Fig. 3: RNA analyses.

A RT-PCR and subsequent Sanger sequencing (left panel) on cDNA derived from RNA of individual P2 and both parents (F1-m, F1-f) to determine the effect of the c.498G>A variant. In P2 and the heterozygous carrier father a smaller product is present (middle panel). Right panel: skipping of exon 4 (r.199_498del) causes an in-frame deletion on protein level (p.Leu67_Lys166del). WT wildtype, C control, E empty. B Coverage and Sashimi-plot (left panel) of RNA-seq from family 3 for the exon 2–exon 3 region. The c.151G>C variant is indicated with a green line. Coverage plot for P4 and the heterozygous carrier father (F3-f) (highlighted in red), indicating intron 2 retention (r.151_152ins151+1_152-1). This is confirmed by the FPKM values shown as box and scatter plots (right side), where P4 is the only outlier. C Coverage and Sashimi-plot (left panel) of RNA-seq data from family 3 for the exon 10–exon 12 region. The position of the c.1029+2T>C variant is indicated with a green line. The Sashimi-plot shows skipping of exon 11 (r.937_1029del; highlighted in red) in P4 and the heterozygous carrier mother (F3-m), which is predicted to cause an in-frame deletion (p.Phe313_Pro343del). In addition, the coverage plot indicates retention of intron 10 (r.936_937ins936+1_937-1; red highlight) in P4 and F3-m, which is confirmed by the FPKM values for this exon (right panel and predicted to cause a truncated protein (p.Leu312_Phe313ins*18).