The Search For'

CANCER RESEARCH 54. 6374-6382, December IS. 19941 Special Lecture

The Search for'

Lori S. Friedman,2 Elizabeth A. Ostermeyer, Eric D. Lynch, Csffla I. Szabo, Lee A. Anderson, Patrick Dowd, Ming K. Lee, Sarah E. Rowell, Jeff Boyd, and Mary-Claire King3

Department of Molecular and Cell Biology and School of Public Health University of @alsfornia.Berkeley. california 94720 IL S. F., E. A. 0., E. D. L, C'. I. S., L A. A., P. D., M. K. L. S. E. R., M.-C. K.], and Depart@nent of Obstetrics and Gynecology, University of Pennsylvania School ofMedicine. Philadelphia. Pennsylvania 19104 ii. B.]

Abstract breast cancer and may not be representative of all families with BRCAJ mutations. That is, there may exist other families with BRCAJ, a gene predisposing to breast and ovarian cancer, was breast cancer linked to less severe mutations in BRCA1. Now that mapped to chromosome 17q21 by linkage analysis. Loss of heterozy the BRCAJ gene is cloned, it will be possible to identify women gosity In breast and ovarian tumors from BRCAJ-linked patients al ways involved loss of wild-type alleles from chromosome 17q21, sug carrying variant alleles and to begin to evaluate the risk associated gesting that BRCAI acts as a tumor suppressor gene. Melotic with each mutant sequence. recombinatlon in linked families constrained the BRCA1 region to an Inherited breast cancer is not restricted to early-onset disease. estimated physical size of 650 kilobases. Twenty-two candidate genes Inherited predisposition influences a higher proportion of breast were isolated by screening complementary DNA libraries with yeast cancer at younger ages, accounting for an estimated 30% of breast artificial chromosomes and cosmids from the critical region. Of these, cancer diagnosed before age 40 years, compared to less than 5% of 8 were known human genes, 7 were homologues of genes identified in breast cancer diagnosed after age 60 years (8, 11). However, it is other species, and 7 encoded novel transcripts. Each gene was Se quenced and analyzed for variation, revealing 44 varIants, Including likely that the absolute number of patients with inherited disease is two missense mutations in two genes which segregated with breast greater among older patients. Reanalysis of the original BRCAJ cancer and were not found in controls. However, no frame-shift, linkage data with a different liability class structure suggested that nonsense, or regulatory mutations were found. inherited breast cancer occurs in all age classes and that linkage of BRCAJ to early-onset disease was easier to identify because far Genetic Epidemiology of Breast and Ovarian Cancer fewer sporadic cases appear (12). This hypothesis can now be Linkage of early-onset breast cancer to chromosome l7q2l was tested directly. discovered 4 years ago ( 1). The existence of a gene that increases Other genes responsible for inherited breast cancer have been susceptibility to breast and ovarian cancer was verified with sub identified. BRCA2, on chromosome 13ql2â€”l3, is linked to breast sequent work, with odds >1026: 1 (2, 3). BRCAJ has recently been cancer in families with both females and males affected, as well as in cloned (4, 5), and experiments were performed to verify that the some families with only female breast cancer (13). In our series, announced candidate is BRCAJ (6). In what follows, we present breast cancer is linked to BRCA2 in approximately 10% of families our approach to the search for BRCAJ, the 22 genes cloned and (i.e., â€”30%of high-risk families with breast cancer not linked to sequenced in the BRCAJ region, the mutations identified in these BRCAJ), including families with breast cancer in males and females genes, and the implications of the cloning of BRCAJ for breast and families with ovarian cancer (14). The first gene identified for cancer research. inherited breast cancer was p53, which is responsible for the Li BRCAJ appears to explain most of inherited breast cancer and Fraumeni syndrome (15). In addition to female breast cancer, families inherited ovarian cancer. Of the 104 families in our series, predispo with Li-Fraumeni syndrome have extremely high rates of brain tumors sition to breast and/or ovarian cancer are linked to BRCAJ in 57% of and adrenocortical cancers among children with a mutant p53 allele. families with five or more affected relatives (14). Similarly, in a series About 1% of women diagnosed with breast cancer before age 30 years of 214 families from Europe and America, 60% of families with at have germline mutations in p53. Additionally, a rare point mutation in least three breast cancer cases and more than 90% of families with the androgen receptor gene on the X chromosome can lead to breast multiple relatives with breast and ovarian cancer trace susceptibility to cancer and androgen insufficiency among males (16). Breast can BRCAJ (3). Furthermore, among families with at least three cases of cer in other families with multiple cases is probably due to other, ovarian cancer and no early-onset breast cancer, the proportion with as yet unidentified, predisposing genes. However, given the very cancer linked to BRCAJ is 78% if risk is assumed to be restricted to high rate of noninherited breast cancer, many remaining families ovarian cancer, and 100% if predisposition to both breast and ovarian with multiple cases of breast cancer may represent coincidental cancer is assumed (7). occurrences of breast cancer, rather than inherited predisposition Risks of breast cancer to women inheriting BRCAJ are extremely due to other genes. high, exceeding 50% before age 50 years and reaching 80% by age 65 years (3, 8â€”10)However,families who have participatedin The great majority of breast and ovarian cancers are due solely to genetic studies were selected because multiple relatives developed acquired mutations: only 5 to 10% of breast cancer patients and 8 to 15% of ovarian cancer patients have inherited mutations leading to the disease. However, although inherited breast cancer is a small fraction Received 9/29/94; accepted I 1/14/94. I Based upon the Thirty-fourth G. H. A. Clowes Memorial Award Lecture presented at of the breast cancer burden, it is a common genetic disease (17). Five the 85th Annual Meeting of the American Association for Cancer Research, San Fran % of a diseaseaffecting1in 10womenoverthe life spanmeansthat cisco, CA. April 10. 1994. This research was supported by grants from the National Cancer Institute (NIH ROl CA27632), the Breast Cancer Fund, and the Ensign and Lewis roughly 1 in 200 women, or more than 600,000 women in the United Foundation. States, will develop breast cancer by reason of inherited susceptibility. 2 Susan G. Komen Breast Cancer Foundation Fellow. Therefore, as an inherited trait, breast cancer is one of the most 3 American Cancer Society Professor of Genetics and Epidemiology. To whom requests for reprints should be addressed. common genetic diseases in the world. 6374

