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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 18  |  Issue : 3  |  Page : 330-341

The association between vitamin D receptor gene polymorphism (FokI), type 2 diabetes, and microvascular/macrovascular complications in postmenopausal women


1 Department of Medical Biochemistry, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt
2 Department of Internal Medicine, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt

Date of Submission16-May-2019
Date of Decision11-Jun-2019
Date of Acceptance10-Jul-2019
Date of Web Publication30-Oct-2020

Correspondence Address:
Shimaa A Mohamed
Department of Medical Biochemistry, Faculty of Medicine for Girls, Al-Azhar University, Cairo, 71111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AZMJ.AZMJ_76_19

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  Abstract 


Background Vitamin D has a wide range of biological functions. The presence of vitamin D receptors (VDRs) in many tissues explains its diverse actions. The VDR gene is highly polymorphic, with many single nucleotide polymorphisms. The FokI polymorphism in the VDR gene is the only polymorphism that influences the size of the translated protein. Type 2 diabetes mellitus (T2DM) is a growing problem worldwide, it results from a complex of inheritance and environment interactions. Vitamin D deficiency and VDR gene polymorphism FokI have been linked to T2DM and diabetic microvascular and macrovascular complications.
Aim The aim of this study is to determine the association of VDR gene polymorphism FokI with T2DM and diabetic microvascular and macrovascular complications in postmenopausal women.
Participants and methods This study was carried out on 200 postmenopausal Egyptian women (50 healthy controls, 50 patients with T2DM without microvascular or macrovascular complications, 50 T2DM patients with microvascular complications, and 50 T2DM patients with macrovascular complications). VDR FokI genotypes were determined by PCR-restriction fragment length polymorphism analysis and hydroxy vitamin D (25OHD) levels were measured by enzyme linked immunosorbent assay.
Results The prevalence of the polymorphic genotypes ff and the f allele was statistically significantly increased in diabetic patients than in controls (P<0.001), and the odds ratio was 7.84 (95% confidence interval: 1.75–35.09). There was a statistically significant increase in the polymorphic genotypes ff and the f allele in T2DM with microvascular or macrovascular complications compared with the control group (P=0.05). Plasma vitamin D (25OHD) levels were significantly lower in diabetic patients than in the control participants (P=0.032). On comparing the studied groups in terms of the plasma vitamin D (25OHD) levels, there was a statistically significant decrease in the levels in patients with microvascular and macrovascular complications in comparison with the control group (P<0.001).
Conclusion The ff genotype and the f allele of VDR polymorphism FokI may represent a significant genetic molecular marker to predict the risk of diabetes and diabetic microvascular and macrovascular complications in postmenopausal women.

Keywords: diabetic microvascular and macrovascular complications, type 2 diabetes mellitus, vitamin D receptor FokI polymorphism, vitamin D


How to cite this article:
Mohamed SA, Shipl WM, Mohamed Sarhan OH, Sakar HE, Arfa AE. The association between vitamin D receptor gene polymorphism (FokI), type 2 diabetes, and microvascular/macrovascular complications in postmenopausal women. Al-Azhar Assiut Med J 2020;18:330-41

How to cite this URL:
Mohamed SA, Shipl WM, Mohamed Sarhan OH, Sakar HE, Arfa AE. The association between vitamin D receptor gene polymorphism (FokI), type 2 diabetes, and microvascular/macrovascular complications in postmenopausal women. Al-Azhar Assiut Med J [serial online] 2020 [cited 2020 Nov 25];18:330-41. Available from: http://www.azmj.eg.net/text.asp?2020/18/3/330/299580




