Oncogene (2001) 20, 4150 ± 4160 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Identi®cation of candidate genes on chromosome band 20q12 by physical mapping of translocation breakpoints found in myeloid leukemia cell lines

Donal MacGrogan*,1,3, Sara Alvarez1,3, Tony DeBlasio1, Suresh C Jhanwar2 and Stephen D Nimer1

1Laboratory of Molecular Aspects of Hematopoiesis, Sloan Kettering Institute for Cancer Research, New York, NY 10021, USA; 2Cytogenetics Service, Department of Human Genetics at Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA

Deletions of the long arm of chromosome 20 have been Introduction reported in a wide range of myeloid disorders and may re¯ect loss of critical tumor suppressor gene(s). To Several lines of evidence point to the importance of one identify such candidate genes, 65 human myeloid cell line or more gene(s) located on the long arm of chromo- DNAs were screened by polymerase chain reaction some 20 whose loss of function contributes to the (PCR) for evidence of allelic loss at 39 highly pathogenesis of multiple myeloid malignancies. Dele- polymorphic loci on the long arm of chromosome 20. A tion of the long arm of chromosome 20 is a common mono-allelic pattern was present in eight cell lines at cytogenetic ®nding in myeloproliferative diseases multiple adjacent loci spanning the common deleted (MPDs), especially in polycythemia vera (p. vera) regions (CDRs) previously de®ned in primary hematolo- patients, where it is often the sole change (Mertens et gical samples, suggesting loss of heterozygosity (LOH) al., 1991; Aatola et al., 1992; Dewald and Wright, at 20q. Fluorescence in situ hybridization (FISH) was 1995; Mitelman, 1995; Kurtin et al., 1996). The 20q then performed using a series of yeast arti®cial deletion is also identi®ed cytogenetically in approxi- chromosomes (YACs) ordered in the CDR, and in ®ve mately 5% of myelodyplastic syndrome (MDS) of eight cell lines, the deletions resulted from cytogen- patients, and in a fraction of acute myeloid leukemia etically detectable whole chromosomal loss or large (AML) cases, generally in association with other interstitial deletion, whereas in another cell line deletion abnormalities (Kurtin et al., 1996, Mitelman, 1995). was associated with an unbalanced translocation. LOH Deletion of 20q has been rarely observed in lympho- in the CMK megakaryocytic cell line, which has a proliferative disorders (Mitelman, 1995) and in lym- hypotetraploid karyotype, was associated with a phoblastoid cell lines derived from MDS or Ph-positive der(20)t(1;20)(q32;q12)x2 leading to complete deletion acute lymphoblastic leukemia patients (Hollings et al., of the CDR. Three additional unbalanced translocations 1995) indicating that the deletion may have occurred in were found within the CDR and all three breakpoints a progenitor cell with both myeloid and lymphoid mapped to a single YAC. We then used a series of P1 potentiality (White et al., 1994; Hollings et al., 1994; arti®cial chromosomes (PACs) spanning this YAC clone, Knuutila et al., 1994). and two PACs produced `split' signals suggesting that Allelic deletion of microsatellite loci on 20q has been they each span one of these breakpoints. Exon trapping found in only 8% of cytogenetically normal patients using PACs that overlap the breakpoint regions yielded with p. vera or myelo®brosis (Asimakopoulos et al., portions of six genes and evaluation of these genes as 1996), suggesting that loss of heterozygosity (LOH) is candidate tumor suppressor genes is underway. The principally due to cytogenetically visible deletions in p. limited information available about these genes suggests vera. In chronic myelogenous leukemia (CML), LOH that the h-l(3)mbt gene is the most attractive candidate. of 20q was found at microsatellite loci in 29% of 30 Oncogene (2001) 20, 4150 ± 4160. blast crisis patients, comparing DNA from blast crisis phase cells to DNA from chronic phase cells (Mori et Keywords: myeloid leukemia; 20q deletion; loss of al., 1997); this suggests that LOH may involve heterozygosity recombination or chromosome loss followed by duplication in these cases. These observations suggest a recessive model of myeloid neoplasia pathogenesis whereby inactivation of a putative suppressor gene(s) on 20q disrupts normal hematopoietic stem cell regulation (Asimakopoulos and Green, 1996). Deletion mapping studies using conventional cyto- genetics (Nacheva et al., 1995), FISH (Roulston et al., *Correspondence: D MacGrogan 1993) and microsatellite PCR/Southern analysis (Asi- 3These authors contributed equally to this work Received 28 November 2000; revised 9 April 2001; accepted 12 makopoulos et al., 1994), have delineated a common April 2001 deleted region (CDR) within the proximal two-thirds Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4151 of band 20q12. Wang et al. (1998) narrowed this segment to an area of 8 Mb ¯anked by a proximal boundary between markers D20S206 and D20S107, and a distal boundary between D20S119 and D20S424. Allowing for the possibility of multiple culprit genes on 20q, Bench et al. (1998) combined their own data with the Wang data to delineate two overlapping CDRs in patients with MDS or MPD that are 7 ± 8 and 8 ± 9 Mb in size, respectively. A distal boundary common in both diseases was mapped between D20S119 and D20S481, whereas the proximal boundary was found between D20S174 and D20S465 in MPD samples and D20S465 and PLC1 in MDS samples (Bench et al., 1998). To further re®ne the commonly deleted region in myeloid malignancies (in particular AML and CML), and identify candidate genes located in the 20q12 region, we obtained and screened a large collection of myeloid leukemia cell lines for large-scale mutations (i.e. homozygous deletion or chromosomal transloca- tion), that can inactivate gene function. Because tumor suppressor gene inactivation often involves mutation (nucleotide substitution, small insertion or deletion) in Figure 1 PCR-based analysis of ®ve chromosome 20 q micro- one copy of the gene, followed by loss of the remaining satellite loci in myeloid leukemia cell lines. Cell lines demonstrat- copy by deletion (Cavenee et al., 1983), we performed ing a mono-allelic pattern of ampli®cation are CMK, HEL, KBM-5, KBM-7, MEG-O1, MOLM-1, MONO-MAC-1, and PCR analysis of highly informative microsatellite di- U937. Examples of cell lines showing a bi-allelic pattern of and tetranucleotide loci along the long arm of ampli®cation are K562, MO-7e, KASUMI-1, ML-1, HL60, chromosome 20, to identify LOH. Cells exhibiting a NB4.306, and TF-1. The background band seen at D20S119 mono-allelic pattern of ampli®cation were screened for and D20S481 in KBM-7 might be due to the presence of a minor additional deletions and rearrangements by Southern population in this cell line blot and FISH analyses and three translocation breakpoints were found by FISH in cells from one line (CMK) and were mapped to PAC clones. D20S174 and D20S481 (see Figure 2). This phenom- Subsequent exon trapping experiments using these enon is most readily explained by the presence of only PAC clones identi®ed six candidate genes which could one chromosome 20 homologue (the presence of two be involved in the pathophysiology of hematologic bands at D20S481 in MOLM-1 and at D20S465 in malignanices that contain 20q12 abnormalities. U937 is likely a PCR artifact, as numerous contiguous markers proximal or distal to these loci were found to be homozygous). Four cell lines (CMK, HEL, MONO- MAC-1 and U937) demonstrated interstitial or term- Results inal deletions, with breakpoints mapping centromeric to marker D20174, which is located at the proximal Microsatellite and Southern blot analysis of myeloid end of the CDR de®ned using MPD patient samples leukemia cell lines (Figure 2). A mono-allelic pattern was found at all 20q Sixty-one of 65 myeloid leukemia cell line DNAs were and 20p loci in the KBM-5 and KBM-7 cell lines and analysed by PCR at 43 microsatellite loci on chromo- for all but one marker in MEG-O1 and MOLM-1 cells, some 20, whereas four cell lines (AS-E2, MR-87, NS- suggesting reduction to monosomy for chromosome 20. MEG, TMM) were evaluated at eleven 20q loci only. Microsatellite instability (indicated by the laddering of A typical PCR generates one or two bands at PCR products) was detected at many di€erent loci in polymorphic loci in the majority of cell lines, MKB-1 and RC-2A cells, whereas it was occasionally suggesting the presence of both chromosome 20 observed in KCL-22 cells (data not shown). homologues (see Figure 1, e.g. HL-60 or TF-1). Allelic PCR-based ampli®cation failed repeatedly at imbalance was noted at several scattered heterozygous D20S110 in CMK cells, at D20S836 in U937 cells, loci in K562, LAMA-84, SAM-1, TF-1 and HEL cells, and at D20S881 in MEG-O1 cells, however new sets which are all erythroid cell lines; the absence of of primers in nested or semi-nested ampli®cation ampli®cation bias involving the smaller alleles at these reactions (see Materials and methods), led to success- loci suggests that the allelic imbalance re¯ects an ful ampli®cation at D20S110 and D20S836; however unequal dosage of chromosome 20. Eight cell lines ampli®cation at D20881 failed repeatedly, suggesting (including CMK and U937, shown in Figure 1) showed the presence of a homozygous deletion at this locus, ampli®cation of only one allele at multiple adjacent which was con®rmed by Southern blot analysis loci, including 19 loci mapping to the CDR de®ned by (Figure 3).

