Disease Genes

The first identified ARVC/D gene in a dominant form was ryanodine receptor-2, involved in ARVD2 [25]. In ARVD2, there is fibro-fatty substitution of the myocardial tissue, though much less pronounced than in the typical ARVC/D. The distinctive feature of this form is the presence of polymorphic, effort-induced arrhythmias. RYR2 is one of the largest human genes (105 exons), encoding a 565Kda protein located in the membrane of smooth sarcoplasmic reticulum. The homo-tetrameric structure known as cardiac ryanodine receptor plays a pivotal role in in-tracellular calcium homeostasis and excitation-contraction coupling in cardiomyocytes [27, 28]. All RYR2 mutations detected in ARVD2 patients were missense resulting in substitutions involving amino acids highly conserved through evolution in critical domains of the protein [25, 29].

Mutations in the human RYR2 gene have also been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT; OMIM 604772) [30,31] and familial polymorphic ventricular tachycardia (FPVT); OMIM 604772) [32, 33]. Putative pathogenic mutations in RYR2 have been reported in

Table 1.1 • Known ARVC/D loci and disease-genes.










Cytokine stimulating fibrosis and modulating cell adhesion

Regulatory mutations in 5' and 3' UTRs





Calcium homeostasis

Missense mutations



































Cell-cell adhesion

Missense, nonsense and splice site mutations





Cell-cell adhesion

Missense, nonsense, insertion/deletion and splice site mutations





Cell-cell adhesion

Missense, nonsense, insertion/deletion and splice site mutations


20 out 240 patients referred for long-QT syndrome genetic testing [34].

All RYR2 mutations described to date cluster in three specific domains: the N-terminal amino-acid residues 176-433, the centrally located residues 22462504, and the C-terminal residues 3778-4959. Detection of RYR2 mutations in both ARVD2 and CPVT patients raises the question of the possible existence of a single genetic defect, different pheno-types of which might be simply due to variable expression and incomplete penetrance. Both ARVD2 and CPVT-RyR2 missense mutations would alter the ability of the calcium channel to remain closed. Intense adrenergic stimulation due to emotional or physical stress can lead to calcium overload, thus triggering severe arrhythmias. The functional role of mutations R176Q, L433P, N2386I, and T2504M, previously detected in ARVD2 patients [25], was recently investigated [35]. RyR2 mutants N2386I and R176Q/T2504M exhibited enhanced sensitivity to caffeine activation and increased Ca2+ release, in agreement with the current hypothesis that defective RyR2 causes Ca2+ leak. In contrast, RyR2 L433P mutation showed reduced response to caffeine activation. This mutation might be interpreted as a "loss-of-function." Therefore, RyR2 mutations might be either "gain-of-function" or "loss-of-function," thus suggesting heterogeneity in functional consequences of RyR2 mutations. Even with this additional information, the question of whether ARVD2 and CPVT are different diseases due to different mutations of the RYR2 gene still remains unsettled.

The first disease gene linked to autosomal dominant ARVC/D showing typical right ventricular phenotype was Desmoplakin (DSP) [22]. In 2002, genome scan in a family with ARVC/D indicated a linkage with a region of chromosome 6 short arm including DSP gene. DNA sequencing of all DSP exons in the affected persons of this family revealed a missense mutation in exon 7 (C1176G; AGC^AGG) (Fig. 1.1).The involved amino acid (Ser299Arg) is at the center of a coiled, charged region, separating the two short helices of DSP subdomain Z. The amino acid substitution suppresses a putative phosphorylation site, which, on the other hand, is fully conserved in related proteins belonging to the same family. This mutation is thought to disrupt a protein kinase C phosphorylation site which is involved in plakoglobin binding and in clustering of desmoso-mal cadherin-plakoglobin complexes.

Desmoplakin, together with plakoglobin, anchors to desmosomal cadherins, forming an ordered array of nontransmembrane proteins, which bind to keratin intermediate filaments (Fig. 1.2) [36]. The primary structure of desmoplakin contains three functional domains: the N-terminal, which binds to the desmo-some via connection with plakoglobin and plakophilin; the rod segment, which is predicted to form a dimeric coil; and the C-terminal domain, which binds intermediate filaments [37]. Alternative splicing of the protein produces two isoforms, desmo-plakin I and desmoplakin II. The cDNAs encoding these two highly related proteins differ in a 1.8 Kbase sequence that is missing in DSPII, most likely due to differential splicing of a longer transcript [38].

