The association of Foxo3a gene polymorphisms with serum Foxo3a levels and oxidative stress markers in vitiligo patients Ummuhani Ozel Turkcu, Nilgun Solak Tekin, Tuba Gokdogan Edgunlu, Sevim C¸elik Karakas, Setenay Oner PII: DOI: Reference:
S0378-1119(13)00039-5 doi: 10.1016/S0378-1119(13)00039-5 GENE 38
To appear in:
Gene
Accepted date:
19 December 2013
Please cite this article as: Ozel Turkcu, Ummuhani, Tekin, Nilgun Solak, Edgunlu, Tuba Gokdogan, Karakas, Sevim C ¸ elik, Oner, Setenay, The association of Foxo3a gene polymorphisms with serum Foxo3a levels and oxidative stress markers in vitiligo patients, Gene (2013), doi: 10.1016/S0378-1119(13)00039-5
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The association of Foxo3a gene polymorphisms with serum Foxo3a levels and oxidative stress markers in vitiligo patients
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b a a Ummuhani Ozel Turkcu , Nilgun Solak Tekin , Tuba Gokdogan Edgunlu ,
c d Sevim Çelik Karakas , Setenay Oner
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a Mugla Sitki Kocman University, MuglaSchool of Health Sciences, Department of Nutrition and
b
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Dietetics, Mugla, Turkey
Bulent Ecevit University, Faculty of Medicine, Department of Dermatology, Zonguldak, Turkey
Osmangazi University, Faculty of Medicine, Department of Biostatistic, Eskisehir, Turkey
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Corresponding Author:
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d
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c Bulent Ecevit University, Faculty of Medicine, Department of Medical BiologyZonguldak, Turkey
Ummuhani OZEL TURKCU
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Mugla Sıtkı Koçman Üniversitesi, Mugla Sağlık Yüksekokulu, S Blok, 1 Kat 48000 Kotekli/MUGLA/TURKEY Tel: +90 252 2113208; Fax: +90 252 2111880; E-mail:[email protected]
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Abstract
Vitiligo is an acquired epidermal pigment loss the skin with destruction of melanocytes. Oxidative
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stress is one of the major theories in the pathophysiology of vitiligo. FOXO3a is the forkhead members of the class O (FOXO) transcription factors, and plays important role in cell cycle regulation,
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apoptosis, oxidative stress, and DNA repair. The aim of our study was to investigate, for the first time,
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FOXO3a gene polymorphisms and FOXO3a protein levels, activities of superoxide dismutase (SOD) and catalase antioxidant enzymes in vitiligo patient and healthy controls. Moreover, plasma advanced oxidation protein products (AOPP) levels in subjects was evaluated to understand the possible role of protein oxidation in disease etiology. Studies groups included 82 vitiligo patients and 81 unrelated
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healthy controls. FOXO3a polymorphisms were determined using polymerase chain reaction-
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restriction fragment length polymorphism method. FOXO3a levels and catalase activity were measured by ELISA whereas AOPP levels and SOD activity were measured spectrophotometric
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analyses. We found a significant relationship between rs4946936 polymorphism of FOXO3a gene and
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vitiligo/active vitiligo patients (p=0.017; p=0.019 respectively), but not for rs2253310 (p>0.05). SOD activity and AOPP levels of vitiligo patient were increased compared with control group, whereas FOXO3a levels and catalase enzyme activity of vitiligo patient were decreased compared with control group (p0.05,
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data not shown).
FOXO3a rs2253310 and rs4946936 haplotypes and their distribution in vitiligo or active/stable vitiligo
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patients and healthy controls were also analyzed. The distributions of FOXO3a haplotypes with an
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estimation of odds ratios in the patients and controls are presented in Tables 6 and 7. Haplotype analysis of FOXO3a did not reveal any significant differences between the vitiligo or stable vitiligo patients and the controls (p>0.05). The frequencies of the GT and CT haplotypes were higher in the
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vitiligo and active vitiligo patients than in the control group.
