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Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress
GENE-41664; No. of pages: 5; 4C: Gene xxx (2016) xxx–xxx
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Research paper
Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress Negin Soroush a, Mania Radfar a,b,⁎, Armita Kakavand Hamidi c, Mohammad Abdollahi d, Mostafa Qorbani e, Farideh Razi f, Ensieh Nasli Esfahani f, Mahsa M Amoli c,⁎ a
Department of Clinical Pharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran Metabolic Disorders Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran d Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran e Department of Community Medicine, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran f Diabetes Research Center, Endocrinology and Metabolism Clinical Science Institute, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 13 July 2016 Received in revised form 3 November 2016 Accepted 7 November 2016 Available online xxxx Keywords: VDR gene polymorphism Diabetic foot ulcer Type 2 diabetes Oxidative stress
a b s t r a c t Purpose: The patient's suffering and financial costs affiliated with Diabetic Foot Ulcer (DFU), as one of the most important complications of diabetes, are highly undesirable and this highlights the importance of preventive medicine about this disorder. Furthermore hyperglycemia causes generation of free radicals which leads to oxidative stress (OS). Hence, this study aims to examine the association between vitamin D receptor (VDR) gene FokI polymorphism and DFU in Iranian population and also its correlation with OS biomarkers. Materials and methods: In a case-control study, a total of 212 patients with type 2 diabetes with and without diabetic foot ulcer were included. Genotyping was conducted by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Samples were analyzed for thiobarbituric reactive substances (TBARS) and ferric reducing ability of plasma (FRAP) as markers of OS. Results: The results indicated a significant difference in genotype frequencies of VDR gene FokI polymorphism in patients with diabetic foot ulcer in comparison to those without diabetic foot ulcer (TT + TC vs. CC p = 0.04; OR = 1.76; 95% CI = 1.02–3.05). Moreover, the patients carrying the T allele had a significantly higher level of TBARS (p = 0.01). Conclusions: We found a significant association between FokI functional variant of VDR gene and diabetic foot ulcer in an Iranian population. Increased levels of TBARS in patients carrying the T allele of FokI polymorphism indicate an association between this variant and OS in patients with diabetes. © 2016 Published by Elsevier B.V.
1. Introduction Diabetic foot ulcer (DFU) is known as one of the serious vascular complications of diabetes. Several factors contribute to its progression including peripheral neuropathy, peripheral vascular disease, poor Abbreviations: AMPs, antimicrobial peptides; BMI, body mass index; bp, base pair; CI, confidence intervals; DFU, diabetic foot ulcer; DN, diabetic nephropathy; DR, diabetic retinopathy; EDTA, ethylenediamine tetra acetic acid; FFA, free fatty acid; FPG, fasting plasma glucose; FRAP, ferric reducing ability of plasma; HbA1C, glycated hemoglobin; IQR, interquartile range; MDA, malondialdehyde; OR, Odds Ratio; OS, oxidative stress; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; SD, standard deviation; SNP, single nucleotide polymorphisms; TBARS, thiobarbituric reactive substances; TPTZ, tripyridyltriazine; T2DM, type 2 diabetes mellitus; VDR, vitamin D receptor. ⁎ Corresponding authors at: EMRI, Dr. Shariati Hospital, North Karegar St, Tehran 1411413137, Iran. E-mail addresses: [email protected] (M. Radfar), [email protected] (M.M. Amoli).
glycemic control and immune-suppression. It is estimated that up to 15% of patients with diabetes develop DFU and thus inflicted with serious consequences such as severe disability and decline in quality of life in addition to soaring costs (Aumiller & Dollahite, 2015). Several factors including age, oxidative stress (OS), and chronic inflammation play important roles in the development of DFU. It is assumed that stimuli such as hyperglycemia increase levels of circulating free fatty acid (FFA), and chronic inflammation might be associated with an increase in the production of reactive oxygen species (ROS) and a decrease in antioxidant activity, leading to OS (GarciaBailo et al., 2011). Oxidative stress has proven to be a leading mechanism in endothelial dysfunction, insulin resistance, and reducing pancreatic β-cells function causing diabetic microvascular and macrovascular complications (Oyenihi et al., 2015). Antioxidant vitamins have been shown to present several biological activities such as immune system stimulation and resistance against genetic changes. They prevent DNA damage induced by ROS (Oyenihi et
http://dx.doi.org/10.1016/j.gene.2016.11.012 0378-1119/© 2016 Published by Elsevier B.V.
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
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al., 2015). The antioxidant activity of vitamin D was elucidated by its ability to modulate free radical formation and upregulate glutathione peroxidase and superoxide dismutase in OS (Hamidieh et al., 2015). The association of vitamin D deficiency and increase in some OS biomarkers in patients with diabetes was shown in some studies (Gradinaru et al., 2012; Codoñer-Franch et al., 2012). Several lines of evidence on the positive effects of vitamin D in epithelial protection and healing process have been investigated in the last decade (Wang et al., 2004; Antal et al., 2011). Additionally, in vitro healing assays in primary cells from DFU patients showed that 1,25(OH)2D3 acts as an improving agent by restoring the production and gene expression of antimicrobial peptides (AMPs). However, there has been no statistically significant difference in vitamin D receptor (VDR) expression in healthy individuals compared to biopsies from DFU patients. Therefore the low responsiveness in a number of in vitro studies could be related to single nucleotide polymorphisms (SNPs) in VDR gene (Gonzalez-Curiel et al., 2014). The vitamin D nuclear receptor gene is located on chromosome 12q13 (Uitterlinden et al., 2004). A single nucleotide polymorphism of VDR gene is a so-called FokI (rs2228570 CN T), in the exon 2, consisting of a T allele to C change. The polymorphism is located in a start codon (ATG) and at the presence of the C variant. An alternative start site causes production of a protein with a different size (Valdivielso, 2006). This is the only known VDR polymorphism which is distinctively translated to two different VDR protein products compared to the other variants. In addition, this is the only polymorphism within VDR gene that is not linked to the other VDR variants, and thus it might have a unique role (Neyestani et al., 2013). It has also been verified that FokI polymorphism is significantly associated with diabetic complications. FokI T allele was shown to be associated with increased risk of Diabetic Retinopathy (DR) and has been considered as a biomarker which might predict the risk of DR in Han Chinese with T2DM (Zhong et al., 2015). A meta-analysis study has indicated that FokI polymorphism has a role in the level of susceptibility to Diabetic Nephropathy (DN) in Caucasian population (Liu et al., 2014). It has been observed that patients with diabetes and DFU have a higher prevalence of severe 25(OH)D deficiency in comparison to patients with diabetes without DFU (Zubair et al., 2013; Tiwari et al., 2013). To the best of our knowledge, there has not been a study to examine the association of VDR polymorphisms with diabetic foot ulcer, a life threatening complication of diabetes. This study was carried out based on previous findings that showed an association between VDR gene FokI polymorphism and diabetic complications such as retinopathy and nephropathy. Hence, this study aims to examine the association between vitamin D receptor (VDR) gene FokI polymorphism and DFU in Iranian population and also its correlation with OS biomarkers. 2. Materials and methods 2.1. Study design and study population This case-control study includes 212 participants, recruited from the Diabetes and Metabolic Disorders Clinic of Dr. Shariati hospital, Tehran University of Medical Sciences (TUMS). All subjects were Iranian and belonged to the same ethnic group. Patients diagnosed as T2DM according to the American Diabetes Association (ADA) guidelines (Association AD, 2014) were screened and selected for this study. Various Information on demographic features, anthropometric measures as body mass index (BMI), medical history, drug usage, family history, fasting plasma glucose (FPG), HbA1C, and serum creatinine were gathered using a questionnaire and face to face interview. The inclusion criteria were: willingness to participate, volunteers aged 30 to 65 years old and duration of diabetes N 5 years in both case and control groups, and also Wagner grade of 2 or 318 for foot ulcers in the case group.
