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Association of VDR-gene variants with factors related to the metabolic syndrome, type 2 diabetes and vitamin D deficiency
Gene 542 (2014) 129–133
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Association of VDR-gene variants with factors related to the metabolic syndrome, type 2 diabetes and vitamin D deficiency Nasser M. Al-Daghri a,b,c,⁎, Omar S. Al-Attas a,b,c, Khalid M. Alkharfy a,b,d, Nasiruddin Khan b, Abdul Khader Mohammed b,c, Benjamin Vinodson b,c, Mohammed Ghouse Ahmed Ansari b, Amal Alenad e, Majed S. Alokail a,b,c a
Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia Biomarkers Research Program, Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia c Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, King Saud University, Riyadh, Saudi Arabia d Clinical Pharmacy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia e School of Biological Sciences, Life Science Building 85, University of Southampton, Southampton SO17 1BJ, UK b
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 16 March 2014 Accepted 21 March 2014 Available online 25 March 2014 Keywords: Polymorphism Haplotype Obesity 25-Hydroxyvitamin D Cardiovascular diseases
a b s t r a c t The prevalence of metabolic syndrome (MetS) is rising alarmingly in the Saudi Arabian population. This study was conducted to assess the association between vitamin D receptor (VDR) polymorphisms and genetic susceptibility to components of the metabolic syndrome, type 2 diabetes mellitus (T2DM), and vitamin D deficiency in the Saudi Arabian population. Five-hundred-seventy Saudi individuals (285 MetS and 285 controls) were enrolled in this cross-sectional study. TaqI, BsmI, ApaI and FokI single nucleotide polymorphisms (SNPs) of the VDR gene were genotyped. The CT genotype and allele T of BsmI were associated with lower HDL-C levels [OR 0.60 (0.37, 0.96), p = 0.03] and obesity [OR 1.4 (1.0, 1.90), p = 0.04], respectively. The CT genotype and the dominant model CT + TT of BsmI were associated with increased risk of diabetes [OR 1.7 (1.2, 2.4), p = 0.007], and [OR 1.5 (1.1, 2.2), p = 0.01], respectively. On the contrary, the CT and CT + CC genotypes of FokI exhibited an association with a reduced risk of diabetes [OR 0.70 (0.49, 0.99), p = 0.05] and [OR 0.67 (0.48, 0.94), p = 0.02], respectively. The allele C of FokI was associated with lower risk of developing T2DM [OR 0.73 (0.56, 0.95), p = 0.02]. The prevalence of vitamin D deficiency was lower in subjects with the AC genotype of ApaI [OR, 0.34 (0.14, 0.80), p = 0.01]. Components of the MetS such as obesity, low HDL and T2DM were associated with the VDR gene. FokI and BsmI have protective and facilitative effects on the risk for T2DM, while the ApaI genotype was associated with reduced vitamin D deficiency. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The components of metabolic syndrome (MetS) (dyslipidemia, hyperglycemia, hypertension and obesity), individually and cumulatively, increase the risk of developing T2DM and cardiovascular diseases (CVD) (Alkharfy et al., 2012). Moreover, the involvement of vitamin D in the prevalence of metabolic syndrome has also been suggested (Ford et al., 2005). The mechanism of action regarding the effect of vitamin D either includes binding of the active metabolite 1,25 (OH)2D3 with the cytosolic/nuclear VDR or via non-genomic pathways (Lips, 2006). Abbreviations: MetS, metabolic syndrome; VDR, vitamin D receptor; T2DM, type 2 diabetes mellitus; single SNPs, nucleotide polymorphisms; IDF, International Diabetes Federation; HDL, high density lipoprotein; PHCC, Primary Health Care Centers; OR, odds ratio. ⁎ Corresponding author at: Prince Mutaib Bin Abdullah Chair for Osteoporosis, Biochemistry Department, College of Science, King Saud University, PO Box, 2455, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (N.M. Al-Daghri).
http://dx.doi.org/10.1016/j.gene.2014.03.044 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Cytosolic/Nuclear VDR is a member of the steroid/thyroid hormone receptor family that functions as a transcriptional activator of many genes (Uitterlinden et al., 2004b). Polymorphisms in the VDR gene that produce variation in the activity of the VDR have been described in various populations (Valdivielso and Fernandez, 2006). Polymorphisms in the VDR gene have also been shown to be associated with the components of MetS, obesity and T2DM in different populations (Bid et al., 2009; Filus et al., 2008). In addition, increased susceptibility to type 1 diabetes has also been associated with allelic variations of the VDR gene (McDermott et al., 1997; Pani et al., 2000). Most VDR gene polymorphisms, including the BsmI, ApaI and TaqI restriction fragment length polymorphisms, are located at the 3′ untranslated region (3′ UTR) of the gene (Panierakis et al., 2009), while the FokI polymorphism, is localized within the 5′ end of the gene, near the promoter region (Uitterlinden et al., 2004b). Recently, we demonstrated cosegregation between VDR and HLA alleles in T2DM patients in the Saudi Arabian population (Al-Daghri et al., 2012a). Numerous crosssectional studies have noted significant negative associations among
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circulating levels of 25-hydroxyvitamin D and cardiometabolic risk factors, highlighting potential extra skeletal functions of this sterol hormone (Al-Daghri et al., 2012b). However, there are still very limited studies that combine VDR gene polymorphisms with the components of the MetS, T2DM, and vitamin D deficiency in this part of the world. Therefore, the aim of this study was to examine the association of four single nucleotide polymorphisms (SNPs) in intron 8 (BsmI, ApaI) exon 9 (TaqI) and exon 2 (FokI) of the VDR gene with components of MetS, T2DM, and vitamin D deficiency in the Saudi Arabian population.
rs10735810) were evaluated by allelic discrimination Real-time PCR using pre-designed TaqMan probes (Applied Biosystems, Foster City, CA, USA). The PCR consisted of a hot start at 95 °C for 10 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. Fluorescence detection takes place at a temperature of 60 °C. All assays were performed in 10 μl reactions, using TaqMan Genotyping Master Mix on 96-well plates using an ABI 7000 instrument (Applied Biosystems, Foster City, CA, USA). Control samples representing all possible genotypes and a negative control were included in each reaction.
2. Materials and methods
2.5. Statistical analyses
2.1. Study design
Data were analyzed using the Statistical Package for the Social Sciences for Windows (SPSS version 16.0, Chicago, IL, USA) and are expressed by mean ± standard deviation (SD). Data was checked for normality using Kolmogorov–Smirnov test. All non-Gaussian variables were either log or square root transformed. Independent sample t-test was used to test control and metabolic syndrome groups. Analysis of variance (ANOVA) was performed between genotypes for each parameters followed by Bonferroni post-hoc test. A chi-square test was used to show the allele and genotype frequency for each SNP in patients and controls. Odds ratios (ORs) and 95% confidence intervals are calculated by binomial logistic regression for the allele, genotype with metabolic diseases/complications after adjustment for covariates including gender, age and body mass index (BMI). Haplotype frequencies were estimated by the Expectation–Maximization algorithm (EM algorithm) implemented in PROC Haplotype in SAS Genetics statistical software package (SAS institute, Cary, NC, USA). The most common haplotype was used as the reference and rare haplotypes were dropped from the analysis. Tests of departures from LD were performed by using the likelihood ratio test (LR test) of linkage disequilibrium as used in PROC Allele of SAS Genetics. Pairwise LD estimations were performed using Haploview 4.2. A power estimation, based on previous studies (Filus et al., 2008; Velayoudom-Cephise et al., 2011; Ye et al., 2001), showed that our study design for specific aims (BMI, Vit D, HDL-C) had 80% power to detect a similar sized-effect (α = 0.05). Significance was set at p b 0.05.
Five-hundred-seventy Saudi individuals (285 MetS patients and 285 healthy controls) were enrolled in the study. These individuals are part of the Biomarker Screening in Riyadh Project (RIYADH COHORT), a capital-wide epidemiologic study taken from over 17,000 consenting Saudis coming from different Primary Health Care Centers (PHCCs). The MetS includes waist circumference ≥102 cm for men and ≥88 cm for women, triglycerides ≥1.7 mmol/l and HDL-Cholesterol b1.03 mmol/l for men and 1.29 mmol/l for women, blood pressure ≥130/85 and fasting plasma glucose levels ≥5.6 mmol/l. Diagnosis was based on the International Diabetes Federation (IDF), which defines MetS as central obesity plus 2 other factors. Healthy control subjects were those who did not match the criteria employed for the selection of MetS subjects. A generalized questionnaire aimed to seek demographic information, past medical history, current medication and family history was given to all participating subjects. Those with co-morbidities that needed medical attention were excluded from the study. Written and informed consents were taken before inclusion. Ethics approval was granted by the Ethics Committee of the College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia (KSA). 2.2. Anthropometry and blood collection Participating subjects were requested to return to their respective PHCCs after an overnight fast (N10 h) for anthropometry and blood withdrawal. Anthropometry included height (to the nearest 0.5 cm), weight (to the nearest 0.1 kg), waist and hip circumference utilizing a standardized measuring tape in cm, systolic and diastolic blood pressure measurements, and BMI calculated as weight in kg divided by height in square meters. Overweight was defined as having a BMI of 25–29.9 kg/m2, obesity ≥30 b 34.9 and morbid obesity N35. Vitamin D deficiency was defined as 25-(OH)D level b50 nmol/L (Bischoff-Ferrari et al., 2006). Blood was transferred immediately to a non-heparinized tube for centrifugation. Serum was then transferred to a pre-labeled plain tube, stored in ice, and delivered to the Biomarker Research Center in King Saud University on the same day. 2.3. Biochemical analysis Fasting serum samples were stored in a − 20 °C freezer prior to analysis. Fasting glucose (FG), lipid profile, albumin, phosphorus and calcium were measured using a chemical analyzer (Konelab, Vantaa, Finland). Serum 25-hydroxyvitamin D [25-(OH)D] was measured by enzyme linked immunosorbent assays (ELISA) (IDS Ltd, Boldon Colliery, Tyne & Wear, UK). The intra-assay variation was 1.4–7.9% and interassay variation was b21%. 2.4. VDR gene analysis Whole blood was collected in EDTA-containing tubes and genomic DNA was isolated from whole blood by using the blood genomic prep minispin kit (GE healthcare, Piscataway, NJ, USA), stored at − 20 °C until analyzed. The four VDR SNPs (rs731236, rs1544410, rs7975232,
3. Results The anthropometric, epidemiologic and metabolic characteristics of MetS and controls are depicted in Table 1. Anthropometric and
Table 1 Anthropometric, epidemiologic, and metabolic characterization of the MetS and control individuals.
