migraine originated from a greece term meaning  hemicraine  or  half of the head  ( 1 ) , usually associated with episodes of strong unilateral pulsating headache ( 2 ) .
migraine disorder shows a high - related relevancy ; approximately 50% of migraine cases have a first - degree related as a migraine sufferer ( 3 ) .
familial clustering migraine points to importance of genetic factors in this illness , but its inheritance pattern is argumentative and supposed it is likely multifactorial , although autosomal dominant inheritance can not account for it ( 3 ) .
the prevalence of migraine impressed about 10%12% of the white population ( 4 ) , 24% of us populations and 12% of adults ( 5 ) contains both sexes ( 4 ) , affected women more higher than men ( 17.1% in women and 5.6% in men ) ( 5 ) and often involves the middle - aged people ( 6 ) .
migraine is observed as an inherited brain disturbance , specified by neurotransmitter imbalances , especially , serotonin 5-hydroxytryptamine ( 7 ) that contribute to neuronal dysfunctions ( 8) . this disorder is generally characterized by strong and recurrent head pains which typically lasting about 472 h and attended by some symptoms like vomiting , neurological disturbance , photophobia and phonophobia . according to the classification of international headache society ( ihs ) , two main classes of migraine consist : migraine without aura ( mo ) , which included 70% of all migraineurs and migraine with aura ( ma ) , which affected the rest of migraine population ( about 25% ) ( 9 ) ,
both sub - groups have an intense and valid genetic background , but according to recent epidemiological information , the genetic factors in increasing development of ma ( 25%30% ) are stronger than mo ( 11 ) .
the differences between ma and mo refer to the clinical symptoms of ma , it can include signals such as food craving , mood changes , neck stiffness , fatigue , reversible visual system symptoms , sensory and aphasic aura signs , each symptom might last from 5 min to 1 h ( 9 ) . since migraine is a complicated and multifactorial disease , no distinct marker is in available to diagnosis the patient s status , yet . although , studies discovered prostaglandins synthesized by cyclooxygenase ( cox ) pathways , play a significant role in pathogenesis of this disorder , cox enzymes involved in pain mechanisms and migraine attacks , are the most important mediators of inflammation and pain ( 2 ) .
cox plays a noticeable role in prostanoids synthesis from arachidonic acid and arises this chemical reaction in both constitutive ( cox-1 ) and inducible ( cox-2 ) isoforms ( 12 ) which leads to sensibility and chronic pain in neuronal cells ( 13 ) .
prostaglandin e2 ( pge2 ) and prostaglandin i2 ( pgi2 ) are two important prostanoids that effect on pain signal considerably .
some pharmaceutics combination like nonsteroidal anti - inflammatory drugs ( nsaids ) can inhibit cox-1 and cox-2 pathway and degrade pain in migraineur by reducing the production of prostanoids consisting pge2 .
thereby , one of the important factor in migraine therapy is cox-2 modulation , hence , applying non - selective cox suppressors such as acetylsalicylic acid was been high usage .
moreover , a selective cox-2 inhibitor as rofecoxib is so effective in patient suffering from migraine with or without aura to tolerate the pain ( 2 ) . as for the substantial role of cox-2 in synthesize pge2 and pathogenesis of migraine , we hypothesized that cox-2 gene basic polymorphisms may increase susceptibility to migraine .
therefore , we did this research project for the first time in iran ( bushehr province ) and second time in the world .
the aim of study was to determine the association of cox-2 - 765g > c ( rs20417 ) and cox-2 - 1195a > g ( rs689466 ) promoter polymorphisms to migraine susceptibility in iranian migraine patients for the first time in this region using rflp method .
in this case - control study a total of 100 migraine patients ( 79 females , 21 males ) and 100 controls ( 77 females , 23 males ) were collected from july to nov 2013 from bushehr province , eastern iran and examined for promoter polymorphisms of cox-2 gene variant .
controls were chosen among the people volunteered to donate their blood in the bushehr blood transforming center .
