research into the pathogenesis of vitiligo has shown a complex of genetic and environmental factors .
endothelin-1 ( edn1 ) and edn receptor type a ( ednra ) are implicated in the inflammatory process .
vitiligo is an acquired cutaneous disorder with a 0.52% incidence worldwide , and is characterized by a loss of melanocytes from epidermis .
although the etiopathogenesis has not been clearly elucidated yet , it was suggested that this common skin disease is a consequence of genetic and environmental factors . in the etiopathogenesis of the disease
several mechanisms including autoimmune , autotoxicity , and neural mechanisms induced by biochemical and neurochemical mediators have been proposed .
previously , keratinocyte - derived endothelin-1 ( edn1 ) has been shown to control melanocyte growth and function .
it is a potent stimulant on melanocyte proliferation , melanogenesis , and migration ; implying a link with vitiligo .
polymorphisms of edn1 and ednra genes have been investigated in autoimmune diseases such as hashimoto 's thyroiditis ( ht ) , graves disease , scleroderma , primary biliary cirrhosis , and psoriasis as well .
there are only two studies in the literature examining the relationship between edn1 gene polymorphisms and vitiligo with controversial results .
kim et al . have found that some haplotypes ( gt and ag haplotypes of intron 4 g / a and 5665 g / t polymorphic loci ) of edn1 gene are associated with vitiligo , whereas lan et al .
therefore , the aim of this study was to investigate both edn1 gene polymorphisms ( g5665 t [ or lys198asn ] and t-1370 g ) and ednra gene polymorphisms ( c + 70 g and g-231a ) , in the etiopathogenesis of vitiligo .
in addition , genotypes and clinical types of vitiligo found in these patients were also evaluated .
all the patients and controls were recruited from a single center and all provided written informed consent . a total of 100 patients with vitiligo ( 53 women/47 men )
vitiligo patients older than 18 years , showing any clinical picture except segmentary type and who were not on systemic or topical therapy during previous 2 months were included in the study .
the control group consisted of 185 , age and sex - matched , dermatology outpatients ( 95 women/90 men ) , with no past history of any systemic , infectious , autoimmune , genetic , or atopic disease .
peripheral venous blood samples were taken in the morning subsequent to an overnight ( 12 h ) fast in edta - k3 tubes for genotype analysis .
genomic dna was isolated from peripheral blood leukocytes by using high pure polymerase chain reaction template preparation kit ( roche diagnostics gmbh , mannheim , germany ) .
we examined the g5665 t and t-1370 g polymorphisms in edn1 gene as well as c + 70 g and g-231a polymorphisms in ednra gene in our dna samples .
these polymorphisms were selected according to the following criteria : previous association for susceptibility to other diseases , and adequate frequency in caucasian populations to perform the evaluation . for detection of the mentioned polymorphisms ,
the detection of polymorphisms was performed in a lightcycler ( roche diagnostics , mannheim , germany ) .
snp in the edn1 and ednra genes all statistical analyses were performed with spss 15.0 for windows ( ibm spss statistics , chicago , il , usa ) .
differences in genotype distributions and allele frequencies in the cases and the controls were compared using the chi - square test .
bonferroni correction is an adjustment made to p values when several dependent or independent statistical tests are being performed .
the statistical significance for deviations from hardy  weinberg equilibrium ( hwe ) was determined using the pearson chi - square test .
odds ratios ( ors ) were calculated and given with 95% confidence intervals ( cis ) .
linkage disequilibrium ( ld ) and haplotype frequencies were estimated using the haploview 4.2 software ( haploview , mit / harvard broad institute , ma , usa ) and compared between cases and controls using a contingency chi - square test .
in addition , the ncss 2000 statistical software ( ncss , lcc , kaysville , utah , usa ) was used to evaluate the power analysis .
a total of 285 subjects ( 100 vitiligo and 185 controls ) were included in this case control study .