The exclusion criteria for both groups include: type 1 diabetes mellitus, pregnancy, smoking in the past two years and intake of antioxidant supplements in the 3 months prior to start date of this study. The participants were classified into two main groups: Group A “control group” (n = 107): Patients with T2DM and no history of DFU. Group B “case group” (n = 105): Patients with T2DM and DFU. This study was approved by Endocrine and Metabolic Research Institute (EMRI) ethical committee (No.92-6-18:6-37) and was carried out from November 2013 to May 2015. A written informed consent was obtained from all participants before enrollment into the study. 2.2. Blood and serum sample collection Venous blood samples were obtained from the subjects and stored in anticoagulant ethylenediamine tetra acetic acid (EDTA) tubes for DNA extraction. Coagulated blood samples were used to examine OS biomarkers using serum plasma stored at −80 °C. 2.3. DNA extraction and genotyping Genomic DNA was isolated from samples collected in EDTA tubes using phenol chloroform protocol. Quality and quantity of the extracted DNA was observed by Nanodrop [UV–vis spectrophotometer, Thermo scientific, USA]. Genotype frequencies for VDR gene polymorphism of the patients were determined using polymerase chain reaction (PCR) amplification and Restriction Fragment Length Polymorphism (RFLP) analysis. A final volume of 20 μl PCR reaction consisting: 1.5 μl genomic DNA, 9.9 μl H2O, 0.8 μl forward primer (5′-CACTGACTCTGGCTCTGACCGT-3′), 0.8 μl reverse primer (5′-AACACCTTGCTTCTTCTCCCTCC-3′) and 7 μl master mix [Thermo scientific, (EU) Lithvania] was prepared. PCR products were amplified using a thermal master cycler gradient [Eppendorf, Hlnz, GmbH, Germany]. The initial DNA denaturation was at 95 °C for 5 min. Temperature of cycling was set for 34 cycles of denaturation at 95 °C for 30s, annealing at 68.1 °C for 30s and extension at 72 °C for 40s and a final extension at 72 °C for 5 min. PCR product length was 250 base pair (bp) and was loaded on a 2% agarose gel containing SYBR green and visualized. The 3.3 μl PCR product with 0.33 μl FokI restriction enzyme [fast digest enzyme, Thermo scientific, (EU) Lithvania], 0.7 μl buffer [Thermo scientific, (EU) Lithvania] and 5.67 μl H2O was digested overnight at 37 °C. Digest products yield (192, 58 bp) for homozygous (TT) and (250, 192, 58 bp) for heterozygous samples (TC) and homozygous CC remained uncut. All digest products were analyzed by electrophoresis on a 3% agarose gel. 2.4. Lipid peroxidation assay Thiobarbituric acid reactive substances (TBARS) were used to measure Lipid peroxidation in samples. Malondialdehyde (MDA) is considered as the oxidation end product of polyunsaturated fatty acids (PUFA). Concentration of MDA in the medium is a conventional measure of lipid peroxidation. The reaction of MDA with TBA in the samples creates a measurable pink color that has an absorbance at 532 nm (Radfar et al., 2005). [All chemicals were purchased from Sigma-Aldrich, Germany]. 2.5. Total antioxidant power (TAP) assay Total antioxidant power of plasma was determined by measuring Ferric reducing ability of plasma (FRAP). The method is based on the ability of antioxidants present in the medium to reduce Fe3 + tripyridyltriazine (TPTZ) complex (colorless) to Fe2+-TPTZ (blue colored) at low pH (Iris et al., 1999). Maximum absorption of the blue color Fe2 +-TPTZ complex was measured at 593 nm. [All chemicals were purchased from Sigma-Aldrich, Germany].
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
N. Soroush et al. / Gene xxx (2016) xxx–xxx
2.6. Statistical analysis Normal distribution of variables was examined using KolmogrovSmirnov test. Pearson chi-square test was used to evaluate independency of variables. Association in different groups and alleles or genotype frequencies of FokI polymorphism was calculated using odds ratio (OR) and 95% confidence intervals (CI). Association between genotypes of FokI polymorphism and possible confounding variables was evaluated using crude and adjusted logistic regression models. Continuous data were expressed as mean ± standard deviation (SD) of the samples. Since TBARS and FRAP were not normally distributed, the relation between these OS parameters and FokI genotype frequencies were determined using non-parametric MannWhitney U test, and data were expressed as median ± interquartile range (IQR). p-Values of b0.05 were considered statistically significant. All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS, version 16.0, USA). The study power was considered as 80% for the number of samples included in the study.
3. Results 3.1. Baseline characteristics of study participants A total of 456 related subjects were screened and 212 eligible subjects were enrolled in the study. Clinical characteristics of patients are described in Table 1. For parameters such as age, BMI, and family history of diabetes there was an acceptable matching among participants (p N 0.05).
