Elena V. Semina

Retinoic acid receptor beta (RARB) is a transcriptional regulator crucial for coordinating retinoic acid- (RA-) mediated morphogenic movements, cell growth, and differentiation during eye development. Loss- or gain-of-function RARB coding variants have been associated with microphthalmia, coloboma, and anterior segment defects. We identified a de novo variant c.157+1895G>A located within a conserved region (CR1) in the first intron of RARB in an individual with complex microphthalmia and significant global developmental delay. Based on the phenotypic overlap, we further investigated the possible effects of the variant on mRNA splicing and/or transcriptional regulation through in silico and functional studies. In silico analysis identified the possibility of alternative splicing, suggested by one out of three (HSF, SpliceAI, and MaxEntScan) splicing prediction programs, and a strong indication of regulatory function based on publicly available DNase hypersensitivity, histone modification, chromatin folding, and ChIP-seq data sets. Consistent with the predictions of SpliceAI and MaxEntScan, in vitro minigene assays showed no effect on RARB mRNA splicing. Evaluation of CR1 for a regulatory role using luciferase reporter assays in human lens epithelial cells demonstrated a significant increase in the activity of the RARB promoter in the presence of wild-type CR1. This activity was further significantly increased in the presence of CR1 carrying the c.157+1895G>A variant, suggesting that the variant may promote RARB overexpression in human cells. Induction of RARB overexpression in human lens epithelial cells resulted in increased cell proliferation and elevated expression of FOXC1, a known downstream target of RA signaling and a transcription factor whose down- and upregulation is associated with ocular phenotypes overlapping the RARB spectrum. These results support a regulatory role for the CR1 element and suggest that the de novo c.157+1895G>A variant affecting this region may alter the proper regulation of RARB and, as a result, its downstream genes, possibly leading to abnormal development.