Europace Advance Access published July 2, 2015
CLINICAL RESEARCH
Europace doi:10.1093/europace/euv205
Phenotypic expression is a prerequisite for malignant arrhythmic events and sudden cardiac death in arrhythmogenic right ventricular cardiomyopathy Alessandro Zorzi 1, Ilaria Rigato 1, Kalliopi Pilichou 1, Martina Perazzolo Marra 1, Federico Migliore 1, Elisa Mazzotti 1, Dario Gregori 1, Gaetano Thiene 1, Luciano Daliento 1, Sabino Iliceto 1, Alessandra Rampazzo 2, Cristina Basso 1, Barbara Bauce 1, and Domenico Corrado1* 1 Inherited Arrhythmogenic Cardiomyopathy Unit, Department of Cardiac, Thoracic, and Vascular Sciences, University of Padua, Via N. Giustiniani 2, Padua 35121, Italy; and 2Department of Biology, University of Padua, Padua, Italy
Received 17 January 2015; accepted after revision 15 May 2015
Whether a desmosomal (DS)-gene defect may in itself induce life-threatening ventricular arrhythmias regardless of phenotypic expression of arrhythmogenic right ventricular cardiomyopathy (ARVC) is still debated. This prospective study evaluated the long-term outcome of DS-gene mutation carriers in relation to the ARVC phenotypic expression. ..................................................................................................................................................................................... Methods The study population included 116 DS-gene mutation carriers [49% males; median age 33 years (16 –48 years)] without and results prior sustained ventricular tachycardia (VT) or ventricular fibrillation (VF). The incidence of the arrhythmic endpoint, including sudden cardiac death (SCD), aborted SCD, sustained VT, and appropriate implantable cardioverter-defibrillator (ICD) intervention was evaluated prospectively and stratified by the presence of ARVC phenotype and risk factors (syncope, ventricular dysfunction, and non-sustained VT). At enrolment, 40 of 116 (34%) subjects fulfilled the criteria for definite ARVC while the remaining were either borderline or phenotype negatives. During a median follow-up of 8.5 (5– 12) years, 10 patients (9%) had arrhythmic events (0.9%/year). The event rate was 2.3%/year among patients with definite ARVC and 0.2%/year among borderline or phenotype negative patients (P ¼ 0.002). In patients with definite ARVC, the incidence of arrhythmias was higher in those with ≥1 risk factors (4.1%/year) than in those with no risk factors (0.4%/year, P ¼ 0.02). Mortality was 0.2%/year (1 heart failure death and 1 SCD). ..................................................................................................................................................................................... Conclusions The ARVC phenotypic expression is a prerequisite for the occurrence of life-threatening arrhythmias in DS-gene mutation carriers. The vast majority of malignant arrhythmic events occurred in patients with an overt disease phenotype and major risk factors suggesting that this subgroup most benefits from ICD therapy.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Cardiomyopathy † Arrhythmogenic right ventricular cardiomyopathy † Implantable cardioverter-defibrillator † Ventricular arrhythmias † Sudden cardiac death † Primary prevention † Genetics
Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable heart muscle disorder characterized by incomplete penetrance and variable phenotypic expression.1 The natural history
predominantly relates to the ventricular electrical instability which may lead to sudden cardiac death (SCD), mostly in young people and athletes.2,3 The progressive loss of myocardium with fibrofatty replacement underlies clinical electrocardiographic (ECG) and morphofunctional ventricular changes and acts as a substrate for life-threatening
* Corresponding author. Tel: +39 049 8212458; fax: +39 049 8212309, E-mail address: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected].
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Aims
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What’s new? † The long-term (8.5 years) arrhythmic risk of DS-gene mutation carriers, with no prior sustained VT or VF, is strongly related to the ARVC phenotypic expression and the presence of major risk factors such as syncope, ventricular dysfunction, or non-sustained VT. † The study results do not support the concept that DS-gene carriers may experience life-threatening arrhythmic events before and/or regardless of the development of ARVC phenotypic features (‘concealed phase’), as postulated on the basis of experimental studies showing that lethal arrhythmogenic mechanisms may arise at a molecular/cellular level as a result of a cross-talk of altered desmosomes with sodium-channel and gap junction proteins. † Our findings also suggest that the ‘concealed phase’ of the disease may be the result of the low sensitivity of routine clinical tests (i.e. electrocardiogram and echocardiography) for detection of early/minor disease phenotypic manifestations such as isolated epicardial scar involvement of the left ventricle, rather than the expression of a subcellular arrhythmogenic mechanism.
Methods Study population This is a single-centre study which enrolled a series of genotyped ARVC probands and relatives who were evaluated at the Inherited Arrhythmogenic Cardiomyopathy Unit of the Department of Cardiac,
Thoracic and Vascular Sciences of the University of Padua. The initial study cohort composed of 137 consecutive DS-gene mutation carriers (45 probands and 92 family members). All subjects had undergone a comprehensive analysis of all amino acid coding exons and intron boundaries of the five ARVC-susceptible DS genes [plakophilin-2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC2), desmoplakin (DSP), and junction plakoglobin (JUP)] and were carriers of ≥1 DS-gene mutation that was considered pathogenic according to the 2010 International Task Force (ITF) criteria, as previously described in details.12,13 In brief, a pathogenetic mutation was defined as a DNA alteration previously associated with ARVC that was unobserved or rare in a large, non-ARVC, ethnically matched control population and was predicted to alter the structure or the function of the encoded protein or cosegregated with disease phenotype in the family. According to the study design, 14 DS-gene carriers were excluded from the study because they had ‘definite’ ARVC and a history of previous sustained VT or VF at the time of first clinical evaluation. Seven individuals were lost during follow-up. The final study sample included 116 DS-gene carriers [28 probands and 88 family members; 57 (49%) males; median age 33 years (16 – 48 years)], with no history of VT or VF at the time of enrolment. A single pathogenetic mutation was identified in 101 individuals (PKP2 ¼ 33, DSP ¼ 41, DSG2 ¼ 26, and DSC2 ¼ 1) and multiple mutations in 15 (Table 1). Most of these subjects constituted the study population of a previous genotype – phenotype investigation by our group with completely different design (retrospective) and endpoint (risk of arrhythmic events since birth).13 The present study was a clinical follow-up study that prospectively evaluated the arrhythmic outcome of DS-gene carriers after their first clinical evaluation. Accordingly, no analysis combining genotype (specific DS-genes, mutation types, and genotype complexity) and arrhythmic risk was performed because this was not a genotype – phenotype study. The study complies with the Declaration of Helsinki, was approved by the local Ethics Committee and all patients gave informed consent to participate.
Clinical protocol At the time of first evaluation, all subjects underwent a detailed clinical evaluation including family history, physical examination, resting 12-lead ECG, signal-averaged ECG (SAECG), 24-h Holter monitoring, exercise testing, and two-dimensional transthoracic echocardiography. Additional tests were reserved to selected cases (mostly probands) and included contrast-enhanced cardiac magnetic resonance (CE-CMR) (n ¼ 23) and invasive exams such as cardiac catheterization (n ¼ 29), endomyocardial biopsy (n ¼ 3), and programmed ventricular stimulation with electroanatomic voltage mapping (n ¼ 21). According to the 2010 ITF diagnostic criteria, in our study population of carriers of ARVC-causing DS-gene mutations (major diagnostic criterion), the diagnosis of ‘definite’ ARVC was fulfilled in the presence of additional one major criterion or two minor criteria from different categories. The diagnosis was considered ‘borderline’ when one additional minor criterion was fulfilled. Desmosomal-gene mutation carriers showing no disease features, either minor or major, were defined as ‘genotype positive-phenotype negative’.12 All individuals were followed-up regularly in the outpatient clinic at 6- to 12-month intervals with ECG, 24-h Holter monitoring, SAECG, and echocardiogram. According to our protocol, implant of an ICD was offered for primary prevention of SCD to DS-gene carriers with a ‘definite’ ARVC diagnosis and ≥1 major risk factors such as unexplained syncope, non-sustained VT, or greater than or equal to moderate ventricular dysfunction, i.e. right ventricular (RV) fractional area change ,24% or RV ejection fraction , 40%; left ventricular (LV) ejection fraction , 45%.
