Circular RNAs as Potential Blood Biomarkers in Amyotrophic Lateral Sclerosis

Abstract

Circular RNAs (circRNAs) are emerging as a novel, yet powerful player in many human diseases. They are involved in several cellular processes and are becoming a noteworthy type of biomarkers. Among other functions, circRNAs can serve as RNA sponges or as scaffolds for RNA-binding proteins. Here, we investigated a microarray expression profile of circRNAs in leukocyte samples from ALS patients and age- and sex-matched healthy controls to identify differentially expressed circRNAs. We selected 10 of them for a qPCR validation of expression on a larger set of samples, identification of their associations with clinical parameters, and evaluation of their diagnostic potential. In total, expression of 7/10 circRNAs was significant in a larger cohort of ALS patients, compared with age- and sex-matched healthy controls. Three of them (hsa_circ_0023919, hsa_circ_0063411, and hsa_circ_0088036) showed the same regulation as in microarray results. These three circRNAs also had AUC > 0.95, and sensitivity and specificity for the optimal threshold point > 90%, showing their potential for using them as diagnostic biomarkers.

Keywords: Amyotrophic lateral sclerosis; Biomarkers; Circular RNAs; Differential expression; Human blood samples.

Fig. 1

Summary of microarray expression profile. a Hierarchical clustering and heatmap visualization of circRNA expression levels. P, ALS patient; C, healthy control. b Volcano plot representation of differentially expressed circRNAs (red points; p value < 0.05 and fold change > 1.5). Two hundred seventy-four of them were upregulated and 151 were downregulated when comparing ALS patients and healthy controls

Fig. 2

Expression of selected circRNAs in ALS patients and healthy controls. Relative expression levels of each circRNA in ALS patients (n = 60) and healthy controls (n = 15) are represented with box plot. Upregulated circRNAs are shown in green, downregulated circRNAs in red, and circRNAs with non-significant differences in expression in blue; significant difference in expression levels is denoted as * (p < 0.05) or *** (p < 0.001); n. s., non-significant

Fig. 3

ROC curves for the circRNAs with statistically significant difference in expression. Full colored lines represent circRNAs with AUC > 0.950, dashed gray lines represent other circRNAs. a ROC curves for upregulated circRNAs. b ROC curve for downregulated circRNA. c Details of shown ROC curves. AUC, area under the curve, SE, standard error for AUC, 95% C. I., 95% confidence interval for AUC. Optimal threshold point was determined as the point on the curve with minimal distance to the ideal point (sensitivity = 1 and specificity = 1)