Integrated Map of the Chromosome 8p12–p21 Region, a Region Involved in Human Cancers and Werner Syndrome

GENOMICS

32, 29–38 (1996) 0073

ARTICLE NO.

Integrated Map of the Chromosome 8p12–p21 Region, a Region Involved in Human Cancers and Werner Syndrome ALEXANDRA IMBERT,* MAX CHAFFANET,*,† LAURENT ESSIOUX,‡ TETSURO NOGUCHI,† JOSE´ ADE´LAI¨DE,† FABIENNE KERANGUEVEN,† DENIS LE PASLIER,§ CATHERINE BONAI¨TI-PELLIE´,‡ HAGAY SOBOL,†,Ø DANIEL BIRNBAUM,*,†,1 AND MARIE-JOSE`PHE PE´BUSQUE*,1 *Laboratoire d’Oncologie Mole´culaire, U.119 INSERM, 13009 Marseille, France; †Laboratoire de Biologie des Tumeurs, Institut Paoli-Calmettes (IPC), 13009 Marseille, France; ‡ U.351 INSERM, 94805 Villejuif, France; §Fondation Jean Dausset, Centre d’Etude du Polymorphisme Humain, 75010 Paris, France; and ØDe´partement d’Oncoge´ne´tique, IPC, 13009 Marseille, France Received August 7, 1995; accepted November 6, 1995

including bladder, breast, colon, liver, lung, and prostate carcinomas (Spurr et al., 1995), has been inferred from studies of loss of constitutional heterozygosity (LOH), which have used polymorphic genetic and microsatellite markers in this region (Weber and May, 1989). Detailed deletion mapping allows identification of two distinct, commonly deleted subregions, with a proximal site located at 8p11.2–p21.3 and a distal site at 8p22–pter (Cunningham et al., 1993; Fujiwara et al., 1993; Chang et al., 1994; MacGrogan et al., 1994; Trapman et al., 1994; Yaremko et al., 1994); these results suggest the presence, on chromosome arm 8p, of at least two tumor suppressor genes implicated in the development of a broad range of cancers. In breast cancer, allelic loss of 8p loci has not been extensively studied compared to other types of tumors or genomic regions (Devilee et al., 1991; Emi et al., 1992; Pykett et al., 1994). We recently reported LOH at several 8p markers located within the proximal consensus region of deletion in a panel of breast tumors (Kerangueven et al., 1994, 1995). In addition, allelotyping of familial breast tumors (Lindblom et al., 1993) and linkage analyses of breast cancer families (Kerangueven et al., 1995) have both suggested the involvement of chromosome arm 8p in inherited breast cancer. This suggests the presence of one or more tumor suppressor genes (hereafter designated TSG) within the proximal region 8p12–p21 involved in sporadic and/or familial breast cancer. Hereditary forms of breast cancer are associated with recently identified susceptibility genes. The BRCA1 gene (located on chromosome band 17q21) was isolated by positional cloning (Miki et al., 1994); its sequence is altered in a high proportion of cases in the Western world (Shattuck-Eidens et al., 1995). A second breast cancer susceptibility locus, BRCA2, was localized to a 6-cM interval on chromosome region 13q12– q13 (Wooster et al., 1994). The first indication of a putative third susceptibility gene, BRCA3, was inferred from the linkage of the 8p12–p21 region with inherited breast cancer in families unlinked to both BRCA1 and

Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome. To improve the definition of the 8p12–p21 chromosomal region, an integrated physical and genetic map was constructed extending from the genes NEFL to FGFR1. The map comprises a series of contigs (the larger of these being around 9 Mb) of yeast artificial chromosomes (YACs) spanning the proximal region of deletion involved in a broad range of human cancers, including breast carcinomas, and in the Werner syndrome. In addition, losses of heterozygosity at 8p markers and linkage analysis of breast cancer families were also detailed. Finally, several genes potentially involved in 8p-associated diseases, namely GTF2E2, PPP2CB, and HGL, were precisely mapped within the YAC contigs. The reported map and contigs of YACs should facilitate the search for putative genes involved in sporadic and familial breast cancer as well as in the Werner syndrome. q 1996 Academic Press, Inc.

