdlemOre,the difficultyininterpre血g miSsense mutations

precludestheir use in most climiCaldiagnos血S. ¶le SC assaythus provides a rapidand

reliable testthat can be readdy adapted to detect heterozygous truncabng muta丘onsin

Cancer predisposition genesand other genes imphcatedinhuman disease.

Melhods

Plasmid consLT'uCtion. The plasmid PCI-HA(URA3)12 was constructed as

fonows: a fhgment spanming nucleotide-number (nL) 423 to 1239 of the plasmid

pRS31614 (GenBank UO3442), which contains URA3 coding sequence from codon 5 10

the naturalternhadon codon, wasamplified by PCR using a set of primers contalnlng a

BmnHI site or a BgaI site atthe 5. end. The BmnHI/Bgul fragment was insertedin-frame

inbthe BLZmHI site of the plasmid PRS-PGK15 10 produce PCI-HA(URju). This vecto,

was digested withNsltand PstT and religated to produce PCIIHA(URA3)-2 (Fig. 1a)･

¶lr綻fragments spaming nt. 96 to 908 (BRCAla), nL 789 to 4214 (BRCAlb)and nL

4089 to 5708 (BRCAIc) of the BRG4J CDNA (GenBank U14680), wereamplified

and insertedin-frameintDthe BLmHI site of the PCI-HA(URA3)-2 to produ∝ PCI-BRla, PCI-BRlband PCI-BRIG, respecdvely (Fig･ lb)･ Four血agments spanning nt･ 19 to

1977 (APCa), nt 1978 to 5256 (APCb), nt. 1978 to 3570 (APCc)and nL 3571 to 5256

(ApCd) of the APC CDNA (GenBank M74088), were amplified and insertedin-frame

intothe BLmHI site of PCI-HA(URA3)-2 b produce PCI-APCa, PCI-APCb, PCI-APCc

and PCI-APCd, respecdvely (Fig. lb).仙the vectors described above resultina Ura'

phenotype, followingintroduction into YPH499 strain･ The gap vectors, PCI-BRlag,

PCI-BRlbg, PCI-BRIcg, PCI-APCag, PCI-APCbgand PCI-APCcg (Fig･ lb)are

idendcalto PCI-BRla, PCI-BRlb, PCI-BRIG, pCIIAPCa, PCI-APCband PCI-APCc,

respectively, exceptthatthe centralportions oftheinserted fragments, BRCAla･

BRCAlb, BRCAIc, APCa, APCband APCc, between nt. 183 to 827, nt. 888 to 4111,

nL 4215 to 5609 (GenBaJik U14680), nt. 109 to 1899, nt. 2054 to 5201 and nt. 2086 to

3489 (GenBank M74088), were replaced bythemique resdction sites, BgnI,

SLuuBamHI/Smd, Bg瓜, BgⅢ, Nsll and BguI, respectively･ A皿the gap vectors

except pCIIAPCbg were produced by PCR using ExTaq (TAKARA) andthe original

plasmids widl fd-lengdlinsertion as templates, followed byligation uslngthe unique

restricdon sites described above and transformation of E. coli (DH5α). The PCI-APCbg

was obtained by removingthe centralpordon of APCb丘agment of the PCI-APCb using

two Nsll sites.

PCR. For BRG41and APCanalysis, genomic DNAand/or totalcenular

RNA was isolated from EBV-immortalized cenlines or peripheralblood mononuc血r

cens. CDNA was syndleSized by using a First-Sb･and CDNA SynthesisKit (Pharmacia)･

BRCAla-Cand APCa (see Fig. lb) were amplified from cDNA･ BRCAband APCb-d

were amplified from genomic DNA･ Primers foramplificadon of BRCAl fragments were

5 ㌧GAAAmAmGAACAGAAAGAA-3 - and

5㌧ACCCTGATACmCTGGATG-3' for BRCAla,

5 LCCCAGATCTGCTGCTFGTGAAr I Tl lCTGAG-3 land

5'-CCCAGATCTTjuGTrrGAATCCATGCTITG-3- for BRCA 1 b,and

5 I-ATGAGGC ATCAGTCTGAAAGC-3 - and

5 I-GTAGTGGCTGTGGGGGATCT-31 for BRCA Ic.

Primers for amplificadon of APCfragments were

5 I- ATGGCTGCAGCTrCATATGAT-3 I

and 5 LCTGTGGTCCTCAmGTAGC-3.for Ape 1 a,

5 LCAAATCCTjuGAGAGAACAACl3 land

5'-GTCCATrATCITITfTCACACG-3'for APCb,

5 ㌔ CAAATCCrAAGAGAGAACAA-3'and

5'-GGCATATITrAAACrATAATC-31 for APCc,and

5 '-ACAGATATFCCTrCATCACAG -3'and

5'- GTCCATrATCITITIICACACG-3- for APCd. A皿PCR fhgments were obtained by

using ExTaq polymerase CrAKjuA). btails of the PCR parametersare avahble from

the authors upon request.

