Discovery and Verification of Extracellular miRNA Biomarkers for Non-invasive Prediction of Pre-eclampsia in Asymptomatic Women

Abstract

Development of effective prevention and treatment strategies for pre-eclampsia is limited by the lack of accurate methods for identification of at-risk pregnancies. We performed small RNA sequencing (RNA-seq) of maternal serum extracellular RNAs (exRNAs) to discover and verify microRNAs (miRNAs) differentially expressed in patients who later developed pre-eclampsia. Sera collected from 73 pre-eclampsia cases and 139 controls between 17 and 28 weeks gestational age (GA), divided into separate discovery and verification cohorts, are analyzed by small RNA-seq. Discovery and verification of univariate and bivariate miRNA biomarkers reveal that bivariate biomarkers verify at a markedly higher rate than univariate biomarkers. The majority of verified biomarkers contain miR-155-5p, which has been reported to mediate the pre-eclampsia-associated repression of endothelial nitric oxide synthase (eNOS) by tumor necrosis factor alpha (TNF-α). Deconvolution analysis reveals that several verified miRNA biomarkers come from the placenta and are likely carried by placenta-specific extracellular vesicles.

Figure 1.. Study Design and Characteristics of Discovery and Verification Sets

(A) Overall study design. Maternal serum samples were collected at both UCSD site (n = 48) and Sera Prognostics site (n = 164). The samples were split into a discovery set (n = 141) and verification set (n = 71) with matching gestational ages and disease severity. Within each set, there were roughly 2× the number of controls as cases. (B) Distribution of cases (49 in discovery and 24 in verification) and controls (92 in discovery and 24 in verification). (C) Distribution of GABD. (D) Correlation between discovery and verification AUCs. Scatterplot of the discovery and verification AUCs for candidate predictors for pre-eclampsia is shown. Best-fit linear trendline and r² value are shown. See also Figure S1.

Figure 2.. Heatmaps of Reversals Selected in Discovery for Each GABD Window

All heatmaps show normalized reversal scores for both discovery and verification subjects, for the 50 reversals selected in discovery. Trackbars above the heatmaps indicate pre-eclampsia cases (orange) and controls with normal pregnancy outcomes (turquoise) and membership in the discovery (red) or verification (blue) set. The color bar is in units of standard deviations of reversal score distributions. Asterisks indicate reversals that passed verification. (A) Data for 50 reversals identified in discovery for the early GABD window, for the early GABD subjects (n = 67; discovery and verification sets). (B) Middle GABD window for the middle GABD subjects (n = 93). (C) Late GABD window for the late GABD subjects (n = 132). (D) Full GABD window for all subjects (n = 212). See also Tables S3 and S4.

Figure 3.. Cell and Tissue miRNA Expression and Deconvolution Analysis

(A) PCA plot showing unsupervised clustering of cell and tissue types by miRNA profiling data. (B) Hierarchical clustering of cell and tissue types with heatmap of differentially expressed miRNAs (q < 10⁻¹²). (C) Box-and-whisker plot of deconvolution results, indicating the percent contribution of each cell/tissue type to the extracellular miRNA profiles of each of the maternal serum samples in this study (both discovery and verification cohorts; n = 212). (D) Bar graphs showing, for each cell/tissue type, the number of numerators/univariate predictors (top) and denominators (bottom) for which that cell/tissue type was a major contributor. See also Table S5.

Figure 4.. miRNAs Associated with Different Carrier Subclasses

Heatmap showing eight sets of co-expressed miRNAs in pooled third-trimester control sample (n = 1; technical replicates = 3) identified by hierarchical clustering. Each set of miRNAs is labeled with the associated carrier subclasses. See also Table S6.