3.2. Comparison of allele and genotype frequencies of FokI variant of VDR gene between case and control groups The allele and genotype frequencies for distribution of VDR gene FokI polymorphism among patients with DFU and control group without DFU are shown in Table 2. The result of genotyping showed that the TT and TC genotypes were significantly more frequent than genotype CC in the case group compared to controls in the T dominant crude model (p = 0.04; OR = 1.76; 95% CI = [1.02–3.05]). There was a significant association between gender and DFU in patients with TT and TC genotypes (p = 0.01). Additionally, The results of Allele frequency (T vs C) showed that T allele was significantly more frequent in the case group compared to the control (p = 0.02; OR = 1.65; 95% CI = [1.075–2.55]). Using Logistic regression analysis, we determined that considering the effects of potential confounding factors including diabetes duration, HbA1c and gender, there was not a significant association between FokI polymorphism and DFU (Table 3).
Table 1 Demographic and disease characteristics of the study groups.
Variables
Case (n = 105)
Control (n = 107)
Gendera (male) Ageb (yr) HgbA1Cb (%) FHa (yes) Duration of diabetesb (yr) BMIb (kg/m2)
62(59) 55.96(7.8) 8(1.2) 98(91.5) 15.4(6.9) 28.9(4.3)
36(33.6) 55.88(6.6) 7.7(1.2) 88(82.2) 13.1(6.4) 28.5(4.6)
HbA1C: Glycated hemoglobin; FH: Family history of diabetes; BMI: body mass index. a Categorical variables are expressed as number (%). b Continuous variables are expressed as mean (SD).
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Table 2 Distribution of genotype and allele frequencies of VDR gene polymorphism at FokI (rs2228570 CNT) in patients with diabetes in study groups.
VDR (FokI) Genotype CC TC TT Allele C T
Cases n (%)
Controls n (%)
49 (46.7) 44 (41.9) 12 (11.4)
65 (60.7) 36 (33.6) 6 (5.6)
142 (67.6) 68 (32.3)
166 (77.5) 48 (22.4)
TT + TC vs. CC; p = 0.04;OR = 1.76; 95% CI = 1.02–3.05.
3.3. Allele and genotype frequencies of VDR gene polymorphism in patients with different grades of DFU As indicated in Table 4, there was a correlation between the Wagner's grade of foot ulcers and genotype and allele frequencies. A significant difference in frequencies of TT and CT genotypes between patients with grade 2 and 3 ulcers was observed (p = 0.04; OR = 2.34; 95% CI: [1.01–6.14]). Also, the frequency of T allele was significantly higher in patients with grade 3 ulcers, compared to other groups (p = 0.03; OR = 1.92; 95%CI: [1.01–3.68]). 3.4. Oxidative stress parameters Table 5 shows OS parameters, TBARS and FRAP levels expressed as median ± IQR. A significant augmentation in lipid peroxidation and TBARS levels were observed in the plasma of patients with diabetes carrying TT or TC genotypes of FokI polymorphism compared to those with CC genotype (p = 0.01). Our results showed no statistically significant difference in FRAP levels in patients with different FokI genotypes (p = 0.31). We also measured the level of OS parameters in 41 healthy subjects. The results showed no significant association between TBARS and FRAP levels with neither TT + TC nor CC genotypes in healthy subjects (respectively p = 0.76, p = 0.29). p b 0.05 is considered statistically significant. 4. Discussion In this study, we found an association between FokI polymorphism of VDR gene and DFU. The results revealed that the frequencies of both T allele and TT and TC genotypes were significantly higher in the case group compared to the controls. Furthermore we observed a significant correlation between T allele and Wagner grade of ulcers showing a high frequency of T allele in patients with grade 3 ulcers. It has been reported that vitamin D deficiency as a nutritional factor may influence glucose intolerance, distorted insulin secretion, insulin resistance and T2DM (Mezza et al., 2012; Jain et al., 2014). The potential role of Vitamin D in progression of T2DM has been speculated to be through the activation of its nuclear receptor (Xuan et al., 2013). The genetic contribution of vitamin D in diabetes has been demonstrated in
Table 3 Adjusted p value for diabetic foot ulcer risk factors. Risk factors
OR
95% CI
a
Diabetes Duration HbA1c Gender FokI genotypes
1.07 1.30 0.31 0.56
(1.02–1.12) (1.02–1.66) (0.17–0.57) (0.31–1.02)
0.004 0.031 0.000 0.061
a
p value
Logistic regression.
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
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Table 4 Allele and genotype frequencies of VDR gene polymorphism in patients with DFU grouped according to Wagner classification. FokI (rs2228570 CNT) VDR gene polymorphism Wagner grade
2 3 a b
Genotype n (%)
Allele n (%)
CC
TC
TT
C
T
37 (52.86) 11 (32.35)b
27 (38.57) 17 (50)
6 (8.57) 6 (17.65)
101 (72.14) 39 (57.35)
39 (27.86) 29 (42.65)a
T vs C; p = 0.03. TT + TC vs CC; p = 0.04.
several studies through investigating the correlation between VDR gene polymorphisms and T2DM. The distinct variant of VDR gene, namely FokI has previously shown association with T2DM (especially in the Asian population), (Li et al., 2013; Wang et al., 2012) insulin sensitivity (Chiu et al., 2001), hypertension (Swapna et al., 2011), metabolic syndrome and body mass index (Zhao et al., 2014; Mackawy & Badawi, 2014). Since these studies have been carried out in various populations with different ethnic backgrounds the results are inconclusive. In addition, the relationship between VDR polymorphisms and other important complications of diabetes such as retinopathy (Zhong et al., 2015), and nephropathy (Yokoyama et al., 2012) has been revealed in some recent studies. These findings may highlight the possible role of vitamin D in diabetic foot complication. It would have been valuable if we had further information regarding the vitamin D status of our patients which must be considered in future studies. It has been demonstrated that hyperglycemia induces production of ROS, which finally leads to increased OS in diabetic patients (Johansen et al., 2005). OS leads to increased lipid peroxidation and elevated production of MDA which is the end product of PUFA (Akkuş et al., 1996). OS also contributes to the development and progression of vascular and neural dysfunction in diabetic patients (Yorek, 2003). It has been shown in some studies that patients with diabetes and foot ulcer have higher level of serum MDA (Singh et al., 2008) and lymphocyte MDA (Arya et al., 2011) in comparison to patients without ulcer or healthy controls. On the other hand, it is suggested that vitamin D contributes to OS process (Garcia-Bailo et al., 2011). A significant association between reduced vitamin D level and increase in some OS biomarkers such as MDA has been shown previously (Codoñer-Franch et al., 2012). Furthermore, significant association between severe vitamin D deficiency and elevated inflammatory cytokine concentrations in diabetic patients and particularly patients with foot infection was observed (Tiwari et al., 2014). We observed that TT and TC genotypes were more frequent among males with DFU. Previous study among the Iranian population found that diabetic patients with TT genotype of FokI Polymorphism might be considered low responders to an increase in 25(OH)D levels following vitamin D intake (Neyestani et al., 2013). This result is in concordance with ours, which showed males who carried TT genotypes
Table 5 – Oxidative stress parameters in different genotypes of VDR gene polymorphism.