the ethics board of hospital for migraine approved this study and all individuals providing samples signed informed consents covering aspects of the experiments conducted .
the numbers of 20 patient samples were the individuals suffered from ma and 80 samples from mo .
inclusion criteria were as follows : 1 ) patients with migraine referred to the mentioned clinic ; 2 ) signing informed consents covering by patients itself ; 3 ) diagnosis migraine by consultant .
exclusion criteria were as follows : 1 ) discontentment of migraine patients or control group ; 2 ) unverified of migraine patients or control group by consultant .
both control and patients were interviewed and examined by a specialist neurologist , all of them answered to a complete and perfect questionnaire and expressed their specifications such as their sex , age of onset , inbreeding or outbreeding marriage , related migraine history and pain severity . after obtaining awarded consent ,
the blood samples were transported to the laboratory and stored at 20 c until needed for analysis .
pcr products for cox-2 gene on agarose gel with cox-2 - 765g > c ( rs20417 ) and cox21195a > g ( rs689466 ) primers showed fragments about 309 and 273 bp respectively ( fig . 1 and 2 ) . the anticipated results after restriction for each gene are also mentioned in table 1 .
the agarose gel electrophoresis of pcr products after digested with acii and pvuii enzymes are showed in fig . 3 and 4 , respectively .
agarose gel stained with ethidium bromide , pcr products of cox-2 gene by using the cox-2 - 765g > c ( rs20417 ) primer . line 16 is 309 bp fragments , line m is 100 bp dna ladder agarose gel stained with ethidium bromide , pcr products of cox-2 gene by using the cox21195ag ( rs689466 ) primer .
line 16 is 273 bp fragments , line m is 100 bp dna ladder result of enzyme digestion by acii .
lane m is 100 bp ladder , lane 1 and 2 : heterozygote polymorphic dnas ( gc ) contained 309 , 209 and 100 bp fragments , lane 3 and 6 : 309 bp normal dnas ( gg ) , lane 4 and 5 : homozygote polymorphic dnas ( cc ) contained 209 and 100 bp fragments result of enzyme digestion by pvuii .
lane m is 100 bp ladder , lane 1 , 3 and 4 : 273 bp normal dnas ( aa ) , lane 2 : homozygote polymorphic dnas ( gg ) contained 220 and 53 bp fragments , lane 5 and 6 : heterozygote polymorphic dnas ( ag ) contained 273 , 220 and 53 bp fragments pcr and rflp procedures and products of cox-2 - 765g > c and cox-2 - 1195a > g genes total dna was extracted from blood samples using dna purification kit ( dnp , cinnagen , iran ) according to the manufacturer s recommendation .
the total extracted dna was measured at 260 nm optical density ( 14 ) . in order to amplify the individual s respective gene ,
therefore , the final volume of 25 l pcr reactions in 0.2 ml tubes containing 500 ng/l of dna template , 2 mm of mgcl2 concentration , 2 mm dntps , 10 pmol/l of each primer , 5 l of 10x pcr buffer and 1 u of taq dna polymerase ( fermentas , germany ) was performed .
thermal pcr conditions consisted of denaturation phase for 5 min at 95 c , followed by 30 cycles of 94 c for 1 min , temperature of 59 c for cox-2 - 1195a > g ( rs689466 ) primer and 56 c for cox-2 - 765g > c ( rs20417 ) primer for 1 min , and 72 c for 1 min , with a final extension for 5 min at 72 c .
pcr - amplified products were examined by 2% agarose gel and analysis of pcr products for presence of cox-2 - 1195a > g gene revealed 273 bp fragments ( fig .
after electrophoresis , dna was observed and photographed in ultraviolet imager ( uvi ) doc gel documentation systems ( uk ) .
restriction fragment length polymorphisms ( rflps ) were used for analysis sequence polymorphisms of proliferated dna .