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4152

Figure 2 Summary of PCR analysis conducted on 43 microsatellite repeat loci on chromosome 20. The order of these markers was obtained from the public database at the Whitehead Institute Center for Genome Research, as accessed through the Internet. Common regions of deletion in myeloproliferative disaease (MPD) and myelodysplatic syndrome (MDS) patients are indicated on the right (Bench et al., 1998). Thick and thin lines represent minimum and maximum areas of loss, respectively

were probed using a set of 37 cDNA clones (corresponding to 27 UniGenes and 10 anonymous ESTs) characterized for hybridization to single copy sequences and correct chromosomal mapping (Table 1). The advantage of screening EST markers, compared to STS markers, is that they are more likely to detect functionally signi®cant deletions a€ecting expressed genes. However, this analysis failed to uncover additional homozygous deletions or smaller rearrange- ments.

Figure 3 Southern blot analysis showing homozygous deletion FISH analysis of cell lines of D20S881 in MEG-O1 cells. EcoRI and/or HindIII-digested genomic DNA from myeloid leukemia cells was hybridized with a To identify large rearrangements, FISH was applied to probe speci®c for the D20S881 locus, and control probes for the eight mono-allelic cell lines, using eight YAC clones MYBL2 and thrombomodulin (THBD) genes, which locate telomeric and centromeric to D20S881, respectively. A hybridiza- localizing to the CDR de®ned by ¯anking markers tion signal at D20S881 (using a 341 bp probe generated by PCR D20S174 and D20S119 (808C5, 804C12, 834H3, using D20S881-2f and -2r as primers) is observed in control cells 936E4, 912C3, 777D11, 909F8, 958C12) and one (KBM-5, KBM-7, MOLM-1), but is absent in MEG-O1 cells telomeric YAC clone (845H4). For each cell line, at despite equal loading of DNA least two YAC probes were used along with CEP20 and WCP20. Two cell lines (CMK and MONO-MAC-1) showed rearrangements within the CDR. In CMK cells, two To identify additional homozygous deletions or populations were found with a tetraploid modal intragenic rearrangements contained within the CDR, chromosome number. In population I (60% of the Southern blots containing EcoRI and HindIII-digested cells), ®ve unbalanced rearrangements were identi®ed genomic DNA from the eight mono-allelic cell lines as der(20)t(1;20)(q31;q11)x2, der(1)t(1;20)(p32;q12),