Materials and Methods grids were produced by growing cosmids in an array on LB plates and transferring to GeneScreen Plus hybridization membranes. YACs were grown DNA Samples. Genomic DNA from lymphoblastswas transformedwith in adenine hemisulfate casein liquid cultures, and total yeast and YAC DNA Epstein-Barr virus or extracted directly as described elsewhere (18). Breast or were isolated (as described in Ref. 20), and digested with EcoRI, run on a 1% ovarian tissue embedded in paraffin blocks was extracted using a 2-mm skin HGT agarose gel (20 cm long) in I X TBE, and Southern blot transferred onto biopsy punch. Paraffin was sliced off using a sterile scalpel, and each tissue Hybond N (Amersham) as described in Ref. 19. punch was treated with 100 @.tgofproteinase K in 200 @.tlof100mMTris-4 mM PCR-amplified products of cDNA were used as probes in hybridizations EDTA, pH 8. Proteinase K was inactivated by incubating the sample at 95Â°C against YAC Southern blots, cosmid grids, and transcript grids. Primers for 10 mm, and the sample was centrifuged to pellet cell debris and paraffin. from cDNA clones were used to amplify DNA from somatic cell hybrids Polymorphic markers were typed on families and tumors as described including parts of human chromosome 17 (21), as well as YACs and elsewhere (9). cosmids from the region. cDNA Isolation. Two cDNA libraries were constructedin AgtlO with DNA Sequence Analysis. PCR-amplifiedproductsof cDNA clones were C600 host, using normal ovarian tissue and a nontransformed fibroblast cell either sequenced directly after PCR amplification using a USB PCR Product line. The cDNA libraries were plated at 4 X l0@plaques/plate(as instructed in Sequencing Kit or digested with EcoRl, then subcloned into Ml3 mpl8, and Ref. 19) and transferred to Colony/Plaque Screen Hybridization Transfer sequenced with USB Sequenase Kit version 2.0. Sequences were resolved on Membranes (GeneScreen Plus) per NEN Dupont instructions. 6% denaturing polyacrylamide gels and exposed to KOdak X-ray film for YAC probes were prepared by running 1% HGT agarose pulsed field gel 24â€”72h. Sequencing primers used were the M13 universal primer and Se electrophoresis, cutting out the unique YAC band, purifying YAC DNA with quence-specific internal primers. BLAST was used for nucleotide and amino a Gene Clean II kit (BiolOl Inc., La Jolla, CA), and radioactively labeling the acid searches of databases, including Genbank and EMBL. YAC for use as a probe with a Multiprime DNA Labeling System (Amersham) Northern and Southern Blot Hybridizations. PCR-amplified products of and [a-32P]dCTP and [a-32P]dATP (from NEN Dupont). cDNA clones were radioactively labeled with a Multiprime DNA Labeling Cosmid probes were prepared by digesting the DNA from cosmids with System (Amersham) and [cr-32P]dCTP (from NEN Dupont) and used as probes NotI, running 1% HGT agarosegel electrophoresis in 1X TBE,4 purifying the in hybridizations against Southern blots and Northern blots. insert DNA with a Gene Clean II kit (BiolOl, Inc., La Jolla, CA). Each cosmid Southern blots were made using genomic DNA from lymphoblastoid cell was radioactively labeled with a Multiprime DNA Labeling System (Amer lines of one or more relatives from each of 16 BRCA]-linked families and sham) and [a-32P]dCTP (from NEN Dupont) and combined into pools of less of unaffected controls. Genomic DNA (7â€”10 @.tg)was cut with MspI, than 8 cosmids/pool. BamHI, or EcoRI and run on a 1% agarose gel (20 cm long) in 1X TBE, Hybridization of YACs and cosmids onto the fibroblast and ovarian and Southern blot was carried out according to Ref. 19. Blots were probed cDNA libraries was carried out in a solution of 6X SSC, 1% SDS, 5% with cDNA clones as described, with a wash stringency of 0.2 X SSC and Dextran Sulfate, 0.25 mg/ml human placental DNA (Sigma). Wash strin 0.1% SDS at 65Â°C. gency was 0.1% SDS and 0.1 X SSC at 65Â°Cduplicate filters were aligned, Northern blots including multiple tissues were made from polyadenylated and positive hybridization signals were used to pull plugs which were RNA or purchased from Clontech, probed, and washed up to a stringency of diluted into 1 ml SM (19) plus 20 @lchloroform. Twenty @lof a 1:1000 1X SSC, 0. 