Association of rs142548867 (EEFSEC) and periodontitis Grade C in a young Brazilian population

Abstract Periodontitis Stage III-IV, Grade C (PerioC) is a severe form of Periodontitis. The individual genetic background has been shown to be an important etiopathogenic factor for the development of this disease in young, systemically healthy, and non-smokers patients. Recently, after exome sequencing of families with a history of the disease, PerioC was associated with three single nucleotide variations (SNVs) – rs142548867 (EEFSEC), rs574301770 (ZNF136), and rs72821893 (KRT25) – which were classified as deleterious or possibly harmful by prediction algorithms. Objective Seeking to validate these findings in a cohort evaluation, this study aims to characterize the allele and genotypic frequency of the SNVs rs142548867, rs574301770, and rs72821893 in the Brazilian population with PerioC and who were periodontally healthy (PH). Methodology Thus, epithelial oral cells from 200 PerioC and 196 PH patients were harvested at three distinct centers at the Brazilian Southern region, their DNA were extracted, and the SNVs rs142548867, rs574301770, rs72821893 were genotyped using 5′-nuclease allelic discrimination assay. Differences in allele and genotype frequencies were analyzed using Fisher’s Exact Test. Only the SNV rs142548867 (C > T) was associated with PerioC. Results The CT genotype was detected more frequently in patients with PerioC when compared with PH subjects (6% and 0.5% respectively), being significantly associated with PerioC (odds ratio 11.76, p=0.02). Conclusion rs142548867 represents a potential risk for the occurrence of this disease in the Brazilian population.


Introduction
Periodontitis is an inflammatory disease of multifactorial etiology triggered by the host's immuneinflammatory response to periodontopathogens present in the subgingival biofilm, clinically characterized by bone destruction and loss of attachment. In 2018, a world workshop classification indicated that the disease profile previously defined as Aggressive Periodontitis can now be defined as Periodontitis Stage 3-4, Grade C (PerioC) occurring in systemically healthy patients and non-smokers. 1,2 This is a particularly severe form of the disease, with early onset, rapid progression, precocious edentulism, familial clustering of cases, and poor response to therapeutic approaches. [2][3][4][5] This disease phenotype, although rare in developed countries, appears to be highly detected in undeveloped one, reaching around 5.5% of young population. 6 The literature shows that individual genetic background is an important etiopathogenic factor that leads to this disease profile. [7][8][9][10][11] Genome-wide association studies (GWAS), that is, the sequencing of the whole-genome or whole-exome of PerioC patients, have been performed to associate multiple genetic variants with the occurrence of PerioC. [12][13][14] Although these studies came out with several associations between genes related to the immune response and  15 The SNV rs142548867 is located at chromosome 3, 128264663 position, at eukaryotic elongation factor, selenocysteine-tRNA specific (EEFSEC) gene, encoding eEFSec protein, and is responsible for the inversion of the nitrogenous base C for T (c.668C>T). This variation results in the replacement of the amino acid Proline (Pro) by a Leucine (Leu) residue at protein position 223 (p.Pro223Leu). This is an important translation factor for selenoproteins and selenoenzymes, which are critical for maintaining the tissue homeostasis, and regulation of immune-inflammatory cells. 16,17 Rs574301770 (c.466C>G) is located on zinc finger

Patients eligibility criteria
For the elaboration of this study, the STREGA checklist has been followed by the authors. 18 All the DNA samples used in this study were first collected in a previous study, 19 20 full-mouth bleeding on probing score, 21 and Probing Pocket Depth (PPD) at six points around each tooth.    in a C allele frequency of 99% and 1% of T. The rare allele was detected more frequently in patients with PerioC when compared with PH subjects (4% and 1%, respectively), and, consequently, the CT genotype was more present at the affected population compared with the healthy one (6% and 0.5%, respectively), being significantly associated with PerioC (odds ratio 11.76, p=0.02), making this SNV a potential risk for the occurrence of this disease. Table 3 shows the results of allele and genotype frequencies.

Study Population
Regarding the other two SNVs, rs574301770 and rs72821893, no statistical difference could be detected when comparing the affected and the control group.

Discussion
In this study, the missense SNV rs142548867 (C > T), in EEFSEC gene, was associated with PerioC, since the rare T allele was found more frequently in this population than in PH. This higher frequency, in turn, leads to a higher frequency of the heterozygous genotype (CT) when compared with PH group -6% and 0.65% respectively, resulting in an odds ratio of 11.76 for PerioC occurrence (p=0.02). Meanwhile, SNVs rs574301770 (ZNF136) and rs72821893 (KRT25) weren't associated to PerioC (p>0.05). It is important to notice that this research was conducted based on the previous results of our group. In that study, a familial whole-exome of PerioC patients -two affected probands, their parents (one with and one without a history of this disease), and one sibling (healthy patient) -were carried out, searching for genetic risk factors related to this disease. The   Recent studies have shown that, in certain cases, the control group used to evaluate genetic susceptibility to periodontal disease may be biased, considering that we can have patients carrying the same SNVs associated with the disease but do not develop it because of their good oral hygiene. 39,40 In this study, we evaluated three SNVs that can be associated with PerioC, a disease that mostly affects young patients, around 20-30 years old or even younger. 6 In this case, establishing a control group is always challenging because, at this similar age, we are not certain that the periodontally healthy patients

Conflict of interest
The authors also declare that there are no conflicts of interest in this study.

Data availability statement
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.