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4153 Table 1 Characterization of cDNA probes used for Southern blot analysis of mono-allelic 20q cell line DNA Hybridizing genomic Marker or UniGene IMAGE Insert Size Hybridizing fragment (kb) locus cluster clone Identity or similarity cut (kb) YAC clone(s) HindIII EcoR1

R60956 42506 EST N/E 1.7 942B1 2.3 ND stSG55010 Hs. 9115 965016 Hs MAFB mRNAa N/E 0.6 808C5 4.3 5.1 SGC44273 Hs. 55366 1402861 EST N/E 0.6/0.5 808C5 9/3.5 9.5/2 R98337 201621 EST P/E 0.8 808C5 8.5 5 stSG12833 Hs. 114017 429692 EST P/E 1.3 808C5/804C12 5 ND stSG15092 Hs. 189726 125067 EST P/E 0.6 808C5/804C12 4.5 2 stSG3032 Hs. 167839 341127 Hs KIAA0395 mRNA N/E 1.3 808C5/804C12 3.5 10 TOP1 Hs. 317 112626 Topoisomerase (DNA) I P/E 1.5 808C5/804C12 23/12/7.5 7.5/3 PLCG1 Hs. 993 588719 Phospholipase C gamma 1 E/X 1.9 808C5/804C12 8 4.3 sts-Z40239 Hs. 7918 486173 Hs hypothalamus protein (HSMNP1) N/E 0.8 808C5/834H3 13 21 stSG27969 Hs. 70704 488201 EST N/E 0.4 804C12 20 18 stSG3021 Hs. 91603 49015 Hs KIAA0283 mRNA N/H 1.7 804C12/834H3/936H3 12 10 N31944 272015 EST N/E 0.8 936H3 4.5 10 AA279992 705070 EST E/X 0.9 905D3 1.9 3.4 WI-6622 Hs. 22237 42082 Hs (3)mbt protein homolog mRNA N/H 2.1 912C3 8.5/5 9.5/3 SGC36858 Hs. 24994 1676403 Hs CGI-53 protein mRNA N/E 1.9 912C3 18/12b/8.5/ ND 8b/4/2.5 MYBL2 Hs. 179718 490234 Myb related protein B N/E 1.8 912C3 2.3 8 sts-T63166 79307 EST E/X 0.7 912C3/777D11/909F8 3.5 2 stSG40369 Hs. 26608 41332 Trinucleotide repeat containing 9 N/H 1.8 912C3/777D11/909F8 7 6.5 (TNRC9/CAGF9) stSG34043 Hs. 75798 966288 EST N/H 1.6 912C3 2.4 13 SGC33922 Hs. 119607 123938 EST N/E 0.6 912C3/951F2/909F8/ 22 ND 742F1 sts-AA010225 430239 EST N/E 0.7/0.4 912C3/951F2/742F1/ 20 3 958C12 NIB331 Hs. 6891 362023 Hs splicing factor SRp55-1 (SRp-55) N/E 1.2 951F2/742F1/958C12 4.3c 7c sts-AA025063 365057 EST N/E 0.5 951F2/742F1/958C12 4.6 7 stSG8505 Hs. 76329 52431 Human placenta (Diff33) mRNA N/H 1.8 951F2/742F1/958C12 5/4.5/4/3 6 stSG16479 122269 EST N/E 0.6 951F2/742F1/958C12 20 12 stSG34079 110208 EST N/E 0.5 951F2/742F1/958C12 8.5 5.5 WI-10260 Hs. 250824 40915 EST N/H 1/0.5 958C12 4.8c 6.5c 2703 Hs. 106233 35730 EST N/H 0.9 951F2/909F8 10/8/3.5 9/4.5 stSG10931 Hs. 8060 1639951 EST N/E 0.5 909F8 10 8 SHGC13528 Hs. 172182 418006 PolyA binding protein, cytoplasmic 1 P/E 1.3 909F8/742F1 4.5/2.5/2.1 10 YWHAB Hs. 182238 223240 Brain protein 14-3-3 beta isoform N/E 1.5 909F8/742F1 4.5 2/1.8 PKIG Hs. 3407 133800 Protein kinase inhibitor gamma N/E 1.2 742F1 5/4.5 20 hTOM34 Hs. 76927 Translocase of outer mitochondrial B/S 0.9 909F8/742F1 4/3/1.9 6/4/3.5 membrane 34 hHNF4A Hs. 54424 Hepatocyte nuclear factor 4 alpha E 2.1 909F8/742F1/958C12 21/20/3.5 20/7/6.5 A004C16 23400 EST N/H 2 742F1/958C12 4 ND STK4 Hs. 35149 Serine/threonine kinase 4 B/S 1.4 742F1/958C12/800A11 8.5/7.5/6 12/10/9.5 aHs=Homo sapiens, B=BamH1, E=EcoR1, H=HindIII, N=Notl, P=Pacl, S=Sall, X=Xhol. ND=not done. bVariable fragment. cAdditional smear

