Molecular and Cellular Biochemistry

, Volume 355, Issue 1–2, pp 289–297 | Cite as

Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease

  • Rizwan Masud
  • Irfan Zia Qureshi


Cardiovascular disorders and coronary artery disease (CAD) are significant contributors to morbidity and mortality in heart patients. As genes of the folate/homocysteine pathway have been linked with the vascular disease, we investigated association of these gene polymorphisms with CAD/myocardial infarction (MI) using the novel approach of tetraprimer ARMS-PCR. A total of 230 participants (129 MI cases, 101 normal subjects) were recruited. We genotyped rs1801133 and rs1801131 SNPs in 5′10′ methylenetetrahydrofolate reductase (MTHFR), rs1805087 SNP in 5′ methyltetrahydrofolate homocysteine methyltransferase (MTR), rs662 SNP in paroxanse1 (PON1), and rs5742905 polymorphism in cystathionine beta synthase (CBS). Angiotensin converting enzyme (ACE) insertion/deletion polymorphism was detected through conventional PCR. Covariates included blood pressure, fasting blood sugar, serum cholesterol, and creatinine concentrations. Our results showed allele frequencies at rs1801133, rs1801131, rs1805087 and the ACE insertion/deletion (I/D) polymorphism varied between cases and controls. Logistic regression, after adjusting for covariates, demonstrated significant associations of rs1801133 and rs1805087 with CAD in the additive, dominant, and genotype model. In contrast, ACE I/D polymorphism was significantly related with CAD where recessive model was applied. Gene–gene interaction against the disease status revealed two polymorphism groups: rs1801133, rs662, and rs1805087; and rs1801131, rs662, and ACE I/D. Only the latter interaction maintained significance after adjusted for covariates. Our study concludes that folate pathway variants exert contributory influence on susceptibility to CAD. We further suggest that tetraprimer ARMS-PCR successfully resolves the genotypes in selected samples and might prove to be a superior technique compared to the conventional approach.


Tetra primer ARMS-PCR Homocysteinemia Single nucleotide polymorphisms 



The authors are grateful to Dr Haider Zaigham Baqai, Dr Asad Riaz for patient selection, Professor Dr. Wasim Ahmad, Drs Jawad Hassan, Mohammad Tariq, Salman Chishti, Mohammad Inam for their valuable comments on the manuscript, and to Dr Keyue Ding, Mayo Clinic Rochester, Minnesota USA for help with statistics. The research work was funded through a graduate scholarship grant No. 17-5-2 Ls2-50 HEC/Sch/2004/5145 awarded to the first author by Higher Education Commission (HEC), Islamabad, Pakistan.