the pcr product from second step digested for 4 h at 37 c by acii and pvuii restriction enzymes for cox-2 - 765g > c ( rs20417 ) and cox-2 - 1195a > g ( rs689466 ) snps , respectively , according to the manufacturer s instructions .
the results of enzymatic digestion were identified in a 2% agarose gel by electrophoresis and ethidium bromide staining . molecular weight 100
bp plus marker ( fermentas , co. ) and undigested pcr products was included in each analysis .
the genotypes were deduced from the fragmentation patterns of the amplified dna , observed in uvidoc gel documentation systems .
statistical analyses were performed by the chi - square test using the spss 20 ( spss inc .
chicago , il , usa ) software . related risk at 95% confidence intervals ( ci ) was calculated as the odds ratio ( or ) .
linkage disequilibrium between cox-2 - 765g > c and cox-2 - 1195a > g polymorphisms was performed using shesis software ( http://analysis.bio-x.cn/myanalysis.php ) ( 15 ) .
in this case - control study a total of 100 migraine patients ( 79 females , 21 males ) and 100 controls ( 77 females , 23 males ) were collected from july to nov 2013 from bushehr province , eastern iran and examined for promoter polymorphisms of cox-2 gene variant .
controls were chosen among the people volunteered to donate their blood in the bushehr blood transforming center .
the ethics board of hospital for migraine approved this study and all individuals providing samples signed informed consents covering aspects of the experiments conducted .
the numbers of 20 patient samples were the individuals suffered from ma and 80 samples from mo .
inclusion criteria were as follows : 1 ) patients with migraine referred to the mentioned clinic ; 2 ) signing informed consents covering by patients itself ; 3 ) diagnosis migraine by consultant .
exclusion criteria were as follows : 1 ) discontentment of migraine patients or control group ; 2 ) unverified of migraine patients or control group by consultant .
both control and patients were interviewed and examined by a specialist neurologist , all of them answered to a complete and perfect questionnaire and expressed their specifications such as their sex , age of onset , inbreeding or outbreeding marriage , related migraine history and pain severity . after obtaining awarded consent ,
the blood samples were transported to the laboratory and stored at 20 c until needed for analysis .
pcr products for cox-2 gene on agarose gel with cox-2 - 765g > c ( rs20417 ) and cox21195a > g ( rs689466 ) primers showed fragments about 309 and 273 bp respectively ( fig . 1 and 2 ) . the anticipated results after restriction for each gene are also mentioned in table 1 .
the agarose gel electrophoresis of pcr products after digested with acii and pvuii enzymes are showed in fig . 3 and 4 , respectively .
agarose gel stained with ethidium bromide , pcr products of cox-2 gene by using the cox-2 - 765g > c ( rs20417 ) primer . line 16 is 309 bp fragments , line m is 100 bp dna ladder agarose gel stained with ethidium bromide , pcr products of cox-2 gene by using the cox21195ag ( rs689466 ) primer .
line 16 is 273 bp fragments , line m is 100 bp dna ladder result of enzyme digestion by acii .
lane m is 100 bp ladder , lane 1 and 2 : heterozygote polymorphic dnas ( gc ) contained 309 , 209 and 100 bp fragments , lane 3 and 6 : 309 bp normal dnas ( gg ) , lane 4 and 5 : homozygote polymorphic dnas ( cc ) contained 209 and 100 bp fragments result of enzyme digestion by pvuii .
lane m is 100 bp ladder , lane 1 , 3 and 4 : 273 bp normal dnas ( aa ) , lane 2 : homozygote polymorphic dnas ( gg ) contained 220 and 53 bp fragments , lane 5 and 6 : heterozygote polymorphic dnas ( ag ) contained 273 , 220 and 53 bp fragments pcr and rflp procedures and products of cox-2 - 765g > c and cox-2 - 1195a > g genes
total dna was extracted from blood samples using dna purification kit ( dnp , cinnagen , iran ) according to the manufacturer s recommendation .
in order to amplify the individual s respective gene , pcr test was accomplished using specific primers ( table 1 ) .