der(6)t(6;20)(p11;q13), der(13)t(13;20)(p11;q12)x4 and der(6)t(6;20) and the der(13)t(13;20)x4 rearrangements. a marker chromosome (mar1) (Figure 4a). None of the Population II of CMK (40% of the cells) contained the YACs showed signals in the der(20)t(1;20)x2, suggest- der(20)t(1;20)(q31;q11)x2 and the der(13)t(13;20) ing that this rearrangement was accompanied by a (p11;q12)x4, but lacked the der(1)t(1;20), the der(6)t deletion involving the entire CDR (which may explain (6;20), and the mar1. We used nine PAC clones with the mono-allelic pattern detected by microsatellite markers common to YAC clone 912C3, which we used PCR). The breakpoints of three rearrangements (the as probes for FISH (Table 2). PAC clone dJ1118N11 der(1)t(1;20), the der(13)t(13;20)x4 and the mar1) were spanned the der(1)t(1;20)(p32;q12) breakpoint (see mapped within YAC clone 912C3 (Figure 4b). 777D11 Figure 4c), whereas PAC clone dJ440H8 spanned the and 909F8 are YAC clones that overlap with the der(13)t(13;20)x4 and the mar1 breakpoints. telomeric end of 912C3 and both clones were found to In MONO-MAC-1 cells, a cell line with a span the der(13)(13;20)x4 and the mar1 breakpoints. hyperdiploid modal chromosome number, several Using centromeric YACs 808C5, 834H3, and 936E4, unbalanced rearrangements were seen, with deletion only two cytogenetically normal chromosome 20 were of the proximal portion of the CDR (Figure 4d). YAC detected. The telomeric YAC clone 845H4 detected the clones 808C5 and 804C12 showed only one signal on

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4154

Figure 4 FISH analysis of six cell lines mono-allelic for 20 q. Metaphase cells from CMK population I hybridized with WCP20 (a, and c, left panel), YAC 912C3 (b), and PAC dJ1118N11 (c, right panel). In b, the der(20)t(1;20) is associated with absence of signal (x) suggesting deletion that might explain the LOH observed by microsatellite-PCR in these cells. The presence of positive signals (yellow arrows) in two normal chromosome 20, 4 der(13)t(13;20), a der(1)t(1;20), and a mar1 suggests that YAC 912C3 spans three breakpoints. In c, signals are present in the two normal chromosome 20 and the der(1)t(1;20) but not the der(13)t(13;20), suggesting that dJ1118N11 spans the der(1)t(1;20) breakpoint only. In d, a merge image of a metaphase cell from MONO-MAC-1 hybridized with WCP20 (red) shows rearrangements of chromosome 20 and translocation of YAC 912C3 (green) to a der(4)t(4;20). In e, metaphase cell from HEL hybridized with CEP20 (red), and YAC 936E4 (green) indicating the presence of a del(20)(q11), and other rearrangements involving 20 q. In f, three MEG-O1 interphase cells are hybridized with CEP20 (red) and YAC 804C12 (green). The middle cell belongs to a tetraploid population with four CEP20 signals, and four 804C12 signals. The other two cells belong to the hyperdiploid population with three CEP20 signals and two 804C12 signals, indicating deletion of 20 q material. In g, interphase cell from MOLM-1 demonstrating two CEP20 signals (red) and two YAC 804C12 signals (green), suggesting chromosome 20 loss in this triploid cell line. In h, an interphase cell from U937 shows three CEP20 signals (red), and three YAC 834H3 signals (green), indicating that LOH occurred by chromosome 20 loss and reduplication in this triploid cell line

chromosome 20, whereas all YACs telomeric to 936E4 some 20s and no signal in the del(20)(q11), showed two signals, one at the chromosome 20 and der(20)t(?;20)(?;q11), or r(20) chromosomes (Figure one on a der(4)t(4;20)(q35;q12). Allelic loss was found 3e). MEG-O1 cells consisted of two populations, throughout the length of chromosome arm 20q (see including a 52n+4 population (*66% of the cells) Figure 1), suggesting the der(4)t(4;20)(q35;q12) oc- that contained two normal chromosomes 20 and a curred after chromosomal loss and duplication. del(20)(q11). A 54n4 population (*34% of the Three cell lines (HEL, MEG-O1, MOLM-1) showed cells) with four chromosome 20s was also present complete loss of the CDR. HEL cells (modal number, (Figure 3f). MOLM-1 cells (modal number, 53n4) 53n4) showed hybridization signals in two chromo- (Figure 3g) showed whole chromosome 20 loss. In

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4155 three cell lines (KBM-5, KBM-7, and U937), despite were mapped to several PAC clones that encompass the presence of only one PCR detectable 20q allele, no clone dJ440H8. cytogenetic loss of material was found (Figure 3h). A second approach involved exon-trapping, using Allelic deletion may have occurred by chromosomal overlapping PAC clones at or near the translocation loss followed by duplication of the remaining chromo- breakpoints, to identify sequences previously mapped some in these cases. by Southern, as well as potentially novel expressed sequences. The exon-trap products for PAC clone dJ637C15 (overlapping with dJ1118N11) yielded a Identification and expression of genes at translocation portion of a serum-glucocorticoid regulated kinase breakpoints gene (SGK), in addition to exons from the CGI-53 Two parallel approaches were taken to identify genes and h-l(3)mbt genes. These exon sequences matched located at or near the translocation breakpoints sequences in dJ138B7 (partially overlapping with identi®ed in CMK cells by dJ1118N11 [the dJ1118N11) in a BLAST analysis. Using dJ1028D15 der(1)t(1;20) breakpoint] and dJ440H8 [located at both (which is overlapping with dJ1118N11), exons for the der(13)t(13;20)x4 and mar1 breakpoints]. First, MYBL2 were trapped and using two PAC clones that DNAs from a series of PAC clones that overlap overlap with dJ440H8 (dJ495O3 and dJ1108D11), we dJ1118N11 and dJ440H8 were hybridized by Southern found the previously mapped EST marker (sts- analysis to EST probes previously mapped to YAC T63166), and the TNRC9/CAGF9 gene; the presence clone 912C3 (Table 1). Hence, IMAGE clones of these genes in these PACs was con®rmed by BLAST 42082,1676403, and 490234 containing portions of search. Querying the UniGene database at NCBI human l(3)mbt (h-l(3)mbt), CGI-53, and MYBL2 provided information that four UniGene clusters, genes, respectively, mapped by Southern analysis to a which correspond to the h-l(3)mbt, CGI-53, MYBL2 region that contains several contiguous PACs that and TNRC9/CAGF9 genes, are contained in many overlap with clone dJ1118N11 (Table 3). IMAGE di€erent cDNA libraries demonstrating that they are clones 79307 and 41332, which contain portions of the actively transcribed genes rather than processed sts-T63166 and TNRC9/CAGF9 genes, respectively, pseudogenes. The EST marker sts-T63166 was found

