Document Type: Original Article


1 Department of Molecular Cell Biology,Islamic Azad University,Tonekabon Branch,Iran.

2 Department of Molecular Cell Biology, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran



Background: Thrombosis is the formation of a blood clot in a blood vessel that prevents blood flow in the blood circulatory system. Nowadays thrombosis is one of the causes of mortality in human societies. Thrombosis can be caused by genetic disorders or environmental factors that help the formation of clots. Two common mutations of methylene tetrahydrofolate reductase gene known as C677T and A1298C are realized as the effective factors for thrombophilia. The purpose of this study is to design a proper ARMS-PCR method for the diagnosis of these two mutations of MTHFR gene and the evaluation of its relationship with people suspected of thrombosis.
Methods: first 50 samples suspected of thrombosis were collected from cases that had gone to laboratories for coagulation tests, then the genomic DNA of the samples was extracted using the proper commercial kit. Proper ARMS-PCR primers for C677T and A1298C mutations were designed and then synthesized. After the optimization of the ARMS-PCR reaction on the positive control sample, the reaction was carried out for all 50 samples. A number of mutated and non-mutated samples were studied for confirmation by the PCR-Sequencing method.
Results: the results from the reaction showed that from 50 samples under study, after the optimization, 13 people had the C677T mutation (26%) from which 5 people had the homozygous genotype and 8 people had the Heterozygous genotype and for the A1298C mutation, 12 positive sample were found (24%) from which 7 people had the Heterozygous genotype and 5 people had the homozygous genotype.


Main Subjects

Abraham, R, Joseph John, M, Calton, R, Dhanoa, J. (2006). Raised serum homocysteine levels in patients of coronary artery disease and the effect of vitamin B12 and folate on its concentration. Indian J. Clin. Biochem., 21(1):95–100.

Ananth, CV, Peltier, MR, De Marco, C, Elsasser, DA, Getahun, D, Rozen, R, Smulian, JC, New Jersey–Placental Abruption Study Investigators, (2007). Associations between 2 polymorphisms in the methylenetetrahydrofolate reductase gene and placental abruption. Am. J. obstet. Gynecol., 197(4):385.

Balasubramaniam, A, Kotalawala, S, Amarasekara, R. (2017). Analysis of methylenetetrahydrofolate reductase (MTHFR) Polymorphisms (C677t & A1298c) in recurrent pregnancy loss (RPL). J Gynecol., 1(4):1-5.

Barlik, M, Drews, K, Seremak-Mrozikiewicz, A, Kurzawińska, G, Mrozikiewicz, PM. (2009). Genetic conditioned changes in activity of 5, 10-methylenetetrahydrofolate reductase (MTHFR) and recurrent miscarriages. Ginekol. polska, 80(10):762-7.

Casas, JP, Chua, W, Loukogeorgakis, S, Vallance, P, Smeeth, L, Hingorani, AD, MacAllister, RJ. (2005). Effect of inhibitors of the renin-angiotensin system and other antihypertensive drugs on renal outcomes: systematic review and meta-analysis. Lancet, 366(9502):2026-2033.

Clarke, R, Daly, L, Robinson, K, Naughten, E, Cahalane, S, Fowler, B, Graham, I. (1991). Hyperhomocysteinemia: an independent risk factor for vascular disease. New England J. Med., 324(17):1149-1155.

Cotlarciuc, I, Malik, R, Holliday, EG, Ahmadi, KR, Paré, G, Psaty, BM, Fornage, M, Hasan, N, Rinne, PE, Ikram, MA, Markus, HS. (2014). Effect of genetic variants associated with plasma homocysteine levels on stroke risk. Stroke, 45(7):1920-1924.

Cui, R, Moriyama, Y, Koike, KA, Date, C, Kikuchi, S, Tamakoshi, A, Iso, H, JACC Study group, (2008). Serum total homocysteine concentrations and risk of mortality from stroke and coronary heart disease in Japanese: The JACC study. Atherosclerosis, 198(2):412-418.