Fig. 1.1 • Family pedigree of the ARVC/D index case carrying the S299R DSP mutation and sequence electropherogram showing the heterozygous missense mutation
Fig. 1.2 • Schematic representation of relationships between desmosomal proteins in myocardiocytes.DSC2:desmocollin-2; DSG2: desmoglein-2; DSP: desmoplakin; PKP2: plakophilin-2; PKP4: plakophilin-4; JUP: plakoglobin; DES: desmin

Mutations in the desmoplakin gene have been shown to be responsible for some cases of an autosomal dominant skin disorder (striate palmoplantar keratoderma) without cardiac involvement [39-41]; a dominant form of ARVC/D without skin disease

[22]; an autosomal recessive condition characterized by dilated cardiomyopathy, woolly hair, and keratoderma (so-called Carvajal syndrome) [42], an autosomal recessive condition characterized by ARVC/D, woolly hair, and keratoderma [43] and a left-sided ARVC/D named arrhythmogenic left ventricular car-diomyopathy (ALVc) [44].

Mutations in DSP gene were detected in different families: they include twelve missense, two nonsense, and two splice-site mutations. In our experience, DSP mutations may account for a considerable number of ARVC/D cases.

In 2004, Gerull et al. [23] selected plakophilin-2 (PKP2) as candidate gene because a homozygous deletion caused a lethal cardiac defect in mice [45]. PKP2 gene encodes plakophilin-2, an essential protein of the cardiac desmosome. By sequencing all 14 exons of the PKP2 gene, including flanking intronic splice sequences, the authors identified 25 different heterozygous mutations (twelve insertion-deletion, six nonsense, four missense, and three splice site mutations) in 32 of 120 unrelated ARVC/D probands [23]. Plakophilin-2 is an armadillo-related protein, located in the outer dense plaque of desmosomes. It links desmosomal cadherins to desmoplakin and the intermediate filament system (Fig. 1.2). Plakophilins are also present in the nucleus, where they may play a role in transcription-al regulation [46]. Gerull et al. [23] speculated that lack of plakophilin-2 or incorporation of mutant plakophilin-2 in the cardiac desmosomes might impair cell-cell contacts and, as a consequence, might disrupt association between adjacent car-diomyocytes.

The frequency of PKP2 mutations among ARVC/D cases ranged from 11% to 43% in different studies [47-49]. These differences might be attributed to different geographical origin of cases or simply to selection bias.

Recently, we decided to shift from linkage studies in ARVC/D families to a candidate gene approach. Thus, we screened different genes encoding desmo-somal proteins. When analyzing DSG2 gene (Desmoglein-2, the only isoform expressed in cardiac myocytes), we detected nine heterozygous mutations in eight of 50 unrelated individuals with ARVC/D which proved negative for mutations of DSP, PKP2, and TGF^3 genes [24]. Among these, five were missense mutations, two were insertion-deletions, one was a nonsense and one was a splice site mutation; one patient had two different DSG2 mutations (compound heterozygote). Endomyocardial biopsy, obtained from five patients, showed extensive loss of myocytes with fibro-fatty tissue replacement. In three patients, electron microscopy showed intercalated disc paleness, decreased desmosome number, and intercellular gap widening [24]. Mutations in DSG2 gene were also detected in an independent study [50]. It is interesting to note that, in this study, there was one patient with compound-heterozygous mutations in DSG2 (Fig. 1.3).

