Association of Salivary MicroRNA Changes With Prolonged Concussion Symptoms

Abstract

Importance: Approximately one-third of children who experience a concussion develop prolonged concussion symptoms. To our knowledge, there are currently no objective or easily administered tests for predicting prolonged concussion symptoms. Several studies have identified alterations in epigenetic molecules known as microRNAs (miRNAs) following traumatic brain injury. No studies have examined whether miRNA expression can detect prolonged concussion symptoms.

Objective: To evaluate the efficacy of salivary miRNAs for identifying children with concussion who are at risk for prolonged symptoms.

Design, setting, and participants: This prospective cohort study at the Penn State Medical Center observed 52 patients aged 7 to 21 years presenting for evaluation of concussion within 14 days of initial head injury, with follow-up at 4 and 8 weeks.

Exposures: All patients had a clinical diagnosis of concussion.

Main outcomes and measures: Salivary miRNA expression was measured at the time of initial clinical presentation in all patients. Patients with a Sport Concussion Assessment Tool (SCAT3) symptom score of 5 or greater on self-report or parent report 4 weeks after injury were designated as having prolonged symptoms.

Results: Of the 52 included participants, 22 (42%) were female, and the mean (SD) age was 14 (3) years. Participants were split into the prolonged symptom group (n = 30) and acute symptom group (n = 22). Concentrations of 15 salivary miRNAs spatially differentiated prolonged and acute symptom groups on partial least squares discriminant analysis and demonstrated functional relationships with neuronal regulatory pathways. Levels of 5 miRNAs (miR-320c-1, miR-133a-5p, miR-769-5p, let-7a-3p, and miR-1307-3p) accurately identified patients with prolonged symptoms on logistic regression (area under the curve, 0.856; 95% CI, 0.822-0.890). This accuracy exceeded accuracy of symptom burden on child (area under the curve, 0.649; 95% CI, 0.388-0.887) or parent (area under the curve, 0.562; 95% CI, 0.219-0.734) SCAT3 score. Levels of 3 miRNAs were associated with specific symptoms 4 weeks after injury; miR-320c-1 was associated with memory difficulty (R, 0.55; false detection rate, 0.02), miR-629 was associated with headaches (R, 0.47; false detection rate, 0.04), and let-7b-5p was associated with fatigue (R, 0.45; false detection rate, 0.04).

Conclusions and relevance: Salivary miRNA levels may identify the duration and character of concussion symptoms. This could reduce parental anxiety and improve care by providing a tool for concussion management. Further validation of this approach is needed.

Figure 1.. Discriminant Analysis of Prolonged Concussion Symptom (PCS) and Acute Concussion Symptom (ACS) Groups and 15 MicroRNAs (miRNAs) With Highest Variable Importance

A, Partial least squares discriminant analysis of total miRNA expression in participants with ACS and PCS. The 2-dimensional partial least squares discriminant analysis accounts for 21.5% of the total variance in the miRNA data. The small and large ellipses indicate 95% CIs for the dispersion of participants with PCS and ACS, respectively. B, Variable importance of projection (VIP) score identifying the 15 miRNAs most important for separating participants with ACS from those with PCS. Seven miRNAs were found to be increased in the PCS group (black boxes), and 8 miRNAs were found to be decreased in the PCS group (white boxes). ^aIdentified in previous studies of traumatic brain injury.

Figure 2.. Pearson Correlation Analysis Between Salivary MicroRNA Concentrations and Concussive Symptoms

Pearson correlation with R values depicting associations between salivary concentrations of the 15 microRNAs of interest (at initial assessment) and concussive symptom severity (at 4 weeks after injury) measured via Sport Concussion Assessment Tool (SCAT3) symptom report. Bolded values indicate nominal significance (P < .05), and highlighted boxes indicate significance (P < .05) following multiple testing corrections. Color-scale values indicate Pearson correlation between 2 features, where red indicates an inverse association and green indicates a direct association. PCS indicates prolonged concussion symptom.

Figure 3.. Multivariate Logistic Regression Analyses for Salivary MicroRNAs (miRNAs) and Subjective Symptom Reports

The 5 miRNAs of interest accurately identified prolonged concussion symptoms in a multivariate regression analysis. A, A receiver operator characteristic curve using salivary concentrations of 5 miRNAs (miR-320c-1, miR-133a-5p, miR-769-5p, let-7a-3p, and miR-1307-3p) demonstrated an area under the curve (AUC) of 0.856 (95% CI, 0.822-0.890) for prolonged concussion symptom (PCS) status. B, 10-Fold cross-validation of this tool demonstrated an AUC of 0.812 (95% CI, 0.691-0.893) for identifying PCS status. C, 10-Fold cross-validation of this tool holding out 20% of participants with acute and prolonged concussion symptoms at random demonstrated an AUC of 0.792 (95% CI, 0.673-0.958) in the cross-validation set and an AUC of 0.933 (95% CI, 0.787-0.960) in the holdout set. D, Logistic regression model using the total child Sport Concussion Assessment Tool (SCAT3) severity score demonstrated an AUC of 0.649 (95% CI, 0.388-0.887) for determining PCS status. E, Logistic regression model using total parent SCAT3 severity score demonstrated an AUC of 0.562 (95% CI, 0.219-0.734) for identifying PCS status. F, Modified clinical risk score including sex, age, previous concussion history, headache, fatigue, processing difficulty, and migraine history demonstrated an AUC of 0.625 (95% CI, 0.093-0.848) for determining PCS status.