MDM2SNP309and p53 Codon 72 Genetic Polymorphisms

Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014 Available online at www.annclinlabsci.org MDM2SNP309and p53 Codon 72 Genetic Polymorphisms and Risk of AML: an Egyptian Study Nabil Mohsen El-Danasouri 1, Shadia Hassan Ragab 2, Maha Ameen Rasheed 2, Zainab Ali El_Saadany 1, and Safa Nabil Abd El-Fattah 2 1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, and 2 National Research Center, Egypt Abstract. Background: Acute myeloid leukemia (AML) is a heterogeneous disease with numerous genetic abnormalities corresponding to a variety of subtypes. p53 is involved in multiple cellular pathways including apoptosis, transcriptional control, and cell cycle regulation. A single nucleotide polymorphism (SNP) at codon 72 of the p53 gene is associated with the risk for development of various neoplasms. MDM2 SNP309 is a single nucleotide T to G polymorphism located in the MDM2 gene promoter, which enhances the expression of MDM2 protein and thereby leads to attenuation of the p53 stress response. Objective: The current study aimed to define the roles of MDM2 and p53 genetic polymorphisms with the risk of AML. Methodology: Genotyping for MDM2 was done by AS-PCR technique while p53 codon 72 genotyping was done by PCR- RFLP for 50 patients and 50 controls. Results: The study did not detect any significant differences regarding MDM2 or p53 polymorphisms in AML cases, as compared to controls. A borderline significance was found between cases and controls regarding combined MDM2 T/G and p53 genotyping. MDM2 variant genotype was significantly associated with a younger age group and lower Hb level, while the P53 variant was significantly associated with less frequent CD117 expression. Key words: MDM2, p53 codon 72, AML, AS-PCR, RFLP-PCR. 449 Introduction Acute myeloid leukemia (AML) refers to a group of marrow-based neoplasms characterized by the presence of blasts and/or blast equivalents accumulating in the bone marrow and interfering with the production of normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases with age [1]. The majority of AML cases are associated with nonrandom chromosomal translocations that result in gene rearrangements [2]. Recurrent balanced aberrations have been identified in AML, with the frequencies of the four most common translocations, i.e. PML-RARα, AML1-ETO, CBFβ-MYH11 and MLL-fusions, between 3% and 10%. For others, the prevalence is significantly smaller (1-2%) [3]. These genetic abnormalities were incorporated in the WHO classification for diagnosis of AML [4]. Address correspondence to Zainab Ali El_Saadany, MD, Assistant Professor, Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Egypt; phone/fax: +202 23654480; e mail: zinab573@hotmail.com The p53 protein is a principal mediator of growth arrest, apoptosis, and senescence in response to an array of cellular damage [5-7]. Various types of stress can induce high levels of p53 protein, thus preventing inappropriate propagation of stressed cells. Because of this protein s vital role in maintaining normal cellular function, tumor cells have developed numerous methods to disable its function. Indeed, the p53 protein is inactivated by mutations or deletions in approximately 50% of human cancers [8]. A polymorphism at codon 72 with a single-base change in the p53 gene causes an amino acid replacement [Arg (CGC) with Pro (CCC)] in the transaction domain of the protein. Although the functional differences of these two variants of the p53 protein remain unclear, it has been demonstrated that a single nucleotide polymorphism (SNP) at codon 72 of the p53 gene is associated with the risk for development of various neoplasms. However, in the remainder of human tumor types, the p53 gene remains in a wildtype form, with its activity eradicated by its principal cellular inhibitor, murine double minute 2 protein (MDM2) [9]. 0091-7370/14/0400-449. 2014 by the Association of Clinical Scientists, Inc.

