Vitiligo is an acquired chronic depigmentary disorder with a highly variable course. It is characterized by the appearance of white patches resulting from the loss of functional melanocytes and melanin from the skin, hairs, and mucous membranes. It affects 0.1–2% of the population worldwide 1. The etiology of vitiligo is obscure. Several mechanisms of melanocyte destruction have been postulated including autoimmunity 2, autocytotoxic/metabolic mechanism 3,4, and impaired melanocyte migration and/or proliferation 5. Recently, studies have pointed to a significant role of genetic susceptibility to vitiligo 6. As none of these theories alone is sufficient to fully explain the pathogenesis of the disease and all of the proposed mechanisms are not mutually exclusive, the convergence theory has been formulated combining biochemical, environmental, and immunological events together with the genetic makeup 7.
Genome-wide association studies have identified some of the generalized vitiligo susceptibility genes that involve immune regulation and immune targeting of melanocytes. This has led to the conclusion that generalized vitiligo is a primary autoimmune disease, although the biological triggers of the autoimmune process remain unknown 6. Several candidate genes have been tested for genetic association with generalized vitiligo, although some have yielded marginal significance and several were not replicated by subsequent studies 8. These genes may be responsible for premature death of melanocytes or affecting melanocyte growth either directly or through paracrine factors, and the combined effect of genes controlling the autoimmune phenomenon could be involved 9.
Cytokines are important mediators of immunity. Imbalance or deficiency in the cytokine network may contribute toward the susceptibility or affect the severity of autoimmune disorders. Tumor necrosis factor α (TNFα) is a multifunctional proinflammatory cytokine 10. Circulating TNFα level and the associations between TNFα genetic polymorphisms are involved in many diseases. The reasoning behind the proposed involvement of TNF gene polymorphisms in diseases either/or disease manifestations is that they may influence in-vivo cytokine levels 11. It has been suggested that the death of melanocytes is mediated by apoptosis in the context of autoimmunity and cytokines such as TNFα. In the epidermis, melanocytes are in close interaction with keratinocytes. The keratinocytes synthesize cytokines such as TNFα, interleukin-1α, interleukin-6, and transforming growth factor-β, which are paracrine inhibitors of human melanocyte proliferation and melanogenesis 12. In addition, TNFα affects the apoptotic pathway of melanocytes and its level may play an important role in vitiligo pathogenesis as TNFα induces the expression of ICAM-1 on the surface of melanocytes 13 that enhances T cell/melanocyte attachment in the skin and may play a role in the destruction of melanocytes in vitiligo 14. TNFα also has the capacity to inhibit melanogenesis through an inhibitory effect on tyrosinase and tyrosinase-related proteins. Moreover, TNFα can inhibit melanocyte stem cell differentiation 12.
A number of polymorphisms within the TNFα gene have been described to have the ability to cause structural changes within the regulatory sites of the gene, which can subsequently influence the regulation of TNFα production. Exchange of guanine by adenine at nucleotide 308 of the TNFα promoter region is a gain-of-function mutation associated with increased production and high serum levels of soluble TNFα 11. The aim of the present work was to study the possible association between the TNFα –308 G/A promotor polymorphism and the susceptibility to nonsegmental vitiligo in a cohort of Egyptian women on the basis of previous reports of such findings in Iranian and Indian women 10,15.
Patients and methods
The study included 100 adult female patients with nonsegmental vitiligo recruited from July to December 2009 from the Dermatology Outpatient Clinic and the Phototherapy Unit, Kasr El-Ainy Teaching Hospital, Cairo University. All patients were subjected to a full assessment of history and a thorough clinical examination. Two hundred age-matched healthy female volunteers with no evidence of or a positive family history of vitiligo were included in our study as a control group. The research protocol was approved by the local ethics committee and informed consents were obtained from all participants.
Genotyping of the tumor necrosis factor α −308 (rs1800629) promotor polymorphism
For each participant, 3–4 ml venous blood was obtained under sterile conditions into an EDTA vacutainer. Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instruction. On the basis of the method described by Bel Hadj Jrad et al.16 for TNFα −308 genotyping by the PCR-RFLP assay, the following primer sets were used: forward primer 5′-AGGCAATAGGTTTTGAGGGCCAT-3′ and reverse primer 5′-TCCTCCCTGCTCCGATTCCG-3′ (Fermentas, Vilnius, Lithuania). All PCR reactions were performed in a total volume of 25 μl containing 12.5 μl Master Mix (Bioron GmbH, Ludwigshafen am Rhein, Germany), 1 μl of each forward and reverse primers (25 pmol), and 3 μl genomic DNA. The thermocycler program applied was initial heating at 95°C for 5 min, followed by 29 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s. A final extension step was carried out at 72°C for 10 min. Amplified products were digested with NcoI (Fermentas). The restricted fragments were visualized by 3% agarose gel electrophoresis stained with ethidium bromide under ultraviolet light. The presence of the NcoI restriction site in the polymorphic allele (A allele) was indicated by the cleavage of the 107 bp amplicon into two fragments of 87 and 20 bp (Fig. 1). For quality control, genotyping was repeated with respect to case/control status to confirm our results for 40 samples. The results of genotyping were interpreted blindly by two different observers, and were 100% concordant.
