Clin Epigenetics. 2026 May 25. doi: 10.1186/s13148-026-02131-y. Online ahead of print.
ABSTRACT
BACKGROUND: Atrial fibrillation (AF) is a common arrhythmia affecting millions of patients globally. While epigenetic modifications play a significant role in cardiovascular diseases, their contribution to AF remains incompletely understood. Annexin A4 (ANXA4), a calcium-dependent phospholipid-binding protein, is implicated in inflammation and immune-mediated diseases, but its epigenetic regulation and mechanistic role in AF pathogenesis have not been systematically explored.
METHODS: This study employed a multi-omics approach, integrating Mendelian randomization (MR), DNA methylation quantitative trait loci (mQTL)-mediated analysis, bulk and single-cell transcriptomics, and clinical validation. Two-sample MR utilized plasma proteome quantitative trait loci data from 4759 Icelandic individuals and AF genome-wide association study summary statistics from FinnGen and OpenGWAS databases. mQTL-based mediation analysis leveraged data from 27,750 individuals, and ANXA4 promoter methylation in atrial tissue was assessed in the GSE62727 dataset. Clinical validation involved qPCR for ANXA4 mRNA and CpG sites cg11942603 and cg22792910 in peripheral blood from 130 participants (77 AF patients and 53 sinus rhythm controls). Additionally, ANXA4 protein levels in peripheral blood leukocytes were analyzed using Western Blotting. Machine learning models were used to assess diagnostic performance, and logistic regression analyzed clinical associations.
RESULTS: MR analysis provided evidence consistent with genetically predicted higher ANXA4 levels increasing AF risk (odds ratio = 1.17, 95% CI 1.11-1.23, P = 1.73 × 10-10), with Bayesian colocalization supporting a shared genetic signal at rs17037076. mQTL-based mediation analysis indicated that hypomethylation at cg11942603 and cg22792910 significantly mediated the association between ANXA4 and AF. Peripheral blood and atrial tissue analyses confirmed lower methylation levels at cg11942603 in AF patients. qPCR confirmed significantly elevated ANXA4 mRNA expression in the peripheral blood of AF patients. Western Blot analysis further revealed a significant increase in intracellular ANXA4 protein levels within peripheral blood leukocytes of AF patients (P = 0.01). Single-cell RNA sequencing showed higher ANXA4 expression across various immune cell types in AF. Larger left atrial diameter, log-transformed NT-proBNP, and ANXA4 mRNA were independently associated with AF, while higher ANXA4 methylation was associated with lower odds of AF. Machine learning models demonstrated high discriminative ability for AF diagnosis (AUC 0.949-0.962).
CONCLUSIONS: Our multi-omics approach provides evidence that hypomethylation at specific ANXA4 CpG sites is associated with increased gene expression and elevated AF risk. Increased ANXA4 mRNA and protein levels are associated with an enhanced immune response, particularly in myeloid cells, suggesting its potential as a diagnostic candidate to differentiate AF from sinus rhythm. Machine learning models further support the predictive value of ANXA4. Furthermore, decreased ANXA4 methylation levels were associated with TET2 upregulation. ANXA4 expression and promoter methylation may serve as potential diagnostic indicators and candidate targets for future precision medicine strategies in AF.
PMID:42185861 | DOI:10.1186/s13148-026-02131-y
