it is a chronic inflammatory disorder affecting both the hair follicles and the nail apparatus , with t - lymphocyte infiltration and chemokine expression being characteristic in involved tissue ( 1 ) .
the prevalence rates of aa is 0.1% in the world population ( 2 ) and 0.9 - 6.9% in korea ( 3 ) .
the association of aa with autoimmune diseases including thyroid disorders , pernicious anemia and vitiligo is well established and alopecia areata itself is conventionally regarded as an autoimmune disease ( 4 ) .
the importance of immune - mediated mechanism , but non necessarily autoimmunity , in the pathogenesis of alopecia areata is suggested by the therapeutic efficacy of immunosuppressive drugs such as cyclosporin a , tacrolimus and corticosteroids . it is well known that locally infiltrating lymphocytes
have been implicated as effector cells being responsible for the reversible alteration of the hair follicles and the resulting hair loss ( 5 , 6 ) . in general ,
the directional recruitment of leukocytes is regulated by chemokines and their counteracting chemokine receptors ( 7 ) .
recently , in human models of aa it was shown that the increased expression of chemokines including monocyte chemoattractant protein-1 ( mcp-1 ) , monokine induced by interferon- ( mig ) might play a pivotal role in the attraction of monocytes around the hair bulb ( 1 ) .
mcp-1 is a -chemokine that is thought to be responsible for monocyte and lymphocytes t recruitment in acute inflammatory conditions and may be an important mediator in chronic inflammation .
in fact , it has been proposed that mcp-1 may be responsible for tissue inflammation in autoimmune diseases because its tissue expression in human and experimental autoimmune models ( 8 - 11 ) .
thus , genetic polymorphism in the regulatory regions of the mcp-1 gene could be implicated in the susceptibility or progression of autoimmune disease .
a biallelic a / g polymorphism at position -2518 of the mcp-1 gene has been described .
the polymorphism proved functionally important , as peripheral blood mononuclear cells of individuals with g / g and a / g genotype produced significantly more mcp-1 after stimulation with il-1 than those with caucasian wild - type a / a ( 12 , 13 ) .
an association of the presence of g at position -2518 with the presence of cutaneous vasculitis could be shown in patients with systemic lupus erythematosus ( 14 ) as well as an association with development of coronary artery aneurysms after acute kawasaki disease ( 13 ) .
furthermore , the g / g genotype was identified as a genetic risk factor for severe coronary artery disease ( 15 ) and a correlation between the incidence and severity of asthma and the g allele at position -2518 has been shown .
these findings suggest an important role for the mcp-1 and the a / g polymorphism in its regulatory region in inflammatory processes .
the mcp-1 gene polymorphism in aa patients has not been reported yet . in this study
, we performed a case - control study in 145 alopecia areata patients and 246 matched korean controls to analyze the possible influence of the polymorphism ( a / g ) at position -2518 of the mcp-1 gene in the susceptibility of aa .
a total of 145 korean patients with aa ( 70 men and 75 women ) were examined at the dermatology clinic at the kyung hee university medical center .
a total of 246 healthy subjects ( 80 men and 166 women ) , who had no clinical evidence of aa , were recruited as controls ( table 1 ) .
this study was approved by the ethics review committee of the medical research institute , kyung hee university medical center .
the clinical diagnosis of aa was based on the presence of initially patchy alopecia with exclamation mark hairs and the exclusion of other causes of alopecia .
detailed clinical information was obtained from each patient , including age at onset of first episode , family history , type of disease ( patchy alopecia , alopecia totalis [ at ] and alopecia universalis [ au ] ) , duration of disease , presence of other autoimmune disease , nail involvement , and other body sites hair loss .
subgroup 1 contained patients with patchy disease ( patchy aa ) and subgroup 2 patients with more extensive hair loss ( at and au ) ( 16 ) .
patchy aa can involve the scalp or other body sites such as the beard area . in patchy aa ( subgroup 1 )
there are well demarcated , mainly circular areas of hair loss typically 2 - 6 cm in diameter . in subgroup 2
, hair loss progresses until all the hair on the scalp has been lost ( at ) ; or to complete loss of the entire body hair ( au ) .
we also divided the aa patients into two subgroups on the basis of age at onset of disease ( 17 ) .
the division of these subgroups with a boundary at age 30 yr was in keeping with the distribution of age at onset in our patients , which was bimodal with peaks at approximately age 20 - 30 and 30 - 40 yr .
genomic dna was prepared from heparinized venous blood samples using a nucleospin dna isolation kit ( mache - rey - nagel gmbh & co. , duren , germany ) .
the identification of the polymorphism was carried out using pcr , followed by a restriction fragment length polymorphism ( rflp ) assay , using a pvuii site , which is introduced by the presence of the g nucleotide .
the regulatory region of the mcp-1 gene ( from -2746 at -1817 ) was amplified by polymerase chain - reaction ( pcr ) using the forward primer 5'ccgagatgttcccagcacag3'and the reverse primer 5'ctgctttgcttgtgcctctt3 ' .
