Am.]. Hum. Genet. 52:718-722, 1993
`
`THRA I and D 175 183 Flank an Interval of <4 eM for the
`Breast-Ovarian Cancer Gene (BRCAI) on Chromosome 17qll
`A. M. Bowcock,* L. A. Anderson, t L. S. Friedman, t D. M. Black,t S. Osborne-Lawrence,*
`S. E. Rowell,t j. M. Hall,t· 1 E. Solomon,t and M.-C Klngt
`
`*Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas; tDepartment of Molecular and Cell Biology and School of
`Public Health, University of California, Berkeley; and :j:lmperial Cancer Research Fund, London
`
`Summary
`
`In order to pinpoint the locale of the gene for early-onset familial breast and ovarian cancer (BRCA1),
`polymorphisms were developed within the locus for thyroid hormone receptor alpha (THRA1) and for several
`anonymous sequences at chromosome 17q12-q21. The THRA1 polymorphism is a dinucleotide repeat with 10
`alleles and heterozygosity .79. Gene mapping in extended families with inherited, early-onset breast and ovarian
`cancer indicates that BRCA1 is distal to THRA1 and proximal to D17S183 (SCG43), an interval of <4 eM.
`This locale excludes HER2, THRA1, WNT3, HOX2, NGFR, PHB, COUA1, NME1, and NME2 as candidates
`for BRCA1 but does not exclude RARA or EDH17B. Resolving the remaining recombination events in these
`families by new polymorphisms in the THRA1-D17S183 interval will facilitate positional cloning of the
`breast-ovarian cancer gene on chromosome 17q12-q21.
`
`Introduction
`
`Early-onset familial breast and ovarian cancer is linked
`to a gene, BRCA1 (MIM 113705), on chromosome
`17q12-q21 (Hall et al. 1990; Narod et al. 1991). Poly(cid:173)
`morphisms closely linked to BRCA1 in early-onset fami(cid:173)
`lies have facilitated more precise localization of the dis(cid:173)
`ease gene (Hallet al. 1992; Easton et al. 1993). In this
`report, we identify polymorphisms at the thyroid hor(cid:173)
`mone receptor alpha locus (THRA1) and in several
`(D17S183, D17S190, and
`anonymous sequences
`D17S181). Recombination events in families with in(cid:173)
`herited disease localize BRCA1 to the interval between
`THRA1 and D17S183, a distance of <4 eM.
`
`Material and Methods
`
`In order to localize BRCA1, polymorphic markers
`from chromosome 17q12-q21 were screened on fami-
`
`Received April10, 1992; final revision received December 7, 1992.
`Address for correspondence and reprints: Mary-Claire King, 140
`Warren Hall, University of California, Berkeley, CA 94720.
`1. Present address: Cell Pro, Inc., Bothell, W A.
`© 1993 by The American Society of Human Genetics. Ali rights reserved.
`0002-9297/93/5204-0008$02.00
`
`718
`
`lies in our series with early-onset breast and ovarian
`cancer. Since our most recent report (Hallet al. 1992),
`three more cases of breast cancer have appeared in fam(cid:173)
`ily 1, in individuals 11-3, III-5, and IV-2 (fig. 1). Family
`81, which has not been previously published, includes
`11 women who have developed breast cancer, one of
`whom (11-1) developed ovarian cancer as well (fig. 2).
`The average age at breast cancer diagnosis in family 81
`is 39.6 years.
`Table 1 indicates the polymorphic markers included
`in this analysis. Genomic DNA was prepared according
`to a method described elsewhere for lymphocyte cell
`lines (Hallet al. 1992). Paraffin-embedded tissue from
`pathology specimens was used as a source of both tu(cid:173)
`mor and normal DNA, which were separately obtained
`by dissecting tumor cells from adjacent normal cells in
`paraffin blocks. DNA from each cell type from paraf(cid:173)
`fin-embedded tissues was prepared according to a
`method described elsewhere (Wright and Manos 1990).
