Abstract
Purpose :
Retinoic acid receptor beta (RARB) is a transcriptional regulator crucial for coordinating retinoic acid-mediated morphogenic movements, cell growth and differentiation during eye development. RARB coding variants have been associated with microphthalmia, coloboma and anterior segment defects. Recently, we identified a novel, de novo RARB deep intronic variant (c.157+1895G>A) in a patient with syndromic microphthalmia consistent with RARB disruption. Here we evaluate the impact of the non-coding variant on the transcriptional or post-transcriptional activities of RARB in order to discover the underlying mechanism.
Methods :
Minigene splicing assays and luciferase reporter assays were designed to functionally test the effect of the variant on mRNA splicing and transcriptional regulation of RARB in human lens epithelial cells and human embryonic kidney cells. The effects of pharmacologically or genetically induced RARB overexpression on retinoic acid-related gene expression, cell growth and viability were assessed in human cultured cells using RT-qPCR and trypan blue assay.
Results :
Minigene splicing assays performed in both cell lines showed no effect on RARB mRNA splicing in the presence of the deep intronic variant. Interestingly, the variant is located in a highly conserved intronic region containing a candidate cis-regulatory element identified through ENCODE. Evaluation of this region specifically in human lens epithelial cells showed a significant increase in luciferase reporter activity in the presence of the candidate enhancer harboring the variant when compared to the wild-type enhancer or the promoter alone, suggesting that the variant may promote RARB overexpression during eye development. RARB overexpression in cultured cells resulted in increased cell proliferation and increased expression of FOXC1, a known downstream target of retinoic acid signaling during ocular development.
Conclusions :
These results support a novel role for the candidate cis-regulatory element in modulating RARB gene expression and indicate that the deep intronic variant may disrupt proper regulation of RARB and downstream target gene expression, resulting in aberrant cell proliferation during eye development. Further investigation into mechanisms affected by RARB overexpression will clarify how the deep intronic variant contributes to human ocular dysgenesis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.