DEVELOPMENT OF A RESTRICTION FRAGMENT LENGTH POLYMORPHISM METHOD FOR GENOTYPING THE ATP2B1 RS2681492 POLYMORPHISM IN VIETNAMESE POPULATION

Thi Thuy Nga BUI1, Thi Thanh Huyen NGUYEN2, Anh Ngoc NGUYEN1, Quang Binh TRAN1,
1 National Institute of Nutrition, Vietnam
2 Hanoi University of Pharmacy

Main Article Content

Abstract

Aims: The rs2681492, a single nucleotide polymorphism in the ATP2B1 gene, has been reported to be associated with cardiovascular diseases and traits including blood pressure, chronic heart failure, and hypertension. The study aimed to develop a PCR-RFLP genotyping method for rs2681492 among the Vietnamese population.


Methods and Results: The Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method incorporated optimizations of primers, thermal cycles, and restriction enzymes in a novel restriction fragment length polymorphism (RFLP) approach. To evaluate the accuracy of the PCR-RFLP method, three different genotypes (AA, AG, and GG) were confirmed by gene sequencing. The experimental results determined the annealing temperature of 55°C for PCR and the minimum of 3 units of restriction enzyme HaeII for incubation with the PCR product.


Conclusion: This optimal PCR-RFLP method was successfully applied to genotype ATP2B1 rs2681492 in large populations to investigate the association between the rs2681492 polymorphism and cardiovascular diseases.

Article Details

References

1. World Health Organization. World Health Statistics 2008. Accessed August 5, 2023 at: https://www.who.int/docs/default-source/gho-documents/world-health-statistic-reports/en-whs08-full.pdf
2. The top 10 causes of death. Accessed August 10, 2023 at: https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death.
3. Whelton PK. Epidemiology of hypertension. The Lancet. 1994 ;344(8915):101–106.
4. Levy D, Ehret GB, Rice K, et al. Genome-wide association study of blood pressure and hypertension. Nature Genetics. 2009;41(6):677–687.
5. Palmgren M.G. and Axelsen K.B. (1998). Evolution of P-type ATPases. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1365(1):37–45.
6. Di Leva F, Domi T, Fedrizzi L, et al. The plasma membrane Ca2+ ATPase of animal cells: Structure, function, and regulation. Archives of Biochemistry and Biophysics. 2008;476(1):65–74.
7. Wang Y, Zhang Y, Li Y, et al. Common variants in the ATP2B1 gene are associated with hypertension and arterial stiffness in the Chinese population. Mol Biol Rep. 2013; 40(2):1867–1873.
8. Hamajima N, Saito T, Matsuo K, et al. Competitive Amplification and Unspecific Amplification in Polymerase Chain Reaction with Confronting Two-Pair Primers. The Journal of Molecular Diagnostics. 2002;4(2):103–107.
9. Nguyen Thi Thanh Huyen, Thesis of Master on Pharmacology, Hanoi University of Pharmacy, Vietnam.
10. The 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature. 2015;526:68–74.
11. Althwab SA, Ahmed AA, Rasheed Z, Alkhowailed M, Hershan A, Alsagaby S, Abdulmonem WA. ATP2B1 genotypes rs2070759 and rs2681472 polymorphisms and risk of hypertension in Saudi population. Nucleosides, Nucleotides & Nucleic Acids. 2021;40(11):1075–1089.