University of Groningen Phospholamban p.Arg14del cardiomyopathy te Rijdt, Wouter

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Phospholamban p.Arg14del cardiomyopathy

te Rijdt, Wouter

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Publication date: 2019

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te Rijdt, W. (2019). Phospholamban p.Arg14del cardiomyopathy: Clinical and morphological aspects supporting the concept of arrhythmogenic cardiomyopathy. Rijksuniversiteit Groningen.

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529310-L-bw-Rijdt 529310-L-bw-Rijdt 529310-L-bw-Rijdt 529310-L-bw-Rijdt Processed on: 1-3-2019 Processed on: 1-3-2019 Processed on: 1-3-2019

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145

phospholamban p.Arg14del mutation carriers

detected by echocardiography

CHAPTER 9

Wouter P. te Rijdt, MDa-b_{, Yoran M. Hummel, PhD}a_{, Paul A. van der Zwaag MD, PhD}b_, J. Peter van Tintelen, MD, PhDc_{, Rudolf A. de Boer, MD, PhD}a_{, Maarten P. van den Berg, MD, PhD}a

a_{Department of Clinical and Experimental Cardiology, University of Groningen,}

University Medical Center Groningen, Groningen, the Netherlands

b_{Netherlands Heart Institute (Nl-HI), Utrecht, the Netherlands} c_{Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands} c_{University of Amsterdam, Amsterdam Medical Center, Department of Clinical Genetics, The Netherlands}

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146

Abstract

Background

A large subset of our patients diagnosed with dilated cardiomyopathy (DCM) or arrhythmogenic right ventricular cardiomyopathy (ARVC) carry the pathogenic phospholamban (PLN) c.40_42delAGA (p.Arg14del) founder mutation. The natural course of these mutation carriers is characterized by a presymptomatic phase of variable length, after which many carriers progress to overt disease, manifesting as DCM and/or ARVC.

We aimed to identify subclinical structural and functional cardiac abnormalities in presymptomatic carriers of the identical PLN p.Arg14del mutation using echocardiography including state-of-the-art tissue deformation imaging techniques.

Methods & Results

Twenty-eight presymptomatic PLN p.Arg14del mutation carriers (13 males and 15 females; median age 33.0 (24.0-41.5)), identified by cascade genetic screening, and 28 healthy matched control subjects were included in this cross-sectional study. The median R-wave amplitude (leads I, II and III) was 24.0 mm (carriers 18.5 mm vs. relatives 27 mm, p<0.05).

Both groups underwent comprehensive transthoracic echocardiography including LV and RV myocardial two-dimensional strain measurements. Global longitudinal systolic strain (GLSS) and global LV early diastolic tissue velocity (Global e’) were assessed from 12 LV segments and from 3 RV segments. In the mutation carrier group, LV ejection fraction (LVEF) was preserved (59.0±5.2 vs 58.8±5.3 (%), p=0.788). However, LV mass index was reduced (58.4±13.9 vs 71.5±15.6 (g/m2_{), p=0.002) and we also observed loss of LV diastolic function (MV-e 0.79±0.15 vs 0.95±0.18} (m/sec), p=0.001; MV-a 0.52±0.13 vs 0.60±0.13 (m/sec), p=0.031; Global e’ 6.7±1.3 vs 7.4±1.2 (cm/s), p=0.081). In addition, RV GLSS was less negative in mutation carriers compared with controls (-25.4±4.6 vs -31.4±4.9 (cm/s), p<0.001).

Conclusions

Presymptomatic PLN p.Arg14del mutation carriers already showed subtle but significant structural remodeling (reduced LV mass) as well as loss of LV diastolic function and RV systolic function. These findings may help to recognize early disease development and support the concept of biventricular cardiomyopathy.