4. Discussion
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Oxidative stress and genetic predisposition is thought to play an important role in the pathogenesis of
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vitiligo. To the best of our knowledge, our study is the first to evaluate the association of FOXO3a gene polymorphisms, FOXO3a levels, and protein oxidation with the pathophysiology of vitiligo. We
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also investigated activities of the main antioxidant enzymes in vitiligo patients and healthy subjects by measuring SOD and catalase activities. In this study, we demonstrated that rs4946936 of the FOXO3a gene might be associated with the risk of vitiligo. Interestingly, we found that FOXO3a levels were lower in vitiligo patients than in healthy controls. In addition, we found increased protein oxidation damage and altered activities of the antioxidant enzymes in the vitiligo patients.
It has been suggested that FOXO3a has critical roles in various biological processes, including cell cycle regulation, apoptosis, and oxidative stress, by controlling the expression of target genes (Furukawa-Hibi 2005; Brunet 1999a). Overexpression of FOXO3a has been shown to protect cells
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against oxidative damage via expression of antioxidant enzyme activity or regulation of reactive oxygen metabolism. Olmos et al. (2009) reported that FOXO3a can protect quiescent cells from
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oxidative stress regulating SOD2 and catalase genes. Other studies have indicated that FOXO3a
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directly activates transcription of antioxidant enzymes, namely, MnSOD, catalase, and peroxiredoxin
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3 (Kops 2002; Nemoto 2002; Chiribau 2008). FOXO3a-deficient hematopoietic stem cells have been observed to reduce the expression of genes involved in ROS detoxification, leading to elevated ROS
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levels (Yalcin 2008). Considering the roles of FOXO3a in oxidative stress and apoptosis, we mainly examined the relationship between FOXO3a gene polymorphisms and FOXO3a levels and vitiligo. In our study, we determined that the TT genotype and T allele frequencies in rs4946936 of the FOXO3a
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gene were associated with risk of vitiligo, but we did not find the same results for rs2253310. Moreover, when we evaluated vitiligo patients in terms of stability, TT genotype and T allele
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frequencies in rs4946936 were higher in active vitiligo patients compared to healthy controls. These
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results can be interpreted to mean that rs4946936 SNP might a putative risk factor for genetic
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susceptibility of vitiligo, especially active vitiligo. We also found that FOXO3a levels were lower in vitiligo patients than in healthy controls; the underlying mechanism is difficult to explain, as there are no published studies about serum/plasma FOXO3a levels. However, we think that the low FOXO3a levels might be associated with increased oxidative stress in the patients. In response to various types of cellular stress stimulus or exogenous agents, the transcriptional activity of FOXO3a can be regulated by post-transcriptional modifications, such as phosphorylation, acetylation, and ubiquitylation (Ponugoti 2012; Maiese 2009). The PI3K/AKT signal pathway is a major regulator for FOXO3a activity (Kops 2002). AKT phosphorylates FOXO3a, and then the phosphorylated FOXO3a protein is transported from the nucleus to the cytoplasm, leading to inhibition of its transcriptional
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activities for target genes (Kops 2002). Furthermore, AKT mediated phosphorylated FOXO3a causes ubiquitination and proteasomal degradation in cytoplasm (Maiese 2009; Plas 2003). However, it has
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been reported that increased AKT activity leads to enhance FOXO3a cytosolic localization and a
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decrease in MnSOD levels in prostate cancer cells (Shukla 2009). The increased cytosolic localization
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of FOXO3a protein and its degradation under oxidative stress conditions have also been reported (Nogueira 2008; Yang 2008; Lu 2013). Lu et al. (2013) showed that overproduction of ROS induced
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by a high glucose concentration decreases the expression of MnSOD via the PI3K-Akt-FOXO3a pathway, and they suggested it further aggravates oxidative stress in diabetic nephropathy. There is no data about whether or not which SNPs of FOXO3a have effect on its function. Nevertheless, only a
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study, Banasik et al. (2011) was investigated FOXO3a rs 2802292 and the levels of FOXO3a mRNA expression in skeletal muscle in young and elderly twins. Their results indicated that the minor G-
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allele of FOXO3A rs2802292 is associated with enhanced peripheral and hepatic insulin sensitivity,
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which may be mediated through increased skeletal muscle-FOXO3A mRNA expression. Also, they
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implicated that metabolic findings are consistent with observations of the G-allele being associated with increased longevity. In our study, we investigated the influence of FOXO3a gene variants (rs4946936, rs2253310) on FOXO3a levels as well, but we found no relationship between FOXO3a gene SNPs and FOXO3a levels. As such, our results might indicate that low FOXO3a levels might be associated with increased oxidative stress. However, we suggest that when FOXO3a SNPs, its mRNA and protein levels study in larger samples of vitiligo patients, these results might give more information about for the role FOXO3a levels in vitiligo pathogenesis.
