Germline BRCA1 Mutations and Loss of the Wild-Type Allele in Tumors from Families with Early Onset Breast and Ovarian Cancer1

Vol. 1, 539-544, May 1995 Clinical Cancer Research 539

Sofia D. Merajver,2 Thomas S. Frank, Junzhe Xu, revealed that only the disease-related allele of BRCAJ was Trinh M. Pham, Kathleen A. Caizone, present. These data strongly support the hypothesis that BRCA1 is a tumor suppressor gene. Pamela Bennett-Baker, Jeffrey Chamberlain, Jeff Boyd, Judy E. Garber, Francis S. Collins, INTRODUCTION and Barbara L. Weber Familial early onset breast/ovarian cancer is an autosomal Departments of Internal Medicine [S. D. M., T. M. P.], Pathology dominant disorder manifested as an increased susceptibility to [T. S. F.], and Human Genetics [J. C., P. B-B.], and the Human breast cancer starting in the third decade, with an 85% lifetime Genome Center [J. X., F. S. C.], University of Michigan, Ann Arbor, Michigan 48109; Dana-Farber Cancer Institute, Boston, risk. Breast adenocancinomas and ovarian cystadenoeancinomas Massachusetts 02115 [J. E. G.]; National Center for Human Genome are the predominant histological types. Mucinous ovarian can- Research, NIH, Bethesda, Maryland 20892 [F. S. C.]; and cens seem to be less common in hereditary breast/ovarian fam- Departments of Obstetrics and Gynecology [J. B.], Internal Medicine ilies (1), but in general the familial tumors are phenotypically [K. A. C., B. L. W.], and Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 indistinguishable from histologically matched sporadic tumors. Atypical hyperplasia, a premalignant lesion, has been found in one study to be more prevalent in families at increased risk for ABSTRACT hereditary breast cancer (2). The natural history of the tumors, The BRCA1 gene on human chromosome 17q21 is re- including detectability by radiographic and clinical means, ne- sponsible for an autosomal dominant syndrome of inherited sponsiveness to therapy, and overall prognosis for the patients early onset breast/ovarian cancer. It is estimated that based on stage at presentation appear to be indistinguishable women harboring a germline BRCAJ mutation incur an from age-matched controls (1). 85% lifetime risk of breast cancer and a greatly elevated Since 1990, it has been known that a single locus on risk of ovarian cancer. The BRCA1 gene has recently been chromosome 17q21, termed BRCAJ,3 is responsible for most isolated and mutations have been found in the germline of cases of hereditary early onset breast/ovarian cancer (3, 4). affected individuals in linked families. Previous studies of Approximately 67% of families with breast cancer diagnosed loss of heterozygosity (LOH) in breast tumors have been before age 45 years and 95% of families with breast/ovarian carried out on sporadic tumors derived from individuals cancer appear to be linked to BRCAJ. Easton et a!. (5) have without known linkage to BRCA1 and on tumors from recently reported that the penetrance, by age 70 years, is 87% linked families. Loss of large regions of chromosome 17 has (95% CI: 72-95) for breast cancer and 44% (95% CI: 28-56) been observed, but these LOH events could not be unequiv- for ovarian cancer. The ovarian cancer risk may be an ovenes- ocally ascribed to BRCA1. timate, however, because of a bias favoring ascertainment of We have studied 28 breast and 6 ovarian tumors from families with both breast and ovarian cancer. In addition, Fond families with strong evidence for linkage between breast et a!. (6) have reported a significant increase in the relative risks cancer and genetic markers flanking BRCAJ. These tumors for colon (4.11, 95% CI: 2.36-7.15) and prostate cancer (3.33, were examined for LOH using genetic markers flanking and 95% CI: 1.78-6.20) in BRCAJ mutation carriers. The BRCAJ within BRCAJ, including THRA1, D175856, EDH17B1, gene has recently been reported and disease-associated muta- EDH17B2, and D17S183. Forty-six percent (16/34) of tu- tions have been found in breast and breast/ovarian kindreds mors exhibit LOH which includes BRCA1. In 8 of 16 tumors (7-1 1). The study of tumors in linked families may help eluci- the parental origin of the deleted allele could be determined date how BRCAJ mutations lead to disease. by evaluation of haplotypes of associated family members; Tumor analyses have proven useful not only in narrowing in 100% of these cases, the wild-type allele was lost. In some the candidate region, thereby expediting the search for BRCAJ, of these families germline mutations in BRCA1 have been but have also provided data supporting the hypothesis that determined; analyses of tumors with LOH at BRCA1 have BRCAJ is a tumor suppressor gene (12, 13). Work from several laboratories (12-16) has revealed that sporadic and familial breast and ovarian tumors may exhibit the phenomenon of LOH

