Abstract

Arrhythmogenic right ventricular cardiomyopathy (ARVD/C) is a genetically heterogeneous disease characterized by progressive degeneration of the right ventricular myocardium and increased risk of sudden death. Here, we report on a genome scan in one Italian family in which the disease appeared unlinked to any of the six different ARVD loci reported so far; we identify a mutation (S299R) in exon 7 of desmoplakin (DSP), which modifies a putative phosphorylation site in the N-terminal domain binding plakoglobin. It is interesting that a nonsense DSP mutation was reported elsewhere in the literature, inherited as a recessive trait and causing a biventricular dilative cardiomyopathy associated with palmoplantar keratoderma and woolly hairs. Therefore, different DSP mutations might produce different clinical phenotypes, with different modes of inheritance. Arrhythmogenic right ventricular cardiomyopathy (ARVD/C) is a genetically heterogeneous disease characterized by progressive degeneration of the right ventricular myocardium and increased risk of sudden death. Here, we report on a genome scan in one Italian family in which the disease appeared unlinked to any of the six different ARVD loci reported so far; we identify a mutation (S299R) in exon 7 of desmoplakin (DSP), which modifies a putative phosphorylation site in the N-terminal domain binding plakoglobin. It is interesting that a nonsense DSP mutation was reported elsewhere in the literature, inherited as a recessive trait and causing a biventricular dilative cardiomyopathy associated with palmoplantar keratoderma and woolly hairs. Therefore, different DSP mutations might produce different clinical phenotypes, with different modes of inheritance. Arrhythmogenic right ventricular cardiomyopathy (ARVD/C) is a progressive disease characterized by degeneration of right ventricular myocardium, followed by fibrous-fatty replacement (Thiene et al. Thiene et al., 1988Thiene G Nava A Corrado D Rossi L Pennelli N Right ventricular cardiomyopathy and sudden death in young people.N Engl J Med. 1988; 318: 129-133Crossref PubMed Scopus (1225) Google Scholar). Myocardial degeneration may extend to left ventricle (Gallo et al. Gallo et al., 1992Gallo P D’Amati G Pelliccia F Pathologic evidence of extensive left ventricular involvement in arrhythmogenic right ventricular cardiomyopathy.Hum Pathol. 1992; 23: 948-952Abstract Full Text PDF PubMed Scopus (71) Google Scholar; Pinamonti et al. Pinamonti et al., 1992Pinamonti B Sinagra G Salvi A Di Lenarda A Morgera T Silvestri F Bussani R Camerini F Left ventricular involvement in right ventricular dysplasia.Am Heart J. 1992; 123: 711-724Abstract Full Text PDF PubMed Scopus (132) Google Scholar; Basso et al. Basso et al., 1996Basso C Thiene G Corrado D Angelini A Nava A Valente M Arrhythmogenic right ventricular cardiomyopathy: dysplasia, dystrophy or myocarditis?.Circulation. 1996; 94: 983-991Crossref PubMed Scopus (660) Google Scholar) and, less frequently, to interventricular septum (Basso et al. Basso et al., 1996Basso C Thiene G Corrado D Angelini A Nava A Valente M Arrhythmogenic right ventricular cardiomyopathy: dysplasia, dystrophy or myocarditis?.Circulation. 1996; 94: 983-991Crossref PubMed Scopus (660) Google Scholar). ARVD/C is usually clinically present with arrhythmias of right ventricular origin, ranging from isolated premature ventricular beats to sustained ventricular tachycardia or to ventricular fibrillation leading to sudden death (Marcus et al. Marcus et al., 1982Marcus FI Fontaine GH Guiraudon G Frank R Laurenceau JL Malergue C Grosgogeat Y Right ventricular dysplasia: a report of 24 adult cases.Circulation. 