74 J Contemp Med Sci | Vol. 2, No. 7, Summer 2016: 74–76 Research Objectives According to previous studies, the aim of the presented work is to investigate the possible association between the codon 72 polymorphism (Pro72Arg, rs1042522) of the tumor suppressor gene (p53) with acute myocardial infarction (AMI) in Iraqi patients of Karbala province. Methods A total of 58 smokers and non-smokers patients with AMI diagnosed by clinical history, electrocardiography and troponin I level and another 35 healthy controls were included in this study. The (Pro72Arg) polymorphism of the p53 gene was evaluated by molecular techniques. Results The genotype distribution for the Pro72Arg variant of the p53 gene in AMI non-smokers patients (PP: n = 9, 47.4%; RP: n = 2, 10.5%; RR: n = 8, 42.1%) and controls (PP: n = 9, 50.0%; RP: n = 9, 50.0%; RR: n = 0, 0.0%) was significantly different (p = 0.002). and the genotype distribution for the Pro72Arg variant of the p53 gene in AMI smokers patients (PP: n = 20, 51.3%; RP: n = 1, 2.6%; RR: n = 18, 46.2%) and controls (PP: n = 14, 82.4%; RP: n = 2, 11.8%; RR: n = 1, 5.9%) was significantly different (p = 0.009). Conclusion These findings suggest that the Pro72Arg polymorphism of tumor suppressor p53 gene is associated with AMI patient studies. Keywords AMI, PCR, RFLP, polymorphism Polymorphism of tumor suppressor gene (p53) Codon 72 in Iraqi patients with acute myocardial infarction Fadhil Jawad Al-Tu’maa, Riyadh D. Al-Zubaidib, Abdul-Mutalib Badr Al-Khaleelic, and Hassan M. Abo-Almaalid ISSN 2413-0516 aDepartment of Biochemistry, College of Medicine, University of Karbala, Karbala, Iraq. bDepartment of Internal Medicine, College of Medicine, University of Karbala, Karbala, Iraq. cDepartment of Chemistry, Pure Science Education College, University of Karbala, Karbala, Iraq. dDepartment of Clinical Laboratory Sciences, College of Pharmacy, University of Karbala, Karbala, Iraq. Correspondence to Abdul-Mutalib Badr Al-Khaleeli (email: abd742007@yahoo.com). (Submitted: 25 January 2016 – Revised version received: 7 May 2016 – Accepted: 23 April 2016 – Published online: 26 September 2016) Introduction Different genes including the tumor suppressor (p53) gene have been implicated in the etiology of coronary artery dis- eases.1 By regulating gene expression and other indirect means p53 participates in the regulation of glucose, fatty acid, amino acid and purine metabolism, in addition it influences mito- chondrial integrity and oxidative phosphorylation, insulin sensitivity, antioxidant response.2 Furthermore p53 gene plays an important role in regulating vascular smooth muscle cell growth and may mediate and abnormal occurrence of apop- tosis in atherosclerotic lesions by attenuating or accelerating the apoptotic death process.3 On the other hand p53 gene could regulate cell division and apoptosis within atheroscle- rotic plaque depending on the level of p53 gene expression induced by DNA damage and cell type. Affected mutations in p53 gene can induce dysfunction of p53 and inhibit apoptosis and loss of gene activity could which play a relevant role in the pathogenesis of atherosclerosis.4 A common p53 tumor suppressor gene polymorphisms occur at codon 72 of exon 4, with two alleles encoding either arginine (CGC) or proline (CCC). The distribution of the three genotypes (R/R, R/P and P/P) depends largely on the ethnic composition of the studied population.5 The Pro or Arg of codon 72 has variants reported to differ in functional activity because this polymorphism is located in the proline rich domain of p53, which is necessary for the p53 protein to fully induce apoptosis.6 The aim of the presented work is to investigate the possible association between the codon 72 polymorphism (Pro72Arg, rs1042522) of the tumor suppressor gene (p53) with (smoker and non-smoker) acute myocardial infarction in Iraqi