`Polymorphisms 0175250, HER2, 017578, D175579,
`GIP, and D17S293 were tested according to a method
`described elsewhere. Five other polymorphisms are
`new to this analysis: D17S518, THRA1, D17S183,
`D17S190, and D17S181. A polymorphic CA dinucleo(cid:173)
`tide repeat within THRA1 was developed from a
`
`GeneDX 1029, pg. 1
`
`

`

`Markers <4 eM Apart Flank BRCA1
`
`719
`
`Family 1
`
`Family 81
`
`D11S251
`D11S511
`HER2
`THRAI
`D11S71
`D11SIIS
`D11S511
`D11Sitl
`D11SIII
`GIP
`
`D11S251
`D11S511
`HER2
`THRAI
`D11S11
`D11SIIS
`D11S571
`D11SIII
`D11SIII
`GIP
`D11S2U
`
`D
`BE
`BA
`DG
`BB
`EE
`LE
`CA
`BH
`A
`A
`
`I
`
`•
`
`•
`
`IY
`
`Br31 Br" 43 Br24 Br 27
`
`34
`
`THRA1
`017571
`0175113
`0175571
`0175111
`
`I
`
`~; ~; i ~ ~l
`
`c
`F
`
`G CG
`F
`J
`
`IY
`
`Figure 2
`Linkage between breast cancer and chromosome
`17q21 markers in family 81. Notation is the same as in fig. 1. Geno-
`types of individuals in generation II were reconstructed from their
`surviving relatives. Chromosome phase for individual lll-14 is not
`determinable with certainty. Recombination between THRA1 and
`017578 in individual lll-6 suggests that the disease gene is distal to
`THRAl. Recombination between 0175183 and 0175579 in individ(cid:173)
`ual JJI-11 suggests that BRCA1 is proximal to 0175579. Recombina(cid:173)
`tion between 017578 and 0175183 in individual lll-7 further sug(cid:173)
`gests that BRCA1 is proximal to 0175183.
`
`Figure I
`Linkage between breast cancer (blackened circles;
`with age at diagnosis [dx] indicated) and markers on chromosome
`17q21 in family 1. Boxed haplotypes carry the BRCA1 disease allele.
`Breast cancers in 11-3, lll-5, and IV -2 have appeared since our last
`report of this family (Hall et al. 1992). Genotypes of 11-6, 11-7, and
`lll-7 were reconstructed completely or in part from their surviving
`relatives. 1-1 developed prostatic cancer at age 79 years; 11-8 devel(cid:173)
`oped ovarian cancer at age 48 years. A recombination event between
`THRA1 and 017578 in patient JJI-6, which was inherited by patient
`IV-2, suggests that the disease gene is distal to THRAl.
`
`and for typing on CEPH families, 33 ng total human
`genomic DNA was amplified by PCR. Reactions were
`performed in a 20-ml volume with 0.25 mM of each
`oligonucleotide primer, 0.5 units Taqi polymerase
`
`Table I
`
`Ordered Polymorphic Markers on Chromosome 17qll-qll
`Linked to BRCA I
`
`Gene/Locus
`
`Name
`
`Reference
`
`[CA]18 sequence beginning at bp 2004 in intron 9 of
`the human thyroid hormone receptor alpha locus
`(THRA1, or c-erbA-1) (Laudet et al. 1991). This se(cid:173)
`quence was screened with the program PRIMER (ver(cid:173)
`sion 0.5; E. Lander, M. Daly, and S. Lincoln, White(cid:173)
`head Institute, Boston), and oligonucleotides were
`selected to yield a PCR product of 143 bp. These were
`THRA-L, GGG CAA AAA TGT CTT AAG C; and
`THRA-R, CAG CAT AGC ATT GCC TTC.
`For analysis of THRA1 genotypes, two methods
`were used. For the development of the polymorphism
`
`0175250 .... .
`0175518 .... .
`HER2 ....... .
`
`THRA1 ..... .
`017578 ..... .
`0175183
`0175579
`0175190
`0175181
`GIP ......... .
`0175293 .... .