1982; 65: 384-398Crossref PubMed Scopus (1216) Google Scholar). ARVD/C is the most common cause of sudden cardiac death in juveniles in northeast Italy (Corrado et al. Corrado et al., 1990Corrado D Thiene G Nava A Rossi L Pennelli N Sudden death in young competitive athletes: clinicopathological correlations in 22 cases.Am J Med. 1990; 89: 588-596Abstract Full Text PDF PubMed Scopus (460) Google Scholar). Seven dominant forms of ARVD/C were identified so far: ARVD1 (14q24.3) (Rampazzo et al. Rampazzo et al., 1994Rampazzo A Nava A Danieli GA Buja G Daliento L Fasoli G Scognamiglio R Corrado D Thiene G The gene for arrhythmogenic right ventricular cardiomyopathy maps to chromosome 14q23-q24.Hum Mol Genet. 1994; 3: 959-962Crossref PubMed Scopus (279) Google Scholar [MIM 107970]), ARVD2 (1q42) (Rampazzo et al. Rampazzo et al., 1995Rampazzo A Nava A Erne P Eberhard M Vian E Slomp P Tiso N Thiene G Danieli GA A new locus for arrhythmogenic right ventricular cardiomyopathy (ARVD2) maps to chromosome 1q42-q43.Hum Mol Genet. 1995; 4: 2151-2154Crossref PubMed Scopus (191) Google Scholar [MIM 600996]), ARVD3 (14q11-q12) (Severini et al. Severini et al., 1996Severini GM Krajinovic M Pinamonti B Sinagra G Fioretti P Brunazzi MC Falaschi A Camerini F Giacca M Mestroni L A new locus for arrhythmogenic right ventricular dysplasia on the long arm of chromosome 14.Genomics. 1996; 31: 193-200Crossref PubMed Scopus (160) Google Scholar [MIM 602086]), ARVD4 (2q32) (Rampazzo et al. Rampazzo et al., 1997Rampazzo A Nava A Miorin M Fonderico P Pope B Tiso N Livolsi B Zimbello R Thiene G Danieli GA ARVD4, a new locus for arrhythmogenic right ventricular cardiomyopathy, maps to chromosome 2 long arm.Genomics. 1997; 45: 259-263Crossref PubMed Scopus (146) Google Scholar [MIM 602087]), ARVD5 (3p23) (Ahmad et al. Ahmad et al., 1998Ahmad F Li D Karibe A Gonzalez O Tapscott T Hill R Weilbaecher D Blackie P Furey M Gardner M Bachinski LL Roberts R Localization of a gene responsible for arrhythmogenic right ventricular dysplasia to chromosome 3p23.Circulation. 1998; 98: 2791-2795Crossref PubMed Scopus (167) Google Scholar), ARVD6 (10p12-p14) (Li et al. Li et al., 2000Li D Ahmad F Gardner MJ Weilbaecher D Hill R Karibe A Gonzalez O Tapscott T Sharratt GP Bachinski LL Roberts R The locus of a novel gene responsible for arrhythmogenic right-ventricular dysplasia characterized by early onset and high penetrance maps to chromosome 10p12-p14.Am J Hum Genet. 2000; 66: 148-156Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar [MIM 602087]), and ARVD7 (10q22) (Melberg et al. Melberg et al., 1999Melberg A Oldfors A Blomstrom-Lundqvist C Stalberg E Carlsson B Larsson E Lidell C Eeg-Olofsson KE Wikstrom G Henriksson KG Dahl N Autosomal dominant myofibrillar myopathy with arrhythmogenic right ventricular cardiomyopathy linked to chromosome 10q.Ann Neurol. 1999; 4: 684-692Crossref Scopus (112) Google Scholar). Among them, only the gene involved in ARVD2 has been identified to date (Tiso et al. Tiso et al., 2001Tiso N Stephan DA Nava A Bagattin A Devaney JM Stanchi F Larderet G Brahmbhatt B Brown K Bauce B Muriago M Basso C Thiene G Danieli GA Rampazzo A Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2).Hum Mol Genet. 2001; 10: 189-194Crossref PubMed Scopus (665) Google Scholar). The only known autosomal recessive form of ARVD/C is associated with palmoplantar keratoderma and peculiar woolly hairs (Naxos syndrome [MIM 601214]), caused by a homozygous 2-nucleotide deletion in the plakoglobin (JUP) gene, the product of which is a key component of desmosomes and adherens junctions (McKoy et al. McKoy et al., 2000McKoy G Protonotarios N Crosby A Tsatsopoulou A Anastasakis A Coonar A Norman M Baboonian C Jeffery S McKenna WJ Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease).Lancet. 