Breast-Ovarian Cancer Gene on Chromosome 1 7q 1 2-q2

Transcription

1 Am. J. Hum. Genet. 52: , 1993 Genetic Heterogeneity and Localization of a Familial Breast-Ovarian Cancer Gene on Chromosome 1 7q 1 2-q2 S. A. Smith,* D. F. Eastont D. Ford,t J. Peto,t K. Anderson,t D. Averill,t M. Stratton,4 M. Ponder,* C. Pye,* and B. A. J. Ponder* *Cancer Research Campaign Human Cancer Genetics Research Group, Department of Pathology, University of Cambridge, Cambridge; and Sections of tepidemiology and tmolecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey, England Summary In a study of 31 breast cancer families and 12 breast-ovarian cancer families, we have obtained clear evidence of linkage to markers on chromosome 17q in the families with ovarian cancer (maximum lod score 3.34 at 0 =.04) but only weak evidence in those without ovarian cancer. Recombinant events indicate that the gene lies between D17S588 and D17S250. Introduction The localization of a predisposing gene on chromosome 17q has recently been reported, by Hall et al. (1990), in families with early-onset breast cancer and, by Narod et al. (1991), in families with breast and ovarian cancer. We report the results of a genetic linkage study in a series of 31 breast and 12 breast-ovarian cancer families, using seven polymorphic markers on chromosome 17q. Methods Forty-three families were studied, 31 of which contained breast cancer only and 12 of which contained breast cancer with at least one case of ovarian cancer (table 1). The majority of the families were initially ascertained in the United Kingdom, but eight were from the United States, one was from France, and one was from India. The families were ascertained either in response to national publicity in the United Kingdom Received June 15, 1992; revision received October 30, Address for correspondence and reprints: B. A. J. Ponder, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, England by The American Society of Human Genetics. All rights reserved /93/ $02.00 or by referral, by oncologists or general practitioners, to a cancer family clinic. All families met one of the following minimum criteria: breast cancer in two siblings diagnosed under age 45 years, plus one other with breast cancer; four cases of breast cancer under age 60 years; or three cases of epithelial ovarian cancer. Five of the "breast-ovary" families were initially ascertained because of a family history of breast cancer, and seven because of an ovarian cancer family history. A total of 229 individuals, 141 of whom were affected, were typed at one or more loci. Breast and/or ovarian cancer was confirmed in 155 affected women, including 120/141 (85%) of typed cases, either by pathology report, hospital records, or death certificate. Typing of DNA Polymorphisms Genomic DNA was prepared either directly from peripheral blood lymphocytes or from lymphocytes which had been immortalized by Epstein-Barr viral transformation. DNA (5 jg) was digested with Hinfl, fractionated by electrophoresis through 0.8% agarose, and blotted onto Hybond-N by standard techniques. pcmm86 plasmid DNA (Nakamura et al. 1988) was radiolabeled by random primer extension and was preannealed with human placental DNA before use. Microsatellite Polymorphisms Microsatellite polymorphisms were typed by PCR using 100 ng of target DNA and 20 pmol of each 767

2 Table I Description of Families Breast No. OF CANCERS Ovarian Under Age 45 Under Age 45 LOD SCORE AT Age Years Age Years 0 =.05 POSTERIOR 45 and Bilat- 45 and PROBABILITY No. OF AFFECTED INDIVIDUAL(S) FAMILY Years Older eral Years Older D17S588 D17S579 OF LINKAGE2 INCONSISTENT wrrh LINKAGEb Breast-ovarian cancer families: Breast cancer families: C C 2 0 4d I (5) 0 (4) 1 (5) breast cancer, age 35 years, 0 (2) 0 (4) 0 (3) 0 (4) 0 (4) 1 (4) ovarian cancer, fourthdegree 0 (5) 1 (6) ovarian cancer, age 54 years, fourth-degree 0 (2) (2) breast cancer, age 37 years, (4) breast cancer, age 41 years, ; and breast cancer, age 42 years, seconddegree (3) breast cancer, age 40 years, (3) (3) (4) (3) (3) breast cancer, age 46 years, (3) (4) breast cancer, age 36 years, (2).11 2 (4) breast cancer, age 77 years, ; and breast cancer, age 35 years, firstdegree.16 1 (2) breast cancer, age 46 years, (continued) 768

