Severity of coronary artery disease is associated with diminished circANRIL expression: A possible blood based transcriptional biomarker in East Africa

Abstract

Antisense Noncoding RNA in the INK4 Locus (ANRIL) is the prime candidate gene at Chr9p21, the well-defined genetic risk locus associated with coronary artery disease (CAD). ANRIL and its transcript variants were investigated for the susceptibility to CAD in adipose tissues (AT) and peripheral blood mononuclear cells (PBMCs) of the study group and the impact of 9p21.3 locus mutations was further analysed. Expressions of ANRIL, circANRIL (hsa_circ_0008574), NR003529, EU741058 and DQ485454 were detected in epicardial AT (EAT) mediastinal AT (MAT), subcutaneous AT (SAT) and PBMCs of CAD patients undergoing coronary artery bypass grafting and non-CAD patients undergoing heart valve surgery. ANRIL expression was significantly upregulated, while the expression of circANRIL was significantly downregulated in CAD patients. Decreased circANRIL levels were significantly associated with the severity of CAD and correlated with aggressive clinical characteristics. rs10757278 and rs10811656 were significantly associated with ANRIL and circANRIL expressions in AT and PBMCs. The ROC-curve analysis suggested that circANRIL has high diagnostic accuracy (AUC: 0.9808, cut-off: 0.33, sensitivity: 1.0, specificity: 0.88). circANRIL has high diagnostic accuracy (AUC: 0.9808, cut-off: 0.33, sensitivity: 1.0, specificity: 0.88). We report the first data demonstrating the presence of ANRIL and its transcript variants expressions in the AT and PBMCs of CAD patients. circANRIL having a synergetic effect with ANRIL plays a protective role in CAD pathogenesis. Therefore, altered circANRIL expression may become a potential diagnostic transcriptional biomarker for early CAD diagnosis.

Keywords: 9p21.3; ANRIL; circANRIL; coronary artery disease; hsa_circ_0008574; peripheral mononuclear blood cells; polymorphism; transcriptional biomarker; visceral adipose tissue.

FIGURE 1

Relative expression levels of ANRIL, NR003529, EU741058, DQ485454 and cicrANRIL in EAT, MAT, SAT and in PBMCs among subgroup includes 25 CAD patients undergoing CABG and 25 non‐CAD patients undergoing valve placement. (A) Relative expression levels of ANRIL in CAD and non‐CAD patients; (B) Relative expression levels of NR003529 in CAD and non‐CAD patients; (C) Relative expression levels of EU741058 in CAD and non‐CAD patients; (D) Relative expression levels of DQ485454 in CAD and non‐CAD patients and (E) Relative expression levels of circANRIL in CAD and non‐CAD patients. AU, arbitrary unit; PDMC, peripheral blood mononuclear cells. *p < 0.05.

FIGURE 2

Association of expression levels of circANRIL with severity of coronary artery disease. The analyses were done in subgroup CAD patients that were divided according to the severity of the disease.

FIGURE 3

Feature importance analysis of included variables obtained from a tuned random forest model in Tanzanian CAD patients. BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure.

FIGURE 4

ROC curve analyses of the diagnostic value of expression levels of ANRIL and circANRIL. ROC curve comparisons between models. (A) Clinical model and clinical + ANRIL expression model, (B) clinical model and clinical + circANRIL expression model. AUC, area under curve; ROC, receiver operating characteristic.

FIGURE 5

The possible effect mechanism of the genotype of 9p21.3of linear ANRIL and circANRIL functions according to our results. The risk alleles lead to up‐regulation of ANRIL and down‐regulation of circANRIL. Increased ANRIL expression regulated of the expression of adjacent protein‐coding genes, including CDKN2A and CDKN2B leading to pro‐atherogenic cell properties (increased cell adhesion, increased proliferation and decreased apoptosis) through CBX7 and SUZ12 function in the polycomb complex., circANRIL binds PES1 protein and impairs ribosome biogenesis, leading to activation of p53 and a subsequent increase in apoptosis and a decrease in proliferative rate. PES1, pescadillo ribosomal biogenesis factor 1; CBX7, Chromobox homologue 7; p53, tumour protein 53; SUZ12, polycomb repressive complex 2 subunit.