|Year : 2018 | Volume
| Issue : 1 | Page : 32-37
DNA methyltransferases 3A −448 G/A and 3B −149C/T single-nucleotide polymorphisms in primary immune thrombocytopenia
Alaa S Abd-Elkader, Tarek T.H ElMelegy, Eman NasrEldin, Zeinab A Abd-Elhafez
Department of Clinical Pathology, Faculty of Medicine, Assiut University Hospital, Assiut University, Assiut, Egypt
|Date of Submission||20-Jan-2018|
|Date of Acceptance||26-Mar-2018|
|Date of Web Publication||3-Aug-2018|
Tarek T.H ElMelegy
Department of Clinical Pathology, Faculty of Medicine, Assiut University Hospital, Assiut University, Assiut, 71515
Source of Support: None, Conflict of Interest: None
Background Primary immune thrombocytopenia (ITP) is a common hematological disorder of unknown etiology. DNA methylation is a major epigenetic modification of the DNA. It has a golden role in gene expression. It is mediated by DNA methyltransferases (DNMTs). The promoter of DNMT3B gene contains some single-nucleotide polymorphisms (SNPs) including that at position −149 (C/T), which was suggested to be implicated in the genetic susceptibility to ITP. The DNMT3A −448 G/A SNP in the gene promoter was found to have a protective effect against systemic lupus erythematosus.
Aim The aim of the study was to investigate the association between DNMT3A −448 G/A SNP (rs1550117) and DNMT3B −149C/T SNP (rs2424913), and the risk for primary ITP and to evaluate the association between these SNPs and patients’ response to therapy.
Participants and methods This prospective case–control study was conducted on 60 primary ITP patients and 30 healthy age-matched and sex-matched controls. Genotype analysis of DNMT3A −448 G/A and DNMT3B −149C/T was done using PCR-restriction fragment length polymorphism.
Results The frequency of the DNMT3A −448 G/A SNP variant A-allele was significantly decreased in primary ITP patients compared with controls (odds ratio=0.829, 95%CI=0.097–0.964). DNMT3B −149C/T SNP variant T-allele was significantly higher in ITP patients with almost double-fold increase in the risk of ITP in comparison to controls (odds ratio=1.731, 95%CI=1.121–2.582).
Conclusion The DNMT3A −448 SNP variant A-allele might has a protective effect against ITP. Also, the DNMT3B −149 SNP variant T-allele could be considered as a molecular risk factor for ITP.
Keywords: DNMT3A, DNMT3B gene, primary immune thrombocytopenia, single-nucleotide polymorphism
|How to cite this article:|
Abd-Elkader AS, ElMelegy TT, NasrEldin E, Abd-Elhafez ZA. DNA methyltransferases 3A −448 G/A and 3B −149C/T single-nucleotide polymorphisms in primary immune thrombocytopenia. Egypt J Haematol 2018;43:32-7
|How to cite this URL:|
Abd-Elkader AS, ElMelegy TT, NasrEldin E, Abd-Elhafez ZA. DNA methyltransferases 3A −448 G/A and 3B −149C/T single-nucleotide polymorphisms in primary immune thrombocytopenia. Egypt J Haematol [serial online] 2018 [cited 2019 Dec 9];43:32-7. Available from: http://www.ehj.eg.net/text.asp?2018/43/1/32/238538
| Introduction|| |
International guidelines define thrombocytopenia as blood platelet count below 100 000 cells/µl ,. Immune thrombocytopenia (ITP) occurs due to immunologic destruction and/or inadequate generation of platelets. Primary ITP is diagnosed after exclusion of the condition known to cause ITP such as systemic lupus erythematosus (SLE) ,.
Both antibody-mediated and T-cell-mediated destruction of platelets are involved in ITP pathogenesis . DNA methylation regulates gene expression and has an important role in maintaining T-cell function and development . Inhibition of DNA methylation in mature T cells can induce autoreactivity .
DNA methylation is mediated by DNA methyltransferases (DNMTs); de-novo methylation is carried out by DNMT3A and DNMT3B .
DNMT3A gene is encoded on chromosome 2 (2p2.3) . There are many single-nucleotide polymorphisms (SNPs) in the DNMT3A gene. A significant protective role of the DNMT3A −448 G/A SNP (rs1550117) against SLE was reported . DNMT3A −448 G/A SNP is not yet studied in ITP.
The DNMT3B gene was mapped to chromosome 20 (20q11.2) ,. A total of 21 SNPs have been identified in it including DNMT3B −149C/T SNP (rs2424913) .