3 Table I (continued) No. OF CANCERS Breast Ovarian Under Age 45 Under Age 45 LOD SCORE AT Age Years Age Years 0 =.05 POSTERIOR 45 and Bilat- 45 and PROBABILITY No. OF AFFECTED INDIVIDUAL(S) FAMILY Years Older eral Years Older D17S588 D17S579 OF LINKAGEa INCONSISTENT WITH LINKAGEb Breast cancer families: (3) breast cancer, age 59 years, (4) (4) breast cancer, age 41 years, ; and breast cancer, age 47 years, seconddegree (3) (2) (2) (2) breast cancer, age 44 years, second-degree (6) breast cancer, age 61 years, ; and breast cancer, age 63 years, firstdegree (3) (5) breast cancer, age 35 years, second-degree (4) breast cancer, age 53 years, (4) (3) breast cancer, age 54 years, (3) C (3) (2) (3) breast cancer, age 59 years, (4) breast cancer, age 40 years, ; and breast cancer, age 32 years, firstdegree NOTE.-All ages are age at diagnosis. a From the multipoint analysis of Easton et al. (1993). All breast-ovary families were linked. Family 28 was ignored because families containing male breast cancer were excluded from the multipoint analysis. b Nos. in parentheses are total no. of affected individuals typed. ' Excludes one non-germ-line breast cancer. d Includes one male case. 769

4 770 primer. The DNAs were amplified through 30 cycles of 1 min at 940C, 1 min at 550C, and 1 min at 720C, with a final extension at 720C for 10 min. One of the PCR primers was end-labeled using y-32p ATP, and the products were detected by electrophoresis through 6% acrylamide sequencing gels, followed by autoradiography. Six microsatellite polymorphisms were typed. These were mfdl5 (recognizing locus D17S250; Weber et al. 1990); mfdl88 (D17S579; Hall et al. 1992); 6C1 (D17S293, provided by Dr. D. Black); 42D6 (D17S588, provided by Dr. M. Skolnick); NME1 (D17S351, provided by Dr. D. Black); and GH1 (Polymeropoulos et al. 1991). Analysis of DNA from Archival Pathology Blocks Individual 5-im sections were incubated in 0.5 ml of extraction buffer containing 10 mm Tris ph 8.3, 50 mm KCl, 1.5 mm MgCl2, 100 ig BSA/ml, 0.45% Tween-20, 0.45% Nonidet NP40, and 100 gg proteinase K/ml. The sections were incubated for at least 8 h at 550C and then were transferred to a boiling water bath for 10 min and cooled on ice. Aliquots of 1-5 gl were used in PCR. Adjacent sections were mounted on glass slides and were stained with hematoxylin and eosin, for histological examination. Statistical Methods Two-point lod scores for linkage between markers and the disease locus were calculated by assuming an autosomal dominant disease gene with a frequency of.003, conferring a risk of.8 by age 80 years, compared with a risk of.08 in noncarriers. For full details of the genetic model see the article by Easton et al. (1993) in this issue of thejournal. Lod scores have been summarized according to female recombination fractions, on the assumption that the female map is twice the male map. The main analyses assumed equal marker allele frequencies, although for D17S588 we have recomputed lod scores on the basis of our observed allele frequencies; these allele frequencies were estimated by typing 48 unrelated individuals. Evidence for heterogeneity was evaluated using the admixture model, in which a proportion of families are assumed to be linked to 17q and the remainder are assumed to be due to other loci (Smith 1961). Lod scores assuming heterogeneity were computed using the HOMOG program (Ott 1985). Posterior probabilities of linkage for each family were calculated from the prior probability of linkage and the lod scores generated by a multipoint analysis of D17S588 and D17S250 and the disease locus. The prior Smith et al. probabilities of linkage were taken from the multipoint linkage analysis of Easton et al. (1993), which included D17S250, D17S588, and the disease. Under their model, all breast-ovary families and 45% of breast cancer-only families would be linked a priori, with the gene being located 8.0 cm proximal to D17S588. Results Linkage Analysis A summary of two-point lod scores for six markers is shown in table 2 (D17S293 