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ventricular arrhythmias.2 The discovery of desmosomal (DS)-gene mutations involved in the pathogenesis of ARVC has offered the potential of a molecular genetic diagnosis in both affected individuals and relatives.1,4 Clinical manifestations of ARVC usually develop during adolescence and young adulthood and are preceded by a long preclinical phase (‘concealed ARVC’), during which SCD has been reported to occur unexpectedly as the first manifestation of the disease.2 As a consequence, the problem of primary prevention of SCD among DS-gene mutation carriers during pre-clinical/early phases of the disease has emerged and an aggressive management strategy including a prophylactic implantable cardioverter-defibrillator (ICD) of asymptomatic DS-gene carriers has been proposed.5 This management strategy has been supported by recent experimental animal studies which demonstrated that the loss of expression of DS-proteins may ‘per se’ induce electrical ventricular instability by ion-channel dysfunction, which predisposes to lethal ventricular arrhythmias regardless or before the expression of a structural (i.e. ‘pre-histological ’) myocardial substrate.6 – 11 However, the relationship between the presence and severity of the phenotypic manifestations of ARVC and the risk of SCD among DS-gene mutation carriers remains to be elucidated. Thus, the present study prospectively evaluated the long-term arrhythmic outcome of a single-centre cohort of DS-gene mutation carriers without previous sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) in relation to the expression of the ARVC phenotype.
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Phenotypic expression and outcome in ARVC
Table 1 Mutation screening results Locus
Exon
NT
AA
Functional meaning
Number of patients
Plakophilin-2
1
148_151delACAG
T50SfsX61
Frameshift
8 + 2a
1 1
c.175C . T c.184C . A
Q59X Q62K
Nonsense Missense
1 1a
1
c.184_185delCA
Q62DfsX23
Frameshift
2
2 2
c.227A . G c.336G . T
N76S K112N
Missense Missense
2 2
3
c.663C . A
Y221X
Nonsense
1a
7 10
c.1643delG c.2009delC
G548VfsX15 K672RfsX12
Frameshift Frameshift
3 7
10
c.2119C . T
Q707X
Nonsense
6 + 6a
12 1
c.2447_2448delCC c.88 G . A
T816RfsX10 V30M
Frameshift Missense
2 + 1a 6 + 1a
...............................................................................................................................................................................
Desmoplakin
Desmocollin-2
c.273+5G . A
Splicing
3
c.423-1G . A c.817C . T
Q273X
Splicing Nonsense
3 3
7
c.897C . G
S299R
Missense
9
9 11
c.1174G . A c.1372A . T
V392I N458Y
Missense Missense
1a 3
11
c.1408A . G
K470E
Missense
2
23 23
c.3337C . T c.3764G . A
R1113X R1255K
Nonsense Missense
4 1a
23
c.3774C . A
D1258E
Missense
2
23 23
c.4803G . A c.4961T . C
M1601I L1654P
Missense Missense
4 6a
23
c. 4973C . T
S1658F
Missense
1a
23 24
c.3203_3204delAG c.7622G . A
p.E1068VfsX19 R2541K
Frameshift Missense
1a 2 + 1a
24
c.7039A . G
I2347V
Missense
1a
4 6
c.298G . C c.689A . G
G100R E230G
Missense Missense
2 1 + 1a
7
c.797A . G
N266S
Missense
4
8 8
c.880A . G c.991G . A
K294E E331K
Missense Missense
3 2a
9
c.1174G . A
V392I
Missense
5
9 Intron12
c.1250_1253dupATGA c.1880-2G . A
D419X
Frameshift Splicing
2 + 1a 2a
13
c.1912G . A
G638R
Missense
1a
14 15
c.2033G . C c.2491C . T
G678A L831F
Missense Missense
4 4 + 2a
15
c.2990delG
G997VfsX20
Frameshift
1 + 1a
15 8
c.2773C . T c.1034T . C
P925S I345T
Missense Missense
1a 1
a
The number of patients with the mutation in compound or double heterozygosis.
Arrhythmic endpoint The primary study endpoint was the occurrence of any major arrhythmic events defined as SCD, cardiac arrest due to VF, sustained VT, or appropriate ICDs intervention on VT/VF. The occurrence of VT/VF during follow-up was documented by ECG recording at the time of symptoms and, in the subgroup of patients who received an ICD for primary prevention, by device interrogation. The secondary endpoint was
all-cause mortality. All events were adjudicated by the consensus of an independent committee blinded to the disease phenotypic features. Appropriate ICD intervention was defined as a device shock or antitachycardia overdrive pacing delivered in response to a ventricular tachyarrhythmia and documented by stored intracardiac ECG data. Implantable cardioverter-defibrillator was routinely programmed to include a monitoring zone that identified VT with a rate .160 b.p.m.
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Desmoglein-2
Intron2 Intron3 7
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A. Zorzi et al.
All ICD interrogations were identified and adjudicated by two electrophysiologists (D.C. and F.M.) blinded to other clinical data. All subjects gave their informed consent to participate in the study.
Statistical analysis Results are summarized as median (25%, 75% quartiles) or n (%) for continuous and categorical variables, respectively. Categorical differences between groups were evaluated by the x 2 test or the Fisher’s exact test as appropriate. Continuous variables were compared using the Rank Sum test because of the small size of subgroups. Event-free survival curves were drawn with the Kaplan– Meier method and compared by the log rank test. Patients were censored at the time of the first event. Cumulative event-rates were expressed as % and the corresponding 95% confidence intervals were calculated with the method of Blaker, which is more powerful than the more common Clopper – Pearson approach.14 The mean annual event rate was calculated assuming exponential (constant) decay over the follow-up. A P-value of ,0.05 was considered significant. Statistics were analysed with the R System (R Foundation for Statistical Computing, Vienna, Austria) and the ExactCI libraries.15
Results Baseline clinical characteristics
Arrhythmic outcome During a median follow-up of 8.5 (5–12) years, 10 patients (9%), all belonging to different families, reached the composite arrhythmic endpoint with a cumulative rate of major arrhythmic events of 8.6% (95% CI 0–14.2%) and an annual rate of 0.9%. The arrhythmic events consisted of cardiac arrest due to VF (n ¼ 1), sustained monomorphic VT (n ¼ 4), appropriate ICD interventions for VT (n ¼ 3) and VF (n ¼ 1), and SCD (n ¼ 1). The SCD victim was a 15-year-old DSP-gene mutation carrier (exon 7; c.897C . G; S299R), who did not show any ECG and echocardiographic abnormalities at the time of his first clinical evaluation and died suddenly 2 years later while sleeping. Eight months before death he underwent follow-up clinical evaluation which provided unremarkable findings. Autopsy investigation showed predominant LV involvement with a band of myocyte necrosis and fibrous myocardial replacement in the outer mid – subepicardial layer of the postero-lateral wall (Figure 1).
Table 2 Baseline characteristics of the study sample at the time of first clinical evaluation according to the 2010 ITF classification 2010 ITF classification
...................................................................................... Overall (n 5 116)
Phenotype negative (n 5 57)
Borderline ARVC (n 5 19)
Definite ARVC (n 5 40)
............................................................................................................................................................................... Median age (years)
33 (16–48)
23 (15–46)
43 (21– 48)
37 (16– 55)
Gender (male)
57 (49)
25 (44)
12 (63)
20 (50)
Proband status 2010 revised ITF criteria
28 (24)
0
8 (42)
20 (50)
21 (18) 5 (4)
0 0
0 0
21 (53) 5 (25)
19 (16) 8 (7)
0 0
0 3 (16)
19 (48) 5 (25)
4 (3) 36 (31)
0 0
0 10 (53)
4 (20) 26 (65)
Morpho-functional criteria Major Minor ECG repolarization abnormalities Major Minor ECG depolarization abnormalities Major Minor Arrhythmic criteria Major Minor Risk factors Unexplained syncope
0
0
28 (24)
0
0
6 (32)
22 (55)
0
6 (15)
7 (6)
0
1 (5)
Non-sustained ventricular tachycardia Moderate-severe ventricular dysfunction
7 (6) 12 (12)
0 0
0 0
7 (18) 12 (30)
≥1 risk factor
20 (17)
0
1 (5)
19 (48)
Data are presented as n (%) or median (IQR). ECG, electrocardiographic; ITF, International Task Force.
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Baseline characteristics of the study population at the time of the first evaluation are shown in Table 2. Forty (34%) DS-gene mutation
carriers fulfilled the 2010 ITF diagnostic criteria for ‘definite’ ARVC, while the remaining 76 (66%) had ‘borderline’ ARVC (n ¼ 19; 16%) or were phenotype negative (n ¼ 57, 50%). No borderline or phenotype negative DS-gene mutation carrier underwent invasive tests such as endomyocardial biopsy or endocardial voltage mapping. Thirteen patients received an ICD for primary prevention.
