Increased Tumor Necrosis Factor (TNF)-α and its promoter polymorphisms correlate with disease progression and higher susceptibility towards vitiligo

Abstract

Tumor Necrosis Factor (TNF)-α, is a paracrine inhibitor of melanocytes, which plays a critical role in the pathogenesis of several autoimmune diseases including vitiligo, as abnormal immune responses have frequently been observed in vitiligo patients. Moreover, vitiligo patients show higher lesion levels of TNF-α. Genetic polymorphisms in the promoter region of TNF-α are involved in the regulation of its expression. The present study explores TNF-α promoter polymorphisms and correlates them with TNF-α transcript and protein levels in vitiligo patients and controls of Gujarat along with its effect on disease onset and progression. PCR-RFLP technique was used for genotyping of these polymorphisms in 977 vitiligo patients and 990 controls. TNF-α transcript and protein levels were measured by Real time PCR and ELISA respectively. The genotype and allele frequencies for the investigated polymorphisms were significantly associated with vitiligo patients. The study revealed significant increase in TNF-α transcript and protein levels in vitiligo patients compared to controls. In particular, haplotypes: AATCC, AACCT, AGTCT, GATCT, GATCC and AGCCT were found to increase the TNF-α levels in vitiligo patients. Analysis of TNF-α levels based on the gender and disease progression suggests that female patients and patients with active vitiligo had higher levels of TNF-α. Also, the TNF-α levels were high in patients with generalized vitiligo as compared to localized vitiligo. Age of onset analysis of the disease suggests that the haplotypes: AACAT, AACCT, AATCC and AATCT had a profound effect in the early onset of the disease. Moreover, the analysis suggests that female patients had an early onset of vitiligo. Overall, our results suggest that TNF-α promoter polymorphisms may be genetic risk factors for susceptibility and progression of the disease. The up-regulation of TNF-α transcript and protein levels in individuals with susceptible haplotypes advocates the crucial role of TNF-α in autoimmune pathogenesis of vitiligo.

Figure 1. Age of onset of the disease in vitiligo patients.

(A) Comparison of age of onset of the disease (years) with respect to TNF-α promoter haplotypes in 977 vitiligo patients. (B) Comparison of age of onset of the disease (years) with respect to gender differences in 451 male patients and 526 female patients with vitiligo.

Figure 2. Relative gene expression of TNF-α in controls and vitiligo patients.

(A) Expression of TNF-α transcripts in 174 controls, 157 vitiligo patients, 115 generalized vitiligo patients and 42 localized vitiligo patients, as suggested by Mean ΔCp. (B) Expression of TNF-α transcripts with respect to TNF-α promoter haplotypes in 157 vitiligo patients and 174 controls, as suggested by Mean ΔCp. (C) Expression of TNF-α transcripts with respect to activity of the disease in 108 patients with active vitiligo and 49 patients with stable vitiligo, as suggested by Mean ΔCp. (D) Expression of TNF-α transcripts with respect to gender differences in 68 male patients and 89 female patients with vitiligo, as suggested by Mean ΔCp.

Figure 3. Relative gene expression of TNF-α with respect to promoter genotypes in controls and vitiligo patients.

(A) Expression of TNF-α transcripts with respect to −238 G/A genotypes in 157 vitiligo patients and 174 controls, as suggested by Mean ΔCp. (B) Expression of TNF-α transcripts with respect to −308 G/A genotypes in 157 vitiligo patients and 174 controls, as suggested by Mean ΔCp. (C) Expression of TNF-α transcripts with respect to −857 C/T genotypes in 157 vitiligo patients and 174 controls, as suggested by Mean ΔCp. (D) Expression of TNF-α transcripts with respect to −863 C/A genotypes in 157 vitiligo patients and 174 controls, as suggested by Mean ΔCp. (E) Expression of TNF-α transcripts with respect to −1031 T/C genotypes in 157 vitiligo patients and 174 controls, as suggested by Mean ΔCp.

Figure 4. Serum TNF-α levels in controls and vitiligo patients.

(A) Comparison of sTNF-α levels (pg/ml) in 236 controls, 214 vitiligo patients, 158 generalized vitiligo patients and 56 localized vitiligo patients, as determined by ELISA. (B). Comparison of sTNF-α levels (pg/ml) with respect to TNF-α promoter haplotypes in 214 vitiligo patients and 236 controls, as determined by ELISA. (C) Comparison of sTNF-α levels (pg/ml) with respect to activity of the disease in 150 patients with active vitiligo and 64 patients with stable vitiligo, as determined by ELISA. (D) Comparison of sTNF-α levels (pg/ml) with respect to gender differences in 97 male patients and 117 female patients with vitiligo, as determined by ELISA.

Figure 5. Serum TNF-α levels with respect to promoter genotypes in controls and vitiligo patients.

(A) Comparison of sTNF-α levels (pg/ml) with respect to −238 G/A genotypes in 214 vitiligo patients and 236 controls, as determined by ELISA. (B) Comparison of sTNF-α levels (pg/ml) with respect to −308 G/A genotypes in 214 vitiligo patients and 236 controls, as determined by ELISA. (C) Comparison of sTNF-α levels (pg/ml) with respect to −857 C/T genotypes in 214 vitiligo patients and 236 controls, as determined by ELISA. (D) Comparison of sTNF-α levels (pg/ml) with respect to −863 C/A genotypes in 214 vitiligo patients and 236 controls, as determined by ELISA. (E) Comparison of sTNF-α levels (pg/ml) with respect to −1031 T/C genotypes in 214 vitiligo patients and 236 controls, as determined by ELISA.