Oncogene (2000) 19, 3902 ± 3913 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identi®cation of candidate

Anthony J Bench1,4, Elisabeth P Nacheva1,4, Tracey L Hood1, Jane L Holden2, Lisa French2, Soheila Swanton1, Kim M Champion1, Juan Li1, Pamela Whittaker2, George Stavrides2, Adrienne R Hunt2, Brian JP Huntly1, Lynda J Campbell3, David R Bentley2, Panos Deloukas2 and Anthony R Green,*,1 together with the UK Group (UKCCG)

1University of Cambridge, Department of Haematology, Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 2XY, UK; 2The Sanger Centre, Wellcome Trust Campus, Hinxton, Cambridgeshire, CB10 1SA, UK; 3Victorian Cancer Cytogenetics Service, St Vincent's Hospital, Fitzroy, Victoria, Australia 3065

Deletion of the long arm of represents deletion of the long arm of chromosome 20 was found the most common chromosomal abnormality associated in 5% of these samples and represented the second with the myeloproliferative disorders (MPDs) and is also most common structural abnormality after the Phila- found in other myeloid malignancies including myelodys- delphia chromosome. plastic syndromes (MDS) and acute myeloid leukaemia Deletion of the long arm of chromosome 20 is (AML). Previous studies have identi®ed a common observed in 10% of patients with polycythaemia vera deleted region (CDR) spanning approximately 8 Mb. (PV) as well as in other myeloproliferative disorders We have now used G-banding, FISH or microsatellite (MPD) (Bench et al., 1998b). It is also seen in PCR to analyse 113 patients with a 20q deletion approximately 4% of patients with myelodysplastic associated with a myeloid malignancy. Our results de®ne syndromes (MDS) (Fenaux et al., 1996) and 1 ± 2% of a new MPD CDR of 2.7 Mb, an MDS/AML CDR of patients with acute myeloid leukaemia (AML) (Heim 2.6 Mb and a combined `myeloid' CDR of 1.7 Mb. We and Mitelman, 1992). These malignancies are thought have also constructed the most detailed physical map of to result from acquired a€ecting a plur- this region to date ± a bacterial clone map spanning ipotent haematopoietic progenitor . By contrast, 5 Mb of the chromosome which contains 456 bacterial 20q deletions are rarely seen in lymphoid malignancies clones and 202 DNA markers. Fifty-one expressed (Mitelman, 1995) although the 20q deletion has been sequences were localized within this contig of which 37 shown to arise in a progenitor cell which is capable of lie within the MPD CDR and 20 within the MDS/AML giving rise to both myeloid cells and B cells (White et CDR. Of the 16 expressed sequences (six genes and 10 al., 1994). Taken together, these observations suggest unique ESTs) within the `myeloid' CDR, ®ve were that 20q deletions mark the site of one or more tumour expressed in both normal bone marrow and puri®ed suppressor genes, the loss of which perturbs the CD34 positive cells. These data identify a set of genes behaviour of multipotent haematopoietic progenitors. which are both positional and expression candidates for Initial studies by isotopic in situ hybridization the target (s) on 20q. Oncogene (2000) 19, 3902 ± demonstrated retention of the c-src gene in three 3913. patients with a 20q deletion (Le Beau et al., 1985) but loss of c-src in two other patients (Morris et al., Keywords: chromosome 20; tumour suppressor genes; 1989). These results may have been complicated by myeloproliferative disorders; myelodysplastic syn- cross-hybridization of the probe to additional se- dromes quences on 20q, but did suggest that deletions were interstitial rather than terminal. Investigations using high resolution G-banding and reverse chromosome Introduction painting identi®ed two groups of deletions (Nacheva et al., 1995b). Large deletions resulted in loss of both Acquired chromosomal deletions are frequently de- Giemsa dark bands on 20q whilst small deletions tected in both solid tumours and haematological resulted in loss of 20q12 but retention of the more malignancies (Mitelman et al., 1997). Such deletions distal Giemsa dark band at 20q13.2. FISH mapping by are believed to harbour tumour suppressor genes, loss Le Beau and colleagues (Roulston et al., 1993) or inactivation of which contributes to the pathogen- provided the ®rst molecular identi®cation of a common esis of the tumour. In a large study of patients with deleted region (CDR) between RPN2 and D20S17, a haematological malignancies, Dewald et al. (1993) region of approximately 13 Mb. Molecular analysis analysed almost 3000 consecutive bone marrow using microsatellite PCR and Southern blotting re®ned samples with a sole chromosomal abnormality. A the proximal boundary to D20S174 and demonstrated that both the centromeric and telomeric breakpoints were heterogeneous (Asimakopoulos et al., 1994; Hollings, 1994). *Correspondence: AR Green 4The ®rst two authors contributed equally to this work Patients with an MPD or MDS have distinct Received 8 March 2000; revised 5 June 2000; accepted 6 June 2000 biological and clinical characteristics and it is not Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3903 known whether the same 20q target genes will prove to variable chromosome partners (Figure 1c). In all ®ve be involved in the two groups of myeloid disorders. It cases, the der(20) marker was the only translocation is therefore prudent to consider the two categories of product retained in the genome. Furthermore, FISH patients separately. On the basis of published data, the mapping showed that the breakpoint on chromosome CDR in MPD patients is ¯anked by D20S206 and 20 had occurred in a region proximal to D20S107 and D20S481 and the CDR in MDS/AML patients is con®rmed loss of the remainder of the long arm of ¯anked by D20S465 and D20S481 (Bench et al., 1998a; chromosome 20 (data not shown). These results will be Wang et al., 1998). Two yeast arti®cial chromosome reported in detail elsewhere. (YAC) contigs which span these CDRs have been In the remaining 42 cases, chromosome painting was constructed (Bench et al., 1998a; Sto€el et al., 1996). consistent with a small interstitial deletion of 20q These have allowed the physical size of the MPD CDR (Figure 1d). The majority (33 cases) carried a 20q to be estimated as 8 ± 9 Mb and the MDS/AML CDR deletion as the sole karyotypic abnormality. In the as 7 ± 8 Mb (Bench et al., 1998a; Sto€el et al., 1996). remaining nine cases, the 20q deletion was accompa- However, the large size of the CDRs makes the task of nied by various additional chromosomal aberrations target gene identi®cation an arduous one. (Table 1). All 42 cases with a small 20q deletion were The discovery of small submicroscopic deletions subjected to further FISH mapping with speci®c would greatly aid identi®cation of putative target probes. genes. Unfortunately systematic microsatellite PCR analysis of an 11 Mb region spanning the CDRs did Deletion mapping analysis not reveal any small deletions in patients with PV and a normal (Asimakopoulos et al., 1996a). The 42 patients with small 20q deletions were analysed Similarly, in patients with a visible 20q deletion, no by FISH using a centromeric PAC (CEP20), a PAC small deletions have been detected on the normal hybridizing to a distal region of 20q (LSI 20q13) and a chromosome 20 homologue (Bench et al., 1998a). In PAC hybridizing within the CDR (LSI D20S108). In both these studies, the microsatellite markers were each patient, signals from both LSI 20q13 and CEP20 separated by an average of 500 ± 1000 kb and so it is but not LSI D20S108 were observed on the deleted possible that deletions smaller than this may have been chromosome 20 con®rming the presence of an missed. interstitial deletion (summarized in Figure 3). Meta- In order to re®ne the CDR further we have now phases from each patient were then hybridized with examined 113 patients with a myeloid malignancy PACs mapping at the boundaries of the known MPD associated with a 20q deletion. Both the MDS/AML and MDS/AML CDRs ± D20S107 which lies at the CDR and MPD CDR have been considerably reduced proximal boundary of the CDRs and D20S176 which in size. The generation of a detailed PAC and BAC lies telomeric of the distal boundary of the CDRs based physical map spanning the CDR allowed the (Bench et al., 1998a; Wang et al., 1998). positioning of 51 unique expressed sequences. RT ± In 37 patients (25 with MDS or AML, 12 with PCR analysis of bone marrow and puri®ed CD34 MPD) both probes failed to produce a signal on the positive cells has allowed the identi®cation of a set of deleted chromosome 20 demonstrating the presence of genes which are both positional and expression extensive deletions which would not help re®ne the candidates for the target gene(s) on 20q. CDRs. Such samples were not investigated further. However, in four patients with MDS (DB53, MH40, DB122 and DB214) and one with MPD (JH41) one of the two probes gave a signal on the deleted Results chromosome 20 and these samples were analysed in more detail. Chromosome banding and painting In addition to the 42 patients with small 20q Bone marrow chromosome preparations of 107 cases deletions which were analysed by FISH using locus with myeloid disorders were analysed by G banding to speci®c probes, a further six patients with a 20q assess the size of the deletion of the long arm of deletion (four with MDS, two with MPD), from whom chromosome 20. As previously described (Nacheva et bone marrow metaphases were not available, were al., 1995b), two categories of 20q deletion were analysed using microsatellite PCR. DNA from puri®ed identi®ed ± large deletions (59 cases), resulting in loss granulocytes and T cells was analysed using a large of both Giemsa dark bands from 20q (Figure 1a, top), panel of polymorphic markers which spanned the and small deletions (48 cases) in which one Giemsa MPD and MDS/AML CDRs (Table 2). All four dark band remains (Figure 1a, bottom). We next MDS patients had deletions extending beyond the focused on the latter group of patients (summarized in limits of the published CDR. However, in one of the Table 1). two MPD patients (RB42), the centromeric boundary FISH analysis using a chromosome 20 paint was of the deletion considerably reduced the MPD CDR performed on the 48 samples with a small 20q deletion. (Figure 2, Table 2). In six cases, the `20q deletion' was revealed to be due to cryptic rearrangements. In one case, simultaneous Reduction of the common deleted region in MDS/AML application of paints for 15 and 20 patients identi®ed a marker derived from but two normal chromosome 20 homologues (Figure 1b). Preliminary FISH analysis identi®ed four MDS In the remaining ®ve cases, chromosome painting patients with deletions that encroached upon the demonstrated the presence of an unbalanced transloca- MDS/AML CDR. In each case, the 20q deletion was tion with a recurrent breakpoint at 20q11.2 and present in all metaphases examined. In three cases