INTRODUCTION

The p11–p22 region of the short arm of chromosome 8 is involved in several pathologies, in particular malignant tumors with deletions (see for review Spurr et al., 1995), genomic amplification (Adnane et al., 1991; Theillet et al., 1993; Dib et al., 1995) or translocation (Mitelman et al., 1991), and the Werner syndrome, an autosomal recessive disorder associated with premature aging (Goto et al., 1992; Thomas et al., 1993). Candidate genes for these diseases have not yet been identified. The involvement of tumor suppressor genes from chromosome arm 8p in several types of human cancer, 1 To whom correspondence should be addressed at Laboratoire d’Oncologie Mole´culaire, U.119 INSERM, 27 Boulevard Leı¨ Roure, 13009 Marseille, France. Telephone: 33 91 75 84 07. Fax: 33 91 26 03 64. E-mail: [email protected].

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0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BRCA2 (Sobol et al., 1994; Kerangueven et al., 1995). A positive significant cumulative multipoint lod score of 2.51 was obtained with two markers of the 8p12– p21 region. Although tentative, this result is indicative of the possible localization in the region of a breast cancer susceptibility gene. Finally, genomic amplification of the 8p12 region is observed in 10–15% of breast carcinomas (Adnane et al., 1991; Theillet et al., 1993). A putative oncogene selected during this event could be identical to, or close to, the FGFR1 gene (Dib et al., 1995). To refine the progressing consensus map of the short arm of chromosome 8 (Spurr et al., 1995) and thus to help position candidate breast cancer genes, we have recently used two-color fluorescence in situ hybridization (FISH) and reported the most likely order of loci within the region: i.e., tel–NEFL–D8S131–D8S339–[D8S540/GSR]– D8S124–D8S259–D8S87–FGFR1–cen (Chaffanet et al., 1996), in agreement with previously reported maps (Gyaypay et al., 1994; Buetow et al., 1994; Cox Matise et al., 1994; Spurr et al., 1995). We report here the construction of an integrated physical map within the 8p12–p21 region, extending from the genes NEFL to FGFR1, which includes assembly of yeast artificial chromosome (YAC) contigs, a further definition of the deleted region, and genetic analysis of the 8p12–p21 region. MATERIALS AND METHODS Yeast artificial chromosome clones. YAC clones were isolated from the CEPH YAC libraries (Albertsen et al., 1990; Bellanne´-Chantelot et al., 1992). The entire screening procedure, including identification of the final colony, was carried out by a PCR-based pooling method using region-specific sequence-tagged sites (Green and Olson, 1990). The initial screening was performed with the published sequences of primers and PCR conditions for NEFL (Rogaev et al., 1992), D8S131 (Yu et al., 1994), D8S137 (Tomfohrde et al., 1992), D8S259, D8S278, D8S283, D8S535, D8S505 (Gyapay et al., 1994), and D8S87 (Weber et al., 1990) loci. Preparation of total yeast DNA (in agarose and in liquid form) and characterization of the YAC clones have been previously described (Imbert et al., 1994; Dib et al., 1995). Before further characterization, YAC clones were mapped (not shown) to the 8p12–p21 region and tested for chimerism by FISH; the results are summarized in Table 1. For this purpose, total yeast DNA from YAC clones was used as previously described (Chaffanet et al., 1996). Identification of YAC insert ends. YAC insert ends were obtained by PCR amplification from vectorette libraries using linkers complementary to RsaI sites as described (Riley et al., 1990). The first PCR was performed with primers HYAC-C, HYAC-D, and 224. The second PCR was performed with 224 and heminested primers LS-2 and RA2. The sequences of the primers are HYAC-C, 5*-GCTACTTGGAGCCACTATCGACTACGCGAT-3*; HYAC-D, 5*-GGTGATGTCGGCGATATAGGCGCCAGCAAC-3*; LS-2, 5*-TCTCGGTAGCCAAGTTGGTTTAAGG-3* (for the HYAC-C/224 YAC left insert end product); RA2, 5*-TCGAACGCCCGATCTCAAGATTAC-3* (for the HYAC-D/224 YAC right insert end product). Annealing temperatures for the two former and latter primers were 62 and 657C, respectively (Szepetowski et al., 1996). End PCR products generated from the vector/insert junction of YACs were gel-purified and used for hybridizations of Southern blots of EcoRI-digested DNAs from yeast clones and human placenta. YAC ends devoid of repetitive sequences were selected and cloned into the pUC18 vector using the Sure Clone ligation kit and used as probes. DNA sequencing of cloned fragments was performed using the T7 sequencing kit (Pharmacia) as suggested by the manufacturer.