patients of Karbala province. Materials and Methods Subjects The study was conducted during the period from Nov., 2014 till Sep., 2015. Fifty eight patients presented with typical chest pain to the cardiac care unit (CCU) in Al-Hussein Teaching Hospital, Al-Hussein Medical City/ Kerbela Health Directorate and Department of Biochemistry- college of Medicine/University of Karbala. Thirty five persons age – matched healthy volunteers were selected as a control group. Both groups were divided into smokers and nonsmokers (39 AMI smokers patient and 17 smoker control) and (19 non- smokers AMI patient and 18 nonsmoker control). The diag- nosis was based on the clinical history, presentation confirmed by ECG and various investigations of cardiac biomarker. DNA extraction About 10 ml of venous blood sample was drawn from each patient and control groups. Two ml of blood sample collected in EDTA tube for genomic DNA extraction used for molecular analysis. The DNA extraction kit was purchased from BIONEER, South Korea. The remaining sample was transferred into another tube at room temperature and stand for 20 min for clotting and then centrifuged at 3000 rpm for serum collection used for various cardiac biomarkers determinations. Tumor suppress gene P53 polymorphism Four primers (Table 1) were used in a single PCR reaction, P1 and P2 to amplify a 281 bp band (control band) which make sure the success of the amplification, P3 specific to amplify a 193 bp band which indicate the present of proline allele and P4 75J Contemp Med Sci | Vol. 2, No. 7, Summer 2016: 74–76 Research Polymorphism of tumor suppressor gene (p53) Codon 72 in Iraqi patients with acute myocardial infarction Abdul-Mutalib Badr Al-Khaleeli et al. specific to amplify a 131 bp band which indicate the present of arginine allele as shown in (Fig. 1).7 Analysis for different subjects (M = molecular weight marker) PCR premix™ kit was used to amplify. It contained 5 µl of extracted DNA, (0.5 µl from each P1 and P2 and 1µl from each P3 and P4 ) of 10 pmol/µl primers were mix in total volume of 20 µl. Then the mixture was added to lyophilized PCR premix formula. The mixture was heated for 5 min at 94°C and underwent 40 cycles of amplification: annealing (65°C for 30 s), extension (72°C for 30 s) and denaturation (94°c for 30 s). The PCR product was analyzed on 1.5% agarose gel stained with ethidium bromide.7 Result The results showed a significant difference in distribution of p53 (codon 72) allelic polymorphism (P < 0.01) as shown in Table 1. Higher percentage in RR(Arginine/Arginine) allele (42.1%) in non-smoker AMI patient in compared (0%) with non-smoker normal control groups, while low percentage in RP (Arginine/Proline) allele (10.5%) in non-smoker AMI patient in compared (50%) with non-smoker normal control groups (Table 2). Significant difference in distribution of p53 (codon 72) allelic polymorphism (P < 0.01) as shown in Table 3. Higher percentage in RR (Arginine/Arginine) allele (46.2%) in smoker AMI patient in compared to smoker normal control groups (5.9%), while low percentage in PP (Proline/Proline) allele (51.3%) in smoker AMI patient in compared to smoker normal control groups (82.4%) (Table 3). Discussion The results showed a significant difference in distribution of p53 (codon 72) allelic polymorphism, high percentage in RR allele in smoker and non-smoker AMI patient in comparing to smoker and non-smoker normal control group. These findings were agreeing to Chilean study by (José Caamaño et al. 2011). This study showed increasing in RR allele in patients and explained that Apoptosis in endothelial cells have related to plaque instability and thrombus formation. The disturbance in the apoptotic response may lead to accu- mulation of intimal cells through a therogenesis and func- tional consequence of the Pro72Arg polymorphism has related to inhibition of p73 