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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. RAPID COMMUNICATION Cytogenetic and Molecular Delineation of a Region of Chromosome 7 Commonly Deleted in Malignant Myeloid Diseases By Michelle M. Le Beau, Rafael Espinosa 111, Elizabeth M. Davis, James D. Eisenbart, Richard A. Larson, and Eric D. Green Loss ofa whole chromosome 7 ora deletion of the long arm, del(7q1, are recurring abnormalities in malignant myeloid diseases. To determine the location ofgenes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [~OYOI) had proximal breakpoints in bands qll-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a welldefined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation. 0 1996 by The American Society of Hematology. R as a tumor suppressor gene in immature myeloid cells by negatively regulating the ~21‘“’signaling pathway.”” With respect to the myeloid disorders, recurring chromosomal deletions includethedel(5q),del(7q),del(9q), del( l lq), del(l2p), del(l3q), and del(20q).’ A commonly deleted segment has beenidentified by physical mapping with DNA probes forseveral of these rearrangements [del(5q), del(1lq), del( 1 2 ~ ) .del( 134, anddel(20q)].’”’hThesecytogenetic findings implicate a number of discrete regions of the genome that are likely to contain other tumorsuppressor genes that are inactivated during myeloid leukemogenesis. Loss of a whole chromosome 7 (-7) or a deletion of the long arm of this chromosome [del(7q)] are common recurring abnormalities in malignant myeloid disorders, and are observed in threegeneral contexts.” First, -7/de1(7q) is noted in the malignant cellsof 10% of patients with a myelodysplastic syndrome(MDS)or acutemyeloid leukemia (AML) arising de novo.2~iR Second, abnormalities of chromosome 7 are observedatthehighestfrequency in MDS or AML arising after cytotoxictherapy for another disorder, usually a primary malignancy (therapy-related or t-MDS/tAML), or after occupational or environmental exposure to In our recently updated series of 246 consecutive patients with t-MDS/t-AML, 230 (94%) had a clonal chromosomal abnormality (Le Beau et all” and Le Beau et al, unpublished results), and 175 patients (7 1%) had a clonal abnormalityleading to loss or deletion of chromosome 5 and/or 7. Amongthese 175 patients, 87 hada loss of chromosome 7, 18 had a del(7q), 21 had loss of 7q as a result of an unbalancedtranslocation, and 1 patient had abalanced translocation involving 7q22. Fifty-seven patients had abnormalities of both chromosomes 5 and 7. Overall, 105 patients (42%) had abnormalities of chromosome 5, and 127 (52%) had abnormalities of chromosome 7. Third, leukemias arising in individuals with constitutional disorders associated with a predisposition to myeloid leukemias, eg, Fanconi anemia, congenitalneutropenia, and NFI, are frequently characterized by -7/de1(7q), often as the sole cytogeneticabn0rma1ity.I~ Taken together,these dataare consistent with the hypothesis that inactivation of a tumor ECURRING CHROMOSOMAL abnormalities are characteristicof human malignantdiseases,particularly the leukemias and lymphomas.’” To date, the major emphasis of the molecular analysis of the chromosomal abnormalities in hematologic malignant diseases has involved the recurring translocations, in which two genes are juxtaposed, resulting in the activation of an oncogene in a dominant fashion! However, theloss of genetic material inhuman tumorshasreceived considerable attention,particularlyin solid tumors. This genetic change may contribute to the development of human neoplasms by inactivating both alleles of tumor suppressor gene^.^.^ The observation of recurring cytogenetic deletionsin malignant cells hasprovided a starting point for the positional cloning of a number of tumor suppressor genes that are important in the development of solid tumors. The identification of recurring chromosomal deletions in the hematologic malignant diseases suggests that, similar to that of solid tumors, the inactivation of tumor suppressor genes may be involved in the pathogenesis of some leukemias.’ In fact, genetic and biochemical studies of leukemia cells from children with neurofibromatosis 1 (NFI ), and of hematopoietic cellsfrom strains of mice withatargeted disruption of murine Nfl, have shown that NFI functions From the Section of Hematology/Oncology, and the Cancer Research Center,The University of Chicago, Chicago, IL; and the Genome Technology Branch, National Center for Human Genome Research, National Institutes of Health, Bethesda, MD. Submitted May X , 1996; accepted June 25, 1996. Supported by Public Health Service Grant No. POI CA40046 (to M.M.L. and R.A.L.). Address reprint requests to Michelle M . Le Beau. PhD, Section of Hematology/Oncology, The University of Chicago, 5841S Maryland Ave. MC2115, Chicago IL 60637. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with I S U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 000~-4971/96/S806-0054$3.00/0 1930 Blood, Vol 88,No 6 (September 15). 1996: pp 1930-1935 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MOLECULARDELlNlATlON OF THE DEL 1931 (7a) suppressor gene located on 7q is a common event that contributes to the pathogenesis of malignant myeloid disorders in a number of biological and clinical contexts. We and others have proposed that the long arm of chromosome 7 contains a tumor suppressor gene and that this gene is likely to be located within a commonly deleted segment in patients who have a del(7q). By cytogenetic analysis of 81 patients with a del(7q), we have identified two commonly deleted segments, suggesting that 7q may contain more than one myeloid leukemia-related gene. In addition, we have used fluorescence in situ hybridization (FISH) to delineate a commonly deleted segment at 7q22 using molecular probes corresponding to a well-defined yeast artificial chromosome (YAC) contig mapping to this region of the genome. MATERIALS AND METHODS Patients. We examined bone marrow (BM) or peripheral blood (PB) specimens from 81 patients who were diagnosed and treated at the University of Chicago Medical Center or referred to our cytogenetics laboratory between 1970 and 1996 from other metropolitan Chicago hospitals. The diagnosis and subclassification of MDS or AML was based on morphological and cytochemical studies of PB smears, BM aspirates, and biopsy specimens obtained before therapy, according to the French-American-British Cooperative Group criteria. Cytogenetic analysis was performed with quinacrine fluorescence and trypsin-Giemsa banding techniques on BM cells from aspirate or biopsy specimens or on PB cells obtained at the time of diagnosis. We examined metaphase cells from direct preparations or from short-term (24 to 72 hours) unstimulated cultures. Although three cytogeneticists evaluated the deleted homologues in each case, the designation of breakpoints in malignant cells can be somewhat imprecise, particularly when the chromosomes are condensed. Of the 81 patients examined, 8 patients were ascertained before 1982, when Q-banding was in use, and the average number of chromosome bands observed per haploid cell was 350 to 400. The remaining patients were studied using G-banding methods; during this period, the average length of the chromosomes had improved to r 4 5 0 bands per haploid cell. Chromosomal abnormalities are described according to the International System for Human Cytogenetic Nomenclature (1995).’* DNA probes. YAC clones used for FISH studies were selected from a physical map of chromosome 7 constructed by sequencetagged site (STS) content mapping (Green et alz3 and Green et al, manuscript in preparation). Yeast cells containing YACs were grown in acid hydrolyzed casein (AHC) media (inoculated with a single colony from an AHC plate), and DNA was prepared using standard procedure^.'^ DNA was resuspended at a concentration of FJ 100ngl wL, and Inter-Alu polymerase chain reaction was performed using the procedures described by Lengauer et a1 25 and Liu et aLZ6The polymerase chain reaction products were biotin-labeled by nicktranslation using Bio-l l-deoxyuridine triphosphate (dUTP; Enzo Diagnostics, New York, NY).*’ FZSH. Human metaphase cells were prepared from phytohemagglutinin-stimulated PB lymphocytes or from BM or PB cells from patients with MDS or AML. FISH was performed as described previously.*’ Hybridization of biotin-labeled probes was detected with fluorescein-conjugated avidin (Vector Laboratories, Burlingame, CA), and chromosomes were identified by staining with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). In some instances, a centromere-specific probe for chromosome 7 (CEP 7Spectrum Orange; Vysis Inc, Downers Grove, L) was cohybridized with the YAC probes, to facilitate the identification of the chromosome 7 homologs. Cytogenetic map locations were determined by analyzing the signal relative to the Ah-R banding pattern observed on hybridized chromosomes and the DAPI staining pattern observed using a DAPI-fluorescein isothiocyanate dual-pass filter (Chromatechnology, Brattleboro, VT). For the analysis of leukemia samples, 10 to 15metaphase cells with an abnormality of 7q were scored by each of two independent observers; in a few instances, only 5 to 9 metaphase cells were available for analysis. RESULTS Cytogenetic delineation of the commonly deleted segments. To determine the location of genes on 7q that may be involved in myeloid leukemogenesis, we first examined the breakpoints of the deletions in a consecutive series of 81 patients. This analysis