(GSK3-a), was the site of phosphorylation in each phospho(cid:173)
`peptide, both in vitro (Fig. 4b) and in vivo (not shown). The
`32P-labelling of other (more acidic) tryptic phosphopeptides was
`not increased by insulin (Fig. 4d). These peptides have been
`noted previously in GSK3 from A431 cells and shown to contain
`phosphoserine and phosphotyrosine 11 •
`PKC-8. £ and s are reported to be activated by mitogens. and
`PKC-s activity is stimulated in vitro by several inositol phospho(cid:173)
`lipids, including PI(3,4,5)P3 , the product of the PI 3-kinase
`reaction 26 . However, purified PKC-£27, PKC-8 and PKC-s (data
`not shown) all
`failed
`to
`inhibit GSK3-a or GSK3-~
`in vitro. Moreover, although PKC-a, ~I and y inhibit GSK3-~
`in vitro 27 , GSK3-a is unaffected, while their downregulation in
`L6 myotubes by prolonged incubation with phorbol esters abol(cid:173)
`ishes the activation of MAPKAP kinase-! in response to subse(cid:173)
`quent challenge with phorbol esters, but has no effect on the
`inhibition of GSK3 by insulin (not shown).
`Taken together, our results identify GSK3 as the first physio(cid:173)
`logically relevant substrate for PKB. The stimulation of glycogen
`synthesis by insulin in skeletal muscle involves the dephosphory(cid:173)
`lation of Ser residues in glycogen synthase that are phosphoryla(cid:173)
`ted by GSK3 in vitro 2 • Hence the 40-501/"o inhibition of GSK3 by
`insulin, coupled with a similar activation of the relevant glycogen
`synthase phosphatase 28 , can account for the stimulation of gly(cid:173)
`cogen synthase by insulin in skeletal muscle2 or L6 myotubes 29 .
`The activation of glycogen synthase and the resulting stimulation
`of glycogen synthesis by insulin in L6 myotubes is blocked by
`wortmannin, but not by PD 98059 (ref. 29), just like the activa(cid:173)
`tion ofPKB and inhibition ofGSK3. However, GSK3 is unlikely
`to be the only substrate of PKB in vivo, and identifying other
`physiologically relevant substrates will be important because
`PKB-~ is amplified and overexpressed
`in many ovarian
`0
`neoplasms 23 .
`
`Received 4 August; accepted 14 November 1995.
`
`1. Embi, N .. Rylatt, D. B. & Cohen P. fur. J. Biochem. 107, 519-527 (1980).
`2. Parker. P. J. J., Caudwell, F. B. & Cohen, P. fur. J. Biochem. 130, 227-234 (1983).
`3. Welsh, G. I. & Proud, C. G. Biochem. J. 294, 625-629 (1993).
`4. N1kolakaki, E., Coffer, P., Hemelsoet, R., Woodgett, J. & Defize, L. Oncogene 8, 833-840
`(1993).
`5. de Groot, R., Auwerx, J., Bourouis, M. & Sassone-Corsi, P. Oncogene 8, 841-847 (1993).
`6. Fiol, C. eta/. J. bioi. Chem. 269, 32187-32193 (1994).
`7. Siegfried, E., Chou, T.-B. & Perrimon, N. Cell 71, 1167-1179 (1992).
`8. He, X., Saint-Jeannet. J.·P., Woodgett, J. R., Varmus, H. E. & Dawid, I. B. Nature 374,617-
`622 (1995).
`9. Hughes, K., Ramakrishna, S., Benjamin, W. B. & Woodgett, J. R. Biochem. J. 288, 309-
`314 (1992).
`10. Cross, D. A. E. et a/. Biochem. J. 303, 21-26 (1994).
`11. Saito, Y., Vandenheede, J. R. & Cohen, P. Biochem. J. 303, 27-31 (1994).
`12. Sutherland, C., Leighton, I. A. & Cohen, P. Biochem. J. 296, 15-19 (1993).
`13. Sutherland, C. & Cohen. P. FfBS Lett. 338, 37-42 (1994).
`14. Welsh, G. 1., Foulstone, E. J., Young, S. W., Tavare, J. M. & Proud, C. G. Biochem. J. 303,
`15-20 (1994).
