An integeative GWAS followed by functional investigation reveals possible involvement of CNTNAP5 in glaucomatous neurodegeneration

Primary angle closure glaucoma (PACG) is one of the major leading causes of blindness. In India, ~30% of people show a narrow iridocorneal angle (<15°), but out of these only 0.5%–1% people develop PACG. Two comprehensive case–control GWAS identified eight loci where heterogenous controls were included with respect to anatomically predisposed parameters. To exclude heterogeneity, we conducted an haplotype based age-agnostic model of progressive angle closure GWAS early-onset PACG patients (PACG: age ≤50 years) compared with anatomically predisposed (narrow angle) non-glaucomatous older individuals (PACS: age ≥60 years). Further, we performed a dual-luciferase assay for functional follow-up of risk variants of CNTNAP5 and subsequently the role of CNTNAP5 figured out in ocular development and morphology of the retinal nerve in zebrafish. In our GWAS cohort (PACG = 148 and PACS = 92), we identified 13 SNPs of CNTNAP5 that were associated with PACG. Subsequently, the prioritized SNP rs780010 of CNTNAP5 was significantly (p = 0.0024) associated with a higher cup-to-disc ratio, a clinical parameter directly correlated with glaucomatous neurodegeneration. We further validated rs780010 in a separate replication cohort (PACG = 50; PACS = 39) and observed a significant association (odds ratio = 2.307, p = 0.012). According to the Hi-C database, the associated genic region shows higher retinal neuronal expression of CNTNAP5 with active enhancer marks; which were subsequently validated using a dual-luciferase assay. Additionally, immunofluorescence analyses showed significant eye size reductions and retinal nerve thinning in zebrafish upon morpholino mediated knockdown of CNTNAP5. Our GWAS results indicate a genomic association of CNTNAP5 with PACG that it might play a role in glaucomatous neurodegeneration. Further, post-GWAS functionalization led us to believe that CNTNAP5 is an important player to perturb the development of the neural retina that further leads to retinal nerve thinning, thereby increasing the risk of PACG-associated vision loss.