Abstract

INTRODUCTION

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC) has been defined as a primary right ventricular cardiomyopathy initially described by Marcus, Fontaine and colleagues in the early 1980's.¹ It is characterized by fibrofatty infiltration of the right ventricular (RV) myocardium and clinically presents with ventricular arrhythmias, heart failure, syncope, and sudden death.^1,2 The left ventricle (LV) may also be affected.³ In familial cases of ARVC, autosomal dominant inheritance with reduced penetrance has been reported and is believed to account for approximately 30% of cases.⁴ In the remaining sporadic cases, the etiology may be an acquired cause such as myocarditis^5,6 or an unidentified inherited disorder. To date, multiple genetic loci and 7 genes including desmoplakin (DSP),⁷ plakophilin-2 (PKP2),⁸ desmoglein-2 (DSG2),⁹ desmocollin-2 (DSC2), ¹⁰ transforming growth factor β3 (TGF β3),¹¹ ryanodine receptor 2 (RYR2)¹², and Transmenbrane Protein-43 (TMEM43) ¹³ have been identified in ARVC patients. In addition, two complex cardiocutaneous disorders with autosomal recessive inheritance in which cardiomyopathy is associated with woolly hair and palmoplantar keratoderma have been reported. These include Naxos disease with ARVC¹⁴ and Carvajal syndrome associated with an LV cardiomyopathy.¹⁵ The genes identified for these disorders include homozygous mutations in junctional plakoglobin (JUP) in Naxos disease,¹⁶ as well as homozygous mutations in DSP in Carvajal syndrome.¹⁷ The most common gene variants identified in ARVC is PKP2, initially reported by Gerull and colleagues to be mutated in ~25% of patients with autosomal dominant inherited disease.⁸ In the analysis of PKP2, an essential armadillo-repeat protein of the cardiac desmosome, they identified heterozygous mutations in 32 of 120 unrelated individuals with ARVC. ⁸ Other investigations have confirmed these findings.^18-21 The majority of genes identified to date causing ARVC encode desmosomal proteins. Within cardiomyocytes, two types of cell adhesion junctions are responsible for intercellular adhesion: the desmosomes and fascia adherens junctions.²² These cell adhesion junctions are both located at the cardiomyocyte intercalated disk and both contain intracellular proteins that link the cytoplasmic domains of cadherins to components of the cytoskeleton. In cardiomyocytes, a variety of proteins interact to form functional cell-cell junctions. DSGs and DSCs are connected to the desmin cytoskeleton by DSP, JUP (γ-catenin), and PKP2. These latter proteins, JUP and PKP2, are members of the armadillo family of nuclear and junctional proteins.^{23, 24} PKP-2 interacts with DSP, DSG and intermediate filament proteins at sites within its N-terminus. DSP and PKP2 are located only in desmosomes, whereas JUP participates as a linker in both desmosomes and adherens junctions. The adherens junctions are located at the ends of sarcomeres and are linked to sarcomeric actin through intracellular linker proteins, most notably members of the catenin family including JUP, β-catenin, αcatenin and p120 catenin.

In this study, we analyzed probands and family members for ARVC using a standardized clinical protocol either developed as part of the North American ARVD Registry²⁵ or using the standard Task Force criteria (Table 1).²⁶ All individuals were screened for mutations in all of genes encoding proteins involved in desmosomal function, even if a variant were already identified in any of these desmosome-encoding genes. We report identification of multiple mutations in these genes, including autosomal dominant heterozygous mutations in 26% of subjects (52/198), including 38 in PKP2 and 14 in other desmosome-encoding genes.

Additionally, compound heterozygous mutations and digenic mutations were identified in 42% of the subjects (16/38) in whom PKP2 mutations were identified. In addition, we demonstrate that many PKP2 mutations have low penetrance and in many cases may not be the primary cause of the disease, contradicting the previously reported contention that PKP2 is the major ARVC causing gene, accounting for the cause of disease in 25% of ARVC patients.

