oral lichen planus ( olp ) is a chronic inflammatory oral condition whose etiology is not fully elucidated . it is the prototype of oral lichenoid lesions characterized by t - cell mediated immune responses and abnormal epithelial keratinization cycles 
 . olp manifests as white striations , white papules , white plaques , erythema , erosions , or blisters predominantly affecting the buccal mucosa , tongue and gingival . the olp lesions may co - exist with cutaneous and genital lesions , or may be the only disease manifestations . the prevalence of olp ranges between less than 1 and 3% of the general population 
 . the prevalence of olp in arab countries including saudi arabia has been reported ranging from 0.35 to 1.7% 
 . the olp lesions are consistently more persistent than the dermal lesions and have been reported to carry a risk of malignant transformation to oral squamous cell carcinoma of 1 - 2% ( reported range of malignant transformation 012.5% ) 
 . clinically , olp may be divided into three subtypes : reticular , erythematous ( atrophic ) , and erosive 
  and more than one subtype of olp may be present in an individual patient . the pathogenesis of olp is very complex and involves possible antigen presentation by the oral keratinocytes that could be either of an exogenous or an endogenous origin 
 . this antigenic trigger is accompanied by a mixed inflammatory response comprising of mainly t - cells , macrophages , and mast cells , as well as the associated cytokines and cytotoxic molecules 


 . it is a disease associated with middle aged people and is more common among women 
 . cytokines play an important role in the pathogenesis and disease progression of olp and a strong body of evidence suggests that olp is a t - cell - mediated disease . the genetic factors that influence the immune function have been suggested to contribute to the olp 


 . the gene polymorphisms of t - helper cell subtype th1/th2 cytokines , tumor necrosis factor ( tnf)- , and interleukin-10 ( il-10 ) have been reported to affect the susceptibility to , and the progression of olp 


 . ( 2013 ) suggested that proinflammatory cytokines are an important factor in understanding the disease burden of olp and their comorbid factors . however , immunogenetic studies of olp have given controversial results and the precise cause of olp is still unclear . tnf- and tnf- genes are located in tandem on chromosome 6 between the class i and class ii cluster of the major histocompatibility complex ( chromosome 6p21.16p21.3 ) . tnf--308g / apolymorphism ( rs1800629 ) has been reported to be associated with several autoimmune / inflammatory diseases including olp 


 . the genetic variation at position 308 of the tnf- gene results in two allelic forms in which the presence of guanine ( g ) defines the common variant and the presence of adenine ( a ) defines the less common one . the a - allele of tnf-308g / a polymorphism displays increased gene transcription as compared to the common allele g. it has been shown to produce levels of tnf- transcription 67 fold greater 
 . tnf- + 252a / g polymorphism ( rs909253 ) has been reported at position + 252 within the first intron of the tnf- gene , consisting of guanine ( tnf- + 252 g ) on one allele and adenine ( tnf- + 252 ) on the alternate allele 
 . the presence of g at this position defines the mutated allele known as tnf- 1 ( allele-1 ) which is a less frequent allele and is associated with higher tnf- and tnf production 
 . it shifts the th1/th2 balance by down regulating the th1 responses and by suppression of pro - inflammatory cytokines , such as tnf- and interferon gamma ( ifn  ) secretion 
 . three promoter polymorphisms : -1082a / g ( rs18000896 ) , -819t / c ( rs1800871 ) and -592a / c ( rs1800872 ) are reportedly involved in the il-10 transcription rate , thereby directly affecting its production level 
 . the -1082 g , -819c and -592c alleles ( gcc haplotype ) have been associated with elevated levels of il-10 production 
  while acc and ata haplotypes exhibit intermediate and low il-10 gene transcription respectively 
