Abstract
Purpose :
In the trabecular meshwork (TM), the correct extracellular matrix (ECM) composition, organization and remodeling is critical to maintain normotensive intraocular pressure (IOP). In primary open angle glaucoma (POAG), there is an accumulation of disorganized ECM, which hinders aqueous humor outflow and eventually leads to elevated IOP. Here, we hypothesized that missense variants in ECM genes could cause subtle changes to the structure and organization of the TM ECM leading to increased outflow resistance.
Methods :
Whole genome sequencing was performed on five individuals with elevated IOP (> 22 mmHg) from a POAG family (n=151). DNA was isolated from nine human TM (HTM) cell strains derived from cadaver eyes with and without documented glaucoma. Sanger sequencing was performed using primers flanking the identified single nucleotide polymorphisms (SNPs). ECMs from the TM cell strains were analyzed by western immunoblotting, confocal and transmission electron microscopy.
Results :
Three missense SNPs were identified in three or more individuals: THBS1, LAMB2 and COL6A3. Sanger sequencing demonstrated that the THBS1 and COL6A3 variants were significantly increased in family members with elevated IOPs. The THBS1 and LAMB2 variants appear in 11% of Caucasians, but the COL6A3 SNP was rare. Nine unrelated HTM cells strains were screened for these SNPs. One strain harbored both the THBS1 and the LAMB2 variants, while two other strains, from donors with glaucoma, harbored one SNP each. By confocal microscopy, both glaucomatous (GTM) cell strains showed major differences in the distribution of COL6A3 and certain integrins compared to wild-type HTM cells. Western immunoblotting showed that matrix metalloproteinase-14 was increased in GTM cells. Analysis of ultrastructure showed that microfibrils produced by GTM cells had exposed globular domains compared to wild-type HTM microfibrils.
Conclusions :
Here, we identified SNPs in ECM genes that are associated with IOP in a large POAG family. ECM organization and remodeling was disrupted in HTM cell strains containing one or more of the common SNPs. Since the ECM is critical to IOP regulation, investigating the molecular function of IOP-associated SNPs will lead to a better understanding of the outflow resistance and TM cell function.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.