METHODS

RESULTS

GENETIC ANALYSIS: PLAKOPHILIN-2 MUTATIONS AND COMPOUND HETEROZYOSITY

After informed consent, 198 probands (143 North American, 55 Italian) meeting clinical criteria for the diagnosis of ARVC using the Task Force criteria (Table 1) and/or ARVD Registry criteria were enrolled in the genetic analysis study. All subjects were screened for mutations in all desmosome-encoding genes, with complete sequencing of the entire gene performed in all cases. 21 variants in PKP2 were identified in 38 of the 198 probands (19%), a frequency similar to previous reports.^{8, 18-21} The variants identified included 8 missense, 4 nonsense, 2 splice site mutations, and 8 deletion/insertion mutations; the deletion/insertion variants each predict a protein frame shift (Table 2). Several variants were identified in multiple probands, including nonsense and frame-shift variants (Table 2). All of the missense substitutions resulted in changes at residues that are conserved between species (Supplemental Figure 1). Among the variants identified, the splice site substitution detected in intron 10, and the 2509delA in exon 13 have been previously reported.⁸ The remaining variants are novel. Despite the fact that several of these variants were identified in multiple individuals, analysis of 700 ethnically matched control individuals (1,400 chromosomes) identified only one of these variants (A372P) in the control population (1 in 700).

Table 2

PKP2 gene variants identified in patients with ARVC

NT change *	Exon	AA Change**	US Probands	Italian Probands
145_148delCAGA	1	S50fsX110	4	3
419C>T	3	S140F	1	0
627C>G	3	S209R	0	1
630C>T	3	Q211X	1	1
1114G>C	4	A372P	3	0
1162C>T	4	R388W	1	0
1170-2A>G		Intron 4	1	0
1212insT	5	L404fsX409	1	0
1368delA	5	K456fsX458	1	0
1592T>G	7	I531S	3	0
1613G>A	7	W538X	2	0
1643delG	7	G548fsX562	0	1
1760G>A	8	V587I	0	1
1978C>T	10	Q660X	1	0
2009delC	10	N670fsX683	0	1
2119C>T	10	Q707X	0	1
2146-1G>C		Intron 10	3	0
2197_2202insGdelCACACC	11	H733fsX740	1	0
2359C>T	12	L787F	1	0
2447_2448delCC	12	T816fsX825	0	2
2509delA	13	V837fsX930	3	0

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^*NT change: Nucleotide change;

^**AA change: Amino acid change.

Analysis of the family pedigrees shows that there is significantly reduced penetrance of these PKP2 variants, similar to that reported by Gerull et al. (Figure 1A and B).⁸ However, in 9 of the probands with PKP2 variants, we identified 2 distinct PKP2 variants (Table 3, patients 5, 7, 8, 11, 12, 14, 15, 16 and 17), consistent with compound heterozygosity. One of these probands (patient 5, Table 3; Figure 1C) was a member of a family with a history of ARVC in his generation (Figure 1C). DNA was available from the living affected subject (patient 6, Table 3; Figure 1C) and unaffected siblings of the proband, as well as the phenotypically normal parents, paternal uncle and paternal grandparents. All family members were clinically evaluated using the Task Force criteria (Table 1). Only the clinically affected brother carried both variants (Figure 1C). The parents were carriers of individual PKP2 variants and the clinically unaffected (not meeting Task Force criteria) sibling hosted only the variant transmitted by his father and paternal grandfather (Figure 1C). RT-PCR of PKP2 mRNA was performed using samples obtained from five other affected probands and sequencing revealed that in each case the variants were encoded in trans. Thus, these variants were either inherited independently from the phenotypically normal patients or were de novo. As noted in Table 3, disparities between the clinical phenotypes and genetic findings appear somewhat common. Notably, all subjects had RV involvement, several had biventricular disease, and all had associated arrhythmias. Importantly, not all subjects met full Task Force Criteria, thereby pointing to imperfections in these criteria and necessitating modifications of these criteria (Marcus et al, Circulation, in press) ³⁰.

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Figure 1

Pedigrees of three families with PKP2 variants and incomplete penetrance

Panel A: L404fsX409 PKP2 was identified in the proband and unaffected sister and was inherited from the unaffected mother (+; in blue). In addition, a de novo variant, DSP Q90R, was identified in the proband (). The arrow indicates the proband. Panel B: K456NfsX458 was identified in the proband and inherited from the unaffected mother (+). The arrow indicates the proband. NA: Not available for genetic analysis. Panel C: Pedigree of a family with compound heterozygous PKP2 variants. V837fsX930 (+) was identified in the proband and both of his siblings and was inherited from the unaffected father, who in turn inherited it from his unaffected father. R388W () was identified in the proband and his affected brother and was inherited from his phenotypically normal mother. The arrow indicates the proband. NA: Not available for genetic analysis.