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147

Introduction

Phospholamban, a phosphoprotein encoded by the PLN gene, regulates SERCA activity and therefore plays an important role in regulating cardiac calcium homeostasis1_{. Various pathogenic} cardiomyopathy-related mutations have been described in the PLN gene.2-5_{The c.40_42delAGA} (p.Arg14del) founder mutation in the coding region of the PLN gene was identifi ed in a large subset of Dutch DCM and ARVC patients, supporting the concept of arrhythmogenic cardiomyopathy (ACM) as overlap syndrome.6_{This mutation is the most prevalent single cardiomyopathy-related} mutation identifi ed in the Netherlands with over 1000 carriers having been identifi ed in the Netherlands,7_{but the same mutation has also been identifi ed in Canada, the USA as well as in} several other European countries. The PLN p.Arg14del mutation leads to reduced SERCA activity and thereby calcium overload which in turn is deemed to cause cardiomyocyte damage and eventually myocardial fi brosis.4

The natural course of these mutation carriers is characterized by a presymptomatic phase of variable length after which many carriers progress to overt disease (i.e. age-related penetrance), characterized by high rates of malignant ventricular arrhythmias (ARVC phenotype) and end-stage heart failure (DCM phenotype) often necessitating internal cardioverter defi brillator (ICD) therapy and heart transplantation.6,8

Two-dimensional speckle tracking echocardiography (STE) is a sensitive method for assessment of global and regional myocardial deformation and an emerging tool for the assessment of subtle changes in cardiac function in preclinical cardiomyopathies.9,10_Adding these strain analysis measurements to conventional echocardiographic fi ndings might prove to be of incremental diagnostic and prognostic value in PLN p.Arg14del mutation carriers. Early identifi cation of disease development and progression, even before the onset of symptoms, can guide early therapeutic intervention and lifestyle adjustments (i.e. refraining from strenuous sports activity) and subsequently might prevent sudden cardiac death as a result of malignant ventricular arrhythmias and/or slow down progression of heart failure.

In the present study, we aimed to investigate whether subclinical structural and/or functional cardiac abnormalities can already be identifi ed in presymptomatic carriers of the identical PLN p.Arg14del mutation using echocardiography including state-of-the-art tissue deformation imaging techniques.

Materials and Methods

Ethical statement

For the present study we randomly selected a group of PLN p.Arg14del mutation carriers without prior symptoms (‘presymptomatic’) from the PHORECAST registry (PHOspholamban RElated CArdiomyopathy STudy; http://www.phorecast.nl). In this registry all identifi ed Dutch carriers of the PLN p.Arg14del mutation are routinely registered. This registry was reported to the Dutch Data Protection Authority (CBP) which supervises compliance with legislation regulating the use of personal data. In addition, age and sex-matched healthy subjects drawn from a previously collected database,11_{served as controls. The study conformed to principles defi ned in the Helsinki} Declaration and the medical ethics committee of the University Medical center Groningen.

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Echocardiography

All echocardiographic examinations were performed as part of routine patient care. Images are routinely stored in raw DICOM format enabling offline analysis, which was performed by a single operator (Y.M.H) who was blinded for the status of the subjects. Images were acquired according to standard image acquisition protocol on a GE VIVID-7 and E9 (General Electric, Horten, Norway), using a 2.5–3.5 MHz probe for image acquisition, which enabled us to perform additional analyses of the images. All measurements were performed during sinus rhythm and in accordance with current ASE and EAE recommendations.12,13

Cardiac dimensions and volumes were measured on two-dimensional (2D) images and left ventricular (LV) ejection fraction, LV mass and left atrial volumes were calculated and indexed to body surface area where appropriate.

Evaluation of LV diastolic function consisted of transmitral inflow measurements and early diastolic tissue velocities (e’). Early diastolic tissue velocities were measured at the septal and lateral side of the mitral annulus, and averaged (global e’). To characterize myocardial function beyond routine echocardiography techniques we applied STE software which allows direct assessment of the deformation of myocardial tissue (‘strain’) as intrinsic part of myocardial performance.14

Twelve segments were analysed of the LV myocardium (septal, lateral, anterior, and inferior; at the basal, mid, and apical level) and three segments of the RV myocardium (RV free wall; at the basal, mid, and apical level), see figure 1.

Figure 1. Myocardial segments model for both heart chambers in apical four-chamber view. Twelve segments for LV myocardium (septal, lateral, anterior, and inferior; at the basal, mid, and apical level) and three segments for RV myocardium (RV free wall; at the basal, mid, and apical level). The colors for the segments are corresponding to the segmental strain curves (an example with RV segmental strain curves is shown in figure 3).