Davalos, IP, Moran, MC, Martinez-Abundis, E, González-Ortiz, M, Flores-Martínez, SE, Machorro, V, Sandoval, L, Figuera, LE, Mena, JP, Oliva, JM, Tlacuilo-Parra, JA. (2005). Methylenetetrahydrofolate reductase C677T polymorphism and Factor V Leiden variant in Mexican women with preeclampsia/eclampsia. Blood Cell. Mol. Dis., 35(1):66-69.

Dossenbach-Glaninger, A, van Trotsenburg, M, Dossenbach, M, Oberkanins, C, Moritz, A, Krugluger, W, Huber, J, Hopmeier, P. (2003). Plasminogen activator inhibitor 1 4G/5G polymorphism and coagulation factor XIII Val34Leu polymorphism: impaired fibrinolysis and early pregnancy loss. Clin. Chem., 49(7):1081-1086.

Eftychiou, C, Antoniades, L, Makri, L, Koumas, L, Costeas, PA, Kyriakou, E, Nicolaides, E, Papadogiannis, D. (2012). Homocysteine levels and MTHFR polymorphisms in young patients with acute myocardial infarction: a case control study. Hellenic J. Cardiol., 53(3):189-194.

Forges, T, Monnier-Barbarino, P, Alberto, M, Gueant, JM, Gueant-Rodriguez, RM, DAval, JL, GUeant, L. (2007).Impact of folate and homocysteine metabolism on human reproductive health. Human Reproduct. Update, 13(3):225-238. 

García-Frade Ruiz, LF, Cuellar-Gamboa, L. (2016). Actualidades en trombosis. Med. Int. México, 32(1):103-114.

Ghaffari, K, Ghasemi, A, Ghotaslou, A, Mohammadi, M, Salmanpour, Z. (2015). Correlation between C677T and A1298C mutations on the MTHFR gene with plasma homocysteine levels and venous thrombosis in pregnant women at risk of thrombosis. Zahedan J. Res. Med. Sci., 17:0-0

Hanafi, S, Hassan, R, Bahar, R, Abdullah, WZ, Johan, MF, Rashid, ND, Azman, NF, Nasir, A, Hassan, S, Ahmad, R, Othman, A. (2014). Multiplex amplification refractory mutation system (MARMS) for the detection of β-globin gene mutations among the transfusion-dependent β-thalassemia Malay patients in Kelantan, Northeast of Peninsular Malaysia. Am. J. Blood Res., 4(1):33.

Hassan, S, Ahmad, R, Zakaria, Z, Zulkafli, Z, Abdullah, WZ. (2013). Detection of β-globin gene mutations among β-thalassaemia carriers and patients in Malaysia: application of multiplex amplification refractory mutation system–polymerase chain reaction. Malaysian J. Med. Sci., MJMS, 20(1):13.

Holmes, MV, Newcombe, P, Hubacek, JA, Sofat, R, Ricketts, SL, Cooper, J, Breteler, MM, Bautista, LE, Sharma, P, Whittaker, JC, Smeeth, L. (2011). Effect modification by population dietary folate on the association between MTHFR genotype, homocysteine, and stroke risk: a meta-analysis of genetic studies and randomised trials. Lancet, 378(9791):584-594.

Hotoleanu, C, Trifa, A, Popp, R, Fodor, D. (2013). The importance of homozygous polymorphisms of methylenetetrahydrofolate reductase gene in Romanian patients with idiopathic venous thromboembolism. Balkan Med. J., 30(2):197.

Iscfwt, D. (2014). Thrombosis: a major contributor to the global disease burden. J. Thromb Haemost, 12(10):1580-90.

Jamison, RL, Hartigan, P, Kaufman, JS, Goldfarb, DS, Warren, SR, Guarino, PD, Gaziano, JM, Veterans Affairs Site Investigators, (2007). Effect of homocysteine lowering on mortality and vascular disease in advanced chronic kidney disease and end-stage renal disease: a randomized controlled trial. Jama, 298(10):1163-1170.

