Figure 4 | Scientific Reports

Figure 4

From: Identification of an elusive spliceogenic MYBPC3 variant in an otherwise genotype-negative hypertrophic cardiomyopathy pedigree

Figure 4

Sanger sequencing of minigene-derived normal and misspliced MYBPC3 mRNA forms. RT-PCR products of HeLa cells transfected with REF or MUT MYBPC3 c.3331−26T>G minigenes (as described in Fig. 3c) were eluted from agarose gel slices and subjected to Sanger sequencing. Sequencing chromatograms of the expected size (a, 361 bp, derived from the REF MYBPC3 minigene) and the longer (b, 581 bp, derived from the MUT MYBPC3 minigene) RT-PCR bands are shown. The sequence of the 361-bp product corresponds to properly spliced MYBPC3 transcript, with exons (E30-E31-E32) normally ligated and introns (i30 and i31) removed. Sequence analysis of the longer 581-bp band revealed that it corresponds to a misspliced transcript with the complete retention of i30 (cRi30), but not i31. The presence of the MYBPC3 c.3331−26T>G nucleotide substitution in the cRi30 misspliced transcript is indicated with a vertical arrow. The sequence analysis of the intermediate-sized (450 bp) RT-PCR band is shown in Supplementary Fig. S8, and revealed that two consecutive AG dinucleotides (named AG1 and AG2, double underlined in the cRi30 sequence), located in the middle of i30, were used as cryptic acceptor sites to generate two misspliced transcripts with partial retention of the second half of i30, named pRi30/AG1 and pRi30/AG2 (horizontal arrows). PTC premature termination codon.

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