|Year : 2016 | Volume
| Issue : 1 | Page : 46-51
Lack of association between FokI polymorphism in vitamin D receptor gene (VDR) & type 2 diabetes mellitus in the Tunisian population
Imen Mahjoubi1, Amani Kallel1, Mohamed Hédi Sbaï1, Bochra Ftouhi2, Meriam ben Halima1, Zeineb Jemaa1, Moncef Feki1, Hedia Slimane2, Riadh Jemaa1, Naziha Kaabachi1
1 Research Laboratory LR99ES11, Biochemistry Department, University of Tunis El Manar, Tunis, Tunisia
2 Endocrinology Department, Rabta University Hospital, Tunis, Tunisia
|Date of Submission||15-Oct-2014|
|Date of Web Publication||3-Nov-2016|
Laboratoire de Biochimie, Hôpital la Rabta, 1007, Jabbari, Tunis
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: The impact of several environmental and genetic factors on diabetes is well documented. Though the association between the vitamin D receptor (VDR) gene polymorphisms and type 2 diabetes mellitus (T2DM) has been analyzed in different ethnic groups, the results have been inconsistent. The aim of this study was to evaluate the possible association between VDR FokI polymorphism and genetic susceptibility to T2DM in Tunisian population.
Methods: A total of 439 unrelated patients with T2DM and 302 healthy controls were included in the study. Genomic DNA was extracted from blood and genotyped for the single nucleotide polymorphism (SNP) of FokI (T/C: (rs2228570) by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis.
Results: The genotype distribution and the relative allelic frequencies for the FokI polymorphism were not significantly different between T2DM and controls: in T2DM patients the frequencies of the CC, CT, and TT genotypes were 52.6, 41.0, and 6.1 per cent, respectively, and in controls the genotype frequencies were 55.6, 38.7, and 5.6 per cent, respectively. In our study, the TT genotype of the FokI polymorphism was not associated with T2DM (OR =1.19, 95% CI 0.63 - 2.25, P=0.577).
Interpretation & conclusions: Our study showed no significant association of the FokI polymorphism in the vitamin D receptor gene with type 2 diabetes mellitus in Tunisian population.
Keywords: FokI - single nucleotide polymorphism - T allele - type 2 diabetes - VDR
|How to cite this article:|
Mahjoubi I, Kallel A, Sbaï MH, Ftouhi B, ben Halima M, Jemaa Z, Feki M, Slimane H, Jemaa R, Kaabachi N. Lack of association between FokI polymorphism in vitamin D receptor gene (VDR) & type 2 diabetes mellitus in the Tunisian population. Indian J Med Res 2016;144:46-51
|How to cite this URL:|
Mahjoubi I, Kallel A, Sbaï MH, Ftouhi B, ben Halima M, Jemaa Z, Feki M, Slimane H, Jemaa R, Kaabachi N. Lack of association between FokI polymorphism in vitamin D receptor gene (VDR) & type 2 diabetes mellitus in the Tunisian population. Indian J Med Res [serial online] 2016 [cited 2021 Sep 17];144:46-51. Available from: https://www.ijmr.org.in/text.asp?2016/144/1/46/193282
The incidence of type 2 diabetes mellitus (T2DM) is increasing at an alarming rate worldwide. T2DM is a multifactorial metabolic disorder, influenced by both genetic and environmental factors and exhibits a wide range of disparities among different ethnic groups ,,,, . The identification of genes predisposing to T2DM could provide means to better understand the pathogenesis of the disease and result in better prevention and treatment.
Vitamin D deficiency is shown to be associated with glucose intolerance, insulin resistance, metabolic syndrome, and increase risk for diabetes  . Vitamin D exerts its actions on target tissues through its binding to the cytosolic/nuclear vitamin D receptor (VDR), which is a member of the steroid/thyroid hormone receptor family that functions as a transcriptional activator of many genes. The gene encoding the VDR is located on chromosome 12q13.1  , contains 11 exons and spans approximately 75 kb. Several polymorphisms have been identified in the VDR gene, and their functional significance and potential effects on disease susceptibility have been investigated  . Among these polymorphisms, the FokI polymorphism (ATG®ACG) located in the exon2 of the gene is the only known locus affecting the structure of the VDR protein produced. The T allele encodes a 427 amino acid protein while the C allele encodes a 424 amino acid protein ,, . The shorter VDR protein variant seems to function more effectively and further increases its capacity of binding 1,25-dihydroxyvitamin D  , and the relatively higher level of vitamin D, in turn, can reduce the risk of T2DM by enhancing pancreatic b-cell secretion function and improve insulin resistance  . This biological mechanism could explain the association between the T allele of FokI polymorphism and susceptibility to T2D.
