Science
Expression of IL4 (VNTR intron 3) and IL10 (-627)
Genes Polymorphisms in Childhood Immune
Thrombocytopenic Purpura
Manal Mohamed Makhlouf, MD,1 Samah Mohamed Abd Elhamid, MD1*
Lab Med Summer 2014;45:211-219
DOI: 10.1309/LMB0QC5T1RXTTRZQ
ABSTRACT
Objective: Immune thrombocytopenic purpura (ITP) is an acquired
autoimmune disorder caused by the production of antiplatelet
antibodies. These autoantibodies opsonize platelets for splenic
clearance, resulting in low levels of circulating platelets. Interleukin
4 (IL4) and interleukin 10 (IL10) are important immunoregulatory
cytokines mainly produced by macrophages, monocytes, T cells, B
cells, and mast cells. Our study was aimed at detecting the frequency
of IL4 (VNTR intron 3) and IL 10 (-627) gene polymorphisms in
Egyptian ITP children as genetic markers for ITP risk and clarifying
their possible role in the pathogenesis of ITP as well as their
correlation with the clinical presentation and laboratory data.
Methods: IL4 (VNTR intron 3) and IL10 (-627) gene polymorphisms
were studied in 70 ITP patients and 50 age- and sex-matched healthy
Immune thrombocytopenic purpura (ITP), also known
as idiopathic thrombocytopenic purpura, is an immune-
mediated acquired disease of adults and children
characterized by transient or persistent decrease in
the platelet count and, depending upon the degree of
thrombocytopenia, increased risk of bleeding.1 In most
children and some adults, ITP is an acute, self-limited
Abbreviations
ITP, immune thrombocytopenic purpura; IL4, interleukin 4; Th, T helper;
IL10, interleukin 10; VNTR, variable number of tandem repeats; IFN-
gamma, Inferon-gamma; PCR-RFLP, polymerase chain reaction-fragment
length polymorphism; EDTA, ethylene diamine tetra-acetic acid; DNA,
deoxyribonucleic acid; SD, standard deviation; OR, odds ratio; CI,
confidence interval; TNF, tumor necrosis factor; GBIIb/IIIa, Glycoprotein
IIb/IIIa.
Department of Clinical and Chemical Pathology, Faculty of Medicine,
1
Cairo University, Cairo, Egypt
*To whom correspondence should be addressed.
E-mail: samah_cpath@yahoo.com
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controls using polymerase chain reaction-restriction fragment length
polymorphism assay (PCR-RFLP).
Results: IL4 RP2 and IL10 A alleles were detected more frequently
among ITP patients compared to controls. A statistically significant
difference was observed in IL10 and IL4 gene polymorphism
distribution between acute and chronic ITP patients, with higher A
allele and RP2 allele among chronic ITP patients versus acute ITP
patients. Combined polymorphisms of IL4 and IL10 genes were
associated with greater risk of ITP.
Conclusion: IL4 and IL10 gene polymorphisms may contribute to
susceptibility for ITP in children.
Key words: IL4 (VNTR intron 3), IL10 (-627), polymorphisms, PCR-
RELP, ITP.
disease that resolves or improves spontaneously within
months. In a small number of children and in many adults,
ITP may be chronic and poorly responsive to treatment.2
ITP is associated with cytokine response and dysregulation
in the cytokine network. Genetic factors have been
reported to be associated with ITP. Single nucleotide
polymorphisms are the most common DNA sequence
variations associated with genetic diseases. Gene
polymorphisms, including those in cytokine genes, have
been associated with adult and childhood ITP.3
The cytokine genes are polymorphic, which accounts
for the different levels of cytokine production, and
are related to regulation of the immune-mediated
inflammatory process. Cytokine gene polymorphisms
have recently attracted interest because distinct alleles
of cytokine genes have been associated with various
immunoinflammatory diseases.4
Interleukin 4 (IL4) is a highly pleiotropic cytokine coded by
a gene on chromosome 523-q31 that is able to influence
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T-helper (Th) cell differentiation. Early secretion of IL4
leads to polarization of Th cell differentiation toward Th2-
like cells. The Th2-cell secretion of IL4 and interleukin 10
(IL10) leads to the suppression of Th1 responses by down
regulating the production of macrophage-derived IL10 and
inhibiting the differentiation of Th1-type cells. IL4 is a key
cytokine that induces activation and differentiation of B
cells as well as development of Th subset of lymphocytes.
The IL4 gene has a variable number of tandem repeats
(VNTR) polymorphism located in the third intron, consisting
of 3, 70 base pair (bp) repeats, The 70-bp polymorphism in
intron 3 is associated with IL4 production.5
IL10 is the most important anti-inflammatory cytokine in
the human immune response. IL10 is a potent inhibitor
of Th1 cytokines, including both IL2 and interferon
(IFN)-γ. This activity accounts for its initial designation as
cytokine synthesis inhibition factor; it is mainly produced
by macrophages, monocytes, T cells, B cells, dendritic
cells, mast cells, and eosinophils. IL10 also limits the
inflammatory response and regulates the differentiation and
proliferation of T cells, B cells, natural killer cells, antigen-
presenting cells, and mast cells. The gene encoding IL10
has been identified on chromosome 1q31–32.6
Wu et al7 reported that IL4 (VNTR intron 3) and IL10 (-627)
polymorphisms were associated with the pathogenesis of
ITP and contributed to the susceptibility of developing ITP,
and that this might be the basis for immunomodulatory
therapies for ITP and provide a tool for early diagnosis of
susceptibility to ITP.
The present work aims to study the expression of
IL4 VNTR intron 3 (NM_172348.2 GI:391224449) and
IL10 (-627) (GQ847742.1 GI:306977724 C/A) gene
polymorphisms by polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) among
children with ITP, and to define their role in modulating
susceptibility to development of ITP and their correlation
with the clinical presentation and laboratory data.
Materials and Methods
Patients
The present study was conducted with 70 ITP patients
ages 1 to 14 years with a mean (SD) age of 7.0 ±3.5
years. The subjects included 36 males (51.4%) and 34
212 Lab Medicine  Summer 2014  |  Volume 45, Number 3 www.labmedicine.com
females (48.6%). Patients were classified into 2 groups.
Group 1 included 40 chronic ITP patients (clinical
condition lasting >6 months and receiving medical
treatment). Group 2 included 30 acute ITP patients
(clinical condition lasting <3 months and not receiving
medical treatment). Patients were selected from cases
referred to the Haematology Clinic of Elmounira Children
Hospital, Faculty of Medicine, Cairo University, Egypt.
Fifty age- and sex-matched individuals were selected as
a control group.
The diagnosis of ITP was based on medical
history, clinical examination for organomegaly and
lymphadenopathy, and laboratory investigations for
diagnosis of ITP including complete blood count, bone
marrow aspiration biopsy, platelet antibody testing, and
special laboratory investigations (for patients and controls)
for detection of IL4 (VNTR intron 3) and IL10 (-627) genes
polymorphisms using the PCR-RFLP technique according
to the method described by Wu et al.8
Methods
Sample Collection
Ten mL of venous blood were collected from each patient,
and 5 mL were collected from each individual of the control
group, by sterile venipuncture and divided as follows:
5 mL of venous blood from each patient and control in
ethylenediaminetetraacetic acid (EDTA) for the complete
blood count (2 mL), and for the direct platelet antibody test
and direct antiglobulin test (3 mL). Five mL were collected
from each patient and control in a plain sterile vacutainer
for the indirect platelet antibody test (2 mL), and for the
study of genotyping for the polymorphisms of the IL4 and
IL10 genes by PCR-RFLP assay (3 mL).
Platelets Antibody Testing
Platelet antibodies were detected using platelet
immunofluoresence test as described by Von dem Borne
et al.9 This test is based on the ability of antihuman
fluorescine-labeled antibodies to bind the antibodies
present on platelets (direct test) or antibodies present in
serum (indirect test).
Detection of IL4 and IL10 Gene Polymorphisms by
PCR-RFLP
Genomic DNA was extracted from 200 mL of whole EDTA
blood using Biorobot EZ1 DNA isolation kits (Qiagen,
Japan, Clinilab) according to the manufacturer’s protocol.
The volume of eluted DNA was 200 mL.