table 2 depicts the clinical characteristics of the vitiligo patients including the clinical type of disease , duration , family history , leukotrichia , stability within 1 year , and associated diseases .
the mean ages were 38.6  12.0 years ( range 1876 ) and 38.2  9.7 years ( range 1872 years ) for patients and controls , respectively .
there was no significant difference between the study and control groups in terms of mean age and sex distribution .
we had 87% power to detect an effect size ( w ) of 0.20 using a 2 degrees of freedom (  = 0.05 ) .
the genotypic and allelic distributions of edn1 ( g5665 t and t-1370 g ) and ednra ( c + 70 g and g-231a ) polymorphisms for patients and controls are shown in table 3 .
the allelic frequencies of the studied polymorphisms found in our control population were similar to the results in turkish , english , and german populations .
however , when the relationship between these polymorphisms and clinical characteristics of patients were evaluated , we found that ednra + 70 variant g allele and genotypes containing g ( cg , gg and cg + gg ) showed protective effects from presence of associated disease in patients with vitiligo [ table 4 ] .
when the bonferroni correction was performed , the significance still existed . in our vitiligo group , we designated a large spectrum of autoimmune diseases as follows : ht , type 1 diabetes mellitus , rheumatoid arthritis , alopecia areata , idiopathic thrombocytopenic purpura , and multiple sclerosis .
in addition , g allele seems to protect against generalized type of vitiligo when compared to focal type ( or : 0.42 , 95% ci = 0.210.86 , pcorr = 0.03 [ table 5 ] ) .
in addition , frequency of gg genotype was marginally lower in generalized type of vitiligo when compared to focal type ( or : 0.18 , pcorr = 0.057 [ table 5 ] ) . however , there is no significant difference in the distribution of this allele between generalized and acrofacial types [ table 5 ] .
edn1 - 1370 gg genotype was 5-fold more in vitiligo patients with leukotrichia in comparison with healthy controls which may be seen in table 6 .
characteristics of the patients with vitiligo distribution of genotypes and allele frequencies for patients with vitiligo and control group distribution of ednra c + 70 g genotypes and allele frequencies for patients with vitiligo : impact of presence of associated disease distribution of ednra c + 70 g genotypes and allele frequencies for patients with focal , acrofacial and generalized type of vitiligo distribution of genotypes and allele frequencies for vitiligo patients with leukotrichia and control group levontin 's standardized disequilibrium coefficient ( d ) was calculated as a measure for ld between the studied polymorphisms in the edn1 and ednra genes [ figure 1 ] .
edn1 5665 and -1370 were found to be in strong ld ( d = 0.73 , r = 0.405 ) .
in addition , a weak ld between ednra  231 and + 70 was found ( d = 0.22 , r = 0.032 ) .
the most frequent haplotype among the patients and controls were edn1 5665/-1370 gt and ednra  231/+70 gg .
the edn1 5665/-1330 tt haplotype was significantly overrepresented in controls than in patients ( p = 0.04 ) .
there was strong linkage disequilibrium between endothelin-1 5665/-1370 ( d = 0.73 , r = 0.405 ) ; and weak linkage disequilibrium between endothelin receptor type a  231/+70 ( d = 0.22 , r = 0.032 ) haplotype analysis of the edn1 and ednra polymorphisms in patients with vitiligo and control subjects
edn1 is a well - known peptide for its role in epidermal hyperpigmentation by signaling mechanisms of mitogenesis and melanogenesis .
previously , the keratinocyte - derived edn1 has been shown to be decreased in vitiligo .
furthermore , edn1 was found to be increased in patients undergoing puva treatment indicating a role of this peptide in vitiligo pathogenesis . besides , edn1 is a strong vasoconstrictive agent which may contribute to controlling the inflammatory process seen in vitiligo . indeed , blocking the edn1/ednra signaling resulted in the loss of pigmentation in vivo .
