Allergy
ORIGINAL ARTICLE
EPIDEMIOLOGY AND GENETICS
Gene expression analysis predicts insect venom anaphylaxis
in indolent systemic mastocytosis
M. Niedoszytko1,2, M. Bruinenberg3,4, J. J. van Doormaal2, J. G. R. de Monchy2, B. Nedoszytko5,
G. H. Koppelman6, M. C. Nawijn7, C. Wijmenga3, E. Jassem1 & J. N. G. Oude Elberink2
1
Department of Allergology, Medical University of Gdansk, Gdansk, Poland; 2Department of Allergology, University Medical Center
Groningen, University of Groningen, Groningen, the Netherlands; 3Department of Genetics, University Medical Center Groningen, University
of Groningen, Groningen, the Netherlands; 4LifeLines, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 5Department of Dermatology, Medical University of Gdansk, Gdansk, Poland; 6Department of Pediatric Pulmonology and Pediatric
Allergology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 7Laboratory of Allergology and
Pulmonary Diseases, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen,
Groningen, the Netherlands
To cite this article: Niedoszytko M, Bruinenberg M, van Doormaal JJ, de Monchy JGR, Nedoszytko B, Koppelman GH, Nawijn MC, Wijmenga C, Jassem E,
Oude Elberink JNG. Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis. Allergy 2010; DOI: 10.1111/
j.1398-9995.2010.02521.x.
Keywords
anaphylaxis; gene expression; insect venom
allergy; mastocytosis; microarray
assessment; prediction of anaphylaxis.
Correspondence
Marek Niedoszytko, MD, PhD, Department
of Allergology, Medical University of
Gdansk, Debinki 7 80-952, Gdansk, Poland.
Tel.: +48583491626
Fax: +48583491625
E-mail: [email protected]
Accepted for publication 9 November 2010
DOI:10.1111/j.1398-9995.2010.02521.x
Edited by: Thomas Bieber
Abstract
Background: Anaphylaxis to insect venom (Hymenoptera) is most severe in patients
with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene
expression between patients with indolent systemic mastocytosis (ISM) and a history
of insect venom anaphylaxis (IVA) compared to those patients without a history of
anaphylaxis, and to determine the predictive use of gene expression profiling.
Methods: Whole-genome gene expression analysis was performed in peripheral
blood cells.
Results: Twenty-two adults with ISM were included: 12 with a history of IVA and
10 without a history of anaphylaxis of any kind. Significant differences in single gene
expression corrected for multiple testing were found for 104 transcripts (P < 0.05).
Gene ontology analysis revealed that the differentially expressed genes were involved
in pathways responsible for the development of cancer and focal and cell adhesion
suggesting that the expression of genes related to the differentiation state of cells is
higher in patients with a history of anaphylaxis. Based on the gene expression
profiles, a naı̈ve Bayes prediction model was built identifying patients with IVA.
Conclusions: In ISM, gene expression profiles are different between patients with a
history of IVA and those without. These findings might reflect a more pronounced
mast cells dysfunction in patients without a history of anaphylaxis. Gene expression
profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in
patients with ISM. Prospective studies are needed to substantiate any conclusions.
Mastocytosis is an uncommon disease resulting from a pathological increase in mast cells in different tissues including
skin, bone marrow, liver, spleen, and lymph nodes (1). The
clinical presentation of mastocytosis is heterogeneous, varyAbbreviations
Fc, fold change difference; GO, gene ontology; IgE,
immunoglobulin E; ISM, indolent systemic mastocytosis; IVA,
insect venom anaphylaxis; KEGG, Kyoto encyclopedia of genes and
genomes; MAPK, mitogen-activated protein kinase; SPT, skin-prick
test; VIT, venom immunotherapy; Wnt, wingless int pathway.
ª 2010 John Wiley & Sons A/S
ing from sole skin presentation found in urticaria pigmentosa
and mastocytoma, to different forms of systemic disease
including indolent systemic mastocytosis (ISM), smoldering
systemic mastocytosis, aggressive systemic mastocytosis and
mast cell leukemia (1, 2). Of the adult patients with systemic
mastocytosis, the large majority (ca. 90%) has the indolent
form of the disease.
Symptoms of the disease result from skin involvement,
mast cell mediator release and massive mast cell infiltration,
as found in aggressive variants of the disease. Symptoms of
mast cell degranulation may vary from pruritus and flushing
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
to anaphylaxis with profound hypotension and with occasionally even fatal outcome (1–5). The cumulative prevalence
of anaphylaxis in patients with mastocytosis has been
reported to be as high as 50% (3). Anaphylaxis is thought to
be the most important burden for the majority of patients
with mastocytosis.
The most important eliciting factor of anaphylaxis in
patients with mastocytosis is an insect sting (3). It is estimated
that 30% of patients with mastocytosis suffer from insect
venom anaphylaxis (IVA) (3, 6). Thus, patients are advised to
carry epinephrine auto-injectors and often start with venom
immunotherapy (VIT), probably lifelong (6, 7). Because fatal
anaphylaxis also has been reported, even without a history of
previous anaphylactic sting reactions (4, 5), some authors postulate prophylactic treatment for all patients with mastocytosis
(7, 8). However, because less than half of the patients with
mastocytosis will develop anaphylaxis on insect stings during
their lifetime, such a strategy of prophylactic treatment would
inflict a significant burden on a large group of patients that is
in no need for such an intervention. Therefore, it is of great
interest to identify those patients at risk for IVA, ideally using
a minimally invasive and highly sensitive assay. Unfortunately,
it has not been possible so far to predict which patients with
mastocytosis are at risk for anaphylaxis to an insect sting (1–9).