2000; 355: 2119-2124Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). Here, we report on the identification of a causative mutation in one family, in which disease was inherited unlinked to any of the different ARVD/C loci reported so far. The index case of the present study (subject III,9) was a young male who, at 18 years and during physical effort, experienced a cardiac arrest due to ventricular fibrillation. By thorough clinical investigation, he was diagnosed as affected with ARVD/C. His family included 26 members in four generations. All family members gave informed consent to be evaluated and to give blood samples for DNA study. The protocol (Nava el al. Nava et al., 2000Nava A Bauce B Basso C Muriago M Rampazzo A Villanova C Daliento L Buja G Corrado D Danieli GA Thiene G Clinical profile and long-term follow-up of 37 families with arrhythmogenic right ventricular cardiomyopathy.J Am Coll Cardiol. 2000; 36: 2226-2233Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar) included: 12-lead electrocardiogram (ECG), signal-averaged ECG, 24 h Holter ECG, 2D-echocardiogram. Additional procedures (right and left ventricular angiography with coronary arteriography) were performed only on strict clinical indication. Diagnosis was made according to McKenna et al. (McKenna et al., 1994McKenna WJ Thiene G Nava A Fontaliran F Blomstrom-Lundqvist C Fontaine G Camerini F Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy.Br Heart J. 1994; 71: 215-218Crossref PubMed Google Scholar): affection status is established when two major criteria, one major criterion plus two minor criteria, or four minor criteria are fulfilled. Criteria involve genetic, structural, histological, electrocardiographic, and arrhythmological aspects. After clinical evaluation, 11 individuals were identified as being affected with ARVD/C (table 1) on the basis of ECG, echocardiographic, and/or angiographic findings; ventricular arrhythmias with left bundle branch block morphology; and/or presence of late potentials at signal averaged ECG (McKenna et al. McKenna et al., 1994McKenna WJ Thiene G Nava A Fontaliran F Blomstrom-Lundqvist C Fontaine G Camerini F Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy.Br Heart J. 1994; 71: 215-218Crossref PubMed Google Scholar; Nava et al. Nava et al., 2000Nava A Bauce B Basso C Muriago M Rampazzo A Villanova C Daliento L Buja G Corrado D Danieli GA Thiene G Clinical profile and long-term follow-up of 37 families with arrhythmogenic right ventricular cardiomyopathy.J Am Coll Cardiol. 2000; 36: 2226-2233Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Typical ECG and echocardiographical images are shown in fig. 1. Three patients (II,4; II,6; and III,9) manifested a severe form of the disease, three (III,13; IV,3; and IV,6) a moderate form, and the remaining ones displayed a mild form. In this last group of patients, repolarization abnormalities were present at ECG in all cases, accompanied, in two subjects, by isolated PVCs. Ventricular arrhythmias occurred isolated or in couplets/triplets in five patients (III,9; III,10; III,13; IV,4; and IV,6), whereas, in two, they progressed to sustained ventricular tachycardia (II,4 and II,6). In all individuals, ventricular arrhythmias were characterized by left bundle branch block morphology, with left axis deviation in six cases, and with right axis deviation in one. Two patients experienced ventricular fibrillation that, in one case, led to sudden death (IV, 3). During follow-up, which lasted, depending on the patient, for 2–17 years (average 10 years), three individuals (II,4; II,6; and III,9) showed a progression of the disease with left ventricular involvement. In these patients, right ventricle appeared severely dilated at 2D-echo (mean value of right ventricular and diastolic volume=130±10mlm2), and ratio between right and left ventricular volumes was higher than 1.7. The left ventricular kinetic alterations were localized in the posterior wall in two cases and in the anterior wall and apex in the third case. Ejection fraction appeared more severely depressed (33±2) in the right ventricle than in the left (45±5).Table 1Clinical Data for Family Members Fulfilling Diagnostic