|Year : 2016 | Volume
| Issue : 3 | Page : 121-127
DNA methyltransferase 3B gene promotor and interleukin-1 receptor antagonist polymorphisms in Egyptian children with immune thrombocytopenic purpura
Dina Ahmed Ezzat1, Amira Ahmed Hammam MD 2, Waleed Mostafa El Malah1, Sanaa Ahmed Hussein3
1 Pediatrics Department, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt
2 Clinical Pathology Department, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt
3 Pediatrics Department, Ministry of Health Hospitals, Beni-Suef, Egypt
|Date of Submission||21-Feb-2016|
|Date of Acceptance||13-Apr-2016|
|Date of Web Publication||27-Dec-2016|
Amira Ahmed Hammam
4 El Sad Elaali St., Dokki, Giza, 11562
Source of Support: None, Conflict of Interest: None
Background and objectives Idiopathic thrombocytopenic purpura (ITP) is an autoimmune condition characterized by increased platelet destruction. Although the etiology of ITP remains unclear, it is accepted that both environmental and genetic factors play an important role in the development of the disease. Many gene polymorphisms have been reported to be closely associated with the susceptibility to ITP.
Aim of the work The current study aimed to investigate the association between the DNA methyltransferase DNMT3B-46359 C/T promoter and IL-1Ra polymorphism and the risk for acquisition of pediatric ITP in a cohort of Egyptian children.
Patients and methods Genotyping of the studied genes using PCR-RFLP assays was conducted on 40 children with ITP and 20 age-matched and sex-matched normal controls.
Results Our results revealed that DNMT3B-46359 C/T heterotype was higher in patients than in controls but did not reach statistical significance and conferred 2.2-fold increased risk for ITP (odds ratio=2.2, confidence interval = 1.4–2.6). There was no statistically significant difference between ITP patients and controls as regards allele frequency. Moreover, there was no statistically significant difference in the clinical and laboratory data between ITP patients with wild or mutant genotypes. Moreover, there was no statistically significant difference in the distribution of DNMT3B-46359 . C/T genotypes between acute and chronic ITP patients.
Conclusion This genetic polymorphism cannot be considered as a molecular marker for ITP risk among Egyptian children. Moreover, it is not a molecular predictor for chronicity.
Keywords: DNMT3B-46359C/T promoter, IL-1Ra polymorphism, ITP
|How to cite this article:|
Ezzat DA, Hammam AA, El Malah WM, Hussein SA. DNA methyltransferase 3B gene promotor and interleukin-1 receptor antagonist polymorphisms in Egyptian children with immune thrombocytopenic purpura. Egypt J Haematol 2016;41:121-7
|How to cite this URL:|
Ezzat DA, Hammam AA, El Malah WM, Hussein SA. DNA methyltransferase 3B gene promotor and interleukin-1 receptor antagonist polymorphisms in Egyptian children with immune thrombocytopenic purpura. Egypt J Haematol [serial online] 2016 [cited 2019 Dec 8];41:121-7. Available from: http://www.ehj.eg.net/text.asp?2016/41/3/121/196179
| Introduction|| |
Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder caused by the production of antiplatelet antibodies .
Epigenetic changes in gene expression, including DNA methylation and histone modifications, might contribute to autoimmunity. DNA methylation is mediated by a family of DNA methyltransferases. Polymorphisms of the DNA methyltransferase 3B (DNMT3B) gene may influence DNMT3B activity on DNA methylation, thereby modulating the susceptibility to several diseases , especially autoimmune diseases .
The cytokine genes are polymorphic, which accounts for the different levels of cytokine production, and are related to the regulation of the immune-mediated pathogenetic process. Gene polymorphisms have recently attracted considerable interest because distinct alleles of cytokine genes have been discovered to be associated with different immunoinflammatory diseases .
The IL-1Ra polymorphism has been found to be associated with a variety of inflammatory diseases, including alopecia areata, lichen sclerosis, Systemic Lupus Erythematosus (SLE), ankylosing spondylitis, rheumatoid arthritis, and late-cytokine onset psoriasis .
