A Strong Candidate for the Breast and Ovarian Cancer Susceptibility BRCA1 Author(s): Yoshio Miki, Jeff Swensen, Donna Shattuck-Eidens, P. Andrew Futreal, Keith Harshman, Sean Tavtigian, Qingyun Liu, Charles Cochran, L. Michelle Bennett, Wei Ding, Russell Bell, Judith Rosenthal, Charles Hussey, Thanh Tran, Melody McClure, Cheryl Frye, Tom Hattier, Robert Phelps, Astrid Haugen-Strano, Harold Katcher, Kazuko Yakumo, Zahra Gholami, Daniel Shaffer, Steven Stone, Steven Bayer, Christian Wray, Robert Bogden, Pri ... Reviewed work(s): Source: Science, New Series, Vol. 266, No. 5182 (Oct. 7, 1994), pp. 66-71 Published by: American Association for the Advancement of Science Stable URL: http://www.jstor.org/stable/2884716 . Accessed: 03/12/2011 17:29

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cer is often familialin origin, althoughthe A Strong Candidate for the risksin relativesare not as high as those for early-onsetbreast cancer (10, 11). The per- centageof such casesthat aredue to genetic Breast and Ovarian Cancer susceptibilityis unknown. Like many other involved in fa- Susceptibility Gene BRCA1 milial cancer, BRCA1 appearsto encode a tumor suppressor,a that acts as a Yoshio Miki, Jeff Swensen, Donna Shattuck-Eidens, P. Andrew Futreal, negative regulatorof tumor growth. Can- Keith Harshman, Sean Tavtigian, Qingyun Liu, Charles Cochran, cer-predisposingalleles typicallycarry mu- L. Michelle Bennett, Wei Ding, Russell Bell, Judith Rosenthal, tations that cause loss or reductionof gene Charles Hussey, Thanh Tran, Melody McClure, Cheryl Frye, Tom Hattier, function. Predispositionto cancer is inher- Robert Phelps, Astrid Haugen-Strano, Harold Katcher, Kazuko Yakumo, ited as a dominant genetic trait, whereas Zahra Gholami, Daniel Shaffer, Steven Stone, Steven Bayer, Christian Wray, the predisposingallele generallybehaves as Narod, Robert Bogden, Priya Dayananth, John Ward, Patricia Tonin, Steven a recessiveallele in somatic cells. Thus, a Pam K. Bristow, Frank H. Norris, Leah Helvering, Paul Morrison, mutant allele Neuhausen, single inherited copy of the Paul Rosteck, Mei Lai, J. Carl Barrett, Cathryn Lewis, Susan inactiva- Alexander Kamb, causes predisposition,and loss or Lisa Cannon-Albright, David Goldgar, Roger Wiseman, completesone of Mark H. Skolnick* tion of the wild-typeallele the steps in progressiontoward malignancy. A strong candidate for the 17q-linked BRCA1 gene, which influences susceptibility to When loss is observed in breast and ovarian cancer, has been identified by positional cloning methods. Probable breast and ovarian tumors from patients predisposing mutations have been detected in five of eight kindreds presumed to seg- who carryBRCA1 predisposing alleles, the regate BRCA1 susceptibility alleles. The mutations include an 1 1- deletion, a wild-typecopy of BRCA1is invariablylost 1-base pair insertion, a stop codon, a missense substitution, and an inferred regulatory while the presumptivemutant allele is re- mutation. The BRCA1 gene is expressed in numerous tissues, including breast and ovary, tained (12-14). This finding supportsthe and encodes a predicted protein of 1863 amino acids. This protein contains a zinc finger hypothesisthat BRCA1is a tumorsuppres- domain in its amino-terminal region, but is otherwise unrelated to previously described sor gene and suggeststhat the functional . Identification of BRCA1 should facilitate early diagnosis of breast and ovarian BRCA1 protein is presentin normalbreast cancer susceptibility in some individuals as well as a better understanding of breast and ovarianepithelium tissue and is altered, cancer biology. reduced,or absentin some breastand ovar- ian tumors. Genetic analysisof recombinantchromo- somes in membersof largekindreds allowed Breast cancer is one of the most common Genetic factorscontribute to an ill-de- localizationof BRCA1initially to a regionof and important diseases affecting women. fined proportionof breastcancer incidence, 1 to 2 megabaseson chromosome17q (15- Currentestimates indicate that one in eight estimatedto be about 5% of all cases but 17) and, subsequently,to a region of about American women who reach age 95 will approximately25% of cases diagnosedbe- 600 kilobasepairs (kb) (18) betweenmarkers develop breast cancer (1). Treatment of fore age 30 (2). Breast cancer has been D17S1321 and D17S1325 (19). A physical advancedbreast cancer is often futile and subdividedinto two types, early-onsetand mapcomprised of overlappingyeast artificial disfiguring,making early detection a high late-onset, a division that is based on an (YACs), Pl, bacterialartificial priorityin medicalmanagement of the dis- inflection in the age-specific incidence chromosomes(BACs), and cosmid clones was ease.Ovarian cancer, although less frequent curvearound age 50. Mutationof one gene, generatedfor this region(18). than breastcancer, is often rapidlyfatal and BRCA1,is thought to accountfor approxi- Identificationof a strong BRCA1 can- is the fourthmost common cause of cancer mately 