A Common Variant in MIR182 Is Associated With Primary Open-Angle Glaucoma in the NEIGHBORHOOD Consortium

Abstract

Purpose: Noncoding microRNAs (miRNAs) have been implicated in the pathogenesis of glaucoma. We aimed to identify common variants in miRNA coding genes (MIR) associated with primary open-angle glaucoma (POAG).

Methods: Using the NEIGHBORHOOD data set (3853 cases/33,480 controls with European ancestry), we first assessed the relation between 85 variants in 76 MIR genes and overall POAG. Subtype-specific analyses were performed in high-tension glaucoma (HTG) and normal-tension glaucoma subsets. Second, we examined the expression of miR-182, which was associated with POAG, in postmortem human ocular tissues (ciliary body, cornea, retina, and trabecular meshwork [TM]), using miRNA sequencing (miRNA-Seq) and droplet digital PCR (ddPCR). Third, miR-182 expression was also examined in human aqueous humor (AH) by using miRNA-Seq. Fourth, exosomes secreted from primary human TM cells were examined for miR-182 expression by using miRNA-Seq. Fifth, using ddPCR we compared miR-182 expression in AH between five HTG cases and five controls.

Results: Only rs76481776 in MIR182 gene was associated with POAG after adjustment for multiple comparisons (odds ratio [OR] = 1.23, 95% confidence interval [CI]: 1.11-1.42, P = 0.0002). Subtype analysis indicated that the association was primarily in the HTG subset (OR = 1.26, 95% CI: 1.08-1.47, P = 0.004). The risk allele T has been associated with elevated miR-182 expression in vitro. Data from ddPCR and miRNA-Seq confirmed miR-182 expression in all examined ocular tissues and TM-derived exosomes. Interestingly, miR-182 expression in AH was 2-fold higher in HTG patients than nonglaucoma controls (P = 0.03) without controlling for medication treatment.

Conclusions: Our integrative study is the first to associate rs76481776 with POAG via elevated miR-182 expression.

Figure 1

Expression of miR-182 in four different normal ocular tissues with miRNA sequencing (A) and ddPCR (B), including CB, cornea, retina, and TM. miRNA-Seq was done with two CB, two corneal, two retinal, and four TM samples, while ddPCR was done with two CB, two corneal, two retinal, and two TM samples.

Figure 2

Nanoparticle tracking analysis (size and concentration) of exosomes derived from primary human nonglaucoma trabecular meshwork cells, using NanoSight NS500 system. The exosome sample was resuspended in 1X PBS and diluted by 1:500 with PBS. Three separate measurements were performed to calculate the mean, which were used to derive this figure.

Figure 3

Differential expression of miR-182 in human aqueous humor samples from nonglaucoma controls (n = 5) and HTG patients (n = 5). The absolute copy numbers of miR-182 in each sample was measured by using ddPCR. All POAG patients were female, while controls included 3 males and 2 females. The males in the control group were marked with the black arrow.