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DNA probes and hybridizations. The following probes were used: HGL probe, a 0.5-kb HindIII–BamHI fragment from pUC118.hrg Heregulin cDNA (Holmes et al., 1992); GTF2E2 probe, a full-length cDNA from pM10 encoding the 34-Kda small subunit of human TFIIE (Peterson et al., 1991); PPP2CB probe, a 1.6-kb EcoRI fragment in pTZ18 (Hemmings et al., 1990); and human genomic placental DNA. All probes were radiolabeled by oligolabeling, and hybridization analyses were performed as previously described (Pe´busque et al., 1993). Loss of heterozygosity analysis. LOH screening was conducted as previously described (Kerangueven et al., 1994, 1995), except that analysis was performed using an automatic fluorescent sequencing apparatus (Applied Biosystems, Paris, France). Genetic analysis. Eighteen families were acquired from the cancer genetic clinics of Institut Paoli-Calmettes, Institut Curie, and Centre Jean Perrin because they exhibited an aggregation of breast cancer. Families were typed with six markers in their last two or three generations to establish the order and genetic distances (Table 2). These families contained 173 typed individuals, with 96 meioses. Ordering of markers and estimations of genetic distances were achieved using the LINKAGE package (Lathrop et al., 1984). The two-point and three-point analyses were performed using MLINK and ILINK. Three-point orders were compared using Akaı¨ke’s information criterion (Akaı¨ke, 1974). For the lod scores determination, 8 previously described families with inherited breast cancer unlinked to BRCA1 or BRCA2 (Kerangueven et al., 1995) were studied.

RESULTS AND DISCUSSION

The YAC map was anchored to genetic and/or integrated and radiation hybrid maps spanning the 8p12– p21 region (Goto et al., 1992; Schellenberg et al., 1992; Weissenbach et al., 1992; Emi et al., 1993; Wood et al., 1993; Buetow et al., 1994; Cox Matise et al., 1994; Gyapay et al., 1994; Oshima et al., 1994; Spurr et al., 1995) between and including the NEFL (Rogaev et al., 1992) and FGFR1 loci (Lafage et al., 1992). In parallel with this study and to ascertain the involvement of loci of the region in breast cancer, a limited LOH analysis was conducted using the newly ordered set of markers. Isolation and Characterization of YAC Clones The YAC CEPH libraries were screened with nine PCR-based microsatellite markers from the 8p12–p21 region, NEFL (Rogaev et al., 1992), D8S131 (Yu et al., 1994), D8S137 (Tomfohrde et al., 1992), D8S259, D8S278, D8S283, D8S535, D8S505 (Gyapay et al., 1994), and D8S87 (Weber et al., 1990). Possible chimerism of the YACs was assessed by FISH by hybridizing total labeled yeast DNA from various clones to metaphase spreads of normal human chromosomes (not shown). Clones that showed hybridization signals to additional regions were scored as chimeric (Table 1). Sixty-five YAC clones were characterized, and 59% of them were found to be chimeric, with a high proportion identified within the NEFL locus (77%). Table 1 summarizes the relevant information on the YAC clones, including their content in microsatellite markers assessed by PCR analysis with all sets of primers. In addition, D8S513 (Gyapay et al., 1994) was included for subsequent studies. When known, YAC sizes, as determined by PFGE and subsequent Southern blot hybridization with total human placenta DNA (Pe´b-

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MAPPING IN THE 8p12–p21 REGION

TABLE 1 Characterization of YAC Clones 8p12–p21 YAC clones

Size (kb)

46E9 155D10 240G10 686G9 733C10 767G4 769E10 774C1 784E10 802H6 803C1 808F9 926H8 949B8 943C4 981G5 984A3 771G9 791B9 810D6 79F6 154D11 268E1 287E8 443B9 542G2 574A2 688G7 710C12 742F2 888D12 751E7 761A2 978B9 87B11 93A1