Criteria for ARVC/D, According to McKenna et al. (McKenna et al., 1994McKenna WJ Thiene G Nava A Fontaliran F Blomstrom-Lundqvist C Fontaine G Camerini F Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy.Br Heart J. 1994; 71: 215-218Crossref PubMed Google Scholar)Result for CriterionDepolarization/ conduction abnormalitiesRight ventricular kinetic alterationsPatientFamilialHistory (Minor)Repolarization abnormalities (Minor)MajorMinorMajorMinorType of ventricular arrhythmiasaVF=ventricular fibrillation. (Minor)No. of criteria (major/minor)II,4++++++Sustained VT2/4II,6+++++−Sustained VT2/3III,4++−+−+−0/4III,9+++++−VF, PVCs2/3III,10++−+−+PVCs0/5III,12++−+−+−0/4III,13++−+−+PVCs0/5IV,3+++−−−VF1/2IV,4++−−−+PVCs0/4IV,6++−−+−PVCs1/3IV,9++−−+−−1/2Note.—A plus sign (+) indicates presence, and a minus sign (−) indicates absence.a VF=ventricular fibrillation. Open table in a new tab Note.— A plus sign (+) indicates presence, and a minus sign (−) indicates absence. Two patients died suddenly (II,1 and IV,3) at ages 65 years and 15 years, respectively, and another (II,6) died because of heart failure at age 68 years. No affected subjects in the family showed phenotypic peculiarities such as woolly hairs, skin alterations, or additional genetic diseases. Genetic investigation revealed no linkage of the disease running in the family with any DNA marker of known ARVD loci. For this reason, a 5-cM genome-wide scan was performed to map the disease locus. A genome scan was performed using 700 microsatellite markers (Applied Biosystems Prism Linkage Mapping Set Version 2.5). All family members were additionally genotyped for D6S1640, obtained from MWG Biotech. In addition, for assessing marker order and intermarker distances on chromosome 6, data from the Human Genome Browser (April 2002 release) was used. Significantly positive linkage was detected for D6S1640, D6S309, D6S470, and D6S1721 (Zmax=4.32 at θ=0 for marker D6S309), mapped to 6p24 (table 2). All patients shared a common haplotype (fig. 2). Since the novel locus (6p24) described in this paper is the eighth reported so far for dominant ARVDs, it should be named ARVD8. Penetrance, estimated in the former two generations of the family, was ∼50%. ARVD8 is probably infrequent, at least in northeast Italy: among 16 families in which we have firmly established linkage with ARVD/C loci, the family reported here is the only one in which the disease was linked to 6p.Table 2Two-Point LOD Scores for Different Recombination Fractions for 6p24 Markers in the Family Reported in Figure 2LOD Score at θ =Marker.00.01.05.10.20.30.40θmaxZmaxD6S1574−1.601.692.152.131.751.18.52.052.15D6S16403.953.883.583.202.381.49.57.003.95D6S3094.324.253.953.572.741.82.83.004.32D6S4704.274.203.913.532.711.80.82.004.27D6S17213.643.583.322.992.281.50.66.003.64D6S259−4.25−2.28−.82−.23.20.27.17.30.27Note.—LOD scores were calculated by assuming 0.95 penetrance as restrictive criterion. Open table in a new tab Note.— LOD scores were calculated by assuming 0.95 penetrance as restrictive criterion. Recombinations detected in subjects III,12, II,4, and III,13 enabled us to define a critical region between D6S1574 and D6S259, spanning ∼9 Mb, which was confirmed by multipoint analysis (data not shown). Gene encoding desmoplakin (DSP), located between D6S1640 and D6S309, appeared as a candidate, since a homozygous DSP nonsense mutation was reported to cause, in an Ecuadorian family, a biventricular dilative cardiomyopathy associated with keratoderma and woolly hairs (Carvajal-Huerta Carvajal-Huerta, 1998Carvajal-Huerta L Epidermolytic palmoplantar keratoderma with woolly hair and dilated cardiomyopathy.J Am Acad Dermatol. 1998; 39: 418-421Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar; Norgett et al. Norgett et al., 2000Norgett EE Hatsell SJ Carvajal-Huerta L Cabezas JC Common J Purkis PE Whittock N Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.Hum Mol Genet. 