oncogenomics - Nature

Oncogene (2002) 21, 6841 – 6847 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Significant contribution of large BRCA1 gene rearrangements in 120 French breast and ovarian cancer families

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Service de Geńe´tique Oncologique, Institut Curie, Paris, France; 2CHU Arnaud de Villeneuve, Montpellier, France; 3Centre Antoine Lacassagne, Nice, France; 4CHU Dupuytren, Limoges, France; 5Centre Rene´ Gauducheau, Nantes, France; 6Centre Jean Perrin, Clermont-Ferrand, France; 7Hoˆpital Charles Nicolle, Rouen, France; 8Centre Paul Strauss, Strasbourg, France; 9Institut Jean Godinot, Reims, France; 10INSERM U509 Pathologie Molećulaire des Cancers, Institut Curie, Paris, France; 11Laboratoire de Biophysique de l’ADN, Institut Pasteur, Paris, France

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast-ovarian cancers. However, BRCA1 germline mutations are found much less frequently than expected, especially as standard PCR-based mutation detection approaches focus on point and small gene alterations. In order to estimate the contribution of large gene rearrangements to the BRCA1 mutation spectrum, we have extensively analysed a series of 120 French breast-ovarian cancer cases. Thirty-eight were previously found carrier of a BRCA1 point mutation, 14 of a BRCA2 point mutation and one case has previously been reported as carrier of a large BRCA1 deletion. The remaining 67 cases were studied using the BRCA1 bar code approach on combed DNA which allows a panoramic view of the BRCA1 region. Three additional rearrangements were detected: a recurrent 23.8 kb deletion of exons 8 – 13, a 17.2 kb duplication of exons 3 – 8 and a 8.6 kb duplication of exons 18 – 20. Thus, in our series, BRCA1 large rearrangements accounted for 3.3% (4/120) of breast-ovarian cancer cases and 9.5% (4/42) of the BRCA1 gene mutation spectrum, suggesting that their screening is an important step that should be now systematically included in genetic testing surveys. Oncogene (2002) 21, 6841 – 6847. doi:10.1038/sj.onc. 1205685 Keywords: BRCA1 mutations; rearrangements; breastovarian cancer The identification of the BRCA1 and BRCA2 genes was a major advance in the understanding of the familial forms of breast and ovarian cancer, since their

*Correspondence: D Stoppa-Lyonnet, Service de Geńe´tique Oncologique, Institut Curie – Section Me´dicale, 26, rue d’Ulm, 75248 Paris, Cedex 05, France; E-mail: [email protected] Received 7 March 2002; revised 3 May 2002; accepted 20 May 2002

alterations result in a high predisposition (Miki et al., 1994; Wooster et al., 1995). Thus far, analysis of the BRCA1 coding sequence by mutation screening methods based on PCR-sequencing protocols, have allowed the identification of 800 different point or small disease-causing germline alterations (Breast Cancer Information Core, (BIC (http://www.nhgri.nih.gov/ Intramural_research/Lab_transfer/Bic/)). However, hitherto only 63% of the expected mutations could be detected by these approaches and the identification of all disease-causing mutations in at-risk families is still a challenge (Ford et al., 1998). A significant proportion of the 37% undetected BRCA1 mutations could be due to large rearrangements which have escaped current methods used. Indeed, some large BRCA1 rearrangements have been reported, scattered over the whole gene sequence and ranging in size from 0.5 to 23.8 kb. They have been detected by Southern blotting, lymphocyte transcript analysis, or Long Range PCR (Petrij-Bosch et al., 1997; Puget et al., 1997, 1999a,b; Swensen et al., 1997; Carson et al., 1999; Montagna et al., 1999; Rohlfs et al., 2000a,b; Payne et al., 2000; Unger et al., 2000). In order to detect such rearrangements, we have developed a BRCA1 bar code on combed DNA which leads to a panoramic view of the gene and its flanking regions (Michalet et al., 1997; Gad et al., 2001a,b). By using this method, we have studied a series of French patients with a breast-ovarian cancer family history, who were previously found negative for point or small mutations in both the BRCA1 and BRCA2 genes (Stoppa-Lyonnet et al., 1997, and unpublished data; Pages et al., 2001). Taking into account all the breastovarian cancer cases tested for BRCA1 and BRCA2 mutations in our laboratory, from which the present series was extracted and analysed using the BRCA1 bar code approach, we have estimated the contribution of large rearrangements to the BRCA1 mutation spectrum in French breast-ovarian cancer families. From January 1991 to December 1999, 212 women affected with breast and/or ovarian cancer (index