450 Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014 AML was based on morphological, immunophenotyping, and cytochemical criteria of the French American British (FAB) system [12,13] and WHO classification [4], and a cutoff of 20% was used to confirm positivity of cytoplasmic MPO done by flow cytometry [14]. Monoclonal Mouse Anti-Human Myeloperoxidase was used after fixation and permeabilization of blast cells (Dako, Denmark). Figure 1. Genotyping of MDM2 SNP309 and p53 codon 72 polymorphism in the studied AML cases. Upper panel: P53 codon 72 genotyping by PCR-RFLP technique:lanes 3, 4, 6 & 8: Homozygous Arg/Arg genotype.lanes 2 & 5: Homozygous Pro/Pro genotype.lanes 1, 7, 9 & 10: Heterozygous Arg/Pro genotype. Lower panel: MDM2 SNP309 genotyping by AS-PCR:Lanes 3a-3b: Wild TT genotype.lanes 1a-1b, 2a-2b, 4a-4b & 5a-5b: Heterozygous TG genotype.m: DNA molecular weight marker ladder. The MDM2 protein provides negative feedback for the tumor suppressor gene, p53. Transcription of MDM2 is activated by p53 and MDM2, which then acts as an E3 ubiquitin ligase that exports p53 out of the nucleus and promotes its degradation. A normal interaction between the two proteins allows p53 to be maintained at low levels in normally dividing cells [10]. A single nucleotide polymorphism (SNP) at position 309 (SNP 309) in the promoter region of MDM2 results in increased affinity for transcription factor Sp1 and subsequent overexpression of MDM2, downregulation of p53 response, and increased tumor genesis [11]. To determine the role of these two genes in acute leukemias, we studied the risk of MDM2 SNP309 and p53 codon 72 polymorphisms in acute myeloid leukemia. Materials and Methods Study population. This study was comprised of 50 adult Egyptian de-novo AML patients and 50 age and gender matched healthy controls. Cases were selected from Kasr El-Aini Teaching Hospital, Cairo University, Egypt. There were 26 males and 24 females, whose ages ranged from 18-60 years with a mean age of 41.58 years. The research protocol was approved by the research Ethics Committee of the Departments of Clinical Pathology and Medical Oncology, Cairo University. Diagnosis of DNA extraction and genotyping. Genomic DNA extraction from peripheral blood leucocytes was done using QIAamp DNA Blood Mini Kit (QIAGEN Germany), following the manufacturer s instructions. DNA samples were routinely stored at -20 C. Genotyping of p53 codon 72 Arg/Pro polymorphism was determined using PCR-based restriction fragment length polymorphism (RFLP) method [15]. The forward primer used was 5 ATCTACAGTCCCCCTTGCCG-3, whereas the reverse primer was 5 GCAACTGACCGTGCAAGTCA-3. PCR conditions were 94 C for 5min, followed by 35 cycles of 94 C for 30 s, 56 C for 30 s and 72 C for 30 s. The final extension was at 72 C for 7 min. The amplified PCR product (296 bp) was digested by restriction enzyme BstUI (New England Biolabs, UK). The DNA fragments were electrophoresed through a 3% agarose gel and stained with ethidium bromide. The Arg allele produced two small fragments of 169 and 127 bp. The Pro allele produced a single 296-bp band. The heterozygous genotype produced three bands of 296, 169 and 127 bp (Figure 1). Genotyping of MDM2 SNP309 was performed using an allele-specific polymerase chain reaction (AS-PCR) [15]. DNA was amplified by PCR reaction using primer pairs specific for the two alleles: primers F1 (5 GGATTTCGGACGGCTCTC-3 ) and R1 (5 TCCGGACCTCCCGCGCCGA- 3 ) were used to amplify the 121-bp wild-type allele (T), and primers F2 (5 GTTTTGTTGGACTGGGGCTA-3 ) and R2 (5 ATCCGGACCTCCCGCGCCGC-3 ) were used to amplify the 168-bp mutant allele (G). Two independent PCR assays were performed for each allele. The reaction conditions were set as follows: for the wild-type T allele: 94 C for 5 min; 5 cycles of 94 C for 30 s, 62 C for 30 s; 35 cycles of 94 C for 30 s, 58 C for 30 s, and 72 C for 30 s; with a final extension at 