45% of families with significantly didate gene. Severalstrategies were used to mortalityin Americanwomen. high breast cancer incidence and at least developa detailedmap of transcriptsfor the 80%of familieswith increasedincidence of 600-kbregion of 17q2l betweenD17S1321 Y. Miki, J. Swensen, K. Yakumo, C. Lewis, S. Neu- both early-onsetbreast cancer and ovarian and DJ 7S1325. Sixty-five candidate ex- hausen, and D. Goldgar are in the Department of Medical Informatics,University of Utah Medical Center, Salt Lake cancer (3). Intense efforts to isolate the pressedsequences (20) within this region City, UT 84132, USA. D. Shattuck-Eidens, K. Harshman, BRCA1 gene have proceededsince it was were identified. Expressedsequences were S. Tavtigian, 0. Liu, W. Ding, R. Bell, J. Rosenthal, C. first mapped to chromosomearm 17q in characterizedby DNA sequence, database Hussey, T. Tran, M. McClure, C. Frye, T. Hattier, R. expressionpat- Phelps, H. Katcher, Z. Gholami, D. Shaffer, S. Stone, S. 1990 (4, 5). A second , BRCA2, re- comparison,transcript size, Bayer, C. Wray, R. Bogden, P. Dayananth, and A. Kamb cently mappedto chromosomearm 13q (6), tern, genomic structureand, most impor- are at MyriadGenetics, 421 Wakara Way, Salt Lake City, appearsto accountfor a proportionof early- tantly, DNA sequenceanalysis in individu- UT 84108, USA. P. A. Futreal,C. Cochran, L. M. Bennett, als fromkindreds that segregate17q-linked A. Huagen-Strano, J. C. Barrett, and R. Wiseman are at onset breast cancer roughly equal to that the Laboratoryof Molecular Carcinogenesis, National In- resulting from BRCA1. Unlike BRCA1, breast and ovarian cancer susceptibility. stitute of EnvironmentalHealth Sciences, National Insti- however,BRCA2 may not influenceovari- Three expressedsequences eventually were tutes of Health, Research TrianglePark, NC 27709, USA. susceptibility merged into a single transcriptionunit J. Ward and L. Cannon-Albrightare in the Department of an cancerrisk. The remaining InternalMedicine, Universityof Utah Medical Center, Salt to early-onsetbreast cancer is likely attrib- whose characteristicsstrongly suggest that Lake City, UT 84132, USA. P. Tonin and S. Narod are in utable to unmappedgenes for familialcan- it is BRCAI (21). This transcriptionunit is the Department of Medical Genetics, McGill University, rare germline mutations in genes located in the center of the 600-kb region Montreal, Quebec, H3G 1A4, Canada. P. K. Bristow, F. cer and H. Norris, L. Helvering, P. Morrmson,P. Rosteck, and M. such as TP53, which encodes the tumor (Fig. 1) spanningD17S855 and will be re- Lai are at LillyResearch Laboratories, Eli Lillyand Com- suppressorprotein p53 (7). It has also been ferredto herein as BRCAJ. pany, Indianapolis, IN 46285, USA. M. H. Skolnick is in suggestedthat heterozygotecarriers of de- A combination of sequences obtained the Department of Medical Informatics,University of Utah DNA (cDNA) clones, Medical Center, and MyriadGenetics, Salt Lake City, UT fective forms of the gene predisposingto from complementary 84108, USA. ataxia telangiectasiaare at higher risk for hybrid-selected sequences, and amplified *To whom correspondence should be addressed. breastcancer (8, 9). Late-onsetbreast can- polymerase chain reaction (PCR) products

66 SCIENCE * VOL. 266 * 7 OCTOBER 1994 allowed the constructionof a composite, ing is coordinatedwith alternativesplicing BRCAI is conservedin mammals. full-length BRCA1 cDNA. The cDNA farther downstream,and whether all the Germline BRCA1 mutations in 17q- clone extendingfarthest in the 3' direction splice variants produce proteins with an linked kindreds. Identificationof a candi- contains a polyadenylatetract precededby identicalNH2-terminus, are questionsthat date gene as BRCAJrequires a demonstra- a polyadenylationsignal. Conceptual trans- remainto be explored. tion of potentiallydisruptive mutations in lation of the cDNA revealeda single, long We also probedgenomic DNA samples that gene in carrierindividuals from kin- open readingframe with a presumptiveini- from severaldifferent species with BRCA1 dredsthat segregate17q-linked susceptibil- tiation codon flankedby sequencesresem- sequencesdevoid of the zinc finger region. ity to breastand ovariancancer. Such in- bling the Kozakconsensus sequence (22). Low-stringencyblots revealedstrongly hy- dividualsmust contain BRCA1alleles that This reading frame encodes a protein of bridizingfragments in tissuesfrom humans, differfrom the wild-typesequence. The set 1863 amino acids (Fig. 2A). Smith-Water- mice, rats,rabbits, sheep, and pigs, but not of DNA samplesused in this analysiscon- man (23) and BLAST (24) searchesiden- chickens (Fig. 5). These resultssuggest that sisted of DNA from individualsrepresent- tified a sequence near the NH2-terminus that has considerablesimilarity to zinc fin- ger domains(25) (Fig. 2B). This sequence A * v y contains cystine and histidine residues 0 MDLSALRVEEVQNVINAMQKILECPICLELIKEPVSTKCDHIFCKFCMLKLLNQKKGPSQ present in the consensus Cys3-His-Cys4 60 CPLCKNDITKRSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEHLKD (C3HC4) zinc fingermotif and sharesmany V other residueswith zinc finger proteins in 120 EVSIIQSMGYRNRAKRLLQSEPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYI the databases.The BRCA1 gene is com- 180 ELGSDSSEDTVNKATYCSVGDQELLQITPQGTRDEISLDSAKKAACEFSETDVTNTEHHQ posed of 22 coding exons distributedover 240 PSNNDLNTTEKRAAERHPEKYQGSSVSNLHVEPCGTNTHASSLQHENSSLLLTKDRMNVE 300 KAEFCNKSKQPGLARSQHNRWAGSKETCNDRRTPSTEKKVDLNADPLCERKEWNKQKLPC roughly100 kb of genomic DNA (Fig. 3). 