YeasL LTanSfonnaLion. The yeast sb･ainusedindds study was YPH499

(AL4 TL"nag-52, lys2-801andw,.de2-101ochre,坤1A63, his3A200,leu2Al) (Sb,atagene).

Competent yeast Gens were prepared bylithium acetate恥iOAc) b'eatment of the strain

culturedinYPD hquid mediumland were stored at -80oCinthe presence of 5% DMSO

until use. Frozen competent yeast retainhigh transformadon efficiency for at least three

months. Gap repair assays Were performed by cob･ansfomation of unpurified PCR

product (∼200ng)and lineari2ed gap vector (∼30ng) bythe LiOAc method16 withminor

modificadonsl･ Toanaly2ethe PCR fragments, BRCAla, BRCAlb, BRCAIc, APCa,

APCb, APCc,and APCd, linearized gap vectors, PCI-BRlag (Bg皿I digest), pCLBRlbg

(BLmHI/Smd digest), PCI-BRIcg (BgEI digest), pCIIAPCag (Bgnl digest),

pCI-APCbg (NsIT digest), PCI-APCcg (BguI digest), and PCI-APCg (NdeIINsll digest) were

used, resp∝dvely･ Transformants were selected on synthedc complete medium (SC)

lacking leucine (SC-Leu); 25 transformants werethen assayed for die Ura'phenotype by

growdl On SC lacking leucineand uracil (SC-Leu-Ura). Ⅳ morethan 85% of

transformants are Ura',the sample is scored as homozygous wad-type, whereas if an

transformants ale Ura ,the sample is scored as homozygous mutant. If 40-50% of

colomies are Ura+,the samples is scored as heterozygous for a truncation mutant.

Seq〟enctng･ YeGLSt DNA extraction from pooled Ura- tranSformants was

descdbed previously17･ TemplateAPC打agments wereamplined as described above and

were sequenced using CircumVentTM ThermalCycle Dideoxy DNA Sequencing Kit (New

hgland Biohbs)･ Appropriate APC specific oligonucbtides were used as sequencing

primers after end-labeled by l7-33p] dATP.

Ackno wledgmcnts

Weare grateful to our padentsandtheir famines for parbcIPatlnginthis studyand we

thank anthe physicians who provided clinicalSamples foranalysis･ Wealso acknowledge

S･H･ Friendand M･ Vidalfor helpfd discussions叫inthis study and H. Shibata for

usefulcomments onthe manuscript･ This work was supportedinpart by Grant-in-jud for

Scientific Research (C).

Rcfer･ences

1. Ishioka, C. etal. ScrEnnLng Patients for heterozygous p53 mutations

using afunctionalassay in yeast. Nahqe denet. 5, 1241129 (1993).

2.Kinzler, K.W. etal. Identification of PAP luus genes from chromosome

5q21. Science 253, 661-5 (1991).

3. Nishisho, I. et al. Mutations of chromosome 5q21 genes in PAP and

colorectalCancer padents. Science 2 53, 665-9 ( 199 I).

4. Groden, J. etal. Identificationand characterizadon of the famihal

adenomatous polyposis coligene. Cell 6 6, 5891600 (199 1).

5. Joslyn, G. etal. Idemification of deletion mutationsandthrEX: new

genes atthe familialpolyposis locus. Cell 6 6, 601-13 (1991).

6. Miki, Y. etal. A strong candidate forthe breiLStand ovarianCancer

susceptibility gene BRCAl. Science 2伽, 66-71 (1994).

7. Ro鮎t, PAM., Roberts, R.G., Sugino, S., VanOmmen, G.J.B. & bn

Dunnen, J.T. Proteintruncation test (叩for rapid detection of

transladon-terminating mutations. Hutn. Mol. Genet. 2 , 17 19-1721 ( 1993).

8. Powell, S.M. et al., Moleculardiagnosis of血milialadenomatous

polyposis, New Engl. ). Med. 329, 198211987 (1993).

9. Vander Luijt, R. etal. Adenomatous polyposis coli (APC) gene by direct

Protein Truncadon Test. Genomics 2 0, I-4 (1994).

10. Hogervorst, F.B. etal. Rapid detection ofBRCAl mutations bythe

protein加ncadon test. Naive Genet. 1 0, 208-12 (1995).