Kaiser, T, Brennecke, SP, Moses, EK. (2000). Methylenetetrahydrofolate reductase polymorphisms are not a risk factor for pre-eclampsia/eclampsia in Australian women. Gynecol. Obstetric Investigat., 50(2):100-102.

Keat Wei, L, Sutherland, H, Au, A, Camilleri, E, Haupt, LM, Gan, SH, Griffiths, LR. (2015). A potential epigenetic marker mediating serum folate and vitamin B12 levels contributes to the risk of ischemic stroke. BioMed Res. Internat., 167976.

Kerlin, B, Cooley, BC, Isermann, BH, Hernandez, I, Sood, R, Zogg, M, Hendrickson, SB, Mosesson, MW, Lord, S, Weiler, H. (2004). Cause-effect relation between hyperfibrinogenemia and vascular disease. Blood, 103(5):1728-1734.

Kesieme, E, Kesieme, C, Jebbin, N, Irekpita, E, Dongo, A. (2011). Deep vein thrombosis: a clinical review. J. Blood Med., 2:59.

Khaleghparast, A, Morovvati, S, Noormohammadi, Z. (2011). Evaluation of the association between the C677T and A1298C polymorphisms of MTHFR gene and recurrent miscarriage. Scientific J. Iran Blood Trans. Org., 8(2):88-95.

Kim, JO, Park, HS, Ryu, CS, Shin, JW, Kim, J, Oh, SH, Kim, NK. (2017). Interplay between 3′-UTR polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene and the risk of ischemic stroke. Scientific Rep., 7(1):1-10.

Kupferminc, MJ. (2003). Thrombophilia and pregnancy. Reproduct. Biol. Endocrinol., 1(1):111.

Liew, SC, Gupta, ED. (2015). Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: epidemiology, metabolism and the associated diseases. Eur. J. Med. Genet., 58(1):1-10.

Lozano, R, Naghavi, M, Foreman, K, Lim, S, Shibuya, K, Aboyans, V, Abraham, J, Adair, T, Aggarwal, R, Ahn, SY, AlMazroa, MA. (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. lancet, 380(9859):2095-2128.

Mallinow MR. (1990). Hyperhomocysteinemia A common and easily reversible risk factor for occlusive atherosclerosis. Circulation, 81(6):2004–6.

Morelli, VM, De Visser, MCH, Vos, HL, Bertina, RM, Rosendaal, FR. (2005). ABO blood group genotypes and the risk of venous thrombosis: effect of factor V Leiden. J. Thromb. Haemost., 3(1):183-185.

Moretti, R, Caruso, P. (2019). The Controversial Role of Homocysteine in Neurology: From Labs to Clinical Practice. Int. J. Mol. Sci., 20.

Mtiraoui, N, Zammiti, W, Ghazouani, L, Jmili Braham, N, Saidi, S, Finan, RR, Almawi, WY, Mahjoub, T. (2006). Methylentetrahydrofolate reductase C677T and A1298C polymorphism and change in homocystein concentrations in woman with idiopathic recurrent pregnancy losses. Reproduction, 131(2):395-401.

Nadir, Y, Hoffman, R, Brenner, B. (2007). Association of homocysteine, vitamin B 12, folic acid, and MTHFR C677T in patients with a thrombotic event or recurrent fetal loss. Annal. Hematol., 86(1):35-40. 

Nishio, K, Goto, Y, Kondo, T, Ito, S, Ishida, Y, Kawai, S, Naito, M, Wakai, K, Hamajima, N. (2008). Serum folate and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism adjusted for folate intake. J Epidemiol., 18(3):125-31.

Pepe, G, Camacho Vanegas, O, Giusti, B, Brunelli, T, Marcucci, R, Attanasio, M, Rickards, O, De Stefano, GF, Prisco, D, Gensini, GF, Abbate, R. (1998). Hetetogeneity in word distribution of the thermolabile C677T mutation in 5,10-Methylentetrahydrofolate reductase. Am. J. Hum. Genet., 63:917-20.

