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Genetic biomarkers of posttraumatic epilepsy: A systematic review

Open ArchivePublished:February 08, 2017DOI:https://doi.org/10.1016/j.seizure.2017.02.002

      Highlights

      • The field of epilepsy biomarkers remains is in its infancy.
      • All the potential biomarkers discussed are preliminary work requiring validation.
      • The most promising genetic biomarkers are IL-1β rs1143634 and A1AR rs10920573.

      Abstract

      Introduction

      Posttraumatic epilepsy (PTE) is caused by traumatic brain injury (TBI) and is an important contributor to the overall social and economic burden of epilepsy. Epidemiological studies suggest that there is a genetic contribution to the development of PTE. Identification of clinically useful genetic biomarkers is important for advancements in diagnosis and treatment of PTE.

      Methods

      A systematic review was performed on the existing literature of genetic biomarkers of posttraumatic epilepsy (PTE). A multi-database search yielded 4 articles deemed suitable for review. Potential genetic biomarkers were identified and critically evaluated.

      Results & discussion

      Biomarkers identified included single nucleotide polymorphism (SNP) rs1143634 of the interkeukin-1β (IL-1β) gene, SNPs rs3828275, rs3791878, and rs769391 of the glutamic acid decarboxylase 1 (GAD1) gene, SNPs rs3766553 and rs10920573 of the adenosine A1 receptor (A1AR) gene, and the functional variant C677T of the methylenetetrahydrofolate reductase (MTHFR) enzyme. The most promising biomarkers identified were IL-1β rs1143634 and A1AR rs10920573. Both had heterogenous at risk genotypes (CT). Those with IL-1β rs1143634 CT genotype developed PTE in 47.7% of cases (p = 0.008) and those with A1AR rs10920573 CT genotype developed PTE in 19.2% of cases (p = 0.022).

      Conclusion

      The majority of articles were preliminary with a need for validation of results. There is a need for continued high calibre research in order to validate the currently identified genetic biomarkers as well as to discover new genetic biomarkers in PTE.