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Phenotypic expression and outcome in ARVC
Table 3 Clinical characteristics according to the occurrence of the arrhythmic endpoint during follow-up P
Negative follow-up (n 5 106)
Positive follow-up (n 5 10)
Median age (years)
34 (16–48)
34 (16–59)
Gender (male)
49 (46)
8 (80)
0.05
Proband status 2010 revised ITF criteria
23 (22)
5 (50)
0.06
14 (13) 5 (5)
7 (70) 0
,0.001 1.0
6 (60) 0
0.001 1.0
4 (4) 28 (26)
2 (20) 8 (80)
,0.001
0 22 (21)
0 6 (60)
– 0.01
5 (5) 5 (5)
2 (20) 2 (20)
0.1 0.1
5 (5)
7 (70)
0.001
12 (11)
8 (80)
,0.001
................................................................................ 1.0
Morpho-functional criteria Major Minor
ECG repolarization abnormalities
Figure 1 Post-mortem findings in the young victim of SCD carrying a DSP-gene mutation. (A) Histologic view of the posterolateral left ventricular wall showing a subepicardial scar (Trichrome Heidenhain × 3). (B) Close-up of the boxed area showing myocyte necrosis with fibrous myocardial replacement in the outer wall layer (Trichrome Heidenhain × 12).
Major Minor
13 (12) 8 (8)
ECG depolarization abnormalities Major Minor Arrhythmic criteria Major Minor
Disease progression and non-arrhythmic events during follow-up During follow-up, 10 patients (7 who were borderline and 3 who were phenotype negative at first clinical evaluation) developed a
Unexplained syncope Non-sustained ventricular tachycardia Moderate-severe ventricular dysfunction ≥1 risk factor
Data are presented as n (%) or median (IQR). ECG, electrocardiographic; ITF, International Task Force.
1.0 No definite ARVC 0.8 Event-free survival
Clinical characteristics of patients with and without events during follow-up are reported in Table 3. The incidence of the arrhythmic endpoint was significantly higher in patients fulfilling the ITF criteria for definite ARVC (cumulative rate ¼ 22.5%; 95% CI 0 – 36.0%; annual rate ¼ 2.3%) than in borderline or phenotype negative ARVC patients (cumulative rate ¼ 1.3%; 95% CI 0 – 6.1%; annual rate ¼ 0.2%) (Figure 2). In the subgroup of patients who fulfilled the ITC criteria for definite ARVC, the incidence of the arrhythmic endpoint was significantly higher in those with ≥1 major risk factor (cumulative rate ¼ 44.4%; 95% CI 0 – 66% and annual rate ¼ 4.1%) than in those with no risk factors (cumulative rate ¼ 4.5%; 95% CI 0 – 19.8; and annual rate ¼ 0.4%) (Figure 3). The arrhythmic endpoint was reached by 1 of 21 (5%) patients with no risk factors, 6 of 15 (40%) with a single risk factor, and 2 of 4 (50%) with multiple risk factors. There was a higher incidence of arrhythmic events in probands (5 of 28; 18%) compared with family members (5 of 88, 6%), which reached a borderline statistical significance (Figure 4). The incidence of arrhythmic endpoint was similar in ARVC patients with and without frequent (.1000/die) premature ventricular beats at 24-h Holter monitoring (3 of 14, 21% vs. 6 of 26, 23%; P ¼ 1.0). Baseline genetics and clinical characteristics of DS-gene mutations carriers who experienced arrhythmic events during follow-up are detailed in Table 4.
Risk factors
Definite ARVC
0.6
0.4
0.2 P = 0.002 0.0 0
3
No definite ARVC
76
66
45
Definite ARVC
40
39
36
6 9 Follow-up length (years)
12
15
34
27
7
29
16
6
Figure 2 Arrhythmia-free survival in the overall population. Kaplan – Meier analysis of survival from the composite arrhythmic endpoint according to the diagnosis of ‘definite ARVC’ according to the 2010 ITC criteria.
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Phenotypic expression and risk factors
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Discussion 1.0 No risk factors Event-free survival
0.8
0.6 ≥1 risk factors 0.4
0.2 P = 0.02 0.0 0
3
No risk factors
21
21
21
≥1 risk factors
19
18
15
6 9 Follow-up length (years)
12
15
16
10
6
13
6
0
Figure 3 Arrhythmia-free survival in patients with definite ARVC. Kaplan– Meier analysis of survival from the composite endpoint according to the presence of risk factors (unexplained syncope, non-sustained ventricular tachycardia, or greater than or equal to moderate ventricular dysfunction) among the 40 patients fulfilling the criteria for ‘definite ARVC’.
Family members
0.8 Probands 0.6
0.4
0.2 P = 0.07 0.0 0
3
6
9
12
15
Family members
88
79
62
47
36
12
Probands
28
26
19
16
7
1
Figure 4 Arrhythmia-free survival according to proband status. Kaplan – Meier analysis of survival from the composite endpoint according to proband status.
‘definite’ ARVC. All these patients showed a mild disease phenotype at the end of follow-up and none suffered arrhythmic events. One additional asymptomatic DSP-gene mutation (exon 23; c.4803G . A; M1601I) carrier with a normal ECG and echocardiogram underwent CE-CMR because of a family history of left-dominant ARVC. The exam confirmed the absence of morpho-functional abnormalities of both ventricles but revealed a subepicardial ‘scar’ in the infero-lateral LV region (Figure 5). One patient died for heart failure and one received cardiac transplantation for end-stage cardiac dysfunction. The estimated total mortality rate during the follow-up was 0.2%/year.
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1.0
The pathologic hallmark of ARVC is the progressive loss of myocardium with fibrofatty replacement, which underlies clinical ECG and morpho-functional ventricular changes and acts as a substrate for life-threatening ventricular arrhythmias. 2 The natural history of ARVC predominantly relates to the ventricular electrical instability which may lead to SCD any time during the disease course.2,3 Accurate risk stratification is crucial for identifying patients at higher risk of SCD who will benefit the most from prophylactic implantation of an ICD. Although symptomatic ARVC patients who survive an episode of VF or sustained VT are deemed at highest risk of experiencing life-threatening arrhythmic events and need an ICD for secondary prevention, primary prevention of SCD among asymptomatic DS-gene carriers is still an unsolved issue.16 – 19 In a previous retrospective study, we demonstrated the prognostic value of genotype to predict the cumulative probability of experiencing a first major arrhythmic event ‘lifetime’ (i.e. after birth) in DS-gene related ARVC.13 The present study prospectively assessed the arrhythmic outcome of a series of DS-gene mutation carriers, without previous sustained VT or VF, in relation to the presence and severity of ARVC phenotypic expression. We found that during a long-term (8.5 years) follow-up period, the annual rate of first malignant arrhythmic events was 0.9% and included one SCD (0.1%/ year). Phenotypic expression of ARVC was a prerequisite for ventricular electrical instability and the arrhythmic risk of DS-gene mutation carriers was strongly related to the severity of the disease phenotypic manifestations. The vast majority of DS-gene carriers who experienced malignant arrhythmic events had an overt disease phenotype, consisting of ECG or echocardiographic abnormalities, and major risk factors, suggesting that this subset of patients most benefit from ICD implant. Our results also suggest that CE-CMR may improve our screening ability to identify otherwise concealed LV myocardial substrates at risk for SCD. Recent experimental studies have showed that susceptible DS-gene carriers may harbour arrhythmogenic mechanisms at a molecular and cellular level because of a cross-talk between altered desmosomes and both voltage gated sodium-channel and gap junction proteins.6 – 11 These findings have raised concerns that DS-gene mutation carriers may experience life-threatening ventricular arrhythmias before structural abnormalities develop and that SCD may occur during the so-called ‘concealed phase’ of the disease.5 The results of our study do not support the concept that subjects with genetically defective DS proteins may experience lethal arrhythmic events before/regardless of the development of ARVC phenotypic features. Our findings are in agreement with those of a previous study by te Riele et al.,20 showing that arrhythmic events during follow-up occurred in DS-gene mutation carriers with CMR abnormalities which fulfilled morpho-functional ITF diagnostic criteria. In our study, all DS-gene mutation carriers who did not fulfil the ITF criteria for ‘definite’ ARVC had an uneventful clinical course, with the exception of a 15-year-old DSP-gene mutation carrier with previously normal ECG and echocardiographic findings. However, post-mortem evaluation in this SCD victim demonstrated the presence of an epicardial scar in the infero-lateral LV region. Although the classic ARVC disease phenotype is characterized by RV dysfunction, there are phenotypic variant characterized with early and
Number Proband Gender Gene
Age at Diagnosis enrolment of ARVC (2010 ITF criteria)
Morho-functional criteria
PKP2 ex1Q59X DSP ex7c.897C . G MM PKP2/ex1 T50SfsX61; DSG2/ex9 V392I DSP Ex24c.7622G . A DSG Ex7c.797A . G DSP Int2c.273 + 5G . A
23
Definite
–
Minor
Major
Minor
Yes
24
ICD shock
37
Definite
Major
–
Major
Minor
No
43
ICD shock
16
Definite
Major
Minor
Major
–
Yes
32
SVT
14
Definite
–
Minor
Major
Minor
Yes
18
CA
49
Definite
Major
Minor
Major
–
Yes
62
ICD shock
57
Definite
Major
Minor
–
Minor
Yes
67
SVT
ECG depolarization ECG repolarization criteria criteria
Arrhythmic Risk Age at criteria factors first event
Event type
............................................................................................................................................................................................................................................. 3
No
Male
19
Yes
Male
32
No
Male
46
Yes
Male
49
No
Male
56
No
Male
61
Yes
Male
PKP2 c.2009delC
40
Definite
Major
Minor
Major
Minor
Yes
41
ICD shock
89
Yes
Female
Definite
Major
Minor
–
–
Yes
56
SVT
96
Yes
Female
MM 46 DSG2/ex8E331K; DSG2/int12 c.1880-2G . A MM 32 DSG2/ex6 E230G; DSG2/ex8 D297G
Definite
Major
Minor
–
Minor
Yes
40
SVT
112
No
Male
DSP/ex7S299R
–
–
–
–
–
No
15
SCD
12
Phenotypic expression and outcome in ARVC
Table 4 Baseline genetic and clinical characteristics of DS-gene mutation carriers who experienced arrhythmic events during follow-up
CA, cardiac arrest; DSG, desmoglein; DSP, desmoplakin, ECG, electrocardiographic; ICD, implantable cardioverter-defibrillator; ITF, International Task Force; MM, multiple mutations; PKP2, plakophilin-2; SCD, sudden cardiac death; SVT, sustained ventricular tachycardia.
