Gene 487 (2011) 8083

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Short paper

Relation between 3435CNT multidrug resistance 1 gene polymorphism with high dose methylprednisolone treatment of childhood acute idiopathic thrombocytopenic purpura
Mehmet Akin a,, Sebahat Turgut b, 1, Ceylan Ayada b, 1, Yusuf Polat c, 2, Yasemin Isik Balci d, 3, Firat Erdoan e, 4
a

Denizli State Hospital, Department of Pediatric Hematology, Denizli, Turkey Pamukkale University, Faculty of Medicine, Department of Physiology, Turkey c Denizli State Hospital, Department of Microbiology, Denizli, Turkey d Pamukkale University, Faculty of Medicine, Department of Pediatric Hematology, Turkey e Medipol University, Department of Pediatric, Istanbul, Turkey
b

a r t i c l e

i n f o

a b s t r a c t
The current study was conducted to assess 3435CNT multidrug resistance 1 gene polymorphism and the efcacy of high dose methylprednisolone (HDMP) in childhood acute idiopathic thrombocytopenic purpura patients. Methods: A total of 31 childhood acute Idiopathic thrombocytopenic purpura patients (17 females, 14 males) between the ages of 2 and 16 years of age were included in the study. High-dose methylprednisolone was given at a dose of 30 mg/kg/day for 3 days and 20 mg/kg/day for 4 days, consecutively and intravenously. Polymerase chain reactionrestriction fragment length polymorphism was used for the detection of C3435T single nucleotide polymorphism. Fragments obtained were 238 bp to T/T genotype, 172 bp and 60 bp fragments to the C/C genotype, and 238 bp, 172 bp and 60 bp to the C/T genotype. Results: The distribution of CC, CT, and TT genotypes were 19.0%, 61.3%, and 19.4%, respectively. Both allele frequencies of C and T were the same 50%. There was no signicant difference in genotype and allele distribution between the patients with ITP and the control group ( 2 = 0.84 p = 0.65, 2 = 0.2 p = 0.63, respectively). There were no signicant differences in age, gender, and pre- and post-treatment platelet counts between CC, CT, and TT genotypes of the MDR gene. Response to treatment shows no signicant difference between genotype and allele groups. Conclusion: In our study, there was no difference in the HDMP treatment response between MDR1 gene genotypes. However, it should be noted that this study includes a small group of patients. Our data should therefore be considered preliminary, awaiting further conrmatory studies on an expanded patient base. 2011 Elsevier B.V. All rights reserved.

Article history: Accepted 11 June 2011 Available online 21 June 2011 Received by A.J. van Wijnen Keywords: MDR1 Idiopathic thrombocytopenic purpura High dose methylprednisolone

1. Introduction Childhood acute Idiopathic thrombocytopenic purpura (ITP) is a common pediatric hematologic disorder characterized by increased

Abbreviations: HDMP, High dose methylprednisolone; ITP, Idiopathic thrombocytopenic purpura; MDR-1, Multidrug resistance-1; P-gp, P-glycoprotein; SNPs, Single nucleotide polymorphisms; IVIG, Intravenous immune globulin; R, response; NR, No response. Corresponding author. Tel.: + 90 505 3945276; fax: + 90 258 2619206. E-mail addresses: drmehmetakin@yahoo.com.tr (M. Akin), sturgut@pau.edu.tr (S. Turgut), drypolat@gmail.com (Y. Polat), raterdogan@yahoo.com (F. Erdoan). 1 Tel.: + 90 258 2962492; fax: + 90 258 2962433. 2 Tel.: + 90 505 3937160; fax: + 90 258 2619206. 3 Tel.: + 90 505 3136592; fax: + 90 258 2962433. 4 Tel.: + 90 505 6763484. 0378-1119/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.06.019