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3904

Figure 1 Analysis of 20q deletions by G-banding, chromosome painting and locus speci®c probes. (a) G banded partial karyotype of normal (left) and deleted (right) chromosome 20 homologues showing a large deletion (top pair) and a small deletion (bottom pair) of the long arm. (b) Bone marrow metaphase cell hybridized with chromosome 15 (red) and chromosome 20 (green) paints demonstrating that the marker chromosome (arrow) identi®ed by G banding as del(20)(q11) is in fact derived from chromosome 15. (c) Bone marrow metaphase cell hybridized with a chromosome 20 paint (red) demonstrating the presence of a cryptic translocation involving chromosome 20 (arrow) which had been identi®ed as del(20q) by G banding. (d) Bone marrow metaphase cell from a patient with a del (20)(q11.2q13.2) hybridized with a chromosome 20 paint showing a hybridization to both chromosome 20 homologues only. (e) Partial bone marrow metaphase cell from MDS patient DB122 hybridized with a chromosome 20 centromeric probe (red) and PAC dJ620E11 (green) showing hybridization of PAC dJ620E11 to both normal and deleted (arrow) chromosome 20 homologues. (f) Partial bone marrow metaphase cell from patient DB122 hybridized with a chromosome 20 centromeric probe (red) and PAC dJ661I20 (green) showing the absence of the green signal on the del(20)(q11.2q13.1) (arrow). (g) Partial bone marrow metaphase cell of MDS patient DB214 hybridized with a chromosome 20 centromeric probe (red) and PAC dJ242A14 (green). Note the absence of a green signal on the del(20)(q11.2q13.1) (arrow). (h) Partial bone marrow metaphase cell from MDS patient DB214 hybridized with a chromosome 20 centromeric probe (red) and PAC dJ196H17 (green) showing the presence of red and green signals on both normal and deleted (arrow) chromosome 20 homologues

Table 1 Clinical diagnosis and karyotype in 48 cases with a small 20q deletion by conventional G-banding analysis Del 20q as a Del 20q in a complex 20q cryptic Clinical diagnosis sole aberration karyotypea rearrangementsb Total

MDS 18 4 1 23 AML 3 4 3 10 PV 8 1 0 9 CML 2c 0 2 4 uMPD 2 0 0 2 Total 33 9 6 48

MDS, myelodysplastic syndrome (RA, eight patients; RARS, one patient; RAEB, two patients, RAEB-t, one patient; CMML, one patient; unclassi®ed MDS, 10 patients); AML, acute myeloid leukaemia; PV, polycythaemia vera; CML, chronic myeloid leukaemia; uMPD, unclassi®ed myeloproliferative disorder. aAdditional changes were del (10)(p12) (one patient), trisomy 8 (one patient), trisomy 8 and trisomy 21 (one patient), two additional structural abnormalities (three patients) and three or more numerical and structural abnormalities (three patients). bdel 20q, as part of a complex karyotype, was revealed to be t(20;V)(q11.2; V) in ®ve cases and to be der(15)(p?/q?) in one case. cdel(20)(q11.2q13.1) detected as a sole abnormality in addition to t(9;22)(q34.1;q11.2)

