Gene 536 (2014) 430–434
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Short Communication
Vitamin D status and vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Egyptian children Somia H. Abd-Allah a, Heba F. Pasha a,⁎, Hoda A. Hagrass b, Ashgan A. Alghobashy c a b c
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Clinical Pathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Pediatric Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
a r t i c l e
i n f o
Article history: Accepted 13 December 2013 Available online 23 December 2013 Keywords: Vitamin D Vitamin D receptor Type 1 diabetes mellitus Gene polymorphism
a b s t r a c t Background: Type 1 diabetes mellitus (T1DM) is recognized as a T-cell-mediated autoimmune disease. Vitamin D compounds are known to suppress T-cell activation by binding to vitamin D receptor (VDR); and thus, VDR gene polymorphisms may be related to T-cell-mediated autoimmune diseases. The aim of this study was to investigate the association between vitamin D status and VDR gene polymorphisms and T1DM. Materials and methods: One hundred and twenty patients with T1DM and one hundred and twenty controls were enrolled in the study. VDR gene BsmI, FokI, ApaI and TaqI polymorphisms were determined using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum 25-hydroxyvitamin D (25(OH)D) was determined using ELISA. Result: Serum 25(OH)D levels revealed a vitamin D deficiency or insufficiency in 75% of the patients. The mean levels of vitamin D were significantly lower in patients as compared to their controls (P = b0.001). VDR BsmI Bb and bb genotypes and VDR FokI Ff and ff genotypes were associated with increased risk of T1DM (OR = 2.3, 95% CI = 1.3–4.2, P = 0.005; OR = 2.2, 95% CI = 1.1–4.7, P = 0.04; OR = 1.8, 95% CI = 1.03– 3.04, P = 0.04; OR = 4.03, 95% CI = 1.2–13.1, P = 0.01 respectively), while the VDR ApaI and TaqI polymorphisms were not. Conclusion: Our study indicated that vitamin D deficiency and VDR BsmI and FokI polymorphisms were associated with T1DM in Egyptian children. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Type 1 diabetes mellitus (T1DM) is an autoimmune disorder, which leads to the destruction of the insulin producing pancreatic beta cells (Chen et al., 2011). It is characterized by deficiency of insulin secretion and ketosis-prone hyperglycemia (Barrett et al., 2009). It is well known that T1DM is caused by complex interactions between many genetic and environmental factors (Zhang et al., 2012). Previous studies indicated that there is widespread hypovitaminosis D around the world (Bener et al., 2009a; Gordon et al., 2008; Jabbar et al., 2009; Rovner and O'brien, 2008) and that it is involved in the induction of autoimmune destruction of β-cells and onset of T1DM through loss of immunomodulation of vitamin D (vitamin D favors
Abbreviations: T1DM, type 1 diabetes mellitus; VDR, vitamin D receptor; PCR-RFLP, polymerase chain reaction restriction fragment length polymorphism; 25(OH)D, 25hydroxyvitamin D; SNPs, single nucleotide polymorphisms; BMI, body mass index. ⁎ Corresponding author at: Heba F Pasha Lecturer of Medical Biochemistry, Faculty of Medicine, Zagazig University, Egypt. Tel.: +20 105788030; fax: +20 552301523. E-mail address: [email protected] (H.F. Pasha). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.032
Th2 response and protects further β-cell destruction) (Danescu et al., 2009; Holick, 2008; Lemire, 2000; Mathieu et al., 2004). There is also compelling evidence to believe that the risk attributable to hypovitaminosis D of developing diabetes keeps on increasing over the years (Littorin et al., 2006; Pozzilli et al., 2005). It is possible that hypovitaminosis D during childhood predisposes to even adult onset T1DM as one of the several mechanisms of slow β-cell destruction. Because vitamin D exerts its effects through the vitamin D receptor (VDR), the VDR gene has become a candidate gene for T1DM. The human VDR gene is located on chromosome 12q12–q14, and four common polymorphisms have been identified namely BsmI (rs1544410), FokI (rs10735810), TaqI (rs731236) and ApaI (rs7975232) (Zhang et al., 2012). As vitamin D deficiency is a major health problem in many parts of the world, including Africa and Middle East (Danescu et al., 2009; Lemire, 2000; Mathieu et al., 2004) and the role of VDR polymorphisms in T1DM pathogenesis is unclear, as several studies have suggested association between one or more of these single nucleotide polymorphisms (SNPs) and T1DM, but others have failed to confirm this finding (Ban et al., 2001; Lemos et al., 2008; Mc Dermott et al., 1997; Motohashi et al., 2003; Turpeinen et al., 2003). The aim of this study
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was to assess the contribution of vitamin D status and VDR polymorphisms to the susceptibility to T1DM in the Egyptian children.
Table 1 Primer sequences and DNA fragment length of VDR gene polymorphisms. SNP
2. Subjects and methods 2.1. Subjects The study group consisted of 120 Egyptian patients with type 1 diabetes mellitus (42 males and 78 females; mean age at time of study ± SD = 11.7 ± 2.8 years) who attended the Pediatrics outpatient clinic, Zagazig University Hospitals (Egypt). Type 1 diabetes mellitus was diagnosed according to the World Health Organization and the American Diabetes Association criteria (Rose, 2011). All patients and controls were subjected to full history taking to exclude individuals with positive family history of diabetes mellitus in order to minimize genetic heterogeneity. Thorough clinical examination was done to all subjects as well as estimation of body mass index (BMI).The control group consisted of 120 non-diabetic children; they were age, sex and ethnic origin matched with the patients. Informed consent was obtained prior to blood sampling. The study protocol was approved by the Ethical Committee of the Zagazig University. 2.2. Biochemical measurements Serum calcium (o-cresolphthalein complexone under alkaline conditions to form a violet colored complex with addition of 8hydroxyquinoline prevents interference by magnesium and iron), phosphate (molybdate UV), fasting plasma glucose (glucose oxidase) and alkaline phosphatase (colorimetric assay using p-nitrophenyl phosphate in the presence of magnesium and zinc ions) were measured. Serum 25-hydroxyvitamin D (25(OH)D) level was measured by ELISA (kit provided by Biosource Europe S.A, Belgium). The serum 25(OH)D level is the best indicator of overall vitamin D status because this measurement reflects total vitamin D from dietary intake and sunlight exposure, as well as the conversion of vitamin D from stores in the liver (Ramos-Lopez et al., 2007). Vitamin D status was classified according to the American Academy of Pediatrics (AAP)/LWEPS's recommendations on the cut-off levels for status of vitamin D. A 25(OH)D level of b 15 ng/mL (b37.5 nmol/L) as deficiency, a level of 15–20 ng/mL (37.5–50 nmol/L) as insufficiency, and a level of N20 ng/mL (N50 nmol/L) as normal (sufficient) (Misra et al., 2008). 