Circulating miR-499a-5p Is a Potential Biomarker of MYH7-Associated Hypertrophic Cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disease with significant genetic and phenotypic heterogeneity. To search for novel biomarkers, which could increase the accuracy of HCM diagnosis and improve understanding of its phenotype formation, we analyzed the levels of circulating miRNAs—stable non-coding RNAs involved in post-transcriptional gene regulation. Performed high throughput sequencing of miRNAs in plasma of HCM patients and controls pinpointed miR-499a-5p as one of 35 miRNAs dysregulated in HCM. Further investigation on enlarged groups of individuals showed that its level was higher in carriers of pathogenic/likely pathogenic (P/LP) variants in MYH7 gene compared to controls (fold change, FC = 8.9; p < 0.0001). Just as important, carriers of variants in MYH7 gene were defined with higher miRNA levels than carriers of variants in the MYBPC3 gene (FC = 14.1; p = 0.0003) and other patients (FC = 4.1; p = 0.0008). The receiver operating characteristic analysis analysis showed the ability of miR-499a-5p to identify MYH7 variant carriers with the HCM phenotype with area under the curve value of 0.95 (95% confidence interval: 0.88−1.03, p = 0.0004); sensitivity and specificity were 0.86 and 0.91 (cut-off = 0.0014). Therefore, miR-499a-5p could serve as a circulating biomarker of HCM, caused by P/LP variants in MYH7 gene.

Keywords: MYH7; MYH7B; RNA-seq; biomarker; hypertrophic cardiomyopathy; miR-499-5p; miRNA; pathogenic genetic variants.

Figure 1

The comparison of the profiles of circulating miRNAs in patients with HCM (n = 9) and controls (n = 6). On the graph, the X-axis represents the values of the fold change (FC) in miRNA levels, the Y-axis represents the p values, all in a logarithmic scale. The dotted line indicates threshold for statistical significance p = 0.05; solid line—threshold for statistical significance adjusted p-value (p.adj) = 0.05; dots—miRNAs.

Figure 2

Plasma levels of miR-208b and miR-499a-5p in 29 HCM patients and 32 healthy controls as determined by RT-qPCR. Here and below, miRNA levels were calculated relative to cel-miR-39-5p using the delta-delta Ct method. Data is presented using a scatter plot (mean with SD). Differences in miRNA levels between HCM patients and healthy controls were insignificant (p > 0.05) according to a Mann-Whitney U test. The green dots define levels of miR-208b and miR-499a-5p in controls, blue dots and red dots—levels of miR-208b and miR-499a-5p, respectively, in patients with HCM. HCM—hypertrophic cardiomyopathy.

Figure 3

Level of circulating miR-499a-5p in each HCM patient depending on sequencing data of HCM-associated genes. ND—patients in whom mutations in sequenced genes were not determined.

Figure 4

Levels of miR-499a-5p in carriers of mutations in MYH7 gene (n = 7), in carriers of mutations in MYBPC3 gene (n = 8), in mixed group of non-MYH7 and non-MYBPC3 mutations carriers and of genotype-negative patients (n = 14); and controls (n = 32) as determined by RT-qPCR. Significant differences were determined by using a Mann-Whitney U test.

Figure 5

The ROC curve analysis of circulating miR-499a-5p for detecting MYH7-positive HCM patients. ROC—receiver operating characteristic; AUC—area under the curve.