Single nucleotide polymorphism in Interleukin 1 alpha gene might be involved in the Oral Herpes recurrent episodes in Brazilian Para-Athletes

The main goal of this study was to investigate if there is an association between Oral Herpes (OH) recurrent episodes and Single Nucleotide Polymorphisms (SNPs) in IL1A, IL10, and IL1RN genes in a group of Brazilian Para-athletes. This transversal study was prepared according to the STrengthening the REporting of Genetic Association Studies (STREGA) guidelines. Oral examination and DNA collection for genotyping were performed in a non-probabilistic convenience sampling composed of Brazilian para-athletes who participated in a Brazilian selective competition. Data referring to the general characterization of sample were collected through a self-reported questionnaire. Candidate genes were chosen with the UCSC Genome Browser and SNPs in IL1A gene (rs17561, rs1304037), IL10 gene Research, Society and Development, v. 10, n. 16, e564101624166, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.24166 2 (rs1800871), and IL1RN gene (rs9005) were selected and investigated in allelic, genotypic, dominant, and recessive models. Hardy-Weinberg equilibrium was evaluated in each SNP. The sample was composed of 273 para-athletes (63 (23.4%) practice swimming, 61 (22.3%) powerlifting and 145 (63.7%) athletics). OH recurrent episodes was related by 47 (17.2%) para-athletes and the presence of T allele in the rs1304037 increased chance of OH. These findings suggest that rs1304037 in IL1A gene is associated with OH recurrent episodes in para-athletes.


Introduction
Para-athletes are individuals who have a physical or intellectual impairment and perform some physical activity regularly and, according to the International Paralympic Committee, this term is applicable for all individuals with impairment that competes in recreational, collegiate, and/or official competitions, regardless of competition level (Tweedy & Vanlandewijck, 2011;Tweedy et al., 2014;Ravensbergen et al., 2016). No doubt, the sports practice reaps significant health benefits to the para-athlete improving their physiological and psychological balance, mental wellbeing, and increasing selfconfidence, self-acceptance, respect, equality and contributing to interpersonal relationships (McConkey et al., 2013;Shapiro & Malone, 2016). On the other hand, despite all benefits of sports practice, when a para-athlete (or athlete) is training intensely, non-rare, an organic imbalance can occur and impacts their general and oral health (Gleeson, 2007).
Many oral conditions can affect individuals of all ages, are prevalent worldwide, and have historically been considered important global health burdens (Piccininni & Fasel, 2005. Oral herpes (OH) is an example: an estimated 3.7 billion people have OH infection globally (James et al., 2016). OH, is a disease caused by a Herpes Simplex Type I virus (HSV-1) and causes extreme discomfort, paresthesia, tenderness, pain, fever, gingivostomatitis, and a burning sensation in the affected (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.24166 3 region (Arduino & Porter, 2008). These lesions are located around the mouth, because of this are also called orolabial, and are very common in stressful moments, like during sports competitions (Liknes, 2011;Collins & O'Connell, 2012) and are usually triggered by stress, exposure to ultraviolet rays and immunosuppression (Minagawa et al., 2004;Peterson et al., 2019).
OH is a complex multifactorial condition, in which many environmental, systemic, and genetic factors play an important role in the final phenotypes (Kriesel et al., 2014). In fact, the recurrence of herpetic lesions, called cold sore, is also dependent on viral factors and mainly on the host's defense response, which involves activation of pro-inflammatory immune cells (Minami et al., 2002;Richards et al., 2003) including interleukins (Hurme et al., 2003). Previous studies showed an association between interleukin and HSV-1 recurrent infections (Minagawa et al., 2004;Richards et al., 2003;Fields et al., 2006). In this context, considering the inflammatory and genetic aspects of the OH, genes that codified interleukin such as interleukin 1 alpha (IL1A), interleukin 1 10 (IL10), and interleukin 1 antagonist receptor (IL1RN) are candidate genes to be studied in OH recurrent episodes susceptibility. So, the main goal of this study was to investigate if there is an association between OH episodes and Single Nucleotide Polymorphisms (SNPs) in IL1A, IL10, and IL1RN genes in a group of Brazilian Para-athletes.

