Abstract
PurposeOcular coloboma arises from genetic or environmental perturbations that inhibit optic fissure (OF) fusion during early eye development. Despite high genetic heterogeneity, 70% to 85% of patients remain molecularly undiagnosed. In this study, we have identified new potential causative genes using cross-species comparative meta-analysis. MethodsEvolutionarily conserved differentially expressed genes were identified through in silico analysis, with in situ hybridization, gene knockdown, and rescue performed to confirm spatiotemporal gene expression and phenotype. Interrogation of the 100,000 Genomes Project for putative pathogenic variants was performed. ResultsNine conserved differentially expressed genes between zebrafish and mouse were identified. Expression of zebrafish ank3a, bmpr1ba/b, cdh4, and pdgfaa was localized to the OF, periocular mesenchyme cells, or ciliary marginal zone, regions traversed by the OF. Knockdown of ank3, bmpr1b, and pdgfaa revealed a coloboma and/or microphthalmia phenotype. Novel pathogenic variants in ANK3, BMPR1B, PDGFRA, and CDH4 were identified in 8 unrelated coloboma families. We showed BMPR1B rescued the knockdown phenotype but variant messenger RNAs failed, providing evidence of pathogenicity. ConclusionWe show the utility of cross-species meta-analysis to identify several novel coloboma disease-causing genes. There is a potential to increase the diagnostic yield for new and unsolved patients while adding to our understanding of the genetic basis of OF morphogenesis.
Highlights
Formation of the vertebrate eye involves coordinated assembly of cell types from different embryological origins and a series of tissue rearrangements
We identified 178 differentially expressed genes (DEGs) at prefusion time point (32 hpf), 78 at perifusion time point (48 hpf), and 62 at postfusion time point (56 hpf) through comparison of tissue isolated from the optic fissure (OF) region and dorsal retina
8 proband presented with an iris coloboma, intellectual disability, and microcephaly, in which we identified a heterozygous variant CDH4 NM_001794.5:c.1291C>T, p.(Arg431Cys), which was classified as likely pathogenic (PS2, PM2, PP2, PP3 and PP4), and the unaffected mother was homozygous for the reference sequence
Summary
Formation of the vertebrate eye involves coordinated assembly of cell types from different embryological origins and a series of tissue rearrangements. This complex process begins with the specification of eye field cells in the anterior neuroectoderm, followed by lateral migration and evagination toward the overlying surface ectoderm to form 2 optic vesicles.[1,2] The interaction between the optic vesicles and the surface ectoderm induces the specification of lens placode, as well as initiates invagination of the optic vesicle to form a double-layered optic cup.[3] The inner and outer layers of the optic cup give rise to the neural retina and the retinal pigment epithelium, respectively. Unlike other epithelial fusion processes, which occur between the apical sides of cells, OF fusion begins with the apposition of the basal cell surface requiring breakdown of the basal lamina.[4,7,8] An increasing number of reports show that the POM cells play a role in OF fusion, and there is no clear mechanistic explanation, abrogation of POM specification or migration can lead to persistence of the OF.[4]
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