No abstract

Exfoliation syndrome (XFS) is a common, age-related, systemic fibrillinopathy. It greatly increases risk of exfoliation glaucoma (XFG), a major worldwide cause of irreversible blindness. Coding variants in the lysyl oxidase-like 1 (LOXL1) gene are strongly associated with XFS in all studied populations, but a functional role for these variants has not been established. To identify additional candidate functional variants, we sequenced the entire LOXL1 genomic locus (∼40 kb) in 50 indigenous, black South African XFS cases and 50 matched controls. The variants with the strongest evidence of association were located in a welldefined 7-kb region bounded by the 3'-end of exon 1 and the adjacent region of intron 1 of LOXL1. We replicated this finding in US Caucasian (91 cases/1031 controls), German (771 cases/1365 controls) and Japanese (1484 cases/1188 controls) populations. The region of peak association lies upstream of LOXL1-AS1, a long non-coding RNA (lncRNA) encoded on the opposite strand of LOXL1. We show that this region contains a promoter and, importantly, that the strongly associated XFS risk alleles in the South African population are functional variants that significantly modulate the activity of this promoter. LOXL1-AS1 expression is also significantly altered in response to oxidative stress in human lens epithelial cells and in response to cyclic mechanical stress in human Schlemm's canal endothelial cells. Taken together, these findings support a functional role for the LOXL1-AS1 lncRNA in cellular stress response and suggest that dysregulation of its expression by genetic risk variants plays a key role in XFS pathogenesis.

PurposeKeratoconus (KC) is the most common corneal ectasia. We aimed to determine the differential expression of coding and long noncoding RNAs (lncRNAs) in human corneas affected with KC.MethodsFrom the corneas of 10 KC patients and 8 non-KC healthy controls, 200 ng total RNA was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50-bp sequencing with Illumina Sequencer. Differential analysis was done using TopHat/Cufflinks with a gene file from Ensembl and a lncRNA file from NONCODE. Pathway analysis was performed using WebGestalt. Using the expression level of differentially expressed coding and noncoding RNAs in each sample, we correlated their expression levels in KC and controls separately and identified significantly different correlations in KC against controls followed by visualization using Cytoscape.ResultsUsing |fold change| ≥ 2 and a false discovery rate ≤ 0.05, we identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas. Pathway analysis indicated the enrichment of genes involved in extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Our correlation analysis identified 296 pairs of significant KC-specific correlations containing 117 coding genes enriched in functions related to cell migration/motility, extracellular space, cytokine response, and cell adhesion. Our study highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis.ConclusionsOur RNA-Seq–based differential expression and correlation analyses have identified many potential KC contributing coding and noncoding RNAs.

Intraocular pressure (IOP), the primary risk factor for primary open-angle glaucoma, is determined by resistance to aqueous outflow through the trabecular meshwork (TM). IOP homeostasis relies on TM responses to mechanical stretch. To model the effects of elevated IOP on the TM, this study sought to identify coding and non-coding RNAs differentially expressed in response to mechanical stretch. METHODS. Monolayers of TM cells from non-glaucomatous donors (n = 5) were cultured in the presence or absence of 15% mechanical stretch, 1 cycle/second, for 24 hours using a computer-controlled Flexcell unit. We profiled mRNAs and lncRNAs with stranded total RNA sequencing and microRNA (miRNA) expression with NanoString-based miRNA assays. We used two-tailed paired t-tests for mRNAs and long non-coding RNAs (lncR-NAs) and the Bioconductor limma package for miRNAs. Gene ontology and pathway analyses were performed with WebGestalt. miRNA-mRNA interactions were identified using Ingenuity Pathway Analysis Integrative miRNA Target Finder software. Validation of differential expression was conducted using droplet digital PCR. RESULTS. We identified 219 mRNAs, 42 miRNAs, and 387 lncRNAs with differential expression in TM cells upon cyclic mechanical stretch. Pathway analysis indicated significant enrichment of genes involved in steroid biosynthesis, glycerolipid metabolism, and extracellular matrix-receptor interaction. We also identified several miRNA master regulators (miR-125a-5p, miR-30a-5p, and miR-1275) that regulate several mechanoresponsive genes. CONCLUSIONS. To our knowledge, this is the first demonstration of the differential expression of coding and non-coding RNAs in a single set of cells subjected to cyclic mechanical stretch. Our results validate previously identified, as well as novel, genes and pathways.

Exfoliation glaucoma (XFG) is the most common identifiable secondary form of open-angle glaucoma in the world. It is an ocular manifestation of exfoliation syndrome (XFS), an age-related systemic disease characterized by deposition of extracellular fibrillar material in various tissues and organs. XFS/XFG has been studied in populations around the world, which has led to identification of genetic factors that play a significant role in disease pathogenesis. Here, we summarize current knowledge of the genetics of XFS/XFG and identify areas for future research.