N Age (years) BMI (kg/m2) Hips (cm) Waist (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Cholesterol (mmol/l) Glucose (mmol/l) Triglycerides (mmol/l) HDL (mmol/l) LDL (mmol/l) Pi (mmol/l) Ca (mmol/l) Corr. Ca (mmol/l) Albumin (g/l) Vitamin D (nmol/l)
Control
MetS
p-Value
285 42.2 ± 16.2 28.7 ± 7.2 92.6 ± 25.6 79.4 ± 21.7 123.2 ± 16.4 76.8 ± 10.1 5.2 ± 1.7 7.4 ± 0.73 1.5 ± 0.35 0.91 ± 0.32 3.9 ± 1.0 1.1 ± 0.31 2.5 ± 0.34 2.4 ± 0.35 45.5 ± 7.7 32.5 ± 14.2
285 49.5 ± 12.7 32.6 ± 6.2 112.7 ± 12.4 105.3 ± 11.7 127.9 ± 13.5 79.7 ± 7.8 5.5 ± 1.1 9.0 ± 0.65 2.0 ± 0.34 0.89 ± 0.32 4.3 ± 1.0 1.2 ± 0.23 2.5 ± 0.24 2.5 ± 0.21 44.4 ± 5.2 26.8 ± 13.2
– b0.001 b0.001 b0.001 b0.001 0.002 0.001 0.02 b0.001 b0.001 0.54 b0.001 0.39 0.56 0.01 0.18 b0.001
Data are presented as mean ± standard deviation; statistical significance is shown. Individuals with metabolic syndrome as defined by IDF; controls: Individuals free from metabolic syndrome; BP: blood pressure; HDL: high density lipoproteins; LDL: low density lipoproteins; Pi: phosphate ion; Corr. Ca: corrected calcium.
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metabolic parameters according to all SNPs of VDR's polymorphisms are shown in Table 2. The individual comparison between VDR SNPs and components of MetS, including HDL, dyslipidemia and hypertension, is presented in Table 3. There was no significant difference between controls and MetS subjects in the distribution of alleles or genotypes of the four SNPs. In addition, the allele T of BsmI SNP was significantly associated with obesity [OR 1.4 (1.0, 1.90), p = 0.04]. The CT genotype and the dominant model CT + TT of BsmI were associated with increased risk of diabetes [OR 1.7 (1.2, 2.4), p = 0.007] and [OR 1.5 (1.1, 2.2), p = 0.01], respectively. Analyses also revealed that CT and CT + CC genotypes of FokI were associated with a reduced risk of diabetes [OR 0.70 (0.49, 0.99), p = 0.05] and [OR 0.67 (0.48, 0.94), p = 0.02], respectively. Moreover, allele C was associated with lower risk of developing T2DM [OR 0.73 (0.56, 0.95), p = 0.02]. Additionally, the CT genotype of BsmI was significantly correlated with lower HDL-C levels [OR 0.60 (0.37, 0.96), p = 0.03]. Our results also demonstrated that the association of vitamin D deficiency was lower in subjects carrying the AC and AC + CC genotypes of ApaI [OR, 0.33 (0.14, 0.80), p = 0.02; OR, 0.39 (0.17, 0.89)], respectively (Table 3). 3.1. Haplotype frequency and linkage disequilibrium The haplotype A–C–C–C significantly increased the risk of vitamin D deficiency [OR 3.9 (1.0, 17.0), p = 0.04]. This effect was not detected in any other common haplotypes (Table 4). Linkage disequilibria values were subsequently generated to examine associations among the four studied polymorphisms. Thus, TaqI was in strong linkage disequilibrium with the BsmI (R2 = 0.84, D′ = 0.95, p b 0.001), and the variant ApaI
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was moderately associated with BsmI and TaqI (R2 = 0.41, D′ = 0.98, p b 0.001 and R2 = 0.46, D′ = 0.81, p b 0.001 respectively). The FokI variant was in weak linkage disequilibrium with all others (Fig. 1).
4. Discussion Polymorphisms in the VDR gene have been linked with the increased susceptibility to obesity in subjects with early-onset T2DM (Nosratabadi et al., 2010; Speer et al., 2001a; Ye et al., 2001). Speer et al. (2001b) have demonstrated a link between diabetes risk and BsmI site in Caucasian patients with type 2 diabetes. Our results favor the study performed by Speer et al. showing the presence of increased VDR polymorphism frequency of BsmI genes (CT and CT + TT) in T2DM patients. We further demonstrated that the CT and CT + CC genotypes of FokI and one of its alleles, C, were associated with reduced risk of diabetes. However, a recent study has demonstrated FokI polymorphism of the VDR gene as a possible risk factor for T2D (Neyestani et al., 2013). On the contrary, there are studies showing no relations between FokI, ApaI, BsmI and TaqI VDR polymorphism and T2DM in different populations (Bid et al., 2009; Malecki et al., 2003; Oh and Barrett-Connor, 2002). From this discussion, it is clear that the association of VDR FokI polymorphism with T2DM is contradictory and needs further evidence for its association with T2DM. However, a possible reason for the divergence between our result and these studies could be explained by the heterogeneity of the studied populations and, thus, further studies are needed to elucidate the genetic differences among various ethnic groups.
Table 2 Anthropometric and metabolic parameters according to genotypes of VDR polymorphisms. VDR rs731236 (TaqI)
N Age BMI (kg/m2) Hips (cm) Waist (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Cholesterol (mmol/l) Glucose (mmol/l) Triglyceride (mmol/l) HDL (mmol/l) Pi (mmol/l) Ca (mmol/l) Corr. Ca (mmol/l) Albumin (g/l) Vitamin D (nmol/l)
VDR rs1544410 (BsmI)
AA
AG
GG
183 43.2 ± 14.8 30.0 ± 6.3 106.7 ± 19.4 95.2 ± 19.7 125.9 ± 16.7 77.1 ± 9.6 5.3 ± 1.7 7.7 ± 1.6 1.7 ± 0.13 0.86 ± 0.31 1.2 ± 0.28 2.5 ± 0.33 2.5 ± 0.26 45.0 ± 6.2 33.5 ± 15.1
277 47.4 ± 15.2* 31.0 ± 7.7 103.1 ± 21.8 95.1 ± 21.0 126.4 ± 14.0 79.3 ± 8.3 5.5 ± 1.3 7.9 ± 1.6 1.7 ± 0.11 0.90 ± 0.33 1.2 ± 0.20 2.6 ± 0.23 2.5 ± 0.28 45.4 ± 4.5 33.4 ± 13.7
110 46.8 ± 14.2 31.3 ± 6.2 104.9 ± 22.8 96.0 ± 21.3 124.5 ± 14.8 78.5 ± 9.2 5.3 ± 1.2 7.8 ± 1.6 1.7 ± 0.15 0.85 ± 0.29 1.2 ± 0.30 2.5 ± 0.20 2.4 ± 0.30 45.2 ± 4.9 32.1 ± 12.6
p-Value
CC
CT
TT
p-Value
0.01 0.26 0.28 0.94 0.63 0.10 0.46 0.70 0.90 0.64 0.61 0.65 0.52 0.66 0.52
200 42.3 ± 14.5 29.7 ± 6.5 104.9 ± 19.9 93.9 ± 21.2 126.1 ± 16.5 77.2 ± 9.6 5.3 ± 1.5 7.7 ± 1.6 1.6 ± 0.10 0.85 ± 0.34 1.2 ± 0.26 2.6 ± 0.25 2.5 ± 0.21 44.7 ± 7.5 33.1 ± 15.1
263 48.5 ± 15.0* 31.1 ± 7.6 104.3 ± 21.5 96.1 ± 19.8 126.2 ± 14.1 79.2 ± 8.4 5.5̉ ± 1.3 8.3 ± 1.6 1.8 ± 0.11 0.95 ± 0.34* 1.2 ± 0.20 2.5 ± 0.33 2.5 ± 0.31 44.9 ± 6.1 33.9 ± 13.7
107 46.7 ± 14.4* 31.4 ± 6.2 104.9 ± 23.2 96.0 ± 21.6 125.0 ± 14.9 78.6 ± 9.1 5.2 ± 1.2 8.0 ± 1.5 1.7 ± 0.13 0.87 ± 0.31 ‡ 1.2 ± 0.42 2.5 ± 0.17 2.4 ± 0.28 44.6 ± 5.0 31.6 ± 12.4
b0.001 0.08 0.95 0.57 0.84 0.16 0.19 0.15 0.12 0.005 0.68 0.65 0.42 0.94 0.54
p-Value
VDR rs7975232 (ApaI)
N Age BMI (kg/m2) Hips (cm) Waist (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Cholesterol (mmol/l) Glucose (mmol/l) Triglyceride (mmol/l) HDL (mmol/l) Pi (mmol/l) Ca (mmol/l) Corr. Ca (mmol/l) Albumin (g/l) Vitamin D (nmol/l)
VDR rs10735810 (FokI)
AA
AC
CC
229 46.3 ± 14.7 31.2 ± 7.1 103.7 ± 24.6 95.1 ± 22.5 125.4 ± 15.2 78.9 ± 8.5 5.4 ± 1.2 7.9 ± 1.6 1.7 ± 0.12 0.88 ± 0.32 1.2 ± 0.33 2.5 ± 0.33 2.4 ± 0.33 45.4 ± 5.2 30.0 ± 1.5
256 46.8 ± 15.2 30.6 ± 7.0 105.1 ± 18.6 95.7 ± 18.9 126.8 ± 13.8 79.1 ± 8.7 5.5 ± 1.7 8.2 ± 1.6 1.8 ± 0.11 0.93 ± 0.36 1.2 ± 0.21 2.6 ± 0.27 2.5 ± 0.25 44.2 ± 7.7 31.0 ± 1.6
85 42.4 ± 14.7 29.5 ± 6.6 105.8 ± 19.3 94.7 ± 21.1 124.7 ± 17.6 75.2 ± 10.0‡ 5.1 ± 1.1 7.6 ± 1.6 1.7 ± 0.11 0.86 ± 0.30 1.2 ± 0.27 2.5 ± 0.18 2.4 ± 0.18 45.0 ± 4.9 27.4 ± 0.47
p-Value
TT
CT
CC
0.08 0.18 0.73 0.93 0.59 0.01 0.06 0.42 0.64 0.18 0.97 0.56 0.47 0.22 0.43
300 46.5 ± 14.6 30.4 ± 6.8 102.6 ± 22.4 93.6 ± 21.8 125.5 ± 14.7 78.3 ± 8.8 5.4 ± 1.4 8.2 ± 1.6 1.8 ± 0.12 0.91 ± 0.33 1.2 ± 0.26 2.5 ± 0.28 2.5 ± 0.26 44.2 ± 5.2 30.5 ± 1.7
228 45.4 ± 15.6 30.8 ± 6.9 106.4 ± 20.8 96.5 ± 19.3 126.3 ± 14.8 78.7 ± 9.1 5.4 ± 1.4 7.8 ± 1.6 1.6 ± 0.10* 0.89 ± 0.35 1.2 ± 0.29 2.5 ± 0.28 2.4 ± 0.30 45.0 ± 7.3 28.4 ± 1.6
42 44.6 ± 15.6 31.89 ± 8.4 109.9 ± 10.8 101.1 ± 18.4 126.9 ± 17.2 78.4 ± 9.3 5.2 ± 1.4 7.8 ± 1.7 1.8 ± 0.19 0.86 ± 0.34 1.2 ± 0.24 s 2.6 ± 0.31 2.5 ± 0.27 47.7 ± 8.2* 32.0 ± 1.5
0.60 0.42 0.07 0.08 0.83 0.88 0.56 0.30 0.04 0.67 0.49 0.29 0.13 0.03 0.37
Data are presented as mean ± standard deviation; statistical significance is shown. *Indicates that the group is significantly different from the first genotype; ‡Indicates that the group is significantly different from the second genotype.
