## 4 Discussion
To our knowledge, our pilot study is one of the first to examine peripheral global DNA methylation in association with mTBI, which could contribute to better understanding of underlying biological mechanisms of post-injury symptoms. We found that individuals with mTBI had significantly higher levels of blood global methylation ratio (5-mC%) than healthy controls and the significance remains when controlling for age. Because of changes in the blood-brain barrier permeability and the entire gliovascular unit after mTBI, blood biomarkers may be developed into clinical monitoring tools to detect brain damage [10]. Even when repetitive or multiple head injuries over time are involved,which might not be accurately assessed, epigenetic changes, such as in global DNA methylation, can be the best biomarker to detect pathologic process after mTBI because epigenetic mechanisms have long-lasting effects within mature neurons without altering the DNA code [71]. Usually located in a gene promoter, DNA methylation typically acts to repress gene transcription [72]. DNA methylation is essential for normal development and is associated with a number of key processes including aging and carcinogenesis (i.e., global hypomethyllation) [73; 74]. Because our participants were cancer-free, their increased global methylation ratio could be a unique biomarker related to mTBI and/or post-injury functional declines. This pilot study has limitations, although the main findings support the changes in cognitive, psychosocial, and global DNA methylation after mTBI. The sample size is small in the present study. Based on the current findings, a larger sample should be examined in a future study to confirm the effectiveness of our comprehensive functions screening and peripheral epigenetic test. Past mTBI events were assessed based on the college student participants' self-report. Although we used a structured interview questionnaire including the GCS and post-traumatic amnesia information, many participants were either unaware of those clinical characteristics/measures or incompetent with recalling memory on such information. However, this is not an unusual situation with individuals who suffered from mTBI, as the majority appears to avoid necessary post-injury screening in a clinical setting [35; 36], especially among college student athletes and student veterans who consider the mTBI diagnosis a stigma and/or a hindrance to their return to play or duty and therefore, avoid screening [37; 38]. In addition, the King-Devick and accommodation visual tests and force-platform postural stability test could not identify any differences between mTBI cases and controls. As discussed before, those measures may not be sensitive enough to detect mTBI-specific functional changes. In future studies, with increased sample sizes, unsupervised and more systemic approaches to detect a wider range of functional variability--such as wearable sensor devices [65] and an eye tracking method [66; 67]--is recommended. Regardless, our main findings revealed several functional deficits long after mTBI, which could contribute to early detection of mTBI symptoms for ensuring early intervention and rehabilitation. In particular, college student athletes and veterans with untreated mTBI face the unique pressures of academic success, coupled with the demands of reintegration to the civilian life, juggling young families and jobs, and/or rigorous training schedules. Navigating any of these issues with a brain injury, diagnosed or undiagnosed, can be profoundly challenging, but very important [39]. There are no available diagnostic tests to assess recovery and no proven treatments to reduce functional damages from mTBI.