Group
Oxidative stress parameters
VDR (FokI) genotype TT + TC
(n = 81) Patients with diabetes TBARSa (μmol/L) 28.89 (9.73) a FRAP (mmol/L) 295.57 (85)
Healthy subjects
CC
p value
(n = 106) 27.10 (5.90) 0.01 273.73 (68.75) 0.31
(n = 15) (n = 26) TBARSa (μmol/L) 15.94 (5.01) 15.8 (3.96) 0.76 a FRAP (mmol/L) 234.90 (49.66) 246.06 (43.17) 0.29
TBARS: Thiobarbituric acid reactive substances; FRAP: Ferric reducing ability of plasma. a Variables are expressed as median (IQR).
might be low responders and therefore more frequently present DFU than females. In the current study we evaluated the relationship between FokI variant of VDR gene and OS biomarkers in diabetic patients. The results demonstrated that there was a significant higher concentration of MDA using TBARS assay in the serum of patients with TT and TC genotypes in comparison to patients with CC genotype. Although the total antioxidant capacity of plasma was higher in TT and TC genotypes, no statistically significant association between FRAP levels and FokI polymorphisms were observed. This higher capacity, although insignificant, can be the result of hedging against OS and compensating by increasing FRAP. In a recent study in Iranian population, it has been shown that there was an association between FokI variants and OS biomarkers (ShabBidar et al., 2015). Similarly an association between TBARS OS biomarker and FokI polymorphism in patients with diabetes was demonstrated in our results, however no significant association was observed between OS biomarkers and FokI genotypes in healthy population in our study. In summary, in our study we observed a significantly higher frequency of T allele of VDR gene FokI polymorphism among Iranian patients with DFU and also among patients with higher level of TBARS. Therefore this allele might be used as a candidate biomarker in preventive medicine to reduce the risk of this life threatening complication of diabetes. However, more investigation is needed to support our finding especially in separated groups of males and females, to diminish the potential confounding effect of gender. Furthermore, considering the possible effect of vitamin D supplementation in reduction of insulin resistance in T2DM (Talaei et al., 2013) and also its effect on reducing inflammatory markers and increasing the total antioxidant capacity (Cavalcante et al., 2015), these findings might pave the way of designing a new generation of treatment for DFU, using vitamin D supplementation. The number of study population was the limitation of our study. Further studies on different ethnic populations and also examination of other genetic polymorphisms of VDR gene are required to confirm our results in this study. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgement This study has been funded by EMRI, Tehran University of Medical Sciences (1392-01-86-1550). References Akkuş, İ., Kalak, S., Vural, H., Çaglayan, O., Menekşe, E., Can, G., et al., 1996. Leukocyte lipid peroxidation, superoxide dismutase, glutathione peroxidase and serum and leukocyte vitamin C levels of patients with type II diabetes mellitus. Clin. Chim. Acta 244, 221–227. Antal, A.S., Dombrowski, Y., Koglin, S., Ruzicka, T., Schauber, J., 2011. Impact of vitamin D3 on cutaneous immunity and antimicrobial peptide expression. Dermatoendocrinol 3, 18–22. Arya, A.K., Pokharia, D., Tripathi, K., 2011. Relationship between oxidative stress and apoptotic markers in lymphocytes of diabetic patients with chronic non healing wound. Diabetes Res. Clin. Pract. 94, 377–384. Association AD, 2014. Diagnosis and classification of diabetes mellitus. Diabetes Care 37, S81–S90. Aumiller, W.D., Dollahite, H.A., 2015. Pathogenesis and management of diabetic foot ulcers. JAAPA 28, 28–34. Cavalcante, I.G., Silva, A.S., Costa, M.J., Persuhn, D.C., Issa, C.I., Freire, T.L., et al., 2015. Effect of vitamin D3 supplementation and influence of BsmI polymorphism of the VDR gene of the inflammatory profile and oxidative stress in elderly women with vitamin D insufficiency: vitamin D3 megadose reduces inflammatory markers. Exp. Gerontol. 66, 10–16. Chiu, K.C., Chuang, L.-M., Yoon, C., 2001. The vitamin D receptor polymorphism in the translation initiation codon is a risk factor for insulin resistance in glucose tolerant Caucasians. BMC Med. Genet. 2, 2. Codoñer-Franch, P., Tavárez-Alonso, S., Simó-Jordá, R., Laporta-Martín, P., Carratalá-Calvo, A., Alonso-Iglesias, E., 2012. Vitamin D status is linked to biomarkers of oxidative
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Shab-Bidar, S., Neyestani, T.R., Djazayery, A., 2015. The interactive effect of improvement of vitamin D status and VDR FokI variants on oxidative stress in type 2 diabetic subjects: a randomized controlled trial. Eur. J. Clin. Nutr. 69, 216–222. Singh, S.K., Sahay, R.K., Krishna, A., 2008. Oxidative stress in diabetic foot ulcer. Diabetes & Metabolic Syndrome: Clinical Research & Reviews 2, 109–113. Swapna, N., Vamsi, U.M., Usha, G., Padma, T., 2011. Risk conferred by FokI polymorphism of vitamin D receptor (VDR) gene for essential hypertension. Indian J Hum Genet 17, 201–206. Talaei, A., Mohamadi, M., Adgi, Z., 2013. The effect of vitamin D on insulin resistance in patients with type 2 diabetes. Diabetol. Metab. Syndr. 5, 8. Tiwari, S., Pratyush, D.D., Gupta, B., Dwivedi, A., Chaudhary, S., Rayicherla, R.K., et al., 2013. Prevalence and severity of vitamin D deficiency in patients with diabetic foot infection. Br. J. Nutr. 109, 99–102. Tiwari, S., Pratyush, D.D., Gupta, S.K., Singh, S.K., 2014. Vitamin D deficiency is associated with inflammatory cytokine concentrations in patients with diabetic foot infection. Br. J. Nutr. 112, 1938–1943. Uitterlinden, A.G., Fang, Y., Van Meurs, J.B., Pols, H.A., Van Leeuwen, J.P., 2004. Genetics and biology of vitamin D receptor polymorphisms. Gene 338, 143–156. Valdivielso, J.M., Fernandez, E., 2006. Vitamin D receptor polymorphisms and diseases. Clin Chim Acta 371, 1–12. Wang, Q., Xi, B., Reilly, K.H., Liu, M., Fu, M., 2012. Quantitative assessment of the associations between four polymorphisms (FokI, ApaI, BsmI, TaqI) of vitamin D receptor gene and risk of diabetes mellitus. Mol. Biol. Rep. 39, 9405–9414. Wang, T.T., Nestel, F.P., Bourdeau, V., Nagai, Y., Wang, Q., Liao, J., et al., 2004. Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immuno 173, 2909–2912. Xuan, Y., Zhao, H.Y., Liu, J.M., 2013. Vitamin D and type 2 diabetes mellitus (D2). J diabetes 5, 261–267. Yokoyama, K., Nakashima, A., Urashima, M., Suga, H., Mimura, T., Kimura, Y., et al., 2012. Interactions between serum vitamin D levels and vitamin D reiceptor gene FokI polymorphisms for renal function in patients with type 2 diabetes. PLoS One 7, e51171. Yorek, M.A., 2003. The role of oxidative stress in diabetic vascular and neural disease. Free Radic. Res. 37, 471–480. Zhao, Y., Liao, S., He, J., Jin, Y., Fu, H., Chen, X., et al., 2014. Association of vitamin D receptor gene polymorphisms with metabolic syndrome: a case-control design of populationbased cross-sectional study in North China. Lipids Health Dis. 13, 129. Zhong, X., Du, Y., Lei, Y., Liu, N., Guo, Y., Pan, T., 2015. Effects of vitamin D receptor gene polymorphism and clinical characteristics on risk of diabetic retinopathy in Han Chinese type 2 diabetes patients. Gene 566 (2), 212–216. Zubair, M., Malik, A., Meerza, D., Ahmad, J., 2013. 25-Hydroxyvitamin D [25(OH)D] levels and diabetic foot ulcer: is there any relationship? Diabetes & Metabolic Syndrome: Clinical Research & Reviews 7, 148–153.
