Development and application of low-cost T-ARMS-PCR assay for AGT and CYP11B1 gene polymorphisms

  • Misbah Hussain
  • Haq Nawaz Khan
  • Fazli Rabbi AwanEmail author
Original Article


Angiotensin II (Ang II: a truncated octapeptide of angiotensinogen, AGT) and 11-β-hydroxylase influence regulation of blood pressure. Dysregulation of Ang II and 11-β-hydroxylase can lead to hypertension and elevate aldosterone levels. Polymorphisms in AGT (encodes AGT) and CYP11B1 (encodes 11-β-hydroxylase) shift the paradigm from physiological to pathological. Currently, various high-throughput techniques are used to genotype these polymorphisms. These techniques require expensive infrastructure and reagents. However, in developing countries, where cost is the main limiting factor, it is not feasible to use expensive techniques. So, the aim of current study was to develop efficient low-cost method for genotyping of cardiovascular disease and hypertension associated polymorphisms of AGT (rs4762, rs5051) and CYP11B1 (rs6410). For this, tetra amplification-refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was developed and optimized for aforementioned AGT and CYP11B1 gene polymorphisms. Efficiency of T-ARMS-PCR was tested by genotyping 776 human samples. These T-ARMS-PCR assays were also validated by Sanger DNA sequencing, where 100% concordance was found, allowing the efficient use of these T-ARMS-PCR assays for polymorphism genotyping in AGT and CYP11B1 in resource limited settings. T-ARMS-PCR is low-cost, efficient and reliable assay for genotyping of AGT and CYP11B1 gene polymorphisms.


PCR assay T-ARMS-PCR Genotyping RAAS AGT CYP11B1 



This study was supported by the Higher Education Commission (HEC), Pakistan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the institutional (National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan) ethics review committee.

Human and animal rights

All procedures were in accordance with the guidelines of Declaration of Helsinki for working with human subjects.

Informed consent

Informed consent was obtained from all individual participants included in this study.

Supplementary material

11033_2018_4493_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 19 KB)


