Blood, 15 May 2002, Vol. 99, No. 10, pp. 3848-3850
BRIEF REPORT
Unlike AML1, CBF
gene
is not deregulated by point mutations in acute myeloid leukemia and in
myelodysplastic syndromes
Hugues Leroy,
Christophe Roumier,
Nathalie Grardel-Duflos,
Elizabeth Macintyre,
Pascale Lepelley,
Pierre Fenaux, and
Claude Preudhomme
From the Laboratoire d'Hématologie A,
Hôpital Calmette, and Service des Maladies du Sang, CHU Lille;
Unité 524 INSERM, Institut de Recherche sur le Cancer de Lille;
and Laboratoire d'Hématologie, Hôpital Necker enfants
malades, Paris, France.
 |
Abstract |
The core-binding factor (CBF) complex is a heterodimeric
transcription factor composed of 2 subunits, CBF
and CBF
, that play a major role in hematopoiesis. Both members of the CBF complex are
frequently altered in acute myeloid leukemia (AML) by translocation, most commonly t(8;21), t(12;21), and t(3;21) for CBF, located in
21q22, and inv16(p13;q22) for CBF, located on
16q22. Recently, a new mechanism of alteration of CBF, by point
mutation, has been reported in myeloid malignancies, particularly in
M0 AML. In the present study, we found no point mutation of the
CBF gene in 30 myelodysplastic syndromes and 100 AMLs, suggesting a limited role, if any, of CBF point mutations in
those disorders.
(Blood. 2002;99:3848-3850)
© 2002 by The American Society of Hematology.
| |
Introduction |
The core-binding factor (CBF) complex is a
heterodimeric transcription factor composed of 2 subunits, and .
CBF is encoded by a family of genes with homology to the
Drosophila runt gene, which contains an evolutionarily
conserved protein motif of 128 amino acids1 that is
responsible for DNA binding, transactivation, and heterodimerization
with the subunit .2 Unlike CBF, the CBF subunit
does not bind DNA but enhances, by a factor of 10 to 20, the DNA
binding of CBF.3 The CBF complex plays a major role in
hematopoiesis4 through regulation of the expression of
various genes, including granulocyte-colony-stimulating factor (G-CSF)
receptor, macrophage-colony-stimulating factor (M-CSF) receptor,
myeloperoxidase, interleukin-3, and T-cell-receptor gene.4-6 CBF (AML1), located on 21q22, and CBF,
located on 16q22, are the 2 genes most frequently deregulated in
leukemia, generally through translocations that produce
chimeric RNA and protein.7 For the AML1 gene,
the major chimeric proteins are AML1-ETO in the t(8;21)(q22;q22)
translocation,8 AML1-ETV6 in the t(12;21)(p13;q22)
translocations, and, less often, AML1-EVI1, AML1-MDS1, and AML1-EAP in
the t(3;21)(q26;q22) translocations.9-11 For the
CBF gene, only one chimeric protein has been
described
CBF-MYH11 in inv16(p13;q22) or
t(16;16)(p13;q22)present in almost all patients with the M4 E0
subtype of AML.12 In most of these cases, fusion proteins
act to repress transcription of the CBF target genes, which have a
promoter consensus sequence for AML1.11,12 In addition,
homozygous knockout mice for AML1 or CBF have the same phenotype
(absence of fetal hematopoiesis).5,6,13 Recently, point
mutations of CBF (AML1) have been reported in acute myeloid leukemia
(AML) and myelodysplastic syndromes (MDS).14-16 In this work, we looked for point mutations of the CBF gene that
could also potentially inactivate it in AML and MDS.
| |
Study design |
Analysis of point mutations of the CBF gene was
made in 25 healthy subjects (bone marrow donors) and 130 leukemia
patients100 patients with AML (40 patients with M0 AML, 17 patients
with M1 AML, 22 patients with M2 AML, 2 patients with M3 AML, 10 patients with M4 AML including 2 patients with M4 E0, and 9 patients with M5 AML according to the French-American-British [FAB]
classification17) and 30 patients with MDS (5 refractory
anemia, 6 refractory anemia with ring sideroblasts, 7 refractory anemia
with excess of blasts, 6 refractory anemia with excess of blasts in
transformation, and 6 chronic myelomonocytic leukemia according to FAB
classification). A large number of patients with M0 AML were included
in this study because a high frequency of AML1 mutations is observed in
this type of AML.16 All patients had given informed consent.