Image 1
PCR-RFLP analysis of IL-4 (VNTR intron 3) gene polymorphism.
Lane 1 indicates DNA molecular weight marker (50–1000 bps);
lanes 2, 6, and 7 show heterozygous RP1/RP2 genotype (183 and
253 bps); lanes 3, 4, 8, 9, 10, and 11 show wild type RP1/RP1 (183
bp); lane 5 shows homozygous RP2/RP2 genotype (253 bp).
Image 2
PCR-RFLP analysis of IL10 (-627) gene polymorphism. Lane 1
indicates DNA molecular weight marker (50–1000bps); lanes 2,
3, 4, and 5 show homozygous A/A genotype (176 and 236 bps);
lanes 6, 7, and 8 showheterozygous C/A genotype (176, 236, and
412 bps); lanes 9 and 10 show wild type C/C genotype (412 bp).
The 25 µl reaction mixture consisted of 5 µL genomic
DNA, 12.5 µL of PCR master mix (0.1 units/μL Taq DNA
Polymerase, 32 mM (NH4)2 SO4, 130 mM Tris HCl, 5.5 mM
MgCl2, and 0.4 mM of each dNTP), 1 µL of each primer
and 6.5 µL distilled water. Primers were prepared to a
concentration of 25 pmol each.
For IL4 (VNTR intron 3) polymorphism the following primers
were used:
Forward primer: 5′-AGGCTGAAAGGGGGAAAGC-3′; reverse
primer: 5′- CTGTTCACCTCAACTGCTCC-3′. An initial heat-
activation step at 94°C for 5 minutes was followed by 35
cycles of denaturation at 94°C for 20 seconds, annealing at
58°C for 20 seconds, and extension at 72°C for 20 seconds,
then a final extension at 72°C for 10 minutes.
For IL10 (-627) polymorphism the following primers were
used:
Forward primer: 5′-CCTAGGTCACAGTGACGTGG-3′;
reverse primer: 5′-GGTGAGCACTACCTGACTAGC-3′.
An initial heat activation step at 94°C for 3 minutes was
followed by 35 cycles of denaturation at 94°C for 1 minute,
annealing at 50°C for 1 minute, and extension at 70°C for 1
minute, then a final extension at 70°C for 3 minutes.
The amplification products were then separated in
parallel using gel electrophoresis on a 4% agarose
gel (electro-4, Thermal Hybaid, Promega, USA) and
visualized with a UV transilluminator (wavelength 312 nm)
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to detect the presence or absence of DNA bands. For the
IL4 (VNTR intron 3) and IL-10 (-627) polymorphisms, 183-
bp and 412-bp fragments, respectively, were amplified.
In individuals lacking the IL4 gene polymorphism, a
183-bp band was detected and was designated RP1/
RP1 genotype (ie, wild type). But if 1 253-bp band was
detected due to a 70-bp insertion, it was designated
RP2/RP2 (ie, homozygous RP2 allele). If 2 bands, 183 bp
and 253 bp, were detected, it was designated RP1/RP2
(ie, heterozygous for RP2 allele) as shown in Image 1.
For IL10 gene polymorphism, the amplified PCR
products (412 bp) were digested at 37°C overnight with
1 µL (10 units) RsaI conventional restriction enzyme
in the manufacturer’s buffer (Fermentas AM, Egypt).
In individuals lacking this polymorphism, 1 band at
412 bp appeared and was designated C/C (genotype
(ie, wild type). The original 176-bp, 236-bp bands and
the cleaved 1 412-bp band were seen in individuals
heterozygous for this polymorphism and were
designated heterozygous C/A genotype. If 2 bands, 176
bp and 236 bp, emerged, it was designated A/A (ie,
homozygous A allele) as shown in Image 2.
Statistical Analysis
Data were statistically described by range, mean,
standard deviation (SD), frequencies, percentages,
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and odds ratio (OR) with 95% confidence interval (CI)
when appropriate. Quantitative variables between the
study groups were compared using Student’s t-test for
independent variables that were normally distributed, and
the Mann-Whitney U test for independent variables that
were not normally distributed. For comparing categorical
data, the Chi-square (c2) test was used, but Fisher’s
exact test was used when the expected frequency
was less than 5. A probability (P) of less than 0.05 was
considered statistically significant. Statistical calculations
were performed using Microsoft Excel 2003 (Microsoft
Corporation, NY, USA) and SPSS (Statistical Package for
the Social Science; SPSS Inc., Chicago, IL, USA) version
15 for Microsoft Windows.
Results
The patient and the control group characteristics are
summarized in Table 1. IL4 (VNTR intron 3) and IL10 (-627)
gene polymorphisms in ITP patients and the control group