72 C for 7min in the last cycle. For the mutant G allele: 94 C for 5 min; 5 cycles of 94 C for 30 s, 65 C for 30 s; 35 cycles of 94 C for 30s, 62 C for 30 s, and 72 C for 30 s; with a final extension at 72 C for 7 min in the last cycle. The amplification products were run by electrophoresis on a 3% agarose gel stained with ethidium bromide (Figure 1). Statistical Analysis. The data were analyzed using Statistical Package for Social Science (SPSS) program version 15. Numerical data were summarized as mean and standard deviation or median and range as appropriate. Non-parametric test (Mann Whitney U test) was

Table 1. Clinical and laboratory characteristics of AML patients. Character used for analysis of quantitative data, as data were not symmetrically distributed. Chi Square test and Fisher s exact test were used for analysis of qualitative data (Fisher s exact test was used when > 20% of variables were <5 or when one variable was <1). Logistic regression was used for calculation of odds ratio (OR) with 95% confidence interval (CI) for risk estimation. A P-value < 0.05 was considered significant. Results No. Sex Male 26 Female 24 Age Mean ± SD 41.58±13.96 Laboratory data WBC count (x10x 9 /L) Median (range) 91.7 (1.30-262.20) Hb (g/dl) Median (range) 6.8 (4.50-9.90) Platelet count (x10x 9 /L) Median (range) 55.5 (7.0-136.0) PB blasts (%) Median (range) 40 (3.0-90.0) BM blasts (%) Median (range) 60 (8.0-95.0) FAB subtypes M0 2 M1 14 M2 14 M3 8 M4 8 M5 4 Immunophenotyping (done by flow cytometry) CD34 19 HLA-DR 31 Cyt-MPO 48 CD33 47 CD13 40 CD117 11 CD14 12 PB: Peripheral blood; BM: bone marrow The clinical and laboratory characteristics of AML patients are presented in Table 1. The frequency of the studied genes among AML patients and controls are presented in Table 2. There was no significant difference in the distribution of MDM2 SNP309 variant genotypes between AML cases and controls (p=>0.99). The frequencies of wild and variant genotypes in cases versus controls was 2%/4% and 98%/96% respectively. Regarding the p53 codon 72 polymorphism, there was also no significant difference in the distribution of variant genotype among cases and controls (p= 0.08), as the frequency of wild and variant genotype in cases versus controls was 40%/28% and 60%/72% respectively. Studying the combined genotyping of MDM2 and p53 genes and risk of AML, a borderline significant difference was found between AML patients and controls: the combined hetero MDM2 TG and p53 Pro/Pro variants were higher in AML patients (p=0.049) Table 3. Further analysis of the influence of the studied genetic polymorphisms on the clinical and laboratory characteristics of the patients revealed a significant younger AML age of onset (mean 32 years) and lower Hb level (median 6.2 g/dl) associated with the MDM2 SNP309 GG genotype (p=0.02 and 0.001 respectively), while the p53 variant genotype showed significance for a lower number of AML patients expressing CD117 (p=0.004). They were either M1 or M2 patients. There was no significant difference in regards to other clinical and laboratory data or FAB subtypes concerning the two genes. However, M1 and M2 subtypes showed a higher frequency of MDM2 309 GG genotype than other subtypes. Discussion MDM2 and p53 polymorphism in AML 451 At the National Cancer Institute of Cairo University, Egypt, acute leukemias accounted for 7% of the newly diagnosed malignant cases registered in the time period between January 2002 and December 2003. Acute myeloid leukemia accounted for approximately 41.5% of these newly diagnosed acute leukemias and for 4.6% of all cancer cases [16]. The triennial report of 2000-2002 Gharbiah population-based cancer registries (GPCR) reported similar data [17]. Several genetic polymorphisms have been identified as risk factors for leukemia by meta-analyses