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Sophie Gad1,10, Virginie Caux-Moncoutier1, Sabine Page`s-Berhouet1, Marion Gauthier-Villars1, Isabelle Coupier1, Pascal Pujol2, Marc Freńay3, Brigitte Gilbert4, Christine Maugard5, Yves-Jean Bignon6, Annie Chevrier7, Annick Rossi7, Jean-Pierre Fricker8, Tan Dat Nguyen9, Liliane Demange9, Alain Aurias10, Aaron Bensimon11 and Dominique Stoppa-Lyonnet*,1,10

Contribution of BRCA1 rearrangements S Gad et al

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cases), ascertained in French genetic clinics (Institut Curie and eight other centers), were referred to our laboratory for BRCA1/2 analysis. Familial criteria for genetic testing were: (i) at least one case of ovarian cancer and two cases of breast or ovarian cancer at any age in the same lineage; or (ii) two first-degree relatives, affected with breast or ovarian cancer, with at least one ovarian cancer case; or (iii) women affected with both primary breast and ovarian cancers whatever the familial history. Each of the index cases was an affected family member with the exception of one woman who was regarded as an obligate carrier based on family history. The probability of being a mutation carrier was calculated as previously described (Claus et al., 1991; Stoppa-Lyonnet et al., 1997). The pathological and medical records were verified for all index cases, and also for 32 and 43% of the relatives affected with breast and ovarian cancer respectively. After informing index cases of the aims and limits of genetic testing, blood samples were collected with their written consent for DNA extraction and lymphoblastoid cell line establishment, required for DNA combing and RNA studies. DNAs were tested for point mutations in both the BRCA1 and BRCA2 genes using combination of DGGE or DHPLC techniques, and FAMA, PTT (exons 10 – 11) or DHPLC techniques respectively (Stoppa-Lyonnet et al., 1997, and unpublished data; Wagner et al., 1999; Pages et al., 2001). A lymphoblastoid cell line was successfully established in 120 among the 212 cases (Table 1). In this subset, a BRCA1 point or small mutation resulting in a truncated protein was identified in 36 cases; in addition, two missense mutations (Cys47Tyr and Arg1751Gln) were regarded as disease-causing, so that a total of 38 BRCA1 point mutations (31.7%) were detected (Table 1). A BRCA2 point or small truncating mutation was identified in 14 cases (11.7%). Thus, a total of 68 cases (56.6%) remained to be studied for large BRCA1 rearrangements (Table 2). However, one of these cases was already known as carrier of a large BRCA1 deletion (IC568: 23.8 kb deletion of exons 8 – 13) detected by Southern blotting and used for designing the BRCA1 bar code (Puget et al., 1999a; Gad et al., 2001a). The mean probability of being a

Table 1 Synthesis of the 120 French consecutive cases with breastovarian cancer family history with cell line available Number Cases studied BRCA1 point mutations identified BRCA2 point mutations identified Cases negatively testeda BRCA1 rearrangements identifiedb Total number of BRCA1 alterations Contribution of rearrangements to the BRCA1 alteration spectrum a

120 38 (31.7%) 14 (11.7%) 68 (56.6%) 4 (3.3%) 42 (35.0%) 4/42 i.e. 9.5%

Prior probability 82.1% 89.3% 82.6% 78.0% 71.8% 87.0%

(9 – 99%) (28 – 99%) (24 – 98%) (9 – 99%) (9 – 98%) (9 – 99%) –

For point mutations in both BRCA1 and BRCA2. bIncluding three rearrangements detected by BRCA1 bar code in 68 cases negatively tested (the present study), and IC568 (del exons 8 – 13, 23.8 kb) already reported (Puget et al., 1999a)