The mechanism(s) responsible for development of anaphylaxis in some and the lack of such reactions in other patients
with mastocytosis are mainly unknown (2, 3, 6, 9–11). It has
been hypothesized that the predisposition to anaphylaxis is
related to activation of mast cell kinase pathways or calcium
ion channels (2, 3, 8–11). Although severe anaphylaxis may
occur in patients with all variants of mastocytosis (1–3), they
are most frequent in the indolent form of mastocytosis. The
mast cells in mastocytosis display several phenotypic abnormalities, among which the activating somatic mutation of the
KIT receptor that also affects histamine release (2). However,
the contribution of these intrinsic alterations to the sensitivity
for developing (insect venom) anaphylaxis is unknown.
Better understanding of the molecular differences between
mastocytosis patients with an IgE mediated anaphylaxis to
insect venom and mastocytosis patients without such reactions
might be helpful to predict the risk for anaphylaxis in those
patients with mastocytosis who have not been stung yet. Analysis of gene expression allows for the direct recognition of gene
activation. Whole-genome expression analysis has the potential
to reveal differences in activation of gene networks involved in
anaphylaxis between patients with and without IVA.
The aim of this study therefore was to analyze whether in
ISM differences in gene expression profiles can be identified
in peripheral blood cells between patients with a history of
anaphylaxis to insect venom who never received VIT and
those patients without anaphylaxis of any kind.
Materials and methods
Patients
Patients with ISM from the Department of Allergology, University Medical Center Groningen (UMCG) were included.
Niedoszytko et al.
ISM has been diagnosed in all patients according to the
WHO criteria (1) by serum tryptase measurement and bone
marrow investigation including histopathological, cytological,
CD2 and CD25 flow cytometry and genetic examinations.
None of these patients had been treated with VIT in the past.
Subjects suffering from other severe chronic or/and malignant diseases and pregnant women were excluded.
Patients were divided into two groups:
Group 1: patients with a history of anaphylaxis caused by
Hymenoptera venom. The diagnosis of IVA was based on
medical history (grade IV reaction according to Mueller (12)
and a positive skin tests and/or sIgE.
Group 2: patients who did not experience any allergic reactions after insect stings (they were stung at least once after
ISM has been diagnosed) or by other known or unknown
factors in the past during at least 10 years of follow-up after
the diagnosis of ISM has been made.
The study was approved by the Medical Ethical Committee of the UMCG (METc 2008/340).
Collection of blood samples
From all patients, RNA was isolated from whole blood using
the PAXgene Blood RNA Tubes (Qiagen, Valencia, CA,
USA). All tubes were immediately frozen and stored in
)20C till RNA isolation (maximal period 2 months). RNA
was isolated using PAXgene Blood RNA Kit CE (Qiagen,
Venlo, the Netherlands). All RNA samples were stored in
)80C till labeling and hybridization.
The quality and concentration of RNA was determined
using the 2100 Bioanalyzer (Agilent, Amstelveen, the Netherlands) with the use of Agilent RNA 6000 Nano Kit. Samples
with a RNA integrity number >7.5 were used for further
analysis on expression arrays.
Gene expression
For amplification and labeling of RNA with Illumina, TotalPrep 96 RNA Amplification kit was used (Applied Biosystems, Nieuwerkerk ad IJssel, the Netherlands). For each
sample, we used 200 ng of RNA. The Human HT-12_V3_
expression arrays (Illumina, San Diego, CA, USA) were processed according to the manufacturer’s protocol. Slides were
scanned immediately using an Illumina BeadStation iScan
(Illumina).
Image and data analysis
First-line check, background correction, and quantile normalization of the data were performed with Genomestudio Gene
Expression Analysis module v 1.0.6 Statistics. Entities containing at least 75% of samples with a signal intensity value
above the 20th percentile in 100% of the samples in at least
2 groups were included for the further analysis.
Data analysis was performed using genespring package
version 10.0.0 (Agilent Technologies, Santa Clara, CA,
USA). Genes for which expression was significantly different
between both groups were chosen based on a log2 fold
ª 2010 John Wiley & Sons A/S
Niedoszytko et al.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
change >2 in gene expression, t-test P-value <0.05 and
a Benjamini–Hochberg false discovery rates <0.05. The
P-values presented in the text were corrected for multiple
testing. A naı̈ve Bayes prediction model was used to build a
prediction model assessing the risk of anaphylaxis in patients
with ISM (13). A naı̈ve Bayesian classifier is a mathematical
process computing the probability of classifying the patient
in a group of risk of anaphylaxis or without risk based on
the results of gene expression (13). The expression of each
particular gene is regarded as an independent predictor classifying patient to the particular group.