Received 9/16/94; accepted 2/17/95.

1 Supported in part by NIH Grants GCRC-MCAP 3M01RR00042-34S1 (S. D. M.), ROl CA57601-01 (B. L. W.), and P30 HG00209 to the

Genome Center at the University of Michigan. 3 The abbreviations used are: BRCAJ, breast cancer gene; LOH, loss of 2 To whom requests for reprints should be addressed, at Department of heterozygosity; CI, confidence interval; SSCP, single-strand conforma- Internal Medicine, 5510 MSRB I, University of Michigan Medical tion polymorphism; LOD score, base 10 logarithm of the odds favoring Center, Ann Arbor, MI 48109-0680. linkage.

in large and diverse ehnomosomal regions, including 17q21, neextraction of tumor/normal DNA from regions equivalent which supports the hypothesis that BRCAJ is likely to be a to those of the original extraction. It was then possible to tumor suppressor gene. In other words, functional loss of both apply uniform criteria for scoring LOH at all markers, even if BRCAJ alleles in the affected tissue(s) may be necessary for more than one extraction was necessary. malignant transformation to occur. In this model, affected mdi- PCR Amplification of Polymorphic Markers. PCR ne- viduals are presumed to be born with a genmline defect in the actions were performed on each tumor/normal pain, a normal BRCAJ gene, which constitutes the ‘ ‘first hit’ ‘ in Knudson’s human DNA control, and two on more samples from CEPH model for tumonigenesis by a tumor supressor gene. The second parents known to be informative for the marker being ana- defect is acquired somatically in the wild-type allele in the lyzed. DNA extraction reagents and water served as negative breast on ovarian tissue of these patients, thereby contributing to control template. The reaction volume was 35 jil. The neac- the development of cancer. One mechanism for disruption of the tion mixture consisted of 8 p.1 DNA template, 3.5 p.1 lOX Taq second allele can be an interstitial chromosomal deletion, de- polymenase buffer, 4.25 p.1 1.25 msi deoxynucleotide tniphos- teeted through loss of alleles at flanking polymorphic markers. phate mixture, 0.2 p.1 Taq polymenase, a variable amount of Thus, LOH is widely held to be an indicator of the presence of forward and reverse primer ranging from 0.8-1.5 jil of a 10 a tumor suppressor gene adjacent to the deleted markers. The ng/jil solution of each, and the remainder in double-distilled observations of LOH presented here support a tumor suppressor water. One of the primers was end labeled with [32P]dATP mechanism for BRCAJ. using T4 kinase. Annealing temperatures ranged between 53#{176}Cand 65#{176}C,depending on the primers, with an extension MATERIALS AND METHODS temperature of 72#{176}C.The annealing time was 1 mm whereas the extension time varied from 1 mm to 40 s + 5 s/cycle for Specimen Selection. Thirty-four paraffin-embedded breast 35 cycles. Optimization of conditions proceeded as follows: tumors from families likely to harbor BRCA1 germline mutations were obtained from the institutions where the patients had under- if amplification was not detected with an annealing temper- atune of 53#{176}C,the amount of template was increased to 12 p.1. gone surgical treatment, in accordance with guidelines from the University of Michigan Human Use Committee for the procure- Nonspecific signals were suppressed by increasing the annealing temperature and/or decreasing the primer concentra- ment of archival materials for research. Living family members tion. The PCR products were analyzed in 0.4-mm thick 8% provided informed consent to proceed with the analyses of their tumors; the results were not shared with the patients. All specimens denaturing polyacrylamide gels, which were subsequently dried and autonadiognaphed by standard techniques. underwent histological examination to confirm the diagnosis and to determine whether both neoplastie and normal components were Although it is widely acknowledged that the