ARVC is a cardiac muscle disorder that is associated with arrhythmias and heart failure of predominantly RV origin. Molecular genetics postulate ARVC to be a “disease of the desmosome.”17
However, the underlying molecular pathogenesis and clinical characteristics, such as variable disease expression and incomplete penetrance, are poorly understood.
Here we report two novel missense mutations in the ICS, a conserved region in the cytoplasmic domain of DSG2 (DSG2 G812S and DSG2 C813R) identified in two ARVC probands. The first mutation affects the same residue as the previously reported DSG2 G812C mutation, which was found in an American family,11
while the second is in the subsequent amino acid. Co-segregation of the missense mutation DSG2 G812S with disease expression supports its causative role for ARVC in family A (). The second family with the DSG2 C813R mutation was too small for co-segregation analysis.
Other mutations in DSG2 have been associated with ARVC.11,18-20
Most of the mutations are located in the extracellular portion of the protein, but no clear correlation has been observed between specific mutations and clinical features. Due to the small number of affected individuals in our study and the observed variable disease expression, we currently cannot draw any conclusions regarding differences in phenotypes of individuals bearing extracellular or cytoplasmic DSG2 mutations.
A few functional studies on the molecular pathology of DSG2 mutations in ARVC have been reported. A DSG2 N266S transgenic mouse model, which mimics the human mutation DSG2 N271S in the extracellular domain of the protein, has been studied.20
This animal showed some typical features of ARVC, such as ventricular arrhythmias and sudden cardiac death. At the histologic level, necrosis was observed in the transgenic hearts; however, desmosomal structure in electron micrographs and immunohistochemical staining for PG, PKP2, DSP, and Cx43 at the intercalated disk appeared to be normal in this animal model.
This finding contrasts with the molecular phenotype observed in our patients with a novel mutation in the C-terminus of DSG2. The reduction of immunoreactive signal specific for the desmosomal proteins DSP, PG, and PKP2 from cardiac intercalated disks in two individuals with the DSG2 G812S mutation (), but not in the mouse model, may potentially be explained by differences between murine ARVC models and human disease. Similarly, the heterozygous PG knockout mice reflected only certain aspects of human disease (arrhythmias and reduced RV function), whereas no fatty fibrotic replacement of cardiomyocytes was evident in this animal model for ARVC.21
The electric isolation of cardiomyocytes by surrounding scar tissue documented in individual A.1 may promote reentrant excitation (Online Supplement Figure 1
). Moreover, the gap junction remodeling observed in the endomyocardial biopsy samples (indicated by reduced immunoreactive signal for Cx43, ) may act synergistically with the histologic abnormalities characteristic of ARVC to enhance conduction heterogeneity and increase the risk of arrhythmia.7
Because loss of PKP2 was evident in the samples (), PKP2-associated conduction slowing (via Cx4322
or sodium channels23
) may also contribute to the development of arrhythmia.
Our functional studies on three ARVC-associated mutations in the cytoplasmic domain of DSG2 suggest that these mutant proteins act in a dominant negative manner. The localization and stability of the mutant proteins were identical to DSG2 wild-type (), suggesting that the mutant proteins are likely to be incorporated into the cardiac desmosomes of the patients. The fact that all mutations cluster in the ICS implies an important role of this domain for DSG2 function. The affected residues are completely conserved among desmosomal cadherins (A), and the ICS domain links DSG2 to the plaque proteins PKP2 and PG9
(B). Contrary to our hypothesis (see also Awad et al.11
), none of the three mutations in the ICS affected binding to PG in binding assays (C). This suggests that the loss of PG immunoreactivity from the intercalated disk, as seen in our ARVC patients (), is a result of a more complex mechanism than just a simple change in binding affinity caused by the missense mutation.
In particular, posttranslational modifications may be important in mediating the binding of DSG2 to PKP2. We observed striking differences in the binding properties between bacterially expressed DSG2 ICS protein and its counterpart expressed in mammalian COS-1 cells. The former bound both PG and PKP2 from rat heart lysates, whereas the latter did not bind to PKP2, even though PKP2 is abundantly expressed in this cell line. Posttranslational modifications of the DSG2 ICS region may occur in mammalian cells, which could change its affinity to PKP2. In agreement with this hypothesis, bacterially expressed DSG2 ICS was able to bind PKP2 from COS-1 cells, whereas COS-1 cell-derived DSG2 ICS did not bind to PKP2 from rat heart extracts (Online Supplement Figure 2
). In addition, changes in the ICS of DSG2 may affect the structure of the adjacent regions of the cytoplasmic domain, thereby modulating their binding properties to desmosomal components.24
Our finding that the DSG2 C813R ICS protein fragment shows abnormal migration behavior in gel electrophoresis further supports a role for unknown functions beyond linking DSG2 to PG and PKP2 (E). This is only evident when the cysteine residue is mutated to arginine but not if it is changed to alanine. This suggests that the observed change of electrophoretic properties is caused by the presence of the arginine at this position rather than by the lack of cysteine (“gain of function”). Currently, the nature of this phenomenon is unclear. Phosphatase treatment did not alter electrophoretic mobility of the proteins (Online Supplement Figure 3
), hence an involvement of protein phosphorylation is unlikely. Alternatively, the GCCS motif is a consensus site for protein palmitoylation,25
and this posttranslational modification may modulate the functions of DSG2.26
Finally, a direct modification of this arginine at position 813 (e.g. by methylation27
) may occur.
Future investigations are needed to identify these additional functions of the DSG2 molecule and thereby provide novel insights into ARVC. This study suggests that the loss of PG immunoreactivity at the intercalated disk is the result of complex molecular alterations in the myocardium, which is consistent with the fact that the immunoreactive signal for PG is reduced in the vast majority of ARVC cases, even in those where no mutation in a known ARVC disease gene could be identified.4
This observation renders the redistribution of PG part of a final common pathway in ARVC pathogenesis,28
not merely stemming from disruptions in binding affinities within the desmosomal plaque.
The elucidation of these pathologic mechanisms is the next step in defining pathogenesis and developing novel therapeutic targets for patients with ARVC.