350 550 450

1100

1000 1200 750 360 420 420 440 360

1750 1020 1180 1730 1100 1700 440

Chimerisma 0 0 0 / / 0 / / / / / / / / / / / 0 0 0 0 0 0 0 0 / / / / 0 0 / 0 0 / 0

Markersb NEFL NEFL NEFL NEFL, D8S540 NEFL NEFL NEFL NEFL NEFL NEFL NEFL NEFL NEFL NEFL NEFL NEFL NEFL D8S131, D8S137 D8S131, D8S137, GTF2E2 D8S131 D8S278 D8S278 D8S278 D8S278 D8S278 D8S278 D8S278 D8S278 D8S278 D8S278, HGL D8S278, HGL D8S278, D8S259 D8S278, D8S259, HGL D8S278, D8S259, HGL D8S283, D8S535 D8S283, D8S535

YAC clones

Size (kb)

169C5 245A4 248C9 726G12 783C11 802F7 808C7 845G8 849C2 173C12 492A3 574E7 613B12 615E2 627D5 716G9 49A2 336A6 466H9 671E1 725B12 828A12 898G11 909A11 951G11 756E1 798G8 847B12 908C1 896F4c

500

814E11c 763A7c 936G4c 953H12c 866E2c

Chimerisma

Markersb

640 490 460 1430 800 860 960 1100 1600 1200 1190

0 / / / / / / 0 0 / / / / / / / 0 / / / / / 0 0 / 0 0 0 / 0

1040 910 770 1420 1720

0 0 0 0 0

D8S283, D8S535 D8S283 D8S283 D8S283 D8S283, D8S535, D8S513 D8S283 D8S283, D8S535 D8S283, D8S513 D8S283, D8S535, D8S513, D8S259 D8S535 D8S535 D8S535 D8S535 D8S535 D8S535 D8S535 D8S505 D8S505 D8S505 D8S505 D8S505 D8S505 D8SS505, D8S87 D8S505 D8S505 D8S87 D8S87 D8S87 D8S87 D8S339, D8S540, D8S124, GSR, PPP2CB D8S339, D8S540, GSR, PPP2CB D8S540, D8S124, GSR, PPP2CB D8S540, GSR, PPP2CB D8S540, D8S124, GSR, PPP2CB D8S339

780 1590 1660 710 340 1500

250 720 460

a

Chimerism (0) chromosomal location at the assigned site associated with more than 90% of the signals by FISH; (/) location at 8p12– p21 and at least one other site with more than 10% of the signal. b The presence of the microsatellites as well as that of NEFL was assessed only by PCR amplification and that of GTF2E2, HGL, GSR, and PPP2CB only by hybridization. c YAC clones previously isolated and characterized for their marker contents (Chaffanet et al., 1996), except for PPP2CB (this work).

usque et al., 1993; Imbert et al., 1994), are indicated. All chimeric clones were discarded from the subsequent analyses. Integrated Physical Map Covering the Proximal Deletion Region Most of the YAC clones thus characterized were shown to contain more than one 8p12–p21 marker (Table 1), thus allowing us to construct contigs within the region defined by the NEFL and FGFR1 genes. Sixteen nonchimeric YACs that form unlinked subcontigs around five starting points were initially identified: NEFL, D8S131, D8S259, D8S283, and D8S87. Marker content was used to obtain direct information concerning linkage of markers and overlaps of YACs. Additional overlaps between YAC groups were identified by YAC end analyses. In total, 18 end fragments with a

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size up to 1.1 kb were generated from 12 YACs using the vectorette PCR method (Riley et al., 1990). Assembly of all the mapping data, including a previously reported contig centered on D8S339 (Chaffanet et al., 1996) and a contig centered on the FGFR1 gene (Dib et al., 1995), led to the integrated physical map presented in Fig. 1. The two contigs centered on NEFL and D8S131 remain unlinked. Detailed mapping showed that within the NEFL contig, YACs 155D10 and 240G10 were included in YAC 767G4 (1100 kb). Both the right and the left end probes generated from the latter used to characterize additional clones did not allow us to extend the contig, due to the high level of YAC chimerism found in this subregion. The proximal contig comprised the D8S137 and D8S131 markers, and the orientation tel–D8S137–D8S131–cen was based on the published radiation hybrid map of chromosome 8p11.2–p21.3 (Oshima et al., 1994).