  Introduction Top


Vitamin D is a fat-soluble vitamin that belongs to the family of steroid hormones. Vitamin D is unique because it can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2), and the body can also synthesize it (from cholesterol) when sun exposure is adequate [1]. In humans, ∼95% of vitamin D is produced through sunlight-dependent processes that occur in the skin. Only a small amount of vitamin D is obtained through diet and dietary supplements [2]. Active vitamin D binds to vitamin D-binding protein and is transported to target cells. Once the complex reaches the target cell, vitamin D is released from the vitamin D-binding protein and 1, hydroxy vitamin D [25(OH)2D3] binds to vitamin D receptors (VDRs), which is present in the cytoplasm [3]. VDR transports vitamin D into the nucleus. Here, it undergoes conformational changes to interact with transcriptional factors. The activated VDR joins with the retinoid X receptor to form a transcriptional regulatory unit that binds to the vitamin D response element (VDRE) in the promoter region of genes. This binding results in the regulation of gene expression [4]. The VDR is a member of the nuclear hormone receptor superfamily that modulates the transcription of target genes and mediates vitamin D genomic actions [5]. VDR is expressed in many tissues, including those involved in the regulation of glucose metabolism, such as pancreatic β-cells [6]. VDR, located on human chromosome 12 (12q12–q14), is a member of the steroid hormone receptor superfamily [7]. The VDR gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) have been recognized such as BsmI, ApaI, TaqI, and FokI [8], as shown in [Figure 1]. The FokI is an independent polymorphic site in the VDR gene with thymine (T) to cytosine (C) substitution in the start codon ATG (methionine), affecting the structure and the function of the encoded protein. The allelic variants of the FokI polymorphism code result in two structurally different VDR proteins: the wild-type 424 amino acids (F allele, C) and the 427 amino acids (f allele, T) protein [9]. In contrast to other VDR, SNPs FokI is the only known VDR polymorphism resulting in two different VDR protein products [10].
Figure 1 Structure of the genomic region of the vitamin D receptor and location of FokI and other BsmI, ApaI, and TaqI restriction sites.

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Type 2 diabetes mellitus (T2DM) is a multifactorial metabolic disorder that is influenced by genetic and environmental factors including a sedentary lifestyle and poor dietary management [11]. Patients with T2DM constitute ∼90–95% of all patients with diabetes worldwide making T2DM an increasing epidemic disease [12]. The International Diabetes Federation has listed Egypt among the world’s top 10 countries in terms of the number of patients with diabetes. In Egypt, the prevalence of diabetes is around 15.56% among adults between 20 and 79 years of age [12].

Vitamin D has been identified to play a crucial role in diabetes, where it regulates the insulin receptor gene and controls the nuclear peroxisome proliferator-activated receptor, an important factor in regulating the fatty acid metabolism in skeletal muscles and adipose tissue, which plays a crucial role in insulin sensitivity [13]. Vitamin D affects insulin secretion and insulin sensitivity. The effect of vitamin D on insulin secretion may be mediated by changes in the intracellular calcium concentration in beta cells [14]. Vitamin D improves insulin sensitivity by its anti-inflammatory activity. Vitamin D attenuates the expression of proinflammatory cytokines involved in insulin resistance such as interleukins (ILs), IL-1, IL-6, and tumor necrosis factor (TNF-α), also downregulates nuclear factor-κB activity [15]. Diabetic microvascular complications (DMI) involve the smallest blood vessels, the capillary and the precapillary arteries, resulting in thickening of the capillary basement membrane, mainly in the kidneys and retina. Therefore, DMI are common and include retinopathy and nephropathy [16]. There is evidence to suggest that vitamin D may play a role in the pathogenesis of diabetic microvascular complications through its effects on the immune system [17]. Vitamin D decreases the production of several proinflammatory cytokines, such as IL-2, IL-6, IL-8, IL-12, and TNF-α. Vitamin D also exerts an anti-inflammatory effect by decreasing the proliferation of helper T-cells, cytotoxic T-cells, and natural killer cells [18]. Moreover, vitamin D deficiency has been implicated as an independent risk factor for the prospective development of cardiovascular diseases (CVD) [19]. Endothelial cells express VDR, which is upregulated under stress; VDR activation modulates response elements in the vascular endothelial growth factor promoter and affects calcium influx across the cell membrane, as well as endothelium-dependent vascular smooth muscle contractions [20].