are shown in Table 2.
Statistical comparison between chronic ITP, acute ITP
and the control group with regard to demographics,
clinical, laboratory data, and IL4 and IL10 polymorphisms
were studied. Comparison revealed a statistically
significant difference between the 3 groups with respect
to splenomegaly, hemoglobin concentration, and platelet
count, with more frequent splenomegaly and higher
hemoglobin levels among acute ITP patients and higher
platelet count among control group (P <0.001). There
was a statistically significant difference in age and total
leukocyte count with higher age among acute ITP patients
and higher total leukocyte count among chronic ITP
patients (P =0.02 and 0.01 respectively). A borderline
significant difference between the 3 groups was found
in IL10 polymorphism distribution, with more frequent A
allele among chronic ITP patients (P =0.05. Although no
statistically significant difference was revealed between
the three groups regarding IL4 gene polymorphism
distribution, the RP2 allele was more frequent in chronic
ITP patients. Also, no statistically significant difference
was observed in the male versus female prevalence of ITP.
The relationship between IL4 and IL10 genotypes and
demographic, clinical, and laboratory data in chronic
versus acute ITP patients was examined. In ITP patients,
a significant liability was observed between IL4 and
214 Lab Medicine  Summer 2014  |  Volume 45, Number 3 www.labmedicine.com
IL10 genotypes with regard to hemoglobin level, platelet
count, and antiplatelet antibodies (P <0.001); lower
hemoglobin levels, platelet counts, and more frequent
antiplatelet antibodies were found among homozygous
RP2/RP2 IL4 and A/A IL10 ITP patients. There was
no statistically significant difference found among
demographic data, the presence of splenomegaly, or
the total leukocytic count when these genotypes were
compared. In chronic ITP patients, statistical comparison
revealed a significant difference between IL10 genotypes
in hemoglobin levels and antiplatelet antibodies
(P = 0.002 and 0.005 respectively); lower hemoglobin
levels and more frequent antiplatelet antibodies
occurred among homozygous A/A and heterozygous
C/A patients, respectively; there was no statistically
significant difference in demographic parameters, the
presence of splenomegaly, or platelet counts among
the A/A and C/A patients. No statistically significant
differences were observed between IL4 genotypes in
demographic, clinical, and laboratory data. In acute
ITP patients, significant difference were found between
IL10 genotypes in hemoglobin levels, platelet counts,
and antiplatelet antibodies (P <0.001); lower hemoglobin
levels, platelet counts, and more frequent antiplatelet
antibodies occurred among homozygous A/A patients
compared to heterozygous C/A patients; demographic
data, the frequency of splenomegaly, and total leukocytic
counts were not significantly different in these groups.
Finally, a significant difference was found between IL4
genotypes in antiplatelet antibodies (P = 0.01), which
were more frequent among heterozygous RP1/RP2
patients; no statistically significant differences were
observed in demographic parameters, or clinical and
laboratory data
Table 3 displays the statistical comparison between IL4
and IL10 gene polymorphisms in ITP patients, whether
chronic or acute, and the control group with regard to the
risk of developing ITP. There was no statistically significant
difference between ITP patients and the control group
in their susceptibility to ITP, and no apparent liability of
patients with IL4 and IL10 polymorphism to develop ITP
(P >0.05; OR 1.07 and 1.03, 95% CI 0.82–3.74 and 0.65–
2.71, respectively). Compared to controls, chronic ITP
patients expressing IL4 and IL10 polymorphism were at
significantly greater riskfor developing the chronic form of
ITP (P =0.03 and 0.04, OR 2.98 and 2.14, 95% CI 1.93–6.15
and 1.62–4.95 respectively). Patients with the IL4 and IL10
polymorphisms were not at greater risk for ITP
(P >0.05, OR 1.02 and 1.11, 95% CI 0.51–3.46 and 0.46–
2.73 respectively).
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Table 1. Characteristics of ITP Patients and the Control Group

