Unraveling the enigma of DSG2 p.(N266S): a culprit in arrhythmogenic cardiomyopathy

Abstract Background Desmoglein-2 (DSG2, MIM#610193) is a transmembrane desmosomal glycoprotein in which primarily truncating variants are thought to cause arrhythmogenic cardiomyopathy (ACM) (OMIM#107970; ORPHA247). ACM pathognomonic features include electrical instability and fibro-fatty replacement of the myocardial tissue. The rare DSG2 missense variant encoding for p.(N266S), identified in an Italian heart transplanted ACM proband, represents one of the pioneering findings linking this gene to the biventricular ACM. Although this missense variant was later recapitulated also in an animal model, its clinical significance remains an open question. Purpose The purpose of this study is to explore pathognomonic features of the DSG2 p.(N266S)-related cardiomyopathy through family studies and experimental evidence. Methods Cardiologic and genetic evaluation followed current ESC and ACMG recommendations, respectively. An in vitro system based on HL-1 cells and 2 geneticin resistant-GFP plasmids, carrying either DSG2 Wild-type (Wt) or the p.(N266S) variant fused to FLAG tag, were used to established HL-1 Wt and HL-1 Mut cell lines. Transfection was obtained by 4-D Nucleofector System (Lonza©), optimizing the manufacturer’s protocol, and isolating transfected beating clones. Moreover, to generate a patient-tailored in vitro system, CD34+ hematopoietic progenitors were isolated from blood of patients and healthy donors and then reprogrammed into induced pluripotent stem cells (iPSCs). Later, patient-derived iPSCs were differentiated into cardiomyocytes. Functional analysis and cell characterization were performed by Sanger sequencing, Real-time PCR, electron transmission microscopy (TEM), confocal microscopy, calcium imaging, and patch clamp. Results DSG2 p.(N266S) missense variant was identified in four families of our patient cohort exhibiting ACM phenotypes with biventricular involvement, arrhythmias, and ECG abnormalities (Fig.1). HL-1 cells were able to continuously divide and spontaneously contract while maintaining a differentiated cardiac phenotype. Antibiotic selection, Real-Time PCR, and Sanger sequencing confirmed the ability of transfected HL-1 cells to express the plasmid carrying the DSG2 p.(N266S) variant. Confocal imaging revealed the insertion of the DSG2 mutated protein into cardiac desmosomes while TEM imaging showed evidence of gap widening in HL-1 Mut. Moreover, calcium imaging and patch clamp revealed that the calcium flux and the beating phenotype were altered in HL-1 Mut. Desmosomal widening and electrophysiological alterations were confirmed in iPSCs-derived cardiomyocytes. Conclusions We provided further evidence to support DSG2 p.(N266S) pathogenicity in arrhythmogenic cardiomyopathy through a combination of clinical observations in four independent affected families and experimental findings, demonstrating desmosomal abnormalities and electrophysiological alterations.