360 SENPRDTEDVPWITLNSSIQKVNEWFSRSDELLGSDDSHDGESESNAKVADVLDVLNEVD Hybridizationof RNA blots to labeled 420 EYSGSSEKIDLLASDPHEALICKSDRVHSKSVESNIEDKIFGKTYRKKASLPNLSHVTEN fragmentsof BRCA1cDNA revealeda sin- 480 LIIGAFVSEPQIIQERPLTNKLKRKRRPTSGLHPEDFIKKADLAVQKTPEMINQGTNQTE 540 QNGQVMNITNSGHENKTKGDSIQNEKNPNPIESLEKESAFKTKAEPISSSISNELELNIM gle transcriptof 7.8 kb. This transcriptis 600 HNSKAPKKNRLRRKSSTRHIHALELWSRNLSPPNCTELQIDSCSSSEEIKKKKYNQMPV most abundantin testis and thymus,but is 660 RHSRNLQLMEGKEPATGAKKSNKPNEQTSKRHDSDTFPELKLTNAPGSFTKCSNTSELKE also present in breast and ovary (Fig. 4). 720 FVNPSLPREEKEEKLETVKVSNNAEDPKDLMLSGERVLQTERSVESSSISLVPGTDYGTQ 780 ESISLLEVSTLGKAKTEPNKCVSQCAAFENPKGLIHGCSKDNRNDTEGFKYPLGHEVNHS The cDNA clones derivedfrom the 5' one- 840 RETSIEMEESELDAQYLQNTFKVSKRQSFAPFSNPGNAEEECATFSAHSGSLKKQSPKVT third of BRCA1 transcriptsdisplay a com- 900 FECEQKEENQGKNESNIKPVQTVNITAGFPVVGQKDKPVDNAKCSIKGGSRFCLSSQFRG plex pattem of altemative splicing. Four 960 NETGLITPNKHGLLQNPYRIPPLFPIKSFVKTKCKKNLLEENFEEHSMSPEREMGNENIP 1020 STVSTISRNNIRENVFKEASSSNINEVGSSTNEVGSSINEIGSSDENIQAELGRNRGPKL altemative splices were observed down- 1080 NAMLRLGVLQPEVYKQSLPGSNCKHPEIKKQEYEEVVQTVNTDFSPYLISDNLEQPMGSS stream of the start codon as independent 1140 HASQVCSETPDDLLDDGEIKEDTSFAENDIKESSAVFSKSVQKGELSRSPSPFTHTHLAQ 1200 GYRRGAKKLESSEENLSSEDEELPCFQHLLFGKVNNIPSQSTRHSTVATECLSKNTEENL cDNA clones (P3, P4, B31, and B21 in Fig. * 3); three of these pattemswere detectedin 1260 LSLKNSLNDCSNQVILAKASQEHHLSEETKCSASLFSSQCSELEDLTANTNTQDPFLIGS breastcDNA (P3, B31, and B21) and two V 1320 SKQMRHQSESQGVGLSDKELVSDDEERGTGLEENNQEEQSMDSNLGEAASGCESETSVSE in ovarycDNA (P3 and B21). In addition, V PCR analysis of cDNA samples prepared 1380 DCSGLSSQSDILTTQQRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALE from breast,ovary, testis, and lymphocyte messenger RNA (mRNA) indicates that 1440 DLRNPEQSTSEKVLQTSQKSSEYPISQNPEGXSADKFEVSADSSTSKNKEPGVERSSPSK there is considerableheterogeneity in splice 1500 CPSLDDRWYMHSCSGSLQNRNYPPQEELIKVVDVEEQQLEESGPHDLTETSYLPRQDLEG 1560 TPYLESGISLFSDDPESDPSEDRAPESARVGNIPSSTSALKVPQLKVAESAQSPAAAHTT junction usage near the 5' end of BRCAI v transcripts,upstream of the presumptive 1620 DTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGLTPEEFMLVYKFARKHHITLTNLI initiationcodon. How this alternativesplic- 1680 TEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVTQSIKERKMLNEHDFEVRGDV +c V R V 1740 VNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQLCGASVVKELSSFTL V V / jD17S250 1800 GTGVHPIVVVQPDAWTEDNGFHAIGQMCEAPVVTREWVLDSVALYQCQELDTYLIPQIPH 1860 SHY | / 4tD1 7S800

* / -D17S-1321 B / BRCA1 -D17S855 BRCA1 CPICLELIKEPVSTK-CDHIFCKFCMLKLLNQKK--- GPSQCPLCK RPT1 CPICLELLKEPVSAD-CNHSFCRACITLNYESNRNTDGKGNCPVCR .// RIN1 CPICLDMLKNTMTTKECLHRFCSDCIVTALRS-----GNKECPTCR -D17S1327 RFP1 CPVCLQYFAEPMMLD-CGHNICCACLARCWGTAC---TNVSCPQCR - CA125 C3HC4 motif C--C------C-H--C--C------C--C -RNU2 Fig. 2. Predicted amino acid sequences for BRCAl (46). (A) Conceptual translation of the BRCA1 open reading frame, indicating the approximate positions of introns (triangles above sequence) and the D1 7S1325 locations of germline mutations (boldface residues). The -1 1 bp deletion in kindred 1901 is shown by an asterisk; the nonsense mutation in kindred 2082 is shown by a star; the frameshift in kindred 1910 is | shown by "+c"; and the missense mutation in kindred 2099 is shown by "R". The BRCAl nucleotide _ X - "~~\, D~~D17S579 sequence is deposited in GenBank with accession number Ul 4680. PCR primersequences are available via anonymous FTP at the following internetaddress: morgan.med.utah.edu in the directorypub/BRCA1; or by fax at the following number: 801-584-3650. (B) Alignment of the BRCA1 zinc finger domain with Fig. 1. Schematic map of human chromosome three other zinc finger domains that scored highest in a Smith-Waterman alignment. RPT1 is a protein 1 7. The pertinentregion containing BRCA 1 is ex- that appears to be a negative regulator of the interleukin-2receptor in mice (47). RINMis a DNA binding panded to indicatethe relativepositions of two protein that includes a RINGfinger motif related to the zinc finger (48). RFP1 is a putative transcription previously identifiedgenes, CA125 (34) and factor comprising the NH2-terminaldomain of the REToncogene product (49). The C3HC4 motif shows RNU2 (45). D1 7S855 is locatedwithin BRCA 1. the positions of the cystines and the histidine that form the zinc binding pockets.