      Keywords

      1. Introduction

      Traumatic brain injury (TBI) is a complex and heterogeneous condition that results directly from an external mechanical force to the head. Causes of TBI vary considerably, ranging from motor accidents to gunshot wounds and explosions. TBI is associated with a broad spectrum of lifelong disorders, such as epilepsy, and is cited as a leading cause of death and disability worldwide, placing a considerable economic and social burden on society [
      • Gupta P.K.
      • Sayed N.
      • Ding K.
      • Agostini M.A.
      • Van Ness P.C.
      • Yablon S.
      • et al.
      Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features.
      ].
      Post-traumatic epilepsy (PTE) is a direct consequence of TBI, occurring in 3-5% of moderate TBIs and in as many as 25–50% of severe TBIs [
      • Gupta P.K.
      • Sayed N.
      • Ding K.
      • Agostini M.A.
      • Van Ness P.C.
      • Yablon S.
      • et al.
      Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features.
      ,
      • Agrawal A.
      • Timothy J.
      • Pandit L.
      • Manju M.
      Post-traumatic epilepsy: an overview.
      ]. PTE is a heterogeneous condition that can vary tremendously in time from injury to onset, from weeks to years. Three classifications of post-traumatic seizures exist : immediate seizures occurring less than 24 h post-injury, early seizures occurring less than 1 week post-injury, and late seizures occurring more than 8 days post-injury [
      • Agrawal A.
      • Timothy J.
      • Pandit L.
      • Manju M.
      Post-traumatic epilepsy: an overview.
      ]. Of those with PTE, 50–66% will experience their first seizure within the first year and more than 75% in the first two years of TBI [
      • Englander J.
      • Bushnik T.
      • Duong T.T.
      • Cifu D.X.
      • Zafonte R.
      • Wright J.
      • et al.
      Analyzing risk factors for late posttraumatic seizures: a prospective, multicenter investigation.
      ]. Diagnosis of PTE is defined as the occurrence of two or more unprovoked seizures seven or more days after injury [
      • Gupta P.K.
      • Sayed N.
      • Ding K.
      • Agostini M.A.
      • Van Ness P.C.
      • Yablon S.
      • et al.
      Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features.
      ,
      • Beghi E.
      • Carpio A.
      • Forsgren L.
      • Hesdorffer D.C.
      • Malmgren K.
      • Sander J.W.
      • et al.
      Recommendation for a definition of acute symptomatic seizure.
      ].
      Acquired epilepsies develop in a relatively predictable pattern that includes three phases. The initial brain insult will usually lead to epileptogenesis during the latency period, defined as the seizure-free period directly following the cerebral insult, which in turn results in recurring and unprovoked epileptic seizures [
      • Lamar C.D.
      • Hurley R.A.
      • Rowland J.A.
      • Taber K.H.
      Post-traumatic epilepsy: review of risks, pathophysiology, and potential biomarkers.
      ]. The time from injury to seizure onset is very important in determining prognosis and treatment. Immediate and early post-traumatic seizures (PTS) appear to be low risk in terms of seizure recurrence because they are usually attributed to cortical inhibition resulting directly from the injury as opposed to epileptic events, whereas late seizures are more highly correlated with the development of PTE [
      • Lamar C.D.
      • Hurley R.A.
      • Rowland J.A.
      • Taber K.H.
      Post-traumatic epilepsy: review of risks, pathophysiology, and potential biomarkers.
      ,
      • Perron A.D.
      • Brady W.J.
      • Huff J.S.
      Concussive convulsions: emergency department assessment and management of a frequently misunderstood entity.
      ]. Most individuals who experience a single posttraumatic seizure are started on anti-epileptic medications following the first seizure event, although there is no evidence this prophylactic treatment works to prevent the subsequent development of PTE [
      • Diamond M.L.
      • Ritter A.C.
      • Failla M.D.
      • Boles J.A.
      • Conley Y.P.
      • Kochanek P.M.
      • et al.
      IL-1β associations with posttraumatic epilepsy development: a genetics and biomarker cohort study.
      ].
      PTE occurs in both children and adults, particularly children under 7 years of age and adults over 65 years of age. In the general population, PTEs account for 5% of all epilepsy cases and 20% of symptomatic epilepsy [
      • Agrawal A.
      • Timothy J.
      • Pandit L.
      • Manju M.
      Post-traumatic epilepsy: an overview.
      ]. This number is understandably higher in the military population where almost 50% of TBI victims, more specifically those who sustain penetrating head injuries, go on to develop PTE [
      • Aarabi B.
      • Taghipour M.
      • Haghnegahdar A.
      • Farokhi M.
      • Mobley L.
      Prognostic factors in the occurrence of posttraumatic epilepsy after penetrating head injury suffered during military service.
      ,
      • Salazar A.M.
      • Jabbari B.
      • Vance S.C.
      • Grafman J.
      • Amin D.
      • Dillon J.D.
      Epilepsy after penetrating head injury: I. Clinical correlates: a report of the Vietnam head injury study.
      ,
      • Aarabi B.
      • Kaufman H.
      Missile wounds of the head and neck.
      ]. Many risk factors have been identified, including but not limited to severity of initial TBI, length of posttraumatic amnesia, presence of intracranial haemorrhage, penetrating head injuries, depressed skull fractures, retention of metal fragments, location of lesion(s), cerebral contusions, age, and comorbid chronic alcoholism [
      • Englander J.
      • Bushnik T.
      • Duong T.T.
      • Cifu D.X.
      • Zafonte R.
      • Wright J.
      • et al.
      Analyzing risk factors for late posttraumatic seizures: a prospective, multicenter investigation.
      ,
      • Annegers J.F.
      • Grabow J.D.
      • Groover R.V.
      • Laws Jr., E.R.
      • Elveback L.R.
      • Kurland L.T.
      Seizures after head trauma: a population study.
      ,
      • Askenasy J.J.M.
      Association of intracerebral bone fragments and epilepsy in missile head injuries.
      ,
      • Haltiner A.M.
      • Temkin N.R.
      • Dikmen S.S.
      Risk of seizure recurrence after the first late posttraumatic seizure.
      ,
      • Weiss George H.
      • Caveness William F.
      Prognostic factors in the persistence of posttraumatic epilepsy.
      ]. Importantly, etiological studies indicate a strong genetic basis for the development of epilepsy [
      • Kjeldsen M.J.
      • Kyvik K.O.
      • Christensen K.
      • Friis M.L.
      Genetic and environmental factors in epilepsy: a population-based study of 11900 Danish twin pairs.
      ].
      Mechanisms of posttraumatic epileptogenesis are incompletely understood. Studies suggest that hippocampal and dentate gyrus involvement and early and selective cell loss in the CA3 (Cornu Ammonis sub-region 3) of the hippocampus and in the hilus of the dentate gyrus are key to the epileptogenic process [
      • Golarai G.
      • Greenwood A.C.
      • Feeney D.M.
      • Connor J.A.
      Physiological and structural evidence for hippocampal involvement in persistent seizure susceptibility after traumatic brain injury.
      ]. In addition, significant hippocampal mossy fibre sprouting as well as neurodegeneration, axonal injury, astrocytosis, and dysfunction of the blood-brain barrier appear to play a role [
      • Kovacs S.K.
      • Leonessa F.
      • Ling G.S.
      Blast TBI models, neuropathology, and implications for seizure risk.
      ,
      • Kharatishvili I.
      • Nissinen J.P.
      • McIntosh T.K.
      • Pitkanen A.
      A model of posttraumatic epilepsy induced by lateral fluid-percussion brain injury in rats.
      ].
      Biomarkers that may be clinically useful in the prediction, diagnosis, prognosis, and treatment of posttraumatic epilepsy are currently under-investigated. Methods employed to discover clinically relevant biomarkers include imaging and diagnostic methods as well as transcriptional profiling, proteomic, genomic, and metabolomic approaches [
      • Dash P.K.
      • Zhao J.
      • Hergenroeder G.
      • Moore A.N.
      Biomarkers for the diagnosis, prognosis, and evaluation of treatment efficacy for traumatic brain injury.
      ]. There is no current method of preventing PTE, with no viable anti-epileptogenic modification identified to date [
      • Christensen J.
      • Pedersen M.G.
      • Pedersen C.B.
      • Sidenius P.
      • Olsen J.
      • Vestergaard M.
      Long-term risk of epilepsy after traumatic brain injury in children and young adults: a population-based cohort study.
      ]. Consequently, all that can be offered is symptomatic management once a pattern of unprovoked seizures is established. Biomarkers offer a potential way of predicting the onset of PTE, a better understanding of the molecular mechanisms associated, and identification of possible preventative measures. The purpose of this review is to investigate and synthesise the literature on the subject of potential genetic biomarkers of PTE with the aim to evaluate the strength of evidence for individual biomarkers in addition to highlighting areas of potential for advancement or areas where evidence is currently lacking.

