Buccal mucosa cells as a potential diagnostic tool to study onset and progression of arrhythmogenic cardiomyopathy
Authors: Van Der Voorn SM , Driessen HE , Bourfiss M , Van Lint FHM , Mirzad F , El Onsri L , Vos MA , Van Veen TAB 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Cardio Vascular Research Initiative (CVON) and 'Stichting Vriendenloterij'

Background
Arrhythmogenic cardiomyopathy (ACM) is predominantly caused by pathogenic variants in genes encoding desmosomal proteins (most often plakophilin-2). However, such variants are also found in genes encoding non-desmosomal proteins, such as phospholamban (PLN, p.Arg14del variant). Previous research showed that plakoglobin protein levels and localization in cardiac tissue of ACM patients, and p.Arg14del patients diagnosed with an ACM phenotype, are disturbed and this could be an additional tool to identify variant carriers at risk of developing ACM. Information about plakoglobin status can only be obtained via endocardial biopsies, however, this technique has major drawbacks, and therefore an alternative is needed. A promising new non-invasive tool is the use of buccal mucosa cells (BMC), as it has been reported that effects of pathogenic variants in desmosomal genes are reflected in non-cardiac tissues like BMC smears that also express those genes.

Purpose
To investigate the clinical usability of BMC as a tool to classify patients at risk of developing ACM by comparing controls with preclinical variant carriers and symptomatic ACM patients, and patients carrying the PLN p.Arg14del variant being preclinical, diagnosed with ACM or dilated cardiomyopathy (DCM).

Methods
BMC of 33 ACM patients (19 males, 28 with a PKP2 mutation), 17 PLN p.Arg14del patients (6 males, 3 diagnosed with ACM, 7 DCM and 7 preclinical carriers) and 34 controls (14 males), were collected on glass slides, fixed and labelled with anti-plakoglobin antibodies diluted 1:5.000, 1:10.000, 1:20.000 and 1:40.000, and scored for their membrane labelling.

Results
Data revealed that for each dilution, plakoglobin protein levels at the membrane were significantly reduced in BMC obtained from ACM patients (both in symptomatic patients and in preclinical variant carriers) when compared to controls. This effect was independent from age and sex of the patients. Dilutions 1:5.000 and 1:10.000 showed a moderate to strong correlation with the revised 2010 Task Force Criteria Score (TFC) which is commonly used for ACM diagnosis (rs = -0.67, n=64, p&lt;0.0001 and rs = -0.71, n=64, p&lt;0.0001 resp.). In contrast, plakoglobin scores of PLN p.Arg14del patients were comparable to controls (p&gt;0.209).

Conclusion
There is a significant reduction of plakoglobin protein in BMC of ACM patients and carriers with variants predisposing to ACM, but not for patients carrying the PLN p.Arg14del variant when compared to controls. However, for clinical diagnosis of the individual ACM patient, this method is not discriminative enough to distinguish true patients from preclinical variant carriers and controls, because of high interindividual variability.