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Table 3 Comparison among different SNPs of the VDR gene, metabolic syndrome and its components along with diabetes mellitus type 2 and vitamin D deficiency in the study population. Metabolic syndrome
Obesity
Odds ratio (95% CI)
Odds ratio (95% CI)
p
rs731236 (TaqI) AA Reference AG 0.90(0.62, 1.3) GG 1.1(0.67, 1.7) GG + AG 0.95(0.67, 1.3) A Reference G 1.0(0.80, 1.3)
0.63 0.81 0.85
rs1544410 (BsmI) CC Reference CT 1.0(0.71, 1.50) TT 1.1(0.71, 1.8) CT + TT 1.1(0.75, 1.5) C Reference T 1.1(0.84, 1.4)
0.92 0.63 0.79
rs7975232 (ApaI) AA Reference AC 1.1(0.74, 1.5) CC 1.2(0.68, 1.8) AC + CC 1.1(0.77, 1.5) A Reference C 1.1(0.83, 1.4) rs10735810 (FokI) TT Reference CT 0.93(0.66, 1.3) CC 1.5(0.76, 2.8) CT + CC 1.0(0.72, 1.4) T Reference C 1.1(0.82, 1.4)
0.90
0.63
0.78 0.70 0.73 0.66
0.72 0.25 0.99 0.64
Reference 1.0(0.61, 1.6) 1.5(0.75, 2.9) 1.1(0.70, 1.8) Reference 1.2(0.85, 1.6)
Reference 1.3(0.80, 2.2) 2.0(0.99, 4.0) 1.4(0.92, 2.3) Reference 1.4(1.0, 1.90)
Reference 0.83(0.50, 1.4) 0.72(0.38, 1.4) 1.2(0.78, 2.0) Reference 0.83(0.60, 1.2)
Reference 0.48(0.15, 1.5) 0.43(0.14, 1.3) 0.45(0.15, 1.3) Reference 0.79(0.55, 1.1)
Diabetes p
0.98 0.31 0.62 0.32
0.31 0.06 0.11 0.04
0.52 0.32 0.35 0.27
0.23 0.16 0.17 0.23
Odds ratio (95% CI) Reference 1.3(0.90, 1.9) 1.2(0.72, 1.8) 1.3(0.89, 1.8) Reference 1.1(0.87, 1.4)
Reference 1.7(1.2, 2.4) 1.3(0.80,0.20) 1.5(1.1, 2.2) Reference 1.2(0.96, 1.5)
Reference 1.3(0.93, 1.9) 0.69(0.41, 1.2) 1.1(0.81, 1.6) Reference 0.92(0.73, 1.2)
Reference 0.70(0.49,0.99) 0.57(0.29, 1.1) 0.67(0.48,0.94) Reference 0.73(0.56,0.95)
Several common chronic diseases are associated with a variation in vitamin D endocrine system (Lips, 2006; Valdivielso and Fernandez, 2006). In addition to being expressed in adipocytes and pancreatic beta cells (Ogunkolade et al., 2002; Reis et al., 2005), the gene for VDR is also expressed ubiquitously in different types of tissues (Grundberg et al., 2004; Uitterlinden et al., 2004a). In the current study, we focused on the association between VDR SNPs and components of MetS and T2DM. The MetS components including obesity, hypertension, and low HDL-C levels were assessed both separately and in clusters. Alicja et al. demonstrated that the BsmI VDR polymorphism influenced BMI, while the FokI VDR polymorphism was associated with lower serum HDL-C level (Filus et al., 2008). On the other hand, a study performed by Urszula and co-workers showed that the BsmI polymorphism in the VDR gene had no association with susceptibility to obesity and insulin resistance, while it was related to a higher LDL-C level (TworowskaBardzinska et al., 2008). Our study results are in agreement with Filus et al.'s findings demonstrating that allele T of BsmI SNP carries positive influence on obesity. There could be distinct mechanisms by which vitamin D–VDR axis could affect lipid profiles. Firstly, vitamin D induces the suppression of PTH secretion, and it has been reported that PTH could reduce lipolysis (Ford et al., 2005). Another, vitamin D might cause improvement in insulin secretion and insulin sensitivity, thereby indirectly shaping lipid metabolism (Pittas et al., 2007). Our result also shows that there is an
Low HDL p
Odds ratio (95% CI)
0.18 0.54 0.20 0.40
0.007 0.33 0.01 0.11
0.12 0.16 0.49 0.58
0.05 0.10 0.02 0.02
Reference 0.83(0.52, 1.3) 1.5(0.77, 2.9) 0.96(0.62, 1.5) Reference 1.1(0.84, 1.5)
Reference 0.60(0.37, 0.96) 1.3(0.66, 2.5) 0.74(0.47, 1.2) Reference 1.0(0.75, 1.4)
Reference 0.62(0.39, 1.1) 1.4(0.65, 2.7) 0.73(0.47, 1.1) Reference 0.97(0.72, 1.3)
Reference 1.0(0.66, 1.6) 1.0(0.45, 2.4) 1.0(0.67, 1.5) Reference 1.0(0.73, 1.4)
Hypertension p
0.48 0.26 0.91 0.40
0.03 0.50 0.22 0.94
0.06 0.48 0.16 0.87
0.99 0.99 0.91 0.93
Odds ratio (95% CI) Reference 1.5(0.89, 2.6) 1.4(0.72, 2.7) 1.5(0.89, 2.4) Reference 1.2(0.87, 1.6)
Reference 1.7(0.97, 2.8) 1.5(0.74, 2.6) 1.6(0.96, 2.7) Reference 1.2(0.89, 1.70)
Reference 1.2(0.72, 1.98) 0.64(0.30, 1.4) 1.0(0.65, 1.6) Reference 0.89(0.64, 1.2)
Reference 1.1(0.69, 1.8) 1.1(0.51, 2.6) 1.1(0.71, 1.7) Reference 1.1(0.77, 1.5)
Vitamin D deficiency p
0.14 0.38 0.13 0.25
0.06 0.28 0.08 0.19
0.54 0.28 0.90 0.55
0.71 0.82 0.64 0.64
Odds ratio (95% CI) Reference 1.0(0.46, 2.1) 2.0(0.65, 6.3) 1.18(0.56, 2.5) Reference 1.3(0.79, 2.2)
Reference 0.93(0.42, 2.0) 2.5(0.73, 8.5) 1.15(0.53, 2.4) Reference 1.36(0.82, 2.2)
Reference 0.34(0.14, 0.80) 0.61(0.19, 1.9) 0.39(0.17, 0.89) Reference 0.70(0.42, 1.16)
Reference 1.8(0.82, 4.0) 0.80(0.24, 0.25) 1.5(0.73, 3.0) Reference 1.16(0.66, 2.1)
p
0.91 0.21 0.64 0.28
0.80 0.14 0.69 0.25
0.01 0.41 0.02 0.19
0.17 0.70 0.28 0.67
association between lower HDL-C and the genotype CT of BsmI SNP. There is no direct evidence for this relation but the results of Urszula and colleagues showed higher LDL-C level associated with genotype CT of BsmI SNP. The pancreatic beta cell expression of VDR (Lee et al., 1994) supports the idea that the VDR polymorphism studies exert genomic actions possibly influencing insulin secretion (Christakos et al., 1996; Pike, 1991). Additionally, as explained earlier, the vitamin D deficiency and chronic disease connection may depend on the presence of VDR in various cells and tissues (Howard et al., 1995; Shi et al., 2001). There are limited studies relating VDR polymorphism and vitamin D (25(OH)D) deficiency in different populations. For instance, BsmI polymorphism was shown to be insignificantly associated with 25(OH)D levels in an adolescent population (d'Alesio et al., 2005), whereas, TaqI polymorphism exhibited an association with higher 25(OH)D levels in healthy adults (25 to 60 years) (Bhanushali et al., 2009). On the other hand, different SNPs of the VDR
Table 4 Haplotype frequency of VDR variants in vitamin D deficient patients versus controls. Haplotype
Odds ratio (95% CI)
p-Value
A–C–A–C A–C–A–T A–C–C–C A–C–C–T G–T–A–C G–T–A–T
1.5 (0.24, 10.2) 3.1 (0.81, 12.1) 3.9 (1.0, 17.0) 1.5 (0.55, 3.9) 2.5 (0.70, 9.0) Reference
0.98 0.11 0.04 0.45 0.21 –
Fig. 1. Pairwise linkage disequilibrium (LD) for VDR gene SNPs. The plot shows the pairwise correlation for the studied SNPs (TaqI, BsmI, ApaI and FokI).