 . in this study we examined the association of five single nucleotide polymorphisms ( snps ) of tnf- , tnf- , and il-10 genes in the etiopathogenesis of olp in saudi patients . a total of 253 saudi subjects visiting prince sultan military medical city ( psmmc ) , riyadh , saudi arabia were recruited for this study . forty two ( 16 male , 26 female ) unrelated patients with oral lichen planus , ages ranging from 27 to 72 years , and 211 ( 140 male , 71 female ) unrelated , healthy patients matched voluntary blood donors with ages ranging from 20 to 65 years from the same population were studied for polymorphisms in tnf- , tnf- and il-10 genes . this study was approved by the research and ethical committee of psmmc and written informed consent was obtained from each subject before recruitment . olp was diagnosed according to the clinical manifestations and histopathological criteria of the world health organization . all patients were diagnosed through a review of the patient s history , physical examination and histological findings by an oral pathologist . a 4 mm punch biopsy of lesional tissue that extends into the submucosa was performed . symptoms were bilateral , more or less symmetrical lesions , and a lace - like network of slightly raised gray - white lines ( reticular pattern ) . erosive , atrophic , bullous , and plaque - type lesions were only accepted as a subtype in the presence of reticular lesions elsewhere in the oral mucosa . histopathological criterium were the presence of a well - defined band - like zone of cellular infiltration that was confined to the superficial part of the connective tissue , consisting mainly of lymphocytes , signs of  liquefaction degeneration  in the basal cell layer , and the absence of epithelial dysplasia 
 . no patient was suspected to have drug or restoration related lichenoid lesions , and no patient showed histologic signs of dysplasia . in cases where the clinical and histological findings were inconclusive , direct immunofluorescence ( dif ) microscopy of perilesional mucosa was used to exclude autoimmune vesiculobullous disorders . genomic dna was extracted from the peripheral blood of olp patients and controls using the qia amp dna mini kit ( qiagen hilden , north rhine - westphalia , germany ) . tnf- , tnf- and il-10 genes were amplified using amplification refractory mutation systems ( arms)-pcr methodology 
  to detect polymorphisms at positions -308 of tnf- , + 252 in intron1 of tnf- and at loci -592 , -819 , and -1082 of il-10 genes . pcr amplification was carried out using ready to go pcr beads ( ge healthcare , little chalfont , buckinghamshire , uk ) . reactions consisted of 10 denaturation temperature cycles for 15 s at 94c , annealing for 50 s at 65c and extension for 40 s at 72c . then 25 denaturation cycles of 20 s at 94c , annealing for 50 s at 59c and then extension of 50 s at 72c . a positive control was included in the pcr assay by amplification of the human growth hormone gene . the amplified products for the various samples were separated on the 1.5% agarose gel , stained with ethidium bromide and photographed . hardy - weinberg equilibrium was determined using hardy - weinberg equilibrium calculator for 2 alleles ( http//www.had2know.com / academics / hardy - weinberg - equilibrium - calculator-2alleles.html ) . the bayesian method of the phase program ( version 2.1 ) was applied to reconstruct the haplotype ( http://stephenslab.uchicago.edu/software.html ) . the differences in allele / genotype frequencies between patients and controls were analyzed by fisher s exact test using the calcfisher software 
 . the strength of the association of the disease with respect to a particular allele / genotype is expressed by odd ratio interpreted as relative risk ( rr ) following the woolf s method as out lined by schallreuter , et al . it was calculated only for those alleles / genotypes which were increased or decreased in olp patients when compared to the control group . the rr was calculated for all the subjects using the formula given below : a = number of patients with expression of allele or genotype b = number of patients without expression of allele or genotype c = number of controls with expression of allele or genotype d = number of controls without expression of allele or genotype . ef was calculated for a positive association only where rr>1 using the following formula 
