Identification of biomarkers of hypertrophic cardiomyopathy development and progression in Dutch MYBPC3 founder mutation carriers

HCM is characterized by an, usually asymmetric, hypertrophied, non-dilated left
ventricle (especially septal wall thickness of >= 15 mm) in absence of other
disorders that may induce cardiac hypertrophy, like hypertension and aortic
valve stenosis7. The inheritance pattern of HCM is autosomal dominant, and most
mutations have been identified in genes encoding sarcomeric proteins8. HCM can
be an innocent condition and not cause any (sub)clinical symptoms throughout
life in some patients. It is estimated that approximately one quarter of
mutation carriers remain asymptomatic up to their seventies5. At the other end
of the spectrum, however, HCM can lead to severe and even lethal cardiac
events4,5,9-11. For instance, HCM can be diagnosed as a result from cardiac
arrest in adolescence in a proband. Subsequently, an asymptomatic parent or
even grandparent, who may not even show signs of HCM on ECG or cardiac
ultrasound, can turn out to be a carrier of the causal mutation that was
detected in the severely affected proband. As yet, there is no solid
explanation for how an identical mutation can lead to extreme phenotypes at
both ends of the disease spectrum. In the Netherlands, DNA-diagnostics for HCM
is offered since 1996, and in about halve of the index cases a pathogenic
mutation is identified3. The majority of these mutations (approximately 70%) is
located in the MYBPC3 gene, encoding the cardiac myosin-binding protein C
(cMyBP-C)3. There are three Dutch MYBPC3 founder mutations accounting for more
than half of the identified MYBPC3 mutations, which are termed c.2373dupG,
c.2827C>T p.Arg943X and c.2864_2865delCT3. It estimated that in the Netherlands
more than 6000 MYBPC3 founder mutation carriers are present (32.000 x 20%).
Studying these carriers with different cardiac phenotypes offers an unique
cohort with a similar genetic background in which disease penetrance and
factors involved can be assessed. The reduced penetrance and variable
expression of HCM is a very challenging aspect in the counseling of
(asymptomatic) mutation carriers and determination of cardiac surveillance
schemes and medical treatment. Initially, learning about carriership which
predisposes to a potential lethal condition can cause a lot of distress and
insecurity about one*s future perspectives. Annual to biannual cardiologic
examinations are now recommended for asymptomatic mutation carriers, but
approximately 25% of these *patients* will undergo these tests throughout life
without developing any clinical signs or symptoms of the disease5. Moreover,
once the onset of HCM has been documented, it remains impossible to predict the
course of disease. Therefore, there is an urgent need to develop prediction
models which that enables quantification of the risk of asymptomatic mutation
carries on disease development. In addition, biomarkers that may predict the
progression of disease in mutation carriers in whom the onset of disease has
been documented are required to improve personal medical treatment.

Age and gender have been shown to be major disease determinants in MYBPC3
mutation carriers6. A potential source of biomarkers with additional predictive
value for HCM development and progression are exosomes. Extracellular vesicles,
including exosomes are nanovesicles secreted into the extracellular
environment, like blood and saliva, upon internal vesicle fusion with the
plasma membrane12. Extracellular vesicles contain cytosolic components and are
expected to serve as excellent sources for biomarkers of different forms of
disease as they reflect a *liquid biopsy* of pathological tissues. Indeed,
exosomal markers are already used in clinics for diagnosis and prognosis of
several tumors12. My collaborators have recently observed the presence of
sarcomeric proteins in extracellular vesicles isolated from blood of patients
with atherosclerotic disease (unpublished data). The founder mutations in
MYBPC3 all occur >50 nucleotides away from the last exon-intron boundary within
the mRNA, targeting them for nonsense medicated RNA decay (NMD)13. Reduced mRNA
of mutated MYBPC3 alleles has been observed in (end-stage) cardiac
tissues14,15. Efficiency of the NMD machinery differs between individuals and
upregulation of the wild type allele, or variation in degradation of toxic
cMyBP-C peptides by the ubiquitin-proteosome system has also been proposed to
explain the variability of HCM in mutation carriers16. Furthermore, recent
murine studies have shown that stress induces a reduction in cMyBP-C content in
mice with a heterozygous MYBPC3 truncating mutation, leading to an exacerbation
of cardiac dysfunction17. This supports the haploinsufficiency hypothesis,
which implies that a reduction in amount of cMyBP-C levels in MYBPC3 mutation
carriers induces cardiac deterioration13. Therefore, the regulation of total
cMyBP-C content could very well be partly responsible for the variable
expression and reduced disease penetrance in mutation carriers. cMyBP-C levels
in exosomes isolated from blood from MYBPC3 mutation carriers may reflect
disease
onset and/or severity.

Another potential source of biomarkers for HCM development and progression are
energy metabolites. Energy depletion has been proposed to be a major
pathophysiological mechanism driving HCM18. In vitro and in vivo studies have
shown that HCM causing mutations induce a higher Ca2+ sensitivity of the
contractile unit18. Subsequently, inefficient cross-bridging leads to more ATP
usage, thereby compromising the energy level of the cardiomyocyte18. Indeed,
using magnetic resonance spectroscopy, a reduction of phospho-creatine to ATP
ratio, which reflects energy status, has been observed in the myocardium of HCM
mutation carriers, even prior to left ventricle hypertrophy occurred19.
Furthermore, ATPase activity during force
development within the cardiomyocyte was shown to be higher in MYBPC3 mutation
carriers20. Perhexiline treatment, which is proposed to shift myocardial
metabolism from fatty acid to glucose utilization, was shown to improve energy
capacity in symptomatic HCM patients21. In keeping with this, metabolic
analyses of heterozygous MYBPC3 knock-in mice were indicative of an altered
fatty acid transport into mitochondria upon perhexiline treatment, suggestive
of a reduction of fatty acid beta oxidation22. This all suggests that a shift
in plasma energy metabolites may represent an early stage of HCM development
which may be exemplified during successive stages of the disease. However, no
study of metabolic energy biomarkers in blood of HCM patients has been reported
to date.

The objective of this study is to identify biomarkers predictive for the
development of HCM in asymptomatic MYBPC3 mutation carriers and to identify
prognostic biomarkers in mutation carriers in whom the disease has already been
revealed. In order to establish the predictive value of these potential
biomarkers, the generation of a nation-wide prospective cohort is required.
This prospective cohort will include plasma samples of MYBPC3 founder mutation
carriers with and without a phenotype at baseline, and plasma will be
additionally collected at different time points and, thereby, at different
stages of the disease. Plasma will be taken during (bi)annual control visits of
all participating mutation carriers in the cohort, preferably prior and after
exercise tests. Rationale behind this is that exercise may increase the level
of a plasma biomarkers (specifically energy metabolites) because of the stress
exposed to the cardiomyocyte during extreme exercise. We aim to include 1000
MYBPC3 founder mutation carriers in the prospective cohort. Follow-up time will
be for indefinite duration (until cardiac transplant, death or withdrawal for
other (personal) reasons).

We will perform a case-control study at the baseline of the creation of our
prospective cohort to identify potential biomarkers. We will analyze potential
biomarkers in 100 included HCM patients with a severe phenotype at baseline
versus 100 included mutation carriers with no cardiac phenotype at baseline (as
determined by ECG and cardiac ultrasound). The prediction models for HCM
development and progression, using the potential biomarkers identified in the
case control setting, will subsequently be developed and validated using
samples of the prospective cohort.

For future studies, this prospective cohort will be an excellent source to
study (epi)genetic and environmental factors involved in HCM development and
progression as well as for large-scale hypothesis-free metabolic and proteomic
approaches.

1. Inclusion of MYBPC3 founder mutation carriers

For the prospective cohort, we aim to included 1000 mutation carriers (with and
without documented HCM). The major exclusion criterium will be cardiac
transplant, as the disease heart is then no longer present to deposit
biomarkers in blood.
For our case-control study at baseline, we will select 100 MYBPC3 founder
mutation carriers with no cardiac phenotype (as determined by recent ECG and
cardiac ultrasound) and 100 MYBPC3 founder mutation carriers with a severe
cardiac phenotype. A severe phenotype will be defined as one or more of the
following: septal thickness of >= 20 mm, cardiac arrest due to ventricular
arrhythmia, LVEF < 40% or indication for myectomy or cardiac transplant.
Since the pitfall of the case control setting for predictive research is that
a difference in biomarker may be a consequence of the cardiac disease rather
than a cause, we aim to select patients that have just been documented with a
severe cardiac phenotype. Furthermore, since advanced age is such a major
determinant of HCM development and progression, the controls will be in a
similar age group.
Mutation carriers will be selected from our own outpatient clinic and from the
outpatient clinics from our collaborators from the AMC (dr. I.Christiaans, dr.
P. van Tintelen, Prof. Dr. A.A. Wilde), Erasmus MC (dr. M. Michels, dr. R.A.
Oldenburg), UMCG (Prof. dr. R.A. de Boer, dr. Y.M. Hoedemakers) and UMCN (dr.
E. Cramer, dr. C. Marcelis).

2. Extracellular vesicle analysis of MYBPC3 founder mutation carriers
The laboratory of my collaborators Prof. Dr. G. Pasterkamp and Dr. H. den
Ruijter in the UMCU is pioneering in biomarker research of exosomes in
cardiovascular disease. Therefore, this part of the research proposal will be
conducted in their laboratory. Extracellular vesicles of MYBPC3 founder
mutation carriers will be isolated from blood using the Xtractt protocol.

For the case control study at baseline, cMyBP-C levels in extracellular
vesicles will be determined in all 200 mutation carriers using an
electrochemiluminescence immunoassay. This sensitive method of cMyBP-C protein
detection was recently developed by my collaborator dr. D.W. Kuster (VUMC)24.

3. Metabolic analysis of MYBPC3 founder mutation carriers
The metabolic laboratory within my department, led by my collaborators Dr. J.J.
Jans and Prof. Dr.N.M. Verhoeven-Duif has a longstanding expertise in metabolic
investigations both in diagnostic and research settings.
For the case control study at baseline, we will focus specifically on plasma
metabolites in the total 200 MYBPC3 mutation carriers that reflect energy
regulation and consumption (creatine/guanidine acetic acid and acylcarnitine
profiles). Levels of these plasma metabolites will be compared between carriers
with (n=100) an without (n=100) a cardiac phenotype. Dedicated tandem mass
spectrometry methods have been developed in the metabolic laboratory to
quantitatively assess these metabolites.

4. Development of a prediction model
In order to identify the potential biomarkers for HCM development and/or
progression, metabolites and exosomal proteins will be analyzed in a case
control setting at the baseline of our prospective cohort of MYBPC3 founder
mutation carriers. Differences between the groups will be evaluated using the
student T-test (continuous variables) or chi square analysis (dichotomous
variables).
We will subsequently apply multivariable logistic regression analysis to
relate the potential predictors (age at determining carriership, gender,
biomarker values) to the presence or absence of HCM and its progression in our
prospective cohort.

5. Ancillary studies
In order to study the effect of environmental risk factors, a questionnaire has
been generated together with collaborators from the UMCG (Prof. Dr. R.A. de
Boer en dr. Y. Hoedemaekers). The questionnaire was based on the study of James
et al. in patients with arithmogenic cardiomyopathy (ref), The questionnaire
will be sent to the participants that have agreed to receive a questionnaire in
the consent form.
If we are able to obtain the financial means, in future studies we will perform
hypothesis-free approaches to identify potential novel predictive biomarkers
for HCM development in MYBPC3 mutation carriers, using large scale metabolomics
and proteomics studies.
In addition, we intend to study genetic modifiers of HCM development and
progression in MYBPC3 mutation carriers using geonome-wide-association studies
and NGS-based techniques (funding already obtained from CVON: DOSIS, by Dr. F
Asselbergs and co-workers). Therefore, during the first blood withdrawal, 10 ml
extra blood will be sampled for storage of leucocytes (from which DNA can be
isolated). Blood spots that will allow future additional metabolic analyses,
will be obtained from blood already derived from plasma sampling.

To study the full spectrum of HCM, we will also include HCM patients with other
mutations in MYBPC3 or mutations in other genes (mainly, but not exclusively,
MYH7, MYL2,TPM1, TNNC1, TNNT2, TNNI3) and asymptomatic relatives that are
carriers of these mutations. These patients and asymptomatic mutation carriers
will receive a slightly altered patient information letter and informed consent.

Items for ancillary research will be addressed in the current patient
information letter and informed consent (IC). Separate amendments to the
current study will be submitted when applicable.

A user-committee will be established which will consist of all principal
investigators of each participating center. This committee will determine for
what purposes the biosamples for the BIO FOr CARe study can be used.

Incidental findings found in the described future studies will be reported to
the TC bio of the UMC Utrecht. If the TC bio decides that the incidental
finding is clinically relevant and needs to be reported to the patient,
appropriate action will be undertaken (consultation with the general
practitioner or treating specialist, in order to get the information in the
appropriate manner to the patient). In the IC, the patient needs to declare
that he is aware that he can be informed about incidental findings.

Burden and risks are minimal (once or biannual blood withdrawal). If we are
able to predict the disease course in MYBPC3 founder mutation carriers, this
will benefit those carriers in the future. Prevention and pharmaceutical
therapies may be designed based upon the acquired results. This will probably
not benefit the participating mutation carriers directly, but may benefit their
progeny.