`15. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. & Sa/tiel, A. L. J. bioi. Chem. 270, 27489-
`27494 (1995).
`16. Kuo, C. J. et al. Nature 358, 70-73 (1992).
`17. Stambolic, V. & Woodgett, J. R. Biochem. J. 303, 701-704 (1994).
`18. Eldar-Finkelman, H., Seger, R., Vandenheede, J. R. & Krebs, E. G. J. bioi. Chem. 270, 987-
`990 (1995)
`19. Vlahos, C. J., Matter. W. F., Hui, K. Y. & Brown, R. F. J. bioi. Chem. 269, 5241-5248 (1994).
`20. Coffer, P. J. & Woodgett, J. R. Eur. J. Biochem. 201, 4 75-481 (1991).
`21. Jones, P. F., Jakubowicz, T., Pitossi, F. J., Maurer, F. & Hemmings, B. A. Proc. natn. Acad.
`Sci. U.S.A. 88, 4171-4175 (1991).
`22. Ahmed, N. N. et a/. Malec. cell. Bioi. 15, 2304-2310 (1995).
`23. Cheng, J. Q. eta/. Proc. natn. Acad. Sci. U.S.A. 89, 9267-9271 (1992).
`24. Franke, T. F. et al. Cell 81, 727-736 (1995).
`25. 8urgering, B. M. Th. & Coffer, P. J. Nature 376, 599-€02 (1995).
`26. Palmer, R. H. eta/. J. bioi. Chem. 270, 22412-22416 (1995).
`27. Goode, N., Hughes, K .. Woodgett, J. R. & Parker, P. J. J. J. bioi. Chem. 267, 16878-16882
`(1992).
`28. Dent, P. eta/. Nature 348, 302-308 (1990).
`29. Lazar, D. F. et al. J. bioi. Chem. 270, 20801-20807 (1995).
`
`ACKNOWLEDGEMENTS. We thank A. Klip for providing L6 cells, and H. McDowell for advice
`and help 1n growing them; A. Salt1el for PD 98059; J. Vandenheede for anti-GSK3 antibodies;
`C. Marshall for anti~p42 MAP kinase antibodies; P. Parker for PKC-8 and ~; N. Morrice for
`providing GSK3; B. Caudwell for peptide sequencing; M. Frech for the PH domain antibodies;
`and J. Thorner for critical reading of the manuscript. D.A.E.C. is the recipient of a CASE stu(cid:173)
`dentship from the U.K. Biotechnology and Biological Sciences Research Council and SmithKiine
`Beecham Pharmaceuticals. This work was supported by the British Diabetic Association. the
`U.K. Medical Research Council, and The Royal Society.
`
`NATURE · VOL 378 · 21/28 DECEMBER 1995
`
`LETTERS TO NATURE
`
`Identification of the
`breast cancer
`susceptibility gene BRCA2
`Richard Wooster*, Graham Bignell*,
`Jonathan Lancastert, Sally Swiftt, Sheila Seal*,
`Jonathan Mangion*, Nadine Collins*, Simon Gregory§,
`Curtis Gumbs!!, Gos Micklem§, Rita Barfoot*,
`Rifat Hamoudi*, Sandeep Patel*, Catherine Rice§,
`Patrick Biggs*, Yasmin Hashim*, Amanda Smitht,
`Frances Connort, Adalgeir Arason•j,
`Julius Gudmundsson~~. David Ficenec~***
`David Kelsall#, Deborah Ford"', Patricia Tonln**,
`D. Timothy Bishoptt, Nigel K. Spurr#,
`Bruce A. J. PonderU, Rosalind Eeles*, Julian Peto"',
`Peter Devilee§§, Cees Cornelisse§§, Henry Lynch II II,
`Steven Narod***·", Gilbert Lenoir~!~!.