Table 3

Clinical Demographics of ARVC Subjects with Desmosomal Gene Mutations. Note that most, but not all subjects with mutations meet full Task Force Diagnostic Criteria. All subjects have right ventricular involvement and arrhythmias and several subjects also have left ventricular involvement.

Pt	Gender	Age onset	FHX	Affected parents	Nationality	Palpitations	Syncope / SCD	CHF/ NYHA class	Arrhythmia	ECG abnl	SAECG abnl	ICD	Transplant	Exer VT	RV inyvol	LV invol	MRI abn	DX Criteria M/m	Histology	gene	Nt Change	aa Change
1	F	25	N	No	US	Yes	No/No	No/I	VTapex RVOT	Yes	Yes	No	No	No	Yes	No	Yes	Yes Yes	Yes	PKP2 DSP	1212insT 269A>G	L404fsX409 Q90R
2	F	22	Y	No	US	Yes	Yes/No	No/I	VTapex RVOT	Yes	Yes	Yes	No	No	Yes	No	Yes	Yes Yes	Yes	PKP2 DSP	1592T>G 620G>A	I531S W207X
3	F	23	Y	No	US	Yes	Yes/No	No/I	VT RVOT	Yes	Yes	Yes	No	Yes	Yes	Yes	Yes	Yes Yes	Yes	PKP2 DSG2	1592T>G 146G>A	I531S R49H
4	M	29	N	No	US	Yes	No/No	No/I	VT apex	Yes	Yes	No	No	Yes	Yes	No	Yes	Yes Yes	No	PKP2 DSG2	145_148delCAGA 1003A>G	S50fsX110 T335A
5* 6	M	16	Y	No	US	Yes	Yes/No	No/I	VT RVOT	Yes	Yes	Yes	No	No	Yes	No	Yes	Yes Yes	Yes	PKP2 PKP2	1162C>T 2509delA	R388W V837fsX930
	M	12	Y		US	Yes	Yes/No	No/I	VT RVOT	Yes	Yes	Yes	No	No	Yes	No	Yes	Yes Yes	Yes
7	M	32	Y	No	US	Yes	Yes/No	No/I	VT RVOT apex	Yes	Yes	Yes	Yes	Yes	Yes	No	Yes	Yes Yes	Yes	PKP2 PKP2	145_148delCAGA 1592T>G	S50fsX110 I531S
8	M	52	N	No	US	Yes	No/No	No/I	VT RVOT	Yes	Yes	Yes	No	No	Yes	Yes	Yes	Yes Yes	No	PKP2 PKP2	1592T>G 2359C>T	I531S L787F
9** 10	M	26	Y	No	US	Yes	No/No	No/I	VT RVOT	Yes	Yes	Yes	No	Yes	Yes	No	Yes	Yes Yes	Yes	PKP2 DSC2	1613G>A 1914G>C	W538X Q63SH
	F	25	Y		US	Yes	No/No	No/I	VT RVOT	Yes	No	Yes	No	No	Yes	No	Yes	No Yes	Yes
11	F	30	N	No	US	Yes	No/No	No/I	NSVT	Yes	No	Yes	No	Yes	Yes	No	Yes	No Yes	No	PKP2 PKP2 DSP	1613G>A 1914G>C 88G>A	W538X Q638H V30M
12	M	41	N	No	US	Yes	Yes/No	No/I	VT	Yes	Yes	Yes	No	Yes	Yes	No	Yes	Yes No	No	PKP2 PKP2 DSP	1114G>C 2145-1G>C 4609C>T	A372P R1537C
13	M	30	Y	No	US	Yes	Yes/No	No/I	VT	No	Yes	Yes	Yes	Yes	Yes	No	Yes	Yes No	Yes	PKP2 PKP4	2146-1G>C 2786A>G	D929G
14	M	50	Y	No	US	Yes	Yes/Yes	No/I	VT-RV origin	Yes	Yes	Yes	No	Yes	Yes	No	Yes	Yes Yes	Yes	PKP2 PKP2 DSG2	419C>T 2146-1G>C 166 G>A	S140F V56M
15	M	44	Y	No	Italian	Yes	Yes/Yes	No/I	VF	Yes	Yes	Yes	No	No	Yes	No	Not perf	1/3	No	PKP2 PKP2 DSG2	627C>G 2447_2448delCC 437G>A	S209R T816fsX825 R146H
16	M	40	Y	No	Italian	Yes	Yes/Yes	No/I	VF	Yes	No	Yes	No	Yes	Yes	No	Yes	1/3	No	PKP2 PKP2 DSP	630C>T 2333T>C 3764G>A	Q211X I778T R1255K
17	F	31	Y	No	Italian	Yes	No/No	No/I	NSVT	Yes	Yes	No	No	No	Yes	Yes	Not perf	1/4	Yes	PKP2 PKP2 DSP	2119C>T 184C>A 4961T>C	Q707X Q62K L1654P
18	M	34	Y	One	Italian	Yes	No/No	No/I	NSVT	Yes	Yes	No	No	No	Yes	No	Not perf	1/4	No	PKP2 DSG2	145_148delCAGA 1115G>A	S50fsX110 V391I