was only typed in "recombinant" families). Evidence of linkage was obtained with D17S579 (lod score 2.42 at 0 =.16, P <.001) and with D17S588 (lod score 1.21 at 0 =.23, P =.02). When the lod scores from D17S588 were recomputed using allele frequencies generated from our own data, the maximum lod score increased to 1.55 at 0 =.21. Maximum lod scores for each of the other markers were below Table 3 presents a breakdown of the results for D17S588 and D17S579, by family type. Breast-ovary families, defined as those containing at least one case of ovarian cancer, have a cumulative lod score of 3.34 for D17S579 at 0 =.04, while for families containing only female breast cancer cases the lod score is maximized at a much larger 0 (.36) and is not significant. This suggests that only a proportion of the breast cancer families result from mutations at the breast-ovarian cancer locus on chromosome 17q. Evidence for linkage to D17S588 is also restricted to breast-ovary families. Analysis using the program HOMOG and based on the D17S579 results estimates the proportion of linked breast-ovary families to be 1.0 (lower limit of lod - 1 support region 0.47) at 0 =.04. The proportion of linked breast cancer families at 0 =.04 is estimated to be.22 (lod - 1 support limits 0-.67), although the evidence for linkage is very weak (lod score 0.32). Among the breast cancer families, evidence for linkage was no stronger among families with at least five cases under age 60 years or among families with a mean age at onset under 45 years. There was, however, stronger evidence of linkage among families with a mean age under 40 years (lod score 1.03 at 0 =.001, for D17S588). An alternative way of analyzing the data, which is model independent, is to look at the number of haplotypes shared by pairs of affected s. The D17S579-D17S588 haplotypes for the families that contain four or more cases of breast cancer under age

5 Familial Breast-Ovarian Cancer on 1 7q 771 Table 2 Two-Point Lod Scores for Linkage of Breast and Ovarian Cancer to Markers on Chromosome 1 7q, under Homogeneity LOD SCORE AT FEMALE RECOMBINATION FRACTION OF2 MAXIMUM MARKER LOD SCORE ESTIMATED 0 D17S D17S D17S NME D17S GH a Under the assumption that the female distance equals twice the male distance. 60 years at diagnosis and for all of the breast-ovary cancer cases in the family, and thus they have a substanfamilies are shown in figure 1. Analysis of the complete tial probability of being sporadic cases. In contrast, data set (table 4) shows that, in the breast-ovary fami- there is little evidence of excess haplotype sharing in the lies, all but three pairs of affected s up to third other breast cancer families, and 31/65 (48%) of reladegree share a haplotype. The three discordant pairs all tive pairs do not share any putative disease haplotype. occur in family 128, where one breast cancer case age There is, however, some suggestion of excess haplotype 35 years at diagnosis does not share the putative dis- sharing in the families with at least five breast cancer ease-bearing chromosome with her affected sisters (see cases under age 60 years. Fifteen of the families contain fig. 2). Two women with ovarian cancer, in families 845 at least one breast cancer under the age of 60 years that and 871, do not share the putative disease haplotype is inconsistent with linkage (see table 1), and 9 contain (see table 1). However, both individuals were at least at least one case under the age of 45 years that is inconfourth-degree s to all other breast or ovarian sistent with linkage. Three families contain two cases Table 3 Lod Scores for Linkage between Breast and Ovarian Cancer and Markers DI and DI 7S588, by Family Type, under Homogeneity LOD SCORE AT FEMALE RECOMBINATION FRACTION OF2 MARKER AND FAMILY TYPE (no. of families typed) D17S588: Breast-ovary (12) Breast: Cases under age 60 years: Fewer than five cases (20) Five or more cases (9) Includes male (1) D17S579: Breast-ovary (10) Breast: Cases under age 60 years: Fewer than five cases (20) Five or more cases (9) Includes male (1) a Under the assumption that the female distance is twice the male distance.