scoring of LOH is a qualitative assessment, we have used the following present. DNA Extraction. With the assistance of a surgical guidelines to ensure consistency of scoring. In experiments where the band intensity of the alleles varied between the pathologist (T. S. F.), each paraffin block was cut in 4-jim tumor and normal samples, but the wild-type allele was still sections and mounted on glass slides. Normal and neoplastic visible, the samples were neextracted from immediately tissue fractions were dissected with a single-use disposable razor blade under a dissecting microscope. This procedure adjacent sections at least two times and independent expeni- ments were performed on the tumor/normal pains. A de- minimized mixing of normal and tumor subpopulations, and crease in intensity of a band of oven 75% was required yielded tumor samples which were estimated to be at least for scoring LOH. The criteria for scoring LOH was uni- 90% free from contamination with normal cells. A typical form for all markers analyzed on samples derived from a sample contained DNA from 100-1000 cells. The samples given extraction, i.e., if the wild-type allele was entirely lost were depanaffinized with 100 pA xylene. An equal volume of 100% ethanol was added and the samples were pelleted for 10 in one marker, to scone LOH on another marker using template from the same extraction, a qualitatively similar de- mm at 15,000 X g, vacuum dried, and digested overnight crease in the wild-type allele intensity was required. The with 200 ng/p.l proteinase K in 100 jil 50 mrvi Tnis (pH 8.3). The samples were boiled for 8 mm, iced, and centrifuged signal intensity was evaluated visually by at least three independent observers. again to remove proteinaceous and other debris. Samples SSCP. PCR was carried out as outlined above in 10-pA were extracted in groups of 8-12, each extraction containing reactions. Direct incorporation of [a-32P]dCTP was used instead a tube without template as a negative control. This method of end labeling of primers. The PCR products were denatured avoids the use of ionic detergents, sonication, on phenol/ chloroform separation, all of which yield samples which may and analyzed on MDE SSCP gels (AT Biochem) run at 6 W for be suboptimal for PCR experiments. 16 h at room temperature. The gels were dried and autoradio- All tumors were dissected and extracted at least twice in graphed by standard methods. The sequences for exon 5 primers are (7): forward, 5’-CTCTFAAGGGCAGlTGTGAG and re- separate batches for verification of results. Each tumor/normal extraction yielded two 100-j.tl samples with a total DNA verse, 5’-TTCCTACTGTGG’LTGTTCC; their annealing tem- mass of the order of a femtognam each. Approximately 8-10 peratune is 60#{176}C. reactions could be performed with template from each extraction. Whenever further extractions were needed for a RESULTS given block, these were carried out on sections immediately Twenty-eight breast and 6 ovarian tumors from 14 families adjacent to the original slide. This procedure resulted in the were studied. Six families had evidence for linkage between

ii- L___ BRCA1 Tissue Hlslologb Amountol Ageat D17S250 THRAI D17S856 D17S1185EDH1781 EDH17B2 D17S183 D17S579 I I se mullon Ipe tp&BRg stoma dx I

- 11 - PS + p p n w #{149} n i- ir r- D + 36 w #{149} n n n n n w#{149} iT- ir D ++ w#{149} w#{149} 0 0 n wS n w#{149} i: U- u + #{247} . . o o 768 15 B D/3 ++ 38 0 0 n n n

- 1____ 1.89 3875de14 B D/2 + + + 33 n n n 0 0 n 0 4__ 27 B D/3 + + 33 0 0 w #{149} n w #{149} 0 0

- 27 ;E MD + + 42 0 n 0 w S w #{149} 0 4w_ 27 B ME + 35 n 0 n n n 1 7 B D/3 +++ 31 fl n n n n

- - 2.74 Cys64Gly 0 D/3 + 36 w#{149} n w#{149} wS n n 0

- 46 B + 41 0 n 0 0 74 46 B D/2 +++ 46 fl n 0 95O 46 B D/3 + 35 0 fl 0 n 800_ 46 2.91 NA B D/2 ++++ 31 0 n 0 62r 3o + 37158 w #{149}w #{149} n 0 n n 61 301 1.i fl7BlnM B + 39 0 n n - - B + + + + 35 fl n n 219 0.41 NA B/B I ++++ 39/43 n n