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mutation carrier was 82.1% (9 – 99) in the 120 cases, 87.5% (24 – 99) in the BRCA1/2 point mutation group and 78.0% (9 – 99) in the negatively tested case group (Tables 1 and 2). The 67 cases tested negatively for BRCA1/2 point mutations for whom a cell line was available were studied using the BRCA1 bar code approach (Figure 1a,b; Gad et al., 2001a,b). Qualitative analysis of the BRCA1 bar code allowed the detection of three different rearrangements. Quantitative analysis, i.e. fragment measures, did not show any double peaks on histograms and therefore no indication of additional rearrangements. The first rearrangement identified, i.e. the duplication of exons 3 – 8 in IC827 patient, was briefly presented in a previous report (Gad et al., 2001a). As shown in Figure 1c, using the optimized bar code recently reported (Gad et al., 2001b), the duplication was clearly detected by the existence of two LR3 – 5 probes. RNA analysis revealed that exon 8 was adjacent to a duplicated exon 3 resulting in a premature stop codon at position 186 and truncation of the BRCA1 protein (data not shown). In order to characterize the duplication boundaries at the genomic level, we designed primers in exons 3 and 8. We observed the fusion of an Alu Sp in intron 8 with an Alu Sq in intron 2, suggesting an unequal recombination event between these two Alu sequences that share 83% homology. Recombination breakpoints were located between nucleotides 11967 and 29213, leading to a 17 247 bp duplication of exons 3 – 8 (GenBank accession number L78833; data not shown). The use of the duplication specific primers will allow further genetic testing in relatives. Microscope analysis of IC657 DNA revealed BRCA1 signals without LR9 – 12 and with half of the LR13 – 15 probes (Figure 1d). This deletion looked similar to the previously reported 23.8 kb deletion of exons 8 – 13 of family IC568 (Puget et al., 1999a; Gad et al., 2001a). A long range PCR of IC657 DNA, performed with primers located in exons 7 and 14, led to a similar PCR product as compared to that obtained with IC568 DNA (4 kb product, data not shown). In addition, the use of the primers specifically designed for genetic testing of IC568 family members by standard PCR allowed the detection of a similar product in IC657 patient, suggesting that these two deletions are identical. This hypothesis was confirmed by the sequencing of the 923 bp PCR product. We then examined whether these two families had a common haplotype by using five microsatellite markers within and surrounding the BRCA1 locus, in two carrier and one non carrier relatives for each family. The haplotype associated with the carrier status was common in the two families namely: tel – D17S1327, 131 bp – D17S1323, deleted – D17S1322, 121 bp – D17S855, 141 bp – D17S1185, 216 bp – cen, strongly suggesting the existence of a common ancestral mutant chromosome (data not shown). A third rearrangement was detected in the IC1712 DNA. Signals with two LR18 – 20 probes were


Table 2 The 68 French families negatively tested for BRCA1/2 point mutations and screened for BRCA1 rearrangements Family IC

Prior probability

Age at diagnosis of the tested person (years)

16 99% ovarian 45 24 91% breast 48+ovarian 50 51 73% breast 40 – 44+ovarian 48 102 90% unaffected, 85 148 89% breast 49 183 98% breast 32 441 86% breast 43+ovarian 49 488 90% breast 39 503 90% breast 31 519 94% ovarian 37 542 77% breast 34 552 16% breast 27+ovarian 30 557 90% breast 48+ovarian 52 568* 9% breast 49+ovarian 61 657* 90% breast 60 659 73% breast 48+45 707 92% breast 53 789 76% breast 48 827* 98% ovarian 49 852 90% breast 52 887 92% breast 32 902 95% breast 35 964 33% breast 58 1083 80% ovarian 53 1084 52% breast 35 1092 90% breast 35 1139 49% breast 29 1158 96% breast 34+41 1165 98% breast 29 1182 96% breast 36 1208 10% breast 74 1244 67% breast 44 1245 84% breast 40 1275 81% breast 61 1283 94% breast 48 1350 12% breast 43+ovarian 44 1389 77% breast 59 1424 70% breast 39 1521 77% ovarian 55 1541 94% ovarian 57 1559 74% breast 60 1587 61% breast 49+ovarian 53 1610 90% breast 34 1625 70% ovarian 44 1642 52% breast 59 1654 90% breast 55 1712* 90% ovarian 61 1725 71% breast 43 1774 90% ovarian 55 1803 68% breast 44 1818 60% breast 48 1891 90% ovarian 49 1910 84% ovarian 55 1929 90% breast 39+43 1962 76% breast 47 1966 92% ovarian 48 1990 78% breast 43 1997 62% breast 47 2015 62% breast 43 – 46+ovarian 49 2018 86% breast 26 2027 96% ovarian 38 2028 95% ovarian 42 2108 96% breast 35 2133 85% breast 45 2221 79% breast 44 2276 82% breast 24 2286 50% breast 52+ovarian 59 2336 96% breast 50 68 families, mean probability=78.0% (9 – 99%)

