Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 31  |  Issue : 2  |  Page : 557--563

Vitamin D receptor (BsmI) gene polymorphism and type 2 diabetes mellitus in an Egyptian population


Waleed M Fathy1, Gehan A Tawfeek1, Ahmed R Tawfeek2, Shimaa M Aboelyazeid Ellayen3,  
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Tala Central Hospital, Tala, Menoufia, Egypt

Correspondence Address:
Shimaa M Aboelyazeid Ellayen
Department of Clinical Pathology, Tala Central Hospital, Tala, Menoufia
Egypt

Abstract

Objective The aim of this study was to evaluate the association between vitamin D receptor (VDR) (BsmI) gene polymorphism and genetic susceptibility to type 2 diabetes mellitus (T2DM) in an Egyptian population. Background The VDR gene is a candidate gene for susceptibility to several diseases. Studies on the association between VDR polymorphisms and T2DM in different ethnic populations are yet inconclusive. Patient and methods The present study was a case–control study conducted at the Department of Clinical Pathology, College of Medicine, Menoufia University, during the period from May 2015 to December 2016. We included 120 patients, who were divided into two groups: group A included 40 healthy patients, which served as the control group, and group B included 80 T2DM patients. Participants were subjected to the following: complete history taking and clinical examination, assessment of BMI, fasting blood sugar, 2-h postprandial, glycated haemoglobin, and lipid profile, renal function tests and PCR-restriction fragment length polymorphism for determining the genotype of BsmI gene polymorphism. Results Genotype distribution and allele frequencies of the VDR gene polymorphism differed significantly between patients and controls. The Bb genotype was higher in the T2DM group (42.5%) than in the control group (12.5%), and the bb genotype was higher in the T2DM group (7.5%) than in the control group (5%), but without statistical significance. The b allele was higher in the T2DM group (28.8%) than in the control group (11.3%). The Bb allele was found to be more risky than the BB allele by 5.610 [confidence interval (CI)=1.97–15.96], whereas the bb allele was found to be more risky than the BB allele by 2.475 (CI = 0.46–13.09). The b allele was found to be more risky than the B allele by 3.18 (CI = 1.47–6.90). Conclusion Our data suggest that VDR (BsmI) gene polymorphism genotype is associated with the risk of T2DM



How to cite this article:
Fathy WM, Tawfeek GA, Tawfeek AR, Aboelyazeid Ellayen SM. Vitamin D receptor (BsmI) gene polymorphism and type 2 diabetes mellitus in an Egyptian population.Menoufia Med J 2018;31:557-563


How to cite this URL:
Fathy WM, Tawfeek GA, Tawfeek AR, Aboelyazeid Ellayen SM. Vitamin D receptor (BsmI) gene polymorphism and type 2 diabetes mellitus in an Egyptian population. Menoufia Med J [serial online] 2018 [cited 2020 Apr 10 ];31:557-563
Available from: http://www.mmj.eg.net/text.asp?2018/31/2/557/239751


Full Text



 Introduction



Type 2 diabetes mellitus (T2DM) is a chronic disease with rapidly increasing prevalence in recent years. According to the latest estimation in 2015, there are more than 415 million cases of T2DM worldwide, and this number is expected to reach 642 million by 2040 [1]. T2DM is threatening the health of people worldwide and is not easy to cure completely at present. Therefore, prevention and control of T2DM is a tough task for healthcare providers and relevant scientists. T2DM is a complex metabolic disorder of multiple etiologies caused by changes in pancreatic β-cell function, insulin sensitivity and systemic inflammation. Recently, many studies have indicated strong involvement of genetic factors in T2DM – for example, familial inheritance and many gene variants have found to be associated with this disease [2]. Thus, screening genes to find genetic variants and evaluating their combined and additive effects on the risk of T2DM and insulin function could help identify individuals who are at risk and ultimately lead to new therapies for the prevention and treatment of T2DM [3].

