Vitiligo, characterized by milky-white patches on the skin, is an acquired disease of unknown etiology. Histologically speaking, it is caused by a loss of melanocytes [1]. Several theories have been proposed to explain the pathogenesis; these include the autoimmune, the neural and the theory of self-destruction. Most current studies have focused on the genetic etiology, with genes related to the autoimmunity, melanin, and the biological reply process to the oxidative stress, being the most thoroughly investigated [2, 3, 4, 5, 6].
With regard to the pathogenic role of oxidative stress in vitiligo, the previous studies were directed toward changes in antioxidant enzyme activity in the blood and tissue. Although some conflicting results were attained, all studies did point to an impact of oxidative stress on vitiligo [7, 8, 9, 10, 11, 12]. Superoxide dismutase (SOD), one of the primary antioxidant enzymes, was previously studied in relation to vitiligo and to date, three isoforms of SOD have been identified in humans. They are coded by three different genes: copper-zinc SOD (CuZn-SOD), manganese SOD (Mn-SOD), and extracellular SOD. The diversity in their form stems from amino acid alignment, active metal zone and their intra cell location. Copper-zinc SOD, defined as SOD1, is a cytosolic enzyme, Mn-SOD, namely SOD2, exists in the mitochondria; while the expression of the SOD3 enzyme, is limited to only plasma, lymphoid tissue or cerebrospinal liquids [13, 14].
Variations of SOD1 have previously been studied in relation to several clinical manifestations including amyo-trophic lateral sclerosis (ALS) and diabetes mellitus (DM) [15, 16, 17]. However, to the best of our knowledge, no studies considering associations between either gene polymorphism SOD1 35 A/C (rs2234694) or SOD2 A16V (C/T) (rs4880) and vitiligo is currently available in the literature. The aim of this study was to investigate the polymorphisms of the SOD1 and SOD2 and their impact on Turkish vitiligo patients.
The study included 101 vitiligo patients, with a control group of 99. The diagnosis of vitiligo was based on clinical findings and the Wood lamp examination, while a vitiligo-free family history, and adequate general health, qualified probands for the control group. Types and volumes of vitiligo in relation to the study group are summarized in Table 1 (focal, segmental, acrofacial, generalized universal and mixed). Other factors, such as disease duration, family history and comorbidities including DM or Hashimoto’s thyroiditis were also noted. Written informed consent was obtained from all subjects. The study was approved by the Ethics Committee of Ege University Medical Faculty, Izmir, Turkey.
Demographic and clinical parameters of vitiligo patients. (Values are presented as mean.) F: females; M: males.Clinical Type Gender Age % F M F M Focal 46 45.55 25 24.76 22 21.78 35.62; F: 32.16; M: 39.39 Acrofacial 25 24.75 13 12.87 11 10.89 32.15; F: 31.26; M: 33.42 Generalized 21 20.79 13 12.87 8 7.92 38.78; F: 41.34; M: 34.62 Segmental 4 3.96 2 1.98 2 1.98 27.35; F: 27.50; M: 27.20 Universal 3 2.97 3 2.97 – – 37.56 Mixed 2 1.98 2 1.98 – – 36.15 Total 101 100.00 58 57.43 43 43.57
Two milliters of peripheral blood were extracted from each participant, collected into EDTA tubes and stored at –20 °C. Genomic DNA was then isolated from the peripheral blood using standard techniques (DNA Isolation Blood Mini Kit; Invitek, Berlin, Germany).
For the polymorphism analysis of SOD1 35 A/C and SOD2 A16V (C/T), polymerase chain reaction-restriction fragment length polymorphim (PCR-RFLP) was performed on both occasions. For SOD1 gene 35 A/C polymorphism, PCR was initiated using forward (5′-CTA TCC AGA AAA CAC GGT GGG CC-3′) and reverse (5′-TCT ATA TTC AAT CAA ATG CTA CAA AAC-3′) primers. For
The 278 bp PCR products for the
A 267 bp amplicon of the
As a safeguard, Sanger sequencing of the PCR product was used to confirm the presence of the
The SOD1 35 A/C and SOD2 A16V (C/T) polymorphism allele frequencies of Vitiligo patients were compared to those of a control group using the χ2 test; the application of statistical program SPSS 9.0.
A total of 101 vitiligo patients; 58 women, (57.4%) and 43 men (42.6%) were enrolled in the study. The mean age was 37.4 ± 15.2; range 7-68 years. The control group consisted of 99 healthy volunteers; 50 women (50.5%) and 49 men (49.5%), with a mean age of 36.6 ± 12.6, range 18-69. In terms of age and gender, no statistical difference existed between the groups.
The median duration of vitiligo was 9.0 years (range 1–35 years). A family history of vitiligo was noted in first-degree relatives of 23 patients (22.7%).
