Figure 3

mRNA expression analysis of HeLa cells transfected with MYBPC3 c.3331−26T>G minigenes. (a) Structure of the 767-bp MYBPC3 minigene cloned in-frame to both epitope tags, 3XFLAG (F) and Myc-tag (M). Exons (E30, E31 and E32) and introns (i30 and i31) are represented with white boxes and black lines, respectively. Location of the variant in i30 is indicated. (b) Sanger DNA sequencing chromatograms of reference (REF, PL. 1446) and mutant (MUT, PL. 1447) plasmids carrying a T (REF) or alternative G (MUT) nucleotide corresponding to the MYBPC3 c.3331−26T>G variant that were used for transfection experiments. The reference and mutant MYBPC3 minigenes were generated by molecular cloning of the allelic copy of genomic DNA isolated from patient III.8, heterozygous carrier of the c.3331−26T>G variant, as described in the Methods section. Full insert DNA sequencing of REF and MUT plasmids are presented in Supplementary Figs. S5 and S6, respectively. (c) RT-PCR analysis of HeLa cells transiently transfected with MYBPC3 REF or MUT plasmids. Samples were derived from the same experiment and processed in parallel. Agarose gel electrophoresis of RT-PCR products from triplicate transfections is shown. The lower, most abundant, 361-bp bands of the REF lanes (1–3) correspond to normal MYBPC3 mRNA (see Fig. 4a). Normal MYBPC3 mRNA was not detected in the MUT lanes (4–6), but two other larger bands were detected. In the MUT lanes, the longer bands (581 bp) correspond to a misspliced transcript with the complete retention of intron 30 (cRi30, see Fig. 4b). Sequence analysis of the intermediate-sized weaker band of about 450 bp revealed that it actually contained two transcripts, with partial retention of intron 30, named pRi30/AG1 (453 bp) and pRi30/AG2 (450 bp) (see Supplementary Fig. S8). Control samples were loaded in lane 7 (non-transfected cells), lane 8 (RT −) and lane 9 (non-template). A DNA ladder was loaded in lanes L. Endogenous RPL19 was amplified as a control for the synthesis of cDNA. Full-size RT-PCR gel image is shown in Supplementary Fig. S7. (d) Schematic representation of normal spliced and the three misspliced transcripts. Location of c.3331−26T>G (red line) and the two preexisting cryptic acceptor sites AG1 and AG2 (cyan lines) in intron 30 are indicated. PTC premature termination codon, FS frameshift.