In addition to its role in calcium and phosphate homoeostasis, there is increasing evidence that vitamin D is also involved in the pathogenesis of diabetes mellitus [4] and that variants in key genes in its metabolic pathway, such as the ubiquitously expressed nuclear vitamin D receptor (VDR), will deteriorate its function in vivo and increase the risk of diabetes mellitus. The VDR gene is located on human chromosome 12. Four common single nucleotide polymorphisms of the VDR gene have been postulated to be associated with T2DM in different ethnic populations – namely, FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236). The full-length human VDR gene is ~63.5 kb. However, whether other single nucleotide polymorphisms are involved in the process of T2DM has not been completely elucidated [5]. The aim of this study was to evaluate the association between VDR gene (BsmI) polymorphism and genetic susceptibility to T2DM in Egyptians.

 Patients and Methods



The present study was a case–control study conducted at the Department of Clinical Pathology, College of Medicine, Menoufia University, during the period from May 2015 to December 2016. The study included 120 patients who were divided into two groups: group A included 40 healthy patients, which served as the control group (11 males, 29 females), with their ages ranging from 36 to 65 years, and group B included 80 T2DM patients (22 males, 58 females), with their ages ranging from 35 to 66 years.

The present study received ethics approval from the Menoufia University Medical Ethics Committee, and written informed consent was obtained from all participants.

For all participants, the following were carried out: complete history taking and clinical examination, assessment of BMI, fasting blood sugar, 2-h postprandial (2HPP), glycated haemoglobin (HbA1c), and lipid profile, renal function tests, and PCR-restriction fragment length polymorphism (RFLP) for determining the genotype and allele frequencies of VDR BsmI (A/G) (rs1544410) gene polymorphism.

Sampling

A volume of 5-ml venous blood was collected from the cubital vein by sterile venipuncture after 8 h of overnight fasting and divided into three parts. First, 2 cm of blood was transferred to a plain vacationer tube and left to clot at 37°C. Serum was separated by centrifugation and used for immediate assay of fasting blood glucose, kidney function tests, and lipid profile. Second, 1.5 cm of blood was transferred to a potassium ethylene diamine tetra-acetic acid-containing tube for HbA1c determination. Finally, 1.5 ml of blood was transferred to an ethylene diamine tetra-acetic acid tube and frozen to extract DNA for PCR. First-voided mid-stream morning urine samples were collected for determining the albumin/creatinine ratio. Samples were centrifuged at 3000 rpm for 10 min before analysis to remove any cells or other debris.

Laboratory methods

Biochemical tests for detecting blood glucose, kidney function, total cholesterol, triglycerides, and high-density lipoprotein-cholesterol (HDL-C) were performed using autoanalyzer SYNCHRON CX9 from Beckman (Beckman Coulter Inc., Fullerton, California, USA). Low-density lipoprotein-cholesterol (LDL-C) was calculated according to the Friedwald equation. Quantitative colorimetric measurement of glycohaemoglobin as a percentage of total haemoglobin was obtained. Estimated creatinine clearance (Cockroft and Gault Formula) value was determined, and the albumin/creatinine ratio was obtained. For DNA analysis, the PCR-RFLP method was used to determine the distribution of genotype and allele frequencies of the VDR gene (BsmI) polymorphism. DNA was extracted using a commercially available spin-column technique kit for DNA extraction from human whole blood (Gene JET Whole Blood Genomic DNA Purification Mini Kit Medical Sciences Building on University of Toronto's St. George Campus, Canada), and was stored in –20°C until PCR was performed.

PCR amplification was performed in a total volume of 25-μl mixture containing 8.5 μl of template DNA (100–500 ng), 12.5 μl 2 × PCR Master Mix, 1 μl H2O, and 1.5 μl (0.5 μmol) of each primer. The forward primers were CAA CCA AGA CTACAA GTA CCG CGT CAG TGA and the reverse primers were AAC CAG CGG GAAGAG GTC AAG. The mixture was incubated for 1 min at 94°C for initial denaturation, followed by 29 cycles of 60 s at 94°C, 60 s at annealing temperature 65°C, 60 s at 72°C and 5 min at 72°C for final extension. The PCR fragments (825 bp) were digested using restriction enzyme BsmI (New England Biolabs, Beverly, MA, USA). The digestion mixture was incubated for 16 h at 37°C. The digested samples were separated by electrophoresis on 2% agarose gel stained with ethidium bromide, and were visualised on a UV transilluminator. The following results for the three genotypes were identified: BB yields (825 bp) not divided; bb divided into two fragments (650 and 175 bp), and Bb divided into three fragments (825, 650, and 175 bp).

