Figure 4

Mutants IVS4A-1T and IVS5-11A>G splice aberrantly. A. Analysis of transfected-cell RNA by RT-PCR. RNA from HEK293 cells (lanes 2–4) or human cornea stromal cells (lanes 6–8) transfected with GFP and the indicated minigene was analyzed by RT-PCR and products were resolved on an agarose gel. The position of correctly spliced minigene product (585 bp) and GFP is shown at the right. M, 100 bp size standards with selected sizes shown. WT, RNA from wild type minigene; -1G>T, RNA from mutant IVS4-1G>T minigene; -11A>G, RNA from mutant IVS5-11A>G minigene. B. Schematic of the 3'ss used in the IVS4-1G>T RT-PCR products from (A) that were cloned and sequenced. C. Schematic of the RNA sequence of patient IVS5-11A>G RT-PCR products from (A) that were cloned and sequenced. In B and C, exon sequences are boxed and in uppercase, intron sequences are in lowercase, and an arrow points to the patient mutation.