(DB53, DB214 and MH40), the 20q deletion was speci®c probes in order to ascertain the deletion present as a sole abnormality. In patient DB122, the boundaries. 20q deletion was the original sole abnormality with two The proximal boundary of the MDS/AML CDR subclones containing trisomy 21 or trisomy 8 and was re®ned by patients DB53, MH40 and DB122. The trisomy 21 in addition to the 20q deletion also present. proximal boundary of the deletion in DB53 lay These four cases were studied using additional locus between D20S107 and WI-11538 (Figure 3), a distance

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3905 Table 2 Deletion mapping by microsatellite PCR in patient DB122 lay between sts-R52161 and Diagnosis RAEB-t MDS MDS RAEB MF PV stSG25449 (Figure 1e,f), a distance of less than (Patient) AM12 AS22 CW28 VF33 JP16 RB42 200 kb (Figures 3 and 4). This represents a large Marker re®nement of the proximal boundary of the MDS/ D20S106 1 1 1 1 2 AML CDR. D20S884 1 2 Patient DB214 allowed considerable re®nement of D20S174 U the distal boundary of the MDS/AML CDR. The D20S908 U U distal boundary of this deletion lay between WI-12515 D20S44 U U D20S438 U and stSG40369 (Figure 1g,h), a distance of less than D20S206 1 U 100 kb (Figures 3 and 4). D20S465 1 1 1 U These data therefore considerably reduce the MDS/ D20S107 1 1 U U 2 AML CDR to a region lying between PAC dJ620E11, D20S850 1 U U 2 D20S855 1 U 1 2 which contains sts-R52161, and PAC dJ196H17 which PLC1 1 U 1 2 contains WI-12515 (Figures 3 and 4). The bacterial D20S170 U U U 1 2 clone contig presented below demonstrates the physical D20S108 1 2 size of this region to be approximately 2.6 Mb. D20S858 1 D20S466 1 1 1 1 1 D20S46 1 1 1 Reduction of the common deleted region in MPD patients D20S110 1 U 1 1 S20S899 1 1 1 1 In patient JH41 (diagnosis PV), the proximal boundary D20S55 1 U of the deletion had previously been determined by D20S43 U U 1 AFMa343xb1 1 1 microsatellite PCR to lie between D20S438 and D20S119 1 1 1 1 1 D20S107 (Bench et al., 1998a). PAC dJ155H19, which PPGB 1 contains D20S107 (Figure 4), consistently gave rise to a D20S838 U reduced signal consistent with the presence of the D20S856 U U proximal deletion breakpoint within this PAC (Figure D20S17 1 1 D20S836 U 1 1 3). D20S197 1 U 1 1 U 1 Granulocytes and T cells from two additional D20S109 1 1 1 1 2 1 patients were investigated using microsatellite PCR (Table 2). One patient with PV (RB42) demonstrated RAEB, Refractory anaemia with excess of blasts; RAEB-t, Refractory anaemia with excess of blasts in transformation; MDS, retention of heterozygosity at D20S108 but loss at unclassi®ed myelodysplastic syndrome; MF, myelo®brosis; PV, D20S858 (Figure 2, Table 2). The proximal breakpoint polycythaemia vera. 2, two alleles were detected in granulocyte of the deletion in this patient lay between D20S108 and DNA at an informative marker; 1, one allele was detected at an D20S858, a distance of less than 200 kb (Figures 3 and informative marker; U, marker uninformative. The order of markers 4). is from proximal to distal These data greatly reduce the MPD CDR whicih is now ¯anked by D20S108 (this study) and D20S481 (Wang et al., 1998). The estimated physical size of this region is 2.7 Mb, using data from the bacterial clone contig presented below. The region of overlap between the new MDS/AML and MPD CDRs de®ned a combined `myeloid' CDR of 1.7 Mb (Figure 3).

A bacterial clone contig spanning the MPD and MDS/AML common deleted regions We had previously constructed an 11 Mb YAC contig of this region of chromosome 20 (Bench et al., 1998a). In order to produce a more detailed physical map, we have now constructed a contiguous PAC and BAC based map. PAC and BAC clones were identi®ed by hybridization of STSs to genomic libraries (Ioannou et Figure 2 Microsatellite PCR results which de®ne the proximal al., 1994; Osoegawa et al., 1998). Clones were boundary of the new MPD common deleted region. Microsatellite overlapped using restriction ®ngerprinting (Gregory PCR was performed using the indicated markers on DNA et al., 1997) and STS content mapping allowing clones extracted from puri®ed granulocytes (G) and T cells (T) from patient RB42. Arrowheads indicate the upper band of each allele. to be grouped into contigs. To bridge contigs, new Open arrowheads indicate the allele that is deleted in granulo- STSs and DNA probes, developed from the ends of cytes. Two alleles are retained at D20S108 whereas one allele is contigs, were used for further library screening. New lost at D20S858 and D20S46 PACs and BACs were then amalgamated into contigs in the same way until a contiguous map was produced. of approximately 400 kb. For patient MH40, a PAC The bacterial clone contig contains a total of 456 containing WI-11538 (dJ357E14) consistently gave rise bacterial clones of which 376 are PACs and 80 are to a reduced signal consistent with the presence of the BACs. It extends from D20S607 to SEMG1 and proximal deletion breakpoint within this PAC (Figure includes 202 DNA markers of which 185 are STSs 3). Furthermore, the proximal boundary of the deletion and 17 are DNA probes. The size of the contig was