Different studies had investigated the association between cytokine gene polymorphisms and different immune-mediated inflammatory disease. A major member of receptor antagonists (Ra) IL-1 family (consisting of 11 members in total) is the IL-1, a natural anti-inflammatory molecule that neutralizes the effects of IL-1Ra.
The balance between IL-1 and IL-1Ra polymorphisms may lead to changes in this IL-1 and IL-1Ra balance and may be associated with susceptibility to a variety of autoimmune diseases, such as rheumatoid arthritis, SLE, ankylosing spondylitis, and ITP .
| Patients and methods|| |
The current study was conducted on 40 Egyptian children suffering from ITP.
Patients were classified on the basis of duration into two groups: the first group (22 patients) (55%) was termed ‘acute (newly diagnosed) ITP’ (within 3 months from diagnosis) and the second group (18 patients) (45%) was termed ‘persistent ITP’ (between 3 and 12 months from diagnosis, including patients not achieving spontaneous remission or not maintaining complete response after stopping treatment during this period). The different phases and the severity of ITP were defined by the Vicenza Consensus Conference ,.
These patients were diagnosed with primary ITP based on proper history taking, physical examination, and laboratory assessment. Laboratory assessment included the following: complete blood count, revealing platelet count below 100 000/μl, normal Hb concentration, and normal white blood cells; bone marrow aspiration revealed increased number of megakaryocytes with defective nuclear lobulation, cytoplasmic granulation, and platelet budding and antiplatelet antibodies. Exclusion criteria included infants below 6 months of age, recent manifestation of active infection, splenomegaly, and secondary causes of ITP. As regards the 22 acute ITP patients, there were 10 male (45.5%) and 12 female patients (54.5%). All acute cases were newly diagnosed and did not receive any treatment. As regards the 18 persistent ITP patients, there were eight male (44.4%) and 10 female patients (55.6%). The patients were selected from the pediatric hematology clinic (Beni-Suef University Hospital) between February 2013 and June 2013.
The control population consisted of 20 age-matched and sex-matched healthy volunteers. There were 12 male and eight female volunteers. All participants were subjected to complete blood picture and showed normal platelet count. They were selected from the children who underwent a health examination in our outpatient department and from the hospitalized patients who had been admitted for elective surgery with no history of recent viral infection, atopy, or medication at the time of taking blood samples.
Both patients and controls were enrolled into the study after obtaining formal written consent from their parents/guardians, in accordance with Helsinki Declaration of Bioethics .
Polymorphism of DNA methyltransferase 3B gene promoter and interleukin-1 receptor antagonist was detected using the PCR-RFLP assay.
Peripheral blood samples were drawn from every patient and healthy volunteers into sterile EDTA vacutainers and then genomic DNA was extracted using Gene Jet Genomic DNA purification kit (cat#K0721, #K0722; Fermentas Life Sciences, Germany) according to the manufacturer's instructions.
A volume of 5 μl genomic DNA was added to a final PCR reaction mixture of 25 μl polymerase in reaction buffer (MgCl2 and dNTPs), and 1 μl of 25 pmol of each of forward and reverse specific primers of DNMT3B and IL-1Ra gene.
The primers used were as follows:
The following PCR cycle was performed for primer amplification of DNMT3B:
- PCR was run for 35 cycles and each cycle was performed at 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s.
- The following PCR cycle was performed for primer amplification of IL-1Ra:
- PCR was run for 35 cycles and each cycle was performed at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s.
- Subsequently, one cycle of final extension step was carried out at 72°C for 5 min.
The amplified products of DNMT3B were then digested with 5 μl of AvrII restriction enzyme (Fermentas, Germany).
Twenty microliter aliquots of the product were fractionated on a 2% agarose gel and visualized using ethidium bromide staining and then photographed. The size of the PCR-fragments was estimated using a 100-bp ladder (Fermentas).
DNMT3B gene polymorphism was interpreted as follows:
- A clear sharp band at 380 bp indicated DNMT3B wild type CC gene.
- Two clear sharp bands at 207 and 173 bp indicated homozygous mutant TT DNMT3B gene.
- Three clear sharp bands at 207 173 and 380 bp indicated heterozygous mutant CT DNMT3B gene.
