Pediatrics International (2015) 57, 870–874
doi: 10.1111/ped.12634
Original Article
Vitamin D receptor gene polymorphisms and type 1 diabetes mellitus in a Korean population Chong-Kun Cheon,1* Hyo-Kyoung Nam,2* Kee-Hyoung Lee,2 Su Yung Kim,1 Ji Sun Song3 and Choongrak Kim4 Department of Pediatrics, Pediatric Endocrinology, Pusan National University Children’s Hospital, Pusan National University School of Medicine, Yangsan, 2Department of Pediatrics, College of Medicine, Korea University, Seoul, 3Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Gyeongnam and 4 Department of Statistics, Pusan National University, Pusan, South Korea 1
Abstract
Background: Vitamin D receptor (VDR) has been suggested to play a role in the pathogenesis of type 1 diabetes mellitus (T1DM). There has been no case–control study examining the association between VDR polymorphisms and T1DM among Korean subjects with a low incidence of T1DM. Methods: Eighty-one T1DM patients and 113 unrelated healthy controls with no history of DM or other autoimmune diseases were investigated at either Pusan National University Children’s Hospital or Korea University Anam Hospital between March 2009 and September 2013. Polymerase chain reaction–restriction fragment length polymorphism was utilized to genotype single nucleotide substitutions at TaqI, BsmI, and ApaI alleles. Results: All frequencies in T1DM and control subjects were in Hardy–Weinberg equilibrium, although ApaI in controls and TaqI in T1DM showed relatively weak equilibrium. TaqI and BsmI differences were significant (P = 0.045 and P = 0.012, respectively) after applying Bonferroni correction. The TT genotype carrier frequency among controls was higher than among the T1DM patients (P = 0.015; OR, 2.98; 95%CI: 1.19–7.42). T allele frequency was higher among controls than T1DM patients (P = 0.019; OR, 2.78; 95%CI: 1.15–6.72). The frequency of bb genotype carriers among controls was higher than among T1DM patients (P = 0.004; OR, 4.13; 95%CI: 1.4–12.10). The frequency of the b allele among controls was higher than that among T1DM patients (P = 0.016; OR, 3.20; 95%CI: 1.19–8.60). Conclusions: T and b TaqI and BsmI alleles are protective against T1DM in Korean subjects.
Key words polymorphism, type 1 diabetes mellitus, vitamin D receptor. Type 1 (insulin-dependent) diabetes mellitus (T1DM) is an autoimmune disease that results in the destruction of pancreatic beta cells due to interactions between susceptibility genes and environmental exposure.1 Most T1DM in humans is considered to be a T-cellmediated autoimmune disease.2 There is evidence for worldwide epidemics of both vitamin D deficiency and T1DM,1 and vitamin D has been identified as a contributing factor to DM.3 Vitamin D, among its many roles, acts as a modulator of the immune system by promoting monocyte differentiation and inhibiting lymphocyte proliferation and cytokine secretion.4 Four single-nucleotide polymorphisms (SNP) in the vitamin D receptor (VDR) gene have been studied: FokI F>f (rs10735810, NM_000376.2: c.2T>C), BsmI B>b (rs1544410, NM_000376.2: c.1024+283G>A), ApaI A>a (rs7975232, NM_000376.2: c.1025-49G>T), and TaqI T>t (rs731236, NM_000376.2: c.1056T>C).5 Allele F of the FokI Correspondence: Chong-Kun Cheon, MD PhD, Department of Pediatrics, Pediatric Genetics and Metabolism, Pusan National University Children’s Hospital, Pusan National University School of Medicine, Geumo-ro, Yangsan-si, Gyeongnam 602-739, South Korea. Email: [email protected] * Chong-Kun Cheon and Hyo-Kyoung Nam are co-first authors. [Correction added on 28 July 2015, after first online publication: The above statement has been added to indicate co-first authorship.] Received 26 July 2014; revised 27 January 2015; accepted 19 February 2015. © 2015 Japan Pediatric Society
SNP creates an alternative ATG initiation codon in exon 2, leading to a VDR protein that is three amino acids longer than the wild type. ApaI, BsmI and TaqI polymorphisms are located near the 3′ end of the VDR gene; BsmI and ApaI SNP are both located in intron 8, and TaqI is a silent SNP in exon 9.5 To date, a large number of studies have suggested that VDR is involved in the pathogenesis of T1DM.6 Several studies have suggested an association between one or more of the aforementioned SNP and T1DM, but others have failed to confirm these findings.7,8 This study therefore assessed the contribution of VDR polymorphisms to TIDM susceptibility in a genetically homogenous population in Korea.
