Figure 6

RT‐PCR analysis of MYBPC3 expression in patients’ blood. (a) Agarose gel electrophoresis of RT‐PCR products of blood RNA isolated from non‐carriers (lane 1‐III.7, lane 2‐III.1) and carriers (lane 3‐III.4, lane 4‐III.6) of the MYBPC3 c.3331−26T>G variant in intron 30. This splicing assay targeted the MYBPC3 mRNA region between exons 30–31 using the primer pair 543–542. Negative controls were loaded in lane 5 (RT −) and 6 (non-template). The lower bands (328 bp) correspond to normal MYBPC3 mRNA (Supplementary Fig. S13). The longer bands from both III.4 and III.6 correspond to a misspliced transcript with the complete retention of intron 30 (548 bp, cRi30‐MYBPC3, Supplementary Fig. S13). Sequence analysis revealed that the intermediate‐sized RT‐PCR bands actually contained two transcripts, with partial retention of intron 30 (Supplementary Fig. S14), named pRi30/AG1 (420 bp) and pRi30/AG2 (417 bp), as previously shown by the minigene expression assay (see Fig. 3). (b) An additional RT-PCR assay targeting the MYBPC3 mRNA region between exons 2–3 with the primer pair 601–603 and the same cDNA samples was performed. This second assay was designed to analyze mRNA splicing in the presence of the MYBPC3 c.292+177C>T variant, located in intron 2, in carriers (lane 1‐III.7, lane 2‐III.1) and non-carriers (lane 3‐III.4, lane 4‐III.6). A single RT-PCR band of the expected size (292 bp) was detected in all lanes. Sanger sequencing of the purified 292-bp band confirmed that it corresponds to normal MYBPC3 mRNA (Supplementary Fig. S15). Full‐size RT‐PCR images are presented in Supplementary Fig. S12. L—DNA ladder.