INTRODUCTION
Several theories have been proposed to explain the heterogeneity in recovery rates and outcome following traumatic brain injury (TBI). Given the complexity of the sequelae of these injuries, there does not yet appear to be a ready explanation that can account for the widespread variability in outcome observed after TBI. However, genetic factors may hold clues. There is growing evidence that the apolipoprotein E (APOE) gene may be linked to outcome following brain injury (Dardiotis et al., Reference Dardiotis, Fountas, Dardioti, Xiromerisiou, Kapsalaki, Tasiou and Hadjigeorgiou2010; Jordan, Reference Jordan2007). APOE is a protein that is predominantly involved in the transportation of lipid molecules across tissues such as the central nervous system. A central function of APOE is to maintain and restore neuronal membranes and tissue after they have been compromised (Dardiotis et al., Reference Dardiotis, Fountas, Dardioti, Xiromerisiou, Kapsalaki, Tasiou and Hadjigeorgiou2010). The APOE gene is polymorphic, meaning that it is comprised of three primary alleles (ϵ2, ϵ3, and ϵ4; Eisenberg, Kuzawa, & Hayes, Reference Eisenberg, Kuzawa and Hayes2010). Possession of at least one ϵ4 allele has been associated with unfavorable outcome following brain injury (for a review, see Dardiotis et al., Reference Dardiotis, Fountas, Dardioti, Xiromerisiou, Kapsalaki, Tasiou and Hadjigeorgiou2010).
To date, many efforts have focused on establishing associations between the ϵ4 allele and global outcomes following TBI (Chiang, Chang, & Hu, Reference Chiang, Chang and Hu2003; Liaquat, Dunn, Nicoll, Teasdale, & Norrie, Reference Liaquat, Dunn, Nicoll, Teasdale and Norrie2002; Teasdale, Murray, & Nicoll, Reference Teasdale, Murray and Nicoll2005). However, few studies have examined more precise measures of outcome. Given that physical, cognitive, and affective difficulties have consistently been reported following mild TBI (Lovell et al., Reference Lovell, Iverson, Collins, Podell, Johnston, Pardini and Maroon2006; Silver, McAllister, & Arciniegas, Reference Silver, McAllister and Arciniegas2009), genetic factors could be illuminating.
Studies that have evaluated the relationship between the APOE gene and symptom reporting following TBI have been mixed. Ariza et al. (Reference Ariza, Pueyo, del M Matarín, Junqué, Mataró, Clemente and Sahuquillo2006) examined post-concussion symptoms in a sample of patients with moderate to severe TBI and found that ϵ4+ patients endorsed more symptoms at 6 months post-injury compared to ϵ4- patients. In contrast to Ariza and colleagues’ (Reference Ariza, Pueyo, del M Matarín, Junqué, Mataró, Clemente and Sahuquillo2006) findings, Chamelian, Reis, and Feinstein (Reference Chamelian, Reis and Feinstein2004) reported no symptom score differences between ϵ4+ and ϵ4- participants with mild to moderate TBI at 6 months post-injury. Moran et al. (Reference Moran, Taylor, Ganesalingam, Gastier-Foster, Frick, Bangert and Wright2009) also evaluated the relationship between the ϵ4 allele and post-concussion symptom reporting in children between the ages of 8 and 15 who had sustained mild TBIs. Symptoms were evaluated at “baseline” (within 2 weeks following the injury), and at 3 and 12 months post-injury. Moran et al. (Reference Moran, Taylor, Ganesalingam, Gastier-Foster, Frick, Bangert and Wright2009) reported no group differences at any of the time points assessed post-injury.
This brief review shows that the current literature regarding the APOE gene and post-concussion symptom reporting is still in its infancy. Thus far, heterogeneous samples have been studied, making it difficult to draw conclusions about the precise role that the APOE gene has on symptom reporting outcome. Furthermore, examination of genetic factors on specific outcomes following sports-related concussions, in particular, has largely gone unexplored. With these considerations in mind, the main objective of this study was to evaluate the relationship between the APOE ϵ4 allele and symptom reporting patterns following sports-related concussion in a sample of collegiate athletes. It was hypothesized that participants with the ϵ4 allele would show greater symptomatology following concussion than participants without the ϵ4 allele.
METHOD
Participants and Procedures
Participants included 42 college athletes who participated in a clinically based university sports-concussion management program. All participants in the present study sustained a mild TBI, or concussion, as defined by the following criteria: loss of consciousness lasting 30 min or less, loss of memory for events immediately before or after the injury lasting less than 24 hr, or any alteration in mental state at the time of injury (i.e., disorientation, confusion, etc.; Ruff, Iverson, Barth, Bush, & Broshek, Reference Ruff, Iverson, Barth, Bush and Broshek2009). Team physicians determined TBI status, and concussed athletes were referred for neuropsychological testing as soon as possible following the injury.