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Patient 5 (proband) and 6 are from same family;

^**Patient 9 (proband) and 10 are from same family.

Pt= Patient number; FHx= Family History of ARVC; SCD= Sudden Cardiac Death; CHF=Congestive Heart Failure; NYHA= New York Heart Association; ECG abnl= Electrocardiographic abnormalities; SAECG= Signal Averaged ECG; ICD= Implantable Cardioverter Defibrillator; Dx Criteria= Diagnostic Criteria; M/m= Major/minor criteria; Nt= Nucleotide; aa= Amino Acid

These findings led us to consider the possibility that ARVC may be due to another form of “compound heterozygosity” with mutations/genetic variants in two desmosomal protein encoding genes required for clinical disease, called “digenic heterozygosity”.

GENETIC ANALYSIS: DIGENIC HETEROZYOSITY

In all 198 probands, the desmosome-encoding genes DSP, DSC2, DSG2, JUP, DSC2,DSG2, PKP4, and DES were sequenced in addition to PKP2 sequencing. This sequencing identified 13 variants in second desmosomal genes in 13 subjects with PKP2 variants, including DSP in 6 cases, DSG2 in 5 cases, PKP4 in 1 subject, and DSC2 in 1 subject (Table 3). None of these variants were identified in at least 700 ethnic-matched controls (>1,400 chromosomes). Thus, of the 38 probands with PKP2 variants, compound or digenic heterozygosity was identified in 16 (42%), including 6 probands with 3 variants: in two of these cases, one of the PKP2 variants was the A372P polymorphism identified in the control population. Five probands had additional family members available for clinical and genetic evaluation including the following probands: patient 1 with the Q90R DSP variant (Table 3), the DSP W207X variant (patient 2, Table 3), the DSG2 V56M variant (patient 14, Table 3), the DSG2 R146H variant (patient 15, Table 3) and the DSP R1255K variant (patient 16, Table 3). The DSP Q90R variant was a de novo substitution and therefore could be pathogenic alone or in combination with the PKP2 variant (Figure 1A). In patient 12, a 4609C>T DSP substitution (R1537C) was also identified, a variant previously reported as a single nucleotide polymorphism (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?type=rs&rs=rs28763967). Whether this variant becomes clinically relevant in combination with the PKP2 variants and is needed for the development of clinical features is speculation until animal models are completed. In the family of Patient 2 (Figure 2A), the PKP2 I531S variant was identified in three of the four siblings available for study. All family members were clinically evaluated using the Task Force criteria (Table 1) and one of the three siblings was clinically unaffected (and also has twin carrier daughters) while the other two siblings were clinically affected. Only the proband hosted the second variant, DSP W207X. In this case, we propose three potential interpretations of these data. Since the proband had the most severe phenotype (sudden death at age 25 years and severe ARVC on autopsy), it is possible that the DSP W207X mutation was required to manifest severe clinical disease. Secondly, this mutation may have no involvement in the development of disease and another, as yet identified mutation, is carried by the affected siblings but not by the unaffected sibling. Thirdly, the I531S mutation may be disease-causing alone with extremely reduced penetrance, as has been described in other forms of cardiomyopathy, and requires other factors, such as acquired agents, to manifest and contribute to clinical expression. In this regard, it should be noted that the proband developed mononucleosis two years prior to death.