IL-1Ra gene polymorphism was interpreted as follows:
- Type I allele: at 410 bp.
- Type II allele: at 240 bp.
- Type III allele: at 325 bp.
- Type IV allele: at 500 bp.
Treatment of ITP patients
Prednisone at a dose of 2 mg/kg/day for 3–7 days, maximum dose 60 mg/day was the initial therapeutic option.
Response was defined as platelet count between 30 000 and 100 000/μl or doubling of the baseline count. Any platelet count lower than 30 000/μl or less than doubling of the baseline count was described as ‘no response’ .
Data were analyzed using IBM SPSS advanced statistics version 20 (SPSS Inc., Chicago, Illinois, USA). Numerical data of scores were expressed as mean and SD or median and range, as appropriate. Qualitative data were expressed as frequency and percentage. The χ2-test (Fisher's exact test) was used to examine the relation between qualitative variables. For non-normally distributed quantitative data, comparison between two groups was made using the Mann–Whitney test (nonparametric t-test). Comparison between three groups was made using the Kruskal–Wallis test (nonparametric analysis of variance), and then post-hoc ‘Scheffe test’ on rank of variables was used for pair-wise comparison. The Spearman-ρ method was used to test correlation between numerical variables. Survival analysis was carried out using the Kaplan–Meier method and comparison between two survival curves was made using the log-rank test. Odds ratio (OR) with 95% confidence interval (CI) was used for risk estimation. The receiver operating characteristic curve was used for the prediction of cutoff values. The κ-test was used to evaluate agreement between two diagnostic methods. A P-value less than 0.05 was considered significant.
| Results|| |
Our ITP patients included 22 acute cases and 18 chronic ones. Among the acute cases, there were 10 male (45.5%) and 12 female patients (54.5%) with ages ranging from 0.9 to 13 years. Their mean age was 6.6 ± 3.4 years. The mean duration of illness was 4.7 ± 7 months.
Among the chronic cases, there were eight male (44.4%) and 10 female patients (55.6%) with ages ranging from 0.8 to 11 years. Their mean age was 5.7 ± 2.9 years. The mean duration of illness was 16.7 ± 7 months.
The control group included eight male (40%) and 12 female participants (60%), with ages ranging from 0.4 to 11 years. Their mean age was 4.8 ± 3.5 years.
During history taking we excluded the following patients: those with a history of recent vaccination or drug intake, to exclude drug-induced thrombocytopenia; those with a history suggesting collagen vascular disorder such as arthritis and rash to exclude secondary causes of thrombocytopenia; those with a history of atypical cases such as recurrent fever, bone pain, or lymphadenopathy; those with a history suggesting viral hepatitis such as jaundice, to exclude hepatitis C virus and hepatitis B virus; and those with a history of splenectomy for chronic ITP patients.
Eleven patients (27.5%) had positive history of consanguinity. Purpuric eruption and ecchymotic patches were the main presenting symptoms as they were reported in 36 (90%) and 24 (60%) patients, respectively. Eleven patients (27.5%) presented with a history of bleeding gums, nine (22.5%) patients had epistaxis, seven (17.5%) patients presented with hematemesis, four (10%) patients presented with hematochezia, and six (15%) patients presented with a history of hematuria.
As regards treatment of ITP patients, acute patients with direct platelet count above 30 000/μl were kept under observation for spontaneous recovery. Acute patients with direct platelet count below 30 000/μl were started on corticosteroid therapy with 2 mg/kg/day oral dose prednisone for 7–10 days, and then gradual tapering was carried out by 5 mg/week. Severe cases that had serious bleeding started treatment with pulse steroid as initial approach in the form of intravenous methylprednisolone 30 mg/kg/day for 3 days.
In our study, four cases with acute ITP received treatment with intravenous methylprednisolone for 3 days and then continued on oral steroids. None of the included patients required splenectomy for treatment. Two patients received only Intravenous immunoglobulin G (IVIG) for treatment.
As regards chronic patients, all received oral steroids with a mean duration of 7.9 ± 3.5 months. Four of them reached complete remission and complete withdrawal of steroids was carried out, whereas nine patients were still on oral steroids (five cases were on gradual withdrawal and four cases were on full dose). The five patients who did not respond properly to steroids and/or developed steroid toxicity had received azathioprine as immunosuppressive drug.
