Novel Truncating Variant c.1222DupC in RBM20 Causes Cardiomyopathy Consistent With Haploinsufficiency.

Figures

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  Figure 1.. Identification of the RBM20-c.1222DupC variant. A, Family pedigree of the proband with the RBM20-c.1222DupC variant. Proband is marked by arrow. B, Schematic visualization of RBM20 (RNA binding motif protein 20) protein structure, numbers represent exons and known functional domains are denoted in different colors. Top diagram represents the full-length RBM20 and bottom diagram represents truncated RBM20-c.1222DupC protein with the position of the variant marked with a red vertical line. DCM indicates dilated cardiomyopathy; MR, mitral regurgitation; MVP, mitral valve prolapse; and SCD, sudden death.

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  Figure 2.. The truncated RBM20 (RNA binding motif protein 20)-DupC protein is less stable. A, Sanger sequencing of the mouse (left) and human (right) wild-type (WT; top), and DupC (bottom) RBM20 constructs. Amino acid sequence is added underneath the nucleotide sequence. The red line in the bottom panels indicates the site of c.1222DupC variant. B, Expression of mouse and human RBM20 mRNA transcript in HEK293 cells transfected with equal amounts of plasmid with mouse or human WT-RBM20 or RBM20-DupC (n=6/condition). C, Representative immunoblotting of FLAG-tagged full-length RBM20 and RBM20-DupC in HEK293 cells. D, Densitometric analysis of the FLAG-tagged RBM20 and FLAG-tagged RBM20-DupC normalized to GAPDH intensity (n=6/condition). **P<0.01, ****P<0.0001. Significance was tested using a 2-tailed t test. E, Immunoblotting of FLAG-tagged WT RBM20 and RBM20-DupC following a cycloheximide (CHX) pulse-chase assay. F, Quantification of FLAG-tagged protein levels normalized to α-tubulin (n=4/timepoint). Protein amounts at 0 hour are set to 100% for both WT RBM20 and RBM20-DupC. Data collected from 2 independent experiments.

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  Figure 3.. RBM20 (RNA binding motif protein 20)-DupC shows both nuclear and cytoplasmic localization. A, Immunofluorescence of neonatal rat cardiomyocytes (NRCMs) transfected with mouse FLAG-tagged Rbm20 or FLAG-tagged Rbm20-DupC. FLAG is stained in green, α-actinin in red, and DAPI (4′,6-diamidino-2-phenylindole) in blue. Scale bar is 10 µm. B, Immunofluorescence of NRCMs transfected with human FLAG-tagged RBM20 or FLAG-tagged RBM20-DupC. FLAG is stained in green, α-actinin in red, and DAPI in blue. Scale bar is 10 µm.

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  Figure 4.. RBM20 (RNA binding motif protein 20)-DupC does not have residual splicing regulatory activity and does not act as a dominant negative. A, Schematic drawing of the TTN splicing reporter. B, Ratio of Firefly luciferase to Renilla luciferase in HEK293 cells transfected with empty vector control, mouse Rbm20, mouse Rbm20-DupC, or both (n=8/condition). C, Ratio of Firefly luciferase to Renilla luciferase in HEK293 cells transfected with empty vector control, human RBM20, human RBM20-DupC, or both (n=8/condition). ****P<0.0001. Significance was tested using a 1-way ANOVA with Bonferroni post hoc test.

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  Figure 5.. RBM20 c.1222DupC human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) present with splicing and calcium-handling abnormalities. A, Schematic representation of the workflow used to differentiate hiPSCs to cardiomyocytes. B, Sanger sequencing of the RBM20-DupC (±) hiPSC-CM. In the DupC (±) panel, the top nucleotide sequence represents the wild-type (WT) sequence, and the bottom nucleotide sequence represents the mutated sequence with the duplicated C marked in yellow. Amino acid sequence is added underneath the nucleotide sequence. C, Immunofluorescence of WT and Dupc (±) hiPSC-CM stained using anti-RBM20 (RNA binding motif protein 20; blue), anti-α-actinin (green), and anti-TNNT2 (red). Scale bar=30 µm. D, Immunoblotting of the cell lysate from WT and DupC (±) lines using anti-RBM20 antibody, the densitometric quantification presented as the bar graph (n=3/condition). E, Quantitative polymerase chain reaction (qPCR) analysis of RBM20 in WT and DupC (±) hiPSC-CM (n=3/condition). F, Percentage of WT and DupC transcripts in RNA-seq of WT and DupC iPSC-CM. G, Reverse Transcription polymerase chain reaction (RT-PCR) of RBM20 splice targets, glyceraldehyde-3-phosphate…

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  Figure 6.. Transcriptomic analysis on RBM20 c.1222DupC human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM). A, Principal component analysis (PCA) plot of the reads from wild-type (WT) and DupC (±) hiPSC-CM. B, Heat map showing the differentially expressed genes (DEGs) in WT and DupC (±) hiPSC-CM. C, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs of DupC (±) hiPSC-CM. D, Gene set enrichment analysis (Cellular component) of the DEGs of DupC (±) hiPSC-CM. E, Number of splice events detected in WT and DupC (±) hiPSC-CM events with an false discovery rate (FDR) <0.01 and delta percent spliced in (ΔPSI) >0.1 were included. F, Heatmap of top 50 differential splicing events between WT and DupC (±) hiPSC-CM. A3SS indicates alternative 3′ starting site; A5SS, alternative 5′ starting site; MXE, mutually exclusive exons; RI, intron retention; and SE, exon skipping.