Abstract 4141501: 9p21.3 variants drive coronary calcification by suppressing statherin expression

Background: Genetic variants on chromosome 9 at p21.3 confer a heightened risk of atherosclerosis and coronary artery calcification. The 9p21.3 variants cause the selective expression of non-coding RNA transcripts (ANRIL) that regulate many genes through ALU element mediated silencing. However, to date, none of these regulated genes account for the effect of 9p21.3 variants on coronary calcification. Methods and Results: To identify genes differentially regulated by the 9p21.3 risk variants, primary human aortic smooth muscle cells (HAoSMCs) from 11 different donors genotyped for 9p21.3 risk variants (n=3-4/genotype) were analyzed for whole-genome expression using microarrays. We identified the mRNA for the hydroxyapatite-binding protein statherin as the most impacted by 9p21.3 genotypes—its mRNA was silenced in carriers homozygous for risk variants. Transfection of ANRIL with an expression plasmid bearing the ALU element from the first intron of the Statherin gene silenced statherin expression. HAoSMCs also express another hydroxyapatite binding protein called matrix GLA protein (MGP). Mice lack a native statherin gene, and if they lose the Mgp gene, they develop severe arterial calcification and die of aortic rupture. In contrast, humans with MGP deficiency (aka Keutel syndrome) rarely develop calcified arteries, suggesting that statherin might compensate for MGP loss. As has been reported for MGP, statherin was found to inhibit bone morphogenic protein 4 (BMP4)-dependent activation of a BMP-responsive luciferase reporter, suggesting functional redundancy of statherin and MGP to inhibit arterial calcification. Lentiviral vectors expressing human statherin blocked calcification of primary aortic smooth muscle cells from Mgp KO mice. Furthermore, expression of human statherin from a transgene conditionally recombined only in smooth muscle cells prevented arterial calcification in Mgp KO mice. Conclusion: The 9p21.3 risk variants expedite arterial calcification by suppressing statherin expression. Since microcalcifications hasten atherosclerosis, targeting this statherin-dependent mechanism could reduce atherosclerotic burden and benefit up to 75% of individuals of European and Asian ancestry that carry the 9p21.3 risk variants.