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Figure 5 Contrast-enhanced cardiac magnetic resonance findings of an asymptomatic 23-year-old female carrying a DSP-gene mutation. Four-chamber (A) and short axis (B) views showing late gadolinium enhancement mostly involving the subepicardial layer of the postero-lateral LV wall at mid-basal level (white arrows), in the absence of other morpho-functional ventricular abnormalities. The electrocardiogram and echocardiogram of this patient were normal.
Study limitations The present study has limitations linked predominantly to relatively low ARVC prevalence and low event rate. Moreover, sequence alterations were divided into pathogenic mutations and polymorphisms according to generally accepted criteria.12 The pathogenetic potential of DS-gene mutations should be interpreted with great caution in the absence of in vitro functional analysis or animal studies that would have been required to conclusively proof the causative nature of DNA variants. However, it is important to stress that
these limitations are exactly the same of all studies previously published in the literature on clinical course of DS-gene related ARVC. Although the inclusion of related family members might influence the survival analysis of freedom from the composite arrhythmic endpoint, events were not clustered into specific families and occurred in patients belonging to different families. A unique feature of our patient cohort was the high prevalence of DSP-gene mutations which may render our results not applicable to a more common population of patients with predominat PKP2-gene mutations. Finally, we performed CE-CMR at the time of enrolment (median 8.5 years ago) in a minority of our patients and this may have limited our ability to identify patients with an overt ARVC phenotype.
Conclusions Clinical management for primary SCD prevention in DS-gene carriers is still controversial. Current recommendations on treatment, including the indications for prophylactic ICD therapy, rely on experts’ opinions with a low level of evidence because of the paucity of data.26 The present study provides scientific evidence that the arrhythmic risk in ARVC-causing DS-gene mutation carriers is significant when the ARVC phenotype becomes overt. These findings support the strategy to (i) manage conservatively DS-gene mutations carriers with regular follow-up evaluations aimed to timely detect the disease onset and stratify the arrhythmic risk; and (ii) to reserve ICD implantation to patients with a clinical diagnosis of ‘definite’ ARVC and arrhythmic risk factors. Further studies are needed to assess whether systematic evaluation of DS-gene mutation carriers (particularly those carrying desmoplakin gene mutations) with CE-CMR in addition to traditional ECG and echocardiogram may improve our ability to identify and stratify the risk of patients with otherwise concealed LV myocardial substrates at risk for arrhythmic SCD.
Funding This study was funded by Fondazione Cariparo, Padova and Rovigo; University of Padua Research Grant TRANSAC, Padova; Veneto Region Target Research, Venice; and PRIN Ministry of Education, University and Research, Rome, Italy. A.Z. research fellowship is in part supported by F.I.G.C., Rome, Italy. Conflict of interest: none declared.
References 1. Corrado D, Basso C, Pilichou K, Thiene G. Molecular biology and clinical management of arrhythmogenic right ventricular cardiomyopathy/dysplasia. Heart 2011; 97:530 – 9. 2. Basso C, Corrado D, Marcus F, Nava A, Thiene G. Arrhythmogenic right ventricular cardiomyopathy. Lancet 2009;373:1289 –300. 3. Migliore F, Zorzi A, Silvano M, Rigato I, Basso C, Thiene G et al. Clinical management of arrhythmogenic right ventricular cardiomyopathy: an update. Curr Pharm Des 2010;16:2918 –28. 4. Sen-Chowdhry S, Syrris P, McKenna WJ. Role of genetic analysis in the management of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy. J Am Coll Cardiol 2007;50:1813 –21. 5. Patel HC, Calkins H. Arrhythmogenic right ventricular dysplasia. Curr Treat Options Cardiovasc Med 2010;12:598 –613. 6. Cerrone M, Lin X, Zhang M, Agullo-Pascual E, Pfenniger A, Chkourko Gusky H et al. Missense mutations in plakophilin-2 cause sodium current deficit and associate with a brugada syndrome phenotype. Circulation 2014;129:1092 –103.
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dominant LV involvement.2,4,21,22 At variance with ‘classic’ RV lesions, ECG and echocardiography may not be enough sensitive to detect segmental and non-transmural epicardial LV scar and a proportion of death during the so-called concealed phase may be the result of such silent substrates undetectable by routine examination. It has been reported that CE-CMR reveals the presence of LV myocardial fibrosis in the majority of ARVC patients.22,23 Another living asymptomatic family member with a family history of left-dominant ARVC showed an epicardial LV scar involvement on CE-CMR postcontrast sequences, despite normal ECG and echocardiographic findings at routine clinical screening. It is noteworthy that both the young SCD victim and the living family member with a LV scar carried a DSP-gene mutation. This finding is in keeping with previous genotype– phenotype correlation studies showing early and greater LV involvement in DSP-gene mutations carriers.24,25 Because current ITF guidelines do not include among diagnostic criteria demonstration of ventricular ‘late gadolinium enhancement’ by CE-CMR, they provide low sensitivity for identification of patients with predominant LV involvement, particularly DSP-gene mutation carriers, who show a LV subepicardial scar as first sign of disease.22 These findings suggest that the ‘concealed phase’ of the disease may be the result of the low sensitivity of routine clinical tests (i.e. ECG and echocardiography) for detection of early/minor disease phenotypic manifestations such as isolated epicardial scar involvement of the left ventricle, rather than expression of a subcellular arrhythmogenic mechanism.
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Phenotypic expression and outcome in ARVC
18.