Table 2 Summary of FISH results using 20q12 PAC clones for analysis of CMK cells Metaphases Modal number WCP 20 dJ138B7 dJ637C15 dJ1118N11 dJ453010 dJ622K8 dJ259J9 dJ11N4 dJ440H8 dJ1108D11 analysed

Population I (88 ± 92)54n-4 chr.20 + + + + + + + +w 7 114 (60%) chr.20 + + +w 77777 7 der(20)(1;20)62 77 7 777777 der(13)(13;20)64 77 7 7777++ der(1)(1;20) 77 +++++77 der(6)(6;20) 77 7 777777 mar(1) 77 7 7777++

Population II (88 ± 92)54n-4 chr.20 + + + + + + + +w 7 76 (40%) chr.20 + + + + + + + +w 7 der(20)(1;20)62 77 7 777777 der(13)(13;20)64 77 7 7777++ aPer cent of each population based on WCP 20 metaphase analysis, +: positive signal, 7: absence of signal, +w: weak positive

Table 3 Genes located at or near translocation breakpoints in band 20q12 Expression Transcript Breakpoint Hybridizing PAC clonea Exon trapping Sequenceb Pc BM CMK h-l(3)mbt der(1)t(1;20) dJ138B7, dJ637C15 dJ637C15 dJ138B7, dJ862K6 + + + SGK dJ637C15 dJ138B7 + (+) + CGI-53 dJ138B7, dJ637C15, dJ1028D15 dJ637C15 dJ138B7, 1028D15 + + + MYBL2d dJ1028D15, dJ453O10 dJ1028D15 dJ1028D15 7 ND + sts-T63166 der(13)(13;20)64 dJ11N4, dJ495O3 dJ495O3 dJ495O3 (+) 77 TNRC9/CAGF9 and mar (1) dJ575O1, dJ1108D11 dJ1108D11 dJ1108D11 + + + aRepresentative PACs are indicated. bAccession nos Z98752 (dJ138B7); AL031681 (dJ862K6); AL121886 (dJ1028D15); AL1211587 (dJ495O3); AL034419 (dJ1108D11). cP=placenta; BM=bone marrow; CMK=CMK cells; + product present; 7 absence of product; (+) low level expression; ND not done. dExpression by Northern blot. All others by RT ± PCR

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4156 in only one library, and may not represent a true gene. tic/erythrocytic (four of 25) cell lines. Three of the four Four of the candidate genes (h-l(3)mbt, CGI-53, SGK allelic losses seen in CML occurred by mitotic non- and TNRC9/CAGF9) were found to be expressed in disjunction, with or without reduplication, which is human placenta, in bone marrow (BM) and in CMK thought to be the principal mechanism of 20q- cells by RT ± PCR (Table 4). MYBL2 expression was associated LOH in CML patients (Mori et al., 1997). found in CMK cells by Northern blot analysis, and sts- In contrast, LOH was associated with cytogenetically T63166 expression was detected in placenta but not visible rearrangements or deletions of 20q in AML BM or CMK cells by RT ± PCR. cells, similar to the abnormalities found in p. vera (Asimakopoulos et al., 1994) and MDS or AML patients (Wang et al., 1998). The variable nature of Discussion allelic losses found in p. vera, MDS, AML and blast crisis of CML may re¯ect the existence of separate The large size and complexity of 20q deletions in target gene(s) on 20q (Asimakopoulos and Green, primary samples and the availability of hundreds of 1996). EST markers and genomic clones mapped to chromo- The unique homozygous deletion found at D20S881 some band 20q12, prompted us to screen a large in MEG-O1 cells is, to our knowledge, the ®rst collection of cell lines derived from AML and CML homozygous deletion to be identi®ed on chromosome patients in blast crisis for deletions and rearrangements arm 20q in any neoplastic cell type. The ®nding of that would help narrow the consensus region of homozygous deletions in cultured cells or cultured deletion and facilitate the identi®cation of candidate xenografts derived from patient samples, has greatly genes. The frequency and nature of allelic deletions of facilitated the detection and cloning of tumor suppres- 20q in the present set of myeloid leukemia cell lines is sor genes critical to the pathogenesis of solid tumors largely consistent with previous studies of primary (Nobori et al., 1994; Schutte et al., 1995). Homozygous samples, which serves to validate our approach. Allelic deletions are rare in hematological malignancies, losses were detected in four of 20 CML blast crisis cell although they have been found at sites of frequent lines (20%), which is slightly lower than the 29% allelic loss, including 9p21 in acute lymphoblastic incidence of allelic loss detected in CML blast crisis leukemia (Hirama and Koe‚er, 1995) and 13q14 in patient samples (Mori et al., 1997). Among 42 AML- B-Cell chronic lymphoblastic leukemia (Liu et al., derived cell lines, allelic losses were found in one AML 1995), both of which are known tumor suppressor loci. M5 (MONO-MAC-1), one AML M6 (HEL), and one Homozygous deletions have been found also in regions AML M7 (CMK) cell line, indicating no association of inherent DNA instability or at fragile sites with any particular FAB subtype. Consistent with these associated with chromosomal rearrangements in cancer observations, deletions of 20q were represented in cells (Paige et al., 2000), neither of which have been myelo-monocytic (four of 18) as well as megakaryocy- reported for chromosome band 20q12.