2000; 9: 2761-2766Crossref PubMed Scopus (572) Google Scholar), now referred as “Carvajal syndrome” (MIM 605676). Mutation screening was performed by direct sequencing. Flanking intronic sequences of each DSP exon were determined by comparing cDNA sequence (GenBank accession number M77830) with genomic sequence, available from public databases. PCR primers flanking each exon of the human DSP gene were designed by PRIMER3. All primer sequences are available at our Web site ARVDnet. Each exon was amplified from patient genomic DNA, purified (PCR Product Presequencing kit; USB), and sequenced using the BIG DYE dideoxy-terminator chemistry (Perkin Elmer) on an ABI 377 DNA sequencer. DNA sequencing of all DSP exons in the index case revealed a missense mutation in exon 7, (C1176G; AGC→AGG). This mutation was detected in all clinically affected individuals and in some relatives whose clinical status is still unknown, since they were very recently recruited to the study. It was detected as well by SSCP analysis (fig. 3). SSCP analysis was performed under the following conditions: 5 μl of a 35-cycle PCR mixture was denatured and separated on a nondenaturing 10% polyacrylamide gel (29:1 acrylamide:bisacrylamide, 5% glicerol), in 1 × TBE buffer at room temperature. Single-strand conformations were detected by silver staining. C1176G mutation could not be detected in any of 240 control subjects (480 alleles) from the same population, thus suggesting such nucleotide substitution is pathogenic. Ser residue (S299R) mutated in patients was placed at the center of a coiled, charged region, separating the two short helices of DSP subdomain Z. Such a coiled region is predicted to be a surface region, possibly involved in protein-protein interactions. The S299R amino acid substitution suppresses a putative phosphorylation site that is fully conserved in related proteins belonging to the same family (fig. 4), supporting the hypothesis that the amino acid change might cause a functional alteration. DNA sequencing revealed an additional variant in exon 7: an A→T transversion resulting in the substitution of an isoleucin with a phenilalanin residue (I305F). Subsequent analysis revealed that I305F is a relatively common (∼8% in frequency) polymorphism in the control population. A single-base insertion before the ATG start codon of DSP (at position –1), cosegregating with the disease in the family, was also detected. Because this insertion does not alter the optimal context of the initiation codon (Kozak et al. Kozak, 1984Kozak M Compilation and analysis of sequence upstream from the translation start site in eukaryotic mRNAs.Nucleic Acid Res. 1984; 12: 857-872Crossref PubMed Scopus (2364) Google Scholar), no pathogenic effect is expected for such a variant. DSP, together with JUP, anchors to desmosomal cadherins, forming an ordered array of nontransmembrane proteins, which then bind to keratin intermediate filaments (IF) (Kowalczyk et al. Kowalczyk et al., 1997Kowalczyk AP Bornslaeger EA Borgwardt JE Palka HL Dhaliwal AS Corcoran CM Denning MF Green KJ The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.J Cell Biol. 1997; 139: 773-784Crossref PubMed Scopus (189) Google Scholar; Smith et al. Smith and Fuchs, 1998Smith EA Fuchs E Defining the interactions between intermediate filaments and desmosomes.J Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (198) Google Scholar; Leung et al. Leung et al., 2002Leung CL Green KJ Liem RK Plakins: a family of versatile cytolinker proteins.Trends Cell Biol. 2002; 12: 37-45Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Desmosomes are major cell-cell junctions, particularly abundant in epidermal cells and in cardiomyocytes (Gallicano et al. Gallicano et al., 1998Gallicano GI Kouklis P Bauer C Yin M Vasioukhin V Degenstein L Fuchs E Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage.J