      2. Methods

      A multi-database search was performed using the following search terms: epilep* (epilepsy, epileptogenesis, epilepsies), seizure, convulsions, posttraumatic epilep*, traumatic brain injur* (injury, injuries), acquired brain injur*, and genetic biomarkers. Databases utilised were PubMed, Science Direct, Cochrane Library, Web of Science, and Google Scholar. The references of each relevant review were manually screened to ensure no important articles were missed (n = 8). Exclusion criteria were defined as follows: papers addressing general epileptogenesis not specifically related to TBI, papers addressing the pathophysiology of epileptogenesis as opposed to biomarkers, papers addressing treatment of epilepsy, animal studies, and reviews. The database search was not limited by language or time period, but only English studies from the years 1980–2015 were included in this review due to the constraints of the exclusion criteria. This review adhered to PRIMSA guidelines. This review was originally part of a much larger and broader body of work, a Master’s dissertation detailing the CSF/serum, genetic, imaging, neurophysiologic, and clinical biomarkers of PTE, reflected by Fig. 1.

      3. Results

      Many factors are at play in individuals who develop PTE after a TBI versus those who do not, including but not limited to predisposing genetic traits. With the completion of the Human Genome Project (HGR) in 2003 as well as recent improvements in genotyping technology, the investigation of genetic factors and their role in the development of disease has become progressively more accessible to researchers, resulting in the increase of genetic association studies and their reliability. In the field of PTE, most genetic associations examined have been single-nucleotide polymorphisms (SNPs), a common variation within a population that can be defined as a variation of a single nucleotide, adenine (A), guanine (G), thymine (T), or cytosine (C), between individuals of the same species. Within each SNP there is a protective and an at-risk genotype for PTE development (Table 1).
      Table 1Summary of genetic biomarkers as reported in the current literature.
      BiomarkerReferenceKey FindingsNMain StrengthsMain Limitations
      rs1143634Diamond et al., 2014
      • serum IL-1β significantly different based on rs1143634 genotype
      • CSF/serum IL-1β ratio showed a trend based on rs1143634 genotype
      • heterozygous (CT) genotype associated with PTE, lower IL-1β serum levels, and higher CSF/serum IL-1β ratio
      256
      • -
        only late-onset PTS were included, increasing the likelihood that an epileptogenic process was taking place as opposed to investigating non-epileptic seizures
      • -
        small sample size
      GAD1 gene

       rs3828275

       rs769391

       rs3791878
      Darrah et al., 2013
      • rs3828275: homozygous wild-type (CC) is defined as the protected genotype and has significantly higher odds of not developing PTS <1 week
      • rs3791878: homozygous wild-type (GG) is defined as the at-risk genotype and has significant associations with PTS occurring 1 week – 6 months
      • rs769391: homozygous wild-type (AA) is defined as the at-risk genotype but is not significantly associated with PTS occurring 1 week – 6 months
      257
      • -
        well-controlled
      • -
        clearly defined variables
      • -
        seizure assessment exclusion criteria, time-specific groupings, and outcome measures are such that incidence of PTE is, if anything, understated
      • -
        small sample size
      MTHFR C677TScher et al., 2011
      • TT is defined as the at-risk genotype and is significantly associated with PTE compared to the CC group
      800 (experi-mental)

      800 (control)
      • -
        well-designed with sufficient controls and variable parameters
      • -
        samples from all subjects were genotyped twice
      • -
        sample population was randomly selected, relatively large, and multi-ethnic
      • -
        sample size is technically too small
      • -
        exclusion of medical encounters while subjects were deployed to combat zones (authors cite inability to access records)
      • -
        possible additional misclassification of seizure status due to use of the ICD-9-CM diagnostic codes without further verification from a clinician (however, this is likely to have weakened rather than strengthened the results)
      Adenosine A1 receptor gene:

      rs3766553

      rs10920573
      Wagner et al., 2010
      • rs3766553: significantly associated with early, late, and delayed onset PTS – AA genotype associated with early PTS and GG genotype associated with both late and delayed onset PTS
      • rs10920573: CT heterozygous genotype significantly associated with late and delayed onset PTS
      • risk variants for both SNPs were independently associated with late and delayed onset PTS and had a cumulative effect on susceptibility to PTS
      206
      • -
        only used subjects who had sustained a severe TBI (GCS of 8 or less), increasing the likelihood of PTE development as severity of TBI is a known clinical predictor
      • -
        all subjects were monitored in a controlled hospital environment, reducing the possibility of confounding factors
      • -
        appropriately controlled DNA collection, genotyping methods, outcome measures, and statistical analysis
      • -
        small sample size
      • -
        penetrating head injury is listed as an exclusion criteria when it is a risk factor for PTE
      CSF – cerebrospinal fluid; GCS – Glasgow Coma Scale; ICD-9–International Statistical Classification of Diseases and Related Health Problems Version 9; PTE – post-traumatic epilepsy; PTS – post-traumatic seizures; TBI – Traumatic Brain Injury.