Table 4 Leukemia cell line screening panel, by type and origin Lymphoid Myelo-monocytic Erythroid (continued) BV173 CML-lymBC AML-193 AML M5 JK-1 CML-myBC CML-T1 CML-lymBC EOL-1 AML M4 K-562 CML-myBC GDM-1 AML KMOE-2 AML M6 Myeloid KBM-3 AML M4 KU 812 CML-myBC AML5q- AML M2 KBM-5 CML-myBC LAMA-84 CML-myBC AML14 AML M2 KBM-7 CML-myBC TF-1 AML M6 EM-2 CML-myBC ML-1 AML M4 SAM-1 AML M6 HL-60 AML M2 ML-2 AML M4 KASUMI-1 AML M2 MONO-MAC-1 AML M5 Megakaryocytic KCL-22 CML-myBC MUTZ-3 AML M4 CHRF-288-11 AML M7 KG-1 AML M2 MV-4-11 AML M5 CMK AML M7 KYO-1 CML-myBC NB4.306 AML M3 HIMeg-1 CML-megBC GF-D8 AML OCI-AML2 AML M4 HU-3 AML M7 MKB-1 AML OCI-AML5 AML M4 MB-02 AML M7 MO-91 AML M0 SKH-1 CML-myBC MC3 CML-my/megBC MR-87 CML-myBC THP-1 AML M5 MEG-O1 CML-megBC MUTZ-1 RAEB RC-2A AML M4 MO-7e AML M7 MUTZ-2 AML M2 U937 `Histiocytic MOLM-1 CML-megBC PL-21 AML M2 lymphoma' MKPL-1 AML M7 RW-leu4 CML-myBC M-MOK AML M7 TMM AML Erythroid NS-MEG CML-megBC UCSD/AML1 AML AP-217 AML M6 RS-1 AML M7 Z-43a RAEB-t AS-E2 AML M6 T-33 CML-megBC Z-55a AML HEL AML M6 UT-7 AML M7

AML, acute myeloid myeloid leukemia; CML, chronic myeloid leukemia; -myBC, myeloid blast crisis; -megBC, megakaryocytic blast crisis; -lymBC, lymphoid blast crisis; RAEB, refractory anemia with excess blasts; RAEB-t, RAEB in transformation. aLymphoblastoid cell line

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4157 The signi®cance of the homozygous deletion identi- by the der(1)t(1;20), it will be necessary to quantitate ®ed here is uncertain because it was found to involve the level of candidate gene expression. only one microsatellite marker locus, that is located During the preparation of this study, Bench et al. outside of the consensus region of deletion de®ned (2000) and Wang et al. (2000) have reported narrowing using patient samples (Wang et al., 1998; Bench et al., the 20q- CDR using primary patient samples. Bench et 1998). Bench and colleagues examined 28 patient al. (2000) de®ned a new region in MDS of 2.6 Mb and samples with 20q loss by PCR at 32 STS markers in MPD of 2.7 Mb with an overlapping (`myeloid') located within the CDR and they did not uncover any region of 1.7 Mb. Interestingly this new `myeloid' CDR homozygous deletions (Bench et al., 1998). Our encompasses both breakpoint regions found in CMK Southern blot analysis of this region in eight mono- cells, and includes all six candidate genes identi®ed in allelic 20q cell lines did not detect any additional our study. A new *250 kb MDS/AML region (over- deletions or intragenic rearrangements at 37 genes/ lapping with the new MPD region of Bench et al., ESTs. Given that 20q12 is a Giesma-staining dark 2000), was identi®ed by Wang et al. (2000) but it is band, which is predicted to be gene-poor (Strachan and located distal to the MDS region de®ned by Bench et Read, 1996), the average EST density of 4 ± 5 Mb used al. (2000). This di€erence may re¯ect the fact that the in our screen may have been sucient to cover most new MDS/AML CDRs were established on the basis genes located within the CDR, although smaller of two cases of MDS in the Bench series and two cases deletions may still be missed. in the Wang series (one of AML-M2 and one of The analysis of CMK cells revealed allelic loss due to MDS); these events may be associated with separate an unbalanced rearrangement accompanied by a series genes. of unbalanced translocations in the remaining allele. In summary, we screened numerous myeloid leuke- The translocation breakpoints in the remaining allele mia cell lines and we identi®ed one cell line, CMK, that map within the minimally deleted region in myeloid contains a series of translocations within the CDR malignancies as de®ned by LOH and cytogenetic found in patients with 20q deletions. We have deletion, and as such, may have disrupted one copy identi®ed several candidate genes, including a human of a putative myeloid leukemia tumor suppressor gene. homologue of a Drosophila tumor suppressor gene A precedent for a two-hit model involving transloca- (h-l(3)mbt), that may be relevant to myeloid leukemia tion and deletion of a key gene was found in cells from pathogenesis. Further analysis of the role of these neuro®bromatosis type 1 (NF-1) patients, where candidate genes in the growth of leukemia cells and the balanced translocations a€ecting a 17q11.2 breakpoint regulated process of normal hematopoiesis is war- is accompanied by LOH at the other allele (Fountain ranted. et al., 1989). Subsequent molecular analysis identi®ed a complete loss of expression of the NF-1 tumor suppressor gene, which was interrupted by the translocation (Wallace et al., 1990; Viskochil et al., Materials and methods 1990). The translocations identi®ed here are unba- lanced and found in a cell line that has a highly Cell lines and DNA extraction complex chromosomal pattern. However they are The cell lines listed in Table 4 were obtained from various located in the region of recurrent deletion in primary laboratories around the world, or were purchased from either samples, and may be of pathological relevance. the Deutsche Sammlung von Mikroorganismen und Zellk- We have identi®ed a set of candidate genes that lie uluren GmbH (Braunswheig, Germany) or the American within the 20q- breakpoint regions using Southern- Type Culture Collection (Rockville, MD, USA). Cell lines based mapping, exon-trapping, and database searches. were grown in RPMI1640 medium supplemented with 10 ± These include known and previously mapped genes 20% fetal bovine serum. A list of culture conditions and (MYBL2, TNRC9/CAGF9, h-l(3)mbt) (Noben-Trauth origin of each cell line will be provided upon request. High molecular weight genomic DNA was prepared from each cell et al., 1996; Margolis et al., 1997; Koga et al., 1999), a line using standard methods (Sambrook et al., 1989). mapped UniGene (CGI-53) (Lai et al., 2000), an unknown mapped EST (sts-T63166), and an exon- trapped transcript not previously mapped (SGK). The PCR-based microsatellite analysis h-l(3)mbt gene encodes a member of the polycomb A set of 43 microsatellite markers on chromosome 20 (42 group family of proteins and is the human homologue dinucleotide and one tetranucleotide) with a mean hetero- of a tumor suppressor gene in Drosophila (Koga et al., zygosity value of 0.76 (range 0.5 ± 1), was identi®ed at the 1999). Based on known and putative functions, the Whitehead Institute/MIT Center for Genome Research h-l(3)mbt is an attractive candidate gene and will be Human Physical Mapping project (Dib et al., 1996) (http:// prioritized for mutational and functional analyses. The www-genome.wi.mit.edu/). Premade pairs of oligonucleotides were obtained from Research Genetics (Huntsville, AL, candidate genes expressed in BM were found tran- USA) (ftp://ftp.resgen.com/pub/mappairs/human/human.txt) scribed in CMK cells by RT ± PCR or Northern blot and additional primers were designed for the following loci: analysis, indicating that the rearrangements do not D20S110 (http://gdbwww.gdb.org/gdb/, sequence accession completely eliminate gene expression. However, due to no. Z16791; D20S110-2f, 5'-AGGTTTGACAGCCAAATG- the presence of potentially normal candidate genes on 3' and D20S110-2r, 5'-TTACCACAAACTTGTGAACTA- the remaining chromosome 20 which are not disrupted GA-3'), D20S881 (accession Z53346; D20S881-2f, 5'-CAG-

