Absence of NPM1 promoter hypermethylation in human myelodysplastic syndrome.

Abstract

Npm1(+/-) heterozygous mice develop a haematological disorder with features resembling human myelodysplastic syndrome (MDS). Promoter hypermethylation of the NPM1 gene may lead to suppressed gene transcription and hence functional haploinsufficiency, which contributes to the development of MDS. Thirty-one patients with MDS and eight normal individuals were studied for promoter methylation and mRNA expression of NPM1. Methylation-specific PCR (MSP), COBRA and bisulfite sequencing were used to examine the NPM1 methylation status. Quantitative PCR was used to assess the expression of NPM1. NPM1 DNA methylation was rare, occurring in one of 31 cases as determined by MSP. There was no significant difference in NPM1 mRNA expression between MDS and normal blood samples. In conclusion, the finding suggests that NPM1 methylation is rare in MDS and does not play a major role in its pathogenesis.

Full text

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Absence of NPM1 promoter hypermethylation in
human myelodysplastic syndrome
Yuen-Yee Cheng,1,2David Chau,1Thomas Chan,1Harinder Gill,1Raymond Liang,1
Yok-Lam Kwong,1Eric Tse1
ABSTRACT
Npm1+/?heterozygous mice develop a haematological
disorder with features resembling human myelodysplastic
syndrome (MDS). Promoter hypermethylation of the
NPM1 gene may lead to suppressed gene transcription
andhencefunctionalhaploinsufficiency,whichcontributes
tothedevelopmentofMDS.Thirty-onepatientswithMDS
and eight normal individuals were studied for promoter
methylationandmRNAexpressionofNPM1.Methylation-
specificPCR(MSP),COBRAandbisulfitesequencingwere
used to examine the NPM1 methylation status.
Quantitative PCR was used to assess the expression of
NPM1.NPM1DNAmethylationwasrare,occurringinone
of 31 cases as determined by MSP. There was no
significantdifferenceinNPM1mRNAexpressionbetween
MDS and normal blood samples. In conclusion, the finding
suggests that NPM1 methylation is rare in MDS and does
not play a major role in its pathogenesis.
INTRODUCTION
Epigenetic changes including alterations in DNA
methylation and histone modifications contribute
to the pathogenesis of myelodysplastic syndrome
(MDS), by inhibiting the expression of genes
encoding tumour suppressors, cell-cycle regulators
and other important proteins.1
hypermethylation of the CpG islands in the
promoter region of these genes is a key component
of the myelodysplastic process, and the reversal of
hypermethylation has been shown to be of thera-
peutic benefit. Recent clinical trials have also
confirmed the effectiveness of the hypomethylating
agents, azacitidine and decitidine, in the manage-
ment of MDS.2 3
Mutations of the nucleophosmin gene (NPM1)
have recently been identified in about 50% of cases
of acute myeloid leukaemia with normal karyo-
type.4NPM1 mutations typically result in an
aberrant localisation of nucleophosmin to the
cytoplasm. Nonetheless, the exact mechanisms by
which this cytoplasmic mutant nucleophosmin
contributes to leukaemogenesis are still poorly
understood. Interestingly, NPM1 mutations have
not been observed in MDS, a leukaemic precursor.
Based on the findings in a mouse model, haploin-
sufficiency of NPM1 has been proposed as a patho-
genetic mechanism for the development of MDS.5
We hypothesised that epigenetic alterations in
NPM1 may lead to NPM1 haploinsufficiency,
thereby contributing to the development of MDS.
In this study, we described a comprehensive
methylation analysis of NPM1 in MDS using
methylation-specificPCR
In particular,
(MSP),a combined
bisulfite restriction analysis technique (COBRA)
and bisulfite sequencing.
MATERIALS AND METHODS
Patients
Peripheral blood and/or bone marrow samples from
31 patients with MDS were obtained for analysis
with informed consent. Eight peripheral blood
samples from normal blood donors were also
included as normal controls.
Bisulfite modification of DNA
Genomic DNA was extracted from peripheral blood
or bone marrow aspirates using the QIAamp DNA
Blood Mini Kit (Qiagen, Valencia, California, USA)
according to the manufacturer’s instructions. For
methylation analysis, genomic DNA extracted from
clinical samples was processed for bisulfite modifi-
cationusingtheZymoDNAmodificationkit(Zymo
Research, Orange, California, USA) according to the
manufacturer’s protocol with minor modification.