included 26 patients with t-MDSI t-AMLand 55 patients who had primary MDS or AML de novo (Fig 1). Our analysis suggested that the deletion breakpoints were heterogeneous and that the deletions were interstitial. In contrast to other recumng deletions in myeloid disorders, eg, de1(5q),I3 there may be two distinct deleted segments of chromosome 7. The majority of patients (65 of 81 [80%]) had proximal breakpoints in q l l (25 patients) or q22 (27 patients); the remaining 13 patients in this group had a proximal breakpoint in 7q21. Thirty-five patients had distal breakpoints in q36; less often, distal breakpoints were observed in q22 (10 patients) and q34 (10 patients; bands q31, q32, q33, or q35 were involved in the remaining 10 patients). The identification of patients who had a proximal breakpoint in 7q22 (27 patients) and of other patients who had a distal breakpoint in this band (10 patients) allowed us to refine this commonly deleted segment of 7q to band q22. We have also identified 9 patients with myeloid leukemias who had balanced translocations involving 7q22, providing further support that a commonly deleted segment of 7q may be localized to this chromosomal band (Le Beau et al, unpublished results). The remaining 16 patients had interstitial deletions involving the distal long arm of chromosome 7. The proximal breakpoints were in q31 (5 patients) or q32 (11 patients). The majority of patients had a distal breakpoint in q36 (10 patients), but distal breakpoints were also observed in q33 (3 patients), q34 (1 patient), and q35 (2 patients). The commonly deleted segment for this group of patients consisted of 7q32-33. Molecular delineation of the commonly deleted segment at 7q22. To delineate the commonly deleted segment at 7q22 at the molecular level, we performed FISH of 13 YACs distributed along 7q, including 7 YACs within 7q22, to metaphase cells with a del(7q). Because of the limited amount of material available for some patients, we were unable to hybridize each probe to metaphase cells from all of the patients. The YACs were hybridized to leukemia cells from 15 patients who had either a proximal breakpoint (6 patients) or a distal breakpoint (9 patients) within 7q22. The majority of patients had a myeloid neoplasm (MDS/AML de novo, 5 patients; t-MDS/t-AML, 5 patients; chronic myelogenous leukemia in myeloid blast crisis, 1 patient); however, we also examined 4 patients with lymphoid diseases characterized by a del(7q) (acute lymphoblastic leukemia, 2 patients; nonHodgkin’s lymphoma, 1 patient; and chronic lymphocytic From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LE BEAU ET AL 1932 p 22 21 15 14 13 12 11 2 3 1 4 1 1 5 6 1 2 1 1 2 2 1l 2 4 1 1 2 2 1 6 1 l 1 t"DSI l 1-AML 5 3 1 de novo 2 commonly the 21 q 22 31 deletions the (t-MDSlt-AML, 32 33 34 35 36 the leukemia, 1 patient), who were not included in our cytogenetic analysis of the deletions described above. The genomic origin of each YAC clone on 7q was confirmed by hybridization to normal metaphase cells.23These studies showed that both the proximal and distal breakpoints were heterogeneous at the molecular level. YAC yWSS1668 and all clones centromeric of this YAC were proximal to the commonly deleted segment and were not deleted in all patients, whereas YAC yWSS3710 and all clones telomeric to this YAC were distal to the commonly deleted segment and were notdeleted in all patients (Figs 2 and 3). In all cases, hybridization of the YACs was observed on the normal chromosome 7, suggesting that submicroscopic deletions had not occurred on the normal homolog. Cloned coverage of commonly deleted region in YACs. The FISH analysis described above indicated that the commonly deleted region of 7q22 was located between YACs yWSS1668 and yWSS3710. As part of a global effort to assemble a fully integrated physical map of human chromosome 7,'"28"0 these two YACs have been mapped to a welldefined, highly redundant YAC contig. The most relevant portion of this contig is shown schematically in Fig 4. The interval defined bythe flanking clones yWSS 1668 and yWSS3710 (Fig 4) is roughly 2 to 3 Mb in size (E.D. Green, unpublished data). Importantly, this region is fully contained Fig 1. Cytogenetic of delineation deleted segments. Diagram of the bending pattern of chromosome 7 shows the breakpoints and extent of in 81 patients with myeloid leukemias 26 patients; MDSlAML de novo, 55 patients). The numbers above each vertical bar indicate number thisof patients with deletion. The shaded boxes delineate the smallest commonly deleted segments at 7q22 and 7q32-33. within a set of overlapping clones, with the overlap relationships established by STS content mapping. Among the STSs mapping to this interval are a number of established genetic markers (D7S I503 [sWSS35171, D7S2509 [sWSS10981, D7S2504 [sWSS2506], D7S818 [sWSS2539], D7S796 [sWSS1636], D7S658 [sWSSl097], D7S2446 [sWSS2492]. D7S2494 [sWSS3045], D7S1530 [sWSS3516], D7S2545 [sWSS1763], and D7S1841 [sWSS3256]) and one known gene (PSMC2 or MSSI [sWSS1845;see GenBank D1 10941). Of note, relevant genes known to reside nearby on7q," such as the genes encoding erythropoietin (EPO) and plasminogen activator inhibitor 1 (PLANHI) map to other regions on chromosome 7 and not to the contig shown in Fig 4. Thus, the commonly deleted region of 7q22 is fully accessible in cloned form, with the corresponding YACs and STSs representing valuable starting reagents for pursuing the isolation of the myeloid leukemia tumor suppressor gene. DISCUSSION Defining a commonly deleted segment on 7q in myeloid leukemias is of particular interest because alterations of this chromosome are common, andthey arise in a variety of clinical contexts. Our results from cytogenetic and molecular mapping of the deletions of chromosome 7 in malignant myeloid disorders suggest that there are two distinct regions Proximal deletions ~~ Patient No. Disease Fig 2. Molecular delineation of the commonlydeleted segmentat 7q22. Diagram of the banding pattern of chromosome 7 shows the results of FISH analysis of 15 patients with a del(7q) involving either a proximal (6 patients) or distal (9 patients) breakpoint in 7q22. The YAC clones (indicated with their prefix"yWSS" followed bya number) and their cytogenetic locations are shown on the rightof the chromosome. Hybridization results for the deleted homologs are indicated bya "+" (positive signal) or "-"(no signal) sign. A positive signal was observed for each YAC on the nondeleted homolog. The genomic segment containing YAC yWSS1269 (shaded box) was deletedin each patient examined. 1 2 CML cu 3 AU Distal deletions ~~~~ 4 5 ~~ 6 ~ M D S NHL m s 7 8 9 ms w a r n s 10 11 12 13 14 15 AMLAMLIAMLAU~MDS IAML + +- . + From www.bloodjournal.org by guest on June 18, 2017. For personal use only. MOLECULAR DELlNlATlON OF THE DEL (70) 1933 Fig 3. FISH of YAC clones t o leukemia cells with a del(7q). (A) Biotin-labeled YAC yWSS1668 DNA was hybridizedt o metaphase cells from patient no. l 1 with a de1(7)(q22q34). Hybridization signal was detected on both the normal (arrow) and deleted (arrowhead) homologs and, thus, defines the proximal boundary of the commonly deleted segment. (B) Hybridization signal for biotin-labeled YACyWSS3710 DNA (detected with fluorescein isothiocyanate-avidin) was observed on the normal homolog (arrow)and on the de1(7)lqllq22) in patient no. 4, defining the distal boundary of the commonly deletedsegment (arrowhead). interstitial. Moreover, wehave delineated the centromeric commonly deleted segment at 7q22. This region is flanked by YAC yWSS1668 on the proximal side and yWSS3710 on the distal side, with the complete interval covered by a well-defined YAC contig. The delineation of a commonly deleted segment on7q has been more difficult than that for deletions of other chromosome, such as 5q. This is largely because loss of a whole chromosome 7 represents the mostcommon abnormality, and relatively few patients with a del(7q) have been available of 7q, 7q22 and 7q32-33, that are likely to contain genes involved in the pathogenesis of these disorders. Furthermore, these results suggest that genes located within the same cytogenetic intervals are involved in the pathogenesis of both de novo and therapy-related myeloid diseases. Band 7q22 also appears to be involved in patients who developed myeloid disorders in the context of a constitutional predisposition (Fanconi anemia or NFI), although the cytogenetic data are limited." We have usedFISH analysis of malignant cells characterized by a del(7q) to confirm that the deletions are S W S S 3 5 1 7 F W S S 3 ; 0 F S W S S W S S 2 I 4 4 2 n 9 0 F S W S S 6 8 2 W S S 1 2 I 7 S W S S I 0 9 8 S W S S I 8 4 5 S S W S S 9 5 S W S 4 2 2 0 6 F W S S 2 5 3 9 S S S W S S I 6 W S S I 6 7 9 W S S I if 0 9 7 S W S S 2 4 9 2 S S W S S 3 0 W S S 4 5 2 9 6 8 S S S W S W S 6 3 8 S S 9 2 8 S W S S 7 4 6 S W S S W S S 5 I 6 6 3 3 1 7 F S S W S S 3 W S S W S S 3 5 8 6 7 9 2 2 5 Fig 4. YAC contig containing the commonly deleted region of 7q22. A highly redundant YAC-based STS-content map of the region of chromosome 7q22 commonly deleted in myeloid leukemia has been constructed (E.D. Green, manuscript in preparation). For simplicity, the minimal set of YACs from this contig that provide cloned coverage across the interval flanked by clones yWSS1668 and yWSS3710 is depicted schematically. The STSs (named with theprefix "sWSS" followed bya unique number) mappingt o these YAC clones are listed along the top, whereas the YACs (named with the prefix "yWSS" followed by a unique number) are shown as horizontal bars. The measured size of each YAC (in kilobasesl is indicated below its name. The presence of an STS in a YAC is indicated bya darkened circle at theappropriate position. All information about the STSs is available in Genbank and the Genome Data Base, whereas information about theYACs is available in the Genome Data Base. The established order of STSs from the centromeric (leftward)t o 7q telomeric (rightward) ends is indicated. Note that the limited set of YACs shown in this figure do not provide sufficient mapping information t o deduce the indicatedorder of STSs; however, this order is well supported by the complete data set. