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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2372-2377
NEOPLASIA
Delineation of a minimal interval and identification of 9 candidates for a tumor suppressor gene in malignant myeloid disorders
on 5q31
Stephen K. Horrigan,
Zarema H. Arbieva,
Hong Yan Xie,
Jelena Kravarusic,
Noreen C. Fulton,
Haley Naik,
Tiffany T. Le, and
Carol A. Westbrook
From the Department of Pediatrics, Lombardi Cancer Center,
Georgetown Medical Center, Washington, DC, and the Section of
Hematology/Oncology, Department of Medicine, University of Illinois at
Chicago, Chicago, IL.
 |
Abstract |
Interstitial deletion or loss of chromosome 5 is frequent in
malignant myeloid disorders, including myelodysplasia (MDS) and acute
myeloid leukemia (AML), suggesting the presence of a tumor suppressor
gene. Loss of heterozygosity (LOH) analysis was used to define a
minimal deletion interval for this gene. Polymorphic markers on 5q31
were identified using a high-resolution physical and radiation hybrid
breakpoint map and applied to a patient with AML with a subcytogenetic
deletion of 5q. By comparing the DNA from leukemic cells to buccal
mucosa cells, LOH was detected with markers D5S476 and D5S1372 with
retention of flanking markers D5S500 to D5S594. The D5S500-D5S594
interval, which covers approximately 700 kb, thus represents a minimal
localization for the tumor suppressor gene. Further refinement of the
physical map enabled the specification of 9 transcription units within
the encompassing radiation hybrid bins and 7 in flanking bins. The 9 candidates include genes CDC25, HSPA9, EGR1, CTNNA1, and 5 unknown ESTs. Reverse-transcription polymerase chain
reaction confirms that all of them are expressed in normal human bone
marrow CD34+ cells and in AML cell lines and thus
represent likely candidates for the MDS-AML tumor suppressor gene at 5q31.
(Blood. 2000;95:2372-2377)
© 2000 by The American Society of Hematology.
| |
Introduction |
Interstitial loss of the long arm of chromosome 5, del(5q), or complete loss of the entire chromosome  5 is a
frequent finding in malignant myeloid disorders, including acute
monocytic leukemia (AML) and myelodysplastic syndrome
(MDS).1-4 These chromosomal losses are especially prevalent
in AML arising after prodromal MDS or in MDS and AML arising after
previous cancer treatment with alkylating agents or radiotherapy. In
these therapy-related myeloid disorders, chromosome 5 deletions occur
in approximately 40% of patients.5,6 Chromosome 5 deletions are less prevalent in de novo AML and occur in an estimated
5% to 7% of patients, but they are increased in the
elderly.1 AML with del(5q) usually shows trilineage
involvement, pronounced dysplasia, and characteristic megakaryocytic
abnormalities. Additional cytogenetic abnormalities are frequent; the
most common is del(7q) or 7.6,7 Regardless of the
etiology, del(5q) is among the worst prognostic indicators in AML
because it is characterized by poor response to chemotherapy, low
complete remission rate (less than 30%), and a median survival time of
4 months.7,8
The prognostic significance of chromosome 5 deletions in MDS, however,
is variable. In therapy-related MDS, or in RAEB and RAEB-t arising de
novo, del(5q) or 5 generally implies a rapid progression to
leukemia and poor outcome.5,6 On the other hand, a
relatively indolent form of MDS is also associated with the loss of 5q.
This disorder, called 5q syndrome, is characterized by
transfusion-dependent anemia with little or no cytopenia, usually shows
an FAB subtype of RA or RARS, and rarely progresses to
leukemia.3,9,10 It is clear that the 5q syndrome is
a distinct clinical entity with a different natural history than the
more aggressive forms of MDS-AML. Thus, the impact of the 5q deletion
in myeloid disorders must be interpreted in light of other clinical information.
The recurrent nature of these chromosomal deletions suggests that 5q
contains tumor suppressor genes important to hematologic transformation. To localize these genes, others and we11-14
have attempted to define a consistently deleted region using
cytogenetic and molecular methods to map the extent of deletions in
clinical samples. These studies suggest that the critical tumor
suppressor gene for AML and some forms of MDS is located at band 5q31.