DNMT3B −149C/T SNP was investigated in ITP but with conflicting results ,,.
| Participants and methods|| |
This prospective case–control study was approved by the institutional ethics committee in accordance with the Helsinki Declaration, 2000. All participants or their guardians provided an informed consent before being included in the study.
Immune thrombocytopenia patients
This study was conducted on 60 primary ITP patients, who were recruited from patients referred to the hematology laboratory with provisional diagnosis of ITP in the period from July 2013 till February 2016. Their age ranged from 6 month to 50 years (21 men and 39 women).
Egyptian patients with isolated thrombocytopenia, no organomegaly or lymphoadenopathy, no constitutional symptoms (bone pains, weight loss, and night sweats) and no history of preceding drug intake (quinine, heparin) were included.
Conditions/diseases associated with secondary ITP were as follows: patients with positive antinuclear antibody test results and/or direct Coombs test, adult patients with positive test results for antihepatitis C antibody, HIV Ag/Ab, anticytomegalovirus immunoglobulin M and/or anti-Helicobacter pylori antibody, and pediatric patients with immunoglobulin deficiency.
Follow-up data (platelet count, presence of bleeding, and therapy) of the study patients were collected. The target follow-up period was 1 year after diagnosis.
If platelet count persists below 100 000 cells/µl for more than 12 months, this is defined as chronic ITP. If thrombocytopenia persists for more than 3 months and for less than 12 months, this is defined as persistent ITP which also includes patients not reaching spontaneous remission or not maintaining complete response to therapy .
The patients’ response to therapy may be classified into complete response (platelet count >100 000 cells/µl and absence of bleeding), response (platelet count between >30 000 cells/µl and <90 000 cells/µl with absence of bleeding), no response (platelet count <30 000 cells/µl and presence of bleeding), loss of complete response and loss of response .
The control group consisted of 30 age-matched and sex-matched apparently healthy participants, aged 9 months to 49 years (13 men and 17 women).
Venous blood samples (2 ml) were collected under aseptic precautions into EDTA containing tubes at the time of diagnosis and were stored frozen (−70°C) to be used later for DNA extraction.
Genomic DNA preparation
Gene JET Whole Blood Genomic DNA Purification Mini Kits (catalog no. K0781; Thermo Scientific, Waltham, Massachusetts, USA) were used to extract DNA from the blood samples according to the manufacturer’s instructions.
Gene polymorphism studies
DNMT3A −448 G/A single-nucleotide polymorphism genotyping
This was done by PCR-restriction fragment length polymorphism using the previously described modified method . A total volume of 25 µl was used for PCR amplification of 358 bp products including 2 µl of DNA (50–100 ng), 1 µl of 20 µmol each of forward: 5’-ACACACCGCCCTCACCCCTT-3’ and reverse: 5’-TCC AGCAATCCCTG CCCACA-3’ primers with 12.5 µl of master mix [MyTaqRed Mix (2×); Bioline Reagents Ltd, London, UK]. The following cycling conditions were applied (Veriti 96-Well Thermal Cycler; Applied Biosystems, Waltham, Massachusetts, USA), initial denaturation for 5 min at 95°C followed by 35 cycles (95°C for 20 s, 65°C for 20 s, and 72°C for 30 s), and a final extension step of 5 min at 72°C. The PCR products were checked by 2% agarose gel electrophoresis together with no-template control and DNA markers.
Digestion was performed by incubating (in Veriti 96-Well Thermal Cycler; Applied Biosystems, Waltham, Massachusetts, USA) 25 μl reaction of 5 μl of the PCR product with 1 μl HpyCH4III (5 U/µl), 2.5 μl of 10× NEBuffer 4 (New England Biolabs, Ipswich, Massachusetts, USA), and 16.5 μl sterile distilled water, for 1 h at 37°C followed by 20 min at 65°C to stop the reaction. Then, 10 μl of the digestion mixture was loaded into 5% agarose gel, followed by electrophoresis to separate the restriction fragments and visualized by ultraviolet illumination. Genotyping interpretation was done by three independent laboratory specialists ([Figure 1]).
