Arrhythmogenic cardiomyopathy: in search of unifying genetic theory

This editorial refers to ‘A novel murine model for arrhythmogenic cardiomyopathy points to a pathogenic role of Wnt signalling and miRNA dysregulation’ by M. Calore et al., pp. 739–751.

Arrhythmogenic cardiomyopathy (AC) remains one of the most difficult cardiomyopathies to diagnose. AC is generally characterized by impairment of ventricular systolic function, prominent ventricular arrhythmias, and fibrofatty myocardial replacement and has been linked to a number of genetic mutations, most of which induce desmosomal dysfunction.¹ AC occurs with an estimated population frequency between 1:1000 and 1:5000, though most clinicians who specialize in AC tend to believe the prevalence is closer to 1:5000.¹ This discrepancy is likely due to the frequent misdiagnosis of AC, an artefact of an over-reliance on MRI observation of intramyocardial fat/wall thinning, incomplete diagnostic testing, and an overall lack of awareness.²

An alternative to these imperfect diagnostics is genetic testing. AC’s high heritability,¹ and recent technological progress in genome sequencing³ creates the potential for genetic diagnosis of AC as an alternative to these otherwise error-prone diagnostic tools. Genetic diagnosis of AC remains challenging, however as the genetic picture of AC pathogenesis is still indistinct.⁴ Though AC is considered to be a disease of the cardiac desmosome, there exists high natural variability of allelic pathogenicity within even control populations. For genetic diagnosis of AC to be of value in routine clinical practice, a deeper understanding of the relationship between specific AC mutations and the AC disease phenotype is essential. It is in this context that the work by Calore et al. is a significant step forward. Not only do the authors look at genotype-phenotype linkage for a clinically relevant DSG2 variant, they link DSG2 dysfunction to the down-regulation of the Wnt/B catenin signalling pathway, a finding that hints at a higher level of dysfunction that may be amenable to therapy.

In this work, Calore et al.⁵ took a common AC associated pathogenic missense mutation in the desmoglein gene, DSG2 Q558*, and examined it for both structural and epigenetic effects on cardiac function and morphology. Calore et al. generated a novel murine model characterized by the overexpression of FLAG-tagged mutant DSG2 in the normal murine heart. This overexpression was accompanied by fibrous replacement, reduction in desmosomal size and suppression of the Wnt/B catenin signalling pathway in mutant hearts. Deep sequencing of mutant hearts revealed a number of differentially expressed miRNAs, which give clues regarding the underlying genetic mechanisms of AC.

Calore et al.’s contributes to the AC field in a number of different ways. Though the pathogenicity of DSG2 mutations was already established, the structural and genetic consequences of the Q558* mutation was not well characterized. Understanding the underlying molecular factors that can drive AC lays the groundwork for future mechanistic studies with therapeutic potential.

One of the more interesting findings from Calore et al. is the down-regulation of the Wnt/B catenin pathway. This down-regulation became apparent in the mutant mice as early as 6 months, preceding the observation of histological indicators of AC which manifested at 12 months. As Wnt/B catenin down-regulation has not been previously associated with DSG2 mutations, this finding adds to the growing body of evidence implicating Wnt/B catenin down-regulation as a possible causative factor in AC.⁶ If Wnt/B-catenin signalling can be confirmed as a unifying causative factor for AC, it represents an attractive therapeutic target for the treatment of AC.

In the short term, this work details a number of miRNAs that should be the subject of future studies. miR-217-5p, miR-708-5p, and miR-499-5p were all found to be differentially regulated in this study and as such provide clues as to the molecular pathogenesis of AC. Future studies surveying the pathogenic effects of the dysregulation of the micro RNAs is a very clear next step that could arise from this work.

In a more abstract sense, however, this transcriptomic work offers an important glimpse into the RNA expression profile of AC hearts that will both guide and colour future research in this area. Transcriptomic analysis of complex genetic disorders is becoming increasingly widespread. Indeed, the advent of single cell RNA seq is resulting in a glut of transcriptomic studies that offer broad insights into the pathogenesis of disease.⁷ Calore et al.’s miRNA expression profile generated in the context of a pathogenic DSG2 overexpression work will function as a roadmap for the future of AC transcriptomic studies. The advent of Mime-seq, a newly described miRNA specific iteration of single cell RNA seq,⁸ suggests future higher resolution RNA profiling studies that will rely on this work as a historical landmark against which to compare results.

AC’s uncertain genetics make widespread study of all associated genes in expensive model organisms a risky scientific proposition. DSG2 has been previously shown to be pathogenic, and as such the transition to a mouse model carried with it less risk. In a funding limited research environment, however, many of the less studied mutations will be deemed too risky to be studied in vivo. In this environment, an approach that differs from the one used by Calore et al. may be fiscally feasible and thus worth pursuing. Dual Integrase Cassette Exchange (DICE)⁹ is a rapid method for functional annotation of genetic variants that could prove useful in the context of ACs uncertain genetic landscape. In brief, DICE functions by using CRISPR/Cas9 in iPSCs to edit in a ‘landing pad’ that allows for irreversible exchange of a normal gene sequence with one containing a variant of unknown significance. These iPSCs can then be differentiated into cardiomyocytes and analysed for pathogenic phenotypes. Indeed, in work recently published by Lv et al.,¹⁰ the DICE system was used to determine the likely pathogenicity of a variant of unknown significance in the TNNT-2 gene on a clinical timescale. Studies in this vein, either via DICE or some other method, could rapidly reduce the uncertainty inherent in AC genetics and create an environment that favours high quality in vivo studies.

The work in this study is a significant step forward in understanding AC pathogenesis. As the functional understanding of AC increases, newer, more targeted therapeutic strategies will emerge with the potential to improve the lives of patients worldwide who suffer from this disease.

Conflict of interest: none declared.

Funding

This work was supported by NIH (RO1HL125881 and RO1HL140061) to R.A. (PI).