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TABLE 2 Families Typed with 8p Markers Families

NEFL D8S259 D8S133 D8S505 D8S535 D8S283

IPC143/20 IPC268/21 IPC304/22 IPC305/23 IPC314/24 IPC339/25 IPC343/26 CLB2/2 CLB3/3 CLB6/6 CLB8/8 CLB9/9 F14/301 F19/303 F20/305 F148/311 00301/202 00003/207

/ / / / / / / / / / / / / / / / / /

/ / / / / /

Total

18

17

/ / / / / / / / / / /

/ / / / / / / / / / / /

/ / / /

/ / / /

/ / / /

/ / / /

/ / / /

/ / / /

12

8

8

8

We next focused our attention on the D8S259 locus, frequently deleted in tumors, and the D8S283 locus, shown to be close to it (Oshima et al., 1994). In total, 13 YACs were identified, and a continuous YAC contig spanning over 3 Mb was thus constructed (Fig. 1). Based on these results, the ordering of markers in this interval was resolved to D8S278–D8S259–[D8S513/ D8S283]/D8S535. As shown in Fig. 1, this contig was linked by its distal and proximal outer limits to that previously described around D8S339 (Chaffanet et al., 1996) and to a more centromeric contig of approximately 2.5 Mb constructed around the D8S87 locus, respectively. Thus, a physical coverage of the region putatively involved in breast cancer was developed over 9 Mb. These physical map results are in accordance with data from our previous analysis using somatic cell hybrids, FISH, and physical mapping (Chaffanet et al., 1996). Localization of Genes within the YAC Contigs The construction of YAC contigs in the 8p12–p21 region helped refine the localization of three genes, namely GTF2E2, encoding the p34 small subunit of the general transcription factor TFIIE (Peterson et al., 1991) and recently localized to 8p12 (Purrello et al., 1994); PPP2CB, formely PP2AB, encoding protein phosphatase-2 (Hemmings et al., 1990) and mapped to 8p12–p11.2 (Jones et al., 1993); and HGL (Heregulin, also called neu differentiation factor) (Holmes et al., 1992), encoding a ligand for members of the ERBB family of tyrosine kinase receptors (Carraway and Cantley, 1994) and assigned to 8p22–p11 (Lee and Wood, 1993). The respective probes for these genes were hybridized to PFGE blots containing all of the nonchimeric YACs from the various contigs. YAC clone 791B9, which contains D8S131 and D8S137,

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was also found to contain the GTF2E2 locus, whereas YACs 771G9 and 810D6 did not (data not shown). Positions of the markers within this contig were thus determined as follows: tel–GTF2E2–D8S137–D8S131–cen. GTF2E2 encodes TFIIE, which regulates TFIIH activity (Ohkuma and Roeder, 1994). The latter is involved in the eukaryotic nucleotide excision-repair pathway (Jaspers and Hoeijmakers, 1995). Since the Werner syndrome is sometimes considered the result of a defect in chromosome stability and DNA repair, GTF2E2 may be considered as a candidate gene for this disease. The exact position of the PPP2CB gene was determined within all the YACs previously characterized except 844E2 (Chaffanet et al., 1996) (Table 1; Fig. 2A). More precisely, using the rare-cutting restriction enzyme map of each YAC, PPP2CB was found to be closely linked to the GSR gene. Figure 2B shows the results of a representative experiment from YACs 896F4 and 953H12. The NotI and SacII patterns revealed by the PPP2CB probe showed fragments of 150 and 60 kb, respectively. Fragments of similar size were visible after hybridization with the GSR probe. Double digests using NotI and MluI gave two fragments of 90 and 60 kb revealed by the GSR and PPP2CB probes, respectively, demonstrating a MluI site within the common NotI fragment. Thus, from the integrated results, the following order, tel–D8S339–[D8S540/ GSR–PPP2CB]–D8S124–cen, could be established. Finally, hybridizations with the HGL probe demonstrated its presence within YACs 978B9, 888D12, 761A2, and 742F2 (Fig. 2A). Given the microsatellite marker content of YACs 888D12 and 742F2 (D8S278) and YACs 761A2 and 978B9 (D8S278 and D8S259) (Table 1; Fig. 1), the HGL gene was localized between D8S278 and D8S259, with the latter being in a centromeric position. HGL and PPP2CB were thus precisely localized. In agreement with our map, HGL has been closely linked to the Werner syndrome locus (Thomas et al., 1993). Furthermore, we have previously shown that these two genes present a low incidence of amplification in breast cancer (Ade´laı¨de et al., 1994; Dib et al., 1995) and thus are probably located at the very end of the 8p12 amplification units, whereas the GSR gene is never found amplified (Dib et al., 1995) and should lie outside the 8p amplicon. Thus, the distal outer limit for the 8p12 amplification was precisely established between GSR and PPP2CB. The integrated map establishes the likely linear order of a large collection of genes and markers within the 8p12–p21 region (Fig. 1), and the following order, tel–NEFL–GTF2E2–D8S137–D8S131–D8S339– [D8S540/GSR–PPP2CB]–D8S124–D8S278–HGL– D8S259– [D8S513/D8S283]/D8S535–D8S505– D8S87–ADRB3– FGFR1–D8S255–cen, could be established. Genetic Map and Linkage Analysis The order of markers D8S133, NEFL, D8S259, and D8S505 was derived from the existing consensus map (Gyapay et al., 1994; Spurr et al., 1995), our previous FISH analysis (Chaffanet et al., 1996), and the physical map reported here and was confirmed by genetic analy-