`Valdgardur Egilsson'], Rosa Bjork Barkadottir•j,
`Douglas F. Easton##, David R. Bentley§,
`P. Andrew Futrealjj, Alan Ashwortht
`& Michael R. Stratton*
`Sections of * Molecular Carcinogenesis and * Epidemiology, and
`t CRC Centre for Cell and Molecular Biology, Institute of Cancer
`Research, Haddow Laboratories, 15 Cotswold Road, Sutton,
`Surrey SM2 5NG, UK, and Chester Beatty Laboratories, Fulham Road,
`London SW3 6JB, UK
`t Laboratory of Molecular Carcinogenesis, National Institute of
`Environmental Health Sciences, National Institutes of Health,
`Research Triangle Park, North Carolina 27709, USA
`§The Sanger Centre, Hinxton Hall, Hinxton,
`Cambridgeshire CBlO lRQ, UK
`II Duke University Medical Centre, Departments of Surgery and
`Genetics, and Division of Gynaecologic Oncology, Research Drive,
`Medical Sciences Research Building, Room 363, Durham,
`North Carolina 27710, USA
`"I Laboratory of Cell Biology, University Hospital of Iceland,
`P.O. Box 1465, IS-121 Reykjavik, Iceland
`# ICRF Clare Hall Laboratories, Blanche Lane, South Mimms,
`Potters Bar EN6 3LD, UK
`** Division of Medical Genetics and Division of Human Genetics,
`Dept of Medicine, McGill University, 1650 Cedar Avenue, Montreal,
`H3G 1A4, Canada
`tt ICRF Genetic Epidemiology Laboratory, 3K Springfield House,
`Hyde Terrace, Leeds LS2 9LU, UK
`H CRC Human Cancer Genetics Research Group, Level 3,
`Laboratories Block, Box 238, Addenbrookes Hospital, Hills Road,
`Cambridge CB2 2QQ, UK
`§§ Department of Human Genetics and Pathology, Leiden University,
`Wassenaarseweg 72,
`P. 0. Box 9503, 2300 RA, Leiden, The Netherlands
`1111 Department of Preventive Medicine and Public Health, Creighton
`University School of Medicine, Omaha, Nebraska 68178, USA
`•'•[International Agency for Research on Cancer, 150 Cours Albert(cid:173)
`Thomas, 69372 Lyon Cedex 08, France
`## CRC Genetic Epidemiology Group, Department of Community
`Medicine, Institute of Public Health, University of Cambridge,
`University Forvie Site, Robinson Way, Cambridge CB2 2SR, UK
`Women's College Hospital, Toronto, Ontario, Canada
`***Genome Sequencing Centre, Washington University in St Louis,
`School of Medicine, St Louis, MO, USA
`
`IN Western Europe and the United States approximately 1 in 12
`women develop breast cancer. A small proportion of breast cancer
`cases, in particular those arising at a young age, are attributable
`to a highly penetrant, autosomal dominant predisposition to the
`disease. The breast cancer susceptibility gene, BRCA2, was
`recently localized to chromosome 13q12-q13. Here we report the
`identification of a gene in which we have detected six different
`germline mutations in breast cancer families that are likely to be
`due to BRCA2. Each mutation causes serious disruption to the
`open reading frame of the transcriptional unit. The results indicate
`that this is the BRCA2 gene.
`
`789
`
`© 1995 Nature Publishing Group
`
`GeneDX 1023, pg. 1
`
`

`

`LETTERS TO NATURE
`
`a
`
`b
`
`t t
`
`FIG. 1 Detection of the BRCA2 gene mutation in family \ARC 2932.
`Mutation screening by migration shift assays. The arrows indicate
`abnormally migrating bands in two early onset breast cancer cases from
`\ARC 2932.
`METHODS. A 32P-Iabelled, 271-bp genomic fragment was amplified from
`lymphocyte DNAs from affected individuals in 46 breast cancer families.
`The PCR product was denatured in 50% formamide and electrophor(cid:173)
`esed through a, 4.5% non-denaturing polyacrylamide gels and b, 6%
`denaturing polyacrylamide gels.
`
`Abnormalities of several genes are known to confer suscept(cid:173)
`ibility to breast cancer. The BRCA 1 gene accounts for the large
`majority of families with both breast and ovarian cancer cases,
`but only half of families with site-specific breast cancer 1• Using
`families with multiple cases of early-onset breast cancer showing
`evidence against linkage to BRCA I we recently demonstrated
`the existence of a second major breast cancer susceptibility locus,
`BRCA2, on chromosome 13q12-q13 (ref. 2). Preliminary studies
`indicate that mutations in BRCA2 confer a similar risk of female
`breast cancer to BRCA I. However, the risk of ovarian cancer
`appears to be lower and the risk of male breast cancer substan(cid:173)
`tially higher. Risks of other cancers, including prostate and
`laryngeal cancer, may also be elevated in carriers of BRCA2
`mutations (unpublished data).