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Figure 2

Pedigree of two U.S. families with compound desmosomal mutations (digenic heterozygosity)

Panel A: PKP2 I531S (+) was identified in the proband and two of her siblings. DSP W207X () was identified in the proband who experienced sudden death at 25 years of age. Her affected living brother was diagnosed by cardiac MRI and is still alive at the age of 42 years. Her unaffected sister carrying the I531S variant is now 51years of age and has twin unaffected daughters, aged 19 years, both of whom carry the variant. Individuals without a definitive diagnosis of ARVC, but in whom signs or symptoms compatible with this diagnosis were reported, are identified in blue. The arrow indicates the proband. Panel B: PKP2 S140F (+), PKP2 IVS10-1G>C () and DSG2 V56M (♣) were detected in the proband. The oldest child of the proband, who is 19 years old, does not meet full Task Force criteria of ARVC but has arrhythmias and symptoms. The arrow indicates the proband. SD: Sudden death; ABN Echo: Abnormal echocardiogram; CHF: Congestive Heart Failure; PM: Pacemaker

As previously noted, Patient 14 hosted a DSG2 V56M variant; in addition, however, this subject also had two additional PKP2 variants (Figure 2B). Based upon evaluation of the pedigree, it is difficult to be certain as to the role of each of these variants; however, it appears that the combination of PKP2 S140F and DSG2 V56M, which was only detected in the proband, led to a more severe clinical phenotype (Figure 2B).

Patient 15, a 51 year old male, was diagnosed with a severe form of ARVC at the age of 39 years after an episode of ventricular fibrillation. He was found to host three different variants (PKP2 S209R, PKP2 T816fsX825 and DSG2 R146H) (Figure 3A). The PKP2 T816fsX825 was inherited from his mother (II,2). Since his father's DNA was not available, it was not possible to establish if the two missense variations (PKP2 S209R and DSG2 R146H) were inherited from the father or are de novo mutations. His mother and maternal aunt, who both carried the PKP2 frameshift variant, were clinically unaffected by Task Force criteria and asymptomatic.

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Figure 3

Pedigree of two Italian families with compound desmosomal mutations

Panel A: PKP2 T816fsX825 () was identified in the proband and two family members. PKP2 S209R (+) and DSG2 R146H (♣) were also identified in the proband. Panel B: PKP2 Q211X (+), PKP2 I778T (♣) and DSP R1255K () were detected in the proband. All family members carrying different mutations were completely asymptomatic. The probands are indicated by the arrows. SD: Sudden death; aSD: aborted sudden death.

Patient 16, a 40 year old male, was diagnosed with a severe form of ARVC after an episode of ventricular fibrillation that occurred during a sports activity. After resuscitation, he received an implantable cardioverter defibrillator (ICD). His family history included a male cousin who died suddenly at 30 years of age while hiking. No autopsy was performed. The proband (II,1) inherited the DSP missense mutation and PKP2 stop mutation from his mother (I,2), and the missense PKP2 mutation from the father (I,1) (Figure 3B). At least one mutation was also detected in the proband's sons. In this family, all mutation carriers, with the exception of the proband, were completely asymptomatic and clinically unaffected by Task Force criteria.

Overall, there was more VF and exercise-induced VT in those subjects having compound or digenic heterozygosity compared to those subjects with single heterozygous PKP2 mutations. In addition, the age of onset of symptomatic ARVC, most commonly ventricular arrhythmias or syncope, was earlier in those subjects with multiple genetic variants in PKP2 or in PKP2 plus other desmosomal genes (mean age 31.5 years) compared to those subjects with single heterozygous gene mutations (mean age 39.5 years). Therefore, it appears that compound and digenic heterozygosity leads to more clinically apparent and severe ARVC with an earlier age of onset than those hosting heterozygous mutations in these genes.

FUNCTIONAL ANALYSIS

Myocardial autopsy samples were available from two individuals with PKP2 variants, including one patient (PG) with a single heterozygous PKP2 W528X nonsense mutation (no second variant has been identified to date) and a second patient (M686) with digenic heterozygosity including the PKP2 I531S variant as well as a DSP W207X mutation. These myocardial samples were sectioned and stained for a variety of junctional proteins, including PKP2, DSP, JUP, N-cadherin, and DSC2/3, as well as for Cx43 and DES (Figure 4). Staining of samples from patient PG was very weak or absent for each of these proteins except for DSP, which was normal. In contrast, staining of samples from patient M686 was essentially normal for each antibody, except for Cx43, which was absent (Figure 4), and DSP, which appeared to stain weakly. These data suggest that the DSP nonsense mutation in patient M686 does not effect DSP localization and that these variants do not grossly affect the desmosomal junctions. In contrast, both the desmosomal and cadherin junctions appear to be affected in patient PG, with loss of staining for N-cadherin and JUP, in addition to PKP2. Signal intensities were comparable in samples obtained from both left ventricle and right ventricle and in areas relatively unaffected by fibrofatty replacement.