2.3. Isolation of DNA Genomic DNA was extracted from EDTA whole blood using a spin column method according to the protocol (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany). 2.4. VDR genotyping Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed for genotyping of SNPs at positions FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) of VDR gene. The sequences of primers are shown in Table 1. The PCR amplifications were carried out in a total volume of 25 μL including 10 μg genomic DNA, 50 ng of each primer (Promega, Madison, WI) and 1 × PCR mix (Taq PCR Master Mix Kit, Qiagen, GmbH, Hilden, Germany) containing (200 μmol/L of each dNTP, 5 μL of 10 × reaction buffer, and 1.25 U Taq Gold Polymerase, and 4 mmol/L MgCl2). The reaction included 29 cycles for BsmI, TaqI and ApaI and 35 cycles for FokI, consisting of 60 s at 94 °C (denaturation); and 60 s at 65 °C (BsmI and TaqI), 60 °C (FokI), 54 °C (ApaI), 1 min at 72 °C (annealing and extension) and 5 min at 72 °C (final extension) in thermal cycler (Biometra, Göttingen, Germany). The PCR products were analyzed on 2% agarose gels containing ethidium bromide and visualized under a
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BsmI Forward Reverse FokI Forward Reverse ApaI Forward Reverse TaqI Forward Reverse
PCR primer sequences (5′–3′)
Fragment size
CAA CCA AGA CTACAA GTA CCG CGT CAG TGA AAC CAG CGG GAAGAG GTC AAG GG
B: 825 bp b: 650 bp,175 bp
AGC TGG CCCTGG CAC TGA CTC TGC TCT ATG GAA ACA CCTTGC TTC TTC TCC CTC
F: 265 bp f: 196 bp, 69 bp
CAG AGC ATGGAC AGG GAG CAA GCA ACT CCT CATGGC TGA GGT C CTC
A: 740 bp a: 530 bp, 210 bp
CAG AGC ATGGAC AGG GAG CAA GCA ACT CCT CATGGC TGA GGT C CTC
T: 495 bp, 245 bp t: 290 bp, 245 bp and 205 bp
UV transilluminator. A 100 bp ladder (Fermentas, Germany) was used as a marker. The amplified products were digested using restriction enzymes; BsmI, FokI, ApaI and TaqI (Fermentas, Germany), according to the manufacturer's instruction. Briefly, 5 μL of each related PCR product was mixed with 1 μL of each restriction enzyme, 2 μL of 10 × buffers and 12 μL H2O and then incubated 16 h at 37 °C for BsmI and ApaI, 3 h at 55 °C for FokI and 3 h at 65 °C for TaqI. Digested samples were run on 2% agarose gels containing ethidium bromide and visualized under a UV transilluminator. Genotypes were determined according to the presence or absence of an appropriate restriction site. The usual nomenclature for restriction fragment length polymorphism alleles was used in this study (Guo et al., 2006; Györffy et al., 2002). The lowercase allele represents the presence of the restriction site (b, f, a, or t) and the uppercase allele represents the absence of the restriction site (B, F, A, or T). Genotypes were scored blindly. Moreover, 10% of the samples were amplified twice for checking accuracy of results. 2.5. Statistical analysis The results for quantitative variables were expressed as means ± SD. Student's t test was used to ascertain the significance of differences between mean values of two continuous variables. Qualitative data were compared by the chi-squared-test. Genotype frequencies in cases and controls were tested for Hardy–Weinberg equilibrium, and any deviation between the observed and expected frequencies was tested for significance using the chi-squared-test. In addition, the crude odds ratios (ORs) and multivariate ORs adjusted for body mass index, blood glucose, calcium, phosphate and vitamin D levels and 95% confidence intervals (CIs) were calculated as a measure of the association of the VDR genotypes with type 1 diabetes. An exact P value (two-tailed) b 0.05 with Bonferroni adjustment (threshold P value is 0.05/n, where n is number of all independent tests at the same time) was considered statistically significant (Bland and Altman, 1995). All data were analyzed using SPSS version 19.0 of the windows. Power calculation was analyzed using the program Power and Sample Size Calculations (Version 2.1.30). 3. Results 3.1. Demographic and laboratory data of the studied children (Table 2) Levels of 25(OH)D and calcium were significantly decreased in type 1 diabetic patients compared to control group (Table 2). Furthermore, levels of phosphate were significantly increased in patients compared to control group. 25% (n: 30) of the patients withT1DM had sufficient vitamin D levels (N50 nmol/L), 5% (n: 6) were vitamin D insufficient (37.5–50 nmol/L), whereas 70% (n: 84) were vitamin deficient (b 37.5 nmol/L).
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Table 2 Characteristics of the studied groups. Parameter Age (years) Mean ± SD Range Male sex Body mass index Blood glucose Calcium(mg/dL) Phosphate (mg/dL) Alkaline phosphatase (U/L) Vitamin D (nmol/L) Sufficient(N50 nmol/L) Insufficiency(37.5–50 nmol/L) Deficiency(b37.5 nmol/L)
Control n = 120
Type 1 diabetes n = 120
P
11.1 ± 2.6 7–17 48 (40%) 21.6 ± 1.5 99.4 ± 10.3 9.9 ± 0.98 5.3 ± 2.2 594.9 ± 227 46.6 ± 13.5 66 (55%) 18 (15%) 36 (30%)
11.7 ± 2.8 7–18 42 (35%) 18.5 ± 4.3 255.3 ± 81.1 8.9 ± 0.43 9.2 ± 5.8 599.9 ± 234.8 35.5 ± 12.5 30 (25%) 6 (5%) 84 (70%)
0.074 0.4 b0.001 b0.001 b0.001 b0.001 0.86 b0.001 – 0.6 b0.001
Quantitative data were presented as mean ± standard deviation; qualitative data were presented as numbers and percent.
3.2. VDR genotype and allele distributions and risk of type 1 diabetes (Table 3) The genotype frequencies of the VDR BsmI, FokI, ApaI and TaqI were in agreement with Hardy–Weinberg equilibrium in all groups (Table 3). In type 1 diabetic patients, the frequencies of bb and Bb genotypes of VDR BsmI and ff and Ff genotypes of VDR FokI were significantly increased compared to control group and subjects with Bb and bb genotypes of VDR BsmI and Ff and ff genotypes of VDR FokI significantly more likely to develop type 1 diabetes (OR = 2.3, 95% CI = 1.3–4.2, P = 0.005; OR = 2.2, 95% CI = 1.1–4.7, P = 0.04; OR = 1.8, 95% CI = 1.03–3.04, P = 0.04; OR = 4.03, 95% CI = 1.2–13.1, P = 0.01 respectively). Regarding the VDR ApaI and TaqI polymorphisms, there were no significant differences in genotype and allele frequencies between cases and controls (P N 0.05). After adjustment for covariates subjects carrying Bb and bb genotypes of VDR BsmI and Ff and ff genotypes of VDR FokI were still more likely to develop type 1 diabetes. 4. Discussion The molecular mechanisms underlying T1DM are only partly understood. It develops as a result of a complex interaction of many genetic and environmental factors leading to the immune destruction of the