I 1. FitzGerald, M.G. etal. Germ-line BRCAl mutationsinJewishand

non-Jewish women withearly-onset breast cancer. New Engl. ). Med. 3 3 4,

143-9 (1996).

12. Nakamura, Y. ''GenetiCanalysis of hereditary cancer syndrome... New

Strategies for T陀atment Of Hereditary ColorectalCancer. Ed. S. Baba

Churchill Livingstone, 1996) 93-98.

13. Miyoshi, Y. etal. Germ-line mutadons oftheAPC genein53血Ililial

adenomatous polyposIS Patients. PT10C. Nail. Acad Sci. USA 8 9, 4452-6

(1992).

14. Sikorski, R.S. & Hieter, P.A. A system of shude vectorsand yeast

host stmins designed for efrICient mmipulationof DNAinSaccharomyces

cerevisiae. Geneh'cs 1 22, 19127 (1989).

15. Ishioka, C. etal. Afuncdonalassay for heterozygous mutationsinthe

GTPase activating proteinrelated domainofthe neurofibromatosis type 1

gene. Oncogene 1 0, 841-847 (1995).

16. Ito, H., Fukuda, K., Nurata, K. &Kimura, A. Transformation of intact

yeast cells treated withabline cations･ J･ Bacteliol･ 1 53･ 163- 168

(1983).

17. Ishioka, C. etal. Mutadonalanalysis of the carboxy-teminalpordon

of p53 using bothyeastand mammaliancen assays in vivo･ Oncogene 1 O･

1485-92 (1995).

Figwe legends

Fig. 1 Schematic representation of SC assay.

a. PCI-HA(URA3)-2 vector. In-frameinsertion of a coding sequence of interest

intothe unique BamHI由te resultsinconstitutive expression ofanHA-tagged URA3

fusion protein, driven fromthe PGK (3-phosphoglycerate kinase) promoter. h addition,

the vector containSthe PGK terminator downstreiLm Ofthe URA3鉦agment,the LEU2

gene as a second selectable marker, and CBNand ARS for stable low copy number

rephcation. The derived gap vector lacksthe ∝ntralpordon oftheinserted fragment,

which can be replaced by a PCR-generated fragmentinserted by homologous

recombinadon, usingthe remainingflanking sequences.

b. Sequences of BRG41and APC chosenforanalysis. CDNA or genomic

fragments denoted BRCAla,Cand APCa-d wereinserted in-frame intothe BamHI site

of PCI-HA(URA3)-2, producing plasmids PCI-BRla, band c, and PCI-APCa, b, c and d.

Plasmids, pCIIBRlag, bg and cg,and PCI-A陀ag, bland cg, are gap vectors.

C. Schemadc representation of SC assay. 1. PCR amplification of CDNA or

genomic fhgment containing w止d-type (W¶ or hncated mutaJlt (mt; nonsense Or

frameshift) is combined widlthe appropriate gap vector, Which contains l00bpflanking

the PCRfragment to anow 氏)r efficient homologous recombinadon. 2. transformation

of leu2 and war defident yeast withthe PCR productand gap vector yields leucine

prototropic transformantsthat haveundergone recirculari2adon of the plasmid fouowing

homologous recombinadon･ 3･ selecdon ofLeu'prototroph fonowing repla血ginthe

absen∝ of uracu distinguishes Ura'prototrophs (Wild-typeinserted sequence)from

Ura-auxotrophs (truncatiOn mutation). OMPD, orotidine-5■-phosphate (OMP) decarboxylase.

4. Representadve SC assay for BRCAI (fragment BRCAlb) and APC

(&agment APCb), showing growthof yeast b･anSformantsinthe absence of uracil･ In

bothcases, die left half-plate represents a specimen derived from a pabent witha

heterozygous trunca血g mutation,andthe dght half-plate is a control sample･

Fig･ 2 Distribution of tJra+ colomies deriyed from specimenswithwiLd･

type BRCAL 帥d Arc or containing a heterozygous truncating

mutation.

Results of the SC assay are shown for 75 specimens for which presence (closed

bar) or absence (open bar) of a truncating mutadon was connmed by nucleotide

sequenclng･ Values in parenthesisindicate mean ± Standard deviation･

a

pC l-H A(U RA3>2

AFLTか'0慧FA'山J

eBEA1a BRCAlb 避退』 .-pcRbagments

pCH泊1a FCトBMb FCトBRIc ーfuHbngthⅥ触 FC柑Rlag pC相Rlbg pCLBRIcg一gapvcch

ATB A PC TAA

l eKOnl･14 l mlS - APCa APCb

A望｣1__止聖コpcRfragmerds

FCLAPCa ,CLA,CcScトAPCbAトA,C.コhd hqh vecton

pcLAPCq FCLA昔篭慌恕触

C ◎,cR

WT l¶t

/へ

NonserLSe Or baTneShift rrLItatbn gap V●OtOr ◎ TrA'dqnad仰 SC･Lou

d BFICAl b

Pt. 99 Contl℃l Truncded protein ◎ Asii･y

Family A control

(lト2)