Item ITP (n=70) Chronic ITP (n= 40) Acute ITP (n=30) Control Group (n=50)

Age (years) 7.0 ± 3.5 (1.0–14.0)a 6.0 ± 3.2 (1.0–12.0) 8.4 ± 3.5 (2.0–14.0) 7.2 ± 3.4 (1.0–13.0)
Sex
Male 36 (51.4%)b 24 (60%) 12.0 (40%) 29 (58%)
Female 34 (48.6%) 16 (40%) 18.0 (60%) 21 (42%)
Clinical data
Hepatomegaly 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Splenomegaly 30 (42.9%) 14 (35%) 16 (53.3%) 0 (0%)
Lymphadenopathy 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Splenectomy 12 (17.1%) 3 (7.5%) 9 (30%) 0 (0%)
Laboratory data
Hb (g/dL) 9.9 ± 2.2 (7–14.5) 9.6 ± 2.1 (7–14.3) 10.3 ± 2.4 (7–14.5) 12.7 ±1.3 (11.0–15.8)
TLC x109/L 8.7 ± 2.8 (4.0–15.0) 9.2 ± 2.6 (4.6–14.9) 8.1 ± 2.9 (4.6–14.9) 7.7 ± 1.9 (4.3–11.0)
Platelet x109/L 49.2 ± 39.5 (4–130) 37.4 ± 30.0 (5–100) 65.0 ± 45.3 (4–130) 285.5±88.6 (155.0–440.0)
Antiplatelet Abs 38 (54.3%) 18 (45%) 20 (66.7%) 0 (0%)
Direct antiglobulin test 0(0%) 0(0%) 0(0%) 0(0%)
Management
Corticosteroids 70 (100%) 40 (100%) 30 (100%) 0 (0%)
IV Ig 2 (2.9%) 2 (5%) 0 (0%) 0 (0%)
Anti D 3 (4.3%) 1 (2.5%) 2 (6.6%) 0 (0%)

