Protein haploinsufficiency drivers identify MYBPC3 variants that cause hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Variants in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are the leading cause of HCM. However, the pathogenicity status of hundreds of MYBPC3 variants found in patients remains unknown, as a consequence of our incomplete understanding of the pathomechanisms triggered by HCM-causing variants. Here, we examined 44 nontruncating MYBPC3 variants that we classified as HCM-linked or nonpathogenic according to cosegregation and population genetics criteria. We found that around half of the HCM-linked variants showed alterations in RNA splicing or protein stability, both of which can lead to cMyBP-C haploinsufficiency. These protein haploinsufficiency drivers associated with HCM pathogenicity with 100% and 94% specificity, respectively. Furthermore, we uncovered that 11% of nontruncating MYBPC3 variants currently classified as of uncertain significance in ClinVar induced one of these molecular phenotypes. Our strategy, which can be applied to other conditions induced by protein loss of function, supports the idea that cMyBP-C haploinsufficiency is a fundamental pathomechanism in HCM.

Keywords: CD; alternative splicing; bioinformatics; cardiac myosin-binding protein C; hypertrophic cardiomyopathy; minigene; protein stability; variants of uncertain significance.

Figure 1

cMyBP-C haploinsufficiency drivers induced by HCM-linked and nonpathogenic MYBPC3 variants.A, left, scheme of the location of cMyBP-C (in yellow) in the sarcomere. Middle, most HCM-causing MYBPC3 variants lead to truncated polypeptides and protein haploinsufficiency. Right, the remaining variants are nontruncating and result in full-length mutant proteins (mutant domain is represented in red). B, workflow to identify cMyBP-C haploinsufficiency drivers in a curated database of HCM-linked and nonpathogenic MYBPC3 variants. Bioinformatics predictions of RNA splicing alteration and protein destabilization are made for the variants building up this database. Positive hits are then further assessed experimentally to identify protein haploinsufficiency drivers induced by these variants. cMyBP-C, cardiac myosin-binding protein C; HCM, hypertrophic cardiomyopathy.

Figure 2

Experimental characterization of RNA splicing alterations induced by MYBPC3 variants.A, prediction of alterations in RNA splicing. Each bar corresponds to a single variant and is colored according to the predicted effect. Predictions for HCM-linked variants are shown in the upper half of the panel, whereas results for nonpathogenic variants appear in the lower half of the panel. Blue triangles indicate exon–exon boundaries. Domain boundaries in cMyBP-C are indicated at the top of the panel. B, location of two HCM-linked variants in exon 17 of MYBPC3 that are predicted to induce alterations of splicing. The positions of donor, d, and acceptor, a, splicing sites are indicated. C, experimental determination of RNA splicing by RT-PCR analysis of mRNA isolated from peripheral blood of carriers. CTRL+, mRNA obtained from healthy myocardium. CTRL–, mRNA isolated from HeLa cells, which do not express MYBPC3. The theoretical size of the amplified region if splicing is correct is 692 bp. In some samples, including CTRL–, a nonspecific band is detected at a mobility between 300 and 400 bp. We could not identify the origin of this band. D, prediction of RNA splicing alteration for mutant c.1624G>C (p.E542Q). E, Sanger sequencing result for c.1624G>C (p.E542Q) amplification product, which identifies the skipping of exon 17. Note that the sequences of exons 17 and 18 are detected from the last nucleotide of exon 16, as expected from the presence of WT and mutant alleles in the heterozygous donor. F, Sanger sequencing result for the WT amplification product showing normal splicing. G, prediction of RNA splicing alteration for mutant c.1505G>A (p.R502Q). H, Sanger sequencing result for the c.1505G>A (p.R502Q) amplification product. I, minigene strategy to study splicing defects in nonpathogenic variant c.492C>T (p.G164G), which is located in exon 4 of MYBPC3. J, results from RT-PCR amplification of mRNA. CTRL− is a nontransfected control. K and L, prediction of RNA splicing alteration and Sanger sequencing result for variant c.492C>T (p.G164G). In panels (C) and (J), “n” and “s” indicate bands corresponding to native splicing or skipping of exons, respectively. In panels (E), (H), and (L), the blue boxes show the sequences resulting from the predicted change in splicing because of the variants. M corresponds to 1 kb plus DNA ladder (Invitrogen), and base pairs are indicated. See File S2 for experimental details. cMyBP-C, cardiac myosin-binding protein C; HCM, hypertrophic cardiomyopathy.

