RESEARCH ARTICLE EDITORS’ PICKProtein haploinsufficiency drivers identify MYBPC3 variants
that cause hypertrophic cardiomyopathy
Received for publication, April 5, 2021, and in revised form, May 21, 2021 Published, Papers in Press, June 5, 2021,
https://doi.org/10.1016/j.jbc.2021.100854
Carmen Suay-Corredera1,‡ , Maria Rosaria Pricolo1,2,‡ , Elías Herrero-Galán1, Diana Velázquez-Carreras1,
David Sánchez-Ortiz1, Diego García-Giustiniani3, Javier Delgado4, Juan José Galano-Frutos5,6 ,
Helena García-Cebollada5,6 , Silvia Vilches7,8 , Fernando Domínguez1,7,8,9, María Sabater Molina8,10,
Roberto Barriales-Villa9,11 , Giulia Frisso2,12, Javier Sancho5,6,13, Luis Serrano4,14,15, Pablo García-Pavía7,8,9,16,
Lorenzo Monserrat3 , and Jorge Alegre-Cebollada1,*
From the 1Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; 2Dipartimento di Medicina Molecolare e
Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; 3Health in Code, A Coruña, Spain; 4EMBL/CRG Systems
Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona,
Spain; 5Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza,
Spain; 6Biocomputation and Complex Systems Physics Institute (BIFI). Joint Units BIFI-IQFR (CSIC) and GBs-CSIC, Universidad de
Zaragoza, Zaragoza, Spain; 7Heart Failure and Inherited Cardiac Diseases Unit. Department of Cardiology. Hospital Universitario
Puerta de Hierro, Madrid, Spain; 8European Reference Network for Rare and Low Prevalence Complex Diseases of the Heart (ERN
GUARD-HEART), Madrid, Spain; 9Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CIBERCV),
Madrid, Spain; 10Hospital C. Universitario Virgen de la Arrixaca, El Palmar, Murcia, Spain; 11Unidad de Cardiopatías Familiares,
Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña, Servizo Galego de
Saúde (SERGAS), Universidade da Coruña, A Coruña, Spain; 12CEINGE Biotecnologie Avanzate, scarl, Naples, Italy; 13Aragon
Health Research Institute (IIS Aragón), Zaragoza, Spain; 14Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona,
Spain; 15Universitat Pompeu Fabra (UPF), Barcelona, Spain; and 16Universidad Francisco de Vitoria (UFV), Pozuelo de Alarcón,
Madrid, Spain
Edited by Enrique De La CruzHypertrophic 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 un-
known, as a consequence of our incomplete understanding of
the pathomechanisms triggered by HCM-causing variants.
Here, we examined 44 nontruncatingMYBPC3 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 mo-
lecular 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 path-
omechanism in HCM.
Hypertrophic cardiomyopathy (HCM) is the most frequent
inherited cardiac muscle disease, with an estimated prevalence‡ These authors contributed equally to this work.
* For correspondence: Jorge Alegre-Cebollada, jalegre@cnic.es.
© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).of at least 0.5% (1–3). HCM is a frequent cause of sudden
cardiac death in the young and can result in several cardio-
vascular complications including heart failure and thrombo-
embolism (1). Identification of HCM-causing variants has
transformed clinical management of HCM families. Today,
genetic testing can confirm a clinical suspicion, help differ-
ential diagnosis, and forms the basis of family cascade
screening to allow reproductive and professional counseling.
However, as with many other genetic conditions, testing is
limited by the rarity of HCM genetic variants (4, 5). Because
the number of individuals affected by some of these variants is
so low, detailed segregation studies are seldom possible, leav-
ing numerous variants classified as variants of uncertain sig-
nificance (VUS). This situation has motivated efforts to assess
pathogenicity of VUS using functional and population genetics
approaches (6–10).
Variants of MYBPC3, the gene encoding cardiac myosin-
binding protein C (cMyBP-C), are a leading cause of HCM
(Fig. 1A) (4, 11, 12). Most well-established pathogenic variants
inMYBPC3 are frameshift, nonsense, or conserved RNA splice
site mutations that result in truncated polypeptides, which are
more prone to degradation leading to lower total cMyBP-C
protein levels (haploinsufficiency) (13–16). Indeed, cMyBP-C
haploinsufficiency results by itself in the development of
HCM (Fig. 1A, middle) (17–19). Intriguingly, a number of
familial HCM cases are caused by variants in exon regions of
MYBPC3 that do not lead to truncations (Fig. 1A, right) (17,
19, 20), including the most common pathogenic variant inJ. Biol. Chem. (2021) 297(1) 100854 1
Biochemistry and Molecular Biology. This is an open access article under the CC
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsHCM (c.1504C>T, p.R502W) (13). As of February 2021,
ClinVar lists 919 nontruncating MYBPC3 variants potentially
linked to HCM.
Nontruncating MYBPC3 variants currently pose a major
challenge to genetic diagnosis of HCM. Despite recent
encouraging developments, for most variants, it remains
impossible to assign pathogenicity just from their location in
the gene or the nature of specific amino acid changes (10, 21,
22). Interestingly, the clinical manifestations of truncating and
nontruncating pathogenic variants are similar, suggesting that
pathogenicity in some nontruncating variants could also result
from cMyBP-C haploinsufficiency (15, 21, 23, 24). The two
most frequent mechanisms of protein haploinsufficiency
induced by putative nontruncating variants in monogenic
diseases are RNA splicing defects that result in the appearance
of premature stop codons (25) and protein destabilization (26).
Both these haploinsufficiency drivers have been reported in
MYBPC3 variants linked to HCM (7, 8, 19, 20, 27–30).