  Participants and methods Top


This study was carried out during the period from October 2016 till November 2017. Samples were collected from Alzahraa University Hospital in Cairo (Department of Internal Medicine) and investigations were carried out at the Medical Biochemistry Department, Faculty of Medicine for Girls, Al-Azhar University. The protocol of this study was approved by the medical ethics committee in the faculty and oral informed consent was obtained from all patients and healthy controls. This study included 150 female patients with physiological menopause and a diagnosis of T2DM according to the American Diabetes Association criteria [21]. A total of 50 postmenopausal women with physiological menopause, but without diabetes, were enrolled as the control group ([Figure 2] and [Figure 3]).
Figure 2 Agarose gel electrophoresis showing PCR product-based restriction fragment length polymorphism analysis of vitamin D receptor gene amplification guided by the marker in the first lane.

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Figure 3 Agarose gel electrophoresis showing PCR product-based restriction fragment length polymorphism analysis of the vitamin D receptor gene polymorphism digested by FokI guided by the marker in the first lane, performed on 3% agarose, visualized by ultraviolet light transillumination.

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The participants were classified into the following four groups:
  1. Group I (control group): included 50 postmenopausal women with no symptoms or signs of diabetes mellitus, who were selected as a control group, and confirmed by normal blood glucose level. Their mean age was 63.16±5.33 years.
  2. Group II (diabetic group without microvascular or macrovascular complications): included 50 postmenopausal female patients with T2DM. Their mean age was 64.76±5.32 years.
  3. Group III (diabetic group microvascular complications): included 50 postmenopausal female patients with T2DM with microvascular complications such as nephropathy, neuropathy, and retinopathy. Their mean age was 63.84±6.21 years.
  4. Group IV (diabetic group with macrovascular complications): included 50 postmenopausal

    female patients with T2DM with macrovascular complications e.g. cardiovascular complications. Their mean age was 63.76±5.52 years.


Exclusion criteria were as follows: patients with type 1 diabetes; end-stage renal disease; use of glucocorticoids, vitamin D replacement therapy, and hormone replacement therapy; primary hyperparathyroidism; malabsorption syndrome; and active neoplasia. The patients and controls were subjected to a full assessment of history, clinical examination with measurement of blood pressure, and calculation of BMI and routine laboratory investigations including complete blood count, blood glucose level (fasting and postprandial), blood glycated hemoglobin (HbA1c), serum urea and creatinine, and serum lipid profile.

Specimen collection

Morning venous blood samples (10 ml) after fasting for 8 h were withdrawn under complete aseptic conditions from all participants. Three milliliter was collected in a sterile EDTA vacutainer and was used for molecular testing of the VDR gene by lymphocyte separation and extraction of DNA for PCR. DNA was extracted and stored at −20°C until assay. Two milliliter was collected in sterile ‘EDTA’ (vacutainer) tubes and used for the measurement of plasma 25OH vitamin D3 using the enzyme linked immunosorbent assay (ELISA) technique. Two milliliter was collected in a sterile ‘EDTA’ vacutainer for the measurement of glycated hemoglobin, the rest was collected in a plain vacutainer, and the serum obtained was used for the estimation of fasting serum glucose, serum urea, and serum creatinine. Another venous blood sample (2 ml) after fasting for 12 h was withdrawn, collected in a plain vacutainer, and the serum obtained was used for the estimation of the lipid profile [total cholesterol (TC), triglyceride, high-density lipoproteins (HDL), and low-density lipoproteins (LDL)].

Another venous blood sample (2 ml) was withdrawn 2 h after breakfast in a fluoride vacutainer for the measurement of 2 h postprandial serum glucose.


  Methods Top


Determination of serum glucose level (fasting and postprandial) [22], serum lipid profile including (TC [23], triglyceride [24], HDL [25], LDL [26], HbA1c [27], serum urea [28], and serum creatinine [29] was performed. estimation of 25OH vitamin D3 in the plasma by ELISA measured by a commercially available ELISA kit supplied Epitope Diagnostic Inc. (San Diego, California, USA) [30]. Genomic DNA extraction from blood leukocytes was analyzed for the VDR FokI gene polymorphism using the PCR-restriction fragment length polymorphism technique.