ITP, immune thrombocytopenic purpura; Hb, hemoglobin;

a
Mean ± SD (range).
b
No. (%).

Table 2. IL4 and IL10 Gene Polymorphisms in ITP Patients and Control Group

Item ITP (n=70) Chronic ITP (n=40) Acute ITP (n=30) Control Group (n=50)

IL4 gene polymorphism (No., %)

Wild (RP1/RP1) genotype 42 (60%) 22 (55%) 20 (66.7%) 36 (72%)
RP2 allele 28 (40%) 18 (45%) 10 (33.3%) 14 (28%)
Heterozygous(RP1/RP2) 23 (32.9%) 14 (35%) 9 (30%) 13 (26%)
Homozygous(RP2/RP2) 5 (7.1%) 4 (10%) 1 (3.3%) 1 (2%)
IL10 gene polymorphism (No., %)
Wild (C/C) genotype 32 (45.7%) 16 (40%) 15 (50%) 26 (52%)
A allele 38 (54.3%) 24 (60%) 15 (50%) 24 (48%)
Heterozygous (C/A) 25 (35.7%) 19 (47.5%) 6 (20%) 18 (36%)
Homozygous (A/A) 13 (18.6%) 4 (12.5 %) 9 (30%) 6 (12%)

ITP, immune thrombocytopenic purpura; IL 4, interleukin 4; IL 10, interleukin 10.

In Figure 1, ITP patients are compared with the control

group with regard to single and combined gene
polymorphisms and their risk of ITP. A statistically
significant difference was obsereved between the ITP
patients, whether chronic or acute, and the control
group; patients with combined gene polymorphisms
were more prone to develop ITP, either chronic or
acute, than the control group (P =0.004, 0.02, 0.003;
OR 6.43, 5.14, 9.52; CI 2.75–24.06, 2.26–20.73, 3.14–
43.35 respectively).
Discussion
ITP is an acquired autoimmune disorder that is the
most common cause of isolated thrombocytopenia in
children.10 ITP results from the production of antiplatelet
autoantibodies. These autoantibodies opsonize platelets
for splenic clearance, resulting in thrombocytopenia.11 The
disease may be mediated by autoreactive B-lymphocytes,
which produce antiplatelet antibodies. Also, increased
activation of T cells and cytokine response suggest

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Table 3. Comparison Between IL4 and IL10 Gene Polymorphisms in ITP Patients and the Control
Group as Regarding the Risk of ITP

Polymorphism ITP Control OR 95% CI P Value

IL4 gene (No., %)

RP1/RP1 genotype 42 (60%) 36 (72%) 1.07 0.82 - 3.74 0.17
RP2 allele 28 (40%) 14 (28%)
IL10 gene (No., %)
C/C genotype 32 (45.7%) 27 (54%) 1.03 0.65 - 2.71 0.49
A allele 38 (54.3%) 23 (46%)

Polymorphism Chronic ITP Control OR 95% CI P Value

IL4 gene (No., %)

