Deoxyribonucleic acid methylation in the enhancer region of the CDKN2A/2B and CDKN2B-AS1 genes in blood vessels and cells in patients with carotid atherosclerosis

Aim. Comparative analysis of the deoxyribonucleic acid (DNA) methylation level in the enhancer region of the CDKN2A/2B and CDKN2B-AS1 genes (9p21.3 locus) in vessels with/without atherosclerotic lesions, as well as in leukocytes of patients with clinically relevant carotid artery (CA) atherosclerosis and healthy individuals.

Material and methods. The group of patients with clinically relevant atherosclerosis included 22 individuals with severe stenosis (>80%) of CA. Samples of atherosclerotic plaques, presenting CA regions, and great saphenous veins, as well as peripheral blood samples (leukocytes) were obtained from patients. The control group consisted of 14 individuals with the mild CA stenosis (£24%) and without hemodynamically relevant changes; peripheral blood samples were obtained from each of them. DNA methylation level was assessed by targeted bisulfite sequencing of amplicons.

Results. The tissue-specific methylation of 31 CpG-site in the CDKN2A/2B and CDKN2B-AS1 gene enhancer was established: the vascular tissues significantly differed from the peripheral blood leukocytes. At the same time, there was an increase in the methylation level of both certain CpG sites and whole analyzed CA region affected by atherosclerosis (48,6 [34,8; 62,0]%), compared with intact vessels, both arteries (25,2 [23,1; 41,60]%, p=0,0001) and veins (35,0 [31,6; 40,0]%, p=0,0039). Patients had lower methylation levels in all CpG sites in blood leukocytes compared to blood vessel samples (8,7 [6,1; 9,7]%; p<0,05). At the same time, the level of DNA methylation in the blood leukocytes of atherosclerotic patients does not differ from that in healthy individuals (9,3 [8,3; 13,6]%; p>0,8).

Conclusion. In the present study, the relationship between an increase in the DNA methylation in the enhancer of the CDKN2A/2B and CDKN2B-AS1 genes in CA and their atherosclerotic lesions was revealed, as well as the tissue-specific DNA methylation between vessels and peripheral blood leukocytes.

1. GWAS Catalog. Region: 9p21.3. https://www.ebi.ac.uk/gwas/regions/9p21.3 (9 August 2020)

2. Motterle A, Pu X, Wood H, et al. Functional analyses of coronary artery disease associated variation on chromosome 9p21 in vascular smooth muscle cells. Human molecular genetics. 2012;21(18)4021-9. doi:10.1093/hmg/dds224.

3. Almontashiri NAM. The 9p21.3 risk locus for coronary artery disease: A 10-year search for its mechanism. Journal of Taibah University Medical Sciences. 2017;12(3):199-204. doi:10.1016/j.jtumed.2017.03.001.

4. Dechamethakun S, Muramatsu M. Long noncoding RNA variations in cardiometabolic diseases. Journal of human genetics. 2017;62(1):97-104. doi:10.1038/jhg.2016.70

5. Navarro E, Mallén A, Cruzado JM, et al. Unveiling ncRNA regulatory axes in atherosclerosis progression. Clinical and translational medicine. 2020;9(1):5. doi:10.1186/s40169-020-0256-3.

6. Aavik E, Babu M, Ylä-Herttuala S. DNA methylation processes in atherosclerotic plaque. Atherosclerosis. 2019;281:168-179. doi:10.1016/j.atherosclerosis.2018.12.006.

7. Clermont PL, Parolia A, Liu HH, et al. DNA methylation at enhancer regions: Novel avenues for epigenetic biomarker development. Frontiers in bioscience (Landmark edition). 2016;21:430-46. doi:10.2741/4399.

8. Nazarenko MS, Markov AV, Lebedev IN, et al. [Methylation profile of INK4B-ARF-INK4A locus in atherosclerosis]. Genetika. 2013;49(6):783-7. (In Russ.) doi:10.7868/s0016675813060076.

9. Zhou S, Cai B, Zhang Z, et al. CDKN2B Methylation and Aortic Arch Calcification in Patients with Ischemic Stroke. Journal of atherosclerosis and thrombosis. 2017;24(6):609-620. doi:10.5551/jat.36897.

10. Zhou S, Zhang Y, Wang L, et al. CDKN2B methylation is associated with carotid artery calcification in ischemic stroke patients. Journal of translational medicine. 2016;14(1):333. doi:10.1186/s12967-016-1093-4.

11. GeneCards®: The Human Gene Database. http://www.genecards.org

12. MethPrimer. http://www.urogene.org/cgi-bin/methprimer2/MethPrimer.cgi

13. Helgeland Ø, Hertel JK, Molven A, et al. The Chromosome 9p21 CVD- and T2D-Associated Regions in a Norwegian Population (The HUNT2 Survey). International journal of endocrinology. 2015;2015:164652. doi:10.1155/2015/164652.

14. Xu JJ, Jiang L, Xu LJ, et al. Association of CDKN2B-AS1 Polymorphisms with Premature Triple-vessel Coronary Disease and Their Sex Specificity in the Chinese Population. Biomedical and environmental sciences. 2018;31(11):787-796. doi:10.3967/bes2018.106.

15. Angeloni A, Bogdanovic O. Enhancer DNA methylation: implications for gene regulation. Essays Biochem. 2019;63(6):707-715. doi:10.1042/EBC20190030.

16. Aavik E, Lumivuori H, Leppänen O, et al. Global DNA methylation analysis of human atherosclerotic plaques reveals extensive genomic hypomethylation and reactivation at imprinted locus 14q32 involving induction of a miRNA cluster. European heart journal. 2015;36(16):993-1000. doi:10.1093/eurheartj/ehu437.

17. Murray R, Bryant J, Titcombe P, et al. DNA methylation at birth within the promoter of ANRIL predicts markers of cardiovascular risk at 9 years. Clinical epigenetics. 2016;8(1):90. doi:10.1186/s13148-016-0259-5.

18. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arteriosclerosis, thrombosis, and vascular biology. 1995;15(9):1512-31. doi:10.1161/01.atv.15.9.1512.

19. Zaina S, Gonçalves I, Carmona FJ, et al. DNA methylation dynamics in human carotid plaques after cerebrovascular events. Arteriosclerosis, thrombosis, and vascular biology. 2015;35(8):1835-42. doi:10.1161/ATVBAHA.115.305630.

20. Nazarenko MS, Markov AV, Lebedev IN, et al. A comparison of genome-wide DNA methylation patterns between different vascular tissues from patients with coronary heart disease. PLoS One. 2015;10(4):e0122601. doi:10.1371/journal.pone.0122601.

21. Zaina S, Heyn H, Carmona FJ, et al. DNA methylation map of human atherosclerosis. Circulation. Cardiovascular genetics. 2014;7(5):692-700. doi:10.1161/CIRCGENETICS.113.000441.

22. Istas G, Declerck K, Pudenz M, et al. Identification of differentially methylated BRCA1 and CRISP2 DNA regions as blood surrogate markers for cardiovascular disease. Scientific reports. 2017;7(1):5120. doi:10.1038/s41598-017-03434-0.

23. Soriano-Tárraga C, Jiménez-Conde J, Giralt-Steinhauer E, et al. Epigenome-wide association study identifies TXNIP gene associated with type 2 diabetes mellitus and sustained hyperglycemia. Human molecular genetics. 2016;25(3):609-19. doi:10.1093/hmg/ddv493.