Genomic DNA analysis for the determination of FokI genotype was carried out by detecting a single nucleotide polymorphism at the position of the 265 bp fragment in the start codon (exon 2) in the coding region of the VDR gene using the PCR technique and restriction fragment length polymorphism. The FokI polymorphism occurs in the start codon of VDR and its polymorphic form (T- or ‘f’ allele) leads to the translation of a longer VDR protein variant (427 amino acid); it functions less effectively than the shorter VDR protein (424 amino acid) encoded by the(C- or ‘F’ allele). Therefore, there are two translation initiation start sites (ACG): the wild form and (ATG) the polymorphic form. The test was carried out in five main steps:
  1. Extraction of genomic DNA from peripheral blood leukocytes of EDTA anti-coagulated blood including
    1. Lymphocyte separation.
    2. DNA extraction: DNA was extracted using the whole blood genomic DNA Purification Miniprep Kit #D3024.
  2. Amplification of the extracted DNA (PCR) by primers for Fok1 in exon 2 of the VDR gene using the 265-bp fragment.

    The VDR FokI genotype was determined by PCR according to the protocol published by Harris et al.[31]: amplification was performed using Taq Red PCR Master Mix supplied by Bioline Reagents Ltd (Bio-25043, Lodon, United Kingdom):
  3. Primer sequence:
    1. Forward primer: 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′.
    2. Reverse primer: 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′.

      for the genotyping of the FokI polymorphism of the VDR gene.

      The amplified fragment was 265 bp.

      The computerized thermocycler (Techne Progene, Cambridge, United Kingdom) was programmed for the following conditions:
      1. An initial cycle of 95°C for 1 min (for initial denaturation), 34 cycles under the following conditions: denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 10 s. The final extension cycle was 72°C for 7 min.
  4. Detection of PCR amplification products using 2% agarose gel electrophoresis stained by ethidium bromide and ultraviolet light transillumination as bands at 265 bp.
  5. The VDR FokI gene polymorphism was identified using a specific restriction enzyme: the amplified products were digested with the FokI (rs2228570) restriction endonuclease enzyme provided by (New England Biolabs), and then the digested products were separated by 3% agarose gel electrophoresis stained with ethidium bromide and visualized using a ultraviolet light transilluminator. Where the homozygote CC (FF: wild genotype) produced one band (265 bp long), the homozygote TT (ff) produced two bands (196, 69 bp long) and the heterozygote CT (Ff) produced three bands (265, 196, and 69 bp long).


Statistical methods

Statistical analysis of the results was carried out using the statistical package for social sciences (IBM SPSS), version 23 (New York, USA). The quantitative data were presented as mean, SD, and ranges when parametric. Also, qualitative variables were presented as number and percentages. The comparison between groups in terms of qualitative data was performed using the χ2-test and/or the Fisher exact test when the expected count in any cell was found to be less than 5. The comparison between more than two independent groups with quantitative data and a parametric distribution was performed using an independent t-test. The comparison between more than two independent groups with quantitative data and a parametric distribution was performed using one-way analysis of variance, followed by post-hoc analysis using the least significant difference (LSD) test. Logistic regression analysis was used to assess the odds ratio (OR) with a 95% confidence interval (CI) for genotypes and alleles between groups. The CI was set to 95% and the margin of error accepted was set to 5%. Therefore, P values up to 0.05 were considered statistically significant [32].


  Results Top


In terms of the clinical data of the groups studied, no statistically significant differences were found in age (P=0.556), whereas there was a statistically significant increase in BMI in group II, group III, and group IV compared with group I (P<0.001). Also, comparison of the frequency of hypertension between the studied groups showed a highly statistically significant increase in group II, group III, and group IV compared with group I (P<0.001). Also, comparison of HbA1c, fasting blood glucose (FBG), PPBG, TC, HDL, LDL, serum urea. and serum creatinine between the groups studied showed a highly statistically significant increase in group II, group III, and group IV compared with group I (P<0.001; [Table 1]).
Table 1 Demographic and clinical characteristics of the groups studied