Functional annotation of genes was described by the Go
Process Analysis and pathways of the Kyoto encyclopedia of
genes and genomes (KEGG) (14) with Genecodis functional
annotation web-based tool (15).
Clinical data of this study were analyzed with statistica
8.0 (StatSoft, Tulsa, OK, USA). The chi-square was used to
compare the number of patients with IVA in the studied
groups. The mean difference of serum tryptase and urinary
excretions of methylhistamine and methylimidazole acetic
acid was analyzed using the U-Mann–Whitney test. P-values
<0.05 were considered statistically significant.
Results
A total of 22 patients with ISM were included. Twelve
patients (two men) had a history of IVA (group 1) and 10
patients (five men) had no history of anaphylaxis caused by
insect venom or other factors (group 2). In group 2, one
patient was asymptomatically sensitized to wasp and bee
venom. No significant differences in clinical characteristics
were found between both groups, except for sensitization to
insect venom (P = 0.0001) (Table 1). Ages at diagnosis of
ISM and levels of serum tryptase and urinary excretions of
methylhistamine and methylimidazole acetic acid were in
medians and ranges in group 1: 49 (25-70) years, 29.4 (5.13112) lg/l, 335 (153-1024) lmol/mol creatinine, and 3.3 (1.47.7) mmol/mol creatinine; and in group 2: 50 (34-64) years,
48.4 (4.62-155) lg/l, 452 (77-1046) lmol/mol creatinine, and
4.5 (1.4-10.9) mmol/mol creatinine, respectively.
Comparison of gene expression profiles
Whole-genome gene expression analysis was performed on
RNA samples isolated from peripheral blood cells. From all
48.804 probes present on the array, 48.676 transcripts passed
the quality control and were included for further analysis.
Of all analyzed transcripts, 1951 showed a log2 > 2-fold
difference in gene expression: 967 (49%) genes were increased
and 984 (51%) were decreased in patients with IVA.
To assess whether the differentially expressed genes were
enriched for a specific cellular function, we performed genome ontology (GO) analysis using the Genecodis website.
The main processes to which the differentially expressed
genes map are signal transduction, multicellular organism
development, transcription, cell differentiation, metabolic
process, and ion transport (Table 2). Using the KEGG database (14), we found that the pathways that are most signifi-
ª 2010 John Wiley & Sons A/S
cantly enriched for in our list of log2 > 2-fold differentially
expressed genes are pathways involved in cancer, focal adhesion, cell adhesion, ubiquitin mediated proteolysis, Wnt signaling, and calcium signaling (Table 3).
Subsequently, we analyzed the 104 transcripts that were
significantly different between the two patient groups in single gene expression corrected for multiple testing (P < 0.05)
revealing a log2 > 3-fold difference in expression. In patients
with a history of IVA, 37 transcripts (36%) were increased,
whereas 67 transcripts (64%) were decreased in expression
(Table 4). A hierarchical clustering of the differentially
expressed genes is presented in Figure 1. The most significant
differences (P corrected for multiple testing <0.002) were
found in eight transcripts: HS 552770, HS 41192,
RBMY1A3P, DVL1, G0S2, HS 540329, HS 546027, and
LOC283487. Except for HS 552770, which was up-regulated
in patients with a history of IVA, the remaining seven transcripts were up-regulated in patients without anaphylaxis. Of
these genes, it is known that DVL1 and G0S2 are related to
the development of cancer, the function of the remaining
transcripts is still unknown. GO analysis using the KEGG
database on this list of 104 transcripts revealed the enrichment of genes mapping to two processes: pathways in cancer
and the mitogen-activated protein kinase (MAPK) signaling
pathway (Table 3). The function of 46 (39%) genes is
unknown yet.
Subsequently, we used a prediction model that uses a naı̈ve
Bayes (NB) classifier, based on the 104 most significant differentially expressed genes. This model was able to differentiate ISM patients with IVA from those without anaphylaxis
with a sensitivity and specificity of 100%.
To relate the whole-genome expression results to specific
cell expression profiles, we analyzed the expression profiles of
leukocyte-specific genes expressed in dendritic cells, B cells,
memory T cells, mast cells, and basophils as described by
Liu et al. (16). A statistically significant difference in expression comparing both patient groups was found for the mast
cell–specific genes CTTNBP2 encoding cortactin-binding protein 2, a central regulator of cytoskeletal rearrangement in
mitosis (17), which was up-regulated in patients with IVA
(P = 0.03) and SIGLEC6 encoding sialic acid–binding Ig-like
lectin 6, playing a role in cell–cell interactions (18), which
was down-regulated in patients with IVA (P = 0.04).
Discussion
The results of our study demonstrate that we are able to
identify in ISM genes that are differentially expressed
between the peripheral blood cells from patients with IVA
and those from patients without a history of anaphylaxis.
GO analysis reveals that the differentially expressed genes are
enriched for genes that function in several pathways that
regulate the balance between proliferation versus terminal
differentiation: Wnt signaling pathway, focal and cell adhesion, calcium signaling, extracellular matrix interactions,
pathways in cancer and MAPK signaling (Tables 3 and 4).
