Background Cardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography. Surprisingly, despite well-established clinical indications, up to 40% of the one million invasive cardiac catheterizations return a result of ‘no blockage’. The present studies employed RNA sequencing of whole blood to identify an RNA signature in patients with angiographically confirmed CAD. Methods Whole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by single-molecule sequencing of RNA (RNAseq) to identify transcripts associated with CAD (TRACs) in a discovery group of 96 patients presenting for elective coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs). Results Surprisingly, 98% of DEGs/TRACs were down-regulated ~ 1.7-fold in patients with mild to severe CAD (> 20% stenosis). The TRACs were independent of comorbid risk factors for CAD, such as sex, hypertension, and smoking. Bioinformatic analysis identified an enrichment in transcripts such as FoxP1, ICOSLG, IKZF4/Eos, SMYD3, TRIM28, and TCF3/E2A that are likely markers of regulatory T cells (Treg), consistent with known reductions in Tregs in CAD. A validation cohort of 80 patients confirmed the overall pattern (92% down-regulation) and supported many of the Treg-related changes. TRACs were enriched for transcripts associated with stress granules, which sequester RNAs, and ciliary and synaptic transcripts, possibly consistent with changes in the immune synapse of developing T cells. Conclusions These studies identify a novel mRNA signature of a Treg-like defect in CAD patients and provides a blueprint for a diagnostic test for CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.

RATIONALE: Cardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography. Surprisingly, despite well-established clinical indications, up to 40% of the one million invasive cardiac catheterizations return a result of ‘no blockage’. OBJECTIVE: The present studies employed RNA sequencing of whole blood to identify an RNA signature in patients presenting with a clinical suspicion of CAD.METHODS AND RESULTS: Whole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by single-molecule sequencing of RNA (RNAseq) to identify transcripts associated with CAD (TRACs) in a discovery group of 96 patients presenting for elective coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs). Surprisingly, 98% of DEGs/TRACs were down-regulated ~1.7-fold in patients with mild to severe CAD (>20% stenosis). The TRACs were independent of comorbid risk factors for CAD, such as gender, hypertension, and smoking. Bioinformatic analysis identified an enrichment in transcripts such as FoxP1, ICOSLG, IKZF4/Eos, SMYD3, TRIM28, and TCF3/E2A that are likely markers of regulatory T cells (Treg), consistent with known reductions in Tregs in CAD. A validation cohort of 80 patients confirmed the overall pattern (92% down-regulation) and supported many of the Treg-related changes. TRACs were enriched for transcripts associated with stress granules, which sequester RNAs, and ciliary and synaptic transcripts, possibly consistent with changes in the immune synapse of developing T cells.CONCLUSIONS: These studies identify a novel mRNA signature of a Treg-like defect in CAD patients and provides a blueprint for a diagnostic test for CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.