This is in contrast to the common deletion interval for the 5q
syndrome, which has been localized to 5q32.15 The
specification of 2 genomic intervals implies that different genes are
responsible for each of these 2 myeloid disorders, as clinical findings
suggest. Identification of each gene will provide valuable insight into
the process of malignant myeloid transformation and useful markers for
diagnosis, prognosis, and targets of therapy.
Previously our group proposed an interval of approximately 1.5 Mb
within 5q31 as a tumor suppressor gene site based on overlapping deletions in clinical samples of MDS and AML, which included a patient
with a submicroscopic chromosomal deletion.13 In the current study, we reexamined this case to further narrow the deletion interval, using additional polymorphic markers and a corrected marker
order based on a high-resolution physical, genetic, and transcript map
of 5q31.16 Here we report the result of this analysis,
showing that the minimal deletion interval spans approximately 700 kb
of 5q31 and thus would be expected to be deleted in all patients with
AML with del (5q). Further refinement of the existing physical map by
radiation hybrid (RH) analysis led to the identification of 9 transcription units that lie within this interval and that represent
candidates for the MDS-AML tumor suppressor gene.
| |
Materials and methods |
Polymerase chain reaction analysis with microsatellite markers
Bone marrow and buccal smear samples were collected as sources of
tumor DNA and normal DNA, respectively. Polymerase chain reaction (PCR)
was performed as described,13 using 0.1 µg sample DNA or
5 µL buccal smear extract under the following conditions: 10 mmol/L
Tris-HCl, pH 8, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L dATP, dGTP, and dTTP, 50 µmol/L dCTP, 1 µCi
32P dCTP (3000 Ci/mmol), 5% glycerol, 0.1% Triton X-100,
0.5 U Taq polymerase (Perkin-Elmer/Cetus, Norwalk, CT),
and 100 ng each primer in a 20 µL reaction. The reactions were run in
a Perkin-Elmer model 480 thermocycler using the following parameters:
initial denaturing at 94°C for 3 minutes; 35 cycles at 94°C (40 seconds), 55°C (30 seconds), and 72°C (30 seconds); final
extension at 72°C for 5 minutes. Then 1 to 5 µL each PCR reaction
was separated on a 6% polyacrylamide gel with 8 mol/L urea and exposed
to x-ray film (Eastman Kodak, Rochester, NY). Loss of heterozygosity
(LOH) was scored visually. The criteria for scoring LOH was as follows: retention of 2 alleles in normal tissue was considered informative, whereas the presence of only 1 allele in normal tissue was not informative. Therefore, LOH was considered when there was a loss of 1 allele in the tumor DNA when compared with the informative control DNA
of 2 alleles.
Radiation hybrid analysis
Markers were tested by PCR on the selected radiation hybrids from
the Whitehead-Genebridge4 (GB4)17 and the Stanford G3 (G3)18 panels as previously described.16
Radiation hybrid panels were purchased from Research Genetics
(Hunstville, AL).
Expression studies in myeloid tissues
Expression of candidate genes was examined in normal human bone
marrow CD34+ cells and 3 human myeloid leukemia cell lines,
KG-1, HL-60, and AML-193 obtained from ATCC (Rockville, MD). The
CD34+ cells were isolated and purified, as previously
described,19 from bone marrow obtained from normal donors
with informed consent under the guidelines provided by the University
of Illinois. Briefly, low-density marrow cells were prepared using
Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) centrifugation and
were further purified using a CD34+ Progenitor Cell
Isolation Kit (Miltenyi Biotec, Auburn, CA). Purity of
CD34+ cells was confirmed by FACS analysis using anti-CD34
antibodies. Total RNA was extracted from approximately 107
cells using TRIZol Reagent (Gibco Technologies, Gaithersburg, MD)
according to the recommended protocol.