AP-Genomics

FIG. 1. Map of chromosome 8p12 – p21, extending from the genes NEFL to FGFR1 and integrating genetic distances and YAC contigs. (A) A portion of a published genetic map (Gyapay et al., 1994) of the 8p12 – p21 region showing selected microsatellite markers and their genetic separations (in cM). (B) The markers, genes (italics), and YAC ends are listed in physical order. The order NEFL, D8S339, D8S259, and D8S87 has been confirmed by two-color FISH experiments (Chaffanet et al., 1996). YAC ends are indicated as YAC addresses followed by R or L, for the URA (right) and TRP (left) ends, respectively. GAP indicates absence of overlap. (C) The nonchimeric YACs assembled in contigs are shown. YACs are not drawn to scale, and positions of markers are shown with uniform distances not reflecting actual physical separation. Solid dots indicate the confirmed marker content of each YAC; internal deletions are indicated by interrupted lines within square brackets. Not all YACs are shown. (D) Order and genetic distances (in cM) determined in the present study.

MAPPING IN THE 8p12–p21 REGION

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TABLE 4 Results of the Three-Point Analysis Akaı¨ke’s information criteriona

Markers

Distance estimation (cM)

NEFL, D8S259, D8S505 NEFL–D8S259–D8S505 NEFL–D8S505–D8S259 D8S259–NEFL–D8S505

1543.5 1548.5 1547.7

14; 6.4 15; 5 12; 14

NEFL, D8S259, D8S283 NEFL–D8S259–D8S283 NEFL–D8S283–D8S259 D8S259–NEFL–D8S283

1536.0 1534.8 1547.2

16; 4 14; 4 14; 14

D8S133, NEFL, D8S259 D8S133–NEFL–D8S259 FIG. 2. Localization of genes within the YAC contigs. (A) Pulsedfield gel electrophoresis and Southern hybridization of nondigested YACs. YACs 742F2 (lane 1) and 888D12 (lane 2) were hybridized with the HGL probe. A Southern blot filter containing YACs 844E2 (lane 3), 896F4 (lane 4), and 953H12 (lane 5) was sequentially hybridized with the PPP2CB and GSR probes; note the positivity of YACs 896F4 and 953H12 for both probes. Yeast chromosome marker, used as size standard, is indicated to the left. (B) Restriction mapping of YACs 896F4 (lanes 4) and 953H12 (lanes 5). Restriction enzyme digests of YACs were analyzed by PFGE and sequentially hybridized with the PPP2CB (top) and GSR (bottom) probes. The migration of a l DNA ladder, used as a molecular weight marker, is indicated to the left. Note the same hybridization pattern of both probes within 150-kb NotI and 60-kb SacII fragments. N, NotI; M, MluI; N / M, double digestion NotI / MluI; S, SacII.

