Differential expression profiles analysis of DNA methylation between “disease” and “syndrome” in coronary heart disease-induced unstable angina patients with Qi deficiency and blood stasis syndrome

Abstract

Objective

To explore the differential expression profiles of DNA methylation sites/regions and potential molecular mechanisms in the peripheral blood of coronary heart disease (CHD)-induced unstable angina pectoris patients with or without Qi deficiency and blood stasis syndrome, and to provide scientific evidence for the conbination of disease and syndrome.

Methods

According to the pre-determined inclusion and exclusion criteria, the study subjects were enrolled and divided into two groups namely CHD-induced unstable angina group (G group) and healthy control group (J group) to conduct “disease” analysis, while G group was further divided into Qi deficiency and blood stasis syndrome group (case group) and non-Qi deficiency blood stasis syndrome group (control group) to perform “syndrome” analysis. The general data and clinical information of the study subjects were collected. The peripheral venous blood was extracted on an empty stomach, and the Illumina Infinium MethylationEPIC BeadChip (850K methylation chip) was used to detect the differential expression profiles of DNA methylation in each group, ChAMP software (V 2.14.0) was used for the differential methylation data analysis, with a threshold of the adjusted P value (adj.P.val) < 0.01. Gene Ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) were employed for the functional and pathway enrichment analyses of related mapped genes.

Results

A total of 263 differentially methylated CpG positions (DMPs) were screened out between G and J groups, including 191 hypermethylated positions such as cg05845204 and cg08906898, and 72 hypomethylated positions such as cg26919182 and cg13149459. These positions were mainly mapped to 148 genes encompassing RNA binding motif protein 39 (RBM39), acetyl-CoA acyltransferase 2 (ACAA2), protein phosphatase 1 regulatory subunit 12B (PPP1R12B), and the dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2). GO functional enrichment analysis revealed that the genes of the DMPs were primarily enriched in protein localization to chromosomes, regulation of cell morphogenesis, negative regulation of calcium-mediated signals, etc. KEGG pathway analysis suggested that the genes were mainly enriched in fatty acid metabolism and endocytosis pathways. In addition, a total of 23 differential methylation regions (DMRs) were identified, with overlapping genes such as transmembrane protein 232 (TMEM232), ribosomal protein large P1 (RPLP1), peroxisomal biogenesis factor 10 (PEX10), and forkhead box N3 (FOXN3) recognized. It was found that GO functions were mainly enriched in the negative regulation of Ras protein signal transduction, small GTPase-mediated signal transduction, negative regulation, etc. A total of 1 703 differential methylation sites were screened out between case and control groups, including 444 increased methylation positions such as cg05573767 and 1 259 decreased methylationpositions such as cg19938535, and cg03893872. These positions were mapped to 1108 genes such as ribosomal protein S6 kinase A2 (RPS6KA2), leucine rich repeat containing 16A (LRRC16A), and hedgehog acyltransferase (HHAT). According to the GO functional enrichment analysis, the genes relating to the DMPs were mainly enriched in biological functions such as transmembrane receptor protein serine/threonine kinase signaling pathway and axonogenesis. The KEGG pathway enrichment analysis suggested the involvement of Rap1 signaling pathway, adenosine 5’-monophosphate-activated protein kinase (AMPK) signaling pathway, etc. A total of 21 DMRs were identified, including 22 overlapping genes such as mucin 4 (MUC4), three prime repair exonuclease 1 (TREX1), and LIM homeobox 6 (LHX6). GO analysis demonstrated that the genes primarily participated in molecular functions such as positive regulation of transmembrane transport, regulation of fatty acid metabolism, and copper ion binding.

Conclusion

This study reveals the methylation patterns of DMPs and DMRs in patients with Qi deficiency and blood stasis syndrome caused by CHD-induced unstable angina pectoris. Potential epigenetic regulation of fatty acid metabolism, Rap1 signaling, and other molecular functions are involved in the development of CHD between the "disease" and "syndrome".