Multiple mutations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia

Article Outline

• Abstract
• Introduction
• Methods
  • Clinical evaluation
  • Mutation screening
• Results
  • Family #137
    • Genetic findings
    • Clinical findings
  • Family #100
    • Genetic findings
    • Clinical findings
  • Family #104
    • Genetic findings
    • Clinical findings
    • LV involvement
    • Single-mutation versus multiple-mutation carriers
• Discussion
• Acknowledgements
• References
• Copyright

Background

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a progressive cardiomyopathy showing a wide clinical spectrum in terms of clinical expressions and prognoses.

Objective

This study sought to estimate the occurrence of compound and double heterozygotes for mutations in desmosomal proteins encoding genes in a cohort of ARVC/D Italian index cases, and to assess the clinical phenotype of mutations carriers.

Methods

Fourty-two consecutive ARVC/D index cases who fulfilled the International Task Force diagnostic criteria were screened for mutations in PKP2, DSP, DSG2, DSC2, and JUP genes by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing.

Results

Three probands (7.1%) showing a family history of sudden death carried multiple mutations. Family screening identified an additional 7 multiple-mutation carriers. Among the 7 double heterozygotes for mutations in different genes, 2 were clinically unaffected, 2 were affected, and 3 showed some clinical signs of ARVC/D even if they did not fulfill the diagnostic criteria. Two compound heterozygotes for mutations in the same gene and 1 subject carrying 3 different mutations showed a severe form of the disease with heart failure onset at a young age. Moreover, multiple-mutation carriers showed a higher prevalence of left ventricular involvement (P = .025) than single-mutation carriers.

Conclusion

Occurrence of compound and double heterozygotes in ARVC/D index cases is particularly relevant to mutation screening strategy and to genetic counseling. Even if multiple-mutation carriers show a wide variability in clinical expression, the extent of the disease is higher compared to that in single-mutation carriers.

Keywords: Arrhythmia, Arrhythmogenic cardiomyopathy, Sudden death, Cell adhesion molecules, Gene mutations, Compound genotypes

Abbreviations: ARVC/D, arrhythmogenic right ventricular cardiomyopathy/dysplasia, ECG, electrocardiogram, DHPLC, denaturing high-performance liquid chromatography, ICD, implantable cardioverter-defibrillator, RPD, repeat domains, RV, right ventricular, VT, ventricular tachycardia

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Introduction 

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a heart muscle disease characterized by a progressive myocardial atrophy followed by fibrofatty replacement, involving mostly the right ventricular (RV) myocardium.¹, ², ³ The pathologic changes offer the substrate for the onset of ventricular arrhythmias that in some cases can lead to sudden death.⁴ Moreover, the disease is clinically heterogeneous, with interfamilial and intrafamilial variability, ranging from benign to malignant forms with a high risk of sudden cardiac death. The mode of inheritance is mostly autosomal dominant with incomplete penetrance.

Twelve genetic loci have been discovered so far, and mutations were documented in 8 different genes. Different mutations have been detected in genes encoding desmosomal proteins: desmoplakin⁵, ⁶ (DSP, ARVD8), plakophilin-2⁷ (PKP2, ARVD9), desmoglein-2⁸, ⁹ (DSG2, ARVD10), desmocollin-2¹⁰, ¹¹, ¹² (DSC2, ARVD11), and plakoglobin¹³ (JUP, ARVD12). Mutations of JUP and DSP have also been shown to cause recessively inherited syndromic forms of ARVC manifesting with palmoplantar keratoderma and woolly hair, named Naxos and Carvajal syndrome, respectively.¹⁴, ¹⁵, ¹⁶ Moreover, mutations in TGFβ3 and TMEM43 are involved in ARVD1¹⁷ and ARVD5,¹⁸ respectively. The cardiac ryanodine receptor gene RyR2 causes a distinct clinical entity, ARVD2, characterized by juvenile sudden cardiac death and effort-induced polymorphic ventricular tachycardia (VT).¹⁹ Whether this is a variant of ARVC/D is a controversial issue.

We report here on genetic and clinical data of 3 ARVC/D families in which mutation screening of desmosomal ARVC/D genes detected the simultaneous presence of different mutations in 1 or 2 different genes.