 . preventive fraction ( pf ) indicates the hypothetical protective effect of one specific allele/ genotype for the disease . values < 1.0 indicate the protective effect of the allele/ genotype against the manifestation of disease . a total of 253 saudi subjects visiting prince sultan military medical city ( psmmc ) , riyadh , saudi arabia were recruited for this study . forty two ( 16 male , 26 female ) unrelated patients with oral lichen planus , ages ranging from 27 to 72 years , and 211 ( 140 male , 71 female ) unrelated , healthy patients matched voluntary blood donors with ages ranging from 20 to 65 years from the same population were studied for polymorphisms in tnf- , tnf- and il-10 genes . this study was approved by the research and ethical committee of psmmc and written informed consent was obtained from each subject before recruitment . olp was diagnosed according to the clinical manifestations and histopathological criteria of the world health organization . all patients were diagnosed through a review of the patient s history , physical examination and histological findings by an oral pathologist . a 4 mm punch biopsy of lesional tissue that extends into the submucosa was performed . symptoms were bilateral , more or less symmetrical lesions , and a lace - like network of slightly raised gray - white lines ( reticular pattern ) . erosive , atrophic , bullous , and plaque - type lesions were only accepted as a subtype in the presence of reticular lesions elsewhere in the oral mucosa . histopathological criterium were the presence of a well - defined band - like zone of cellular infiltration that was confined to the superficial part of the connective tissue , consisting mainly of lymphocytes , signs of  liquefaction degeneration  in the basal cell layer , and the absence of epithelial dysplasia 
 . no patient was suspected to have drug or restoration related lichenoid lesions , and no patient showed histologic signs of dysplasia . in cases where the clinical and histological findings were inconclusive , direct immunofluorescence ( dif ) microscopy of perilesional mucosa was used to exclude autoimmune vesiculobullous disorders . genomic dna was extracted from the peripheral blood of olp patients and controls using the qia amp dna mini kit ( qiagen hilden , north rhine - westphalia , germany ) . tnf- , tnf- and il-10 genes were amplified using amplification refractory mutation systems ( arms)-pcr methodology 
  to detect polymorphisms at positions -308 of tnf- , + 252 in intron1 of tnf- and at loci -592 , -819 , and -1082 of il-10 genes . pcr amplification was carried out using ready to go pcr beads ( ge healthcare , little chalfont , buckinghamshire , uk ) . reactions consisted of 10 denaturation temperature cycles for 15 s at 94c , annealing for 50 s at 65c and extension for 40 s at 72c . then 25 denaturation cycles of 20 s at 94c , annealing for 50 s at 59c and then extension of 50 s at 72c . a positive control was included in the pcr assay by amplification of the human growth hormone gene . the amplified products for the various samples were separated on the 1.5% agarose gel , stained with ethidium bromide and photographed . hardy - weinberg equilibrium was determined using hardy - weinberg equilibrium calculator for 2 alleles ( http//www.had2know.com / academics / hardy - weinberg - equilibrium - calculator-2alleles.html ) . the bayesian method of the phase program ( version 2.1 ) was applied to reconstruct the haplotype ( http://stephenslab.uchicago.edu/software.html ) . the differences in allele / genotype frequencies between patients and controls were analyzed by fisher s exact test using the calcfisher software 
 . the strength of the association of the disease with respect to a particular allele / genotype is expressed by odd ratio interpreted as relative risk ( rr ) following the woolf s method as out lined by schallreuter , et al . it was calculated only for those alleles / genotypes which were increased or decreased in olp patients when compared to the control group . the rr was calculated for all the subjects using the formula given below : a = number of patients with expression of allele or genotype b = number of patients without expression of allele or genotype c = number of controls with expression of allele or genotype d = number of controls without expression of allele or genotype . ef was calculated for a positive association only where rr>1 using the following formula 