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8 774 Smith et al. Table 4 Haplotype Sharing in Pairs of Individuals Affected with Ovarian Cancer at Any Age or with Breast Cancer at Age under 60 Years PROPORTION OF INDIVIDUALS SHARING "DISEASE" CHROMOSOME No. OF HAPLOTYPES SHARED BETWEEN AFFECTED SISTERSa First-Degree Second-Degree Third-Degree FAMILY TYPE Relatives Relatives Relatives Breast-ovary... 16/19 11/11 7/7 Breast: Two to four cases under age 60 years... 5/9 3/8 2/ Five or more cases under age 60 years /7 2/ Total... 29/36 19/26 11/ a"disease" chromosome is unknown. under the age of 60 years that are inconsistent with linkage. Critical Recombinants for Mapping the Disease Locus The disease trait can be localized on chromosome 17q by following the inheritance of markers in relation to breast and ovarian cancers in recombinant families. Since none of the families with breast cancer alone shows convincing evidence of linkage, we have restricted attention to breast-ovary families where the posterior probability of linkage is high. Three families have been identified which contain interesting recombinations proximal to the disease locus and two families contain recombinations on the distal side (see fig. 2). Recombinations Proximal to the Disease Locus Family The affected individuals in this family include a sibship of four women each diagnosed with either breast cancer or ovarian cancer. Three of the affected sisters inherited the same chromosome from their affected mother. The fourth (individual 305), who is an ovarian cancer case, is recombinant for D17S250. The precise location of the recombination is unclear, since it was not possible to reconstruct the complete haplotype of the mother; however, if this is a linked family, then the gene must be located distal to D17S250. Family 128.-In this family, linkage to chromosome 17q has been tested by typing five affected sisters. As noted above, individual 301 does not share the maternal 17q haplotype (presumed to be the disease-bearing chromosome) with her affected sisters. In light of the fact that 301 is affected with breast cancer at a young age (35 years) and is therefore unlikely to be sporadic, this suggests that family 128 may be unlinked. If the family were linked, then individual 307 would provide a recombinant placing BRCA1 distal to D17S250. Family This family contains six affected individuals, including one ovarian cancer case. The haplotypes indicate that the disease in this family is linked to chromosome 17q. A breast cancer case (individual 300) diagnosed at age 72 years, however, is recombinant for each of the markers above D17S588. If this case is due to the inheritance of a gene on chromosome 17q, then the disease locus must be distal to D17S293. However, the old age at diagnosis suggests that the individual concerned may be a phenocopy and that conclusions drawn from this recombinant should be treated with extreme caution. Recombinations Distal to the Disease Locus Family 925.-In this breast-ovarian cancer family, a recombinant in individual 2, one of three young affected sibs, suggests that the disease gene is proximal to D17S588. Family 871.-The cancer in this family extends over three generations, with five affected women typed. The haplotypes demonstrate that, while the disease in this family is linked in the interval D17S250-D17S293, individual 413, an ovarian cancer case, is recombinant with each of the markers below D17S293. This family adds to the evidence that the disease locus is proximal to D17S588. These recombinant families therefore suggest that

