Identification of Novel Coloboma Candidate Genes through Conserved Gene Expression Analyses across Four Vertebrate Species

Abstract

Ocular coloboma (OC) is a failure of complete optic fissure closure during embryonic development and presents as a tissue defect along the proximal–distal axis of the ventral eye. It is classed as part of the clinical spectrum of structural eye malformations with microphthalmia and anophthalmia, collectively abbreviated to MAC. Despite deliberate attempts to identify causative variants in MAC, many patients remain without a genetic diagnosis. To reveal potential candidate genes, we utilised transcriptomes experimentally generated from embryonic eye tissues derived from humans, mice, zebrafish, and chicken at stages coincident with optic fissure closure. Our in-silico analyses found 10 genes with optic fissure-specific enriched expression: ALDH1A3, BMPR1B, EMX2, EPHB3, NID1, NTN1, PAX2, SMOC1, TENM3, and VAX1. In situ hybridization revealed that all 10 genes were broadly expressed ventrally in the developing eye but that only PAX2 and NTN1 were expressed in cells at the edges of the optic fissure margin. Of these conserved optic fissure genes, EMX2, NID1, and EPHB3 have not previously been associated with human MAC cases. Targeted genetic manipulation in zebrafish embryos using CRISPR/Cas9 caused the developmental MAC phenotype for emx2 and ephb3. We analysed available whole genome sequencing datasets from MAC patients and identified a range of variants with plausible causality. In combination, our data suggest that expression of genes involved in ventral eye development is conserved across a range of vertebrate species and that EMX2, NID1, and EPHB3 are candidate loci that warrant further functional analysis in the context of MAC and should be considered for sequencing in cohorts of patients with structural eye malformations.

Cell adhesion marker expression dynamics during fusion of the optic fissure

“…For SNAI2 RNA fluorescence in situ hybridisation we used RNAScope (ACD, Biotechne) following the RNAscope Multiplex Fluorescent V2 Assay protocol on 14 μm cryosections prepared as above and processed as described previously (Hardy et al, 2019;Trejo-Reveles et al, 2023) with the probe ID #1172211-C1. This probe was designed to target Gallus gallus snail family transcriptional repressor 2 (SNAI2) transcript variant X1 mRNA [NCBI GeneID:432368].…”

FarmGTEx TWAS-server: an interactive web server for customized TWAS analysis in both human and farm animals

“…In addition, translating genetic findings across species is also important in the field of evolution, biology, and genetics. For instance, previous studies demonstrated the conserved functional impacts of orthologous variants on gene expression and complex traits between livestock and humans (23)(24)(25)(26)(27)(28). Liang et al (29) also proposed polygenic transcriptomic risk scores (PTRS), which paved the way to translate polygenic signals across human ancestry groups.…”