Several epidemiologic studies have examined the association of the FokI polymorphism of the VDR gene and T2DM, and the results are inconsistent across different populations ,,,,, . The aim of the present study was to analyze the association between the FokI polymorphism of the VDR gene and TD2M in a sample of the Tunisian population.
| Material & Methods|| |
A total of 741 individuals were included in this case-control study. Four hundred thirty nine unrelated patients with T2DM (263 women and 176 men) with mean age of 55.9 ± 9.7 yr were enrolled at the Department of Endocrinology of Rabta University Hospital of Tunis, Tunisia, from January 2007 to July 2009. Diabetes was diagnosed according to the World Health Organization (WHO) criteria  . Diabetes was defined as hyperglycaemia, requiring antidiabetic drugs or testing blood glucose level ≥ 7.0 mmol/l or a 2-h post-challenge glucose level ≥ 11.1 mmol/l. The control group consisted of 302 unrelated individuals (190 women and 112 men) with a mean age of 49.3 ±
9.6 yr, who underwent a medical checkup in our hospital. The exclusion criteria included fasting blood glucose levels of more than 100 mg/dl or a family history of diabetes. Diabetic patients with complications of malignancies were also excluded. Patients and controls were homogeneous Tunisian Arab lineage from Northern Tunisia. Informed written consent was obtained from all participants and the design of the study was approved by the local ethics committee.
Infromation on demography, socio-economic status, education, lifestyle and mental health was collected. Weight and height were measured. Body mass index (BMI; kg/m  ) was calculated and obesity was defined as BMI ≥ 30 kg/m  for both genders  . Hypertension was defined as systolic blood pressure (SBP) ≥ 140 mm Hg and/or diastolic blood pressure (DBP) ≥ 90 mm Hg, or the use of antihypertensive drug treatment or a combination of these. Dyslipidaemia was defined as a total cholesterol (TC) level > 6.47 mmol/l and /or triglyceride level (TG) > 2.26 mmol/l. Smoker definition included both ex-smokers and active smokers. A daily consumption of more than five cigarettes was considered a smoking habit  .
Laboratory analysis : Blood samples (10 ml) were obtained after an overnight fast. Blood glucose, TC, TG and high-density lipoprotein cholesterol (HDL-C) were measured in the hospital laboratory on a Hitachi 912 analyzer (Roche Diagnostics, Mannheim, Germany) using the respective reagent kits. Low-density lipoprotein cholesterol (LDL-C) was calculated according to the equation of Friedwald et al .
DNA analysis: Genomic DNA was prepared from white blood cells by phenol extraction  . Genotyping of the FokI (T/C) (rs2228570) VDR polymorphism was performed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as described previously  . Briefly, a fragment of 265 bp including the Fok1 polymorphism was amplified using two oligonucleotides  :
The PCR product was digested by the restriction enzyme FokI (MBI Fermentas, Vilnius, Lithuania) followed by electrophoresis on a 3 per cent agarose gel stained with ethidium bromide and visualized using ultraviolet illumination. The wild type homozygote (CC), heterozygote (CT) and mutant homozygote (TT) showed one band (265 bp), three bands (265, 196 and 69 bp) and two bands (196 and 69 bp), respectively [Figure 1]. The genotype of each sample was determined by two technicians working independently and for quality control, 20 per cent of the samples were selected at random for repeated genotyping and concordance was 100 per cent.
|Figure 1. The FokI digestion profiles of the 265 bp PCR product of the VDR gene homozygous (CC) individuals showing an undigested 265 bp product (lands 1, 2, 5 and 6); heterozygous (CT) individuals showing 265, 196, and 69 bp fragments (lane 3); homozygous (TT) showing 196 and 69 bp fragments (lane 7). GeneRuler DNA ladder (1000-50 bp) (lane 4)|
Click here to view
Statistical analysis: Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS 15.0 for windows, SPSS Inc., Chicago, IL, USA). Distributions of continuous variables in groups were expressed as mean ± SD, and compared with unpaired Student's t test. c  test was used to test for departures from Hardy-Weinberg equilibrium and to compare genotype distributions between groups. Odds ratio (OR) at 95% confidence interval (CI) was determined to describe the strength of association by logistic regression model.