SCIENCE * VOL.266 * 7 OCTOBER1994 67 ing eight differentBRCAJ kindreds (Table 6A). Kindred1910 containsa single nucle- The missensemutation observed in kindred 1). The lod scores (likelihood ratios for otide insertionin codingexon 20 (Gln1756 2099 is potentially disruptiveas it substi- linkage) in these kindredsrange from 9.49 frameshiftto 1829 Stop) (Fig. 6B), and tutes a large,charged amino acid (Arg) for to -0.44 for a set of markersin 17q2l. Four kindred2099 containsa missensemutation a small, hydrophobicamino acid (Met). of the familieshave convincing lod scores in coding exon 21 (Metl775Arg). The Five common polymorphismswere also for linkage, and four have low positive or frameshiftand nonsensemutations are like- identified in the BRCAI coding sequence negative lod scores. The latter kindreds ly to disruptthe function of the BRCA1 (Table 3). were included because they demonstrate proteins.The proteinencoded by the inser- The individualstudied in kindred2035 haplotypesharing at chromosome17q21 for tion allele in kindred 1910 would contain is likely to carrya regulatorymutation in at least three affected members.Further- an altered sequence beginning 107 amino BRCAI. In her cDNA, two polymorphic more, all kindredsin the set displayearly- acids residuesfrom the wild-typeCOOH- sites (PM1 and PM7) appearedhomozy- onset breastcancer, and fourof the kindreds terminus.The effect of the 11-bp deletion gous, whereasher genomic DNA revealed includeat least one case of ovariancancer, in kindred 1901 would be even more dra- heterozygosityat these positions (Fig. 6C). both hallmarksof BRCAJ kindreds.Kin- maticbecause it occursat the twenty-fourth One possible explanationfor this observa- dred 2082 has nearly equal incidence of codon. This deletion removesthe last 11 bp tion is that mRNA from her mutant breastand ovarian cancer, an unusualoc- of exon 2 and begins at the first cystine of BRCAI allele is absent becauseof a muta- currencegiven the relativerarity of ovarian the zinc fingermotif, therebyremoving the tion that affectsRNA productionor stabil- cancer in the population(17). All but two zinc finger domain. The mutant allele in ity. We exploredthis possibilityfurther by of the kindredswere ascertainedin Utah. kindred2082 would encode a proteinmiss- examining three additional polymorphic Kindred2035 is fromthe midwesternUnit- ing 548 residuesfrom the COOH-terminus. sites (PM6, PM7, and PM2) in the BRCAI ed States. Kindred 2099 is an African Americankindred from the southernUnit- ed States;all other kindredsare Caucasian. Table 1. Kindreddescriptions and associated lod scores (50). Br, breast cancer; Br <50, breast cancer diagnosed under age 50; Ov, ovarian cancer. In the initial screenfor predisposingmu- tations in BRCAJ,DNA fromone individ- Cases (n) Sporadic Lod ual carrying the predisposinghaplotype Total Br casest score Markers from each kindredwas tested. The 21 cod- Kindred Total Br ~ Ov (n)scr ing exons and associatedsplice junctions Br <50 were amplifiedfrom either genomic DNA samplesor cDNA preparedfrom lympho- 2082 31 20 22 7 9.49 D1 7S1327 2099 22 14 2* 0 2.36 D1 7S800 and cyte mRNA (26). When the amplified D1 7S855t DNA sequenceswere compared to the wild- 2035 10 8 1* 0 2.25 D1 7S1327 type sequence, four of the eight kindred 1901 10 7 1* 0 1.50 D1 7S855 samples were found to contain sequence 1925 4 3 0 0 0.55 D17S579 variants(Table 2). All four sequencevari- 1910 5 4 0 0 0.36 D1 7S579 and ants are heterozygous,and each appearsin D1 7S250t 1911 8 5 0 1 -0.20 D1 7S250 only one of the kindreds. Kindred 1901 1927 5 4 0 1 -0.44 D1 7S250 contains an 11-base pair (bp) deletion in exon 2 frameshiftto 36 Stop). Kin- *Kindredcontains one individualwho had both breastand ovariancancer; this individualis countedas both a breast (Cys24 cancercase and as an ovariancancer case. tNumberof womenwith breast cancer (diagnosed under age 50) or dred2082 containsa nonsensemutation in ovariancancer (diagnosed at anyage) who do not sharethe BRCA1-linked haplotype. tBoth markerswere used to coding exon 11 (Gln1313 to Stop) (Fig. calculatemultipoint lod scores.

Fig. 3. Diagramof BRCA1 BRCA1 3 12 13 114 1 1 21 2 2 2 . ;I 15 16 17 18 19' 20 24 mRNA,mRNA,~ showingshowing~ ~~~~~~~~ the6h loca-ATTG7oa ' t ' i ' 12 13 14, '21' 22,23 iA tions of introns and the vari- P3 - ants of BRCA1 mRNA pro- P4; duced by alternative splic- B cDNA ing. The top cDNA (BRCA1) is the composite used to OvarycDNA. generate the protein se- TY3- <- quence in Fig. 2. Intronloca- TE2 tions are shown by filled tri- 2 angles, and the exons are Bi1 numbered below the com- B31. posite cDNA. Alternative TY4r mRNAs identified as cDNA F103 clones or in hybrid-selection TY6 experiments are shown be- low the composite. The start Fl 91 - codon (ATG)and stop codon F3. (TGA)are indicated.The zinc B9. finaerreaion is denoted noted by a double line. "V" lines connecting exons indicate the absence of internal TY3 and TE2 are 5' RACEclones from thymus and testis, respectively; B21 exons. All exons are drawn proportionallyexcept exon 11 (indicated with a and B9 are cDNA clones from a normal breast cDNA library;B31 is a hybrid- dotted line). Upward-pointing unfilledtriangles show the position of a single selected cDNA clone from breast cDNA; TY4 and TY6 are cDNA clones codon (CAG)found at the start of exons 8 and 14 in some cDNAs. Leftward- isolated from a thymus cDNA library;and F191, F103, and F3 are cDNA and rightward-pointing unfilled triangles represent partial exons in some clones isolated from a fetal brain library.The BRCA1 variants labeled breast cDNAs. P3 and P4 are cDNA clones isolated from a placental cDNA library; cDNA and ovary cDNA are the majorforms detected in these tissues by PCR.

