The Diagnostic Potential of miRNAs in Pulmonary Arterial Hypertension

Rationale
Pulmonary Arterial Hypertension (PAH) is a rare life limiting and difficult to diagnose orphan disease and a common complication of systemic sclerosis. Micro-RNAs (miRNAs) are post transcriptional regulators of gene expression detectable in circulating fractions and have been reported as potential biomarkers in multiple diseases. Circulating miRNA indicators of survival in PAH have been published, however, diagnostic miRNA panels have yet to be explored.

Objective
To determine if circulating miRNAs can be used to identify the presence of PAH in patients with systemic sclerosis who are at a known risk of developing PAH.

Methods
A miRNA microarray screen was used to determine if plasma or whole blood was more suitable for biomarker detection in a case/control cohort (n = 28/17) of healthy volunteers (HV) and PAH.

A further microarray screen in HV/PAH (n = 33/47) was used to develop a multiple biomarker panel discovery process using ROC determined cut-offs.

Finally, potential miRNA biomarkers were identified in a microarray screen of SSc / SSc-PAH (n = 10/18) and validated using a larger cohort (n = 14/29) by qPCR.

Results
Plasma was selected as the source of miRNAs to use in further investigations. Differential expression analysis identified 90 plasma miRNAs vs 1 whole blood miRNA as significantly differentially expressed (p < 0.05). Additionally, in a feature selection task 0 miRNAs from whole blood were identified by the Boruta algorithm compared to 56 miRNAs from plasma.

11 miRNAs were selected for qPCR verification as constituents of potential biomarker panels. Mir-483-5p, miR-584-5p and miR-638 had acceptable amplification profiles and were tested in the larger SSc/SSc-PAH verification cohort. From those, miR-483-5p correlated with the microarray findings, r = 0.55, p < 0.01 and expression was increased in disease p = 0.02, adjusted p = 0.056. Furthermore, when all 3 miRNAs were used in a panel an increase in predictive power was seen from a maximum individual AUC of 0.69 to a panel AUC of 0.72.

Conclusion
This work highlights the potential utility of miR signatures as a diagnostic tool in PAH. MiRNA-483-5p was identified as a miRNA with relevance to PAH in patients with SSc, this miRNA has not previously been reported in PAH literature but has links to lung cancer and warrants further investigation.