however , the exact role of edn1 in vitiligo has not been clearly elucidated yet .
edn1 gene polymorphisms have been investigated and incriminated in the etiopathogenesis of various diseases before .
the mutant t allele of a g5665 t polymorphism in edn1 gene was found as a risk factor for some diseases and responsible for disease progression in others .
some studies were shown that the presence of t allele was associated with higher edn1 levels , raised systolic blood pressure , and predisposed patients to preeclampsia , suggesting the functional importance of this polymorphism in pregnancy .
confirmed the impact of the g5665 t polymorphism on vascular reactivity . in the present study ,
the lack of association between g5665 t with ht is also noteworthy since ht is a close counterpart of vitiligo .
two former studies investigating the role of g5665 t polymorphism in vitiligo did not show a significant relationship as well .
considering the potential regulatory effects of promoter region polymorphisms on gene expression and plasma protein levels , we also studied t-1370 g polymorphism which is located in the promoter region of edn1 gene .
recently , it has been shown that the edn1 mrna and the secreted edn1 levels in primary osteosarcoma hcell culture obtained from tt homozygotes were significantly higher than those from gg homozygotes .
interestingly , the gg genotype frequency of t-1370 g polymorphism , related with the low production of edn1 , was increased 5-fold in vitiligo patients with leukotrichia in comparison to controls .
however , this subset of patients is relatively therapy resistant , and this information may be valuable in determining therapy options . however , in the present study we did not detect a relationship between this polymorphism and vitiligo in general .
lack of association between g5665 t and t-1370 g polymorphisms and vitiligo indicates that these polymorphisms alone may not play a major role in the pathogenesis of this autoimmune disease .
on the other hand , our haplotype analysis revealed that there was strong ld between g5665 t and t-1370 g polymorphisms .
the edn1 5665/-1370 tt haplotype was significantly less presented in vitiligo patients in comparison with controls .
t alleles in both 5665 and -1370 are known to be related with the high production of edn1 protein which causes melanocyte proliferation and melanogenesis .
actually , many studies have shown that vitiligo skin expresses low levels of edn1 . consequently , our haplotype results are in line with what is expected ; namely , high levels of edn1 means increased amount of melanin protection against vitiligo .
lan et al . and kim et al . also did haplotype analyses between g5665 t and two different intronic polymorphisms in edn1 gene ( intron 2 a / g and intron 4 g / a , respectively ) .
lan et al . could not find a significant difference between cases and controls in haplotype frequencies of g5665 t and intron 2 a / g .
however , kim et al . reported that gt and ag haplotype frequencies at intron 4g / a and g5665 t loci of edn1 gene in focal and segmental clinical types differed significantly from healthy controls .
these findings suggest that edn1 polymorphisms may not be a direct risk factor alone , but their presence , especially with g5665 t may contribute as additional / cumulative factors in the development of vitiligo .
regarding polymorphisms of ednra gene , the functional consequence of ednra g-231a and c + 70 g polymorphisms are unknown , since the functional studies are not yet available .
the c + 70 g and g-231a polymorphisms of edra gene are within the 3-untranslated region ( utr ) and the 5- utr of the gene , respectively .
3- utrs of eukaryotic mrnas are usually implicated in the posttranslational regulation and have a pivotal role in message stability .
the functional consequences of 5- utr polymorphisms might be related to the regulatory sequences of gene transcriptions and associated with altered gene expression .
in addition , such polymorphisms may create novel splice sites influencing the function of the receptor and receptor - ligand interactions .
therefore , these polymorphisms could have significant effects on mrna expression and message stability , and thus affecting the total amount and activity of the functional receptor . on the other hand
our study showed the variant g allele of ednra + 70 polymorphism was protective against the generalized type of vitiligo .
it was previously demonstrated that that carrying variant allele protects also from early onset disease of ht .
