Investigating regulatory mechanisms for COL5A1 expression in the human cornea in health and disease

Abstract

Keratoconus is a complex corneal condition that is characterised by progressive corneal thinning and significant irregular astigmatism and eventual visual loss. The aetiology of keratoconus is not known although genetics is likely to play a large role in individual risk. The underlying biological process that leads to progressive stromal loss and corneal thinning in keratoconus may reflect a pathological extreme of the biological processes controlling healthy corneal thickness.

Genome wide association studies (GWAS) have identified several loci significantly associated with central corneal thickness (CCT) and keratoconus risk. Of particular interest are the single nucleotide polymorphisms (SNPs) rs4841899, rs1536482, rs3132303 and rs7032489 positioned in an intergenic region of chromosome 9 between RXRA and COL5A1 and in the first intron of COL5A1 respectively. COL5A1 encodes the alpha chain of Type V Collagen, a regulatory fibril-forming collagen, and the second most abundant collagen in corneal stroma. Thus, I hypothesised that these variants lie in regulatory elements that affect COL5A1 transcription thereby influencing corneal thickness. Functional studies were performed to elucidate whether these variants have regulatory impact and to identify the underlying mechanisms of how they exert their effects. Luciferase assays showed that the CCT-reducing haplotypes around SNPs rs3118515, rs3132303 and rs1536482 lower enhancer activity compared to the alternate haplotypes, in human telomerase-immortalised keratocye cells (hTK). Rs3118515 was prioritised as a likely causal variant and modulates enhancer activity specifically in hTK. DNA binding factors cFos and PRDM5, respectively a component of the transcription factor AP1 and a regulator implicated in collagen gene regulation, were identified as candidates to bind near rs3118515.

Chromatin immunoprecipitation confirmed PRDM5 and cFos binding to the genomic region encompassing rs3118515, in hTK cells. Inhibition of AP1 was found to significantly disrupt COL5A1 gene expression and protein expression in vitro. Small deletions introduced in hTK using CRISPR-Cas9 genome editing in the region flanking rs3118515 influenced COL5A1 expression.

The role of Prdm5 in the cornea was further investigated by procuring Prdm5-/- mice. Those were found to have significantly thinner corneas at 1 and 3 months old. Primary corneal keratocytes isolated from wild type and homozygous mutant mice showing reduced expression of Col1a1, Col5a1 and Fn1 (encoding fibronectin) in mutant keratocytes. Overall, these results supported a role for the variant rs3118515 in altering expression of COL5A1 in corneal stroma cells by a mechanism involving transcription factors AP1 and PRDM5.