 . preventive fraction ( pf ) indicates the hypothetical protective effect of one specific allele/ genotype for the disease . . values < 1.0 indicate the protective effect of the allele/ genotype against the manifestation of disease . there was no significant difference in clinical manifestation or prognosis comparing men to women in our study . thirty - five patients ( 83.333% ) had lesions on the buccal mucosa , three ( 7.14 % ) each on the tongue and gingival while one patient ( 2.39% ) had lesions on the palate . reticular type olp was the commonest ( 80.95% ) followed by erosive ( 23.81% ) and then atrophic types ( 4.76% ) . the genotype and allele frequencies of tnf- ( -308g / a ) and tnf- ( + 252a / g ) promoter polymorphisms are presented in table 1 . in both the lichen planus patients and control groups the genotype distributions were in hardy - weinberg equilibrium . the frequency of the heterozygous genotype ga was significantly higher in olp patients than in the control ( p<0.001 ) whereas the frequency of homozygous genotypes gg was significantly lower in olp than controls ( p<0.001 ) . the frequency of allele - a was significantly higher in olp patients than control subjects ( p<0.001 ) . on the other hand , allele - g was significantly lower in olp patients when compared to the control ( p<0.001 ) . the difference in frequency of the aa genotype between the two groups was not statistically significant . table 1genotype and allele frequencies of tnf- and tnf- variants in oral lichen planus patients and matched controlsgenotype/ alleleolp ( n=42)control ( n=200 )  n%n%p - valuerref/ pf-308 g / a  gg511.911055<0.001 * 0.110.257ga3685.727638<0.001 * 9.7890.288 
 aa12.381470.4790.3240.121 g allele4654.7629674<0.001 * 0.4250.153a allele3845.2410426<0.001 * 2.3510.153 
 + 252 a / g  gg49.5228140.6160.6470.063ga3890.48148740.025 * 6.7670.173 
 aa0024120.010 * --g allele4654.76204510.551.1630.026 
 a allele3845.24196490.550.8590.026n = number of subjects,*statistically significant , rr = relative risk , ef = etiologic fraction , pf = preventive fraction 
  n = number of subjects,*statistically significant , rr = relative risk , ef = etiologic fraction , pf = preventive fraction the frequency of the ga genotype of tnf- ( + 252a / g ) promoter polymorphism was significantly higher in patients group than in the controls . homozygous aa genotype was completely absent in olp patients whereas it was present in 12% of the controls . the frequencies of alleles of tnf- ( + 252a / g ) polymorphism were not significantly different between olp patients and healthy controls ( table 1 ) . albeit , the frequencies of allele - g were slightly higher in the olp patients than they were in the control subjects . the results of snps for il-10(-1082g / a ) , il-10(-592c / a ) , il-10(-819c / t ) , and corresponding alleles and genotypes are summarized in table 2 . the genotype distributions of il-10 polymorphisms in patient and controls were in hardy - weinberg equilibrium . the frequencies of -1082gg and 1082ga genotypes were slightly higher while the frequency of -1082aa genotype was lower in olp patients when compared to the control subjects , however , the differences were not statistically significant . table 2genotype and allele frequencies of il-10 variants in oral lichen planus patients and matched controlsgenotype/ alleleolp ( n=42)control ( n=186 )  n%n%p - valuerref*/pf-1082g / a  gg511.9147.530.3571.60.098*ga3378.514075.270.8411.20.032 
 aa49.53212.20.2510.510.095g - allele4351.216845.160.3341.30.047*a - allele4148.820454.840.3340.780.048 - 819c / t  cc1228.58841.710.1220.5590.086ct2559.510248.340.2391.50.065*tt511.9219.950.781.20.032 
 c - allele496227865.880.5210.8460.026t - allele303814434.120.5211.1820.172 * -592c / a  cc1228.58841.710.1220.5590.086ca2559.510248.340.2391.50.065*aa511.9219.950.781.20.032*c - allele496227865.880.5210.8460.026a - allele303814434.120.5211.1820.172*n = number of subjects,*statistically significant , rr = relative risk , ef = etiologic fraction , pf = preventive fraction 
  n = number of subjects,*statistically significant , rr = relative risk , ef = etiologic fraction , pf = preventive fraction similarly the frequencies of alleles and genotypes of il-10 ( -819c / t ) and il-10 ( -592c / a ) polymorphisms did not differ significantly between olp patients and controls ( p>0.05 ) . albeit , the frequencies of genotypes -819 ct , -819 tt , 592 ca and 592 aa were higher in olp patients while -819 cc and -592 cc genotypes showed a reverse pattern with lower frequency in olp patients when compared to the controls ( table 2 ) . the frequencies of haplotypes extracted from 1082g / a , -819c / t , -592c / a polymorphisms of il-10 are shown in table 3 . the haplotype ata was significantly increased in olp patients when compared to controls ( p=0.008 ) . table 3haplotype analysis of il-10 -1082g / a , -819t / c and -592c / a polymorphisms in olp and control groupshaplotypeolp ( n=42)controls ( n=186 )  n ( frequency)n ( frequency)p - valuegcc7 ( 0.166)42 ( 0.225)0.533acc13 ( 0.309)82 ( 0.440)0.165ata20 ( 0.476)48 ( 0.258)0.008*aca2 ( 0.047)14 ( 0.075)0.742n = number of subjects,*statistically significant 