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In terms of the plasma 25OHD level, there was a statistically significant decrease in the 25OHD level in the group of diabetic patients compared with the control group (P=0.032). Using cut-offs for plasma vitamin D levels below 30 ng/ml, the prevalence of vitamin D deficiency was 28% in control participants and 74.7% in diabetic patients, and this was statistically significant (P<0.001; [Table 2]).
Table 2 25 hydroxy vitamin D level in the control and diabetic patient groups

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The correlation between plasma 25OHD (ng/ml) and demographic and clinical data of all patients showed a statistical negative correlation between plasma vitamin D (25OHD) levels and postprandial blood sugar (PPBS) (r=−0.168, P=0.04). There was no statistically significant correlation between plasma vitamin D (25OHD) levels and other variables (age, BMI, FBS, HbA1c, triacyle glycerol (TAG), TC, HDL, LDL, serum creatinine, and urea) ([Table 3]).
Table 3 Correlation between plasma 25 hydroxy vitamin D (ng/ml) and demographic and clinical data of all patients

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In this study, there was a statistically significant increase in the ff genotype (polymorphic form) in the diabetic patient group on comparing them with the control group (P<0.001) and the OR was 7.8448 (95% CI 1.7536–35.0942). Also, there was an increased frequency of the f allele (polymorphic allele) in the diabetic patient group compared with the control group (P<0.001), and the OR was 2.0894 (95% CI: 1.2646–3.4521; [Table 4]).
Table 4 FokI genotypic and allelic frequencies in controls and all diabetic

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Comparison of the frequency of the FokI genotype in the groups studied showed no statistically significant difference in FF and Ff genotypes (P>0.05). There was a statistically significant increase in the ff genotype in group II, group III, and group IV compared with group I (P=0.04). In terms of the allele frequency in the groups studied, there was a statistically significant increase in the f allele in group II, group III, and group IV compared with group I (P<0.001; [Table 5]).
Table 5 FokI genotypic and allelic frequencies in the groups studied

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Comparison between FOKI genotypes of clinical data showed a statistically highly significant increase in the TAG levels in the ff genotype compared with the FF and Ff genotypes (P=0.009). There was no statistically significant difference between FokI genotypes and the other clinical data studied such as age, BMI, PPBS, HbA1c, TC, TAG, HDL, LDL, urea, and creatinine (P>0.05). Comparison between FokI genotypes and 25OHD levels in the groups studied showed no statistically significant difference (P=0.7; [Table 6]).
Table 6 Comparison between FokI genotypes in terms of the clinical data of the groups studied

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  Discussion Top


T2DM is a multifactorial metabolic disorder that is influenced by genetic and environmental factors [13]. The prevalence of T2DM is increasing at an alarming rate worldwide. According to the recent International Diabetes Federation report (2017), it was estimated that there are 425 million individuals living with diabetes and, by 2045, this number is expected to increase by 629 million individuals [11].

Vitamin D is a fat-soluble vitamin that acts as a steroid hormone. Vitamin D exists in two major forms: vitamin D2 and vitamin D3, which are both inactive. Vitamin D3 accounts for more than 90% of the total vitamin D content in the body. Exposure to the sun is the main source of vitamin D3 [19].

Vitamin D initiates its biological responses by binding to the VDR. VDR, located on human chromosome 12 (12q12–q14), is a member of the steroid hormone receptor superfamily. VDR is an intracellular hormone receptor that binds to the biologically active form of vitamin D, affecting cell growth and differentiation, embryonic development, and metabolic homeostasis [7].

The VDR gene is highly polymorphic, and many SNPs have been recognized such as FokI, BsmI, ApaI, and TaqI. The FokI polymorphism is located on exon 2 at the fifth end. Moreover, it is the only polymorphism that is not linked to any of the other VDR variants [33].

The FokI is an independent polymorphic site in the VDR gene with a thymine (T) to cytosine (C) substitution in the start codon ATG (methionine), affecting the structure and the function of the encoded protein. The allelic variants of the FokI polymorphism code result in two structurally different VDR proteins: the wild-type 424 amino acids (F allele, C) and the 427 amino acids (f allele, T) protein [8].

