GENES, CHROMOSOMES & CANCER 10:71-76 (1994)
`
`Localisation of the Breast-Ovarian Cancer Susceptibility
`Gene (BRCAI) on 17q 12-2 I to an Interval of 5 I cM
`
`Simon A. Smith, Richard A. DiCioccio, Jeffery P. Struewing, Douglas F. Easton, Holly H. Gallion. Hans Albertsen,
`Sylvie Mazoyer, Bertil Johansson, Elisabeth Steichen-Gersdorf, Mike Stratton, Debbie Ford, Gill Marshall,
`Raymond L. White, M. Steven Piver, and Bruce A. J. Ponder
`CRC Human Cancer Genetics Research Group, Department of Pathology, University of Cambridge, Tennis Court Rd, Cambridge
`CB2 I QP. UK (S.A.S.. H.H.G.. S.M., B.J.. E.5-G., B.A.J.P.); Roswell Park Cancer Institute, Department of Gynecologic Oncology,
`Buffalo, New York 14263, USA (R.A.D., M.S.P.); National Institutes of Health, Genetic Epidemiology Branch, Bethesda, Maryland
`20892, USA (J.P.S.); Sections of Epidemiology and Molecular Carcinogenesis, Institute of Cancer Research, Belrnont, Sutton. Surrey
`SM2 SNG, UK (D.F.E., M.S., D.F., G.M.); Eccles Institute of Human Genetics, Howard Hughes Medical Institute & University of Utah,
`Salt Lake City, Utah 84 I 12, USA (H.A., RL.W.)
`
`A breast-ovarian cancer susceptibility gene, BRCAI, which is responsible for disease in approximately 45% of breast cancer
`families and most families that contain breast and ovarian cancer, has been assigned by genetic linkage to 17q I 2-2 I. Here, we
`report the analysis of three marker-disease recombinants in families that contain breast and ovarian cancer, two of which
`strongly suggest a location for BRCAI telomeric to D17S702, a microsatellite polymorphism, and a third which suggests a
`location centromeric to EDHl78. the gene encoding estradiol- 17B dehydrogenase. If the interpretation of these recombinants
`is correct, the results localise BRCAI to an interval of 5 IcM. Genes Chrorn Cancer 10:71-76 (1994).
`0 1994 Wiley-Liss. Inc.
`A susceptibility locus for breast cancer has been
`mapped by genetic linkage to the long arm of chro-
`mosome 17, in the interval 17q12-21 (Hall et al.,
`1990). Predisposition at this locus, named BRCAl,
`is responsible for the disease in approximately 45%
`of families that contain multiple cases of breast
`cancer only and most families that contain breast
`cancer and at least one case of epithelial ovarian
`cancer (Easton et al., 1993). T h e cloning of BRCAl
`will help identify those individuals who are at risk
`of cancer through the inheritance of predisposing
`mutations and is the first step towards elucidating
`the role of this gene in the pathogenesis of breast
`and ovarian cancer. T h e BRCAl gene has already
`been mapped to a region of approximately 2cM
`defined proximally by D17S857 and distally by
`D17S78 (Anderson et al., 1993; Kelsell et al., 1993;
`Simard et al., 1993). Here, we report the further
`genetic mapping of BRCAl, based upon recombi-
`nation events in three breast-ovarian cancer fami-
`lies.
`Fifteen microsatellite polymorphisms (Table 1)
`spanning 16cM were typed by PCR using standard
`techniques (Smith et al., 1993). T h e genetic order
`of the markers is as described by Anderson et al.
`(1993). In addition, D17S702 has been mapped on
`a YAC contig of the region to lie between D17S800
`and D17S846 (S. Mazoyer et al., unpublished re-
`sults). Lod scores were calculated assuming an au-
`tosomal dominant disease gene with a risk to car-
`riers of 0.8 by age 80 years and a risk of 0.08 in
`
`non-carriers as previously described (Easton et al.,
`1993).
`T h e families and the most likely haplotypes con-
`structed by typing markers in the region D17S250-
`NMEZ are shown in Figures 1-3. In family 8101
`(Figure l), five of the six affected individuals for
`whom we have typings share a common haplotype.
`T h e sixth case, who was diagnosed with ovarian
`cancer aged 41 years, appears to inherit a recombi-
`nant chromosome from her affected mother that
`contains the “linked” haplotype distal to D17S702,
`suggesting that the disease locus, BRCAl, is lo-
`cated telomeric to D17S702. A recombinant in an-
`other ovarian cancer case, diagnosed at 51 years in
`a second family (family 801, Figure Z), also sug-
`gests that BRCAl is located telomeric to D17S702.
`T h e third family, family 64 (Figure 3), contains a
`recombination in an individual diagnosed with
`breast cancer at age 34 years, which suggests that
`BRCAl is located centromeric to EDHl 7B, the
`gene encoding estradiol-17B dehydrogenase. T h e
`polymorphism in EDHZ7B which is recombinant in
`family 64, HSD-DEL, occurs in the 5‘ untrans-
`
`Received December 7, 1993, accepted January 12, 1994.
`Address reprints to, Dr. S. A. Smith, CRC Human Cancer Ge-
`netics Research Group, Department of Pathology, University of
`Cambridge, Tennis Court Rd, Cambridge CB2 IQP, UK.
`Present addresses: Department of Obstetrics and Gynecology,
`University of Kentucky, Lexington, Kentucky 40536, USA
`(H.H.G. ); Univ. Kinderklinik, Anichstrasse 35, A-6020 Innsbruck,
`Austria ( E S G . ) .
`
`0 1994 Wiley-Us. Inc.
`
`GeneDX 1033, pg. 1
`
`