insulin-producing β-cells (Pugliese, 2004). Vitamin D could play a role in the immunopathogenesis of autoimmune diseases including type 1 diabetes mellitus. Vitamin D and its metabolites could inhibit T-cell proliferation and suppress production of interleukin 1, interleukin 2, tumor necrosis factor-α and interferon-β (Muller and Bendtzen, 1992; Rigby et al., 1984; Sandler et al., 1994; Tsoukas et al., 1984). Moreover, during the last decade, several VDR gene polymorphisms have been shown to be associated with autoimmune diseases (Ban et al., 2001; Chang et al., 2000; Györffy et al., 2002; Yokota et al., 2002). To clarify the contribution of vitamin D status and VDR polymorphisms in susceptibility to T1DM among Egyptian children, we conducted this study aiming to assess vitamin D status and analyze four wellcharacterized VDR polymorphisms. The present study revealed that vitamin D deficiency was considerably higher in T1DM children (70%) compared to non-diabetic children (30%). The prevalence of vitamin D deficiency was lower in our cohort than in previous study by Bener et al. (2009b) who found that 90.6% of T1DM children have vitamin D deficiency. In contrast, the prevalence of vitamin D deficiency was 60.5% in a study in Switzerland (Jannera et al., 2010), 43% in an Australian study (Greer et al., 2007), about 25% in an Italian study (Pozzilli et al., 2005) and 15% in a study in US East Coast youth (Svoren et al., 2009). Interestingly, in this study, vitamin D deficiency was prevalent in diabetic children. In the same line with our result Bener et al. (2009b) reported that vitamin D deficiency was prevalent in T1DM and healthy children, but it was more deficient in diabetic children. On the contrary, Bierschenk et al. (2009) did not find significant differences in 25(OH)D levels among healthy control subjects and type 1 diabetic patients. This study demonstrated that VDR gene polymorphisms were associated with susceptibility to T1DM in the Egyptian population, which can be explained by differences in VDR BsmI and FokI genotype distributions between T1DM and control subjects. VDR BsmI and FokI polymorphisms and T1DM are closely correlated. In patients with T1DM, VDR Bb genotype, bb genotype and b allele frequencies were significantly higher than in control individuals. Also VDR Ff genotype, ff genotype and f allele frequencies were significantly higher in patients with T1DM than in control individuals. In agreement with our results, Mory et al. (2009) and Panierakis et al. (2009) found significant difference in VDR BsmI genotype frequencies between T1DM and control subjects, while Ban et al. (2001),
Table 3 VDR genotype and allele distributions and risk of type 1 diabetes. Control (N = 120) N (%) BsmI genotypes BB Bb bb b allele FokI genotypes FF Ff ff f allele ApaI genotypes AA Aa aa a allele TaqI genotypes TT Tt tt t allele
Type 1 diabetes (N = 120) N (%)
OR
Confidence interval
P
OR
Confidence interval
P⁎
48 (40) 52 (43.3) 20 (16.7) 92 (38.2)
27 (22.5) 68 (56.7) 25 (20.8) 118 (49.2)
2.3 2.2 1.6
(1.3–4.2) (1.1–4.7) (1.1–2.2)
0.005 0.04 0.02
2.1 1.7 1.5
(1.1–3.2) (1.1–2.5) (1–1.9)
0.008 0.005 0.04
78 (65) 38 (31.7) 4 (3.3) 46 (19.2)
58 (48.3) 50 (41.7) 12 (10) 74 (30.8)
1.8 4.03 1.9
(1.03–3.04) (1.2–13.1) (1.2–2.9)
0.04 0.01 0.003
1.7 3.8 1.5
(1–2.7) (1.2–9.4) (1.1–2.7)
0.04 0.02 0.006
36 (30) 68 (56.7) 16 (13.3) 100 (41.7.7)
44 (36.7) 65 (54.2) 11 (9.1) 87 (36.2)
0.8 0.5 0.8
(0.5–1.4) (0.2–1.4) (0.6–1.1)
0.4 0.2 0.2
0.6 0.6 0.7
(0.5–1) (0.2–1.1) (0.7–0.9)
0.6 0.25 0.2
33 (27.5) 69 (57.5) 18 (15) 105 (43.7)
42 (35) 66 (55) 12 (10) 90 (37.5)
0.7 0.5 0.8
(0.4–1.3) (0.2–1.2) (0.5–1.1)
0.3 0.5 0.2
0.6 0.7 0.6
(0.4–1.2) (0.4–1.3) (0.4–0.8)
0.5 0.74 0.2
⁎ Adjusted for body mass index, blood glucose, calcium, phosphate and vitamin D levels then corrected by Bonferroni test.
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Panierakis et al. (2009) and Zemunik et al. (2005) reported that VDR FokI polymorphism correlates with the susceptibility to and development of T1DM. Moreover a recent meta-analysis conducted by Zhang et al. (2012) suggests that BsmI polymorphism is associated with increased risk of T1DM, especially in Asians. On the contrary, Mory et al. (2009) found no association between T1DM patients and controls in the allele and genotype frequencies in VDR FokI gene polymorphism. VDR ApaI and TaqI polymorphisms were not correlated with T1DM in Egyptian population. The VDR ApaI and TaqI genotype and allele frequencies showed no statistical difference between the control and type 1 diabetes groups. In agree with our results Lemos et al. (2008) showed that there is no difference between T1DM patients and controls regarding the allele and genotype frequencies in VDR ApaI gene polymorphism in their population. In contrast, Chang et al. (2000) and Panierakis et al. (2009) found that VDR ApaI gene polymorphism was associated withT1DM in Taiwanese population. In the same line with our results, Chang et al. (2000) and Lemos et al. (2008) showed that there is no difference between T1DM patients and controls in the allele and genotype frequencies in VDR TaqI gene polymorphism in Taiwanese and Portuguese populations, while in Greek (Panierakis et al., 2009), Romanian (Guja et al., 2002) and Iranian (Mohammadnejad et al., 2012) populations, polymorphism in VDR TaqI region contributes to the genetic heterogeneity of T1DM. VDR FokI polymorphism has been suggested to possess a functional role in the immune system (Van Etten et al., 2007). Furthermore, Shimada et al. (2008) described that peripheral blood mononuclear cells of T1DM patients with VDR BsmI BB genotype produced higher levels of interferon-gamma, suggesting that this VDR polymorphism could contribute to T-helper 1 response. Taking into consideration that the active form of vitamin D was shown to activate expression of transforming growth factor-beta1 and IL-4 cytokines, thereby inhibiting Th1-type responses and inducing regulatory T-cells, it was suggested that it can also regulate differentiation and maturation of dendritic cells critical in the induction of T-cell-mediated immune responses (Penna and Adorini, 2000). Previous studies (genome-wide association and candidate gene polymorphism) have focused on the association between VDR gene and development of T1DM, but findings have often been inconsistent among different populations worldwide (Chang et al., 2000; Guja et al., 2002; Mohammadnejad et al., 2012; Mory et al., 2009; Panierakis et al., 2009; Zemunik et al., 2005). Generally the discrepancies between studies may be due to false positive finding, replication study lacks power, heterogeneity between studies and heterogeneity across studies (Ioannidis et al., 2004; Lohmueller et al., 2003). There are a few limitations of our study. Firstly, our sample numbers considered relatively small. Secondly, lack of replication studies of the association of VDR gene polymorphisms and T1DM in Egyptian population. Consequently, further studies including larger sample numbers and replication of significant findings are necessary to clarify the role of the VDR gene polymorphism in T1DM. In conclusion, it is evident that vitamin D deficiency has prevailed in Egyptian children with T1DM. Moreover our study documents a correlation between VDR BsmI and FokI gene polymorphisms and susceptibility to T1DM in the Egyptian children.
Conflict of interest The authors declare that they have no conflict of interest.
References Ban, Y., Taniyama, M., Yanagawa, T., 2001. Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population. BMC Med. Genet. 2, 7–13.