`BRCA2 was originally positioned within a 6-cM region
`between DI3S289 and DI3S267 that was defined on the basis
`of meiotic recombinants in early-onset breast cancer cases within
`clearly linked families 2 • (The genetic map in this region is
`centromere- DI3S289- 3cm- DJ3S260- IcM - Dl3Sl71- 2cM (cid:173)
`Dl3S267- telomere3. ) We further mapped the centromeric boun(cid:173)
`dary of the interval within which the gene lies to D /3S260 using a
`set of Icelandic families (unpublished data). Subsequently, using
`recombinants in other families and additional microsatellite mar(cid:173)
`kers isolated from the region, we established that BRCA2 is
`likely to be located in a 600-kb interval centred around D IJS 171.
`An unexpected contribution to the fine localization of BRCA2
`was provided by the detection of a homozygous somatic deletion
`in a single pancreatic cancer4 • The centromeric boundary of this
`deletion is approximately 300 kb centromeric to DI3SJ71 and
`the telomeric boundary close to, but still centromeric of,
`D I3Sl7I (ref. 5) . Despite the ambiguity of the relationship
`between this deletion and BRCA2, we combined the genetic
`recombinant information from families and the physical localiza(cid:173)
`tion from the homozygous deletion, and prioritized analysis of
`the 300-kb region immediately centromeric to D13Sl7I.
`
`Yeast artificial chromosome (YAC) 6 and PI artificial chromo(cid:173)
`some (PAC) 7 contigs extending approximately 700 kb centro(cid:173)
`meric and 300 kb telomeric to Dl3Sl7I were constructed and a
`identified .
`minimally overlapping set of 14 PACs was
`Transcribed sequences located on these genomic contigs were
`identified using two methods : ex on amplification (ex on trap(cid:173)
`ping) from subcloned PAC DNA8 , and direct selection by solu(cid:173)
`tion hybridization of complementary DNA to PAC genomic
`DNA 9 . To identify BRCA2, genomic DNA fragments of less
`than 300 bp containing putative coding sequences were screened
`for mutations. At least one affected member of 46 breast cancer
`families was examined. Each family included in this set either
`shows evidence of linkage to BRCA2, and/ or shows evidence
`against linkage to BRCAI, and/ or has not been found to carry
`a BRCAI mutation , and/ or includes a case of male breast
`cancer. Most, but probably not all, of these families would be
`expected to have cases caused by BRCA2 mutations.
`Disease-associated mutations in most known cancer suscept(cid:173)
`ibility genes usually result in truncation of the encoded protein
`and inactivation of critical functions. In the course of the muta(cid:173)
`tional screen of candidate coding sequences from the BRCA2
`region, the first detected sequence variant that was predicted to
`disrupt translation of an encoded protein was observed in IARC
`2932 (Fig. I). This family is clearly linked to BRCA2 with a
`multipoint LOD score of 3.01 using DI3S260 and DI3S267. A
`deletion of 6 bp removes the last five bases of the ex on examined
`(exon S66), deletes the conserved G of the 5' splice site of the
`intron , and directly converts the codon TTT for phenylalanine
`to the termination codon T AA. By sequencing, this mutation
`has been detected in lymphocyte DNA from two other early(cid:173)
`onset breast cancer cases in this family. The individuals exam(cid:173)
`ined share only the disease-associated haplotype. The mutation
`is absent in more than 500 chromosomes from normal indi(cid:173)
`viduals and in the remaining families and cancers. This finding
`therefore identified a strong candidate for the BRCA2 gene.