      3.1 IL-1β gene: SNP rs1143634

      Interkeukin-1β (IL-1β) is a pro-inflammatory cytokine that is released in the central nervous system (CNS) by activated astrocytes and microglia and in the periphery by macrophages and other immune cells as a response to injury or other pathology in the brain [
      • Diamond M.L.
      • Ritter A.C.
      • Failla M.D.
      • Boles J.A.
      • Conley Y.P.
      • Kochanek P.M.
      • et al.
      IL-1β associations with posttraumatic epilepsy development: a genetics and biomarker cohort study.
      ]. The first of its kind, a study examined the potential link between IL-1β and PTE on the basis of evidence supporting IL-1β involvement in both TBI and other kinds of epilepsies [
      • Zhu G.
      • Okada M.
      • Yoshida S.
      • Mori F.
      • Ueno S.
      • Wakabayashi K.
      • et al.
      Effects of interleukin-1beta on hippocampal glutamate and GABA releases associated with Ca2+-induced Ca2+ releasing systems.
      ,
      • Vezzani A.
      • Moneta D.
      • Conti M.
      • Richichi C.
      • Ravizza T.
      • De Luigi A.
      • et al.
      Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice.
      ,
      • Ravizza T.
      • Lucas S.M.
      • Balosso S.
      • Bernardino L.
      • Ku G.
      • Noé F.
      • et al.
      Inactivation of Caspase-1 in rodent brain: a novel anticonvulsive strategy.
      ,
      • Rijkers K.
      • Majoie H.J.
      • Hoogland G.
      • Kenis G.
      • De Baets M.
      • Vles J.S.
      The role of interleukin-1 in seizures and epilepsy: a critical review.
      ]. This study investigated genetic variations in the gene coding for IL-1β, located in the region of 2q12-13, and their possible role in the prediction of PTE onset in moderate-to-severe TBI [
      • Diamond M.L.
      • Ritter A.C.
      • Failla M.D.
      • Boles J.A.
      • Conley Y.P.
      • Kochanek P.M.
      • et al.
      IL-1β associations with posttraumatic epilepsy development: a genetics and biomarker cohort study.
      ]. Only late PTS (onset one or more weeks post-injury) were included. Of the SNPs investigated, rs1143634 was the only one with a significant association with the eventual development of PTE (Table 2). In addition to being significantly associated with the development of PTE, it seems that rs1143634 also has effects on CSF and serum levels of IL-1β. Serum levels of IL-1β are diminished in the CT genotype (the at-risk genotype), a finding that aligns with decreased levels observed in PTE patients compared to controls [
      • Diamond M.L.
      • Ritter A.C.
      • Failla M.D.
      • Boles J.A.
      • Conley Y.P.
      • Kochanek P.M.
      • et al.
      IL-1β associations with posttraumatic epilepsy development: a genetics and biomarker cohort study.
      ].
      Table 2Summary of results of genetic posttraumatic epilepsy (PTE) biomarker studies.
      GeneSNPAt RiskProtectiveP valueN
      IL-1βrs1143634CT (47.7%)TT (0%)0.008256
      GAD1rs3828275
      Early posttraumatic seizures (PTS) only (within a week of traumatic brain injury (TBI)).
      TT (20%)CC (2.99%)0.014257
      rs3791878
      PTS (1 week-6 months).
      GG (16%)TT (0%)0.05257
      rs769391
      PTS (1 week-6 months).
      AA (14%)AG (3.7%)0.09257
      A1ARrs3766553AA (17.4%)
      Early posttraumatic seizures (PTS) only (within a week of traumatic brain injury (TBI)).
      AG (3.9%) & GG (3.2%)
      Early posttraumatic seizures (PTS) only (within a week of traumatic brain injury (TBI)).
      0.052206
      GG (32.2%)
      PTS (1 week-6 months).
      AA & AG (15.2%)
      PTS (1 week-6 months).
      0.044206
      GG (28.1%)
      Delayed onset PTS.
      AA & AG (7.1%)
      Delayed onset PTS.
      0.005206
      rs10920573CT (19.2%)CC & TT (6.7%)0.022206
      MTHFRC677TTT (39.1%)
      Subjects in this cohort limited to cases with 2+ medical encounters for epilepsy.
      CC (25.7%)
      Subjects in this cohort limited to cases with 2+ medical encounters for epilepsy.
      0.0261600
      * Early posttraumatic seizures (PTS) only (within a week of traumatic brain injury (TBI)).
      ** PTS (1 week-6 months).
      *** Delayed onset PTS.
      **** Subjects in this cohort limited to cases with 2+ medical encounters for epilepsy.
      The results of this study implicate CSF and serum IL-1β levels as well as the SNP rs1143634 in the development of PTE. A significant association between IL-1β CSF and serum levels and rs1143436 is also apparent. As the functionality of the rs1143634 genotype of the IL-1β is unknown, the exact reasons why the CT genotype would be considered the at-risk group while the TT genotype is suggested as protective has not been determined.