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arrhythmogenic right ventricular dysplasia/cardiomyopathy undergoing implantable cardioverter-defibrillator implantation for primary prevention. J Am Coll Cardiol 2011;58:1485 – 96. Corrado D, Calkins H, Link MS, Leoni L, Favale S, Bevilacqua M et al. Prophylactic implantable defibrillator in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia and no prior ventricular fibrillation or sustained ventricular tachycardia. Circulation 2010;122:1144 –52. Roguin A, Bomma CS, Nasir K, Tandri H, Tichnell C, James C et al. Implantable cardioverter-defibrillators in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy. J Am Coll Cardiol 2004;43:1843 –52. te Riele AS, Bhonsale A, James CA, Rastegar N, Murray B, Burt JR et al. Incremental value of cardiac magnetic resonance imaging in arrhythmic risk stratification of arrhythmogenic right ventricular dysplasia/cardiomyopathy-associated desmosomal mutation carriers. J Am Coll Cardiol 2013;62:1761 –9. Basso C, Bauce B, Corrado D, Thiene G. Pathophysiology of arrhythmogenic cardiomyopathy. Nat Rev Cardiol 2012;9:223–33. Sen-Chowdhry S, Syrris P, Prasad SK, Hughes SE, Merrifield R, Ward D et al. Leftdominant arrhythmogenic cardiomyopathy: an under-recognized clinical entity. J Am Coll Cardiol 2008;52:2175 –87. Perazzolo Marra M, Leoni L, Bauce B, Corbetti F, Zorzi A, Migliore F et al. Imaging study of ventricular scar in arrhythmogenic right ventricular cardiomyopathy: comparison of 3D standard electroanatomical voltage mapping and contrast-enhanced cardiac magnetic resonance. Circ Arrhythm Electrophysiol 2012;5:91 –100. Bauce B, Basso C, Rampazzo A, Beffagna G, Daliento L, Frigo G et al. Clinical profile of four families with arrhythmogenic right ventricular cardiomyopathy caused by dominant desmoplakin mutations. Eur Heart J 2005;26:1666 –75. Sen-Chowdhry S, Syrris P, Ward D, Asimaki A, Sevdalis E, McKenna WJ. Clinical and genetic characterization of families with arrhythmogenic right ventricular dysplasia/cardiomyopathy provides novel insights into patterns of disease expression. Circulation 2007;115:1710 –20. Ackerman MJ, Priori SG, Willems S, Berul C, Brugada R, Calkins H et al. HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies. Europace 2011;13:1077 –109.
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7. Delmar M, Liang FX. Connexin43 and the regulation of intercalated disc function. Heart Rhythm 2012;9:835 –8. 8. Gomes J, Finlay M, Ahmed AK, Ciaccio EJ, Asimaki A, Saffitz JE et al. Electrophysiological abnormalities precede overt structural changes in arrhythmogenic right ventricular cardiomyopathy due to mutations in desmoplakin-A combined murine and human study. Eur Heart J 2012;33:1942 –53. 9. Kaplan SR, Gard JJ, Protonotarios N, Tsatsopoulou A, Spiliopoulou C, Anastasakis A et al. Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm 2004;1:3– 11. 10. Rizzo S, Lodder EM, Verkerk AO, Wolswinkel R, Beekman L, Pilichou K et al. Intercalated disc abnormalities, reduced Na(+) current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes. Cardiovasc Res 2012;95:409 –18. 11. Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patino GA, Taffet SM et al. Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res 2009;105:523 –6. 12. Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria. Circulation 2010;121:1533 –41. 13. Rigato I, Bauce B, Rampazzo A, Zorzi A, Pilichou K, Mazzotti E et al. Compound and digenic heterozygosity predicts lifetime arrhythmic outcome and sudden cardiac death in desmosomal gene-related arrhythmogenic right ventricular cardiomyopathy. Circ Cardiovasc Genet 2013;6:533 –42. 14. Blaker H. Confidence curves and improved exact confidence intervals for discrete distributions. Can J Stat 2000;28:783 –9. 15. Fay MP. Two-sided exact tests and matching confidence intervals for discrete data. R J 2010;2:53 –8. 16. Corrado D, Leoni L, Link MS, Della Bella P, Gaita F, Curnis A et al. Implantable cardioverter-defibrillator therapy for prevention of sudden death in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia. Circulation 2003; 108:3084 –91. 17. Bhonsale A, James CA, Tichnell C, Murray B, Gagarin D, Philips B et al. Incidence and predictors of implantable cardioverter-defibrillator therapy in patients with
CLINICAL RESEARCH
Europace doi:10.1093/europace/euv205
Phenotypic expression is a prerequisite for malignant arrhythmic events and sudden cardiac death in arrhythmogenic right ventricular cardiomyopathy Alessandro Zorzi 1, Ilaria Rigato 1, Kalliopi Pilichou 1, Martina Perazzolo Marra 1, Federico Migliore 1, Elisa Mazzotti 1, Dario Gregori 1, Gaetano Thiene 1, Luciano Daliento 1, Sabino Iliceto 1, Alessandra Rampazzo 2, Cristina Basso 1, Barbara Bauce 1, and Domenico Corrado1* 1 Inherited Arrhythmogenic Cardiomyopathy Unit, Department of Cardiac, Thoracic, and Vascular Sciences, University of Padua, Via N. Giustiniani 2, Padua 35121, Italy; and 2Department of Biology, University of Padua, Padua, Italy
Received 17 January 2015; accepted after revision 15 May 2015
Whether a desmosomal (DS)-gene defect may in itself induce life-threatening ventricular arrhythmias regardless of phenotypic expression of arrhythmogenic right ventricular cardiomyopathy (ARVC) is still debated. This prospective study evaluated the long-term outcome of DS-gene mutation carriers in relation to the ARVC phenotypic expression. ..................................................................................................................................................................................... Methods The study population included 116 DS-gene mutation carriers [49% males; median age 33 years (16 –48 years)] without and results prior sustained ventricular tachycardia (VT) or ventricular fibrillation (VF). The incidence of the arrhythmic endpoint, including sudden cardiac death (SCD), aborted SCD, sustained VT, and appropriate implantable cardioverter-defibrillator (ICD) intervention was evaluated prospectively and stratified by the presence of ARVC phenotype and risk factors (syncope, ventricular dysfunction, and non-sustained VT). At enrolment, 40 of 116 (34%) subjects fulfilled the criteria for definite ARVC while the remaining were either borderline or phenotype negatives. During a median follow-up of 8.5 (5– 12) years, 10 patients (9%) had arrhythmic events (0.9%/year). The event rate was 2.3%/year among patients with definite ARVC and 0.2%/year among borderline or phenotype negative patients (P ¼ 0.002). In patients with definite ARVC, the incidence of arrhythmias was higher in those with ≥1 risk factors (4.1%/year) than in those with no risk factors (0.4%/year, P ¼ 0.02). Mortality was 0.2%/year (1 heart failure death and 1 SCD). ..................................................................................................................................................................................... Conclusions The ARVC phenotypic expression is a prerequisite for the occurrence of life-threatening arrhythmias in DS-gene mutation carriers. The vast majority of malignant arrhythmic events occurred in patients with an overt disease phenotype and major risk factors suggesting that this subgroup most benefits from ICD therapy.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Cardiomyopathy † Arrhythmogenic right ventricular cardiomyopathy † Implantable cardioverter-defibrillator † Ventricular arrhythmias † Sudden cardiac death † Primary prevention † Genetics
Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable heart muscle disorder characterized by incomplete penetrance and variable phenotypic expression.1 The natural history
predominantly relates to the ventricular electrical instability which may lead to sudden cardiac death (SCD), mostly in young people and athletes.2,3 The progressive loss of myocardium with fibrofatty replacement underlies clinical electrocardiographic (ECG) and morphofunctional ventricular changes and acts as a substrate for life-threatening
* Corresponding author. Tel: +39 049 8212458; fax: +39 049 8212309, E-mail address: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected].
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Aims
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What’s new? † The long-term (8.5 years) arrhythmic risk of DS-gene mutation carriers, with no prior sustained VT or VF, is strongly related to the ARVC phenotypic expression and the presence of major risk factors such as syncope, ventricular dysfunction, or non-sustained VT. † The study results do not support the concept that DS-gene carriers may experience life-threatening arrhythmic events before and/or regardless of the development of ARVC phenotypic features (‘concealed phase’), as postulated on the basis of experimental studies showing that lethal arrhythmogenic mechanisms may arise at a molecular/cellular level as a result of a cross-talk of altered desmosomes with sodium-channel and gap junction proteins. † Our findings also suggest that the ‘concealed phase’ of the disease may be the result of the low sensitivity of routine clinical tests (i.e. electrocardiogram and echocardiography) for detection of early/minor disease phenotypic manifestations such as isolated epicardial scar involvement of the left ventricle, rather than the expression of a subcellular arrhythmogenic mechanism.