So, our data indicate that in spite of a high number of mast
cells in all patients, the sensitivity to develop anaphylaxis
70
54
64
50
34
41
64
54
50
43
38
63
F
F
Group 2 F
F
M
M
F
M
M
F
M
F
UP
UP
UP
UP
UP
19.7
29.8
28.9
34.1
21.7
48.3
27.4
15.2
74.5
5.13
112
31.3
adult UP
155
adult UP
20.4
adult UP
55.4
juvenile UP 146
adult UP
4.62
neg
28.2
adult UP
52.3
adult UP
36.5
neg
44.4
adult UP
109
neg
adult UP
neg
adult
adult
neg
adult
neg
neg
adult
adult
neg
1046
293
433
470
102
77
530
851
310
823
289
593
153
380
166
604
1024
266
194
190
404
542
10.9
3.0
4.9
4.0
1.6
1.4
6.4
5.9
3.4
6.3
2.7
4.1
1.4
4.8
3.1
6.5
3.4
2.3
2.7
1.7
7.7
6.5
0.49
0.06
0.45
1.61
0.10
0.09
n.d.
0.34
n.d.
n.d.
0.10
n.d.
0.09
0.29
0.11
0.17
n.d.
0.30
0.10
0.13
n.d.
0.17
pos
pos
pos
neg
pos
pos
n.d.
pos
n.d.
n.d.
pos
n.d.
neg
neg
pos
pos
n.d.
neg
pos
pos
n.d.
pos
pos
pos
pos
pos
pos
pos
n.d.
pos
n.d.
n.d.
pos
n.d.
pos
pos
pos
pos
n.d.
pos
pos
pos
n.d.
pos
pos
pos
pos
pos
pos
pos
n.d.
pos
pos
n.d.
pos
pos
neg
pos
pos
pos
n.d.
neg
pos
neg
pos
pos
D816VKIT
mutation
in bone
marrow
cells
pos
pos
pos
pos
neg
neg
pos
pos
pos
pos
neg
pos
neg
pos
neg
pos
pos
pos
pos
pos
pos
pos
‡2
aggregates
of ‡15 MCs
in bone
marrow
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
Abnormal
morphology
of ‡25% of
MCs in
bone
marrow
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
normal
slightly
hyperplastic
normal
normal
Histological
bone
marrow
cellularity
neg
neg
neg
neg
neg
pos
neg
neg
neg
neg
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
neg
neg
neg
neg
neg
pos
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
pos
neg
neg
neg
Sensitization Sensitization
to wasp
to bee
venom venom Abbreviations: M: male; F: female; n.d.: not done; UP: urticaria pigmentosa; juvenile UP: juvenile-onset UP; adult UP: adult-onset UP; MH: methylhistamine; MIMA: methylimidazole acetic
acid; MCs: mast cells.
*Reference values: tryptase 11.4 lg/l (95 percentile); MH 167 lmol/mol creatinine (97.5 percentile); MIMA 1.9 mmol/mol creatinine (97.5 mmol/mol creatinine (97.5 percentile).
Sensitization pos: sIgE >0.35 kU/l or positive skin-prick test.
50
43
64
47
25
61
51
38
37
36
Group 1 M
F
F
F
F
F
F
F
M
F
Group
Age at
diagnosis
Sex (years)
UP
MCs in
bone
Urine
Serum Urine MH MIMA
marrow CD2
CD25
tryptase (lmol/mol (mmol/mol aspirate immunoimmuno(lg/l)*
creat)*
creat)*
(%)
phenotype phenotype
Table 1 Clinical characteristics of the patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis (group 1) and without such a history (group 2)
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
Niedoszytko et al.
ª 2010 John Wiley & Sons A/S
Niedoszytko et al.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
Table 2 Gene co-occurrence annotation found by Genecodis (19, 20) (GOSlim Process Function) for the genes differentially expressed
(log2fc>2) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any
kind
Genes
NGR
NG
Hyp
Hyp*
Annotations
88 genes
50 genes
1700 (37435)
874 (37435)
88 (1022)
50 (1022)
8.37904e-09
9.52928e-07
2.5975e-07
1.47704e-05
73 genes
8 genes
1516 (37435)
43 (37435)
73 (1022)
8 (1022)
2.37117e-06
1.8644e-05
2.45021e-05
0.000144491
29 genes
30 genes
18 genes
471 (37435)
503 (37435)
232 (37435)
29 (1022)
30 (1022)
18 (1022)
4.93153e-05
6.56447e-05
7.74516e-05
0.000305755
0.000339164
0.000343
GO:0007165: signal transduction
GO:0007275: multicellular organismal
development
GO:0006350: transcription
GO:0007165: signal transduction
GO:0030154: cell differentiation
GO:0008152: metabolic process
GO:0006811: ion transport
GO:0006629: lipid metabolic process
Hyp: P-values have been obtained through Hyper geometric analysis; Hyp*: P-values have been obtained through hypergeometric analysis,
corrected by false discovery rate method; NGR: number of annotated genes in the reference list; NG: number of annotated genes in the
input list.