Figure 3

Experimental characterization of protein destabilization induced by MYBPC3 variants.A, CD spectra (presented as mean residue ellipticity [MRE]) obtained for C4 WT (black) and C4-A627V (red) domains. The C4 domain spectrum has been reported elsewhere (30). B, CD spectra obtained for the C1 WT (black) and C1-R177H (green) domains. C, temperature at the midpoint of the thermal transition, T_m, for WT cMyBP-C domains, obtained by performing a sigmoidal fitting to denaturation curves considering a two-state unfolding process. Error bars correspond to the standard deviation of the sigmoidal fittings (Figs. S2 and S6). D, change in T_m induced by nonpathogenic variants. Position of cMyBP-C domains is indicated at the top of the panel. E, fraction of variants preserving or not protein expression and native structure, as indicated by WT-like far-UV CD spectrum at 25 °C. The total number of variants is indicated. CD data for all domains are presented in Figure S2. cMyBP-C, cardiac myosin-binding protein C; HCM, hypertrophic cardiomyopathy.

Figure 4

Landscape of molecular phenotypes induced by putative nontruncating HCM-linked and nonpathogenic variants in MYBPC3.A, identification of cMyBP-C protein haploinsufficiency drivers in a database of nontruncating MYBPC3 variants according to the workflow proposed in Figure 1B. The number of variants positive for predicted alterations in RNA splicing or protein stability is indicated, together with the outcomes of experimental assessment. Some variants could not be tested bioinformatically because of lack of identification of native splicing sites or lack of high-resolution protein structures. Experimental assessment of protein destabilization shown in brackets corresponds to variants with no available in silico predictions. Results for HCM-linked and nonpathogenic variants are indicated in pink and green, respectively. B, pie chart summarizing the proportion of HCM-linked variants in MYBPC3 inducing different types of cMyBP-C protein haploinsufficiency drivers. For this analysis, we only considered the 14 HCM-linked variants in our database (File S1) for which data on both RNA splicing and protein stability were available. Four of these variants show altered RNA splicing. Four of them lead to domain destabilization. Our analysis assumes that bioinformatics predictions are able to capture alterations with 100% sensitivity (i.e., that there are no false negatives) (7, 42). HCM, hypertrophic cardiomyopathy.

Figure 5

Assessment of cMyBP-C haploinsufficiency drivers in MYBPC3 VUS.A, 73 VUS in ClinVar were screened for alterations to RNA splicing and protein stability. B, results from predictions of RNA splicing. Blue triangles indicate exon–exon boundaries. Each bar corresponds to a single variant and is colored according to the predicted effect on RNA splicing. C, experimental assessment of predicted changes. Variants whose effects on splicing could not be tested experimentally are colored light pink (see Table 2). D, predicted protein destabilization of the 73 VUS. Each bar corresponds to a single variant and is colored according to the predicted protein destabilization. The dotted line marks the highest destabilizing change in ΔΔG detected for a nonpathogenic variant (Fig. S1A). E, experimental determination of changes in thermal stability for the 10 VUS with predicted ΔΔG >3 kcal/mol. The green reference line at ΔT_m = 5 °C marks destabilization values that can be found in nonpathogenic variants (Fig. 3D), whereas we consider ΔT_m > 10 °C (pink reference line) to be a signature of HCM-linked variants (corresponding bars are colored red, whereas variants below the 10 °C threshold are shown in gray). Figure S6 shows the CD data. cMyBP-C, cardiac myosin-binding protein C; VUS, variants of uncertain significance.