  1. 1.
    Mathers CD, Loncar D (2006) Projections of global mortality, burden of disease from 2002 to 2030. PLoS Med 3(11):e442PubMedCrossRefGoogle Scholar
  2. 2.
    Yusuf S, Reddy S, Ounpuu S, Anand S (2001) Global burden of cardiovascular diseases: part I: general considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation 104(22):2746–2753PubMedCrossRefGoogle Scholar
  3. 3.
    Jafar TH, Qadri Z, Chaturvedi N (2008) Coronary artery disease epidemic in Pakistan: more electrocardiographic evidence of ischaemia in women than in men. Heart 94(4):408–413PubMedCrossRefGoogle Scholar
  4. 4.
    Yusuf S, Reddy S, Ounpuu S, Anand S (2001) Global burden of cardiovascular diseases: Part II: variations in cardiovascular disease by specific ethnic groups and geographic regions and prevention strategies. Circulation 104(23):2855–2864PubMedCrossRefGoogle Scholar
  5. 5.
    McKeigue PM (1992) Coronary heart disease in Indians, Pakistanis, and Bangladeshis: aetiology and possibilities for prevention. Br Heart J 67(5):341–342PubMedCrossRefGoogle Scholar
  6. 6.
    Hansson GK (2005) Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 352(16):1685–1695PubMedCrossRefGoogle Scholar
  7. 7.
    Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, Hansson GK (1999) Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 145(1):33–43PubMedCrossRefGoogle Scholar
  8. 8.
    Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrom LE, Ueland PM, Palma-Reis RJ, Boers GH, Sheahan RG, Israelsson B, Uiterwaal CS, Meleady R, McMaster D, Verhoef P, Witteman J, Rubba P, Bellet H, Wautrecht JC, de Valk HW, Sales Luis AC, Parrot-Rouland FM, Tan KS, Higgins I, Garcon D, Andria G et al (1997) Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. JAMA 277(22):1775–1781PubMedCrossRefGoogle Scholar
  9. 9.
    Boushey CJ, Beresford SA, Omenn GS, Motulsky AG (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 274(13):1049–1057PubMedCrossRefGoogle Scholar
  10. 10.
    Lima LM, Carvalho MG, Fernandes AP, Sabino Ade P, Loures-Vale AA, da Fonseca Neto CP, Garcia JC, Saad JA, Sousa MO (2007) Homocysteine and methylenetetrahydrofolate reductase in subjects undergoing coronary angiography. Arq Bras Cardiol 88(2):167–172PubMedCrossRefGoogle Scholar
  11. 11.
    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP et al (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 10(1):111–113PubMedCrossRefGoogle Scholar
  12. 12.
    Kluijtmans LA, Whitehead AS (2001) Methylenetetrahydrofolate reductase genotypes and predisposition to atherothrombotic disease; evidence that all three MTHFR C677T genotypes confer different levels of risk. Eur Heart J 22(4):294–299PubMedCrossRefGoogle Scholar
  13. 13.
    Bathum L, Petersen I, Christiansen L, Konieczna A, Sorensen TI, Kyvik KO (2007) Genetic and environmental influences on plasma homocysteine: results from a Danish twin study. Clin Chem 53(5):971–979PubMedCrossRefGoogle Scholar
  14. 14.
    Szczeklik A, Sanak M, Jankowski M, Dropinski J, Czachor R, Musial J, Axenti I, Twardowska M, Brzostek T, Tendera M (2001) Mutation A1298C of methylenetetrahydrofolate reductase: risk for early coronary disease not associated with hyperhomocysteinemia. Am J Med Genet 101(1):36–39PubMedCrossRefGoogle Scholar
  15. 15.
    Laraqui A, Allami A, Carrie A, Raisonnier A, Coiffard AS, Benkouka F, Bendriss A, Benjouad A, Bennouar N, El Kadiri N, Benomar A, Fellat S, Benomar M (2007) Relation between plasma homocysteine, gene polymorphisms of homocysteine metabolism-related enzymes, and angiographically proven coronary artery disease. Eur J Intern Med 18(6):474–483PubMedCrossRefGoogle Scholar
  16. 16.
    Vinukonda G, Shaik Mohammad N, Md Nurul Jain J, Prasad Chintakindi K, Rama Devi Akella R (2009) Genetic and environmental influences on total plasma homocysteine and coronary artery disease (CAD) risk among South Indians. Clin Chim Acta Inter J Clin Chem 405(1–2):127–131CrossRefGoogle Scholar
