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 Table of Contents  
Year : 2014  |  Volume : 34  |  Issue : 1  |  Page : 36-40

Tumor necrosis factor α promoter −308G/A polymorphism in patients with patchy alopecia areata

1 Department of Dermatology and Venereology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Microbiology and Immunology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission17-Jan-2014
Date of Acceptance11-Mar-2014
Date of Web Publication24-Jul-2014

Correspondence Address:
Al-Hasan M El-Hefnawy
MD, Departments of Dermatology and Venereology, Faculty of Medicine, Ain Shams University, Cairo 12311
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1110-6530.137295

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Alopecia areata (AA) is a common recurrent inflammatory nonscarring hair loss disease with a worldwide prevalence ranging from 1 to 2%. Although the exact etiology of AA is unknown, there is evidence for both genetic and autoimmune components. Recently, tumor necrosis factor a (TNF-a) promoter −308 polymorphism was suggested to contribute to the pathogenesis of a wide range of autoimmune and infectious diseases. There is a plausible association between TNF-α polymorphism and AA.
The aim of the study was to determine TNF-α 308 gene polymorphism in patients with patchy AA.
Patients and methods
The present case-control study included 20 patients with patchy AA and 20 age-matched and sex-matched apparently healthy controls. TNF-α 308 gene polymorphism was detected by restricted fragment length polymorphism PCR in all of them.
The mutant TNF-α 308 gene was found in 28.6% of patients with patchy AA and in 4.8% of controls, with statistically nonsignificant difference (P = 0.093). No statistically significant differences were found between patients with normal genotype and those with mutant genotype with respect to age, sex, disease duration, medical associations, family history of autoimmune diseases, or history of previous attacks. Statistically significant difference was found with respect to family history of AA and number of lesions.
TNF-α 308 gene polymorphism was present in small percentage of patients with patchy AA, with statistically nonsignificant difference from controls.

Keywords: Alopecia areata, TNF-α, tumor necrosis factor α polymorphism

How to cite this article:
El Sayed MH, El-Hefnawy AHM, Al-Mashaiky FS, El Sayed SB. Tumor necrosis factor α promoter −308G/A polymorphism in patients with patchy alopecia areata. Egypt J Dermatol Venerol 2014;34:36-40

How to cite this URL:
El Sayed MH, El-Hefnawy AHM, Al-Mashaiky FS, El Sayed SB. Tumor necrosis factor α promoter −308G/A polymorphism in patients with patchy alopecia areata. Egypt J Dermatol Venerol [serial online] 2014 [cited 2023 Mar 20];34:36-40. Available from: http://www.ejdv.eg.net/text.asp?2014/34/1/36/137295

  Introduction Top

Alopecia areata (AA) is a common nonscarring autoimmune hair loss disease with no race or sex predilection [1]. Despite the extensive research on AA, no clear etiological trigger factor has been established [2] and many etiologic factors have been suggested to contribute to the development of AA. These include autoimmune factors, stress, infectious agents, vaccinations, hormonal factors, and genetics. The disease onset and severity are probably determined by multiple factors [1]. Other causes include cytokines, intrinsically abnormal melanocytes or keratinocytes, and neurological factors [1],[2],[3].

Various molecules are produced during the pathogenesis of AA, such as tumor necrosis factors (TNF), granzymes, and Fas ligand. Potentially, these molecules may trigger apoptosis in AA-affected hair follicle cells and generally disrupt the normal functioning [4]. Recent progress in the understanding of AA has shown that regulation of local and systemic cytokines plays an important role in its pathogenesis. TNF-α is one of these cytokines [5]. It has been shown that TNF-α inhibits hair follicle growth in in-vitro studies. The changes in hair follicles incubated with TNF-α are similar to those reported in AA. These changes include condensation and distortion of the dermal papilla, marked vacuolation of the hair follicle matrix, abnormal keratinization of the follicle bulb and inner root sheath, disruption of follicular melanocytes, and the presence of melanin granules within the dermal papilla [3],[6]. In addition, TNF-α induced the formation of a club-like hair follicle, similar to catagen morphology of the hair bulb. It also stimulates intracellular production of mitochondrial Reactive oxygen species [7]. An increase in reactive oxygen species produces cell degeneration and apoptosis, which may be related to oxidative stress in AA [8]. Biological responses to TNF involve signaling complex (DISC), which results in activation of the initiator caspases that initiate apoptosis [9].