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
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Research paper
Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress Negin Soroush a, Mania Radfar a,b,⁎, Armita Kakavand Hamidi c, Mohammad Abdollahi d, Mostafa Qorbani e, Farideh Razi f, Ensieh Nasli Esfahani f, Mahsa M Amoli c,⁎ a
Department of Clinical Pharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran Metabolic Disorders Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran d Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran e Department of Community Medicine, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran f Diabetes Research Center, Endocrinology and Metabolism Clinical Science Institute, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 13 July 2016 Received in revised form 3 November 2016 Accepted 7 November 2016 Available online xxxx Keywords: VDR gene polymorphism Diabetic foot ulcer Type 2 diabetes Oxidative stress
a b s t r a c t Purpose: The patient's suffering and financial costs affiliated with Diabetic Foot Ulcer (DFU), as one of the most important complications of diabetes, are highly undesirable and this highlights the importance of preventive medicine about this disorder. Furthermore hyperglycemia causes generation of free radicals which leads to oxidative stress (OS). Hence, this study aims to examine the association between vitamin D receptor (VDR) gene FokI polymorphism and DFU in Iranian population and also its correlation with OS biomarkers. Materials and methods: In a case-control study, a total of 212 patients with type 2 diabetes with and without diabetic foot ulcer were included. Genotyping was conducted by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Samples were analyzed for thiobarbituric reactive substances (TBARS) and ferric reducing ability of plasma (FRAP) as markers of OS. Results: The results indicated a significant difference in genotype frequencies of VDR gene FokI polymorphism in patients with diabetic foot ulcer in comparison to those without diabetic foot ulcer (TT + TC vs. CC p = 0.04; OR = 1.76; 95% CI = 1.02–3.05). Moreover, the patients carrying the T allele had a significantly higher level of TBARS (p = 0.01). Conclusions: We found a significant association between FokI functional variant of VDR gene and diabetic foot ulcer in an Iranian population. Increased levels of TBARS in patients carrying the T allele of FokI polymorphism indicate an association between this variant and OS in patients with diabetes. © 2016 Published by Elsevier B.V.
1. Introduction Diabetic foot ulcer (DFU) is known as one of the serious vascular complications of diabetes. Several factors contribute to its progression including peripheral neuropathy, peripheral vascular disease, poor Abbreviations: AMPs, antimicrobial peptides; BMI, body mass index; bp, base pair; CI, confidence intervals; DFU, diabetic foot ulcer; DN, diabetic nephropathy; DR, diabetic retinopathy; EDTA, ethylenediamine tetra acetic acid; FFA, free fatty acid; FPG, fasting plasma glucose; FRAP, ferric reducing ability of plasma; HbA1C, glycated hemoglobin; IQR, interquartile range; MDA, malondialdehyde; OR, Odds Ratio; OS, oxidative stress; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; SD, standard deviation; SNP, single nucleotide polymorphisms; TBARS, thiobarbituric reactive substances; TPTZ, tripyridyltriazine; T2DM, type 2 diabetes mellitus; VDR, vitamin D receptor. ⁎ Corresponding authors at: EMRI, Dr. Shariati Hospital, North Karegar St, Tehran 1411413137, Iran. E-mail addresses: [email protected] (M. Radfar), [email protected] (M.M. Amoli).
glycemic control and immune-suppression. It is estimated that up to 15% of patients with diabetes develop DFU and thus inflicted with serious consequences such as severe disability and decline in quality of life in addition to soaring costs (Aumiller & Dollahite, 2015). Several factors including age, oxidative stress (OS), and chronic inflammation play important roles in the development of DFU. It is assumed that stimuli such as hyperglycemia increase levels of circulating free fatty acid (FFA), and chronic inflammation might be associated with an increase in the production of reactive oxygen species (ROS) and a decrease in antioxidant activity, leading to OS (GarciaBailo et al., 2011). Oxidative stress has proven to be a leading mechanism in endothelial dysfunction, insulin resistance, and reducing pancreatic β-cells function causing diabetic microvascular and macrovascular complications (Oyenihi et al., 2015). Antioxidant vitamins have been shown to present several biological activities such as immune system stimulation and resistance against genetic changes. They prevent DNA damage induced by ROS (Oyenihi et
http://dx.doi.org/10.1016/j.gene.2016.11.012 0378-1119/© 2016 Published by Elsevier B.V.