sis. Using two-point and three-point analyses (Tables 3 and 4), we propose the order and distances shown in Fig. 1D. As shown in Table 4, the orders NEFL, D8S283, D8S259 and NEFL, D8S259, D8S283 were similar based on Akaı¨ke’s criterion. Although the order NEFL, D8S283, D8S259 had a better Akaı¨ke’s criterion, the two-point analyses with D8S505 favored the second order. The order of markers D8S133, NEFL, D8S259, and D8S505 was found to be compatible with the physical map. This map is in agreement with the

5; 15

a 2LnL / 2k; where k is the number of parameters and L is the maximum likelihood.

previously reported one (Gyapay et al., 1994) (Fig. 1A), except for the distances between D8S259 and D8S505. Available information does not allow us to discriminate between these two maps. Using this map, a new genetic linkage analysis conducted with eight previously described breast cancer families (Kerangueven et al., 1995), yielded a maximum cumulative two-point lod score of 1.79 with D8S505. The maximum three-point lod score was obtained with NEFL and D8S505 (Z Å 2.73). Negative lod scores were obtained with D8S133 and D8S136. Due to the refinement of the map, these figures were higher than in the previous study (Z Å 2.51), although still not significant. They are suggestive of the possible existence of a putative susceptibility locus and are an incentive for setting up a larger study. Involvement of Genes of the Proximal 8p Region in Breast Cancer We have previously reported the pattern of LOH for several 8p loci in a panel of sporadic breast carcinomas

TABLE 3 Recombination Fraction between Markers (Results of the Two-Point Analysis)

D8S133 NEFL D8S259 D8S283 D8S535 D8S505

a

D8S133

NEFL

D8S259

D8S283

D8S535

0.04 [0.01; 0.11]a 0.21 [0.04; 0.50] 0.45 (0.14, 0.50) 0.50 [0.27; 0.50] 0.45 [0.20; 0.50]

0.12 [0.06; 0.23] 0.11 [0.02; 0.33] 0.18 [0.01; 0.50] 0.16 0.04; 0.35

0.03 [0.01; 0.15] 0.15 [0.01; 0.50] 0.06 [0.03; 0.22]

0.00 [0.00; 0.15] 0.03 [0.01; 0.15]

0.10 [0.03; 0.40]

1-LOD interval.

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FIG. 3. Examples of loss of heterozygosity (LOH) determined by 8p microsatellite marker analyses. (A) Computer representation of the polyacrylamide gel analysis of polymerase chain reaction products from tumors 188 and 466 (lower rows) and corresponding blood samples (upper rows). Microsatellite alleles are represented by two (heterozygote) peaks except in the case of homozygosity (noninformative case as in 466/D8S278). LOH at one allele (arrowhead) corresponds to a significant decrease in one of the two peaks. (B) Schematic representation of the distribution of LOH at 8p in 10 breast tumors. Order (according to Gyapay et al., 1994; Spurr et al., 1995; Chaffanet et al., 1995; this work) of the loci examined is represented (top) from telomere (left) to centromere (right). Regions I, II, and III, discussed in the text, are indicated by roman numerals (see also Fig. 4). Results of LOH analysis are represented by squares, as follows: open squares, retention of heterozygosity; black squares, LOH; cross-hatched squares, noninformative; nd, no data. The identification of the selected tumor samples is indicated to the left.

(Kerangueven et al., 1995). To establish further the presence of deletions in this region with respect to the current map, a selected series of 10 representative tumors previously identified as carrying a deletion was studied with an additional set of markers. The results are represented in Fig. 3. Three chromosomal regions (I to III) were artificially distinguished (see also Figs. 1 and 4). Region I is distal to NEFL. Due to a bias in the selection of the panel (LOH at 8p were initially detected with D8S133), LOH patterns always involved this region. LOH were also observed in proximal region III, at loci D8S259 and D8S283. Unfortunately, the other markers were noninformative. Only tumor 553 did not show LOH in this region (although no firm conclusion can be drawn for D8S259). Some tumors (Fig. 3B, top) presented LOH at markers from region II. This is probably due to the presence of a large deletion encompassing the three regions. In contrast, other tumors (middle) showed LOH in regions I and III but not in region II (in each case, at least two markers of this region, or adjacent markers, were not affected). This distribution is probably due to the presence of two distinct deletions, possibly one on each chromosome 8. The availability of the physical map will greatly help the incoming LOH analyses. It is interesting to note that similar results obtained by others on various types of cancers (Fujiwara et al., 1993; Chang et al., 1994; Yaremko et al., 1994) point to the presence of two TSGs at chromosome arm 8p. Some of the genes that were precisely located on the map represent candidates for TSG, in particular PPP2CB and HGL, and LOH patterns are compatible with this hypothesis. The first encodes a phosphatase, a type of protein often associated with negative regulatory circuits of the cell (Sun and Tonks, 1994). The second encodes a ligand for receptors of the ERBB family (Carraway and Cantley, 1994), and plays an important role in the development of the mammary gland.