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3906

Figure 3 Summary of mapping data. This ®gure presents the FISH and microsatellite PCR results which allow the delineation of the new MDS/AML and MPD CDRs. Patients were analysed by FISH with locus speci®c probes except for patient RB42 for whom microsatellite PCR was performed. Microsatellite PCR had previously been performed for patients MH40 and JH41 (Bench et al., 1998a). STS markers and corresponding PACs are shown on the left. The distance between markers in megabasepairs or centiMorgans is indicated. Bracketed markers are separated by less than 0.1 Mb. The distal boundary of the MPD CDR has previously been reported (Wang et al., 1998). The MDS/AML CDR is ¯anked by PACs dJ620E11 and dJ196H17. The MPD CDR is ¯anked by DS20S108 and D20S481. Patient DB214 also showed retention of PACs dJ149D20 (D20S481), dJ81O8 (D20S454), dJ207N8 (stSG34079) and dJ734B23 (WI-4255) (data not shown)

based on an average of 3.5 kb per HindIII ®ngerprint within the MDS CDR. Evidence was obtained that band (P Deloukas (2000), unpublished results). The six out of these eight putative expressed sequences estimated size of the contig was calculated as 5 Mb by represented bona ®de transcribed genes, rather than multiplying the total number of di€erent ®ngerprint processed or genomic DNA contami- bands within the contig by 3.5. A representation of the nants within the EST database. KCNS1 is a gene map illustrating the minimal tiling path and PACs which encodes a potassium voltage-gated channel which were used for FISH experiments is shown in subunit. ESTs AA053206/AA053121 were subse- Figure 4. A full version of the map can be found at quently found to be derived from the SGK2 gene http://webace.sanger.ac.uk/cgi-bin/ace/pic/20ace?name= (Kobayashi et al., 1999). Alignment of cDNA Chr-20ctg125&class=Map. sequence to genomic sequence demonstrated an exon/intron structure for three of the remaining six ESTs (AI312497, AA568401 and AA910031) with the Identification of expressed sequences splice site consensus sequence present at all exon/ Forty-three unique ESTs were located between intron boundaries. In all three of these cases, the D20S607 and stSG34035 inclusively. Of these, 34 ESTs presence of each exon was con®rmed by exon were mapped by hybridization to a `polygrid' of PACs prediction programs such as GRAIL. The existence and BACs or by colony PCR of bacterial colonies of an exon was also predicted by GRAIL, containing PACs or BACs (Figure 4). An additional GENSCAN and Gene®nder within the region of nine ESTs, previously mapped in this region of PAC dJ1108D11 which aligned to EST AA993161 chromosome 20 (Bench et al., 1998a; Deloukas et al., implying that this EST also represented a transcribed 1998) were positioned upon the contig by BLAST gene. alignment of EST sequence with the available PAC or Sequence analysis of PACs from within the minimal BAC genomic sequence. tiling path also allowed us to establish the genomic In order to identify putative novel expressed structure of known and novel genes in this region. sequences, 23 PAC clones from the minimal tiling Alignment of RPTPrho cDNA to PACs dJ1121H13, path were partially or completely sequenced. The dJ707K17, dJ81G23, dJ230I19, dJ3E5, dJ232N11 and genomic sequence of these clones was analysed using dJ269M15 demonstrated that this gene spans at least the NIX program (Williams et al., 1998) which 1.2 Mb. PAC dJ138B7 contained two genes (h-l(3)mbt allows the simultaneous observation of a number of and SGK2) as well as the 5' portion of the gene gene identi®cation tools including GRAIL, GEN- corresponding to SHGC-36858. In particular, the SCAN and BLAST. Eight previously unmapped h-l(3)mbt gene contains 20 exons with two putative ESTs and genes were identi®ed (Table 3). Six of alternative ®nal exons. Analysis of dJ644L1 demon- these lay within the MPD CDR and seven lay strated that the MafB gene consists of a single

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3907

Figure 4 Summary of bacterial clone contig. This ®gure illustrates a representative sample of PAC and BAC clones which make up the contig. Those clones which are part of the tiling path used for sequencing are shown in normal type. The MDS/AML CDR spans the 2.6 Mb region between dJ620E11 and dJ196H17 whereas the MPD CDR extends from D20S108 to D20S481, a distance of 2.7 Mb. The region of overlap between the two CDRs is 1.7 Mb in size. Not all PACs, BACs and markers are indicated. A portion of the complete map between dJ128O17 and dJ272H18 is shown. The complete map can be viewed at http:// webace.sanger.ac.uk/cgi-bin/ace/pic/20ace?name=Chr_20ctg125&class=Map exon. Analysis of ®nished sequence was also performed mined which of the transcribed sequences were by The Sanger Centre (http://www.sanger.ac.uk/HGP/ expressed in bone marrow and puri®ed CD34 positive Humana) and this can be viewed at http://webace. cells. sanger.ac.uk/cgi-bin/webace?db=acedb20&class=Gen Reverse transcription PCR (RT ± PCR) ampli®ca- omeSequence. tion was performed with primers representing each of These data establish the presence of six genes and a the 51 transcribed sequence (Figure 5). At least two further 14 unqiue ESTs within the new MDS/AML independent RT ± PCR ampli®cations were performed CDR and a total of 14 genes with 23 unique ESTs for each transcribed sequence. Primers corresponding within the new MPD CDR (Table 3). Six genes and 10 to an EST (WI-7685) derived from the 3' UTR of the unique ESTs are present in the region of overlap CD34 gene were used as a positive control (Figure 5). between the two CDRs. Where two or more ESTs corresponded to the same transcribed sequence, the same result was obtained for each EST. Expression profiling The data are presented in Figure 5, Tables 3 and The myeloproliferative disorders, myelodysplastic syn- 4. Of the 37 expressed sequences in the MPD CDR, dromes and acute myeloid leukaemia are thought to 20 were expressed in bone marrow mononuclear cells. result from transformation of a multipotent cell within Of these, 16 were also expressed in CD34 positive the haematopoietic compartment (Bonnet and cells. Within the MDS/AML CDR, 11 out of 20 Dick, 1997; Kralovics and Prchal, 1998). Moreover, it transcribed sequences were expressed in bone marrow has previously been shown that the 20q deletion can mononuclear cells. Of these, eight were also expressed arise in a progenitor cell capable of giving rise to both in CD34 positive cells. Of the 16 transcribed myeloid cells and B cells (White et al., 1994). These sequences which lie within the region of overlap considerations suggest that the gene or genes on between the MPD and MDS/AML CDRs, eight were chromosome 20q which are inactivated in these expressed in bone marrow cells of which ®ve were diseases are likely to be expressed in normal also expressed in CD34 positive cells. These ®ve haematopoietic progenitor cells. We therefore deter- transcribed sequences therefore represent prime

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3908 Table 3 Expression pro®ling of transcribed sequences Expression PAC clone Associated or Accession statusc EST marker (accession number)a similar geneb number Function BM CD34+