  n = number of subjects,*statistically significant the frequencies of alleles and genotypes of all five polymorphisms with gender are summarized in table 4 . no statistically significant difference was found in the frequencies of alleles or genotypes of tnf- , tnf- and il-10 gene polymorphisms between male and female olp patients . table 4genotype and allele frequencies in male and female oral lichen planus patientsgenotype/ allelemale ( n=16)female ( n=26 )  n%n%p - valuetnf- ( -308 )  gg212.5311.541ga1487.52284.611aa0013.850.99g - allele1856.252853.850.99a - allele1443.752446.150.99tnf- ( + 525 )  gg16.25311.541ga1593.752388.461aa0000-g - allele1753.132955.770.82a - allele1546.872344.230.82il-10 ( -1082 )  gg16.25415.380.63ga1593.751869.230.12aa00415.380.27g - allele1753.1326500.82a - allele1546.8726500.82il-10 ( -819 )  cc637.5623.080.48ct8501765.380.35tt212.5311.541c - allele2062.52955.770.65t - allele1237.52344.230.65il-10 ( -592 )  cc637.5623.080.48ca8501765.380.35aa212.5311.541c - allele2062.52955.770.65a - allele1237.52344.230.65 olp has previously been reported to be more prevalent in females than males in american , british , chinese , iranian , italian , japanese and spanish populations 




 . in our patients olp was more frequent in the third to fourth decade of life and the median age of the patients group was 42 years . reticular olp was the most common ( 80.95% ) with the buccal mucosa as the most affected site ( 83.33% ) . there was no significant difference in the clinical manifestation or prognosis when comparing men to women however , in our study , these observations should be taken with caution as the study s sample size is small . our results on tnf- promoter polymorphism in saudi olp patients suggested that genotype ga - positive individuals at position -308 of tnf- are at higher risk for olp , whereas gg genotypes might be negatively associated with the disease . data analysis further demonstrated that allele a ( tnf- 2 allele ) increases the risk of olp , whereas allele g ( tnf- 1 allele ) reduces olp risk . similarly higher frequencies of tnf- 308 a allele and -308 ga genotype have been reported in italian 
 , indian 
 , and chinese patients with an olp 


 . predominance of the aa genotype was also reported in thai 
  and brazilian patients with olp 
 . further , genetic polymorphisms of cytokine - encoding genes are also known to predispose malignant diseases and have been associated with oral precancerous lesions ( opcls ) . tnf- ( -308g / a ) , il-6 and tgf- polymorphisms have been associated with the development of opcls in taiwanese patients 
 . allele a ( tnf- 2 allele ) lies on the extended haplotype hla - a1-b8-dr3-dq2 , which is associated with high tnf- production . the allele a ( tnf- 2 allele ) has been demonstrated to be a much stronger transcriptional activator than the common allele g ( tnf- 1 allele ) 
 . moreover , tnf- ( -308g / a ) promoter polymorphism has a direct effect on tnf- gene regulation ( increased tnf- expression ) and may be responsible for the association of allele a with the high tnf- phenotype and more severe diseases 
 . further populations bearing a higher proportion of the allele a ( tnf- 2 allele ) are reportedly predisposed to several metabolic , degenerative , inflammatory , and autoimmune diseases 
 . the results of this study also show that the ga genotype of tnf- ( + 252 a / g ) was positively associated with olp risk , whereas genotype aa might be protective . this report is the first to show an association between tnf- ( + 252 a / g ) polymorphism and olp . this + 252 a / g polymorphism on intron 1 of tnf- is in linkage disequilibrium with a missense variant ( thr 26 asn ) in exon3 
 . it is also closely linked to tnf- , which is in linkage disequilibrium with hla - a , b8 , and dr3 
  and has been reported to define a high tnf- expressing haplotype in addition to a modifying expression of tnf- itself 
 . tnf- ( + 252a / g ) polymorphism has also been associated with numerous autoimmune diseases 
