ABSTRACT Aims/Introduction: The gene expressing vitamin D receptor

ABSTRACT

Aims/Introduction:  The gene expressing vitamin D receptor (VDR) has been involved in a number of disease conditions, such as cancer, bone diseases, tuberculosis, metabolic syndrome, obesity and diabetes. Association studies between the gene polymorphisms of the VDR gene and the susceptibility of type 2 diabetes mellitus (T2DM) in different populations and different environmental factors like atmospheric temperature are as yet inconclusive.  The role of vitamin is to plays the role of a transcription regulator in controlling the ?-cell insulin secretion by its interaction with its nuclear receptor complex.  This study aimed to examine the association between the VDR FokI and BsmI gene polymorphisms and the risk to T2DM among Saudi people  in Makkah region that has a warm to hot climate. 

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Materials and Methods: Extraction of genomic DNA from peripheral blood leucocytes was performed followed by genotyping for FokI (T/C) and BsmI (A/G) restriction sites using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR-RFLP).

Results: Our data showed that in Makkah region, which has a hot climate throughout the year, there is no significant difference of VDR FokI and BsmI gene polymorphisms between the subjects with T2DM and the control group (p > 0.05).  Thus our results validate and confirm a number of studies previously done in diverse ethnic populations (European, Chinese, and Tunisian subjects) and geographical locations.

Conclusion: The VDR FokI and BsmI gene polymorphisms may not be associated with the risk to T2DM among Saudi population in Makkah region.

 

 Key words

T2DM, Vitamin D Receptor, Gene Polymorphism

 

Abbreviations:   VDR, vitamin D receptor; T2DM, type 2 diabetes mellitus; T1DM,  type 1 diabetes mellitus; SNP, single nucleotide polymorphism; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

 

INTRODUCTION  

Diabetes mellitus (DM) is one of the major and serious health problem all over the world including Saudi Arabia1, 2.  Little is known about the cause of T2DM; nevertheless many of its risk factors have been identified and are investigated. T2DM, like other inflammatory conditions may be prevented if its risk factors are identified as early as possible, and managed3-6.  Therefore an understanding of T2DM risk factors and thereby applying corrective preventive measures is the first step in disease prevention, as this  will allow T2DM patients to make the informed decision that leads to a healthy lifestyle7,8. In Saudi Arabia, there are relatively few studies conducted to determine and evaluate the T2DM risk factors and preventive measures in the Saudi population9.

It is well known that environment and genes both play contributory roles in the development of T2DM.    Although the genetic factors play a critical role in various forms of diabetes mellitus, how the inheritance of these genetic loci contributing to the disease remains obscure.   Furthermore, such genetic factors may also interact with environmental factors such as diet and atmospheric temperature10,11.  In general, predominantly, T2DM and T1DM are polygenic conditions; however, several monogenic forms of diabetes have been identified12.  Such identified genes are grouped into different categories such as those involved in regulation of growth factors, those mediating signal transduction, and those involved in energy metabolism and energy utlization13. A number of association studies have been carried out on the relationship between the genetic variation and diabetes; these studies require confirmation in different racial and ethnic groups and environmental conditions14,15.

The VDR gene is highly poly-morphic and is located on chromosome 12q12-14. There are six commonly studied VDR polymorphisms: FokI polymorphism in exon 2; BsmI, Tru9I, and ApaI polymorphisms located between exons 8 and 9; the TaqI polymorphism present in exon 9; and the poly-A polymorphism downstream of the 3´ un-translated region16,17.  The involvement of vitamin D in the development of T1DM has been investigated in several studies, and it was shown that infants whose diets were supplemented with vitamin D have a lower incidence of T1DM in adulthood18.   In addition, abnormal vitamin D and calcium homeostasis also contributes in the development of T2DM.  High vitamin D status in subjects have been shown to provide protection against T2DM19,20.  

 

Vitamin D Receptor (VDR) is a member belonging to the steroid/thyroid hormone receptor family21.  Vitamin D plays an important role in the regulation of the endocrinal functions of pancreas, particularly in the secretion of insulin22. The mediation of vitamin D action is performed through binding to a specific nuclear receptor (VDR) that is expressed in pancreatic beta (?)-cells21. It has been shown that insulin secretion from the beta (?)-cell is regulated by Vitamin D and its receptor complex. Furthermore, it has been reported that vitamin-D deficiency reduces insulin synthesis and secretion in humans and in animal models of diabetes and vitamin D supplement in diet may increase the secretion of insulin23,24.   Gene polymorphisms have been described in the VDR genomic sequences that are able to alter the activity of VDR protein25.  Although the genetic basis of T2DM is still poorly understood, several studies suggested that the VDR gene is a novel candidate gene contributing to the susceptibility to the diabetes and specifically T2DM26-30.  

Herein, we aimed to investigate the association between VDR gene FokI and BsmI polymorphisms and the susceptibility of T2DM among Saudi population in Makkah region and its environs.  Few studies about gene polymorphism of VDR gene in T2DM have been conducted in Saudi Arabia31. Thus, it is important to confirm the association between VDR polymorphism and the susceptibility of T2DM in a select Saudi population.

