Expression of IL4 (VNTR intron 3) and IL10 (-627) Genes Polymorphisms in Childhood Immune Thrombocytopenic Purpura

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1 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, MD 1 * Lab Med Summer 2014;45: DOI: /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 immunemediated 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; IFNgamma, 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. 1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt *To whom correspondence should be addressed. samah_cpath@yahoo.com 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 Summer 2014 Volume 45, Number 3 Lab Medicine 211

2 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, antigenpresenting cells, and mast cells. The gene encoding IL10 has been identified on chromosome 1q Wu et al 7 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_ GI: ) and IL10 (-627) (GQ GI: 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 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. 212 Lab Medicine Summer 2014 Volume 45, Number 3

3 Image 1 PCR-RFLP analysis of IL-4 (VNTR intron 3) gene polymorphism. Lane 1 indicates DNA molecular weight marker ( 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 ( bps); 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 (NH 4 ) 2 SO 4, 130 mm Tris HCl, 5.5 mm MgCl 2, 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 heatactivation 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. 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 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 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. 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) Statistical Analysis Data were statistically described by range, mean, standard deviation (SD), frequencies, percentages, Summer 2014 Volume 45, Number 3 Lab Medicine 213

4 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 (c 2 ) 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 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 = and 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 and , 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 and 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 and respectively). 214 Lab Medicine Summer 2014 Volume 45, Number 3

5 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 ( ) a 6.0 ± 3.2 ( ) 8.4 ± 3.5 ( ) 7.2 ± 3.4 ( ) 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 ( ) TLC x10 9 /L 8.7 ± 2.8 ( ) 9.2 ± 2.6 ( ) 8.1 ± 2.9 ( ) 7.7 ± 1.9 ( ) Platelet x10 9 /L 49.2 ± 39.5 (4 130) 37.4 ± 30.0 (5 100) 65.0 ± 45.3 (4 130) 285.5±88.6 ( ) 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 , , 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 Summer 2014 Volume 45, Number 3 Lab Medicine 215

6 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%) RP2 allele 28 (40%) 14 (28%) IL10 gene (No., %) C/C genotype 32 (45.7%) 27 (54%) 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%) RP2 allele 18 (45%) 14 (28%) IL10 gene (No., %) C/C genotype 16 (40%) 27 (54%) 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%) RP2 allele 10 (33.3%) 14 (28%) IL10 gene (No., %) C/C genotype 15 (50%) 27 (54%) A allele 15 (50%) 23 (46%) ITP, immune thrombocytopenic purpura; OR: odds ratio, CI: confidence interval; IL4, interleukin 4; IL10, interleukin 10. Figure 1 Comparison between ITP patients and the control group of single and combined gene polymorphism with the risk of ITP. Percentage (%) ITP Chronic ITP Acute ITP Single Combined Control 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 216 Lab Medicine Summer 2014 Volume 45, Number 3

7 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, 6,8 8, including ITP. 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 al 15 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 al 8 and Chen et al 15 (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 al 15 (34.4%). Also, the RP2/RP2 homotype frequency reported by Wu et al 8 and Chen et al 15 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 al 8 in Chinese patients (64%), and it also coincided with that stated by Chen et al 15 (63.6%). The RP1/RP2 heterotype was close to that studied by Wu et al 8 in Chinese patients (28.0%) and by Chen et al 15 (27.3%). Regarding RP2 homotype, Wu et al 8 and Chen et al 15 reported that the 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 al 8 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 al 8 reported that the homotype RP2/RP2 was statistically higher in patients compared to controls (5% versus 3%). On the contrary, Chen et al 15 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. Summer 2014 Volume 45, Number 3 Lab Medicine 217

8 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 al 8 (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 al 8 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 al 8 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 al 8 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 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 al 8 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 al 18 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 , 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 , respectively). Contrary to our findings, Wu et al 8 and Chen et al 15 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 218 Lab Medicine Summer 2014 Volume 45, Number 3

9 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. References 1. Cooper N, Bussel J. The pathogenesis of immune thrombocytopaenic purpura. Br J Haematol. 2006;133: Psaila B, Bussel JB. Immune thrombocytopenic purpura. Hematol Oncol Clin North Am. 2007;21: Wu KH, Peng CT, Li TC, Wan L, Tsai CH, Tsai J. Interleukin-1beta exon 5 and interleukin-1 receptor antagonist in children with immune thrombocytopenic purpura. J Pediatr Hematol Oncol. 2007;29: Lin MT, Storer B, Martin PJ, et al. Relation of an interleukin-10 promoter polymorphism to graft-versus-host disease and survival after hematopoietic-cell transplantation. N Engl J Med. 2003;349: Wu MC, Huang CM, Tsai JJ, Chen HY, Tsai FJ. Polymorphisms of the interleukin-4 gene in chinese patients with systemic lupus erythematosus in Taiwan. Lupus. 2003;12: Núñez C, Alecsandru D, Varadé J, et al. Interleukin-10 haplotypes in Celiac Disease in the Spanish population. BMC Med Genet. 2006;7: Wu KH, Peng T, Li TC, et al. Interleukin 4, interleukin 6 and interleukin 10 polymorphisms in children with acute and chronic immune thrombocytopenic purpura. Br J Haematol, 2005:128: Wu SF, Chang JS, Wan L, Tsai CH, Tsai FJ. Association of IL-1Ra gene polymorphism, but no association of IL-1 ß and IL-4 gene polymorphisms with Kawasaki disease. J Clin Lab Anal. 2005;19: von dem Borne AE, Verheugt FW, Oosterhof F, von Riesz E, de la Rivière AB, Engelfreit CP. A simple immunofluorescence test for the detection of platelet antibodies. Br J Haematol. 1978;39: Bergmann AK, Grace RF, Neufeld EJ. Genetic studies in pediatric ITP: outlook, feasibility, and requirements. Ann Hematol. 2010;89(S1): Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science. 2008;320: Kamradt T, Mitchison NA. Tolerance and autoimmunity. N Engl J Med. 2001;344: Vukmanovic-Stejic M, Vyas B, Gorak-Stolinska P, Noble A, Kemeny DM. Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes. Blood. 2000;95: Ogawara H, Handa H, Morita K, et al. High Th1 Th2 ratio in patients with chronic idiopathic thrombocytopenic purpura. Eur J Haematol. 2003;71: Chen X, Xu J, Chen Z, et al. Interferon-c +874A T and interleukin-4 intron3 VNTR gene polymorphisms in Chinese patients with idiopathic thrombocytopenic purpura. Eur J Haematol. 2007;79: Pravica V, Perrey C, Stevens A, Lee JH, Hutchinson IV. A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN gamma production. Hum Immunol. 2000;61: Buchs N, Silvestri T, di Giovine FS, et al. IL-4 VNTR gene polymorphism in chronic polyarthritis. The rare allele is associated with protection against destruction. Rheumatology (Oxford). 2000;39: Satoh T, Pandey JP, Okazaki Y, et al. Single nucleotide polymorphism of the inflammatory cytokine genes in adults with chronic immune thrombocytopenic purpura. Br J Haematol. 2004;124: Mouzaki M, Cooper N, Jean C, Frissora C, Bussel B. Does Helicobacter pylori initiate or perpetuate immune thrombocytopenic purpura? Blood. 2004;103: Chouhan JD, Herrington JD. Treatment options for chronic refractory idiopathic thrombocytopenic purpura in adults: focus on romiplostim and eltrombopag. Pharmacotherapy. 2010;30: To read this article online, scan the QR code, ascpjournals.org/content/45/3/211. full.pdf+html Summer 2014 Volume 45, Number 3 Lab Medicine 219