Statistical analysis

The collected data were analysed using SPSS software (version 20, IBM SPSS Statistics for Mac, Released 2011; IBM Corp., Armonk, New York, USA). Descriptive statistics in the form of mean ± SD were used for parametric data. The χ2-test was used for qualitative variables, and the Student t-test was used for quantitative variables. Analysis of variance was used for comparing between three or more groups having normally distributed quantitative variables. Fischer's exact test for 2 × 2 tables when the expected cell count of more than 25% of cases was less than five was performed. The Mann–Whitney test (U) (nonparametric test) is a test of significance, and was used for comparison between two groups not having normally distributed quantitative variables. Odds ratio (ORs) and confidence intervals (CI) were calculated. The significance level was set at 0.05 or less.

 Results



In the present study, there were no statistically significant differences between the studied groups regarding age and sex (P = 0.987, 1.00, respectively). The T2DM group had higher BMI compared with the control group (P < 0.001) [Table 1].{Table 1}

There was a highly significant statistical difference between the T2DM group and the control group with regard to mean blood pressure (P < 0.001) [Table 2].{Table 2}

There was a highly significant statistical difference between the T2DM group and the control group with regard to fasting blood sugar, 2HPP blood sugar, and HbA1c (P < 0.001) [Table 3].{Table 3}

There was a highly significant statistical difference between the T2DM group and the control group with regard to total cholesterol, triglycerides, LDL, and HDL (P < 0.001) [Table 4].{Table 4}

There was no significant statistical difference between the T2DM group and the control group with regard to urea and estimated glomerular filtration rate (eGFR) (P = 0.349 and 0.646, respectively). In addition, there was a highly significant statistical difference between the T2DM group and the control group with regard to the albumin/creatinine ratio (P < 0.001) [Table 5].{Table 5}

According to BB as the reference genotype, there was a highly significant statistical difference in the distribution of the Bb genotype between the T2DM group and the control group (P = 0.001). Moreover, this genotype increased the risk of diabetes mellitus by 5.610 fold (OR = 5.610, 95% CI = 1.97–15.96). There was also no significant difference in the distribution of the bb genotype between the T2DM group and the control group (P = 1.200). However, this genotype increased the risk for diabetes mellitus by 2.475 fold (OR = 2.475, 95% CI = 0.46–13.09), but it was not statistically significant. There was significant difference between the T2DM group and the control group with regard to the Bb + bb genotype (P = 0.001). This genotype significantly increased diabetes risk by 4.713 fold (OR = 4.713, 95% CI = 1.87–11.90). With respect to B as the reference allele, there was a significant difference in the distribution of the b allele between the T2DM group and the control group (P = 0.002). Moreover, this allele increase diabetes mellitus risk by 3.18 fold (OR = 3.18, 95% CI = 1.47–6.90) [Table 6].{Table 6}

Agarose gel electrophoresis of the BsmI gene showed that before addition of the restriction enzyme, the gene corresponded to a band size of 825 bp [Figure 1]a. After addition of the restriction enzyme, agarose gel electrophoresis showing PCR-RFLP analysis of the BsmI gene corresponded to the following: lanes 1 (ladder), 2, and 6 (BB) band size 825 bp; lanes 3 and 7 (bb) band size 650 and 175 bp; and lanes 4, 5,8, 9, and 10 (Bb) band sizes 825, 650, and 175 bp [Figure 1]b.{Figure 1}

 Discussion



In the present study, the mean age of participants was not a significant factor in diabetic patients as compared with the control group (P = 0.987). This result is in agreement with another study, as age was found to be unimportant and duration of diabetes was found to be important [6].