destruction of antibody-sensitized platelets with normal to increased megakaryocytes in the bone marrow, as well as the presence of thrombocytopenia with otherwise normal red cells and leukocytes, absence of splenomegaly and the absence of other causes of thrombocytopenia (Cines and Blanchette, 2002; Khne et al., 2003). Multidrug resistance-1 (MDR-1) is characterized by the overfunction of P-glycoprotein (P-gp), a pump molecule that decreases intracellular drug concentration by efuxing them from the intracellular space. P-gp is expressed in the apical membrane of cells with excretory functions, such as those in the liver, kidney, small intestine, stomach, and the bloodbrain barrier (Lum and Gosland, 1995). Functional P-gp is found in several types of human leukocytes and stem cells. Among hematological cells, P-gp expression is highest in natural killer cells, CD4+ and CD8+ lymphocytes, and bone marrow progenitor cells (Klimecki et al., 1994). Single nucleotide polymorphisms (SNPs) of the MDR1 gene have been identied and correlate with P-gp expression (Hoffmeyer et al.,

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2000). Exon 26 3435CNT polymorphism was found to correlate signicantly with intestinal P-gp expression levels as well as the bioavailability of some drugs. Individuals who are homozygous for 3435T have signicantly decreased intestinal P-gp expression and increased digoxin serum levels after oral administration (Wang et al., 2005). Previous studies suggest that the MDR-1 gene may play a role in the development of drug resistance in humans with cancer (Zhenfeng et al., 2004). There is, however, little information regarding the MDR-1 phenotype in autoimmune disorders (Llorente et al., 2000; Levy et al., 2002). The aim of ITP therapy is a rapid reversal of thrombocytopenia that minimizes the risk of intracranial hemorrhage (Watts, 2004). The most appropriate initial therapy for children with apparent ITP remains controversial. In our center, we prefer to use high dose methylprednisolone (HDMP) treatment because it is as efcient as intravenous immune globulin (IVIG) and is inexpensive. The current study was conducted to assess the correlation, if any, between the 3435CNT multidrug resistance 1 gene polymorphism and the efcacy of high dose methylprednisolone (HDMP) in childhood acute idiopathic thrombocytopenic purpura. 2. Patients and methods This study was performed on patients diagnosed with acute ITP. Data regarding 31 children with acute ITP were evaluated. An ethical committee approved this study. Patients less than 2 years old and over 16 years old at initial diagnosis were excluded. Patients with proven secondary ITP were excluded. A total of 31 patients (17 females, 14 males) between the ages of 2 and 16 years of age were included in the study. The ITP diagnosis was made after a detailed physical examination and history, testing for the presence of thrombocytopenia (b150 10 9/L) with otherwise normal red cells and leukocytes, an evaluation of a Giemsa-stained peripheral blood smear and bone marrow aspiration, and serological tests for infectious causes and autoimmune diseases. Denition of newly diagnosed ITP: within 3 months of diagnosis Persistent ITP: between 3 and 12 months from diagnosis. Includes patients not reaching spontaneous remission or not maintaining a complete response from therapy Chronic ITP: persistent thrombocytopenia with or without continued therapy for 12 months following initial diagnosis (Rodeghiero et al., 2009). Numbers of episodes: subjects with b20 10 9/L for one month in the last 12 months and treated. 3. Genotyping Anticoagulated blood samples were obtained from the antecubital vein of the subjects. DNA was extracted from peripheral blood (2 mL) by standard phenol/chloroform extraction method. Polymerase chain reaction (PCR)restriction fragment length polymorphism was used to detect C3435T SNP. The primer design was based on published sequences for genotyping MDR1 polymorphism using genomic DNA. A PCR assay using the forward primer MDR1F 5-TGC TGG TCC TGA AGT TGA TCT GTG AAC-3 and the reverse primer MDR1R 5-ACA TTA GGC AGT GAC TCG ATG AAG GCA-3 was performed with 10 buffer, 1.5 mM MgCl2 and 0.2 mM each dNTP and 1 U Taq DNA polymerase (Turgut et al., 2006). PCR-grade water was added to a nal volume of 50 l. PCR amplication consisted of an initial denaturation for 2 min at 94 C followed by 35 cycles of denaturation at 94 C for 30 s, annealing temperature 60 C for 30 s, and extension at 72 C for 30 s. Terminal elongation was performed at 72 C for 4 min. This was followed by the digestion of a 248-bp PCR product with restriction enzyme MboI for 2 h at 37 C. Digested products were separated on a

70 60 50 40

%
30 20 10 0 CC CT ITP TT C Control T
Fig. 1. Genotype and allele frequencies of the MDR1 gene exon 26 C3435T polymorphism in this study.

