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Am J Hum Genet. Apr 2002; 70(4): 858–865.
Published online Mar 5, 2002. doi:  10.1086/339434
PMCID: PMC379114

Distinct BRCA1 Rearrangements Involving the BRCA1 Pseudogene Suggest the Existence of a Recombination Hot Spot

Abstract

The 5′ end of the breast and ovarian cancer–susceptibility gene BRCA1 has previously been shown to lie within a duplicated region of chromosome band 17q21. The duplicated region contains BRCA1 exons 1A, 1B, and 2 and their surrounding introns; as a result, a BRCA1 pseudogene (ΨBRCA1) lies upstream of BRCA1. However, the sequence of this segment remained essentially unknown. We needed this information to investigate at the nucleotide level the germline deletions comprising BRCA1 exons 1A, 1B, and 2, which we had previously identified in two families with breast and ovarian cancer. We have analyzed the recently deposited nucleotide sequence of the 1.0-Mb region upstream of BRCA1. We found that 14 blocks of homology between the tandemly repeated copies (cumulative length = 11.5 kb) show similarity of 77%–92%. Gaps between blocks result from insertion or deletion, usually of repetitive elements. BRCA1 exon 1A and ΨBRCA1 exon 1A are 44.5 kb apart. In the two families with breast and ovarian cancer mentioned above, distinct homologous recombination events occurred between intron 2 of BRCA1 and intron 2 of ΨBRCA1, leading to 37-kb deletions. Breakpoint junctions were found to be located at close but distinct sites within segments that are 98% identical. The mutant alleles lack the BRCA1 promoter and harbor a chimeric gene consisting of ΨBRCA1 exons 1A, 1B, and 2, which lacks the initiation codon, fused to BRCA1 exons 3–24. Thus, we report a new mutational mechanism for the BRCA1 gene. The presence of a large region homologous to BRCA1 on the same chromosome appears to constitute a hot spot for recombination.

Introduction

More and more germline rearrangements have been identified in the BRCA1 gene (MIM 113705) during the last 4 years, in families with breast and/or ovarian cancer (Petrij-Bosch et al. 1997; Puget et al. 1997; Swensen et al. 1997; Montagna et al. 1999; Puget et al. 1999a, 1999b; Payne et al. 2000; Rohlfs et al. 2000a, 2000b; Unger et al. 2000; Gad et al. 2001a, 2001b). This is due to the fact that a growing number of laboratories screening for mutations in BRCA1 use techniques allowing their identification—that is, Southern blot analysis (Swensen et al. 1997; Montagna et al. 1999; Puget et al. 1999a; Rohlfs et al. 2000b; Unger et al. 2000; Lahti-Domenici et al. 2001), cDNA analysis (Petrij-Bosch et al. 1997; Puget et al. 1997, 1999b; Rohlfs et al. 2000a), DNA combing (Gad et al. 2001a, 2001b), long-range PCR (Payne et al. 2000), and semiquantitative PCR (Robinson et al. 2000). Indeed, 20 different large alterations have been reported, among which 14 are fully characterized. All these rearrangements share several characteristics with the far more numerous point mutations found in BRCA1: they are scattered throughout the gene, >60% are unique, and the recurrence of certain mutations is due to founder effects (Petrij-Bosch et al. 1997; The BRCA1 Exon 13 Duplication Screening Group 2000; Rohlfs et al. 2000b). The breakpoint junction of only one rearrangement falls within an exonic sequence (Payne et al. 2000). In all other cases, they fall within intronic Alu sequences, which represent 41.5% of the BRCA1 gene (Smith et al. 1996).

In a previous study (Puget et al. 1999a), we identified two different germline deletions that involve exons 1 and 2 and whose breakpoints could not be characterized because knowledge of the structure of the region upstream of BRCA1 was not comprehensive. A BRCA1 pseudogene, ΨBRCA1, had been shown to lie ~30 kb upstream of BRCA1 (Barker et al. 1996; Brown et al. 1996), because of the duplication of a region containing BRCA1 exons 1 and 2. The first exons of the NBR2 (next to BRCA1 2) gene, located between BRCA1 and its pseudogene, are homologous to the first exons of the NBR1 (next to BRCA1 1) gene, which lies head-to-head with ΨBRCA1 (Xu et al. 1997). The deletions we identified in the two families with breast and ovarian cancer, F32 and F3514, did not include ΨBRCA1 exons 1 and 2 (Puget et al. 1999a) but were found to totally encompass NBR2 (data not shown). We suspected that the existence of the duplicated regions could represent a recombination hot spot and could be involved in predisposition to breast and ovarian cancer in families F32 and F3514.

