Assessment of TNF‐α (‐857 C/T) gene polymorphism in oral lichen planus disease: A case–control study

Abstract Background and aims Oral lichen planus (OLP) is an inflammatory mucocutaneous disorder with an immune‐mediated pathogenesis. The tumor necrosis factor‐α (TNF‐α) level in the serum of OLP patients is significantly higher than in the control group. TNF‐α‐857 C/T polymorphism can be related to the increased TNF‐α level in blood circulation. This study investigated the relationship between TNF‐α (‐857 C/T) polymorphism and OLP patients in an Iranian population. Methods Saliva samples were taken from 200 people, including 100 patients with OLP and 100 healthy people who did not have significant differences in age and sex. Then, DNA was extracted from them and the TNF‐α (‐857 C/T) genotype was identified using the polymerase chain reaction with confronting two‐pair primers method. Statistical Package for the Social Sciences version 16 software analyzed the results. Results The frequency of C/C, C/T, and T/T genotypes of the TNF‐α‐857 C/T polymorphism in the patient group were 78%, 18%, and 4%, respectively, and in the control group were 72%, 23%, and 5%, respectively. The differences between the two groups regarding allele (χ 2  =  0.97, p  = 0.32) and genotype (χ 2  =  0.96, p  = 0.62) frequency among the studied population were insignificant. Conclusion This study showed that the difference in the frequency of single nucleotide polymorphism TNF‐α‐857 C/T polymorphism in the patient and control group had no significant relationship with the increased OLP incidence. Also, no significant association was observed between allele and genotype frequency of TNF‐α (‐857 C/T) with OLP subtypes.

Lichen planus is a mucocutaneous disorder characterized by abnormal epithelial keratinization cycles and T-cell-mediated immune responses. 1al lichen planus (OLP) appears as white lesions like striations, papules, and plaques, and red lesions like erythema, abrasions, or blisters, mainly affecting the tongue, buccal mucosa, and gingiva.Although it sometimes involves other places.Lesions are usually bilateral and often present as a combination of clinical symptoms. 2,3OLP occurs more than the cutaneous form.It is also more lasting and resistant to treatment. 4P is usually seen in adults' fifth to sixth decade of life; it is rarely seen in children. 5OLP is twice as common in women as in men. 6Clinically, it may be categorized into different types, such as papular, atrophic, plaque-like, reticular, erosive, and bullous. 7e most common forms of OLP are reticular and erosive. 8As OLP has the risk of developing into oral squamous cell carcinoma (OSCC), it is therefore defined as a potentially malignant disease. 9,10rophic and erosive forms of OLP and plaque lesions on the back of the tongue are more potent to become malignant. 5For detecting OSCC, saliva tumor necrosis factor-α (TNF-α) is a better sample.
Salivary TNF-α levels increase in the histological differentiation grade in OSCC. 113][14] OLP is an autoimmune T-cell-mediated disease, but its cause is often unknown.
There is a theory about immune responses mediated by T cells that play a role in it. 15A vital and early condition in OLP is the increased level of TH1 cytokines, which is genetically induced, and the resulting lesions in the mouth and skin appear to be due to genetic polymorphism of the cytokines. 16TNF-α, as a pro-inflammatory cytokine, plays a critical role in the pathogenesis of numerous inflammatory or autoimmune disorders.TNF-α is extensively reported in OLP pathogenesis compared with other inflammationrelated cytokines. 17,18The level of TNF-α in the serum of OLP patients was significantly higher than the control group. 9,19TNF-α expression level and genetic polymorphisms are involved in many diseases. 2Several single nucleotide polymorphisms (SNPs) that play a role in transcriptional regulation have been reported in the promoter region of the TNF-α gene. 20One of these is SNP at position -857.The association between this C/T SNP and autoimmune disorders like Behçet's disease has been shown in several studies. 21The effect of this polymorphism is through the regulation of TNF-α gene transcription and, as a result, its expression level.As a result, this polymorphic change can be related to the high level of TNF-α in the blood circulation. 2,22ported studies confirmed that TNF-α is a highly polymorphic gene; the review of the previous studies on other polymorphisms of TNF-a sites and other cytokines generally suggests a relationship with the occurrence of OLP disease.However, there is no study on the association of TNF-α-857 polymorphism in patients with OLP.
Therefore, our study aims to address this knowledge gap and explore the potential link between TNF-α-857 polymorphism and OLP in the Iranian population (Kermanshah province of Western Iran).The purpose of the research was explained to the subjects and they filled out consent forms.To maximize DNA yield, individuals were told not to eat, drink, smoke, use mouthwash, or brush their teeth for at least 2 h before taking a saliva sample.Without intervention, 3 mL of unstimulated saliva samples were taken from each individual with the spitting method 23 in falcon tubes (15 mL).Then, collected samples were immediately transferred to the laboratory on ice and stored at −80°C until use.