`

`72
`
`Locus
`
`Name
`
`D I75250
`
`Mfd 15
`
`D I 7S857
`
`OF I
`
`D17S800
`
`2OOzf4
`
`D I75702
`
`UT394
`
`D I 7S846
`
`UM8
`
`GAS
`
`D I75856
`
`OF2
`
`EDHI76
`
`HSD-DEL
`
`EDHI76
`
`HSD-A3T
`
`D I75855
`
`248yg9
`
`D I75858
`
`OF3
`
`DI 751 83
`
`scG43
`
`D I75579
`
`Mfd 188
`
`D I75588
`
`42D6
`
`NMEl
`
`Nm 23
`
`SMITH ET AL.
`Table I. Polymorphic Genetic Markers on 17q 12-2 I *
`Primer sequence
`TEMP
`
`GGAAGAATCAAATAGACAAT
`GCT GGC CAT ATA TAT ATT TAA ACC
`TIT GTC TGC AAA CAT GGA GG
`TCA GCT GAA GAG AAA AAT GGC
`GGT CTC ATC CAT CAG G l T TT
`ATA GAC TGT GTA CTG GGC ATT GA
`AGC AAC ACA TAT CAG GGG C
`TGT AGG l T G ACC TTA AGG C
`TGCATACCTGTACTACTTCAG
`TCC TIT G l T GCA GAT l T C TTC
`CAGAGAGGATGGGGGAGAAG (GAS I)
`GATGTACATAAAGCGCCCTG (GAS2)
`TGA GC(C/T)(G/A)(AIT)GAT(C/T)(GlA)
`(C/T(G/A)CCA(C/T)TGCACTCCAGCCTG
`GG (ALU-CON)
`AAG GCA AGA CTT CGT CGA GA
`CAT TCC CTG GTC CTG TGC
`GTGACCCACGAAACACAGG
`CAGAAGGTGAAGAACTCATCCA
`CAG TAC TAA AGG CCC TAT TAT CAA A
`AGG CTG CAGTGA GTC CAG AT
`GGA TGG CCT TIT AGA AAG TGG
`ACACAGACTTGTCCTACTGCC
`GCACTCTGACTAGAATCTGGGG
`TCCAAGTGGGAATGAGTGC
`ACAAACTGATGTGGGCTCTAG
`GTACATAGCATGGGTGCAGCT
`AGTCCTGTAGACAAAACCTG
`CAG TIT CAT ACC AAG TTC CT
`CCTGGTCTAGGAAGAGTGTCA
`GTGTAAGCATCTGTGTATACTAC
`TTGACCGGGGTAGAGAACTC
`TCTCAGTACTTCCCGTGACC
`
`55°C
`
`55°C
`
`55°C
`
`52°C
`
`58°C
`
`65°C
`
`55°C
`
`55°C
`
`55°C
`
`55°C
`
`56°C
`
`55°C
`
`55°C
`
`55°C
`
`55°C
`
`55°C
`
`References
`
`Weber et al., 1990
`
`Anderson et al., 1993
`
`Weissenbach et al.,
`
`I992
`
`Albertsen et al., in press
`
`Flejter et al., 1993
`
`Epstein et al.. 1990
`
`Anderson et al., 1993
`
`Friedman et al., 1993
`
`Friedman et al., 1993
`
`Weissenbach et al..
`
`I992
`
`Anderson et al., 1993
`
`Black et al.. 1993
`
`Hall et al.. 1992
`
`Easton et al., 1993
`
`Easton et al., 1993
`
`*The polymorphitm are ordered as they appear on the chromosome from DI 75250 (centromeric) to NMEl (telomeric). TEMP indicates the optimum
`annealing temperature of the primers during PCR amplification
`
`GeneDX 1033, pg. 2
`
`