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gene were shown to be associated with levels of 25(OH)D (Smolders et al., 2009). Dundar et al. (2009) have also shown that higher bone metabolism and bone mineral density is linked with ApaI gene variant. The genotype, ApaI of the VDR gene has been associated with vitamin D deficiency in Caribbean patients with T2DM (VelayoudomCephise et al., 2011). Our study corroborates this finding showing that the association of vitamin D deficiency was lower in subjects carrying the AC and AC + CC genotypes of ApaI. The probable mechanism for protective association of ApaI genotypes against vitamin D deficiency is also evident by a study showing that ApaI can affect circulating levels of vitamin D (Howard et al., 1995). However, no significant associations between BsmI, FokI and TaqI SNPs and vitamin D deficiency were observed. In conclusion, our results demonstrate that components of the MetS were possibly associated with the VDR gene BsmI. Whereas, FokI and BsmI showed contradictory results related to T2DM. Low prevalence of vitamin D deficiency was related to patients carrying the ApaI gene. Conflict of interest The authors have no financial conflicts of interest. Acknowledgments The authors thank the Center of Excellence in Biotechnology Research, King Saud University, Riyadh, KSA, for funding this study. We acknowledge primary care physicians and nurses for their cooperation in recruiting and collecting the data of the subjects. We thank the Prince Mutaib Chair for Biomarkers of Osteoporosis for technical support. References Al-Daghri, N.M., Al-Attas, O., Alokail, M.S., Alkharfy, K.M., Draz, H.M., Agliardi, C., Mohammed, A.K., Guerini, F.R., Clerici, M., 2012a. Vitamin D receptor gene polymorphisms and HLA DRB1*04 cosegregation in Saudi type 2 diabetes patients. Journal of Immunology 188, 1325–1332. Al-Daghri, N.M., Alkharfy, K.M., Al-Saleh, Y., Al-Attas, O.S., Alokail, M.S., Al-Othman, A., Moharram, O., El-Kholie, E., Sabico, S., Kumar, S., Chrousos, G.P., 2012b. Modest reversal of metabolic syndrome manifestations with vitamin D status correction: a 12month prospective study. Metabolism 61, 661–666. Alkharfy, K.M., Al-Daghri, N.M., Al-Attas, O.S., Alokail, M.S., Mohammed, A.K., Vinodson, B., Clerici, M., Kazmi, U., Hussain, T., Draz, H.M., 2012. Variants of endothelial nitric oxide synthase gene are associated with components of metabolic syndrome in an Arab population. Endocrine Journal 59, 253–263. Bhanushali, A.A., Lajpal, N., Kulkarni, S.S., Chavan, S.S., Bagadi, S.S., Das, B.R., 2009. Frequency of fokI and taqI polymorphism of vitamin D receptor gene in Indian population and its association with 25-hydroxyvitamin D levels. Indian Journal of Human Genetics 15, 108–113. Bid, H.K., Konwar, R., Aggarwal, C.G., Gautam, S., Saxena, M., Nayak, V.L., Banerjee, M., 2009. Vitamin D receptor (FokI, BsmI and TaqI) gene polymorphisms and type 2 diabetes mellitus: a North Indian study. Indian Journal of Medical Sciences 63, 187–194. Bischoff-Ferrari, H.A., Giovannucci, E., Willett, W.C., Dietrich, T., Dawson-Hughes, B., 2006. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. The American Journal of Clinical Nutrition 84, 18–28. Christakos, S., Raval-Pandya, M., Wernyj, R.P., Yang, W., 1996. Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin D3. The Biochemical Journal 316 (Pt 2), 361–371. d'Alesio, A., Garabedian, M., Sabatier, J.P., Guaydier-Souquieres, G., Marcelli, C., Lemacon, A., Walrant-Debray, O., Jehan, F., 2005. Two single-nucleotide polymorphisms in the human vitamin D receptor promoter change protein–DNA complex formation and are associated with height and vitamin D status in adolescent girls. Human Molecular Genetics 14, 3539–3548. Dundar, U., Solak, M., Kavuncu, V., Ozdemir, M., Cakir, T., Yildiz, H., Evcik, D., 2009. Evidence of association of vitamin D receptor Apa I gene polymorphism with bone mineral density in postmenopausal women with osteoporosis. Clinical Rheumatology 28, 1187–1191. Filus, A., Trzmiel, A., Kuliczkowska-Plaksej, J., Tworowska, U., Jedrzejuk, D., Milewicz, A., Medras, M., 2008. Relationship between vitamin D receptor BsmI and FokI polymorphisms and anthropometric and biochemical parameters describing metabolic syndrome. The Aging Male 11, 134–139. Ford, E.S., Ajani, U.A., McGuire, L.C., Liu, S., 2005. Concentrations of serum vitamin D and the metabolic syndrome among U.S. adults. Diabetes Care 28, 1228–1230.
133
Grundberg, E., Brandstrom, H., Ribom, E.L., Ljunggren, O., Mallmin, H., Kindmark, A., 2004. Genetic variation in the human vitamin D receptor is associated with muscle strength, fat mass and body weight in Swedish women. European Journal of Endocrinology 150, 323–328. Howard, G., Nguyen, T., Morrison, N., Watanabe, T., Sambrook, P., Eisman, J., Kelly, P.J., 1995. Genetic influences on bone density: physiological correlates of vitamin D receptor gene alleles in premenopausal women. The Journal of Clinical Endocrinology and Metabolism 80, 2800–2805. Lee, S., Clark, S.A., Gill, R.K., Christakos, S., 1994. 1,25-Dihydroxyvitamin D3 and pancreatic beta-cell function: vitamin D receptors, gene expression, and insulin secretion. Endocrinology 134, 1602–1610. Lips, P., 2006. Vitamin D physiology. Progress in Biophysics and Molecular Biology 92, 4–8. Malecki, M.T., Frey, J., Moczulski, D., Klupa, T., Kozek, E., Sieradzki, J., 2003. Vitamin D receptor gene polymorphisms and association with type 2 diabetes mellitus in a Polish population. Experimental and Clinical Endocrinology and Diabetes 111, 505–509. McDermott, M.F., Ramachandran, A., Ogunkolade, B.W., Aganna, E., Curtis, D., Boucher, B.J., Snehalatha, C., Hitman, G.A., 1997. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians. Diabetologia 40, 971–975. Neyestani, T.R., Djazayery, A., Shab-Bidar, S., Eshraghian, M.R., Kalayi, A., Shariatzadeh, N., Khalaji, N., Zahedirad, M., Gharavi, A., Houshiarrad, A., Chamari, M., Asadzadeh, S., 2013. Vitamin D Receptor Fok-I polymorphism modulates diabetic host response to vitamin D intake: need for a nutrigenetic approach. Diabetes Care 36, 550–556. Nosratabadi, R., Arababadi, M.K., Salehabad, V.A., Shamsizadeh, A., Mahmoodi, M., Sayadi, A.R., Kennedy, D., 2010. Polymorphisms within exon 9 but not intron 8 of the vitamin D receptor are associated with the nephropathic complication of type-2 diabetes. International Journal of Immunogenetics 37, 493–497. Ogunkolade, B.W., Boucher, B.J., Prahl, J.M., Bustin, S.A., Burrin, J.M., Noonan, K., North, B.V., Mannan, N., McDermott, M.F., DeLuca, H.F., Hitman, G.A., 2002. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 51, 2294–2300. Oh, J.Y., Barrett-Connor, E., 2002. Association between vitamin D receptor polymorphism and type 2 diabetes or metabolic syndrome in community-dwelling older adults: the Rancho Bernardo Study. Metabolism 51, 356–359. Pani, M.A., Knapp, M., Donner, H., Braun, J., Baur, M.P., Usadel, K.H., Badenhoop, K., 2000. Vitamin D receptor allele combinations influence genetic susceptibility to type 1 diabetes in Germans. Diabetes 49, 504–507. Panierakis, C., Goulielmos, G., Mamoulakis, D., Petraki, E., Papavasiliou, E., Galanakis, E., 2009. Vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Crete, Greece. Clinical Immunology 133, 276–281. Pike, J.W., 1991. Vitamin D3 receptors: structure and function in transcription. Annual Review of Nutrition 11, 189–216. Pittas, A.G., Harris, S.S., Stark, P.C., Dawson-Hughes, B., 2007. The effects of calcium and vitamin D supplementation on blood glucose and markers of inflammation in nondiabetic adults. Diabetes Care 30, 980–986. Reis, A.F., Hauache, O.M., Velho, G., 2005. Vitamin D endocrine system and the genetic susceptibility to diabetes, obesity and vascular disease. A review of evidence. Diabetes & Metabolism 31, 318–325. Shi, H., Norman, A.W., Okamura, W.H., Sen, A., Zemel, M.B., 2001. 1alpha,25Dihydroxyvitamin D3 modulates human adipocyte metabolism via nongenomic action. FASEB Journal 15, 2751–2753. Smolders, J., Damoiseaux, J., Menheere, P., Tervaert, J.W., Hupperts, R., 2009. Fok-I vitamin D receptor gene polymorphism (rs10735810) and vitamin D metabolism in multiple sclerosis. Journal of Neuroimmunology 207, 117–121. Speer, G., Cseh, K., Winkler, G., Takacs, I., Barna, I., Nagy, Z., Lakatos, P., 2001a. Oestrogen and vitamin D receptor (VDR) genotypes and the expression of ErbB-2 and EGF receptor in human rectal cancers. European Journal of Cancer 37, 1463–1468. Speer, G., Cseh, K., Winkler, G., Vargha, P., Braun, E., Takacs, I., Lakatos, P., 2001b. Vitamin D and estrogen receptor gene polymorphisms in type 2 diabetes mellitus and in android type obesity. European Journal of Endocrinology 144, 385–389. Tworowska-Bardzinska, U., Lwow, F., Kubicka, E., Laczmanski, L., Jedzrzejuk, D., Dunajska, K., Milewicz, A., 2008. The vitamin D receptor gene BsmI polymorphism is not associated with anthropometric and biochemical parameters describing metabolic syndrome in postmenopausal women. Gynecological Endocrinology 24, 514–518. Uitterlinden, A.G., Fang, Y., Van Meurs, J.B., Pols, H.A., Van Leeuwen, J.P., 2004a. Genetics and biology of vitamin D receptor polymorphisms. Gene 338, 143–156. Uitterlinden, A.G., Fang, Y., van Meurs, J.B., van Leeuwen, H., Pols, H.A., 2004b. Vitamin D receptor gene polymorphisms in relation to Vitamin D related disease states. The Journal of Steroid Biochemistry and Molecular Biology 89–90, 187–193. Valdivielso, J.M., Fernandez, E., 2006. Vitamin D receptor polymorphisms and diseases. Clinica Chimica Acta 371, 1–12. Velayoudom-Cephise, F.L., Larifla, L., Donnet, J.P., Maimaitiming, S., Deloumeaux, J., Blanchet, A., Massart, C., Munoz-Bellili, N., Merle, S., Chout, R., Bonnet, F., Foucan, L., 2011. Vitamin D deficiency, vitamin D receptor gene polymorphisms and cardiovascular risk factors in Caribbean patients with type 2 diabetes. Diabetes & Metabolism 37, 540–545. Ye, W.Z., Reis, A.F., Dubois-Laforgue, D., Bellanne-Chantelot, C., Timsit, J., Velho, G., 2001. Vitamin D receptor gene polymorphisms are associated with obesity in type 2 diabetic subjects with early age of onset. European Journal of Endocrinology 145, 181–186.