433
Barrett, J.C., et al., 2009. Genome-wide association study and meta-analysis finds over 40 loci affect risk of type 1 diabetes. Nat. Genet. 42, 703–707. Bener, A., Al-Ali, M., Hoffmann, G.F., 2009a. High prevalence of vitamin D deficiency in young children in a highly sunny humid country: a global health problem. Minerva Pediatr. 61, 15–22. Bener, A., et al., 2009b. High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children. Acta Diabetol. 46, 183–189. Bierschenk, L., Alexande, J., Wasserfall, C., Haller, M., Schatz, D., Atkinson, M., 2009. Vitamin D levels in subjects with and without type 1 diabetes residing in a solar rich environment. Diabetes Care 32, 1977–1979. Bland, J.M., Altman, D.G., 1995. Multiple significance tests: the Bonferroni method. BMJ 310, 170–171. Chang, T.J., et al., 2000. Vitamin D receptor gene polymorphism influences susceptibility to type 1 diabetes mellitus in the Taiwanese population. Clin. Endocrinol. 52, 575–580. Chen, H., et al., 2011. AluYb8 insertion in the MUTYH gene is related to increased 8-OHdG in genomic DNA and could be a risk factor for type2 diabetes in a Chinese population. Mol. Cell. Endocrinol. 332, 301–305. Danescu, L.G., Levy, S., Levy, J., 2009. Vitamin D and diabetes mellitus. Endocrinology 35, 11–17. Gordon, C.M., et al., 2008. Prevalence of vitamin D deficiency among healthy infants and toddlers. Arch. Pediatr. Adolesc. Med. 162, 505–512. Greer, R.M., et al., 2007. Australian children and adolescents with type 1 diabetes have low vitamin D levels. Med. J. Aust. 187, 59–60. Guja, C., et al., 2002. The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population. J. Cell. Mol. Med. 6, 75–81. Guo, S.W., Magnuson, V.L., Schiller, J.J., 2006. Meta-analysis of vitamin D receptor polymorphisms and type 1 diabetes: a huge review of genetic association studies. Am. J. Epidemiol. 146, 711–724. Györffy, B., et al., 2002. Gender-specific association of vitamin D receptor polymorphism combinations with type 1 diabetes mellitus. Eur. J. Endocrinol. 147, 803–808. Holick, M.F., 2008. Diabetes and vitamin D connection. Curr. Diabet. Rep. 8, 393–398. Ioannidis, J.P., Ntzani, E.E., Trikalinos, T.A., 2004. “Racial” differences in genetic effects for complex diseases. Nat. Genet. 36, 1312–1318. Jabbar, Z., et al., 2009. High prevalence of vitamin D deficiency in North Indian adults is exacerbated in those with chronic kidney disease. Nephrology 14, 345–349. Jannera, M., Ballinarib, P., Mullisa, P.E., Flücka, C.E., 2010. High prevalence of vitamin D deficiency in children and adolescents with type 1 diabetes. Swiss Med. Wkly. 140, w13091. http://dx.doi.org/10.4414/smw.2010.13091. Lemire, J., 2000. 1,25 vitamin D3—a hormone with immunodulatory properties. J. Rheumatol. 59, 24–27. Lemos, M.C., et al., 2008. Lack of association of vitamin D receptor gene polymorphisms with susceptibility to type 1 diabetes mellitus in the Portuguese population. Hum. Immunol. 69, 134–138. Littorin, B., et al., 2006. Lower levels of plasma 25-hydroxyvitamin D among young adults at diagnosis of autoimmune type 1 diabetes compared with control subjects: results from the nationwide Diabetes Incidence Study in Sweden (DISS). Diabetologia 49, 2847–2852. Lohmueller, K.E., Pearce, C.L., Pike, M., Lander, E.S., Hirschhorn, J.N., 2003. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat. Genet. 33, 177–182. Mathieu, C., et al., 2004. Vitamin D and 1,25-dihydroxyvitamin D3 as immunomodulators in the immune system. J. Steroid Biochem. Mol. Biol. 89, 449–452. Mc Dermott, M.F., Ramachandran, A., Ogunkolade, B.W., 1997. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians. Diabetologia 40, 971–975. Misra, M., Pacaud, D., Petryk, A., Collett-Solberg, P.F., Kappy, M., Drug and Therapeutics Committee of the Lawson Wilkins Pediatric Endocrine Society, 2008. Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics 122, 398–417. Mohammadnejad, Z., et al., 2012. Association between vitamin D receptor gene polymorphisms and type 1 diabetes mellitus in Iranian population. Mol. Biol. Rep. 39, 831–837. Mory, D.B., Rocco, E.R., Miranda, W.L., Kasamatsu, T., Crispim, F., Dib, S.A., 2009. Prevalence of vitamin D receptor gene polymorphisms FokI and BsmI in Brazilian individuals with type 1 diabetes and their relation to β-cell autoimmunity and to remaining βcell function. Hum. Immunol. 70, 447–451. Motohashi, Y., Yamada, S., Yanagawa, T., 2003. Vitamin D receptor gene polymorphism affects onset pattern of type 1 diabetes. J. Clin. Endocrinol. Metab. 88, 3137–3140. Muller, K., Bendtzen, K., 1992. Inhibition of human T lymphocyte proliferation and cytokine production by 1,25-dihydroxyvitamin D3. Differential effects on CD45RA + and CD45RO+ cells. Autoimmunity 14, 37–43. 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. Clin. Immunol. 133, 276–281. Penna, G., Adorini, L., 2000. 1 Alpha, 25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J. Immunol. 164, 2405–2411. Pozzilli, P., et al., 2005. Low levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in patients with newly diagnosed type 1 diabetes. Horm. Metab. Res. 37, 680–683. Pugliese, A., 2004. Genetics of type 1 diabetes. Endocrinol. Metab. Clin. N. Am. 33, 1–16. Ramos-Lopez, E., Brück, P., Jansen, T., Herwig, J., Badenhoop, K., 2007. CYP2R1 (vitamin D 25-hydroxylase) gene is associated with susceptibility to type 1 diabetes and vitamin D levels in German. Diabetes Metab. Res. Rev. 23, 631–636. Rigby, W.F., Stacy, T., Fanger, M.W., 1984. Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol). J. Clin. Investig. 74, 1451–1455.
434
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Rose, C.J., 2011. Vitamin D insufficiency. N. Engl. J. Med. 364, 248–254. Rovner, A.J., O'brien, K.O., 2008. Hypovitaminosis D among healthy children in the United States: a review of the current evidence. Arch. Pediatr. Adolesc. Med. 162, 513–519. Sandler, S., Buschard, K., Bendtzen, K., 1994. Effects of 1,25-dihydroxyvitamin D3 and the analogues MC903 and KH1060 on inteleukin-1 beta-cell function in vitro. Immunol. Lett. 41, 73–77. Shimada, A., et al., 2008. Evidence for association between vitamin D receptor BsmI polymorphism and type 1 diabetes in Japanese. J. Autoimmun. 30, 207–211. Svoren, B.M., Volkening, L.K., Wood, J.R., Laffel, L.M., 2009. Significant vitamin D deficiency in youth with type 1 diabetes mellitus. J. Pediatr. 154, 132–134. Tsoukas, C.D., Provvedini, D.M., Manolagas, S.C., 1984. 1,25 dihydroxyvitamin D3: a novel immunoregulatory hormone. Science 224, 1438–1440.
Turpeinen, H., Hermann, R., Vaara, S., 2003. Vitamin D receptor polymorphisms: no association with type 1 diabetes in the Finnish population. Eur. J. Endocrinol. 149, 591–596. Van Etten, E., et al., 2007. The vitamin D receptor gene FokI polymorphism: functional impact on the immune system. Eur. J. Immunol. 37, 395–405. Yokota, I., et al., 2002. Association between vitamin D receptor genotype and age of onset in juvenile Japanese patients with type 1 diabetes. Diabetes Care 25, 1244–1250. Zemunik, T., et al., 2005. FokI polymorphism, vitamin D receptor, and interleukin-1 receptor haplotypes are associated with type 1 diabetes in the Dalmatian population. J. Mol. Diagn. 7, 600–604. Zhang, J., et al., 2012. Polymorphisms in the vitamin D receptor gene and type 1 diabetes mellitus risk: an update by meta-analysis. Mol. Cell. Endocrinol. 355, 135–142.