 

MATERIALS AND METHODS

Subjects and sample collection:  This study was performed between 2014 and 2016 and it included 163 subjects (Table 1). Fully informed consent was obtained from the control and diseased groups, and this study was approved by the Umm Al Qura University’s IRB (Internal Review Board).  Seventy eight unrelated type 2 diabetic patients were recruited from health centers and hospitals in Makkah region, Saudi Arabia. The control subjects consisted of eighty five healthy subjects who either attended a routine health check at a general practice or at work. Venous blood was collected from all subjects between 9:00 and 11:00 a.m. after fasting from 10:00-11:00 p.m. the previous day. Each sample was divided into two halves, one half for the serum preparation and the other half was put in sterile K3EDTA (tri-potassium ethylene-diaminetetraacetic acid) coated tubes. Low speed centrifugation was used to isolate blood plasma; white cells were removed from the buffy coat for the isolation of DNA. Samples were kept at –20?C till the time of use.

 

Determination of fasting blood glucose: 

We utilized glucose oxidase method to determine the fasting glucose level, using a kit purchased from Human Diagnostics, Wiesbaden, Germany.  The protocol was as given in the instructions by the kit manufacturer.

 

Determination of hemoglobin A1c (HbA1c): The quantity ofHbA1c was determined using enzymatic HbA1c assay kit according to the manufacturer instructions (Human Diagnostics, Wiesbaden, Germany).

 

Genomic DNA extraction:

Genomic DNA was extracted from peripheral blood leukocytes using Qiagen DNA extraction kit (QIAamp DNA Blood Mini Kits, Qiagen, Hilden, Germany). The extraction was done according to the manufacturer instructions.   For PCR amplification of the DNA, aliquots of genomic DNA were utilized.

 

VDR FokI and BsmI genotyping:

The FokI polymorphic region of exon 2 of VDR gene was performed as described elsewhere with slight modification32. It was amplified using the following oligonucleotide primers: 5′ -AGC TGG CCC TGG CAC TGA CTC TGC TCT-3′ (forward primer) and: 5′ -ATG GAA ACA CCT TGC TTC TTC TCC CTC- 3′ (used as the reverse primer).  In 25 ml PCR mixture, 100 ng of genomic DNA were used for PCR amplification and 2 ´ PCR master mix was used (Thermo Fisher Scientific, Inc, MA USA).  Following conditions were used for the PCR amplification: initial DNA denaturation at 96°C for 4 min, then 30 cycles of denaturation for 30 seconds at 94 °C, annealing for 30 seconds at 57 °C, and extension for 30 seconds at 72 °C. The reaction was terminated following 7 min elongation at 72 °C. PCR product was then digested with FokI restriction endonuclease.  Restriction fragments were analyzed by electrophoresis through a 2 % agarose gel containing ethidium bromide, the bands were visualized under ultraviolet light and photographed.

For the genotyping of VDR BsmI, the following primers were used: the forward primer:  5¢-AAC CAG CGG GAA GAG GTC AAG GG- 3¢ -and the reverse primer: 5¢ -CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA- 3¢.  The PCR reaction was carried out in 25 ml final volume and 2 ´ PCR master mix was used.  The PCR protocol was as follows: initial denaturation step at 95 °C for 3 min, 35 ´ (95 °C for 30 s, 65°C for 30s, 72°C for 30 s) and finally, 72 °C for 10 min. The amplified PCR product was digested using BsmI restriction enzyme.  DNA fragments were separated on 2 % agarose gel by electrophoresis and genotypes were identified as previously described 33.

Statistical Analysis:  We utilized SPSS for Windows version 20.0 (SPSS Inc, Chicago, IL, USA) for data analysis. To compare mean values of continuous variable in cases and control, Student’s t test was used, whereas ?2 analysis was used to compare categorical data.

 

 

RESULTS

 

Table-1 shows the demographic data in studied groups. Table 2 shows the genotype and allele frequencies in both T2DM and control groups. The genotype distribution of FokI polymorphism is in Hardy-Weinberg equilibrium in both the T2DM and control groups. The genotypes FF, Ff and ff were 61.5 %, 24.3 % and 14.1% in subjects with T2DM respectively and were 55.2 %, 36.4 % and 8.2 % respectively in the control group.

 

We did not observe any statistically significant difference in the genotype and allele frequencies between patients with T2DM and healthy controls.  The BsmI genotype and allele distribution, were found to be in Hardy-Weinberg equilibrium in both T2DM and control groups. BsmI genotypes and allele frequencies in T2DM groups and in controls are shown in Table 2.  In the T2DM patients, the genotypes BB, Bb and bb were 29.48 %, 33.33 % and 37.17 % respectively.  However, these genotypes were 29.62 %, 32.1 % and 38.27 % in the control subjects respectively.   There was no statistically significant difference in the distribution of genotypes and allele frequencies of BsmI between patients with T2DM and the matched healthy controls.

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