With regard to vitiligo type, 46 patients were focal (45.5%), 25 acrofacial (24.7%), 21 generalized (20.7%), four segmental (3.9%), three universal (2.9%) and two patients were classified as mixed (1.9%). Results can be seen in Table 1. The comorbidities in vitiligo patients were as follows: Hashimoto’s thyroiditis 12 patients (11.8%), DM 11 patients (10.8%), pernicious anemia two patients (1.9%). When considering the distribution of the comor-bidities between the vitiligo and the control group (
The genotype and allele distribution in the patient and control groups for the SOD1 35 A/C SOD2 Ala-9Val (C/T)
polymorphism. a Patients HWE: 0.0101, b Patients HWE: 2.5320, Genotype Patient Control Total SOD1 AA 96 96.05 97 97.98 193 CA 5 4.95 2 2.02 7 CC 0 0.00 0 0.00 0 0.277 Total 101 100.00 99 100.00 200 35 A/C A 197 97.52 196 98.98 393 C 5 2.48 2 1.02 7 0.214 Total 202 100.00 198 100.00 400 SOD2 CC 26 25.70 35 35.36 61 CT 37 36.60 40 40.40 77 0 047 TT 38 37.60 24 24.24 62 Total 101 100.00 99 100.00 200 Ala9Val (C/T) T 154 76.24 124 62.63 278 C 48 23.76 74 37.37 122 0.460 Total 202 100.00 198 100.00 400
Distributions of SOD1 35 A/C and SOD2 Ala9Val (C/T) polymorphisms in cases and controls and risk of vitiligo. This is a statistically significant This is a statistically significant Genotype/Allele OR 95% CI SOD1 AA 1 – – AC 0.380 0.072-2.005 0.271 A 0.688 0.426-1.110 0.281 C – – – SOD2 CC CT 1 – – TT 1.750 0.889-3.446 0.105 C 2.158 1.044-4.462 0.038 T 0.792 0.648-0.968 0.022
However, when the patient cohort and control group were compared for the presence of the SOD2 Ala9Val (C/T) polymorphism, the distribution of the genotypes [
More common alleles of both polymorphisms were considered as reference alleles. Logistic regression analysis showed no significant association between SOD1 35 A/C polymorphism (AA
The SOD enzymes together with glutathione peroxidase and catalase play a critical role, assuring the cell protection against free radicals. These enzymes contain redox metals in the centers of their catalytic zones that transform superoxide radicals to hydrogen peroxide and oxygen [14, 15, 16, 17, 18].
Oxidative stress can trigger a reaction that causes melanocytes destruction. Thus, the lesions of vitiligo may be brought on by psychological stress or physical events. The trauma and stress cause a catecholamine release, which results in vasoconstriction and hypoxia. Following cellular hypoxia and reoxygenation there is an increase in free oxygen radicals and toxic materials. In a study on malondialdehyde (MDA), level, SOD and glutathione peroxidase activity in the tissues of 114 vitiligo patients, Yildirim
This is the first study to evaluate the relationship between the SOD1 and SOD2 polymorphisms and vitiligo. In terms of the SOD2 Ala-9Val (C/T), the TT genotype frequency in the patient group was statistically and significantly higher than the control group. We observed a 2-fold relative risk increase for the development of vitiligo in subjects with the TT genotype. Thus, while not conclusive, our results do suggest that the SOD2 polymorphism might play a role in vitiligo.
In the literature, studies of the CuZn-SOD, described as the SOD1 enzyme, found the frequency of the Ala4Val mutation in SOD1 to be significantly higher than other mutations [5, 19, 20]. In our study, when we compared the distribution of genotype for the SOD1 35 A/C polymorphism, no significant diversity could be determined (
The SOD1 enzyme is cytosolic, and the antioxidant reactions occur in the mitochondria. This might explain the similarity of genotype and allele frequencies in both the cohort and control group.
The SOD2 enzyme, also known as Mn-SOD, has been studied more extensively than SOD1 due to the fact it is mitochondrial and inducible. The production of this particular enzyme rises
The Ala-9Val polymorphism has been studied in relation to Parkinson’s disease, schizophrenia, urolithiasis, adult-onset obesity, motor neuron disease, non familial idiopathic dilated cardiomyopathy, age-related macular degeneration, non alcoholic fatty liver disease, diabetic neuropathy, various cancers such as breast, prostate, stomach, colon, lung and skin cancer [22, 23, 24, 25, 26, 27, 28, 29]. The SOD2 polymorphism, however, has been generally studied in relation to DM and its associated complications [29, 30, 31, 32].
Previous studies have shown that different polymorphisms from various SOD isoforms can be associated with vitiligo. Additionally, there have also been studies evaluating the expression and enzyme activity of SOD isoforms, in both blood and tissue. Many of these studies have produced conflicting outcomes, especially concerning the peripheral blood samples of vitiligo patients.
Picardo
One of the limitations of this study was our inability to exactly measure the enzyme activities of