      3.2 GAD1 gene: rs3828275, rs3791878, & rs769391

      Many of the gene association studies in the field of PTE are the first to identify their respective SNPs with the occurrence of post-traumatic seizures. One gene in particular that seems to contribute to PTS in general is the glutamic acid decarboxylase gene (GAD), more specifically GAD1 [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ]. One study examined the previously unexplored link between GAD1 and PTE based on multiple studies that confirm its involvement via the GABAergic neurotransmission pathway in TBI pathophysiology and seizure susceptibility individually [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ,
      • Treiman D.M.
      GABAergic mechanisms in epilepsy.
      ,
      • Nilsson P.
      • Hillered L.
      • Pontén U.
      • Ungerstedt U.
      Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats.
      ,
      • Kanthan R.
      • Shuaib A.
      Clinical evaluation of extracellular amino acids in severe head trauma by intracerebral in vivo microdialysis.
      ,
      • Hasegawa D.
      • Matsuki N.
      • Fujita M.
      • Ono K.
      • Orima H.
      Kinetics of glutamate and gamma-aminobutyric acid in cerebrospinal fluid in a canine model of complex partial status epilepticus induced by kainic acid.
      ]. GAD1 SNPs were found to be significantly associated with PTS. One of the three SNPs discussed, rs3828275, was correlated with early PTS only, that is seizures occurring within one week of the TBI (Table 2). It was further found that when TT was defined as the at-risk genotype, the CC genotype had a statistically significant chance of not developing PTS when compared to the other two genotypes (p = 0.055) [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ]. With regard to this particular SNP, it is important to note that seizures occurring within the first week of injury may be a direct result of the injury as opposed to being epileptic in nature.
      The other two SNPs investigated, rs3791878 and rs769391 in the GAD gene, were both significantly associated with PTS within the timeframe of one week to 6 months (Table 2). Although this association was not significant on multivariate analysis comparing all three genotypes, there was a significant difference between the AA genotype compared to either the AG genotype or the GG genotype. Through additional statistical analysis, it was shown that for rs3791878, people with the GG genotype had significantly higher odds of developing PTE when compared to GT and TT genotypes (p = 0.0231) [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ]. The same was true of the rs769391 SNP, with the AA genotype having higher chances of developing PTE when compared to the other two genotypes, demonstrating an insignificant trend (p = 0.058). Due to the apparent involvement of rs3791878 and rs769391 in PTS occurring within the timeframe of 1 week to 6 months as well as the common gene on which they are located (GAD1), the authors performed a multivariate analysis between the two at-risk genotypes, GG (rs3791878) and AA (rs769391). Results from this analysis proved significant when comparing individuals with zero, one, or both risk variants [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ]. Patients with both risk variants (GG and AA) had significantly higher rates of PTS when compared to those with one or none of the risk variants (p = 0.019). Moreover multivariate analysis showed that patients with both risk variants were at increased risk of developing PTS compared to those without any risk variants (p = 0.024) [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ]. Interestingly, no notable differences were uncovered in the multivariate analysis of both risk variants compared to one risk variant or of no risk variants compared to one risk variant [
      • Darrah S.D.
      • Miller M.A.
      • Ren D.
      • Hoh N.Z.
      • Scanlon J.M.
      • Conley Y.P.
      • et al.
      Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
      ].

      3.3 A1AR gene: rs3766553 & rs10920573

      A third gene to be investigated in the search for PTE biomarkers is the adenosine A1 receptor (A1AR) gene and its variants. The A1AR gene is suspected to be involved in the development of PTE due to the location of A1ARs in regions associated with both TBI and seizure pathology, namely the hippocampus and the cortex, and their proximity to N-methyl-D-aspartate (NMDA) receptors [
      • Wagner A.K.
      • Miller M.A.
      • Scanlon J.
      • Ren D.
      • Kochanek P.M.
      • Conley Y.P.
      Adenosine A1 receptor gene variants associated with post-traumatic seizures after severe TBI.
      ]. Not only has adenosine been demonstrated to be an important neuroprotective agent in TBI, but compromises in adenosine synthesis due to secondary TBI pathology such as glial scars has been linked to potential posttraumatic seizure development [
      • Wagner A.K.
      • Miller M.A.
      • Scanlon J.
      • Ren D.
      • Kochanek P.M.
      • Conley Y.P.
      Adenosine A1 receptor gene variants associated with post-traumatic seizures after severe TBI.
      ]. Based on this evidence, a gene association study was performed to determine if genetic variability within this gene could account for an increased susceptibility to PTE in people with TBI. Of the SNPs analysed, two were identified as having significant associations with the development of PTS, both early and late [
      • Wagner A.K.
      • Miller M.A.
      • Scanlon J.
      • Ren D.
      • Kochanek P.M.
      • Conley Y.P.
      Adenosine A1 receptor gene variants associated with post-traumatic seizures after severe TBI.
      ]. Notably, different genotypes of rs3766553 were associated with early (less than one week post injury) PTS as well as late (one or more weeks post-injury) and delayed-onset (6 or more months post-injury) PTS (Table 2).
      The second SNP implicated in the development of late and delayed-onset PTS is rs10920573, where the heterozygous (CT) genotype was defined as the at-risk group compared to the two homozygous genotypes (wild-type (CC) and variant (TT)). As both rs3766553 and rs10920573 are either significantly correlated with or are trending towards a significant correlation with late and/or delayed-onset seizures, additional statistical analysis was performed to determine the risk associated with carrying none, one, or both of the risk genotypes (GG (rs3766553) and CT (rs10920573)). Results indicate significant associations with carrying one or both of the risk genotypes and the development of late or delayed-onset PTS [
      • Wagner A.K.
      • Miller M.A.
      • Scanlon J.
      • Ren D.
      • Kochanek P.M.
      • Conley Y.P.
      Adenosine A1 receptor gene variants associated with post-traumatic seizures after severe TBI.
      ].
      The fact that two different genotypes of the rs3766553 SNP correlate with two different kinds of PTS is noteworthy. This kind of association within a single SNP has certain implications for neuroprotection as well as susceptibility after TBI. It would seem that the rs3766553 heterozygotes are relatively protected against the development of seizures post-TBI. Conversely, wild-type homozygotes (AA) are susceptible to early PTS but apparently not PTE. Variant homozygotes (GG) seem to be the group at highest risk for developing PTE, as they are statistically more likely than AA or AG genotypes to experience late or delayed-onset seizures post-TBI. When examining the second implicated SNP, rs10920573, it is the heterozygotes that are at greatest risk, indicating that neither allele is protective on its own.
      In this study, one of the exclusion criteria was penetrating head injury [
      • Wagner A.K.
      • Miller M.A.
      • Scanlon J.
      • Ren D.
      • Kochanek P.M.
      • Conley Y.P.
      Adenosine A1 receptor gene variants associated with post-traumatic seizures after severe TBI.
      ]. The reasons for this are not given, despite the fact that penetrating head injury is a well-documented risk factor for the development of PTE [
      • Salazar A.M.
      • Jabbari B.
      • Vance S.C.
      • Grafman J.
      • Amin D.
      • Dillon J.D.
      Epilepsy after penetrating head injury: I. Clinical correlates: a report of the Vietnam head injury study.
      ,
      • Weiss G.H.
      • Salazar A.M.
      • Vance S.C.
      • Grafman J.H.
      • Jabbari B.
      Predicting posttraumatic epilepsy in penetrating head injury.
      ,
      • Aarabi B.
      • Taghipour M.
      • Haghnegahdar A.
      • Farokhi M.
      • Mobley L.
      Prognostic factors in the occurrence of posttraumatic epilepsy after penetrating head injury suffered during military service.
      ,
      • Eftekhar B.
      • Sahraian M.A.
      • Nouralishahi B.
      • Khaji A.
      • Vahabi Z.
      • Ghodsi M.
      • et al.
      Prognostic factors in the persistence of posttraumatic epilepsy after penetrating head injuries sustained in war.
      ,
      • Raymont V.
      • Salazar A.M.
      • Lipsky R.
      • Goldman D.
      • Tasick G.
      • Grafman J.
      Correlates of posttraumatic epilepsy 35 years following combat brain injury.
      ,
      • Kendirli M.T.
      • Rose D.T.
      • Bertram E.H.
      A model of posttraumatic epilepsy after penetrating brain injuries: effect of lesion size and metal fragments.
      ]. Aside from this oversight, the A1AR gene seems to show promising links to PTS, early, late, and delayed-onset, and thus could have implications for individual susceptibility to PTE after TBI.