therefore , the final volume of 25 l pcr reactions in 0.2 ml tubes containing 500 ng/l of dna template , 2 mm of mgcl2 concentration , 2 mm dntps , 10 pmol/l of each primer , 5 l of 10x pcr buffer and 1 u of taq dna polymerase ( fermentas , germany ) was performed .
thermal pcr conditions consisted of denaturation phase for 5 min at 95 c , followed by 30 cycles of 94 c for 1 min , temperature of 59 c for cox-2 - 1195a > g ( rs689466 ) primer and 56 c for cox-2 - 765g > c ( rs20417 ) primer for 1 min , and 72 c for 1 min , with a final extension for 5 min at 72 c .
pcr - amplified products were examined by 2% agarose gel and analysis of pcr products for presence of cox-2 - 1195a > g gene revealed 273 bp fragments ( fig .
dna was observed and photographed in ultraviolet imager ( uvi ) doc gel documentation systems ( uk ) .
restriction fragment length polymorphisms ( rflps ) were used for analysis sequence polymorphisms of proliferated dna .
the pcr product from second step digested for 4 h at 37 c by acii and pvuii restriction enzymes for cox-2 - 765g > c ( rs20417 ) and cox-2 - 1195a > g ( rs689466 ) snps , respectively , according to the manufacturer s instructions .
the results of enzymatic digestion were identified in a 2% agarose gel by electrophoresis and ethidium bromide staining . molecular weight 100
bp plus marker ( fermentas , co. ) and undigested pcr products was included in each analysis .
the genotypes were deduced from the fragmentation patterns of the amplified dna , observed in uvidoc gel documentation systems .
statistical analyses were performed by the chi - square test using the spss 20 ( spss inc .
chicago , il , usa ) software . related risk at 95% confidence intervals ( ci ) was calculated as the odds ratio ( or ) .
linkage disequilibrium between cox-2 - 765g > c and cox-2 - 1195a > g polymorphisms was performed using shesis software ( http://analysis.bio-x.cn/myanalysis.php ) ( 15 ) .
the mean age was 33.1610.38 for patients ( 1869 age ) , and 34.2010.16 yr ( 1860 age ) for controls .
there were symmetry between patient and control subjects in terms of sex prevalence and mean age and most of patients experienced intensive pain , which disorganized their functions and activities . table 2 shows the frequencies of cox-2 - 765g > c and cox-2 - 1195a > g genotype distributions in controls , ma and mo patients separately .
there were statistically considerable differences in cox-2 - 765g > c and cox-2 - 1195a > g genotypes between the controls and patients .
carriers of cox-2 - 765 cc and gc genotypes ( polymorph types ) in patients were further than in the controls and frequencies of cox-2 - 765 gg genotype ( the wild type ) in controls were higher than in all the patients ( p<0.001 , :34.03 ) . in ma and mo patients , -765 cc and -765 gc
genotypes were significantly higher than in controls ( p<0.018 , :8.00 and p<0.001 , :36.83 respectively ) , and not marked diversities in -765 cc and -765 gc genotypes between ma and mo patients ( p<0.084 , :4.95 ) .
c and cox-2 - 1195a > g genotype distributions in patients and controls p1 : p value of migraine with aura vs control .
polymorphism , only aa genotype ( the wild type ) was statistically higher in the controls than in the all patients ( p<0.010 , :9.23 ) and determined that cox-2 - 1195 aa genotype had decreased risk for migraine .
conversely , cox-2 - 1195 gg and ag genotypes in patients were higher than in controls considerably ( p<0.010 ,  : 9.23 ) .
we also looked into the prevalence of cox-2 - 765 c+ and cox-2 - 1195 g+ genotype dispensation in ma and mo patients and control subjects , separately ( table 2 and 3 ) . or and 95% ci distributions in ma , mo patients and controls 1 : migraine with aura vs control .