In 2005, our group identified the gene involved in ARVD1 [26]. The large critical interval for ARVD1 included 40 known genes; five of them (POMT2, KIAA0759, KIAA1036, C14orf4, and TAIL1) were unsuccessfully screened for pathogenic ARVC/D mutations [51,52]. Among genes mapped to the ARVD1 critical region and expressed in myocardium, transforming growth factor-beta3 (TGF^3) appeared to be

Fig. 1.3 • Pedigree of the proband carrying two DSG2 mutations (988G>A, 1881-2A>G). Hatched symbol represents an individual of unknown disease status.Presence (+) or absence (-) of the DSG2 mutation is indicated

a good candidate, since it encodes a cytokine stimulating fibrosis and modulating cell adhesion. After previous analyses failed to detect any mutation in the coding region of this gene, mutation screening was extended to the promoter and untranslated regions (UTRs). A nucleotide substitution (c.-36G>A) in 5'UTR of TGF^3 gene was detected in all affected subjects belonging to a large ARVD1 family. After the investigation was extended to 30 unrelated ARVC/D index patients, an additional mutation (c.1723C>T) was identified in the 3' UTR of one proband. In vitro expression assays of constructs containing the mutations showed that mutated UTRs were twofold more active than wild type [26].

TGF^3 is a member of the transforming growth factor superfamily, which includes a diverse range of proteins regulating many different physiological processes. TGF^1, -^2, and -^3 are the prototype of the TGFP superfamily. They inhibit proliferation in most types of cells and induce apoptosis of epithelial cells. Conversely, they stimulate mesenchymal cells to proliferate and produce extracellular matrix and they induce a fibrotic response in various tissues in vivo.

Finding TGF^3 mutations associated with ARVC/D is very interesting, since it is well established that TGF^s stimulate mesenchymal cells to proliferate and to produce extracellular matrix components. Since mutations in UTRs of the TGF^3 gene, detected in ARVC/D, showed enhanced gene expression in vitro, it is likely that they could promote myocardial fibrosis in vivo. Myocardial fibrosis may disrupt electrical and mechanical behavior of myocardium and extracellular matrix abnormalities may predispose to reentrant ventricular arrhythmias. In agreement with this hypothesis, endomyocardial biopsy in the two probands in which TGF^3 UTR mutations were detected showed extensive replacement-type fibrosis. Moreover, it has been shown that TGF^s modulate expression of genes encoding desmosomal proteins in different cell types. cDNA microarray analysis, performed on RNA from cardiac fibroblasts incubated in the presence or in the absence of exogenous TGF^s, revealed increased expression of different genes, including plakoglobin [53].Yoshida et al. [54] reported that TGF^1 exposure of cultured airway epithelial cells increases the content of desmoplakins I and II. This suggests that regulation of cell-cell junctional complexes may be an important effect of TGF^s. Therefore, overexpression of TGF^3, caused by UTRs mutations, might affect cell-to-cell junction stability, thus leading to disease expression similar to that observed in ARVC/D due to mutations of genes encoding desmosomal proteins.

Desmosomes are important cell-cell adhesion junctions, predominantly found in the epidermis and heart. They couple cytoskeletal elements to plasma membrane at cell-cell or cell-substrate adhesions. Whereas adherens junctions are linked with microfilaments at cell-cell interfaces, desmosomes anchor stress-bearing intermediate filaments at sites of strong intercellular adhesion. The resulting scaffold plays a key role in providing mechanical integrity to tissues such as epidermis and heart, which experience mechanical stress. Desmosomes include proteins from at least three distinct gene families: cadherins, armadillo proteins, and plakins (Fig. 1.2). Desmoso-mal cadherins include desmogleins and desmo-collins; members of both subfamilies are single-pass transmembrane glycoproteins, mediating Ca2+-de-pendent cell-cell adhesion. Armadillo proteins include plakoglobin and plakophilins (PKP1-3). The plakin family proteins include desmoplakin, plectin, and the cell envelope proteins envoplakin and periplakin. Desmoplakin (involved in ARVD8), plakophilin-2 (involved in ARVD9), desmoglein-2 (involved in ARVD10), and plakoglobin (involved in Naxos syndrome, the autosomal recessive form of ARVC/D) are desmosomal proteins. Based on present evidence we may conclude that different defects in proteins of desmosomal complex lead to ARVC/D. Therefore, additional components of the desmoso-mal complex may be targets for pathogenic mutations leading to ARVC/D.

Was this article helpful?

0 0

Post a comment