1% SDS at 65Â°C.Lymphoblastoid polyadenylated RNA from 15 dilution of the plug in SM were plated for secondary screening. Lifts of BRCA]-linked patients and 8 controls were extracted, blotted, probed, and secondary filters were used to pull tertiary positive cDNA clones, which washed at a stringency of up to 0.1X SSC-0.l% SDS. were plated and screened in the same manner. Mutation Searching Methods. Chemical cleavage mismatch of cDNA Constructing a Physical Map and Contig of the Region. Pulsed field gel PCR productsbetween 200 and 300 base pairs long was done according to electrophoresis was done on genomic DNA samples digested with NotI, Miul, the method of Cotton et a!. (22). Analysis of SSCP was carried out using RsrII, NruI, Sac!!, or EcIXI and run on 1% HGT gels in 0.5X TBE. Southern PCR as described above, with the use of 50 ng DNA or cDNA as template, blot filters were probed with cDNA clones from the BRCAJ region. Sequence using 10 @MdCTP,and adding 0.5 pCi [a-32PJdCTP (NEN Dupont). DNA tagged sites and polymorphic markers on chromosome l7q2l were used to template for PCR was lymphoblastoid cDNA or genomic DNA from at identify YACs from the Centre d'Etude du Polymorphisme Humain and least 15 unrelated BRCAJ-linked patients. Controls included normal breast, Washington University libraries.5 Cosmids were identified by hybridizing normal ovary, and cell lines MCF7 and HeLa, and untransformed IMR9O Alu-PCR products of YACs to a flow-sorted chromosome 17 cosmid library.6 fibroblast. PCR-amplified samples were diluted 1:10 in formamide buffer Identification and Amplification of cDNA Clones. PCR amplification of (98% formamide, 10 mM EDTA, pH 8â€”0.05%bromophenol blue-0.05% clones isolated from bacteriophage AgtlO fibroblast cDNA library screened xylene cyanol). Diluted samples were then held at 95Â°Cfor 5 mm, cooled with YAC or cosmid probes was done in 50-pA reactions, containing 1 pi rapidly to 4Â°Cand held for 5 mm. For each sample, 5 pi were loaded onto phage solution (a single phage plaque in 0.5 ml SM buffer), 200 @Mconcen a SSCP gel run at 6 W (constant power) for 14 h in 0.6X TBE at room trations of each deoxynucleotide triphosphate (Perkin-Elmer), I X PCR buffer temperature. An 80 ml gel solution contains 0.5X MDETM (AT Biochem), (Perkin-Elmer), 50 pmol each primer, and 1.25 units AmpliTaq DNA polym 0.6X TBE, 160 @.tl25% ammonium persulfate, and 38 @.tlN,N,N',N', erase (Perkin-Elmer). Thermocycling was done for 35 cycles at 94Â°Cfor 1 tetramethylethylenediamine. Gels were dried on a vacuum gel dryer and mm, 55Â°Cfor1 mm, and 72Â°Cfor3 mm. Primer sequences used were: AgtlO exposed to film for 24â€”48 h. Variant bands were cut out of the gel, Left primer -40: 5' GCT CTA TAG ACT GCT OCX)TAG TC 3', and AgtlO rehydrated in 100 p.1 water overnight at 4Â°C,amplified with appropriate Right primer -38: 5' AAG AU GGG GOT AAA TAA CAG AG 3'. SSCP PCR primers, and directly sequenced with the USB PCR product Amplified products of A clone inserts were run on 1% HGT agarose gel in sequencing kit. 1X TBE to ascertain the insert sizes, and the DNA was purified with Gene Clean II kit for use in hybridizations against Southern blot membranes, Results and Discussion Northern blot membranes, transcript grid membranes, and cosmid grid membranes. Indirect Evidence That BRCAJ Is a Tumor Suppressor Gene Hybridization of eDNA Clones to Determine Redundancy and Chro Analysis of tumors from families with BRcAJ-linked breast and mosomal Location. Transcript grid membranes were made by growing A cDNA clones identified in the library screen in an array on LB top agarose ovarian cancer suggests that BRCAJ may act as a tumor suppressor plates and transferring to GeneScreen Plus hybridization membranes. Cosmid gene. Tumors from BRcAJ-linked cases in families 3 and 82 were evaluated for loss of heterozygosity at markers near BRCAJ. Of the seven tumors with LOH in the linked region, loss was invariably from 4 The abbreviations used are: ThE, 90 msi Tris-borate, 2 msi EDTA, pH 8; SSCP, single strand conformational polymorphisms; LOH, loss of heterozygosity; cDNA, complementary the chromosome 17 carrying the normal allele at BRCAJ. Fig. 1 DNA; YAC, yeast artificial chromosome; HGT, high gelling temperature;PCR, polymerase illustrates pedigrees for BRCAJ-linked families 3 and 82 and results chain reaction; LB. Luria broth; FISH, fluorescence in situ hybridization. 5 L. Friedman et al., unpublished data. from PCR amplification of informative markers from tumor and 6 F. Couch et al., unpublished data. adjacent normal cells from the same paraffin-embedded biopsy 6375

