A De Novo Noncoding RARB Variant Associated with Complex Microphthalmia Alters a Putative Regulatory Element

Figure 3

c.157+1895G>A variant has no apparent effect on the splicing of the RARB transcript. (a) Schematic of RARB minigene design. Exonic sequences are represented as open boxes; intronic sequences are represented as dashed lines; exon and intron sizes are indicated in base pairs. The RARB minigene contains the exon 1 coding sequence and 2,144 bp of the 5 end of intron 1-2 ligated to 435 bp of the 3 end of intron 1-2 and the exon 2 coding sequence. Positions of primers used for amplification of minigene-specific and endogenous RT-PCR products are shown (arrowheads). RT-PCR analysis of RNA isolated from transfected (b) human lens epithelial or (c) HEK293 cells showing endogenous and minigene-specific products. Cells were either mock transfected (-, lane 1) or transfected with wild-type (WT, lane 2) or variant (G>A, lane 3) minigene DNA. RNA was analyzed using primers that amplify minigene RNA only (top) or both minigene and endogenous RARB RNA (middle) or cellular GAPDH (bottom). Specific sizes of 1 kb standard marker (std) and expected band sizes for correctly spliced products are indicated.

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