Ethical Approval, Type of Study, and Sampling
This is a transversal study that was approved by the local Ethics and Research Committee (#3.261.377) following resolution 466/12 of the National Health Commission and was prepared according to the STrengthening the REporting of Genetic Association Studies (STREGA) guidelines (Little et al., 2005). Appropriate written informed consent was obtained from all participants and legal guardians when the para-athlete is underage. A non-probabilistic convenience sampling composed by para-athletes was used. The para-athletes were selected during Brazilian selective competition organized by the Brazilian Paralympic Committee in April 2019, in Curitiba, Paraná, Brazil. The criteria of grouping para-athletes in each sport were determined by the Brazilian Paralympic Committee according to International Standard for Eligible Impairments guidelines and, in this study, we selected para-athletes who participated in athletics, powerlifting, and swimming. Additional exclusion criteria were para-athletes who have a diagnosis of mental disorder or severe intellectual disability that non-enables them to understand and answer a questionnaire or that makes DNA sample collection impossible and genetic syndromes.

Non-clinical data collection
Data referring to the general characterization of the para-athletes were collected through a self-reported questionnaire in the Portuguese language that was previously described in von Held et al. (2021). The questionnaire addressed gender, age, ethnicity, school education, sport and inquired the participant about OH lesions and OH recurrent episodes. All individuals that were not sure about their answers were excluded from the survey.

Clinical data collection
A team composed of previously trained dentists and note-takers performed the para-athletes' oral examination. The calibration process was coordinated by an experienced examiner in epidemiological surveys (Gold-Standard) who guided the conduct of the theoretical and practical training steps. Kappa value after calibration was higher than 0.90 indicating a high degree of data reproducibility. The examination was conducted with the para-athletes seated in a chair, using natural light, tongue depressors, and gauze. Research, Society and Development, v. 10, n. 16, e564101624166, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.24166

DNA sample collection and Genotyping
Genomic DNA for genotyping analysis was extracted from buccal cells isolated from saliva as previously described and established (Küchler et al., 2012). The amount and purity of the DNA were determined by a spectrophotometer (Nanodrop 1000; Thermo Scientific, Wilmington, DE). Candidate genes were chosen with the UCSC Genome Browser website to identify previously characterized SNPs for each candidate gene, according to their possible function regulation and alleles frequency. A total of 4 SNPs in IL1A gene (rs17561, rs1304037), IL10 gene (rs1800871) and IL1RN gene (rs9005) were selected and investigated. The characteristics of the selected SNPs are presented in table 1.
Genotyping was performed using TaqMan SNP Genotyping Assays (Life Technologies TM) in Stratagene Mx3005P (Agilent Technologies). The real-time PCR reactions were performed in a total volume of 3 mL (4 ng DNA/reaction, 1.5 mL Taqman PCR master mix, and 0.075 SNP assay, Applied Biosystems). The thermal cycling was performed by starting with a hold cycle of 95 o C for 10 minutes followed by 45 amplification cycles of 92 o C for 15 seconds and 60 o C for 1 minute. Assays and reagents were supplied by Applied Biosystems (Foster City, CA, USA). All examiners at the laboratory were blinded to the samples' group assignment. Hardy-Weinberg equilibrium was evaluated using the chi-square test within each SNP in each population and only the results that were in Hardy-Weinberg equilibrium were further analyzed.

Statistical analysis
Data were analyzed using the Epi Info 3.5.7 and Stata software (StataCorp, College Station, TX, USA, version 11).
The study-dependent variable was oral herpes. The odds ratio (OR) and the Chi-square test were used to assess whether the allelic and/or genotypic profile in the conventional, dominant, or recessive model. An alpha of 0.05 was considered statistically significant.