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Association of VDR-gene variants with factors related to the metabolic syndrome, type 2 diabetes and vitamin D deficiency Nasser M. Al-Daghri a,b,c,⁎, Omar S. Al-Attas a,b,c, Khalid M. Alkharfy a,b,d, Nasiruddin Khan b, Abdul Khader Mohammed b,c, Benjamin Vinodson b,c, Mohammed Ghouse Ahmed Ansari b, Amal Alenad e, Majed S. Alokail a,b,c a
Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia Biomarkers Research Program, Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia c Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, King Saud University, Riyadh, Saudi Arabia d Clinical Pharmacy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia e School of Biological Sciences, Life Science Building 85, University of Southampton, Southampton SO17 1BJ, UK b
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 16 March 2014 Accepted 21 March 2014 Available online 25 March 2014 Keywords: Polymorphism Haplotype Obesity 25-Hydroxyvitamin D Cardiovascular diseases
a b s t r a c t The prevalence of metabolic syndrome (MetS) is rising alarmingly in the Saudi Arabian population. This study was conducted to assess the association between vitamin D receptor (VDR) polymorphisms and genetic susceptibility to components of the metabolic syndrome, type 2 diabetes mellitus (T2DM), and vitamin D deficiency in the Saudi Arabian population. Five-hundred-seventy Saudi individuals (285 MetS and 285 controls) were enrolled in this cross-sectional study. TaqI, BsmI, ApaI and FokI single nucleotide polymorphisms (SNPs) of the VDR gene were genotyped. The CT genotype and allele T of BsmI were associated with lower HDL-C levels [OR 0.60 (0.37, 0.96), p = 0.03] and obesity [OR 1.4 (1.0, 1.90), p = 0.04], respectively. The CT genotype and the dominant model CT + TT of BsmI were associated with increased risk of diabetes [OR 1.7 (1.2, 2.4), p = 0.007], and [OR 1.5 (1.1, 2.2), p = 0.01], respectively. On the contrary, the CT and CT + CC genotypes of FokI exhibited an association with a reduced risk of diabetes [OR 0.70 (0.49, 0.99), p = 0.05] and [OR 0.67 (0.48, 0.94), p = 0.02], respectively. The allele C of FokI was associated with lower risk of developing T2DM [OR 0.73 (0.56, 0.95), p = 0.02]. The prevalence of vitamin D deficiency was lower in subjects with the AC genotype of ApaI [OR, 0.34 (0.14, 0.80), p = 0.01]. Components of the MetS such as obesity, low HDL and T2DM were associated with the VDR gene. FokI and BsmI have protective and facilitative effects on the risk for T2DM, while the ApaI genotype was associated with reduced vitamin D deficiency. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The components of metabolic syndrome (MetS) (dyslipidemia, hyperglycemia, hypertension and obesity), individually and cumulatively, increase the risk of developing T2DM and cardiovascular diseases (CVD) (Alkharfy et al., 2012). Moreover, the involvement of vitamin D in the prevalence of metabolic syndrome has also been suggested (Ford et al., 2005). The mechanism of action regarding the effect of vitamin D either includes binding of the active metabolite 1,25 (OH)2D3 with the cytosolic/nuclear VDR or via non-genomic pathways (Lips, 2006). Abbreviations: MetS, metabolic syndrome; VDR, vitamin D receptor; T2DM, type 2 diabetes mellitus; single SNPs, nucleotide polymorphisms; IDF, International Diabetes Federation; HDL, high density lipoprotein; PHCC, Primary Health Care Centers; OR, odds ratio. ⁎ Corresponding author at: Prince Mutaib Bin Abdullah Chair for Osteoporosis, Biochemistry Department, College of Science, King Saud University, PO Box, 2455, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (N.M. Al-Daghri).
http://dx.doi.org/10.1016/j.gene.2014.03.044 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Cytosolic/Nuclear VDR is a member of the steroid/thyroid hormone receptor family that functions as a transcriptional activator of many genes (Uitterlinden et al., 2004b). Polymorphisms in the VDR gene that produce variation in the activity of the VDR have been described in various populations (Valdivielso and Fernandez, 2006). Polymorphisms in the VDR gene have also been shown to be associated with the components of MetS, obesity and T2DM in different populations (Bid et al., 2009; Filus et al., 2008). In addition, increased susceptibility to type 1 diabetes has also been associated with allelic variations of the VDR gene (McDermott et al., 1997; Pani et al., 2000). Most VDR gene polymorphisms, including the BsmI, ApaI and TaqI restriction fragment length polymorphisms, are located at the 3′ untranslated region (3′ UTR) of the gene (Panierakis et al., 2009), while the FokI polymorphism, is localized within the 5′ end of the gene, near the promoter region (Uitterlinden et al., 2004b). Recently, we demonstrated cosegregation between VDR and HLA alleles in T2DM patients in the Saudi Arabian population (Al-Daghri et al., 2012a). Numerous crosssectional studies have noted significant negative associations among
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circulating levels of 25-hydroxyvitamin D and cardiometabolic risk factors, highlighting potential extra skeletal functions of this sterol hormone (Al-Daghri et al., 2012b). However, there are still very limited studies that combine VDR gene polymorphisms with the components of the MetS, T2DM, and vitamin D deficiency in this part of the world. Therefore, the aim of this study was to examine the association of four single nucleotide polymorphisms (SNPs) in intron 8 (BsmI, ApaI) exon 9 (TaqI) and exon 2 (FokI) of the VDR gene with components of MetS, T2DM, and vitamin D deficiency in the Saudi Arabian population.
rs10735810) were evaluated by allelic discrimination Real-time PCR using pre-designed TaqMan probes (Applied Biosystems, Foster City, CA, USA). The PCR consisted of a hot start at 95 °C for 10 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. Fluorescence detection takes place at a temperature of 60 °C. All assays were performed in 10 μl reactions, using TaqMan Genotyping Master Mix on 96-well plates using an ABI 7000 instrument (Applied Biosystems, Foster City, CA, USA). Control samples representing all possible genotypes and a negative control were included in each reaction.
2. Materials and methods
2.5. Statistical analyses
2.1. Study design
Data were analyzed using the Statistical Package for the Social Sciences for Windows (SPSS version 16.0, Chicago, IL, USA) and are expressed by mean ± standard deviation (SD). Data was checked for normality using Kolmogorov–Smirnov test. All non-Gaussian variables were either log or square root transformed. Independent sample t-test was used to test control and metabolic syndrome groups. Analysis of variance (ANOVA) was performed between genotypes for each parameters followed by Bonferroni post-hoc test. A chi-square test was used to show the allele and genotype frequency for each SNP in patients and controls. Odds ratios (ORs) and 95% confidence intervals are calculated by binomial logistic regression for the allele, genotype with metabolic diseases/complications after adjustment for covariates including gender, age and body mass index (BMI). Haplotype frequencies were estimated by the Expectation–Maximization algorithm (EM algorithm) implemented in PROC Haplotype in SAS Genetics statistical software package (SAS institute, Cary, NC, USA). The most common haplotype was used as the reference and rare haplotypes were dropped from the analysis. Tests of departures from LD were performed by using the likelihood ratio test (LR test) of linkage disequilibrium as used in PROC Allele of SAS Genetics. Pairwise LD estimations were performed using Haploview 4.2. A power estimation, based on previous studies (Filus et al., 2008; Velayoudom-Cephise et al., 2011; Ye et al., 2001), showed that our study design for specific aims (BMI, Vit D, HDL-C) had 80% power to detect a similar sized-effect (α = 0.05). Significance was set at p b 0.05.
Five-hundred-seventy Saudi individuals (285 MetS patients and 285 healthy controls) were enrolled in the study. These individuals are part of the Biomarker Screening in Riyadh Project (RIYADH COHORT), a capital-wide epidemiologic study taken from over 17,000 consenting Saudis coming from different Primary Health Care Centers (PHCCs). The MetS includes waist circumference ≥102 cm for men and ≥88 cm for women, triglycerides ≥1.7 mmol/l and HDL-Cholesterol b1.03 mmol/l for men and 1.29 mmol/l for women, blood pressure ≥130/85 and fasting plasma glucose levels ≥5.6 mmol/l. Diagnosis was based on the International Diabetes Federation (IDF), which defines MetS as central obesity plus 2 other factors. Healthy control subjects were those who did not match the criteria employed for the selection of MetS subjects. A generalized questionnaire aimed to seek demographic information, past medical history, current medication and family history was given to all participating subjects. Those with co-morbidities that needed medical attention were excluded from the study. Written and informed consents were taken before inclusion. Ethics approval was granted by the Ethics Committee of the College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia (KSA). 2.2. Anthropometry and blood collection Participating subjects were requested to return to their respective PHCCs after an overnight fast (N10 h) for anthropometry and blood withdrawal. Anthropometry included height (to the nearest 0.5 cm), weight (to the nearest 0.1 kg), waist and hip circumference utilizing a standardized measuring tape in cm, systolic and diastolic blood pressure measurements, and BMI calculated as weight in kg divided by height in square meters. Overweight was defined as having a BMI of 25–29.9 kg/m2, obesity ≥30 b 34.9 and morbid obesity N35. Vitamin D deficiency was defined as 25-(OH)D level b50 nmol/L (Bischoff-Ferrari et al., 2006). Blood was transferred immediately to a non-heparinized tube for centrifugation. Serum was then transferred to a pre-labeled plain tube, stored in ice, and delivered to the Biomarker Research Center in King Saud University on the same day. 2.3. Biochemical analysis Fasting serum samples were stored in a − 20 °C freezer prior to analysis. Fasting glucose (FG), lipid profile, albumin, phosphorus and calcium were measured using a chemical analyzer (Konelab, Vantaa, Finland). Serum 25-hydroxyvitamin D [25-(OH)D] was measured by enzyme linked immunosorbent assays (ELISA) (IDS Ltd, Boldon Colliery, Tyne & Wear, UK). The intra-assay variation was 1.4–7.9% and interassay variation was b21%. 2.4. VDR gene analysis Whole blood was collected in EDTA-containing tubes and genomic DNA was isolated from whole blood by using the blood genomic prep minispin kit (GE healthcare, Piscataway, NJ, USA), stored at − 20 °C until analyzed. The four VDR SNPs (rs731236, rs1544410, rs7975232,
3. Results The anthropometric, epidemiologic and metabolic characteristics of MetS and controls are depicted in Table 1. Anthropometric and
Table 1 Anthropometric, epidemiologic, and metabolic characterization of the MetS and control individuals.