Reverse transcription (RT)-PCR was performed using either the Titan 1 Tube RT-PCR System (Boehringer Mannheim, Indianapolis, IN) or the
Enhanced Avian RT-PCR kit (Sigma, St. Louis, MO) according to the
manufacturer's protocol. Primers were designed based on published or
publicly available database sequences. Optimal Mg2+
concentrations and annealing temperatures were determined for each primer pair. All reactions were performed in duplicate, including control reactions that did not include RT or RNA. RT-PCR products were
visualized on 1.5% agarose gels stained with EtBr.
The following  2-microglobulin primers were used as
positive controls for RT-PCR: forward 5'TGCCTGTACACTGCTCTTTATG
3'; reverse 5'GAATGTTTGGTGAACCTTCCT 3'. Because
primers span an intron, the expected size of the RNA-derived PCR
product was 245 bp whereas that from genomic DNA was 860 bp.
| |
Results |
Microsatellite analysis of AML with a minimal deletion
Our previously published high-resolution physical map spanning 6 Mb
of 5q3116 was used to identify polymorphic markers for this
study. This map includes all PCR-formatted polymorphisms that could be
identified in existing databases; marker order was independently
confirmed by typing on a CEPH meiotic breakpoint panel.20 Selected microsatellite markers from
5q31 were used to continue the analysis of a patient with AML whom we
previously reported to have a submicroscopic deletion of chromosome
5.13 This clinical sample, designated case 24 in that
study, is a diagnostic bone marrow sample from a 48-year-old man who
had a relapse of AML of FAB M5a morphology and a white blood cell count
of 104 000/µL. The karyotype was t,6;11
del7(q31q36),+18, showing no visible abnormalities of
chromosome 5, and allelotype analysis revealed LOH limited to a small
interval on 5q31.
In the current study, LOH was similarly assessed by comparing the
amplification patterns of the selected polymorphic markers in the
leukemia sample to that of the buccal smear. Figure
1 depicts a diagrammatic summary of these
results relative to marker order. The study revealed loss of single
alleles in 2 contiguous markers, D5S476 and D5S1372, with retention of
both alleles in flanking markers, D5S500 and D5S594 (Figure
2). Note that LOH with D5S476 and D5S1372
was reported in the previous study, but the placement of the markers
within the map was in error; they were thought to be proximal to
D5S500.13 The corrected marker order presented here is
based on a high-resolution integrated map13,20 and is
consistent with that reported elsewhere.14 Note also that marker AFMB350YB1, which had previously been interpreted as LOH in case
24, was found to be noninformative on further testing. LOH results thus
led us to conclude that the minimal deletion in case 24 results in the
loss of internal markers D5S476 and D5S1372 and is completely contained
within the flanking markers D5S500 and D5S594. The size of this minimal
interval is estimated to be approximately 700 kb, based a radiation
hybrid distance between flanking markers of 29 cR and using the
approximation of 1 cR10,000 = 29 kb for the Stanford G3
radiation hybrid panel.18
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| Fig 1.
Allelotype results for AML case 24.
Markers are shown in order from centromere (top) to telomere (bottom),
with their approximate cytogenetic band locations indicated on the
chromosome 5 histogram. Filled oval, marker is informative with loss of
heterozygosity; open oval, marker is informative with no loss; hatched
oval, marker is not informative.
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| Fig 2.
PCR amplification patterns for critical markers in case
24.
Marker names indicated on the top of the figure are shown in order from
telomere (left) to centromere (right). PCR analysis is shown for DNA
isolated from normal buccal smear (B) compared with DNA from leukemia
cells (L) isolated from the patient's bone marrow sample. For each
marker, the 2 alleles in the buccal DNA are indicated by arrows.