RP1/RP1 genotype 22 (55%) 36 (72%) 2.98 1.93 - 6.15 0.03
RP2 allele 18 (45%) 14 (28%)
IL10 gene (No., %)
C/C genotype 16 (40%) 27 (54%) 2.14 1.62 - 4.95 0.04
A allele 24 (60%) 23 (46%)

Polymorphism Acute ITP Control OR 95% CI P Value

IL4 gene (No., %)

RP1/RP1 genotype 20 (66.7%) 36 (72%) 1.02 0.51 - 3.46 0.82
RP2 allele 10 (33.3%) 14 (28%)
IL10 gene (No., %)
C/C genotype 15 (50%) 27 (54%) 1.11 0.46 - 2.73 1.00
A allele 15 (50%) 23 (46%)

ITP, immune thrombocytopenic purpura; OR: odds ratio, CI: confidence interval; IL4, interleukin 4; IL10, interleukin 10.

Single
Figure 1 Combined
Comparison between ITP patients and the control group of single 100 91.4
and combined gene polymorphism with the risk of ITP. 90
80
Percentage (%)

67.7
70 62.5
60 52.9
47.1
50
37.5
40 32.3
30
20
8.6
10
0
ITP Chronic Acute Control
ITP ITP ITP

that T-lymphocytes could play an important role in this
autoimmune process.3 Genetic factors such as gene
polymorphisms, including those in cytokine genes, have
been reported to be associated with ITP.7
Naive (Th) cells differentiate into Th1 or Th2 cells, and
the balance of Th1 and Th2 cells regulates the immune
response under normal conditions and is critical in the
pathogenesis of autoimmune diseases, including ITP.
Th1 predominance can induce autoimmunity, whereas
Th2 predominance can inhibit the immune response.12
Th1 cells promote cellular immunity by secreting
interferon (IFN)-γ, interleukin (IL) 2 and tumor necrosis
factor (TNF)-α. In contrast, Th2 cells mostly induce
humoral immunity by producing IL4, IL5, IL6, IL10, and
IL13. Allelic variations in regulatory regions of cytokine
genes have been shown to affect the expression of some
cytokines. ITP has been associated with dysregulation