  17. 17.
    Mendonca MI, Dos Reis RP, Freitas AI, Sousa AC, Pereira A, Faria P, Gomes S, Silva B, Santos N, Serrao M, Ornelas I, Freitas S, Freitas C, Araujo JJ, Brehm A, Cardoso AA (2009) Gene–gene interaction affects coronary artery disease risk. Rev Port Cardiol 28(4):397–415PubMedGoogle Scholar
  18. 18.
    Mohamed RH, Mohamed RH, Karam RA, Abd El-Aziz TA (2010) The relationship between paraoxonase1–192 polymorphism and activity with coronary artery disease. Clin Biochem 43(6):553–558PubMedCrossRefGoogle Scholar
  19. 19.
    Zintzaras E, Raman G, Kitsios G, Lau J (2008) Angiotensin-converting enzyme insertion/deletion gene polymorphic variant as a marker of coronary artery disease: a meta-analysis. Arch Intern Med 168(10):1077–1089PubMedCrossRefGoogle Scholar
  20. 20.
    Tsai MY, Welge BG, Hanson NQ, Bignell MK, Vessey J, Schwichtenberg K, Yang F, Bullemer FE, Rasmussen R, Graham KJ (1999) Genetic causes of mild hyperhomocysteinemia in patients with premature occlusive coronary artery diseases. Atherosclerosis 143(1):163–170PubMedCrossRefGoogle Scholar
  21. 21.
    Lai E (2001) Application of SNP technologies in medicine: lessons learned and future challenges. Genome Res 11(6):927–929PubMedCrossRefGoogle Scholar
  22. 22.
    Ye S, Dhillon S, Ke X, Collins AR, Day IN (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29(17):E88PubMedCrossRefGoogle Scholar
  23. 23.
    Rigat B, Hubert C, Corvol P, Soubrier F (1992) PCR detection of the insertion\letion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res 20(6):1433PubMedCrossRefGoogle Scholar
  24. 24.
    Yang YG, Kim JY, Park SJ, Kim SW, Jeon OH, Kim DS (2007) Apolipoprotein E genotyping by multiplex tetra-primer amplification refractory mutation system PCR in single reaction tube. J Biotechnol 131(2):106–110PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang X, Miao X, Guo Y, Tan W, Zhou Y, Sun T, Wang Y, Lin D (2006) Genetic polymorphisms in cell cycle regulatory genes MDM2 and TP53 are associated with susceptibility to lung cancer. Hum Mutat 27(1):110–117PubMedCrossRefGoogle Scholar
  26. 26.
    Christen WG, Ajani UA, Glynn RJ, Hennekens CH (2000) Blood levels of homocysteine and increased risks of cardiovascular disease: causal or casual? Arch Intern Med 160(4):422–434PubMedCrossRefGoogle Scholar
  27. 27.
    Wald DS, Law M, Morris JK (2002) Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ (Clinical research Ed) 325(7374):1202CrossRefGoogle Scholar
  28. 28.
    Antoniades C, Antonopoulos AS, Tousoulis D, Marinou K, Stefanadis C (2009) Homocysteine and coronary atherosclerosis: from folate fortification to the recent clinical trials. Eur Heart J 30(1):6–15PubMedCrossRefGoogle Scholar
  29. 29.
    Klerk M, Verhoef P, Clarke R, Blom HJ, Kok FJ, Schouten EG (2002) MTHFR 677C→T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA 288(16):2023–2031PubMedCrossRefGoogle Scholar
  30. 30.
    Lewis SJ, Ebrahim S, Davey Smith G (2005) Meta-analysis of MTHFR 677C→T polymorphism and coronary heart disease: does totality of evidence support causal role for homocysteine and preventive potential of folate? BMJ (Clinical research) 331(7524):1053CrossRefGoogle Scholar
  31. 31.
    Laraqui A, Allami A, Carrie A, Coiffard AS, Benkouka F, Benjouad A, Bendriss A, Kadiri N, Bennouar N, Benomar A, Guedira A, Raisonnier A, Fellati S, Srairi JE, Benomar M (2006) Influence of methionine synthase (A2756G) and methionine synthase reductase (A66G) polymorphisms on plasma homocysteine levels and relation to risk of coronary artery disease. Acta Cardiol 61(1):51–61PubMedCrossRefGoogle Scholar
  32. 32.
    Lakshmi VS, Naushad SM, Rupasree Y, Rao SD, Kutala VK (2011) Interactions of 5′-UTR thymidylate synthase polymorphism with 677C→T methylene tetrahydrofolate reductase and 66A→G methyltetrahydrofolate homocysteine methyl-transferase reductase polymorphisms determine susceptibility to coronary artery disease. J Atheroscler Thromb 18(1):56–64CrossRefGoogle Scholar