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Methods 

Clinical evaluation 

A cohort of 42 consecutive index cases with a typical form of ARVC/D was investigated at the Department of Cardiothoracic and Vascular Sciences of the University of Padua, Italy. All of these patients gave informed consent to be evaluated and to give blood samples for DNA study. The study protocol²⁰ included: physical examination, family history, 12-lead electrocardiogram (ECG), signal-averaged ECG, 24-hour Holter ECG, 2-dimensional echocardiogram. As far as ECG evaluation, the term RV conduction delay was used to define both the presence of a QRS duration in V1 to V3 of 110 to 120 ms and of incomplete bundle branch block (QRS <120 ms, presence of a secondary R wave in V1, or S wave notched with a prolonged duration in V1 or an S1 to S2 to S3 aspect with a secondary R wave in lead aVR).²¹, ²² Measurements of the QRS voltages in the precordial leads were calculated summing the amplitude of the different QRS components in each of the precordial leads.²² Moreover, premature ventricular complexes were defined as abnormal when more than 1,000/24 h were present. Diagnosis was made according to Task Force criteria.²³ Moreover, family members of these 42 subjects were also analyzed using the same clinical protocol. The clinical and instrumental findings of multiple mutations were compared with those of single-mutation carriers. All continuous variables were expressed as the mean value ± SD. An unpaired Student t-test was used for comparison of normally distributed data. To compare noncontinuous variables, the Fisher exact test was performed to contingency tables.

Mutation screening 

Forty-two consecutive index case patients were screened for mutations in PKP2, DSP, DSG2, DSC2, JUP, and TGFβ3 genes by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. The analysis was performed using the Wave Nucleic Acid Fragment Analysis System 3500HT with DNASep HT cartridge technology (Transgenomic Inc, Omaha, USA). The temperatures for sample analysis were predicted using the Wave Navigator software. Samples showing a change in DHPLC pattern were directly sequenced using the Big Dye dideoxy-terminator chemistry (Perkin Elmer, Waltham, USA) on an ABI 377 DNA sequencer (PE Applied Biosystems, Foster City, USA). Chromas 1.5 software (Technelysium) and Lasergene package computer programs (DNASTAR) were used to edit, assemble, and translate sequences. DSP sequences were compared with reference sequence NM_004415, PKP2 sequences to NM_001005242, DSG2 sequences to NM_001943, DSC2 sequences to NM_024422, JUP sequences to NM_002230, and TGFβ3 to NM_003239.

A control group of 250 healthy and unrelated subjects (500 alleles) from the Italian population was used to exclude that the detected mutations were DNA polymorphisms. Identified mutations altering restriction sites were confirmed by restriction digest according to the manufacturer's protocol (New England Biolabs, Ipswich, USA). Mutation screening was performed in all living family members of index cases in whom a mutation was detected.

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Results 

Among 42 unrelated ARVC/D index cases, 7 (16.6%) carried a PKP2 mutation, 5 (11.9%) a DSP mutation, 4 (9.5%) a DSG2 mutation, 2 (4.8%) a DSC2 mutation, and 2 (4.8%) a TGFβ3 mutation. No one carried JUP mutations. Overall, 3 index cases (7.1%) carried multiple mutations in the same gene or in different genes.

Family #137 

Index case (IV,5) showed 2 different mutations in the DSP gene (V30M and R2541K) (Figure 1A). Substitution c.88G>A, leading to a replacement of a valine by methionine (V30M), was previously reported.²⁴ Methionine in position 30 of DSP does not show phylogenetic conservation in vertebrates (Figure 1B). On the contrary, strong conservation is present in the upstream residues. Mutation R2541K (c.7622G>A) occurred in a highly conserved residue (Figure 1B), placed in a region linking repeat domains (RPD) of DSP C-terminal. None of such nucleotide changes were detected in 250 control subjects (500 chromosomes) from the same population. Restriction digestion with BstEII was used as an independent genotyping method to validate sequence analysis results for DSP V30M. No additional mutations were detected in PKP2, DSG2, DSC2, and JUP genes.

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• Figure 1. 

  A: Sequence electropherograms showing the detected mutations. Numbering of the nucleotides starts at the ATG codon. B: Evolutionary conservation of the missense mutations identified in desmoplakin, desmoglein-2, and plakophilin-2 genes, among different species. The mutated amino acids are in red.

Analysis of family members available to the study showed that the index case inherited V30M from her mother (III,3) and R2541K from the paternal side. V30M was detected as well in subjects IV,2; V,1; V,3; V,4; V,5; and V6, whereas R2541K was detected in subjects IV,7; V,7; V,8; and V,9 (Figure 2).