No breast tumor

No ovarian tumor

No breast and ovarian tumors

3 3 5 3 3 8 3 3 5 1 1 1 1 0 3 5 4 2 1 7 3 3 2 3 2 3 1 2 1 3 1 1 3 3 7 0 4 1 4 3 2 1 2 1 1 1 1 1 5 1 1 0 0 4 2 2 3 2 2 1 3 1 2 2 2 3 2 1

1 0 0 1 1 2 2 1 1 2 1 0 1 0 2 1 1 2 2 1 1 1 1 1 1 1 1 1 2 2 1 0 1 0 2 0 4 2 1 1 1 0 0 1 1 1 3 2 1 1 1 2 2 1 0 1 2 1 0 1 1 4 0 1 1 1 0 2

0 1 1 0 0 0 1 0 0 0 0 1 1 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 1 2 1 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 1 0 0 0 1 0

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*Families with a BRCA1 rearrangement, IC568 identified by Puget et al., 1999a, and the three others by the present study Oncogene


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Figure 1 Color bar code of the BRCA1 gene on combed DNA. (a) BRCA1 locus and probes used for the bar code on combed DNA (not drawn to scale, adapted from Brown et al. (1996) and Xu et al. (1997)). The BRCA1 gene is spread over 81 kb (Smith et al., 1996) and has a common promoter with the NBR2 gene which covers 19 kb (Puget et al., unpublished data). The BRCA1 pseudogene (cBRCA1) lies next to NBR2 and corresponds to a partial duplication of BRCA1, from the promoter region to intron 2 (17 kb; Barker et al., 1996; Brown et al., 1996; Puget et al., unpublished data). The NBR1 gene is located 5’ to cBRCA1. The probes used for the bar code of BRCA1 are presented in the color of their revelation step (five layers of antibodies): PAC 103O14 insert (approximately 120 kb long) covers the region between the first exons of NBR1 and the 3’ BRCA1 UTR (exon 24) (Brown et al., 1995, 1996; Smith et al., 1996; Xu et al., 1997); cosmid ICRFc105D06121 clone is rearranged since the insert Oncogene


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observed, indicating a duplication of at least exons 18 – 20 (Figure 1e). RT – PCR analysis using primers 15a/22b from the patient sample showed a 1040 bp fragment, that may correspond to duplicated exons 18 – 20. Sequencing of this large PCR product indeed revealed that exon 20 was adjacent to a duplicated exon 18, leading to a premature stop codon at position 1769 (data not shown). In order to define the duplication boundaries, primers were designed around the putative breakpoints and led to the detection of a 1404 bp PCR product. Sequencing of this fragment showed the fusion of an Alu Jb in intron 20 with an Alu Y in intron 17 sharing 73% of homology. Recombination breakpoints were located between nucleotides 63859 and 72516, resulting in a 8658 bp duplication (data not shown). Besides, in the series of 92 cases with no available cell line, in which 38 BRCA1/2 point mutations (41.3%) had been detected, the 54 cases negatively tested were examined for the frequency of the three different rearrangements described above, using the primers specifically designed for genetic testing. In addition, we also tested the exon 13 duplication which is the most frequent rearrangement reported so far (Puget et al., 1999b; The BRCA1 Exon 13 Duplication Screening Group, 2000). None of these four different rearrangements tested was detected in these 54 cases, suggesting that they are not frequent in the French population. The aim of the present study is to estimate the frequency of large BRCA1 rearrangements in French breast-ovarian cancer families fulfilling current criteria for genetic testing, and their contribution to the BRCA1 mutation spectrum. A set of 120 cases defined by the availability of a lymphoblastoid cell line among a series of 212 cases consecutively referred to our laboratory, was extensively examined for both BRCA1/ 2 point mutations, and BRCA1 large rearrangements by bar code on combed DNA. Four BRCA1 rearrangements were identified, i.e. 3.3% (4/120), including two duplications (IC827, IC1712), and a deletion detected twice (IC568, IC657). Regarding this deletion, its absence in the set of 92 families, although the common haplotype identified in IC568 and IC657 families, suggests that this deletion is not associated with a high frequency in French families. No inversions