Markers D5S500 and D5S594 show retention of both alleles in the
leukemia sample, whereas loss of 1 allele is apparent for markers
D5S476 and D5S1372.
|
|
Refinement of the physical map and identification of expressed
sequences within the deletion interval
Expressed sequences within the minimal deletion interval were
identified with reference to our previously published RH breakpoint map. The construction of this map resulted in the placement of 122 expressed sequences within 5q31, including both mapped expressed sequence tags (ESTs) from the Human Gene Map and unmapped ESTs from the
Radiation Hybrid Database (RHdb). To localize more accurately the
expressed sequences within 5q31 and to order them relative to the
candidate gene interval, it was necessary to improve the resolution of
the RH breakpoint map. This was done by additional PCR typing of ESTs
on the Stanford G3 RH panel. The G3 panel has a higher resolution (more
radiation hybrid breaks per given genomic distance) than the GB4 panel,
where most of the ESTs were typed. Our RH breakpoint panel of 5q31 as
published consisted of 13 hybrids from both the G318 and
the GB417 panels; from this panel of 13 hybrids, 8 of them
had breakpoints within the interval and 4 contained the
interval within its entirety. Selected PCR markers were typed or
confirmed on this panel as necessary (Figure
3). Radiation hybrid "bins" were then
defined by the breakpoints on G3 and GB4 hybrids, and markers were
placed in different bins based on visual inspection of their PCR typing
results relative to the breakpoints (Figure 3). The list of ESTs and
their localizations has been updated from the previous
publication.16
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| Fig 3.
Radiation hybrid breakpoint map of 5q31 interval.
Vertical bars indicate the boundaries of the RH bins based on the
presence of a breakpoint in any hybrid in the panel. The extent of the
minimal deletion interval is labeled and indicated by bold vertical
bars. Markers used in the study are listed in the row above the panel,
from centromere (left) to telomere (right). Expressed sequences are
italicized; known genes are italicized in bold and appear next to an
identifying EST, if known. Polymorphic markers are shown in
nonitalicized bold script. Identifying numbers for radiation hybrids
are listed in the left column for the Stanford G3 panel (upper half)
and the Genebridge 4 panel (lower half). Shaded squares (+) indicate
that the marker scored positive; () indicates that the marker
scored negative; (2) indicates an ambiguous signal; blanks
indicate the marker has not been tested.
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The relative order of the RH bins is fixed, but the order
of markers within each bin cannot be further resolved by the RH panel.
The breakpoint map was compared to the deletion interval, and all ESTs
within bins 4 to 6, encompassing the flanking markers and
intervening sequences, were considered to be primary candidate genes
(Figure 3). ESTs from adjacent bins 3 and 7, which lie outside the
deletion interval, were considered secondary candidates because their
corresponding coding sequences could potentially overlap into the
deletion interval. A summary of candidate genes or ESTs and secondary
candidates in flanking bins is provided in Table 1.
Within the deletion interval, 9 expressed sequences were found,
including 4 known genes, CTNNA1, CDC25C, early growth response gene-1 (EGR1), and HSPA9. CTNNA1 ( E-catenin)
encodes a cadherin-associated protein that plays an important role in
cell-cell association and cell differentiation.21,22
CTNNA1 is a promising candidate for a tumor suppressor gene; it
has recently been shown to contain mutations in colon cancer cell
lines, and it can function to suppress invasion.23
CDC25C encodes a cell cycle regulatory protein required for
cell entry into mitosis.24 EGR1 encodes a
zinc-finger protein essential for the differentiation of monocytic
cells.25 Both CDC25C and EGR1 have been
evaluated as tumor suppressor genes but have not been found to contain
mutations in clinical AML samples.14 HSPA9
(Mortalin) encodes a cellular immortalization protein that is
implicated in the control of cell proliferation and cellular senescence.26 It is also a good candidate for the AML
because since its loss may promote cellular immortalization. Among the EST candidates, WI-15469 is linked to the Unigene cluster that represents Matrin 3, Unigene cluster Hs.198992. The
assignment of WI-15469 to Hs.198992 was made since our
previous map was completed,20 and we believe that this
association is a database error because WI-15469 shares no
sequence homology with the complete cDNA sequence of Matrin 3 (Genbank M63483). Additionally, WI-15469 does not enter the
TIGR cluster for the Matrin 3 gene but is instead assigned to
the cluster THC252032, an unidentified partial cDNA. We are trying to
obtain additional sequence and resolve this inconsistency, which
probably resulted from an incorrect or chimeric cDNA sequence in
Unigene. The other 4 ESTs have no known homologies.