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149 For calculation of global longitudinal systolic strain (GLSS) for the LV as a whole the mean of the 12 individual segments was calculated and for the RV as a whole the mean of the three individual segments was calculated. Peak systolic strain was measured as the maximum value independent of aortic valve closure timing. Global longitudinal systolic strain is expressed as percentage and global e’ is expressed in cm/s. In case >2 segments were not analyzable by the 2D speckle tracking software the analysis was discarded.

ECG analysis

Standard 12-lead resting ECGs, recorded around the time of echocardiography for usual patient care, were collected. R-wave amplitudes of the precordial leads were measured. Low voltage ECG was as previously defi ned as QRS peak-to-peak amplitude in leads I, II, and III <5 mm, the sum of the amplitudes <15 mm, and amplitude in all precordial leads <10 mm.8_{Inverted T-waves were} determined and considered present if inverted in the right precordial leads (V1 as well as V2) and/ or in at least two adjacent lateral leads (V4, V5 or V6).

Statistical analysis

Normally distributed variables were tested through Student’s t-tests and expressed as mean ± standard deviation, while non-normally distributed variables were tested through Mann-Whitney U- tests and expressed as the median with interquartile range. Normality of distribution was tested through probability plots. Correlations were analysed using Pearson’s test. Two-tailed p-values of less than 0.05 were considered statistically signifi cant. Statistical analyses were performed in SPSS (version 24.0, 2016 release 24.0.0.0, IBM SPSS Inc., Chicago, IL, USA).

Results

Study subjects

Twenty-eight presymptomatic heterozygous carriers (46% males) of the identical PLN p.Arg14del mutation, identifi ed by cascade genetic screening, were included in this cross-sectional study. The control group consisted of 28 age- and gender-matched healthy subjects drawn from a previously collected database.11_{Median age at echocardiographic evaluation in the carriers was} 33.0 (24.0-41.5) versus 28.5 (21.3-36.0) years (p = 0.129) for the healthy controls. Heart rate, blood pressure and body surface area were comparable in the mutation carriers and controls (Table 1). In addition, serum N- proBNP levels were measured and tended to be slightly higher in the carriers but this did not reach statistical signifi cance.

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150

Table 1. Overview of general characteristics and echocardiographic measurements_{Table 1 Overview of general characteristics and echocardiographic measurements}

Variable PLN mutation carriers (n=28) Controls (n=28) P-value General characteristics Age (yrs) 33 (24-41.5) 28.5 (21.3-36) .129 Sex (male, %) 13 (46%) 13 (46%) 1.00 BSA (m2) 1.94 ± 0.16 1.94 ± 0.22 .978 HR (bpm) 70.0 (61.3-79.5) 64.0 (55-77) .129 BPs (mmHg) 117.0 ± 19.8 117.4 ± 9.4 .911 BPd (mmHg) 73.6 ± 10.9 78.2 ± 7.2 .066 Serum BNP (pg/ml) 44.5 (9-1545) 37.5 (17-164) .776 Median R wave amplitude (mm) 18.5 (6.0-38.0) 27 (12.0-48.0) .008

LV and LA dimensions, volumes and mass

EDDi (cm/m2) 2.4 ± 0.2 2.5 ± 0.3 .058 EDVi (mL/m2) 56.3 ± 11.7 62.7 ± 13.5 .071 ESDi (cm/m2) 1.6 ± 0.2 1.7 ± 0.3 .081 ESVi (ml/m2) 23.4 ± 6.3 25.8 ± 6.4 .167 LVMI (g/m2) 58.4 ± 13.9 71.5 ± 15.6 .002 IVS (mm) 7.8 ± 1.4 8.6 ± 1.2 .031 LVPW (mm) 7.5 ± 1.3 8.0 ± 1.1 .151 LAVi (ml/m2) 24.6 (22.4-30.9) 27.3 (24.5-29.9) .167

