Association Analysis of Polymorphic Variants of the BDNF Gene in Athletes

As BDNF is one of the group of neurotrophins highly influencing the processes happening in the brain, such as the processes of learning and personality creation, we decided to look closer at its genetic variations in association with the personality of a group of athletes and their controls. The study group consisted of 305 volunteers: martial arts athletes (n = 153; mean age = 24.06) and healthy non-athletes as controls (n = 152; mean age = 22.23). Thirty-eight percent of the martial arts group achieved the championship level. Both the martial arts and control subjects were examined using the NEO Five-Factor Personality Inventory (NEO-FFI) and the State-Trait Anxiety Inventory (STAI) scales. The results of the NEO-FFI and STAI inventories were given as sten scores. The conversion of the raw score to the sten scale was performed according to Polish norms for adults. Genomic DNA was extracted from blood leukocytes and then genotyped using a PCR method for the following polymorphisms: BDNF rs10767664 and BDNF rs2030323. We observed statistical significance for both polymorphisms when comparing martial arts athletes with the control group in relation to the conscientiousness and extraversion scales. However, since few extant articles consider this association, our results still require further analysis, probably by considering a larger group.


Introduction
Brain-derived neurotrophic factor (BDNF) is one of the most considered neurotrophins due to its importance in homeostasis, health, and disease. However, being one of the most widely studied neurotrophins, numerous questions concerning BDNF's connection to molecular biology and signaling pathways still remain. Although there is a lot of information concerning BDNF's gene structure, peptide composition, signaling pathways, and the functional effects of genetic variations, the biology of BDNF is diverse and varied [1,2]. BDNF is, as a rule, expressed in both developing and adult mammalian brains where it is critical factor in neuronal survival, morphogenesis, brain plasticity, synaptic function, morphological change, and in the differences in neuronal response [3]. The mammalian nervous system presents 2 of 11 highly experience-dependent changes in synapse structure and function and is considered an important element in memory formation [4], influencing the long-term potentiation (LTP) of hippocampal synapses, especially in the CA1 region. The sequence of plasticity formation is connected with some steps. The first step, known as early-LTP, is transcription-and translation-independent and lasts for 1-2 h; the second step, late-LTP, can last for hours or days and is connected to transcription and translation activity [5][6][7]. BDNF is considered a key mediation of activity-induced LTP in the hippocampus and other brain regions [8][9][10]. The modulatory effect of BDNF is connected to the modification of components already available at the synapse in case of early effects, while the long-term results from the modification of translation activity at the synapse changes in transcription. Such changes can be of high importance for behavioral plasticity that, as a result, modifies human personality [2].
In this aspect, changes in BDNF expression, release, and neuromodulatory activity can be mediated by both epigenetic and post-translational mechanisms. The association between many pathological conditions and developmental experiences has been emphasized in connection with plasticity in the hippocampus, which is traditionally considered an element in learning and memorizing processes; however, other important structures should also be considered, such as the amygdala, which is also induced by BDNF changes. Nonetheless, BDNF is shown to be a biochemical integrator of convergent cellular signals and a central element of neural plasticity [11][12][13].
Considering the importance of BDNF in learning processes and personality traits, we decided to concentrate on an association between physical activity, personality traits, and polymorphic differences. Some studies emphasize that environmental enrichment in a form of environmental variation [14], physical exercise [15], or social enrichment [16] can induce higher a level of BDNF in the brain, which in turn results in increased memorization and learning abilities [17]. Animal models also show an upregulation of BDNF gene expression, especially in the hippocampal area [18,19]; however, other structures of the nervous system were also influenced, among them the inferior temporal areas of the brain [20].
As all these areas and pathways are of high importance in the creation of personality, we considered it reasonable to search for an association between them. A direct relation exists between the brain-derived neurotrophic factor gene and personality traits. BDNF expression, being an important factor in learning processes, is influenced by genetic polymorphisms. One of the examples indicated on nucleotide polymorphism at nucleotide 196G/A, which results in the substitution of valine by methionine at codon 66 (val66met) of the pro-BDNF molecule, and therefore the presence of the met allele, was linked to the decreased activity-dependent secretion of BDNF [21]. However, the authors suggest that the existence of such an association has not been deeply investigated [22][23][24]. Taking this finding into consideration, we decided to direct our study to an investigation of the association between personality traits and polymorphisms in the BDNF gene.
The martial arts and control subjects were examined using the NEO Five-Factor Personality Inventory (NEO-FFI) and State-Trait Anxiety Inventory (STAI) scales.
Sten scores were used to present the results of the NEO-FFI and STAI inventories. The obtained raw scores were converted using the sten scale according to Polish norms for adults with the assumption that 1-2 indicated very low scores; 3-4 indicated low scores; 5-6 indicated average scores; 7-8 indicated high scores; 9-10 indicated very high scores.