RH bins 3 and 7 contain the secondary candidates, including thyroid
hormone receptor coactivator protein (THRCP). This protein is a
tyrosine kinase and nuclear receptor coactivator, which regulates the
expression of target genes resulting from thyroid hormone and other
cellular proliferation signals.27 Gene CDC23
(located in the flanking bin 3) was recently proposed as a
candidate tumor suppressor gene, but it has been excluded because of
the lack of mutations in leukemia cells with the loss of
5q.28 The EST stSG30881 encodes a protein
that shows some homology to murine kinesin-like protein
RabKinesin. Kinesins are microtubule-dependent molecular motors
involved in intracellular transport and mitosis.29
Expression of candidate genes and ESTs in myeloid tissue
As a first step in the evaluation of these 9 genes as AML tumor
suppressor gene candidates, their expression in myeloid cells was
investigated. RNA was isolated from 3 human myeloid leukemia cell lines
and CD34+ cells obtained from normal human bone marrow. The
HL-60 cell line was established from the peripheral blood cells of a
patient with acute promyelocytic leukemia,30 KG-1 from the
bone marrow of a patient with acute erythroblastic
leukemia,31 and AML-193 from a patient with acute monocytic
leukemia.32 Both KG-133 and HL-6030
are reported to have chromosome 5 deletion or loss and consistently
show single alleles on allelotyping (data not shown). AML-193, on the
other hand, has retained both chromosome 5 alleles by
karyotype32 and allelotype assessment (data not shown).
Primers were designed using available cDNA and EST sequences (Table
2) and were tested on the RNA by RT-PCR.
The analysis showed that all sequences were expressed in the cell lines
studied (Figure 4), confirming that these
ESTs originated from expressed sequences and were appropriately
expressed in myeloid tissues, as would be expected of a leukemia tumor
suppressor gene.
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| Fig 4.
Expression analysis of candidate genes/ESTs.
RT-PCR was performed on human bone marrow CD34+ cells and
myeloid cell lines, AML-193, KG-1, and HL-60, as indicated, and was
visualized by agarose gel electrophoresis and ethidium bromide
staining. The left column lists the gene or EST that was tested,
using the PCR primers indicated in Table 2. The right column
indicates the product size in base pairs. The PCR results are
displayed for each primer set, with or without the addition of RT,
indicated by (+) or (), respectively. Negative controls for
template pairs (no RNA added) is not shown.
2-Microglobulin primers were used as the positive
control for RT-PCR.
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| |
Discussion |
In this study we present results that define a minimal
deletion interval for the proposed tumor suppressor gene in 5q31, which is implicated in AML and some forms of MDS. In previous work, others
and we11 specified a region on 5q31 that is consistently deleted in patients with AML and MDS with del(5q). The smaller interval, which we define here, is contained within this larger region;
thus it would be expected to be deleted in all patients with AML with
del(5q). We have identified 9 expressed sequences within the interval
and 7 in adjacent regions as primary and secondary candidates,
respectively, for this tumor suppressor gene.
The minimal deletion interval of approximately 700 kb in size was
specified based on the application of a high-density panel of
polymorphic markers that enabled the identification of more closely
spaced flanking markers, D5S500 and D5S594. It should be noted that the
assignment of this genomic location for the tumor suppressor gene is
based on a marker order that differed from that in our previous
article.13 The resultant minimal deletion interval is not
only smaller, it has been transposed telomerically from IL9-D5S414 to
D5S500-D5S594. This transposition is the result of an error in our
previous marker order, which was based on an unpublished YAC contig.
