Mis-splicing is not unique to DUX4.V5 cDNAs.

<p>(A) The myotilin cDNA fused to V5 creates a functional splice donor that was fused to splice acceptors resident in the DUX4 3’ UTR and that of an unrelated gene, MDM2. In both circumstances, sequence chromatograms showed that the V5 tag was deleted, similar to the event that occurred in the original DUX4.V5 transgene arising from the AAV D4Z4.V5.pLAM vector. (B) The humanized renilla luciferase cDNA contained 5 predicted splice donor sites. When attached to the DUX4 3’ region (exon 1 UTR, intron 1, exon 2, intron 2, exon 3), one site (SD5; boxed in red) was utilized and fused to DUX4 exon 2. PCR of 3’ RACE products produced the SD5 transcript (1,268 bp) and some smaller products on an ethidium bromide stained gel. Only the 1,268 bp band contained Renilla luciferase sequences, and chromatograms confirmed the SD5-mediated mis-splicing event. This construct showed significantly reduced luciferase enzyme activity in vitro (indicated by **, p<0.0001; ANOVA with Tukey’s multiple comparison test, n = 3 replicates). Mutating the SD5 T nucleotide (boxed) destroyed the SD5 splice donor and restored luciferase activity to above normal levels (indicated by #, p = 0.0076; ANOVA with Tukey’s multiple comparison test, n = 3 replicates). (C) Comparison of splice donor sequences identified in this study to the consensus splice donor site.