Abstract Body (Do not enter title and authors here): Objective: To elucidate the molecular mechanisms by which CD73 variants contribute to inflammation and atherosclerosis in humans.
Approach and Results: To explore this knowledge gap, we conducted phenotype-wide association analysis (PheWAS) in a cohort of 25,000 patients to identify ASCVD-associated variants in the NT5E (CD73) gene in a non-biased manner. PheWAS analysis of coding variants identified that the rs200648774 single nucleotide polymorphisms in ecto-5’-nucleotidase (NT5E; CD73) that was associated with atherosclerosis (ICD-9; 440.x codes) in this cohort. The SNP rs200648774 (C-->T; MAF 0.000188) results in an Arginine (R) to Cysteine (C) substitution at amino acid 354 within the active site of CD73 (OR: 10.18, SE: 1.1; P=0.035). This Arginine residue is evolutionarily conserved across species. The variant protein is expressed but displays a loss of function (LOF; CD73 Cys354 variant). To examine the impact of the CD73 Cys354 variant, we generated a murine model using Efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) gene editing. CD73-Arg354/Arg354 (cd73^wt/wt) and CD73-Cys354/Cys354 (cd73^Δ/Δ) mice in an Ldlr^-/- background were placed on HFD for 12 weeks then sacrificed for quantification of aortic root atherosclerosis. Representative images of oil red O staining of aortic valve from CD73-Arg354/Arg354- Ldlr^-/- (cd73^wt/wt,Ldlr^-/-) mice (Fig. A; Bar= 100 um) and CD73-Cys354/Cys354 Ldlr^-/- (cd73^Δ/Δ, Ldlr^-/-) mice (Fig. B; Bar= 100 um). Quantified area of atherosclerosis in CD73-Cys354/Cys354 (cd73^Δ/Δ) aortic roots were significantly higher in CD73-Cys354/Cys354 (cd73^Δ/Δ) than in CD73-Arg354/Arg354 (cd73^wt/wt) (Fig. C; p=0.0096). The Percentage of Oil Red O staining in aortic root atherosclerotic lesions was also significantly higher in CD73-Cys354/Cys354 (cd73^Δ/Δ) aortic roots compared to CD73-Arg354/Arg354 (cd73^wt/wt) aortic roots. Together these data demonstrate that the rs200648774 single nucleotide polymorphisms resulting in cysteine substitution for arginine in the active site of CD73 results in a loss of CD73 function that also associates with increased atherosclerosis in genetically susceptible mice.
Conclusion: The LOF rs200648774 single nucleotide polymorphisms associates with atherosclerosis in humans and generation of the CD73-Cys354/Cys354 polymorphism in Ldlr^-/- mice results in increased HFD induced atherosclerosis