Methods Study population This is a single-centre study which enrolled a series of genotyped ARVC probands and relatives who were evaluated at the Inherited Arrhythmogenic Cardiomyopathy Unit of the Department of Cardiac,
Thoracic and Vascular Sciences of the University of Padua. The initial study cohort composed of 137 consecutive DS-gene mutation carriers (45 probands and 92 family members). All subjects had undergone a comprehensive analysis of all amino acid coding exons and intron boundaries of the five ARVC-susceptible DS genes [plakophilin-2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC2), desmoplakin (DSP), and junction plakoglobin (JUP)] and were carriers of ≥1 DS-gene mutation that was considered pathogenic according to the 2010 International Task Force (ITF) criteria, as previously described in details.12,13 In brief, a pathogenetic mutation was defined as a DNA alteration previously associated with ARVC that was unobserved or rare in a large, non-ARVC, ethnically matched control population and was predicted to alter the structure or the function of the encoded protein or cosegregated with disease phenotype in the family. According to the study design, 14 DS-gene carriers were excluded from the study because they had ‘definite’ ARVC and a history of previous sustained VT or VF at the time of first clinical evaluation. Seven individuals were lost during follow-up. The final study sample included 116 DS-gene carriers [28 probands and 88 family members; 57 (49%) males; median age 33 years (16 – 48 years)], with no history of VT or VF at the time of enrolment. A single pathogenetic mutation was identified in 101 individuals (PKP2 ¼ 33, DSP ¼ 41, DSG2 ¼ 26, and DSC2 ¼ 1) and multiple mutations in 15 (Table 1). Most of these subjects constituted the study population of a previous genotype – phenotype investigation by our group with completely different design (retrospective) and endpoint (risk of arrhythmic events since birth).13 The present study was a clinical follow-up study that prospectively evaluated the arrhythmic outcome of DS-gene carriers after their first clinical evaluation. Accordingly, no analysis combining genotype (specific DS-genes, mutation types, and genotype complexity) and arrhythmic risk was performed because this was not a genotype – phenotype study. The study complies with the Declaration of Helsinki, was approved by the local Ethics Committee and all patients gave informed consent to participate.
Clinical protocol At the time of first evaluation, all subjects underwent a detailed clinical evaluation including family history, physical examination, resting 12-lead ECG, signal-averaged ECG (SAECG), 24-h Holter monitoring, exercise testing, and two-dimensional transthoracic echocardiography. Additional tests were reserved to selected cases (mostly probands) and included contrast-enhanced cardiac magnetic resonance (CE-CMR) (n ¼ 23) and invasive exams such as cardiac catheterization (n ¼ 29), endomyocardial biopsy (n ¼ 3), and programmed ventricular stimulation with electroanatomic voltage mapping (n ¼ 21). According to the 2010 ITF diagnostic criteria, in our study population of carriers of ARVC-causing DS-gene mutations (major diagnostic criterion), the diagnosis of ‘definite’ ARVC was fulfilled in the presence of additional one major criterion or two minor criteria from different categories. The diagnosis was considered ‘borderline’ when one additional minor criterion was fulfilled. Desmosomal-gene mutation carriers showing no disease features, either minor or major, were defined as ‘genotype positive-phenotype negative’.12 All individuals were followed-up regularly in the outpatient clinic at 6- to 12-month intervals with ECG, 24-h Holter monitoring, SAECG, and echocardiogram. According to our protocol, implant of an ICD was offered for primary prevention of SCD to DS-gene carriers with a ‘definite’ ARVC diagnosis and ≥1 major risk factors such as unexplained syncope, non-sustained VT, or greater than or equal to moderate ventricular dysfunction, i.e. right ventricular (RV) fractional area change ,24% or RV ejection fraction , 40%; left ventricular (LV) ejection fraction , 45%.
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ventricular arrhythmias.2 The discovery of desmosomal (DS)-gene mutations involved in the pathogenesis of ARVC has offered the potential of a molecular genetic diagnosis in both affected individuals and relatives.1,4 Clinical manifestations of ARVC usually develop during adolescence and young adulthood and are preceded by a long preclinical phase (‘concealed ARVC’), during which SCD has been reported to occur unexpectedly as the first manifestation of the disease.2 As a consequence, the problem of primary prevention of SCD among DS-gene mutation carriers during pre-clinical/early phases of the disease has emerged and an aggressive management strategy including a prophylactic implantable cardioverter-defibrillator (ICD) of asymptomatic DS-gene carriers has been proposed.5 This management strategy has been supported by recent experimental animal studies which demonstrated that the loss of expression of DS-proteins may ‘per se’ induce electrical ventricular instability by ion-channel dysfunction, which predisposes to lethal ventricular arrhythmias regardless or before the expression of a structural (i.e. ‘pre-histological ’) myocardial substrate.6 – 11 However, the relationship between the presence and severity of the phenotypic manifestations of ARVC and the risk of SCD among DS-gene mutation carriers remains to be elucidated. Thus, the present study prospectively evaluated the long-term arrhythmic outcome of a single-centre cohort of DS-gene mutation carriers without previous sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) in relation to the expression of the ARVC phenotype.
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Phenotypic expression and outcome in ARVC
Table 1 Mutation screening results Locus
Exon
NT
AA
Functional meaning
Number of patients
Plakophilin-2
1
148_151delACAG
T50SfsX61
Frameshift
8 + 2a
1 1
c.175C . T c.184C . A
Q59X Q62K
Nonsense Missense
1 1a
1
c.184_185delCA
Q62DfsX23
Frameshift
2
2 2
c.227A . G c.336G . T
N76S K112N
Missense Missense
2 2
3
c.663C . A
Y221X
Nonsense
1a
7 10
c.1643delG c.2009delC
G548VfsX15 K672RfsX12
Frameshift Frameshift
3 7
10
c.2119C . T
Q707X
Nonsense
6 + 6a
12 1
c.2447_2448delCC c.88 G . A
T816RfsX10 V30M
Frameshift Missense
2 + 1a 6 + 1a
...............................................................................................................................................................................
Desmoplakin
Desmocollin-2
c.273+5G . A
Splicing
3
c.423-1G . A c.817C . T
Q273X
Splicing Nonsense
3 3
7
c.897C . G
S299R
Missense
9
9 11
c.1174G . A c.1372A . T
V392I N458Y
Missense Missense
1a 3
11
c.1408A . G
K470E
Missense
2
23 23
c.3337C . T c.3764G . A
R1113X R1255K
Nonsense Missense
4 1a
23
c.3774C . A
D1258E
Missense
2
23 23
c.4803G . A c.4961T . C
M1601I L1654P
Missense Missense
4 6a
23
c. 4973C . T
S1658F
Missense
1a
23 24
c.3203_3204delAG c.7622G . A
p.E1068VfsX19 R2541K
Frameshift Missense
1a 2 + 1a
24
c.7039A . G
I2347V
Missense
1a
4 6
c.298G . C c.689A . G
G100R E230G
Missense Missense
2 1 + 1a
7
c.797A . G
N266S
Missense
4
8 8
c.880A . G c.991G . A
K294E E331K
Missense Missense
3 2a
9
c.1174G . A
V392I
Missense
5
9 Intron12
c.1250_1253dupATGA c.1880-2G . A
D419X
Frameshift Splicing
2 + 1a 2a
13
c.1912G . A
G638R
Missense
1a
14 15
c.2033G . C c.2491C . T
G678A L831F
Missense Missense
4 4 + 2a
15
c.2990delG
G997VfsX20
Frameshift
1 + 1a
15 8
c.2773C . T c.1034T . C
P925S I345T
Missense Missense
1a 1
a
The number of patients with the mutation in compound or double heterozygosis.
Arrhythmic endpoint The primary study endpoint was the occurrence of any major arrhythmic events defined as SCD, cardiac arrest due to VF, sustained VT, or appropriate ICDs intervention on VT/VF. The occurrence of VT/VF during follow-up was documented by ECG recording at the time of symptoms and, in the subgroup of patients who received an ICD for primary prevention, by device interrogation. The secondary endpoint was
all-cause mortality. All events were adjudicated by the consensus of an independent committee blinded to the disease phenotypic features. Appropriate ICD intervention was defined as a device shock or antitachycardia overdrive pacing delivered in response to a ventricular tachyarrhythmia and documented by stored intracardiac ECG data. Implantable cardioverter-defibrillator was routinely programmed to include a monitoring zone that identified VT with a rate .160 b.p.m.
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Desmoglein-2
Intron2 Intron3 7
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A. Zorzi et al.
All ICD interrogations were identified and adjudicated by two electrophysiologists (D.C. and F.M.) blinded to other clinical data. All subjects gave their informed consent to participate in the study.