Table 3 Gene co-occurrence annotation found by Genecodis (Kyoto encyclopedia of genes and genomes [KEGG] pathways) (17–20) for the
genes differentially expressed (log2fc>2) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis
and without anaphylaxis of any kind
Genes
NGR
NG
Hyp
Hyp*
Annotations
25 genes
17 genes
13 genes
5 genes
320 (37435)
194 (37435)
131 (37435)
19 (37435)
25 (1022)
17 (1022)
13 (1022)
5 (1022)
3.05203e-06
2.65238e-05
6.47261e-05
0.000126934
0.000378452
0.00164448
0.00267535
0.00393495
12 genes
13 genes
14 genes
9 genes
15 genes
9 genes
9 genes
7 genes
129 (37435)
150 (37435)
176 (37435)
82 (37435)
206 (37435)
96 (37435)
98 (37435)
62 (37435)
12 (1022)
13 (1022)
14 (1022)
9 (1022)
15 (1022)
9 (1022)
9 (1022)
7 (1022)
0.000225735
0.000254129
0.000362833
0.000400788
0.000580926
0.00126496
0.00146319
0.00147513
0.00559822
0.00525199
0.00642733
0.00621222
0.00800387
0.0130712
0.0139566
0.0130655
16 genes
262 (37435)
16 (1022)
0.0024577
0.0190472
(KEGG) 05200: Pathways in cancer
(KEGG) 04510: Focal adhesion
(KEGG) 04514: Cell adhesion molecules (CAMs)
(KEGG) 05200: Pathways in cancer (KEGG) 04120:
Ubiquitin mediated proteolysis
(KEGG) 04120: Ubiquitin mediated proteolysis
(KEGG) 04310: Wnt signaling pathway
(KEGG) 04020: Calcium signaling pathway
(KEGG) 04512: ECM-receptor interaction
(KEGG) 04810: Regulation of actin cytoskeleton
(KEGG) 04912: GnRH signaling pathway
(KEGG) 04916: Melanogenesis
(KEGG) 00980: Metabolism of xenobiotics by
cytochrome P450
(KEGG) 04010: MAPK signaling pathway
Hyp: P-values have been obtained through hypergeometric analysis; Hyp*: P-values have been obtained through hypergeometric analysis,
corrected by false discovery rate method; NGR: number of annotated genes in the reference list; NG: number of annotated genes in the
input list.
might be correlated to the differentiation state of the mast
cells. The functional annotation of the four most significantly
differentially expressed genes between the two patient groups
confirms this observation (Table 5). Together, these data
indicate that ISM patients with a more differentiated mast
cell phenotype are at risk for developing of IVA.
Further, our data indicate that gene expression profiling
on peripheral blood cells from patients with ISM enables to
differentiate patients with IVA from ISM patients without
anaphylaxis in their medical history. It indicates that this
procedure might be adapted to identify mastocytosis patients
at risk for anaphylaxis to benefit from prophylactic treatment.
ª 2010 John Wiley & Sons A/S
It is assumed that baseline serum tryptase concentrations
reflect the mast cell mass of the body. In patients without
mastocytosis, an increased tryptase concentration is associated
with an increased risk for the occurrence of insect venom
allergy (28), and in addition, it is also associated with the
more severe reactions (28). In patients with mastocytosis,
higher basal tryptase values were also associated with a
greater risk of anaphylaxis (3). In our ISM group, however,
we found in patients with IVA a trend toward lower levels of
tryptase in serum and lower urinary excretion of the histamine
metabolites methylhistamine and methylimidazole acetic acid,
which are also thought to reflect mast cell burden. This difference might be because of the fact that we only included
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
Niedoszytko et al.
Table 4 List of genes that were differentially expressed (log2 fc > 3, P < 0.05 corrected for multiple testing by Benjamini–Hochberg method
P < 0.05) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any
kind
Gene symbol
Gene name
P-value
Corrected
P-value
R* 1/2
R* 2/1
ABI3BP
ANKRD6
B3GAT1
C14ORF115
C14ORF118
C14ORF165
C18ORF56
C20ORF106
C20ORF132
CCDC134
CDC42BPA
CEP57
COL9A1
CYORF15A
CYORF15B
CYORF15B
DHX9
DVL1
EIF1AY
EWSR1
FAM110A
FBLN1
FBXL21
FLJ39660
G0S2
GLYAT
HGD
HLA-DRB4
HOXA6
HRB
HS,107801
HS,124514
HS,134088
HS,189987
HS,41192
HS,436654
HS,527174
HS,537553
HS,540329
HS,540415
HS,541520
HS,544017
HS,545032