The mean duration of steroid therapy was 2.8 months in acute patients and 7.9 months in chronic patients.
Statistical comparison between acute and chronic cases as regards hematological laboratory data revealed that direct platelet count in ITP children was significantly lower among acute ITP children in comparison with chronic ITP children (P < 0.001). However, there was no statistically significant difference as regards other parameters (P > 0.05).
Case–control association study of DNMT3B polymorphism in the studied groups is shown in [Table 1].
|Table 1 Comparison of genotypes and alleles of DNMT3B gene between acute ITP cases, chronic ITP cases, and controls|
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There was no statistically significant difference (P > 0.05) between different studied groups as regards different genotypes and between ITP cases and controls as regards allelic frequencies ([Table 1]).
There was no statistically significant difference (P > 0.05) between different genotypes of DNMT3B gene as regards clinical signs and symptoms (including ecchymosis, purpura, epistaxis, etc.) and laboratory findings (including Hb, Total Leucocytic Count (TLC), and platelet count).
There was no statistically significant difference (P > 0.05) between different genotypes as regards steroid use, steroid response, or azathioprine treatment. However, there was a statistically significant difference (P = 0.04) between different genotypes as regards steroid duration, with a high mean in homozygous mutant genotype (TT). In contrast, there was no statistical significance as regards disease duration and number of recurrences (P > 0.05) ([Table 2]).
|Table 2 Comparison of disease character and treatment between different DNMT3B gene genotypes|
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As regards the other genetic study in our work, the IL1-Ra, there was no statistically significant difference (P > 0.05) between acute and chronic ITP cases and controls as regards IL1-Ra genotypes or allelic frequencies ([Table 3] and [Table 4]).
|Table 3 Comparison of IL-1Ra alleles between acute ITP cases, chronic ITP cases, and controls|
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There was no statistically significant difference (P > 0.05) between different genotypes of IL1-Ra gene as regards clinical signs and symptoms (including ecchymosis, purpura, epistaxis, etc.) and laboratory findings (including Hb, TLC, and platelet count).
There was no statistically significant difference (P > 0.05) between different genotypes as regards steroid use, steroid response, azathioprine treatment, or steroid duration.
Correlation of different genotypes of both DNMT3B and IL1-Ra percentage among ITP cases did not show significance (P > 0.05). ([Table 5]).
| Discussion|| |
Most researchers believe that platelet destruction is immune mediated and may also involve the inhibition of platelet release by megakaryocytes .
DNA methylation is a key epigenetic modification of the genome. The term methylation is the DNA modification after its synthesis, which is responsible for modulation of gene expression . It is mediated by a family of DNA methyltransferases (DNMTs), which includes at least four independent methyltransferases: DNMT1, DNMT3A, DNMT3, and DNMT3L.
IL-1Ra, a major member of the IL-1 family (consisting of 11 members in total), is a natural anti-inflammatory molecule that neutralizes the effect of IL-1. The balance between IL-1 and IL-1Ra is important in maintaining the homeostasis of the immune system. As a result, IL-1Ra polymorphisms may lead to changes in this IL-1 and IL-1Ra balance and be associated with susceptibility of a variety of autoimmune diseases such as rheumatoid arthritis, SLE, and ankylosing spondylitis .
In the current study, we investigated the association of DNMT3B-46359 C/T polymorphism and IL-1Ra gene polymorphism and the susceptibility to childhood ITP in Egyptian population.
DNMT3B-46359 C/T genotyping in ITP patients revealed that 62.5% had CC wild type, 35% of patients had the CT heteromutant type, and 2.5% had the TT homomutant type. Statistical analysis revealed that the incidence of heteromutant genotype (CT) was higher in ITP patients compared with controls, although not reaching the significant value, and conferred 2.2-fold increased risk for ITP among our patients (OR = 2.2, CI = 1.4–2.6).
As regards the frequency of alleles, C allele was found in 80% of ITP cases and in 90% of controls (OR = 0.9, CI = 0.9–3.1). However, no statistically significant difference (P > 0.05) between different genotypes in ITP patients and controls was reported.