Study participants were selected from a sample of athletes who had sustained concussions between 2002 and 2014 and were subsequently referred for post-concussion neuropsychological testing. Briefly, to be included in the study, participants must have sustained a mild TBI or concussion, according to the criteria described above, and completed post-concussion testing within three months following their injury. The 3-month time frame was chosen because we were interested in examining the relationship between genetic factors and relatively acute outcome following concussion, while maintaining a sufficient sample size. Additionally, concussed athletes must have provided a buccal (cheek cell) sample that was successfully analyzed for their APOE genotype. Our clinically based concussion management program was initiated in 2002, but the genetics arm of the program did not begin until 2011. At the time of participant selection, 34 athletes had been recruited prospectively (i.e., at the time of their post-concussion assessment) for the genetics portion of the study, and another 31 athletes who had previously participated in post-concussion testing were contacted by phone or email and offered participation in the genetics portion of the study. Among these 31 participants, 18 declined participation due to lack of interest in participating in a follow-up study, and 13 consented; however, 5 of these 13 were eventually excluded because they had completed post-concussion testing more than 3 months after their concussive injury.
The final sample (n=42) was comprised of mostly male athletes (83.3%) who had completed, on average, 13.5 years (SD=1.3) of education. The average time tested post-injury was 9.8 days (SD=14.6; Mdn=4.0; Range=0–72 days), and 73.8% of the athletes were tested within the first week following their concussion. All participants had sustained relatively mild concussions, as only 14.3% of the entire sample reported loss of consciousness.
This study was approved by our university’s institutional review board, and eligible participants signed an informed consent form before participation in research.
Laboratory Procedures
DNA extraction was performed on buccal samples using methods and materials as described by Freeman et al. (Reference Freeman, Smith, Curtis, Huckett, Mill and Craig2003). The APOE genotype for each participant was determined by using two Taqman® Single Nucleotide Polymorphism (SNP) assays for the SNPs APOE112 and APOE158. The procedures outlined in Christensen et al. (Reference Christensen, Batterham, Mackinnon, Jorm, Mack, Mather and Easteal2008) and Ingelsson et al. (Reference Ingelsson, Shin, Irizarry, Hyman, Lilius, Forsell and Graff2003) were used to define the different genotypes. Genotyping results could be any pair of ϵ2, ϵ3, and ϵ4 alleles.
Measures
The Post-Concussion Symptom Scale (PCSS) was used to evaluate athletes’ self-reported symptoms following concussion. The PCSS (Lovell et al., Reference Lovell, Iverson, Collins, Podell, Johnston, Pardini and Maroon2006) is a 22-item measure designed to evaluate the severity of commonly experienced post-concussion symptoms. Athletes are asked to rate their current symptoms using a 0–6 scale, where 0 represents no symptoms and 6 represents severe symptoms. The PCSS is administered through the ImPACT computer program, and all athletes individually completed the PCSS under the supervision of a trained doctoral student or undergraduate research assistant. The internal consistency of the PCSS is excellent, ranging from 0.89 to 0.94 (Lovell et al., Reference Lovell, Iverson, Collins, Podell, Johnston, Pardini and Maroon2006).
The PCSS was used to calculate several symptom-related outcome indices, including a total symptom score and four symptom clusters. Briefly, the total symptom score was calculated by adding the ratings of all 22 items on the PCSS (higher scores represent greater symptoms), and the symptom clusters were derived from previous factor analytic work (Merritt & Arnett, Reference Merritt and Arnett2014) and were calculated as follows: physical symptoms (7 items; α=.85; possible range=0–42), cognitive symptoms (4 items; α=.94; possible range=0–24), affective symptoms (4 items; α=.76; possible range=0–24), and sleep symptoms (4 items; α=.80; possible range=0–24).
RESULTS
Descriptive Statistics
Participants were divided into two groups based on ϵ4 allele status: 15 athletes (35.7%) were ϵ4 positive and 27 (64.3%) were ϵ4 negative. Descriptive statistics, including basic demographic and injury severity variables, are presented in Table 1 by group. As indicated in the table, the allele groups were well-matched, as there were no significant differences between the two groups on any of the demographic or injury severity variables examined.
Table 1 Sample characteristics by ϵ4 allele group.
a There was an outlier in the negative ϵ4 allele group (one athlete was tested 72 days post-injury). When the outlier was removed, the results did not significantly change; thus, the outlier was used in the analyses.
b Contact sports include football, hockey, lacrosse, and rugby; limited contact sports include basketball, golf, and soccer.