Data were analyzed using the SPSS statistical package (version 17; SPSS Inc., Chicago, Illinois, USA). For numerical data, parametric data were expressed as mean, SD, and range, whereas nonparametric data were expressed as median and interquartile range. Qualitative data were expressed as frequency and percentage. The χ2-test or Fisher exact test was used to examine the relation between qualitative variables. Nonparametric numerical data were analyzed using the Mann–Whitney U-test. Correlation analysis was carried out using Spearman’s rank correlation. Unconditional logistic regression analysis was used to calculate odds ratios and 95% confidence intervals for risk estimation. A P-value of less than 0.05 was considered significant.
Demographic data of the studied patients are presented in Table 1, whereas the results of TNFα −308 A/G genotyping are presented in Table 2. There are no units for the alleles (G allele and A allele). G and A alleles represent the frequency of the wild type (common allele) and the polymorphic alleles in the groups studied. The frequency of the alleles expressed in Table 2 is not presented as percentage. It is calculated by dividing the number of the alleles, whether wild or polymorphic, by the total number of alleles. For example, in the patient group, the frequency of the genotypes is as follows: GG 28%, GA 53%, and AA 19%. G allele=GG (28×2)+GA (53)/200 (genotypes=100; thus, the alleles=100×2)=56+53/200=109/200=0.545. The prevalence of the polymorphic genotypes was higher in vitiligo patients than in controls and conferred an almost three folds increased risk of vitiligo.
Statistical analysis of the influence of the studied genetic polymorphism on the demographic characteristics of the disease showed that there was no statistically significant difference between patients harboring the wild type or the polymorphic genotypes in their age, presenting symptoms, or clinical data (Table 3). Further analysis showed that there was no statistical difference between vitiligo patients with heteromutant or homomutant genotypes in terms of their clinical characteristics.
TNFα is produced by lymphocytes, keratinocytes, and macrophages and can inhibit melanocyte proliferation and melanogenesis. It can cause melanocyte death through apoptosis in the context of autoimmunity 17. Therefore, it is possible that individuals who naturally produce higher levels of TNFα will show different susceptibility or severity toward vitiligo 16. Several single nucleotide polymorphisms have been reported in the promotor region of the TNFα gene. Previous in-vitro studies have reported that a polymorphism at nucleotide 308 that involved substituting G for A, was associated with a higher rate of TNF gene transcription than the wild-type allele, G allele. Considering the role of TNFα in vitiligo pathogenesis, the diallelic polymorphisms within the gene for this cytokine might play a role in vitiligo induction or the clinical characteristics of the disease 18.
The rationale behind this study was the previous reports on the association of the TNFα −308 A allele (polymorphic allele) with vitiligo in Iranian and Indian women 10,15. It was suggested that estrogen may play an important role in the depigmentation process of vitiligo as initiation or progression of the disease is reported in pregnancy, during the postpartum period, or after the use of oral contraceptive pills. On the basis of this suggestion, women in the child-bearing period can be considered more prone to vitiligo and by considering the role of TNFα in vitiligo development, the inheritance of a high producer allele of TNFα −308 (A allele) in women could provide the required microenvironment for development of vitiligo; therefore, the association of the TNFα −308 A allele with vitiligo in women is expected.
Our results showed that the frequency of TNFα −308 polymorphic genotypes was significantly higher in vitiligo patients than in the controls and conferred an increased risk of acquisition of vitiligo. This is in agreement with the study of Namian et al.15 in Iran and Laddha et al.10 in India. In contrast, the study carried out by Yazici et al.18 reported that there was no association between TNFα −308 promotor polymorphisms and vitiligo in a Turkish population. The lack of association in the Yazici and colleagues study could be attributed to the relatively small sample size as their study included 30 female patients. The variation in the results of these studies may be attributed to differences in the genetic background, along with differences in the environmental factors in the populations studied. This variation further indicates that vitiligo is inherited as a multifactorial or a complex trait, although heritable, it is not due to a single gene defect, but it is determined by interaction of multiple genetic factors, both among themselves and with the environment 19.
Further analysis of the influence of the studied genetic polymorphism on the characteristics of the disease showed that there was no statistically significant difference between patients harboring the wild or the polymorphic genotypes in their age of onset, presenting symptoms, clinical data, or their response to therapy. Unlike the present study, Namian et al.15 reported a significant association between the presence of leukotrichia and the TNFα −308 G/A promoter polymorphism. The presence of leukotrichia in the lesions of vitiligo was associated with disease chronicity and also poor response to treatment. Therefore, they reported that carriers of the TNFα −308 A allele might show a lower probability of response to repigmenting therapies in vitiligo. In addition, the study of Laddha et al.10 suggested that female patients harboring the polymorphic alleles had an early onset of vitiligo. This discrepancy again stresses the effect of genetic variation of the different populations studied on the results and the need for inclusion of larger number of cases per study when feasible or carrying out meta-analysis of data as an alternative. In conclusion, the TNFα −308 promotor polymorphism can be considered as a molecular marker for the risk of vitiligo in Egyptian women. Further exploration of additional variants in TNFα and its receptors may provide further insight into the pathogenesis and ultimately the prevention and treatment of nonsegmental vitiligo.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 Egyptian Women's Dermatologic Society
Egypt; females; nonsegmental vitiligo; tumor necrosis factor α –308 G/A