The genes coding for TNF-α are located within a 7-kb DNA locus on the chromosomal region 6p21.3-21 and are closely linked to HLA-B locus within a high polymorphic region of the major histocompatibility complex, telomeric to the class II and centromeric to the class I region [10]. Genetic alterations in the TNF-α locus are now known to be involved directly in high TNF-α production. Several polymorphisms have been identified inside the TNF-α promoter. Among these variants, a polymorphism that directly affects TNF-α expression is located at nucleotide position −308. A single base polymorphism within the promoter of the gene for TNF-α results in two allelic forms, one in which guanine defines the common TNF-G allele and the other in which guanine is substituted by adenosine, forming the rarer TNF-A allele. The presence of the rarer TNF-A allele has been found to correlate with enhanced spontaneous or stimulated TNF-α production both in vitro and in vivo [11].

Regarding TNF-α gene polymorphism and AA, Galbraith and Pandey [12] have found associations between the presence of TNF-α 308G/A and the development of AA with different distribution of TNF-α phenotypes between groups of patients with AA. The TNF G/G and A/G phenotypes were associated with patchy AA but not with alopecia totalis (AT)/universalis (AU), which suggests that the two forms of disease are genetically distinct. Moreover, Cristina et al. [2] have recently reported the association between TNF-α promoter −308G/A polymorphism and Mexican patients with patchy AA. Given the role of TNF-α in AA and the paucity of studies on TNF-α gene polymorphism in AA and the absence of previous studies on Egyptian patients, the present study aimed to assess the presence of TNF-α promoter −308G/A polymorphism in Egyptian patients with patchy AA.

  Patients and methods Top

The present study represents a case-control study. It included 20 patients with patchy AA, with no history of previous treatment (newly diagnosed) or who did not receive treatment for the previous 3 months, and 20 age-matched and sex-matched healthy volunteers as controls. The patients were enrolled from those attending the Dermatology outpatient clinic of Ain Shams University Hospitals during the period from January 2013 to May 2013.

Exclusion criteria included patients with other types of alopecia and patients experiencing spontaneous regrowth of terminal hair at the time of presentation. Patients having any disease that may affect the outcome of the study (such as psoriasis and autoimmune diseases) were also excluded. Before initiation, every participant was informed about the aim of the study and gave consent.

All patients were subjected to full history taking and general and dermatological clinical examination. Three milliliters venous blood was then taken from each individual participating in the study under complete aseptic conditions. Blood was then collected in sterile EDTA vacutainer tubes. Samples were either stored at −20°C until used or used directly within 24 h for DNA extraction.

Detection of tumor necrosis factor a promoter −308 gene polymorphism

This was performed by Restricted Fragment Length Polymorphism PCR, which included DNA extraction, and RNA was extracted from the PBMCs using MagNA pure compact Nucleic Acid Isolation Kit I (Cat. No. 03730964001; Roche, Mannheim, Germany). Amplification of the extracted DNA for detection of TNF-α −308 genetic polymorphisms was then performed. The amplified product was restricted by the restriction enzyme NcoI. The amplified products were visualized (after incubation with NcoI) on 2% agarose gel electrophoresis. The test primers for TNF-α gene at position −308 were used according to Seidemann et al. [10]. Primers were provided by Fermentas (Lithuania, Pittsburg, USA).


Reverse primer: 3'-TCCTCCCTGCTCCGATTCCG-5'.

The primers were added to the Master Mix supplied by Bioron GmbH (cat. No: 101005; Heidelberg, Germany), and the reaction mixture was centrifuged by the Eppendorf microcentrifuge 5410 followed by the DNA thermal cycler Perkin Elmer No. 9600. The presence of NcoI restriction sites was indicated by the cleavage of the 142 bp amplified product to yield two fragments of 126 and 2 bp. The two allelic forms of TNF, corresponding to the presence or absence of the NcoI site, are referred to as TNF-α G and TNF-α A, respectively.

