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

Abstract

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.

Keywords: brain; concussion; contact sports; diagnosis; trauma.

Figure 1

Study profile. Participants were divided into concussion confirmed (HIA+) or ruled out (HIA−) after their head injury assessment (HIA), or controls, represented by players who played in the same game but were uninjured or had had a musculoskeletal injury. Twenty-three HIAs were excluded from the analysis owing to insufficient information to confirm the diagnosis from the HIA records and/or video footage after independent review. Twenty-four further HIAs were ineligible for inclusion, as the samples failed quality control checks.

Figure 2

Heat map representing the average value of the concentrations of the miRNAs across different groups. Hierarchical clustering was performed across groups and miRNAs to check the similar behaviour of the miRNAs. HIA, head injury assessment; MSK, musculoskeletal.

Figure 3

Plots of comparisons between players with a head injury (HIA+; n=106) and non-concussed groups (uninjured (n=102), HIA− (n=50) and musculoskeletal (n=66) groups)) at all time points. The y-axis represents the area under the curve (AUC) and the x-axis the log 2 expression of fold change of significantly differentially expressed sncRNAs between HIA+ and the other groups at each time point. The colour identifies the p values of the t-test analysis of each sncRNA (red ≤0.001, green ≤0.01 and >0.001, and blue ≤0.05 and >0.01). The full analysis results (AUC, CI, count, ΔCq average, SD, ΔΔcq, fold change and t-test p value) are available in the online supplemental materials. MSK, musculoskeletal; sncRNAs, small non-coding RNAs.

Figure 4

Longitudinal analysis. Thrty-two biomarkers selected as differentially expressed in the comparison HIA+ versus HIA− were used for the longitudinal analysis. Analysis of variance was performed in HIA+ and HIA− groups over time (T1, T2 and T3) and compared with baseline. Comparisons across multiple time points were evaluated using post-hoc Tukey’s honestly significant difference test. *Significantly different from baseline p<0.05; **significantly different form baseline p<0.001. HIA, head injury assessment.

Figure 5

Receiver operating characteristic curve results for a panel of 14 combined small non-coding RNAs differentiating players with confirmed concussion after the head injury assessment (HIA+) from other groups, including players who underwent a head injury assessment but had concussion ruled out (HIA−, orange line); Uninjured players from the same game who played a comparable number of minutes to those of the HIA players (uninjured, green line); players who were removed from the game due to musculoskeletal injuries (MSK, blue line); HIA−, uninjured and MSK groups combined (all combined, black line); and preseason values for concussed players (baseline, red line). The curves are shown for all time points (T1=during the game; T2=immediately after the game), and T3=36–48 hours after the game). Season 1 represents the training dataset and season 2 the test dataset (HIA+, HIA−, uninjured and MSK groups, as well as HIA−, uninjured and MSK groups combined) for the logistic regression model. Samples were not collected during the game in season 2.