68 SCIENCE * VOL. 266 * 7 OCTOBER 1994 RESEARCH ARTICLES coding region, which are separatedby as including both carriersand noncarriersof were based on 39 samplesused in linkage much as 3.5 kb in the BRCA1transcript. In the predisposinghaplotype (Fig. 6). In each studies and on samples from 20 African all caseswhere her genomicDNA appeared kindred, the correspondingmutant allele Americansfrom Utah (28). None of the heterozygous for a polymorphism, her was detected only in individualscarrying fourpotential predisposing mutations tested cDNA appearedhomozygous. In individuals the BRCAI-associatedhaplotype. In the was found in the appropriatecontrol popu- from other kindredsand in nonhaplotype case of the potentialregulatory mutation in lation, indicatingthat they are rare in the carriersin kindred2035, these polymorphic kindred 2035, cDNA and genomic DNA general population (Table 2). Thus, both sites appearedheterozygous in cDNA, im- fromcarriers in the kindredwere compared importantrequirements for BRCA1suscep- plying that amplificationfrom cDNA was for heterozygosityat polymorphicsites. In tibility alleles are fulfilledby the candidate not biasedin favorof one allele. This anal- every instance, the extinguishedallele in predisposingmutations: cosegregation of ysis indicates that a BRCA1 mutation in the cDNA samplewas shown to lie on the the mutant allele with diseases and ab- kindred2035 either preventstranscription chromosomethat carriesthe BRCAI pre- sence of the mutant allele in controls, or causesinstability or aberrantsplicing of disposingallele. indicating a low frequencyin the general the BRCA1transcript. To exclude the possibilitythat the mu- population (29). Cosegregation of BRCA1 mutations tations were simply common polymor- Phenotypic expressionof BRCA1 mu- with BRCA1 haplotypes and population phisms in the population,we used allele- tations. The effect of the mutationson the frequencyanalysis. In additionto potential specific oligonucleotides(ASOs) for each BRCA1 protein correlateswith differences disruptionof protein function, a sequence mutation to screen a set of control DNA in the observedphenotypic expressionin variant must meet two other criteria to samples(27). The actual mutation in kin- the BRCAI kindreds.Most BRCA1 kin- qualify as a candidate predisposingmuta- dred 2035 has not been identified, so we dredshave a moderatelyincreased ovarian tion: It must be presentin membersof the could not determine its frequencyin the cancerrisk, and a smallersubset have a high kindredwho carrythe predisposinghaplo- general population. Gene frequencyesti- risk of ovarian cancer comparableto that type and absentfrom other membersof the mates in Caucasianswere based on random for breastcancer (3). Fourof the five kin- kindred,and it must be rarein the general samplesfrom the Utah population.Gene dredsin which BRCA1mutations were de- population. frequencyestimates in African Americans tected fall into the formercategory, and the To test for cosegregationof mutations fifth (kindred 2082) falls into the group with the correspondingBRCA1 susceptibil- with high ovariancancer risk. The BRCAI ity allele, we screened several individuals C nonsense mutationfound in kindred2082 E fromkindreds 1901, 1910, 2082, and 2099, _ has an interestingphenotype. Kindred 2082 has a high incidenceof ovariancancer, and the mean age of breastcancer diagnosisis older than that in the other kindreds(17). This differencein age of onset could be due to an ascertainmentbias in the smaller, 0 U) E o Co more highly penetrantfamilies, or it could U)D) 0 E reflect tissue-specificdifferences in the be- haviorof BRCAImutations. The otherfour 0 _-9.5 0 9.50- -7.50 kindredsthat segregateknown BRCA1mu- tations have, on average,1 ovariancancer 7.50- -4.40 for every 10 casesof breastcancer, but have a high proportionof breast cancer cases -2.40 diagnosedat an earlyage (late 20s or early 4.40- Fig. 5. Blot showing hybridizationof a BRCA1 30s). Kindred1910, which has a 1-bpinser- Fig. 4. Tissue expression pattern of BRCA1. The probe to genomic DNAfragments from various tion mutation,is noteworthybecause three blots were obtained from Clontech (Palo Alto, CA) species. DNAwas digestedwith Eco RI,subject- of the fouraffected individuals had bilateral and contain RNA from the indicated tissues. Hy- ed to electrophoresisthrough a 0.65% agarose breastcancer, and in each case the second bridizationconditions were those recommended gel, and transferredto a nylonmembrane, which tumor was diagnosedwithin a year of the by the manufacturer,and the probe was a BRCA1 was then hybridized(32) to a probe of consisting first occurrence.Kindred which cDNA fragment corresponding to nucleotides random-primed,at-32 P-labeled BRCA 1 cDNAse- 2035, seg- 3575 to 3874. Note that these tissues are heter- quences comprisinga totalof 4.6 kb. The probe regates the potential regulatoryBRCA1 ogeneous and the percentage of relevant epithe- excluded the zinc fingerregion. The finalwash mutation,might also be expectedto have a lial cells in breast and ovary can be variable. Size was at 550C in x2 SSPE and 1% SDS for20 min. in Size standards are in kilobases. standards are kilobases. Table 3. Neutral polymorphisms in BRCA 1. For the frequency in control chromosomes, the number of chromosomes with a particular base Table 2. Predisposing mutations in BRCA1. NA indicates not applicable, as the regulatory mutation is at the indicated polymorphic site is shown (A, C, inferredand the position has not been identified. G, orT).