|Figure 1 DNMT3A −448 A/G genotyping patterns on 5% agarose gel after HpyCH4III restriction enzyme digestion. Nucleotide substitution from G to A at DNMT3A −448 leads to loss of one restriction site. GG genotype produces three bands (153, 94, and 87 bp). AA genotype produces two bands (247 and 87 bp). AG genotype produces four bands (247, 153, 94, and 87 bp).|
Click here to view
DNMT3B −149C/T single-nucleotide polymorphism genotyping
Genotyping was studied by PCR-restriction fragment length polymorphism as previously described . Two primer sequences, forward one: 5′-TGCTGTGACAGGCAGAGCAG-3′ and reverse one: 5′-GGTAGCCGGGAACTCCACGG-3′, were used to generate PCR product of 380 bp of DNMT3B promoter. The total volume used for PCR amplification was 25 µl including 2 µl of DNA (50–100 ng), 1 µl of 20 µmol each of forward and reverse primers with 12.5 µl of master mix [MyTaqRed Mix (2×); Bioline Reagents Ltd, London, UK].
For PCR amplification, Veriti 96-Well Thermal Cycler (Applied Biosystems, Waltham, Massachusetts, USA) was used, with initial denaturation for 5 min at 95°C, followed by 35 cycles (95°C for 30 s, 65°C for 30 s, and 72°C for 30 s) and final extension step at 72°C for 5 min. Prior to enzyme digestion, 2% agarose gel electrophoresis was used to check the PCR products including a no-template control.
The 380 bp PCR products were digested using AvrII enzyme (Catalog no. R0174S; New England Biolab, Ipswich, Massachusetts, USA). Digestion was performed by incubating (Veriti 96-Well Thermal Cycler; Applied Biosystems, Waltham, Massachusetts, USA) 25 μl reaction of 3 μl of the PCR product with 1 μl AvrII (5 Ul/µl), 2.5 μl of 10× NEBuffer 4 (New England Biolabs, Ipswich, Massachusetts, USA) and 18.5 μl sterile distilled water at 37°C for 45 min and then 5 μl of glycerol loading dye was added to stop the reaction. Then, 10 μl of the digestion mixture was loaded into 3% agarose gel followed by electrophoresis to separate the restriction fragments and visualized by ultraviolet illumination. Three independent laboratory specialists interpreted the genotype patterns from the gel ([Figure 2]).
|Figure 2 DNMT3B −149C/T genotyping pattern on 3% agarose gel after AvrII restriction enzyme digestion. Nucleotide substitution from C to T at DNMT3B −149 creates an AvrII restriction site, whereas wild-type C-allele lacks this restriction site. C-allele produces a single band (380 bp). Variant T-allele is cleaved into two bands (207 and 73 bp).|
Click here to view
The data were analyzed using IBM SPSS version 21 (SPSS Inc., Chicago, Illinois, USA). The χ2-test was used to examine the relation between qualitative variables. Odds ratio with 95%CI was used for the relative risk estimation of both genotypic and allelic frequencies. A P value less than or equal to 0.05 was considered statistically significant. Allele frequency was calculated by direct counting and then dividing by the number of chromosomes. Genotype frequency was calculated by direct counting and then dividing by the number of participants. The genotype data were tested for their fit to Hardy–Weinberg equilibrium (HWE) by calculating the expected frequency of each genotype and comparing it to the observed value using χ2-test ; the χ2 value indicates the difference between expected and observed values for genotype counts. The genotype distribution is significantly deviated from HWE at a χ2 value of greater than or equal to 3.84.
| Results|| |
Disease fate and outcome
The 60 primary ITP patients were categorized into 16 patients with chronic ITP, six patients with persistent ITP, and the remaining 38 patients recovered within 3 months from diagnosis (acute ITP).
Six pediatric patients out of the 60 recovered spontaneously without treatment within 15 days of diagnosis. Oral corticosteroid therapy with an average dose of 10 mg/day and/or intravenous immunoglobulin therapy were used by 54 patients. Out of the 54 patients who received therapy, 32 patients showed complete response, seven patients had response, 11 patients showed no response, and four patients had loss of complete response. However, for statistical analysis, the three groups (response, no response, and loss of complete response) were grouped together into one group called ‘no complete response group’.