Superoxide dismutase and catalase are well-known first line defense antioxidant enzymes that protect the cell against oxidative stress. Antioxidant enzyme activities have been investigated in clinical
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studies to assess the oxidative stress status in the epidermis and blood of vitiligo patients. Yildirim et al. (2003) reported significantly higher SOD activity in erythrocytes in generalized vitiligo patients
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than in controls. Similarly, Hazneci et al. (2005) and Jain et al. (2011) observed that blood SOD
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activity in vitiligo patients was significantly higher than in healthy controls. However, some studies
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have shown decreased SOD activity in the blood of patients with vitiligo (Koca 2004). In the present study, we observed that SOD activity was significantly higher in the vitiligo patients than in the
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healthy subjects. This increase might be due to overexpression of the SOD enzyme against increased superoxide anions in the vitiligo patients. In addition, we found that catalase activity in the vitiligo patients was significantly lower than in the healthy controls. In agreement with our study, several
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researchers have found decreased catalase activity in the skin and blood of vitiligo patients (Hazneci 2005; Sravani 2009). In these studies, different reasons were suggested for the decreased catalase
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activity. Some of the authors suggested that an accumulation of H2O2 might lead to inhibition of
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catalase activity (Schallreuter 1999d), while others proposed that catalase SNPs and its mutant alleles
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might affect both catalase activity and its expression in patients with vitiligo (Courtney 2002; Liu 2010). However, these results are insufficient to explain the decreased catalase activity in vitiligo patients definitively. In the present study, the increased SOD and decreased catalase activities could be due to a systemic accumulation of H2O2 in the body as a result of an inability to eliminate it efficiently. Consistent with the other studies, our results clearly support that oxidative stress might be implicated in the pathophysiology of vitiligo, due to alterations in the antioxidant defenses.
H2O2 is also a substrate of the nicotinamide adenine dinucleotide phosphate oxidase complex, and along with chloride ions, is converted into hypochlorous acid (HOCl) by the myeloperoxidase enzyme in activated neutrophils/leucocytes. As potent oxidants, HOCl and chloramine derivatives react with
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plasma proteins during oxidative stress, resulting in the formation of dityrosine, which contains crosslinked protein products known as advanced oxidation protein products (AOPP) (Hanasand 2012,
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Witko-Sarsat 1994; Alagozlu 2013). AOPP have been widely used as reliable biomarkers to assess
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oxidative protein damage (Witko-Sarsat 1994; Alagozlu 2013). However, according to the literature,
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AOPP levels have not been investigated in patients with vitiligo. In this study, we found that plasma AOPP levels in vitiligo patients were higher than in healthy controls. Thus, we suggest that oxidative
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protein damage might play a critical role in vitiligo pathogenesis, and that AOPP levels could be used for determining oxidative stress in vitiligo patients.
To the best of our knowledge, this is the first study to investigate the influence of FOXO3a genetic
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variants, FOXO3a levels, and protein oxidation in vitiligo patients. In conclusion, our results
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demonstrate, first, that rs4946936 in the FOXO3a gene might be associated with susceptibility to vitiligo, especially active vitiligo, and second, that decreased FOXO3a levels in vitiligo patients might
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be associated with oxidative stress. Our observations confirm that oxidative stress is implicated in the
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pathophysiology of vitiligo, through increased protein oxidation and impaired antioxidant defenses. Therefore, we suggest that additional treatments might be useful for reducing the oxidative stress status of vitiligo patients. However, further studies with larger samples are required to elucidate the molecular mechanism responsible for the relationship between FOXO3a and oxidative stress and vitiligo.