`
`\ARC 2932
`\ARC 3594
`CRC 8211
`CRC 8196
`Montreal 681
`Montreal 440
`
`FBCs
`15
`6
`5
`17
`3
`2
`
`TABLE 1 BRCA2 mutations in breast cancer families
`LOD score
`at BRCA1
`- 2.38
`nd
`- 0.48
`- 2.21
`nd
`nd
`
`FBCs < 50
`10
`5
`3
`12
`2
`2
`
`OvCs
`0
`0
`4
`0
`0
`0
`
`MBCs
`0
`0
`0
`0
`1
`2
`
`LOD score
`at BRCA2
`3.01
`nd
`0.49
`0.92
`nd
`nd
`
`BRCA2 mutation
`CCC.TTT.CGgtaa
`CAT.AAC.TCT.CTA
`AGT.CTI.CAC
`AAA.ACT.GAA.ACT
`GCA.AGT.GGA
`GAT.AAACAA.GCA
`
`LOD scores at BRCA1 were calculated using the markers 0175250 and 0175579; those at BRCA2 were calculated using the markers 0135260
`and 0135267. Exon sequence is denoted by upper case, intron sequence by lower case; Codons are indicated by stops. The underlined letters
`indicate the deleted bases in each family. Abbreviations: FBCs, female breast cancers; OvCs, ovarian cancers, MBCs, male breast cancers.
`
`790
`
`NATURE · VOL 378 · 21/28 DECEMBER 1995
`
`© 1995 Nature Publishing Group
`
`GeneDX 1023, pg. 2
`
`

`

`FIG. 2 Predicted amino acid sequence of the BRCA2
`gene. The positions of the frameshift mutations indica(cid:173)
`ted in Table 1 are boxed, and the positions of intron(cid:173)
`exon boundaries are arrowed above the amino acid
`sequence.
`METHODS. Exon 566 and others that had been trapped
`in association with it were used to isolate segments of
`the candidate eDNA by hybridization to normal human
`fetal brain, placental, monocyte and breast cancer eDNA
`libraries. Additional fragments were isolated by PCR
`amplification from known exon sequences to vector
`ends. In the course of these analyses, other previously
`trapped exons and cDNAs selected by solution hybrid(cid:173)
`into an extended eDNA
`ization were
`incorporated
`sequence. In addition, the exon prediction program
`Genemark was used to define the location of adjacent
`candidate transcribed sequences from the genomic
`sequence. Putative intron-exon boundaries were con(cid:173)
`firmed by amplification from eDNA and direct sequenc(cid:173)
`ing of amplification products. Northern analysis
`indicates that the transcript from the BRCA2 gene is
`large (approximately 10-12 kb), and hence theN term(cid:173)
`inus of the BRCA2 protein may well be missing from the
`above sequence.
`
`LETTERS TO NATURE
`
`HIGKSMPNVLEDEVYETVVDTSEEDSFSLCFSKCRTKNLQKVRTSKTRKKIFHEANADEC
`
`60
`
`EKSKNQVKEKYSFVSEVEPNDTDPLDSNVANQKPFESGSDKISKEVVPSLACEWSQLTLS
`
`120
`
`GLNGAQMEKIPLLHISSCDQNISEKDLLDTENKRKKDFLTSENSLPRISSLPKSEKPLNE
`
`ETVVNKRDEEQHLESHTDCILAVKQAISGTSPVASSFQGIKKSIFRIRESPKETFNASFS
`
`GHMTDPNFKKETEASESGLEIHTVCSQKEDSLCPNLIDNGSWPATTTQNSVALKNAGLIS
`TLKKKTNKFIYAIHDETSYKGKKIPKDQKSELINCSAQFEANAFEAPLTFANAD~LLHS
`SVKRSCSQNDSEEPTLSLTSSFGTILRKCSRNETCSNNTVISQDLDYKEAKCNKEKLQLF
`
`180
`
`240
`
`300
`
`360
`
`420
`
`ITPEADSLSCLQEGQCENDPKSKKVSDIKEEVLAAACHPVQHSKVEYSDTDFQSQKSLLY
`
`480
`
`DHENASTLILTPTSKDVLSNLVMISRGKESYKMSDKLKGNNYESDVELTKNIPMEKNQDV
`
`540
`
`CALNENYKNVELLPPEKYMRVASPSRKVQFNQNTNLRVIQKNQEETTSISKITVNPDSEE
`600
`LFSDNENNFVFQVANERNNLALGNTKELHETDLTCVNEPIFKNSTMVLYGDTG~TQV 660
`
`SIKKDLVYVLAEENKNSVKQHIKMTLGQDLKSDISLNIDKIPEKNNDYMNKWAGLLGPIS