On the basis of a color bar code of the BRCA1 region on combed DNA in carriers, we show evidence in favor of this hypothesis. The recent availability of the full sequence of the 1-Mb region upstream of BRCA1 allowed us (1) to describe its precise organization, which revealed its high potential of recombination and (2) to use PCR to characterize the deletions’ breakpoints. This showed that both recombinations occurred between intron 2 of the pseudogene and intron 2 of the gene, at close, but not identical, sites. These findings strengthen the hypothesis that the presence of a large region homologous to BRCA1 and on the same chromosome may constitute a hot spot for recombination.

Subjects and Methods

Sequence Data Analyses

The sequence of BAC RP11-242D8 (GenBank accession number AC060780) was identified by performing a BLAST search with BRCA1 intron 2, using a “human repeats” filter in the High-Throughput Genome Sequences database. The sequence was, at the time this manuscript was written, a “working draft” consisting of three contigs and not yet annotated. Because the first 39 kb of the first contig (152,199 bp) is the only one to contain BRCA1 sequences (from exon 1A to most of intron 12), it is certain to be a clone end. Therefore, coordinates given in the present report will not change when the sequence is updated. Exons of NBR1 and NBR2 were positioned within the sequence by use of the SIM4 program, while “exons” 1A, 1B, and 2 of ΨBRCA1 were defined by means of homology with BRCA1 exons, using the BLAST program. The extent of the similarity between gene and pseudogene was investigated using the LALIGN program. Blocks were arbitrarily defined as regions of homology within which gaps did not exceed 50 nucleotides. RepeatMasker was run, to identify interspersed repeat sequences. The sequence between the ends of ΨBRCA1 and NBR2 (nucleotides 59596–67624, referring to the AC060780 sequence) was analyzed by use of the NIX analysis program (nucleotide identification of unknown sequences, U.K. Medical Research Council Human Genome Mapping Project), a Web-based package of gene-analysis software (including GRAIL, GENSCAN, Fgenes, Fex, Hexon, Genemark, Genefinder, BLAST, Polyah, RepeatMasker, and TRNAscan).

Color Bar Coding the BRCA1 Region on Combed DNA from Families F32 and F3514

DNA samples were extracted from lymphoblastoid cell lines of BRCA1 carriers belonging to families F32 and F3514, as reported elsewhere (Michalet et al. 1997). Combing relies on homogeneous stretching of DNA molecules at a constant rate of 2 kb/μm (Bensimon et al. 1994; Michalet et al. 1997). FISH was performed on combed DNA, using six probes generated by random labeling (as described by Gad et al. [2001a]) of a PAC covering the whole BRCA1 region (PAC103O14 from the chromosome 17 library of the Lawrence Livermore National Laboratory [Brown et al. 1995]), a rearranged cosmid covering the BRCA1 promoter region (cosmid ICRFc105D06121 from the Ressourcen Zentrum Primary Database [Brown et al. 1996; Gad et al. 2001a]), and four long-range (LR) PCR products (Expand Long Template PCR System, Roche Diagnostics). Three PCR products covered exons of the BRCA1 gene, and the remaining one covered exons 2–4 of the NBR2 gene (Gad et al. 2001a; primers used are available at the Institut Curie Web site).

Full signals were observed under an epifluorescence DMRB microscope (Leica) and were captured with IPLab Spectrum-SU2 software (Vysis), using an NU 200 CCD camera (Photometrics). Image analyses were performed with CartographiX software (X. Michalet, Institut Pasteur, Paris), which permits the measurement of fragments of full signals with respect to the constant stretching rate of 2 kb/μm (Michalet et al. 1997; Gad et al. 2001a).

Cloning of Breakpoints in Families F32 and F3514

PCRs were performed using the Expand Long Template PCR System (Roche) according to the manufacturer’s recommendations. Among BRCA1 carriers in families F32 and F3514, forward primer pintron2.2F (ACC TAA AAT TCC TTC TGC TGG AC), which is located within intron 2 of the pseudogene at the beginning of block 11 (nucleotides 71699–71677), and reverse primer intron2.R2 (CCT TGG GCT AAC CAC TCT ACC), which is located in intron 2 (nucleotides 33948–33968), gave products of ~800 bp for F32 and F3514 carriers' DNA only, the normal fragment being far too large (37.8 kb) to be amplified. These fragments were then sequenced.