Acknowledgements: This study was supported by Research Fund of University Mugla Sitki Kocman (No: 2011/68). We would also like to thank Dr. Seda Sevinc KAYA for her assistance in collecting patient samples.
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Declaration of Interest: The authors report that there is no conflict of interests.
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Table I. Clinical characteristics of vitiligo patients and healthy controls
Age
Vitiligo (n=82) Median (Min.-Max.) 36.68 (32.96-40.40)
Control (n=81) Median (Min.-Max.) 34 (18-80)
n (%) 37 (0.45)
n (%) 43 (0.53)
Gender, Female
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Clinical Characteristics
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Family History
− −
Yes No
64 (0.78) 18 (0.22)
Generalize Segmental Localize Acrofacial Vulgaris Focal
39 (0.48) 2 (0.02) 23 (0.28) 13 (0.16) 2 (0.02) 3 (0.04)
Stable Active (Unstable)
17 (0.21) 65 (0.79)
− −
0-15 16-30 31-45 46-70
17 (0.21) 29 (0.35) 16 (0.20) 20 (0.24)
− − − −
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Stability
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3
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Age onset
Duration
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Vitiligo Types
31 (0.38) 17 (0.21) 34 (0.41)
− −
− − −
p-value 0.919
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Table II. FOXO3a rs2253310 and rs4966936 single-nucleotide polymorphisms (SNP), primer sequences, annealing temperatures, restriction enzymes, and allele sizes Primer
Annealing Temp. (0C)
Restriction Enzymes Dpnl
F: 5’-GAGCTTGCTTTGGAGATGCA-3’ R: 5’- CCCAGTCACTCACATAGTCCT-3’
530C
rs4966936
F: 5’-GGGTCCTGAGAACTTCTGAGT-3’ R: 5’-GACATTCTGTAAGACATTCTGCCT-3’
530C
SC MA NU ED PT CE AC
Sfcl
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rs2253310
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SNP
Allele Size (bp) GG: 321 GC: 321, 127, 194 CC: 127, 194 TT:224 TC:224, 152, 72 CC:152, 72
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Table III. FOXO3a and AOPP levels and SOD and catalase activities in vitiligo patients and healthy controls Control (n=81) Median (Min.-Max.) 29.97 (5.49-56.33)
534.49 (214.39-1248.04)
368.83 (147.10-901.31)
S0378-1119(13)00039-5 doi: 10.1016/S0378-1119(13)00039-5 GENE 38
To appear in:
Gene
Accepted date:
19 December 2013
Please cite this article as: Ozel Turkcu, Ummuhani, Tekin, Nilgun Solak, Edgunlu, Tuba Gokdogan, Karakas, Sevim C ¸ elik, Oner, Setenay, The association of Foxo3a gene polymorphisms with serum Foxo3a levels and oxidative stress markers in vitiligo patients, Gene (2013), doi: 10.1016/S0378-1119(13)00039-5
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The association of Foxo3a gene polymorphisms with serum Foxo3a levels and oxidative stress markers in vitiligo patients
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b a a Ummuhani Ozel Turkcu , Nilgun Solak Tekin , Tuba Gokdogan Edgunlu ,
c d Sevim Çelik Karakas , Setenay Oner
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a Mugla Sitki Kocman University, MuglaSchool of Health Sciences, Department of Nutrition and
b
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Dietetics, Mugla, Turkey
Bulent Ecevit University, Faculty of Medicine, Department of Dermatology, Zonguldak, Turkey
Osmangazi University, Faculty of Medicine, Department of Biostatistic, Eskisehir, Turkey
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Corresponding Author:
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d
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c Bulent Ecevit University, Faculty of Medicine, Department of Medical BiologyZonguldak, Turkey
Ummuhani OZEL TURKCU
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Mugla Sıtkı Koçman Üniversitesi, Mugla Sağlık Yüksekokulu, S Blok, 1 Kat 48000 Kotekli/MUGLA/TURKEY Tel: +90 252 2113208; Fax: +90 252 2111880; E-mail:[email protected]
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Abstract
Vitiligo is an acquired epidermal pigment loss the skin with destruction of melanocytes. Oxidative
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stress is one of the major theories in the pathophysiology of vitiligo. FOXO3a is the forkhead members of the class O (FOXO) transcription factors, and plays important role in cell cycle regulation,
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apoptosis, oxidative stress, and DNA repair. The aim of our study was to investigate, for the first time,
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FOXO3a gene polymorphisms and FOXO3a protein levels, activities of superoxide dismutase (SOD) and catalase antioxidant enzymes in vitiligo patient and healthy controls. Moreover, plasma advanced oxidation protein products (AOPP) levels in subjects was evaluated to understand the possible role of protein oxidation in disease etiology. Studies groups included 82 vitiligo patients and 81 unrelated
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healthy controls. FOXO3a polymorphisms were determined using polymerase chain reaction-
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restriction fragment length polymorphism method. FOXO3a levels and catalase activity were measured by ELISA whereas AOPP levels and SOD activity were measured spectrophotometric
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analyses. We found a significant relationship between rs4946936 polymorphism of FOXO3a gene and
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vitiligo/active vitiligo patients (p=0.017; p=0.019 respectively), but not for rs2253310 (p>0.05). SOD activity and AOPP levels of vitiligo patient were increased compared with control group, whereas FOXO3a levels and catalase enzyme activity of vitiligo patient were decreased compared with control group (p0.05,
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data not shown).