Family 3

Ov 0V44 7S 75 1@JBRB @NB 1@Je DIA D17525S @ @i- â€˜J'G BIG â€˜Jill F'F @ I4IJA 4* 14. 11501 DID DID IDlE E@C DI7SSSS IEIE EJE kfr c@c DI7S1$3 L- NE GIE LYG t4i D17S571 @ ___ 45 B@ OvA Sr42 Br Ov 0v4$ 0v51 BID 1@- F@- i@o @- HIF â€˜J'- IJI- â€˜J'F Iji- â€˜@ 1@1DIE@DIJIFI F1F ala I@4- IAI- N@ EJE ID@ IDI- IDlE dc â€˜El-El- Idc E dcldE GJKN-N-kJM Fig. 1. A, LOH in tumors revealed by polymor phic markers from chromosome 17q21 on paired normal and tumor DNA samples from family 3. D17S250 THRA1 HSDI D175e58 D17S183 D17S579 @ @31 3S 2$ Filled symbols, breast or ovarian cancer; speckled NT NT NT NT NT NT symbols, tumors. Below each individual is their age .@ f@JD1@JDiNEF@E at cancer diagnosis, or current age or age at death if IJIF IjIr rip IJIF unaffected. Persons with reconstructed genotypes are denoted with brackets. For each tumor with 4.H@ale14. LOH, wild-type alleles are lost and mutant BRCAJ loic IDICEIEIDlE alleles were retained. B, LOH in tumors from :1 1 1 :@ :@:IU@E@E[@ Family 82. The chromosome 17 carrying the wild typeBRCAIalleleis lostin the ovariantumorof individual 11-2and the small intestinal tumor of individual 11-7. * Tumor DNA Illustrated In photograph.

Family 82 B

D17S856 D1SS8SS D17S858 D17S579

Br42@47 OvSO 47 45 47 44 IN 46 57 40 Br 29,33 BE BEB- BE BE BIE BE BEB-BB BB BB BB DB DE D- CB CE DID DD DD DA DD DD DF CB DC DB @]C EC CDC-ED ED CD CB CF CEC- BF BE @]D ED CDC-EB EF CF CF

specimen. In family 3, LOH was observed throughout the BRCAJ deletion too small to be detectable by genotyping markers several mega region in two breast tumors and three ovarian tumors from three bases apart. The observation from these families that WH is always of patients with cancer linked to BRCAJ. In all five tumors, the chro the chromosome carrying the normal BRCAJ allele confirms data previ mosome carrying the mutant BRCAJ was retained. In family 82, the ously reported for other BRCAJ-linked families (23, 24). normal BRCAJ allele was lost in the ovarian tumor of one patient and BRCAJmay alsoactasatumorsuppressorinthefarmorecommon in the tumor of the small intestine of a male relative, both of whom cases of sporadic, rather than inherited, breast or ovarian cancer. carry BRCAJ germline mutations. Loss of heterozygosity was not seen Breast and ovarian tumors from patients not selected for family in one tumor sample from family 3, which may reflect no deletion, or history frequently lack the BRCAJ region of 17q21. The frequency of amplification of contaminating normal tissue, or the presence of a LOH in the BRCA1region ranges from 40 to 80% among sporadic 6376