Screening for BRCA1 Rearrangements Involving ΨBRCA1 in Families with Breast and Ovarian Cancer

An original set of ~100 families, ascertained by H. T. Lynch (Creighton University School of Medicine), were screened for mutations in the coding region and splice sites of the BRCA1 and BRCA2 genes by use of heteroduplex analysis and the protein truncation test (Mazoyer et al. 1996; Serova et al. 1996; Puget et al. 1997, 1999a, 1999b; Serova et al. 1997). Of the families that remained negative for mutations, 13 were used in this analysis. Families were selected according to the following criteria: (1) at least 5 cases of breast cancer, of which at least 4 were diagnosed at <60 years of age; (2) at least 4 cases of breast cancer, of which at least 2 were diagnosed at <45 years of age; (3) at least 1 case of ovarian cancer diagnosed at any age and 3 cases of breast cancer diagnosed at <60 years of age; or (4) at least 2 cases of ovarian cancer diagnosed at any age and 1 case of breast cancer diagnosed at <45 years of age or 2 cases of breast cancer diagnosed at any age. Among these families, seven are affected by breast cancer alone (one includes one case of male breast cancer), whereas the remaining six are families with breast and ovarian cancer (one includes one case of male breast cancer).

Screening for rearrangements involving ΨBRCA1 was conducted with the following primer pairs: NBR1.1AF (CCT GAG GCC TGA ATA TCA GC)/intron2.R4 (CTA TCC TCT CAA CGA CAC CGA T) to amplify from blocks 5–8; pintron2.7F (TCA AGG AAA TTT TCT TTT GTG C)/intron2.R7 (TGT GGA GTT TCC CCC ATT CT) to amplify blocks 10–14.

The expected size of the mutant product in the presence of a rearrangement is 2.4–2.9 kb for the first pair and ~7 kb for the second one. All screened DNA samples were subjected to a preliminary PCR with primers giving rise to a 7.2-kb fragment, in order to check their quality. A positive control—that is, carriers of BRCA1 from F32 and F3514, could only be used with primers pintron2.7F and intron2.R7. More information about screening protocols, including the choice of primers, can be obtained upon request from the corresponding author.

Results

Structure of the Region Upstream of the BRCA1 Gene

The complete genomic sequence of the human BRCA1 gene (GenBank accession number L78833), which begins 2,965 nucleotides upstream of exon 1A and therefore contains NBR2 exons 1A and 1B, was made available in 1996, 2 years after the BRCA1 cDNA was cloned. Comparison of this sequence with available NBR1 and ΨBRCA1 sequences (U72483 and U77841) showed an overall similarity of 90.3%, scattered over 3,865 nucleotides. All attempts to amplify the region between ΨBRCA1 and BRCA1 by long-range PCR with a wide collection of primers have been unsuccessful, either because this region was larger than the ~30 kb proposed or because it contained sequences difficult to amplify or for both reasons. In the same way, we were unable to amplify the region between ΨBRCA1 and NBR2 or that between NBR2 exon 2 and BRCA1.

To obtain more information on the structure of this region, we developed a restriction map of a 60-kb P1 clone (P1 1141 [Swensen et al. 1997]), from which we deduced that most of BRCA1 intron 2 was likely to be contained within the duplicated region (data not shown). The deduction was later confirmed with the recently deposited sequence of BAC RP11-242D8 (GenBank accession number AC060780), which, at the present time, consists of three contigs, among which a 152,199-nucleotide piece comprises NBR1, ΨBRCA1, NBR2, and 39,879 nucleotides of BRCA1. Analysis of this sequence revealed that BRCA1 exon 1A and ΨBRCA1 exon 1A are 44,490 nucleotides apart. The duplicated segments extend over 14,491 and 24,970 bp in the NBR2/BRCA1 and the NBR1/ΨBRCA1 regions, respectively, and are fragmented in 14 blocks of homology (fig. 1 and table 1), ranging from 77 to 1,555 bp (total 11,429 bp). The percentage of identity between gene and pseudogene within blocks varies from 76.9 to 92.1 (table 1). Gaps between blocks (61–7,457 bp) result from insertion or deletion mostly of repetitive elements in BRCA1 or ΨBRCA1 (fig. 1), with two exceptions: (1) a 3,794-bp region containing repetitive elements, nonspecific sequences, and NBR1 exon 2 (between blocks 1 and 2) and (2) a large P1 ribosomal protein pseudogene in the ΨBRCA1 region (between blocks 5 and 6). The sequence between ΨBRCA1 and NBR2 is 8,029 nucleotides and was analyzed by the use of NIX, which incorporates a number of independent gene-prediction tools. Although a couple of genes were predicted (data not shown), no known transcribed sequence was identified. RepeatMasker indicates that interspersed repeats account for 56.5% of this region.