However, how these molecular features cause disease remainsFigure 1. cMyBP-C haploinsufficiency drivers induced by HCM-linked and n
(in yellow) in the sarcomere. Middle, most HCM-causing MYBPC3 variants lead to
variants are nontruncating and result in full-length mutant proteins (muta
loinsufficiency drivers in a curated database of HCM-linked and nonpathogeni
protein destabilization are made for the variants building up this database.
haploinsufficiency drivers induced by these variants. cMyBP-C, cardiac myosin
2 J. Biol. Chem. (2021) 297(1) 100854unknown because of the lack of systematic comparison with
nonpathogenic variants. For instance, mild changes in splicing
may be better tolerated than full native splicing abrogation by
a conserved splice site mutation (20). Ito et al. (7) showed that
putative nontruncating MYBPC3 variants appearing in car-
diomyopathy gene databases more frequently lead to RNA
splicing alterations than variants in control databases; how-
ever, interpretation of results is complicated by the presence of
HCM variant carriers in the general population (31). In a
recent report, Thompson et al. (22) showed that 32% of
pathogenic and 7% of nonpathogenic missense MYBPC3 var-
iants are predicted bioinformatically to induce protein domain
destabilization, suggesting that protein destabilizing features
can provide evidence supporting variant pathogenicity. These
encouraging computational results call for experimental
assessment of protein destabilization phenotypes induced by
MYBPC3 variants.
Here, in quest of molecular features that identify patho-
genicity, we examined cMyBP-C haploinsufficiency driversonpathogenic MYBPC3 variants. A, left, scheme of the location of cMyBP-C
truncated polypeptides and protein haploinsufficiency. Right, the remaining
nt domain is represented in red). B, workflow to identify cMyBP-C hap-
c MYBPC3 variants. Bioinformatics predictions of RNA splicing alteration and
Positive hits are then further assessed experimentally to identify protein
-binding protein C; HCM, hypertrophic cardiomyopathy.
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsin missense and synonymous MYBPC3 variants. Our strategy
builds on the characterization of variants for which cose-
gregation and population genetics data are available,
providing information about their pathogenicity. We pro-
pose that experimentally testable variant-induced RNA
splicing alteration and extensive protein destabilization
provide functional evidence of pathogenicity in 11% of pu-
tative nontruncating MYBPC3 variants currently classified as
VUS in ClinVar.
Results
A database of HCM-linked and nonpathogenic MYBPC3
variants
We built a database containing 20 HCM-linked and 24
nonpathogenic putative synonymous and missense MYBPC3
variants covering the entire coding sequence of the gene (see
Experimental procedures section, File S1). We classified as
HCM-linked those variants showing minor allele frequency
(MAF) <10−4 in the Genome Aggregation Database and
showing evidence of pathogenicity based exclusively on
cosegregation and population genetics criteria validated by
the American College of Medical Genetics (ACMG) (32).
Our approach enables agnostic investigation and quantifi-
cation of molecular pathomechanisms, since pathogenicity is
not assigned according to functional or bioinformatics
criteria. Interestingly, the majority of HCM-linked variants
in our database target domains C3, C6, and C10, as observed
in the Sarcomeric Human Cardiomyopathy Registry (21). All
nonpathogenic variants have MAF >10−4, which is incom-
patible with HCM prevalence (13). We next investigated
whether altered RNA splicing and protein stability are spe-
cific to HCM-linked variants, under the premise that mo-
lecular features related to disease should not appear in
nonpathogenic variants. We first triaged variants using bio-
informatics predictors and then assessed positive hits
experimentally (Fig. 1B).
Alteration of RNA splicing by HCM-linked variants
RNA splicing is the process by which noncoding introns
are removed from precursor mRNA, leading to exon-only–
containing mature mRNA. Splicing involves specific recog-
nition of the strictly conserved two first (donor site) and last
(acceptor site) intron nucleotides. Mutations in these se-
quences impair canonical splicing, resulting in alternative
mRNAs that contain premature stop codons, or lead to
insertion/deletions in the polypeptide (33). Indeed, muta-
tions in MYBPC3 that affect conserved splicing sites are
considered to lead to truncations and are classified as
pathogenic (8, 13). However, the correct functioning of the
splicing machinery also involves recognition of sequence
features in the exons, particularly in regions close to exon–
exon boundaries. As a consequence, variants in exonic se-
quences of MYBPC3 traditionally classified as missense can
result in RNA splicing alterations that lead to truncated
polypeptides (7, 8, 20, 34). Indeed, we found that four HCM-
linked variants in our database are predicted in silico toinduce splicing site losses (Fig. 2A and Table 1). These
variants target the first or last nucleotide of an exon (Note
S1). Predictions also identified two HCM-linked variants
that can lead to activation of new splicing sites. In contrast,
all nonpathogenic variants target nucleotides outside exon–
exon boundaries, and none of them is predicted to cause loss
of native splicing sites. For two nonpathogenic variants, the
appearance of a new splicing site is suggested (Fig. 2A,
Table 1, and Note S1).
To validate predicted RNA splicing alterations, we mined
data available in the literature and/or examined RNA
splicing experimentally. RNA splicing of the MYBPC3
transcript is best studied from myocardial samples; however,
the scarcity of genotyped myocardium has limited analysis to
a few variants (8, 19, 20). We employed two alternative and
more accessible strategies whose results are so far in excel-
lent agreement with experiments using myocardial samples.
These strategies are analysis of mRNA from the leukocyte
fraction of peripheral blood from variant carriers, which has
the advantage of immediate translational potential (34–39),
and in vitro experiments using minigene constructs (7, 38,
40) (Table S1). In both cases, isolated mRNA was amplified
by RT-PCR using specific pairs of primers, and results were
analyzed by electrophoresis and Sanger sequencing (Fig. 2
and File S2).
