A homozygous DSC2 deletion associated with arrhythmogenic cardiomyopathy is caused by uniparental isodisomy.

AIMS: We aimed to unravel the genetic, molecular and cellular pathomechanisms of DSC2 truncation variants leading to arrhythmogenic cardiomyopathy (ACM). METHODS AND RESULTS: We report a homozygous 4-bp DSC2 deletion variant c.1913_1916delAGAA, p.Q638LfsX647hom causing a frameshift carried by an ACM patient. Whole exome sequencing and comparative genomic hybridization analysis support a loss of heterozygosity in a large segment of chromosome 18 indicating segmental interstitial uniparental isodisomy (UPD). Ultrastructural analysis of the explanted myocardium from a mutation carrier using transmission electron microscopy revealed a partially widening of the intercalated disc. Using qRT-PCR we demonstrated that DSC2 mRNA expression was substantially decreased in the explanted myocardial tissue of the homozygous carrier compared to controls. Western blot analysis revealed absence of both full-length desmocollin-2 isoforms. Only a weak expression of the truncated form of desmocollin-2 was detectable. Immunohistochemistry showed that the truncated form of desmocollin-2 did not localize at the intercalated discs. In vitro, transfection experiments using induced pluripotent stem cell derived cardiomyocytes and HT-1080 cells demonstrated an obvious absence of the mutant truncated desmocollin-2 at the plasma membrane. Immunoprecipitation in combination with fluorescence measurements and Western blot analyses revealed an abnormal secretion of the truncated desmocollin-2. CONCLUSION: In summary, we unraveled segmental UPD as the likely genetic reason for a small homozygous DSC2 deletion. We conclude that a combination of nonsense mediated mRNA decay and extracellular secretion is involved in DSC2 related ACM.

The aberrant expression or disruption of desmocollin2 in human diseases.

The desmosome is a member of intercellular junctions that named 'anchoring junction', which contributes to the integrity and homeostasis of tissue structure and function of multicellular living organisms. As an important component of the desmosome and the most widely distributed isoform of desmocollins (DSCs), desmocollin2 (DSC2) has been demonstrated to be essential for the unity of epithelial cells, and served as a vital regulator in signaling processes such as epithelial morphogenesis, differentiation, wound healing, cell apoptosis, migration, and proliferation. Recent studies suggested that the aberrant expression or disruption of DSC2 might lead to some disorders, including heart disorders, certain cancers, and some other human diseases. The distinctions in expression and regulation mechanisms of DSC2 in different human diseases provided a potential target for diagnosis and individualized treatment. Further research is required to certify the signaling capacity of DSC2 and to shed light on the down-stream consequences of the signaling for us to understand the new area of DSC2 biology and the development of certain diseases. This review summarizes the molecular structure and dynamics of desmosome and DSC2, the relationship between the disruption or aberrant expression of DSC2 and human diseases and related molecular mechanisms, as well as the possible prospects.

Structural basis of adhesive binding by desmocollins and desmogleins.

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.

Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins.

The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell-cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pull-down assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins.

Desmosomal adhesion in vivo.

Desmosomes are intercellular junctions that provide strong adhesion or hyper-adhesion in tissues. Here, we discuss the molecular and structural basis of this with particular reference to the desmosomal cadherins (DCs), their isoforms and evolution. We also assess the role of DCs as regulators of epithelial differentiation. New data on the role of desmosomes in development and human disease, especially wound healing and pemphigus, are briefly discussed, and the importance of regulation of the adhesiveness of desmosomes in tissue dynamics is considered.

Homozygous founder mutation in desmocollin-2 (DSC2) causes arrhythmogenic cardiomyopathy in the Hutterite population.

BACKGROUND: Dominant mutations in cellular junction proteins are the major cause of arrhythmogenic cardiomyopathy, whereas recessive mutations in those proteins cause cardiocutaneous syndromes such as Naxos and Carvajal syndrome. The Hutterites are distinct genetic isolates who settled in North America in 1874. Descended from <100 founders, they trace their origins to 16th-century Europe. METHODS AND RESULTS: We clinically and genetically evaluated 2 large families of the Alberta Hutterite population with a history of sudden death and found several individuals with severe forms of biventricular cardiomyopathy characterized by mainly left-sided localized aneurysms, regions of wall thinning with segmental akinesis, in addition to typical electric and histological features known for arrhythmogenic right ventricular cardiomyopathy. We identified a homozygous truncation mutation, c.1660C>T (p.Q554X) in desmocollin-2 (DSC2), in affected individuals and determined a carrier frequency of this mutation of 9.4% (1 in 10.6) among 1535 Schmiedeleut Hutterites, suggesting a common founder in that subgroup. Immunohistochemistry of endomyocardial biopsy samples revealed altered expression of the truncated DSC2 protein at the intercalated discs but only minor changes in immunoreactivity of other desmosomal proteins. Recombinant expressed mutant DSC2 protein in cells confirmed a stable, partially processed truncated protein with cytoplasmic and membrane localization. CONCLUSIONS: A homozygous truncation mutation in DSC2 leads to a cardiac-restricted phenotype of an early onset biventricular arrhythmogenic cardiomyopathy. The truncated protein remains partially stable and localized at the intercalated discs. These data suggest that the processed DSC2 protein plays a role in maintaining desmosome integrity and function.