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4158 CACAAAAGGCAGAGGA-3' and D20S881-2r, 5'-AGA- Fish CTTTGAGGTTGGGGTGA-3'), and D20S836 (accession Z52210; D20S836-2f, 5'-CTAAGAGCAGCCTCCCCATC-3' YAC and PAC DNAs were labeled by nick translation with and D20S836-2r, 5'-ACTAGATGCCAGCAGCACAC). One Bio-14-dATP (Gibco ± BRL). Metaphase preparations from oligonucleotide for each primer pair was end-labeled using T4 control normal human lymphocyte and leukemic cell lines polynucleotide kinase (New England Biolabs) and [g33P]dATP were pretreated with 26SSC at 378C for 1 h and dehydrated (Sambrook et al., 1989), and the PCR reactions were in a graded ethanol series. Slides were denatured in 70% performed in 10 ml volumes containing 50 ng of template formamide, 26SSC at 758C for 2 min, followed by DNA, 25 ng of each primer, Taq DNA polymerase and other dehydration in a cold ethanol series. Labeled probe DNA constituents in the bu€er supplied (Gibco ± BRL). Conditions (300 ng/ml), along with 12 ml of hybridization mixture (50% for ampli®cation were 948C for 2 min, followed by 30 cycles formamide/26SSC, 10% dextran sulfate, and Denhardt's) of 948C for 15 s, 558C for 15 s and 728C for 30 s, followed by and 2.5 mg of Cot-1 DNA (Gibco ± BRL), was denatured at a ®nal extension reaction at 728C for 8 min. The ampli®ed 708C for 7 min, pre-annealed at 378C for 10 min and applied products were separated by 6% denaturing polyacrylamide to the slide. Each probe was co-hybridized along with the gel electrophoresis. centromeric (CEP20) or painting probe for chromosome 20 (WCP20) (Vysis, Downers Grove, IL, USA). Hybridization was carried out overnight at 378C followed by washing with 50% formamide/26SSC (10 min) and 26SSC (10 min) at YAC and PAC clones 428C. Labeled probes were detected with avidin-conjugated Sixteen mega YAC clones spanning the interval between ¯uorescein isothyocyanate (avidin-FITC) and biotinylated D20S174 and D20S119 were obtained from commercial anti-avidin (Oncor, Gaithersburg, MD, USA). Slides were sources (Research Genetics, Genome Systems, St Louis, counter-stained with 4'6'-diamideno-2-phenylindole (DAPI, MO, USA) and from the Centre d'Etude du Polymorphisme Oncor). Analysis was performed using a Nikon ¯uorescence humain (CEPH, Paris, France), and sized by pulse ®eld gel microscope equipped with a CCD camera with Smartcapture electrophoresis. The order of the YACs has been reported View Point multicolour imaging station software (Digital as q-cen-942B1-808C5-804C12-834H3-925D5-889A-936H3- Scienti®c). Seventeen to 74 metaphases and 400 interphases 936E4-777D11-905D3-912C3-951F2-909F8-742F1-958C12- were scored for each cell line. 800A11- q-tel (Sto€el et al., 1996; Bench et al., 1998) and was largely con®rmed by us by STS/probe mapping. Eleven PAC Exon trapping clones were obtained from the BAC/PAC Resource Center at Roswell Park Cancer Institute and PAC DNA was prepared Exon trapping was performed using the Exon Trapping using a Qiagen Plasmid Midi kit (Qiagen, Chatsworth, CA, System (Gibco ± BRL) essentially as described by the USA). The order of the PAC clones was determined at the manufacturer. Brie¯y, PAC DNA was restriction digested Sanger Centre as q-cen-dJ138B7-dJ637C15-dJ1118N11- with HindIII and BamHI and products from each reaction dJ1028D15-dJ453O10-dJ622K8-dJ11N4-dJ495O3-dJ440H8- were ligated into pSPL3. Ligation products were transfected dJ1118N6-dJ1108D11-q-tel (http://webace.sanger.ac.uk/cgi- into COS-7 cells using Lipofectamine (Gibco ± BRL), and bin/ace/pic/20ace?name=Chr 20ctg125&class=Map). after 24 h, total RNA was isolated by the guanidine- isothiocyanate method (Sambrook et al., 1989). Reverse- transcribed RNA was generated using Superscript Reverse Transcriptase (Gibco ± BRL) and the products PCR-ampli®ed Characterization of cDNA clones and digested with BstXI in order to eliminate vector-derived A set of 90 Expressed Sequence Tags (ESTs) derived from 73 products. After a second round of ampli®cation, PCR UniGene clusters was identi®ed at the National Center for products were separated on 2% agarose gels and individual Biotechnology Information (NCBI) Human Gene Map bands excised and subcloned into pAMP10. Inserts were Project (Deloukas et al., 1998) (http://www.ncbi.nlm.nih.- sequenced and analysed using the BLAST program (Altschul gov/genemap/) using D20S174 and D20S119 as proximal and et al., 1990) against the non-redundant database at NCBI. distal interval endpoints, respectively. Plasmid clones were obtained from the IMAGE collection from commercial Reverse transcription-polymerase chain reaction and Northern sources (Research Genetics, Genome Systems). Complemen- blot analysis tary DNA (cDNA) inserts were radiolabeled with a32P-dCTP, by the random priming method, and serially hybridized in Total RNA was extracted from CMK cells using Tri-reagent RapidHyb bu€er to nylon membranes containing genomic (Sigma) per the manufacturer's instructions. Total RNA from DNA from the 15 YAC clones and control DNA obtained human placenta was obtained from Research Genetics and from mouse A9/ human chromosome 20 hybrid cells bone marrow RNA was obtained from Clontech, (Palo Alto, (GM13260), from parental A9 cells (GM00346) (both CA, USA). Reverse transcription and PCR were performed obtained from NIGMS Human Genetic Mutant Cell using a GeneAmp kit (Roche Molecular Systems, Branch- Repository, Coriell Institute, Camden, NJ, USA), and from burg, NJ, USA), under conditions recommended by the human placenta DNA (Sigma, St Louis, MO, USA). Final manufacturer (with minor modi®cations). Brie¯y, reactions washes were in 0.16SSC, 0.5% SDS at 688C, and were carried out using 1 mg of RNA in a 20 ml volume membranes were then exposed to Kodak Biomax MS ®lms containing 16PCR bu€er II, 2.5 mM oligodT, 1 mM of each overnight. Characterization of the IMAGE clones by these dNTP, 5 mM MgCl2, 20 U Rnase inhibitor and 50 U reverse methods yielded 34 single copy probes, mapping to the transcriptase, at 428C for 20 min. In each case, a reaction was D20S174-D20S119 interval (*26% of the clones tested). The performed in the absence of reverse transcriptase to control identities of several clones were also con®rmed by single for genomic DNA contamination. Samples were heated at passage DNA sequencing. Three cDNA clones were obtained 988C for 5 min then chilled on ice; 2 ml aliquots were PCR- for hTOM34 (SD Nuttall), hHNF4A (I Talianidis), and ampli®ed in 20 ml reactions consisting of 16PCR bu€er II, STK4 (CL Creasy). 2mM MgCl2, and 0.5 U AmpliTaq DNA polymerase for 33