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of the cytokine response.13 It has been proposed that
chronic ITP is a Th1 polarization disease characterized
by a decrease in Th2 cell counts; a high Th1 ⁄ Th2 ratio
was closely related to the etiology and disease status of
chronic ITP.14 In ITP, contact between macrophages,
dendritic cells, and foreign antigens may initiate a
proinflammatory cytokine cascade characterized by IL1,
TNFα, IL6, and IL8 production followed by a chemokine
burst and a counter-response of anti-inflammatory
cytokines such as IL1Ra, tumor growth factor-B, and
IL10.3
The IL4 gene has a 70-bp VNTR polymorphism in intron
3 associated with IL4 production. It has been proposed
that increased responsiveness of the RP1 allele to
transcriptional activation may lead to overexpression of
IL4.15 IL4 intron 3 VNTR polymorphisms account for the
overproduction of this cytokine, which in turn affects the
magnitude and duration of the immune response, perhaps
predisposing the individual to autoimmune disorders.16
An IL10 A/C polymorphism at position -627 in the IL10
gene promoter has been identified (IL10 C-627A) and the
-627*A allele is associated with low IL10 expression, which
may favor inflammatory and immune-mediated diseases,
including ITP.6,8 8,
In our study, genotyping detected IL4 VNTR intron 3
RP1/RP1, RP1/RP2, and RP2/RP2 in 60.0%, 32.9%,
and 7.1% of ITP patients, respectively. The RP1/RP1
genotype frequency matched that reported by Chen et
al15 in Chinese patients (62.7%), while RP1/RP2 and RP2/
RP2 genotypes were lower than those reported in the
same study (34.7% and 8.0% respectively). In chronic ITP
patients, IL4 VNTR intron 3 RP1/RP1, RP1/RP2, and RP2/
RP2 genotypes were detected in 55%, 35%, and 10%
respectively. The frequency of the RP1/RP1 genotype was
lower than that found by Wu et al8 and Chen et al15 (86.7%
and 64.1% respectively), while the RP1/RP2 heterotype
was higher than that reported in Chinese patients (13.3%),
but close to that reported by Chen et al15 (34.4%). Also,
the RP2/RP2 homotype frequency reported by Wu et al8
and Chen et al15 was lower than our findings Versus ours.
In acute ITP patients, IL4 VNTR intron 3 genotypes were
detected in 66.6%, 30%, and 3.3% respectively. The RP1/
RP1 genotype was similar to that reported by Wu et al8
in Chinese patients (64%), and it also coincided with that
stated by Chen et al15 (63.6%). The RP1/RP2 heterotype
was close to that studied by Wu et al8 in Chinese patients
(28.0%) and by Chen et al15 (27.3%). Regarding RP2
homotype, Wu et al8 and Chen et al15 reported that the
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RP2/RP2 homotype was higher than our findings, detected
in 8.0% and 9.1% of acute ITP patients respectively.
In our study, the frequency of the IL4 RP1/RP1 genotype
was lower in ITP patients compared to controls (60%
vs 72%); however, the difference was not statistically
significant. On the other hand, Wu et al8 found that
the wild type RP1/RP1 was statistically more frequent
in patients compared to controls (72.5% vs 64.0%).
The difference between their results and ours may be
explained by the large number of control subjects enrolled
in their study or it may be more highly expressed in
Chinese people compared to Egyptians. The frequency
of the IL4 RP1/RP2 heterotype was higher in ITP patients
compared to controls (32.9% vs 26.0%), in contrast to
the findings by Wu et al,8 in which the RP1/RP2 allele was
more frequent in control group than in ITP (33.0% versus
13.3%). Also, the frequency of IL4 RP2/RP2 genotype
was higher in ITP patients in our study compared to
controls (7.1% versus 2.0%); however, the difference was
not statistically significant. Wu et al8 reported that the
homotype RP2/RP2 was statistically higher in patients
compared to controls (5% versus 3%). On the contrary,
Chen et al15 reported that the frequency of the homotype
RP2/RP2 in ITP patients (2.6%) was lower than that of
the control group (5.5%). This discrepancy may be due
to ethnic differences, the geographic distribution, or the
different sample sizes used in these studies.
Although the function of the intron 3 polymorphism of the
IL4 gene is not known, it is possible that distinct numbers
of VNTR might affect the transcriptional activity of the IL4
gene.5 Some studies have provided evidence suggesting
that the RP1 allele induces higher expression of IL4 than
the RP2 allele, and that the RP1 allele may be a protective
factor in some diseases.17
Comparison between the chronic ITP, acute ITP, and
control groups with regard to demographic, clinical, and
laboratory parameters revealed statistically significant
differences between the 3 groups for splenomegaly,
hemoglobin levels, and platelet counts, with more frequent
splenomegaly and higher hemoglobin levels among acute
ITP patients and higher platelet counts among the control
group. Also, there was a statistically significant difference
between the 3 groups regarding age and total leucocyte
count with higher age among acute ITP patients and higher
total leucocytic count among chronic ITP patients.
Among ITP patients, IL10 C/C, C/A, and A/A genotypes
were detected in 45.7%, 35.7%, and 18.6% respectively.
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When comparing our results with previous studies,
the C/C genotype was lower than that reported in
Chinese patients (52.5%), whereas previously reported
frequencies of the C/A heterotype and A/A homotype
(32.5% and 15.0% of ITP patients, respectively, reported
by Wu et al.8) were similar to our results. In our chronic
ITP patients, IL10 C/C, C/A, and A/A genotypes were
detected in 40.0%, 47.5%, and 12.5% respectively. These
results were lower than that reported in Chinese patients
