Comparison of serum and saliva miRNAs for identification and characterization of mTBI in adult mixed martial arts fighters

Abstract

Traumatic brain injury (TBI) is a major cause of death and disability worldwide, with mild TBI (mTBI) accounting for 85% of cases. mTBI is also implicated in serious long-term sequelae including second impact syndrome and chronic traumatic encephalopathy. mTBI often goes undiagnosed due to delayed symptom onset and limited sensitivity of conventional assessment measures compared with severe TBI. Current efforts seek to identify accurate and reliable non-invasive biomarkers associated with functional measures relevant to long-term outcomes. Here we evaluated the utility of serum and salivary microRNAs (miRNAs) to serve as sensitive and specific peripheral biomarkers of possible mTBI. Our primary objectives were to establish the relationship between peripheral measures of miRNA, objective quantification of head impacts, and sensitive indices of balance and cognitive function in healthy young adult athletes. A secondary objective was to compare the sensitivity of miRNA versus commonly used blood-based protein biomarkers. 50 amateur mixed martial arts (MMA) fighters participated. 216 saliva and serum samples were collected at multiple time points, both pre- and post-fight. Levels of 10 serum proteins were compared in a subset of the fighters (n = 24). Levels of miRNAs were obtained by next generation sequencing. Functional outcomes were evaluated using a computerized assessment system that measured cognitive performance, body sway, and combined cognitive performance and body sway during dual task completion. Data were analyzed using multivariate logistic regression for predictive classification, analysis of variance, correlation analysis and principal component analysis. We identified a subset of salivary and serum miRNAs that showed robust utility at predicting TBI likelihood and demonstrated quantitative associations with head impacts as well as cognitive and balance measures. In contrast, serum proteins demonstrated far less utility. We also found that the timing of the responses varies in saliva and serum, which is a critical observation for biomarker studies to consider.

Diagnosing mild traumatic brain injury using saliva RNA compared to cognitive and balance testing

“…Balance was also assessed for a portion of participants (n = 321; mTBI = 176 and control = 145) using the validated ClearEdge system 42,43 (Quadrant Biosciences Inc, Syracuse, NY), as we have previously reported. 39…”

“…RNA was isolated from each saliva sample using the miRNeasy Kit (Qiagen, Inc, Germantown, MD) according to the manufacturer's instructions, as we have previously described. 39 RNA quality was assessed using the Agilent Technologies Bioanalyzer on the RNA Nanochip. RNA-sequencing libraries were prepared using the TruSeq Stranded Small RNA Kit (Illumina) according to manufacturer instructions.…”

“…38 Finally, we showed that miRNA levels change within hours of mTBI, and this response occurs in saliva before serum. 39 To date, the diagnostic potential of salivary ncRNAs has not been assessed in a large mTBI cohort with adequate controls (ie, individuals with orthopedic injury [OI], chronic headache, recent exercise, and neuropsychological comorbidities). Further, the diagnostic accuracy of ncR-NAs relative to current standard-of-care assessments has not been tested.…”

Saliva microRNA Biomarkers of Cumulative Concussion

“…Previously, we showed that perturbation in miRNA signaling after traumatic brain injury results in overlapping miRNA profiles in cerebrospinal fluid and saliva [ 31 ]. Further, we demonstrated that disruptions in salivary miRNA levels can persist for weeks after initial injury [ 32 ], and predict the occurrence of persistent post-concussive symptoms [ 33 ]. The purpose of the current study was to determine whether non-invasive salivary measurement of miRNAs could provide insights into the pathobiology of repetitive concussions among professional football athletes.…”

Towards the Development of an Integrative, Evidence-Based Suite of Indicators for the Prediction of Outcome Following Mild Traumatic Brain Injury: Results from a Pilot Study

“…The temporal stability of blood-based biomarkers is increasingly being recognised as a potential limitation to their routine clinical use. Thus, non-protein based biomarkers such as microRNAs may be better suited for the prediction of outcome following mTBI [116][117][118][119].…”