Genotype-dependent metabolic and inflammatory regulation at the ANRIL locus in human primary macrophages

The long non-coding RNA ANRIL and several single-nucleotide polymorphisms have been strongly implicated in genetic susceptibility to coronary artery disease (CAD). However, how these variants influence gene expression remains incompletely understood. This study aims to investigate genotype-dependent differences in linear and circular ANRIL isoform expression and selected CAD- and diabetes-related genes in monocyte-derived macrophages from healthy adult males carrying rs1333049, rs10757274, and rs564398 variants. Peripheral blood monocytes from healthy male donors with specific ANRIL genotypes were isolated and differentiated into macrophages using macrophage colony-stimulating factor. Monocyte identity and macrophage differentiation were confirmed by flow cytometry and confocal imaging. Gene expression levels of CDKN2A, CDKN2B, ABCA1, PRKAA1, PPARGC1A, TNFA, TNFAIP3, and circular and linear ANRIL transcripts were quantified by qRT-PCR. Expression differences across genotype groups and genotype-dependent correlations were assessed. The rs10757274/rs1333049 AA/CC genotype was associated with reduced expression of PPARGC1A and ABCA1, accompanied by lower TNFA and higher TNFAIP3 expression. In contrast, carriage of the G allele at rs10757274/rs1333049 was associated with increased expression of CDKN2A and linear ANRIL. The rs564398 CC genotype was associated with reduced PPARGC1A expression and altered PRKAA1 levels. Correlation analyses revealed genotype-specific associations between ANRIL isoforms and selected genes. These findings suggest that ANRIL variants are associated with genotype-dependent differences in macrophage gene expression across pathways of cell cycle regulation, inflammation, lipid metabolism, and energy homeostasis. Although exploratory, the results support the possibility that CAD- and diabetes-associated 9p21.3 variants may partly exert their effects through such transcriptional alterations.