The results of this study showed that the polymorphic genotype of VDR Fok1 polymorphism (ff), was statistically significantly increased in diabetic patients compared with the control group (P<0.001) and the OR was 7.84 (95% CI: 1.75–35.09). There was no statistically significant difference in (FF and Ff) genotypes in diabetic patients compared with the control group (P=0.066) and the OR was 1.16 (95% CI: 0.59–2.28). Also, the allele frequency of FokI (f allele) was higher in the T2DM patient group compared with the control group (P=0.004) and the OR was 2.089 (95% CI: 1.26–3.45).

In studies of Egyptian populations, the VDR FokI gene polymorphism showed a significant increase in the (ff) genotype and the (f) allele in patients with T2DM compared with the control group [13],[34]. This finding was in accordance with Angel et al., 2018 in Chilean nationality [35],[36], and also in Saudi individuals and in East Asians [37].

It has been assumed that genetic variations in the VDR play a significant role in the pathogenesis of T2DM by altering calcium (Ca) metabolism, adipocyte function, insulin release, and cytokine expression [13].

Also, the effects of vitamin D on glucose metabolism are mainly because of the distribution of its receptors (VDR) on pancreatic β-cells, skeletal muscle, and adipose tissue. Also, the presence of 1α hydroxylase in pancreatic B cells and the presence of the VDRE in the human insulin receptor gene promoter play an important role in glucose metabolism [38].

Activation of vitamin D through VDRs plays an important role in regulating insulin secretion from the pancreatic β cells. The calcitriol directly activates the transcription of the human insulin receptor gene and activates the peroxisome proliferator activator receptor, an important factor in regulating the fatty acid metabolism in skeletal muscles and adipose tissue, which plays a crucial role in insulin sensitivity [39].

VDR gene polymorphisms have been identified to affect the activity of VDR protein. The FokI can suppress the first translation initiation site, resulting in a peptide lacking three amino acids; the FF genotype of FokI polymorphism was shown to be associated with higher VDR mRNA copy numbers and increased transcriptional activity of VDR [7].

However, in a study of postmenopausal Brazilian women with T2DM [40], no significant differences were found in the genotype and allele frequency distribution of VDR FokI compared with the controls. Also, other studies showed no association between the VDR FokI polymorphism and the risk of diabetes [41],[42].

In addition, an association has been found between the FokI polymorphism and T2DM among Emirates population [43]. However, our results are not in agreement with the findings that the F allele and FF and Ff genotypes of the FokI polymorphism are more frequent in T2DM patients. The F allele of the FokI polymorphism encodes a 424 amino acid protein. The authors explained that the shorter protein has a higher transcriptional activity, which further increases its capacity to bind to 1,25 dihydroxyvitamin D [9]. This can reduce the risk of T2DM by enhancing pancreatic β-cell secretion function and improving insulin resistance [44]. Therefore, their study showed that the F allele of the FokI polymorphism is more frequent in T2DM patients, possibly as a reaction of the body against insulin resistance.

In contrast, a study carried out by Malecki et al. [45] on a Polish population showed no association between the VDR FokI polymorphism and the risk of diabetes. The reason for the discrepancy between our result and these studies could be explained by the genetic differences in the populations studied or their exposure to different environmental factors.

In this study, there was a statistically significant increase in the polymorphic genotype (ff) in T2DM with microvascular complications compared with the controls (P=0.05). There was no statistically significant difference in (FF and Ff) genotypes between diabetic patients with microvascular complications and the control group (P=0.233 and 0.865) for FF and Ff genotypes, respectively. In terms of the allele frequency of the VDR (FokI) polymorphism, the f allele was higher in T2DM with microvascular complications than the controls (P=0.007).

In agreement with our results, Zhong et al. [46] showed that the (FF) genotype and the (F) allele of the FokI polymorphism have been found to be associated with increased transcriptional activity of VDR and greater activation of vitamin D in target cells. Any beneficial effect of vitamin D in the retina would be greater in individuals carrying the F allele than in those carrying the f allele. This would account for the significant association between the presence of the f allele and increased risk of diabetic retinopathy in T2DM.