`
`720
`
`NHSFGGSFRTASNKEIKLSEHNIKKSKMFFKDIEEQYPTSLACVEIVNTLALDNQKKLSK
`
`780
`
`PQSINTVSAHLQSSVVVSDCKNSHITPQMLFSKQDFNSNHNLTPSQKEQITELSTILEDS
`
`840
`
`GSQFEFTQFRKPSYILQKSTFEVPENQMTILKTTSEECRDADLHVIMNAPSIGQVDSSKQ
`
`900
`
`FEGTVEIKRKFAGLLKNDCNKSASGYLTDENEVGFRGFYSAHGTKLNVSTEALQKAVKLF
`
`960
`
`SDIENISEETSAEVHPISLSSSKCHDSVVSMFKIENHNDKTVSEKNNKCQLILQNNIEMT 1020
`
`TGTFVEEITENYKRNTENEDNKYTAASRNSHNLEFDGSDSSKNDTVCIHKDETDLLFTDQ 1080
`
`HNICLKLSGQFMKEGNTQIKEDLSDLTFLEVAKAQEACHGNTSNKEQLTATKTEQNIKDF 1140
`
`ETSDTFFQTASGKNISVAKESFNKIVNFFDQKPEELHNFSLNSELHSDIRKNKMDILSYE 1200
`
`ETDIVKHKILKESVPVGTGNQLVTFQGQPERDEKIKEPTLLGFHT~GKKVKIAKESLDK 1260
`
`VKNLFDEKEQGTSEITSFSHQWAKTLKYREACKDLELACETIEITAAPKCKEMQNSLNND 1320
`
`KNLVSIETVVPPKLLSDNLCRQTENLKTSKSIFLKVKVHENVEKETAKSPATCYTNQSPY 1380
`
`SVIENSALAFYTSCSRKTSVSQTSLLEAKKWLREGIFDGQPERINTADYVGNYLYENNSN 1440
`
`STIAENDKNHLSEKQDTYLSNSSMSNSYSYHSDEVYNDSGYLSKNKLDSGIEPVLKNVED 1500
`
`QKNTSFSKVISNVKDANAYPQTVNEDICVEELVTSSSPCKNKNAAIKLSISNSNNFEVGP 1560
`
`PAFRIASGKIVCVSHETIKKVKDIFTDSFSKVIKENNENKSKICQTKIMAGCYEALDDSE 1620
`
`DIL~DNDECSTHSHKVFADIQSEEILQHNQNMSGLEKVSKISPCDVSLETSDICKCS 1680
`
`IGKLHKSVSSANTCGIFSTASGKSVQVSDASLQNARQVFSEIEDSTKQVFSKVLFKSNEH 1740
`
`SDQLTREENTAIRTPEHLISQKGFSYNVVNSSAFSGFSTASGKQVSILESSLHKVKGVLE 1800
`
`EFDLIRTEH~HYSPTSRQNVSKILPRVDKRNPEHCVNSEMEKTCSKEFKLSNNLNVEGG
`SSENNHSIKVSPYLSQFQQDKQQLVLGTKVSLVENIHVLGKEQASPKNVKMEIG~FS
`DVPVKTNIEVCSTYSKDSENYFETEAVEIAKAFMEDDELTDSKLPSHATHSLFT~ENEE
`MVLSNSRIGKRRGEPLILJtEPSIKRNLLNEFDRIIENQEKSLKASKSTP~TIKDRRLF
`VHHVSLEPITCV~TKERQEIQNPNFTAPGQEFLSKSHLYEHLTLEKSSSNLAVSGHP
`FYQVSGNKNGKMRKLITTGRPTKVFVPPFKTKSHFHRVEQCVRNINLEGNRQKQNIDGHG
`SDDSKNKINDNEIHQFNKNNSNQAAAVTFTKCEEEP~LITSLQNARDIQDMRIKKKQRQ
`RVFPQPGSLYLAKTSTLPRISLKAAVGGQVPS~SHKQLYTYGVSKHCIKINSKNAESFQ
`FHTEDYFGKESLWTGKGIQLADGGWLIPSNDGKAGKEEFYRILCDVKAT
`
`1860
`
`1920
`
`1980
`
`2040
`
`2100
`
`2160
`
`2220
`
`2280
`
`2329
`
`To characterize this gene further, exon S66 was used to isolate
`a series of eDNA clones which represented segments of the
`BRCA2 candidate (see Fig. 2 legend). At this stage the initial
`shotgun sequence data from a 900-kb region thought to contain
`BRCA2 was completed at the Sanger Centre and Washington
`University and became available to us through the public release
`of the assembled sequence (at ftp://ftp.sanger.ac.uk/pub/
`human/sequences/13q and ftp ://genomc.wustl.edu/pub/gscl I
`brca2 from 23 November 1995). From alignment of the eDNA
`and genomic sequence data, the candidate BRCA2 gene was
`found to lie in three sequence contigs which also contained other
`previously isolated transcribed sequences. The exon and open
`reading frame prediction program Genemark was used to define
`putative additional 5' exons of the gene. Contiguity of the
`transcription unit was confirmed by reverse-transcription-poly(cid:173)
`merase chain reaction (RT -PCR) on eDNA and sequence ana(cid:173)
`lysis. The availability of extensive sequence information at the
`eDNA and genomic level allowed mutational analysis of further
`coding regions of the putative BRCA2 gene in samples from
`breast cancer families.