  33. 33.
    Liaugaudas G, Jacques PF, Selhub J, Rosenberg IH, Bostom AG (2001) Renal insufficiency, vitamin B(12) status, and population attributable risk for mild hyperhomocysteinemia among coronary artery disease patients in the era of folic acid-fortified cereal grain flour. Arterioscler Thromb Vasc Biol 21(5):849–851PubMedCrossRefGoogle Scholar
  34. 34.
    Robertson J, Iemolo F, Stabler SP, Allen RH, Spence JD (2005) Vitamin B12, homocysteine and carotid plaque in the era of folic acid fortification of enriched cereal grain products. CMAJ 172(12):1569–1573PubMedCrossRefGoogle Scholar
  35. 35.
    Franco RF, Elion J, Lavinha J, Krishnamoorthy R, Tavella MH, Zago MA (1998) Heterogeneous ethnic distribution of the 844ins68 in the cystathionine beta-synthase gene. Hum Hered 48(6):338–342PubMedCrossRefGoogle Scholar
  36. 36.
    Dutta S, Sinha S, Chattopadhyay A, Gangopadhyay PK, Mukhopadhyay J, Singh M, Mukhopadhyay K (2005) Cystathionine beta-synthase T833C/844INS68 polymorphism: a family-based study on mentally retarded children. Behav Brain Funct 1:25PubMedCrossRefGoogle Scholar
  37. 37.
    Koubaa N, Nakbi A, Hammami S, Attia N, Mehri S, Ben Hamda K, Ben Farhat M, Miled A, Hammami M (2009) Association of homocysteine thiolactonase activity and PON1 polymorphisms with the severity of acute coronary syndrome. Clin Biochem 42(9):771–776PubMedCrossRefGoogle Scholar
  38. 38.
    Wheeler JG, Keavney BD, Watkins H, Collins R, Danesh J (2004) Four paraoxonase gene polymorphisms in 11212 cases of coronary heart disease and 12786 controls: meta-analysis of 43 studies. Lancet 363(9410):689–695PubMedCrossRefGoogle Scholar
  39. 39.
    Saeed M, Perwaiz Iqbal M, Yousuf FA, Perveen S, Shafiq M, Sajid J, Frossard PM (2007) Interactions and associations of paraoxonase gene cluster polymorphisms with myocardial infarction in a Pakistani population. Clin Genet 71(3):238–244PubMedCrossRefGoogle Scholar
  40. 40.
    Mendonca MI, Dos Reis RP, Freitas AI, Sousa AC, Pereira A, Faria P, Gomes S, Silva B, Santos N, Serrao M, Ornelas I, Freitas S, Araujo JJ, Brehm A, Cardoso AA (2008) Human paraoxonase gene polymorphisms and coronary artery disease risk. Rev Port Cardiol 27(12):1539–1555PubMedGoogle Scholar
  41. 41.
    Pizza V, Bisogno A, Lamaida E, Agresta A, Bandieramonte G, Volpe A, Galasso R, Galasso L, Caputo M, Tecce MF, Capasso A (2010) Migraine and coronary artery disease: an open study on the genetic polymorphism of the 5,10 methylenetetrahydrofolate (MTHFR) and angiotensin I-converting enzyme (ACE) genes. Cent Nerv Syst Agents Med Chem 10(2):91–96PubMedGoogle Scholar
  42. 42.
    Agerholm-Larsen B, Nordestgaard BG, Tybjaerg-Hansen A (2000) ACE gene polymorphism in cardiovascular disease: meta-analyses of small and large studies in whites. Arterioscler Thromb Vasc Biol 20(2):484–492PubMedCrossRefGoogle Scholar
  43. 43.
    Giusti B, Saracini C, Bolli P, Magi A, Sestini I, Sticchi E, Pratesi G, Pulli R, Pratesi C, Abbate R (2008) Genetic analysis of 56 polymorphisms in 17 genes involved in methionine metabolism in patients with abdominal aortic aneurysm. J Med Genet 45(11):721–730PubMedCrossRefGoogle Scholar
  44. 44.
    Tietjen GE, Herial NA, Utley C, White L, Yerga-Woolwine S, Joe B (2009) Association of von Willebrand factor activity with ACE I/D and MTHFR C677T polymorphisms in migraine. Cephalalgia 29(9):960–968PubMedCrossRefGoogle Scholar
  45. 45.
    Bentley P, Peck G, Smeeth L, Whittaker J, Sharma P (2010) Causal relationship of susceptibility genes to ischemic stroke: comparison to ischemic heart disease and biochemical determinants. PloS one 5(2):e9136PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  1. 1.Laboratory of Animal and Human Physiology, Department of Animal Sciences, Faculty of Biological SciencesQuaid-i-Azam UniversityIslamabadPakistan

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