Genome-Wide DNA Methylation Profiling Reveals Enrichment of Developmental Vascular and Muscle Programs in Peripheral Artery Disease: A Pilot Epigenome-Wide Association Study

Abstract Background: Peripheral artery disease (PAD) is a common atherosclerotic condition with incompletely understood molecular drivers. DNA methylation is a key epigenetic regulator influenced by smoking, aging, and metabolic risk factors, yet genome-wide methylation patterns specifically associated with PAD remain understudied. Objective: To characterize genome-wide DNA methylation patterns associated with peripheral artery disease (PAD) using the Infinium Methylation EPIC v2.0 platform. Design: Single-center pilot epigenome-wide association study using pooled whole blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Ten adults patients diagnosed with PAD and 22 healthy controls. Interventions: None. Methods: In this prospective single-center pilot study, we performed genome-wide DNA methylation profiling on blood DNA from 10 clinically diagnosed patients with PAD (mean age 72.7 {+/-} 11.2 years; 50% female) and 22 age- and sex ratio-matched controls (mean age 74.6 {+/-} 7.8) using the Illumina Infinium MethylationEPIC v2.0 BeadChip (>935,000 CpG sites). Data was processed with the SeSAMe pipeline. Differentially methylated loci (DMLs) and regions (DMRs) were identified at FDR [≤] 0.01 and |{Delta}{beta}| [≥] 0.10. Enrichment analysis of associated genes was performed using clusterProfiler (Gene Ontology, GO, and Kyoto Encyclopedia of Genes and Genomes, KEGG, computational methods). Results: Following quality control, 933,942 probes were analyzed. We observed that a majority of significant DMLs exhibited hypomethylation in PAD. Intersection of DML- and DMR-supported genes yielded 16,854 high-confidence genes. GO analysis revealed a strong enrichment for muscle system processes, regulation of membrane potential, embryonic organ development, DNA-binding transcription activator activity, actin binding, and metal ion transmembrane transporter activity. Cellular component terms highlighted cell leading edge, focal adhesion, and cell cortex. KEGG pathways were dominated by calcium signaling, cadherin signaling, MAPK signaling, and cytoskeleton in muscle cells. Conclusions: Blood DNA methylation patterns in a cohort of PAD patients are enriched in developmental vascular, muscle, and cell-motility programs that are characteristically reactivated in adult vascular pathology (endothelial dysfunction, vascular smooth muscle cell (VSMC) phenotypic switching, pathological angiogenesis). These findings generate a hypothesis for biomarker development and epigenetic therapeutic targeting in PAD. Larger longitudinal and multi-ethnic validation studies are warranted.