R98337 dJ409O10 (AL031256) 7 7 stSG30600 dJ690O1 (AL118521) 7 7 129564d dJ644L1 (AL035665) M.m. kreisler L36435 Myeloid transcription factor + + G.g. MafB D28600 Inhibits erythroid differentiation SGC44273 dJ644L1 (AL035665) 7 7 stSG15092 dJ1J6 (AL035652) + + stSG12833 dJ1J6 (AL035652) + + WI-7849 dJ1J6 (AL035652) TOP1 J03250 DNA metabolism. + + SR kinase sts-M34667 dJ511B24 (AL022394) PLCG1 M34667 Second messenger production + + during signal transduction stSG3032 dJ511B24 (AL022394) + 7 sts-R52161 dJ620E11 (AL031557) 7 7

" stsSG9889 dJ620E11 (AL031557) H.s. CHD3 AF006515 Modification of structure. + + Chromodomain containing protein AI312497e dJ661I20 (AL031669) + + AA568401e dJ661I20 (AL031669) + +

" stSG3021 dJ1121H13 (AL049812) RPTPrho AF043644 Receptor protein tyrosine phosphatase 7 7 SGC32867 dJ862K6 (AL031681) SFRS6 U30828 Pre-mRNA splicing factor + + WI-6622 dJ138B7 (Z98752) h-l(3)mbt U89358 Chromosome condensation during + + AA053121e dJ138B7 (Z98752) SGK2 AF169034 Serum- and glucocorticoid-induced 7 7 protein kinase SHGC-36858 dJ399F1 CGI-53 AF151811 Gene of unknown function downregulated + +

CDR M.m NGD5 L38481 by d-opioid agonist WI-7535 dJ399F1 MYBL2 X13293 Transcription factor involved in cell cycle + + stSG53114 dJ399F1 X.1. D7 X13856 Oocyte maturation in Xenopus + 7 AA910031e dJ1030M6 (AL035089) 7 7 MDS stSG8966 dJ495O3 (AL121587) 7 7 sts-T63166 dJ495O3 (AL121587) + 7 stSG20384 dJ1108D11 (AL034419) 7 7 stSG40369 dJ1108D11 (AL034419) CAGF9 U80736 Glutamine rich protein + 7 AA716165e dJ1108D11 (AL034419) 7 7 AA993161e dJ1108D11 (AL034419) 7 7

" AI081352e dJ1183I21 (AL035447) 7 7 A002H21d dJ1183I21 (AL035447) + + stSG4525 dJ881L22 (AL117382) H.s. GIDAP1 Y17849 Ganglioside-induced differentiation 7 7 associated protein 1 SGC33922 dJ881L22 (AL117382) 7 7 A004C16 dJ881L22 (AL117382) 7 7 stSG34079 dJ1013A22 7 7 HNF4A/Bd dJ1013A22 HNF4A X76930 Liver specific transcription factor 7 7 CDR sts-AA010225 dJ179M20 (Z97053) + + WI-15802 dJ179M20 (Z97053) + 7 sts-AA025063 dJ179M20 (Z97053) + +

MPD SGC32730 dJ179M20 (Z97053) PL33 U49188 Serpentine membrane receptor + + WI-10228 dJ179M20 (Z97053) PKIG AB019517 Protein kinase inhibitor + + SHGC-11905 dJ179M20 (Z97053) ADA X02994 Adenosine deamination + + stSG3056 dJ78181 (ALI18522) 7 7 stSG20411 dJ81O8 7 7 WI-9624 dJ781B1 (AL118522) 7 7 stSG3015 dJ148E22 (AL008725) YWHAB X57346 14-3-3b protein + + SHGC-13528 dJ1069P2 (AL109839) H.s. PABPL1 Y00345 mRNA polyA binding + + SGC31967 dJ1069P2 (AL109839) hTOM34 U58970 Mitochondrial membrane translocase + + U18297d dJ211D12 (Z93016) STK4 U18297 Serine/threonine kinase + +

" H98683 dJ211D12 (Z93016) + + WI-10260 dJ211D12 (Z90316) + + KCNS1e dJ211D12 (Z93016) KCNS1 AF043473 K+ channel alpha subunit 7 7 stSG34035 dJ172H20 (AL049767) PI3 Z18538 Epidermal serine protease inhibitor 7 7

EST markers are listed centromeric to telomeric. Transcribed sequences between stSG3021 and KCNS1 lie within the MPD CDR whereas those between sts-R52161 and A002H21 lie within the MDS/AML CDR. aOne PAC containing each EST marker is shown. Where a PAC accession number is listed, this indicates that the presence of an EST within a PAC has been con®rmed by BLAST alignment. bTranscribed sequences corresponding to known genes are indicated. Genes which are underlined indicate similarity of the EST to the known gene. H.s., Homo sapeins; M.m. Mus musculus; G.g., Gallus gallus; X.l., Xenopus laevis. cExpression status; +, RT ± PCR product obtained on at least two separate occasions; 7, no RT ± PCR product obtained on at least two separate occasions. Additional STSs were available for a number of expressed sequences (see Materials and methods). In each case, an identical result was with the alternative STS. dSTS which corresponds to a known EST shown in Figure 4: 129564 corresponds to WI-11538; A002H21 to WI-12515; HNF4A/B to stSG38333 and U18297 to stSG33865. eEST marker represents expressed sequences, previously unmapped, identi®ed by analysis of PAC genomic sequence

candidate genes since they lie within both the MPD include two unknown genes corresponding to ESTs and MDS/AML CDRs and are expressed within the SHGC-36858 and WI-12515 as well as three genes, haematopoietic progenitor cell compartment. They SFRS6, h-l(3)mbt and MYBL2.

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3909

Figure 5 Expression analysis of genes within the contig. RT ± PCR data are shown for three EST markers within the contig (AI312497, stSG3032, AA716165) and for WI-7685, an EST derived from the 3' UTR of the CD34 gene. RT ± PCR was performed using RNA derived from bone marrow cells of two normal individuals (BM) or from CD34 positive cells of a further normal individual (CD34+). Sizes of PCR products are indicated. M, FX174/HaeIII digest; +RT, reverse transcription performed in the presence of reverse transcriptase; 7RT, mock reverse transcription performed without reverse transcriptase; 7ve, PCR set up without DNA; +ve, PCR set up using genomic DNA