The study of Liu et al. [16] showed that the VDR gene FokI polymorphism may be associated with an increased risk of diabetic nephropathy in diabetic patients. However, Yin et al. [7] and Cyganek et al. [47] showed no association between the VDR FokI polymorphism and diabetic microvascular complications. Also, there were no significant differences in genotypes and alleles of the FokI polymorphism in relation to retinopathy, nephropathy, and neuropathy in postmenopausal Brazilian women with T2DM [40].

The result of this study showed a statistically significant increase in the polymorphic genotype (ff) of the FokI polymorphism in T2DM with macrovascular complications compared with the control group (P=0.05). There was no statistically significant difference in (FF and Ff) genotypes in patients with macrovascular complications compared with the control group (P=0.233 and 0.865) for the FF genotype and the Ff genotype, respectively. The (f) allele of FokI was higher in T2DM with macrovascular complications than in controls (P=0.007).

The study by Maia et al. [40] found a significant association between the FokI polymorphism in the recessive model (Ff+ff) and coronary artery disease (CAD). They found that the (Ff) genotype of VDR FokI SNPs is probably a protective factor for CAD in postmenopausal Brazilian women with T2DM. In addition [48], in a study of a Chinese Han population, it was found that the FokI-FF genotype showed a significant decrease in T2DM patients with CVD (36.1%) compared with the controls (47.5%). Most of the experiments conducted so far point to the fact that the protein (424 AA) of the short form is more active than that of the long form (427 AA) in terms of its transactivation activity as a transcription factor. However, it seems to be gene and cell type specific. Thus, certain genes and cell types will be more sensitive to the polymorphism than others [9].

In contrast to our study, Pan et al. [49] found that no significant differences were observed in the genotype and allele frequencies of the FokI polymorphism between patients with CAD and controls in a Chinese population. They reported that FokI in exon 2 consists of a T to C change, which seems to cause gene-specific and cell type-specific effects. Some genes and some cell types will be more sensitive to the effect of the polymorphism than others.

In this study and in agreement with previous studies [50],[51], no significant difference was observed between (HDL, LDL, TC, FBG, PPBS, and BMI) and FokI genotypes. However, Filus et al. [52] found lower serum HDL in the VDR FokI polymorphism.

There could be a distinct mechanism by which the VDR axis could affect the lipid profile. First, vitamin D induces the suppression of PTH secretion, and it has been reported that PTH could reduce lipolysis [53]. Second, vitamin D might lead to an improvement in insulin secretion and insulin sensitivity, thereby indirectly affecting lipid metabolism [54].

However, we found a significant increase in TAG in the ff genotype than in the other FokI genotypes (FF and Ff) (P=0.001). This was in agreement with Mackawy and Badawi [34], who found that lipid profile parameters showed a significant association with the VDR (FokI) polymorphism in diabetic patients. They reported higher TC, TG, and LDL and lower HDL levels in Ff and ff genotype carriers.

In this study, the plasma vitamin D (25OHD) level was significantly lower in diabetic patients (25.96±10.75 ng/ml) than in the control group (35.51±13.17 ng/ml) (P=0.032).

This result was consistent with the work of Gendy and colleagues [13],[35],[40]. They showed that vitamin D activates the transcription of the human insulin gene and the VDRE found in the insulin gene promoter area. Also, it increases the sensitivity of cells to insulin by increasing the expression of insulin receptors and by maintaining an adequate supply of the calcium pool. Alterations in the Ca supply lead to peripheral insulin resistance because of impaired insulin transduction, which decreases glucose transporter 4, the main player in glucose metabolism that maintains glucose homeostasis [34]. In addition, vitamin D improves insulin sensitivity by its anti-inflammatory activity. Vitamin D attenuates the expression of pro inflammatory cytokines involved in insulin resistance such as ILs, IL-1, IL-6, and TNF-α, and also downregulates nuclear factor-κB activity [55].

This study showed significant decrease in plasma 25OHD in the groups studied when compared with the control group, this was in agreement with the study of Sun et al. [19], who reported that patients with vitamin D deficiency experienced the highest risk of CVD when compared with the control group. They explained that the potential mechanism of vitamin D deficiency leading to CVD may be a result of different factors. First, vitamin D could regulate vascular smooth muscle cells and local intercellular adhesion molecule 1 (ICAM1) to protect the vascular endothelium. Second, vitamin D could regulate immune cells and inhibit the release of inflammatory cytokines, thereby playing a role in the protection of blood vessels. Third, vitamin D is an important regulator of the renin–angiotensin system, which inhibits the release of renin and reduces levels of angiotensin II (Ang II), which play an important role in lowering blood pressure. The same results were also reported by [20],[56], who found that vitamin D deficiency is associated with CVD morbidity and mortality.

In addition, Yin et al. [7] and Xiaoyan et al. [57] reported that vitamin D deficiency was associated with the severity and progression of diabetic nephropathy. They explained that vitamin D is a suppressor of renin biosynthesis; thus, deficiency of vitamin D has been shown to be associated with chronic kidney disease progression. Also, John et al. [17] reported that vitamin D may play a role in the pathogenesis of diabetic retinopathy through its effects on the immune system. Vitamin D decreases the production of several proinflammatory cytokines, such as IL-2, IL-6, IL-8, IL-12, and TNF-α. Vitamin D also exerts an anti-inflammatory effect by decreasing the proliferation of helper T-cells, cytotoxic T-cells, and natural killer cells. Vitamin D may also contribute to diabetic retinopathy by angiogenesis mechanisms. Vitamin D is a potent inhibitor of retinal neovascularization.

In addition, Guang et al. [58] found that T2DM patients with vitamin D deficiency are 1.22 times to suffer from diabetic peripheral neuropathy than those with normal vitamin D levels in Asia. Vitamin D deficiency is an important causative factor for the development of diabetic peripheral neuropathy in White with diabetes mellitus. Vitamin D deficiency has been shown to be associated with low levels of neurotrophins (especially nerve growth factor) and defective neuronal calcium homeostasis. Vitamin D, through its receptor, modulates neuronal differentiation as well as neuronal growth and functions [59]. In addition, vitamin D deficiency has been linked to a lower pain threshold, which increases when vitamin D deficiency is corrected [60]. Also, Soderstrom et al. [61] found an association between vitamin D insufficiency (<30 ng/ml) and self-reported peripheral neuropathy symptoms (numbness, loss of feeling, pain, or tingling in the hands or the feet) in a representative population of US adults with diabetes.

In this study, we found a significant negative correlation between plasma vitamin D (25OHD) levels and PPBS (r=−0.168, P=0.04), but there was no significant correlation between plasma vitamin D (25OHD) levels and other variables (age, BMI, FBS, HbA1c, TAG, TC, HDL, LDL, serum creatinine, and urea).

The same result was also reported by Gendy et al. [13], who did not find any significant correlations between serum 25OHD and FBS, HbA1c, lipid profile, and BMI in T2DM patients. Also, Halaw et al. [62] found a significant negative correlation between PPBS and vitamin D levels, and also found no significant correlation between vitamin D levels and BMI in diabetic patients. In contrast to our study, they found a significant correlation between vitamin D levels and (age, FBS, and HbA1c).

We found no significant association between plasma vitamin D level (25OHD) and different genotypes of VDR FokI polymorphism (P=0.7). The same result was reported by Gendy and colleagues [13],[36],[63],[64], who found no significant association between 25OHD and VDR polymorphism FokI.


  Conclusion Top


Our study suggests that the FokI polymorphism in the VDR gene may represent a significant genetic molecular marker to predict the risk of diabetes and diabetic microvascular and macrovascular complications in postmenopausal women in its polymorphic form (ff genotype).

Recommendations

Studies of other polymorphisms in the VDR gene and their relationships with diabetes and its microvascular and macrovascular complications should be carried out. Also, further researches are required in different ethnic populations and on large numbers of patients to indicate its usefulness as a potential new genomic indicator and biomarker to screen populations for diabetes and diabetic microvascular and macrovascular complications.

Acknowledgements

The authors thank members of Al-Zahra University Hospital (Department of Internal Medicine) and Medical Biochemistry Department, Faculty of Medicine for Girls, Al-Azhar University, for help with the preparation of this manuscript.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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