Figure  1
Schematic representation of the duplicated regions showing the homology between NBR1 and NBR2, and between ΨBRCA1 and BRCA1. Blackened boxes represent exons, and shaded boxes represent blocks of homology, which were arbitrarily numbered. Repetitive ...
Table 1
Blocks of Homology between NBR1/NBR2 and ΨBRCA1/BRCA1

Cloning of the Breakpoints of the Deletions in Families F32 and F3514

In a previous study, we analyzed 78 DNA samples by quantitative Southern blot hybridization, to assess the proportion of rearrangements in the BRCA1 mutation spectrum (Puget et al. 1999a). Densitometric analysis of the blots showed, in two American families with breast and ovarian cancer (F32 and F3514), deletions of BRCA1 exons 1 and 2 but not of exon 3 or ΨBRCA1 exons 1 and 2 (Puget et al. 1999a). Allelotyping with three BRCA1 intragenic markers revealed that these two families do not share a haplotype (Puget et al. 1999a). Further Southern blot analysis with more probes showed that these deletions totally encompass NBR2 (data not shown). To estimate the extent of the deleted regions in F32 and F3514, the BRCA1 gene region was analyzed using the dynamic molecular-combing technique in association with FISH (Bensimon et al. 1994; Michalet et al. 1997). Homogeneously stretched DNA molecules were visualized with a four-color bar code, allowing the analysis of the whole BRCA1 and upstream region (Gad et al. 2001a). As shown in figure 2, two signal patterns were observed in the F32 and F3514 DNA samples: the abnormal allele showed a similar BRCA1 bar code in both cases, with absence of hybridization of the NBR2 LR2–4 and of the cosmid probe, which normally allows visualization of both ΨBRCA1 and BRCA1 exons 1 and 2 because of cross-hybridization, on one of its targets. Taken together with the Southern blot results, these data suggested that a deletion comprising the region between ΨBRCA1 and BRCA1 had occurred in F32 and F3514 and had led to the replacement of BRCA1 exons 1 and 2 by ΨBRCA1 exons 1 and 2.

Figure  2
BRCA1 color bar code of DNA samples from families F32 and F3514. Two allele populations could be visualized under a microscope when combed DNA samples were hybridized with fluorescent probes generated from PAC 103O14 (green) and cosmid D06121 (red) and ...

In the same way that we were unable to amplify the region between ΨBRCA1 and BRCA1 by long-range PCR in control individuals, we were unable to amplify the mutant allele with various combinations of forward primers located in ΨBRCA1 exon 2 and reverse primers located in BRCA1 exon 3 by long-range PCR, using a wide range of conditions, although the expected fragment was estimated to be <15 kb.

When sequence information concerning intron 2 of the BRCA1 pseudogene became available, it gave us the opportunity to design primers that specifically hybridized the pseudogene. Such forward primers were used in conjunction with reverse primers specifically hybridizing the BRCA1 gene. Primers pintron2.2F and intron2.R2 gave rise to an ~800-bp fragment when they were used with DNA from members of families F32 and F3514 who carried the deletion. No product was obtained when these primers were used with DNA from control individuals. Sequencing of this fragment revealed that a 36,934-bp fragment, comprising BRCA1 exons 1 and 2 and the region located between ΨBRCA1 and BRCA1, is deleted in both families. In F32, the breakpoint junctions occurred in two regions of 237 bp within block 11 with 100% similarity (nucleotides 34439–34675 in BRCA1 and nucleotides 71374–71610 in ΨBRCA1, referring to the RP11-242D8 BAC sequence). The breakpoint junctions in F3514 occurred in 23-bp regions of perfect homology, located 77 bp apart (nucleotides 34,339–34,361 in BRCA1 and nucleotides 71,274–71,296 in ΨBRCA1). These regions do not contain any repetitive element.

