Fig. 3

Minigene assay for the targeted variant. a Minigene construction; the vertical dashed red line shows the position of OTOF on chromosome 2p23.3. The next dashed underline represents OTOF, including its introns and exons. The minigene target sequence is from exon 25 to exon 29; this sequence was cloned in a CAGGS plasmid with internal ribosome entry sites (IRES), and enhanced green fluorescent protein (EGFP). b Sanger sequencing of the wild type (WT) and the mutant for splicing variants 1 and 2; the dashed line indicates the exon junction between exons 26 and 27. Splicing variant 1 deleted 1–10 bp from exon 27, and splicing variant 2 deleted 1–13 bp from the same exon. c The protein translation of the WT sequence and splicing variants 1 and 2. The splicing acceptor site mutation produced truncated proteins of 1141 amino acids (p.Ile1097Glnfs45) in splicing variant 1 and 1140 amino acids (p.Ile1097Glyfs44) in splicing variant 2, instead of the normal mature protein of 1998 amino acids. Ile = isoleucine, Gln = glutamine, Gly = glycine