      3.4 MTHFR C677T

      Methylenetetrahydrofolate reductase (MTHFR) is an enzyme key to the metabolic processes of methionine, an essential amino acid, and is encoded by the MTHFR gene, located on chromosome 1p36.3 [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. Recent studies suggest that the C677T genotype, one of two common functional variants, is associated with the onset of PTE. Homocysteine is a sulfur-containing amino acid and can result from compromised methionine metabolism [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. Evidence exists implicating homocysteine in lower seizure thresholds and in increased risk of the development of alcohol withdrawal seizures [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. Additionally, there is a suggestion that the TT genotype of the MTHFR C677T gene variant may be disproportionately represented in individuals with epilepsy, although no study has investigated this phenomenon in relation to PTE [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ].
      This study investigated the presence of this variant in an age-matched case-control military study (n = 1600). In this study, the authors were investigating epilepsy prevalence and medical encounters suggestive of TBI via anonymous military medical databases. Results of this study suggest that, regardless of genotype, epilepsy was more prevalent in those who had medical encounters that were indicative of TBI (17%) compared to controls (4%) [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. Of those who experienced a possible TBI, 85% of cases were diagnosed as having PTE [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. When the authors genotyped subjects for MTHFR C677T, it was shown that the homozygous wild-type (CC) was relatively protective while the homozygous variant (TT) was defined as the at-risk group [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. The chances of being diagnosed with any kind of epilepsy, including PTE, was significantly higher in the TT genotype than in the CC genotype, a result that was enhanced when the analysed cases were limited to those cases who had two or more medical encounters for epilepsy [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ].
      This study was well-designed and included sufficient controls and variable parameters, such as a randomly selected, relatively large, and multi-ethnic sample population. Due to the lack of clinical verification of seizure assessment inherent in a study using an anonymous database, researchers opted to restrict epilepsy cases to people with two or more medical encounters for epilepsy. Samples from all subjects involved in the study were genotyped twice, with inconsistent results excluded. The exclusion of medical encounters while subjects were deployed to a combat zone is an obvious limitation, due to the fact that subjects would have been at higher risk for TBI (especially penetrating TBI) whilst in a combat zone. The authors cite inability to access these records as the reason for having excluded this data [
      • Scher A.I.
      • Wu H.
      • Tsao J.W.
      • Blom H.J.
      • Feit P.
      • Nevin R.L.
      • et al.
      MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
      ]. This limitation is likely to have resulted in an underestimation of PTE cases, or the incorrect classification of PTE cases as non-traumatic epilepsy cases and therefore a potential source of selection bias.