MSP
Bisulfite-modified DNA was used as a template for
MSP (ABI 9700 thermocycler; Applied Biosystems,
Foster City, California, USA). OLIGO 6 software
(Molecular Biology Insights) was used to design
primers specific for methylated and unmethylated
alleles (figure 1A), and the sequences of the primers
used are listed in table 1. Briefly, the unmethylated
allele-specific primers should include sequences
complementing thymines converted from unme-
thylated cytosines in the allele after bisulfite
treatment, whereas the methylated allele-specific
primers should include sequences complementing
the unconverted methylated cytosines. CpGenome
universal methylated DNA (Chemicon Interna-
tional Inc, Temecula, California, USA) was used as
a positive control for methylation. PCR products
were then analysed by gel electrophoresis.
COBRA
The methylation status of the CG-rich regions
around the transcription initiation site of NPM1
between nucleotides ?548 and +36 spanning 55
CpG dinucleotides was examined by COBRA
(figure 1A). Bisulfite-modified DNA was PCR
amplified using the primers listed in table 1. The
PCR products were then digested with BstUI (New
England Biolabs, Beverly, Massachusetts, USA) and
analysed by gel electrophoresis.
Bisulfite sequencing
For bisulfite DNA genomic sequencing, 2 ml bisul-
fite-modified DNA was amplified using the primers
1Division of Haematology and
Medical Oncology, Department
of Medicine, University of Hong
Kong, Queen Mary Hospital,
Hong Kong
2Asbestos Diseases Research
Institute (ADRI), Bernie Banton
Centre, University of Sydney,
Concord, Australia
Correspondence to
Dr Eric Tse, Division of
Haematology and Medical
Oncology, Department of
Medicine, University of Hong
Kong, Queen Mary Hospital,
Hong Kong;
ewctse@hku.hk
Y-Y C and DC contributed
equally to this work.
Accepted 13 August 2010
Published Online First
5 October 2010
1008J Clin Pathol 2010;63:1008e1011. doi:10.1136/jcp.2010.080465
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listed in table 1. The PCR products were cloned into the
pCR2.1eTOPO cloning vector (Invitrogen, Carlsbad, California,
USA). Eight to ten colonies were randomly chosen for plasmid
DNA extraction with the Qiaprep Spin Mini kit (Qiagen).
Plasmid DNA was sequenced using the ABI PRISM BigDye
Terminator Cycle Sequencing Kit and ABI 3100 sequencer
(Applied Biosystems). Sequence analysis was performed by
SeqScape software (Applied Biosystems).
Real-time reverse transcription (RT)-PCR and statistical analysis
Total RNA was extracted from 11 MDS and eight normal blood
samples using Trizol reagent (Invitrogen). RT reaction was
performed using 2 mg total RNA with a first-strand cDNA kit
(Promega, Madison, Wisconsin, USA). NPM1 mRNA expression
level was determined by quantitative real-time PCR (Light-
Cycler; Roche Diagnostic, Mannheim, Germany). Probe Design
software (Roche Diagnostic) was used to design PCR primers
Table 1
Primer sequences
Primer typeGene SequenceProduct sizeTm (8C)
Real-time RT-PCRNPM1 forward
NPM1 reverse
GAPDH forward
GAPDH reverse
NPM1 M forward
NPM1 M reverse
NPM1 U forward
NPM1 U reverse
NPM1 BS forward
NPM1 BS reverse
59GTACAGCCAACGGTTT 39
59AGACCGCTTTCCAGAT 39
59GGAGTCAACGGATTTGGT 39
59GTGATGGGATTTCCATTGAT 39
59GTGCGTATAGGCGATAGTAGC 39
59AACCCTAAAAATCTCGCGAA 39
59GTGTGTATAGGTGATAGTAGTGG 39
59TTATATATAAACCCTAAAAATCTCACAAA 39
59TAGGGGATGGGGAAAGGTGAA 39
59TCAAAAAACTCATATCCATATCCAT 39
16860
206 60
MSP*
21757
22652
COBRA and bisulfite
sequencing
585 57
*M, methylated; U, unmethylated.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MSP, methylation-specific PCR; RT, reverse transcription.
Figure 1
methylation status of the NPM1 promoter was determined by MSP. M, methylation; U, unmethylation. MSP results show the methylation status in the
representative myelodysplastic syndrome (MDS) samples. Universal methylated DNA (IVD) was used as a methylation-positive control. (C)
Representative gel image of the NPM1 methylation status in MDS samples and universal methylated DNA sample (IVD). The undigested fragment
(upper band) represents the unmethylated DNA (U). The digested fragments (lower bands, indicated by the arrows) represent the methylated DNA (M).
