Vitiligo susceptibility is a complex genetic trait that may include genes involved in melanin biosynthesis, response to oxidative stress and regulation of autoimmunity. The importance of genetic factors for vitiligo susceptibility is evident by reports of its significant familial association 
. Our previous study suggests that 22% of Gujarat vitiligo patients exhibit positive family history and 14% patients have at least one first-degree relative affected 
. Autoimmunity has been suggested to play a major role in the pathogenesis of vitiligo. Destruction of melanocytes due to an autoimmune response in vitiligo can be either through cellular and/or humoral immune response 
. We have also shown that 66% of vitiligo patients possessed anti-melanocyte antibodies in their circulation as compared to control population 
. Recently, we have shown positive association of HLA-A*33:01, HLA-B*44:03, and HLA-DRB1*07:01 with vitiligo patients from North India and Gujarat suggesting an autoimmune link of vitiligo in these cohorts 
. The genotype-phenotype correlation of CTLA
-4 and IL
-4 gene polymorphisms also supported the autoimmune pathogenesis of vitiligo in Gujarat population 
, whereas our earlier studies on MBL
-2, ACE, PTPN
22 polymorphisms did not show significant association 
Cytokines are important mediators of immunity and there is now convincing evidence that cytokines also have an important role in the pathogenesis of autoimmunity 
. The cytokines mRNA and protein levels depend on both genetic and environmental factors. Analysis of cytokine gene polymorphisms would be able to detect genetic abnormality of cytokine regulation and hence establishment of genotype-phenotype correlation may be important in unraveling the disease pathogenesis. The promoter polymorphisms of TNF
-α are reported to be involved in modulating expression of TNF
-α gene which may be responsible for melanocyte death.
TNF-α, is an important multifunctional cytokine secreted by macrophages, T-lymphocytes, fibroblasts and keratinocytes with wide-ranging biological effects of protection from infection, surveillance against tumors and stimulation of inflammatory responses. In the epidermis, the epidermal melanin unit consists of the close interaction of a melanocyte and an associated pool of keratinocytes. Close relationship between these two cell types is important for melanocyte survival and differentiation mainly as keratinocyte-derived cytokines act on melanocytes via specific receptors 
. Keratinocytes synthesize cytokines, such as TNF-α, IL-1a, IL-6, and transforming growth factor-b (TGF-b), which are paracrine inhibitors of human melanocyte proliferation and melanogenesis 
. However, primary role of TNF-α is in the regulation of immune cells and its overproduction has been implicated in a variety of human diseases including autoimmune disorders and cancer 
. In vitro
, direct analysis of skin T cells from margins of vitiliginous skin show that polarized type-1 T cells (CD4+ and particularly CD8+), which predominantly secrete interferon (IFN)-γ and TNF-α are associated with the destruction of melanocytes during active vitiligo 
. In vitiligo affected skin, a significantly higher expression of TNF-α 
, IL-6 
, IFN-γ 
was detected compared with healthy controls and perilesional, non-lesional skin 
indicating that cytokine imbalance plays an important role in the depigmentation process of vitiligo.
It has been reported that cytokines such as IFN-γ and TNF-α can initiate apoptosis and thus lead to melanocyte death in the context of autoimmunity 
. In addition, IFN-γ and TNF-α induce the expression of intercellular adhesion molecule-1 (ICAM-1) on the cell-surface of melanocytes 
. The increased expression of ICAM-1 on the melanocytes enhances T cell/melanocyte attachment in the skin and thus may result in destruction of melanocytes in vitiligo 
. TNF-α also has the capacity to inhibit melanogenesis through an inhibitory effect on tyrosinase and tyrosinase related proteins 
Thus, it becomes pertinent to study all TNF-α promoter polymorphisms in adequate number of vitiligo patients and controls to elucidate the role of these polymorphisms in vitiligo susceptibility and to analyze the possible genotype - phenotype correlation. Here, we report that TNF-α −238, −308, −857, −863 and −1031 promoter polymorphisms are significantly associated with Gujarat vitiligo patients. Our results clearly suggest the important role of TNF-α in pathogenesis of vitiligo. Vitiligo patients showed significant increase in TNF-α transcript and protein levels as compared to controls suggesting that melanocyte death in patients could be triggered due to the increased TNF-α levels.
For the first time we report that generalized vitiligo has significantly higher TNF
-α transcript and protein levels as compared to localized vitiligo patients which indicate involvement of autoimmunity in precipitation of generalized vitiligo. Our results also indicate that active vitiligo patients have significantly higher TNF
-α transcript and protein levels as compared to the patients with stable vitiligo which signifies the role of TNF-α in disease progression. Our results also suggest that there are significantly higher transcript and protein levels of TNF-α in female patients as compared to male patients. Moreover, female patients have an early onset as compared to male patients suggestive of the fact that females have increased susceptibility towards vitiligo as compared to males, implicating gender biasness in the development of autoimmunity 
-α −308 G/A and −238 G/A polymorphisms were found to influence serum TNF-α levels in patients with sarcoidosis of Asian Indian population 
and our results are in line with this study. Interestingly, we found that the five promoter polymorphisms influence TNF
-α expression wherein TNF
-α −238, −308, −857 and −1031 were found to increase whereas −863 was found to decrease the expression. Furthermore, a genotype-phenotype study carried out on SLE patients showed increased TNF
-α transcript levels with −238 AA and GA genotypes as compared to GG genotypes 
. In particular, in the present study when combined effect of various genotypes was analyzed in the form of haplotypes, AATCC haplotype was found to be the highest risk combination observed for the disease. Intrestingly, it has all susceptible alleles except −863A which is reported to decrease the levels of TNF-α. The −863 C/A polymorphism was associated with serum TNF-α levels, carriers of the rare ‘A’ allele having a significantly lower TNF-α levels in Swedish population 
. The −863A allele was associated with 31% lower transcriptional activity in chloramphanicol acetyltransferase (CAT) reporter gene studies in human hepatoblastoma (HepG2) cells 
. Moreover, the haplotype analysis revealed the degree of susceptibility to the disease as predicted by the odds ratio with generalized vitiligo: AATCC >AATCT >AACCT >AACAT >AGCCT and AATCC >AATCT >AACCT >AACAT >AGCCC for localized vitiligo. Also, the age of onset analysis of the disease suggested the haplotypes involved in the early age of onset in patients with vitiligo are those involved in high degree of susceptibility of the disease: AATCC >AATCT >AACCT >AACAT >AGCCC >AGCCT.