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`
`

`

`74
`
`201 L b
`
`dx52
`3
`
`3
`3
`5
`
`1
`
`3
`
`307
`
`BILAT dx46,50
`
`I -
`1;
`1:
`
`SMITH ET AL.
`D17S800 has been unresolved (Anderson et al.,
`1993). A recombination on a non-disease chromo-
`some in individual 413 of family 8101 (Figure l),
`indicates
`that D17S857 maps centromeric to
`D17S800. Having mapped D17S800 and D17S702
`on a YAC contig of the region (S. Mazoyer et al.,
`unpublished results) we have determined the order
`of these markers to be cen-Dl7S857-Dl7SSOO-
`D17S702-qter, which, in conjunction with the re-
`combinants in families 801 and 8101, defines
`D17S702 as the new proximal boundary for the
`region containing BRCAI.
`The recombinant in family 64 suggests a new
`distal boundary for the BRCAZ region, defined by
`the locus for EDHI7B. T h e interpretation of this
`result depends upon the evidence firstly, that fam-
`ily 64 is linked to BRCAI and secondly, that the
`recombinant individual is not a sporadic case. The
`formal evidence for linkage to 17q from the lod
`scores is not very strong (multipoint lod score
`0.12). However, the presence in this family of an
`
`
`ovarian cancer case indicates a prior probability of
`
`linkage of at least 80% (Easton et al., 1993) and the
`posterior probability of linkage is thus at least 84%.
`An allele loss for the marker D17S2.50, in the tu-
`mour of individual 500, provides further evidence
`that the family is linked to BRCAZ, because it af-
`fects the wild-type chromosome (Figure 4). In pre-
`vious studies of tumours from 17q-linked families,
`each of sixteen allele losses that were detected af-
`fected the wild-type chromosome, consistent with
`BRCAl being a tumour suppressor gene (Smith et
`al., 1992; Kelsell et al., 1993). Finally, the young
`age of breast cancer diagnosis in the recombinant
`individual in family 64, at 34 years, argues against
`it being a phenocopy. Thus, although not conclu-
`sive, the evidence favours the interpretation that
`BRCAl lies proximal to EDHl7B and distal to
`D17S702. This is an interval of 1cM or less, and is
`a significant narrowing of the region over that re-
`ported previously (Bowcock et al., 1993; Simard et
`al., 1993; Kelsell et al., 1993).
`
`309
`
`
`
`
`
`
`
`
`-
`3
`3
`
`-
`2
`2
`1
`3
`
`1
`
`1
`
`1
`1
`3
`
`2
`2
`1 -
`6
`-
`
`2
`
`-
`
`3
`
`3
`3
`
`2
`1
`3
`
`1
`
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`
`1 1 3
`
`m 7 s m
`~ 7 5 8 5 7
`m7sm
`~ 7 5 7 0 2 I
`
`~ 7 ~ 8 4 6
`GAS
`~ 7 5 8 5 6
`HSD-DEL
`HSD3AT
`m 7 s w
`~ 7 5 8 5 8
`m 7 s m
`r n 7 s 5 n
`m 7 s m
`NMM
`
`1
`2
`
`2
`1
`
`
`
`
`-
`2 1
`
`-
`2
`
`
`
`
`Figure L Pedigree of family 80 I. Symbols and legend as for Figure I ,
`except that -, indicates alleles not amplified by PCR from archival
`Pa*w
`rampler
`
`lated region of the gene (Friedman et al., 1993).
`However, since the orientation of EDHl7B on the
`chromosome is not known, EDHl7B cannot be ex-
`cluded with certainty as the BRCAI gene.
`Thus, we report two observations, both in
`breast-ovarian cancer families, which
`identify
`D17S702 as the closest proximal marker which
`flanks BRCAI. Since each of these two families
`contain ovarian as well as breast cancer, they have
`a high prior probability of being linked to BRCAZ
`(Easton et al., 1993), and the recombinant individ-
`uals, being ovarian cancer cases, are unlikely to be
`phenocopies. The closest previously reported
`proximal marker is stated to be D17S857 (Kelsell et
`al., 1993), based upon an observation in a breast
`cancer case in which both D17S857 and D17S800
`were recombinant with BRCAI. However, the rel-
`ative order of the two markers D17S857 and
`
`NOTE ADDED IN PROOF
`Subsequent analysis of the offspring of individ-
`ual 309 in family 64 has indicated that the ovarian
`cancer case did not inherit the putative linked hap-
`lotype. This suggests that either the ovarian cancer
`is a sporadic case or that the family is not linked to
`17q12-21.
`
`ACKNOWLEDGMENTS
`This work was supported by the Cancer Re-
`search Campaign (SAS, DFE, MS, DF, GM &
`
`GeneDX 1033, pg. 4
`
`