      4. Conclusions

      From the results of these genetic association studies, it is reasonable to conclude that there is a genetic element to the development of PTE, however understudied. Most promising seems to be the IL-1β SNP rs1143634, as the individuals with TT genotype seemed to be completely protected from developing PTE (Table 2). However, the functionality of the T allele is not fully understood, as it is also part of the at-risk genotype (CT). Similarly, the A1AR SNP rs10920573 seems to be relatively promising and is comparable to the IL-1β SNP rs1143634, with the at-risk genotype being the heterogeneous one (CT). The other genes and their respective SNPs discussed in this review were more powerfully associated with PTS, defined as early seizures that cannot be definitively categorised as PTE.
      The subject of genetic biomarkers for PTE is vastly understudied. The majority of potential biomarkers identified have been examined in, on average, one to three primary studies.
      The studies that do exist are heterogeneous in their discussion of PTE versus PTS. As previously mentioned, PTS can occur as a direct result of the TBI and are not necessarily epileptogenic while PTE is an epileptogenic process. A single occurrence of a seizure after a traumatic event does not constitute a diagnosis of epilepsy, as there are many seizure-inducing factors that can cause provoked seizures in individuals who do not have epilepsy, or who will not go on to develop epilepsy (PTE). Epilepsy is operationally defined as the occurrence of two or more unprovoked seizures following a cascade of neurochemical changes in the brain, wherein seizures are a symptom of the disorder. The distinction between these two categories of seizures is significant, as it has direct implications for the diagnosis, prognosis and management of the seizures.
      Additionally, genetic association studies require very large sample sizes, in the order of 10,000 patients or more to have real significance, which requires collaboration between multiple groups [
      • Dardiotis E.
      • Fountas K.N.
      • Dardioti M.
      • Xiromerisiou G.
      • Kapsalaki E.
      • Tasiou A.
      • et al.
      Genetic association studies in patients with traumatic brain injury.
      ,
      • Zintzaras E.
      • Lau J.
      Synthesis of genetic association studies for pertinent gene-disease associations requires appropriate methodological and statistical approaches.
      ]. This general lack of research could be a result of the relatively unknown nature of PTE pathophysiology. However, PTE is a well-recognised clinical consequence of TBI, and as such should be treated with priority due to the projected economic and social burden that may be produced from TBI and PTE.

