A microRNA Signature for the Diagnosis of Statins Intolerance

Abstract

Atherosclerotic cardiovascular diseases (ASCVD) are the leading cause of morbidity and mortality in Western societies. Statins are the first-choice therapy for dislipidemias and are considered the cornerstone of ASCVD. Statin-associated muscle symptoms are the main reason for dropout of this treatment. There is an urgent need to identify new biomarkers with discriminative precision for diagnosing intolerance to statins (SI) in patients. MicroRNAs (miRNAs) have emerged as evolutionarily conserved molecules that serve as reliable biomarkers and regulators of multiple cellular events in cardiovascular diseases. In the current study, we evaluated plasma miRNAs as potential biomarkers to discriminate between the SI vs. non-statin intolerant (NSI) population. It is a multicenter, prospective, case-control study. A total of 179 differentially expressed circulating miRNAs were screened in two cardiovascular risk patient cohorts (high and very high risk): (i) NSI (n = 10); (ii) SI (n = 10). Ten miRNAs were identified as being overexpressed in plasma and validated in the plasma of NSI (n = 45) and SI (n = 39). Let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p were overexpressed in the plasma of SI patients. The receiver operating characteristic curve analysis supported the discriminative potential of the diagnosis. We propose a three-miRNA predictive fingerprint (let-7f, miR-376a-3p and miR-376c-3p) and several clinical variables (non-HDLc and years of dyslipidemia) for SI discrimination; this model achieves sensitivity, specificity and area under the receiver operating characteristic curve (AUC) of 83.67%, 88.57 and 89.10, respectively. In clinical practice, this set of miRNAs combined with clinical variables may discriminate between SI vs. NSI subjects. This multiparametric model may arise as a potential diagnostic biomarker with clinical value.

Keywords: atherosclerotic cardiovascular diseases; biomarkers; circulating microRNAs; statin intolerance; statins-adverse myalgia symptoms.

Figure 1

Color heatmap based on raw miRNA expression values where each column represents a patient, and each row represents a miRNA. The color scale illustrates the relative expression level of miRNAs (red and yellow represent low expression and blue and purple represent high expression). MiRNA expression levels were normalized to miR-148a-3p and let-7b-5p. MiRNA: microRNA; NSI: non-statin intolerant; SI: statin intolerant.

Figure 2

Boxplots of miRNA expression levels in NSI and SI cohorts. The analysis was carried out using qPCR. Data are presented in log2. Data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005. Error bars represent SDs. NSI, non-statin intolerant; SI: statin intolerant.

Figure 3

ROC curves for evaluating the predictive performance of differentially expressed miRNAs to discriminate between SI vs. NSI. (A) ROC curves for let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p. (B) The ROC curve of the 5-miRNA panel combination value of let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p. AUC: area under the curve; miRNA: microRNA; NSI: non-statin intolerant; SI: statin intolerant.

Figure 4

ROC curves for evaluating the predictive performance of clinical factors with differentially expressed miRNAs. (A) Box plot of DLP in NSI (n = 45) and SI (n = 39) subjects. (B) Box plot of non-HDLc levels in NSI (n = 45) and SI (n = 39) subjects. (C) ROC curves for each clinical parameter, DLP and non-HDLc, and for the association of DLP plus non-HDLc. (D) The ROC curve of the combined value of the 3-miRNA panel (let-7f-5p, miR-376a-3p and miR-376c-3p), DLP and non-HDLc plasmatic concentration. DLP: years of dyslipidemia SI: statin intolerant; NSI: non-statin intolerant; non-HDLc: non-high-density lipoprotein cholesterol. * p < 0.05; *** p < 0.005.

Figure 5

ROC curve of 10-fold cross validation test for 3-miRNA panel + DLP + non-HDLc model (Ada Boost M1).

Figure 6

KEGG and GO analysis of differentially expressed miRNAs. (A) miRNA-gene network for of let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p. (B) Venn diagram showing overlap of gene targets of let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p. (C) GO and KEGG functional enrichment analysis of let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p.

Figure 7

The flowchart of the study design. This figure illustrates the experimental workflow of the study including screening and validation. MiRNA: microRNA; NSI: non-statin intolerant; SI: statin intolerant.