452 Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014 Table 2. The frequency of the studied genes among AML patients and controls. Genotypes Cases Controls OR (95% CI) P value (n=50), no. (%) (n=50) no. (%) MDM2 309 T>G Wild TT 1(2) 2 (4) 1.0 (Ref.) Hetero TG 42 (84) 42 (84) 2 (0.17-22.9) >0.99 Homo GG 7 (14) 6 (12) 2.33 (0.17-32.58) >0.99 TG + GG 49 (98) 48 (96) 2.04 (0.18-23.27) >0.99 P53 codon 72 Arg>Pro Arg/Arg 20 (40) 14 (28) 1.0 (Ref.) Arg/Pro 20 (40) 31 (62) 0.45 (0.19-1.09) 0.12 Pro/Pro 10 (20) 5 (10) 1.4 (0.39-4.99) 0.75 Arg/Pro + Pro/Pro 30 (60) 36 (72) 0.58 (0.25-1.35) 0.34 Fisher s exact test [18-20]. Nevertheless, despite the importance of genetic factors, only a few gene polymorphisms associated with leukemia susceptibility have been identified so far [21]. Loss of p53 plays an important role in the pathogenesis of cancer, and its negative regulator MDM2 has also been suggested to be mutated in a variety of cancers [22]. In the current study, we examined fifty de novo AML patients and fifty age and sex matched control subjects to explore whether the common polymorphisms in MDM2 SNP309 and p53 codon 72 would affect the risk for AML in the Egyptian population. We observed no association between MDM2 SNP309 and risk of AML, which was consistent with another Egyptian study done by Ebid et al. [23]. Our study is somewhat similar to that reported by Ellis et al. [24], who studied associations between t-aml and two common functional P53- pathway variants, the MDM2 SNP309 and the P53 codon 72 polymorphism. They showed that the MDM2 SNP309G allele was associated with a modest increased risk in de novo AML but not in therapy-related AML. Phang et al. [25] claimed that the MDM2 SNP309G allele reduced the risk of the disease in a Singaporean Chinese population. Our results are inconsistent with a case-control study from a northern Chinese population. They observed a nearly 3.52-fold increase in AML risk associated with the MDM2 GG genotype compared with the MDM2 TT genotype (p=0.001) [15]. Many publications also showed a significant association of the MDM2 T309G polymorphism with leukemia risk. The ORs of this meta-analysis ranged from 1.2 to 1.47, thus indicating that individuals carrying a homozygous G allele may have an increased leukemia risk compared with those bearing a wild-type T allele [21]. This difference in results may be due to the larger number of patients in the Chinese study. A larger number of patients should be incorporated in a study to determine whether the difference is actually due to the number of patients or merely due to the ethnic and/or geographic variations of the frequency of these alleles in different healthy populations [26,27]. Also proposed is the possibility that carcinogen exposure in different populations, combined with genetic background, may affect the frequency of aforementioned alleles [15]. In our study, no association between the p53 codon 72 polymorphism and risk of AML was found; there was no significant difference between p53 genotypes (Arg/Arg, Arg/Pro and Pro/Pro) in the AML cases and control groups, with p value of 0.08. This is consistent with a case-control study from a northern Chinese population (p=0.25) [15]. Similarly, several studies (23,24,28,29) reported no association of p53 genotypes with risk of de novo AML. A meta-analysis of thirteen case-control studies showed significant associations between the p53 Arg72Pro polymorphism and lymphoma