Oncogene Mapping of genes at translocation breakpoints in 20q12 D MacGrogan et al 4159 cycles under conditions described above. Primer sequences Acknowledgments and PCR product sizes were as follows: h-l(3)mbt, (accession We are grateful to the following investigators for providing U89358), 5'-CCCACACTAGGACCTCCAAA-3' and 5'- cell lines: B Andersson, M Baumanne, H Ben-Bassat, R ACACAAGGTCCCCAAGCTG-3', 582 and 701 bp; SGK, Berthier, B Clarkson, J Cossman, W Dai, Z Estrov, C (accession Z98752), 5'-CCAATGGGAACATCAACCTG-3' Gambacorti, J Goldman, H Hirai, J Kao, N Komatsu, I and 5'-ACCTCAGCAGCGTAGAACCT-3', 344 bp; CGI- Kubonishi, T Kudo, M Kurosawa, M Lieberman, Y 53, (accession AF151811), 5'-GCGTCAGGTAACCATG- Matsuo, I Miyoshi, D Morgan, J Okamura, A Rimbaldi, GAG-3' and 5'-CGGCTAATTTCCCTGCTCAA, 438 bp; L Skinnider, T Tange, T Takahashi, E Thompson, M TNRC/CAGF9, (accession AL034419), 5'-AGGAAACAG- Tomonaga. We thank Hong Wu for technical assistance, CTGCAAACGAC-3' and 5'-AAAACATTTTGTGGGGG- Frederic Gilles and Andrew Zelenetz for their assistance AAA-3', 338 bp; sts-T63166, (accession T63166), 5'-TCC- and advice in pulse-®eld gel electrophoresis, and Ellie Park ATCGATAGAGGATTGGA-3' and 5'-TCACACATTGCT- for her assistance in preparing the manuscript. This work CCAGCTTC-3', 294 bp. Northern analysis was performed was supported by the Gabrielle Rich Leukemia Research using standard methods (Sambrook et al., 1989) and Fund, the Renny Saltzman Leukemia Research Fund and hybridizations carried out as described above. NIHgrantRO1DK43025(SDNimer)

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Oncogene