DNMT3A −448 G/A single-nucleotide polymorphisms and primary immune thrombocytopenia
Genotype distribution of DNMT3A −448 G/A polymorphism was in accordance with HWE (χ2=2.57). Homozygous AA genotypes were not present neither in patients nor controls. The frequency of A-allele was significantly decreased in ITP patients compared with controls (P=0.020). Also, there was statistically significant decrease in heterozygous genotype in ITP patients versus controls [P=0.033, odds ratio (OR)=0.862, 95%CI=0.056–0.982; [Table 1]. The presence of A-allele was significantly associated with decreased risk of ITP (OR=0.829, 95%CI=0.097–0.964).
|Table 1 Genotype and allele frequencies of DNMT3A −448 G/A in primary immune thrombocytopenia patients and controls|
Click here to view
Regarding response to therapy, there was no significant difference in the genotype and allele distribution between ITP patients with complete response and those without complete response ([Table 2]).
|Table 2 Genotype and allele frequencies of DNMT3A −448 G/A in study patients in relation to response to treatment|
Click here to view
DNMT3B −149C/T single-nucleotide polymorphism and primary immune thrombocytopenia
Genotype distribution of DNMT3B −149C/T SNP was in accordance with HWE (χ2=2.97).
The frequency of DNMT3B −149 variant T-allele was significantly higher in ITP patients compared with controls (P=0.041). Its presence was associated with statistically significant nearly double-fold increase in the risk of ITP (OR=1.731, 95%CI=1.121–2.582; [Table 3]). There was no significant association between DNMT3B −149C/T genotype and allele distribution and response to therapy in ITP patients ([Table 4]).
|Table 3 Genotype and allele frequencies of DNMT3B −149C/T in primary immune thrombocytopenia patients and controls|
Click here to view
|Table 4 Genotype and allele frequencies of DNMT3B −149C/T in the study patients in relation to response to treatment|
Click here to view
| Discussion|| |
To date, this is the first report to investigate the association of DNMT3A −448 G/A gene polymorphism with primary ITP. The presence of the variant A-allele was significantly associated with decreased risk of ITP. This finding is in agreement with the study of Piotrowski et al.  on SLE in Polish population who found that the frequency of DNMT3A −448 variant A-allele was significantly decreased in SLE patients compared with controls and reported that its presence was associated with decreased risk of SLE.
The protective role of DNMT3A variant A-allele in autoimmune diseases (ITP and SLE) could be attributed to its association with increased DNMT3A expression in hematopoietic stem cells and/or mature immune cells, and thus adequate DNA methylation pattern of genes of these cells, maintaining their normal gene expression and normal cell functions. It was previously stated that DNA hypomethylation in mature immune cells can induce autoreactivity in vitro and autoimmunity in vivo , and that failure to maintain DNA methylation patterns can modify gene expression and hence immune function, contributing to the development of lupus-like diseases and other forms of autoimmune diseases such as ITP .
This hypothesis is supported by the study of Wang et al. , who found that DNMT3A mRNA expression was highest in −448 SNP AA genotype carriers (−448 AA > −448 GA > −448 GG). Also, it was found that the DNMT3A mRNA expression was significantly decreased in Egyptian ITP patients when compared with that in Egyptian controls . The association of DNMT3A variant A-allele with increased DNMT3A expression could be explained by that specificity protein 1 (SP1) possesses a higher binding affinity to −448 SNP G-allele than to A-allele. SP1 is a recently revealed transcription repressor of the DNMT3A gene. Therefore, compared with the G-allele, the A-allele would decrease the SP1 binding affinity leading to an increased expression of DNMT3A .The presence of DNMT3B −149C/T variant T-allele was associated with a statistically significant nearly double-fold increase in the risk of primary ITP. This was in agreement with two previous studies ,.
However, in another study , no significant difference in the genotype and allele distribution of DNMT3B −149 SNP was found between ITP pediatric patients and controls. This may be due to the patient selection criteria being restricted to pediatric patients only.
The mechanism by which DNMT3B −149 SNP variant T-allele could be involved in the pathogenesis of ITP is that it may result in alteration of methylation status. This defective methylation produces autoimmunity by inducing overexpression of methylation-sensitive genes that are engaged in autoreactive immune response in lymphocytes, including cytokines that induce differentiation of lymphocytes into immunoglobulin-producing cells ,. This is supported by the findings that DNMT3A and DNMT3B mRNA expressions were significantly lower in ITP patients than in healthy controls, suggesting that the aberrant DNA methylation pattern (hypomethylation) is possibly involved in the pathogenesis of ITP .
To conclude, the DNMT3A −448 G/A SNP variant A-allele was significantly associated with decreased risk of primary ITP. On the other hand, the DNMT3B −149C/T SNP variant T-allele was significantly associated with nearly double-fold increase in the risk of primary ITP.