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. BRCAI AND CLONING OF 22 GENES FROM CHROMOSOME17q21 breast carcinomas (25â€”27)and from 30 to 60% among sporadic translocations were detected in the three patients karyotyped thus far. ovarian carcinomas (28â€”30). We tried the same idea in reverse, screening records of amniocenteses Patients with no evident family history of breast or ovarian cancer for alterations in chromosome 17q and then interviewing parents of may nevertheless have germline BRCAJ mutations (5). Breast and pregnancies with 17q alterations to determine family history of breast ovarian carcinomas from 25 patients with primary tumors at both sites cancer. Two families were identified who had both balanced translo were evaluated for LOH using 21 dinucleotide repeat markers in the cations involving chromosome 17q21 and family history of breast BRCAJ region (31). Of the ovarian tumors, 20 (80%) had lost part or cancer. The informative relatives in both families were sampled and all of chromosome 17q. The loss always included at least the entire karyotyped, but in neither family did the balanced translocation BRCAJ region. Of the breast tumors from the same patients, 40% had segregate with breast cancer. lost the BRCAJ region. Of the 10 patients whose breast and ovarian A Possible Contiguous Gene Syndrome. An extendedkindredin tumors both revealed LOH, losses were always of the same chromo which breast and ovarian cancer were coinherited with palmoplantar some 17q. These patients may have inherited a mutant BRCAJ allele keratoderma was reported 6 years ago (36). Last year, palmoplantar on the retained chromosome, even in the apparent absence of a family keratoderma was mapped to chromosome 17q12â€”q21(37). Hence, the history of breast or ovarian cancer. family with both conditions might carry an inversion or deletion involv An alternative to the hypothesis that BRCAJ is a tumor suppressor ing BRCAJ. Pulsed field filters and Southern blots with DNA from both is the possibility that BRCAJ is a dominant predisposing gene located affected and unaffected members of the family were probed with keratin very near a still-unknown tumor suppressor. If this were true, tumor genes, cosmids, and other genes in the BRCA1 region. No rearrangements development would require that acquired alterations in the tumor were detected on chromosome 17q by Southern or by further karyotyping suppressor be associated with selection for retention of the mutant or FISH analysis. Subsequently, a point mutation in keratin 9 was BRCAJ in the tumor. However, the observation that most BRCAJ identified as the cause of palmoplantar keratoderma in this family (38). mutations observed thus far appear to lead to loss of function (4â€”6) This family probably carries independent mutations on chromosome 17q, suggests that this alternative hypothesis is unlikely. one leading to breast and ovarian cancer and the other to palmoplantar keratoderma, certainly not impossible given the estimated frequency of 1 Hopeful Digressions in 200 for carriers of BRCAJ mutations. FISH Analysis of Possible Chromosomal Rearrangements in In our experience, positional cloning involves two types of activi Tumors. Two ovarian cancer patients whose tumors had possible rear ties in parallel. One pathway is physical mapping, contig construction, rangements of chromosome 17q were identified.5 The patients were library screening, and characterization of candidate genes. The other diagnosed at ages 68 and 79 years, with no family history of breast or pathways are â€œhopefuldigressions,â€•searches for critical patients or ovarian cancer. Both tumors were aneuploid, with 3 and 4 copies of tumors that would provide shortcuts to the gene if they can be found. chromosome 17, respectively. In one tumor, a translocated 17 was oh Although none of these shortcuts revealed BRCAJ, the same ap served in one preparation only. In the other tumor, one of the four copies proaches may reveal other cancer genes. of chromosome 17 was consistently observed to carry a translocation, Identification and Characterization of Triplet Repeats. More which by FISH analysis was proximal to the region of linkage. recent generations of women in breast cancer families develop breast cancer at younger ages than did their ancestors. This obser Narrowing the Linked Region by Meiotic Recombination vation could represent ascertainment bias, a cohort effect due to changes in prevalence of noninherited risk factors, and/or biolog The original linkage data mappedBRCAJ to a 50-cM region. In ical anticipation. The discovery that unstable triplet repeats might order to close in on the gene, new polymorphisms were developed at play an important role in inherited disease (32, 33) made a search multiple loci (39â€”41), and a high density genetic map was con for triplet repeats in the BRCAJ region irresistible. CAG, CCG, structed for chromosome 17q12â€”q21(21). Transformed lymphocyte GAG, AAG, and AAT triplets were identified from cosmids in the lines were established for 104 families with multiple cases of breast or region, sequenced, and screened for polymorphism. A few repeats ovarian cancer, and all informative relatives were genotyped. The detected by this method were polymorphic, but none underwent region of 17q21 linked to breast and ovarian cancer was first refined large expansions. to 8 cM (9), then to 4 cM (40), then to 1 megabase bounded by the Instabifity of Short Repeats. Genomic instability characterizes markers D17S856 and D17S78 (42, 43). tumors from families with inherited colon and endometrial cancer (34, Positional cloning of candidate genes began when the linked region 35).TumorDNAsamplesfrompatientsinfamilieswithcancerlinked was 1 megabase long. However, the linked region was further refined to BRCAJ were screened for 20 dinucleotide repeats on various by an informative recombination event in Family 1 (Fig. 2). Recom chromosomes. No instability was found, although occasional new bination between breast cancer and D17S1141 (44) constrains the alleles did appear in markers with high heterozygosities. BRCAJ interval to an estimated physical region of about 650 kilobases Search for Chromosome 17q Alterations in Patients. A patient (Fig. 3). In principle, either or both breast cancer patients whose with severe developmental disabilities and early-onset breast cancer recombination events defme the 650-kilobase interval could be spo might have a chromosomal rearrangement involving both BRCA1 and radic cases. However, the critical proximal recombinant occurred in a developmentally important genes. Three patients were identffied with patient diagnosed with breast cancer at age 37 years with a daughter severe developmental disabilities and breast cancer at ages 28, 32, and (also recombinant, of course) diagnosed at age 27 years and with 15 33 years, respectively. The three patients had chromosomal aberra other relatives with breast and/or ovarian cancer in the family. This tions on three different chromosomes, none of which involved chro recombinant excluded the gene for 17@-hydroxysteroid dehydrogen mosome 17. We will return to these alterations to search for other ase as a BRCAJ candidate. breast cancer genes. A balanced translocation involving BRCAJ might be revealed as Isolating and Mapping Candidate Genes in the BRCA1 Region both breast cancer and a history of miscarriage. Six patients were identified who were diagnosed with breast cancer before age 30 years Candidate genes in the BRCAJ region were isolated by screening and who had, or whose mothers had, frequent miscarriages. No fibroblast and ovarian cDNA libraries directly with eight YACS and four 6377