Our results show that there was no NPM1 methylation detected in MDS samples using COBRA.
(A) Methylation-specific PCR (MSP) region and COmbined Bisulfite Restriction Analysis (COBRA) of the NPM1 promoter. (B) The
J Clin Pathol 2010;63:1008e1011. doi:10.1136/jcp.2010.0804651009
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(table 1). GAPDH was used as an internal control of RNA
integrity. The ratio of NPM1 to GAPDH expression was deter-
mined with commercial software (Relative Quantification
Software; Roche Diagnostic). Student t test was used to calcu-
late the difference in mRNA expression level between MDS
samples and normal blood samples.
RESULTS
DNA methylation of NPM1 was evaluated in 18 men and 13
women with MDS. The median age of the patients was 61 years
(range 19e86 years). The diagnoses of MDS were confirmed by
bone marrow biopsies and classified according to the World
Health Organization (WHO) classification. The patient charac-
teristics are described in table 2. NPM1 methylation was
detected in one of 31 MDS samples using MSP (figure 1B). The
sensitivity and specificity of MSP were verified by amplification
of the universal methylated DNA control. Because MSP analysis
only examined a limited number of CpG dinucleotides, we
Figure 2
syndrome (MDS) and normal blood samples as determined by
quantitative real-time PCR (normal, n¼8; cancer, n¼11).
mRNA expression level of NPM1 in myelodysplastic
Table 2
Patient characteristics
PatientSex/age WHO diagnosisHaemoglobin (g/dl)WBC (3109/l) Platelet (3109/l) Karyotype
1 M/83Refractory cytopenia with unilineage
dysplasia
Refractory anaemia with excess blasts-2
Refractory anaemia with excess blasts-2
Refractory anaemia with excess blasts-2
Refractory anaemia with excess blasts-1
Myelodysplastic syndrome, unclassifiable
Refractory anaemia with excess blasts-2
Refractory anaemia with multilineage
dysplasia
Refractory anaemia with excess blasts-1
11.2 5.854NA
2
3
4
5
6
7
8
F/66
M/38
F/53
F/34
M/50
F/76
M/73
8.2
6.5
9
12.6
7.8
6.8
10
2.7
0.6
12.13
3.5
4.6
1.2
1.7
77
15
43
130
273
19
112
NA
Dup (1) (q21q32), +8
Normal
47XX, +8, 46, XX
45, X, ?Y
Normal
NA
9 F/207.57 70 Complex 43e49, XX, dup (1) (q21, 2q42),
+8, del (13) (q12q13), e18, +19, +21,
+22
45, XY, e7
Normal
10
11
M/65
M/67
Refractory anaemia with excess blasts-2
Refractory anaemia with ringed
sideroblasts
Refractory anaemia with multilineage
dysplasia
Myelodysplastic syndrome, unclassifiable
Refractory anaemia
Refractory anaemia with multilineage
dysplasia
Refractory anaemia with excess blasts-1
Myelodysplastic syndrome, unclassifiable
Refractory cytopenia with unilineage
dysplasia
Refractory anaemia with excess blasts-2
9.4
7.6
15
2.9
210
91
12M/76 5.21.142 Normal
13
14
15
F/46
M/86
M/37
12.7
3.4
7.3
2
3.21
6.3
130
36
29
Trisomy 8
Normal
Trisomy 8
16
17
18
M/75
M/70
F/50
8.3
9.9
10.4
5.35
3.41
2.4
44
214
45
Normal
Normal
Normal
19 M/46 9.73.2 88 Complex 39e47, XY, del(5)(q31), e7,
e8, e13, e17
Normal
NA
20
21
F/60
M/61
Refractory anaemia with excess blasts-2
Refractory anaemia with ringed
sideroblasts
Refractory cytopenia with unilineage
dysplasia
Refractory anaemia with excess blasts-2
Refractory anaemia with excess blasts-1
Refractory anaemia with multilineage
dysplasia
Refractory anaemia with multilineage
dysplasia
Chronic myelomonocytic leukaemia
Refractory anaemia with excess blasts-1
Refractory anaemia with excess blasts-1
Refractory anaemia with excess blasts-1
Refractory anaemia with multilineage
dysplasia
9.3
6.7
1.18
6.0
45
287
22 M/62 12.81.9 54Normal
23
24
25
F/80
M/56
M/61
9.7
8.4
10.7
10
2.57
2.0
37
322
130
NA
45, XY, +1, der(1;7)(q10;p10)
NA
26M/784.6 9.0118 NA
27
28
29
30
31
F/56
M/19
F/54
F/71
F/76
9.0
12.6
10.5
10.9
7.7
2.6
2.7
2.5
6.1
2.8
72
32
59
65
32
Normal
Trisomy 8
Normal
NA
NA
WBC, white blood cell count.