However, the sample size in this study was small, due to financial issues; so, it can be considered as a pilot study. It is recommended to validate these results through their confirmation in another independent study with a larger sample size and perhaps more restricted patient selection criteria such as a certain age group.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Provan D, Stasi R, Newland A, Blanchette V, Maggs P, Bussel JB et al.
International consensus report on the investigation and management of primary immune thrombocytopenia. Blood
Nomura S. Advances in diagnosis and treatments for immune thrombocytopenia. Clin Med Insights Blood Disord
Neunert C, Lim W, Crowther M, Cohen A, Solberg L, Crowther A. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood
Zufferey A, Kapur R, Semple W. Pathogenesis and therapeutic mechanisms in immune thrombocytopenia (ITP). J Clin Med
Sekigawa I, Okada M, Ogasawara H, Kaneko H, Hishikawa T, Hashimoto H. DNA methylation in systemic lupus erythematosus. Lupus
Ballestar E, Esteller M, Richardson BC. The epigenetic face of systemic lupus erythematosus. J Immunol
Wu H, Tao J, Sun Y. Regulation and function of mammalian DNA methylation patterns: a genomic perspective. Brief Funct Genomics
Chen T, Ueda Y, Xie S, Li E. A novel DNMT3A isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J Biol Chem
Piotrowski P, Grobelna M, Wudarski M, Olesińska M, Jagodziński P. Genetic variants of DNMT 3A and systemic lupus erythematosus susceptibility. Mod Rheumatol
Xie S, Wang Z, Okano M, Nogami M, Li Y, He W et al.
Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene
Robertson D, Uzvolgyi E, Liang G, Talmadge C, Sumegi J, Gonzales A et al.
The human DNA methyltransferases (DNMTs) 1, 3A and 3B: coordinate mRNA expression in normal tissues and over expression in tumors. Nucleic Acids Res
Zhang Y, Xu H, Shen Y, Gong Z, Xiao T. Association of DNMT3B −283 T/C and −579 G/T polymorphisms with decreased cancer risk: evidence from a meta-analysis. Int J Clin Exp Med
Pesmatzoglou M, Lourou M, Goulielmos N, Stiakaki E. DNA methyltransferase 3B gene promoter and interleukin-1 receptor antagonist polymorphisms in childhood immune thrombocytopenia. Clin Dev Immunol
Gouda H, Kamel N, Meshaal S. Association of DNA methyltransferase 3B promotor polymorphism with childhood chronic immune thrombocytopenia. Lab Med
Ezzat D, Hammam A, El Malah W, Hussein S. DNA methyltransferase 3B gene promotor and interleukin-1 receptor antagonist polymorphisms in Egyptian children with immune thrombocytopenic purpura. Egypt J Haematol
Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold M et al.
Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. The American Society of Hematology. Blood
Fan H, Liu D, Qiu X, Qiao F, Wu Q, Su X et al.
A functional polymorphism in the DNA methyltransferase-3A promoter modifies the susceptibility in gastric cancer but not in esophageal carcinoma. BMC Med
Wang Y, Wang R, Wen D, Li Y, Guo W, Wang N et al.
Single nucleotide polymorphism in DNA methyltransferase 3B promoter and its association with gastric cardiac adenocarcinoma in North China. World J Gastroenterol
Rodriguez S, Gaunt R, Day M. Hardy-Weinberg equilibrium testing of biological ascertainment for Mendelian randomization studies. Am J Epidemiol
Wilson B, Makar W, Shnyreva M, Fitzpatrick R. DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin Immunol
Wang J, Li C, Wan F, Li Z, Zhang J, Zhang J et al.
The rs1550117 A>G variant in DNMT3A gene promoter significantly increases non-small cell lung cancer susceptibility in a Han Chinese population. Oncotarget
El-Shiekh H, Bessa S, Abdou M, El-Refaey A. Role of DNA methyltransferase 3A mRNA expression in Egyptian patients with idiopathic thrombocytopenic purpura. Int J Lab Hematol
Liu Y, Chen Y, Richardson B. Decreased DNA methyl-transferase levels contribute to abnormal gene expression in ‘Senescent’ CD4+CD28-Tcells. Clin Immunol
Li H, Xuan N, Yang R. DNA methylation and primary immune thrombocytopenia. Semin Hematol
Tao J, Yang M, Chen Z, Huang Y, Zhao Q, Xu J et al.
Decreased DNA methyltransferase3A and 3B mRNA expression in peripheral blood mononuclear cells and increased plasma SAH concentration in adult patients with idiopathic thrombocytopenic purpura. J Clin Immunol
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]