Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2981-2983
CORRESPONDENCE
Hypermethylation of p15INK4B Gene in a
Patient With Acute Myelogenous Leukemia Evolved From Paroxysmal
Nocturnal Hemoglobinuria
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LETTER |
To the Editor:
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disease
resulting from somatic mutations in the PIG-A gene involving primitive hematopoietic stem cells. The PNH clones may have growth or
survival advantages relative to normal clones that may promote their
expansion, resulting in the development of overt PNH. However, little is known about how PNH clones gain growth advantage. Recent studies demonstrated preferential hematopoiesis by PNH clones in
vivo.1,2 However, proliferation may be affected similarly in PIG-A-deficient clones and in normal clones,3
suggesting that PIG-A abnormalities alone may not be sufficient to
confer a growth advantage on PNH clones.
A proportion of PNH patients terminate in severe pancytopenia with
dysplasia, ie, myelodysplastic syndrome (MDS), and rarely progress to
acute leukemia. We previously reported in BLOOD that specific
p15INK4B gene inactivation by promoter
hypermethylation may be associated with the development of
MDS,4 because it may confer a growth advantage on cells.
One overt leukemia patient analyzed in this study in whom PNH evolved
through MDS (PNH/MDS-OL) showed intense hypermethylation of the
p15INK4B gene. Surface marker analysis of his
leukemic blasts showed low levels of expression of CD59,
suggesting that leukemic blasts were derived from the PNH clone.
So, to clarify at what point the p15INK4B gene was
densely methylated and inactivated in this PNH/MDS-OL patient and
whether this p15INK4B gene methylation is
related to the expansion of PNH clones, we analyzed this patient and an
additional 17 PNH patients.
We obtained clinical samples after receiving informed consent from a
total of 18 patients (4 men and 14 women) who were positive for sugar
water test and/or acidified serum (Ham) test and were diagnosed
as PNH based on clinical manifestations. They included 12 female
patients (unique patient no. [UPN] 1 through 12) analyzed in our
previous study in which we demonstrated the existence of monoclonal
populations with PNH phenotype by clonality analysis using X-chromosome
inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins by flow
cytometry.5 We extracted DNA from polymorphonuclear cells
(PMNCs) and mononuclear cells (MNCs) (or T lymphocytes), as described
previously,5 and analyzed the methylation status of the
p15INK4B gene by the simple and sensitive
methylation-specific polymerase chain reaction (MSP)
method.6 Because MSP can detect 10
3
methylated alleles among unmethylated alleles,6 changes of the methylation status in PNH clones could be detected even if they
are present at a very low incidence. After bisulfite
modification, we amplified their DNAs with each primer set specific for
unmethylated and methylated DNA in a thermal cycler (TAKARA, Kyoto,
Japan).
DNA from the PNH/MDS-OL patient (UPN 14) in the leukemic state
showed densely methylated p15INK4B gene,
consistent with the results of Southern blotting. However, the
p15INK4B gene in both his PMNCs and MNCs
obtained in the PNH state was not methylated (Fig
1A). The p15INK4B
gene of PMNCs in which we showed the monoclonal PNH clone in our previous report5 was also not methylated (Fig 1B, UPN
10 and UPN 11). The strong methylation band of PMNCs
similar to that of MNCs in another MDS-OL patient (Fig 1C)
supported our previous Southern blotting result, indicating that the
p15INK4B gene methylation is not limited to
leukemic blasts but is also present in PMNCs of MDS
origin.4 Therefore, the methylation band may be able to
be detected in the PMNC population of PNH if
p15INK4B gene methylation occurs in PNH
clones. Finally, we could not detect methylation bands in PMNC or
MNC populations in any patient with PNH. This sensitive method also
detected no methylated band in healthy volunteers (Fig 1C).
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| Fig 1.
Methylation status of the p15INK4B
gene in patients with PNH. (A) Methylation status of the
p15INK4B gene in a patient (UPN 14) with acute
myelogenous leukemia (AML) evolved from PNH. (a) Unmethylated
DNA-specific and methylated DNA-specific MSP primers produced 162-bp
and 154-bp products, respectively. Bone marrow (BM) samples at PNH
(BM-P, polymorphonuclear cells; BM-M, mononuclear cells), his colon
cancer, and normal colon tissue resected 1 year after PNH presentation
showed unmethylated p15INK4B gene. In contrast,
leukemic blasts showed hypermethylation of the
p15INK4B gene. (b) Southern blotting also showed
the methylated status in leukemic blasts4; lanes 1 and 2, control B lymphocytes. Lane 3, patient's leukemic blasts. (B)
Methylation status of the p15INK4B gene in patients
with PNH. All samples obtained from peripheral blood (PB) showed
unmethylated pattern. PB-P, polymorphonuclear cells; PB-M, mononuclear
cells; PB-T, T lymphocytes; SM, size marker (ØX174/HaeIII).
UPNs are common to those in our previous report.5 (C)
Methylation status of the p15INK4B gene in control
samples. Two healthy volunteers (HV) showed unmethylated pattern. A
patient with overt leukemia evolved from MDS (MDS-OL) showed intense
methylation in both PB-M and PB-P populations, whereas a patient with
refractory anemia (RA) showed faint methylation. ML1 and HL60 were
completely methylated and unmethylated, respectively, as previously
reported.4
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|
Our results indicated that p15INK4B gene
inactivation is not related to the growth advantage of PNH clones and
may not play any role in their expansion. These observations also
support our4 and other investigator's7
suggestion that p15INK4B gene methylation promotes
the progression of MDS to overt leukemia.
Toshiki Uchida
Haruhiko Ohashi
Tomohiro Kinoshita
Hidehiko Saito
First Department of Internal Medicine
Nagoya
University School of Medicine
Nagoya, Japan
Ryo Taguchi
The Faculty of Pharmaceutical Sciences
Nagoya City
University
Nagoya, Japan
Tomomitsu Hotta
Fourth
Department of Internal Medicine
Tokai University School of
Medicine
Isehara, Japan
Takashi Murate
Nagoya University
School of Health Sciences
Nagoya, Japan
| |
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