Cell Biol. 1998; 143: 2009-2022Crossref PubMed Scopus (249) Google Scholar; Smith et al. Smith and Fuchs, 1998Smith EA Fuchs E Defining the interactions between intermediate filaments and desmosomes.J Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (198) Google Scholar). DSP consists of 2,871 amino acids, and it is predicted to be a homodimer containing two globular end domains joined by a central alpha-helical coiled-coil rod domain (O’Keefe et al. O'Keefe et al., 1989O'Keefe EJ Erickson HP Bennett V Desmoplakin I and desmoplakin II: purification and characterization.J Biol Chem. 1989; 264: 8310-8318PubMed Google Scholar; Green et al. Green et al., 1990Green KJ Parry DA Steinert PM Virata ML Wagner RM Angst BD Nilles LA Structure of the human desmoplakins: implications for function in the desmosomal plaque.J Biol Chem. 1990; 265: 2603-2612Abstract Full Text PDF PubMed Google Scholar; Virata et al. Virata et al., 1992Virata ML Wagner RM Parry DA Green KJ Molecular structure of the human desmoplakin I and II amino terminus.Proc Natl Acad Sci USA. 1992; 89: 544-548Crossref PubMed Scopus (81) Google Scholar). A carboxy-terminal domain of DSP interacts with IF (Stappenbeck et al. Stappenbeck et al., 1993Stappenbeck TS Bornslaeger EA Corcoran CM Luu HH Virata ML Green KJ Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.J Cell Biol. 1993; 123: 691-705Crossref PubMed Scopus (146) Google Scholar; Kouklis et al. Kouklis et al., 1994Kouklis PD Hutton E Fuchs E Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.J Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (239) Google Scholar; Meng et al. Meng et al., 1997Meng JJ Bornslaeger EA Green KJ Steinert PM Ip W Two-hybrid analysis reveals fundamental differences in direct interactions between desmoplakin and cell type-specific intermediate filaments.J Biol Chem. 1997; 272: 21495-21503Crossref PubMed Scopus (132) Google Scholar), whereas amino-terminal domain including 2-subdomain is required for DSP localization to the desmosomal plaque (Stappenbeck et al. Stappenbeck et al., 1993Stappenbeck TS Bornslaeger EA Corcoran CM Luu HH Virata ML Green KJ Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.J Cell Biol. 1993; 123: 691-705Crossref PubMed Scopus (146) Google Scholar) and is binding to JUP (Kowalczyk et al. Kowalczyk et al., 1997Kowalczyk AP Bornslaeger EA Borgwardt JE Palka HL Dhaliwal AS Corcoran CM Denning MF Green KJ The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.J Cell Biol. 1997; 139: 773-784Crossref PubMed Scopus (189) Google Scholar). Mutated Ser residue in the Z subdomain represents the only PKC phosphorylation site conserved in the whole N-terminal of all PKC-regulated plakins (data not shown). Such residue is likely to represent a crucial site for PKC-mediated regulation of DSP interactions with desmosomal components at the plasma membrane side. Moreover, the first 584 amino acids of the N-terminal domain, where S299R mutation was detected, are known to be involved in JUP binding and in clustering of desmosomal cadherin-JUP complexes (Kowalczyk et al. Kowalczyk et al., 1997Kowalczyk AP Bornslaeger EA Borgwardt JE Palka HL Dhaliwal AS Corcoran CM Denning MF Green KJ The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.J Cell Biol. 1997; 139: 773-784Crossref PubMed Scopus (189) Google Scholar). Nonsense DSP mutations, leading to functionally null alleles, were reported to have produced striate palmoplantar keratoderma in heterozygotes, thus demonstrating that dosage of DSP is critical in maintaining epidermal integrity (Armstrong et al. Armstrong et al., 1999Armstrong DK McKenna KE Purkis PE Green KJ Eady RA Leigh IM Hughes AE Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.Hum Mol Genet. 1999; 8: 143-148Crossref PubMed Scopus (209) Google Scholar; Whittock et al. Whittock et al., 1999Whittock NV Ashton GH Dopping-Hepenstal PJ Gratian MJ Keane FM Eady RA McGrath JA Striate palmoplantar keratoderma resulting from desmoplakin haploinsufficiency.J Invest Dermatol. 