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Figure 4

Immunostaining of myocardial sections from a control heart and from the hearts of ARVC patients PG (PKP2 W538X) and M686 (PKP2 I531S and DSP W207X)

The panels show staining for PKP2 (top), DSP (middle), and CX43 (bottom). Bars = 20μm

DISCUSSION

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has emerged as a significant cause of sudden death, heart failure and the need for heart transplantation over the past decade.^{2, 31}The incidence was initially thought to be particularly high in the Veneto region of Italy and in other parts of Europe, but low elsewhere.² More recently, the disease has been increasingly recognized throughout the world.^3,32,33 It is believed to be inherited in a moderate percentage of cases, with autosomal dominant inheritance predominating.⁴ Multiple genes, mostly those encoding desmosomal proteins, have been identified as causative in ARVC, with PKP2 reported to be responsible for approximately 25% of all cases.^{8, 18-21} In the work presented herein, however, interpretation of these data are questioned and the concepts of low penetrance, as well as compound and digenic heterozygosity are proposed as potential determinants of the clinical presentation in subjects carrying mutations and in their family members. In addition, identification of novel heterozygous mutations in other desmosome-encoding genes, including novel mutations in JUP, further supports the notion that the “final common pathway” for ARVC is the cell-cell junctions. ³⁴

In this report, we demonstrate that, while variants in PKP2 are relatively common (identified in 38/198 or 19% of probands), harboring one PKP2 variant may not by itself be sufficient to determine overt clinical disease. In at least 16/38 (42%) cases, concomitant causes such as either a “second hit” in the same gene (compound heterozygosity) or in a second desmosome-encoding gene (digenic heterozygosity) or an acquired disruption of these proteins or environmental factors, has been shown to be required for the overt clinical phenotype to develop or for modification of disease severity. Hence, while PKP2 “mutations” are relatively common, a second variant in PKP2 or in DSP, DSC2, DSG2, PKP4, JUP or other interacting junction protein-encoding gene appears to be important for the disease and its clinical consequences to be manifest. Other “second hit” genes or interactors are likely to be discovered in the future. In addition, a variety of heterozygous mutations in all of the known desmosome-encoding genes were identified to cause ARVC and its associated clinical signs and symptoms.

