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Figure 5 (A) Schematic representation of the KCNH2 exon 7 minigene construct. The human KCNH2 exon 7 was cloned into the Nde I site of the -globin fibronectin EDB minigene (black shaded and white boxes, respectively, with intervening sequences [IVS] shown as thin lines). The intronic mutation T1945+6C is shown with exonic sequence in upper case and intronic sequence in lower case. The superimposed arrows indicate the primers used in the reverse transcription-polymerase chain reaction (RT-PCR) assay. (B) Agarose gel electrophoresis of the RT-PCR products generated from the splicing assay: T1945+6 (WT) generates a single band of approximately 637 base pairs (bp) (lane 2) while T1945+6C generates three different sized bands (lane 3), whose specific identity was established by cloning and sequencing. On the right of the gel, there is a graphic representation illustrating the DNA content of each band. The 239 bp band contains the fibronectin exons, the 637 bp band contains the fibronectin exons with exon 7 inserted between them, and the 1,302 bp band contains the fibronectin exons, exon 7, as well as the intron 3' to this exon. Note that the mutation causes intron retention and some exon skipping. The modified snRNA (A>G-U1) rescues this splicing defect (lane 4). (C) Upper panel: Base pairing between U1 snRNA (WT-U1) and the 3' end of KCNH2 exon 7. The nucleotide change T1945+6C reduces the base pairing between the RNA and the WT-U1 snRNA. Lower panel: The variant snRNA (A>G-U1) modified to complement the nucleotide change seen in the patient's pre-mRNA, and restored the appropriate base pairing.