Pseudoexfoliation associated protective variant, rs7173049, reveals a novel regulatory region downstream of LOXL1

Abstract

Purpose : To identify and functionally characterize regulatory variants in or nearby the LOXL1 gene, which is known to be the main genetic risk factor for PEX syndrome/glaucoma.

Methods : Deep sequencing of LOXL1 was performed on a total of 3281 PEX cases and 3280 controls from Japan and Italy. Genotype data from a German cohort (755 PEX cases and 1243 controls) were imputed. Transcription factor binding affinities were analyzed by electrophoretic mobility shift assays (EMSA) using nuclear extracts from human Tenon`s capsule fibroblasts (hTKF), trabecular meshwork cells (hTMC) and nonpigmented ciliary epithelial cells (hNPE). LOXL1 promoter activity was measured using dual luciferase reporter assays in hTKF, hTMC and hNPE. Genome editing of human embryonic kidney cells 293T (HEK293T) was done by CRISPR/Cas9 technique using a dual cut approach. Transcript levels were determined by qPCR in ocular tissues obtained from donor eyes without and with manifest PEX syndrome.

Results : Deep sequencing and data imputing revealed a common variant, rs7173049, downstream of LOXL1 with minor allele G enriched in German, Italian, Japanese and South African controls (p <2.6x10^-5), translating to a protective effect. Specific DNA-protein interaction was demonstrated with nuclear extracts from disease-relevant cell types by EMSA using 30bp probes spanning rs7173049. However, LOXL1 promoter activity was not affected by a 200bp DNA fragment flanking rs7173049 in various ocular cell types. Nevertheless, deletion of this region demonstrated a significant effect on expression levels of LOXL1 and on two other neighboring genes within ±1Mb of rs7173049. Both genes were found to be differentially expressed (p <0.05) in ocular tissues of PEX and control patients.

Conclusions : These findings indicate that the region surrounding rs7173049 contributes to the regulation of PEX-relevant genes, including LOXL1, and suggest two novel genes to be involved in PEX pathogenesis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.