In contrast, the study by Pesmatzoglou et al. (2012)  in Crete children reported that there was a significant difference in the frequency of alleles between ITP patients and controls, with a high frequency of T allele in ITP patients, which seems to increase the relative risk for ITP; the frequency of T/T genotype was very low in Crete population. However, they reported that, as we did, there was no significant difference in the genotypes between ITP patients and controls.
However, in the study by Chen et al. (2008)  in Chinese population, there was no association between the DNMT3B promoter polymorphism and the susceptibility to ITP. They reported distinct prevalence of the T/T genotype and absence of C/C genotype; the frequency of the C/T genotype was very low among ITP patients. This finding highlights the importance of the racial origin in this type of studies, thus implying probably the different methylation status in different races.
The study by Pesmatzoglou et al. (2012) , which was conducted on DNMT3B-579 G/T (another part of our studied gene) promoter polymorphism, reported that the C/C wild genotype was not detected in ITP patients or in controls. In healthy controls, the frequencies of the T/T and C/T genotypes and the T and C alleles were 93.9, 6.1, 97, and 3%, respectively, whereas their frequencies in patients were 91.5, 8.5, 95.8, and 4.2%, respectively. Therefore, they concluded that there was no significant difference in either genotypes or allelic distribution between ITP patients and controls, and no statistically significant difference could be detected between acute and chronic ITP patients.
Chen et al. (2008)  also could not detect such statistically significant difference between the studied acute and chronic ITP patients.
However, our findings suggest that the single-nucleotide polymorphism in the promoter of DNMT3B gene may not contribute to the pathogenesis of ITP and may not be considered as a stratification marker to predict the susceptibility to childhood ITP in Egypt.
Statistical comparison between ITP patients harboring the wild genotype or the mutant genotypes revealed that there was no statistically significant difference between the two groups as regards their age, sex, clinical data, or laboratory data. This is in accordance with the study by Pesmatzoglou et al. (2012) .
On the basis of the disease course and the duration of illness, patients were further subdivided into two groups: acute and chronic ITP patients. However, there was no statistically significant difference in the distribution of the DNMT3B-64359 C/T genotypes between the two patient groups. Similar results were reported by Pesmatzoglou et al. (2012) .
As regards IL-1Ra polymorphism in our study, we found that the frequency of I/I, I/III, and II/I genotype was 68.2, 0, and 13.6% in acute patients, 55.5, 11.1, and 22.2% in chronic patients, and 80, 10, and 10% in controls, respectively.
As regards the frequency of alleles: allele III and IV were not present in acute cases and were of low levels (5.6%) in chronic cases, while allele I was 75% in acute cases, 77.7% in chronic cases and 90% in controls, while allele II level was 25% in acute cases, 11.1% in chronic cases and 5% in controls. However, there was no statistically significant difference (P > 0.05) between ITP children and controls as regards genotype and frequency of alleles of IL-1Ra.
In contrast the study by Wu et al. (2005)  found that there were significant differences in genotype distribution (P = 0.02) and the allelic frequencies (P = 0.007) found among children with acute ITP, chronic ITP, and controls for IL-1Ra. Their study found that the IL-1Ra but not IL-1β gene polymorphism was associated with childhood ITP. They concluded that the IL-1Ra gene polymorphism is implicated in the pathophysiology of childhood ITP.
Pesmatzoglou et al. (2012)  in their study on Crete children found that IL-1Ra polymorphism is associated with childhood ITP. They detected genotype I/II more frequently in children with ITP than in controls. More specifically, they found that the presence of allele II seems to increase the risk for the development of ITP 2.12 times.
From our point of view, searching for genes and pathways that contribute to ITP will allow us to identify at-risk population and will be of value in choosing the therapeutic regimen that track or block these pathways so as to improve the future clinical practice.
| Conclusion|| |
Our results provide that the single nucleotide polymorphism in the promoter DNMT3B-46359 C/T gene and IL-1Ra polymorphism may not contribute to the pathogenesis of ITP and may not be considered as a stratification marker to predict the susceptibility to childhood ITP in Egypt.
However, further large-scale studies are needed to prove this conclusion.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]