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• Figure 2. 

  Pedigrees of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) probands carrying multiple mutations. Arrows indicate index cases. + and – denote the presence or absence of a desmosomal gene mutation.

Among the 28 family members, 7 died before the time of the study. The index case (IV,5) required cardiac evaluation because of palpitations at the age of 36, and she was diagnosed as carrying a biventricular form of ARVC/D (left ventricular [LV] end diastolic volume = 75 ml/m², LV ejection fraction = 50%, RV end diastolic area = 32 cm², RV fraction shortening = 30%). At the age of 40, the patient showed symptoms and signs of heart failure, and at 45 she received an implantable cardioverter-defibrillator (ICD) because of episodes of sustained VT. Finally, at age 49 she underwent a heart transplantation because of refractory heart failure. Gross and histologic examination of the heart removed at transplantation revealed a moderate biventricular dilatation with wall thinning caused by diffuse myocardial atrophy and fibrofatty replacement of the RV and LV free walls; marked cardiomyopathic changes of the myocytes were also evident with dysmetric and dysmorphic nuclei (Figure 3). Analysis of the family tree showed that subjects I,2; II,3, and III,4 had died suddenly at the age of 46, 47, and 45 years, respectively (no clinical data available) (Figure 2). Subject III,5 was affected by a biventricular form of ARVC and had died of refractory heart failure at the age of 64, whereas subject IV,4 was affected by a severe form of the disease and had died suddenly at the age of 43. Autopsy confirmed the clinical diagnosis of ARVC/D by showing a biventricular dilatation and fibrofatty replacement with patchy inflammatory infiltrates. Moreover, myocytes showed dysmetric and dysmorphic nuclei. Among the remaining family members, 2 (IV,7; V,4) fulfilled ARVC/D diagnostic criteria, whereas additional 2 (V,1; V,6) showed some clinical signs of the disease, although they did not fulfill the diagnostic criteria. None of the patients showed woolly hairs or palmoplantar keratoderma.

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• Figure 3. 

  Clinical findings of index case (IV,5) of family #137. A: Twelve-lead electrocardiogram with presence of intraventricular conduction delay, negative T waves in precordial and inferior leads, and low QRS voltages. B: Two-dimensional echocardiogram showing a severe RV dilatation with moderate systolic depression. The LV is also dilated. C: Histological section of the RV free wall at high magnification; residual hypertrophied myocytes are entrapped within fibrous and fatty tissue (Heidenhain trichrome). D: Severe myocyte abnormalities with bizarre dysmorphic and dysmetric nuclei and perinuclear halo (hematoxylin-eosin). LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle.

Family #100 

Mutation screening performed in the proband's DNA (IV,16) identified a G to A transition (c.1174G>A) at codon 392 of DSG2, leading to substitution of a isoleucine for valine (Figure 1A). The involved amino acid is highly conserved among mammals (Figure 1B). Mutation c.1174G>A was previously reported.²⁵ DHPLC analysis of 500 unrelated normal chromosomes was negative.

DHPLC mutation screening of all PKP2 exons, followed by sequencing of abnormal profiles, succeeded in identifying a 4-base pair deletion in exon 1 (c.145_148delCAGA), producing a frame shift and premature termination of the protein (T50SfsX60, previously named S50fsX110 or T50fsX60) (Figure 1A). This mutation has been previously reported by different groups.⁷, ²⁶, ²⁷, ²⁸ DHPLC analysis of DSP, DSC2, and JUP identified no mutations in these genes. DSG2 V392I mutation was inherited from the paternal side and PKP2 T50SfsX60 mutation from the maternal side of the family.

Seven family members carried the PKP2 mutation (IV,13; IV,19; IV,21; IV,24; V,7; V,9; V,10), 9 subjects carried the DSG2 mutation (III,9; IV,2; IV,13; IV,15; V,1; V,2; V,4; V,5; V,6) (Figure 2). Subject IV,13 was heterozygous for both mutations.