Statistical analysis Results are summarized as median (25%, 75% quartiles) or n (%) for continuous and categorical variables, respectively. Categorical differences between groups were evaluated by the x 2 test or the Fisher’s exact test as appropriate. Continuous variables were compared using the Rank Sum test because of the small size of subgroups. Event-free survival curves were drawn with the Kaplan– Meier method and compared by the log rank test. Patients were censored at the time of the first event. Cumulative event-rates were expressed as % and the corresponding 95% confidence intervals were calculated with the method of Blaker, which is more powerful than the more common Clopper – Pearson approach.14 The mean annual event rate was calculated assuming exponential (constant) decay over the follow-up. A P-value of ,0.05 was considered significant. Statistics were analysed with the R System (R Foundation for Statistical Computing, Vienna, Austria) and the ExactCI libraries.15
Results Baseline clinical characteristics
Arrhythmic outcome During a median follow-up of 8.5 (5–12) years, 10 patients (9%), all belonging to different families, reached the composite arrhythmic endpoint with a cumulative rate of major arrhythmic events of 8.6% (95% CI 0–14.2%) and an annual rate of 0.9%. The arrhythmic events consisted of cardiac arrest due to VF (n ¼ 1), sustained monomorphic VT (n ¼ 4), appropriate ICD interventions for VT (n ¼ 3) and VF (n ¼ 1), and SCD (n ¼ 1). The SCD victim was a 15-year-old DSP-gene mutation carrier (exon 7; c.897C . G; S299R), who did not show any ECG and echocardiographic abnormalities at the time of his first clinical evaluation and died suddenly 2 years later while sleeping. Eight months before death he underwent follow-up clinical evaluation which provided unremarkable findings. Autopsy investigation showed predominant LV involvement with a band of myocyte necrosis and fibrous myocardial replacement in the outer mid – subepicardial layer of the postero-lateral wall (Figure 1).
Table 2 Baseline characteristics of the study sample at the time of first clinical evaluation according to the 2010 ITF classification 2010 ITF classification
...................................................................................... Overall (n 5 116)
Phenotype negative (n 5 57)
Borderline ARVC (n 5 19)
Definite ARVC (n 5 40)
............................................................................................................................................................................... Median age (years)
33 (16–48)
23 (15–46)
43 (21– 48)
37 (16– 55)
Gender (male)
57 (49)
25 (44)
12 (63)
20 (50)
Proband status 2010 revised ITF criteria
28 (24)
0
8 (42)
20 (50)
21 (18) 5 (4)
0 0
0 0
21 (53) 5 (25)
19 (16) 8 (7)
0 0
0 3 (16)
19 (48) 5 (25)
4 (3) 36 (31)
0 0
0 10 (53)
4 (20) 26 (65)
Morpho-functional criteria Major Minor ECG repolarization abnormalities Major Minor ECG depolarization abnormalities Major Minor Arrhythmic criteria Major Minor Risk factors Unexplained syncope
0
0
28 (24)
0
0
6 (32)
22 (55)
0
6 (15)
7 (6)
0
1 (5)
Non-sustained ventricular tachycardia Moderate-severe ventricular dysfunction
7 (6) 12 (12)
0 0
0 0
7 (18) 12 (30)
≥1 risk factor
20 (17)
0
1 (5)
19 (48)
Data are presented as n (%) or median (IQR). ECG, electrocardiographic; ITF, International Task Force.
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Baseline characteristics of the study population at the time of the first evaluation are shown in Table 2. Forty (34%) DS-gene mutation
carriers fulfilled the 2010 ITF diagnostic criteria for ‘definite’ ARVC, while the remaining 76 (66%) had ‘borderline’ ARVC (n ¼ 19; 16%) or were phenotype negative (n ¼ 57, 50%). No borderline or phenotype negative DS-gene mutation carrier underwent invasive tests such as endomyocardial biopsy or endocardial voltage mapping. Thirteen patients received an ICD for primary prevention.
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Phenotypic expression and outcome in ARVC
Table 3 Clinical characteristics according to the occurrence of the arrhythmic endpoint during follow-up P
Negative follow-up (n 5 106)
Positive follow-up (n 5 10)
Median age (years)
34 (16–48)
34 (16–59)
Gender (male)
49 (46)
8 (80)
0.05
Proband status 2010 revised ITF criteria
23 (22)
5 (50)
0.06
14 (13) 5 (5)
7 (70) 0
,0.001 1.0
6 (60) 0
0.001 1.0
4 (4) 28 (26)
2 (20) 8 (80)
,0.001
0 22 (21)
0 6 (60)
– 0.01
5 (5) 5 (5)
2 (20) 2 (20)
0.1 0.1
5 (5)
7 (70)
0.001
12 (11)
8 (80)
,0.001
................................................................................ 1.0
Morpho-functional criteria Major Minor
ECG repolarization abnormalities
Figure 1 Post-mortem findings in the young victim of SCD carrying a DSP-gene mutation. (A) Histologic view of the posterolateral left ventricular wall showing a subepicardial scar (Trichrome Heidenhain × 3). (B) Close-up of the boxed area showing myocyte necrosis with fibrous myocardial replacement in the outer wall layer (Trichrome Heidenhain × 12).
Major Minor
13 (12) 8 (8)
ECG depolarization abnormalities Major Minor Arrhythmic criteria Major Minor
Disease progression and non-arrhythmic events during follow-up During follow-up, 10 patients (7 who were borderline and 3 who were phenotype negative at first clinical evaluation) developed a
Unexplained syncope Non-sustained ventricular tachycardia Moderate-severe ventricular dysfunction ≥1 risk factor
Data are presented as n (%) or median (IQR). ECG, electrocardiographic; ITF, International Task Force.
1.0 No definite ARVC 0.8 Event-free survival
Clinical characteristics of patients with and without events during follow-up are reported in Table 3. The incidence of the arrhythmic endpoint was significantly higher in patients fulfilling the ITF criteria for definite ARVC (cumulative rate ¼ 22.5%; 95% CI 0 – 36.0%; annual rate ¼ 2.3%) than in borderline or phenotype negative ARVC patients (cumulative rate ¼ 1.3%; 95% CI 0 – 6.1%; annual rate ¼ 0.2%) (Figure 2). In the subgroup of patients who fulfilled the ITC criteria for definite ARVC, the incidence of the arrhythmic endpoint was significantly higher in those with ≥1 major risk factor (cumulative rate ¼ 44.4%; 95% CI 0 – 66% and annual rate ¼ 4.1%) than in those with no risk factors (cumulative rate ¼ 4.5%; 95% CI 0 – 19.8; and annual rate ¼ 0.4%) (Figure 3). The arrhythmic endpoint was reached by 1 of 21 (5%) patients with no risk factors, 6 of 15 (40%) with a single risk factor, and 2 of 4 (50%) with multiple risk factors. There was a higher incidence of arrhythmic events in probands (5 of 28; 18%) compared with family members (5 of 88, 6%), which reached a borderline statistical significance (Figure 4). The incidence of arrhythmic endpoint was similar in ARVC patients with and without frequent (.1000/die) premature ventricular beats at 24-h Holter monitoring (3 of 14, 21% vs. 6 of 26, 23%; P ¼ 1.0). Baseline genetics and clinical characteristics of DS-gene mutations carriers who experienced arrhythmic events during follow-up are detailed in Table 4.
Risk factors
Definite ARVC
0.6
0.4
0.2 P = 0.002 0.0 0
3
No definite ARVC
76
66
45
Definite ARVC
40
39
36
6 9 Follow-up length (years)
12
15
34
27
7
29
16
6
Figure 2 Arrhythmia-free survival in the overall population. Kaplan – Meier analysis of survival from the composite arrhythmic endpoint according to the diagnosis of ‘definite ARVC’ according to the 2010 ITC criteria.
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Phenotypic expression and risk factors
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A. Zorzi et al.
Discussion 1.0 No risk factors Event-free survival
0.8
0.6 ≥1 risk factors 0.4
0.2 P = 0.02 0.0 0
3
No risk factors
21
21
21
≥1 risk factors
19
18
15
6 9 Follow-up length (years)
12
15
16
10
6
13
6
0
Figure 3 Arrhythmia-free survival in patients with definite ARVC. Kaplan– Meier analysis of survival from the composite endpoint according to the presence of risk factors (unexplained syncope, non-sustained ventricular tachycardia, or greater than or equal to moderate ventricular dysfunction) among the 40 patients fulfilling the criteria for ‘definite ARVC’.
Family members
0.8 Probands 0.6
0.4
0.2 P = 0.07 0.0 0
3
6
9
12
15
Family members
88
79
62
47
36
12
Probands
28
26
19
16
7
1
Figure 4 Arrhythmia-free survival according to proband status. Kaplan – Meier analysis of survival from the composite endpoint according to proband status.