In Figure 2, B–H, we show results from amplification of the
exon 15–exon 21 region using mRNA obtained from blood
samples that carry different MYBPC3 variants. If splicing is
correct, amplification results in a fragment of 692 bp, as shown
for the WT individual (Fig. 2C). We observed that variant
c.1624G>C (p.E542Q) leads to a higher electrophoretic
mobility band at 500 bp, marking skipping of exon 17, in
agreement with predictions that the mutation perturbs a native
donor site (Fig. 2, C–E). In contrast, the prediction that variant
c.1505G>A (p.R502Q) causes an acceptor site gain was not
validated, as splicing proceeded as for WT (Fig. 2, C and F–H).
Equivalent results have been obtained before using other
experimental approaches (Table 1).
In Figure 2, I–L, we present the results of a minigene
strategy to test whether the nonpathogenic variant c.492C>T
(p.G164G) induces the appearance of a new donor site, as
suggested bioinformatically. RT-PCR amplification generated
two bands both in the WT and the mutant sample (Fig. 2J).
The 850 bp band corresponds to the skipping of the MYBPC3
insert, a scenario that is not uncommon in minigene assays.
The 1100 bp band results from the correct inclusion of
MYBPC3 exons 4 and 5 in the minigene transcript, which was
further verified by Sanger sequencing (Fig. 2L). No other band
was amplified in the c.492C>T sample. Hence, the prediction
in Figure 2K of a new splicing site for this variant was not
validated experimentally.
Combining our findings with data from the literature, we
were able to collect results for all predicted RNA splicing al-
terations (Table 1). Four HCM-linked variants were confirmed
to induce aberrant splicing leading to premature stop codons
(Table S2). The validation rate in this set of variants was 4/4
for splicing site losses and 0/2 for splicing site gains.J. Biol. Chem. (2021) 297(1) 100854 3
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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 cor-
responding 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.
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsImportantly, none of the predicted alterations of splicing in
nonpathogenic variants was confirmed experimentally
(Table 1). Hence, our results indicate that alteration of RNA4 J. Biol. Chem. (2021) 297(1) 100854splicing occurs in 20% of HCM-linked variants and is associ-
ated with HCM at 100% specificity (p = 0–0.016; Table 1 and
Note S2).
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EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsHCM-linked variants induce extensive protein destabilization
A reduction in protein stability leads to more frequent
unfolding, which can result in degradation-sensitive poly-
peptides and reduced total protein levels (26, 41). To analyze
protein destabilization induced by the variants in our database,
we first used FoldX (CRG) (42). This software can predict
protein destabilization if the high-resolution 3D structures of
targeted domains are known. Hence, we examined the stability
of the variants affecting domains C0, C1, C2, C3, and C5 of
cMyBP-C, for which high-resolution structures are available
(Protein Data Bank codes: 2K1M, 3CX2, 1PD6, 2MQ0, and
1GXE, respectively). Surprisingly, the distribution of ΔΔG
values for these ten HCM-linked mutants is indistinguishable
from that of nonpathogenic variants. Indeed, the HCM-linked
mutant with the highest destabilization is C2-F448S (ΔΔG =
2.2 kcal/mol), but nonpathogenic variants C3-G507R and C3-
A522T are predicted to induce at least the same level of
destabilization (Fig. S1A and File S1). These results are in
apparent contradiction with those reported by Thompson
et al. (22), who predicted protein destabilization in 32% of
HCM-linked variants. This discrepancy probably originates
from the fact that most destabilizing mutations identified by
Thompson et al. target domains C6 and C10, which we did not
screen because their high-resolution structures remain un-
known. Hence, we extended analysis of protein destabilization
to the variants in our database with no FoldX prediction by
examining experimentally recombinant WT and mutant
cMyBP-C domains. We also included in this analysis the three
nonpathogenic variants with the highest predicted destabili-
zation. This approach allowed us to obtain information about
the extent of protein destabilization that is not expected to be
associated with development of HCM. Unfortunately, variants
targeting domains C6 and C8 could not be studied because the
corresponding WT domains were refractory to recombinant
expression (see Experimental procedures section).
Protein expression was induced in Escherichia coli, and
purified domains (Fig. S1B) were analyzed by far-UV CD
spectroscopy, a technique that reports protein secondary
structure (43, 44). We found that three of the four HCM-
linked mutants (C4-D610N, C10-G1206D, and C10-Y1251H)
could not be produced in soluble form in the most favorable
expression conditions, suggesting strong destabilization (see
Experimental procedures section, File S1 and Fig. S1C). The
CD spectrum of the remaining HCM-linked mutant C4-
A627V had features not present in the WT domain, suggest-
ing structural alterations in this mutant (Fig. 3A). Of the
nonpathogenic variants, eight of nine preserve protein struc-
ture (Figs. 3B and S2). To characterize the impact of mutations
on domain stability, we tracked CD signals at increasing
temperatures. As protein domains transition between the
native and the unfolded states, the CD signal varies (Figs. S2
and S3), and the temperature at the midpoint of the dena-
turing transition, or melting temperature (Tm), informs on the
thermal stability of the domain (44). We determined that
nonpathogenic variants induce maximum ΔTm (Tm [WT] −
Tm [mutant]) = 4.3 
C (Fig. 3, C and D and File S1), suggestingJ. Biol. Chem. (2021) 297(1) 100854 5
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsthat limited changes in Tm up to 5 
C are generally well
tolerated and cannot be linked to pathogenicity. There was
only one nonpathogenic variant that could not be produced
(C9-R1138H) (Fig. S1C). Using molecular dynamics simula-
tions, we obtained evidence that mutant domains that could
not be expressed in native form are indeed destabilized
(Table S3).