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LE BEAU ET AL 1934 for analysis. Kereet al’* usedrestriction fragment-length polymorphisms and pulsed-field gel electrophoresis to localize the proximal breakpoints on 7q in leukemia cells from four adults with MDS or AML and a del(7q). They observed lossofheterozygosity forcDNAprobes immediatelytelomeric of the EPO gene at7q21.3-22,including the PLANHl gene, which is located -3 centimorgans (CM)distal to EPO. Although cytogenetic analysis of their patients suggested that the entire distal q arm was deleted, analysis with DNA markers showed that the deletions were interstitial.” Lewis et a174examined five patients with MDS and a del(7q) and found that the deletions were interstitial in each case; constitutionalheterozygosity of theTCRB locusat 7q34 was retained in three patients. By using FISH analysis, Fischer et a1” defined a 3- to 4-Mb commonly deleted segment that includes the PLANHl and CUTLl genes in 7q22, a region that may be close to the commonly deleted segment identified in our studies.Tosi et a1j6 examined amyeloid leukemia cell line with a del(7q) and determined that the breakpoint at 7q22 was proximal to GNB2, which is centromeric of EPO and PLANHI. Althoughthestudiesdescribedabove suggestthat the commonly deleted segment in 7q22 is close to or contains the PLANHl gene,several groups of investigators have identified other regions of 7q22 or even 7q31 as potential locations for amyeloid tumor suppressor locus. For example, Kiuru-Kuhlefelt et al’7 used microsatellite markers to examine leukemia cells with a del(7q); the allele loss observed in four patients delineated a segment between markers D7S51S and D7S685 that was lost. This region overlaps slightly with the distal end of our commonly deleted segmentbut extends into q3 1. Other studiesof a few patients with -7 and unidentified marker chromosomes suggestedthatloss of a small segment including D7S522 (7q31) may be a critical event in the pathogenesis of some myeloid disorders.3x Johnson et al” recentlyreported on afamily in which multiple members in a 3-generation pedigree had a constitutional inversion of 7q, inv(7)(q22.lq34). One individual developed MDS, and anotherhad BM hypoplasia; both had the inv(7q). The breakpoint at 7q22.1 was mapped to a YAC containing the asparagine synthetase gene (ASNS). Although these results are intriguing, their significance is unclear because the incidence of myeloid disorders is low in family members with the inversion and because the ASNS gene has been mapped proximal toEPO and COLlA2, in aregion that retains heterozygosity in many patients with MDS/AML and a del(7q).””’ Moreover, this region is proximal to the commonlydeletedsegment that wehave defined in this study. It is possible that there aremultiple genes in 7q21.3-22 that are involved in the pathogenesis of malignant myeloid disorders or thatthe myeloid diseases in thisfamily are unrelated to the constitutional inversion. The commonly deletedregion of 7q22 defined in our studies is contained in a well-defined contig; the corresponding YAC clones and STSs represent valuable reagents for the isolation of a myeloid leukemia tumor suppressor gene. ACKNOWLEDGMENT We thank the manyphysicians from other hospitals in the Chicago area who provided clinical and laboratory information on some of the patients studied. We are grateful to the technologists in the Hematology/Oncology Cytogenetics Laboratory for assistance in the cytogenetic studies and to M. Isaacson for management of the cytogenetics database. We thank Dr K. Shannon for helpful discussions. REFERENCES 1. Le Beau MM, Rowley JD: Cytogenetics, in Beutler E, Lichtman MA, Coller BS, Kipps TJ (eds): Hematology. New York, NY. 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For personal use only. 1996 88: 1930-1935 Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases MM Beau, R 3rd Espinosa, EM Davis, JD Eisenbart, RA Larson and ED Green Updated information and services can be found at: http://www.bloodjournal.org/content/88/6/1930.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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