pcr was performed using buffer 10 ( 10 mm tris - hcl ph 9 , 2.0 mm mgcl2 , 50 mm kcl ) , 200 m dntps , 2.5 pmoles of each primer , 0.5 l of dna , 0.5 u taq polymerase ( pharmacia ) and ddh2o up to a final volume of 10 l .
the following thermal profiles were run : 94 for 1 min , 55 for 1 min , and 72 for 1.5 min . after a final extension of 10 min at 72 ,
7 l of the pcr products were resolved in 2% agarose gels stained with ethidium bromide previous dilution in blue juice buffer to check the expected 930-bp band .
after checking , 8 l of the pcr products were digested with 10 u of pvuii in 10 buffer and h2o up to a final volume of 20 l at 37 for 2 hr 30 min .
the resulting products were separated by gel - electrophoresis in 1.5% agarose gels , containing ethidium bromide in a final concentration of 0.5 g / ml . samples showing only a 930 bp band were assigned as a / a , samples showing two bands of 708 and 222 bp were considered g / g and samples showing three bands at 930 , 708 and 222 bp were typed a / g .
the distribution of the mcp-1 a / g genotypes for patients and control subjects were compared by the chi - square (  ) test .
a total of 145 korean patients with aa ( 70 men and 75 women ) were examined at the dermatology clinic at the kyung hee university medical center .
a total of 246 healthy subjects ( 80 men and 166 women ) , who had no clinical evidence of aa , were recruited as controls ( table 1 ) .
this study was approved by the ethics review committee of the medical research institute , kyung hee university medical center .
the clinical diagnosis of aa was based on the presence of initially patchy alopecia with exclamation mark hairs and the exclusion of other causes of alopecia .
detailed clinical information was obtained from each patient , including age at onset of first episode , family history , type of disease ( patchy alopecia , alopecia totalis [ at ] and alopecia universalis [ au ] ) , duration of disease , presence of other autoimmune disease , nail involvement , and other body sites hair loss .
subgroup 1 contained patients with patchy disease ( patchy aa ) and subgroup 2 patients with more extensive hair loss ( at and au ) ( 16 ) .
patchy aa can involve the scalp or other body sites such as the beard area . in patchy aa ( subgroup 1 )
there are well demarcated , mainly circular areas of hair loss typically 2 - 6 cm in diameter . in subgroup 2
, hair loss progresses until all the hair on the scalp has been lost ( at ) ; or to complete loss of the entire body hair ( au ) .
we also divided the aa patients into two subgroups on the basis of age at onset of disease ( 17 ) .
the division of these subgroups with a boundary at age 30 yr was in keeping with the distribution of age at onset in our patients , which was bimodal with peaks at approximately age 20 - 30 and 30 - 40 yr .
genomic dna was prepared from heparinized venous blood samples using a nucleospin dna isolation kit ( mache - rey - nagel gmbh & co. , duren , germany ) .
the identification of the polymorphism was carried out using pcr , followed by a restriction fragment length polymorphism ( rflp ) assay , using a pvuii site , which is introduced by the presence of the g nucleotide .
the regulatory region of the mcp-1 gene ( from -2746 at -1817 ) was amplified by polymerase chain - reaction ( pcr ) using the forward primer 5'ccgagatgttcccagcacag3'and the reverse primer 5'ctgctttgcttgtgcctctt3 ' .
pcr was performed using buffer 10 ( 10 mm tris - hcl ph 9 , 2.0 mm mgcl2 , 50 mm kcl ) , 200 m dntps , 2.5 pmoles of each primer , 0.5 l of dna , 0.5 u taq polymerase ( pharmacia ) and ddh2o up to a final volume of 10 l .
the following thermal profiles were run : 94 for 1 min , 55 for 1 min , and 72 for 1.5 min . after a final extension of 10 min at 72 ,
7 l of the pcr products were resolved in 2% agarose gels stained with ethidium bromide previous dilution in blue juice buffer to check the expected 930-bp band . after checking ,
8 l of the pcr products were digested with 10 u of pvuii in 10 buffer and h2o up to a final volume of 20 l at 37 for 2 hr 30 min .
the resulting products were separated by gel - electrophoresis in 1.5% agarose gels , containing ethidium bromide in a final concentration of 0.5 g / ml . samples showing only a 930 bp band were assigned as a / a , samples showing two bands of 708 and 222 bp were considered g / g and samples showing three bands at 930 , 708 and 222 bp were typed a / g .
the distribution of the mcp-1 a / g genotypes for patients and control subjects were compared by the chi - square (  ) test .
in the case - control study , genotype distributions and allele frequencies of the mcp genetic marker in both aa and the control population was analyzed ( table 2 ) . in the aa patients
the frequency of the a and g alleles was 40.3 and 59.7% , respectively and the distribution of the a / a , a / g and g / g genotypes was 19.3 , 42.1 and 38.6% , respectively . amongst the controls the frequency of the a and g alleles was 39.8 and 60.2% , and the distribution of the a / a , a / g , g / g genotypes in the same group was 17.5 , 44.7 and 37.8% , respectively . the observed genotype frequencies of the aa patient ( p=0.129 ) and the control population ( p=0.574 ) did not show significant difference predicted by the hardy - weinberg equation .