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10 776 Smith et al. the locus which predisposes to breast and ovarian cancer is located in the genetic interval between D17S250 and D17S588. However, it is not clear whether the disease is proximal or distal to D17S293. Discussion Our results provide further strong evidence that a high proportion of breast-ovarian cancer families are the result of a disease gene on chromosome 17ql2-q21, confirming the results of Hall et al. (1990) and Narod et al. (1991). Of the 12 breast-ovarian cancer families, only 3 provided any evidence against linkage. The most convincing of these was family 128, where a 35-year-old breast cancer case did not share the putative diseasebearing chromosome with her affected sisters. Two other ovarian cancer cases, in families 845 and 871, did not appear to share the disease chromosome, but these were fourth-degree s of other affected cases. Key recombinants in two breast-ovarian cancer families suggest a location for the disease gene proximal to D17S588, while one other recombinant indicates the locus to be distal to D17S250. These recombinants may be helpful in further localizing the disease locus by using other markers. In contrast to the results of Hall et al. (1990), we find little or no evidence of linkage in breast cancer families which are not also segregating ovarian cancer. Hall et al. (1990) suggested that genetic heterogeneity was explicable in terms of age at onset; all families with mean age at onset under 46 years were linked in their study. This does not appear to explain our results; for families falling in this category, the lod score with D17S579 was at 0 =.05, although the six families with average age under 40 years did show some evidence of linkage. It should be noted that the majority of families in our study were ly small. There was some suggestion of linkage among families with at least six breast cancer cases. A critical exception in our set was family 114, which contained seven cases diagnosed at age 40 years or under. The evidence against linkage in this family derives primarily from two affected individuals not sharing the putative disease haplotype. These individuals were, however, the oldest affected individuals in the family (ages 61 and 63 years at diagnosis) and are thus candidates for sporadic cases. The smaller families all contain at least three cases of breast cancer and thus (on the basis of epidemiological studies) are unlikely to be chance aggregations of sporadic cases. Our results suggest, therefore, that other genes contribute significantly to breast cancer susceptibility and may be mappable by linkage studies. Acknowledgments This work was funded by the Cancer Research Campaign. We are very grateful to the many individuals who have taken part in this study. We also thank the following people who collected blood samples and allowed us access to families: Dian Donnai, D. G. R. Evans, G. Fraser, J. O'D. McGee, I. Scott, P. Hendy-Ibbs, J. M. Andrieu, E. Steiner, T. Powles, A. McKinna, I. Smith, D. F. Hayes, J. M. Russell, R. Levi, M. Adams, A. J. Rostrom, J. Ford, B. Alhejazi, J.-C. Gazet, P. Trowbridge, A. Evans, R. de Mars, and R. Love. We thank Rosemary Booth for her work in documenting these families; Jean Miller for preparing the manuscript; P. Harrington, L. Giddings, and J. Mangion for technical assistance; and Dr. I. Jacobs and Dr. E. Steichen-Gersdorf for useful discussion. The collection of U.K. families was initiated by a publicity campaign organized by Elizabeth Skinner at the Cancer Research Campaign. References Easton DF, Bishop DT, Ford D, Crockford GP, the Breast Cancer Linkage Consortium (1993) Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families. Am J Hum Genet 52: Hall JM, Friedman L, Guenther C, Lee MK, Weber JL, Black DM, King M-C (1992) Closing in on a breast cancer gene on chromosome 17q. Am J Hum Genet 50: Hall JM, Lee MK, Newman B, Morrow J, Anderson L, Huey B, King M-C (1990) Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250: Nakamura Y, Lathrop M, O'Connell P, Leppert M, Barker D, Wright E, Skolnick M, et al (1988) A map set of DNA markers for human chromosome 17. Genomics 2: Narod SA, Feunteun J, Lynch HT, Watson P, Conway T, Lynch J, Lenoir GM (1991) Familial breast-ovarian cancer locus on chromosome 17q Lancet 338:82-83 Ott J (1985) Analysis of human genetic linkage. Johns Hopkins University Press, Baltimore Polymeropolous MH, Rath DS, Xiao H, Merril CR (1991) A simple sequence repeat polymorphism at the human growth hormone locus. Nucleic Acids Res 19:689 Smith CAB (1961) Homogeneity test for linkage data. Proc Soc Int Congress Hum Genet 1: Weber JL, Kwitek AE, May PE, Wallace MR, Collins FS, Ledbetter DH (1990) Dinucleotide repeat polymorphisms at the D17S250 and D17S261 loci. Nucleic Acids Res 18:4640