HS,545163
HS,545866
HS,546019
HS,546027
HS,549742
HS,552354
HS,552770
HS,562039
HS,562265
HS,563189
Abi gene family, member 3 (nesh) binding protein
Ankyrin repeat domain 6
Beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase p)
Chromosome 14 open reading frame 115
Chromosome 14 open reading frame 118
Chromosome 14 open reading frame 165
Chromosome 18 open reading frame 56
Chromosome 20 open reading frame 106
Chromosome 20 open reading frame 132
Hypothetical protein flj22349
cdc42-binding protein kinase alpha (dmpk-like)
Centrosomal protein 57 kda
Collagen, type ix, alpha 1
Chromosome y open reading frame 15a
Chromosome y open reading frame 15b
0.0018
0.0099
0.0102
0.0025
0.0034
0.0108
0.0212
0.0035
0.0073
0.0095
0.0044
0.0147
0.0089
0.0162
0.0080
0.0417
0.0068
0.0001
0.0294
0.0258
0.0088
0.0081
0.0035
0.0038
0.0002
0.0014
0.0012
0.0184
0.0168
0.0037
0.0090
0.0060
0.0071
0.0107
0.0000
0.0024
0.0022
0.0007
0.0001
0.0072
0.0003
0.0060
0.0084
0.0061
0.0020
0.0353
0.0001
0.0106
0.0015
0.0000
0.0287
0.0004
0.0007
0.0094
0.0158
0.0158
0.0094
0.0104
0.0159
0.0269
0.0104
0.0138
0.0153
0.0112
0.0204
0.0148
0.0217
0.0141
0.0477
0.0138
0.0020
0.0347
0.0317
0.0148
0.0141
0.0104
0.0104
0.0023
0.0089
0.0089
0.0238
0.0221
0.0104
0.0148
0.0138
0.0138
0.0159
0.0009
0.0094
0.0094
0.0055
0.0023
0.0138
0.0036
0.0138
0.0143
0.0138
0.0094
0.0409
0.0023
0.0159
0.0089
0.0006
0.0346
0.0044
0.0055
0.26
0.46
0.42
0.25
3.08
0.34
0.42
3.13
2.36
0.42
0.39
1.98
0.23
0.19
0.22
0.11
2.73
0.22
0.41
0.36
2.12
0.55
0.24
0.27
0.13
0.40
0.40
0.46
2.65
5.05
0.36
0.28
0.33
3.23
0.20
0.37
2.61
0.24
0.21
0.23
4.50
2.35
2.22
0.24
3.43
3.31
0.27
0.24
0.39
4.81
2.80
4.82
2.48
3.81
2.15
2.41
4.05
0.33
2.96
2.41
0.32
0.42
2.36
2.56
0.51
4.29
5.31
4.59
9.07
0.37
4.46
2.42
2.79
0.47
1.81
4.19
3.64
7.74
2.49
2.52
2.20
0.38
0.20
2.80
3.55
3.04
0.31
4.90
2.68
0.38
4.20
4.87
4.31
0.22
0.43
0.45
4.15
0.29
0.30
3.68
4.15
2.54
0.21
0.36
0.21
0.40
Deah (asp-glu-ala-his) box polypeptide 9
Dishevelled, dsh homolog 1 (drosophila)
Eukaryotic translation initiation factor 1a, y-linked
Ewing sarcoma breakpoint region 1
Chromosome 20 open reading frame 55
Fibulin 1
F-box and leucine-rich repeat protein 21
Hypothetical protein dkfzp434p055
G0/g1switch 2
Glycine-n-acyltransferase
Homogentisate 1,2-dioxygenase (homogentisate oxidase)
Major histocompatibility complex, class ii, dr beta 1
Homeobox a6
Hiv-1 rev-binding protein
ª 2010 John Wiley & Sons A/S
Niedoszytko et al.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
Table 4 (Continued)
Gene symbol
HS,563982
HS,565704
HS,566231
HS,572999
HS,576804
HS,580555
HS,581365
HS,581671
HS,581933
HS,583304
HS,583989
HS3ST4
IIP45
INOC1
JARID1D
JUP
KLRC1
LBH
LBP
LIPJ
LOC283487
LOC387885
LOC388588
LOC391025
LOC641742
LOC648897
LOC650227
LOC652418
LOC653308
LOC731682
LRTM1
LTK
MAP2K3
MAP7
MEGF8
N4BP2L1
PDGFA
PKIB
PRKY
RBM3
RBMY1A3P
RGMB
RHD
RP11-45B20,2
SPAG17
SPN
SUDS3
TBPL2
TMSB4Y
TNFRSF4
TRAF4
Gene name
Heparan sulfate (glucosamine) 3-o-sulfotransferase 4
Invasion inhibitory protein 45
Ino80 complex homolog 1 (s, cerevisiae)
Smcy homolog, y-linked (mouse)
Junction plakoglobin
Killer cell lectin-like receptor subfamily c, member 1
Hypothetical protein dkfzp566j091
Lipopolysaccharide-binding protein
Lipase-like, ab-hydrolase domain containing 1
Hypothetical protein loc283487
Hypothetical loc387885
Hypothetical gene supported by bc035379; bc042129
Similar to protein tyrosine phosphatase, receptor type,
u isoform 2 precursor
Hypothetical protein loc641742
Similar to atp-binding cassette sub-family d member
1 (adrenoleukodystrophy protein) (aldp)
Similar to mucin 6, gastric
Similar to hypothetical protein flj36492
Similar to n-acylsphingosine amidohydrolase 2
Leucine-rich repeats and transmembrane domains 1
Leukocyte tyrosine kinase
Mitogen-activated protein kinase kinase 3
Microtubule-associated protein 7
Egf-like domain, multiple 4
Hypothetical gene cg018
Platelet-derived growth factor alpha polypeptide
Protein kinase (camp-dependent, catalytic) inhibitor beta