- 3.4__ 0__ EN + + 33 0 n 0 n 847 34 221 NA 0 D12 + + + 51 0 0 0 S 0 B LD ++ 27 0 S n n n 0 0

- 1!i NA NA B Di2 + + 54 S n n n 0 441 30 1.63 NA BiB D/2 + 48/54 0 n 0 n S n n o- iw- N a i’U + + 0 S 0 S S n 0

-.- 5!__ __ kg13ler B 0/2 + #{247}+ 53 0 n 0 n n 2!_ 2_ B + + 35 0 0 0 n n 29_ B + + + 33 0 0 0 n n 2003300 NA NA B D/2 +++ 32 0 0 0 n n n

- 1 - G Mn + 70 0 0 0 0 S 0

- S + 0 0 0 0 i 1 #{149} 0 957 178 NA NA BIB ID/2 ++#{247}+ 33135 0

2ii #{241}irii + + 0 0 0 #{149} 0 0 0 _i 1 :: Bil ID/2 + + + 81 0 0 0 n 0

Fig. I Clinical, pathological, genetic (LOD scone), LOH, and BRCAI mutation data on 34 tumors from beast/ovarian families analyzed with markers within and flanking the BRCA I candidate region. Mutations are reported following the convention of Beaudet and Tsui (16). For patients affected by more than one tumor, the histological type of the tumor analyzed is listed: #{149},LOH; w #{149},LOH, wild-type allele is lost; LI, indeterminate, LOH cannot be scored; 0, retention of heterozygosity; n, not informative; blank, untested markers; B, breast; 0, ovary; E, endometnium; G, stomach; AxLN, axillary lymph node; ID, infiltrating duetal carcinoma; IL, infiltrating lobular carcinoma; ILD, infiltrating lobular and ductal carcinoma; ME, medullary carcinoma; PS, papillary serous carcinoma; EN, endometnioid carcinoma; MD, metastatie duetal carcinoma; A/rn, moderately differentiated adenocancinoma; B-Rg, Bloom-Richardson grade of the ductal carcinoma; NA, not available; +, <10%; + +, 10-25%; + + +, 25-50%; + + + +, 50-75%.

breast/ovarian cancer and genetic markers flanking BRCA 1 Fig. 1 summarizes the results of the LOH experiments for (LOD score range, 1.18-2.91), one family has a LOD scone of a subset of representative markers in the BRCA 1 region, pro-

0.41, one family was probably unlinked (LOD scone, - 1.3), and vides clinical data on the patients, histological data on the for six families the LOD scores are not available due to the tumors, and genetic and mutation data on the families. Several limited number of affected individuals available for sampling. distinct patterns of LOH are seen in these families. In family 15, The tumors were examined for LOH using polymorphic markers four tumors (tumors 209, 191, 157, and 224) have LOH eneom- flanking BRCAJ including THRA1, D17S856, EDH17B1, passing all scorable markers, whereas tumors 768 and 763 EDH17B2, and D17S183. Sixteen (47%) of 34 tumors exhibited showed no LOH for any of the markers tested. In family 27, detectable LOH using the genetic markers described. In a related tumors 422 and 532 exhibit LOH in a region containing the independent study, BRCAJ mutations were identified in some of BRCAJ gene. Tumor 484 belongs to a family found to be the kindreds from which these tumors derive (8). unlinked to BRCAJ; we did not observe LOH for the markers