`
`Mfd15
`
`pl31
`B43, 5CG43
`Mfd188
`C54,5CG41
`5CG40
`
`6C1
`
`Weber et al. 1990
`Couch et al. 1991
`Hall and King 1991;
`Papewalis et al. 1991
`Present report
`Hallet al. 1990
`Black et al. 1993
`Hall et al. 1992
`Black et al. 1993
`Black et al. 1993
`Johnson et al. 1990
`Hall et al. 1992
`
`GeneDX 1029, pg. 2
`
`

`

`720
`
`Bowcock et al.
`
`(Promega), 0.25 mM spermidine, and 200 mM each of
`dA TP, dGTP, dTTP, and dCTP was added to the stan(cid:173)
`dard PCR buffer. One 5'-labeled oligonucleotide (104
`cpm) was included in the PCR reaction. Samples were
`overlaid with mineral oil (ONase and RNase-free;
`Sigma) and amplified for 30 cycles of 30 sat 94°C, 30 s
`at 55°C, and 1 min at 72°C, followed by 7 min at 7rc.
`Products were electrophoresed in 4.5% denaturing
`polyacrylamide gels containing 35% formamide. Sizes
`of alleles were determined by comparing the most in(cid:173)
`tense band, for each allele, with an M13 sequencing
`ladder. Allele frequencies were estimated from 210
`unrelated CEPH parents and grandparents. Alterna(cid:173)
`tively, and for typing the breast cancer families, we fol(cid:173)
`lowed the procedure described elsewhere (Hall et al.
`1992), with the addition of 2 mg BSA/ml to the ampli(cid:173)
`fication reaction.
`The smallest interval including BRCA1 was defined
`by identifying critical recombinants among breast and
`ovarian cancer patients in families with early-onset dis(cid:173)
`ease. Only genotypes obtained directly or recon(cid:173)
`structed unambiguously were considered.
`
`Results
`
`Allele sizes and frequencies for THRA1 are as fol(cid:173)
`lows: A = 157 bp (frequency .01); B = 155 bp (fre(cid:173)
`quency .01); C = 153 bp (frequency .08); 0 = 151 bp
`(frequency .13); E = 149 bp (frequency .04); F = 147 bp
`(frequency .31); G = 145 bp (frequency .08); H = 143
`bp (frequency .29); I = 141 bp (frequency .02); and J =
`135 bp (frequency .04).
`Figures 1, 2, and 3 indicate linkage of breast and
`ovarian cancer to this interval of chromosome 17 q 12-
`q21, for families 1, 81, and 4, respectively. In family 1,
`recombination between THRA1 and 017S78 in breast
`cancer patient III-6 suggests that BRCA1 is distal to
`THRA1 (fig. 1). This suggestion was strengthened when
`patient IV -2, who inherited the recombinant haplotype
`from her mother, was diagnosed with breast cancer at
`age 27 years this year (as the consequence of going to
`her physician to have blood drawn for this study). How(cid:173)
`ever, breast cancer diagnosed at age 71 years in individ(cid:173)
`ual II-3 and at age 45 years in individualiii-5 does not
`appear linked to 17q, although the possibility of a dou(cid:173)
`ble recombination or gene conversion cannot be ex(cid:173)
`cluded.
`In family 4, breast cancer patient II-9, whose geno(cid:173)
`type was obtained from normal cells from paraffin-em(cid:173)
`bedded tissue, carries the alleles linked to susceptibility
`in the family (fig. 3). Also in family 4, recombination
`
`Figure 3
`Linkage between breast cancer and chromosome
`17q21 markers in family 4. Notation is the same as in figs. 1 and 2.
`III-10 had a prophylactic mastectomy (pm) at age 31 years, prior to
`the beginning of this study. Since our previous report on this family
`(Hallet al. 1990), DNA extraction from paraffin-embedded tissue has
`enabled us to type PCR-based markers for deceased individual 11-9.
`The genotype of 11-2 was reconstructed from her spouse and chil(cid:173)
`dren. Recombination between THRA1 and D17S579 in patient III-3
`suggests that the disease gene is distal to THRAl.
`
`between THRA1 and 017S579 in patient III-3 places
`BRCA1 distal to THRAl. 017S78 and 017S183 are
`uninformative in this family.