Protein kinase, y-linked
Rna-binding motif (rnp1, rrm) protein 3
RNA-binding motif protein, Y-linked, family 1, member A3 pseudogene
rgm domain family, member b
rh blood group, ccee antigens
Similar to hypothetical protein mgc48915
Sperm associated antigen 17
Sialophorin (gpl115, leukosialin, cd43)
Suppressor of defective silencing 3 homolog (s, cerevisiae)
Tata box-binding protein like 2
Thymosin, beta 4, y-linked
Tumor necrosis factor receptor superfamily, member 4
Tnf receptor-associated factor 4
P-value
Corrected
P-value
R* 1/2
R* 2/1
0.0114
0.0018
0.0133
0.0014
0.0038
0.0002
0.0041
0.0015
0.0027
0.0023
0.0076
0.0131
0.0064
0.0073
0.0216
0.0106
0.0054
0.0004
0.0077
0.0027
0.0002
0.0020
0.0007
0.0035
0.0166
0.0094
0.0187
0.0089
0.0104
0.0026
0.0110
0.0089
0.0094
0.0094
0.0139
0.0186
0.0138
0.0138
0.0271
0.0159
0.0129
0.0042
0.0140
0.0094
0.0023
0.0094
0.0055
0.0104
0.20
0.43
2.51
0.46
3.77
0.25
0.46
3.51
0.31
0.36
0.38
0.26
0.34
0.46
0.11
2.46
0.44
2.94
0.22
2.90
0.32
0.35
3.57
5.55
4.90
2.30
0.40
2.17
0.27
4.03
2.18
0.28
3.23
2.76
2.66
3.91
2.96
2.17
8.92
0.41
2.30
0.34
4.48
0.34
3.13
2.87
0.28
0.18
0.0063
0.0064
0.0138
0.0138
0.29
3.67
3.48
0.27
0.0291
0.0033
0.0101
0.0253
0.0069
0.0160
0.0025
0.0433
0.0036
0.0156
0.0024
0.0165
0.0276
0.0189
0.00001
0.0304
0.0051
0.0016
0.0069
0.0116
0.0071
0.0052
0.0026
0.0081
0.0043
0.0347
0.0104
0.0158
0.0314
0.0138
0.0217
0.0094
0.0492
0.0104
0.0214
0.0094
0.0218
0.0336
0.0243
0.0017
0.0356
0.0128
0.0092
0.0138
0.0167
0.0138
0.0128
0.0094
0.0141
0.0112
3.79
2.36
0.39
0.10
2.97
2.54
0.22
2.46
4.98
0.32
0.26
0.33
0.11
0.41
0.24
2.43
3.00
0.27
0.18
0.50
0.34
0.37
0.19
4.05
0.40
0.26
0.42
2.57
9.90
0.34
0.39
4.54
0.41
0.20
3.16
3.84
3.03
8.87
2.47
4.20
0.41
0.33
3.71
5.45
1.99
2.96
2.69
5.30
0.25
2.51
*Ratio of the expression levels for each individual gene when comparing patients with indolent systemic mastocytosis with a history of
insect venom anaphylaxis and without anaphylaxis of any kind.
ª 2010 John Wiley & Sons A/S
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
Patients with
mastocytosis and IVA
Patients with
mastocytosis without
anaphylaxis
Niedoszytko et al.
Figure 1 Hierarchical clustering dendrogram of differentially expressed genes
that were differentially expressed (log2
fc>3, P < 0.05 corrected for multiple
testing by Benjamini–Hochberg method
P < 0.05) between patients with indolent
systemic mastocytosis with a history of
insect venom anaphylaxis and without
anaphylaxis of any kind. Each column
represents a patient sample, each row an
individual gene. For each gene green color
represents underexpression, red color
overexpression, and black signal missing
data.
Table 5 Function of the most differently expressed genes with P < 0.01
Gene
Known function of the gene
DVL 1
Wnt signaling pathway (19, 20)
Up-regulation of DVL1 was found in inflammatory bowel disease mucosa and also in colon cancer cells (19)
DVL1 is a key molecule in Wnt/planar cell polarity signaling pathway controlling tissue polarity and cell movement (19, 20)
and is up-regulated in various types of human cancers like melanoma, gastric and lung cancer, and neuroblastoma (19, 20)
Up-regulation of both PDGFA and PDGFRA was found in patients with gastrointestinal stromal tumors forming
autocrine-paracrine loop (21), and in prostate cancer, where the over expression was found not only in cancer but also in
stromal cells (22)
MAPK signaling pathway (http://www.genome.jp/kegg/pathway.html)
Expressed i.e. in lymphocytes and monocytes during the switch from G0 to G1 phase of the cell cycle (23, 24)
Up-regulation was found also in patients with vasculitis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus (23)
Product of this gene MAP kinase kinase 3 activates p38 MAP kinase that induces IL1a, IL1b IL12 production (25), B-cell
proliferation (26), and probably also enhances murine mast cell survival (27)
PDGFA
G0S2
MAP2K3
These genes were over expressed in patients with indolent systemic mastocytosis without a history of anaphylaxis of any kind.
