ABCA1 and ABCG1 DNA methylation in epicardial adipose tissue of patients with coronary artery disease

Results DNA methylation analysis

DNA was isolated from ~ 50 mg of EAT (Epicardial Adipose Tissue) and SAT (Subcutaneous Adipose Tissue) samples using standard proteinase K digestion and phenol-chloroform extraction method. DNA quality and quantity were analyzed using the NanoDrop 8000 UV–Vis spectrophotometer (Thermo Scientific; Waltham, MA). DNA methylation status of ABCA1 and ABCG1 CpG-sites was assessed by pyrosequencing using PyroMark Q24 (Qiagen) according to the manufacturer’s instructions. Biotin-labeled single-stranded amplicons were retrieved and subjected to pyrosequencing with use of 0.3 μM sequencing primer, according to the manufacturer’s protocol. The percent of methylation for each of the CpG sites within the target sequence was calculated using PyroQ CpG Software (Qiagen). Non-CpG cytosine residues were used as built-in controls to verify bisulfite conversion. Each sample was tested in two replicates and their average was used in the statistical analysis.

Genes and primers for pyrosequencing

Gene Genomic position of CpG sites* (GCRh37/hg19) Primer sequence ABCA1*

chr9:107,690,762; chr9:107,690,770 (cg14019050); chr9:107,690,773; chr9:107,690,791; chr9:107,690,797 5’-AACAAATTCCACTAATACCCTTAACT-3’ 5’-biotin-GGGTGGAGGGTATAGTAGGT-3’ Seq 5’-AACAAATTCCACTAATACCCTTAACT-3’ [1]

ABCG1

chr21:43,642,336; chr21:43,642,354;chr21:43,642,367 (cg27243685) 5’-TGAGTTTAGGAGGTTAAGGAGAAATT-3’ 5’-biotin-CAAATAAACCAACAACAAAACAATAC-3’ Seq 5’-TGAGTTTAGGAGGTTAAGGA-3’

chr21:43,656,587 (cg06500161); chr21:43,656,590 5'-GTAAGGTTTGGGGTTATTTTAGTGG-3’ 5’-biotin-AAAACAAACCCTTAAATCTCTTTACAT-3’ Seq 5’-GAGATTAGGGTTTTTTTTAGATA-3’ [2]

*For ABCA1 promoter region average methylation level of promoter 5 CpG-sites was calculated.

Methylation_ABCA1_ABCG1.xlsx -- DNA methylation status of ABCA1 and ABCG1 CpG-sites

Pyro_Results (folder) -- Examples of pyrograms for each locus

[1]. Guay SP, Brisson D, Munger J, Lamarche B, Gaudet D, Bouchard L ABCA1 gene promoter DNA methylation is associated with HDL particle profile and coronary artery disease in familial hypercholesterolemia. Epigenetics. 2012 May;7(5):464-72. doi: 10.4161/epi.19633.

[2].Tasnim Dayeh, Tiinamaija Tuomi, Peter Almgren, Alexander Perfilyev, Per-Anders Jansson, Vanessa D. de Mello, Jussi Pihlajamäki, Allan Vaag, Leif Groop, Emma Nilsson, and Charlotte Linga DNA methylation of loci within ABCG1 and PHOSPHO1 in blood DNA is associated with future type 2 diabetes risk. Epigenetics. 2016 Jul; 11(7): 482–488. doi: 10.1080/15592294.2016.1178418