Because of the deletions, carriers in families F32 and F3514 harbor a chimeric gene that consists of ΨBRCA1 exons 1A, 1B, and 2 fused to BRCA1 exons 3–24 (fig. 3). As a consequence, the BRCA1 promoter is absent from this mutant allele. It is not known yet whether the BRCA1 pseudogene is transcribed, and therefore we do not know whether the mutant gene might be expressed. Nevertheless, all attempts to identify a mutant transcript were unsuccessful (data not shown). In any case, and although both exons 2 are highly similar, the methionine codon used as the BRCA1 translation initiation codon is lost in the chimeric gene, because it is changed to an isoleucine codon in ΨBRCA1. Missense mutations changing this methionine codon to a valine or an isoleucine codon have been reported in five families with breast and ovarian cancer (Breast Cancer Information Core). It implies that even if other methionine codons can be used for translation initiation; as has been suggested elsewhere (Liu et al. 2000), the resulting BRCA1 isoform cannot substitute for the tumor-suppressor function of the wild-type protein in breast and ovarian tissues.

Figure  3
Representation of the 37-kb deletion in F32 and F3514. Drawn-to-scale schema of the normal and mutant alleles at the NBR1 and BRCA1 loci, showing the location of the 36,934-bp deletion. The size of the NBR1, ΨBRCA1, NBR2, and BRCA1 genes are given ...

Screening for Rearrangements Involving the ΨBRCA1 in Families with Breast and Ovarian Cancer

The identification of two different recombination events involving homologous regions located respectively in the BRCA1 gene and ΨBRCA1 leads to the possibility that these regions represent a strong hot spot for recombination. We therefore screened additional candidate families for the presence of such deletions, with two different combinations of forward primers located in NBR1 or ΨBRCA1 and reverse primers located in BRCA1. The 13 tested families with breast and ovarian cancer were chosen on the basis of strict criteria and were negative in a mutation screening analysis of the BRCA1 and BRCA2 coding sequence and splice sites. The BRCA1 promoter lies between exons 1A of NBR2 and BRCA1. Therefore, rearrangements involving blocks 1–5 would disrupt the NBR1 and the NBR2 genes but would not disrupt any part of the BRCA1 gene, including its promoter. We thus focused our analysis on screening for rearrangements within blocks 6–14. Because of difficulties, as mentioned above, in amplifying the region between NBR1 and BRCA1, we split the screened interval into two segments: blocks 5–8 and blocks 10–14 (see fig. 1). No other large deletions resulting from recombination between BRCA1 and its pseudogene were observed.

Discussion

We show here that the first exons of the BRCA1 gene were replaced, in carriers from two families with breast and ovarian cancer, by those of the BRCA1 pseudogene, ΨBRCA1 (fig. 3). Among the 14 blocks of homology identified between the NBR1/ΨBRCA1 and the NBR2/BRCA1 regions, the breakpoint junctions of the 37-kb deletions identified in families F32 and F3514 were both found to be located in block 11 (fig. 1), at close but distinct sites within segments of 624 bp that are 98% identical. These segments contain the longest stretches of complete sequence identity found among any of the 14 blocks: 237 and 188 bp, compared with <115 bp. The mutant alleles harbor a chimeric gene that consists of ΨBRCA1 exons 1A, 1B, and 2 fused to BRCA1 exons 3–24 (fig. 3). This chimeric gene lacks the BRCA1 promoter and the BRCA1 translation initiation codon. The concomitant absence of the NBR2 gene in the mutant alleles does not appear to lead to a contiguous-gene-deletion syndrome, because carriers from families F32 and F3514 do not show any remarkable phenotypic trait apart from being predisposed to breast and ovarian cancers (10 cases of breast cancer and 2 cases of ovarian cancer in family F32; 2 cases of breast cancer, 1 case of ovarian cancer, and 1 case of bilateral breast cancer and ovarian cancer in family F3514). It should be noted that nothing is known, as yet, about the potential protein encoded by NBR2.