we think it may be possible that + 70 wild c allele carrying receptors bind their ligands with decreased affinity , resulting in at least weakening of signals at the membrane level , or slowing internalization of signal in the melanocytes with subsequent ceasing of melanocyte proliferation or melanocyte destruction . in another study , examining the differences of edn1 and ednra expressions , aly et al .
however , in that study the patients were few in numbers ( n = 10 ) and we think a larger number of cases and advanced methods of evaluation could show different results .
altogether , it can be concluded that ednra does not contribute to vitiligo process , rather its + 70 g polymorphism is protective against both generalized type of vitiligo and associated diseases .
although most vitiligo patients are otherwise healthy , vitiligo may be associated with a large spectrum of autoimmune diseases .
this is one of the few studies examining the edn1 and ednra polymorphisms in vitiligo .
our results suggest that edn1 ( g5665 t and t-1370 g ) and ednra ( c+70 g and g-231a ) polymorphisms may not have a causative effect in vitiligo .
furthermore , ednra + 70 polymorphism which is protective against generalized type of vitiligo and associated diseases , may indicate that there should be other causes , yet unknown , disrupting the normal function of keratinocyte - melanocyte interaction , in the course of vitiligo .
the studied endothelin.1 ( edn1 ) ( g5665 t and t.1370 g ) and edn receptor type a ( c+70 g and g-231a ) polymorphisms may not have a causative effect in vitiligo .
in fact , haplotype analyses denoting protective effects against generalized vitiligo and associated diseases suggest that there should be other causes disrupting melanogenesis in the course of vitiligo .
the studied endothelin.1 ( edn1 ) ( g5665 t and t.1370 g ) and edn receptor type a ( c+70 g and g-231a ) polymorphisms may not have a causative effect in vitiligo .
in fact , haplotype analyses denoting protective effects against generalized vitiligo and associated diseases suggest that there should be other causes disrupting melanogenesis in the course of vitiligo .
there are no conflicts of interest . the studied endothelin.1 ( edn1 ) ( g5665 t and t.1370 g ) and edn receptor type a ( c+70 g and g-231a ) polymorphisms may not have a causative effect in vitiligo .
in fact , haplotype analyses denoting protective effects against generalized vitiligo and associated diseases suggest that there should be other causes disrupting melanogenesis in the course of vitiligo .
 background : endothelin-1 ( edn1 ) and edn receptor type a ( ednra ) are implicated in melanocyte functions.aim and objectives : this study examines the role of edn1 ( g5665 t and t-1370 g ) and ednra ( c + 70 g and g-231a ) polymorphisms as a risk factor for vitiligo , and evaluates the relationship between genotypes and clinical characteristics of vitiligo patients.materials and methods : we analyzed genotype / allele distributions of edn1 and ednra polymorphisms in 100 patients with vitiligo and 185 healthy controls by real - time polymerase chain reaction.results:there was no notable risk for vitiligo afflicted by studied polymorphisms . 
 however , the presence of ednra + 70 variant g allele was found to be related with decreased risk for development of generalized type of vitiligo ( odds ratio [ or ] : 0.42 , 95% confidence interval [ ci ] = 0.210.86 , pcorr = 0.03 ) and showed protective effect against associated diseases seen in vitiligo ( or : 0.49 , 95% ci = 0.270.88 , pcorr = 0.034 ) . 
 haplotype analysis demonstrated a strong ( disequilibrium coefficient = 0.73 , r2 = 0.405 ) linkage disequilibrium between edn1 g5665 t and t-1370 g polymorphisms . 
 the edn1 5665/-1330 tt haplotype was over represented significantly in controls than in patients ( p = 0.04).conclusion : the studied polymorphisms do not seem to be a major risk for vitiligo . 
 haplotype analysis denoting protective effects against vitiligo may indicate an indirect interaction in the course of vitiligo . 
 in addition , ednra + 70 polymorphism is protective against generalized type of vitiligo and associated diseases . 