patients with ISM and excluded of patients with solely cutaneous mastocytosis (i.e. mastocytosis in the skin without systemic involvement). This is in contrast to the findings of
Brockow who analyzed patients with both cutaneous and systemic mastocytosis. Our data might indicate that in patients
with mastocytosis, the total mast cell load does not contribute
to the particular risk for IVA or anaphylaxis of any kind. In
fact, we postulate that the absence of anaphylaxis might be
because of a more pronounced mast cell dysfunction in the
patients with nonanaphylactic mastocytosis . This assumption
is supported by the observation that in our group of Dutch
and Polish patients with a more advanced (i.e. smoldering,
aggressive, or leukemic) form of mastocytosis (n = 10 + 13),
none of the subjects is suffering from IVA (Kluin-Nelemans
H., de Monchy J.G.R., van Doormaal J.J., Oude Elberink
J.N.G. and Niedoszytko M. unpublished observation). The
differences in expression of the described genes were not
found in patients with IVA without mastocytosis as described
previously by our group (29). Therefore, these effects seem to
be specific for patients with mastocytosis.
ª 2010 John Wiley & Sons A/S
Niedoszytko et al.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis
The observation that the risk of anaphylaxis is related to
more differentiated phenotype of mast cells may also fit very
well to the recently developed concept of a mast cell activation syndrome (MMAS) (30–32). Patients with mast cell activation syndrome suffer from repeated and severe anaphylaxis
and show some criteria of systemic mastocytosis, but fail to
meet sufficient criteria to be classified as mastocytosis. It
might be possible that the mast cell phenotype in these
patients is yet more differentiated compared to patients with
mastocytosis, and it would be interesting to evaluate whether
the findings of our study might also be of relevance in this
new patient group (30–32).
Using a prediction model that uses naı̈ve Bayes (NB) classifier based on the 104 most significantly and most differentially expressed genes, it was possible to differentiate ISM
patients with IVA from those without anaphylaxis. The value
of single gene expression was regarded as an independent
predictor of IVA. This differentiation was not possible using
well-known measurements, such as serum tryptase, urinary
histamine metabolites, and KIT mutation analysis.
It is known that the prevalence of Hymenoptera sensitization in the general population is higher than the prevalence
of IVA. Sensitization to insect venom as evidenced by skinprick tests and specific IgE was also found in one ISM
patient without a history of IVA. In some patients with IVA,
especially with co-existing mastocytosis, it is difficult to detect
the presence of specific IgE (6, 29, 33). Here, it has been
speculated that other mechanisms, besides specific IgE, might
be involved in insect venom-associated activation of mast
cells. As we have found no differences in other factors that
could be related to the level of specific IgE (i.e. time elapsed
since the last sting, number of stings, atopic status), the
observed differences in gene expression may be related to the
regulation of the production, secretion, binding to cells, and/
or clearance of IgE.
Our data indicate that it might be possible to construct a
simple method based on expression values of several genes in
peripheral blood cells that can be used in clinical practice to
assess the risk of IVA by a minimally invasive technique in a
similar way to predicting the response to chemotherapy in
oncology (34, 35). Our results need to be replicated in independent populations, especially in a prospective way evaluating the natural history of anaphylaxis in patients with ISM.
So far, it is not recommended to perform prophylactic
VIT in mastocytosis patients, although this has been recently
suggested by Rueff et al. (7, 8) who reported a patient with
ISM and urticaria pigmentosa who died of an anaphylaxis
after a yellow jacket sting without a history of previous anaphylactic sting reactions. Our study indicates that it might be
possible to identify patients with mastocytosis at risk for
anaphylaxis and might enable to define criteria for the selection of mastocytosis patients eligible for ‘prophylactic’ VIT
as has been suggested elsewhere (7, 8).
The analysis of peripheral whole-blood cells was chosen
because sampling is less invasive for patients compared to
bone marrow aspiration. Furthermore, the standardized
method of RNA isolation and analysis we used reduces
potentially deleterious effects of sample handling on the
result and allows reliable clinical application. Because of this
methodology, the observed differences in expression were
detected in other cell lines of peripheral blood than mast
cells. The analysis of whole-blood cells might be justified
because recent data show that KIT mutation can be present
not only in mast cells but also in myeloid and lymphoid cell
lineages (36).
We can only speculate whether the higher expression of
genes related to the neoplastic transformation of cells is
clinically relevant. At the one side, it is known that patients
with ISM may develop haematological malignancies, but at
the other side this phenomenon has proven to be very rare
in our patient population that is presenting mainly with
urticaria pigmentosa, osteoporosis, and/or anaphylaxis. The
presenting symptom in all our patients with mastocytosis
who have a haematological malignancy was that malignancy.
In conclusion, we demonstrate that in ISM (1), differentially expressed genes in patients with IVA are annotated to
act in pathways favouring cellular differentiation over proliferation, indicating that the differentiation state of the mast
cell might be a critical determinant of the sensitivity to anaphylaxis, and (2) gene expression profiling might be a useful
tool in identifying patients who are at risk for IVA by a
minimally invasive technique. Further studies in larger groups
of patients are required to validate our approach for the
development of a predictive tool to be used in clinical practice.
Acknowledgment
The research was supported by the Foundation for Polish
Science and a grant of the Polish Ministry of Science and
Higher Education N40201031/0386 and N402085934.
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