The index case (IV,16) was a 22-year-old man who was diagnosed to be affected because of palpitations and nonsustained VT recorded on 24-hour Holter ECG. One sister (IV,10) and 1 brother (IV,11) suffered sudden death during physical activity at the ages of 21 and 26, respectively, as first manifestation of the disease. Autopsy investigation revealed transmural myocardial atrophy with fibrofatty replacement of the RV myocardium and diffuse myocyte abnormalities. The mother (III,10) had died of heart failure. Among the remaining family members, 5 (III,9; IV,2; IV,15; IV,21; V,7) were found to be affected by ARVC/D. Of these, subject V,7 experienced an episode of sustained VT at the age of 30.

Family #104 

Mutation detection analysis of all DSP exons in the index case (II,7) DNA identified a T-to-C mutation (c.4961T>C) at codon 1654 leading to replacement of a leucine by proline (Figure 1A). This change was absent in 500 unrelated normal chromosomes. The involved amino acid is highly conserved among mammals (Figure 1B). Leucine residue in position 1654 is placed in the “core” of the alpha-helical coiled-coil rod-domain of DSP. Leucine is a hydrophobic amino acid, which acts as structural element on the inner side of proteins (alpha-helix or beta-sheet). Therefore, this mutation could severely affect the alpha-helix structure. Surprisingly, 1 of the 2 affected subjects did not carry this mutation.

DHPLC screening of all PKP2 exons followed by sequencing of abnormal profiles identified a missense mutation in exon 1 (c.184C>A, Q62K) and a nonsense mutation in exon 10 (c.2119C>T, Q707X) (Figure 1A). None of the detected nucleotide changes was found in 500 control chromosomes. The missense variation Q62K has been previously reported.²⁷, ²⁹

Analysis of all of the available family members showed that the index case inherited the DSP L1654P mutation and PKP2 Q62K from the father (I,1) and the PKP2 Q707X mutation from the mother (I,2). Five subjects carried the DSP L1654P mutation (I,1; II,4; II,9; III,2; III,3), 4 subjects carried the PKP2 Q62K variant (I,1; II,5; II,8; III,4), and 8 carried the PKP2 Q707X mutation (I,2; II,4; II,5; II,9; III,1; III,2; III,3; III,5). Subjects I,1; II,4; II,5; II,9; III,2, and III,3 were heterozygous for 2 mutations (Figure 2).

The family consisted of 16 subjects. Of these, 2 died suddenly at the age 22 and 24 years, respectively, under emotional and physical stress. No clinical or autopsy data are available. The index case (II,7) is a 31-year-old woman who underwent cardiological screening because of palpitations and nonsustained ventricular tachycardia recorded at 24-hour Holter ECG. A 2-dimensional echocardiogram revealed a biventricular form of ARVC/D. Endomyocardial biopsy confirmed the diagnosis of ARVC/D by showing a significant amount of fibrous and fatty tissue and spotty T-lymphocytes. At the age of 42 years, she showed an episode of heart failure; 2 years later she was put on the waiting list for a heart transplant because of refractory heart failure, and received an ICD for primary prevention. The proband's father (I,1) was found to be affected by a mild form of the disease, whereas the mother (I,2) was clinically unaffected. The brother (II,5) had a severe form of the disease with biventricular involvement and sustained VT that required an ICD.

It is noteworthy that a total of 6 patients (4 multiple-mutation and 2 single-mutation carriers) showed LV involvement. In detail, 2 subjects (family 100, V-7 and family 104, III,7) presented a severely depressed LV function (LV ejection fraction <30%) with diffuse kinetic abnormalities. In the remaining patients, the LV was mildly dilated (LV end diastolic volume >70 and <80 ml/m² at 2-dimensional echocardiogram) and the function was mildly reduced (LV ejection fraction >45% and <55%) with localized kinetic abnormalities in 3 cases (2 on the inferior and 1 on the anterior walls).

Multiple-mutation carriers showed a higher prevalence of ECG abnormalities (P = .018), of major RV abnormalities (P = .051), and of LV involvement (P = .025) when compared with single-mutation carriers (Table 1).