In summary, in four (20%) of the HCM-linked variants, our
protein stability analysis workflow was able to capture domain
destabilization, as indicated by lack of expression of mutant
domains or by alterations in CD spectra at 25 
C. These results
indicate that domain destabilization associates with pathoge-
nicity with 94% specificity (p = 0.01–0.075; Fig. 3E and Note
S2).
Prevalence of cMyBP-C haploinsufficiency drivers
Our results in the previous sections show that 40% of the
HCM-linked variants in our database induce altered RNA
splicing or protein destabilization (Fig. 4A). The extent of these
cMyBP-C molecular phenotypes among pathogenic variants
may be higher since we could not obtain information on
splicing for three mutants because of limited bioinformatics
predictions and on protein destabilization for six mutants
because of our inability to produce WT domains. Hence, we
analyzed the distribution of cMyBP-C haploinsufficiency
drivers in the subset of variants for which we have information
for both RNA splicing and protein stability. This analysisA
250240230220210200
Wavelength (nm)
M
RE
(d
eg
·c
m
2 ·d
m
ol
-1
) x
 1
0-
3
 C4 WT
 C4-A627V
0
-2
-4
-6
2
M
RE
(d
eg
·c
m
2 ·d
m
ol
-1
) x
 1
0-
3
B
220210200
Wavele
8
6
4
2
0
-2
-4
D
Δ 
T m
 (º
C)
-1
0
1
2
3
4
Number of amino acid in cMyBP-C sequence
10008006004002000
C0 P C1 M C2 C3 C4 C5 C6 C7 C8
Figure 3. Experimental characterization of protein destabilization induced
[MRE]) obtained for C4 WT (black) and C4-A627V (red) domains. The C4 domain
C1 WT (black) and C1-R177H (green) domains. C, temperature at the midpoin
forming a sigmoidal fitting to denaturation curves considering a two-state u
sigmoidal fittings (Figs. S2 and S6). D, change in Tm induced by nonpathogenic
fraction of variants preserving or not protein expression and native structure,
variants is indicated. CD data for all domains are presented in Figure S2. cMyB
6 J. Biol. Chem. (2021) 297(1) 100854showed that >55% of HCM-linked variants induce RNA
splicing alteration or protein destabilization (Fig. 4B). Strik-
ingly, several variants show no evidence of either of these
molecular phenotypes, as observed previously for p.R502W
(19, 45), the most common pathogenic variant in HCM (13).
Molecular phenotyping of VUS
According to our data, defects in RNA splicing and protein
destabilization are associated with pathogenicity with close to
100% specificity. Prompted by this observation, we investigated
the extent to which assessment of protein haploinsufficiency
drivers might contribute to assigning pathogenicity to
MYBPC3 variants currently classified as VUS. For this, we
studied putative nontruncating VUS reported in ClinVar with
MAF <10−4. To be able to triage domain destabilization bio-
informatically, we restricted our analysis to the 73 VUS tar-
geting cMyBP-C domains whose high-resolution structures are
known (Fig. 5A and File S3). RNA splicing was predicted to be
altered in 14 of 68 variants in which native splice sites were
detected, and a new splicing site was predicted in one of the
five remaining variants (Fig. 5B and File S3). We managed to
gather experimental information for ten of these, leading to
confirmation of RNA splicing alterations in two of two pre-
dicted splice site losses and in three of nine predicted site
gains, all of them generating premature stop codons (Fig. 5C,
Table 2, Fig. S4, and Table S2). For the five untested variants,
we could not recruit carriers, and the minigene assays did not250240
ngth (nm)
230
HCM-linked Nonpathogenic
Preserving
Not preserving
0
100
80
60
40
20
%
 v
ar
ia
nt
s
1/9
8/94/4
C
T m
 (º
C)
60
50
40
30
C0
 W
T
C1
 W
T
C2
 W
T
C3
 W
T
C4
 W
T
C5
 W
T
C7
 W
T
C9
 W
T
C1
0 
W
T
1200
C9 C10
E
 C1 WT
 C1-R177H
by MYBPC3 variants. A, CD spectra (presented as mean residue ellipticity
spectrum has been reported elsewhere (30). B, CD spectra obtained for the
t of the thermal transition, Tm, for WT cMyBP-C domains, obtained by per-
nfolding process. Error bars correspond to the standard deviation of the
variants. Position of cMyBP-C domains is indicated at the top of the panel. E,
as indicated by WT-like far-UV CD spectrum at 25 
C. The total number of
P-C, cardiac myosin-binding protein C; HCM, hypertrophic cardiomyopathy.
Preserved RNA splicing
and protein stability
(43%)
Alteration of
RNA splicing
(29%)
Decreased
protein
stability
(29%)
A
Alteration of
RNA splicing
0/10
0/12
5/17
2/22
Alteration of
protein stability
DATABASE OF PUTATIVE
NONTRUNCATING
MYBPC3 VARIANTS 
BIOINFORMATICS
PREDICTIONS
EXPERIMENTAL
ASSESSMENT
(+4/4)
0/3; (+1/6)
4/4 site loss; 0/2 new site
0/2 new site20 HCM-linked variants
24 nonpathogenic variants
B
Figure 4. Landscape of molecular phenotypes induced by putative nontruncating HCM-linked and nonpathogenic variants in MYBPC3. A, iden-
tification 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.