Genotyping
Blood for the genetic assays was collected in tubes with EDTA (anticoagulant). Blood leukocytes were also used to obtain genomic DNA. We used a High Pure Polymerase Chain Reaction (PCR) Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) to isolate the cell DNA. The process of extraction was conducted in accordance with the manufacturer's instructions. The extracted DNA samples were stored at 4 • C until further analysis.
Venous blood collected according to standard procedures was the source of genomic DNA. The PCR method was used to genotype the samples. We used the set of Search TaqMan ® Assays from Thermo Fisher Scientific to identify the polymorphisms distribution.

Statistical Analysis
The concordance between the genotype frequency distribution and Hardy-Weinberg equilibrium (HWE) was checked with HWE software (https://wpcalc.com/en/equilibriumhardy-weinberg/ (accessed on 3 June 2021)). The associations between BDNF rs10767664 and BDNF rs2030323, the martial arts and control subjects, and the NEO Five-Factor Inventory (NEO-FFI) were analyzed with a multivariate analysis of the factor effects of ANOVA (NEO-FFI/ scale STAI/ × genetic feature × control and martial arts × (genetic feature × control and martial arts)). The homogeneity of the variance condition was satisfied (Levene test p > 0.05). The normality of distribution was not fulfilled in the case of the analyzed variables. The NEO Five-Factor Inventory (neuroticism, extraversion, openness, agreeability, and conscientiousness) was analyzed and compared with the usage of the Mann-Whitney U test. A chi-square test was applied to compare genotype frequencies between healthy control subjects and martial arts athletes for BDNF rs10767664 and BDNF rs2030323 polymorphism. All calculations were performed using STATISTICA 13 (Tibco Software Inc, Palo Alto, CA, USA) for Windows (Microsoft Corporation, Redmond, WA, USA).

Results
The frequency distributions were in accordance with the HWE. No statistical difference was found between martial arts participants and people from the control group (Table 1). The BDNF rs10767664 and BDNF rs2030323 genotypes and alleles frequencies in the studied sample did not differ in the analyzed subject groups (Table 2). The means and standard deviations for the NEO Five-Factor Inventory results in the martial arts subject and control subject groups are presented in Table 3. In comparison with the controls, the case group subjects had significantly higher scores on the extraversion/scale (M 6.89 vs. M 6.43, p = 0.0405) and conscientiousness/scale (M 7.23 vs. M 5.89, p < 0.0001). Conscientiousness/scale and BDNF rs10767664. The results of 2 × 3 factorial ANOVA illustrated a statistically significant effect of the combined factor BDNF rs10767664 genotype of martial arts/control (F 2275 = 3.94, p = 0.0205, η 2 = 0.028) (Table 4, Figure 1). Our sample had more than 71% power to detect the combined factor of martial arts/control × BDNF rs10767664 and their interaction effect (about 2.8% of the phenotype variance). The post hoc analysis is shown in Table 4. The results of the post hoc test are included in Table 6.   The results of 2 × 3 factorial ANOVA illustrated a statistically significant effect of the combined factor BDNF rs2030323 genotype of martial arts/control (F2,247 = 4.08, 0.0181, η 2 = 0.032) (Table 5, Figure 2.). Our sample had more than 72% power to detect the combined factor of martial arts/control × BDNF rs2030323 and their interaction effect (about 3.2% of the phenotype variance). The post hoc analysis is shown in Table 4. The results of the post hoc test are included in Table 6.  Conscientiousness/scale and BDNF rs2030323. The results of 2 × 3 factorial ANOVA illustrated a statistically significant effect of the combined factor BDNF rs2030323 genotype of martial arts/control (F 2,247 = 4.08, 0.0181, η 2 = 0.032) (Table 5, Figure 2.). Our sample had more than 72% power to detect the combined factor of martial arts/control × BDNF rs2030323 and their interaction effect (about 3.2% of the phenotype variance). The post hoc analysis is shown in Table 4. The results of the post hoc test are included in Table 6.