ci : confidence intervals in despite of being considerable differences in frequencies of cox-2 - 765 c+ and cox-2 - 1195 g+ genotypes between migraine cases and healthy people ( p<0.001 , :25.41 , or:4.93 ci= 2.59- 9.39 and p<0.002 , :9.23 , or:0.408 ci= 0.228- 0.732 respectively ) , but there were no significant diversity between mo and ma patients in cox-2 - 765 c+ and cox-2 - 1195 g+ genotypes ( p<0.6 , :4.89 or:0.296 ci= 0.097- 0.905 and p<1 , :0 , or:1 ci= 0.375- 2.664 respectively ) . on the other hand , carriers of cox-2 - 1195 g+ genotype ( the mutant type ) in patient subjects were higher than in all the migraine- free cases ( p<0.002 , :9.23 , or : 0.408 95% ci= 0.2280.732 ) .
cox-2 - 765 gg and cox-2 - 1195 aa genotypes decrease risk for migraine and cox-2 - 765 c+ and cox-2 - 1195 g+ genotypes contribute to increase risk of migraine susceptibility .
table 2 and 3 also show the details of significant differences in frequencies of c allele and g allele from -765g > c polymorphism ( p<0.001 , :37.9 , or : 3.62 ci= 2.39- 5.5 ) and an allele and g allele from 1195a > g polymorphism ( p<0.003 , :8.76 , or : 0.489 ci= 0.303- 0.789 ) between control and migraine cases .
the association of migraine disorder with cox-2 gene polymorphisms was confirmed by haplotype analysis and specified that the frequencies of cox-2 - 765 g : 1195a haplotype were significantly lower in patients as compared with those of controls ( p<0.001 ) , also revealed that frequencies of cox-2 - 765c : 1195a and cox-2 - 765c : 1195 g haplotype frequencies were significantly higher in patients as compared with those of controls ( p<0.002 and p<0.001 ) ( table 4 ) .
there was high linkage disequilibrium between cox-2 - 765g > c and cox-2 - 1195a > g polymorphisms . in this analysis , no significant difference was observed in frequencies of cox-2 - 765 g : 1195 g haplotype in patients as compared with controls ( p<0.886 ) .
the frequencies of haplotypes of cox-2 gene in patients and controls we also looked into the frequencies of cox-2 - 765g > c and cox-2 - 1195a > g genotype distributions in patients with related or unrelated parents separately ( table 5 ) .
frequencies of cox-2 - 765g > c and cox-2 - 1195a > g genotype distributions in patients with related and unrelated marriage parents p1 : p value of patients with related parent vs control .
c , quantity of cox-2 - 765 gg genotype ( the wild type ) in controls were higher as compared with the patients with related parents and unrelated parents , however , quantity of cox-2 - 765 cc and cox-2 - 765 gc genotypes in patients with related and unrelated parents were more than in migraine- free subjects ( p<0.001 ,  : 44.67 and p<0.004 ,  : 11.30 respectively ) .
likewise , it can be perceived from the date in table 5 that there are significant discrepancies between patients with related parents and patients with unrelated parents in cox-2 - 765 cc and cox-2 - 765 gc genotypes ( p<0.001 ,  : 17.16 ) . moreover , cox-2 - 1195 aa genotype ( the wild type ) in controls were higher than in patients with related parents and unrelated parents and besides , frequencies of cox-2 - 1195 gg and cox-2 - 1195 ag genotypes in control cases were lower than patients with related parents and unrelated parents ( p<0.001 ,  : 39.76 and p<0.047 ,  : 6.12 respectively ) .
moreover , considerable differences were achieved in frequencies of cox-2 - 1195 gg and cox-2 - 1195 ag genotypes between patients with related parents and patients with unrelated parents ( p<0.001 ,  : 49.94 ) , consanguineous marriage would heighten the risk of susceptibility to migraine illness .
we also looked into the quantity of g allele , c allele , a allele and g allele distributions in patients with related parents , patients with unrelated parents and controls .