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsrecapitulate native splicing in WT sequences (File S2 and
Fig. S5). Overall, we were able to detect splicing alterations in
four variants (5.5% of all VUS retrieved from ClinVar).
Regarding protein stability, FoldX predicted that ten vari-
ants have higher destabilization than the most destabilized
nonpathogenic variant (Fig. 5D and File S3). We experimen-
tally verified extensive domain destabilization, as evidenced by
Tm changes >10 
C, in C1-G155V, C1-G169S, C1-L199P, and
C2-V385M (Figs. 5E and S6) (46). We also observed slight
changes in the CD spectra of mutants C0-R44H and C5-
A686P, although these were not as pronounced as those
induced by pathogenic variant C4-A627V (Figs. 3A and S6).
Hence, our workflow captured at least four variants that
induce strong domain destabilization.
Discussion
The clinical management of HCM families has benefited
from the discovery of causative genes over the last two
decades (1). Currently, genetic testing is a class I recom-
mendation in both the European and American HCM
clinical practice guidelines. Thanks to the advent of next-
generation DNA sequencing techniques, the number of
genetic variants found in HCM patients has increasedconsiderably, creating new challenges in variant interpre-
tation (4, 37, 47–49). Many genetic variants are present in
only a few patients, limiting the power of cosegregation
analyses to assess pathogenicity. Alternatively, functional
deficits linked to disease progression can enable classifica-
tion of rare pathogenic variants, as implemented in the
ACMG guidelines (32, 47, 50–52).
Here, we have phenotyped MYBPC3 variants in the search
for protein haploinsufficiency drivers that can sustain patho-
genicity. Importantly, we compared HCM-linked and
nonpathogenic variants, which allowed us to link molecular
properties to pathogenicity. Although our approach leads to
small sample sizes, our results demonstrate that around 50% of
putative nontruncating HCM-linked variants in MYBPC3
significantly and specifically induce altered RNA processing or
extensive protein destabilization (Fig. 4B). Furthermore, we
have identified these pathogenicity drivers in 11% of MYBPC3
variants currently classified as VUS in ClinVar (Fig. 5).
Considering these results, we estimate that close to 20% of
missense and synonymous variants in MYBPC3 currently
classified as VUS in ClinVar may show functional evidence of
pathogenicity based on the induction of protein hap-
loinsufficiency drivers.J. Biol. Chem. (2021) 297(1) 100854 7
73 nontruncating
VUS in MYBPC3
RNA splicing Protein stability
PR
ED
IC
TI
O
N
A
SS
ES
SM
EN
T
Splicing site lossNo alteration of RNA splicing
New splicing site Both new splicing siteand splicing site loss 
Number of amino acid in cMyBP-C sequence
0 200 400 600 800
Experimentally confirmed
change in RNA splicing 
Predicted change in RNA
splicing (not assessed) 
0 200 400 600 800
Number of amino acid in cMyBP-C sequence
10
5
0''
G
 (k
ca
l/m
ol
)
8006004002000
Number of amino acid in cMyBP-C sequence
VUS with predicted
ΔΔG > 3 Kcal/mol
VUS with predicted
ΔΔG < 3 Kcal/mol
16
14
12
10
8
6
4
2
0
8006004002000
Number of amino acid in cMyBP-C sequence
Δ 
T m
 (º
C)
B
C
D
E
A
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
ΔTm = 5 
C marks destabilization values that can be found in nonpathogenic variants (Fig. 3D), whereas we consider ΔTm > 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.
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsThe first step of our workflow to assess protein hap-
loinsufficiency drivers in MYBPC3 variants uses in silico tools
to predict alterations in RNA splicing and protein stability,
which are then validated experimentally (Fig. 5). Hence, it is
important to consider the sensitivity and specificity of these
bioinformatics tools. For example, experimental analysis
following negative predictions is discouraged because the
sensitivity of predictions is generally high (7, 42). Direct
experimental testing would only be indicated if algorithms fail
to capture native splicing sites, or when there is no high-
resolution protein structural information. Regarding speci-
ficity of predictions of RNA splicing alterations, we observed
0% (0/6) false positives for the prediction of site losses,8 J. Biol. Chem. (2021) 297(1) 100854contrasting with a 77% (10/13) false-positive rate for the pre-
diction of new sites, in agreement with previous observations
(7, 37) (Tables 1 and 2). Hence, experimental validation of
alterations of RNA splicing, particularly for prediction of new
sites, is a strict requirement for the identification of true
positives, which in the clinical setting can be implemented
easily by analyzing the peripheral blood of carriers. We also
recommend validation of protein destabilization predictions,
since we could only verify extensive protein destabilization in
40% of FoldX-triaged VUS variants (Fig. 5, D and E).
Our study supports the emerging view that genetic variants
that affect RNA splicing are pathogenic (7, 8, 53, 54). Inter-
estingly, these RNA-splicing–altering mutants can be found in
Table 2
Experimental assessment of predicted RNA splicing alterations in MYBPC3 VUS using minigenes
VUS (cDNA) VUS (protein) Predicted splicing alteration Experimental splicing alteration Reference
39C>T S13S DG — File S2
194C>T T65M AG — File S2
479G>A R160Q AG No Fig. S4, B and C
505G>A G169S DL, DG Yes (DL, AG) Fig. S4, B and C
659A>G Y220C DG No (7)
1213A>G M405V DG Yes (7, 40)
1287G>A A429A AG — File S2
1309G>A V437M DG — File S2
1466A>T D489V DG — File S2
2155T>C C719R DG No Fig. S4, B and C
2217G>A E739E AG No Fig. S4, B and C
2234A>G D745G DG No (7)
2249C>T T750M AG No Fig. S4, B and C
2288A>G N763S DG Yes Fig. S4, B and C
2308G>C D770H DL Yes Fig. S4B
Abbreviations: AG, acceptor gain; DG, donor gain; DL, donor loss; VUS, variants of uncertain significance.