  1. 1.
    Navar LG (2014) Physiology: hemodynamics, endothelial function, renin–angiotensin–aldosterone system, sympathetic nervous system. JASH 8(7):519PubMedGoogle Scholar
  2. 2.
    WHO (2013) A global brief on hypertension: silent killer, global public health crisis. 2:1–40Google Scholar
  3. 3.
    Hussain M, Awan FR (2018) Hypertension regulating angiotensin peptides in the pathobiology of cardiovascular disease. Clin Exp Hypertens 40:344–352CrossRefGoogle Scholar
  4. 4.
    Jia E-Z, Xu Z-X, Guo C-Y, Li L, Gu Y, Zhu T-B, Wang L-S, Cao K-J, Ma W-Z, Yang Z-J (2012) Renin–angiotensin–aldosterone system gene polymorphisms and coronary artery disease: detection of gene–gene and gene–environment interactions. Cell Physiol Biochem 29(3–4):443–452CrossRefGoogle Scholar
  5. 5.
    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):1077–1089CrossRefGoogle Scholar
  6. 6.
    Srivastava K, Chandra S, Bhatia J, Narang R, Saluja D (2012) Association of angiotensinogen (M235T) gene polymorphism with blood pressure lowering response to angiotensin converting enzyme inhibitor (Enalapril). J Pharm Pharm Sci 15(3):399–406CrossRefGoogle Scholar
  7. 7.
    Wu C, Luo J-L, Tsai C, Huang Y, Cheng C, Lee J, Lin L, Lin J, Hwang J, Chiang F (2010) Demonstrating the pharmacogenetic effects of angiotensin-converting enzyme inhibitors on long-term prognosis of diastolic heart failure. Pharmacogenom J 10(1):46CrossRefGoogle Scholar
  8. 8.
    Simonyte S, Kuciene R, Medzioniene J, Dulskiene V, Lesauskaite V (2017) Renin-angiotensin system gene polymorphisms and high blood pressure in Lithuanian children and adolescents. BMC Med Genet 18(1):100CrossRefGoogle Scholar
  9. 9.
    Ji L, Li J, Yao B, Cai X, Shen Q, Xu J (2017) Are genetic polymorphisms in the renin–angiotensin–aldosterone system associated with essential hypertension? Evidence from genome-wide association studies. J Hum Hypertens 31(11):695CrossRefGoogle Scholar
  10. 10.
    Collins A, Ke X (2012) Primer1: primer design web service for tetra-primer ARMS-PCR. Open Bioinform J 6(1):55–58CrossRefGoogle Scholar
  11. 11.
    Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 35(suppl_2):W43–W46CrossRefGoogle Scholar
  12. 12.
    Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO (2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36(suppl_2):W163–W169CrossRefGoogle Scholar
  13. 13.
    Medrano RFV, de Oliveira CA (2014) Guidelines for the tetra-primer ARMS–PCR technique development. Mol Biotechnol 56(7):599–608Google Scholar
  14. 14.
    Hussain M, Awan FR, Gujjar A, Hafeez S, Islam M (2018) A case control association study of ACE gene polymorphism (I/D) with hypertension in Punjabi population from Faisalabad, Pakistan. Clin Exp Hypertens 40(2):186–191CrossRefGoogle Scholar
  15. 15.
    Rajput-Williams J, Wallis S, Yarnell J, Bell G, Knott T, Sweetnam P, Cox N, Miller N, Scott J (1988) Variation of apolipoprotein-B gene is associated with obesity, high blood cholesterol levels, and increased risk of coronary heart disease. Lancet 332(8626–8627):1442–1446CrossRefGoogle Scholar
  16. 16.
    Timasheva YR, Nasibullin TR, Tuktarova IA, Erdman VV, Mustafina OE (2018) CXCL13 polymorphism is associated with essential hypertension in Tatars from Russia. Mol Biol Rep 45:1557–1564CrossRefGoogle Scholar
  17. 17.
    Bayram B, Sayın E, Güneş HV, Değirmenci İ, Türkoğlu Z, Doganer F, Coşan DT (2011) DD genotype of ace gene I/D polymorphism is associated in a Turkish study population with osteoarthritis. Mol Biol Rep 38(3):1713–1716CrossRefGoogle Scholar
  18. 18.
    Liu X, Jiang C, Yang P (2017) Association of single nucleotide polymorphisms in the 5′-upstream region of the C4BPA gene with essential hypertension in a northeastern Han Chinese population. Mol Med Rep 16(2):1289–1297CrossRefGoogle Scholar
  19. 19.
    Mahdieh N, Rabbani B (2013) An overview of mutation detection methods in genetic disorders. Iran J Pediatr 23(4):375PubMedPubMedCentralGoogle Scholar
  20. 20.
    Martín V, Perales C, Fernández-Algar M, Dos Santos HG, Garrido P, Pernas M, Parro V, Moreno M, García-Pérez J, Alcamí J (2016) An efficient microarray-based genotyping platform for the identification of drug-resistance mutations in majority and minority subpopulations of HIV-1 quasispecies. PLoS ONE 11(12):e0166902CrossRefGoogle Scholar
  21. 21.
    Matsuda K (2017) PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement. In: Advances in clinical chemistry, vol 80. Elsevier, pp 45–72Google Scholar
  22. 22.
    Li X, Huang Y, Guan Y, Zhao M, Li Y (2007) A novel one cycle allele specific primer extension—molecular beacon displacement method for DNA point mutation detection with improved specificity. Anal Chim Acta 584(1):12–18CrossRefGoogle Scholar
  23. 23.
    Islam M, Awan FR, Baig SM (2014) Development of ARMS-PCR assay for genotyping of Pro12Ala SNP of PPARG gene: a cost effective way for case–control studies of type 2 diabetes in developing countries. Mol Biol Rep 41(9):5585–5591CrossRefGoogle Scholar
  24. 24.
    Fazelnia S, Farazmandfar T, Hashemi-Soteh SMB (2016) Significant correlation of angiotensin converting enzyme and glycoprotein IIIa genes polymorphisms with unexplained recurrent pregnancy loss in north of Iran. Int J Reprod BioMed 14(5):323CrossRefGoogle Scholar
  25. 25.
    Heidari MM, Sheikholeslami M, Yavari M, Khatami M, Seyedhassani SM (2017) The association of renin–angiotensinogen system genes polymorphisms and idiopathic recurrent pregnancy loss. Hum Fertil. CrossRefGoogle Scholar
  26. 26.
    Khatami M, Heidari MM, Hadadzadeh M, Scheiber-Mojdehkar B, SANI MB, Houshmand M (2017) Simultaneous genotyping of the rs4762 and rs699 polymorphisms in angiotensinogen gene and correlation with Iranian CAD patients with novel hexa-primer ARMS-PCR. Iran J Public Health 46(6):811PubMedPubMedCentralGoogle Scholar
  27. 27.
    Bru D, Martin-Laurent F, Philippot L (2008) Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appl Environ Microbiol 74(5):1660–1663CrossRefGoogle Scholar
  28. 28.
    Huang M-M, Arnheim N, Goodman MF (1992) Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res 20(17):4567–4573CrossRefGoogle Scholar
  29. 29.
    Simsek M, Adnan H (2000) Effect of single mismatches at 3′-end of primers on polymerase chain reaction. J Sci Res Med Sci 2(1):11PubMedPubMedCentralGoogle Scholar
  30. 30.
    Kwok S, Kellogg D, McKinney N, Spasic D, Goda L, Levenson C, Sninsky J (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18(4):999–1005CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Diabetes and Cardio-Metabolic Disorders Laboratory, Health Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  2. 2.Pakistan Institute of Engineering and Applied Sciences (PIEAS)NilorePakistan

Personalised recommendations