Table 1. Cross-comparison of clinical data of ARVC/D single- and multiple-mutation carriers

Characteristic	Mutation carriers		P value
	Multiple	Single
Total subjects (n)	10	30
Male/female	4(40%)/6(60%)	14(47%)/16(53%)	1
Age (yrs)	51±21	43±24	.35
Age at first evaluation (yrs)	34±21	31±20	.68
Age at diagnosis (yrs)	40±21	34±19	.40
Actively involved in sport activity	0	8(27%)
Symptomatic, n (%)	5(50%)	7(23%)	.13
Palpitations	5	5
Syncope	1	1
Chest pain	1	1
Subjects fulfilling diagnostic criteria	5(50%)	7(23%)	.13
12-lead ECG abnormalities, n (%)	7(70%)	7(23%)	.02
RV conduction delay	2	7
Localized QRS prolongation V1–V3⁎	2	6
Negative T-wave V2–V3⁎	2	3
Negative T-wave beyond V3⁎	3	1
Negative T-wave inferior leads	1	2
Epsilon wave⁎	2	0
Low QRS voltages	4	2
Signal-averaged ECG, n (%)⁎	5(50%)	8(26%)	.25
Ventricular arrhythmias, n (%)⁎	5(50%)	7(23%)	.13
PVCs	1	4
Nonsustained VT	3	3
Sustained VT/VF	1	0
RV alterations (major)⁎	4(40%)	3(10%)	.05
RV alterations (overall)†	5(50%)	8(26%)	.25
LV involvement n (%)	4(40%)	2(7%)	.02

Data shown as mean ± SD or n (%).

ARVC/D = arrhythmogenic right ventricular cardiomyopathy/dysplasia; ECG = electrocardiogram; LV = left ventricular; PVC = premature ventricular complexes; RV = right ventricular; VF = ventricular fibrillation; VT = ventricular tachycardia.

None of the patients carrying double mutations had participated in competitive sports activity. Among the 30 single-mutation carriers, 8 (7 male, 1 female) were engaged in competitive sports. Of these, 1 fulfilled and 2 did not fulfill the diagnostic criteria even if they showed some ARVC/D abnormalities (presence of premature ventricular complexes and minor RV abnormalities, respectively).

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Discussion 

Among 42 ARVC/D index cases screened for mutations in PKP2, DSP, DSG2, DSC2, and JUP genes, 3 (7.1%) carried multiple mutations leading to double or compound heterozygote genotype. Similar observations of patients carrying multiple mutations were reported in hypertrophic cardiomyopathy, where multiple-mutation carriers account approximately for 2.5% to 5% of the total.³⁰, ³¹, ³², ³³

Until now, only 2 compound heterozygous ARVC/D cases were reported in the literature.⁸, ⁹ Both patients carried in trans different mutations in DSG2 gene. Because members of their families carrying a single mutation appeared nonaffected, the question arises of whether such mutations could be considered dominant or recessive. In the present study, mutations detected in family #100 (DSG2 V392I and PKP2 T50SfsX60) and in family #137 (DSP V30M and DSP R2541K) are clearly inherited as dominant, whereas in family #104 the dominant inheritance can be clearly established only for 1 mutation (PKP2 Q62K).

All but 1 variant detected by the present study alter a residue conserved across mammals and cause a change in the amino acid sequence. Only V30M missense mutation affects an amino acid not conserved during evolution; however, in vitro functional studies showed that the N-terminus V30M affects the localization of DSP, probably because of loss of binding to JUP.²⁴ All the above variants were never found in a series of 500 control chromosomes. The Q62K substitution was previously reported in patients carrying an additional mutation in the same gene.²⁷, ²⁹ Van Tintelen et al²⁷ speculated the possibility that the Q62K allele may contribute to pathogenesis of ARVC/D in co-occurrence of another mutant allele. In our study, in family #104 only patients carrying the Q62K substitution with an additional one fulfilled the diagnostic criteria for ARVC/D (I,1; II,5, II,7).

In the 3 families, at least 1 variation could be considered pathogenic (in family #137 DSP V30M, in family #100 PKP2 T50SfsX60, and in family #104 PKP2 Q707X) because of functional data or mutation type. However, detected pathogenic mutations do not segregate with ARVC/D phenotype in these families. Thus, another mutation in a known or still unknown ARVC/D gene should be involved. Additional missense variations have been detected in desmosomal encoding genes and in 2 of the present families, each of the 2 distinct mutations was detected in affected family members. At present, the possibility that the additional missense variations could represent benign polymorphisms or modifier mutations cannot be ruled out.

Family #100 belongs to a huge family tree in which we previously established significant linkage with 14q24.3.³⁴ Mutation screening of the TGFβ3 gene, considered as the ARVC/D gene mapped to such locus, was negative in family #100, thus indicating that probably the large family was made by different branches characterized by segregation of different mutations.