Five variants could not be studied. We experimentally studied the variants for which no information was available in the literature. Experimental results were obtained from the
literature and/or this study, as indicated.
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsboth exonic and intronic regions that are far from splice sites if
the consequence of the mutation is the activation of a new
splice site (Table 2) (7, 37). Interestingly, some nonpathogenic
variants are found in regions close to splice sites; however, they
are not predicted to alter splicing to the same extent as
pathogenic variants (8). Our data are consistent with this
observation (Note S1). To date, there have been no reports of
nonpathogenic MYBPC3 variants causing altered RNA
splicing.
To the best of our knowledge, our study is the first to
experimentally characterize the frequency of protein destabi-
lization induced by HCM-linked missense MYBPC3 variants
(Fig. 4B). Our results are in good agreement with previous
estimates suggesting that 32% of pathogenic variants could
induce protein destabilization (22). However, we acknowledge
that there are challenges associated with the functional
assessment of MYBPC3 variants based on protein stability.
First, there is no high-resolution structural information for
many cMyBP-C domains, which may hamper effective triaging
of potentially disruptive variants.
A second challenge stems from experimental validation of
protein destabilizing phenotypes using recombinant domains,
which in the case of highly destabilizing mutations can be
difficult or even impossible. Our interpretation that strong
destabilization hampers mutant domain production is sup-
ported by molecular dynamics simulations (Table S3) and is
consistent with similar observations in similar settings (55);
however, recombinant production of mutant domains may fail
for reasons other than reduced stability, including toxicity to
host or codon bias (56). In our set of experiments, we could
not produce the nonpathogenic C9 domain carrying variant
R1138H. The WT C9 domain has the lowest Tm value among
all domains assayed (42.2 
C; Fig. 3C). However, C9 is a
fibronectin-III type domain, a protein fold whose typical Tm
values are well above 50 
C (57). We speculate that our re-
combinant C9 domain may not recapitulate native stability and
that the impact of minor, nonpathogenic changes on stability
may be higher in this context. It is also possible that the native
environment of the sarcomere limits the destabilizing effect ofthe mutation via post-translational modifications or protein–
protein interactions.
The fact that protein stability is not binary poses a third
challenge to protein destabilization–guided functional classi-
fication of variants. We observed that nonpathogenic variants
can cause slight protein destabilization (ΔTm < 5 
C; Fig. 3D).
Based on the typical distribution of ΔTm in unselected single
amino-acid polymorphisms across different proteins (46), we
considered that ΔTm > 10 
C is a signature of pathogenic
protein destabilization. The confidence of this threshold could
be increased by measuring the stability of additional
nonpathogenic variants. In addition, more refined thresholds
could be obtained by assessing the stability of multidomain
cMyBP-C constructs, although the highly similar thermal
stability of consecutive cMyBP-C domains (Fig. 3C) can
complicate interpretation of results.
From amechanistic point of view, both the alteration of RNA
splicing and the destabilization of cMyBP-C domains can lead to
reduced cMyBP-C levels, similar to the situation induced by
truncating variants (18, 28, 58, 59). It is remarkable though that
many HCM-linkedMYBPC3 variants do not alter RNA splicing
or protein stability (Fig. 4B). The pathogenicity triggers in those
cases, which include well-established HCM variants such as
c.1504C > T, p.R502W, remain unknown. A tempting hy-
pothesis is that some of them can induce protein hap-
loinsufficiency by alternative mechanisms, including decreased
rates of transcription and translation (60–62), increased
recognition bymRNA and protein degradationmachineries (21,
60, 63, 64), and defective incorporation of cMyBP-C in the
sarcomere (65). Alternatively, in the absence of protein hap-
loinsufficiency, variants can lead to perturbed binding to protein
partners, resulting in altered sarcomere function (66–70).
Tantalizingly, both protein haploinsufficiency and altered
binding could result in converging misregulation of the super-
relaxed state of myosin, inducing sarcomere hypercontractility
typical of HCM (71).
In summary, we propose that identification of protein
haploinsufficiency drivers in MYBPC3 variants provides
functional evidence of pathogenicity (PS3 criterion in theJ. Biol. Chem. (2021) 297(1) 100854 9
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variantsACMG guidelines (32)) and can contribute to the assessment
of pathogenicity of putative nontruncating variants in the
MYBPC3 gene. RNA splicing alterations and protein destabi-
lization can be validated using readily available laboratory as-
says, addressing the urgent need for methods to assign
pathogenicity of genetic variants associated with HCM (51,
72). Our results increase the number of actionable variants in
MYBPC3, thus having the potential to improve clinical man-
agement of HCM families, and can be extended to other dis-
eases caused by protein loss of function.
Experimental procedures
Human samples
Human samples were obtained from patients with informed
consent according to Declaration of Helsinki guidelines. Research
involving humans was authorized by the Comité de Ética de
Investigación of Instituto de Salud Carlos III (ISCIII; PI 39_2017).
Selection of genetic variants
We retrievedMYBPC3 variants from the Health in Code-Mu-
tations database, which includes information about >155,000
individuals obtained from>50,000 articles in the literature, aswell
as from Health in Code’s own clinical reports. Variants were
included in theHCM-linked group if they showMAF<10−4 in the
Genome Aggregation Database and are enriched in HCM in-
dividuals (ACMGPS4criterion), cosegregatewithdisease (ACMG
PP1 criterion), or arede novomutations in the context of no family
history of disease (ACMG PS2 criterion). All nonpathogenic var-
iants satisfy MAF >10−4. To retrieve VUS in MYBPC3 from
ClinVar, we considered all nontruncating variants associated with
HCM, excluding those with conflicting interpretations of patho-
genicity or showing MAF >10−4. We restricted our VUS analysis
to variants targeting domains for which there is high-resolution
structural information.
Bioinformatics prediction of haploinsufficiency drivers
We used Alamut Visual (Interactive Biosoftware) to predict
RNA splicing alterations induced by all variants in our HCM-
linked/nonpathogenic and VUS databases. Alamut implements
analyses of four RNA splicing prediction algorithms (SSF,
MaxEnt, NNSPLICE, and GeneSplicer). We first determined if
at least two of the algorithms identified the canonical splicing
site around the mutation site. We then calculated the percent
change in splicing score for native sites and potential new
splicing sites. Positive hits result in at least two tools predicting
>10% decrease (for native sites) or increase (for new sites) in
the splicing score, in agreement with published guidelines (73).
Changes in protein thermodynamic stability were estimated by
FoldX. This software estimates changes in free energy (ΔΔG)
upon mutation from empirically and statistically derived en-
ergy functions (42).
Analysis of RNA splicing
For experimental determination of RNA splicing, we
analyzed blood samples from variant carriers or used10 J. Biol. Chem. (2021) 297(1) 100854engineered minigene constructs (File S2). In the case of blood
samples, total RNA was extracted from leukocytes of carriers
using Trizol (Thermo Fisher Scientific). RNA retro-
transcription was done with random primers by SuperScript
IV VILO Master Mix (Thermo Fisher Scientific), and the re-
gion of interest was PCR amplified using specific oligos (File
S2). RT-PCR products were purified using the QIAquick
PCR Purification Kit (Qiagen) and then Sanger sequenced
when necessary. Correct RNA splicing generates readable
electropherograms, obtained for both primers, and whose
sequence matches the sequence of the canonical comple-
mentary DNA (cDNA) for cMyBP-C. For minigene experi-
ments, the WT and mutant genomic DNA fragments were
obtained from Integrated DNA Technologies, including at
least the exon of interest and the 50 and 30 intronic flanking
regions (File S2). The constructs were cloned into the β-glo-
bin’s intron 2 of the pMGene vector (74) using KpnI. Alter-
natively, pMGene vectors including the inserts of interest were
obtained from GeneArt Gene Synthesis (from Thermo Fisher
Scientific). In both cases, the resulting constructs were
expressed in human embryonic kidney 293 monolayers
cultured in Dulbecco’s modified Eagle’s medium (Gibco)
supplemented with 10% fetal bovine serum and 1% penicillin/
streptomycin at 37 
C and 5% CO2. Cells were transiently
transfected with 1 μg of WT or mutant pMGene using
FuGENE HD (Promega) according to the manufacturer’s
protocol. About 24 to 48 h after transfection, cells were
collected and mRNA was extracted and retrotranscribed, and
PCR products were purified and sequenced to compare
splicing of WT and mutant constructs as aforementioned.
Alternatively, purified PCR products were loaded onto 2%
preparative agarose gels to isolate specific bands of interest
(Fig. S4). These bands were purified using the QIAquick Gel
Extraction Kit (Qiagen) prior to Sanger sequencing.
Protein expression and purification
The cDNAs encoding the cMyBP-C domains and their
mutants were cloned from myocardial RNA, produced by PCR
mutagenesis, or acquired commercially from Integrated DNA
Technologies. Sequences are available in File S4. cDNAs were
cloned into a custom-modified pQE80L expression plasmid
(Qiagen) using BamHI and BglII enzymes. Final expression
plasmids were verified by Sanger sequencing. Domains were
expressed in E. coli BLR(DE3). Cultures at an absorbance of 0.6
to 1 at 600 nm were induced with IPTG (specific induction
conditions can be found in Files S1, S3, and S4). In general, we
found that expression at lower concentrations of IPTG and
temperature ≤25 
C resulted in better yield of purified
challenging-to-express domains, so these conditions were
preferred for the expression of mutant domains. Purification of
His-tagged domains was achieved by metal affinity and gel
filtration chromatographies (75). Although different expres-
sion conditions were assayed, WT domains C6 and C8 could
not be produced (File S4), so variants targeting these domains
could not be analyzed. Proteins were eluted from the final size-
exclusion chromatography in 20 mM NaPi, pH 6.5, and
EDITORS’ PICK: Molecular phenotyping of MYBPC3 variants63.6 mM NaCl. Proteins were stored at 4 
C. Results of the
expression and purification procedures were evaluated by
SDS-PAGE and Western blotting following standard proced-
ures (Fig. S1B).
CD
CD spectra were collected using a Jasco J-810 spec-
tropolarimeter. Purified proteins were tested in 20 mM NaPi,
pH 6.5, and 63.6 mM NaCl at protein concentrations ranging
from 0.1 to 0.5 mg/ml in 0.1-cm-pathlength quartz cuvettes.
Protein concentration was obtained from absorbance at 280
nm values using theoretical extinction coefficients (Files S1,
S3, and S4). Spectra were recorded at 50 nm/min scanning
speed and a data pitch of 0.2 nm. Four scans were averaged to
obtain the final spectra. The contribution of the buffer was
subtracted, and spectra were normalized by peptide bond
concentration. Major changes in the shape of the CD spec-
trum that could not be explained by concentration inaccur-
acies were considered a signature of domain destabilization.
