TNF- α -308G/A gene polymorphism in bullous pemphigoid and alopecia areata

1. Introduction

Bullous pemphigoid (BP) and alopecia areata (AA) are two organ-specific autoimmune diseases of the skin and hair follicle, respectively. BP is a difficult-to-treat autoimmune disease characterized by the presence of autoantibodies directed against collagen XVII and structural components of the skin which presents with blisters and erythema, frequently in conjunction with urticarial plaques [1]. The clinical presentation of AA is characterized by patches of non-scarring hair loss with a wide range of a single patch of hair loss on the scalp to complete loss of all body hair in severe cases [2]. Various pathogenic factors such as the release of cytokines are essential for induction of autoantibodies and clinical presentation of these diseases [3, 4, 5, 6, 7]. Tumor necrosis factor-alpha (TNF- α ), a multifunctional proinflammatory cytokine, was originally reported as a circulating factor which results in tumor necrosis but has since also been suggested as a key regulator of inflammatory responses [8]. Elevated serum and tissue levels of TNF- α have been shown in many inflammatory conditions, including BP and AA [9, 10, 11, 12].

Cytokine gene polymorphisms, including TNF- α , may influence the induction and release of cytokines, contribute to the disease-associated cytokine imbalance, and thereby affect the susceptibility to autoimmune diseases [13, 14]. While TNF- α cytokine gene polymorphisms have been studied substantially in several autoimmune diseases, few studies have been performed in patients with AA [15, 16, 17] and only one study in BP [18]. Nevertheless, no results have been reported so far on the association between TNF- α gene polymorphism and development of AA or BP in Iranian population. Since TNF- α polymorphism might be associated with susceptibility to the diseases, the present study designed to examine the frequency of -308G/A TNF- α polymorphism in Iranian BP and AA patients comparing with healthy individuals. Due to different gene expression of TNF- α as a result of polymorphism, the RNA expression of TNF- α was evaluated in the patients with different allelic variation and furthermore, compared with control subjects.

2. Materials and methods

3. Results

4. Discussion

Bullous Pemphigoid (BP) is a rare autoimmune skin disease characterized by autoantibodies against structural components of the dermal-epidermal junction (BP180 and BP230) which cause blisters and erosions on skin [1]. These components of basement membrane zone are similarly expressed in the anagen hair follicles of human scalp [24, 25]. Although the role of cytokines has been shown in the pathogenesis of BP and alopecia areata (AA) [3, 4, 5, 6, 7], there are controversies about the major cytokine profile in these diseases. Various significant alleles have been proposed in several studies due to the different ethnic background of populations. Herein, beside HLA genes which are obviously associated with autoimmune diseases, TNF- α is one of the most studied genes in their pathogenesis. Despite positive associations, no specific gene variant has yet been identified as a cause of Bullous pemphigoid (BP) or alopecia areata (AA), and associated alleles should be identified in each particular population. In the present study, no association was found neither between the presence of the -308G/A polymorphism in the promoter region of the TNF- α gene and BP patients, nor AA patients in Iranian population.

TNF- α gene, due to encoding one of the most potent proinflammatory cytokines and the autoimmune modulator, is considered as a significant candidate determining genetic susceptibility to the development autoimmune diseases. This gene is located in HLA class III (6p21) which is a highly polymorphic region [26]. The location of TNF- α , as well as it’s biological activities, make it a good candidate in which polymorphisms within this region influence the pathogenesis of several autoimmune diseases. In contrast to other TNF- α gene polymorphisms with little or no association, a single nucleotide substitution at -308 nucleotides in the TNF- α promoter is contributed to transcription elevation of the gene, higher plasma TNF- α levels, and disease susceptibilities [27, 28]. Nevertheless, contradictory findings have so far been reported. In a previous study, it has been shown that the plasma levels of TNF- α are significantly higher in patients with acute myocardial infarction compared with healthy individuals [21]. In addition, these results showed a significantly high expression of TNF- α mRNAs and proteins in the patients with ‘A’ allele comparing with ‘G’ allele in TNF- α gene variation which tends to be related to increased transcription of TNF- α gene [21]. Although BP and AA share the common features with autoimmune diseases [29], the contribution of TNF- α -308G/A polymorphism in the disease process, in particular, its effect on transcription levels remains largely unclear.

Although previous findings have been shown an association between TNF- α -308G/A polymorphism and AA [15], other report failed to reach significance when corrected for multiple contrasts [16]. To our knowledge in the case of BP, there is lacking data regarding the investigation of cytokine gene polymorphisms in BP patients and it has yet been conducted only in one study in a Chinese population [18]. To date, there is no available report investigating TNF- α -308G/A variant in Iranian ethnicity, neither in AA nor in BP. In this study, frequencies were found to be similar for either genotypes or alleles in patients and controls which suggest no association of this gene variant with susceptibility to these autoimmune diseases. This polymorphism at position nucleotide -308G/A in the promoter region affect the expression of TNF- α gene [27]. Herein, our findings showed discrepant results concerning TNF- α gene expression in BP and AA diseases. While there was a significant difference in gene expression between patients and control individuals in BP disease, our data did not achieve statistical significance in expression levels of TNF- α in patients with AA compared to the control subjects. On the other hand, our results confirmed that the TNF- α gene has similar activity in the patients with ‘A’ allele comparing with ‘G’ allele in both diseases. Indeed, the frequencies of ‘A’ allele in TNF- α -308G/A polymorphism differ from 4% in the Mexican population [15] to 24% in Australians [30]. Based on our results, the ‘A’ allele frequency in TNF- α -308G/A polymorphism in BP and AA patients are 7.5 and 20%, respectively. This frequencies are in agreement with our previous study [21] as well as other Iranian reports [31]. Nevertheless, Iranian population is one of the most heterogeneous populations in the world [32] and case-control studies in the Iranian Kurdish or Turkish ethnic groups indicate an ‘A’ allele frequencies of 8 and 9.5%, respectively [33, 34]. These controversies may be due to different factors such as multifactorial nature of different types of diseases, variations in designing different studies, and genetic heterogeneity within and between the studied populations. In addition to this, activation and presence of other molecules that interact with the TNF- α promotor region can also take into account. Of note, genetic polymorphism investigations have shown a wide difference in allele frequencies in the various ethnic groups [35, 36]. Herein, the difference of risk allele frequency between populations may explain the variations in disease prevalence among those populations [37]. Natural selection and certain environmental conditions can also modulate the balance in allele frequencies across populations [38].

In Conclusion, a common gene polymorphism in the TNF- α locus was investigated as a risk factor for the development of two autoimmune diseases. Our findings suggest that polymorphism in the TNF- α promoter might not be contributed to the susceptibility of BP and AA. Although this polymorphism has an effect on promoter hyperactivity and in turn increased TNF- α transcription in BP patients, this effect was not seen in AA patients. To our knowledge, this is the first study evaluating these factors in Iranian patients with BP and AA. Given the presence of various ethnic groups in Iran, screening of several populations from different regions and ethnicities especially with larger sample size could help to validate these results and draw any representative picture of Iranian population. In the present study, we did not investigate the relationship between polymorphism and TNF- α secretion. Future studies may identify this association.