function, a member of the p53 family of nuclear transcription factors, implicated in tumor suppression, Arg polymorphic allele is more efficient in binding to p73, blocking its action and facilitating the prolif- eration of vascular smooth muscle cells. By this mechanism Arg variant of the P53 Pro72Arg polymorphism is more sus- ceptible to suffer deregulation of apoptosis during athero- sclerosis progression.8 Also another Brazilian study by (Disciplina de Genética et al 2007) suggested that Arg72 allele is associated with cardi- ovascular disease.9 In additional type 2 diabetes mellitus T2DM which is risk factors for AMI and the fact that the Carriers of genotypes containing Arg72 allele previously asso- ciated with susceptibility to type 2 diabetes mellitus T2DM than pro72 genotype carriers.10 Conclusion In this study, we ascertain that a significant association bet- ween the codon 72 polymorphism (Pro72Arg, rs1042522) of the tumor suppressor gene (p53) with acute myocardial infarction (AMI). n Table 3. Comparison between smoker myocardial infarction patients and smoker control groups in the distribution of p53 (codon 72) allelic polymorphism Codon 72 allelic polymorphism p- value RR (n =), % PP (n =), % RP (n =), % Smoker Patient (18) 46.2% (20) 51.3% (1) 2.6% p < 0.01 Control (1) 5.9% (14) 82.4% (2) 11.8% *RR: Arginine/Arginine, PP: Proline/Proline, RP: Arginine/Proline. Table 1. Sequence of primers used for PCR of p53 gene polymorphism Primer Sequence P1 5’-GCCGTCCCAAGCAATGGATGATT-3’ P2 5’-GGCAACTGACCGTGCAAGTCACAG-3’ P3 5’-AGAATGCCAGAGGCTGCTCCACC-3’ P4 5’-CTTCTGGTGCAGGGGCCAAGC-3’ Fig. 1 Agarose gel electrophoretogram of the p53 Arg72Pro polymorphism. Table 2. Comparison between non-smoker myocardial infarc- tion patients and non-smoker control groups in the distribution of p53 (codon 72) allelic polymorphism Codon 72 allelic polymorphism p- value RR (n =), % PP (n =), % RP (n =), % Non- smoker Patient (8) 42.1% (9) 47.4% (2) 10.5% p < 0.01 Control (0) 0.0% (9) 50.0% (9) 50.0% *RR: Arginine/Arginine, PP: Proline/Proline, RP: Arginine/Proline. 76 J Contemp Med Sci | Vol. 2, No. 7, Summer 2016: 74–76 Polymorphism of tumor suppressor gene (p53) Codon 72 in Iraqi patients with acute myocardial infarction Research Abdul-Mutalib Badr Al-Khaleeli et al. References 1. Geng YJ. Molecular signal transduction in vascular cell apoptosis. Cell Res. 2001;11:253–264. 2. Maddocks OD, Vousden KH. Metabolic regulation by p53. J Mol Med (Berl). 2011;89:237–245. 3. Geng YJ. Biologic effect and molecular regulation of vascular apoptosis in atherosclerosis. Curr Atheroscler Rep. 2001;3:234–242. 4. Mercer J, Mahmoudi M, Bennett M. DNA damage, p53, apoptosis and vascular disease. Mutat Res. 2007;621:75–86. 5. Omori S, Yoshida S, Kennedy S. Polymorphism at codon 72 of the p53 gene is not associated with endometriosis in a Japanese population. Gynecol Investing. 2004;11:232–236. 6. Baptiste N, Friedlander P, Chen X, Prives C. The proline-rich domain of p53 is required for cooperation with anti-neoplastic agents to promote apoptosis of tumor cells. Oncogene. 2002;21:9–21. 7. Bassam L, Amal A. Detection of Arg72pro polymorphism of tumor suppressors gen TP53 by a rapid one-step amplification refractory mutation system-PCR. Am J Biochem Mol Biol. 2011;2: 231–236. 8. Caamaño J, Saavedra N, Jaramillo PC, Lanas C, Lanas F, Salazar LA. TP53Codon 72 Polymorphism is associated with coronary artery disease in Chilean subjects. Med Princ Pract. 2011;20:171–176. 9. Smith MA, Silva MD, Cendoroglo MS, Ramos LR, Araujo LM, Labio RW, et al. TP53codon 72 polymorphism as a risk factor for cardiovascular disease in a Brazilian population. Braz J Med Biol Res. 2007;40:1465– 1472. 10. Katarina K, Lukas P, Veronika D, Katerina K. Association of the Arg72Pro polymorphism in p53 with progression of diabetic nephropathy in T2DM subjects. J Nephrol Ther. 2014;4:153.