BRCAJ AND CLONING OF 22 GENES FROM CHROMOSOME 17q21

Family I

E:JO@Ã˜8782Pr779289Pr79RB 0

ED A i@@A IAA A@A A@A I BA DE Afr 1DB DA BR IDE EB 1KBIB ElIrBB [LiBIC .!:IEIB ELrBs L1@ FE O@o 53. Pr5759 7977Br7lBr3lBr4SBr3S0v48 17q210v61haplotypeFig. 2. Alleles defining the chromosome linked to breast and ovarian cancer in E B BIC B@B BIC B B B B BD B@D EBE B E BD Family 1 (outlined). Individual 111-12 is recombi A i-lA A AlA AlA AlA A A A A AlA AlA ABAB â€˜-IA B A A nantatDl7SII4l.HerdaughterlV-2,diagnosedat DB @iA DD @iA @lE 1ADD AC DB lD @lB DE DE Di DE EB age 27 years, also mhented the recombinant chro I mosomel7.B II KEB LK LI a@JG LILK EL LE EE KEB LF LF DF LFKE Ã³@O Br28BIB48BE Br33 Br43 Br3942Ã³OÃ¶Ã©ciÃ³OÃ³ 37 34 Br28D@E Br29 Br45 Br37BIA

BB BB BB BD FB BD DIE DIE EIA EIB BB A i@@A A AAAAAAAAAA@AA@A BAA@AAA DB A@E DC DA DC DF DC DF BD BD DF EB LICIB hG LL LE IL LE EELE EEEE IF EJ ID

Br 25 Br 27

D17S856 BR ER D1IS1 141 A BA D17S855 DC DR D17S579 LE U pools of cosmids from a partial contig of the 1-megabase BRCAJ region. binding protein (E1A-F). Additional sequence and analysis of E1A-F More than 400 independent cDNA clones were isolated from the libraries is described elsewhere.5 after the first round of screening. The 17q21 chromosomal locations of Seven genes are newly cloned human homologues of genes known the cDNA clones were con.firrned by hybridization to multiple chromo in other species; Genbank accession numbers for the human genes are some 17 YACS, to chromosome 17 cosmids, and to hybrid somatic cell shown in Fig. 3. cDNA clones B66 and B60 encode a protein homol lines carrying Critical portions of chromosome 17q (21). About 40% of ogous to proton pump cloned from rat (M58758) and Caenorhabditis cDNAs identified with YACS did not map back to chromosome 17q21. elegans (Zi 1115), as well as to bovine vacuolar H+ ATPase cDNA clones that mapped back to 17q21 were cross-hybridized with (L31770) and a mouse immunosuppressor factor (M31226). cDNA each other and fell into clusters that defined 22 genes (Fig. 3). The order clone LF26 encodes a protein homologous to an acyl-CoA desaturase of genes on the chromosome was determined by their position in the cloned from mouse (M21285), rat (J02585), and yeast (J05676). contig. The BRCAJ gene appears to map to a gap; i.e., a region not cDNA clones B52 and LF96 encode a protein homologous to the represented in the YACS that made up the contig. Physical distances were Drosophila melanogaster transcription factor Enhancer of zeste estimated by analysis of Southern blots from pulsed field gels of genomic (U00180).7 cDNA clone B7F appears to encode a pseudogene of the DNA samples of BRCAJ-linked patients and controls. high mobility group protein HMG17 (M12623), which maps to chro mosome ip and may have more than 50 pseudogenes (49). cDNA Definition of 22 Genes in the BRCAJ Region by Sequence clone LF1 13 encodes the human homologue of Pacific electric ray Analysis vesicle amine transport protein VAT1 (P19333) and is described cDNA clones representing each gene that were sequenced and the elsewhere.5 cDNA clone EL1O7 encodes a homologue of human databases searched for nucleotide and amino acid homologies (45). endogenous retrovirus-related pol and protease polyproteins The locations of these genes are shown in Fig. 3. Eight genes (sp P10266, M14123) and mouse gag polyprotein (pir 531034). identified by positional cloning were known human genes: cDNA B154 and cross-hybridizing clones encode the human homo Ras-related protein Rab5c (46); 17@3-estradiol dehydrogenase; a-N- logue of Saccharomyces cerevisiae PRP22 (X58681), a helicase in acetylglucosaminidase (47); -y-tubulin; Ki nuclear autoantigen; a B- box protein IAI3B (48); vaccinia virus VH1-related dual-specificity tyrosine/serine phosphatase (VHR); and adenovirus E1A enhancer 7 K. Abel et aL unpublished data. 6378

kb YACS :lones and markers Transcript______@!!@! Accessionnumber

t.J (,,J

@ woo@o.â€”@[email protected] rn ei â€˜C I'.. 1000 EL46 1.5kb Rab5C U11293 Dl75856

LF26 ACyI-COAdesaturase homolog new

366/B60 3.5kbnew3.2 Proton pump homolog HSD.AAAT LF77 kbM844722.3kb@-N-acetyIgIucosarnInIdase HSD.del 750 LF78 LF91 2.5 kb Novel new 1179 2.2 kb Novel new 1174 4.0kb Novel new B105 1.5 kb Gamma tubulin M61764 B1021LF98 6.4 kbEDH17BNo@mIM84472 new 650 D17S1141 B5Z/LF96 62 kb of zeste homolog B7F HP4G17pseudogene new Fig. 3. The BRCAJ region of chromosome EL76 1.0 kb Novel gene new 17q21, defined by meiotic recombinants at Nowlgene new D17S856 and D17S78. The proximal boundary was B32 2.2kb subsequently defined by D17S1141, narrowing the B555.0,A60537151 2.5kbEnhancer Klantlgennew linked region to â€”650 kilobases (shown between the horizontal lines), and excluding 10 candidate homolognewDl132.8 kbVAT1 genes.cDNAclonesrepresentinggenesisolatedin 550 our library screens were mapped to the partial YAC 75855BRCA1U14680EL894.3 contig (vertical lines on the left) and are listed in orderfromcentromeretotelomere.Alsoshownis MOX1, which we did not find (57). Transcript sizes, homology, and Genbank accession numbers are listed to the right of each clone. proteInX76952EL1O7u2 kbIAI3B,CA1 25-related