FOXO3a rs2253310 and rs4946936 haplotypes and their distribution in vitiligo or active/stable vitiligo
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patients and healthy controls were also analyzed. The distributions of FOXO3a haplotypes with an
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estimation of odds ratios in the patients and controls are presented in Tables 6 and 7. Haplotype analysis of FOXO3a did not reveal any significant differences between the vitiligo or stable vitiligo patients and the controls (p>0.05). The frequencies of the GT and CT haplotypes were higher in the
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vitiligo and active vitiligo patients than in the control group.
4. Discussion
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Oxidative stress and genetic predisposition is thought to play an important role in the pathogenesis of
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vitiligo. To the best of our knowledge, our study is the first to evaluate the association of FOXO3a gene polymorphisms, FOXO3a levels, and protein oxidation with the pathophysiology of vitiligo. We
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also investigated activities of the main antioxidant enzymes in vitiligo patients and healthy subjects by measuring SOD and catalase activities. In this study, we demonstrated that rs4946936 of the FOXO3a gene might be associated with the risk of vitiligo. Interestingly, we found that FOXO3a levels were lower in vitiligo patients than in healthy controls. In addition, we found increased protein oxidation damage and altered activities of the antioxidant enzymes in the vitiligo patients.
It has been suggested that FOXO3a has critical roles in various biological processes, including cell cycle regulation, apoptosis, and oxidative stress, by controlling the expression of target genes (Furukawa-Hibi 2005; Brunet 1999a). Overexpression of FOXO3a has been shown to protect cells
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against oxidative damage via expression of antioxidant enzyme activity or regulation of reactive oxygen metabolism. Olmos et al. (2009) reported that FOXO3a can protect quiescent cells from
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oxidative stress regulating SOD2 and catalase genes. Other studies have indicated that FOXO3a
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directly activates transcription of antioxidant enzymes, namely, MnSOD, catalase, and peroxiredoxin
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3 (Kops 2002; Nemoto 2002; Chiribau 2008). FOXO3a-deficient hematopoietic stem cells have been observed to reduce the expression of genes involved in ROS detoxification, leading to elevated ROS
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levels (Yalcin 2008). Considering the roles of FOXO3a in oxidative stress and apoptosis, we mainly examined the relationship between FOXO3a gene polymorphisms and FOXO3a levels and vitiligo. In our study, we determined that the TT genotype and T allele frequencies in rs4946936 of the FOXO3a
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gene were associated with risk of vitiligo, but we did not find the same results for rs2253310. Moreover, when we evaluated vitiligo patients in terms of stability, TT genotype and T allele
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frequencies in rs4946936 were higher in active vitiligo patients compared to healthy controls. These
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results can be interpreted to mean that rs4946936 SNP might a putative risk factor for genetic
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susceptibility of vitiligo, especially active vitiligo. We also found that FOXO3a levels were lower in vitiligo patients than in healthy controls; the underlying mechanism is difficult to explain, as there are no published studies about serum/plasma FOXO3a levels. However, we think that the low FOXO3a levels might be associated with increased oxidative stress in the patients. In response to various types of cellular stress stimulus or exogenous agents, the transcriptional activity of FOXO3a can be regulated by post-transcriptional modifications, such as phosphorylation, acetylation, and ubiquitylation (Ponugoti 2012; Maiese 2009). The PI3K/AKT signal pathway is a major regulator for FOXO3a activity (Kops 2002). AKT phosphorylates FOXO3a, and then the phosphorylated FOXO3a protein is transported from the nucleus to the cytoplasm, leading to inhibition of its transcriptional
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activities for target genes (Kops 2002). Furthermore, AKT mediated phosphorylated FOXO3a causes ubiquitination and proteasomal degradation in cytoplasm (Maiese 2009; Plas 2003). However, it has