`A TG deletion and a TT deletion were detected in families
`CRC 8196 and CRC 8211 respectively (Table I). In both famil-
`
`NATURE · VOL 378 · 21/28 DECEMBER 1995
`
`ies the mutation has been detected by sequencing other indi(cid:173)
`viduals with early onset breast cancer who share only the
`haplotype of 13q microsatellite markers that segregates with the
`disease. Therefore, the mutations are on the disease-associated
`chromosomes. ACT deletion was detected in family IARC 3594.
`This mutation has arisen within a short repetitive sequence
`(CTCTCT), a feature that is characteristic of deletion/insertion
`mutations in many genes, and which is presumed to be due to
`slippage during DNA synthesis. Finally, a T deletion and an
`AAAC deletion have been found in Montreal 681 and 440,
`respectively. Both these families include a male breast cancer
`case, and previous analyses have indicated that the large major(cid:173)
`ity of such families will have BRCA2 mutations 10. All these mut(cid:173)
`ations are predicted to generate frameshifts leading to premature
`termination codons. None of the mutations have been found in
`over 500 chromosomes from healthy women and are therefore
`unlikely to be polymorphisms. The identification of several
`different germline mutations that truncate the encoded protein in
`breast cancer families that are highly likely to be due to BRCA2
`strongly suggests that we have identified the BRCA2 gene.
`Northern analysis has demonstrated that BRCA2 is encoded
`by a transcript of I 0-12 kb (data not shown), which is present
`
`791
`
`© 1995 Nature Publishing Group
`
`GeneDX 1023, pg. 3
`
`

`

`LETTERS TO NATURE
`
`in normal breast epithelial cells, placenta and the breast cancer
`cell line MCF7. This suggests that our present contig of cDNAs
`covering approximately 7.3 kb (including 300 bp of 3' untrans(cid:173)
`lated sequence) may not include the whole BRCA2 coding
`sequence. The known sequence of2,329 amino acids encoded by
`the BRCA2 gene does not show strong homology to sequences in
`the publicly available DNA or protein databases, and therefore
`we have no clues to its functions. However, some weak matches
`were detected including, intriguingly, a very weak similarity to
`the BRCA I protein over a restricted region (amino acids 1394-
`1474 in BRCAJ, and I 783-1863 in the portion of BRCA2 shown
`in Fig. 2). The significance of this is unclear.
`Loss of heterozygosity on chromosome 13q has been observed
`in sporadic breast and other cancers, suggesting that there is a
`somatically mutated tumour suppressor gene in this region ''-' 3
`BRCA2 is a strong candidate for this gene, and the analysis of
`a large series of cancers is underway to investigate if BRCA2 is
`somatically mutated during oncogenesis.
`The identification of BRCA2 should now allow more compre(cid:173)
`hensive evaluation of families at high risk of developing breast
`cancer. However, the roles of environmental, lifestyle or genetic
`factors in modifying the risks of cancer in gene carriers are
`unknown, and further studies will be required before routine
`diagnosis of carrier status can be considered.