N T N1N2 T1T2 NT NT

. ‘#{225} 140 50 210 223 267227 4G 1 I in 119

A B CD

Fig. 2 Autoradiographs of PCR products from normal (N) and tumor Key: Breast cancer (7) cell fractions of sample 422, family 27. A, retention of heterozygosity at D17S856; B, LOH at EDH17B2; C, LOH at D17S1185; and D, retention of heterozygosity at D17S183 (SCG43). Ovanan cancer 422 Oood T41 140 162 140 162 140 162 D17S1186 219 211 219 - 219 211 EDH17B2 217 255 267 - 26? 255 BAa4I 4G *1 4G - 4G wt tested. The minimum region of overlap of LOH among all of the D17S579 In 119 123 119 123 119 tumors tested lies between D17S856 (OF2) and D17S183 Fig. 3 Section ofthe pedigree of family 27 (LOD score, 2.74) showing (SCG43), which includes BRCAI. All of the samples which affected (bold type) and nonaffeeted haplotypes for markers THRA1, showed LOH had less than 50% normal stromal infiltration on D17S1185, EDH17B2, BRCAI, and D17S579. Individual marked with the slides used for DNA extraction. No relationship between the an arrow is 422 from Fig. 2. wt, wild-type; NA, not available; ----, deleted allele. presence of LOH and tissue of origin, histological type, or grade was observed. Fig. 2 displays data from experiments on a single tumor/ normal pair (sample 422) using primers which flank Dl75856 (a BGT tetnanueleotide repeat), EDH17B2 (a 12-base pair insertion- deletion polymorphism in 173-hydroxysteroid dehydrogenase; Ref. I 7), Dl 75 1 185 (a tetnanueleotide repeat), and Dl 75183 (a dinucleotide repeat). The EDHI7B complex consists of two

loci, one of which is a pseudogene, arranged in tandem on J 17q21 (18). The 5’ region of EDH17B2 contains a 12-base pain insertion-deletion polymorphism (17), around which we have designed PCR primers that yield a product of 267 and/on 255 _*4M base pains suitable for amplification from paraffin-derived DNA Fig. 4 SSCP of exon 5 of BRCAJ of normal control (non-BRCAI templates; PCR products less than 300 base pains in length are mutation carrier) DNA (B), germline (G), and tumor (7) DNA for more consistently amplified from archival material than larger sample 422. All samples were run on the same gel and autoradiographed segments, perhaps due to the DNA being nicked on alkylated. simultaneously. Arrow, variant band arising from the Cys64Gly muta- We encountered rare blocks which would not yield amplifiable tion. DNA for any markers, possibly due to chemical modification of the DNA during fixation. In Fig. 2, A and D show retention of heterozygosity for 17q2l. We note however that the mother of 422 is a nonpen- D17S856 and D17S183, respectively; B shows consistent etrant carrier. Fig. 3 summarizes the BRCA I haplotype data for LOH at EDH17B2 for two different DNA extractions of the constitutional and tumor DNA of sample 422 derived from adjacent mienotomed slides; and Cshows LOH at D17S1 185. Fig. 4. The assays in A, the N1/Tl normal/tumor pain in B, and C Fig. 4 depicts SSCP analyses of blood DNA from a normal were carried out on the same DNA sample; the assays for the control (B) and the genmline (G) and tumor (7) DNA from the N2/T2 normal/tumor pain of B and those in D were performed paraffin block of individual 422. The variant band marked with on template extracted from a slide immediately adjacent to an arrow is present in the germline and in the tumor of 422, but the previous one. not in the normal contol. The decrease in the relative intensity of Haplotypes of additional family members for several mark- the normal to the variant band is consistent with LOH in this ens flanking and within the BRCAI region confirmed that the sample. allele lost in the tumor was the wild-type allele, as shown by the Other tumors in Fig. 1 have a region of LOH which is pedigree in (Fig. 3). The individual marked with an arrow in likely to be entirely contained within the flanking markers Fig. 3 is 422, whose tumor assays are depicted in Fig. 2. For tested, such as samples 704 and 532. Fig. S shows examples of clarity, we do not present the full haplotype here, as it has been LOH at EDH17B1 and EDH17B2 for samples 532 (A) and 704 reported previously (19) and supports linkage to BRCA1 on (B), and LOH at D17S856 for sample 623 (C).