`

`LOCAUSATION OF BRCAl ON 17q12-21 TO A REGION OF s l d .
`
`75
`
`303 Ja
`
`400
`
`2
`3
`3
`1
`4
`-
`3
`1
`2
`1
`2
`2
`2
`2
`2
`1
`
`3
`2
`1
`2
`4
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`2
`1
`1
`1
`1
`2
`1
`1
`3
`3
`
`
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`
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`
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`
`
`age 67
`
`2
`3
`
`1
`1
`1
`2
`1
`3
`1
`
`1
`2
`
`6
`
`6
`
`2
`3
`3
`1
`4
`3
`1
`1
`
`2
`2
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`1
`1
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`3
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`2
`1
`
`2
`2
`
`2
`1
`
`2
`1
`
`1
`2
`Figure 3. Pedigree of family 64. Symbols and legend as for Figure I.
`
`GeneDX 1033, pg. 5
`
`

`

`1.3
`
`293
`
`2-3
`
`23 13
`Figure 4. Analysis of loss of heterozygosity in the breast tumun of
`Family 64 using the marker D 175250. Alleles were amplified from con-
`stitutional DNA either from blood (6) or normal breast stroma (S), and
`from the wmwr (T) for the affected individuals. Alle1otype.s are indi-
`cated at the bottom of the figure and the arrow indicates the Iw of
`allele 2 in the wmwr of individual 500.
`
`SMITH ET AL.
`White R Genetic mapping of the BRCAl region on chromosome
`17q21. Am J Hum Genet (in press).
`Anderson LA, Friedman L. Osborne-Lawrence S, Lynch E, Weis-
`senbach J. Bowcock A, King M-C (1993) High density genetic
`map of the BRCAl region of chromosome 17q12-21. Genomics
`17:618-623.
`Black DM, Nicolai H, Borrow J, Solomon E (1993) A somatic cell
`hybrid map of the long arm of chromosome 17 containing the
`familial breast cancer locus (BRCAl). Am J Hum Genet 52702-
`710.
`Bowcock AM, Anderson LA, Friedman LS, Black DM, Oshorne-
`Lawrence SM, Rowell SE, Hall JM, Solomon E, King M-C
`(1993) THRAl and D17S183 flank a <4cM interval for the
`breast-ovarian cancer gene (BRCAl) on chromosome 17q12-21.
`Am J Hum Genet 52:718-722.
`Easton DF, Bishop DT, Ford D, Crockford GP (1993) Genetic
`linkage analysis in familial breast and ovarian cancer: Results from
`214 families. Am J Hum Genet 52678-701.
`Epstein N, Nahor 0, Silver J (1990) The 3’ ends of alu repeats are
`highly polymorphic. Nucleic Acids Res 184634.
`Flejter WL, Kukowska-Latallo JF, Kioussis S, Chandrasekharappa
`SC, King SE, Chamberlain JS (1993) Tetranucleotide repeat