N Age (years) BMI (kg/m2) Hips (cm) Waist (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Cholesterol (mmol/l) Glucose (mmol/l) Triglycerides (mmol/l) HDL (mmol/l) LDL (mmol/l) Pi (mmol/l) Ca (mmol/l) Corr. Ca (mmol/l) Albumin (g/l) Vitamin D (nmol/l)
Control
MetS
p-Value
285 42.2 ± 16.2 28.7 ± 7.2 92.6 ± 25.6 79.4 ± 21.7 123.2 ± 16.4 76.8 ± 10.1 5.2 ± 1.7 7.4 ± 0.73 1.5 ± 0.35 0.91 ± 0.32 3.9 ± 1.0 1.1 ± 0.31 2.5 ± 0.34 2.4 ± 0.35 45.5 ± 7.7 32.5 ± 14.2
285 49.5 ± 12.7 32.6 ± 6.2 112.7 ± 12.4 105.3 ± 11.7 127.9 ± 13.5 79.7 ± 7.8 5.5 ± 1.1 9.0 ± 0.65 2.0 ± 0.34 0.89 ± 0.32 4.3 ± 1.0 1.2 ± 0.23 2.5 ± 0.24 2.5 ± 0.21 44.4 ± 5.2 26.8 ± 13.2
– b0.001 b0.001 b0.001 b0.001 0.002 0.001 0.02 b0.001 b0.001 0.54 b0.001 0.39 0.56 0.01 0.18 b0.001
Data are presented as mean ± standard deviation; statistical significance is shown. Individuals with metabolic syndrome as defined by IDF; controls: Individuals free from metabolic syndrome; BP: blood pressure; HDL: high density lipoproteins; LDL: low density lipoproteins; Pi: phosphate ion; Corr. Ca: corrected calcium.
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metabolic parameters according to all SNPs of VDR's polymorphisms are shown in Table 2. The individual comparison between VDR SNPs and components of MetS, including HDL, dyslipidemia and hypertension, is presented in Table 3. There was no significant difference between controls and MetS subjects in the distribution of alleles or genotypes of the four SNPs. In addition, the allele T of BsmI SNP was significantly associated with obesity [OR 1.4 (1.0, 1.90), p = 0.04]. The CT genotype and the dominant model CT + TT of BsmI were associated with increased risk of diabetes [OR 1.7 (1.2, 2.4), p = 0.007] and [OR 1.5 (1.1, 2.2), p = 0.01], respectively. Analyses also revealed that CT and CT + CC genotypes of FokI were associated with a reduced risk of diabetes [OR 0.70 (0.49, 0.99), p = 0.05] and [OR 0.67 (0.48, 0.94), p = 0.02], respectively. Moreover, allele C was associated with lower risk of developing T2DM [OR 0.73 (0.56, 0.95), p = 0.02]. Additionally, the CT genotype of BsmI was significantly correlated with lower HDL-C levels [OR 0.60 (0.37, 0.96), p = 0.03]. Our results also demonstrated that the association of vitamin D deficiency was lower in subjects carrying the AC and AC + CC genotypes of ApaI [OR, 0.33 (0.14, 0.80), p = 0.02; OR, 0.39 (0.17, 0.89)], respectively (Table 3). 3.1. Haplotype frequency and linkage disequilibrium The haplotype A–C–C–C significantly increased the risk of vitamin D deficiency [OR 3.9 (1.0, 17.0), p = 0.04]. This effect was not detected in any other common haplotypes (Table 4). Linkage disequilibria values were subsequently generated to examine associations among the four studied polymorphisms. Thus, TaqI was in strong linkage disequilibrium with the BsmI (R2 = 0.84, D′ = 0.95, p b 0.001), and the variant ApaI
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was moderately associated with BsmI and TaqI (R2 = 0.41, D′ = 0.98, p b 0.001 and R2 = 0.46, D′ = 0.81, p b 0.001 respectively). The FokI variant was in weak linkage disequilibrium with all others (Fig. 1).
4. Discussion Polymorphisms in the VDR gene have been linked with the increased susceptibility to obesity in subjects with early-onset T2DM (Nosratabadi et al., 2010; Speer et al., 2001a; Ye et al., 2001). Speer et al. (2001b) have demonstrated a link between diabetes risk and BsmI site in Caucasian patients with type 2 diabetes. Our results favor the study performed by Speer et al. showing the presence of increased VDR polymorphism frequency of BsmI genes (CT and CT + TT) in T2DM patients. We further demonstrated that the CT and CT + CC genotypes of FokI and one of its alleles, C, were associated with reduced risk of diabetes. However, a recent study has demonstrated FokI polymorphism of the VDR gene as a possible risk factor for T2D (Neyestani et al., 2013). On the contrary, there are studies showing no relations between FokI, ApaI, BsmI and TaqI VDR polymorphism and T2DM in different populations (Bid et al., 2009; Malecki et al., 2003; Oh and Barrett-Connor, 2002). From this discussion, it is clear that the association of VDR FokI polymorphism with T2DM is contradictory and needs further evidence for its association with T2DM. However, a possible reason for the divergence between our result and these studies could be explained by the heterogeneity of the studied populations and, thus, further studies are needed to elucidate the genetic differences among various ethnic groups.
Table 2 Anthropometric and metabolic parameters according to genotypes of VDR polymorphisms. VDR rs731236 (TaqI)
N Age BMI (kg/m2) Hips (cm) Waist (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Cholesterol (mmol/l) Glucose (mmol/l) Triglyceride (mmol/l) HDL (mmol/l) Pi (mmol/l) Ca (mmol/l) Corr. Ca (mmol/l) Albumin (g/l) Vitamin D (nmol/l)
VDR rs1544410 (BsmI)
AA
AG
GG
183 43.2 ± 14.8 30.0 ± 6.3 106.7 ± 19.4 95.2 ± 19.7 125.9 ± 16.7 77.1 ± 9.6 5.3 ± 1.7 7.7 ± 1.6 1.7 ± 0.13 0.86 ± 0.31 1.2 ± 0.28 2.5 ± 0.33 2.5 ± 0.26 45.0 ± 6.2 33.5 ± 15.1
277 47.4 ± 15.2* 31.0 ± 7.7 103.1 ± 21.8 95.1 ± 21.0 126.4 ± 14.0 79.3 ± 8.3 5.5 ± 1.3 7.9 ± 1.6 1.7 ± 0.11 0.90 ± 0.33 1.2 ± 0.20 2.6 ± 0.23 2.5 ± 0.28 45.4 ± 4.5 33.4 ± 13.7
110 46.8 ± 14.2 31.3 ± 6.2 104.9 ± 22.8 96.0 ± 21.3 124.5 ± 14.8 78.5 ± 9.2 5.3 ± 1.2 7.8 ± 1.6 1.7 ± 0.15 0.85 ± 0.29 1.2 ± 0.30 2.5 ± 0.20 2.4 ± 0.30 45.2 ± 4.9 32.1 ± 12.6
p-Value
CC
CT
TT
p-Value
0.01 0.26 0.28 0.94 0.63 0.10 0.46 0.70 0.90 0.64 0.61 0.65 0.52 0.66 0.52
200 42.3 ± 14.5 29.7 ± 6.5 104.9 ± 19.9 93.9 ± 21.2 126.1 ± 16.5 77.2 ± 9.6 5.3 ± 1.5 7.7 ± 1.6 1.6 ± 0.10 0.85 ± 0.34 1.2 ± 0.26 2.6 ± 0.25 2.5 ± 0.21 44.7 ± 7.5 33.1 ± 15.1
263 48.5 ± 15.0* 31.1 ± 7.6 104.3 ± 21.5 96.1 ± 19.8 126.2 ± 14.1 79.2 ± 8.4 5.5̉ ± 1.3 8.3 ± 1.6 1.8 ± 0.11 0.95 ± 0.34* 1.2 ± 0.20 2.5 ± 0.33 2.5 ± 0.31 44.9 ± 6.1 33.9 ± 13.7
107 46.7 ± 14.4* 31.4 ± 6.2 104.9 ± 23.2 96.0 ± 21.6 125.0 ± 14.9 78.6 ± 9.1 5.2 ± 1.2 8.0 ± 1.5 1.7 ± 0.13 0.87 ± 0.31 ‡ 1.2 ± 0.42 2.5 ± 0.17 2.4 ± 0.28 44.6 ± 5.0 31.6 ± 12.4
b0.001 0.08 0.95 0.57 0.84 0.16 0.19 0.15 0.12 0.005 0.68 0.65 0.42 0.94 0.54
p-Value
VDR rs7975232 (ApaI)
N Age BMI (kg/m2) Hips (cm) Waist (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Cholesterol (mmol/l) Glucose (mmol/l) Triglyceride (mmol/l) HDL (mmol/l) Pi (mmol/l) Ca (mmol/l) Corr. Ca (mmol/l) Albumin (g/l) Vitamin D (nmol/l)
VDR rs10735810 (FokI)
AA
AC
CC
229 46.3 ± 14.7 31.2 ± 7.1 103.7 ± 24.6 95.1 ± 22.5 125.4 ± 15.2 78.9 ± 8.5 5.4 ± 1.2 7.9 ± 1.6 1.7 ± 0.12 0.88 ± 0.32 1.2 ± 0.33 2.5 ± 0.33 2.4 ± 0.33 45.4 ± 5.2 30.0 ± 1.5
256 46.8 ± 15.2 30.6 ± 7.0 105.1 ± 18.6 95.7 ± 18.9 126.8 ± 13.8 79.1 ± 8.7 5.5 ± 1.7 8.2 ± 1.6 1.8 ± 0.11 0.93 ± 0.36 1.2 ± 0.21 2.6 ± 0.27 2.5 ± 0.25 44.2 ± 7.7 31.0 ± 1.6
85 42.4 ± 14.7 29.5 ± 6.6 105.8 ± 19.3 94.7 ± 21.1 124.7 ± 17.6 75.2 ± 10.0‡ 5.1 ± 1.1 7.6 ± 1.6 1.7 ± 0.11 0.86 ± 0.30 1.2 ± 0.27 2.5 ± 0.18 2.4 ± 0.18 45.0 ± 4.9 27.4 ± 0.47
p-Value
TT
CT
CC
0.08 0.18 0.73 0.93 0.59 0.01 0.06 0.42 0.64 0.18 0.97 0.56 0.47 0.22 0.43
300 46.5 ± 14.6 30.4 ± 6.8 102.6 ± 22.4 93.6 ± 21.8 125.5 ± 14.7 78.3 ± 8.8 5.4 ± 1.4 8.2 ± 1.6 1.8 ± 0.12 0.91 ± 0.33 1.2 ± 0.26 2.5 ± 0.28 2.5 ± 0.26 44.2 ± 5.2 30.5 ± 1.7
228 45.4 ± 15.6 30.8 ± 6.9 106.4 ± 20.8 96.5 ± 19.3 126.3 ± 14.8 78.7 ± 9.1 5.4 ± 1.4 7.8 ± 1.6 1.6 ± 0.10* 0.89 ± 0.35 1.2 ± 0.29 2.5 ± 0.28 2.4 ± 0.30 45.0 ± 7.3 28.4 ± 1.6
42 44.6 ± 15.6 31.89 ± 8.4 109.9 ± 10.8 101.1 ± 18.4 126.9 ± 17.2 78.4 ± 9.3 5.2 ± 1.4 7.8 ± 1.7 1.8 ± 0.19 0.86 ± 0.34 1.2 ± 0.24 s 2.6 ± 0.31 2.5 ± 0.27 47.7 ± 8.2* 32.0 ± 1.5
0.60 0.42 0.07 0.08 0.83 0.88 0.56 0.30 0.04 0.67 0.49 0.29 0.13 0.03 0.37
Data are presented as mean ± standard deviation; statistical significance is shown. *Indicates that the group is significantly different from the first genotype; ‡Indicates that the group is significantly different from the second genotype.