NT BNT NT NT N T tumor suppressor genes other than BRC’AI on chromosome 17 makes interpretation of LOH data derived from widely spaced markers difficult; these tumor suppressor genes may also be involved in the progression of the malignant pheno- type. In this regard, carrying out LOH studies on samples ,.-. obtained from families with documented BRCAJ linkage provides additional critical information: if LOH affects BRCAI, the wild-type allele should be the one lost. The development of techniques for the extraction of (a) (b) (a) (b) relatively pure normal and tumor DNA fractions from panaf- fin-embedded tissue has allowed us to study 34 tumors from A B C linked families. The success of the extraction and, ultimately, of the experiments performed with the samples hinges on the Fig. 5 Autoradiographs of PCR products from normal (N) and tumor careful manual separation of tumor from normal cells under (7) cell fractions and blood (B). A, sample 532, and B, sample 704, show the dissecting microscope using surgical blades. This was LOH at markers EDH17B2 (a) and EDHI7B2 (b). C, sample 623 with LOH at D17S856. accomplished in our study by a skilled surgical pathologist (T. S. F.). The experiments were repeated as many as five times before a definitive scone was assigned. In spite of great cane being taken in separating tumor from surrounding non- DISCUSSION mal tissue when dissecting specimens, some samples could The phenomenon of LOH has proven to be a useful not be definitively scored as exhibiting LOH, although di- indicator of the involvement of tumor suppressor genes in the minished intensity of the wild-type allele in the tumor sample development of breast and other tumors. Following the cx- suggested LOH. peniments of Feanon and Vogelstein (20) and Vogelstein et a!. A heterogeneous pattern of LOH was observed in these (21) in colon cancer, Sato et a!. (14) demonstrated that at tumors. Some tumors exhibited no LOH for any of the least four distinct regions on chromosomes 13q (D13S52), markers tested; others showed loss of the wild-type allele 16q22-23, and Ylpl3.l-l3.3 exhibit LOH in sporadic breast flanking BRCAI. LOH might have been present and not tumors, suggesting that as many as four different tumor detected in our experiments if the tumor were heavily infil- suppressor genes may be implicated in sporadic carcinogen- trated with stromal elements, and thus not separable. Also, if sis. Chen (15) selected probes on chromosomes 22q, l8p, the interstitial deletion spanned an interval smaller than the Yip, 1 lp, 13q, and lq, all of which are candidate regions for distance between the markers we used, LOH would not be tumor suppressor genes, to ascertain whether LOH at these observed. The minimum region of overlap for the LOH seen locations is present above a background level. They showed in our familial tumors contains the now known BRCAJ gene that, with the exception of 1 lplS, all locations tested exhib- and excludes the region between D17S846 and D17S746 ited LOH at well above background levels. Smith et a!. (13) described by Cnopp et a!. (22), which may contain another analyzed 13 (2 breast and 1 1 ovarian) tumors in families tumor suppressor gene. thought to be linked to BRCAJ; they found LOH for two Genmline mutations in BRCAJ were found in four of the markers flanking BRCAI (D17S250 and D17S855) in 9 tu- families shown in Fig. 1, designated by numbers 15, 27, and mors, all of which had lost the wild-type allele. The region 51 (8). Affected members in families 15 and 27 had tumors encompassed by these two markers is greaten than 10 eM and which showed LOH. The individuals from family 27 whose includes other genes potentially involved in breast cancer tumors were analyzed and listed in Fig. 1 have been con- such as NME1 and pnohibitin. Both genes were subsequently firmed to canny the Cys64Gly genmline mutation, except 532 excluded from the BRCAI candidate region by genetic link- who is diseased. Those tumors from family 27 which did not age. More recently, Kelsell et a!. (12) have reported LOH at show LOH were also found to have abundant normal stromal marker D17S855 (248yg9) within BRCAJ in five familial infiltration within the tumor sections. This is a potential breast tumors and one sporadic ovarian tumor. In all cases, reason why we failed to detect LOH in these eases. When the wild-type allele was shown to be lost. The marker closest BRCAI mutations were analyzed in a subset of familial to the present limits of the BRCAJ region which retained tumors which exhibit LOH such as the one depicted in Fig. 4, hetenozygosity was located near TP53 on Yip and therefore the disease-related allele was the most prominent in the did not contribute to narrowing the BRCAI candidate region. tumor fraction (8), providing further confirmation of the The observation of LOH in 17q21 in sporadic breast tumors LOH results by a different method. Our data thus confirm has led to the hypothesis that BRCAI is implicated in spo- that LOH does, in some tumors, occur specifically within the radie carcinogenesis (15). BRCAJ region and virtually always deletes the wild-type Recently, Cnopp et a!. (22) studied 130 sporadic breast allele, thereby strongly supporting the hypothesis that BRCAJ tumors and found LOH in a relatively small 120-150-kilo- is a tumor suppressor gene. base region of minimum overlap between D17S846 and The role that BRCAJ plays in sporadic eaneinogenesis is, D17S746. However, BRCAJ is located distal to D17S746, in contrast, not clear at present. The two-hit tumor suppressor thereby excluding the minimum region of overlap by LOH mechanism predicts that somatic mutations of BRCA I would observed by Cnopp et a!. (22). The presence of at least three be present in the genomic DNA of sporadic tumors. However,