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Short Communication
Vitamin D status and vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Egyptian children Somia H. Abd-Allah a, Heba F. Pasha a,⁎, Hoda A. Hagrass b, Ashgan A. Alghobashy c a b c
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Clinical Pathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Pediatric Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
a r t i c l e
i n f o
Article history: Accepted 13 December 2013 Available online 23 December 2013 Keywords: Vitamin D Vitamin D receptor Type 1 diabetes mellitus Gene polymorphism
a b s t r a c t Background: Type 1 diabetes mellitus (T1DM) is recognized as a T-cell-mediated autoimmune disease. Vitamin D compounds are known to suppress T-cell activation by binding to vitamin D receptor (VDR); and thus, VDR gene polymorphisms may be related to T-cell-mediated autoimmune diseases. The aim of this study was to investigate the association between vitamin D status and VDR gene polymorphisms and T1DM. Materials and methods: One hundred and twenty patients with T1DM and one hundred and twenty controls were enrolled in the study. VDR gene BsmI, FokI, ApaI and TaqI polymorphisms were determined using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum 25-hydroxyvitamin D (25(OH)D) was determined using ELISA. Result: Serum 25(OH)D levels revealed a vitamin D deficiency or insufficiency in 75% of the patients. The mean levels of vitamin D were significantly lower in patients as compared to their controls (P = b0.001). VDR BsmI Bb and bb genotypes and VDR FokI Ff and ff genotypes were associated with increased risk of T1DM (OR = 2.3, 95% CI = 1.3–4.2, P = 0.005; OR = 2.2, 95% CI = 1.1–4.7, P = 0.04; OR = 1.8, 95% CI = 1.03– 3.04, P = 0.04; OR = 4.03, 95% CI = 1.2–13.1, P = 0.01 respectively), while the VDR ApaI and TaqI polymorphisms were not. Conclusion: Our study indicated that vitamin D deficiency and VDR BsmI and FokI polymorphisms were associated with T1DM in Egyptian children. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Type 1 diabetes mellitus (T1DM) is an autoimmune disorder, which leads to the destruction of the insulin producing pancreatic beta cells (Chen et al., 2011). It is characterized by deficiency of insulin secretion and ketosis-prone hyperglycemia (Barrett et al., 2009). It is well known that T1DM is caused by complex interactions between many genetic and environmental factors (Zhang et al., 2012). Previous studies indicated that there is widespread hypovitaminosis D around the world (Bener et al., 2009a; Gordon et al., 2008; Jabbar et al., 2009; Rovner and O'brien, 2008) and that it is involved in the induction of autoimmune destruction of β-cells and onset of T1DM through loss of immunomodulation of vitamin D (vitamin D favors
Abbreviations: T1DM, type 1 diabetes mellitus; VDR, vitamin D receptor; PCR-RFLP, polymerase chain reaction restriction fragment length polymorphism; 25(OH)D, 25hydroxyvitamin D; SNPs, single nucleotide polymorphisms; BMI, body mass index. ⁎ Corresponding author at: Heba F Pasha Lecturer of Medical Biochemistry, Faculty of Medicine, Zagazig University, Egypt. Tel.: +20 105788030; fax: +20 552301523. E-mail address: [email protected] (H.F. Pasha). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.032
Th2 response and protects further β-cell destruction) (Danescu et al., 2009; Holick, 2008; Lemire, 2000; Mathieu et al., 2004). There is also compelling evidence to believe that the risk attributable to hypovitaminosis D of developing diabetes keeps on increasing over the years (Littorin et al., 2006; Pozzilli et al., 2005). It is possible that hypovitaminosis D during childhood predisposes to even adult onset T1DM as one of the several mechanisms of slow β-cell destruction. Because vitamin D exerts its effects through the vitamin D receptor (VDR), the VDR gene has become a candidate gene for T1DM. The human VDR gene is located on chromosome 12q12–q14, and four common polymorphisms have been identified namely BsmI (rs1544410), FokI (rs10735810), TaqI (rs731236) and ApaI (rs7975232) (Zhang et al., 2012). As vitamin D deficiency is a major health problem in many parts of the world, including Africa and Middle East (Danescu et al., 2009; Lemire, 2000; Mathieu et al., 2004) and the role of VDR polymorphisms in T1DM pathogenesis is unclear, as several studies have suggested association between one or more of these single nucleotide polymorphisms (SNPs) and T1DM, but others have failed to confirm this finding (Ban et al., 2001; Lemos et al., 2008; Mc Dermott et al., 1997; Motohashi et al., 2003; Turpeinen et al., 2003). The aim of this study
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was to assess the contribution of vitamin D status and VDR polymorphisms to the susceptibility to T1DM in the Egyptian children.
Table 1 Primer sequences and DNA fragment length of VDR gene polymorphisms. SNP
2. Subjects and methods 2.1. Subjects The study group consisted of 120 Egyptian patients with type 1 diabetes mellitus (42 males and 78 females; mean age at time of study ± SD = 11.7 ± 2.8 years) who attended the Pediatrics outpatient clinic, Zagazig University Hospitals (Egypt). Type 1 diabetes mellitus was diagnosed according to the World Health Organization and the American Diabetes Association criteria (Rose, 2011). All patients and controls were subjected to full history taking to exclude individuals with positive family history of diabetes mellitus in order to minimize genetic heterogeneity. Thorough clinical examination was done to all subjects as well as estimation of body mass index (BMI).The control group consisted of 120 non-diabetic children; they were age, sex and ethnic origin matched with the patients. Informed consent was obtained prior to blood sampling. The study protocol was approved by the Ethical Committee of the Zagazig University. 2.2. Biochemical measurements Serum calcium (o-cresolphthalein complexone under alkaline conditions to form a violet colored complex with addition of 8hydroxyquinoline prevents interference by magnesium and iron), phosphate (molybdate UV), fasting plasma glucose (glucose oxidase) and alkaline phosphatase (colorimetric assay using p-nitrophenyl phosphate in the presence of magnesium and zinc ions) were measured. Serum 25-hydroxyvitamin D (25(OH)D) level was measured by ELISA (kit provided by Biosource Europe S.A, Belgium). The serum 25(OH)D level is the best indicator of overall vitamin D status because this measurement reflects total vitamin D from dietary intake and sunlight exposure, as well as the conversion of vitamin D from stores in the liver (Ramos-Lopez et al., 2007). Vitamin D status was classified according to the American Academy of Pediatrics (AAP)/LWEPS's recommendations on the cut-off levels for status of vitamin D. A 25(OH)D level of b 15 ng/mL (b37.5 nmol/L) as deficiency, a level of 15–20 ng/mL (37.5–50 nmol/L) as insufficiency, and a level of N20 ng/mL (N50 nmol/L) as normal (sufficient) (Misra et al., 2008). 