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151 Abbreviations: BSA = Body surface area; HR = Heart rate; BPs = Blood pressure (systolic); BPd = Blood pressure (diastolic); BNP=N-terminal pro-brain natriuretic peptide; IVS = Interventricular septum thickness; LVPW = Left ventricular posterior wall thickness; EDDi = LV end diastolic diameter index; EDVi = LV end diastolic volume index; ESDi = LV end systolic diameter index; ESVi = LV end systolic volume index; LAVi = LA end systolic volume index; LVSV = LV stroke volume; LVSVi = LV stroke volume index; CO = cardiac output; RVEDD = Right ventricular end diastolic diameter; LVMi = LV mass index; LVEF = LV ejection fraction; MV-e = LV early diastolic infl ow; MV-a = LV late diastolic infl ow; E/A ratio = Ratio MV-e and MV-a; DecT = Deceleration time of MV-E; Global e’ = global LV early diastolic tissue velocity (mean of 12 segments); GLSS = LV Global Longitudinal Systolic Strain (mean of 12 segments); TAPSE = Tricuspid Annular Plane Systolic Excursion; RV e’ = RV early diastolic tissue velocity (mean of 3 segments); RV GLSS = RV Global Longitudinal Systolic Strain (mean of 3 segments)

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LV systolic parameters LVEF (%) 59 ± 5.2 58.8 ± 5.3 .788 GLSS (%) -20.5 ± 2.3 -21.4 ± 1.7 .109 LVSV 61.5 (52.8-67.5) 66 (54-86) .188 LVSVi 31.1 (29.4-35.6) 35.6 (29.7-44.3) .109 CO 4.3 (3.8-5.1) 4.2 (3.6-5.5) .965 LV diastolic parameters MV-e (m/s) 0.79 ± 0.15 0.95 ± 0.18 .001 MV-a (m/s) 0.52 ± 0.13 0.60±0.13 .031 E/A ratio 1.6±0.5 1.6±0.4 .707 DecT (ms) 192.5 (177.8-235.5) 194.5 (173-220.1) .678 Global e’ (cm/s) 6.7 (5.6-7.7) 7.4 (6.7-8.1) .081 RV parameters TAPSE (mm) 24 (21-26) 24 (22.3-26.8) .393 RVEDD (mm) 37.0 ± 5.5 35.5 ± 4.3 .275 RV e’ (cm/s) 7.4 (5.4-8.8) 7.3 (6.3-8.0) .840 RV GLSS (%) -25.4±4.6 -31.4±4.9 .001

Abbreviations: BSA = Body surface area; HR = Heart rate; BPs = Blood pressure (systolic); BPd = Blood pressure (diastolic); BNP=N-terminal pro-brain natriuretic peptide; IVS = Interventricular septum thickness; LVPW = Left ventricular posterior wall thickness; EDDi = LV end diastolic diameter index; EDVi = LV end diastolic volume index; ESDi = LV end systolic diameter index; ESVi = LV end systolic volume index; LAVi = LA end systolic volume index; LVSV = LV stroke volume; LVSVi = LV stroke volume index; CO = cardiac output; RVEDD = Right ventricular end diastolic diameter; LVMi = LV mass index; LVEF = LV ejection fraction; MV-e = LV early diastolic inflow; MV-a = LV late diastolic inflow; E/A ratio = Ratio MV-e and MV-a; DecT = Deceleration time of MV-E; Global e’ = global LV early diastolic tissue velocity (mean of 12 segments); GLSS = LV Global Longitudinal Systolic Strain (mean of 12 segments); TAPSE = Tricuspid Annular Plane Systolic Excursion; RV e’ = RV early diastolic tissue velocity (mean of 3 segments); RV GLSS = RV Global Longitudinal Systolic Strain (mean of 3 segments)

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152

Echocardiographic measurements

Detailed echocardiographic results are also presented in Table 1.

General measurements:

There was a trend towards lower LV size in the PLN mutation-carrier group (LVEDDi: 2.4 ± 0.2 vs 2.5 ± 0.3 (cm/m2); LVEDVi: 56.3 ± 11.7 vs 62.7 ± 13.5 (mL/m2)) but this did not reach statistical significance (p= 0.058 and p=0.071, respectively).