the first reported analyses of 35 breast and 12 ovarian spo- L. C., Collins, F. S., and Weber, B. L. Mutations in the BRCA1 gene in nadie tumors by Futreal et a!. (11) failed to demonstrate such families with early-onset breast and ovarian cancer. Nature (Genet.), 8: 387-391, 1994. somatic BRCAI mutations in the tumor samples; the four mutations described were present in both the tumor and the 9. Simand, J., Tonin, P., Durochen, F., Morgan, K, Rommens, J., Gingras, S., Samson, C., Leblanc, J-F., Belanger, C., Dion, F., Lb. 0., Skolnick, M., genmline. From this experience it might be predicted that Goldgar, D., Shattuek-Eidens, D., Labrie, F., and Narod, S. Common thorough searches of the BRCAI coding and regulatory re- origins of BRCAI mutations in Canadian breast and ovarian cancer families. gions in many samples of breast, ovarian, and prostate tumors Nature (Genet.), 8: 392-398, 1994. may be necessary to identify a role for BRCAJ in sporadic 10. Friedman, L. S., Ostermeyen, E. A., Szabo, C. I., Dowd, P., Lynch, eaneinogenesis. E. D., Rowell, S. E., and King, M-C. Confirmation of BRCAI by In some familial and sporadic tumors which do not analysis of germline mutations linked to breast and ovarian cancer in 10 exhibit LOH or point mutations, loss of BRCAI function may families. Nature (Genet.), 8: 399-404, 1994. be caused by derangement of its transcription or translation 11. Futneal, P. A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harsh- mechanisms due to alterations in regulatory elements or in man, K., Tavtigian, S., Bennett, L., Haugen-Strano, A., Swensen, J., Miki, Y., Eddington, K., McClure, M., Frye, C., Weaven-Feldhaus, other genes. One such phenomenon is postulated by Brown et J., Ding, W., Gholami, Z., Sodenkvist, P., Terry, L., Jhanwan, S., a!. (23) in their study of the genomic structure of the 5’ end Berchuck, A., Inglehant, J., Marks, J., Ballinger, D., Barrett, J., of BRCA 1 . They showed that exon 1 of BRCA 1 is centromenic Skolniek, M., Kamb, A., and Wiseman, R. BRCAI mutations on to the JAI-3B gene and that the two genes lie only 295 base primary breast and ovarian carcinomas. Science (Washington DC), pains apart. The JAJ-3B gene had been isolated from a eDNA 266: 120-122, 1994. ovarian library with CA125 antisera. The intervening se- 12. Kelsell, D. P., Black, D. M., Bishop, D. T., and Spurn, N. K. Human quence between BRCAJ and JAI-3B contains putative genetic analysis of the BRCA1 region in a large breast/ovarian family: refinement of the minimal region containing BRCAI. Hum. Mol. Genet., ‘‘ CAT’ ‘ boxes which may conegulate the dis-coordinate tran- 2: 1823-1828, 1993. seniption of these genes. These data suggest that future stud- 13. Smith, S. A., Easton, D. F., Evans, D. G. R., and Ponder, B. A. 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Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. Germline BRCA1 mutations and loss of the wild-type allele in tumors from families with early onset breast and ovarian cancer.

S D Merajver, T S Frank, J Xu, et al.

Clin Cancer Res 1995;1:539-544.

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