‘definite’ ARVC. All these patients showed a mild disease phenotype at the end of follow-up and none suffered arrhythmic events. One additional asymptomatic DSP-gene mutation (exon 23; c.4803G . A; M1601I) carrier with a normal ECG and echocardiogram underwent CE-CMR because of a family history of left-dominant ARVC. The exam confirmed the absence of morpho-functional abnormalities of both ventricles but revealed a subepicardial ‘scar’ in the infero-lateral LV region (Figure 5). One patient died for heart failure and one received cardiac transplantation for end-stage cardiac dysfunction. The estimated total mortality rate during the follow-up was 0.2%/year.
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1.0
The pathologic hallmark of ARVC is the progressive loss of myocardium with fibrofatty replacement, which underlies clinical ECG and morpho-functional ventricular changes and acts as a substrate for life-threatening ventricular arrhythmias. 2 The natural history of ARVC predominantly relates to the ventricular electrical instability which may lead to SCD any time during the disease course.2,3 Accurate risk stratification is crucial for identifying patients at higher risk of SCD who will benefit the most from prophylactic implantation of an ICD. Although symptomatic ARVC patients who survive an episode of VF or sustained VT are deemed at highest risk of experiencing life-threatening arrhythmic events and need an ICD for secondary prevention, primary prevention of SCD among asymptomatic DS-gene carriers is still an unsolved issue.16 – 19 In a previous retrospective study, we demonstrated the prognostic value of genotype to predict the cumulative probability of experiencing a first major arrhythmic event ‘lifetime’ (i.e. after birth) in DS-gene related ARVC.13 The present study prospectively assessed the arrhythmic outcome of a series of DS-gene mutation carriers, without previous sustained VT or VF, in relation to the presence and severity of ARVC phenotypic expression. We found that during a long-term (8.5 years) follow-up period, the annual rate of first malignant arrhythmic events was 0.9% and included one SCD (0.1%/ year). Phenotypic expression of ARVC was a prerequisite for ventricular electrical instability and the arrhythmic risk of DS-gene mutation carriers was strongly related to the severity of the disease phenotypic manifestations. The vast majority of DS-gene carriers who experienced malignant arrhythmic events had an overt disease phenotype, consisting of ECG or echocardiographic abnormalities, and major risk factors, suggesting that this subset of patients most benefit from ICD implant. Our results also suggest that CE-CMR may improve our screening ability to identify otherwise concealed LV myocardial substrates at risk for SCD. Recent experimental studies have showed that susceptible DS-gene carriers may harbour arrhythmogenic mechanisms at a molecular and cellular level because of a cross-talk between altered desmosomes and both voltage gated sodium-channel and gap junction proteins.6 – 11 These findings have raised concerns that DS-gene mutation carriers may experience life-threatening ventricular arrhythmias before structural abnormalities develop and that SCD may occur during the so-called ‘concealed phase’ of the disease.5 The results of our study do not support the concept that subjects with genetically defective DS proteins may experience lethal arrhythmic events before/regardless of the development of ARVC phenotypic features. Our findings are in agreement with those of a previous study by te Riele et al.,20 showing that arrhythmic events during follow-up occurred in DS-gene mutation carriers with CMR abnormalities which fulfilled morpho-functional ITF diagnostic criteria. In our study, all DS-gene mutation carriers who did not fulfil the ITF criteria for ‘definite’ ARVC had an uneventful clinical course, with the exception of a 15-year-old DSP-gene mutation carrier with previously normal ECG and echocardiographic findings. However, post-mortem evaluation in this SCD victim demonstrated the presence of an epicardial scar in the infero-lateral LV region. Although the classic ARVC disease phenotype is characterized by RV dysfunction, there are phenotypic variant characterized with early and
Number Proband Gender Gene
Age at Diagnosis enrolment of ARVC (2010 ITF criteria)
Morho-functional criteria
PKP2 ex1Q59X DSP ex7c.897C . G MM PKP2/ex1 T50SfsX61; DSG2/ex9 V392I DSP Ex24c.7622G . A DSG Ex7c.797A . G DSP Int2c.273 + 5G . A
23
Definite
–
Minor
Major
Minor
Yes
24
ICD shock
37
Definite
Major
–
Major
Minor
No
43
ICD shock
16
Definite
Major
Minor
Major
–
Yes
32
SVT
14
Definite
–
Minor
Major
Minor
Yes
18
CA
49
Definite
Major
Minor
Major
–
Yes
62
ICD shock
57
Definite
Major
Minor
–
Minor
Yes
67
SVT
ECG depolarization ECG repolarization criteria criteria
Arrhythmic Risk Age at criteria factors first event
Event type
............................................................................................................................................................................................................................................. 3
No
Male
19
Yes
Male
32
No
Male
46
Yes
Male
49
No
Male
56
No
Male
61
Yes
Male
PKP2 c.2009delC
40
Definite
Major
Minor
Major
Minor
Yes
41
ICD shock
89
Yes
Female
Definite
Major
Minor
–
–
Yes
56
SVT
96
Yes
Female
MM 46 DSG2/ex8E331K; DSG2/int12 c.1880-2G . A MM 32 DSG2/ex6 E230G; DSG2/ex8 D297G
Definite
Major
Minor
–
Minor
Yes
40
SVT
112
No
Male
DSP/ex7S299R
–
–
–
–
–
No
15
SCD
12
Phenotypic expression and outcome in ARVC
Table 4 Baseline genetic and clinical characteristics of DS-gene mutation carriers who experienced arrhythmic events during follow-up
CA, cardiac arrest; DSG, desmoglein; DSP, desmoplakin, ECG, electrocardiographic; ICD, implantable cardioverter-defibrillator; ITF, International Task Force; MM, multiple mutations; PKP2, plakophilin-2; SCD, sudden cardiac death; SVT, sustained ventricular tachycardia.
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Figure 5 Contrast-enhanced cardiac magnetic resonance findings of an asymptomatic 23-year-old female carrying a DSP-gene mutation. Four-chamber (A) and short axis (B) views showing late gadolinium enhancement mostly involving the subepicardial layer of the postero-lateral LV wall at mid-basal level (white arrows), in the absence of other morpho-functional ventricular abnormalities. The electrocardiogram and echocardiogram of this patient were normal.
Study limitations The present study has limitations linked predominantly to relatively low ARVC prevalence and low event rate. Moreover, sequence alterations were divided into pathogenic mutations and polymorphisms according to generally accepted criteria.12 The pathogenetic potential of DS-gene mutations should be interpreted with great caution in the absence of in vitro functional analysis or animal studies that would have been required to conclusively proof the causative nature of DNA variants. However, it is important to stress that
these limitations are exactly the same of all studies previously published in the literature on clinical course of DS-gene related ARVC. Although the inclusion of related family members might influence the survival analysis of freedom from the composite arrhythmic endpoint, events were not clustered into specific families and occurred in patients belonging to different families. A unique feature of our patient cohort was the high prevalence of DSP-gene mutations which may render our results not applicable to a more common population of patients with predominat PKP2-gene mutations. Finally, we performed CE-CMR at the time of enrolment (median 8.5 years ago) in a minority of our patients and this may have limited our ability to identify patients with an overt ARVC phenotype.
Conclusions Clinical management for primary SCD prevention in DS-gene carriers is still controversial. Current recommendations on treatment, including the indications for prophylactic ICD therapy, rely on experts’ opinions with a low level of evidence because of the paucity of data.26 The present study provides scientific evidence that the arrhythmic risk in ARVC-causing DS-gene mutation carriers is significant when the ARVC phenotype becomes overt. These findings support the strategy to (i) manage conservatively DS-gene mutations carriers with regular follow-up evaluations aimed to timely detect the disease onset and stratify the arrhythmic risk; and (ii) to reserve ICD implantation to patients with a clinical diagnosis of ‘definite’ ARVC and arrhythmic risk factors. Further studies are needed to assess whether systematic evaluation of DS-gene mutation carriers (particularly those carrying desmoplakin gene mutations) with CE-CMR in addition to traditional ECG and echocardiogram may improve our ability to identify and stratify the risk of patients with otherwise concealed LV myocardial substrates at risk for arrhythmic SCD.
Funding This study was funded by Fondazione Cariparo, Padova and Rovigo; University of Padua Research Grant TRANSAC, Padova; Veneto Region Target Research, Venice; and PRIN Ministry of Education, University and Research, Rome, Italy. A.Z. research fellowship is in part supported by F.I.G.C., Rome, Italy. Conflict of interest: none declared.
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