Abstract
Purpose :
Keratoconus is characterised by progressive corneal thinning and significant irregular astigmatism. The process that leads to stromal loss is poorly understood, but may reflect a pathological extreme of biological mechanisms controlling healthy central corneal thickness (CCT), a highly heritable trait. Genome wide association studies (GWAS) have identified multiple variants associated with decreased CCT and increased keratoconus risk. One set of such variants lies in the intergenic region between RXRA and COL5A1 on chromsome 9. We hypothesize that this locus lies in a putative enhancer for COL5A1, encoding the a1 isoform chain of type 5 collagen, a major component of the corneal stroma.
Methods :
Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed on a human keratocyte cell line derived from corneal stroma (hTK) and a human corneal epithelial cell line (hTCEpi). The COL5A1 gene was expressed in hTK but not hTCEpi and both expressed the RXRA gene. Dual luciferase reporter assays were performed by transient transfection of pGL4 plasmids to test associated haplotypes in hTK and in a human skin fibroblast cell line (RITVA). Site directed mutagenesis was used to change the CCT decreasing haplotype to the CCT increasing haplotype to dissect the contribution of associated SNPs to enhancer activity.
Results :
ATAC-seq shows the associated variants are located in regions of accessible chromatin in hTK cells, but not in hTCEpi, consistent with a cell type specific regulatory mechanism being disturbed by the associated variants. Transfection with the CCT reducing haplotype construct displayed significantly lower (p=0.004) normalised luminescence compared to reference haplotype in hTK cells, whereas no significant difference was seen in RITVA cells. When the CCT reducing allele of a single variant, rs3118515, was changed to the reference allele, a significant increase in relative luciferase activity was recorded, consistent with variation at rs3118515 influencing enhancer activity.
Conclusions :
We showed that the SNP rs3118515 is a functional variant in a transcriptional regulatory element that disrupts a putative enhancer region in a cell type specific manner. The gene this enhancer is influencing is being investigated. We are also using chromatin immunoprecipitation to confirm transcription factors predicted to bind in this region.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.