multiple sclerosis ( ms , omim 126200 ) is the most common cause of nontraumatic chronic neurological disability of the central nervous system ( cns ) , characterized by demyelination , axonal degeneration , and inflammation ( 1 - 5 ) .
the disease onset usually occurs in young adults and it is more common in females , with prevalence rates varying across ethnic groups and depending on geographic latitude ( 6 - 8 ) .
most ms patients ( 85% ) present a relapsing - remitting ms ( rrms ) clinical course at the onset , while the remaining groups of patients present primary - progressive ms ( 9 ) .
epidemiological analysis shows that ms results from unknown environmental factors , acting on genetically susceptible individuals ( 10 ) .
susceptibility to ms is held to have a strong genetic component in a large degree , as shown by the increased risk of the disease seen among relatives of affected individuals ( 11 ) .
genetic contributions are clearly included at least in 20% of affected individuals , indicating the family background of the disease .
concurrence rates comprise 26% monozygotic twins compared to 3% in dizygotic twins ( 12 ) .
although the etiology of ms is unknown , epidemiological data recommend that predisposition to the disease is approximately genetically found out and linked to a number of genetic loci .
an association with human leukocyte antigen ( hla ) class ii is well recorded ( 13 ) .
tumor necrosis factor  ( tnf- ) gene is located between the hla - b ( class i ) locus and the class  region in tandem in the major histocompatibility complex ( mhc ) region .
the location of the tnf locus within the mhc region has generated notion about the emphasis of tnf in the etiology of mhc - associated diseases , especially those with an inflammatory or autoimmune history .
ms is not figured as a hereditary disease ; although , a lot of genetic variations have been exhibited to increase the risk of developing the disease ( 8 , 13 ) .
the role of tnf- in a demyelinating disease such as experimental allergic encephalomyelitis ( eae ) is well understood and increasing evidence exists to recommend a role for tnf in the pathogenesis of ms ( 13 ) .
considering the significant differences between the tnf- microsatellite polymorphism gene in patients with ms and healthy controls in europeans , we studied the role of tnf- genotype in ms by determining the correlation between the tnf- microsatellite located in the hla region and ms in patients of two southern provinces of iran ( hormozgan and fars ) .
from february to november 2012 , all individuals involved in this comparative case - control study gave written informed consents for the genetic analysis according to the iran medical committee .
the 81 unrelated patients with ms ( 26 males and 55 females ) considered in this study lived in these two southern areas of iran ( hormozgan and fars provinces ) and had iranian origins dating back at least three generations from both maternal and parental sides .
they were classified according to the poser criteria ( 14 ) and diagnosed with either clinically definite ( 90% ) , laboratory supported definite ( 7% ) or clinically probable ( 3% ) ms .
all the controls were free from acute or chronic internal and neurological diseases , determined by physical examinations .
hla typing had been previously performed for hla class ii alleles : increase of drb1 * 15 allele ( p < 0.005 ) in the patients was the most important point .
genomic dna was extracted from peripheral blood using dna extraction kit ( dnptm , cinnagen co. , iran ) according to the manufactures protocol and stored at -20c until used . using spectrophotometry , the dna quantity was evaluated in each sample .
the microsatellite marker used in this study contained ac / gt repeats and had 13 alleles .
the amplification was carried out in a pcr reaction using 5'-gcctctagatttcatccagccaca-3 ' and 5'-cctctctcccctgcaacacaca-3 ' primers . of the genomic dna , a 100 ng sample
was amplified in a total volume of 10 l containing 10 mm tris - hcl , 50 mm kcl , 1.5 mm mgcl2 , 250 m of each dntp , 1 m of each primer and 0.5 u of taq dna polymerase ( fermentas , germany ) .
the following thermal - cycling conditions were used : initial denaturation at 94c for two minutes , 30 cycles of 94c for 45 seconds , 65c for 30 seconds and 72c for 45 seconds , and final extension at 72c for 10 minutes .
the amplified products were loaded into a 9% polyacrylamide gel with a standard x174/hinf1 marker ( 15 ) .
chicago , il , usa ) in which all the averages were reported as mean  sd and frequencies as percentage .
allele , genotype and haplotype the frequencies were analyzed using gene pop program ( http://genepop.curtin.edu.au ) and the comparison of intergroup differences were conducted by the chi - square and t tests .
test of hardy - weinberg ( hw ) equilibrium was carried out for tnf- microsatellite gene in the case and control populations to check any significant deviation .
our study was performed in a case - control design consisting of 163 people ( 81 patients with ms and 82 hcs ) .
basic demographic details of the patients were unremarkable with mean age [ mean  sd ] of 31.20  9.11 years , mean expanded disability statue score ( edss ) of 4 , mean disease duration of 8.55 years , mean age at diagnosis of 27.49  9.07 years , and female : male ratio of 2.12 . in total , 83.95% of the patients had rrms , 11.11% had secondary progressive disease ( spms ) , and 4.94% had primary progressive multiple sclerosis ( ppms ) at the time of assessment .
a total of 82 unrelated healthy controls ( 27 males and 55 females ; mean age 34  4.7 years ) with almost the same ages , sexes and geographical origins as cases were selected for this study .
most of the case group had a family history of ms ( 89.9% , p < 0.005 ) .
all the 13 alleles were identified for tnf- microsatellite and genotypic distribution of tnf- microsatellite differed significantly between the two population ( p < 10 - 4 ) ( table 2 ) .
tnf - a*11 and tnf-*10 alleles increased significantly in patients with ms ( 0.25 , p < 0.005 and 0.19 ,
p < 0.005 , compare to 0.06 and 0.08 , p < 0.005 , respectively ) .