when the observed control and patient genotype frequencies were compared with expected values using 32 contingency table in a standard chi - square test ( table 2 ) , there was no significant difference ( p=0.848 ) . also , there was no significant difference in the allele frequencies between the patients and the controls ( p=0.889 ) .
we then divided the patients into two subgroups ( subgroup 1 [ patchy aa ] and subgroup 2 [ alopecia totalis and alopecia universalis ] ) according to disease severity and also into two further subgroups on age at onset of disease ( subgroup a [ onset age 30 ] and subgroup b [ onset age > 30 ] ) .
comparison of the genotype distributions and the allele frequencies between each subgroup and the control population revealed no significant difference ( table 3a , b ) .
although mcp-1 has been proposed as the main chemokine responsible for initiating autoimmune tissue damage ( 8 , 11 , 18 , 19 ) , the association of polymorphisms in the regulatory regions of the mcp-1 gene with alopecia areata has not been studied until now .
recently , in human models of aa it was shown that the increased expression of mcp-1 might play a pivotal role in the attraction of monocytes around the hair bulb ( 1 ) .
in addition , the mcp-1 -2518 g allele was found to increase the mcp-1 expression ( 12 ) .
therefore , we initially hypothesized that the development of aa might have been related to g allele in the mcp-1 gene polymorphism in aa patients .
the results of this study , however , did not support this assumption . in this case - control study ,
the genotype distribution and allelic frequencies showed no significant difference between the patients population and the controls ( p=0.848 , p=0.889 , respectively ) .
there are several potential reasons why there was no association between the -2518a / g polymorphism and alopecia areata .
it is noteworthy that there are considerable differences in the frequencies of these polymorphisms among the different races .
indeed , it is well known that ethnicity and population structure may strongly influence the role of genetic risk factors in chronic inflammatory diseases including various autoimmune diseases .
the distribution of several gene variations may be greatly different between various countries , as well as among different area in the same countries .
the dominance of the g - type allele in koreans , which were observed in this present study , is contrasting to what has been reported from caucasians and afroamericans , where the proportion of g allele was 29 and 22% , respectively ( 12 ) .
such reversed ratio was also observed in japanese ( 13 ) , suggesting that the genotypic profile of mcp-1 -2518 polymorphism varies greatly among ethnic groups . and
previously , it was shown that allelic frequency of -2518 polymorphism among patients with other autoimmune diseases with increased serum mcp-1 level , such as rheumatoid arthritis , systemic lupus erythematosus and adult - onset still 's disease , is also similar to that of normal controls ( 20 ) .
in addition , in study about the chemokine expression pattern of aa , benoit et al . have demonstrated that mcp-1 expression does not appear to be a particular feature of aa due to showing mcp-1 expression by keratinocytes of the inner root sheath but only weak expression around the hair follicles .
and chemokine expression patterns did not appear to correlate with clinical severity such as extent of hair loss , number of aa episodes , or presence of associated diseases ( 1 ) .
these findings argue against a role of mcp-1 -2518a / g polymorphism for susceptibility to alopecia areata . in conclusion , this study found no association of -2518a / g polymorphism in the distal regulatory region of the mcp-1 with alopecia areata susceptibility or severity in a korean population . however , it is noteworthy that there are considerable differences in the frequencies of these polymorphisms among the different races .
this variation may contribute to the pathogenesis of alopecia areata and must be considered in gene - association studies in different ethnic populations .
 monocyte chemoattractant protein-1 ( mcp-1 ) levels are increased in scalp lesions of patients with alopecia areata ( aa ) , suggesting a role in the development of aa . 
 recently , a biallelic a / g polymorphism in the mcp-1 promoter at position -2518 has been found , influencing the level of mcp-1 expression in response to an inflammatory stimulus . 
 we investigated whether the presence of these polymorphisms were associated with aa in korean population . 
 145 korean patients with aa , 246 healthy subjects without clinical evidence of aa were screened for genotype with a pcr - based assay . in the aa patients 
 the frequency of the a and g alleles was 40.3 and 59.7% , respectively and the distribution of the a / a , a / g and g / g genotypes was 19.3 , 42.1 and 38.6% , respectively . amongst the controls the frequency of the a and g alleles was 39.8 and 60.2% , and the distribution of the a / a , a / g , g / g genotypes in the same group was 17.5 , 44.7 and 37.8% , respectively . 
 there was no significant difference in the allele frequencies and genotype distributions between the patients and the controls ( p=0.889 , p=0.848 , respectively ) . 
 our data indicates that no association exists between the -2518a / g polymorphism of the mcp-1 gene and susceptibility to alopecia areata . 
INTRODUCTION
MATERIALS AND METHODS
Patients and controls
MCP-1 genotyping
Statistical analysis
RESULTS
DISCUSSION