2.3. Isolation of DNA Genomic DNA was extracted from EDTA whole blood using a spin column method according to the protocol (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany). 2.4. VDR genotyping Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed for genotyping of SNPs at positions FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) of VDR gene. The sequences of primers are shown in Table 1. The PCR amplifications were carried out in a total volume of 25 μL including 10 μg genomic DNA, 50 ng of each primer (Promega, Madison, WI) and 1 × PCR mix (Taq PCR Master Mix Kit, Qiagen, GmbH, Hilden, Germany) containing (200 μmol/L of each dNTP, 5 μL of 10 × reaction buffer, and 1.25 U Taq Gold Polymerase, and 4 mmol/L MgCl2). The reaction included 29 cycles for BsmI, TaqI and ApaI and 35 cycles for FokI, consisting of 60 s at 94 °C (denaturation); and 60 s at 65 °C (BsmI and TaqI), 60 °C (FokI), 54 °C (ApaI), 1 min at 72 °C (annealing and extension) and 5 min at 72 °C (final extension) in thermal cycler (Biometra, Göttingen, Germany). The PCR products were analyzed on 2% agarose gels containing ethidium bromide and visualized under a
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BsmI Forward Reverse FokI Forward Reverse ApaI Forward Reverse TaqI Forward Reverse
PCR primer sequences (5′–3′)
Fragment size
CAA CCA AGA CTACAA GTA CCG CGT CAG TGA AAC CAG CGG GAAGAG GTC AAG GG
B: 825 bp b: 650 bp,175 bp
AGC TGG CCCTGG CAC TGA CTC TGC TCT ATG GAA ACA CCTTGC TTC TTC TCC CTC
F: 265 bp f: 196 bp, 69 bp
CAG AGC ATGGAC AGG GAG CAA GCA ACT CCT CATGGC TGA GGT C CTC
A: 740 bp a: 530 bp, 210 bp
CAG AGC ATGGAC AGG GAG CAA GCA ACT CCT CATGGC TGA GGT C CTC
T: 495 bp, 245 bp t: 290 bp, 245 bp and 205 bp
UV transilluminator. A 100 bp ladder (Fermentas, Germany) was used as a marker. The amplified products were digested using restriction enzymes; BsmI, FokI, ApaI and TaqI (Fermentas, Germany), according to the manufacturer's instruction. Briefly, 5 μL of each related PCR product was mixed with 1 μL of each restriction enzyme, 2 μL of 10 × buffers and 12 μL H2O and then incubated 16 h at 37 °C for BsmI and ApaI, 3 h at 55 °C for FokI and 3 h at 65 °C for TaqI. Digested samples were run on 2% agarose gels containing ethidium bromide and visualized under a UV transilluminator. Genotypes were determined according to the presence or absence of an appropriate restriction site. The usual nomenclature for restriction fragment length polymorphism alleles was used in this study (Guo et al., 2006; Györffy et al., 2002). The lowercase allele represents the presence of the restriction site (b, f, a, or t) and the uppercase allele represents the absence of the restriction site (B, F, A, or T). Genotypes were scored blindly. Moreover, 10% of the samples were amplified twice for checking accuracy of results. 2.5. Statistical analysis The results for quantitative variables were expressed as means ± SD. Student's t test was used to ascertain the significance of differences between mean values of two continuous variables. Qualitative data were compared by the chi-squared-test. Genotype frequencies in cases and controls were tested for Hardy–Weinberg equilibrium, and any deviation between the observed and expected frequencies was tested for significance using the chi-squared-test. In addition, the crude odds ratios (ORs) and multivariate ORs adjusted for body mass index, blood glucose, calcium, phosphate and vitamin D levels and 95% confidence intervals (CIs) were calculated as a measure of the association of the VDR genotypes with type 1 diabetes. An exact P value (two-tailed) b 0.05 with Bonferroni adjustment (threshold P value is 0.05/n, where n is number of all independent tests at the same time) was considered statistically significant (Bland and Altman, 1995). All data were analyzed using SPSS version 19.0 of the windows. Power calculation was analyzed using the program Power and Sample Size Calculations (Version 2.1.30). 3. Results 3.1. Demographic and laboratory data of the studied children (Table 2) Levels of 25(OH)D and calcium were significantly decreased in type 1 diabetic patients compared to control group (Table 2). Furthermore, levels of phosphate were significantly increased in patients compared to control group. 25% (n: 30) of the patients withT1DM had sufficient vitamin D levels (N50 nmol/L), 5% (n: 6) were vitamin D insufficient (37.5–50 nmol/L), whereas 70% (n: 84) were vitamin deficient (b 37.5 nmol/L).
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Table 2 Characteristics of the studied groups. Parameter Age (years) Mean ± SD Range Male sex Body mass index Blood glucose Calcium(mg/dL) Phosphate (mg/dL) Alkaline phosphatase (U/L) Vitamin D (nmol/L) Sufficient(N50 nmol/L) Insufficiency(37.5–50 nmol/L) Deficiency(b37.5 nmol/L)
Control n = 120
Type 1 diabetes n = 120
P
11.1 ± 2.6 7–17 48 (40%) 21.6 ± 1.5 99.4 ± 10.3 9.9 ± 0.98 5.3 ± 2.2 594.9 ± 227 46.6 ± 13.5 66 (55%) 18 (15%) 36 (30%)
11.7 ± 2.8 7–18 42 (35%) 18.5 ± 4.3 255.3 ± 81.1 8.9 ± 0.43 9.2 ± 5.8 599.9 ± 234.8 35.5 ± 12.5 30 (25%) 6 (5%) 84 (70%)
0.074 0.4 b0.001 b0.001 b0.001 b0.001 0.86 b0.001 – 0.6 b0.001
Quantitative data were presented as mean ± standard deviation; qualitative data were presented as numbers and percent.
3.2. VDR genotype and allele distributions and risk of type 1 diabetes (Table 3) The genotype frequencies of the VDR BsmI, FokI, ApaI and TaqI were in agreement with Hardy–Weinberg equilibrium in all groups (Table 3). In type 1 diabetic patients, the frequencies of bb and Bb genotypes of VDR BsmI and ff and Ff genotypes of VDR FokI were significantly increased compared to control group and subjects with Bb and bb genotypes of VDR BsmI and Ff and ff genotypes of VDR FokI significantly more likely to develop type 1 diabetes (OR = 2.3, 95% CI = 1.3–4.2, P = 0.005; OR = 2.2, 95% CI = 1.1–4.7, P = 0.04; OR = 1.8, 95% CI = 1.03–3.04, P = 0.04; OR = 4.03, 95% CI = 1.2–13.1, P = 0.01 respectively). Regarding the VDR ApaI and TaqI polymorphisms, there were no significant differences in genotype and allele frequencies between cases and controls (P N 0.05). After adjustment for covariates subjects carrying Bb and bb genotypes of VDR BsmI and Ff and ff genotypes of VDR FokI were still more likely to develop type 1 diabetes. 4. Discussion The molecular mechanisms underlying T1DM are only partly understood. It develops as a result of a complex interaction of many genetic and environmental factors leading to the immune destruction of the
