pterygium is a common wing - shaped and oriented fibrovascular lesion coating the surface of the eye . according to the population - based studies , its prevalence rate varies from 0.7% to 33% . this abnormality arises from the conjunctiva and extends into the cornea and can result in remarkable cosmetic problems , visual impairment , recurrent inflammation , and mild irritation . surgery is needed for cases when lesion expands to the central part of the cornea   ( figure 1 ) . there are countless theories regarding the causes of pterygium including uv light exposure , viruses , oxidative stress , dna methylation , apoptotic and oncogenic proteins , loss of heterozygosity , microsatellite instability , inflammatory mediators , extracellular matrix modulators , lymphangiogenesis , cell epithelial - mesenchymal transition , and alterations in cholesterol metabolism . therefore , we focused on lats1 and lats2 genes which are the common tumor suppressor genes in the uv - induced dna damage response ( ddr ) signaling pathways . lats ( large tumor suppressor ) gene , a ser / thr kinase , belongs to the ndr / lats subfamily of agc ( protein kinase a / pkg / pkc ) kinases originally isolated from drosophila melanogaster . two mammalian homologs of fly lats , lats1 and lats2 , are located in 6q25.1 and 13q12.11 chromosomes , respectively . one of the ddr signaling pathways , which facilitate apoptosis following high levels of uv - induced damage , is chk1-lats2-p21 axis . and chk1-lats2-(14 - 3 - 3 ) regulates the p - body formation as a unique signaling pathway in response to uv - induced dna damage . lats1 and lats2 are also engaged in the regulation of cell cycle through g2-m arrest and g1-s arrest , respectively [ 8 , 9 ] . after dna damage the integrity of genome is warranted through rassf1a - lats1/2-mdm2-p53 signaling pathway . in addition , a large amount of literature has reported the function of these genes in morphogenesis , cell division , and apoptosis . epigenetic modifications such as dna methylation of cpg islands in promoter regions are the main cause of tumor suppressor gene silencing and can result in tumor development . some tumors such as breast cancer and astrocytoma have shown downregulation of lats1 and lats2 mrna expression through promoter methylation . to our knowledge for the first time , this study highlights the status of lats1 and lats2 promoter methylation and mrna expression profiles in pterygium . this case - control study was performed from 2010 to 2013 consisting of 70 primary pterygium tissues ( 35 males and 35 females with a mean age of 52.44  20.611 ) and 70 normal conjunctiva tissues of the patients who had undergone cataract surgery ( 35 males and 35 females with a mean age of 50.67  23.318 ) . all procedures in this study were approved by the ethical board at the zahedan university of medical sciences . then 1 - 2  g of isolated dna was diluted in 20  l of water and used for bisulfite treatment by wizard dna clean - up system ( promega ) kit which converts unmethylated cytosine to uracil and leaves methylated cytosine unaltered . according to the manufacturer 's instructions of promega , the treated dna should be diluted in 20  l of water and kept at 20c for using in the further experiments . to carry out the msp analysis , promoters of the genes were recognized through online data analysis ( http://www.ensembl.org ) and then the preferred sequences were used to design methylated and unmethylated primers by methprime online software . our selection for the site of methylated and unmethylated was consistent with the related literature . each pcr reaction contained 1  l of modified dna and 0.5  l of each primer which is dissolved in the lyophilized blue pellet of accupower hotstart pcr premix reached a final volume of 20  l with water . the msp amplification was set as follows : 95c for 5  min , followed by 40 cycles ( 95c for 40  s , the annealing temperature for lats1 : m = 53 , u = 57 ; lats2 : m = 55.5 , u = 55 for 40  s and extension at 72c for 40  s ) . pcr products were detected by electrophoresis in 2% agarose gel , 80100 volts for an hour until being well separated ( figure 2 ) . total rna was extracted from pterygium and control tissues using the rnx - plus solution ( cat . number : k1621 ) was used to reverse - transcribe 1  mg of rna to cdna in a final volume of 20  l . real - time pcr was performed using sybr green in abi 5700 sequence detection system ( applied biosystems ) . pcr efficiencies ( e ) were calculated for all used primers from the given slopes of standard curves , generated from serial dilutions of positive controls , according to the following equation : e = 2 . the effect of lats1 and lats2 genes methylation on the risk of pterygium formation was detected by estimating odds ratios ( or ) and 95% confidence intervals ( 95% ci ) , using logistic regression . avoiding bias in estimating or , we calculated confidence intervals by three methods including exact , cornfield , and woolf . the stata se ( version 13.1 ) the methylation frequency of lats1 gene was 66 ( 94.28% ) for cases and 54 ( 77.14% ) for healthy controls . lats2 gene showed 98.57% ( n = 69 ) methylation in cases and 82.86% ( n = 58 ) in the controls group . comparison of methylated versus unmethylated indicated significant difference between cases and controls in last1 ( or = 4.9 ; 95%ci : 1.54 to 15.48 , p = 0.003 ) and lats2 ( or = 7.1 ; 95%ci : 1.53 to 33.19 , p = 0.004 ) ( table 3 ) . decreased expression in case group of both candidate genes ( 0.42  0.030 in case versus 0.57  0.068 for controls in lats1 and 0.44  0.028 in cases versus 0.57  0.061 controls in lats2 ) was detected . comparison of mean between cases and controls revealed a statistically significant difference in both genes ( p < 0.05 ) ( table 4 ) . pterygium is a benign lesion that extends from conjunctiva to the cornea where it may interfere with vision . since the current treatment of pterygium is invasive , mainly based on surgery , studies for new markers should be conducted . environmental factors such as uv radiation play an important role in tumor promotion through the epigenetic dysregulation of the cell cycle genes . lats1 and lats2 as a part of ddr signaling pathways are putative serine / threonine kinase proteins that localize to the mitotic apparatus and constitute a complex with cell cycle control system [ 9 , 18 ] . lats1 acts as a negative regulator of cdc2/cyclina , which reduces h1 histone kinase activity of cdc2 and results in a g2-mcell - cycle arrest . also , lats1 is activated by rassf1a ( ras association domain family 1 isoform a ) that stimulates response to dna damage . the activation of lats1 promotes genomic stability via stabilizing replication forks by restricting cdk2-mediated phosphorylation of brca2 . this modulation not only has a fundamental role in error - free dna repair but also maintains nucleofilament formation at stalled replication forks . it interacts with the centrosomal proteins aurora - a and ajuba and also is required for the accumulation of gamma - tubulin and spindle formation at the onset of mitosis . it also interacts with a negative regulator of p53 and may function in a positive feedback loop with p53 that responds to the cytoskeleton damage . the lats2-mdm2-p53 axis thus constitutes an innovative checkpoint pathway critical for the maintenance of proper chromosome number . both lats1 and lats2 as tumor suppressors are part of hippo signaling pathway which has profound effects on normal cell fate and tumorigenesis . therefore , silencing of lats1 and lats2 putative tumor suppressor genes through promoter methylation may cause the development of pterygium . in addition , promoter hypermethylation mediates decreased expression of lats1 and lats2 in human astrocytoma . downregulation of lats1 and lats2 mrna expression by promoter hypermethylation has been reported in breast cancer . consistent with the abovementioned studies , our results confirmed the significant relationship between reduced expression of the lats1 and lats2 through methylation and the risk of pterygium formation . besides our data , the literature reviews showed the aberrant dna methylation and decreased expression of p16 , ecadherin , tgm 2 , mmp2 , and cd24 genes in pterygium [ 2628 ] . exploring pterygium methylation markers and their subsequent effects on mrna expression paves the road for better therapy such as discovering drugs with the regulation of methylation characteristic . further studies are required to identify the exact molecular function of lats1/lats2 genes in pterygium in various and larger genetic populations using advanced molecular techniques .
purpose . 
 pterygium is a serious eye problem in countries with high exposure to uv . however , despite numerous studies , the molecular etiology of pterygium is unclear . 
 recent studies have indicated that lats1 and lats2 genes are involved in ddr signaling pathways against continuous uv exposure . 
 our aim was to evaluate the lats1 and lats2 promoter methylation with the risk of pterygium formation . 
 methods . 
 we evaluated the promoter methylation status of lats1 and lats2 using methylation - specific pcr technique . 
 also , mrna expression of lats1 and lats2 was assessed in 14 cases of pterygium and 14 normal specimens by real - time pcr . 
 results . 
 promoter methylation of lats1 and lats2 was detected significantly between pterygium tissues and normal tissues [ lats1 ; or = 4.9 ; 95% ci : 1.54 to 15.48 , p = 0.003 ; lats2 ; or = 7.1 ; 95% ci : 1.53 to 33.19 , p = 0.004 ] . 
 the gene expression analysis showed a statistically significant difference between pterygium tissues and healthy controls for both lats1 and lats2 ( p < 0.05 ) . 
 conclusions . 
 the data of this study is the first report regarding the effect of promoter methylation of the lats1 and lats2 in the pterygium . to confirm these data , 
 doing further studies in various genetic populations with large sample sizes using advanced molecular techniques is proposed .