in the study of Wu et al8 (60.0%, 13.3%, and 26.7%
respectively). In our acute ITP patients, IL10 genotypes
were detected in 50%, 20%, and 30% respectively. The
C/C genotype was similar to that reported in Chinese
patients (48%); the C/A heterotype was higher than ours,
being found in (44%); and the A/A homotype was lower
(8%) than that stated by Wu et al.8
The frequency of IL10 (-627) C/C genotype was lower in
ITP patients compared to controls (45.7% versus 52%)
with a statistical difference between the two groups.
On the other hand, Wu et al8 stated that the wild type
C/C was statistically higher in patients than controls
(52.5% versus 41.0%). The difference between their
results and ours may be explained by the large number
of control subjects enrolled in their study or due to racial
differences; the C/C genotype may be overexpressed
in Egyptians versus Chinese people. In our study, the
frequency of the IL10 (-627) C/A heterotype was similar in
ITP patients compared to controls (35.7% versus 36.0%).
In contrast, Wu et al8 found that the heterotype C/A was
statistically lower in ITP patients compared to controls
(32.5% versus 44.0%) whereas the frequency of IL-10
(-627) A/A homotype was higher in ITP patients than
controls (18.6% versus 12.0%). However, Wu et al8 found
that the homotype A/A was statistically the same in ITP
patients compared to controls (15%).
In chronic ITP patients, a significant difference was
observed in hemoglobin levels and antiplatelet antibodies
between IL10 genotypes. No significant differences in
demographic, clinical, and laboratory data were observed
between IL4 and IL10 genotypes; in acute ITP patients,
significant differences in these parameters occurred
between IL10 genotypes. Most people have B cells
directed to make autoantibodies as well as detectable
peripheral blood T cells reactive to glycoprotein IIb/
IIIa. Therefore, the immunological machinery does not
need to be created de novo but merely turned on. The
limited reports illustrating genomic associations suggest
that patients with ITP may have a more general genetic
susceptibility towards antiplatelet antibody production. The
218 Lab Medicine  Summer 2014  |  Volume 45, Number 3 www.labmedicine.com
associations with other diseases including autoimmune
thyroid disease and SLE support this hypothesis.1
We did not observe a statistical difference in the presence
of the A allele of IL10 gene polymorphism, when acute
ITP patients were compared with the control group. In
contrast, Wu et al8 reported that occurrence of the A
allele of IL10 was significantly different in ITP compared
to control patients. However, our results revealed a
statistically significant difference in the occurrence of this
allele between acute and chronic ITP.
In our study, there was a statistically significant correlation
between chronic ITP patients demonstrating IL10 gene
polymorphism and low platelet count, suggesting that
IL10 gene polymorphism reflects the severity of chronic
ITP. This finding is in concordance with Satoh et al18
who showed that the immune response in ITP patients
is towards Th1 polarization and that IL10 is an important
factor regulating Th1 and Th2 cytokine synthesis, has a
significant role in autoimmunity and tumorigenesis, and that
patients with chronic ITP and the IL10 gene polymorphism
have lower platelet counts compared to patients without
polymorphism. According to Mouzaki et al,19 raised IL10
levels occur in children with chronic ITP, further suggesting
Th1 involvement in the pathology of ITP illustrated by an
increase in the Th1 cytokines; these findings may be related
to ongoing immune activation related to autoimmunity.
In our study, IL4 RP2 alleles and IL10 A alleles were
associated with the development of ITP when compared
with the control group (OR 2.98, 2.14 and 95% CI 1.93-
6.15, 1.62–4.95 respectively). Further, when combined
IL4 and IL10 gene polymorphisms in acute and chronic
ITP cases were studied, these polymorphisms were
more prone to genetic risk of ITP development compared
to controls (OR 9.52, 5.14 and 95% CI 3.14–43.35,
2.26–20.73 respectively). Contrary to our findings, Wu
et al8 and Chen et al15 found that the RP2 allele was not
associated with increased susceptibility of developing
ITP. The discrepancy may result from both genetic
and environmental factors that play important roles in
development of ITP, and the fact that Th2 cells mostly
induce humoral immunity by producing IL4, IL5, IL6 , IL10
and IL13, whereas the polarization of the immune system
towards either cellular (Th1) or humoral (Th2) immunity
depends on the level of secreted cytokines at the site of
immune activation, while allelic variations in regulatory
regions of cytokine genes have been reported to be
associated with cytokine response and dysregulation.13
Discovery of other polymorphisms in IL4 and IL10 genes

may facilitate further exploration of association between
mutations and pathogenesis of ITP. Approximately 60%
of adult ITP patients develop therapy-refractory chronic
disease within 12 months, and this has necessitated the
development of novel therapeutic strategies. ITP is a Th1
cell predominant disease. Shifting the cytokine patterns
from Th1 to Th2 may be a beneficial therapeutic approach
for ITP. Identification of expression pattern of IL4 and IL10
and their polymorphisms should aid the development of
new and promising therapeutic modalities. 20 LM
Acknowledgments
This study was processed in Kasr Al-Aini Hospital. We
thank our patients for their willing participation in our
research.
Declaration of Ethics
Oral and written informed consent was obtained from all
patients according to the Declaration of Helsinki.
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