F･taure i

20  40  60  80 100

tJra+ Fraction (%)

Fi3㌦e 2/

0 L O O         5

2

1

1

p 翁 L P ! t n S u a t t Z P a d s J o J 3 q t t m Z

Table 1

Table 1 SC assay for βFICA7 in women with early･onSet breast cancer

SC assay (% Ura'cdonles)      Mutatjon

BRCAl fragment 1 a 1 b 1 c Sequenco Location

codonS       1 I 263    224 - 1365  1324. 1863

PCR analysis CDNA gDNA CDNA CDNA Patient Pt. 43 Pt. 79 Pt. 84 Pt. 99 Pl. 103 Pt. 118 Pt. 231 Pt. 250 Pt. 253 Pt. 364 92    N D    88    92 ND    92    ND ND 92     91    92    96 96  【垂]  国   88 88    N D    88    96 88     94     94    88 【垂]  N D   88   92 96   ND BB   @ 【垂]  ND   88  1 00 96    N D ri71   92

WT

WT

WT

2bp dolotjon (frameshift)

WT

WT

2bp deletion (frameshjft) C insenjon (frarneshift) 2bp deletion (frameshift)

CGA to T丘A -nonsonso1

codon 327 (Oxon 1 1)

codon 23 (Oxon 2) codon 1 756 (Oxon 20)

codon 23 (Oxon 2) codon 563 Eexcln ll)

AJl pabnts aro women with broast cancer boforo tho ago 30 and havo been charactedzed for BF7CAl mutadons previou$ly

(ref･1 1)･ Lmdon o‖〕RCAl fragment was hldicatod ln Fig･ 1 ･ Bc･xed number indicates existence of trunca蜘1g mutation in

examined DNA fragment by SC assay･ gDNA･ genomlc DNA; ND, not detormjned; WT. dld-type jn the entire codjng

Sequenco. The mutations detocted ln Pt.231 and Pt. 253 aro known aS 185delAG. The mutation detected in Pt. 250 is known

Table 2

Table 2 SC assay for APC mutations in 6 FAPfamilies･

SC as組y (% Ura'colonies)

APC fragment b c d Sequ ence

Codon$        654- 1748 654- 1184 1185 ･ 1748

PCR anaLysIS gDNA gDNA gDNA

Farnlly & lndivisuats

FamiLyA H11 1 00 Fami一y a Fami一y C Family E FamHy F ll1 園 11･1  96 日･2  88

H ND

ト2  ND ト3  ND I-7 圃 Ilo ND

tH ND

lト2  ND lト3  ND Mutation 92    ND WT

[垂I   92   4bp deletion (frameshift)

固   ND   4bp deletion (frameshlft) [蚕】 1 00   T insonion (frameshiq 92     N D WT

1 00 ND WT

ND 1 OO         Ⅵ汀

ND 1 00 WT

ND     96      WT 92  [頭   2bp delotjon (frameshift) N D     92      WT ND  [垂l   2bp deletion (framoshift) ND  [垂】  2bp deletion (frameshift) ND   固   2bp delotion (frameshjft) I-1 @   92

1H ND ND

lト2  ND ND lト3  ND ND ト1 国   96

1H ND ND

lト2  ND ND H-3  ND ND L-1 匿]  92 1ト1  ND ND [垂l  5bp doleぬn (framoshift) 92      WT 96      WT 88      WT

[垂I Sbp doletjon (frameshift)

92      WT

固   5bp doletjon (framoshift)

92      WT

[垂I A deletion (frameshift)

92      WT Location ccdon 929 - 930 (Exon 15) codon 929 - 930 (Exon 15) codon 938 (Exon 1 5) codon 1249 I 1250 (Exon15) codon 1249 - 1250 (Exon15) codon 1249 - 1250 (Exon15) codon 1249 I 1250 (Exon15) codon 1309 -1311 (Exon 15) codon 1309 -131 1 (Exon 15) codon 1309 -1311 (Exon 15) codon 1322 (Exon 15)

AJHndividuals in the 6 FAPfarnllies aro Japaneso. Locaぬn of Ape fragmont was indicated in Fjgl 1･ Boxod numbor

indbates existenco of truncating mutation in examined DNA fragmont by SC assay. gDNA. gonomic DNA; ND, not

dotomlined; WT, wild-typo ln tho examined ceding sequence.

D

_ > ∩