Detection of CBF gene mutations was made on DNA extracted
from bone marrow cells, by single-stranded conformation polymorphism (SSCP) analysis of the 6 exons corresponding to the entire coding region of the CBF gene. Primers for polymerase chain
reaction (PCR) were chosen upstream and downstream of each exon to
include the detection of mutations in acceptor and donor splicing
sites. PCR was performed in a total reaction volume of 50 µL
containing 50 ng DNA, 0.3 µM each primer (primer names and sequences
are given in Table 1), 1 to 3.5 mM
MgCl2 (Table1), 10 mM Tris HCl, 50 mM KCl, 200 µM each
dNTP (Pharmacia, Stockholm, Sweden), 2.5 U Taq DNA polymerase (Quiagen,
Courtaboeuf, France), and 0.037 MBq 32P-labeled
deoxycytidine triphosphate. Samples were heated for 5 minutes at 94°C
and then underwent 34 cycles for 1 minute at 94°C, 1 minute at 55 to
57°C (Table 1), and 1 minute at 72°C, followed by a final
elongation of 7 minutes at 72°C. After amplification, PCR products
were loaded on a 2% agarose gel stained with ethidium bromide. The
size of the PCR products was 163 to 276 bp (Table 1). For SSCP
analysis, 4 µL PCR product was diluted in 16 µL solution containing
0.1% sodium dodecyl sulfate and 20 mM EDTA; 3 µL this mixture was
mixed with 3 µL solution of 95% formamide, 20 mM EDTA, 0.05%
bromophenol blue, and 0.05% xylene cyanol and then was heated at
95°C for 2 minutes and cooled in ice. Finally, 3 µL
was loaded on an MDE gel (Tebu, Le Perray en Yvelines, France). Our
preliminary experiments showed the best SCCP profiles were observed
with a gel containing 5% glycerol, 1× TBE, and
electrophoresis run for 12 hours at room temperature.
| |
Results and discussion |
No abnormal SSCP profile for exons 1 to 6 of the CBF
gene was observed in healthy subjects and in 130 patients with MDS and AML studied (Figure 1). These results
appear to differ from results observed with the AML1 gene.
Indeed, others and we14-16 reported a new mechanism of
inactivation of AML1 in hematologic malignancies, through point
mutations of the gene in AML and, less frequently, in MDS and
myeloproliferative disorders. All mutations of AML1 were located in the
runt domain, and most of them were observed in M0 AML or in hematologic
malignancies with acquired trisomy 21. In M0 AML, most of the patients
had bi-allelic alteration of the AML1 gene either by
mutation of the 2 alleles or duplication of the mutated allele with
deletion of the wild-type allele or by loss of heterozygosity for AML1.
In all patients, point mutations of AML1 gene were acquired,
and functional analysis of these abnormal proteins showed that
transactivation properties of AML1 on the promoter of the MCSF-receptor
gene were abolished. Inactivation of those properties seemed to result
from absent binding of the mutated protein on the DNA consensus site of
AML1 target genes. In addition, the missense mutations appeared to act
in a dominant manner by higher affinity binding of CBF. Therefore, we
looked for point mutations of the second partner of the CBF complex, CBF, in MDS and AML.
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| Figure 1.
SSCP profile of exons 1 to 6 of the CBF
gene.
Patient 2 was a healthy subject used as negative control. Patients 7, 14, and 31 had, respectively, M0 AML, M1 AML, and RAEB.
|
|
Our negative results suggest that, unlike AML1, CBF is probably not
inactivated by point mutations in AML and MDS. Because, except in M0
AML, the number of patients with each FAB subtype was low, we cannot
completely exclude a low incidence of mutations in some specific FAB
subtypes of AML or MDS. In addition, despite using optimal conditions
for SSCP, including small PCR products, we could not exclude the
presence of some mutations undetectable by the SSCP technique, which
generally is 50% to 100% accurate. The apparent absence of
CBF mutations in AML and MDS could be linked with the function of
the CBF protein. Indeed, CBF acts as a cofactor, and only
bi-allelic mutations or mono-allelic mutations with dominant-negative
effects could inactivate the CBF complex, inducing the loss of
transactivation of HIS target genes. This kind of alteration of
CBF could have drastic effects and induce cell death. In that
context, mutations with a dominant-negative effect could lead to a
phenotype closed to that of CBF KO
/ mice with an
absence of fetal hematopoiesis. On the other hand, mutations inducing
lack of the transcription of only 1 of 2 alleles could lead to a
phenotype close to that of KO+/ CBF heterozygous mice,
which have few abnormalities. In conclusion, unlike AML1 mutations,
CBF mutations are probably not an important event in the
pathogenesis of AML or MDS.
| |
Acknowledgments |
We thank P. Liu for giving us the intronic sequence of the
CBF gene, and we thank B. Vaast, M. Crepin, C. Denis, and
the Institut Fédératif de recherché no. 22 for their
excellent technical assistance and support in molecular biology.
| |
Footnotes |
Submitted September 20, 2001; accepted January 3, 2002.
Supported by the Centre Hospitalier Universitaire of Lille (PHRC 1997),
the Ligue Nationale contre le cancer (Comité du Nord et de
l'Aisne), and the Fondation de France (Comité Leucémie).
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.
Reprints: Claude Preudhomme, Unité 524 INSERM,
Institut de Recherche sur le Cancer de Lille, 1 place de Verdun, 59045 Lille Cedex, France; e-mail:
cpreudhomme{at}chru-lille.fr.
| |
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Abstract
Introduction