400 RNA newB1684.1 EndogenousretrovlrusX02665

phosphataseL05147B169/B213kbSenne-tyroslne

3.8 kbNovel gene 200 D17S859 U10492Qi4.1 MOX1new D17S8583.0,

2765B1544.2, kbE1A-FDl

6.5 kbPRP22 homolognew

0 D17S78 PPY

volved in pre-mRNA splicing. Human PRP22 is described in more garter Notch, human TAN-i, a human trithorax-homologous protein, detail elsewhere.8 and rat neurexin. EL76 encodes a 1.0-kilobase message with no Seven apparently novel genes were identified. B102, LF98, and significant similarities to known genes. B32 and several cross-hybrid other cross-hybridizing clones encode a 6.4-kilobase message with izing clones encode a protein with coiled coils and similarity to patches of sequence similarity to Xenopus laevis Xotch, D. melano tropomyosin, myosin, nuclear mitotic apparatus protein, ezrin, and merlin. B169 and B213 encode a novel gene with two transcript sizes 8 E. Ostermeyer et aL, unpublished data. (3.0 and 3.8 kilobases) in lymphoblasts and no similarity to known 6379

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. BRCAJ AND CLONING OF 22 GENES FROM CHROMOSOME 17q2l genes. LF91, LF79, and LF74 encode messages of 2.5, 2.2, and 4.0 FamIly 74 kilobases, respectively, with no significant similarities to known genes, although the sequence for these three genes is incomplete. These genes have been submitted to Genbank. BISS 8r37 Br40 Pr$S Br4Ss C4O Analysis of Genes for Mutations in BRCAJ-linked Families

The genes identified by positional cloning were analyzed for mu tations, with thorough analysis of genes between D17S1141 and DI 7S78. All variants found by Southern blot, by analysis of single strand conformation polymorphism (50, 51), and by chemical mis 3$ Br match cleavage (22) are summarized in Table 1. No aberrant tran D17S$55 C AD C IrAl CB scripts appeared on Northern blots of RNA from lymphoblast lines of E1AF.02B C CC lid D17S579 CA NB CB LCJ c

Table 1. Variants in Genes in the BRCA1 Region t@. Br31 Gene Clone Variant Br31 Variants segregating with breast cancer in families and not obseri'ed in controls â€œa E1A-F QI SSCP Hisâ€”'Asn â€” PRP22 homologue B154 2 RFLP:MspI SSCP Asnâ€”Ser Polymorphisms found in cases and controls Proton pump homologue B66/B60 SSCP Novel LF77 2 SSCP Fig. 5. A missense mutation in the adenovirus E1A enhancer binding protein, which is EDH17@3â€• LF78 11 SSCP (cf Ref. 58) linked to breast cancer in family 74. In the sequences illustrated, Lanes from left to right Novel LF91 SSCP are C, T, A, and G. The sequence on the left is that of individual 111-1and has both C and Novel LF79 sSCP A nucleotides at base pair 457 of E1A-F (Genbank No. D12765), whereas the wild-type Novel B1021LF98 2 SSCP sequence (right) has only a C. The Câ€”'Avariant appeared in all relatives in family 74 RFLP:Mspl with BRCAJ-linked breast cancer chromosomes. E(z) homologue B52/LF96 RFLP:Mspl Novel EL76 CCM Novel B32 2 SSCP RFLP:EcoRI/Hindlll BRCAJ-linkedpatients. RFLPs were found in nine of ten genes Ki antigen B55 RFPL:Mspl screened by Southern blot. Fourteen genes contained point mutations, VAT1 homologue LF113 2 SSCP RFLP:HindIII which were identified and sequenced. JAJ3B EL89 SSCP Three mutations, one in the PRP22 homologue and two in E1A-F, RFLP:Mspl changed the predicted amino acid sequence. In the PRP22 homologue, RFLP:EcoRI/HindIII VHR phosphatase B168 5 SSCP (2 also CCM) SSCP analysis detected a variant that cosegregated with breast cancer RFLP:EcoRI/HindIII in family 94 (Fig. 4). Sequence analysis of this variant revealed a Novel B169 SSCP missense mutation (asparagine to serine) in the PRP22 homologue RFLP:EcoRIJHindIIl E1A-F 01 SSCP that was not seen in 100 control chromosomes.8 A missense mutation aEDH17j3,17j3-estradioldehydrogenase. at nucleotide 457 of the published E1A-F sequence (D12765) changed cytosine to adenine and hence histidine to asparagine. This Family 94: B154 SSCP mutation cosegregated with breast cancer in family 74 (Fig. 5) and was not seen in 80 control chromosomes.5 The third missense muta 1 2345 8 tion (arginine to cysteine) at nucleotide 1243 in E1A-F changed cytosine to thymine and cosegregated with breast cancer in family 95 but was a polymorphism with a frequency of 0.03. The effect of these missense mutations on the protein product is not yet known. a;j@1@; Relevance of BRCAJ to the Control of Breast Cancer Incidence rates of breast cancer have increased 25% in the past 20 years and are now 10% by age 80 years (52). Mortality rates have not increased, because survival of breast cancer patients has improved. The increase in breast cancer risk almost certainly has nothing to do with changes in frequencies of susceptibility alleles. Rather, breast cancer risk has probably increased as the result of gradual changes in the prevalence of established risk factors for breast cancer, particu larly the length of the interval between menarche and first full-term pregnancy (53). At the beginning of this century, the average age at menarche was 14.5 years and the average maternal age at first birth dx34 dx38 36 dx3l was 21 years (54). Among young women in the United States now, the Fig. 4. A missense mutation (Asnâ€”@Ser)in a RNA helicase which is linked to breast average age at menarche is 11 years, and child-bearing frequently cancer in family 94. The mutation was identified by SSCP analysis using primers specific begins in the late 20s or 30s. Therefore, the interval of exposure of to a 200-base pair product of the B154 clone, a PRP22 homologue. The variant appeared dividing breast ductal cells to hormonal stimulation is twice as long in the three sisters with breast cancer (individuals 1, 2, and 5) and their mother (individual 8), but not in >100 control chromosomes. now as a century ago. Epidemiological studies of women exposed to 6380