In our study, the number of females was higher than the number of males between groups, but with no statistically significant difference (P = 1.00). In disagreement with our result, another study showed that male sex is an independent risk factor for diabetes [7]. In addition, there was a strong association between male sex and increased incidence of diabetes in the study by Ahmedani et al. [8].

The mean value of blood pressure was significantly higher than normal in the patient group (P < 0.001) when compared with the control group. These results are in agreement with Zhang et al. [9], who reported that diabetic patients had higher blood pressure than the control group.

In our study, there was a highly significant difference between the studied groups with regard to BMI (P < 0.001). In agreement with our results, another study demonstrated that patients with diabetes had higher BMI values compared with controls [10],[11]. However, Zhang et al. [9] showed that there was no significant difference between T2DM patients and controls with regard to BMI.

Fasting blood glucose levels were significantly higher in diabetic patients compared with controls (P < 0.001). In agreement with our results, another study demonstrated that patients with diabetes mellitus had high fasting plasma glucose levels [12].

In our study, the mean postprandial blood glucose level was significantly higher in diabetic patients when compared with the control group (P < 0.001).

In accordance, a study by Tanaka [12] showed that diabetic patients had high 2HPP plasma glucose levels than control patients.

HbA1c was significantly higher in diabetic patients than controls (P < 0.001). A study by Lind et al. [13] showed that HbA1c was high in diabetic patients than in controls.

Total cholesterol and triglyceride levels were significantly higher in the patient group in comparison with the control group. In consistence with these results, another study showed that cholesterol and triglyceride levels were significantly higher in diabetic patients than in healthy controls [14]. In contrast, a study by Buch Archana et al. [15] showed that there was no significant difference in the lipid profile of diabetic patients when compared with healthy controls.

LDL and HDL were significantly higher in the studied group when compared with controls. In agreement with our results, patients had higher LDL-C, but lower HDL-C levels, than the controls in the studies by Yu et al. [10] and Xiao et al. [11]. In disagreement with our results, another study showed that there was no difference in HDL and LDL levels between the diabetic group and the control group [9].

The mean blood urea levels were not significant in diabetic patients (P = 0.349) when compared with controls. In contrast with our results, another study showed that patients with diabetes had significant high values of blood urea [15].

Serum creatinine levels were significantly higher in diabetics in comparison with the control group (P < 0.001). In accordance, a study by Amin et al. [16] showed that serum creatinine levels were higher than the normal range in diabetic patients.

The mean of eGFR rate was lower in diabetics when compared with controls but was not statically significant (P = 0.646). In accordance, a study by Xiao et al. [11] showed that eGFR had a tendency to decrease with diabetes progression.

In the present study, the urine albumin/creatinine ratio was highly significant in diabetic patients when compared with controls (P < 0.001). In accordance, a study by Abdulrahaman et al. [17] showed that the urine albumin/creatinine ratio was increased in diabetic patients as compared with controls.

The vitamin D endocrine system is associated with various diseases including diabetes, cancer, cardiovascular disorder, metabolic syndrome, autoimmune disorders, and tuberculosis [18]. Active vitamin D mediates its biological effects by binding to the VDR, which is located in the nuclei of target cells. VDR is associated with insulin secretion and sensitivity, and is also expressed in the pancreas. These results show that VDR have a possible role in the development of diabetes [18].

To date, more than 25 different polymorphisms have been mapped to the VDR locus. The human VDR gene is located on chromosome 12q13.1. It has at least five promoter regions, eight protein-coding exons, and six untranslated exons, which are alternatively spliced into FokI (in exon 2), BsmI and ApaI (both in intron 8), and TaqI (in exon 9). There are several reports that these VDR polymorphisms are associated with type 2 diabetes and insulin secretion [9],[19]. In addition, VDR polymorphisms are related to metabolic syndrome, metabolic changes related with obesity [20], and diabetic retinopathy [21]. These studies suggested that VDR polymorphism may play a possible role in the development of diabetic complications [22].