insulin-producing β-cells (Pugliese, 2004). Vitamin D could play a role in the immunopathogenesis of autoimmune diseases including type 1 diabetes mellitus. Vitamin D and its metabolites could inhibit T-cell proliferation and suppress production of interleukin 1, interleukin 2, tumor necrosis factor-α and interferon-β (Muller and Bendtzen, 1992; Rigby et al., 1984; Sandler et al., 1994; Tsoukas et al., 1984). Moreover, during the last decade, several VDR gene polymorphisms have been shown to be associated with autoimmune diseases (Ban et al., 2001; Chang et al., 2000; Györffy et al., 2002; Yokota et al., 2002). To clarify the contribution of vitamin D status and VDR polymorphisms in susceptibility to T1DM among Egyptian children, we conducted this study aiming to assess vitamin D status and analyze four wellcharacterized VDR polymorphisms. The present study revealed that vitamin D deficiency was considerably higher in T1DM children (70%) compared to non-diabetic children (30%). The prevalence of vitamin D deficiency was lower in our cohort than in previous study by Bener et al. (2009b) who found that 90.6% of T1DM children have vitamin D deficiency. In contrast, the prevalence of vitamin D deficiency was 60.5% in a study in Switzerland (Jannera et al., 2010), 43% in an Australian study (Greer et al., 2007), about 25% in an Italian study (Pozzilli et al., 2005) and 15% in a study in US East Coast youth (Svoren et al., 2009). Interestingly, in this study, vitamin D deficiency was prevalent in diabetic children. In the same line with our result Bener et al. (2009b) reported that vitamin D deficiency was prevalent in T1DM and healthy children, but it was more deficient in diabetic children. On the contrary, Bierschenk et al. (2009) did not find significant differences in 25(OH)D levels among healthy control subjects and type 1 diabetic patients. This study demonstrated that VDR gene polymorphisms were associated with susceptibility to T1DM in the Egyptian population, which can be explained by differences in VDR BsmI and FokI genotype distributions between T1DM and control subjects. VDR BsmI and FokI polymorphisms and T1DM are closely correlated. In patients with T1DM, VDR Bb genotype, bb genotype and b allele frequencies were significantly higher than in control individuals. Also VDR Ff genotype, ff genotype and f allele frequencies were significantly higher in patients with T1DM than in control individuals. In agreement with our results, Mory et al. (2009) and Panierakis et al. (2009) found significant difference in VDR BsmI genotype frequencies between T1DM and control subjects, while Ban et al. (2001),
Table 3 VDR genotype and allele distributions and risk of type 1 diabetes. Control (N = 120) N (%) BsmI genotypes BB Bb bb b allele FokI genotypes FF Ff ff f allele ApaI genotypes AA Aa aa a allele TaqI genotypes TT Tt tt t allele
Type 1 diabetes (N = 120) N (%)
OR
Confidence interval
P
OR
Confidence interval
P⁎
48 (40) 52 (43.3) 20 (16.7) 92 (38.2)
27 (22.5) 68 (56.7) 25 (20.8) 118 (49.2)
2.3 2.2 1.6
(1.3–4.2) (1.1–4.7) (1.1–2.2)
0.005 0.04 0.02
2.1 1.7 1.5
(1.1–3.2) (1.1–2.5) (1–1.9)
0.008 0.005 0.04
78 (65) 38 (31.7) 4 (3.3) 46 (19.2)
58 (48.3) 50 (41.7) 12 (10) 74 (30.8)
1.8 4.03 1.9
(1.03–3.04) (1.2–13.1) (1.2–2.9)
0.04 0.01 0.003
1.7 3.8 1.5
(1–2.7) (1.2–9.4) (1.1–2.7)
0.04 0.02 0.006
36 (30) 68 (56.7) 16 (13.3) 100 (41.7.7)
44 (36.7) 65 (54.2) 11 (9.1) 87 (36.2)
0.8 0.5 0.8
(0.5–1.4) (0.2–1.4) (0.6–1.1)
0.4 0.2 0.2
0.6 0.6 0.7
(0.5–1) (0.2–1.1) (0.7–0.9)
0.6 0.25 0.2
33 (27.5) 69 (57.5) 18 (15) 105 (43.7)
42 (35) 66 (55) 12 (10) 90 (37.5)
0.7 0.5 0.8
(0.4–1.3) (0.2–1.2) (0.5–1.1)
0.3 0.5 0.2
0.6 0.7 0.6
(0.4–1.2) (0.4–1.3) (0.4–0.8)
0.5 0.74 0.2
⁎ Adjusted for body mass index, blood glucose, calcium, phosphate and vitamin D levels then corrected by Bonferroni test.
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Panierakis et al. (2009) and Zemunik et al. (2005) reported that VDR FokI polymorphism correlates with the susceptibility to and development of T1DM. Moreover a recent meta-analysis conducted by Zhang et al. (2012) suggests that BsmI polymorphism is associated with increased risk of T1DM, especially in Asians. On the contrary, Mory et al. (2009) found no association between T1DM patients and controls in the allele and genotype frequencies in VDR FokI gene polymorphism. VDR ApaI and TaqI polymorphisms were not correlated with T1DM in Egyptian population. The VDR ApaI and TaqI genotype and allele frequencies showed no statistical difference between the control and type 1 diabetes groups. In agree with our results Lemos et al. (2008) showed that there is no difference between T1DM patients and controls regarding the allele and genotype frequencies in VDR ApaI gene polymorphism in their population. In contrast, Chang et al. (2000) and Panierakis et al. (2009) found that VDR ApaI gene polymorphism was associated withT1DM in Taiwanese population. In the same line with our results, Chang et al. (2000) and Lemos et al. (2008) showed that there is no difference between T1DM patients and controls in the allele and genotype frequencies in VDR TaqI gene polymorphism in Taiwanese and Portuguese populations, while in Greek (Panierakis et al., 2009), Romanian (Guja et al., 2002) and Iranian (Mohammadnejad et al., 2012) populations, polymorphism in VDR TaqI region contributes to the genetic heterogeneity of T1DM. VDR FokI polymorphism has been suggested to possess a functional role in the immune system (Van Etten et al., 2007). Furthermore, Shimada et al. (2008) described that peripheral blood mononuclear cells of T1DM patients with VDR BsmI BB genotype produced higher levels of interferon-gamma, suggesting that this VDR polymorphism could contribute to T-helper 1 response. Taking into consideration that the active form of vitamin D was shown to activate expression of transforming growth factor-beta1 and IL-4 cytokines, thereby inhibiting Th1-type responses and inducing regulatory T-cells, it was suggested that it can also regulate differentiation and maturation of dendritic cells critical in the induction of T-cell-mediated immune responses (Penna and Adorini, 2000). Previous studies (genome-wide association and candidate gene polymorphism) have focused on the association between VDR gene and development of T1DM, but findings have often been inconsistent among different populations worldwide (Chang et al., 2000; Guja et al., 2002; Mohammadnejad et al., 2012; Mory et al., 2009; Panierakis et al., 2009; Zemunik et al., 2005). Generally the discrepancies between studies may be due to false positive finding, replication study lacks power, heterogeneity between studies and heterogeneity across studies (Ioannidis et al., 2004; Lohmueller et al., 2003). There are a few limitations of our study. Firstly, our sample numbers considered relatively small. Secondly, lack of replication studies of the association of VDR gene polymorphisms and T1DM in Egyptian population. Consequently, further studies including larger sample numbers and replication of significant findings are necessary to clarify the role of the VDR gene polymorphism in T1DM. In conclusion, it is evident that vitamin D deficiency has prevailed in Egyptian children with T1DM. Moreover our study documents a correlation between VDR BsmI and FokI gene polymorphisms and susceptibility to T1DM in the Egyptian children.
Conflict of interest The authors declare that they have no conflict of interest.
References Ban, Y., Taniyama, M., Yanagawa, T., 2001. Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population. BMC Med. Genet. 2, 7–13.