Desmosomal cadherins in zebrafish epiboly and gastrulation.

BACKGROUND: The desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg), are the adhesion molecules of desmosomes, intercellular adhesive junctions of epithelia and cardiac muscle. Both the DCs and desmosomes have demonstrably essential roles in mammalian development. In order to initiate their study in a more tractable developmental system we have characterised zebrafish DCs and examined their roles in early zebrafish development. RESULTS: We find that zebrafish possess one Dsc, the orthologue of mammalian Dsc1, which we designate zfDsc. Unlike mammalian Dscs, zfDsc exists only as the "a" form since it lacks the alternatively-spliced mini-exon that shortens the cytoplasmic domain to produce the "b" form. Zebrafish possess two Dsgs, designated zfDsgα and zfDsgß, orthologues of mammalian Dsg2. They show 43.8% amino acid identity and the α form has a 43 amino acid glycine-rich sequence of unknown function in its extracellular domain. Both zfDsc and zfDsgα were present as maternal and zygotic transcripts whereas zfDsgß was first expressed from 8 hours post-fertilisation (hpf). All three transcripts were present throughout subsequent stages of development. Morpholino knockdown of both zfDsc and zfDsgα expression produced similar defects in epiboly, axis elongation and somite formation, associated with abnormal desmosomes or reduced desmosome numbers. CONCLUSIONS: These results demonstrate an important role for DCs and desmosomes in the early morphogenesis of the zebrafish embryo, provide a basis for more detailed analysis of their role and raise interesting questions relating to the evolution and functional significance of DC isoforms.

The p.A897KfsX4 frameshift variation in desmocollin-2 is not a causative mutation in arrhythmogenic right ventricular cardiomyopathy.

Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), an autosomal-dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 112 ARVC/D probands for mutations in desmocollin-2 (DSC2) gene and detected two different amino-acid substitutions (p.E102K, p.I345T) and a frameshift variation (p.A897KfsX4) in 7 (6.2%) patients. DSC2a variant p.A897KfsX4, previously reported as a p.E896fsX900 mutation, was identified in five unrelated probands. Four of them were found to carry one or two mutations in different ARVC/D genes. Unexpectedly, p.A897KfsX4 variation was also found in 6 (1.5%) out of 400 control chromosomes. In vitro functional studies showed that, unlike wild-type DSC2a, this C-terminal mutated protein was localised in the cytoplasm. p.A897KfsX4 variation affects the last five amino acids of the DSC2a isoform but not of DSC2b. In contrast with what we found in other human tissues, in the heart DSC2b is more expressed than DSC2a, suggesting that relative deficiency of DSC2a might be compensated by isoform b. In conclusion, DSC2 gene mutations are not frequently involved in ARVC/D. The p.A897KfsX4 variation, identified in several Italian healthy control subjects, which affects only one of the two DSC2 isoforms, may be considered a rare variant, though possibly affecting phenotypic expression of concomitant ARVC/D mutations.

Comprehensive desmosome mutation analysis in north americans with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited disorder typically caused by mutations in components of the cardiac desmosome. The prevalence and significance of desmosome mutations among patients with ARVD/C in North America have not been described previously. We report comprehensive desmosome genetic analysis for 100 North Americans with clinically confirmed or suspected ARVD/C. METHODS AND RESULTS: In 82 individuals with ARVD/C and 18 people with suspected ARVD/C, DNA sequence analysis was performed on PKP2, DSG2, DSP, DSC2, and JUP. In those with ARVD/C, 52% harbored a desmosome mutation. A majority of these mutations occurred in PKP2. Notably, 3 of the individuals studied have a mutation in more than 1 gene. Patients with a desmosome mutation were more likely to have experienced ventricular tachycardia (73% versus 44%), and they presented at a younger age (33 versus 41 years) compared with those without a desmosome mutation. Men with ARVD/C were more likely than women to carry a desmosome mutation (63% versus 38%). A mutation was identified in 5 of 18 patients (28%) with suspected ARVD. In this smaller subgroup, there were no significant phenotypic differences identified between individuals with a desmosome mutation compared with those without a mutation. CONCLUSIONS: Our study shows that in 52% of North Americans with ARVD/C a mutation in one of the cardiac desmosome genes can be identified. Compared with those without a desmosome gene mutation, individuals with a desmosome gene mutation had earlier-onset ARVD/C and were more likely to have ventricular tachycardia.

Interactions of plakoglobin and beta-catenin with desmosomal cadherins: basis of selective exclusion of alpha- and beta-catenin from desmosomes.

Plakoglobin and beta-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with alpha-catenin. Plakoglobin, but normally not beta-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of beta-catenin and alpha-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between beta-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of beta-catenin. Binding of plakoglobin and beta-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and beta-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, beta-catenin binds to desmoglein-1 more weakly than does plakoglobin. beta-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal beta-catenin "tails" that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and alpha-catenin compete directly for binding to plakoglobin, consistent with the absence of alpha-catenin in desmosomes.