Interestingly, the subjects harboring more than one variant in PKP2 and/or PKP2 plus other desmosome-encoding genes appear to have earlier onset of disease and more clinical severity than those individuals harboring heterozygous mutations alone. Interpretation of these data, however, is confounded by the small number of large pedigrees available for analysis. In order to track segregation of the genotype with disease, large families with clinical ARVC are required but, in our study, small families and sporadic cases were predominantly seen. However, in the families in which multiple individuals were enrolled and genetically screened, a single genetic variant commonly did not lead to overt clinical disease using the Task Force criteria (Table 1) or the stringent diagnostic evaluation used by the ARVD Registry. It will be interesting to determine if the carriers previously phenotyped as unaffected will be found to have any signs of early ARVC upon detailed examination (MRI, echocardiogram, etc) or whether later development of clinical signs due to an otherwise concealed pathological process will occur over time. These genetic findings should encourage physicians caring for patients with ARVC to screen as many families as possible, irrespective of clinical presentation. Clearly, longitudinal follow-up of patients and carrier family members will be critical in determining the influence of these gene variants and, more importantly, will be critical in providing excellent preventive care. As noted, we have only identified second variants in about 40% of the probands in whom PKP2 variants were identified. What about the remainder? Firstly, many genes that encode proteins that either directly or indirectly contributes to the function and integrity of cell adhesion junctions remain to be screened, such as the genes encoding α- or β-catenin. Mutations in proteins of the myocyte cytoarchitecture which are linked to these proteins, such as actin, α-actinin-2, metavinculin, or Z-disk proteins could be important and will also be evaluated. Secondly, some mutations could result in dominant negative proteins giving rise to autosomal dominant inheritance, the mode of inheritance widely held as the most common in patients suffering from ARVC. In addition to the compound heterozygous and digenic mutations noted, we also identified apparent isolated heterozygous mutations in desmosome-encoding genes in another 7% of subjects. We would speculate that other relevant genes have yet to be discovered. An interesting observation from these studies is that nonsense or frame-shift mutations alone in PKP2 often do not result in clinical disease in these families. These data would suggest that haploinsufficiency for PKP2 is not critically important or even sufficient or that compensatory mechanisms occur. Only when the remaining copy of PKP2 is mutated or there is a mutation in another gene, does overt clinical disease develop. It also suggests that the description of these genetic variants as “disease-causing mutations” may be inaccurate and that defining affectation status by genetic analysis alone may not be entirely appropriate. For instance, on face value, the I531S variant would appear to be a polymorphism. However, the detection of this variant in 5 of the 143 probands (from different regions of the U.S.), but in none of the 700 controls, would suggest that this variant is linked to disease. On the other hand, nonsense mutations in PKP2 would be expected to result in overt clinical disease but, in many cases, does not. For this reason, we would caution potential “fee-for-service” laboratories offering “clinical testing” for ARVC from definitive statements regarding cause-and effect relationships, particularly in subjects in whom PKP2 variants are identified, especially if the remaining genes are not screened. In this circumstance, not only is the affected subject at risk to have a mistaken causative gene assigned, but “at-risk” family members may be either mistakenly diagnosed with a causative mutation or, more concerning, be given a negative result. In the latter case, this could lead to discharge from follow-up despite actually carrying a disease causing mutation in another gene not analyzed. Thia could lead to tragic outcomes. Grossman et al.³⁵ ablated the PKP2 gene in mice, causing lethal alteration in heart morphogenesis at mid-gestation characterization by reduced trabeculations, disarrayed cytoskeleton, rupture of the cardiac wall, and hemopericardium. In the absence of PKP2, the cytoskeletal linker protein desmoplakin dissociates from the junctional plaques that connect cardiomyocytes, resulting in reduced architectural stability of intercalated disks. Constant mechanical stress, as seen in the contracting heart, is likely to be deleterious to the weakened intercellular junctions, leading to disruption, dilation and dysfunction. However, heterozygous mice were healthy and fertile. This is consistent with our notion that haploinsufficiency for PKP2 is not sufficient to cause disease. The right ventricle, based on its geometry, architecture, and role in cardiac function, dilates to a variety of volume and pressure abnormalities and would likely develop structural and functional disease earlier than the left ventricle. This was clearly shown in the DSP transgenic mouse model of Yang et al, who demonstrated early right ventricular dilation with later left ventricular dilation, as well as intercalated disk disruption and loss of desmosomes.²⁹ In addition, further support is provided by the fact that the left ventricle develops late-onset disease in some patients with ARVC.^{3, 36} In addition to the genetic mutations, mechanical stress and stretch forces on the disturbed desmosome and intercalated disk, likely plays a role in disease development and severity.^34,37

Together, the genetic and functional data provided here should help to define the nature of the genetic and clinical basis of ARVC. This work could significantly impact on clinical genetic screening, as simple single gene analysis (particularly for PKP2) would be inappropriate and the potential for inaccurate interpretation based on single (or even multiple) gene analysis appears to be high. More detailed information regarding the true clinical relevance of PKP2 mutations is needed. Further, the moderate number of mutations in the other desmosome-encoding genes strongly suggests that all genes in this pathway should be screened in all subjects. Identification of heterozygous mutations in JUP, as well as the potential mutation within the PKP4 gene adds to the spectrum of affected genes involving the desmosome. Further studies of PKP4 in subjects with ARVC, as well as functional analysis of models with mutant PKP4 will be needed to clearly state that this is a potential disease-causing gene in ARVC.

The lack of functional studies of the variants described is a limitation of the study. Cellular and animal models incorporating these mutations individually and together could help to resolve these issues to some extent but these approaches may not necessarily recapitulate the human condition. In attempt to better understand the mechanisms involved in the development (or lack of development) of the clinical phenotype associated with these variants, we are in the process of developing these models for study. In addition to the models, the addition of mechanical stretch and stress on the cells and animals could facilitate the development of phenotypic differences from non-stressed models and provide greater insight.

Supplementary Material

Footnotes

REFERENCES