Unique diagnostic signatures of concussion in the saliva of male athletes: the Study of Concussion in Rugby Union through MicroRNAs (SCRUM)

Objective To investigate the role of salivary small non-coding RNAs (sncRNAs) in the diagnosis of sport-related concussion. Methods Saliva was obtained from male professional players in the top two tiers of England's elite rugby union competition across two seasons (2017-2019). Samples were collected preseason from 1028 players, and during standardised head injury assessments (HIAs) at three time points (in-game, post-game, and 36-48 hours post-game) from 156 of these. Samples were also collected from controls (102 uninjured players and 66 players sustaining a musculoskeletal injury). Diagnostic sncRNAs were identified with next generation sequencing and validated using quantitative PCR in 702 samples. A predictive logistic regression model was built on 2017-2018 data (training dataset) and prospectively validated the following season (test dataset). Results The HIA process confirmed concussion in 106 players (HIA+) and excluded this in 50 (HIA-). 32 sncRNAs were significantly differentially expressed across these two groups, with let-7f-5p showing the highest area under the curve (AUC) at 36-48 hours. Additionally, a combined panel of 14 sncRNAs (let-7a-5p, miR-143-3p, miR-103a-3p, miR-34b-3p, RNU6-7, RNU6-45, Snora57, snoU13.120, tRNA18Arg-CCT, U6-168, U6-428, U6-1249, Uco22cjg1,YRNA_255) could differentiate concussed subjects from all other groups, including players who were HIA- and controls, immediately after the game (AUC 0.91, 95% CI 0.81 to 1) and 36-48 hours later (AUC 0.94, 95% CI 0.86 to 1). When prospectively tested, the panel confirmed high predictive accuracy (AUC 0.96, 95% CI 0.92 to 1 post-game and AUC 0.93, 95% CI 0.86 to 1 at 36-48 hours). Conclusions SCRUM, a large prospective observational study of non-invasive concussion biomarkers, has identified unique signatures of concussion in saliva of male athletes diagnosed with concussion.