VDR gene polymorphisms have been identified to be associated with type 1 diabetes [23], T2DM, and insulin secretion [24].

In our study, the BsmI gene was statistically significant between cases and controls. In agreement with our study, many other studies have shown a significant association between BsmI gene polymorphism and susceptibility to T2DM [9],[22]. In contrast, other studies have shown no significant differences between T2DM patients and controls with regard to BsmI at rs1544410 [5],[10].

There were three genotypes detected at the BsmI site: BB (825 bp), bb (650 and 175 bp), and Bb (825, 650 and 175 bp) [9]. In our study, Bb and Bb + bb genotypes were significant in T2DM patients and increased the risk of T2DM by 5.610 fold and 4.713 fold, respectively. In addition, the b allele was significant in T2DM patients (P = 0.002). However, Zhang et al. [9], showed that BB + Bb phenotype and the frequency of the B allele in the T2DM group were higher than in the control group.

In our study, the bb phenotype was not significant in T2DM patients (P = 0.691). However, Zhang et al. [9], showed that the BB phenotype was significant in T2DM patients.

From these results, we concluded that the Bb genotype and the b allele increase diabetes risk, and BsmI gene polymorphism is associated with increased risk for T2DM.

An Egyptian study on patients with T1DM showed that the VDR gene polymorphisms (BsmI and FokI) were associated with increased risk of T1DM [25]. It also showed that Bb genotype, bb genotype, and b allele frequencies were significantly higher in T1DM than in control individuals [25].

This study demonstrated that VDR gene polymorphism (BsmI) is associated with susceptibility to T2DM in an Egyptian population, which can be explained by differences in VDR BsmI genotype distributions between cases and controls. Therefore, the BsmI polymorphism in the VDR gene could be used as a susceptibility marker for diabetes. However, further large studies will be needed to confirm this.