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Table 3 Comparison among different SNPs of the VDR gene, metabolic syndrome and its components along with diabetes mellitus type 2 and vitamin D deficiency in the study population. Metabolic syndrome
Obesity
Odds ratio (95% CI)
Odds ratio (95% CI)
p
rs731236 (TaqI) AA Reference AG 0.90(0.62, 1.3) GG 1.1(0.67, 1.7) GG + AG 0.95(0.67, 1.3) A Reference G 1.0(0.80, 1.3)
0.63 0.81 0.85
rs1544410 (BsmI) CC Reference CT 1.0(0.71, 1.50) TT 1.1(0.71, 1.8) CT + TT 1.1(0.75, 1.5) C Reference T 1.1(0.84, 1.4)
0.92 0.63 0.79
rs7975232 (ApaI) AA Reference AC 1.1(0.74, 1.5) CC 1.2(0.68, 1.8) AC + CC 1.1(0.77, 1.5) A Reference C 1.1(0.83, 1.4) rs10735810 (FokI) TT Reference CT 0.93(0.66, 1.3) CC 1.5(0.76, 2.8) CT + CC 1.0(0.72, 1.4) T Reference C 1.1(0.82, 1.4)
0.90
0.63
0.78 0.70 0.73 0.66
0.72 0.25 0.99 0.64
Reference 1.0(0.61, 1.6) 1.5(0.75, 2.9) 1.1(0.70, 1.8) Reference 1.2(0.85, 1.6)
Reference 1.3(0.80, 2.2) 2.0(0.99, 4.0) 1.4(0.92, 2.3) Reference 1.4(1.0, 1.90)
Reference 0.83(0.50, 1.4) 0.72(0.38, 1.4) 1.2(0.78, 2.0) Reference 0.83(0.60, 1.2)
Reference 0.48(0.15, 1.5) 0.43(0.14, 1.3) 0.45(0.15, 1.3) Reference 0.79(0.55, 1.1)
Diabetes p
0.98 0.31 0.62 0.32
0.31 0.06 0.11 0.04
0.52 0.32 0.35 0.27
0.23 0.16 0.17 0.23
Odds ratio (95% CI) Reference 1.3(0.90, 1.9) 1.2(0.72, 1.8) 1.3(0.89, 1.8) Reference 1.1(0.87, 1.4)
Reference 1.7(1.2, 2.4) 1.3(0.80,0.20) 1.5(1.1, 2.2) Reference 1.2(0.96, 1.5)
Reference 1.3(0.93, 1.9) 0.69(0.41, 1.2) 1.1(0.81, 1.6) Reference 0.92(0.73, 1.2)
Reference 0.70(0.49,0.99) 0.57(0.29, 1.1) 0.67(0.48,0.94) Reference 0.73(0.56,0.95)
Several common chronic diseases are associated with a variation in vitamin D endocrine system (Lips, 2006; Valdivielso and Fernandez, 2006). In addition to being expressed in adipocytes and pancreatic beta cells (Ogunkolade et al., 2002; Reis et al., 2005), the gene for VDR is also expressed ubiquitously in different types of tissues (Grundberg et al., 2004; Uitterlinden et al., 2004a). In the current study, we focused on the association between VDR SNPs and components of MetS and T2DM. The MetS components including obesity, hypertension, and low HDL-C levels were assessed both separately and in clusters. Alicja et al. demonstrated that the BsmI VDR polymorphism influenced BMI, while the FokI VDR polymorphism was associated with lower serum HDL-C level (Filus et al., 2008). On the other hand, a study performed by Urszula and co-workers showed that the BsmI polymorphism in the VDR gene had no association with susceptibility to obesity and insulin resistance, while it was related to a higher LDL-C level (TworowskaBardzinska et al., 2008). Our study results are in agreement with Filus et al.'s findings demonstrating that allele T of BsmI SNP carries positive influence on obesity. There could be distinct mechanisms by which vitamin D–VDR axis could affect lipid profiles. Firstly, vitamin D induces the suppression of PTH secretion, and it has been reported that PTH could reduce lipolysis (Ford et al., 2005). Another, vitamin D might cause improvement in insulin secretion and insulin sensitivity, thereby indirectly shaping lipid metabolism (Pittas et al., 2007). Our result also shows that there is an
Low HDL p
Odds ratio (95% CI)
0.18 0.54 0.20 0.40
0.007 0.33 0.01 0.11
0.12 0.16 0.49 0.58
0.05 0.10 0.02 0.02
Reference 0.83(0.52, 1.3) 1.5(0.77, 2.9) 0.96(0.62, 1.5) Reference 1.1(0.84, 1.5)
Reference 0.60(0.37, 0.96) 1.3(0.66, 2.5) 0.74(0.47, 1.2) Reference 1.0(0.75, 1.4)
Reference 0.62(0.39, 1.1) 1.4(0.65, 2.7) 0.73(0.47, 1.1) Reference 0.97(0.72, 1.3)
Reference 1.0(0.66, 1.6) 1.0(0.45, 2.4) 1.0(0.67, 1.5) Reference 1.0(0.73, 1.4)
Hypertension p
0.48 0.26 0.91 0.40
0.03 0.50 0.22 0.94
0.06 0.48 0.16 0.87
0.99 0.99 0.91 0.93
Odds ratio (95% CI) Reference 1.5(0.89, 2.6) 1.4(0.72, 2.7) 1.5(0.89, 2.4) Reference 1.2(0.87, 1.6)
Reference 1.7(0.97, 2.8) 1.5(0.74, 2.6) 1.6(0.96, 2.7) Reference 1.2(0.89, 1.70)
Reference 1.2(0.72, 1.98) 0.64(0.30, 1.4) 1.0(0.65, 1.6) Reference 0.89(0.64, 1.2)
Reference 1.1(0.69, 1.8) 1.1(0.51, 2.6) 1.1(0.71, 1.7) Reference 1.1(0.77, 1.5)
Vitamin D deficiency p
0.14 0.38 0.13 0.25
0.06 0.28 0.08 0.19
0.54 0.28 0.90 0.55
0.71 0.82 0.64 0.64
Odds ratio (95% CI) Reference 1.0(0.46, 2.1) 2.0(0.65, 6.3) 1.18(0.56, 2.5) Reference 1.3(0.79, 2.2)
Reference 0.93(0.42, 2.0) 2.5(0.73, 8.5) 1.15(0.53, 2.4) Reference 1.36(0.82, 2.2)
Reference 0.34(0.14, 0.80) 0.61(0.19, 1.9) 0.39(0.17, 0.89) Reference 0.70(0.42, 1.16)
Reference 1.8(0.82, 4.0) 0.80(0.24, 0.25) 1.5(0.73, 3.0) Reference 1.16(0.66, 2.1)
p
0.91 0.21 0.64 0.28
0.80 0.14 0.69 0.25
0.01 0.41 0.02 0.19
0.17 0.70 0.28 0.67
association between lower HDL-C and the genotype CT of BsmI SNP. There is no direct evidence for this relation but the results of Urszula and colleagues showed higher LDL-C level associated with genotype CT of BsmI SNP. The pancreatic beta cell expression of VDR (Lee et al., 1994) supports the idea that the VDR polymorphism studies exert genomic actions possibly influencing insulin secretion (Christakos et al., 1996; Pike, 1991). Additionally, as explained earlier, the vitamin D deficiency and chronic disease connection may depend on the presence of VDR in various cells and tissues (Howard et al., 1995; Shi et al., 2001). There are limited studies relating VDR polymorphism and vitamin D (25(OH)D) deficiency in different populations. For instance, BsmI polymorphism was shown to be insignificantly associated with 25(OH)D levels in an adolescent population (d'Alesio et al., 2005), whereas, TaqI polymorphism exhibited an association with higher 25(OH)D levels in healthy adults (25 to 60 years) (Bhanushali et al., 2009). On the other hand, different SNPs of the VDR
Table 4 Haplotype frequency of VDR variants in vitamin D deficient patients versus controls. Haplotype
Odds ratio (95% CI)
p-Value
A–C–A–C A–C–A–T A–C–C–C A–C–C–T G–T–A–C G–T–A–T
1.5 (0.24, 10.2) 3.1 (0.81, 12.1) 3.9 (1.0, 17.0) 1.5 (0.55, 3.9) 2.5 (0.70, 9.0) Reference
0.98 0.11 0.04 0.45 0.21 –
Fig. 1. Pairwise linkage disequilibrium (LD) for VDR gene SNPs. The plot shows the pairwise correlation for the studied SNPs (TaqI, BsmI, ApaI and FokI).