1010J Clin Pathol 2010;63:1008e1011. doi:10.1136/jcp.2010.080465
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performed COBRA analysis to obtain an overview of the
methylation status of other CpG islands of the NPM1 promoter.
The results from COBRA supported the finding of MSP (figure
1B), showing a low extent of promoter methylation of NPM1 in
MDS (figure 1C). In addition, we performed bisulfite sequencing
in four MSP-negative and the one MSP-positive cases to confirm
the NPM1 methylation level quantitatively. No evidence of
significant methylation was found in the MSP-negative cases. In
the MSP-positive case (MD5 in figure 1B), there were only two
CpG sites that showed 10% of methylation (data not shown).
Finally, to determine if promoter hypermethylation might
decrease NPM1 expression and hence lead to a functional
haploinsufficiency, we analysed NPM1 mRNA level in 11 MDS
and eight normal blood samples. Real-time quantitative RT-PCR
showed that there was no significant difference in NPM1 mRNA
expression level between MDS and normal blood samples
(Student t test, p¼0.523, figure 2). Although NPM1 protein level
was not assessed in this study, our results are compatible with
the findings of Ishikawa et al6that significant reduction of
NPM1 protein rarely occurs in human MDS.
DISCUSSION
In this study, we examined the DNA methylation level of NPM1
in MDS. Our result indicates that only one out of 31 cases
showed promoter methylation using MSP, but no significant
methylation was found using COBRA and bisulfite sequencing.
Furthermore, there was no significant difference in NPM1
mRNA expression level between MDS and normal blood
samples. Oki et al7has examined DNA methylation of NPM1 in
50 MDS samples using bisulfite DNA pyrosequencing, and
found no NPM1 promoter methylation. Pyrosequencing is
a sequencing-by-synthesis technology that relies on the lumi-
nometric detection of pyrophosphate release upon nucleotide
incorporation througha cascade
However, one limitation of this method is that it gives an esti-
mate of methylated cytosine content in only 1e6 CpG sites
simultaneously.8e10In order to be able to perform a more
extensive analysis of the NPM1 promoter methylation, we used
conventional bisulfite sequencing which covered a total of 55
CpG sites.
NPM is a multifunctional protein, but its role in oncogenesis
remains undefined.TheNPM1geneisacommontargetofgenetic
alterations, primarily in haematopoietic malignancies. Somatic
mutations ofNPM1have beencommonlyfoundinacutemyeloid
leukaemia with normal karyotype.4 11However, NPM1 muta-
tions have not been observed in MDS, chronic myeloid leukaemia
or lymphoid malignancies. Previous studies have shown that
mice heterozygousforNpm1
developed a haematological syndrome with features simulating
human MDS. In this study, we examined a well-characterised
series of MDS samples for evidence of NPM1 methylation. The
results show that NPM1 methylation was not common, and that
ofenzymatic reaction.
nullmutation (Npm1+/?)
there was no difference in NPM1 expression between MDS and
normal peripheral blood samples. Promoter methylation and
hence functional haploinsufficiency of NPM1 therefore do not
play a major role in the pathogenesis of human MDS.
Funding The project was supported by Small Project Funding (200807176120),
University of Hong Kong.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Queen Mary
Hospital, University of Hong Kong.
Contributors YYC performed the experiments, and wrote and approved the
manuscript. DC performed the experiments. TC and HG collected and characterised all
the patient samples and data. RL and YLK coordinated the research and approved the
manuscript. ET conceived the project, designed the experiments, and wrote and
approved the manuscript.
Provenance and peer review Not commissioned; externally peer reviewed.
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Take-home messages
< Haploinsufficiency of NPM1 has been found to be associated
with myelodysplasia in a mouse model.
< Hypermethylation of the NPM1 gene promoter is absent in
human myelodysplastic syndrome (MDS), and there is no
difference in the mRNA expression level of NPM1 between
normal human bone marrow and MDS cells.
J Clin Pathol 2010;63:1008e1011. doi:10.1136/jcp.2010.0804651011
Original article
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doi: 10.1136/jcp.2010.080465
2010
2010 63: 1008-1011 originally published online October 5,J Clin Pathol

Yuen-Yee Cheng, David Chau, Thomas Chan, et al.
syndrome
myelodysplastic hypermethylation in human
promoter
NPM1
Absence of

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