| Genetic analysis
DNA extraction from saliva samples was performed using the standard kit for extracting DNA from the saliva of Azma Elixir Pajoh Company according to its protocol.Extracted DNA was evaluated on 1% agarose gel electrophoresis.Also, the quantity and purity of the extracted DNA were analyzed using a NanoDrop (Thermo-2000/ 2000c). 24Polymerase chain reaction (PCR) with confronting two-pair primer technique was used to determine the genotype and all frequencies of the TNF-α-857 C/T.PCR amplified TNF-α gene with a set of primers that are shown in Table 1. 25 PCR parameters were as follows: one cycle at 95°C for 5 min, 30 cycles including 95°C for 1 min, 66°C for 1 min, 72°C for 1 min, and a final at 72°C for 5 min.Finally, the PCR products were electrophoresed on the 2% agarose gel.In the obtained electrophoresis pattern, the CC genotype is 219 and 336 bp, the mutant genotype (TT) is 336 and 160 bp fragment, and TC as heterogeneous genotype is 336, 219, and 160 bp (Figure 1).

| Ethical considerations
The Ethics Committee at Kermanshah University of Medical Sciences, Iran, approves the study design and protocols (IR.KUMS.REC.1401.386).
All procedures performed were in accordance with the ethical standards and relevant guidelines and regulations in the Declaration of Helsinki.All participants were required to sign a written consent form and were provided with a participant information statement that thoroughly explained the consent process.TNF-α-857 in OLP patients and control groups were in Hardy-Weinberg equilibrium.The distribution of TNF-α-857 alleles and genotypes in OLP and control groups are depicted in Table 2.

| RESULTS
The frequencies of C/C, C/T, and T/T genotypes of the TNF-α-857 in the patient group were 78%, 18%, and 4%, respectively, and in the control group, were 72%, 23%, and 5%, respectively (Table 2).The results indicated no significant difference in the genotype frequencies of the codominant, dominant, overdominant, and recessive genetic models between the OLP patient and controls.As demonstrated in Table 3, no positive association was found between the frequency distribution of TNF-α-857 alleles and genotypes and OLP severity.