`In family 81, eight of the nine genotyped breast
`cancer patients carry the same alleles for the critical
`region of chromosome 17q12-q21 (fig. 2). Patient IV-2
`was typed from paraffin-embedded tissues; she carries
`the alleles linked to susceptibility in the family. Breast
`cancer in patient III-4 does not appear linked to
`BRCAl. This patient's breast cancer occurred at age 57
`years, the oldest age at diagnosis in the family. Three
`patients in family 81 have informative recombination
`events in the BRCA1 region. In patient III-11, recombi(cid:173)
`nation between 017S183 and 017S579 suggests that
`BRCA1 is proximal to 017S579. In patient III-7, recom(cid:173)
`bination between 017S78 and 017S183 suggests that
`BRCA1 is proximal to 017S183 as well. In patient III-5,
`recombination between THRA1 and 017S78 strength(cid:173)
`ens the conclusion, based on families 1 and 4, that
`BRCA1 is distal to THRAl. 017S190 is uninformative
`in family 81.
`Several other families in our series carry recombina(cid:173)
`tion events placing BRCA1 between HER2 and
`
`GeneDX 1029, pg. 3
`
`

`

`Markers <4 eM Apart Flank BRCA1
`
`721
`
`D17S293. However, the recombination events de(cid:173)
`scribed above represent all those we have detected be(cid:173)
`tween THRA1 and D17S579.
`
`markers THRA1, D17S579 (Mfd 188), and D17S181
`(SCG40) provide an informative and easily screened set
`of polymorphisms.
`
`Discussion
`
`Multiple recombination events reported by the
`Breast Cancer Linkage Consortium place BRCA1 in the
`interval between D17S250 and D17S588 (Easton et al.
`1993). However, as far as we know, the recombinants
`in families 1, 4, and 81 are the first to place BRCA1
`distal to THRA1 and proximal to D17S183. There are
`at least two other reports of convincing recombinants
`placing BRCA1 proximal to D17S579 (Chamberlain et
`al. 1993; Devilee et al. 1993).
`The recombination events placing BRCA1 distal to
`THRA1 in families 1 and 4, as well as those placing it
`proximal to D17S183 and D17S579 in family 81, ap(cid:173)
`peared in patients with young ages at diagnosis who are
`members of extended, early-onset, 17q-linked families.
`These patients are not likely to be sporadic cases. Breast
`cancer in patient III-5 in family 81 was diagnosed at age
`50 years, so it might be sporadic disease. However, pa(cid:173)
`tient III-5 represents the third THRA1 recombinant in
`this series and is therefore less critical.
`The distance between THRA1 and D17S183 is <4
`eM. This rough estimate is based on the 4.1-cM genetic
`distance between THRA1 and D17S509 (data from the
`Chromosome 17 Workshop, March 11-12, 1992), the
`absence of recombination between D17S509 and
`D17S579 in more than 30 informative CEPH families
`(CEPH, version 5; Anderson et al., in press), and recom(cid:173)
`bination between D17S183 and D17S579 in both
`CEPH and breast cancer families, placing D17S183
`;;::0.3 eM proximal to D17S579 (authors' unpublished
`data).
`This locale for BRCA1 excludes HER2, THRA1,
`WNT3, HOX2, NGFR, PHB, COLIA1, NM£1, and
`NME2 as candidates for BRCA1 (Lemons et al. 1990;
`Anderson et al., in press). On the other hand, RARA
`and EDH17B remain candidates in the linked interval.
`Clearly, the gene-mapping approach to identifying
`BRCA1 has not yet been exhausted. Within the
`THRA 1-D17S183 interval, four informative recombina(cid:173)
`tion events remain to be resolved in these three families
`alone. Additional polymorphisms in this interval will
`enable us to pinpoint the BRCA1locale more precisely
`by genetic mapping (Anderson et al., in press). Finally,
`as a means to test whether breast and/ or ovarian cancer
`is/are attributable to inherited alterations of BRCA1 in
`newly ascertained, multiply affected families, the three
`
`Acknowledgments
`
`This work was supported by NIH grant R01 CA27632 to
`M.-C.K. and A.M.B. We thank Katrina Swanson for technical
`assistance, and we thank Eric Lynch and Ming Lee for advice
`and discussion.
