Abstract
Purpose :
To gain further insights into genes and pathways that could be related to pseudoexfoliation syndrome/glaucoma we performed RNA-seq analysis using ocular tissues from PEX patients and healthy subjects.
Methods :
Iris (n=16) and lens epithelial-capsule (n=16) specimens were obtained from human donor eyes with PEX syndrome and healthy subjects within 12 hours post mortem. Total RNA was extracted using the RNeasy Mini kit (Quiagen), and RNA quality was analyzed using the 2100 Bioanalyzer system (Agilent). Libraries were prepared from 100 ng of total RNA using Illumina’s TruSeq stranded total RNA kit and were subjected to single-end sequencing (101 bp) on a HighSeq-2500 platform (Illumina). After quality-trimming of the ends with fqtrim v0.9.5, reads were mapped onto the human reference genome (Ensembl GRCh37) using the STAR aligner v. 2.5.2b (Dobin 2013), and a STAR genome directory created by supplying the Ensembl gtf annotation file (release 87) for GRCh37. Read counts per gene were obtained using subread’s featureCounts program v1.5.1 (Liao 2014) and the Ensembl gtf annotation file. Differential expression analysis was performed with the DESeq2 package v.1.12.3 (Love 2014). Ingenuity® Pathway Analysis (IPA®) software was used for data interpretation.
Results :
Analysis of RNA-seq data showed 793 and 993 genes that were differentially expressed in iris specimens and lens specimens, respectively, of PEX and control eyes (p<0.01). Pathway analyses were first separately performed for iris and lens samples, followed by a comparative pathway analysis for both tissue types, assuming common pathogenetic mechanisms. In this comparison, several pathways, including cAMP-signaling, ERK/MAPK-signaling, Gαs-signaling and LXR/RXR activation, were consistently dysregulated in tissues of PEX patients compared to controls. Within these pathways, a number of overlapping genes related to connective tissue disorders (e.g. MMP9, LTBP2, FBN1, IL1B, SERPIN1F, TGFBR2, RXRG, EDN, NFKB) and upstream regulators (e.g. TGFB1, tretinoin) showed significant differential expression between PEX and control samples. Differentially expressed candidate genes were confirmed by qPCR assays.
Conclusions :
Genes and pathways identified in this study provide new insights into the molecular pathophysiology underlying PEX syndrome/glaucoma. Thus, RNAseq is a powerful tool to identify novel genes involved in PEX disease.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.