Symptom Reporting and ϵ4 Allele Status
Given that the post-concussion PCSS symptom scores were not normally distributed, non-parametric statistics were used to compare the various symptom indices across the two ϵ4 allele groups. Figure 1 displays the means and standard errors of each symptom variable by ϵ4 allele group. Mann-Whitney U tests showed that athletes with the APOE ϵ4 allele reported greater symptoms than athletes without the ϵ4 allele on all of the symptom indices evaluated, with significant differences found on the following indices: the PCSS total symptom score, the physical symptom cluster, and the cognitive symptom cluster (all p at least <.05; see Figure 1).
Fig. 1 Means and standard errors of each symptom variable are presented in the figure, according to ϵ4 allele group. Total symptom score comparisons are illustrated in Figure 1a and symptom cluster comparisons are illustrated in Figure 1b. The total symptom score is comprised of all 22 items on the PCSS, and the symptom clusters were derived from a previous factor analysis of the PCSS (Merritt & Arnett, Reference Merritt and Arnett2014). *p<.05, **p<.01. Total symptom score: Cohen’s d=0.73, medium-large effect; physical symptom cluster: Cohen’s d=0.87, large effect; cognitive symptom cluster: Cohen’s d=0.60, medium effect.
Logistic regression analyses were then used to further examine the relationship between ϵ4 allele status and post-concussion symptom reporting. Specifically, the physical and cognitive symptom clusters were each dichotomized into “symptoms present” versus “symptoms absent” groups, and each served as a criterion variable in separate logistic regressions. Symptom groups were calculated as follows: athletes with a physical symptom score of “0” were classified into the “symptoms absent” group (n=27; 64.3%), and athletes with a physical symptom score of “≥1” were classified into the “symptoms present” group (n=15; 35.7%). Similarly, athletes with a cognitive symptom score of “0” were classified into the “symptoms absent” group (n=24; 57.1%), and athletes with a cognitive symptom score of “≥1” were classified into the “symptoms present” group (n=18; 42.9%). Table 2 shows the breakdown of participants who were classified as “symptoms present” and “symptoms absent” by ϵ4 allele group for the physical and cognitive symptom clusters.
Table 2 Participants classified as “symptoms present” and “symptoms absent” by ϵ4 allele group
With respect to physical symptoms, ϵ4 allele status was a significant predictor of the “symptoms present” group, such that ϵ4+ athletes were more likely to endorse physical symptoms than ϵ4- athletes, χ2(1, N=45)=5.95, p=.015 (Nagelkerke’s R 2=.18; odds ratio=5.25; 95% CI=1.33–20.76). As for cognitive symptoms, ϵ4 allele status was also a significant predictor of the “symptoms present” group, such that ϵ4+ athletes were more likely to endorse cognitive symptoms than ϵ4- athletes, χ2(1, N=45)=5.45, p=.020 (Nagelkerke’s R 2=.16; odds ratio=4.75; 95% CI=1.23–18.41).
DISCUSSION
Given that several previous studies have evaluated the relationship between the APOE ϵ4 allele and gross outcome following TBI, the main purpose of our study was to narrow the focus and evaluate whether there may be an association between the ϵ4 allele and more specific outcomes following TBI. To our knowledge, this is the first study to have specifically examined the relationship between the APOE ϵ4 allele and post-concussion symptom reporting patterns among concussed collegiate athletes. The PCSS was used as the primary outcome measure, and several symptom-related variables were derived from the PCSS, including a total symptom score and four symptom clusters (physical, cognitive, affective, and sleep). It was hypothesized that ϵ4 positive participants would show greater symptomatology following concussion as compared to ϵ4 negative participants, and the results largely supported our hypothesis. Specifically, ϵ4 positive athletes reported significantly more symptoms than ϵ4 negative athletes across the following symptom indices: the PCSS total symptom score, the physical symptom cluster, and the cognitive symptom cluster, indicating that ϵ4 positive participants may be at greater risk for experiencing poorer post-concussion outcomes.
When placing our findings within the context of the broader TBI literature, as noted previously, there are few studies available for comparison. Ariza et al. (Reference Ariza, Pueyo, del M Matarín, Junqué, Mataró, Clemente and Sahuquillo2006) examined adult patients with moderate to severe TBI and reported that ϵ4+ patients reported greater symptoms than ϵ4- patients at 6 months post-injury. Findings from Ariza et al. (Reference Ariza, Pueyo, del M Matarín, Junqué, Mataró, Clemente and Sahuquillo2006) are consistent with our results, as both studies indicate that APOE ϵ4 allele carriers show greater symptomatology after sustaining a brain injury than do non-ϵ4 allele carriers. However, in contrast to our findings and the results of Ariza et al. (Reference Ariza, Pueyo, del M Matarín, Junqué, Mataró, Clemente and Sahuquillo2006), other studies have found no differences between ϵ4+ and ϵ4- patients with respect to symptom reporting when assessing patients with mild to moderate TBI (Chamelian et al., Reference Chamelian, Reis and Feinstein2004; Moran et al., Reference Moran, Taylor, Ganesalingam, Gastier-Foster, Frick, Bangert and Wright2009).