However, LV mass index was significantly reduced (58.4±13.9 vs 71.5±15.6 (g/m2), p=0.002) in mutation-carriers vs. the controls. An example of reduced LV mass and wall thickness in a PLN mutation-carrier in comparison to the control is shown in Figure 2.

Figure 2. Example of reduced LV wall thickness and mass in a PLN mutation-carrier (left) in comparison to a healthy control (right). In this example (parasternal long axis view) the LV posterior and septal wall as well as LV mass are substantially lower in the PLN carrier.

Left ventricular systolic function:

In the mutation carrier group, LV ejection fraction (LVEF) was preserved (59.0±5.2 vs 58.8±5.3 (%), p=0.788) while LV GLSS was less negative although not significantly different when comparing both groups (-20.5 ± 2.3 vs -21.4 ± 1.7 (%), p=0.109).

Left ventricular diastolic function:

We observed loss of LV diastolic function in mutation-carriers shown by a lower MV-e (0.79±0.15 vs 0.95±0.18 (m/sec), p=0.001) and lower MV-a (0.52±0.13 vs 0.60±0.13 (m/sec), p=0.031) and a trend towards reduced global e’ (6.7±1.3 vs 7.4±1.2 (cm/s), p=0.081).

Right ventricular function:

No differences were found when comparing RVEDD and RV e’ between both groups. However, RV GLSS was significantly less negative in mutation carriers compared with controls (-25.4±4.6 vs - 31.4±4.9 (%), p<0.001). A representative example of abnormal RV deformation in a PLN mutation- carrier versus the control is shown in Figure 3.

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153 Figure 3. Representative example of abnormal deformation in a PLN mutation-carrier (left) in comparison to a healthy control (right). The colored tracings represent the speckle tracking-derived left ventricular longitudinal strains per segment (see fi gure 1 for RV myocardial segments model). The dashed curve represents the average strain which is lower in the PLN carrier.

ECG fi ndings

Inverted T-waves in the right precordial leads were seen in 3 (11%) mutation carriers and in the lateral leads in 3 (11%) mutation carriers, of which 2 carriers had inverted T-waves in the right as well as the lateral leads. In the control group, no inverted T-waves were observed (p=0.18 for both right as well as lateral leads). Four carriers had a low voltage ECG while in the control group no person had a low voltage ECG (p=0.12). The median R-wave amplitude (leads I, II and III) was 24.0 mm (carriers 18.5 mm vs. relatives 27 mm, p<0.05).

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154

Discussion

In the present study we investigated cardiac structure and function in presymptomatic PLN p.Arg14del mutation carriers using STE. The main findings are that presymptomatic carriers already have signs of LV structural remodeling and decreased LV diastolic function when compared to healthy controls while LV systolic function was (still) preserved. In addition to these LV findings we found RV systolic dysfunction, indicating early biventricular myocardial dysfunction in mutation carriers. These observed differences in cardiac structure and function cannot be explained by age, sex, body mass index or blood pressure as the mutation carrier and the control group were well matched and no significant differences were present in these characteristics.

Previous studies showed a high risk for end-stage heart failure (DCM phenotype) and malignant ventricular arrhythmias (ARVC phenotype) in carriers of the PLN p.Arg14del mutation.6, 8 High mortality and a poor prognosis are present from late adolescence onwards.8_{Interestingly,} a significant clinical overlap between DCM and ARVC phenotypes was found in a substantial number of mutation carriers in which left- or right-sided forms may predominate.

The underlying pathophysiological mechanisms responsible for these phenotypes are not fully understood, but at the basis is the disturbed calcium homeostasis as a consequence of intrinsic functional consequences of the PLN p.Arg14del mutation, namely intracellular calcium overload. This is the result of superinhibition of the sarcoplasmic reticulum Ca2+_{-ATPase (SERCA2a)} by the mutated phospholamban protein.4_{Eventually this disbalance leads to cardiomyocyte} damage, cardiomyocyte loss and fibrosis. More in general, i.e. irrespective of the specific underlying etiology, the development of heart failure is accompanied by extensive remodeling of the entire myocardium. Following cardiomyocyte loss, formation of fibrous tissue takes place in an attempt to preserve the structural integrity. Later remodeling processes include fibrotic reorganization that eventually leads to cardiac failure. Changes in the collagen network usually impair both systolic and diastolic function.15

In present study we did not look at advanced disease but instead we investigated possible derangements in the early stage of the disease process, i.e. the presymptomatic stage. Somewhat to our surprise, already in this early stage this remodeling process could already be observed by conventional echocardiography. Left ventricular wall thickness and mass were significantly lower in the carriers than the controls.