Discussion Table 4 Summary of mapping and expression pro®ling of genes and ESTs within the MDS/AML, MPD and combined myeloid CDRs We analysed metaphase chromosome samples from MPD MDS/AML Myeloid 107 patients with a myeloid malignancy and a 20q Size 2.7 Mb 2.6 Mb 1.7 Mb deletion. Patients with a large deletion, as determined Number of genes 14 6 6 by G-banding were not analysed further since it was Number of ESTs 23 14 10 unlikely that these patients would re®ne the known Total expressed sequences 37 20 16 CDR. Of the 48 patients whose G-banding demon- Expressed in BM 20 11 8 Expressed in CD34+ 16 8 5 strated a small deletion of chromosome 20, ®ve (10%) actually possessed cryptic unbalanced translocations involving the long arm of chromosome 20 as part of a complex karyotype. Given the banding structure and compared to previous estimates of 8 ± 9 Mb (Bench et small size of chromosome 20, such subtle rearrange- al., 1998a; Wang et al., 1998). The region of overlap ments are beyond the resolution of G-banding and between the MDS/AML and MPD CDRs spans a can only be detected by molecular cytogenetic distance of only 1.7 Mb compared to previous techniques. In one further patient, chromosome estimates of 7 ± 8 Mb (Bench et al., 1998a; Wang et painting demonstrated that the `20q deletion' was al., 1998). actually derived from chromosome 15. These results The bacterial clone contig presented here represents emphasize the importance of using both G-banding the most detailed physical map of this region to date. It and chromosome painting to identify chromosome 20 includes 456 clones and 202 DNA markers and spans a deletions. distance of 5 Mb from D20S607 to SEMG. This map Myelodysplastic syndromes and myeloproliferative provided a large number of clones for FISH mapping disorders represent clinically and biologically distinct which enabled us to de®ne deletion boundaries very diseases. Di€erent target genes may well be involved in precisely in a number of patients. Furthermore, it the two groups of disorders and it is therefore prudent forms the basis of a minimal tiling path for the to distinguish two separate CDRs on 20q (Table 4). elucidation of the complete genomic sequence of the Our data markedly reduce the MDS/AML CDR to a common deleted region. Indeed, partial and complete region of 2.6 Mb compared to previously published sequence of a number of PACs have assisted in the estimates of 7 ± 8 Mb (Bench et al., 1998a; Wang et al., identi®cation of novel genes. The complete genomic 1998). The MPD CDR has also been greatly reduced sequence of all clones within the tiling path will not to a region ¯anked by D20S108 (this study) and only provide resources for the identi®cation of novel D20S481 (Wang et al., 1998), a distance of 2.7 Mb genes but will also be vital for the development of a

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3910 screening strategy of individual exons and reveal any small deletions on the normal chromosome promoters of candidate genes. 20 homologue (Bench et al., 1998a). A second It has been estimated that approximately 70 000 explanation is that breakpoints are clustered at speci®c genes (Fields et al., 1994) are present within the human sites which are hotspots for recombination. However, a haploid genome of 3000 Mb, giving an average gene number of investigators have reported that both density of approximately 23 genes per megabase. centromeric and telomeric breakpoints of 20q deletions However, genes are non-randomly distributed within are very heterogeneous (Asimakopoulos et al., 1994; the genome between gene poor Giemsa dark bands, Bench et al., 1998a; Hollings, 1994; Roulston et al., gene rich R'-bands and very gene rich sub-telomeric T- 1993; Wang et al., 1998) arguing against this bands which are estimated to contain 9.3, 20 and 78 possibility. We therefore favour a third possibility, genes/Mb respectively (Bernardi, 1993). The 5 Mb namely that more than one target gene exists on 20q. bacterial clone contig contains 20q12 which is a This would be consistent with the observation of large Giemsa dark band (gene poor) together with portions deletions in the majority of patients. Furthermore, of the ¯anking Giemsa light bands (gene rich). The di€erent combinations of target genes may account for Giemsa dark band contains the 1.7 Mb region between the di€erent biological characteristics of the myeloid D20S170 and stSG25486 within which only a single disorders associated with 20q deletions. If this large gene (RPTPrho) has been identi®ed so far. Since hypothesis is true, it has important consequences for 20q12 is cytogenetically visible at the 550-band stage, the design of experimental strategies to identify target its physical size can be estimated to be of the order of genes. at least 3 Mb. These considerations allow us to calculate that the 5 Mb contig is likely to contain approximately 68 genes. The 51 transcribed sequences reported here would therefore represent approximately Materials and methods 75% of the total number of genes. A number of genes previously thought to be Patient material positional candidates lie outside the CDRs de®ned Bone marrow samples from 107 patients with myeloid here. These include MafB, TOP1 and PLCG1. Indeed, disorders (73 with MDS or AML and 34 with MPD) and analysis of MafB in 25 MDS/AML patients with 20q deletions of the long arm chromosome 20 were analysed by deletions failed to identify any mutations (Wang et al., cytogenetic techniques. Diagnoses were established by the 1999). TOP1 has recently been reported to be involved referring clinicians. Samples were collected with the help of in a fusion with the NUP98 gene on in the UK Cancer Cytogenetics Group and cytogenetics laboratories from Europe, USA and Australia. Two patients t-MDS patients (Ahuja et al., 1999), but mutation (MH40, JH41) had previously been analysed using micro- screening of TOP1 and PLCG1 in patients with 20q satellite PCR (Bench et al., 1998a). Peripheral blood was deletions has not been reported. obtained from an additional six patients with a chromosome Within the MPD CDR our results have identi®ed 16 20 deletion (four with MDS, two with MPD) for whom a genes and ESTs that are expressed in both bone bone marrow sample was not available. marrow and puri®ed CD34 positive cells. These include SFRS6, h-l(3)mbt, MYBL2, PL33, PKIG, ADA, Chromosome banding and chromosome painting YWHAB, hTOM34 and STK4. The SFRS6, h- l(3)mbt and MYBL2 genes as well as two ESTs also Bone marrow samples were subjected to chromosome lie within the MDS/AML CDR. SFRS6 (also termed banding analysis using conventional methods, digital imaging and karyotyping as previously described (Nacheva et al., SRp55) encodes an SR protein important in the 1995b). Chromosome painting with whole chromosome regulation of of mRNA (Screaton paints (WCP) was performed according to the manufacturer's et al., 1995). MYBL2 (also termed B-myb) is a member instructions (Vysis, Downers Grove, IL, USA; Cambio, of the myb family of transcription factors and is Cambridge, UK). believed to play a role in cell cycle control (Saville and Watson, 1998). The h-l(3)mbt gene is the human FISH mapping homologue of a Drosophila tumour suppressor gene and regulates chromatin structure during mitosis (Koga FISH mapping was performed using the dual colour/dual et al., 1999). These genes, along with the genes probe approach as previously described (Asimakopoulos et corresponding to ESTs SHGC-36858 and WI-12515, al., 1996b; Nacheva et al., 1995a) with one probe being a PAC derived from the centromeric region of chromosome 20 will be prioritized during mutation screening of (CEP20). PACs which mapped to the long arm of candidate genes upon 20q. chromosome 20 were identi®ed from the PAC/BAC contig Despite considerable e€ort, small deletions of 20q or obtained from Vysis (LSI D20S108 and LSI 20q13). have not yet been identi®ed (Asimakopoulos et al., DNA was isolated and nick translated using directly 1996a; Bench et al., 1998a). The smallest deletion labelled dUTP (SpectrumGreen-dUTP or SpectrumOrange- reported to date is approximately 5 ± 6 Mb (Wang et dUTP, Vysis) or hapten labelled nucleotides (biotin-dATP, al., 1998) and most are larger than 10 Mb. Deletions of Life Technologies, Paisley, UK; digoxigenin-dUTP, Boehrin- 20q are therefore many fold larger than the size of the ger-Mannheim, Mannheim, Germany). Chromosome bone CDRs. What might be the reason for this? One marrow preparations were pre-treated with pepsin/0.1 M explanation is that small deletions on 20q have been HCl/PBS, followed by 26SSC at 378C for 60 min and ®nally with 1% paraformaldehyde. Slides were denatured in missed. This seems unlikely since systematic micro- 26SSC/formamide solution at 728C for 45 ± 360 s. Labelled satellite PCR analysis of 48 cytogenetically normal PV probes were denatured along with COT-1 DNA (Life patients failed to detect any small deletions (Asimako- Technologies) at 758C for 10 min, pre-annealed at 378C for poulos et al., 1996a). Moreover, a similar investigation 30 ± 45 min and applied to the slide. Each probe was of 28 patients with a visible 20q deletion also did not cohybridized along with the centromeric probe, CEP20,