3% agarose gel with ethidium bromide. Afterwards, restriction fragments were identied using the UVI Gel Documentation system. Fragments obtained were 238 bp to T/T genotype, 172 bp and 60 bp fragments to the C/C genotype, and 238 bp, 172 bp and 60 bp to the C/ T genotype. 4. Treatment Patients with PC b20 10 9/L and/or bleeding symptoms at the initial diagnosis were treated. High-dose methyl prednisolone (HDMP) was the rst choice in patients under 2 years old. HDMP was given at a dose of 30 mg/kg/day for 3 days and 20 mg/kg/day for 4 days, consecutively and intravenously. For children under 6 years old, HDMP was given either orally or intravenously at the same dosage (Ozer et al., 2000). The maximum dose given was 1 g. A complete response to therapy is dened as a return to normal platelet count (greater than 100 109/L) during or after therapy. A response (R) is dened as any platelet count between 30 and 100 109/L. No response (NR) is dened as any platelet count lower than 30 109/L or less than double the baseline count (Rodeghiero et al., 2009). 5. Statistical analysis Quantitative variables were reported in the text as mean standard deviation (SD). Independent sample t-tests were used to analyze the differences in continuous variables between two alleles groups. MannWhitney U-tests were used to analyze differences in continuous variables between two genotypes. Kruskal Wallis tests were used to analyze the differences in continues variables among three genotypes. A chi-square test was used to compare nominal variables between groups. P values b0.05 were accepted as statistically signicant. Statistical signicance of the observed genotype frequencies was evaluated according to the HardyWeinberg rule compared to the expected genotype frequencies. HardyWeinberg equilibrium was evaluated by the chi-square test. All analyses were carried out using SPSS 10.0 software (Statistical Package for Social Sciences, SPSS Inc., IL). 6. Results The frequency of the genotype MDR1 C3435T gene in patients did not show a signicant deviation from the HardyWeinberg equilibrium. Observed and expected frequencies for p-glycoprotein were in HardyWeinberg equilibrium in both the control group and the patients, respectively, ( 2 = 1.39 P N 0.05). The distribution of

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Table 1 Association between clinicopathological parameters and MDR1 genotypes in patients with ITP. CC genotypes All patients (n: 31) Age (year) Gender Male (n = 14) Female (n = 17) Platelet count (initial diagnosis) Pre-treatment (109/L) Post-treatment (109/L) N 12 months Chronic Non-chronic Number of episode No episode One episode Two episode HDMP 1CR 2CR 3R 1CR, 1R 1CR, 2R 6 6.6 5.1 2 4 6.3 1.2 260.6 34.3 2 4 3 2 1 3 1 1 1 CT genotypes 19 7.6 4.4 8 11 5.5 2.1 289.3 137.5 9 10 10 5 4 10 3 1 4 1 TT genotypes 6 8.0 4.4 4 2 8.1 6.5 346.2 149.7 3 3 3 2 1 2 1 1 1 p value p = 0.826 2 = 1.5 p = 0.46

p = 0.674 p = 0.675 2 = 0.43 p = 0.805

2 = 0.21 p = 0.995

2 = 1.98 p = 0.98

CC, CT, and TT genotypes were 19.0%, 61.3%, 19.4%, respectively. Both allele frequencies of C and T were the same, 50% (Fig. 1). Studying genotype and allele distribution in patients with ITP, which we have done previously in a healthy population (Turgut et al., 2006), we compared the results of the studiesand there was not a signicant difference between them ( 2 = 0.84 p = 0.65, 2 = 0.2 p = 0.63, respectively). Clinical parameters of the patients were shown according to genotype and allele distribution in Tables 1 and 2. There were no signicant differences in age, gender, and pre- and post-treatment platelet counts between CC, CT, and TT genotypes of the MDR gene. After a 1-year follow-up, chronic course rates in CC, CT and TT genotypes were 33.3%, 47.3%, and 50.0%, respectively. The differences between these groups were not statistically signicant ( 2 = 0.20 df = 4 p = 0.99).The number of C and T alleles was equal in ITP patients. There was no signicant difference in age, gender, and platelet count between these two alleles (Table 1). In C and T allele carriers, those developing the chronic disease after 12 months were 41.9% and 51.6%, respectively. The distribution of genotype,