`D
`
`M. Ponder, H. Vasen, J. Feuntein, 0. Serova, R. Gwilliam, S. Humphray, M. Leversha, Y. Ramsey,
`H. King, D. Cain, D. Averill, P. Mitchell, M. Crompton, C. Cochran, J. Marks, D. Iglehart, R.
`Wiseman. J. Lancaster and the members of the Human Sequencing teams at the Sanger Centre
`and the Genome Sequencing Centre at Washington University for making their data publicly
`available; M. Schutte and S. Kern for discussions; and C. Marshall, C. Cooper and P. Garland for
`encouragement. This work was supported by the Cancer Research Campaign, BREAKTHROUGH
`Breast Cancer Charity and the Jean Rook Appeal, the Institute of Cancer Research, the Wel!come
`Trust, the Medical Research Council, the Imperial Cancer Research Fund, the U.S. Army, Duke
`University SPORE in Breast Cancer, the National Cancer Institute, the Icelandic Cancer Research
`Fund, the Nordic Cancer Union, the Ligue National contre !e Cancer, the Dutch Cancer Society,
`the Cancer Research Society, the Canadian Breast Cancer Foundation, the Fonds de !a recher
`che en Sante du Quebec, and the Canadian Genetic Diseases Network. Access to BRCA2
`sequences can be obtained from rich w@icr.ac.uk.
`
`RETRACTION
`
`Cloning and functional
`expression of a
`rat heart KATP channel
`M. L. J. Ashford, C. T. Bond, T. A. Blair
`& J. P. Adelman
`
`Nature 370, 456-459 (1994)
`
`Received 5 December; accepted 7 December 1995.
`
`1. Easton, D. F .. Bishop, D. T., Ford, D. & Crockford, G. P. Am. J. hum. Genet. 52, 678-701
`(1993).
`2. Wooster, R. eta/. Science 265, 2088-2090 (1994).
`3. Gyapay, G. eta/. Nature Genet. 7, 248-339 (1994).
`4. Schutte, M. et a/. Proc. natn. Acad. Sci. U.S.A. 92, 5950-5954 (1995).
`5. Schutte, M. eta/. Cancer Res. 55, 4570-4574 (1995).
`6. Cohen, D., Chumakov, I. & Weissenbach, J. Nature 366, 698-701 (1993).
`7. Ioannou, P. A. eta/. Nature Genet. 6, 84-89 (1994).
`8. Nehls, M., Pfeifer, D. & Boehm, T. Oncogene 9, 2169-2175 (1994).
`9. Tagle, D. A., Swaroop, M., Lovett, M. & Collins, F. S. Nature 361, 751-753 (1993).
`10. Stratton, M. R. eta/. Nature Genet. 7, 103-107 (1994).
`11. Devilee, P. & Cornelisse, C. J. Biochim. biophys. Acta 1198, 113-130 (1994).
`12. Lundberg, C., Skoog, L., Cavenee, W. K. & Nordenskjold, M. Proc. natn. Acad. Sci. U.S.A.
`84, 2372-2376 (1987).
`13. Cleton-Jansen, A.-M. et al. Br. J. Cancer 72, 1241-1244 (1995).
`
`IN this letter we described the cloning and expression of an
`inward rectifier potassium-channel subunit from rat heart (Kir
`3.4) which, when transfected into HEK293 and BHK21 cells,
`endowed them with A TP-sensitive potassium channels. Since this
`paper appeared, we have not been able regularly to reproduce
`those findings. In addition, the data presented by Krapivinsky
`eta!. 1 presents a compelling argument that Kir 3.4 is an intrinsic
`component of the channel underlying IKAch in atrium, and that
`it does not contribute to the channel underlying cardiac IKATP·
`Therefore, we cannot support our previous statement that Kir
`3.4 represents a subunit of cardiac KATP channels.
`D
`
`ACKNOWLEDGEMENTS. We thank the families for their continuing help and encouragement in
`this work and the many clinicians who have referred families; F. Dion, R. Carter, K. Anderson,
`
`1. Krap1vmsky, G. et a/. Nature 374, 135-141 (1995).
`
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`NATURE · VOL 378 · 21(28 DECEMBER 1995
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`© 1995 Nature Publishing Group
`
`GeneDX 1023, pg. 4
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