`
`References
`
`Anderson LA, Friedman LS, Osborne-Lawrence S, Lynch E,
`Weissenbach j, Bowcock AM, King MC. A high-density
`genetic map of the BRCA1 region of chromosome 17q12-
`q21. Genomics (in press)
`Black DM, Nicolai H, Borrow J, Solomon E (1993) A somatic
`cell hybrid map of the long arm of human chromosome 17,
`containing the familial breast cancer locus (BRCA1). Am J
`Hum Genet 52:702-710
`Chamberlain JS, Boehnke M, Frank TS, Kiousis S, Xu J, Guo
`S-W, Hauser ER, et al (1993) BRCA1 maps proximal to
`D17S579 on chromosome 17q21 by genetic analysis. Am J
`Hum Genet 52:792-798
`Couch Fj, McCarthyTV,GreggRG, Hogan K(1991) Dinucle(cid:173)
`otide repeat polymorphism at the D17S518 locus. Nucleic
`Acids Res 19:5093
`Devilee P, Cornelis RS, Bootsma A, Bardoel A, van Vliet M,
`van Leeuwen I, Cleton Fj, et al (1993) Linkage to markers
`for the chromosome region 17q12-q21 in 13 Dutch breast
`cancer kindreds. Am J Hum Genet 52:730-735
`Easton DF, Bishop DT, Ford D, Crockford GP, the Breast
`Cancer Linkage Consortium (1993) Genetic linkage analy(cid:173)
`sis in familial breast and ovarian cancer: results from 214
`families. Am J Hum Genet 52:678-701
`HalljM, 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 50:1235-1242
`Hall JM, King MC (1991) PCR detection of an Mbol poly(cid:173)
`morphism in the ERBB2 (HER2; NEU) gene on chromo(cid:173)
`some 17q11.2-q12. Nucleic Acids Res 19:2515
`Hall JM, Lee MK, Morrow j, Newman B, Morrow j, Ander(cid:173)
`son L, Huey B, et al (1990) Linkage of early-onset familial
`breast cancer to chromosome 17q21. Science 250:1684-
`1689
`johnson TL, Reus BE, Culpepper AL, Naylor SL, Leach Rj
`(1990) Detection of a length polymorphism for human GIP
`gene by polymerase chain reaction. Nucleic Acids Res
`19:4312
`Laudet V, Begue A, Henry-Duthoit C, joubel A, Martin P,
`Stehelin D, Saule S (1991) Genomic organization of the
`
`GeneDX 1029, pg. 4
`
`

`

`722
`
`Bowcock et al.
`
`human thyroid hormone receptor a (c-erbA-1) gene. Nu(cid:173)
`cleic Acids Res 19:1105-1112
`Lemons RS, Eilender D, Waldmann RA, Rebentisch M, Frej
`AK, Ledbetter DH, Willman C, et al (1990) Cloning and
`characterization of the t(15;17) translocation breakpoint
`region in acute promyelocytic leukemia. Genes Chromo(cid:173)
`som Cancer 2:79-87
`Narod SA, Feunteun J, Lynch HT, Watson P, Conway T,
`Lynch J, Lenoir GM (1991) Familial breast-ovarian cancer
`locus on chromosome 17q12-q23. Lancet 338:82-83
`Papewalis J, Nikitin A Y, Rajewsky MJ (1991) G to A poly-
`
`morphism at amino acid codon 655 of the human erbB-2/
`HER2 gene. Nucleic Acids Res 19:5452
`Weber JM, 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
`Wright DK, Manos MM (1990) Sample preparation from par(cid:173)
`tissues. In: Innis MA, Gelfand DH,
`affin-embedded
`Sninsky JJ, White TJ (eds) PCR protocols: a guide to meth(cid:173)
`ods and applications. Academic Press, San Diego, pp 153-
`158
`
`GeneDX 1029, pg. 5
`
`