Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington's disease

Despite the advances in the understanding of Huntington's disease (HD), there is the need for molecular biomarkers to categorize mutation-carriers during the preclinical stage of the disease preceding functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are downregulated early in mutation-carriers and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p, and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR-21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species highlighting the progression and cognitive changes occurring at the premanifest stage. Overall design: To evaluate the biomarker potential of plasma extracellular vesicles (EVs) and their sRNAs in Huntington's disease, we first analyzed the the sRNA distribution in the different plasma subfractions obtained through Size-Exclusion Chromatography (SEC) using samples from healthy individuals. Plasma samples from healthy non-mutation carriers and Huntington's disease patients, both at the premanifest and manifest stages, were subjected to SEC and ultrafiltration to isolate EVs. RNA from the vesicles was obtained and 10 samples per group were used for smallRNAseq. Small RNAseq data were processed and analyzed using two bioinformatic tools: ExceRpt and SeqCluster. Comparative univariate and multivariate analyses were performed to identify differentially expressed small RNAs between Huntington's disease patients and healthy subjects. Results from smallRNAseq were further validated using qRT-PCR in 20 samples per group.