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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
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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
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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
+
+-
.
+
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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
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7
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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.
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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.
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From www.bloodjournal.org by guest on June 18, 2017. 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
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