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been reported that increased AKT activity leads to enhance FOXO3a cytosolic localization and a
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decrease in MnSOD levels in prostate cancer cells (Shukla 2009). The increased cytosolic localization
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of FOXO3a protein and its degradation under oxidative stress conditions have also been reported (Nogueira 2008; Yang 2008; Lu 2013). Lu et al. (2013) showed that overproduction of ROS induced
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by a high glucose concentration decreases the expression of MnSOD via the PI3K-Akt-FOXO3a pathway, and they suggested it further aggravates oxidative stress in diabetic nephropathy. There is no data about whether or not which SNPs of FOXO3a have effect on its function. Nevertheless, only a
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study, Banasik et al. (2011) was investigated FOXO3a rs 2802292 and the levels of FOXO3a mRNA expression in skeletal muscle in young and elderly twins. Their results indicated that the minor G-
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allele of FOXO3A rs2802292 is associated with enhanced peripheral and hepatic insulin sensitivity,
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which may be mediated through increased skeletal muscle-FOXO3A mRNA expression. Also, they
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implicated that metabolic findings are consistent with observations of the G-allele being associated with increased longevity. In our study, we investigated the influence of FOXO3a gene variants (rs4946936, rs2253310) on FOXO3a levels as well, but we found no relationship between FOXO3a gene SNPs and FOXO3a levels. As such, our results might indicate that low FOXO3a levels might be associated with increased oxidative stress. However, we suggest that when FOXO3a SNPs, its mRNA and protein levels study in larger samples of vitiligo patients, these results might give more information about for the role FOXO3a levels in vitiligo pathogenesis.
Superoxide dismutase and catalase are well-known first line defense antioxidant enzymes that protect the cell against oxidative stress. Antioxidant enzyme activities have been investigated in clinical
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studies to assess the oxidative stress status in the epidermis and blood of vitiligo patients. Yildirim et al. (2003) reported significantly higher SOD activity in erythrocytes in generalized vitiligo patients
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than in controls. Similarly, Hazneci et al. (2005) and Jain et al. (2011) observed that blood SOD
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activity in vitiligo patients was significantly higher than in healthy controls. However, some studies
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have shown decreased SOD activity in the blood of patients with vitiligo (Koca 2004). In the present study, we observed that SOD activity was significantly higher in the vitiligo patients than in the
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healthy subjects. This increase might be due to overexpression of the SOD enzyme against increased superoxide anions in the vitiligo patients. In addition, we found that catalase activity in the vitiligo patients was significantly lower than in the healthy controls. In agreement with our study, several
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researchers have found decreased catalase activity in the skin and blood of vitiligo patients (Hazneci 2005; Sravani 2009). In these studies, different reasons were suggested for the decreased catalase
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activity. Some of the authors suggested that an accumulation of H2O2 might lead to inhibition of
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catalase activity (Schallreuter 1999d), while others proposed that catalase SNPs and its mutant alleles
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might affect both catalase activity and its expression in patients with vitiligo (Courtney 2002; Liu 2010). However, these results are insufficient to explain the decreased catalase activity in vitiligo patients definitively. In the present study, the increased SOD and decreased catalase activities could be due to a systemic accumulation of H2O2 in the body as a result of an inability to eliminate it efficiently. Consistent with the other studies, our results clearly support that oxidative stress might be implicated in the pathophysiology of vitiligo, due to alterations in the antioxidant defenses.
