Molecular mechanisms in Primary Glaucoma: POAG-associated variant disrupts microRNA regulation of CDKN2B expression

Abstract

Purpose: To evaluate functional consequences of SNP rs3217992—which is associated with increased susceptibility to optic nerve degeneration in glaucoma in the NEIGHBOR study. One of 17 such SNPs in chromosome 9p21, its odds ratio indicates risk. It occurs in the 3’ untranslated region (UTR) of CDKN2B, encoded in an intron of CDKN2B-AS. CDKN2B inhibits cell-cycle progression via CDK4. We hypothesize the minor allele disrupts binding of a microRNA which regulates CDKN2B expression and modulates cell-cycle progression, with effect in glaucoma.

Methods: With our microRNA (miR) seed site detection algorithm ZoomMiR, we identified microRNA candidates. ZoomMiR finds all 6 to 8 base complementary matches to initial bases of every miR archived in miRbase. In CDKN2B, it found 5 seeds. SNP rs3217992 is in a predicted seed sequence for miR-138-2*. HapMap showed the SNP is in separate haplotypes from protective SNPs. GENSAT, dbEST, GEO and miRbase queries showed CDKN2B is expressed in eye and glia; and miR-138-2*, in brain. To determine to what extent the alleles and a disrupted seed sequence affect miR-138-2* ability to target CDKN2B, we used a dual-luciferase reporter assay. We used quantitative PCR to measure CDKN2B, CDKN2A, and miR levels in human neural progenitors (NP), in neurons derived from human induced pleuripotent stem cells, and in selected mouse tissues.

Results: miR-138-2* effectively targets a luciferase reporter transcript engineered to contain 100 bases surrounding the risk SNP in its 3’ UTR. Disrupting the seed increased reporter activity, demonstrating this mutation/change negatively affected miR-138-2* ability to target the reporter transcript. Importantly, introducing the minor allele into the construct likewise increased reporter activity, strongly suggesting this risk allele—through the action of miR-138-2*—alters the stability of CDKN2B transcript. Furthermore, CDKN2B and miR-138-2*, but not CDKN2A, are expressed in human NP, neurons and mouse retina. As revealed by database queries, CDKN2B is expressed in glial cells and miR-138 precursor, in glial stem cells, and this SNP in CDKN2B has been shown as protective in glioma.

Conclusions: Our results support a model in which CDKN2B regulation by miR-138-2* is important, especially in glia, and is impaired by SNP rs3217992. This molecular mechanism may underlie primary glaucoma and have implications in glia-related diseases.

Keywords: 629 optic nerve • 540 glia • 738 transcription