| DISCUSSION
In this study, we investigated TNF-α gene polymorphism in the saliva sample of OLP disorder.In genomic applications, there is a need for suitable samples and efficient methods for obtaining a high yield of DNA.Saliva is an attractive source of genomic DNA because it has many advantages compared to blood, wound tissue, and different body fluid samples, such as easy access, noninvasiveness, safety, lower infection rate, and cost-efficiency. 26Zhang et al. 27 evaluated the nuclear factor-κB-dependent pro-inflammatory cytokines, including TNF-α, interleukin-6 (IL-6), and IL-8 in serum and the saliva samples of OLP patients and healthy individuals; they suggested that saliva-based test can be used as a cost-effective adjunctive tool for measuring pro-inflammatory cytokines in OLP patients.
In our study, the age range of 45-65 years was the most significant number of patients, which confirms the previous findings that the incidence of OLP was higher in the fourth decade of life and women. 6,28Consistent with our results, Al-Mohaya et al. 29 have found that Saudi OLP patients were more frequent in the third to fourth decade of life with a median age of 42 years.In our study, 71% of OLP patients were women.This data is compatible with previous studies that show a higher prevalence of OLP among women in different populations. 29,30 has been demonstrated that gene polymorphism in regulatory sequences of the TNF-α gene can change its expression. 17Several studies have shown the association between TNF-α (-857 C/T) and autoimmune or inflammatory disorders. 31However, the relationship between this polymorphism and OLP disease has not yet been investigated. 20,21Limited studies with contradictory results have been done on other TNF-α gene polymorphisms with inflammatory diseases.The association between TNF-α-857 polymorphism and ulcerative colitis and Crohn's disease has been investigated, and a significant relationship with Crohn's disease was observed. 32A systematic review and meta-analysis by Zhou and Vieira 33 showed that TNF-α -308 G/A polymorphism in TNF-α is a potential genetic marker for OLP.Kimkong et al. 34 found no significant correlation between TNF-α polymorphisms (-863 and -238) and OLP development in a Thai population; however, a higher proportion of TNF-α-308 AA genotype) among OLP patients compared to healthy controls was reported.In addition, we studied the effect of TNF-α (-857 C/T) gene polymorphism on OLP severity was studied.Our results indicate no significant difference between the TNF-α (-857 C/T) allele and genotype frequency with OLP subtypes.Our result is consistent with a previous report, which found no significant association between TNF-α gene polymorphism (-863 and -238) and the development of OLP in the Thai population. 34In another study, Carrozzo et al. 35 investigated 13 cytokine genes with 22 SNPs on sensitivity to Caucasian OLP.They suggested that the increased frequency of the -308 A TNF-α allele and genetic polymorphism of the first intron of the interferon-γ gene may contribute to the development of OLP lesions.
T A B L E 3 Frequencies of TNF-α-857 polymorphism in erosive and non-erosive subtypes of OLP.

2 | METHODS 2 . 1 |
Study group and sampling A total of 200 samples, including 100 patients with OLP (29 men and 71 women; 65 erosive and 35 non-erosive) and 100 healthy individuals (28 men and 72 women) without any history of the disease, were included in our study.All studied individuals were from the Kermanshah province of Western Iran.Our groups were matched by gender and age.The average age of our study population in both groups was 48 years and most patients were 45-65 years old.The OLP diagnosis was based on the World Health Organization's definition of OLP and confirmed by pathology reports and clinical manifestations detected by an oral medicine specialist.We excluded pregnant women, OLP patients with accompanying malignancies, autoimmune/inflammatory or systemic disorders, and patients who received any relevant treatment over the past 3 months.Additionally, patients with amalgam lichenoid reaction lesions were excluded.A family history of OLP was not considered in the selection of subjects.

Using
Statistical Package for the Social Sciences software (version 16), the χ 2 test investigated the significance of the difference in the frequency distribution of alleles and genotypes between the control and patient groups.The 95% confidence interval and the odds ratio of the logistic regression model were used.Disparities in demographic, anthropometric, and clinical characteristics between the case and control groups were assessed.Data related to quantitative characteristics are expressed as mean ± SD.The results were considered statistically significant if p < 0.05.
Case and control groups were matched by gender and age.The most significant number of patients was in the age range of 45-65 years (average age of 48 years).Among the OLP patients, there were 71 females and 29 males.Among 100 OLP patients, 655 were erosive and 355 of them were non-erosive.The genotype distributions of F I G U R E 1 Electrophoresis pattern of polymerase chain reaction with confronting two-pair primer (PCR-CTPP) products for position -857 of TNF-α gene.Lane 1: sample with T/T genotype; Lane 2: sample with C/C genotype; Lane 3: sample with T/C genotype; Lane M: DNA size marker (50 bp).T A B L E 2 Comparison of the frequency distribution of TNF-α-857 genotypes and alleles in OLP patients and control group.
Abnormal expression of TNF-α in lesions, saliva, or serum of OLP patients indicates that the elevated expression of TNF-α may be associated with OLP immunopathogenesis.In our study, we investigated the TNF-α (-857 C/T), because it is one of the most frequent polymorphisms in the TNF-α gene that can affect the expression level of TNF-α.The current study showed that the difference in the allele frequency of SNP TNF-α-857 C/T polymorphism in the control and patient group had no significant relationship with the increase in the incidence of OLP disease in the population of western Iran.Al-Mohaya et al.29 concluded that TNF-α (-308 G/A) polymorphism is associated with OLP susceptibility among Saudi patients; also, their results support the role of genetic factors in OLP disease.