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3911 which was labelled with a di€erent hapten. Hybridization was ftp://ftp.sanger.ac.uk/pub/human/sequences/Chr_20/un®nished carried out overnight at 378C in a humidi®ed chamber, sequence (un®nished sequences). Genomic sequence followed by washing with 0.16SSC at 728C for 3 ± 5 min. analysis was performed upon the sequence of 23 PACs Indirectly labelled probes were detected with the biotin- using the NIX suite of programs (Williams et al., 1998). avidin-FITC or mouse antidigoxigenin/rabbit antimouse/goat These were dJ644L1, dJ970A17, dJ620E11, dJ661I20, antirabbit-TRITC systems according to the manufacturer's dJ128O17, dJ1121H13, dJ707K17, dJ81G23, dJ230I19, instructions (Sigma, St Louis, MO, USA). dJ3E5, dJ730D4, dJ232N11, dJ753D4, dJ269M15, Slides were inspected by ¯uorescence microscopy to dJ862K6, dJ138B7, dJ1030M6, dJ1108D11, dJ1183I21, establish that hybridization had been successful. The slides dJ995J12, dJ179M20, dJ148E22 and dJ211D12. were then screened under DAPI excitation (360 nm) at low I.M.A.G.E. consortium cDNA clones (Lennon et al., 1996) magni®cation (206) to make a random selection of between corresponding to seven transcribed sequences were obtained 20 and 50 metaphases across the hybridization ®eld. Cells from the UK HGMP-RC, Hinxton, UK. CloneIDs were without a centromeric signal from both chromosome 20 129564 (which corresponds to EST marker WI-11538), homologues were not analysed. The selected cells were 429692 (stSG12833), 31051 (WI-6622/SHGC-15668), 49827 automatically imaged in a single batch at three or four (WI-12515/A002H21), 123938 (SGC33992), 23400 (A004C16) excitation frequencies. Captured images were analysed by two and 202552 (WI-15802). Clones were fully sequenced and new independent researchers using the SmartCapture View Point STS primers were designed from the 5' end of clones 129564, multicolour imaging station (Digital Scienti®c Ltd, Cam- 49827 and 123938 using the web interface of Primer3 (Rozen bridge, UK). and Skaletsky, 1998). STS primers were also designed from nine ESTs and genes corresponding to eight previously unmapped transcribed sequences. Primer sequences and Microsatellite polymerase chain reaction PCR product sizes were: 129564 CCTGGCTTTCTGAACT- The deletion in a further six patients was mapped by TTGCAA and CCAGAGTCCCTCTCCTTTCC 104 bp; microsatellite PCR. Granulocytes and T cells were prepared 49827 CGCCAGAACTCTGTCTGGCTC and GCCATGT- from the peripheral blood of these patients and microsatellite GGGACATTTTCTT 135 bp; 123938 GGTGGAGGCAC- PCR ampli®cation was performed on DNA samples as CAAAGATAA and TCATTCTGCCTCCATTTTCC 101 bp; previously described (Asimakopoulos et al., 1996a). AA564801 TCACTCCCCAATGTCTGATG and GGGTA- GCAGCTTCCTCTTCA 154 bp; AI312497 CTCAGGACT- CCGCAGAGATG and CAAGGAGCAAGGACCAACC, Construction of a bacterial clone contig 121 bp; AA053121 CCCTATGAGCTTGTTACATCTCTG STSs and ESTs were imported from GDB (http:// and AGAGGGCCTCCTGGTGTTT 81 bp; AA053206 AG- www.gdb.org/), the Whitehead Institute (http://www.geno CCTGAAGACACCACATCC and GCATCTCGTAGAG- me.wi.mit.edu/), the human gene map (http://www.ncbi.nlm. GACTGCC 127 bp; AA910031 CTGAGAAGGCGGAAG- nih.gov/genemap/) or the Sanger Centre (http://www.sanger. GCTAT and TGTCTAAAAGGGCAGGAGGA 78 bp; ac.uk/). These were supplemented with STSs derived from a AA716165 AGACTGCAGCATCCTGAACC and CCCA- ¯ow sorted chromosome 20 speci®c library (Ross and CAGCTCTGCATCTACA 233 bp; AI081352 TCTACCC- Langford, 1997). A radiation hybrid map of chromosome CATCCACAGCAAT and ACCTCCCTCCCGTGTAACTT 20 with an average density of 20.7 STSs per Mb was 206 bp; AA993161 TTCCAGGTGCCCTCATACTT and constructed (http:www.sanger.ac.uk/cgi-bin/rhtop?chr=20). CTTTGAGGTGGCAGGTGTCT 198 bp and KCNS1 GT- STSs were labelled by PCR (Bentley et al., 1992), pooled GTGGCCTACACAGCTGAA and CTGAGAGGTTTCTC- and hybridized to the RPCI P1 arti®cial chromosome (PAC) GGGATG 341 bp. Primers corresponding to AA053206 span and the RPCI-11 bacterial arti®cial chromosome (BAC) an intron and therefore give a PCR product in cDNA only. libraries (Ioannou et al., 1994; Osoegawa et al., 1998). STS primers corresponding to the HNF4 gene were obtained Positive clones were gridded to produce a `polygrid' to which from Kritis et al. (1996). individual STSs were then hybridized. In addition, clones were analysed by restriction ®ngerprinting (Gregory et al., Reverse-transcription polymerase chain reaction 1997). Brie¯y, DNA was digested with HindIII and Sau3AI. HindIII sites were end-labelled with a ¯uorescent dye and To purify CD34 positive cells, peripheral blood stem cells fragments were electrophoresed through a 4.5% denaturing from a normal individual were obtained after informed polyacrylamide gel on an ABI 377 automated DNA consent. RNase free DNase I (HT Biotechnology Ltd, sequencer. The landmark content and ®ngerprint data were Cambridge, UK) and heparin were added to 100 and 20 imported into FPC (http://www.sanger.ac.uk/Users/cari/ units/ml in order to reduce clotting and viscosity. Cells were fpc.shtml) and used to assemble the clones into contigs washed several times in phosphate-bu€ered saline (PBS) (Soderlund et al., 1997). In order to bridge the gaps between containing 2% foetal calf serum. An initial puri®cation of contigs, probes and STSs were developed from the ends of CD34 positive cells was carried out using the CEPRATE1 clones at the border of each contig. Further PAC and BAC SC Stem Cell Concentration System (CellPro, Bothell, WA, clones were then identi®ed by hybridization of these new USA) according to the manufacturer's instructions. Approxi- markers to genomic libraries and were incorporated into mately 108 cells were recovered at a purity of 80%, as contigs by ®ngerprinting and STS content. PCR conditions determined by ¯ow cytometry. CD34 positive cells were for each STS can be found at http://www.sanger.ac.uk/cgi- further puri®ed by ¯ow cytometry using 6 mg FITC- bin/ace/simple/20ace using the AltaVista search option. conjugated anti-CD34 (Anti-HPCA-2, Becton Dickinson, San Jose, CA, USA) giving a yield of 3.56106 cells and a purity, as determined by analytical ¯ow Identification of transcribed sequences cytometry, of 98.5%. The majority of ESTs were placed upon the bacterial clone Bone marrow cells were obtained from two normal contig in the same was as for other STSs. Other ESTs were individuals after informed consent. Mononuclear cells were localized on speci®c PACs and BACs by BLAST analysis of puri®ed using density gradient centrifugation with Histopa- PAC and BAC genomic sequence with the sequence of the que 1077 (Sigma) and washed with PBS. EST (Altschul et al., 1990). After puri®cation, cells were pelleted and resuspended in A number of clones within the minimal tiling path were fully Tri-reagent (Sigma) at a concentration of 107 cells per ml. or partially sequenced. These can be found at ftp://ftp.sanger. Total RNA was prepared from cells according to the ac.uk/pub/human/sequences/Chr_20 (®nished sequences) or manufacturer's instructions. One mg RNA was treated with