1999; 113: 940-946Crossref PubMed Scopus (113) Google Scholar, Whittock et al., 2002Whittock NV Wan H Morley SM Garzon MC Kristal L Hyde P McLean WH Pulkkinen L Uitto J Christiano AM Eady RA McGrath JA Compound heterozygosity for non-sense and mis-sense mutations in desmoplakin underlies skin fragility/woolly hair syndrome.J Invest Dermatol. 2002; 118: 232-238Crossref PubMed Scopus (100) Google Scholar). Conversely, heterozygotes for DSP truncated at its C-terminal domain (Carvajal syndrome) showed no keratoderma; it was suggested that, in this case, either DSP binding to IF is reduced but not lost or that loss of desmosomes-IF binding via DSP could be compensated for by other desmosomal proteins (Norgett et al. Norgett et al., 2000Norgett EE Hatsell SJ Carvajal-Huerta L Cabezas JC Common J Purkis PE Whittock N Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.Hum Mol Genet. 2000; 9: 2761-2766Crossref PubMed Scopus (572) Google Scholar). It is possible that absence of skin defects in heterozygous carriers of DSP missense mutation S299R can be explained by considering that this mutation does not affect DSP-IF binding, which, on the contrary, is targeted by other mutations producing a keratoderma phenotype. In heterozygotes for the S299R mutation, the majority of desmosomal cadherin-JUP complexes would be defective because of the dimeric nature of DSP functional molecules. This would explain the dominant pattern of inheritance of the disease caused by such mutation. The involvement of two desmosomal proteins (DSP and JUP) in two different ARVD/C clinical phenotypes (ARVD8 and Naxos disease) suggests that some ARVD/C might result from defects in intercellular connections. According to present knowledge, mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels via cadherins' mechanical intracellular signaling (Ko et al. Ko et al., 2001Ko KS Arora PD McCulloch CAG Cadherins mediate intracellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium permeable channels.J Biol Chem. 2001; 276: 35967-35977Crossref PubMed Scopus (126) Google Scholar). Moreover, stretching of cardiomyocytes is known to modulate the elementary calcium release process from ryanodine receptor release channels (Petroff et al. Petroff et al., 2001Petroff MGV Kim SH Pepe S Dessy C Marban E Balligand JL Sollott SJ Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes.Nat Cell Biol. 2001; 3: 867-873Crossref PubMed Scopus (260) Google Scholar). Therefore, a genetically impaired response to mechanical stress might adversely affect intracellular calcium concentration and the excitation-contraction coupling. In addition, it might induce apoptosis and cellular necrosis, which, in turn, would promote fibrosis and adipose substitution, as in several muscular dystrophies. The almost selective affection of the right ventricle might be in relation to its extensibility, in comparison with that of left-ventricular free wall. It is interesting to notice that mutations in RYR2 (cardiac ryanodine receptor) cause dominant ARVD/C type 2, thus supporting the hypothesis of a key pathogenic role played by altered intracellular calcium concentration in these diseases. The authors are grateful to all family members who kindly participated in this study and to Mrs. Paola Marcon for help in collecting familial data. This publication is based on the project performed pursuant to Baylor College of Medicine grant 1 U01 HL 65652 from the National Institutes of Health. This work was supported also by TELETHON, Italy (Grant 1288, to G.A.D.), MURST 40%, ARVC EC Contract QLG1-CT-2000-01091, and Fondazione Cassa di Risparmio, Padova e Rovigo, Italy. B.B. and A.R. are recipients of a research assistant temporary position, pursuant to ARVC EC Contract QLG1-CT-2000-01091.