| Results|| |
The clinical characteristics of the study population are shown in [Table 1]. There was significant differences for age (P<0.001), and BMI (P<0.001), and the frequencies of dyslipidaemia (P<0.001), hypertension (P<0.001) and cigarette smoking (P<0.01) between T2DM and control groups. The baseline TG (P<0.05) and LDL-C concentrations were higher in the T2DM patients (P<0.01). In addition, T2DM patients presented lower HDL-C levels (P<0.001). The genotype distribution and the relative allele frequency of the FokI polymorphism at the VDR gene in T2DM patients and controls are shown in [Table 2]. Genotype frequencies did not deviate from the Hardy-Weinberg equilibrium in control individuals and T2D patients. The frequencies of the CC, CT and TT genotypes among control group were 55.6, 38.7, and 5.6 per cent, respectively whereas the corresponding frequencies among the patients were 52.6, 41.0 and 6.4 per cent, respectively.
|Table 1. Demographic and clinical characteristics of the study population |
Click here to view
|Table 2. Genotype distribution and allele frequencies of VDR-FokI polymorphism |
Click here to view
No significant difference in polymorphism, genotype distribution and allele frequency was observed between patients and controls [Table 2]. When the samples were subgrouped by gender, no significant association was noted between T2DM patients and the controls (data not shown).
| Discussion|| |
Type 2 diabetes mellitus is a complex disease caused by complex interplay between environmental and genetic factors. Vitamin D is essential for the function of the endocrine pancreas, and the VDR gene may be involved in the pathogenesis and progression of T2DM. Several studies have examined the association of various VDR genetic variants and T2DM, and the results are inconsistent , . Our data showed that the FokI VDR polymorphism was not associated with T2DM in Tunisian population. The T allele frequency of FokI VDR polymorphism was similar between T2DM and control subjects. The FokI polymorphism, either singly or in combination with other VDR polymorphisms, has been investigated in a few studies on diabetes risk assessment and results were controversial [Table 3]. Bid et al using FokI, BsmI, and TaqI demonstrated that there was no link between polymorphisms in the VDR gene and T2DM. Malecki et al reported no significant difference in the distribution of the allele and genotype frequencies of the FokI polymorphism between 308 T2DM patients and 239 healthy controls from Poland similar to our study. However, in an Egyptian study, involving 63 patients with T2DM and 60 controls, the FokI variant was significantly associated with risk of T2DM only in patients with metabolic syndrome  . A meta-analysis of 10 studies involving 1562 cases and 1461 controls, showed that the FokI polymorphism was associated with an increased risk of T2DM (T vs. C: OR = 1.30, 95% CI = 1.17 - 1.45, P<0.001), especially in East Asians  . This was confirmed in another meta-analysis including five studies (1101 cases and 969 controls) of Li et al , which showed that allele T and variant homozygote TT of FokI variant were significantly associated with T2DM, especially in an Asian population (T vs. C: OR = 1.25, 95% CI = 1.10 - 1.41; TT vs. CC: OR = 1.48, 95% CI = 1.13 - 1.94). A meta-analysis by Yu et al including 12 studies showed significant associations of FokI polymorphism with T2DM for three genetic models (TT vs. CC: OR = 1.57, 95% CI = 1.28 - 1.93, P<0.001; CT vs. CC: OR = 1. 54, 95% CI = 1.31 - 1.81, P<0.001; TT + CT vs. CC: OR = 1.57, 95% CI = 1.35 - 1.83 , P<0.001), especially in Chinese population. On the other hand, a protective effect of the FokI T allele against T2DM was reported by Errouagui et al in a case-control study involving 176 patients with T2DM and 177 healthy controls subjects (OR = 0.35, 95% CI = 0.14 - 0. 83, P=0.018) in Moroccan population.
|Table 3. Associations with type 2 diabetes mellitus of the VDR gene in various sample populations |
Click here to view
The discrepancies between the studies may be explained by the different allelic frequencies observed in different ethnic groups. For example, the C allele frequency was lower in Africans when compared to Caucasians and Asians  . Other explanation for the diversity of the results are selection criteria adopted for patients and controls, in particular age, ethnicity, extent of disease, concomitant environmental risk factors like differences in the lifestyles (smoking, diet and physical activity) and the gene-gene and gene-environment interactions.
The present study had some limitations. First, the small size of patients and controls groups which may limit the power (60%) to detect the FokI polymorphism effect on T2DM. Second, our results were limited by the absence of both dietary information and plasma vitamin D concentrations for our study participants. Studies have shown that the association between VDR polymorphisms and disease can vary by either past sun exposure or vitamin D level  . Our study was based on estimates without adjusting for sun exposure or vitamin D intake. Third, polymorphisms of other VDR genotypes, i.e., TaqI, ApaI, and BsmI, and their possible interactions with FokI variants were not evaluated.
