Biopolym. Cell. 2024; 40(4):314-318.
Short Communications
Modified method for analysis of InDel ACE1 gene polymorphism using the temperature melting curves of PCR products
- Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143 - Taras Shevchenko National University of Kyiv
64, Volodymyrska Str., Kyiv, Ukraine, 01601 - Ukrainian center of мaternity and childhood, NAMS of Ukraine
8, Platona Mayborody Str., Kyiv, Ukraine, 04050 - D. F. Chebotarev Institute of Gerontology, NAMS of Ukraine
67, Vyshgorodska Str., Kyiv, Ukraine, 04114
Abstract
Aim. The ACE1 gene is a key to the renin–angiotensin system, as it encode the ACE1 enzyme, which regulates blood pressure and water–electrolyte balance. The InDel (I/D) polymorphism of the ACE1 gene causes a different level of enzyme synthesis and is associated with various pathological conditions, including the course of COVID–19. The aim of our study was to develop an efficient method for the analysis of I/D polymorphism of the ACE1 gene by analyzing the melting temperature curves of specific PCR products. Methods. Genotyping was carried out by the PCR method using DNA isolated from peripheral blood leukocytes. The primers were designed using BLAST SEARCH and synthesized at METABION (Germany). The reaction was carried out using the "HOT FIREPol EvaGreen qPCR Mix Plus" mixture. Amplification was performed on iQ5TM Multicolor Real–Time PCR Detection System (BIO–RAD), melting curves were analyzed using BIO–RAD iQ5 Optical System Software V 2.0. Results. The proposed method, using one pair of primers, turned out to be effective for detecting genotypes based on the melting temperature curves of PCR products: the peak at 83–85°C corresponded to deletion, at 86–89°C to insertion, and in the presence of both peaks, the genotype was defined as heterozygous. Conclusions. The proposed method of genotyping the I/D polymorphism of the ACE1 gene is simple and reliable, and can be used for the analysis of genetic markers of cardiovascular pathology and the prognosis of the course of COVID–19.
Keywords: АСЕ1, InDel, melting temperature curve analysis
Full text: (PDF, in English)
References
[1]
"Physiology, Renin-Angiotensin System." John H. Fountain., Jasleen Kaur., Sarah L. Lappin 2023.
[2]
Zheng H, Cao JJ. Angiotensin-Converting Enzyme Gene Polymorphism and Severe Lung Injury in Patients with Coronavirus Disease 2019. Am J Pathol. 2020; 190(10):2013-7.
[3]
Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990; 86(4):1343-6.
[4]
Stroke genetics. Pankaj Sharma, James F. Meschia : Springer-Verlag London Ltd. 2013. 320p 10.1007/978-0-85729-209-4 ISBN (Print)0857292080, 9780857292087
[5]
Nissen PH, Campbell NB, Højskov CS, Fløe A, Hoffmann HJ, Hilberg O, Ladefoged SA, Møller HJ. Development of a high-resolution melting genotyping assay for the angiotensin I converting enzyme insertion/deletion polymorphism and establishment of genotype-specific reference intervals in a Danish population. Ann Clin Biochem. 2015; 52(Pt 1):105-12.
[6]
Chen KY, Xu JX, Wang MM, Hu D, Xie F, Huang D, Chen J, Yang T, Zhang J, Song F, Huang S, Zhong T. Single probe PCR melting curve analysis MTHFR C677T SNP sites. Anal Biochem. 2021; 619:114102.
[7]
Zobel CM, Kuhn H, Schreiner M, Wenzel W, Wendtland J, Goekeri C, Scheit L, Oltmanns K, Rauschning D, Grossegesse M, Hofmann N, Wirtz H, Spethmann S. Impact of ACE I gene insertion/deletion, A-240T polymorphisms and the renin-angiotensin-aldosterone system on COVID-19 disease. Virol J. 2024; 21(1):15.
[8]
Harashchenko TA, Umanets TR, Mohylevets AO, Gorodna OV, Krasnenkov DS, Antypkin YuH, Livshyts LA. Association of rs4646994 polymorphism of the ACE1 gene with the severity of COVID-19 in children from Ukraine. Cytol Genet. 2025. In press.