The genotypes were identified as follows:

  1. Normal genotype harboring the wild-type alleles GG (TNF-α G/G) if two bands 126 and 16 bp were detected.
  2. Heterozygous mutant (heterotype; TNF-α A/G) if three bands appeared at 142, 126, and 16 bp.
  3. Homozygous mutant (homotype; TNF-α A/A) if a single band of 142 bp was detected.

Statistical analysis

Statistical analysis was carried out using statistical package for social sciences (SPSS Inc., Chicago, Illinois, USA) version 15. Quantitative data were presented as mean±SD, whereas qualitative data were presented as number (n) and percentage (%). The comparison between quantitative data was performed using the independent Student t-test, whereas the Mann-Whitney U-test was used only with nonparametric data. The comparison between qualitative data was performed using the χ2 -test. The Fisher exact test was used instead of the χ2 -test when the expected count in any cell was found less than 5. P values of 0.05 or less were considered statistically significant and of 0.001 or less were highly significant.

  Results Top

The present study included 20 patients with patchy AA and 20 age-matched and sex-matched controls. The age of the patients ranged from 14 to 45 years with a mean age of 29.1 ± 8.5 years. The mean disease duration was 2.38 ± 2.2 months. Single patch of AA was found in 14 of the 20 patients (66.7%). Patients having negative family history of autoimmune disease and negative family history of AA represented 66.7 and 81% of included patients, respectively. In all, 67.2% of patients had no previous attacks of AA.

Regarding TNF-promoter −308G/A gene polymorphism, genotype assessment by PCR-RPLP revealed normal TNF-α G/G genotype in 71.4% of the patients, TNF A/G genotype (heterotype) in 23.8%, and TNF A/A genotype (homotype) in 4.8% of the patients [Figure 1]. Accordingly, overall mutant gene was detected in 28.6% of patients with patchy AA. Regarding controls, genotyping of the TNF −308G/A gene by PCR-RPLP revealed normal TNF G/G genotype in 95.2%, TNF A/G in 4.8%, and TNF A/A genotype in none of the controls. Although there was a difference between TNF genotyping of patients and controls, it did not meet statistical significance between both groups regarding the frequency of the mutant gene, as 28.6% of patients had mutant gene, whereas 4.8% of controls had the mutant gene (P = 0.093) [Figure 2].
Figure 1:

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Figure 2:

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Within the patients with patchy AA, no statistically significant differences were found between patients with normal genotype and those with mutant genotype with respect to their age (P = 0.309), sex (P = 0.336), medical associations (P = 1.00), family history of autoimmune diseases (P = 0.354), duration of illness (P = 0.237), or history of previous attacks (P = 0.258). However, statistically significant difference was found with respect to family history of AA (P = 0.003), where 100% of patients with mutant TNF genotypes had family history of AA compared with only 11.8% of patients with normal genes. In addition, statistically significant difference was found with respect to the number of lesions (P = 0.025). Multiple patches of AA were found in patients with mutant TNF genotype, whereas only single patch was found among patients with normal genes.

  Discussion Top

In the present study, mutant TNF-α promoter −308 gene was detected in 28.6% of patients with patchy AA compared with 4.8% of healthy age-matched and sex-matched controls. Jime'nez Morales et al. [13] reported in their series that there is a great deal of evidence that points to the association of TNF-α gene as a common genetic factor in the pathogenesis of diseases that are caused by inflammatory and/or autoimmune etiologies. As reported in the literature, TNF-α −308G/A polymorphism has been observed in several diseases, and many studies in the Egyptian population had described the association between this polymorphism and autoimmune diseases such as rheumatoid arthritis [14], pemphigus [15], type 1 diabetes mellitus [16], and chronic obstructed pulmonary disease [17].