The screening of 13 additional highly selected families with breast and ovarian cancer, negative after intensive searches for BRCA1 and BRCA2 mutations, failed to identify any more large deletions resulting from homologous recombination between BRCA1 and ΨBRCA1. More such families should be tested with the primers we describe, to determine the frequency of these alterations in the BRCA1 mutation spectrum. Notably, two families with breast and ovarian cancer that were reported in the literature to present an abnormal pattern in Southern blot experiments, with probes covering BRCA1 exons 1 and 2 (Unger et al. 2000), might carry a recombination event involving ΨBRCA1.

Duplication of genes, gene segments, and repeat-gene clusters generated by the evolution of the mammalian genome provides substrates for homologous recombination that results in DNA rearrangements. When rearrangements alter the genome by causing complete loss or gain of a gene(s) sensitive to a dosage effect or by disrupting the structural integrity of a gene, they are responsible for many diseases called “genomic disorders.” These include notably α- and β-thalassemia, hemophilia A, growth hormone deficiency, Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP) and Smith-Magenis syndrome (for review, see Lupski 1998). Several common features associated with the mechanisms that lead to DNA rearrangements were apparent when analyzing such diseases: (1) long regions of high similarity appear to be required for recombination, and (2) the greater the distance between repeats, the greater the repeat length required for efficient recombination. This is exemplified by unequal crossing-over events causing α-thalassemia, which involve repeated segments of ~4 kb located 3.7 or 4.2 kb apart within the α-globin locus; causing hemophilia A, which involve 98% identical repeats of 9.6 kb located ~500 kb apart within the factor VIII gene locus; and causing CMT1A and HNPP, which involve repeats (CMT1A-REPs) of 24 kb >98% identical located 1,500 kb apart. DNA sequence analysis of the junctions of the recombinations responsible for genomic disorders showed those crossing-over events took place in shorter regions within the repeats where the sequence identity is the strongest. Alternatively, specific elements that may stimulate recombination, such as MITE (χ- and mariner-like transposable element) and a χ-like sequence, were found in or close to the 1.4-kb CMT1-REP hot spot (Reiter et al. 1996) and the 2-kb NF1-REP hot spot (Lopez-Correa et al. 2001), respectively.

In the case of the BRCA1 locus, the duplicated region is >10 kb and is located <50 kb upstream of BRCA1, which makes homologous recombination events likely to occur frequently. However, the requirement of an overall high similarity between the repeated segments is not met, because of numerous interruptions generated by insertions and/or deletions of repetitive sequences (see fig. 3). The fact that the duplicated copy and the original one diverged from one another to such an extent might be related to the unusually high content of repetitive elements in the BRCA1 gene (Smith et al. 1996). In the case of the disorders mentioned above, genome rearrangements represent a high proportion of mutations. Contrary to this, it seems clear from the data obtained in our panel of ~100 families with breast and ovarian cancer that rearrangements between BRCA1 and ΨBRCA1 are heavily outnumbered by point mutations, small insertions, small deletions, and splice-site mutations. This might be explained by the overall low similarity of the repeats (as mentioned above), associated with the large size of the BRCA1 coding sequence, which provides more opportunities for mutations during replication. Nevertheless, it is reasonable to expect more families with breast and/or ovarian cancer worldwide to carry large deletions, like the ones described here. Therefore, screening protocols should include PCR assays, such as those described in the present article, to detect this new type of BRCA1 rearrangement in families with breast and ovarian cancer that have no mutation in BRCA1/2 coding sequences and splice sites.

Acknowledgments

We thank the family members who collaborated in this study. We also thank H. T. Lynch, for a long-term collaboration in the study of these families, C. Bonnardel and C. Snyder, for their expert assistance, and M. Zody for communicating helpful information concerning the progress of the RP11-242D8 BAC sequencing by the Whitehead Institute/MIT Center for Genome Research. This work was supported by program grants from le Comité Départemental de l’Ain et du Rhône de la Ligue contre le Cancer and by the Institut Curie Programme Incitatif et Coopératif: Génétique et Biologie des Cancers du Sein. N.P., S.G., and L.P.-V. are supported by fellowships from the Ligue contre le Cancer de Haute-Savoie; the Ministère de l'Education National, de la Recherche et de la Technologie; and the Ligue Nationale contre le Cancer, respectively.

Electronic-Database Information

Accession numbers and URLs for data in this article are as follows:

GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for BAC sequence [accession number AC060780] and BRCA1 [accession number L78833])
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for BRCA1 [MIM 113705])

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