Table 3. Comparison of combined genotype analysis of MDM2 SNP309 and p53 codon 72 between AML patients and controls. Genotypes Case Controls P value MDM2 P53 no. (%) no. (%) Arg/Arg 2 (28.6) 1 (16.7) Homo GG Pro/Pro 1 (14.3) 1 (16.7) > 0.99 Arg/Pro 4 (57.1) 4 (66.6) Arg/Arg (40.5) 11 (26.2) Hetero TG Pro/Pro 9 (21.4) 4 (9.5) 0.049 Arg/Pro 17 16 (38.1) 27 (64.3) Arg/Arg 1 (100) 2 (100) Wild TT Pro/Pro 0 0 > 0.99 Arg/Pro 0 0 Chi Square test (non-hodgkin lymphoma) risk, while no significant association was found between this polymorphism and leukemia risk [30]. A single study conducted on acute leukemia cases found a significant association between homozygous Arg genotype and AML (p < 0.001), indicating that Arg/Arg genotype might confer increased risk to development of acute myeloid leukemia [31]. Present work shows that the MDM2 309 GG genotype is significantly associated with an earlier age of onset of AML. Many studies of leukemia are controversial, showing this association in Caucasian and Black populations but not in Hispanic and Singaporean populations [25,32]. Phillips et al. also showed that the variant SNP309 influences susceptibility to pediatric AML, where the GG genotype was associated with an increased susceptibility to AML [33]. Xiong et al. [15] showed that the SNP309 has no effect on the timing of acute leukemia onset in the northern Chinese population, which further supported the idea that the SNP309 has different ethnic effects. Regarding association of MDM2 SNP309 polymorphism with gender, clinical parameters, or hematological findings in the studied AML cases, a MDM2 and p53 polymorphism in AML 453 highly significant correlation was found only between the MDM2 SNP309 TG, GG genotypes, and the Hb level (p=0.001). Other than the Hb level, we did not observe any correlation between MDM2 SNP309 polymorphism and the other studied parameters. Xiong et al. did not identify any correlation between MDM2 SNP309 and gender, clinical parameters, and hematological findings [15]. In our study, we observed that the p53 codon 72 polymorphism did not affect the age onset of AML. We also did not associate the p53 codon 72 polymorphism with gender, clinical parameters or hematological findings of the studied cases, besides a correlation between p53 genotypes and the myeloid marker CD117 (p=0.004). Our results are consistent with the results of the Xiong et al. [15] study in the northern Chinese population and those of Nakano et al. [34]. In our study, the combination of the MDM2 SNP309 and p53 codon 72 polymorphisms among the studied groups was examined, and a borderline significant correlation drawn between the MDM2 TG and p53 genotypes (p=0.049). This suggested a possible interactive effect between the MDM2 309TG and the p53 genotypes in increasing the risk of AML. Similarly, Ellis et al. [24] reported that individuals carrying both an MDM2 G allele and a P53 Pro allele were at increased risk of t-aml. A meta-analysis of six studies conducted on 27,813 cases with various tumor types and 30,295 controls indicated a possible compounding effect between the MDM2 309GG and p53 Pro/Pro genotype that lead to a significantly increased risk of cancer [35]. However, the Xiong et al. study [15] showed no multiplicative effect of interaction between MDM2 SNP309 and p53 codon 72 polymorphisms and risk of AML. Again, this difference may be due to ethnic and/or geographic variations of the frequency of these alleles in different populations [26,27].

454 In conclusion, our study suggested that the interactive effect between the MDM2 SNP309 and p53 codon 72 polymorphisms may increase the risk of acute myeloid leukemia. Neither the MDM2 SNP309 nor p53 codon 72 polymorphisms increased the risk of acute myeloid leukemia. Our results showed a significant correlation between the MDM2 SNP309 variant genotypes, age of onset of AML, and Hb level. A significant correlation between p53 genotypes and the myeloid marker CD117 was also found. Acknowledgements We thank our patients for their willing participation in our research. There is no conflict of interest. References Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014 1. Deschler B and Lubbert M: Acute myeloid leukemia: epidemiology and etiology. Cancer 2006; 107: 2099 2107. 2. Look AT. Oncogenic transcription factors in the human acute leukemias. Science 1997; 278(5340):1059 64. 3. Martens JH and Stunnenberg HG. 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