were observed despite the BRCA1 bar code strategy could detect them, suggesting that if inversions occur, their frequency is very low. The three different rearrangements reported here (dup ex3 – 8, dup ex18 – 20 and del ex8 – 13) combined with the 18 different rearrangements characterized so far underline the broad diversity of the BRCA1 gene rearrangements. Such a diversity is likely to result from the high density of Alu sequences located in the intronic sequences of the BRCA1 gene (Smith et al., 1996). In addition, this diversity, similar to the one observed with point and small mutations, is not unexpected when a disease process results from gene inactivation. This observation stresses the need to analyse the whole BRCA1 gene for large rearrangements rather than only the previously reported ones. The rearrangements identified in our series were found in three probands from families with a high predisposition probability (90 – 98%), and in one case (IC568) in a woman affected with both primary breast and ovarian cancers but no first to third degree affected relatives, resulting in a low predisposition probability (9%; Table 2). This observation underlines that while large rearrangements are obviously expected in families with high predisposition probabilities that are negative for BRCA1/2 point mutations, they may occur in families with a low predisposition probability and supports the recommendation to systematically include large rearrangement screening once BRCA1 genetic testing has been decided. By taking into account the 38 BRCA1 point mutations in the series of the 120 cases with cell line available for the bar code approach, we can estimate that the contribution of rearrangements to the BRCA1 mutation spectrum in French breast-ovarian cancer families is 4/42, i.e. 9.5% (Table 1). We paid particular attention not to introduce any bias for this estimation by avoiding to select families with a very high predisposition probability for BRCA1 bar code analyses among those ascertained for genetic testing. Such a bias would have overestimated the contribution. Since the frequency of BRCA1 point mutations (35%) in the 120 cases, was similar to the frequency of BRCA1 point mutations (38%) in the remaining 92 cases with no available cell line, we can consider the first subset as representative of the 212 patients

was found to be 6 kb long (Brown et al., 1996; Xu et al., 1997) and cross-hybridized with cBRCA1; PAC 44B1 insert has been located from intron 13 to the 3’ BRCA1 region (Brown et al., 1995). The other probes used are Long Range PCR products (primers available at http://www.curie.net/genetique) covering several exons and generated from PAC 103O14 (NBR2-4, LR3-5, LR9-12, LR13-15 and LR18-20) or from PAC 44B1 (LR24-3’). (b) Control DNA: Two full signals showing the BRCA1 bar code. Total human genomic DNA was extracted from lymphoblastoid cell line using agarose blocks as described previously (Michalet et al., 1997; Gad et al., 2001a). Combing was performed using the Molecular Combing ApparatusTM (Institut Pasteur, Paris). Hybridization and probe detection were performed as previously described (Michalet et al., 1997; Gad et al., 2001a). Full signals, corresponding to the BRCA1 gene, were observed under an epifluorescence Leica DMRB microscope and captured with IPLab Spectrum-SU2 software (Vysis, Downers Grove, IL, USA) using an NU 200 CCD camera (Photometrics, Tucson, AZ, USA). This first view of full signal images is regarded as a qualitative analysis. After screening the slide of the control DNA, 50 full signals were captured. To facilitate their view and analyses, full signals were aligned. Measurement of probes on each full signal was performed with CartographiX software with the use of the constant stretching rate of 2 kb/mm as described previously and was considered as quantitative analysis (Michalet et al., 1997; Gad et al., 2000a). (c,d,e) Patients IC827 (c), IC657 (d) and IC1712 (e) DNA. Full BRCA1 signals are shown for the normal (N) and mutant (dup, del) alleles. Rearrangements have been identified as 17.2 kb duplication of exons 3 – 8 (C, Gad et al., 2001a), 23.8 kb deletion of exons 8 – 13 (d) and 8.6 kb duplication of exons 18 – 20 (e) Oncogene