Discussion
In this transversal study we tested the hypothesis that SNPs in genes that codified interleukin such as IL1A, IL10, and IL1RN are associated with OH recurrent episodes. For that, we collected DNA samples from 273 Brazilian para-athletes that were in a classificatory competition organized by the Brazilian Paralympic Committee and selected 4 SNPs in candidate genes to be studied. We observed that the prevalence of the OH recurrent episodes in the studied population was lower than the global estimative. Even so, the rs1304037 in the IL1A gene showed a marginally significant influence on OH episodes in allelic and in dominant models.
Regarding the prevalence of self-reported OH recurrent episodes, while the global prevalence is around 66.6% in general population (James et al., 2016), in our study, only 17.2% of the para-athletes reported OH recurrent episodes. Although interesting, this information should be interpreted cautiously, because our study provides a snapshot picture of the OH prevalence at that moment, before an important competition. It is plausible that, despite the care that the researchers had in conducting the exams and applying the questionnaires, at that moment, the focus of the para-athletes was their performance in the competition and they did not make the association with perceiving signs and symptoms of the OH. So it is important to clarify that it could be an underestimate prevalence. Even with these limitations, an important result was observed: we found an association between rs1304037 in the IL1A gene with OH recurrent episodes.
At this point, it is important to mention that HSV is a human pathogen that exists in two forms: type 1 and type 2.
Transmission of HSV type 2 is mainly sexual and causes genital herpes (Pinninti & Kimberlin, 2013). Despite HSV type 1 also causes genital herpes, its mainly transmitted by oral-to-oral contact (Higgins et al., 1993) and causes infection in the skin and mucosal epithelial cells, inhibiting inflammatory pathways (Milora et al., 2014). In the active phase, causes vesicle ulcerative lesions that are usually 1-2 mm blisters and affects mainly lips and rapidly break down produces classic symptoms (Arduino & Porter, 2008). Infected keratinocytes showed high expression of interleukins that are believed could be a signal to the recruitment of the inflammatory cells to the damaged tissue (Dinarello, 2009). After resolution of a primary infection that persist 3-10 days, the virus migrates to the trigeminal nerve ganglion, where it is capable of remaining in a latent state.
Recurrent infection occurs when the individual is exposed to environmental factors (Stoopler & Greenberg, 2003) and, in fact, even during latent state HSV-1 induces persistent expression of interleukin genes by varying a continuous source of antigenic stimulation (Baker et al., 1999), so it is natural that genes that encoding interleukin might be candidates to be studied.
In this sense, we decided to evaluate the association between genes that codified interleukin and selected SNPs rs17561, rs1304037, rs1800871, and rs9005. The first one, rs17561, represents a missense mutation leading to aminoacid change (Ala114Ser) and is a SNP target to be studied once have been associated with various inflammatory conditions (Berger et al., 2002;Liu et al., 2013). The rs1800871 and rs9005 are SNPs extensively investigated in abnormal cell proliferation and cancer development in different stages of disease progression (Tindall et al., 2010;Niu et al., 2015;Moghimi et al., 2018).
Despite involvement in the inflammatory response, in our study, it was not possible to prove the association between rs17561, rs1800871, and rs9005 with OH episodes in any of the analyzed genetic models. Thus, the primary conclusion is that these polymorphisms do not appear to be associated with OH but is important to mention that sometimes the analysis of a single allele could be inappropriate.
Regarding rs1304037, in contrast with the other studied polymorphisms, the results indicated that rs1304037 was associated with OH episodes. In fact, our result suggests that allele T in affected para-athletes increased the risk of OH episodes and TT+CT genotypes are borderline for this condition. Similarly, this allelic variation was associated with chronic spontaneous urticaria (Moghimi et al., 2018). Is important to mention that this SNP is located in the 3′-untranslated region of the gene and SNPs in this region could be involved in gene expression variations consequently with important biological impacts (Johnsen et al., 2008;Badie et al., 2020) but to the best of our knowledge, this seems to be the first research paper linking the genetic polymorphism in the IL-1 in the context of the pathogenesis of OH. Furthermore, another point that needs to be considered is the possible interactions of these four SNPs as contributed for OH. In fact, an analysis based on haplotypes or diplotypes in this population remains necessary.

Conclusion
This study provides new insight into the genetics contribution for OH episodes and showed that single nucleotide polymorphism 1304037, allele T, in IL1A gene may confer an increased risk for the susceptibility to OH episodes in paraathletes.