Injuries from exposure to explosions rose dramatically during the Iraq and Afghanistan wars, which motivated investigations of blast-related neurotrauma and operational breaching. In this study, military ''breachers'' were exposed to controlled, lowlevel blast during a 10-day explosive breaching course. Using an omics approach, we assessed epigenetic, transcriptional, and inflammatory profile changes in blood from operational breaching trainees, with varying levels of lifetime blast exposure, along with daily self-reported symptoms (with tinnitus, headaches, and sleep disturbances as the most frequently reported). Although acute exposure to blast did not confer epigenetic changes, specifically in DNA methylation, differentially methylated regions (DMRs) with coordinated gene expression changes associated with lifetime cumulative blast exposures were identified. The accumulative effect of blast showed increased methylation of PAX8 antisense transcript with coordinated repression of gene expression, which has been associated with sleep disturbance. DNA methylation analyses conducted in conjunction with reported symptoms of tinnitus in the low versus high blast incidents groups identified DMRS in KCNE1 and CYP2E1 genes. KCNE1 and CYP2E1 showed the expected inverse correlation between DNA methylation and gene expression, which have been previously implicated in noise-related hearing loss. Although no significant transcriptional changes were observed in samples obtained at the onset of the training course relative to chronic cumulative blast, we identified a large number of transcriptional perturbations acutely pre-versus post-blast exposure. Acutely, 67 robustly differentially expressed genes (fold change ‡1.5), including UFC1 and YOD1 ubiquitin-related proteins, were identified. Inflammatory analyses of cytokines and chemokines revealed dysregulation of MCP-1, GCSF, HGF, MCSF, and RANTES acutely after blast exposure. These data show the importance of an omics approach, revealing that transcriptional and inflammatory biomarkers capture acute low-level blast overpressure exposure, whereas DNA methylation marks encapsulate chronic long-term symptoms.

Background: Injuries from exposure to explosions rose dramatically during the Iraq and Afghanistan wars, which motivated investigation of blast-related neurotrauma. We have undertaken human studies involving military "breachers"-exposed to controlled, low-level blast during a 3-days explosive breaching course. Methods: We screened epigenetic profiles in peripheral blood samples from 59 subjects (in two separate U.S. Military training sessions) using Infinium MethylationEPIC BeadChips. Participants had varying numbers of exposures to blast over their military careers (empirically defined as high ≥ 40, and conversely, low < 39 breaching exposures). Daily self-reported physiological symptoms were recorded. Tinnitus, memory problems, headaches, and sleep disturbances are most frequently reported. Results: We identified 14 significantly differentially methylated regions (DMRs) within genes associated with cumulative blast exposure in participants with high relative to low cumulative blast exposure. Notably, NTSR1 and SPON1 were significantly differentially methylated in high relative to low blast exposed groups, suggesting that sleep dysregulation may be altered in response to chronic cumulative blast exposure. In comparing lifetime blast exposure at baseline (prior to exposure in current training), and top associated symptoms, we identified significant DMRs associated with tinnitus, sleep difficulties, and headache. Notably, we identified KCNN3, SOD3, MUC4, GALR1, and WDR45B, which are implicated in auditory function, as differentially methylated associated with self-reported tinnitus. These findings suggest neurobiological mechanisms behind auditory injuries in our military warfighters and are particularly relevant given tinnitus is not only a primary disability among veterans, but has also been demonstrated in active duty medical records for populations exposed to blast in training. Additionally, we found that differentially methylated regions associated with the genes CCDC68 and COMT track with sleep difficulties, and those within FMOD and TNXB track with pain and headache. Conclusion: Sleep disturbances, as well as tinnitus and chronic pain, are widely reported in U.S. military service members and veterans. As we have previously demonstrated, DNA methylation encapsulates lifetime exposure to blast. The current Wang et al. Methylation Pattern of Cumulative Blast data support previous findings and recapitulate transcriptional regulatory alterations in genes involved in sleep, auditory function, and pain. These data uncovered novel epigenetic and transcriptional regulatory mechanism underlying the etiological basis of these symptoms.

Injuries from exposure to explosions rose dramatically during the Iraq and Afghanistan wars, which motivated investigations of blast-related neurotrauma and operational breaching. In this study, military "breachers" were exposed to controlled, low-level blast during a 10-day explosive breaching course. Using an omics approach, we assessed epigenetic, transcriptional, and inflammatory profile changes in blood from operational breaching trainees, with varying levels of lifetime blast exposure, along with daily selfreported symptoms (with tinnitus, headaches, and sleep disturbances as the most frequently reported). Although acute exposure to blast did not confer epigenetic changes, specifically in DNA methylation, differentially methylated regions (DMRs) with coordinated gene expression changes associated with chronic lifetime cumulative blast exposures were identified. The accumulative effect of blast showed increased methylation of PAX8 antisense transcript with coordinated repression of gene expression, which has been associated with sleep disturbance. DNA methylation analyses conducted in conjunction with reported symptoms of tinnitus in the low vs. high blast incidents groups identified DMRS in KCNE1 and CYP2E1 genes. KCNE1 and CYP2E1 showed the expected inverse correlation between DNA methylation and gene expression, which have been previously implicated in noise related hearing loss.Although no significant transcriptional changes were observed in samples obtained at the onset of the training course relative to chronic cumulative blast, we identified a large number of transcriptional perturbations acutely pre-versus post-blast exposure. Acutely, 67 robustly differentially expressed genes (fold change ≥ 1.5), including UFC1 and YOD1, ubiquitin-related proteins were identified. Inflammatory analyses of cytokines and chemokines revealed dysregulation of MCP-1, GCSF, HGF, MCSF, and RANTES acutely following blast exposure. These data show the importance of an omics approach, revealing that transcriptional and inflammatory biomarkers capture acute lowlevel blast overpressure exposure, whereas DNA methylation marks encapsulate chronic long-term symptoms.