Mutation Frequency CodonBase Frequency in control chromosomes Kindred Codon Name loca- Nucleotide Coding chromosomes in tion codon A C G T change effect

1901 24 -11 bp Frameshift or splice 0/180 PM1 317 2 152 0 10 0 2082 1313 C-*T Gln--Stop 0/170 PM6 878 2 0 55 0 100 1910 1756 Extra C Frameshift 0/162 PM7 1 190 2 109 0 53 0 2099 1775 TG Met->Arg 0/120 PM2 1443 3 0 115 0 58 2035 NA ? Loss of transcript NA PM3 1619 1 116 0 52 0

SCIENCE * VOL. 266 * 7 OCTOBER 1994 69 dramatic phenotype. Eighty percent of mutanttumor suppressor genes identifiedto moleculeis very acidic,with a net excess of breast cancer cases in this kindred occur date encode proteinsthat are absent,non- 70 acidic residues. The excess negative underage 50. This figureis as high as any in functional,or reducedin function.The ma- chargeis particularlyconcentrated near the the set, suggestingthat this BRCAJmutant jorityof TP53 mutationsare missense; some COOH-terminus.Thus, one possibility is allele has a high penetrance(Table 1). Kin- of these have been shown to produceab- that BRCA1encodes a transcriptionfactor dred 1901 displays a phenotypic pattern normal p53 molecules that interferewith with an NH2-terminalDNA binding do- similarto that of kindred2035. It is likely the function of the wild-typeproduct (30, main and a COOH-terminal"acidic blob" that the 11-bp deletion beginningat codon 31). A similar dominant-negativemecha- domain with transactivationalactivity. In- 24 carriedin kindred1901 resultsin a loss nism of action has been proposedfor some terestingly,the productof anotherfamilial of gene functionsimilar to the effect of the adenomatouspolyposis coli (APC) alleles tumorsuppressor gene, WTI, also contains regulatorymutation in kindred2035. that producetruncated molecules (32) and zinc finger domains (34), and these are Although the mutations described in for point mutations in the Wilms tumor alteredby many cancer-predisposingmuta- this researcharticle are clearlydeleterious, gene (WTI), which alter DNA binding of tions in the gene (33-35). The WT1 gene causing breast cancer in women at very the WT1 protein(33). The natureof three encodes a transcriptionfactor, and alterna- young ages, each of the four kindredswith mutationsobserved in the BRCAJ coding tive splicing of exons that encode partsof mutationsincludes at least one womanwho sequence is consistent with productionof the zinc finger domains alters the DNA carriedthe mutationbut lived until age 80 either dominant-negativeproteins or non- binding propertiesof WT1 (36). Some al- withoutdeveloping a malignancy.It will be functionalproteins. All threemutations are ternativelyspliced forms of WT1 mRNA of utmost importancein future studies to located in the COOH-terminalhalf of the generate WT1 proteins that act as tran- identify other genetic factors or environ- protein. The regulatorymutation inferred scriptional repressors (37). Differential mental factorsthat may amelioratethe ef- in kindred2035 cannot be dominant-nega- splicingof BRCAI mayalter the zinc finger fects of BRCA1 mutations.In addition, in tive; rather,this mutationlikely causesre- motif (Fig. 3), raisingthe possibilitythat a three of the eight putative BRCAl-linked ductionor completeloss of BRCA1expres- regulatorymechanism similar to that occur- kindreds,potential predisposingmutations sion from the affectedallele. Similarly,the ring in W71 may applyto BRCA1. were not found. All of these kindredshave 11-bp deletion in kindred1901 likely pro- The identificationof a gene that (i) falls lod scoresfor BRCA1-linked markers of less duces a nonfunctionalproduct. within the interval known from genetic than 0.55 and thus may not trulysegregate The BRCA1protein contains a C3HC4 studiesto includeBRCAI and (ii) contains BRCAl-predisposingalleles. Alternatively, zinc fingerdomain similar to domainsfound frameshift,nonsense, and regulatorymuta- the mutationsin these three kindredsmay in numerousnucleic acid bindingproteins. tions that cosegregate with predisposing lie in noncoding regions of BRCAJ and The first 180 amino acids of BRCA1 con- BRCAI alleles stronglyindicates that this thereforehave escapeddetection. tain five more basic residuesthan acidic gene is BRCAI. The observationof poten- The role of BRCAI in cancer. Most residues.In contrast,the remainderof the tial predisposingmutations in individuals

A P a b c d e f g B 2 C 1353- 1078- 872- 603- 1 4

310- 1 234 Fig.~~~~~ PM4,56789 ~~~~~~~~~~~~~~~ Fig. 6. Mutation and cosegregation PM1 4 5 6 7 8 9 analysis in BRCA1 kindreds. Carrierin- A dividuals are represented as filled cir- cles and squares in the pedigree dia- G grams. (A) Nonsense mutation in kin- A dred 2082. P indicates the person orig- inallyscreened; b and c are haplotype G carriers;a, d, e, f, and g do not carrythe BRCA1 haplotype. The C to T mutation ...... - results in a stop codon and creates a site for the restrictionenzyme Avr 11.A PCR amplification products were cut IA -I C-I G I T I with this enzyme, subjected to electro- phoresis through 1.0% agarose gels, and stained with ethidium bromide. The A carriers are heterozygous for the site and, therefore, show three bands. The PCR products of noncarriers remain uncut by Avr IIand, therefore, show one band. Size standards are in base pairs. (B) Frameshift mutation in kindred 1910. Sequencing reactions were loaded side by side as A, C, G, and T reactions. The first three lanes for each nucleotide set contain sequence PM1 and PM7 (Table 3). Samples were examined for heterozygosity in the ladders from noncarriers. Lanes 1 to 3 contain A ladders from carrier germ line and compared to the heterozygosity of lymphocyte mRNA. The individuals. Lane 4 contains the A ladder from a kindred member who does top two rows of each panel contain PCR products amplified from genomic not carry the BRCA 1 mutation. The frameshift resulting from the cytosine DNA, and the bottom two rows contain PCR products amplified from insertion is apparent in lanes 1 through 3. (The diamond shape in the lymphocyte cDNA. "A" and "G" are the two alleles detected by the ASOs. pedigree diagram is used to protect the confidentiality of the transmitting The dark spots indicate that a particular allele is present in the sample. The parent.) (C) Inferred regulatory mutation in kindred 2035. ASO analysis of first three lanes of the lower panel represent the three genotypes from haplotype carriers and noncarriers used two different polymorphisms, unrelated individuals. There is no cDNA for sample 4 in either panel.