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Our previous linkage analyses have suggested the possible presence on 8p of a putative breast cancer susceptibility gene (Kerangueven et al., 1995). However, the number (8) and informativity of the families unlinked to BRCA1 or BRCA2 tested was limited, and the derived cumulative lod scores obtained with markers NEFL, D8S259, and D8S505 did not allow a definitive conclusion. Whether this putative gene is identical to a TSG involved in the deletion detected by LOH analysis is not known. The establishment of the physical map and cloning of the region in YACs will help identify possible candidate genes. One gene that could not be mapped in the YAC contigs was LHRH, although it has been localized to 8p11.2–p21 (Yang-Feng et al., 1986). LHRH, encoding the hypothalamic luteinizing hormone-releasing hormone, may be considered as a candidate gene putatively involved in mammary carcinogenesis. In the absence of mapping information, we sequenced the coding exons of the LHRH gene of breast cancer patients from families CLB9 (Table 2) and IPC268/F507 (Kerangueven et al., 1995) with positive lod scores at 8p markers (respectively 0.23 and 0.53). No variation in the sequences could be evidenced (not shown), thus providing evidence against the participation of LHRH to 8p-associated familial breast cancer. In conclusion, the YAC contigs presented in this study provide the first close to complete coverage of the proximal 8p region, in which genes involved in breast cancer could be located. Data are summarized in Fig. 4. It also represents an essential intermediate step in the detailed analysis of other disease genes, especially the Werner syndrome gene. ACKNOWLEDGMENTS We are most grateful to the following scientists for their kind gift of molecular probes: P. T. Cohen (PPP2CB), R. Tjian (TFIIEGTF2E2), Y. Yarden, W. E. Holmes, and R. Vandlen (HGL). We

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IMBERT ET AL. Allione for occasional help, M. Fernandes for advice, and our colleagues of the Molecular Oncology Laboratory for comments. This work was supported by INSERM, Institut Paoli-Calmettes, and grants from the Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, Comite´s des Bouches-du-Rhoˆne et du Var de la Ligue Nationale Contre Le Cancer, FEGEFLUC, and Groupement de Recherches et d’Etudes sur les Ge´nomes. A.I. is the recipient of a fellowship from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche. L.E. was supported by a grant from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche and from the Institut de Formation Supe´rieure aux Sciences Bio-Me´dicales.

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FIG. 4. Comprehensive results from the various analyses of the 8p11–p21 region. A portion of the genetic map for 8p11–p21 is drawn to scale (according to the various maps) showing the loci of all different genes and markers localized within the region (LOCUS), from telomere (top) to centromere (bottom). The different YAC contigs we established (Dib et al., 1995; this work) are shown (YACs). The distances between the various contigs is better appreciated than in Fig. 1. To the right the results of analyses demonstrating the probable involvement of the region in breast cancer are schematized, taking into account previous analyses. The proximal portion of the region is amplified in 10–15% of breast tumors (Dib et al., 1995) (Amplification). Linkage analyses in breast cancer families have yielded positive lod scores with NEFL, D8S137, D8S259, and D8S505 (Kerangueven et al., 1995; this work) and negative lod scores with D8S133 and D8S136. Loss of heterozygosity (LOH) at markers from the region has been evidenced by several authors (see Spurr et al., 1995, for review), and current data tend to distinguish two affected regions (here designated I and III; see also Fig. 3). Putative localization of genes is shown to the far right: TSG (unidentified tumor suppressor genes, involved in LOH), BRCA3 (putative breast cancer susceptibility gene), ONC (putative oncogene selected in the amplification events), and WRN (gene for the Werner syndrome).

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