Besides lower LV weights, we also found evidence for loss of LV diastolic function in the carriers. Although LVEF was not lower, systolic longitudinal myocardial deformation was reduced (borderline). Subtle functional LV derangement seems a consequence of the abovementioned beginning structural abnormalities. Although no significant differences were found when comparing RVEDD and RV e’ between both groups, we observed early RV systolic dysfunction in terms of GLSS which was significantly less negative (i.e. lower in absolute terms) in the carriers. Indeed, originally ACM was described to predominantly affect the right ventricle.16_However, over the years LV involvement has been demonstrated across a broad spectrum of disease severity. Recent clinical6, 8,17_{and histopathological}18-20_{findings underpin that PLN p.Arg14del} cardiomyopathy is a truly biventricular disease. Our findings show for the first time that already in an early stage there may be involvement of both ventricles.

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155 deterioration of LVEF (<45%) occurs. Cardiomagnetic resonance imaging (CMR) revealed late gadolinium enhancement, indicating fi brosis, in many PLN p.Arg14del mutation carriers with still preserved LV systolic function (LVEF>45%).17_{The early presence of low voltage ECG and} lateral negative T waves, which we also observed previously,6,21_{was indeed related to fi brosis on} CMR.17 Even more, histopathological examination of 20 complete heart specimens (although mostly end-stage cardiomyopathy and therefore not representative for early phase disease) and RV endomyocardial biopsy samples and PLN p.Arg14del mouse hearts4 revealed extensive myocardial fi brosis.18-20_{It is most probable that myocardial fi brosis is the common pathway and} substrate underlying the observed early electrocardiographical and structural abnormalities, i.e. ‘early fi brosis phenotype’, and the primary phenomenon responsible for the further development of heart failure and electrical instability, i.e. ventricular arrhythmias.

Early preventive treatment is therefore essential, yet there is no proven eff ective treatment. We are currently prospectively investing the effi cacy of eplerenone to reduce disease progression and delay the onset of overt disease in presymptomatic carriers of the PLN p.Arg14del mutation in the iPHORECAST-trial (intervention in PHOspholamban RElated CArdiomyopathy Study; NCT 2013- 001067-23).

Limitations

The study was limited by a rather small cohort of subjects included in the study. Results should therefore be interpreted with care and be considered hypothesis generating. A larger study would be needed to confi rm our fi ndings and to be able to reach fi rm conclusions. Furthermore, speckle tracking echocardiography depends highly on image quality and image artifacts can decrease the accuracy of assessment of myocardial deformation, therefore we selected only images of suffi cient quality.

Conclusions

Presymptomatic PLN p.Arg14del mutation carriers already showed subtle but signifi cant structural remodeling as well as loss of LV diastolic function and RV systolic function. These fi ndings support the concept of biventricular cardiomyopathy in PLN p.Arg14del mutation carriers. The underlying mechanism remains to be elucidated but disturbed calcium homeostasis due to the PLN p.Arg14del mutation and subsequent early myocardial fi brosis most probably is the primary cause. Future prospective studies are needed to evaluate the incremental value of our fi ndings in the early diagnostic workup and risk-stratifi cation of presymptomatic mutation carriers.

Funding sources

This work was fi nancially supported by a grant from the Fondation Leducq (CurePLaN) and the Netherlands Cardiovascular Research Initiative, an initiative supported by the Dutch Heart Foundation (The Hague, the Netherlands): CVON2012-10 PREDICT, CVON2014-40 DOSIS and CVON 2015-12 eDETECT projects. Wouter P. te Rijdt is supported by Young Talent Program (CVON PREDICT) grant 2017T001 from the Dutch Heart Foundation.

Disclosures

The authors report no confl ict of interest.

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156

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