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ascertained for genetic testing. In addition, our estimation of the contribution could be slightly underestimated since the bar code approach does not detect rearrangements less than 2 kb in length, which represent 15% of the rearrangements described so far in the literature (BIC). Our estimation of 9.5% is also close to the one reported in the study of 71 American families, i.e. 15% (6/40), that were highly selected for linkage studies and that contributed to the identification of the BRCA1/2 genes (Serova et al., 1996; Puget et al., 1999a). Interestingly, three out of six rearrangements consisted of the exon 13 duplication which is frequent in English-speaking countries (Puget et al., 1999b). A study in the Dutch population has estimated that two deletions account for 36% of BRCA1 mutations (Petrij-Bosh et al., 1997). All in all, the contribution of BRCA1 rearrangements could range from 10 to 30% depending on the presence of frequent mutations due to a founder effect. Thus, screening for large rearrangements is now a step to introduce in BRCA1 genetic testing. Comparative analysis of different screening methods, taking both sensitivity and cost into consideration, are now required. Recent approaches such as quantitative PCR (Laurendeau et al., 1999; Charbonnier et al., 2000), MAPH (Armour et al., 2000) and CGH-array (Bruder et al., 2001) can be promising tools, since they do not require high molecular weight DNA and may be automated. In the 120 cases extensively studied, 46.7% have a BRCA1/2 disease-causing mutation (Table 1). By taking into account the mean predisposition probability of cases (82.1%) and the estimation of the proportion of breast-ovarian cancer families linked to BRCA1 or to BRCA2 locus (95%), a BRCA1/2 mutation was expected in 78% of cases (Ford et al., 1998). The low level of mutation detected (46.7% vs 78%) may be explained by: (i) large BRCA2 rearrangements, which seem, however, infrequent since only two deletions have been reported so far (Nordling et al., 1998; Wang et al., 2001). Indeed, a color bar code of the BRCA2 gene has been performed, and a series is

under screening (Gad et al., in preparation); (ii) undetected mutations in BRCA1/2 coding sequences (including point, small and large alterations); (iii) missense mutations classified as rare variants with unknown biological effect – four BRCA1 and two BRCA2 variants in the present series (Stoppa-Lyonnet et al., unpublished data); (iv) intronic BRCA1/2 mutations, distant from consensus splice sites, relevance of which would not be obvious. Thus, in addition to screening for point mutations and large gene rearrangements, clinical research on hereditary breast-ovarian cancer families is still required in worldwide laboratories, and will surely add complementary approaches to allow reliable genetic testing. Note added in proof Following the submission of this paper, a large BRCA1 rearrangement (IC557) was detected in a collaborative study by Casilli et al. (Human Mutation, in press) ‘Rapid Detection of Novel BRCA1 Rearrangements in High-Risk Breast-Ovarian Cancer Families Using Multiplex PCR of Short Fluorescent Fragments’. Consequently, in our set of 120 French breast and ovarian cancer families, BRCA1 rearrangements account for 4.2% (5/120) of breast-ovarian cancer cases and 11.6% (5/43) of the BRCA1 gene mutation spectrum.

Acknowledgements We wish to thank the patients and their families for their cooperation. We gratefully acknowledge Isabelle Eugene and Sandrine Miglierina for their daily and efficient support in the organization of the genetic clinic. We wish to thank also Isabelle Lambert and Isabelle Bezier for the lymphoblastoid cell line establishment (Geńe´thon, Evry). Thanks also to Stanislas Lyonnet and Mario Tosi for critical reading of the manuscript. This work was supported by the Institut Curie ‘Programme Incitatif et Coope´ratif: Geńe´tique et Biologie des Cancers du Sein’ and the Ligue Nationale Contre le Cancer (A Aurias). S Gad is supported by a fellowship from the Association pour la Recherche contre le Cancer.

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