      Conflict of interest

      None.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      References

        • Gupta P.K.
        • Sayed N.
        • Ding K.
        • Agostini M.A.
        • Van Ness P.C.
        • Yablon S.
        • et al.
        Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features.
        J Neurotrauma. 2014; 31: 1439-1443
        • Agrawal A.
        • Timothy J.
        • Pandit L.
        • Manju M.
        Post-traumatic epilepsy: an overview.
        Clin Neurol Neurosurg. 2006; 108: 433-439
        • Englander J.
        • Bushnik T.
        • Duong T.T.
        • Cifu D.X.
        • Zafonte R.
        • Wright J.
        • et al.
        Analyzing risk factors for late posttraumatic seizures: a prospective, multicenter investigation.
        Arch Phys Med Rehabil. 2003; 84: 365-373
        • Beghi E.
        • Carpio A.
        • Forsgren L.
        • Hesdorffer D.C.
        • Malmgren K.
        • Sander J.W.
        • et al.
        Recommendation for a definition of acute symptomatic seizure.
        Epilepsia. 2009; 51: 671-675
        • Lamar C.D.
        • Hurley R.A.
        • Rowland J.A.
        • Taber K.H.
        Post-traumatic epilepsy: review of risks, pathophysiology, and potential biomarkers.
        J Neuropsychiartry Clin Neurosci. 2014; 26: iv-113
        • Perron A.D.
        • Brady W.J.
        • Huff J.S.
        Concussive convulsions: emergency department assessment and management of a frequently misunderstood entity.
        Acad Emerg Med. 2001; 8: 296-298
        • Diamond M.L.
        • Ritter A.C.
        • Failla M.D.
        • Boles J.A.
        • Conley Y.P.
        • Kochanek P.M.
        • et al.
        IL-1β associations with posttraumatic epilepsy development: a genetics and biomarker cohort study.
        Epilepsia. 2014; 55: 1109-1119
        • Aarabi B.
        • Taghipour M.
        • Haghnegahdar A.
        • Farokhi M.
        • Mobley L.
        Prognostic factors in the occurrence of posttraumatic epilepsy after penetrating head injury suffered during military service.
        Neurosurg Focus. 2000; 8: 1-6
        • Salazar A.M.
        • Jabbari B.
        • Vance S.C.
        • Grafman J.
        • Amin D.
        • Dillon J.D.
        Epilepsy after penetrating head injury: I. Clinical correlates: a report of the Vietnam head injury study.
        Neurology. 1985; 35: 1406-1414
        • Aarabi B.
        • Kaufman H.
        Missile wounds of the head and neck.
        Park Ridge, Ill.: American Association of Neurological Surgeons. 1999
        • Annegers J.F.
        • Grabow J.D.
        • Groover R.V.
        • Laws Jr., E.R.
        • Elveback L.R.
        • Kurland L.T.
        Seizures after head trauma: a population study.
        Neurology. 1980; 30: 683-689
        • Askenasy J.J.M.
        Association of intracerebral bone fragments and epilepsy in missile head injuries.
        Acta Neurol Scand. 1989; 79: 47-52
        • Haltiner A.M.
        • Temkin N.R.
        • Dikmen S.S.
        Risk of seizure recurrence after the first late posttraumatic seizure.
        Arch Phys Med Rehabil. 1997; 78: 835-840
        • Weiss George H.
        • Caveness William F.
        Prognostic factors in the persistence of posttraumatic epilepsy.
        J Neurosurg. 1972; 37: 164-169
        • Kjeldsen M.J.
        • Kyvik K.O.
        • Christensen K.
        • Friis M.L.
        Genetic and environmental factors in epilepsy: a population-based study of 11900 Danish twin pairs.
        Epilepsy Res. 2001; 44: 167-178
        • Golarai G.
        • Greenwood A.C.
        • Feeney D.M.
        • Connor J.A.
        Physiological and structural evidence for hippocampal involvement in persistent seizure susceptibility after traumatic brain injury.
        J Neurosci. 2001; 21: 8523-8537
        • Kovacs S.K.
        • Leonessa F.
        • Ling G.S.
        Blast TBI models, neuropathology, and implications for seizure risk.
        Front Neurol. 2014; 5: 47
        • Kharatishvili I.
        • Nissinen J.P.
        • McIntosh T.K.
        • Pitkanen A.
        A model of posttraumatic epilepsy induced by lateral fluid-percussion brain injury in rats.
        Neuroscience. 2006; 140: 685-697
        • Dash P.K.
        • Zhao J.
        • Hergenroeder G.
        • Moore A.N.
        Biomarkers for the diagnosis, prognosis, and evaluation of treatment efficacy for traumatic brain injury.
        Neurotherapeutics. 2010; 7: 100-114
        • Christensen J.
        • Pedersen M.G.
        • Pedersen C.B.
        • Sidenius P.
        • Olsen J.
        • Vestergaard M.
        Long-term risk of epilepsy after traumatic brain injury in children and young adults: a population-based cohort study.
        Lancet. 2009; 373: 1105-1110
        • Zhu G.
        • Okada M.
        • Yoshida S.
        • Mori F.
        • Ueno S.
        • Wakabayashi K.
        • et al.
        Effects of interleukin-1beta on hippocampal glutamate and GABA releases associated with Ca2+-induced Ca2+ releasing systems.
        Epilepsy Res. 2006; 71: 107-116
        • Vezzani A.
        • Moneta D.
        • Conti M.
        • Richichi C.
        • Ravizza T.
        • De Luigi A.
        • et al.
        Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice.
        Proc Natl Acad Sci U S A. 2000; 97: 11534-11539
        • Ravizza T.
        • Lucas S.M.
        • Balosso S.
        • Bernardino L.
        • Ku G.
        • Noé F.
        • et al.
        Inactivation of Caspase-1 in rodent brain: a novel anticonvulsive strategy.
        Epilepsia. 2006; 47: 1160-1168
        • Rijkers K.
        • Majoie H.J.
        • Hoogland G.
        • Kenis G.
        • De Baets M.
        • Vles J.S.
        The role of interleukin-1 in seizures and epilepsy: a critical review.
        Exp Neurol. 2009; 216: 258-271
        • Darrah S.D.
        • Miller M.A.
        • Ren D.
        • Hoh N.Z.
        • Scanlon J.M.
        • Conley Y.P.
        • et al.
        Genetic variability in glutamic acid decarboxylase genes: associations with post-traumatic seizures after severe TBI.
        Epilepsy Res. 2013; 103: 180-194
        • Treiman D.M.
        GABAergic mechanisms in epilepsy.
        Epilepsia. 2001; 42: 8-12
        • Nilsson P.
        • Hillered L.
        • Pontén U.
        • Ungerstedt U.
        Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats.
        J Cereb Blood Flow Metab. 1990; 10: 631-637
        • Kanthan R.
        • Shuaib A.
        Clinical evaluation of extracellular amino acids in severe head trauma by intracerebral in vivo microdialysis.
        J Neurol Neurosurg Psychiatry. 1995; 59: 326-327
        • Hasegawa D.
        • Matsuki N.
        • Fujita M.
        • Ono K.
        • Orima H.
        Kinetics of glutamate and gamma-aminobutyric acid in cerebrospinal fluid in a canine model of complex partial status epilepticus induced by kainic acid.
        J Vet Med Sci. 2004; 66: 1555-1559
        • Wagner A.K.
        • Miller M.A.
        • Scanlon J.
        • Ren D.
        • Kochanek P.M.
        • Conley Y.P.
        Adenosine A1 receptor gene variants associated with post-traumatic seizures after severe TBI.
        Epilepsy Res. 2010; 90: 259-272
        • Weiss G.H.
        • Salazar A.M.
        • Vance S.C.
        • Grafman J.H.
        • Jabbari B.
        Predicting posttraumatic epilepsy in penetrating head injury.
        Arch Neurol. 1986; 43: 771-773
        • Aarabi B.
        • Taghipour M.
        • Haghnegahdar A.
        • Farokhi M.
        • Mobley L.
        Prognostic factors in the occurrence of posttraumatic epilepsy after penetrating head injury suffered during military service.
        Neurosurg Focus. 2000; 8: e1
        • Eftekhar B.
        • Sahraian M.A.
        • Nouralishahi B.
        • Khaji A.
        • Vahabi Z.
        • Ghodsi M.
        • et al.
        Prognostic factors in the persistence of posttraumatic epilepsy after penetrating head injuries sustained in war.
        J Neurosurg. 2009; 110: 319-326
        • Raymont V.
        • Salazar A.M.
        • Lipsky R.
        • Goldman D.
        • Tasick G.
        • Grafman J.
        Correlates of posttraumatic epilepsy 35 years following combat brain injury.
        Neurology. 2010; 75: 224-229
        • Kendirli M.T.
        • Rose D.T.
        • Bertram E.H.
        A model of posttraumatic epilepsy after penetrating brain injuries: effect of lesion size and metal fragments.
        Epilepsia. 2014; 55: 1969-1977
        • Scher A.I.
        • Wu H.
        • Tsao J.W.
        • Blom H.J.
        • Feit P.
        • Nevin R.L.
        • et al.
        MTHFR C677T genotype as a risk factor for epilepsy including post-traumatic epilepsy in a representative military cohort.
        J Neurotrauma. 2011; 28: 1739-1745
        • Dardiotis E.
        • Fountas K.N.
        • Dardioti M.
        • Xiromerisiou G.
        • Kapsalaki E.
        • Tasiou A.
        • et al.
        Genetic association studies in patients with traumatic brain injury.
        Neurosurg Focus. 2010; 28: E9
        • Zintzaras E.
        • Lau J.
        Synthesis of genetic association studies for pertinent gene-disease associations requires appropriate methodological and statistical approaches.
        J Clin Epidemiol. 2008; 61: 634-645