Discussion
We observed a statistically significant interaction between the occurrence of T/T and A/T genotypes in BDNF rs10767664 among individuals practicing martial arts, and higher results on the NEO FFI sten and conscientiousness scales in comparison with the control group (Figure 1, 7.32 vs. 5.62, p = 0.0000; 7.22 vs. 6.35, p = 0.0420; Table 6).
We also noticed a statistically significant interaction between the frequency of G/G genotypes and higher results on the NEO FFI sten and conscientiousness scales by comparing the martial arts athletes with the control group (Figure 2, 7.37 vs. 5.64, p = 0.0000, Table 6).
The novelty of our scientific assumption is its combination of personality traits with genotype variants of BDNF polymorphism. This assumption is correct when we consider biological level due to the fact that BDNF influences brain neuroplasticity. Conscientiousness as a phenotype expressed with behavior might be conditioned with polymorphic variants of genes connected to neurotransmission [2].
As the level of BDNF is connected to different factors, amongst others, with physical activity, some research has concentrated on such an association. One study noticed the association between 35 min sessions of physical activity, cognitive training, and mindfulness practice in a group of healthy older adults. The results showed that a single bout of physical activity influenced serum BDNF levels more significantly than any of two other forms of activity. The authors emphasized that physical activity influenced serum BDNF by peripheral origin; however, the cognitive functions of an individual can be implied by it [26].
Humans, both younger and older, showed an association between their physical activity level and the BDNF level in the serum [27], even after a singular bout of training [28,29]. The authors noticed that barely any research exists concerning physical activity in their capacity to alter BDNF levels. Nonetheless, they assumed that physical exercises can show immediate effects on the serum levels of BDNF in healthy humans. What is interesting is that they also noticed that mindfulness practice can induce the same reaction as an increased level of BDNF. Few previously conducted longitudinal studies have suggested that that there is an association between meditation practice and cognition [30], and BDNF is suggested to be a key mediator in this situation [31]. Moreover, a combination of the two over a 12-week period improved visuospatial memory, which is probably connected to parallel changes in brain connectivity. Therefore, we can think about both physical activity and mindfulness training as elements with a high potential to change BDNF levels [32].
Polymorphisms in BDNF were found to be a modifying factor in cases of depression in children [33], as well as in incidents of depression in elders [34]. The authors of that research emphasized that BDNF val66met polymorphism did not directly influence ones' vulnerability to depressive disorders, but that its occurrence was more probable following stressful events. The authors also indicated that such a relation exists not only between depression and BDNF polymorphism, but also between other health disorders, such as stroke, cancer, and subsequent depressive states [35,36]. Both these studies and our observations have underlined the fact that BDNF polymorphisms have the potential to influences health environments with long-term effects. However, the authors' observations suggest that the results may differ for various populations [37,38].
Nevertheless, we should also mention some limitations of our research. In our research, we chose only to analyze BDNF; however, we are aware that the phenotypic manifestation of personality traits may also be influenced by other polymorphisms, as it is a polygenic and multifactorial feature. However, an analysis of BDNF is crucial and necessary to understand its biological bases. Nonetheless, further analyses concerning more numerous groups of athletes and higher number of genes are still needed. It also seems justified to analyze methylation in the promotor region of chosen genes.

Conclusions
Since we observed the statistical significance of both polymorphisms when comparing martial arts athletes with the control group in relation to the conscientiousness scale, we contend that it is an element of high importance in the creation of human personality. However, since few extant articles consider this association, our results still require further analysis, probably by considering a more numerous group.