H2O2 is also a substrate of the nicotinamide adenine dinucleotide phosphate oxidase complex, and along with chloride ions, is converted into hypochlorous acid (HOCl) by the myeloperoxidase enzyme in activated neutrophils/leucocytes. As potent oxidants, HOCl and chloramine derivatives react with
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plasma proteins during oxidative stress, resulting in the formation of dityrosine, which contains crosslinked protein products known as advanced oxidation protein products (AOPP) (Hanasand 2012,
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Witko-Sarsat 1994; Alagozlu 2013). AOPP have been widely used as reliable biomarkers to assess
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oxidative protein damage (Witko-Sarsat 1994; Alagozlu 2013). However, according to the literature,
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AOPP levels have not been investigated in patients with vitiligo. In this study, we found that plasma AOPP levels in vitiligo patients were higher than in healthy controls. Thus, we suggest that oxidative
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protein damage might play a critical role in vitiligo pathogenesis, and that AOPP levels could be used for determining oxidative stress in vitiligo patients.
To the best of our knowledge, this is the first study to investigate the influence of FOXO3a genetic
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variants, FOXO3a levels, and protein oxidation in vitiligo patients. In conclusion, our results
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demonstrate, first, that rs4946936 in the FOXO3a gene might be associated with susceptibility to vitiligo, especially active vitiligo, and second, that decreased FOXO3a levels in vitiligo patients might
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be associated with oxidative stress. Our observations confirm that oxidative stress is implicated in the
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pathophysiology of vitiligo, through increased protein oxidation and impaired antioxidant defenses. Therefore, we suggest that additional treatments might be useful for reducing the oxidative stress status of vitiligo patients. However, further studies with larger samples are required to elucidate the molecular mechanism responsible for the relationship between FOXO3a and oxidative stress and vitiligo.
Acknowledgements: This study was supported by Research Fund of University Mugla Sitki Kocman (No: 2011/68). We would also like to thank Dr. Seda Sevinc KAYA for her assistance in collecting patient samples.
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Declaration of Interest: The authors report that there is no conflict of interests.
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Table I. Clinical characteristics of vitiligo patients and healthy controls
Age
Vitiligo (n=82) Median (Min.-Max.) 36.68 (32.96-40.40)
Control (n=81) Median (Min.-Max.) 34 (18-80)
n (%) 37 (0.45)
n (%) 43 (0.53)
Gender, Female
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Clinical Characteristics
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Family History
− −
Yes No
64 (0.78) 18 (0.22)
Generalize Segmental Localize Acrofacial Vulgaris Focal
39 (0.48) 2 (0.02) 23 (0.28) 13 (0.16) 2 (0.02) 3 (0.04)
Stable Active (Unstable)
17 (0.21) 65 (0.79)
− −
0-15 16-30 31-45 46-70
17 (0.21) 29 (0.35) 16 (0.20) 20 (0.24)
− − − −
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Stability
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Age onset
Duration
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Vitiligo Types
31 (0.38) 17 (0.21) 34 (0.41)
− −
− − −
p-value 0.919
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Table II. FOXO3a rs2253310 and rs4966936 single-nucleotide polymorphisms (SNP), primer sequences, annealing temperatures, restriction enzymes, and allele sizes Primer
Annealing Temp. (0C)
Restriction Enzymes Dpnl
F: 5’-GAGCTTGCTTTGGAGATGCA-3’ R: 5’- CCCAGTCACTCACATAGTCCT-3’
530C
rs4966936
F: 5’-GGGTCCTGAGAACTTCTGAGT-3’ R: 5’-GACATTCTGTAAGACATTCTGCCT-3’
530C
SC MA NU ED PT CE AC
Sfcl
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rs2253310
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SNP
Allele Size (bp) GG: 321 GC: 321, 127, 194 CC: 127, 194 TT:224 TC:224, 152, 72 CC:152, 72
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Table III. FOXO3a and AOPP levels and SOD and catalase activities in vitiligo patients and healthy controls Control (n=81) Median (Min.-Max.) 29.97 (5.49-56.33)
534.49 (214.39-1248.04)
368.83 (147.10-901.31)