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. BRCAI AND CLONING OF 22 GENES FROM CHROMOSOME 17q21 radiation as teenagers indicate that the first mutations leading to breast 10. Ford, D., Easton, D. F., Bishop, D. T., Narod, S. A., and Goldgar, D. E. Risks of cancer probably occur in this period (55, 56). An association between cancer in BRCAJ-mutationcarriers. Breast Cancer Linkage Consortium. Lancet, 343: 692â€”695,1994. increased breast cancer risk and an extended interval of menstruation 11. Claus, E. B., Risch, N., and Thompson, W. D. Genetic analysis of breast cancer in the before pregnancy is biologically plausible, because all breast ductal cancer and steroid hormone study. Am. J. Hum. Genet., 48: 232â€”242,1991. cells, whether normal or mutant, are dividing rapidly during this 12. Margarine, P., Bonaiti-Pellie, C., King, M-C., and Clerget-Darpoux, F. Linkage of familial breast cancer to chromosome 17q21 may not be restricted to early-onset period therefore abnormal cells would have every opportunity for disease. Am. J. Hum. Genet., 50: 1231â€”1234,1992. clonal growth. 13. Wooster, R., Neuhausen, S. L., Mangion, J., Quirk, Y., Ford, D., Collins, N., Nguyen, The link between inherited predisposition and increasing risk of K., Seal, S., Tran, T., Averill, D., Fields, P., Marshall, G., Narod, S., Lenoir, G., Lynch, H., Feunteun, J., Devilee, P., Comelisse, C. J., Menko, F. H., Daly, P. A., breast cancer lies in the application of genetics to diagnosis and Ormiston, W., McManus, R., Pye, C., Lewis, C. M., Cannon-Albright, L. A., Peto, J., prevention. Creating molecular tools for earlier diagnosis and devel Ponder, B. A. J., Skolnick, M. H., Easton, D. F., Goidgar, D. E., and Stratton, M. R. oping ways to reverse the first steps of tumorigenesis may be the most Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12â€”13.Science (Washington DC), 265: 2088â€”2090, 1994. effective means of breast cancer control. Now that BRCAJ has been 14. Arena, J. F., Marteili, L, Lynch, E., Morrow, I., Friedman, L, Benigno, A., Dowd, P., cloned, additional critical questions can be addressed. What are the Meza, J., Goode, E., Lee, M., Roweil, S., Donahue, R., Lubs, H., and King, M-C. inherited breast cancer@confirmation of linkage to BRCA2, possible linkage to the normal and mutant products of the gene? What are the biological estrogen receptor,and the clinical implicationsof genetic complexity.JAMA, in press, consequences of these early changes? What are the different BRCAJ 1995. mutations and how does the risk associated with each differ? What 15. Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F., Jr., Nelson, C. E., Kim, D. H., Kassel, J., Gryka, M. 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What are the choices? JAMA, 269: 1975â€”1980,1993. missing gene product be replaced or the effects of an aberrant one 18. Hall, J. M., Zuppan, P. J., Anderson, L. A., Huey, B., Carter, C., and King, M-C. be prevented? Oncogenes and human breast cancer. Am. J. Hum. Genet., 44: 577-584, 1989. 19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.). Molecular Cloning Manual, Ed. 2. Cold Spring Harbor, NY: Cold Spring Harbory Laboratory, 1989. Acknowledgments 20. Dracopoli, N., Haines, J. L, Korf, B. R., Moir, D. T., Morton, C. C., Seidman, C. E., Seidman, J. G., and Smith, D. R. Current Protocols in Human Genetics (Ed. 1), New We thank Jay Ellison for helping us to constructcDNA libraries;Beth York: John Wiley and Sons, Inc., 1994. 21. Anderson, L A., Friedman, L S., Osbome-Lawrence, S., Lynch, E. D., Weissenbach, Newman, Lynn Hartmann, Robert Jenkins, Nicole Schopf, Dorothy Warbur J., Bowcock, A. M., and King, M-C. 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