In family #137, subjects carrying mutation DSP R2541K in a single dose (III,4; III,5) encountered sudden death at age 45 and heart failure at age 64, respectively; patient IV,5 carrying such a mutation and mutation V30M in the same gene presented with heart failure symptoms and signs earlier compared with other affected members of her family. In family #104, among double heterozygotes for DSP and PKP2 mutations, 4 appeared unaffected (II,4; II,9; III,2; III,3) and 1 showed a mild form of the disease (I,1), whereas compound heterozygotes for mutations in the PKP2 gene (II,5; II,7) showed a severe form of the disease with early heart failure onset.

Clinical and instrumental evaluation of different members of family #100 showed that among the 7 adult patients carrying DSG2 mutation, 3 (III,9; IV,2; IV,15) showed a mild form of the disease and 3 (V,1; V,2; V,4; V,5) were unaffected. In the same family, among 6 patients carrying the PKP2 mutation T50SfsX60, 2 were severely affected (IV,21; V,7), whereas the remaining 4 did not fulfill current diagnostic criteria for ARVC/D, although symptomatic. Thus, in family #100, affected subjects carrying the PKP2 mutation showed a more severe form than patients carrying the DSG2 mutation. In the same family, the presence of double mutations in 2 subjects (IV,13; IV,16) seems not associated with a severe phenotype: 1 subject (IV,16) shows a moderate form with nonsustained VT, whereas the other (IV,13), 58 years old, does not show clinical signs and symptoms of the disease. It is noteworthy that despite the wide clinical spectrum of subjects with multiple mutations, ranging from healthy carriers to severe forms, comparison of clinical features between subjects carrying single and those carrying multiple mutations showed in the latter a more severe phenotype in terms of LV (P = .025) and RV dilatation (trend toward statistical significance P = .051). This is similar to what has been described in patients with hypertrophic cardiomyopathy.³⁰, ³²

We know that homozygous mutations of DSP underlie an autosomal recessive cardiocutaneous syndrome characterized by ARVC/D, keratoderma, and woolly hair.¹⁶, ³⁵, ³⁶ All cases of compound heterozygotes for DSP mutations reported so far invariably showed keratoderma without cardiac involvement.³⁷ On the contrary, our case (family #137, patient IV,5) carrying 2 DSP mutations did not show palmoplantar keratoderma or woolly hair.

Variability in clinical expression of the same mutation, alone or in combination with additional mutations, is currently reported as caused by reduced penetrance. This old concept corresponds to individual differences in manifestation of a given trait, caused by differences in genetic background such as modifier genes or to environmental factors. In our case, the hypothesis of environment factors on clinical manifestation cannot be dismissed. We previously hypothesized that a genetically impaired response to mechanical stress caused by desmosomal defects would mostly selectively affect the RV because of its distensibility in comparison with the thicker LV free wall. Therefore, we expect that the more intensively a given subject is involved in physical activity in the teen years, the heavier the workload would be experienced by the heart, and in particular, by the RV free wall, and the earlier would be the manifestation of the disease. Nonetheless, the higher severity of the disease expression in multiple-mutation than in single-mutation carriers cannot be ascribed to sports performance, because none of the former group was involved in competitive sports.

On the other hand, evaluation of penetrance in a given family is difficult because the majority of members usually available to the study are young and those who are asymptomatic now may later develop clinical signs of the disease.

The observation that compound heterozygotes are not infrequent among ARVC/D probands suggests that potentially pathogenic variants might recur in the general population at a higher rate than previously assumed. This is not surprising, because we know that some pathogenic mutations may show either mild or late manifestation in some individuals. Difficulty in estimating penetrance and prevalence of given mutations makes it almost impossible to give a reliable prognosis even when the presence of one or more pathogenic mutations was established.

According to evidence provided by the present study, gene mutation screening should not be stopped after identification of a single mutation, but should be continued on the same gene and performed on at least the 3 major desmosomal ARVC/D genes (PKP2, DSP, and DSG2). Moreover, before genetic counseling and before attempting to establish genotype-phenotype correlations, the genetic status of each family member should be fully investigated to provide better genetic counseling to those families.

Because of the limited number of consecutive index cases screened for mutations in ARVC/D genes, further clarification of the proportion of patients with multiple mutations and of genotype-phenotype correlation is mandatory to understand clinical heterogeneity of ARVC/D patients and for prognostic purposes.

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Acknowledgements 

The authors thank Paola Marcon for her help in collecting ARVC/D families' data.

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