Poursadegh Zonouzi, A, Chaparzadeh, N, Asghari Estiar, M, Mehrzad Sadaghiani, M, Farzadi, L, Ghasemzadeh, A, Sakhinia, M, Sakhinia, E. (2012). Methylenetetrahydrofolate reductase C677T and A1298C mutations in women with recurrent spontaneous abortions in the Northwest of Iran. ISRN obstetr. Gynecol. Internat. Schol. Res. Network, doi:10.5402/1012/945486 .

Prasad, AN, Rupar, CA, Prasad, C. (2011). Methylenetetrahydrofolate reductase (MTHFR) deficiency and infantile epilepsy. Brain Dev., 33:758-769.

Puri, R, Auffret, V, Rodés-Cabau, J. (2017). Bioprosthetic valve thrombosis. J. Am. Coll. Cardiol., 69(17):2193-2211.

Robien, K, Ulrich, CM. (2003). 5,10-Methylenetetrahydrofolate reductase polymorphisms and leukemia risk: a HuGE minireview. Am. J. Epidemiol., 157(7):571-82.

Scifres, CM, Macones, GA. (2008). The utility of thrombophilia testing in pregnant women with thrombosis: fact or fiction?. Am. J. Obstet. Gynecol., 199(4):344.

Soltanpour, MS, Soheili, Z, Shakerizadeh, A, Pourfathollah, AA, Samiei, S, Meshkani, R, Shahjahani, M, Karimi, A. (2013). Methylenetetrahydrofolate reductase C677T mutation and risk of retinal vein thrombosis. Journal of research in medical sciences: the official J. Isfahan University of Medical Sci.18(6), 487.

Spiroski, I, Kedev, S, Antov, S, Arsov, T, Krstevska, M, Dzhekova-Stojkova, S, Bosilkova, G, Kostovska, S, Trajkov, D, Petlichkovski, A, Strezova, A. (2008). Methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genotypes and haplotypes and plasma homocysteine levels in patients with occlusive artery disease and deep venous thrombosis. Acta biochim. Polon., 55(3):587-594.

Tonetti, C, Burtscher, A, Bories, D, Tulliez, M, Zittoun, J. (2000). Methylenetetrahydrofolate reductase deficiency in four siblings: A clinical, biochemical, and molecular study of the family. Am. J. Med. Genet. 91(5):363-367.

Toyoda, K, Uwatoko, T, Shimada, T, Hagiwara, N, Fujimoto, S, Ibayashi, S, Okada, Y. (2004). Recurrent small-artery disease in hyperhomocysteinemia: widowers' stroke syndrome?. Int. med., 43(9):869-872.

Trabetti, E. (2008). Homocysteine, MTHFR gene polymorphism, and cardio-cerebrovascular risk. J. Appl. Genet., 49(3):267-282.

Wei, LK, Sutherland, H, Au, A, Camilleri, E, Haupt, LM, Gan, SH, Griffiths, LR. (2016). Methylenetetrahydrofolate reductase CpG islands: epigenotyping. J. Clin. Laboratory Anal.,  30(4):335-344.

Wu, C, Gong, Y, Sun, A, Zhang, Y, Zhang, C, Zhang, W, Ge, J. (2013). The human MTHFR rs4846049 polymorphism increases coronary heart disease risk through modifying miRNA binding. Nutrit. Metabol. Cardiovascul. Dis., 23(7):693-698.

Yang, L, Ijaz, I, Cheng, J, Wei, C, Tan, X, Khan, MA, Fu, X, Fu, J. (2018). Evaluation of amplification refractory mutation system (ARMS) technique for quick and accurate prenatal gene diagnosis of CHM variant in choroideremia. Applicat. Clin. Genet., 11:1–8.

Ye, S, Dhillon, S, Ke, X, Collins, AR, Day, IN. (2001). An efficient procedure for genotyping single nucleotide polymorphisms. Nucl. Acid. Res., 29(17):E88-8.