DNA Methylation Patterns of Chronic Explosive Breaching in U.S. Military Warfighters

Abstract

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.

Study protocol: Identifying transcriptional regulatory alterations of chronic effects of blast and disturbed sleep in United States Veterans

“…This underscores the utility of DNA methylation not only as a potential risk marker of disease progression, but also as a putative marker of treatment response. Accumulating evidence linking epigenetic mechanisms with response to traumatic stress and the subsequent risk for PTSD [ 47 – 49 ] fits with our observed observations in Service Members with repeated exposures to blast and blast associated sequalae [ 26 , 31 ]. Similarly, we aim to identify DNA methylation patterns associated with chronic exposures to blast in our Service Members in high-risk occupations, which we will then use to determine MRS predictive of blast exposures in our Veterans.…”

“…All study procedures involving human participants for Phase 2 were jointly approved by the Colorado Multiple Institutional Review Board (COMIRB) and the JJPVAMC. Because methods being implemented for Phase 1 have already been described [ 26 , 30 , 31 ], we will focus here on Phase 2. However, Phase 1 methods are briefly described below.…”

“…Prior studies have identified transient changes in transcriptome profiles [ 26 , 27 ] as well as blood-based neurotrauma biomarkers including neuronally-derived Tau, glial fibrillary acidic protein, ubiquitin carboxyl hydrolase (UCH)–L1, amyloid β (Aβ)–40, and Aβ-42 acutely following exposures to LLB during operational training [ 28 – 30 ]. Although DNA methylation studies in the same cohorts did not show alterations acutely following exposures to blast [ 31 ], altered DNA methylation was identified within genes involved in sleep and circadian rhythm chronically in individuals with a history cumulative blast exposures [ 30 , 32 , 33 ]. Specifically, work by Haghighi (author) and colleagues identified differentially methylated regions (DMRs) associated with high (≥40 exposures) versus low cumulative blast exposure [ 30 , 32 ], with DMRs found in genes such as NTSR1 (neurotensin receptor 1), associated with REM and anxiety-like behaviors in animal models [ 34 ].…”

“…Building on results from prior work [ 26 , 31 ], the purpose of this study is to carry out in-depth DNA methylation and functional gene expression experiments and phenotyping of sleep disturbance associated with cumulative blast exposures. This study will be completed in two phases.…”

The potassium channels: Neurobiology and pharmacology of tinnitus

“…Despite BMS‐191011 itself having little solubility in water and oral bioavailability, structure–activity relationship studies have explored analogs and cleavable prodrugs with the aim of enhancing solubility while preserving high brain availability and particular BK channel activity (Romine et al., 2007), which hold potential for tinnitus treatment. Apart from BK channels, a recent study identified that different DNA methylation of the KCNN3 (K Ca 2.3, SK3) gene is associated with self‐reported tinnitus in U.S. military warfighters (Wang, Wilson, Ge, et al., 2020). It provides the first evidence of the association of the KCNN3 gene with the human symptom of tinnitus.…”