`polymorphism at D17S846 maps within 40kb of GAS at 17q12-
`q22. Hum Mol Genet 2: 1080.
`Friedman LS, Lynch ED, King M-C (1993) Two independent poly-
`morphisms at the 17B-hydroxysteroid dehydrogenase (EDHl7B)
`gene (17q21). Hum Mol Genet 2:821.
`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 2501684-1689.
`Hall JM, Friedman L, Guenther C, Lee MK, Weber JL, Black DM,
`King M-C (1992) Closing in on a breast cancer gene on chromo-
`some 17q. Am J Hum Genet 50 1235-1242.
`Kelsell DP, Black DM, Bishop DT, Spun NK (1993) Genetic anal-
`ysis of the BRCAI region in a large breadovarian family: refine-
`ment of the minimal region containing BRCAl. Hum Mol Genet
`2: 1823-1828.
`Simard J, Feunteun J, Lenoir G, Tonin P, Normand T , The VL,
`Vivier A, Lasko D, Morgan K, Rouleau GA, Lynch H, Labrie F,
`Narod SA (1993) Genetic mapping of the breast-ovarian cancer
`syndrome to a small interval on chromosome I7qlZ-21: exclusion
`of candidate genes EDH17B2 and RARA. Hum Mol Genet
`2: 1193-1 199.
`Smith SA, Easton DF, Evans DGR, Ponder BAJ (1992) Allele losses
`in the region 17q12-21 in familial breast and ovarian cancer in-
`volve the wild-type chromosome. Nature Genet 2: 128-131.
`Smith SA, Easton DF, Ford D, Peto J, Anderson K, Stratton M,
`Ponder M, Pye C, Ponder BAJ (1993) Genetic heterogeneity and
`localisation of a familial breast-ovarian cancer gene on chromo-
`some 17q12-21. Am J Hum Genet 52767-776.
`Weber JL, Kwitek AE, May PE, Wallace MR, Collins FS, Ledhet-
`ter DH (1990) Dinucleotide repeat polymorphisms at the
`D17S250 and D17S261 loci. Nucleic Acids Res 184640.
`Weissenbach J, Gyapay G, Dib C, Vignal A, Morissette J, Millas-
`seau P, Vaysseix GA, Lathrop M (1992) A second generation
`linkage map of the human genome. Nature 359794-801.
`
`76
`400
`
`401
`
`404
`
`I
`
`I
`
`502
`
`500
`
`1
`
`B
`
`B
`
`S
`
`T
`
`B
`
`T
`
`B
`
`T
`
`
`
`BAJP) and by the Gilda Radner Familial Ovarian
`Cancer Registry (RAD & MSP). S. Mazoyer is sup-
`ported by an EC fellowship. We thank P. Har-
`rington and R. Gwilliam for technical assistance;
`and M.A. Ponder, C. Pye and M.A. Tucker for
`help in collecting the families. BAJP is a CRC
`Gibb fellow.
`
`REFERENCES
`Albertsen A, Plaetke R, Ballard L, Fujimoto E, Connolly J,
`Lawrence E, Rodriques P, Robertson M, Bradley P, Milner B,
`Fuhrman D, Marks A, Sargent R, Cartwright P, Matsunami N,
`
`GeneDX 1033, pg. 6
`
`