 . high serum and salivary levels of proinflammatory cytokine and tnf have been detected in olp patients 
 . thalidomide , a known tnf suppressant has been used to successfully treat olp showing the implication of high tnf- expressing genotypes of tnf- and - polymorphisms in the pathogenesis of olp 
 . our results show no significant differences in the frequencies of alleles and genotypes of il-10 ( 1082g / a ) , il-10 ( -592c / a ) , il-10 ( -819c / t ) polymorphisms between olp patients and healthy saudi controls . however , haplotype ata from 1082g / a , -819c / t , -592c / a of il-10 had significant association with olp in saudi patients . two other studies dealing with the il-10 gene polymorphisms in olp populations could also not find any difference in the frequencies of il-10 alleles and genotypes between the patients with olp and healthy controls 


 . however , the -1082a/-819t/-592a ( ata ) haplotype of the il-10 polymorphisms , which correlated with a lower serum level of il-10 , has a significant association with olp in a chinese cohort with han ethnicity 
 . thus , this study together with the study from china supports that the low producer ata haplotype of il-10 polymorphisms is linked to the risk of olp . our results also indicated that the frequencies of alleles and genotypes of all the five polymorphisms studied are independent of gender ( table 4 ) . however , these results should be taken with caution as the number of males and females is too low for any significant conclusion . besides tnf- and il-10 , a number of inflammation and immune regulation - related cytokines , including interferon - gamma ( ifn- ) , il-4 , and il-8 , have been suggested to have an important role in the pathogenesis of olp . abnormal expression patterns of various inflammation - related cytokines , including il-10 , and tnf- , in lesions , saliva , serum and peripheral blood mononuclear cells from patients with olp has also been reported , which may reflect the immune dysregulation status of cytokines and emergence as central players in the immunopathogenesis of olp 
 . recently carrozzo 
  ( 2014 ) suggested that the pro inflammatory milieu associated with degranulated mast cells might play a role in helping t cells breach the epidermal basement membrane in olp lesions . the polymorphisms in tnf- ( -308 ) , tnf- ( + 252 ) and il-10 ( -1082 , -819 and -592 ) gene are associated significantly with the risk of olp susceptibility . however , further studies are required that use a larger sample size to confirm this association .
objectives oral lichen planus ( olp ) is a chronic inflammatory oral mucosal disease 
 . cytokines play an important role in the pathogenesis and disease progression of olp . 
 various reports have implicated cytokine gene polymorphisms in susceptibility to develop some immune mediated conditions including olp . 
 the purpose of this study was to investigate the association of tumor necrosis factor ( tnf)- , tnf- and interleukin ( il)-10 gene polymorphisms with the olp risk.material and methods forty two unrelated patients with olp and 211 healthy volunteers were genotyped for tnf- ( -308 g / a ) , tnf- ( + 252a / g ) , il-10 ( -1082g / a ) , il-10 ( -819c / t ) , and il-10 ( -592c / a ) polymorphisms.results the frequencies of allele a and genotype ga of tnf- ( -308g / a ) were significantly higher while allele g and gg genotypes were lower in olp patients as compared to the controls ( p<0.001 ) . 
 the frequency of ga genotype of tnf- ( + 252a / g ) was significantly higher in patients than in controls while the aa genotype was completely absent in olp patients . 
 these results indicated that allele a and genotype ga of tnf- ( -308g / a ) as well as the ga genotype of tnf- ( + 252a / g ) polymorphisms are associated with olp risk . 
 the frequencies of alleles and genotypes of -1082g / a , -819c / t and -592c / a polymorphisms in il-10 gene did not differ significantly between olp patients and controls ( p>0.05 ) . however , haplotype ata extracted from 1082g / a , -819c / t , -592c / a polymorphisms of il-10 were more prevalent in olp patients when compared to controls indicating its possible association with olp susceptibility.conclusion it is concluded that tnf- ( -308g / a ) , tnf- ( + 252a / g ) and il-10 ( -1082g / a , -819c / t and -592c / a ) polymorphisms are associated with the susceptibility of olp , thus giving additional support for the genetic basis of this disease .