When evaluating the above studies, considerable methodological differences are observed across the studies with regard to the sample studied (i.e., adult vs. child, mechanism of injury, severity of TBI), the timing of the post-injury assessment, and the method of evaluating post-concussion symptoms. These methodological differences likely contribute to the disparate findings that have resulted, and suggest a need for a more fine-tuned approach for evaluating the influence of genetic factors on outcome following TBI. For instance, given the proposed pathophysiological differences across mild, moderate, and severe TBI (Blennow, Hardy, & Zetterberg, Reference Blennow, Hardy and Zetterberg2012), it may be beneficial to examine these populations as unique cohorts to more precisely understand how genetics influence response to brain injury. Furthermore, the timing of the post-injury evaluation is another important variable that could impact conclusions. A major advantage to the current study was that our sample was relatively homogeneous—all participants were collegiate athletes with similar ages and levels of education, all had sustained concussions as a result of sports participation, and the majority of the sample was assessed within one week of sustaining a concussion. Thus, our findings extend current knowledge by illustrating how genetic factors impact relatively acute symptom expression following sports-related concussion.
In addition to examining symptom severity differences between ϵ4+ and ϵ4- athletes, we also evaluated the extent to which the ϵ4 allele could predict those individuals who specifically endorsed physical and cognitive symptoms post-concussion. Results showed that the ϵ4 allele significantly predicted the presence of both physical and cognitive symptoms. With respect to physical symptoms, the ϵ4 allele explained 18% of the variance; for cognitive symptoms, it explained 16% of the variance. Importantly, past research has suggested that several pre-morbid and injury-specific variables influence the presence and duration of post-concussion symptoms (Lange et al., Reference Lange, Brickell, French, Ivins, Bhagwat, Pancholi and Iverson2013; Merritt & Arnett, Reference Merritt and Arnett2014). Our findings suggest that, in addition to these variables, genetic factors play a significant role in post-concussion symptom reporting, and that both physical and cognitive symptoms may be especially susceptible to the effects of the ϵ4 allele.
One limitation of the present study is the small sample size. However, there is a precedent in the literature for conducting genetics-related studies using similar sample sizes (Bazarian, Zemlan, Mookerjee, & Stigbrand, Reference Bazarian, Zemlan, Mookerjee and Stigbrand2006; Sundström et al., Reference Sundström, Marklund, Nilsson, Cruts, Adolfsson, Van Broeckhoven and Nyberg2004). Another limitation concerns the generalizability of our findings. In the present study, we specifically focused on collegiate athletes who had sustained concussions, or mild TBIs; thus, our findings may be less generalizable to other populations such as adolescents or older adults, as well as to samples with more severe brain injuries. However, as discussed above, given the disparate findings that have resulted in the literature with respect to the APOE gene and outcome following brain injury, it is necessary to examine more homogeneous TBI samples (i.e., limit sample to a specific group who has sustained similar injury severities such as concussed athletes) so that we may develop a more nuanced understanding of such relationships. Furthermore, given the widespread interest and concern over the effects of sports-related concussions, it is thought that our findings will still be relevant and clinically meaningful to a broad population. Another limitation with respect to the generalizability of our findings is that our results may not be as applicable to athletes who do not have a history of concussion, as approximately two-thirds of the sample had at least one prior concussion. Finally, our study was restricted to the evaluation of self-reported sequelae of concussion. Future studies would benefit from not only evaluating self-reported symptoms, but to also assess the relationship between the ϵ4 allele and other measures of impairment following concussion.
Although these results will need to be replicated in a larger sample, our findings indicate that, compared to athletes without the ϵ4 allele, athletes with the ϵ4 allele have a propensity to report greater symptomatology post-concussion, particularly within the domain of physical and cognitive symptoms. Future studies examining the role of the ϵ4 allele in concussion outcome are warranted.
ACKNOWLEDGMENTS
We thank Dr. Deborah Grove, PhD, Director of the Genomics Core Facility at Penn State, who was instrumental in processing the genetic data for this study. She also was involved in writing the description of the laboratory procedures (under the Method section). We also thank Dr. Wayne Sebastianelli, MD, Director of Athletic Medicine at Penn State, and Penn State Sports Medicine, for their ongoing support of our research. Ms. Merritt has no conflicts of interest to report. Dr. Arnett has served as part of a paid speaker series sponsored by EMD Serono. He has also served as a consultant for Biogen IDEC. Finally, the research described in this manuscript was supported by a grant to the first author from the American Psychological Foundation.