N.M. Al-Daghri et al. / Gene 542 (2014) 129–133
gene were shown to be associated with levels of 25(OH)D (Smolders et al., 2009). Dundar et al. (2009) have also shown that higher bone metabolism and bone mineral density is linked with ApaI gene variant. The genotype, ApaI of the VDR gene has been associated with vitamin D deficiency in Caribbean patients with T2DM (VelayoudomCephise et al., 2011). Our study corroborates this finding showing that the association of vitamin D deficiency was lower in subjects carrying the AC and AC + CC genotypes of ApaI. The probable mechanism for protective association of ApaI genotypes against vitamin D deficiency is also evident by a study showing that ApaI can affect circulating levels of vitamin D (Howard et al., 1995). However, no significant associations between BsmI, FokI and TaqI SNPs and vitamin D deficiency were observed. In conclusion, our results demonstrate that components of the MetS were possibly associated with the VDR gene BsmI. Whereas, FokI and BsmI showed contradictory results related to T2DM. Low prevalence of vitamin D deficiency was related to patients carrying the ApaI gene. Conflict of interest The authors have no financial conflicts of interest. Acknowledgments The authors thank the Center of Excellence in Biotechnology Research, King Saud University, Riyadh, KSA, for funding this study. We acknowledge primary care physicians and nurses for their cooperation in recruiting and collecting the data of the subjects. We thank the Prince Mutaib Chair for Biomarkers of Osteoporosis for technical support. References Al-Daghri, N.M., Al-Attas, O., Alokail, M.S., Alkharfy, K.M., Draz, H.M., Agliardi, C., Mohammed, A.K., Guerini, F.R., Clerici, M., 2012a. Vitamin D receptor gene polymorphisms and HLA DRB1*04 cosegregation in Saudi type 2 diabetes patients. Journal of Immunology 188, 1325–1332. Al-Daghri, N.M., Alkharfy, K.M., Al-Saleh, Y., Al-Attas, O.S., Alokail, M.S., Al-Othman, A., Moharram, O., El-Kholie, E., Sabico, S., Kumar, S., Chrousos, G.P., 2012b. Modest reversal of metabolic syndrome manifestations with vitamin D status correction: a 12month prospective study. Metabolism 61, 661–666. Alkharfy, K.M., Al-Daghri, N.M., Al-Attas, O.S., Alokail, M.S., Mohammed, A.K., Vinodson, B., Clerici, M., Kazmi, U., Hussain, T., Draz, H.M., 2012. Variants of endothelial nitric oxide synthase gene are associated with components of metabolic syndrome in an Arab population. Endocrine Journal 59, 253–263. Bhanushali, A.A., Lajpal, N., Kulkarni, S.S., Chavan, S.S., Bagadi, S.S., Das, B.R., 2009. Frequency of fokI and taqI polymorphism of vitamin D receptor gene in Indian population and its association with 25-hydroxyvitamin D levels. Indian Journal of Human Genetics 15, 108–113. Bid, H.K., Konwar, R., Aggarwal, C.G., Gautam, S., Saxena, M., Nayak, V.L., Banerjee, M., 2009. Vitamin D receptor (FokI, BsmI and TaqI) gene polymorphisms and type 2 diabetes mellitus: a North Indian study. Indian Journal of Medical Sciences 63, 187–194. Bischoff-Ferrari, H.A., Giovannucci, E., Willett, W.C., Dietrich, T., Dawson-Hughes, B., 2006. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. The American Journal of Clinical Nutrition 84, 18–28. Christakos, S., Raval-Pandya, M., Wernyj, R.P., Yang, W., 1996. Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin D3. The Biochemical Journal 316 (Pt 2), 361–371. d'Alesio, A., Garabedian, M., Sabatier, J.P., Guaydier-Souquieres, G., Marcelli, C., Lemacon, A., Walrant-Debray, O., Jehan, F., 2005. Two single-nucleotide polymorphisms in the human vitamin D receptor promoter change protein–DNA complex formation and are associated with height and vitamin D status in adolescent girls. Human Molecular Genetics 14, 3539–3548. Dundar, U., Solak, M., Kavuncu, V., Ozdemir, M., Cakir, T., Yildiz, H., Evcik, D., 2009. Evidence of association of vitamin D receptor Apa I gene polymorphism with bone mineral density in postmenopausal women with osteoporosis. Clinical Rheumatology 28, 1187–1191. Filus, A., Trzmiel, A., Kuliczkowska-Plaksej, J., Tworowska, U., Jedrzejuk, D., Milewicz, A., Medras, M., 2008. Relationship between vitamin D receptor BsmI and FokI polymorphisms and anthropometric and biochemical parameters describing metabolic syndrome. The Aging Male 11, 134–139. Ford, E.S., Ajani, U.A., McGuire, L.C., Liu, S., 2005. Concentrations of serum vitamin D and the metabolic syndrome among U.S. adults. Diabetes Care 28, 1228–1230.
133
Grundberg, E., Brandstrom, H., Ribom, E.L., Ljunggren, O., Mallmin, H., Kindmark, A., 2004. Genetic variation in the human vitamin D receptor is associated with muscle strength, fat mass and body weight in Swedish women. European Journal of Endocrinology 150, 323–328. Howard, G., Nguyen, T., Morrison, N., Watanabe, T., Sambrook, P., Eisman, J., Kelly, P.J., 1995. Genetic influences on bone density: physiological correlates of vitamin D receptor gene alleles in premenopausal women. The Journal of Clinical Endocrinology and Metabolism 80, 2800–2805. Lee, S., Clark, S.A., Gill, R.K., Christakos, S., 1994. 1,25-Dihydroxyvitamin D3 and pancreatic beta-cell function: vitamin D receptors, gene expression, and insulin secretion. Endocrinology 134, 1602–1610. Lips, P., 2006. Vitamin D physiology. Progress in Biophysics and Molecular Biology 92, 4–8. Malecki, M.T., Frey, J., Moczulski, D., Klupa, T., Kozek, E., Sieradzki, J., 2003. Vitamin D receptor gene polymorphisms and association with type 2 diabetes mellitus in a Polish population. Experimental and Clinical Endocrinology and Diabetes 111, 505–509. McDermott, M.F., Ramachandran, A., Ogunkolade, B.W., Aganna, E., Curtis, D., Boucher, B.J., Snehalatha, C., Hitman, G.A., 1997. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians. Diabetologia 40, 971–975. Neyestani, T.R., Djazayery, A., Shab-Bidar, S., Eshraghian, M.R., Kalayi, A., Shariatzadeh, N., Khalaji, N., Zahedirad, M., Gharavi, A., Houshiarrad, A., Chamari, M., Asadzadeh, S., 2013. Vitamin D Receptor Fok-I polymorphism modulates diabetic host response to vitamin D intake: need for a nutrigenetic approach. Diabetes Care 36, 550–556. Nosratabadi, R., Arababadi, M.K., Salehabad, V.A., Shamsizadeh, A., Mahmoodi, M., Sayadi, A.R., Kennedy, D., 2010. Polymorphisms within exon 9 but not intron 8 of the vitamin D receptor are associated with the nephropathic complication of type-2 diabetes. International Journal of Immunogenetics 37, 493–497. Ogunkolade, B.W., Boucher, B.J., Prahl, J.M., Bustin, S.A., Burrin, J.M., Noonan, K., North, B.V., Mannan, N., McDermott, M.F., DeLuca, H.F., Hitman, G.A., 2002. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 51, 2294–2300. Oh, J.Y., Barrett-Connor, E., 2002. Association between vitamin D receptor polymorphism and type 2 diabetes or metabolic syndrome in community-dwelling older adults: the Rancho Bernardo Study. Metabolism 51, 356–359. Pani, M.A., Knapp, M., Donner, H., Braun, J., Baur, M.P., Usadel, K.H., Badenhoop, K., 2000. Vitamin D receptor allele combinations influence genetic susceptibility to type 1 diabetes in Germans. Diabetes 49, 504–507. Panierakis, C., Goulielmos, G., Mamoulakis, D., Petraki, E., Papavasiliou, E., Galanakis, E., 2009. Vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Crete, Greece. Clinical Immunology 133, 276–281. Pike, J.W., 1991. Vitamin D3 receptors: structure and function in transcription. Annual Review of Nutrition 11, 189–216. Pittas, A.G., Harris, S.S., Stark, P.C., Dawson-Hughes, B., 2007. The effects of calcium and vitamin D supplementation on blood glucose and markers of inflammation in nondiabetic adults. Diabetes Care 30, 980–986. Reis, A.F., Hauache, O.M., Velho, G., 2005. Vitamin D endocrine system and the genetic susceptibility to diabetes, obesity and vascular disease. A review of evidence. Diabetes & Metabolism 31, 318–325. Shi, H., Norman, A.W., Okamura, W.H., Sen, A., Zemel, M.B., 2001. 1alpha,25Dihydroxyvitamin D3 modulates human adipocyte metabolism via nongenomic action. FASEB Journal 15, 2751–2753. Smolders, J., Damoiseaux, J., Menheere, P., Tervaert, J.W., Hupperts, R., 2009. Fok-I vitamin D receptor gene polymorphism (rs10735810) and vitamin D metabolism in multiple sclerosis. Journal of Neuroimmunology 207, 117–121. Speer, G., Cseh, K., Winkler, G., Takacs, I., Barna, I., Nagy, Z., Lakatos, P., 2001a. Oestrogen and vitamin D receptor (VDR) genotypes and the expression of ErbB-2 and EGF receptor in human rectal cancers. European Journal of Cancer 37, 1463–1468. Speer, G., Cseh, K., Winkler, G., Vargha, P., Braun, E., Takacs, I., Lakatos, P., 2001b. Vitamin D and estrogen receptor gene polymorphisms in type 2 diabetes mellitus and in android type obesity. European Journal of Endocrinology 144, 385–389. Tworowska-Bardzinska, U., Lwow, F., Kubicka, E., Laczmanski, L., Jedzrzejuk, D., Dunajska, K., Milewicz, A., 2008. The vitamin D receptor gene BsmI polymorphism is not associated with anthropometric and biochemical parameters describing metabolic syndrome in postmenopausal women. Gynecological Endocrinology 24, 514–518. Uitterlinden, A.G., Fang, Y., Van Meurs, J.B., Pols, H.A., Van Leeuwen, J.P., 2004a. Genetics and biology of vitamin D receptor polymorphisms. Gene 338, 143–156. Uitterlinden, A.G., Fang, Y., van Meurs, J.B., van Leeuwen, H., Pols, H.A., 2004b. Vitamin D receptor gene polymorphisms in relation to Vitamin D related disease states. The Journal of Steroid Biochemistry and Molecular Biology 89–90, 187–193. Valdivielso, J.M., Fernandez, E., 2006. Vitamin D receptor polymorphisms and diseases. Clinica Chimica Acta 371, 1–12. Velayoudom-Cephise, F.L., Larifla, L., Donnet, J.P., Maimaitiming, S., Deloumeaux, J., Blanchet, A., Massart, C., Munoz-Bellili, N., Merle, S., Chout, R., Bonnet, F., Foucan, L., 2011. Vitamin D deficiency, vitamin D receptor gene polymorphisms and cardiovascular risk factors in Caribbean patients with type 2 diabetes. Diabetes & Metabolism 37, 540–545. Ye, W.Z., Reis, A.F., Dubois-Laforgue, D., Bellanne-Chantelot, C., Timsit, J., Velho, G., 2001. Vitamin D receptor gene polymorphisms are associated with obesity in type 2 diabetic subjects with early age of onset. European Journal of Endocrinology 145, 181–186.