on one hand table 5 and 6 show significant differences in frequencies of g allele and c allele between patients with related parent and control subjects ( p<0.001 ,  : 46.96 , or : 5.51 ci= 3.329.14 ) , besides , frequencies of c allele in patients with unrelated parent were higher than in controls significantly ( p<0.002 ,  : 9.67 , or : 2.25 ci= 1.34  3.77 ) and considerable diversity was observed between patients with related parents and patients with unrelated parents in frequencies of g allele and c allele ( p<0.002 ,  : 9.44 , or : 2.45 ci= 1.38  4.36 ) .
table 5 and 6 indicate diversities in amount of a allele and g allele in patients with related parents , patients with unrelated parents and controls on the other hand .
information clarify marked discrepancy in frequencies of a allele and g allele between patients with related parents and migraine free cases ( p<0.001 ,  : 35.95 , or : 0.21 ci= 0.130.36 ) , although significant difference between patients with unrelated parents and healthy cases was noted in quantity of a allele and g allele ( p<0.008 ,  : 6.98 , or : 3.48 ci= 1.319.22 ) .
there are considerable diversity in frequencies of these two alleles between patients with related parents and patients with unrelated parents ( p<0.001 ,  : 45.11 , or : 0.61 ci= 0.020.16 ) .
moreover , consanguineous marriage plays a key role in boosting prevalence of migraine . or and 95% ci distributions in patients with related parents , unrelated parents and control 1 :
ci : confidence intervals we also achieved that carries of cox-2 765 c+ and cox-2 765 g+ genotypes in patients with related parents were higher and lower than in control cases respectively ( p<0.001 ,  : 36.56 , or : 28.70 ci= 6.63124.27 and p<0.001 ,  : 27.28 , or : 7.66 ci= 3.1118.89 respectively ) .
meanwhile , considerable difference in cox-2 765 c+ genotype between patients with related parents and patients with unrelated parents has mentioned in table 5 and 6 ( p<0.001 ,  : 17.08 , or : 14.62 ci= 3.1468.07 ) .
table 5 and 6 illustrate that there is considerable difference in cox-2 - 1195 g+ genotype between control and patient with related parents ( p<0.001 ,  : 39.68 , or : 0.091 ci= 0.0400.204 ) although in cox-2 - 1195 g+ genotype distribution , significant difference was observed between subjects with related parents and patients with unrelated parents ( p<0.001 , =49.50 , or= 0.028 95%ci= 0.0090.088 ) .
generally , there were statistically considerable differences in cox-2 - 765g > c and cox-2 - 1195a > g snps between the ma patients , mo patients , patients with related parents , patients with unrelated parents as compared with control individuals , so these variants and consanguineous type of marriage can increase risk of migraine susceptibility .
we have studied the impression of genetic polymorphisms of cox-2 gene and the risk of migraine susceptibility . in this study
, there is a positive relation between the -765 gc , -765 cc , -1195 ag and -1195 gg and being adventured to migraine attacks . in other words
c allele of cox-2 - 765g > c and g allele of cox-2 - 1195a > g play key role in increasing migraine risk and besides -765 gg and -1195 aa have protective effect against migraine .
we showed this association on iranian population as the first study in iran and second in the world .
recent studies are available now and have recognized four new genetic variants related to migraine .
a new variant known as rs1835740 moderates glutamate homeostasis is associated with concepts of neurotransmitter disorders .
this new variant may be more specific for acute sorts of migraine such as ma than mo ( 8) .
another variant exists as rs11172113 , involves the lipoprotein receptor lrp1 which may interact with neuronal glutamate receptors and pathway however the specify function of third variant rs2651889 ( prdm16 ) in migraine is unknown yet ( 8) . the fourth variant that for the first time connected gene impressions on migraine pain pathways , was rs10166942 is in close proximity to trpm8 , which encodes for pain and cold receptors ( 8) .
cox-2 derived prostanoids enumerated as crucial clinical mediators of pain and other inflammatory symptoms of knee osteoarthritis ( 2 ) suggested that cox-2 polymorphisms could contribute susceptibility to sarcoidosis ( 16 ) .