433
Barrett, J.C., et al., 2009. Genome-wide association study and meta-analysis finds over 40 loci affect risk of type 1 diabetes. Nat. Genet. 42, 703–707. Bener, A., Al-Ali, M., Hoffmann, G.F., 2009a. High prevalence of vitamin D deficiency in young children in a highly sunny humid country: a global health problem. Minerva Pediatr. 61, 15–22. Bener, A., et al., 2009b. High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children. Acta Diabetol. 46, 183–189. Bierschenk, L., Alexande, J., Wasserfall, C., Haller, M., Schatz, D., Atkinson, M., 2009. Vitamin D levels in subjects with and without type 1 diabetes residing in a solar rich environment. Diabetes Care 32, 1977–1979. Bland, J.M., Altman, D.G., 1995. Multiple significance tests: the Bonferroni method. BMJ 310, 170–171. Chang, T.J., et al., 2000. Vitamin D receptor gene polymorphism influences susceptibility to type 1 diabetes mellitus in the Taiwanese population. Clin. Endocrinol. 52, 575–580. Chen, H., et al., 2011. AluYb8 insertion in the MUTYH gene is related to increased 8-OHdG in genomic DNA and could be a risk factor for type2 diabetes in a Chinese population. Mol. Cell. Endocrinol. 332, 301–305. Danescu, L.G., Levy, S., Levy, J., 2009. Vitamin D and diabetes mellitus. Endocrinology 35, 11–17. Gordon, C.M., et al., 2008. Prevalence of vitamin D deficiency among healthy infants and toddlers. Arch. Pediatr. Adolesc. Med. 162, 505–512. Greer, R.M., et al., 2007. Australian children and adolescents with type 1 diabetes have low vitamin D levels. Med. J. Aust. 187, 59–60. Guja, C., et al., 2002. The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population. J. Cell. Mol. Med. 6, 75–81. Guo, S.W., Magnuson, V.L., Schiller, J.J., 2006. Meta-analysis of vitamin D receptor polymorphisms and type 1 diabetes: a huge review of genetic association studies. Am. J. Epidemiol. 146, 711–724. Györffy, B., et al., 2002. Gender-specific association of vitamin D receptor polymorphism combinations with type 1 diabetes mellitus. Eur. J. Endocrinol. 147, 803–808. Holick, M.F., 2008. Diabetes and vitamin D connection. Curr. Diabet. Rep. 8, 393–398. Ioannidis, J.P., Ntzani, E.E., Trikalinos, T.A., 2004. “Racial” differences in genetic effects for complex diseases. Nat. Genet. 36, 1312–1318. Jabbar, Z., et al., 2009. High prevalence of vitamin D deficiency in North Indian adults is exacerbated in those with chronic kidney disease. Nephrology 14, 345–349. Jannera, M., Ballinarib, P., Mullisa, P.E., Flücka, C.E., 2010. High prevalence of vitamin D deficiency in children and adolescents with type 1 diabetes. Swiss Med. Wkly. 140, w13091. http://dx.doi.org/10.4414/smw.2010.13091. Lemire, J., 2000. 1,25 vitamin D3—a hormone with immunodulatory properties. J. Rheumatol. 59, 24–27. Lemos, M.C., et al., 2008. Lack of association of vitamin D receptor gene polymorphisms with susceptibility to type 1 diabetes mellitus in the Portuguese population. Hum. Immunol. 69, 134–138. Littorin, B., et al., 2006. Lower levels of plasma 25-hydroxyvitamin D among young adults at diagnosis of autoimmune type 1 diabetes compared with control subjects: results from the nationwide Diabetes Incidence Study in Sweden (DISS). Diabetologia 49, 2847–2852. Lohmueller, K.E., Pearce, C.L., Pike, M., Lander, E.S., Hirschhorn, J.N., 2003. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat. Genet. 33, 177–182. Mathieu, C., et al., 2004. Vitamin D and 1,25-dihydroxyvitamin D3 as immunomodulators in the immune system. J. Steroid Biochem. Mol. Biol. 89, 449–452. Mc Dermott, M.F., Ramachandran, A., Ogunkolade, B.W., 1997. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians. Diabetologia 40, 971–975. Misra, M., Pacaud, D., Petryk, A., Collett-Solberg, P.F., Kappy, M., Drug and Therapeutics Committee of the Lawson Wilkins Pediatric Endocrine Society, 2008. Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics 122, 398–417. Mohammadnejad, Z., et al., 2012. Association between vitamin D receptor gene polymorphisms and type 1 diabetes mellitus in Iranian population. Mol. Biol. Rep. 39, 831–837. Mory, D.B., Rocco, E.R., Miranda, W.L., Kasamatsu, T., Crispim, F., Dib, S.A., 2009. Prevalence of vitamin D receptor gene polymorphisms FokI and BsmI in Brazilian individuals with type 1 diabetes and their relation to β-cell autoimmunity and to remaining βcell function. Hum. Immunol. 70, 447–451. Motohashi, Y., Yamada, S., Yanagawa, T., 2003. Vitamin D receptor gene polymorphism affects onset pattern of type 1 diabetes. J. Clin. Endocrinol. Metab. 88, 3137–3140. Muller, K., Bendtzen, K., 1992. Inhibition of human T lymphocyte proliferation and cytokine production by 1,25-dihydroxyvitamin D3. Differential effects on CD45RA + and CD45RO+ cells. Autoimmunity 14, 37–43. 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. Clin. Immunol. 133, 276–281. Penna, G., Adorini, L., 2000. 1 Alpha, 25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J. Immunol. 164, 2405–2411. Pozzilli, P., et al., 2005. Low levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in patients with newly diagnosed type 1 diabetes. Horm. Metab. Res. 37, 680–683. Pugliese, A., 2004. Genetics of type 1 diabetes. Endocrinol. Metab. Clin. N. Am. 33, 1–16. Ramos-Lopez, E., Brück, P., Jansen, T., Herwig, J., Badenhoop, K., 2007. CYP2R1 (vitamin D 25-hydroxylase) gene is associated with susceptibility to type 1 diabetes and vitamin D levels in German. Diabetes Metab. Res. Rev. 23, 631–636. Rigby, W.F., Stacy, T., Fanger, M.W., 1984. Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol). J. Clin. Investig. 74, 1451–1455.
434
S.H. Abd-Allah et al. / Gene 536 (2014) 430–434
Rose, C.J., 2011. Vitamin D insufficiency. N. Engl. J. Med. 364, 248–254. Rovner, A.J., O'brien, K.O., 2008. Hypovitaminosis D among healthy children in the United States: a review of the current evidence. Arch. Pediatr. Adolesc. Med. 162, 513–519. Sandler, S., Buschard, K., Bendtzen, K., 1994. Effects of 1,25-dihydroxyvitamin D3 and the analogues MC903 and KH1060 on inteleukin-1 beta-cell function in vitro. Immunol. Lett. 41, 73–77. Shimada, A., et al., 2008. Evidence for association between vitamin D receptor BsmI polymorphism and type 1 diabetes in Japanese. J. Autoimmun. 30, 207–211. Svoren, B.M., Volkening, L.K., Wood, J.R., Laffel, L.M., 2009. Significant vitamin D deficiency in youth with type 1 diabetes mellitus. J. Pediatr. 154, 132–134. Tsoukas, C.D., Provvedini, D.M., Manolagas, S.C., 1984. 1,25 dihydroxyvitamin D3: a novel immunoregulatory hormone. Science 224, 1438–1440.
Turpeinen, H., Hermann, R., Vaara, S., 2003. Vitamin D receptor polymorphisms: no association with type 1 diabetes in the Finnish population. Eur. J. Endocrinol. 149, 591–596. Van Etten, E., et al., 2007. The vitamin D receptor gene FokI polymorphism: functional impact on the immune system. Eur. J. Immunol. 37, 395–405. Yokota, I., et al., 2002. Association between vitamin D receptor genotype and age of onset in juvenile Japanese patients with type 1 diabetes. Diabetes Care 25, 1244–1250. Zemunik, T., et al., 2005. FokI polymorphism, vitamin D receptor, and interleukin-1 receptor haplotypes are associated with type 1 diabetes in the Dalmatian population. J. Mol. Diagn. 7, 600–604. Zhang, J., et al., 2012. Polymorphisms in the vitamin D receptor gene and type 1 diabetes mellitus risk: an update by meta-analysis. Mol. Cell. Endocrinol. 355, 135–142.