In conclusion, our study indicated that the FokI variant of the VDR gene was not associated with T2DM in the Tunisian population. It is possible that the effect of FokI variant on T2DM risk is specific to some particular ethnic populations. The present results require confirmation in further and larger studies in the Tunisian population.
| Acknowledgment|| |
This work was supported by a grant from the "Ministry of Higher Education and Scientific Research" of Tunisia.
Conflicts of Interest : None.
| References|| |
Kahn CR, Vincent D, Doria A. Genetics of non-insulin-dependent (type II) diabetes mellitus. Ann Rev Med
Zisser H, Gong P, Kelley CM, Seidman JS, Riddell MC. Exercise and diabetes. Int J Clin Pract Suppl
2011; (170): 71-5.
Prokopenko I, McCarthy MI, Lindgren CM. Type 2 diabetes: new genes, new understanding. Trends Genet
Ferland-McCollough D, Ozanne SE, Siddle K, Willis AE, Bushell M. The involvement of microRNAs in type 2 diabetes. Biochem Soc Trans
Rezende VB, Barboza F Jr, Montenegro MF, Sandrim VC, Gerlach RF, Tanus-Santos JE. An interethnic comparison of the distribution of vitamin D receptor genotypes and haplotypes. Clin Chim Acta
Salekzamani S, Neyestani TR, Alavi-Majid H, Houshiarrad A, Kalayi A, Shariatzadeh N, et al
. Is vitamin D status a determining factor for metabolic syndrome? A case-control study. Diabetes Metab Syndr Obes
Taymans SE, Pack S, Pak E, Orban Z, Barsony J, Zhuang Z. The human vitamin D receptor gene (VDR
) is localized to region 12cen-q12 by fluorescent in situ
hybridation and radiation hybrid mapping: Genetic and physical VDR map. J Bone Mineral Res
Zmuda JM, Cauley JA, Ferrell RE. Molecular epidemiology of vitamin D receptor gene variants. Epidemiol Rev
Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene
Arai H, Miyamoto K, Taketani Y, Yamamoto H, Iemori Y, Morita K, et al
. A vitamin D receptor gene polymorphism in the translation initiation codon : effect on protein activity and relation to bone mineral density in Japanese women. J Bone Miner Res
Nejentsen S, Godfrey L, Snook H, Rance H, Nutland S, Walker NM. Comparative high-resolution analysis of linkage disequilibrium and tag single nucleotide polymorphisms between populations in the vitamin D receptor gene. Hum Mol Genet
Reis AF, Hauache OM, Velho G. Vitamin D endocrine system and the genetic susceptibility to diabetes, obesity and vascular disease. A review of evidence. Diabetes Metab
Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis. J Clin Endocrinol Metab
Wang 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
Malecki MT, Frey J, Moczulski D, Klupa T, Kozek E, Sieradzki J. Viamin D receptor gene polymorphisms and association with type 2 diabetes mellitus in a polish population. Exp Clin Endocrinol Diabetes
Bid HK, Konwar R, Aggarwal CG, Vitamin D receptor (FokI, BsmI,
) gene polymorphisms and type 2 diabetes mellitus: A North Indian study. Indian J Med Sci
Al-Daghri NM, Al-Attas O, Alokail MS, Alkharfy KM, Draz HM, Agliardi C, et al
. Vitamin D receptor gene polymorphisms and HLA DRB1*04 cosegregation in Saudi type 2 diabetes Patients. J Immunol
Vedralova M, Kotrbova-Kozak A, Zeleznikova V, Zoubkova H, Rychlik I, Cerna M. Polymorphisms in the vitamin D receptor gene and parathyroid hormone gene in the development and progression of diabetes mellitus and its chronic complications, diabetic nephropathy and non-diabetic renal disease. Kidney Blood Press Res
Li L, Bo W, Ji-Yong L, Li-Bo Y. Vitamin D receptor gene polymorphisms and type 2 diabetes: A meta-analysis. Arch Med Res
Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus. Provisional report of a WHO consultation. Diabet Med
World Health Organization. Physical status: the use and interpretation anthropometry
: Report of a WHO Expert Committee
, WHO Technical Report Series, no. 854. Geneva: WHO; 1995. p. 321-44.
Wardle H, Mindell J. Adult cigarette smoking and exposure to other's smoke. In: Craig R, Shelton N, editors. Health lifestyles: knowledge, attitudes and behaviour.
London: NHS Information Centre for Health and Social Care; 2008. p. 149-76.
Friedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem
Marcadet A, O'Connel P, Cohen D. Standardized Southern blot workshop techniques. In: Dupont B. editor. Histocompatibility testing.
New York: Springer; 1987. p. 587-90.
Karray EF, Ben Diffalah I, Ben Abdelghani K, Ben Ghorbel I, Khanfir M, Houman H, et al.
Association of FokI and BsmI polymorphisms in vitamin D receptor gene with susceptibility to rheumatoid arthritis and Behçet's disease in the Tunisian population. Rev Rumathol
Nosratabadi R, Arababadi MK, Salehabad VA. Vitamin D receptor polymorphisms in type 2 diabetes in Southeastern Iranian patients. Lab Med
Paul D, Khan I, Faruque O, Choudhury N, Hassan Z, Ali L. Association of vitamin D receptor gene G>T and T>C polymorphisms in young onset diabetes mellitus in a Bangladeshi population. J Mol Pathophysiol
Mackawy AMH, Badawi MEH. Association of vitamin D and vitamin D receptor gene polymorphisms with chronic inflammation, insulin resistance and metabolic syndrome components in type 2 diabetic Egyptian patients. Meta Gene
Errouagui A, Benrahma H, Charoute H, Ghalim N, Barakat A, Kandil M, et al
. Vitamin D receptor gene polymorphisms and vitamin D status and susceptibility to type 2 diabetes mellitus in Moroccans patients. Int J Sci Res Pub
Yu F, Cui L, Ba Y, Wang C, Li W, Wang L. Association of vitamin D receptor gene polymorphism with the risk of type 2 diabetes mellitus: an update meta-analysis. Nutr Diabetes Complications
Nikooyeh B, Neyestani TR, Farvid M, Alavi-Majd H, Houshiarrad A, Kalayi A, et al.
Daily consumption of vitamin D- or vitamin D + calcium-fortified yogurt drink improved glycaemic control in patients with type 2 diabetes: a randomized clinical trial. Am J Clin Nutr
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||Vitamin D Receptor gene polymorphisms and susceptibility to type 2 diabetes: evidence from a meta-regression and meta-analysis based on 47 studies
| ||Surendar Aravindhan,Mohammed Fadhil Mohammed Almasoody,Nihad Abdallah Selman,Alekhina Natalia Andreevna,Sahithya Ravali,Payam Mohammadi,Mohammad Masoud Eslami,Bahman Razi,Saeed Aslani,Danyal Imani |
| ||Journal of Diabetes & Metabolic Disorders. 2021; |
|[Pubmed] | [DOI]|
||Evaluation of association studies and a systematic review and meta-analysis of VDR polymorphisms in type 2 diabetes mellitus risk
| ||Yao Liu,Xin Guo,Shao-Yan Huang,Luan Gong,Jin-Hui Cui,Hu-Wei Shen,Xiang-Hua Ye,Xiao-Feng He |
| ||Medicine. 2021; 100(28): e25934 |
|[Pubmed] | [DOI]|
||Artificial Neural Networks Model for Predicting Type 2 Diabetes Mellitus Based on VDR Gene FokI Polymorphism, Lipid Profile and Demographic Data
| ||Ma’mon M. Hatmal,Salim M. Abderrahman,Wajeha Nimer,Zaynab Al-Eisawi,Hamzeh J. Al-Ameer,Mohammad A. I. Al-Hatamleh,Rohimah Mohamud,Walhan Alshaer |
| ||Biology. 2020; 9(8): 222 |
|[Pubmed] | [DOI]|
||Genotyping of Vitamin D Receptor FOKI Polymorphism as a predictor for Type 2 diabetes Mellitus by a Tetra primer-ARMS-PCR Assay
| ||Shaymaa M. Hadi,Riyadh Al-Zubaidy |
| ||Gene Reports. 2019; |
|[Pubmed] | [DOI]|
||Diminished 25-OH vitamin D
levels and vitamin D receptor variants are associated with susceptibility to type 2 diabetes with coronary artery diseases
| ||Lei Ma,Shujin Wang,Heming Chen,Lin Cui,Xiaoxiang Liu,Hua Yang,Guohong Li,Songfang Liu,Ting Qi,Hongyan Tian |
| ||Journal of Clinical Laboratory Analysis. 2019; |
|[Pubmed] | [DOI]|
||Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy
| ||Farideh Razi,Marzieh Arshadi Meshkani,Fariba Zarrabi,Maryam Sadr,Saeedeh Asgarbeik,Fatemeh Bandarian,Katayoon Forouzanfar,Mahsa Mohammad Amoli |
| ||Gene. 2018; |
|[Pubmed] | [DOI]|