cox-2 - 765g > c and cox-2 - 1195a > g would alter the mrna levels and transcriptional pattern ( 17 ) .
there is a major relevance between cox-2 expression and the risk of vitiligo ( 19 , 20 ) .
correlation of cox-2 functional polymorphisms ( cox-2 - 1195a > g , -765g > c , -8473t > c ) and the risk of vitiligo has been reported ( 21 ) .
furthermore , expression of the two main isoforms of cyclooxygenase ( cox-1 and cox-2 ) in human cancerous prostatic tissues has been studied and overexpression of both of them is reported ( 23 ) . moreover ,
an increased propensity for lung cancer development was observed in individuals who caring the c allele of a polymorphism in the 3-utr of cox-2 ( 24 ) . in a study on iranian population
, there was no significant relation between ptgs2 ( cox-2)-765g > c gene polymorphism and the risk of sporadic colorectal cancer ( 25 ) .
moreover , 75% of patient with poorly differentiated tumors and 84.6% of patient with moderately differentiated tumors had high scale of cox-2 in their primary tumor cells ( 26 ) .
induction of cox-2 in dementia of alzheimer type and down s syndrome may contribute to the integration of free radicals and then may be related to neuronal degeneration ( 27 ) .
selective cox-2 inhibitors can increase the risk of vascular events ( 28 ) myocardial infarction ( 29 ) and cardiovascular occurrences ( 30 ) .
conversely , selective cox-2 inhibitors made a significantly reduction in the risk of breast cancer ( 31 ) , this reality can be related to the finding that elevated cox-2 expression in breast cancers was seen as a common and ordinary point ( 32 ) .
cox-2 may be a target for the prohibition , treatment and abatement the risk of squamous cell carcinoma of the head and neck ( hnscc ) ( 33 ) .
the cox-2 pathway can lead to the growth and apoptosis of pancreatic cancer ( 34 ) and interesting results are in available , pointed to that overexpression of cox-2 in multiple myeloma ( mm ) is correlated with reduced survival ( 3536 ) .
as the first study in iran , demonstrates that cox-2 gene polymorphisms would increase the risk of affliction to migraine disorder .
further studies on other races in the other regions with different climate are necessary to compare various results . regarding effective role of genetic factors in ma and related clustering migraine
, more genetically based studies can be helpful in diagnosing the reasons , symptoms and inheritance prognosis of illness .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
 background : migraine is a common debilitating primary headache disorder with current head pain attacks , which contributes to physical activity dysfunctions in chronic pain phase . pge2 and pgi2 are two important prostaglandins synthesised by cox-2 enzymes , involved in migraine pain signals . 
 cox-2 modulation is essential in treatment and pathogenesis of migraine . 
 this study aimed to investigating the association between cox-2 gene polymorphisms with the risk of migraine susceptibility in migraine patients with related and unrelated parents.methods:this case- control study was based on 100 migraine patients and 100 non - migraine subjects in bushehr province , iran in 2013 . 
 genomic dna of blood samples was extracted and genotyping of cox-2 - 765g > c ( rs20417 ) and cox-2 - 1195a > g ( rs689466 ) gene variants was investigated by pcr - rflp method . 
 statistical analyses were accomplished using the spss software package.results:there was a significant differences in the frequencies of the cox-2 - 765g > c and cox-2 - 1195a > g genotypes between migraine patients and controls ( p0.05).conclusion : cox-2 - 765cc , cox-2 - 765cg , cox-2 - 1195gg and cox-2 - 1195ag genotypes can increase the risk of migraine significantly . as the first study in iran 
 , we are hopeful to achieve greater results about the relevancy of cox-2 gene , migraine and pain signals pathway by repeating these experiments on more samples . 
Introduction
Materials and Methods
Samples
DNA preparation
PCR assay
Polymorphism analysis
Statistical analysis
Results
Discussion
Conclusion
Ethical considerations