Abstract
Purpose :
To investigate the single nucleotide polymorphisms (SNPs) in the LOXL1 locus using Bi-allelic Targeted Self Transcribing Active Regulatory Region Sequencing (BiT-STARR-seq) to identify those that modify risk for exfoliation syndrome (XFS) through alterations in transcriptional regulation.
Methods :
We identified 81 SNPs in linkage disequilibrium (r2>0.4) with rs2165241, the peak associated SNP in Thorleifsson et al. 2007. Oligos were designed for each allele of the 81 SNPs and for several proposed cis-regulatory elements near LOXL1. The oligos were pooled and cloned into the hSTARR-seq_ORI plasmid, which was transfected into HEK293 cells in triplicate. For each replicate, DNA and RNA was isolated and used to create NGS libraries, that were barcoded, pooled, and sequenced, obtaining paired-end 150 nt reads which were aligned to the human reference genome (GRCh38). After normalizing for transfection efficiency and library depth, we compared transcription between each pair of LOXL1 SNP alleles to identify those with enhancer activity that stimulated their own transcription.
Results :
All eighty-one pairs of oligos with LOXL1 SNP alleles were robustly represented in our plasmid libraries. We obtained high-quality sequence of transcripts from the transfected plasmid library. Analysis of the sequence data identified several SNPs that appear to modulate transcription in an allele-specific fashion. The normalized transcription values were highly consistent among replicates (r2=0.82-0.95). The most significant SNPs exhibiting allele-specific transcriptional regulation are rs12441130 (highlighted in Figure 1, below) and rs72745365. The minor alleles of both of these SNPS were seen to increase self-transcription, suggesting they might also increase transcription of LOXL1. We also identified several putative enhancer and repressor elements that did not contain common sequence variations. We were also able to confirm and refine the genomic extent for several of the proposed cis-regulatory elements included in our oligo design.
Conclusions :
BiT-STARR-seq is a powerful technique to study risk loci like LOXL1. This study reports a functional exploration of SNPs near LOXL1 to distinguish risk-altering SNPs. While these SNPs are promising exfoliation syndrome risk factor candidates, further analyses are needed including including BiT- STARR-seq studies in more relevant cell types, i.e., lens cells and/or neurons.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.