 Conclusion



Our data suggest that the VDR (BsmI) gene polymorphism genotype is associated with the risk of T2DM, and thus could be used as a genetic risk marker for T2DM.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1International Diabetes Federation (IDF). 2015 IDF Diabetes Atlas, 7th ed. Brussels: International Diabetes Federation. Available at: http://www.diabetesatlas.org [Last accessed on 2016 Apr 01].
2Tomimoto D, Okuma C, Ishii Y, Kobayashi A, Ohta T, Kakutani M, et al. JTT-553, a novel Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 inhibitor, improves glucose metabolism in diet-induced obesity and genetic T2DM mice. J Pharmacol Sci 2015; 129 51–58.
3Palmer ND, Hester JM, An SS, Adeyemo A, Rotimi C, Langefeld CD, et al. Resequencing and analysis of variation in the TCF7L2 gene in African Americans suggests that SNP rs7903146 is the causal diabetes susceptibility variant. Diabetes 2011; 60 :662–668.
4Al-Daghri NM, Alkharfy KM, Al-Othman A, El-Kholie E, Moharram O, Alokail MS, et al. Vitamin D supplementation as an adjuvant therapy for patients with T2DM: An 18-month prospective interventional study. Cardiovasc Diabetol 2012; 11:85.
5Wang Q, Xi B, Reilly KH, Liu M, Fu M. 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 2012; 39:9405–9414.
6Afkhami-Ardekani M, Modarresi M, Amirchaghmaghi E. Prevalence of microalbuminuria and its risk factors in type 2 diabetic patients. Indian J Nephrol 2008; 18:112–117.
7Alrawahi AH, Rizvi SGA, Al-Riyami D, Al-Anqoodi Z. Prevalence and risk factors of diabetic nephropathy in Omani type 2 diabetics in al-dakhiliyah region. Oman Med J 2012; 27:212–216.
8Ahmedani MY, Hydrie MZ, Iqbal A, Gul A, Mirza WB, Basit A. Prevalence of microalbuminuria in type 2 diabetic patients in Karachi: Pakistan: a multi-center study. J Pak Med Assoc 2005; 55:382–386.
9Zhang H, Wang J, Yi B, Zhao Y, Liu Y, Zhang K, et al. BsmI polymorphisms in vitamin D receptor gene are associated with diabetic nephropathy in type 2 diabetes in the Han Chinese population. Gene 2012; 495:183–188.
10Yu F, Wang C, Wang L, Jiang H, Ba Y, Cui LL, et al. Study and evaluation the impact of vitamin D receptor variants on the risk of type 2 diabetes mellitus in Han Chinese. J Diabetes 2017; 9:275-284.
11Xiao X, Wang Y, Hou Y, Han F, Ren J, Hu Z. Vitamin D deficiency and related risk factors in patients with diabetic nephropathy. J Int Med Res 2016; 44:673–684.
12Tanaka M. Relationship between fasting and 2-hour postprandial plasma glucose levels and vascular complications in patients with type 2 diabetes mellitus. J Int Med Res 2012; 40:1295–1303.
13Lind M, Oden A, Fahlen M, Eliasson B. The true value of HbA1c as a predictor of diabetic complications: Simulations of HbA1c variables. PLoS One 2009; 4:e4412.
14Waheed HJ. A comparative study for cystatin c and some biochemical markers for predicting diabetic nephropathy in Iraqi patients. Int J Curr Microbiol App Sci 2015; 4:108–114.
15Buch Archana CS, Chandanwale S. Study of renal and lipid profile in diabetic patients. Int J Pharm Bio Sci 2015; 5:33–41.
16Amin N, Mohamood R, Asad MJ, Zafar M, Raja AM. Evaluating urea and creatinine levels in chronic renal failure pre and post dialysis: a prospective study. J Cardiovasc Dis 2014; 2:1–4.
17Abdulrahaman A Momin PSN, Gouri MB. Albumin/creatinine ratio, as predictor of microalbuminuria, a risk factor for nephropathy in type 2 diabetes mellitus patients. Int J Health Sci Res 2011; 1:36–40.
18Tamilselvan B, Seshadri KG, Venkatraman G. Role of vitamin D on the expression of glucose transporters in L6 myotubes. Indian J Endocrinol Metab 2013; 17(Suppl 1): S326–S328.
19Ferrarezi DA, Bellili-Munoz N, Dubois-Laforgue D, Cheurfa N, Lamri A, Reis AF, et al. Allelic variations of the vitamin D receptor (VDR) gene are associated with increased risk of coronary artery disease in type 2 diabetics: the diabhycar prospective study. Diabetes Metab 2013; 39:263–270.
20Zhao Y, Liao S, He J, Jin Y, Fu H, Chen X, et al. Association of vitamin D receptor gene polymorphisms with metabolic syndrome: a case-control design of population-based cross-sectional study in North China. Lipids Health Dis 2014; 13:129.
21Hong YJ, Kang ES, Ji MJ, Choi HJ, Oh T, Koong S et al. Association between Bsm1 polymorphism in vitamin D receptor gene and diabetic retinopathy of type 2 diabetes in Korean population. Endocrinol Metab 2015; 30:469–474.
22Al-Daghri NM, Al-Attas OS, Alkharfy KM, Khan Nb, Mohammed A, Vinodson B, et al. Association of VDR-gene variants with factors related to the metabolic syndrome, type 2 diabetes and vitamin D deficiency. Gene 2014; 542:129–133.
23Cooper JD, Smyth DJ, Walker NM, Stevens H, Burren OS, Wallace C, et al. Inherited variation in vitamin D genes is associated with predisposition to autoimmune disease type 1 diabetes. Diabetes 2011; 60:1624–1631.
24Li L, Wu B, Liu JY, Yang LB. Vitamin D receptor gene polymorphisms and type 2 diabetes: a meta-analysis. Arch Med Res 2013; 44:235–241.
25Abd-Allah SH, Pasha HR, Hagrass HA, Alghobashy AA. Vitamin D status and vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Egyptian children. Gene 2013; 536:430–434.