This error has since been corrected, such that the map used in the
current study16 is now in agreement with other physical
maps, including that published by Zhao et al.14
The minimal interval defined in the current study overlaps partially
with that specified by Zhao et al14 and Fairman et al.12 Zhao et al14 used fluorescence in situ
hybridization analysis to define the minimal overlap of cytogenetically
visible deletions in myeloid malignancies; this overlap included D5S479 to D5S500. Fairman et al12 placed the minimal interval
between IL-9 and EGR1, using LOH analysis of a deletion
accompanying a translocation involving 5q. Given the different methods
and clinical samples used and the slight discrepancies in map positions
for some markers, there is fairly good concordance in the resultant deletion intervals. It is therefore reasonable to assume that all these
investigators are localizing the same gene. Combining the data from all
3 reports, it seems most probable that the gene of interest is located
within the centromeric portion of our interval near the EGR1
gene. Continued LOH studies in AML and MDS are underway in our group to
try to identify additional incidences of LOH that further narrow the
deletion interval.
Defining a minimal interval for a disease permits the identification of
candidate genes within the interval. Although there are a number of
positional cloning strategies by which this can be done, the approach
presented here is rapid and does not require additional cloning steps
but instead uses publicly available database information. RHdb is such
a database, supported by the Human Genome Program, that aims to
generate a transcript map of the human genome by typing ESTs on the RH
panels34; the resultant transcript map, Genemap, has been
estimated to have achieved at least 50% coverage of all human genes.
The current version of Genemap has a resolution that is too low for our
purposes, but additional data exist because there are many ESTs in RHdb
that have been typed but have not been positioned on Genemap. Thus,
additional analysis is still required to complete a transcript map of a
small genomic interval such as ours. The analysis presented here
enabled the placement of 9 confirmed ESTs or 1 per 80 kb. It is
reasonable to estimate that we have identified at least half the
transcription units that are present based on the estimated coverage of
RHdb. Additional genes will undoubtedly be discovered by complementary methods such as exon trapping and with continued updating of the various Human Genome Project databases. Of note, a major sequencing effort for 5q31 is already underway at 2 centers (Lawrence Berkeley Laboratory, http://www-hgc.lbl.gov/seq/, and Washington University, http://www.genome.wustl.edu/gsc/) that will help to validate these results and to identify additional genes in the interval.
The candidate genes reported here include 6 known genes (THRCP,
HSPA9, EGR1, CTNNA1, CDC23, and CDC25C) and 1EST homologous to kinesin. The remaining are ESTs of unknown function that have not
yet been characterized or sequenced. All candidate genes identified in
this study were confirmed to be expressed in myeloid tissues and cell
lines; however, it was not possible to evaluate their expression in
case 24, or in the del(5q) cases reported previously by
us13 because only DNA was then available.
Although there is no prior information about the function of this
myeloid malignancy tumor suppressor gene, the minimal criteria possibly
required are that the gene be expressed in normal myeloid tissue, that
1 allele be lost by the deletion of 5q, and that the remaining allele
be mutationally inactivated in AML or MDS cells with del(5q). Thus we
expect that the coding sequences of the gene will harbor mutations in
clinical samples. Such criteria have been successfully applied to
confirm tumor suppressor genes in other diseases and to exclude
candidates at 5q31 (eg, the gene CDC23).28
Alternatively, expression might be decreased sufficiently to inactivate
the gene. Efforts are now underway to further characterize these genes
by obtaining full-length cDNA sequences and evaluating them for
expression and mutations in AML and MDS tumor samples.
| |
Acknowledgments |
We thank Dr Ronald Hoffman for kindly providing CD34+
cells. We also thank Dr Ignatius Gomez and Dr Zhenbo Hu for helpful
discussion and critical reading of the manuscript, and Seby Edassery
and Brent Chyna for invaluable help in the preparation of the manuscript.
| |
Footnotes |
Submitted August 16, 1999; accepted December 1, 1999.
Supported by the W. M. Keck Foundation (C.A.W.) and by Public Health
Service grants R01CA72593 and P01CA75606 (C.A.W.).
Reprints: Carol A. Westbrook, Section of Hematology/Oncology,
Department of Medicine, University of Illinois at Chicago, 900 S
Ashland Ave, M/C 734, Chicago, IL 60607; e-mail: cwcw{at}uic.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction