European Heart Journal (2022) 43, 1901–1916 SPECIAL ARTICLE

https://doi.org/10.1093/eurheartj/ehab895

Interpretation and actionability of genetic

variants in cardiomyopathies: a position
statement from the European Society of
Cardiology Council on cardiovascular

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genomics
Eloisa Arbustini 1*, Elijah R. Behr 2, Lucie Carrier 3,4, Cornelia van Duijn 5,
Paul Evans6, Valentina Favalli7, Pim van der Harst 8, Kristina Hermann Haugaa 9,10,
Guillaume Jondeau 11,12,13†, Stefan Kääb14,15, Juan Pablo Kaski 16,17,
Maryam Kavousi18, Bart Loeys 19,20, Antonis Pantazis21, Yigal Pinto22,
Heribert Schunkert23,24, Alessandro Di Toro 1, Thomas Thum 25,26, Mario Urtis 1,
Johannes Waltenberger 27,28, and Perry Elliott 29,30
1
Transplant Research Area and Centre for Inherited Cardiovascular Diseases, Department of Medical Sciences and Infectious Diseases, Fondazione IRCCS Policlinico San Matteo,
Pavia, Italy; 2Cardiology Research Section and Cardiovascular Clinical Academic Group, Institute of Molecular and Clinical Sciences, St George’s, University of London and St
George’s University Hospitals NHS Foundation Trust, London, UK; 3Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf,
Hamburg, Germany; 4DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Hamburg, Germany; 5Nuffield Department of Population Health,
University of Oxford, Oxford, UK; 6Department of Infection, Immunity and Cardiovascular Disease, and INSIGNEO Institute, University of Sheffield, Sheffield S10 2RX, UK;
7
Genetics and Bioinformatics, 4bases SA, Lugano, CH, Switzerland; 8Department of Cardiology, Division of Heart and Lungs, University Medical Center Utrecht, Utrecht, The
Netherlands; 9ProCardio Center for Innovation, Department of Cardiology, Oslo University Hospital, Rikshospitalet, Postboks 4950 Nydalen, Oslo 0424, Norway; 10University of
Oslo, Boks 1072 Blindern, Oslo 0316, Norway; 11CNMR Syndrome de Marfan et apparentés, Member of VASCERN, AP-HP Hopital Bichat, Service de Cardiologie, 46 rue Henri
Huchard, Paris 75018, France; 12INSERM LVTS U1148, Paris 75018, France; 13Université de Paris, Paris, France; 14Medizinische Klinik und Poliklinik I, LMU University Hospital
Munich, Munich, Germany; 15German Center for Cardiovascular Research, Munich Heart Alliance, Munich, Germany; 16Institute of Cardiovascular Science, University College
London, London, UK; 17Centre for Inherited Cardiovascular Diseases, Great Ormond Street Hospital, London, UK; 18Department of Epidemiology, Erasmus MC, University
Medical Center Rotterdam, Rotterdam, The Netherlands; 19Cardiogenomics, Center for Medical Genetics, Antwerp University Hospital/University of Antwerp, Antwerp,
Belgium; 20Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; 21The Royal Brompton and Harefield Hospitals, Part of Guy’s and St
Thomas’ NHS Foundation Trust, London, UK; 22Department of Experimental Cardiology, University of Amsterdam, Amsterdam University Medical Center, Meibergdreef 15,
Amsterdam 1105 AZ, The Netherlands; 23Department of Cardiology, Deutsches Herzzentrum München, Technische Universität München, München, Germany; 24Deutsches
Zentrum für Herz- und Kreislaufforschung (DZHK), Munich Heart Alliance, Munich, Germany; 25Institute of Molecular and Translational Therapeutic Strategies (IMTTS),
Hannover Medical School, Hannover, Germany; 26Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM), Hannover, Germany; 27Department of Cardiology and
Cardiovascular Medicine, Medical Faculty, University of Münster, Münster, Germany; 28Cardiovascular Medicine, Hirslanden Klinik Im Park, Seestrasse 220, Zürich 8027,
Switzerland; 29Barts Heart Centre St Bartholomew’s Hospital, London, UK; and 30Institute for Cardiovascular Science, University College London, London, UK

Received 22 June 2021; revised 3 December 2021; accepted 20 December 2021; online publish-ahead-of-print 28 January 2022

* Corresponding author. Tel: +39 0382501487, Fax: +39 0382501893, Email: [email protected]
†
www.marfan.fr; www.LVTS.fr.
© Crown copyright 2022.
This article contains public sector information licensed under the Open Government Licence v3.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/).
1902 E. Arbustini et al.

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Graphical Abstract Impact of the cardiologic phenotyping of probands and relatives on ACMG criteria. The ideal ‘drawing’ of the family
pedigree is complete and correct when all available family members have been clinically evaluated and, eventually, longitudinally monitored.
*Cardiologists and geneticists may add their own experience, data, and local population information. oEndomyocardial biopsy - anti-GB3
immuno-stain (positive brown; §Typical ultrastructural pattern. DCM = dilated cardiomyopathy; HCM = hypertrophic cardiomyopathy;
RCM = restrictive cardiomyopathy; ACM = arrhythmogenic cardiomyopathy; ASD = atrial septal defect; VSD = ventricular septal defect;
GB3 = globotriaosylceramide.
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This document describes the contribution of clinical criteria to the interpretation of genetic variants using heritable Mendelian cardiomy-
opathies as an example. The aim is to assist cardiologists in defining the clinical contribution to a genetic diagnosis and the interpretation of
molecular genetic reports. The identification of a genetic variant of unknown or uncertain significance is a limitation of genetic testing, but
current guidelines for the interpretation of genetic variants include essential contributions from clinical family screening that can establish a
de novo assignment of the variant or its segregation with the phenotype in the family. A partnership between clinicians and patients helps to
solve major uncertainties and provides reliable and clinically actionable information.
...............................................................................................................................................................................
Keywords Genetic variant • Pathogenicity • Interpretation • Cardiomyopathies • Variants of uncertain significance (VUS)

mutation is identified in the index case, relatives can be offered pre-

Introduction dictive testing. Given the pivotal role that genetic diagnoses have in
Following major successes in linking cancer genomics to treatment, modern cardiological practice, it is timely and necessary for profes-
cardiovascular disease is one of the next fields in which complex sional organizations like the European Society of Cardiology (ESC)
genetic data have the potential to transform clinical care. There to take the lead in promoting genetic and genomic literacy among
are, however, many barriers delaying translation of new scientific healthcare professionals.
discoveries into direct benefit for patients, including the need for In this statement, we show how a structured and systematic ap-
translational research that bridges the gap between sequence infor- proach to history taking and clinical phenotyping is central to the
mation and treatment, and the development of a workforce with interpretation of genetic test results. In fact, genetic diagnosis is a
the skills to exploit new scientific opportunities. Clinical services fundamental component of precision medicine. Technological ad-
for inherited cardiac conditions need to cater for the social, psycho- vances over the last 20 years have made it possible to move from
logical, and medical needs of patients and relatives of all ages and tests that evaluate single genes to those that analyse multigene pa-
throughout the life course. In particular, they should provide genetic nels, whole exomes, and whole genomes.1 Multigene panels and
counselling to all patients with potentially inheritable cardiovascular whole-exome sequencing (WES) or whole-genome sequencing
conditions by trained healthcare professionals working as part of a (WGS) substantially increase the probability of identifying genetic
multidisciplinary team to help patients understand and manage the variants associated with human diseases2 but also pose challenges
psychological, social, professional, ethical, and legal implications of a with respect to the interpretation. Guidelines from the American
genetic disease. Molecular genetic testing is offered to patients with College of Medical Genetics and Genomics (ACMG) and the
a heritable clinical phenotype and, when a definite causative genetic Association for Molecular Pathology (AMP) provide criteria for
Interpretation and actionability of genetic variants in cardiomyopathies 1903

the assignment of pathogenicity to genetic variants and help to prevalence of the variant in general population is sufficient to con-
prevent false-positive interpretation.3 They are underpinned by sider it benign) to strong (BS1–4), or supporting (BP1–7). The
clinical data from patients and families and bioinformatic analyses ACMG guidelines provide a ‘baseline’ suggested strength (e.g. in
that use population data (allele frequencies in populations such PM1 the suggested strength is moderate) that can be, and often
as 1000G, Exp, ExAC, gnomAD, computational predictors of func- is, modified based on the available evidence. Some criteria have
tional damage, and in silico tools), interpretation of established data been originally formulated to be flexible (PP1); others have been
repositories (e.g. ClinVar, ClinGen, and LOVD3.0), and clinical da- adapted with subsequent studies (PVS1 and BS3).6,7 Over time,
tabases (e.g. OMIM and MedGen).4,5 When available, in vitro and in these adaptations have become both gene-specific and disease-
vivo functional data also contribute to the classification of variants. specific (e.g. MYH7).8
The specific aim of this document from the ESC Council on The general ACMG/AMP scheme is summarized in Table 1. The
Cardiovascular Genomics is to show how clinical and family data combination of each contributor generates a score that corre-
are essential for the correct interpretation of genetic variants. sponds to the five classes of variants: benign (B), likely benign
Indeed, broadening of the sequencing capacity of molecular genet- (LB), variants of uncertain significance (VUS), likely pathogenic

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ic tests calls for an even more stringent clinical assessment (i.e. (LP), and pathogenic (P). In practice, the five classes are often sim-
clinical phenotyping) to provide the correct interpretation of any plified into three categories: benign (B and LB), VUS, and pathogenic
finding. Mendelian cardiomyopathies are used as a reference to (LP and P). For common genetic diseases such as hypertrophic car-
show how detailed, systematic, and iterative evaluation of patients diomyopathy (HCM), VUSs are relatively common. Variants are
and relatives cannot only establish the pathogenicity of genetic var- classified as VUS due to the lack of information and/or the presence
iants but also provide a basis for personalized medicine in heritable of conflicting data on their role in a given phenotype.
cardiovascular conditions.
Variant reinterpretation
Variants classified as VUS or pathogenic at initial evaluation may be
American College of Medical reclassified subsequently based on novel validated biomarkers, the
Genetics and Genomics/ observation of new cases/families confirming or excluding patho-
genicity, new functional studies, or the identification of novel
Association for Molecular causative genes.14,15 On the contrary, the probability that a variant
Pathology guidelines and variant originally interpreted as benign is reinterpreted as pathogenic is
very low and may concern rare synonymous variants introducing
classification, reinterpretation, alternative cryptic splice sites, subsequently identified in functional
and incidental findings studies.
Between 2016 and 2019, of 4501 variants reclassified in ClinVar
In 2013, a working group consisting of members of the ACMG, —the major archive of variant interpretation containing .1 million
AMP, and College of American Pathologists set out to develop re- submissions—41 P variants (0.91%) and 165 LP variants (3.7%)
commendations for a standard terminology for classifying se-
were reclassified as VUSs while only 4 B variants (0.09%) were re-
quence variants with the aim of reducing the substantial number classified as P (n = 1) and LP (n = 3), respectively.16 In children, 71
of variants being reported as ‘causative’ of disease in the absence of 330 variants were reclassified (21.5%); 44 VUSs were reclassi-
of sufficient supporting evidence. The terms ‘mutation’ and ‘poly-
fied, 9 as LP/P and 35 as LB/B, respectively; 25 of 71 (35.2%)
morphism’ were replaced by the term ‘variant’ with modifiers: (i) were reclassified from LP/P to VUSs; 0 LB/B to LP/P.17
benign, (ii) likely benign, (iii) uncertain significance, (iv) likely patho-
genic, or (v) pathogenic.3
Misinterpretation
The system is applicable to variants in all Mendelian genes,
The primary purposes of clinical genetic testing are to support the
whether identified by single-gene tests, multigene panels, exome identification or confirmation of a disease, to guide individualized
sequencing, or genome sequencing. Pathogenicity is determined
treatment decisions, and reliable cascade screening of families.
by the entire body of evidence in aggregate, converging on a single
Misinterpretation may negatively affect not only diagnostic tests
unequivocal interpretation. but also—and even more importantly—predictive tests used in
asymptomatic persons to predict future risk of disease.18 Thus,
Variant classification the reliability of a test is fundamental to the actionability of any
The ACMG/AMP guidelines for variant interpretation utilize 16 findings (positive or negative) in the clinic.
criteria that favour pathogenicity (P) and 12 criteria that support Inconclusive test results can create mistrust of genetic evalu-
benign (B) interpretation.3 They are based on clinical genetics ation and cynicism about the opportunities for targeted manage-
and phenotype information, genetic epidemiology/population ment of heritable diseases.19–22 This emphasizes the importance
data, computational/predictive data, and the characteristics of of clinicians in the assessment of clinical phenotypes and the fram-
the gene and mutation under examination. The strength of each ing of question to geneticists.
contributor to the interpretation of pathogenicity is classified as Continuous reappraisal of genetic data in the light of familial seg-
very strong (PVS1), strong (PS1–4), moderate (PM1–6), or sup- regation and functional data is one of the methods by which uncer-
porting (PP1–5). The evidence grade for each contributor to a be- tainty in the clinics can be reduced. A VUS, as long as it remains so
nign interpretation ranges from stand-alone (BA1, the high classified, is not clinically actionable while a variant, previously
1904 E. Arbustini et al.

interpreted as a VUS, that is proven by new evidence to be patho- performed for any reason. These recommendations may not coin-
genic takes on new clinical significance with practical implications cide with the national guidelines about the reporting of incidental
(e.g. monitoring of healthy carriers, early initiation of medications, and secondary findings.
concealed arrhythmogenic risk, and pre-natal/pre-implantation
diagnosis).9,14,15,23–26 Variants in genes of uncertain clinical
significance
Guidelines for contacting and informing According to the ACMG definition, a ‘gene of uncertain signifi-
variant carriers after reinterpretation cance’ (GUS) corresponds to ‘a gene without validated association
In the past, many genetic variants were discovered and classified in with the patient’s phenotype’.3 A gene is a GUS when it has never
the context of research programmes rather than clinical genetic been associated with any patient phenotype or it has been previ-
evaluation. The American Society of Human Genetics (ASHG) ously associated with a different phenotype from the one under
have provided guidelines on the contacting of research participants consideration. When variants are identified in GUS, the guidelines

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after reinterpretation of genetic and genomic research results27,28 recommend their report as ‘variants in a gene of uncertain signifi-
(see Supplementary material online, Table S1). As a general prin- cance’ that should always be classified as a VUS,3 irrespective of
ciple, it is reasonable to contact participants and offer updated re- fulfilling the ACMG criteria for pathogenicity (an LP/P variant in
sults if the reinterpretation is consistent with the phenotype under a GUS is a VUS).
study and is reasonably expected to affect a research participant’s A ClinGen initiative has recently comprehensively re-evaluated
medical management. Information on a possible reinterpretation all genes involved in HCM, dilated cardiomyopathy, and arrhyth-
of genetic variants should be anticipated in the pre-test counselling mogenic right ventricular cardiomyopathy, and only a minority of
phase, focusing on two issues: (i) diagnostic tests in affected car- them are now considered to have robust evidence for causal
riers vs. risk-predicting tests in healthy carriers and (ii) levels of ac- link with the disease/s. Currently, according to the ClinGen, genes
tionability of the reclassified variants. An LP/P variant downgraded that meet categories of ‘limited or disputed evidence’ are consid-
to a VUS (common) makes the variant non-actionable clinically. ered as the GUS (https://clinicalgenome.org).
Conversely, the implications of upgrading a VUS or a B/LB variant
to an LP/P (rare) can be substantial but depend on the disease (e.g.
severe cardiomyopathies such as Barth syndrome or Danon dis- How clinicians contribute to
ease, pre-natal or pre-implantation diagnosis, potential malignancy: implementation of the ACMG/
poly-ADP ribose polymerase inhibitors or risk-reducing surgery in
hereditary breast and ovarian cancer). For children, a recent scien-
AMP guidelines
tific statement from the American Heart Association highlighted The essential role of clinicians in variant interpretation is the gath-
the role of pre- and post-test counselling, balancing benefits and ering of coherent evidence that supports and reinforces classifica-
harms, but does not mention reclassification,29 which occurred tion of gene variants identified in a laboratory at their request. In
in around 10% of variants before the ACMG guidelines.17 In the fact, of the 16 ACMG/AMP criteria of pathogenicity, at least 8
cardiomyopathy setting, guardians of healthy children, carriers of are based upon clinical and phenotypic data and family segregation
parental pathogenic variants, should be reassured that deep clinical studies3 (Graphical Abstract).
phenotyping and systematic monitoring guarantee the best cardio- Clinical data are informative not only at baseline evaluation but
logic care. also during the follow-up of patients and families.11,12 For most
cardiomyopathy subtypes, clinical screening of relatives can be in-
Incidental or secondary findings conclusive at first evaluation due to age-related expression of dis-
The use of clinical exome and WES/WGS in clinical practice has ease. Depending upon the type of cardiomyopathy, the phenotype
inevitably led to the identification of variants in genes unrelated may fully manifest only in adult life (all cardiomyopathies) or less
to the primary medical reason for testing. These findings have commonly in children (e.g. lethal restrictive troponinopathies,
been defined as incidental30 or secondary.31 The possibility of TNNI3 gene).32
identifying secondary findings should be properly communicated
before testing, and patients should also consent to the receipt of De novo variants (PS2 and PM6 criteria)
such results. The ACMG SF v3.0 has provided recommendations While there are some pathogenic variants that typically occur de
on the reporting of secondary findings of those pathogenic or like- novo (an example is DES p.Arg454Trp), evaluation of both parents
ly pathogenic variants identified in the 73 actionable genes re- of an affected individual is required for confirmation (PS2 is the cri-
ported in Table 1 of Miller et al.31 This list of ACMG SF v3.0 terion used for proven de novo variants). In some cases, variants ap-
genes includes 19 cardiomyopathy genes (MYBPC3, MYH7, pear to be de novo, but paternity and/or maternity cannot be
TNNT2, TNNI3, TPM1, MYL3, ACTC1, PRKAG2, GLA, GAA, MYL2, confirmed (PM6 is the criterion used for assumed but not proven
TTN, FLNC, LMNA, PKP2, DSP, DSC2, TMEM43, and DSG2), as well de novo variants) (Figure 1). If paternity is not established, the
as genes related to non-cardiomyopathy cardiovascular pheno- Cardiomyopathy Expert Panel (CMP-EP) suggests upgrading of
types, cancer, inborn errors of metabolism, and to miscellaneous the PM6 to PS2 when at least three proven de novo occurrences
phenotypes (see Supplementary material online, Table S2).10 have been reported, e.g. MYH7 for cardiomyopathies.8 For other
Variants in these genes have to be returned in genetic test conditions such as genetic hearing loss, the ClinGen variant
Interpretation and actionability of genetic variants in cardiomyopathies 1905

Table 1 American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria for
interpretation of pathogenicity of genetic variants

Criterion Description Clinical role Source of information Notes

........................................................................................................................................................................
PVS1 Null variant (nonsense, frameshift, This criterion does not depend Computational and There are proven null pathogenic
canonical +1 or 2 splice sites, upon clinical evaluation predictive data variants whose interpretation of
initiation codon, single or pathogenicity stands alone. Vice versa,
multi-exon deletion) in a gene other predicted null variants are
where loss of function (LOF) is a non-pathogenic [i.e. DSC2
known mechanism of disease (p.Ala897LysfsTer4); TRPM4
(p.Trp525*)]
PVS1 strength can be downgraded if
appropriate6

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PS1 Same amino acid change as a This criterion does not depend Computational and Interpretation may eventually benefit of
previously established pathogenic upon clinical evaluation predictive data segregation studies, i.e. a cryptic splice
variant regardless of nucleotide site is introduced
change
PS2 De novo (both maternity and Parents should Segregation data If the same variant has been
paternity confirmed) in a demonstrate normal previously published as de novo in
patient with the disease and cardiac evaluation peer-reviewed journals, then it is
no family history standard practices to use this
evidence
PS3 Well-established in vitro or in vivo This criterion does not depend Functional: either in vivo Tissue studies (i.e. tissue biopsy) in
functional studies supportive of a upon direct clinical or in vitro studies certain cardiomyopathies
damaging effect on the gene or evaluation demonstrated the effects of the
gene product variants
PS4 The prevalence of the variant Clinical evaluation in a Genetic epidemiology —
in affected individuals is large at-risk population or population data
significantly increased can support the real
compared with the prevalence of the disease
prevalence in controls
PM1 Located in a mutational hot spot This criterion does not depend Functional, protein Hot spot and functional domain [i.e. GLA
and/or critical and upon clinical evaluation modelling, (p.Asn215Ser)]
well-established functional computational, and
domain (e.g. active site of an predictive data
enzyme) without benign variation Public databases
(ClinVar, ClinGen,
LOVD, HMD)
PM2 Absent from controls (or at This criterion does not depend Prevalence in population Activated when the variant is rare
extremely low frequency if upon clinical evaluation databases enough in population databases to be
recessive) in Exome Sequencing plausibly pathogenic
Project, 1000 Genomes Project,
or Exome Aggregation
Consortium
PM3 For recessive disorders, This criterion may benefit Public databases Parents are healthy carriers with
detected in trans with a from clinical evaluation (OMIM, MedGen) proven normal clinical
pathogenic variant and inheritance model in evaluation. Allelic data from
family screening9–12 sample bioinformatic analysis
and segregation data
PM4 Protein length changes as a result of This criterion does not depend Computational and —
in-frame deletions/insertions in a upon clinical evaluation predictive data.
non-repeat region or stop-loss Predicted protein
variants change
PM5 Novel missense change at an amino This criterion does not depend Public databases Different missense variants affecting the
acid residue where a different upon clinical evaluation (ClinVar, ClinGen, same residue. [i.e. DES (p.Arg454Trp)
missense change determined to LOVD, HMD) vs. DES (p.Arg454Gln) or LMNA
be pathogenic has been seen (p.Arg189Trp), (p.Arg189Gln), and
before (p.Arg189Pro)]

Continued
1906 E. Arbustini et al.

Table 1 Continued

Criterion Description Clinical role Source of information Notes

........................................................................................................................................................................
PM6 Assumed de novo, but without This criterion may benefit Segregation data De novo not proven: one parent is
confirmation of paternity from clinical evaluation not available for testing.
and maternity and family However, if the same variant has
screening9–12 been previously published as de
novo in peer-reviewed journals,
then it is standard practice to use
this evidence
PP1 Co-segregation with disease in Clinical evaluation, genetic Segregation data This criterion is met when the
multiple affected family counselling, and family family members, both affected

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members in a gene screening9–12 and non-affected, had clinical and
definitively known to cause genetic screening. Deductions
the disease from pedigrees risk
misinterpretation of
pathogenicity.
However, if extensive segregation
data for the same variant have
been previously published in
peer-reviewed journals, then it is
standard practice to use this
evidence
PP2 Missense variant in a gene that has a This criterion does not depend Computational and —
low rate of benign missense on clinical evaluation predictive data
variation and in which missense
variants are a common
mechanism of disease
PP3 Multiple lines of computational This criterion does not depend Computational and —
evidence support a deleterious on clinical evaluation predictive data
effect on the gene or gene
product (conservation,
evolutionary, splicing impact,
etc.)
PP4 Patient’s phenotype or family Geno-phenotype Genetic counselling, This criterion strongly relies on the
history is highly specific for a correlation clinical evaluation deep phenotyping of proband
disease with a single genetic of the patient and relatives, particularly for
aetiology cardiomyopathies with red flags
(cardiac and extra-cardiac)
characterizing the phenotype
PP5 Reputable source recently reports This criterion does not depend Public databases To be considered with caution: many
variant as pathogenic, but the on clinical evaluation (ClinVar, ClinGen, variants classified as ‘disease causing’
evidence is not available to the LOVD, HMD) in the past, are now being shifted to
laboratory to perform an PubMed non-pathogenic class and vice versa.
independent evaluation The strength of the criterion may
increase with the number and the
reliability of sources. Recently, the
experts of the ClinGen Sequence
Variant Interpretation Working
Group proposed that laboratories
discontinue the use of criteria PP5 and
BP6 as soon as that is practically
achievable13
BA1 Allele frequency is .5% in Exome This criterion does not depend Genetic epidemiology. —
Sequencing Project, 1000 on clinical evaluation Population genetic
Genomes Project, or Exome databases
Aggregation Consortium

Continued
Interpretation and actionability of genetic variants in cardiomyopathies 1907

Table 1 Continued

Criterion Description Clinical role Source of information Notes

........................................................................................................................................................................
BS1 Allele frequency is greater than This criterion does not depend Genetic epidemiology. —
expected for disorder on clinical evaluation Population genetic
databases
Public databases
(ClinVar, ClinGen,
LOVD, HMD)
BS2 Observed in a healthy adult The clinical screening and Genetic To be considered with caution: for
individual for a recessive deep phenotyping of epidemiology. most CMP genes penetrance can
(homozygous), dominant family members is Population genetic be variable, incomplete and

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(heterozygous), or X-linked essential for this databases age-dependent. BS2 can be
(hemizygous) disorder, with criterion9–12 Public databases activated for conditions such as
full penetrance expected at (ClinVar, ClinGen, XLR Danon disease in male
an early age LOVD, HMD) patients
BS3 Well-established in vitro or in vivo This criterion does not depend Functional studies Functional studies or pathological
functional studies show no on clinical evaluation studies do not show markers that are
damaging effect on protein specifically linked with the
function or splicing cardiomyopathy
BS4 Lack of segregation in affected The clinical screening and Only from This criterion largely depends upon
members of a family deep phenotyping of segregation data the number of relatives available
family members is for clinical and genetic screening,
essential for this and their age. Regular clinical
criterion.9–12 monitoring of relatives may
modify this criterion
BP1 Missense variant in a gene for which This criterion does not depend on Computational and The typical example is provided by most
primarily truncating variants are clinical evaluation predictive data missense TTN gene variants
known to cause disease
BP2 Observed in trans with a pathogenic This criterion does not depend Public databases Allelic data from bioinformatic analysis
variant for a fully penetrant on clinical evaluation (ClinVar, ClinGen, and segregation studies
dominant gene/disorder or LOVD, HMD)
observed in cis with a pathogenic
variant in any inheritance pattern
BP3 In-frame deletions/insertions in a This criterion does not depend Computational and —
repetitive region without a on clinical evaluation predictive data
known function
BP4 Multiple lines of computational This criterion does not depend Computational and —
evidence suggest no impact on on clinical evaluation predictive data
gene or gene product
(conservation, evolutionary,
splicing impact, etc.)
BP5 Variant found in a case with an This criterion does not depend Public databases This criterion has limited value for most
alternate molecular basis for on clinical evaluation (ClinVar, ClinGen, cardiomyopathies that are the
disease LOVD, HMD) paradigm of genetically
heterogeneous diseases
BP6 Reputable source recently reports This criterion does not depend Public databases The impact of the criterion may increase
variant as benign, but the on clinical evaluation (ClinVar, ClinGen, with the number and the reliability of
evidence is not available to the LOVD, HMD) sources
laboratory to perform an PubMed
independent evaluation
BP7 A synonymous (silent) variant for This criterion does not depend Computational and —
which splicing prediction on clinical evaluation predictive data. When
algorithms predict no impact to the possible, RNA-based
splice consensus sequence nor the functional analysis
creation of a new splice site and the
nucleotide is not highly conserved

Criteria based upon clinical evaluation are in bold.

1908 E. Arbustini et al.

A B C D
Proband: onset at 6 yrs Proband: onset at 8 yrs Proband: onset at 17 yrs Proband: onset at 26 yrs
Phenotype: RCM, HTx at 12 Phenotype: RCM, HTx at 14 Phenotype: HCM, ICD at 25 Phenotype: DCM + AVB
TNNT2 (p.Phe97Cys) MYL2 (p.Gly162Arg) MYBPC3 c.2737+5G>T LMNA (p.Leu183Pro)
ACMG Likely Pathogenic ACMG Likely Pathogenic ACMG VUS ACMG Likely Pathogenic
(PM1, PM2, PP2, PP3) (PM1, PM2, PM5, PP2, PP3) (PM2, BP4) (PM1, PM2, PP2, PP3)
The addion of the ACMG criterion PS2 (Proven de novo) shis the class.
ACMG Likely Pathogenic ACMG Likely Pathogenic ACMG VUS ACMG Likely Pathogenic
+ PS2 -> Pathogenic + PS2 -> Pathogenic + PS2 -> Likely Pathogenic + PS2 -> Pathogenic

_ _ _ _

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1 1 2 1 1 2
+ +

1 1 1 1 2

Unaffected female/male Affected female/male + Variant Carrier - Non carrier Proband / Deceased
Same variants: the addion of the ACMG criterion PM6 (assumed de novo but without confirmaon of maternity and
paternity) does not shi the class
ACMG Likely Pathogenic ACMG Likely Pathogenic ACMG VUS ACMG Likely Pathogenic
+ PM6 -> Likely Pathogenic + PM6 -> Likely Pathogenic + PM6 -> VUS +PM6 -> Likely Pathogenic
HTx= Heart transplantaon; HCM= Hypertrophic Cardiomyopathy; RCM= Restricve cardiomyopathy; DCM= Dilated Cardiomyopathy; AVB=
Atrioventricular block; ICD= Implantable Cardioverter Defibrillator

Figure 1 Four examples of proven de novo variants—American College of Medical Genetics and Genomics criterion PS2 contributes to shift
the American College of Medical Genetics and Genomics class from variant of uncertain significance to likely pathogenic.

curation SOP Committee proposed a point-based system for an informed consent by entitled relatives. Given the age-related
modified strength levels of PS2 and PM6 criteria, based on the par- penetrance of most cardiomyopathies, co-segregation of a VUS
ental confirmation, phenotypic consistency, and number of the de (so defined at first detection) in a family may require repeat eva-
novo observations.33,34 luations in multiple individuals over some years (Figure 2).
Therefore, confidence that a de novo variant is pathogenic is in- Therefore, interpretation of a VUS may not be possible at base-
creased by documentation of multiple individual occurrences asso- line family screening but only after family monitoring in the mid or
ciated with the expected phenotype. Nevertheless, a high long term. The evaluation of the descendants of deceased pa-
frequency of the given de novo variant in population databases tients may prove the obligate carrier status of affected family
should raise doubts about pathogenicity, irrespective of the de members who died at a young age (see Supplementary material
novo status of the variant.35,36 For cases with an apparent germinal online, Figure S1).
mosaicism (e.g. affected sibs, offspring of negative parents), pater- The segregation PP1 criterion applies when a variant occurs in a
nity/maternity must be proven for de novo criteria to be applied. gene definitively known to cause the disease and co-segregates in
multiple affected family members (see Supplementary material
online, Figure S2). A quantitative definition of segregation has
Co-segregation (PP1) and been proposed for the PP1 criterion. The computation of segre-
non-segregation (BS2 and BS4) criteria gation evidence proposes cut-off values for three levels of evi-
For cardiomyopathies, confirmation of pathogenicity is greatly en- dence in single family or .1 family: strong, moderate, and
hanced by evidence of co-segregation in families. Ideally, as many supporting.16 Supplementary material online, Figure S3 shows
relatives as possible should be evaluated using deep phenotyping an example of how to evaluate the three levels of evidence.
(defined as the precise and comprehensive analysis of phenotypic Co-segregation studies may shift the ACMG class from uncertain
abnormalities in which the individual components of the pheno- to likely pathogenic or pathogenic (see Supplementary material
type are observed and described)11,37–39 and documentation of online, Figure S4).
disease in deceased individuals. Post-mortem genetic tests can The non-segregation BS4 criterion applies when one family mem-
be performed on a case-by-case basis; most reported studies ad- ber is affected but is a non-carrier of the genetic variant inter-
dress the search for the cause of sudden cardiac death more than preted as disease-causing in the family. However, a digenic
segregation studies. However, post-mortem testing can contrib- contribution to the phenotype or coexistent phenocopies should
ute to variant classification and can be performed when retained be excluded (Figure 3) and the stringency of the phenotypic traits
biological samples of deceased individuals are available and are of each cardiomyopathy (diagnostic criteria) can influence the re-
used within an appropriate ethical/regulatory framework with liability of the segregation study.8
Interpretation and actionability of genetic variants in cardiomyopathies 1909

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Figure 2 The 15-year evolution of medical history; segregation is calculated after 15 years monitoring—American College of Medical Genetics
and Genomics criterion PP1.
1910 E. Arbustini et al.

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Figure 3 An example of non-segregation and non-pathogenic TNNT2 variant in the two sibs II:3 and II:8; both remain genetically uncharacter-
ized. The apparently non-segregating sib (II:4) is affected by Fabry disease; his daughter is obligate carrier. None of the members of the third
generation had TNNT2 test.
Interpretation and actionability of genetic variants in cardiomyopathies 1911

The non-segregation BS2 criterion applies when one family such as mild left ventricular hypertrophy for which there may be
member is a VUS carrier but not affected (Supplementary other explanations such as hypertension, obesity, or athleticism.
material online, Figure S5). The criterion is for adult or older However, the coexistence of additional disease phenotypes does
healthy carriers. Although variable expressivity is often reported not exclude the pathogenicity of a variant (e.g. a TTN stop codon
in cardiomyopathies, this criterion is relevant for Mendelian mono- may predispose a person with alcohol abuse to develop cardiomy-
genic diseases. However, BS2 is reported with low prevalence opathy).43 Clinical markers (or diagnostic ‘red flags’)38 may be of
(0.37%) among variants classified as pathogenic and likely assistance in identifying specific disease phenotypes but need to
pathogenic.40 be interpreted carefully. For example, a short PR interval occurs
in the early phases of storage diseases such as Danon disease
Variants in cis or trans (PM3 and BP2 and, if associated with other traits such as HCM, skeletal myopathy
and cognitive impairment, supports the diagnosis. Other examples
criteria) include the combination of cardiomyopathy with left ventricular
Cis variants (two or more) occur in the same copy of the gene or in
non-compaction, myopathy, leukopenia, skeletal abnormalities,

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two adjacent genes that are jointly inherited (Figure 4A). Trans var-
methylglutaconic aciduria in the X-linked recessive Barth syn-
iants are in different copies of the gene or in two different genes
drome associated with pathogenic variants in TAZ gene;44 atrial
and are independently transmitted (Figure 4B). The key for inter-
and ventricular septal defects, dilated cardiomyopathy, conduction
preting the Cis and Trans rules is the inheritance.3,34 For autosomal
disease skeletal anomalies, limbs in particular—syndactyly, thumb,
recessive (AR) conditions, knowing the allelic configuration of gen-
carpal, radial, ulnar, vertebral, and chest anomalies—in the AD
etic variants helps in their interpretation, particularly in recessive
Holt–Oram syndrome associated with pathogenic variants in the
single-gene disorders. Indeed, in this circumstance, when two het-
TBX5 gene;45 symmetrical HCM, cornea verticillata, angiokerato-
erozygous variants are detected in the same gene [e.g. GAA
mas, renal, brain, and peripheral nervous system involvement in
(Figure 4B), or in rare neonatal dilated cardiomyopathy,41 or in
the X-linked Fabry disease caused by pathogenic variants in the
rare adult HCM]42 and one is known as pathogenic, the determin-
GLA gene.46
ation of the trans status of the second variant (e.g. novel and mis-
sense) can add ‘evidence for pathogenicity’ to the second variant
(PM3). Otherwise, if the two variants are in cis and one is known
as pathogenic, the interpretation of the second variant shifts to be-
Functional criteria (PS3 and BS3):
nign (BP2). For autosomal dominant (AD) conditions, the applica- can pathology and disease-specific
tion of Cis and Trans rules depends upon the effect of
homozygosity on the phenotype (embryonic lethality or very se-
biomarkers contribute to variant
vere disease phenotypes) and is gene-dependent.34 interpretation?
In the case of homozygous variants, a family study confirms the
In vivo, ex vivo, or in vitro functional studies are complex, time-
heterozygous status of the parent (Figure 5A). Typical examples in
consuming, and may require samples from affected tissues. The
the field of cardiomyopathies are desminopathies that are trans-
PS3 and BS3 criteria address the evidence functional studies sup-
mitted both in AR and AD ways, depending on the variant in the
porting (PS3) or excluding (BS3) damaging effects of the mutated
DES gene (Figure 5).
gene. The Sequence Variant Interpretation Working Group
A peculiar case is represented by the trans status of a deleted
(SVI-WG) recommends a four-step framework to determine the
allele associated with a pathogenic variant in the other allele of
strength of evidence of functional studies: (i) define the disease
a recessive cardiomyopathy gene. Deletion of one copy of a
mechanism, (ii) evaluate the applicability of general classes of as-
gene region (hemizygosity) may mimic homozygosity in
says used in the field, (iii) evaluate the validity of specific instances
sequencing-based tests; however, the completion of the test
of assays, and (iv) apply evidence to individual variant interpret-
with copy number variation analysis demonstrates the deletion.
ation.7 With new next-generation sequencing technologies, the
When one of the parents is unavailable for testing, his/her relatives
detection rate of genetic variants is so fast that functional studies
may contribute (maternal and paternal sibs and related offspring) if
for each variant deemed potentially pathogenic is beyond the diag-
they are carriers of the given variants. Finally, a patient can be af-
nostic capacity of most genetic labs. However, some cardiomyop-
fected by two different genetic diseases, a condition whose correct
athies are characterized by specific pathological findings that can
detection should be based on precise phenotypic characterization.
be more informative than in vitro studies. Examples include
An illustrative example is represented by sarcomeric HCM and
Danon disease (loss of LAMP2) (Figure 7), Fabry disease (substrate
Duchenne muscular dystrophy (Figure 6).
accumulation)46 (Figure 3), dystrophinopathies (focal or extensive
loss of dystrophin expression of the cardiomyocyte membrane
Pertinence of the phenotype to the and skeletal muscle),47 myofibrillar cardio-desminopathies (intra-
affected gene/s (the PP4 criterion) cellular deposits of osmiophilic granulofilamentous material that
When evaluating a genetic variant, it is important to consider is immunoreactive with anti-desmin-antibodies in myocardium
whether the phenotype in question is highly specific for a unique and skeletal muscle)48 (Figure 5B), and laminopathies (severe nu-
genetic aetiology (for example, LAMP2 in Danon disease). This is clear damage and loss of myocyte nuclear immunostaining).49,50
rarely the case in cardiomyopathies as most are characterized by In most cardiomyopathies, gene-specific biomarkers do not ex-
genetic heterogeneity and the frequent occurrence of phenotypes ist, and commonly tested biomarkers inform about myocyte
1912 E. Arbustini et al.

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Figure 4 (A) Two variants in cis—the likely pathogenic variant is in MYH7 (p.Arg1250Gly). (B) Late-onset Pompe disease. Two variants in trans
—the second variant does not meet American College of Medical Genetics and Genomics pathogenicity criteria, but is validated as pathogenic
in ClinVar.

damage, or involvement of extra-cardiac organs and tissues but increase the spectrum of functional tests to be explored to
not on their cause. Functional effects of genetic variants may be strengthen the role of functional tests in the interpretation of
obtained in the case of lysosomal diseases involving the heart variants.
(e.g. GAA and GLA)46,51 by measuring blood enzyme level. New Finally, future progress in variant interpretation will benefit
emerging biomarkers to be validated and confirmed will help from new technologies such as single-cell RNA sequencing
Interpretation and actionability of genetic variants in cardiomyopathies 1913

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Figure 5 (A) Autosomal recessive desminopathies—parents are from the same small village. (B) Autosomal dominant restrictive cardiodes-
minopathy—endomyocardial biopsy shows the typical intramyocyte accumulation of granulofilamentous osmiophilic material.
1914 E. Arbustini et al.

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Figure 6 X-linked recessive Duchenne muscular dystrophy and MYH7 likely pathogenic variant inherited from the father who only shows mild
left ventricular hypertrophy.

(RNAseq), assays for transposase-accessible chromatin sequen- responsibility. The need to extend genetic testing to family mem-
cing (ATACseq, exploring open regions of chromatin on a bers should be anticipated in the pre-test counselling, when the
genome-wide scale), integration of ATACseq and RNAseq with reasons and significance of the test as well as the role of the family
epigenomics as well as integration of large data sets collecting gen- study should be explained to the patient.26
etic information, and the contribution of artificial intelligence (e.g. When a test is concluded, the patient may receive three types of
machine learning) tools.52,53 information:

(1) The genetic test is positive. This result provides the opportun-
From the clinic to the laboratory ity to positively impact both patient and family’s health.47,54
and back to the clinic (2) The genetic test is negative. Patients should be informed about
the limits of the test (number of genes analysed and complete-
How to inform patients and families ness of the investigation). In the case of a negative test but clin-
The owners of clinical genetic test results are patients and the ical evidence of familial cardiomyopathy, clinical monitoring of
transmission of information to other family members is their at-risk relatives should be maintained, and efforts should be

Figure 7 Pathological findings in endomyocardial biopsy of a male patient with Danon disease. Endomyocardial biopsy shows the loss of ex-
pression of the LAMP2 protein.
Interpretation and actionability of genetic variants in cardiomyopathies 1915

made to continue with the search for the causative genetic young relatives of the proband) should be scheduled according
mechanism as well as for novel disease- or modifier-genes. to the age, baseline tests, symptoms, and other non-cardiac
(3) The possibility of finding a VUS should be discussed with the traits in the case of syndromic cardiomyopathies.
proband in the pre-test counselling. Informing the patient of • Pre-clinical diagnosis: A gene variant recognized as pathogenic
the result of a test that has identified pathogenic (positive and with eventual complete penetrance should be given special
test) or benign (negative test for the analysed genes) variants attention from the moment of its identification. Very early clin-
is easier than communicating tests that have identified one or ical manifestations can be subtle and recorded only with serial
more VUSs (inconclusive test). Unlike many routine tests, monitoring (e.g. progressive prolongation of the ECG PQ inter-
there is no ‘normal range’ for a VUS.37,55,56 The patient should val, even within normal ranges).26
be helped to understand that the relative importance of some • Pre-symptomatic diagnosis: Early markers of disease or extra-
criteria depends on the study of his/her family and that family- cardiac traits in the case of syndromic cardiomyopathies may
based data are uniquely useful for the correct interpretation of be detected in asymptomatic family members.57–59
the variant. • Early therapy: Administration of treatment to healthy carriers of

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pathogenic variants is evolving. For cardiomyopathies associated
Guidelines for contacting variant carriers with ventricular or atrial remodelling (dilation, hypertrophy),
medications are usually administered when there is an instru-
after reinterpretation mental evidence for disease. This may change in the future
In the past, many genetic variants were discovered and classified in
with the advent of disease-modifying medication. A recent ex-
the context of research programmes rather than clinical genetic
ample is mavacamten in obstructive HCM.60 Other examples in-
evaluation. The ASHG has provided guidelines on the contacting
clude enzyme replacement therapy in some storage disorders
of research participants after reinterpretation of genetic and
or mitogen-activated protein/extracellular signal-regulated ki-
genomic research results28 (see Supplementary material online,
nase inhibitors (i.e. selumetinib) for RASopathies, including
Table S1). As a general principle, it is reasonable to contact parti-
neurofibromatosis type 1, Noonan syndrome, cardiofaciocuta-
cipants and offer updated results if the reinterpretation is consist-
neous syndrome, Costello syndrome, and others.61,62
ent with the phenotype under study and is reasonably expected to
• Personalized treatments: At present, few therapies (in cardio-
affect a research participant’s medical management.
myopathies) are based on genetic data with the exception of pri-
mary prophylactic implantation of cardioverter-defibrillators.
Release of genetic reports However, ongoing trials of new therapeutic strategies, including
Concluding a genetic workup with laboratory information alone small molecules and gene therapies, may transform this
can generate serious consequences for both individual patients landscape.60
and their families. A dynamic bidirectional exchange of information • Lifestyle adjustment: Advice on daily activities and recreational
between laboratory and clinical teams is preferable before releas- sport activity remains uncertain in healthy carriers63 but are
ing diagnostic reports. Moreover, analysis of multigene panels tar- clear in individuals with evidence for disease expression.
geting cardiomyopathies often identifies more than one genetic • Pre-natal diagnosis and pre-implantation diagnoses: Identification
variant potentially associated with the phenotype and the need of a pathogenic variant provides the opportunity for pre-
to issue a formal report within a pre-defined ‘turnaround’ period implantation genetic testing.
means that incomplete interpretation, especially when informative
segregation data are missing, is frequent. In such circumstances,
completion of the clinical and genetic screening of families (where Conclusions
both carriers and non-carriers of given variants contribute to the
computation of the segregation) is essential.16 Engagement with Correct clinical interpretation of genetic variants will be one of the
patients and their families in this endeavour assists in the comple- key contributors to precision cardiology of the future but requires
tion of the segregation study and improves the likelihood of cor- effective partnerships between clinicians, patients, scientists, and
rect interpretation. Clinical screening in the family is generally industry to maximize the benefits of genetic knowledge.
accepted by relatives, who, regardless of the result of the test, Importantly, a genetic test supports and confirms, but does not
can benefit from either clinical exclusion of the disease or early substitute for a clinical diagnosis.
diagnosis or unexpected diagnosis. Conflict of interest: T.T. declares lecture fees from Takeda, Amicus
Therapeutics, and Sanofi-Genzyme. T.T. also reports to be the com-
mittee member for the ESC Council for Cardiovascular Genomics,
Cardiomyopathies and levels of and founder and shareholder of Cardior Pharmaceuticals. Y.P. declares
actionability of genetic variants a contract with Roche Diagnostics, consulting fees from Forbion BV
Netherlands and Daiichi Sankyo, two patents planned on treatment
Variants proven to be pathogenic are clinically actionable. For car- of cardiomyopathy, and a minor stock (,5%) in University Spin-off.
diomyopathies, clinical scenarios currently influenced by genetic J.W. declares consulting fees from Boehringer Ingelheim and
test results include: Siemens Healthineers, and honoraria from Daiichi Sankyo. P.E. reports
consulting fees from Sarepta, Pfizer, DinaQor, Bristol Myers Squibb
• Clinical monitoring according to the phenotype and genotype:26 (BMS), and Astra Zeneca; honoraria for lectures from Pfizer and
The monitoring of affected and healthy carriers (e.g. children or BMS. P.E. is the Chairman of the ESC Council for Cardiovascular
1916 E. Arbustini et al.

Genomics, chair for the ESC Academy, and has participated in the 20. Ouellette AC, Mathew J, Manickaraj AK, Manase G, Zahavich L, Wilson J, et al.
INDORSIA modify ID-069A301 trial (IDMC). A.P. declares payments Clinical genetic testing in pediatric cardiomyopathy: is bigger better? Clin Genet
2018;93:33–40.
for advisory board meetings from Bristol Myers Squibb. J.K. reports a
21. Lawal TA, Lewis KL, Johnston JJ, Heidlebaugh AR, Ng D, Gaston-Johansson FG,
grant from the Medical Research Council and consulting fees from et al. Disclosure of cardiac variants of uncertain significance results in an exome
DiNAqor and Cytokinetics. K.H.H. declares honoraria from cohort. Clin Genet 2018;93:1022–1029.
Boehringer Ingelheim. 22. Mazzarotto F, Olivotto I, Walsh R. Advantages and perils of clinical whole-exome
and whole-genome sequencing in cardiomyopathy. Cardiovasc Drugs Ther 2020;34:
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23. Bennett JS, Bernhardt M, McBride KL, Reshmi SC, Zmuda E, Kertesz NJ, et al.
Supplementary material Reclassification of variants of uncertain significance in children with inherited ar-
rhythmia syndromes is predicted by clinical factors. Pediatr Cardiol 2019;40:
1679–1687.
Supplementary material is available at European Heart Journal online.
24. Tsai GJ, Rañola JMO, Smith C, Garrett LT, Bergquist T, Casadei S, et al. Outcomes
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