Abstract
Introduction. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease characterized by progressive loss of cardiomyocytes and fibro-fatty replacement in the myocardium. Clinically, ARVC manifests with life-threatening ventricular arrhythmias, syncope, heart failure, and sudden cardiac death, which often occurs as the first clinical manifestation, especially in athletes and young adults engaged in effort. The diagnosis of ARVC is difficult to establish and it often occurs postmortem, thus highlighting the necessity of improving the diagnostic tools or to identify early biomarkers for the disease.
Aim of the study. Because of their implication in several cardiac diseases, we sought to identify circulating microRNAs (miRNAs) differentially expressed in ARVC patients compared to healthy controls (HCs), as potential biomarkers for ARVC.
Methods and results. In the pilot study, the expression level of 754 circulating miRNAs was analysed in 21 ARVC patients and 20 HCs using the Taqman Array Human MicroRNA A+B Card Set version 3.0. Both cohorts were divided into four pools. ARVC samples were pooled as follows: samples from 5 patients carrying a frameshift mutation in plakophilin-2 (PKP2) gene were grouped in pool 1; samples from 5 patients showing a severe arrhythmic phenotype were collected in pool 2; samples from 6 patients carrying variants in desmoplakin (DSP) and desmoglein-2 (DSG2) genes were grouped in pool 3, while samples from 5 more ARVC patients were collected in pool 4 without making a distinction based on genotype or phenotype. While no significant differences were detected in the circulating miRNA expression among the different patient pools, 8 circulating miRNAs showed significant differential expression between ARVC patients and HCs. After filtering the miRNAs considering a log fold-change cut-off of ± 1, p-value < 0.05 and the Bonferroni correction, miR-20b and miR-185-5p were selected for a subsequent validation study, based on TaqMan-based real-time PCR in samples from 37 ARVC patients and 30 HCs. Interesting, miR-185-5p resulted significantly upregulated in ARVC patients (2.34 ± 0.19, P < 0.0001) compared to HCs. In contrast, although significantly downregulated (0.55 ± 0.05, P < 0.001), miR-20b was not further considered in the study due to the lower fold change of its expression, compared to the pilot study. Receiver operating characteristic analysis suggested that miR-185-5p may be a predictive factor of ARVC, with an area under the curve of 0.854. Finally, in silico analyses on DIANA mirPath v.3, DIANA-micro T-CDS v.5.0 and MiRTarBase suggested the involvement of miR-185-5p in the regulation of cell adhesion and in Wnt/b-catenin and Hippo pathways, which were previously shown to be altered in ARVC.
Conclusion. This study corroborates the potential of miR-185-5p as a non-invasive circulating biomarker for ARVC, which could help achieve proper diagnosis and risk stratification in ARVC patients.