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
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al., 2015). The antioxidant activity of vitamin D was elucidated by its ability to modulate free radical formation and upregulate glutathione peroxidase and superoxide dismutase in OS (Hamidieh et al., 2015). The association of vitamin D deficiency and increase in some OS biomarkers in patients with diabetes was shown in some studies (Gradinaru et al., 2012; Codoñer-Franch et al., 2012). Several lines of evidence on the positive effects of vitamin D in epithelial protection and healing process have been investigated in the last decade (Wang et al., 2004; Antal et al., 2011). Additionally, in vitro healing assays in primary cells from DFU patients showed that 1,25(OH)2D3 acts as an improving agent by restoring the production and gene expression of antimicrobial peptides (AMPs). However, there has been no statistically significant difference in vitamin D receptor (VDR) expression in healthy individuals compared to biopsies from DFU patients. Therefore the low responsiveness in a number of in vitro studies could be related to single nucleotide polymorphisms (SNPs) in VDR gene (Gonzalez-Curiel et al., 2014). The vitamin D nuclear receptor gene is located on chromosome 12q13 (Uitterlinden et al., 2004). A single nucleotide polymorphism of VDR gene is a so-called FokI (rs2228570 CN T), in the exon 2, consisting of a T allele to C change. The polymorphism is located in a start codon (ATG) and at the presence of the C variant. An alternative start site causes production of a protein with a different size (Valdivielso, 2006). This is the only known VDR polymorphism which is distinctively translated to two different VDR protein products compared to the other variants. In addition, this is the only polymorphism within VDR gene that is not linked to the other VDR variants, and thus it might have a unique role (Neyestani et al., 2013). It has also been verified that FokI polymorphism is significantly associated with diabetic complications. FokI T allele was shown to be associated with increased risk of Diabetic Retinopathy (DR) and has been considered as a biomarker which might predict the risk of DR in Han Chinese with T2DM (Zhong et al., 2015). A meta-analysis study has indicated that FokI polymorphism has a role in the level of susceptibility to Diabetic Nephropathy (DN) in Caucasian population (Liu et al., 2014). It has been observed that patients with diabetes and DFU have a higher prevalence of severe 25(OH)D deficiency in comparison to patients with diabetes without DFU (Zubair et al., 2013; Tiwari et al., 2013). To the best of our knowledge, there has not been a study to examine the association of VDR polymorphisms with diabetic foot ulcer, a life threatening complication of diabetes. This study was carried out based on previous findings that showed an association between VDR gene FokI polymorphism and diabetic complications such as retinopathy and nephropathy. Hence, this study aims to examine the association between vitamin D receptor (VDR) gene FokI polymorphism and DFU in Iranian population and also its correlation with OS biomarkers. 2. Materials and methods 2.1. Study design and study population This case-control study includes 212 participants, recruited from the Diabetes and Metabolic Disorders Clinic of Dr. Shariati hospital, Tehran University of Medical Sciences (TUMS). All subjects were Iranian and belonged to the same ethnic group. Patients diagnosed as T2DM according to the American Diabetes Association (ADA) guidelines (Association AD, 2014) were screened and selected for this study. Various Information on demographic features, anthropometric measures as body mass index (BMI), medical history, drug usage, family history, fasting plasma glucose (FPG), HbA1C, and serum creatinine were gathered using a questionnaire and face to face interview. The inclusion criteria were: willingness to participate, volunteers aged 30 to 65 years old and duration of diabetes N 5 years in both case and control groups, and also Wagner grade of 2 or 318 for foot ulcers in the case group.
The exclusion criteria for both groups include: type 1 diabetes mellitus, pregnancy, smoking in the past two years and intake of antioxidant supplements in the 3 months prior to start date of this study. The participants were classified into two main groups: Group A “control group” (n = 107): Patients with T2DM and no history of DFU. Group B “case group” (n = 105): Patients with T2DM and DFU. This study was approved by Endocrine and Metabolic Research Institute (EMRI) ethical committee (No.92-6-18:6-37) and was carried out from November 2013 to May 2015. A written informed consent was obtained from all participants before enrollment into the study. 2.2. Blood and serum sample collection Venous blood samples were obtained from the subjects and stored in anticoagulant ethylenediamine tetra acetic acid (EDTA) tubes for DNA extraction. Coagulated blood samples were used to examine OS biomarkers using serum plasma stored at −80 °C. 2.3. DNA extraction and genotyping Genomic DNA was isolated from samples collected in EDTA tubes using phenol chloroform protocol. Quality and quantity of the extracted DNA was observed by Nanodrop [UV–vis spectrophotometer, Thermo scientific, USA]. Genotype frequencies for VDR gene polymorphism of the patients were determined using polymerase chain reaction (PCR) amplification and Restriction Fragment Length Polymorphism (RFLP) analysis. A final volume of 20 μl PCR reaction consisting: 1.5 μl genomic DNA, 9.9 μl H2O, 0.8 μl forward primer (5′-CACTGACTCTGGCTCTGACCGT-3′), 0.8 μl reverse primer (5′-AACACCTTGCTTCTTCTCCCTCC-3′) and 7 μl master mix [Thermo scientific, (EU) Lithvania] was prepared. PCR products were amplified using a thermal master cycler gradient [Eppendorf, Hlnz, GmbH, Germany]. The initial DNA denaturation was at 95 °C for 5 min. Temperature of cycling was set for 34 cycles of denaturation at 95 °C for 30s, annealing at 68.1 °C for 30s and extension at 72 °C for 40s and a final extension at 72 °C for 5 min. PCR product length was 250 base pair (bp) and was loaded on a 2% agarose gel containing SYBR green and visualized. The 3.3 μl PCR product with 0.33 μl FokI restriction enzyme [fast digest enzyme, Thermo scientific, (EU) Lithvania], 0.7 μl buffer [Thermo scientific, (EU) Lithvania] and 5.67 μl H2O was digested overnight at 37 °C. Digest products yield (192, 58 bp) for homozygous (TT) and (250, 192, 58 bp) for heterozygous samples (TC) and homozygous CC remained uncut. All digest products were analyzed by electrophoresis on a 3% agarose gel. 2.4. Lipid peroxidation assay Thiobarbituric acid reactive substances (TBARS) were used to measure Lipid peroxidation in samples. Malondialdehyde (MDA) is considered as the oxidation end product of polyunsaturated fatty acids (PUFA). Concentration of MDA in the medium is a conventional measure of lipid peroxidation. The reaction of MDA with TBA in the samples creates a measurable pink color that has an absorbance at 532 nm (Radfar et al., 2005). [All chemicals were purchased from Sigma-Aldrich, Germany]. 2.5. Total antioxidant power (TAP) assay Total antioxidant power of plasma was determined by measuring Ferric reducing ability of plasma (FRAP). The method is based on the ability of antioxidants present in the medium to reduce Fe3 + tripyridyltriazine (TPTZ) complex (colorless) to Fe2+-TPTZ (blue colored) at low pH (Iris et al., 1999). Maximum absorption of the blue color Fe2 +-TPTZ complex was measured at 593 nm. [All chemicals were purchased from Sigma-Aldrich, Germany].