LD analysis suggests that TNF-α −238 G/A & −308 G/A polymorphisms in moderate LD association as compared to the other investigated polymorphisms and are strongly associated with the disease risk in patients as suggested by the odds ratio. Moreover, the haplotype analysis showed the presence of haplotypes involving the susceptible alleles of TNF-α −238 and −308 polymorphisms, having increased levels of TNF-α in patients as compared to controls.
The region between −254 to −230 contains a regulatory sequence that acts as a TNF-
α repressor site 
. Previously, Bayley et al. 
showed that −238A allele increases the TNF
-α expression in transfected B cell line Raji and monocytic cell line U937 with a series of mutant constructs within the repressor region, including one where the –238G allele was replaced by a 10 bp linker sequence containing the –238A mutant allele. U937 and Raji cells containing the –238 minor A allele construct showed consistent increase (1.4 to 1.8-fold) in both basal and inducible promoter activity suggesting that the –238 SNP and the region surrounding it could be important in TNF
-α regulation and a mutation at position –238 could disrupt its regulation 
Kroeger et al. 
first showed that −308A allelic form gave a two-fold higher level of transcription than the −308G form in PMA-stimulated Jurkat and U937 cells using a luciferase reporter gene assay suggesting that the −308 G/A polymorphism plays an important role in the altered TNF
-α gene expression. Furthermore, Wilson et al. 
also showed functional significance of −308G/A polymorphism by investigating its effects on TNF
-α transcription using reporter gene assays suggesting that the −308 minor A allele is a much more powerful transcriptional activator than the common allele in a human B cell line.
The study of TNF
-α −308 G/A polymorphism in Iranian population have revealed significant association of −308A allele with vitiligo patients 
and these results are in line with our study however, a previous study of Turkish population suggested that TNF-α
−308 G/A polymorphism has no significant influence on vitiligo susceptibility 
. These contradictory reports may be because of the differences in ethnicity of the studied populations. However, both the studies involved less sample size and hence the association results needed further confirmation. Furthermore, there are no reports available on the effect of these TNF
-α promoter polymorphisms on its expression in vitiligo patients and the present study revealed the significant role of these promoter polymorphisms on the levels of TNF-α which might be playing a central role in vitiligo pathogenesis.
It has been known that the ROS microenvironment decides the fate of a cell for TNF-α mediated apoptosis 
. Our earlier reports with other studies suggest that the high oxidative environment prevails in vitiligo patients for the melanocyte destruction [51
. The destruction of melanocytes might be due to the increased secretion of TNF-α which further increases ROS and thus may lead to an early/defective apoptosis of the melanocytes via TNF-α mediated pathway. The possiblity of the TNF-α secretion is very high since the keratinocytes (a source of TNF-α) surround these melanocytes forming a melanin epidermal unit and thus affect its proliferation and melanogenesis process.
Disturbances in TNF-α metabolism have been well documented and found to be associated with several other autoimmune and infectious diseases such as rheumatoid arthritis 
, systemic lupus erythematosus 
, crohn’s disease 
, cerebral malaria 
and lesihmaniasis 
. Previously North Indian and Caucasian studies revealed strong association of −308 G/A polymorphism with T1DM 
. A study with psoriatic arthritis patients in Caucasian population for the five promoter polymorphisms suggested significant association of −238 G/A polymorphism with patients being −238 (A) variant, a significant risk factor for the disease 
. The TNF
-α −308 G/A polymorphism was significantly associated with susceptibility to asthma in patients of South Iran and with susceptibility to inflammatory bowel disease in European population 
. A metaanalysis study suggested that TNF
-α −238G/A and −308G/A polymorphisms might be used as biomarkers for psoriasis risk prediction 
. Furthermore, a study involving 22 SNPs in Caucasian patients with Graves’ disease (GD) showed significant association of TNF
-α −238G/A and −308G/A polymorphisms 
Simon and Burgor-Vargas 
described a patient with ankylosing spondylitis (AS) and vitiligo who was treated with infliximab (a chimeric monoclonal anti-TNF antibody), which resulted in gradual fading of vitiligo lesions suggesting that TNF-α
was involved in the pathogenesis of vitiligo. Alghamdi et al. 
also showed the effect of anti TNF-α agents: infliximab, etanercept, and adalimumab in generalized vitiligo patients. The patients did not develop any new depigmented patches during treatment or at the six-month follow-up. These reports signify the involvement of TNF-α in vitiligo pathogenesis.
In conclusion, our findings suggest that the increased TNF-α levels in vitiligo patients could result, at least in part, from variations at the genetic level. For the first time, we show that the promoter polymorphisms of the TNF-α gene influence the expression both at transcriptional as well as translational levels in vitiligo. The study also emphasizes the influence of TNF-α on the disease progression, onset of the disease and gender biasness for developing vitiligo. More detailed studies regarding role of TNF-α in precipitation of vitiligo and the development of effective anti-TNF-α agents may prove to be useful as preventive/ameliorative therapies.