Fig. 4

Functional evaluation of candidate pathogenic 5’UTR variants in the ARL3, MERTK, PAX6, and RDH12 genes. A Various approaches were used for functionally evaluating candidate variants, including in vitro studies (dual luciferase reporter assays and overexpression) and experiments with clinically-accessible tissues (expression analysis in patient-derived lymphocytes). B Results from the luciferase assays for the MERTK:c.-125G>A, PAX6:c.-44T>C, and RDH12:c.-123C>T variants. The bar plot shows, for each variant, the fold change (FC) of the luciferase reporter level relative to the level of their corresponding wild-type (WT) construct luciferase vector (FC = 1). The RDH12:c.-123C>T and MERTK: c.-125G>A variants resulted in significant (p < 0.001) decrease in luciferase activity (~92% and ~99%, respectively). C Relative Renilla luciferase mRNA levels were significantly decreased (~42%, p<0.01) for the MERTK:c.-125G>A variant while they remain the same for the RDH12:c.-123C>T variant when normalized to mRNA of Firefly luciferase and compared to their corresponding wild-type (WT) construct luciferase vectors. D qPCR quantification of ARL3 mRNA abundance in lymphocyte cDNA of two affected siblings carrying the ARL3:c.-88G>A novel variant and six healthy controls. No significant differences were observed in ARL3 mRNA abundance between the affected carriers and controls. Data presented as the mean of biological replicates within each group ± their corresponding standard deviation. E–F Using an overexpression setting, the RDH12:c.-123C>T variant was shown to result in E unaltered mRNA levels but F significantly reduced (~73%, p < 0.01) RDH12 protein levels