TNF-α gene encodes one of the most powerful proinflammatory cytokines, which acts as an autoimmune modulator. Accordingly, this gene may constitute an important candidate for determining genetic susceptibility to the development of different autoimmune diseases including AA. Several genetic studies on AA have shown that immune factors are key genetic components of AA. Early association studies with selected candidate genes revealed that AA is associated with several immune-related genes. In addition, recent genome-wide linkage and association studies have identified several susceptibility loci in AA near or within genes related to autoimmunity [18]. AA has also been proposed to have different characteristics in common with autoimmune diseases [19]. Moreover, Philpott et al. [20] reported that interleukin a (IL-α), IL-1ί, and TNF-α may play an important role in the pathophysiology of AA. They also described alterations in TNF-α levels that led to changes in the normal growth of the hair follicle. Galbraith and Pandey [12] found associations between the presence of TNF-α 308G/A and the development of AA; the distribution of TNF-α phenotypes differed significantly between groups of patients with AA. The TNF G/G and A/G phenotypes were associated with patchy AA but not with AT/AU, which suggests that the two forms of disease are genetically distinct. Cristina et al. [2] also found that the distributions of TNF-α genotypes differed significantly between patients with AA and controls. Shimizu et al. [21] proposed that macrophage migration inhibitory factor (MIF), a cytokine produced by lymphocytes and peripheral blood mononuclear cells, levels are significantly elevated in patients with AA. This molecule stimulates the production of IL-1 and TNF-α by macrophages, whereas the latter exert positive feedback effect on MIF. The cycle endpoint is an on-going inhibition of hair growth. Polymorphisms within the MIF-173C allele confer an increased risk for early onset extensive form of AA below the age of 20 years, as shown in the study by Shimizu et al. [22]. In contrast, Thein et al. [23] examined the cytokine profiles of infiltrating activated T cells from the margins of involved AA lesions and revealed that T-cell clones inhibited the proliferation of neonatal keratinocytes. In examining the cytokine profiles and relating them to regulatory capacity, the authors found that T-cell clones that released high amounts of TNF-α and/or TNF-α inhibited keratinocyte growth. Kasumagic-Halilovic et al. [5] demonstrated that the mean serum levels of TNF-α were significantly elevated in AA patients in comparison with healthy individuals. Conversely, Cristina et al. [2] observed that the TNF1 allele may confer a protective effect when it is distributed in a homozygous manner as for the TNF1/TNF1 genotype, but this effect becomes diluted for TNF1/TNF2 because of the presence of the polymorphic allele; however, the TNF2/TNF2 genotype might be more closely related to the appearance of more seriously disabling or severe autoimmune pathologies. These results were based on their study on 59 Mexican patients affected by patchy AA and 103 control individuals without AA, which investigated for TNF-α −308 promoter gene polymorphism. They concluded that there was a plausible association between the presence of the TNFα −308G/A polymorphism and a higher susceptibility for developing patchy AA. This risk might be due to overproduction of TNFα, which would facilitate an autoimmune response against the hair follicle. In contrast, our results were similar to those reported by Galbraith and Pandey [12] on 50 White patients with AA in southern California, which revealed the presence of a difference in phenotype distribution between patients with AA and controls, but this failed to reach significance. It also revealed that the distribution of TNF-α phenotypes differed significantly between groups of patients with AA.

Meanwhile, TNF-α seems to be a useful indicator of the activity of AA, and it may play an important role in the development of this disease [5]. In our study, multiple patches in AA patients were only found with the mutant TNF-α genotype. The previous finding suggests that the use of TNF inhibitors may be of beneficial effect in treatment of AA [24]. However, there are some reports of failure of these TNF-α inhibitors in controlling AA. Etanercept was ineffective in treating patients with refractory, moderate to severe AA, AT, or AU [25].

The variation of the results between different studies may be because of probable differences in genetic background and the number of patients along with differences in environmental factors. Further studies are recommended on a large number of patients and controls to determine whether the presence of the TNFα −308G/A polymorphism is associated with high susceptibility for developing patchy AA and to assess both serum TNFα level and TNFα -308G/A polymorphism in patients with AA to put firm conclusion.

  Acknowledgements Top

Conflicts of interest

None declared.

  References Top

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