age and gender were not signicant risk factors in logistic regression analysis of chronicity. The odds ratio in individuals with the CC/CT genotype was 1.25 (p N 0.5). Response to treatment shows no signicant difference between genotype (Table 1) and allele groups (Table 2); 33.3% CC, 31.1% CT, and 40% TT genotype of patients have shown R. Treatment R to C or T alleles were 32.2% and 34.5%, respectively. A patient with the TT genotype was not treated due to having a platelet count below 21 10 9/L. Between those three genotypes (Table 1) and two allele groups (Table 2), rst episode rates did not show any signicant differences. 7. Discussion In our study, genotype and allele distribution of childhood acute ITP patients was no different from the healthy population. CC, CT, TT genotypes and C and T alleles of the MDR1 gene did not show a signicant difference in chronicity and relapse rate. Also, in three genotype patient groups, there was no signicantly different HDMP response. We

Table 2 Association between clinicopathological parameters and MDR1 alleles in patients with ITP. C Allele All patients (n: 31) Age (year) Gender Male (n = 14) Female (n = 17) Platelet count (initial diagnosis) Pre-treatment (109/L) Post-treatment (109/L) N 12 months Chronic Non-chronic Number of episode No episode One episode Two episode HDMP 1CR 2CR 3R 1CR, 1R 1CR, 2R 31 7.2 4.5 12 19 5.8 1.8 278.2 109.3 13 18 16 9 6 16 5 1 6 3 T Allele 31 7.8 4.2 16 15 6.5 4.3 308.9 139.0 16 15 16 9 6 14 5 1 6 3 p value p = 0.587 2 = 1.07 p = 0.307 p = 0.085 p = 0.076 2 = 0.55 p = 0.459

p=1

2 = 0.067 p = 0.996

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could not compare our results with other studies since this study is the rst investigation of p-glycoprotein gene polymorphism. However, Drozdzik et al. (2006) reported that in rheumatoid arthritis, patients with CC genotypes had a weaker methylprednisolone response than did patients with the TT genotype. The explanation of different results could be that in our study, high dose methylprednisolone IV is used, whereas in previous studies, low dosages are used and the application route was not stated. Previous studies showed high intestinal expression of glycoprotein in subjects with the CC genotype and decreased oral drug use effectiveness (Wang et al., 2005). In our center, like most of the other centers in Turkey, we prefer to use HDMP; in our study, the HDMP treatment response was 100%. Of our cases, 29 were CR and 1 case was R. In some studies, after the initial diagnosis, regardless of CC, CT, TT genotype, the HDMP treatment response was 75.4%, 83.1%, and 87.4%, respectively. In same studies, the frequency of chronic ITP in children is between 25% and 31% in literature (Akbayram et al., 2010; Demirciolu et al., 2009; Yaprak et al., 2010). Although children older than 10 years old, female adolescents, and an initial PC N20 109/L have been associated with a chronic course, the initial treatment strategy, HDMP, did not show any signicant difference in progression to chronic ITP (Sutor et al., 2001; Kalyoncu et al., 2009). In our study, the chronic ITP rate was 45%; the TT genotype of the MDR1 gene had the highest chronicity and R response of treatment. In previous studies, no signicant differences in MDR1 values before and after the prednisone treatment correlate with the notion that glucocorticoids do not signicantly modify the function and expression of P-gp in adults with ITP. (Lpez-Karpovitch et al., 2008). In our study, there was no difference in the HDMP treatment response between MDR1 gene genotypes. However, it should be noted that this study includes a small group of patients. Our data should therefore be considered preliminary, awaiting further conrmatory studies on an expanded patient base. References
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