70 SCIENCE * VOL. 266 * 7 OCTOBER 1994 SRESEARCH ARTICLES whose early-onsetbreast or ovariancancer bled into contigs composed of several independently ASO (20 ng) was used in an overnight hybridization identified sequences. Candidate genes may com- reaction in the same buffer used for prehybridization. was not ascertainedby family history sup- prise more than one of these candidate expressed Each blot was washed twice in X5 saline sodium ports the view that many early-onsetcases sequences. citrate and 0.1 % SDS for 10 min at room tempera- are due to mutationsat the BRCAI locus 21. Three independent contigs of expressed sequences ture, and then for 30 min at progressively higher (38). The role of BRCAI in cancerprogres- 1141:1 (649 bp), 694:5 (213 bp), and 754:2 (1079 temperatures until nonspecific hybridizationsignals bp) were isolated by hybrid selection and eventually were eliminated. Blots were exposed to x-ray filmfor sion may now be addressedwith molecular shown to represent portions of BRCA1. When ex- 40 min without an intensifyingscreen. precision. The large size and fragmented pressed sequencetags (ESTs)for 1141:1 and 754:2 28. The AfricanAmerican samples from Utah were from nature of the coding sequence will make were used as hybridizationprobes for RNA blots, a a newborn screening program. single transcript of approximately 7.8 kb was ob- 29. A reviewer suggested the possibility that this gene, exhaustivesearches for new mutationschal- served in normal breast mRNA, which suggested which we call BRCA1, may contain frameshift muta- lenging.Nevertheless, the percentageof total that they encode different portions of a single gene. tions other than those detected here at a significant breastand ovariancancer caused by mutant Screens of breast, fetal brain, thymus, testis, lym- frequency in members of the general population. phocyte, and placental cDNA librariesand PCR ex- 30. E. Shaulian, A. Zauberman, D. Ginsberg, M. Oren, BRCAI alleles will soon be estimated,and periments with breast mRNA linked the 1141:1, Mol. Cell. Biol. 12, 5581 (1992). individual mutation frequenciesand pen- 694:5, and 754:2 contigs. 5' RACE experiments 31. S. Srivastava, S. Wang, Y. A. Tong, Z. M. Hao, E. H. etrancesmay be established.This in turnmay with thymus, testis, and breast mRNAs extended Chang, Cancer Res. 53, 4452 (1993). the contig to the putative 5' end, yielding a compos- 32. L. K. Su et al., ibid., p. 2728. permitaccurate genetic screening for predis- ite full-lengthsequence. PCR and direct sequencing 33. M. H. Liftleet al., Hum. Mol. Genet 2, 259 (1993). position to a common, deadlydisease. Al- of Pl s and BACs in the region were used to identify 34. D..A. Haber et al., Cell 61, 1257 (1990). thoughsuch researchrepresents an advance the location of introns and allowed the determination 35. M. H. Littleet al., Proc. Natl. Acad. Sci. U.S.A. 89, in medicaland biologicalknowledge, it also of splice donor and acceptor sites. 4791 (1992). 22. M. Kozak, Nucleic Acids Res. 15, 8125 (1987). 36. W. A. Bickmore et al., Science 257, 235 (1992). raisesnumerous ethical and practicalissues, 23. T. F. Smith and M. S. Waterman, J. Mol. Biol. 147, 37. I. A. Drummond et al., Mol. Cell. Biol. 14, 3800 both scientificand social, that must be ad- 195 (1981). (1994). dressedby the medicalcommunity. 24. S. F. Altschul, W. Gish, W. Miller,E. W. Myers, D. J. 38. P. A. Futrealet al., Science 266,120 (1994). Note addedin proof:Analysis of kindred Lipman,ibid. 215, 195 (1990). 39. M. Lovett, J. Kere, L. M. Hinton, Proc. Natl. Acad. 25. We performed database comparison by using (i) Sc. U.S.A. 88,9628 (1991); P. A. Futrealetal., Hum. 1911 indicates a possible linkage to BLAST alignment algorithms (24) on the National Mol. Genet 3,1359 (1994). BRCA2,suggesting that earlybreast cancer Center for Biotechnology Information(NCBI) data- 40. A. Kamb et al., Nature Genet. 8; 22 (1994). in this kindredis not due to a mutationof bases; (ii) Smith-Waterman alignment algorithms 41. A. Thomas and M. H. Skolnick, IMAJ. Math. Appl. (23) on a MasPar computer to search the SwissProt Med. Biol., in press. BRCAI (51). database (at MasPar and at the European Molecular 42. J. Weaver-Feldhaus et al., Proc. Nati. Acad. Sci. Biology Laboratory);and (iii)Smith-Waterman algo- U.S.A. 91, 7563 (1994). REFERENCESAND NOTES rithms on a Compugen Biocelerator (at the Weiz- 43. J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular mann Institute)to search the GenBank (nucleotide) Cloning: A Laboratory Manual (Cold Spring Harbor 1. American Cancer Society, Cancer Facts & Figures and SwissProt (protein)databases. Laboratory, Cold Spring Harbor, New York, ed. 2, 1994 (AmericanCancer Society, Atlanta, GA, 1994), 26. The templates for PCR were lymphocyte cDNA or 1989). p. 13. genomic DNA from members of BRCA1 kindreds 44. I.G. Campbell etal., Hum. Mol. Genet. 