To study thermal denaturation, the CD signal at a wavelength
at which folded and unfolded protein signals were different
was monitored as the temperature increased from 25 to 85 
C
at a rate of 30 
C/h (File S4). Temperature control was
achieved using a Peltier thermoelectric system. Changes in
CD signal were fitted to a sigmoidal function using IGOR Pro
(Wavemetrics) to estimate Tm.
Molecular dynamics simulations
Full atom molecular dynamics simulations of the WT
domains and their mutants (Table S3) in dodecahedral
boxes (1 nm minimal distance between protein atoms and
box edges) filled with Tip3p water molecules were per-
formed at 410 K in protonation conditions mimicking pH
7.0 using Charmm27 + CMAP force field as described (76).
Homology models were obtained using SWISS-MODEL
(Swiss Institute of Bioinformatics) (77). Mutations were
modeled on the WT structures using Swiss-PdbViewer,
version 4.1.0 (Swiss Institute of Bioinformatics) (77). For
each WT and variant structure, three 1-μs long trajectories
were obtained. Trajectories were analyzed using the model
described in (78) to obtain estimates of ΔΔG upon mutation
at 298 K.
Data availability
All data described in this work is contained within this
article and the supporting information. Sourcing data are
available on reasonable request to the corresponding author.
Supporting information—This article contains supporting
information (79–86).
Acknowledgments—We thank Natalia Vicente for excellent tech-
nical support (through grant PEJ16/MED/TL-1593 from Consejería
de Educación, Juventud y Deporte de la Comunidad de Madrid and
the European Social Fund, European Union). We thank the Spec-
troscopy and Nuclear Magnetic Resonance Core Unit at CNIO foraccess to CD instrumentation. We thank Andrea Thompson and
Sharlene Day for critical reading of the article. Simon Bartlett
(Centro Nacional de Investigaciones Cardiovasculares [CNIC])
provided English editing. We thank all members of the Molecular
Mechanics of the Cardiovascular System team for helpful
discussions.
Author contributions—J. A.-C. conceptualization; D .G.-G., P. G.-P.,
M. R. P., J. A.-C., and L. M. data curation; C. S.-C., M. R. P., E. H.-G.,
D. V.-C., J. D., J. J . G.-F., H. G.-C., J. S., L. S., and J. A.-C. investi-
gation; D. S.-O., S. V., F. D., M. S. M., R. B.-V., G. F., P. G.-P., and
J. A.-C. resources; C. S.-C., M. R. P., and J. A.-C. writing-original
draft; C. S.-C., M. R. P., and J. A.-C. writing-review and editing.
Funding and additional information—J. A.-C. acknowledges fund-
ing from the Ministerio de Ciencia e Innovación (MCIN) through
grants BIO2014-54768-P; BIO2017-83640-P/MINECO/AEI/
FEDER, UE; EIN2019-102966 and RYC-2014-16604, the European
Research Area Network on Cardiovascular Diseases (ERA-CVD/
ISCIII, Spain MINOTAUR, AC16/00045), and the Comunidad de
Madrid (consortium Tec4Bio-CM, S2018/NMT-4443, FEDER). The
CNIC is supported by the ISCIII, MCIN, and the Pro CNIC
Foundation and was a Severo Ochoa Center of Excellence (SEV-
2015-0505). We acknowledge funding from ISCIII to the Centro de
Investigación Biomédica en Red (CB16/11/00425). L. S. acknowl-
edges funding from MCIN (BFU2015-63571-P). J. S. acknowledges
funding from AEI (PID2019-107293GB-I00), Gobierno de Aragón
(E45_17R), and ERDF-InterregV-A POCTEFA, Spain (PIREPRED-
EFA086/15). C. S.-C. is the recipient of an FPI-SO predoctoral
fellowship BES-2016-076638. M. R. P. was the recipient of a PhD
fellowship from the Italian Ministry of Education, Universities and
Research, Italy. H. G.-C. is the recipient of an FPU16/04232 doctoral
contract from MCIN.
Conflict of interest—L. M. holds share in Health in Code. All other
authors declare that they have no conflicts of interest with the
contents of this article.
Abbreviations—The abbreviations used are: ACMG, American
College of Medical Genetics; cDNA, complementary DNA; cMyBP-
C, cardiac myosin-binding protein C; HCM, hypertrophic cardio-
myopathy; MAF, minor allele frequency; VUS, variants of uncertain
significance.
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mation in the membrane. Sci. Rep. 6, 33340Carmen Suay-Corredera is a PhD student at the Molecular Mechanics of the Cardiovascular System group, led by Dr. Jorge
Alegre-Cebollada at Centro Nacional de Investigaciones Cardiovasculares (Madrid, Spain). She studies the molecular mecha-
nisms by which missense mutations in cardiac myosin-binding protein c (cMyBP-C) cause hypertrophic cardiomyopathy
(HCM). By unravelling the pathomechanisms involved in the development of HCM, she aims at providing key insights for the
design of new potential treatments and for the classification of genetic variants in the context of disease. https://www.cnic.es/en/
investigacion/1/7246/miembros.Maria Rosaria Pricolo is a postdoctoral scientist in the Molecular Mechanics of the Cardiovascular System group at Centro
Nacional de Investigaciones Cardiovasculares (Madrid). She obtained a PhD in Molecular Medicine and Medical Biotechnology
from University of Naples–Federico II. Her PhD project dealt with examination of molecular phenotypes of cardiac myosin-
binding protein c missense hypertrophic cardiomyopathy variants, particularly mRNA splicing defects. She is currently work-
ing on the development of novel tools to study pathomechanisms in titin mutations that cause dilated cardiomyopathy and to
examine protein mechanics in living cells. https://www.cnic.es/en/investigacion/1/7246/miembros.