Cell-Free mRNA Sequencing of Human CSF for Molecular Characterization of Severe Traumatic Brain Injury

Introduction: Current methods of characterizing severe traumatic brain injury (sTBI) are limited, limiting advancements of sTBI treatment and prognostication. CSF TBI biomarker research has focused on protein and microRNA due to their stability and ease of isolation. In comparison, messenger RNA (mRNA) holds greater potential to detail the heterogeneous molecular landscape of sTBI. Towards this end, we isolated and analyzed cell-free mRNA (cfRNA) from CSF of individual sTBI patients using next-generation sequencing (NGS, RNA-seq).

Methods: Under an IRB-approved protocol, 2mL CSF aliquots were collected serially from external ventricular drains of sTBI patients using two different pre-analytical conditions. Controls included CSF collected intraoperatively from patients with normal pressure hydrocephalus (NPH) undergoing ventriculoperitoneal shunt placement. Demographic and clinical data were collected prospectively. cfRNA was isolated, converted to cDNA, and sequenced using NGS.

Results: Eighteen samples were collected from seven sTBI patients over the course of seven days after injury. sTBI patients had a median age of 48 years (IQR 33-56), presenting Glasgow Coma Scale score of 7 (IQR 6-8), and 14% were female. CSF was collected from four control NPH patients. All samples had at least 17,285 unique mRNA transcripts detected. Control and sTBI patients clustered separately on principal component analysis. Expression of frequently studied TBI biomarkers, such as UCH-L1, GFAP, S100B and NSE, were detected in all samples. Gene expression varied between paired samples collected using different pre-analytical conditions.

Conclusion : We present the first-in-human pilot study sequencing cfRNA isolated from small volumes of individual sTBI patient CSF as an initial step towards identifying molecular mechanisms underlying brain injury in the acute and subacute phases following sTBI. Importantly, this study has implications for how CSF collection methods in clinical trials and multi-center collaborations can influence future NGS results.