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

Figures

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  Figure 1. Family pedigree of the index patient. Family members diagnosed with HCM are shown with filled circles (females) or squares (males). Arrow indicates the index patient. The index patient's parents (I.1, I.2) died (d.) relatively young without known cardiac disease, and also three brothers of the index patient (II.1, II.2, II.3) have died of other causes without being known to have heart disease. A fourth brother (II.7) of the index patient has died of sudden death. A fifth brother (II.6) of the index patient has been diagnosed with moderate septal hypertrophy, but he could not be included in the genetic testing. In generations III and IV, two sons, four daughters and a granddaughter of the index patient were included in both the clinical evaluation and genetic testing. Massively parallel sequencing analysis of the index patient identified two rare MYBPC3 intronic variants (see Supplementary Fig. S1). Family genetic testing results (see Fig. 2) of these two intronic variants are shown for each family member analyzed. At the last clinical evaluation, the index patient’s daughter III.6 did not meet the criteria for HCM, and she was considered an unaffected carrier of the MYBPC3…

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  Figure 2. Family inheritance analysis of two rare intronic MYBPC3 variants identified in the index patient. Sanger DNA chromatograms testing the MYBPC3 c.292+177C>T variant in intron 2 (a) and the MYBPC3 c.3311−26T>G variant in intron 30 (b) are shown. DNA chromatograms from the index patient (II.5) revealed the presence of both nucleotide substitutions in the heterozygous state, thus independently confirming the massively parallel sequencing results (see Supplementary Fig. S1). Sequence analyses of four family members of the III generation are also shown: one index patient’s son (III.1) and three daughters (III.4, III.6 and III.7). Two of them inherited from their mother the MYBPC3 variant in intron 2 (III.1, III.7) and the other two (III.4, III6) the MYBPC3 variant in intron 30. The complete family testing results are presented in Fig. 1. Arrows indicate the position of the affected nucleotide. The intron–exon boundary (i30-E31) is marked with a dotted line.

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  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…

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  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…

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  Figure 5. Western blot analysis of fusion proteins derived from MYBPC3 c.3331−26T>G minigenes. MYBPC3 reference (REF, Plasmid 1446) or mutant (MUT, Plasmid 1447) minigenes were transiently transfected into HeLa cells in triplicate as indicated on the top of each lane. Cell lysates derived from the same experiment and processed in parallel were analyzed by Western blot to detect the presence of FLAG-MYBPC3 and MYBPC3-Myc fusion proteins with monoclonal anti-FLAG (a) or anti-Myc (b) antibodies. Cells transfected with the REF plasmid expressed the expected normal MYBPC3 fusion protein of about 24 kDa encoded by the minigene, detected with both anti-tag (FLAG and Myc) monoclonal antibodies. The corresponding normal MYBPC3 fusion protein was not detected with either of the two antibodies in cells transfected with the MUT plasmid. A very faint band of about 16 kDa could be observed only in the MUT lanes with anti-FLAG, but not with anti-Myc, which may correspond to a poorly translated product from the misspliced cRi30 transcript. Each PVDF membrane was stained for total protein with Amido Black (AB) after immunostaining. Full size Western blot film and membrane images including the protein…

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  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…