in contrast , tnf-*1 and tnf-*2 allele frequencies decreased in patients ( 0.01 and 0.02 respectively , p < 0.005 ) , compared to hcs ( 0.15 and 0.21 , p < 0.005 ) .
the heterozygosity for the tnf- microsatellite gene was particularly high in this study ( 0.8 ) ; the tnf- genotypes in studied population were in hardy - weinberg equilibrium ( p = 0.002 for cases and p = 0.05 for controls ) .
linkage analysis is a main method for recognition of genetic factors which can cause diseases .
however , this procedure is not enough for finding the etiology of complex diseases such as ms . to date , crohn 's disease is the only one that can be diagnosed successfully using linkage analysis ( 16 ) .
we screened tnf- microsatellite polymorphism in patients with ms to find their association in an iranian population . to our knowledge , this is the first study on tnf- microsatellite performed in a tropical area of iran , showing association with pathogenesis of ms .
the position of tnf- gene within the hla region has led to paying more attention to the role of tnf- alleles in etiology of ms ( 15 ) .
the tnf- gene is located tandemly and maps within the mhc region centromeric to hla - b and telomeric to the class  region on chromosome 6p2l.3 .
its chromosomal region and the immunomodulatory influence of this gene have caused much more speculation about the role of the tnf locus in mhc - linked diseases ( 17 ) .
it has been repeatedly suggested that genetic predisposition to ms might be influenced by tnf gene polymorphism ( 3 , 18 ) .
the investigation of polymorphic markers ( including microsatellites ) within the tnf locus has resulted in a lot of studies which have shown the relationship between tnf haplotypes and pathogenesis of the disease ( 17 ) . to find the association of tnf- polymorphism and ms , goertsches et al . performed a research on 200 patients with ms ( 67 males and 133 females ) and 200 hcs in a spanish caucasian population .
all the patients had clinically definite ms according to poser and mcdonald criteria ( 14 , 19 , 20 ) . performing pcr according to the genetic analysis of ms in europeans ( games ) consortium ( http://www-gene.cimr.cam.ac.uk/msgenetics/games ) , tnf- polymorphism showed a significant correlation , which confirmed the association of this microsatellite marker and the etiology of this disease ( 20 ) .
as a part of the games collaborative project , godde et al . screened microsatellite markers in 198 germans with ms and 198 hcs to identify any susceptible region to ms . performing pcr and statically analysis ,
tnf- marker residing in the hla region on chromosome 6p21 showed the most significant relationship ( 12 ) . comparing gene frequencies of tnf- alleles in a french population of 74 patients with ms and 75 hcs , lucotte et al . showed a strongly significant positive association between tnf-*11 allele and ms ( 15 ) .
our results were consistence with those of a study on europeans patients with ms in the huge games project using 6000 microsatellite markers .
the games collaborative screened 19 case - control cohorts and 10 trio family - based cohorts ( 8 , 21 ) , as described in the original publications : australia ( 22 ) , belgium ( 23 ) , finland ( 24 ) , france ( 25 ) , germany 1 ( 6 ) , germany 2 ( 26 ) , hungary ( 27 ) , iceland ( 28 ) , ireland ( 29 ) , italy ( 30 ) , poland ( 31 ) , portugal 1 ( 32 ) , portugal 2 ( 33 ) , sardinia ( 34 ) , scandinavia ( 35 ) , spain ( 20 ) , turkey ( 36 ) , and uk ( 37 ) .
it was found that some special alleles of these markers were noticed significantly more usual in ms groups , as compared with the hc groups . in the games project altogether involving 13896 individuals , meta - analysis determined by the fisher 's method for the six markers was validated .
three non - mhc markers and three mhc markers ( including tnf- marker ) were identified to be potentially associated with ms ( 10 ) .
our findings also demonstrated that the tnf-*11 allele was more frequent in rrms , ppms and spms groups than the hcs , suggesting a possible genetic predisposition to ms in iranians patients with ms .
a large number of microsatellites from the human mhc region presented the polymorphism information content ( pic ) of around 0.75 . for the tnf- microsatellite locus , pic values around 0.85
have been observed ( 38 ) . in the iranian population ( current study ) ,
the findings indicated the association between tnf- microsatellite polymorphism in the hla region and the risk of developing ms in the native iranian population .
our findings also confirmed the relationship between tnf- microsatellite and predisposition to ms , as reported previously by the games project .
however , achieving the exact results need similar studies in other parts of iran as well as in other parts of asia .
 background : multiple sclerosis ( ms ) is an immune - mediated disease of polygenic etiology . 
 tumor necrosis factor- ( tnf- ) microsatellite as a proinflammatory cytokine is believed to play an important role in the etiology of this disease.objectives:the aim of this study was to investigate the association of tnf- microsatellite sequence variation in patients with ms and its risk factor in the southern iranian population.patients and methods : this polymorphism was investigated in an iranian population of 163 native southern people [ 81 patients with ms according to the poser criteria and 82 healthy controls ( hc ) with the same age , sex , social , ethnical and geographical features ( hormozgan and fars provinces ) ] . 
 all the controls were nonimmunological , neurological patients . 
 all the cases and controls were chosen randomly and genotyped for polymorphism of tnf- microsatellite.results:the frequencies of tnf-*11 ( 0.25 , p < 0.005 ) and tnf-*10 ( p < 0.005 ) alleles increased in patients with ms compared with controls , showing a significant difference among the studied population.conclusions:the current study adds evidence to the association of tnf- gene polymorphism and ms in this southern south iranian population which is consistent with the genetic analysis of ms in europeans ( games ) project reports and these two alleles reported in this study may be one of the genetic risk factor for ms . 
 furthermore , this data can be used to build the iranian gene bank for future studies . 