3,589 (1994). 2. E. B. Claus, W. D. Thompson, N. Risch, Am. J. Hum. who carried the predisposing haplotype. Sequences 45. G. Westin et al., Proc. NatI. Acad. Sc. U.S.A. 81, Genet. 48, 232 (1991). of PCR primers used to amplifyeach exon of BRCA1 3811 (1984). 3. D. F. Easton, D. T. Bishop, D. Ford, B. P. Crockford, are available upon request. The PCR conditions 46. Abbreviationsfor the amino acid residues are: A, Ala; Breast Cancer Linkage Consortium, ibid. 52, 678 were: one cycle at 950C (5 min);four cycles at 950C C, Cys; D, Asp; E, Glu; F, Phe; G, Gly;H, His; I, lie; K, (1993). (10 s), with the annealing temperature (Tanr.)at 680C Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln;R, Arg;S, 4. J. M. Hallet al., Science 250, 1684 (1990). for 10 s, and at 720C for 10 s; four cycles with Tann= Ser; T, Thr;V, Val; W, Trp; and Y, Tyr. 5. S. A. Narod et al., Lancet 338, 82 (1991). 660C; four cycles with Tann= 64?C; four cycles with 47. R. Patarca et al., Proc. Natl. Acad. Sci. U.S.A. 85, 6. R. Wooster et al., Science 265, 2088 (1994). Tann= 62?C; and 30 cycles with Tann= 600C. The 2733(1988). 7. D. Malkinet al., ibid. 250, 1233 (1990). buffer conditions were as described (42). The PCR 48. R. Loveringet al., ibid. 90, 2112 (1993). 8. M. Swift, L. Sholman, M. Perry, C. Chase, Cancer products were purifiedfrom 1.0% agarose gels with 49. M. Takahashi, Y. Inaguma, H. Hiai, F. Hirose, Mol. Res. 36, 209 (1976). Qiaex beads (QIAGEN),analyzed by cycle sequenc- Cell. Biol. 8,1853 (1988). 9. M. Swift, P. J. Reitnauer, D. Morrell,C. L. Chase, N. ing with [oa-32P]deoxy-adenosine triphosphate (43), 50. Two of the kindreds referredto here have been stud- Engl. J. Med. 325,1831 (1991). and subjected to electrophoresis on 6% polyacryl- ied independently [J. Feunteun et al., Am. J. Hum. 10. L. Cannon-Albright et al., Cancer Res. 54, 2378 amide gels. Polymorphisms were initiallydetected by Genet 52, 736 (1993); D. Anderson, unpublished (1994). eye on both strands and subsequently confirmed by results]. 11. C. Mettlin,I. Croghan, N. Natarajan,W. Lane, Am. J. ASO analysis (27). 51. D. Goldgar, unpublished results. Epidemiol. 131, 973 (1990). 27. PCR products were generated as described (26) 52. We are grateful for the cooperation of the individuals 12. H. S. Smith et al., J. Cell. Biochem. Suppl. 14G, 144 and quantified after electrophoresis through 2% from the BRCA1 kindreds and for the assistance of (1993). agarose gels containing ethidium bromide by com- our clinic coordinators P. Fields, L. Steele, M. Mac- 13. D. P. Kelsell, D. M. Black, D. T. Bishop, N. K. Spurr, parison with DNA standards. PCR product (10 pl) Donald, and K. Brown and for the help of C. J. Hum. Mol. Genet. 2,1823 (1993). was added to 110 ,ll of denaturant (7.5 ml of H20, Marshall.We thank D. Ballinger, K. Fournier,W. Gil- 14. S. Neuhausen et al., Cancer Res., in press. 6.0 ml of 1 N NaOH, 1.5 ml of 0.1% bromophenol bert, L. Norton, G. Omen, J. Rine, J. Simard, R. 15. A. M. Bowcock et al., Am. J. Hum. Genet. 52, 718 blue, and 75 ml of 0.5 mM EDTA)and incubated for Williams, and B. Wold fot scientific advice; and F. (1993). 10 min at room temperature. Samples (30 pl) were Bartholomew, H. Brownlee, S. Burgett, J. Collette, 16. J. Simard et al., Hum. Mol. Genet. 2,1193 (1993). then blotted onto Hybond membrane (Amersham) B. S. Dehoff, l. L. Jenkins, A. Leavitt, K. Richardson, 17. D. E. Goldgar et al., J. Natl. Cancer Inst. 86, 200 with a dot-blotting apparatus (Gibco-BRL).The DNA and K. Rowe for technical support. Linkage study (1994). was fixed on the membrane by exposure to ultravio- controls were kindlyprovided by M. Pericak-Vance. 18. S. L. Neuhausen et al., Hum. Mol. Genet., in press. let light (Stratagene). Prehybridizationwas carried Supported in part by NIHgrants CA-55914 (M.H.S.), 19. D. E. Goldgar et al., unpublished results. out at 450C in x5 SSPE (0.75 M NaCI, 0.05 M CA-54936, CA-4871 1, CA-42014, CN-0522, RR- 20. Candidate expressed sequences are defined as NaH2Po4H2O, and 0.005 M EDTA)and 2% SDS 00064, and HG-00571 (D.G.), the National Cancer DNA sequences obtained by (i) direct screening of (34). Wild-type and mutant ASOs were labeled by Instituteof Canada, the Canadian Genetic Diseases breast, fetal brain, lymphocyte, or ovary cDNAs (39) incubation at 370C for 10 min in a reaction that in- Network (S.N.), and the Cedars Cancer Institute of or (ii)random sequencing of genomic DNA (40) and cluded 5 ,uCiof [32P]adenosine triphosphate, 100 ng the Royal VictoriaHospital (P.T.). prediction of coding exons by XPOUND (41). These of ASO, 10 units of T4 polynucleotide kinase (Boe- expressed sequences in many cases were assem- hringer-Mannheim),and kinase buffer (44). Labeled 2 September 1994; accepted 14 September 1994

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SCIENCE * VOL.266 * 7 OCTOBER1994 71