Oncogene Chromosome 20 deletions in myeloid malignancies AJ Bench et al 3912 ampli®cation grade DNase I (Life Technologies) according to Acknowledgments the manufacturer's instructions. The DNase I was inactivated We thank the following cytogeneticists and clinicians for and the RNA was puri®ed by phenol/chloroform extraction, sending us patient samples: Profs P Fenaux (Lille, France), precipitated and resuspended with 4 mg oilgodT, heated to O Haas (Vienna, Austria) and J Prchal (Birmingham, AL, 658C for 5 min and plunged onto ice. Reverse transcription USA) and Drs M Abela (Gateshead, UK), E Blennow was carried out in a 40 ml reaction volume containing 10 mM (Stockholm, Sweden), A Carroll (Birmingham, AL, USA), DTT, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, JA Copplestone (Plymouth, UK), W Finley (Birmingham, 250 mM each dNTP and 40 units RNasIN (Promega, AL, USA), G Lucas (Manchester, UK), K Michalova Madison, WI, USA) using 400 units M-MLV reverse (Prague, Czech Republic), S Raynaud (Nice, France), J transcriptase (Life Technologies) at 428C for 90 min. In each Reilly (Sheeld, UK), A Semple (Epsom, UK) and I case, a duplicate reaction was set up without reverse Wlodarska (Leuven, Belgium). The UKCCG laboratories transcriptase to act as a control for DNA contamination. participating in this study were as follows: the Department The cDNA was diluted 1 in 10 and 2 ml was subjected to of , University of Newcastle upon Tyne PCR ampli®cation in a 20 ml reaction volume containing (Newcastle, UK); the Cytogenetics Laboratory, Depart- 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, ment of Haematology, Royal Free Hospital (London, UK); 250 mM each dNTP and 1 mM of each primer using 0.5 units the Northern Ireland Regional Genetics Centre, Belfast AmpliTaq Gold (PE Biosystems, Foster City, CA, USA) on a City Hospital (Belfast, UK); the Cytogenetics Laboratory, thermal cycler (PTC-200, MJ Research Inc., Watertown, MA, Department of Clinical Haematology, University College USA). Conditions were 948C for 10 min followed by 30 Hospital (London, UK) and the Oxford Medical Genetics cycles of 948C for 50 s, an annealing temperature of between Laboratories, The Churchill Hospital (Oxford, UK). We 488C and 558C for 1 min with a ®nal extension of 728C for thank Drs Jenny Craig, Mike Scott and Kevin Jestice and 10 min. PCR products were analysed on 2% agarose gels. Ray Hicks for assistance with isolation of CD34 positive A number of transcribed sequences were represented by 2 cells. This work was supported by the Leukaemia Research EST markers. Alternative STSs were stSG3011 (equivalent to Fund, The Medical Research Council and The Wellcome stSG3032), SRp55 (SGC32867), SHGC-15668 (WI-6622), Trust. AA053206 (AA053121), 49827 (A002H21), 123938 (SGC33922), HNF4C (HNF4A/B), stSG20535 (stSG34079), stSG8281 (WI-15802), BCD2703 (stSG20411). SHGC-15668 represents an alternative 3' UTR to WI-6622 and HNF4C represents an alternative 3' UTR to HNF4A/B.

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Oncogene