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 13ql2—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., —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, 8—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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. BRCAI AND CLONING OF 22 GENESFROM CHROMOSOMEl7q2l 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 95°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 24—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.