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
N. Soroush et al. / Gene xxx (2016) xxx–xxx
2.6. Statistical analysis Normal distribution of variables was examined using KolmogrovSmirnov test. Pearson chi-square test was used to evaluate independency of variables. Association in different groups and alleles or genotype frequencies of FokI polymorphism was calculated using odds ratio (OR) and 95% confidence intervals (CI). Association between genotypes of FokI polymorphism and possible confounding variables was evaluated using crude and adjusted logistic regression models. Continuous data were expressed as mean ± standard deviation (SD) of the samples. Since TBARS and FRAP were not normally distributed, the relation between these OS parameters and FokI genotype frequencies were determined using non-parametric MannWhitney U test, and data were expressed as median ± interquartile range (IQR). p-Values of b0.05 were considered statistically significant. All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS, version 16.0, USA). The study power was considered as 80% for the number of samples included in the study.
3. Results 3.1. Baseline characteristics of study participants A total of 456 related subjects were screened and 212 eligible subjects were enrolled in the study. Clinical characteristics of patients are described in Table 1. For parameters such as age, BMI, and family history of diabetes there was an acceptable matching among participants (p N 0.05).
3.2. Comparison of allele and genotype frequencies of FokI variant of VDR gene between case and control groups The allele and genotype frequencies for distribution of VDR gene FokI polymorphism among patients with DFU and control group without DFU are shown in Table 2. The result of genotyping showed that the TT and TC genotypes were significantly more frequent than genotype CC in the case group compared to controls in the T dominant crude model (p = 0.04; OR = 1.76; 95% CI = [1.02–3.05]). There was a significant association between gender and DFU in patients with TT and TC genotypes (p = 0.01). Additionally, The results of Allele frequency (T vs C) showed that T allele was significantly more frequent in the case group compared to the control (p = 0.02; OR = 1.65; 95% CI = [1.075–2.55]). Using Logistic regression analysis, we determined that considering the effects of potential confounding factors including diabetes duration, HbA1c and gender, there was not a significant association between FokI polymorphism and DFU (Table 3).
Table 1 Demographic and disease characteristics of the study groups.
Variables
Case (n = 105)
Control (n = 107)
Gendera (male) Ageb (yr) HgbA1Cb (%) FHa (yes) Duration of diabetesb (yr) BMIb (kg/m2)
62(59) 55.96(7.8) 8(1.2) 98(91.5) 15.4(6.9) 28.9(4.3)
36(33.6) 55.88(6.6) 7.7(1.2) 88(82.2) 13.1(6.4) 28.5(4.6)
HbA1C: Glycated hemoglobin; FH: Family history of diabetes; BMI: body mass index. a Categorical variables are expressed as number (%). b Continuous variables are expressed as mean (SD).
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Table 2 Distribution of genotype and allele frequencies of VDR gene polymorphism at FokI (rs2228570 CNT) in patients with diabetes in study groups.
VDR (FokI) Genotype CC TC TT Allele C T
Cases n (%)
Controls n (%)
49 (46.7) 44 (41.9) 12 (11.4)
65 (60.7) 36 (33.6) 6 (5.6)
142 (67.6) 68 (32.3)
166 (77.5) 48 (22.4)
TT + TC vs. CC; p = 0.04;OR = 1.76; 95% CI = 1.02–3.05.
3.3. Allele and genotype frequencies of VDR gene polymorphism in patients with different grades of DFU As indicated in Table 4, there was a correlation between the Wagner's grade of foot ulcers and genotype and allele frequencies. A significant difference in frequencies of TT and CT genotypes between patients with grade 2 and 3 ulcers was observed (p = 0.04; OR = 2.34; 95% CI: [1.01–6.14]). Also, the frequency of T allele was significantly higher in patients with grade 3 ulcers, compared to other groups (p = 0.03; OR = 1.92; 95%CI: [1.01–3.68]). 3.4. Oxidative stress parameters Table 5 shows OS parameters, TBARS and FRAP levels expressed as median ± IQR. A significant augmentation in lipid peroxidation and TBARS levels were observed in the plasma of patients with diabetes carrying TT or TC genotypes of FokI polymorphism compared to those with CC genotype (p = 0.01). Our results showed no statistically significant difference in FRAP levels in patients with different FokI genotypes (p = 0.31). We also measured the level of OS parameters in 41 healthy subjects. The results showed no significant association between TBARS and FRAP levels with neither TT + TC nor CC genotypes in healthy subjects (respectively p = 0.76, p = 0.29). p b 0.05 is considered statistically significant. 4. Discussion In this study, we found an association between FokI polymorphism of VDR gene and DFU. The results revealed that the frequencies of both T allele and TT and TC genotypes were significantly higher in the case group compared to the controls. Furthermore we observed a significant correlation between T allele and Wagner grade of ulcers showing a high frequency of T allele in patients with grade 3 ulcers. It has been reported that vitamin D deficiency as a nutritional factor may influence glucose intolerance, distorted insulin secretion, insulin resistance and T2DM (Mezza et al., 2012; Jain et al., 2014). The potential role of Vitamin D in progression of T2DM has been speculated to be through the activation of its nuclear receptor (Xuan et al., 2013). The genetic contribution of vitamin D in diabetes has been demonstrated in
Table 3 Adjusted p value for diabetic foot ulcer risk factors. Risk factors
OR
95% CI
a
Diabetes Duration HbA1c Gender FokI genotypes
1.07 1.30 0.31 0.56
(1.02–1.12) (1.02–1.66) (0.17–0.57) (0.31–1.02)
0.004 0.031 0.000 0.061
a
p value
Logistic regression.
Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012
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Table 4 Allele and genotype frequencies of VDR gene polymorphism in patients with DFU grouped according to Wagner classification. FokI (rs2228570 CNT) VDR gene polymorphism Wagner grade
2 3 a b
Genotype n (%)
Allele n (%)
CC
TC
TT
C
T
37 (52.86) 11 (32.35)b
27 (38.57) 17 (50)
6 (8.57) 6 (17.65)
101 (72.14) 39 (57.35)
39 (27.86) 29 (42.65)a
T vs C; p = 0.03. TT + TC vs CC; p = 0.04.
several studies through investigating the correlation between VDR gene polymorphisms and T2DM. The distinct variant of VDR gene, namely FokI has previously shown association with T2DM (especially in the Asian population), (Li et al., 2013; Wang et al., 2012) insulin sensitivity (Chiu et al., 2001), hypertension (Swapna et al., 2011), metabolic syndrome and body mass index (Zhao et al., 2014; Mackawy & Badawi, 2014). Since these studies have been carried out in various populations with different ethnic backgrounds the results are inconclusive. In addition, the relationship between VDR polymorphisms and other important complications of diabetes such as retinopathy (Zhong et al., 2015), and nephropathy (Yokoyama et al., 2012) has been revealed in some recent studies. These findings may highlight the possible role of vitamin D in diabetic foot complication. It would have been valuable if we had further information regarding the vitamin D status of our patients which must be considered in future studies. It has been demonstrated that hyperglycemia induces production of ROS, which finally leads to increased OS in diabetic patients (Johansen et al., 2005). OS leads to increased lipid peroxidation and elevated production of MDA which is the end product of PUFA (Akkuş et al., 1996). OS also contributes to the development and progression of vascular and neural dysfunction in diabetic patients (Yorek, 2003). It has been shown in some studies that patients with diabetes and foot ulcer have higher level of serum MDA (Singh et al., 2008) and lymphocyte MDA (Arya et al., 2011) in comparison to patients without ulcer or healthy controls. On the other hand, it is suggested that vitamin D contributes to OS process (Garcia-Bailo et al., 2011). A significant association between reduced vitamin D level and increase in some OS biomarkers such as MDA has been shown previously (Codoñer-Franch et al., 2012). Furthermore, significant association between severe vitamin D deficiency and elevated inflammatory cytokine concentrations in diabetic patients and particularly patients with foot infection was observed (Tiwari et al., 2014). We observed that TT and TC genotypes were more frequent among males with DFU. Previous study among the Iranian population found that diabetic patients with TT genotype of FokI Polymorphism might be considered low responders to an increase in 25(OH)D levels following vitamin D intake (Neyestani et al., 2013). This result is in concordance with ours, which showed males who carried TT genotypes
Table 5 – Oxidative stress parameters in different genotypes of VDR gene polymorphism.
Group
Oxidative stress parameters
VDR (FokI) genotype TT + TC
(n = 81) Patients with diabetes TBARSa (μmol/L) 28.89 (9.73) a FRAP (mmol/L) 295.57 (85)
Healthy subjects
CC
p value
(n = 106) 27.10 (5.90) 0.01 273.73 (68.75) 0.31
(n = 15) (n = 26) TBARSa (μmol/L) 15.94 (5.01) 15.8 (3.96) 0.76 a FRAP (mmol/L) 234.90 (49.66) 246.06 (43.17) 0.29
TBARS: Thiobarbituric acid reactive substances; FRAP: Ferric reducing ability of plasma. a Variables are expressed as median (IQR).
might be low responders and therefore more frequently present DFU than females. In the current study we evaluated the relationship between FokI variant of VDR gene and OS biomarkers in diabetic patients. The results demonstrated that there was a significant higher concentration of MDA using TBARS assay in the serum of patients with TT and TC genotypes in comparison to patients with CC genotype. Although the total antioxidant capacity of plasma was higher in TT and TC genotypes, no statistically significant association between FRAP levels and FokI polymorphisms were observed. This higher capacity, although insignificant, can be the result of hedging against OS and compensating by increasing FRAP. In a recent study in Iranian population, it has been shown that there was an association between FokI variants and OS biomarkers (ShabBidar et al., 2015). Similarly an association between TBARS OS biomarker and FokI polymorphism in patients with diabetes was demonstrated in our results, however no significant association was observed between OS biomarkers and FokI genotypes in healthy population in our study. In summary, in our study we observed a significantly higher frequency of T allele of VDR gene FokI polymorphism among Iranian patients with DFU and also among patients with higher level of TBARS. Therefore this allele might be used as a candidate biomarker in preventive medicine to reduce the risk of this life threatening complication of diabetes. However, more investigation is needed to support our finding especially in separated groups of males and females, to diminish the potential confounding effect of gender. Furthermore, considering the possible effect of vitamin D supplementation in reduction of insulin resistance in T2DM (Talaei et al., 2013) and also its effect on reducing inflammatory markers and increasing the total antioxidant capacity (Cavalcante et al., 2015), these findings might pave the way of designing a new generation of treatment for DFU, using vitamin D supplementation. The number of study population was the limitation of our study. Further studies on different ethnic populations and also examination of other genetic polymorphisms of VDR gene are required to confirm our results in this study. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgement This study has been funded by EMRI, Tehran University of Medical Sciences (1392-01-86-1550). References Akkuş, İ., Kalak, S., Vural, H., Çaglayan, O., Menekşe, E., Can, G., et al., 1996. Leukocyte lipid peroxidation, superoxide dismutase, glutathione peroxidase and serum and leukocyte vitamin C levels of patients with type II diabetes mellitus. Clin. Chim. Acta 244, 221–227. Antal, A.S., Dombrowski, Y., Koglin, S., Ruzicka, T., Schauber, J., 2011. Impact of vitamin D3 on cutaneous immunity and antimicrobial peptide expression. Dermatoendocrinol 3, 18–22. Arya, A.K., Pokharia, D., Tripathi, K., 2011. Relationship between oxidative stress and apoptotic markers in lymphocytes of diabetic patients with chronic non healing wound. Diabetes Res. Clin. Pract. 94, 377–384. Association AD, 2014. Diagnosis and classification of diabetes mellitus. Diabetes Care 37, S81–S90. Aumiller, W.D., Dollahite, H.A., 2015. Pathogenesis and management of diabetic foot ulcers. JAAPA 28, 28–34. Cavalcante, I.G., Silva, A.S., Costa, M.J., Persuhn, D.C., Issa, C.I., Freire, T.L., et al., 2015. Effect of vitamin D3 supplementation and influence of BsmI polymorphism of the VDR gene of the inflammatory profile and oxidative stress in elderly women with vitamin D insufficiency: vitamin D3 megadose reduces inflammatory markers. Exp. Gerontol. 66, 10–16. Chiu, K.C., Chuang, L.-M., Yoon, C., 2001. The vitamin D receptor polymorphism in the translation initiation codon is a risk factor for insulin resistance in glucose tolerant Caucasians. BMC Med. Genet. 2, 2. Codoñer-Franch, P., Tavárez-Alonso, S., Simó-Jordá, R., Laporta-Martín, P., Carratalá-Calvo, A., Alonso-Iglesias, E., 2012. Vitamin D status is linked to biomarkers of oxidative
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Please cite this article as: Soroush, N., et al., Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress, Gene (2016), http://dx.doi.org/10.1016/j.gene.2016.11.012