Profile of plasma microRNAs as a potential biomarker of Wilson's disease

Abstract

Background: Wilson's disease (WD) is a rare condition resulting from autosomal recessive mutations in ATP7B, a copper transporter, manifesting with hepatic, neurological, and psychiatric symptoms. Timely diagnosis and appropriate treatment yield a positive prognosis, while delayed identification and/or insufficient therapy lead to a poor outcome. Our aim was to establish a prognostic method for WD by characterising biomarkers based on circulating microRNAs.

Methods: We conducted investigations across three cohorts: discovery, validation (comprising unrelated patients), and follow-up (revisiting the discovery cohort 3 years later). All groups were compared to age- and gender-matched controls. Plasma microRNAs were analysed via RNA sequencing in the discovery cohort and subsequently validated using quantitative PCR in all three cohorts. To assess disease progression, we examined the microRNA profile in Atp7b^-/- mice, analysing serum samples from 6 to 44 weeks of age and liver samples at three time points: 20, 30, and 40 weeks of age.

Results: In patients, elevated levels of the signature microRNAs (miR-122-5p, miR-192-5p, and miR-885-5p) correlated with serum activities of aspartate transaminase, alanine aminotransferase and gamma-glutamyl transferase. In Atp7b^-/- mice, levels of miR-122-5p and miR-192-5p (miR-885-5p lacking a murine orthologue) increased from 12 weeks of age in serum, while exhibiting fluctuations in the liver, possibly attributable to hepatocyte regenerative capacity post-injury and the release of hepatic microRNAs into the bloodstream.

Conclusions: The upregulation of the signature miR-122-5p, miR-192-5p, and miR-885-5p in patients and their correlation with liver disease progression in WD mice support their potential as biomarkers of WD.

Keywords: Drug monitoring; Prognosis; miR-122-5p; miR-192-5p; miR-885-5p.

Fig. 1

Analysis of the miRNA profile. a Discovery, b validation, and c follow-up cohorts. Comparative analysis of miRNA levels between patients and controls, representing IQR (interquartile range) of log₂FC relative expression (2^−ΔΔCt). ****P value < 0.0001; ***P value < 0.001; **P value < 0.01; *P value < 0.05

Fig. 2

Comparative analysis of the relative expression of miRNA levels by gender from case groups from the a discovery, c validation, and e follow-up cohorts. Correlation matrix including biochemical parameters, age, and levels of miRNAs profile in patients belonging to the b discovery, d validation, and f follow-up cohorts. In each cell, Spearman’s coefficient (R_s) of correlation by pair of variables is indicated. Coloured cells represent significant correlation (*P value < 0.05) in orange when positive, and in blue when negative. ns not significant, AP alkaline phosphatase, AST aspartate aminotransferase, ALT alanine aminotransferase, GGT gamma-glutamyl transferase, L-score Leipzig score

Fig. 3

Validation of the miRNAs profile in serum of wild-type (WT) and Atp7b^−/− mice (WD, Wilson’s disease) samples. a Comparative analysis of miR-122 levels and, b miR-192 levels between groups at different ages, representing IQR (interquartile range) of log₂FC relative expression (2^−ΔΔCt). c Comparative analysis of relative expression miRNA levels from Atp7b^−/− group from a and b by sex. ****P value 0.0001; **P value < 0.01; *P value < 0.05

Fig. 4

Validation of the liver tissue miRNAs profile in wild-type (WT) and Atp7b^−/− (WD) mice samples. a Comparative analysis of miR-122 levels and, b miR-192 levels between groups at different ages, representing IQR (interquartile range) of log₂FC relative expression (2^−ΔΔCt)