Methods Patients
The study group consisted of 81 Korean patients with T1DM (35 boys and 46 girls; mean age, 10.28 ± 3.73 years old). An additional 113 unrelated healthy control group (53 boys and 60 girls; mean age, 9.98 ± 3.56 years old) with no history of DM or other autoimmune diseases was recruited from local schools. Most samples were taken from patients diagnosed and treated at either Pusan National University Children’s Hospital or Korea University Anam Hospital between March 2009 and September 2013. T1DM was diagnosed under 15 years of age, according to the World Health
VDR polymorphisms and T1DM in Korea 871 Organization criteria. All DM patients had typical histories of ketoacidosis and diabetes-associated antibodies and required continuous insulin treatment upon diagnosis. The study was approved by the Ethics Committee of the Yangsan National University Hospital and informed consent was obtained from the participating subjects and/or parents. DNA extraction
Genomic DNA was extracted from whole blood samples using the QIAamp DNA blood kit (Qiagen) according to the manufacturer’s protocol. DNA was stored at 20°C until use. VDR genotyping
Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to genotype single-nucleotide substitutions at TaqI (rs731236), BsmI (rs1544410) and ApaI (rs7975232) polymorphisms in the VDR gene (Fig. 1). Primers were designed based on GenBank cDNA sequences (Table 1). All amplifications were performed using 40 ng DNA, 10 pmol of each primer, polymerase, and PCR master mix (Promega, Madison, WI, USA) in a total volume of 25 μL. Reactions were cycled 29 times for TaqI and 35 times for ApaI, and BsmI, with each cycle consisting of 60 s at 94°C (denaturation), 60 s at 65°C (TaqI), 68°C (BsmI), or 54°C (ApaI), and 1 min at 72°C (annealing and extension), followed by a final extension at 72°C for 5 min. PCR products were run on 2% agarose gels and visualized on ethidium bromide staining. A 100 bp ladder (Bioneer, Alameda, CA, USA) was used as a marker. The amplified products were digested using the restriction enzymes ApaI and TaqI (Fermentas; St Leon-Rot, Germany), according to the manufacturer’s protocol. Briefly, 5 μL of each associated PCR product was mixed with 0.5 μL of each restriction enzyme, 1 μL of buffer, and 3.5 μL H2O, and then
incubated for 3 h at 65°C for TaqI and 16 h at 37°C for ApaI and BsmI. After incubation, the restriction enzymes were inactivated at 65°C for 20 min (BsmI and ApaI; TaqI is not inactivated). Digested samples were separated on 2% agarose gels and visualized with ethidium bromide (0.5 mg/mL). Statistical analysis
Statistical analysis was performed using SPSS for Windows (version 20.0, SPSS Inc., Chicago, IL, USA). We estimated mode of inheritance using Fisher’s exact test for the codominant, dominant, recessive and VDR genotype, and Cochran–Armitage test for the trend model in patients and healthy controls. OR and 95%CI were calculated for each allele, genotype, and haplotype. Two-sided P < 0.05 indicated statistical significance. Hardy–Weinberg equilibrium was assessed using the chi-squared goodness-of-fit test to compare the observed and allele-based expected genotype frequencies.
Results All frequencies in T1DM and control subjects were in Hardy–Weinberg equilibrium (P > 0.05), although ApaI in controls and TaqI in T1DM showed relatively weak equilibrium. In all three polymorphisms tested, the distribution of the two VDR genotype frequencies differed significantly between T1DM patients and controls. We first compared the VDR-TaqI polymorphism between T1DM patients overall and healthy subjects. As shown in Table 2, the frequency of TT genotype carriers among controls was significantly higher than that in T1DM patients (P = 0.015; OR, 2.98; 95%CI: 1.19–7.42). A significantly higher T allele frequency was seen among controls, compared with T1DM patients (P = 0.019; OR, 2.78; 95%CI: 1.15–6.72). The frequency of bb genotype carriers among controls was significantly higher than among T1DM patients (P = 0.004; OR, 4.13; Fig. 1 Genotyping of the vitamin D receptor (VDR) gene. (a) ApaI polymorphism: lane 1, markers; lane 2, uncut product (421 bp); lane 3, genotype aa homozygous (233 and 188 bp); lane 4, genotype Aa heterozygous (421, 233 and 188 bp); lane 5, genotype AA homozygous (421 bp). (b) TaqI polymorphism: lane 1, markers; lane 2, uncut product (716 bp); lane 3, genotype TT homozygous (514 and 202 bp); lane 4, genotype Tt heterozygous (514, 237 and 169 bp). (c) BsmI polymorphism: lane 1, markers; lane 2, uncut product (801 bp); lane 3, genotype bb homozygous (500 and 301 bp); lane 4, genotype Bb heterozygous (801, 500 and 301 bp); lane 5, genotype BB homozygous (801 bp).
© 2015 Japan Pediatric Society
872 C-K Cheon et al. Table 1 PCR primers used to amplify fragments harboring VDR SNP SNP
PCR primer (5’-3’)
Fragment size (bp)
ApaI
Forward GAC GCT GAG GGA TGG Reverse GTC GGC TAG CTT CTG GAT Forward AAC TTG CAT GAG GAG GAG CAT GTC Reverse GGA GAG GAG CCT GTG TCC CAT TTG Forward GGG ACG CTG AGG GAT GGA CAG AGC Reverse GGA AAG GGG TTA GGT TGG ACA GGA
421
BsmI
TaqI
801
716
PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; VDR, vitamin D receptor.
95%CI: 1.41–12.10), and the frequency of the b allele in controls was significantly higher than among T1DM patients (P = 0.016; OR, 3.20; 95%CI: 1.19–8.60). The ApaI polymorphism did not appear to be significantly associated with overall T1DM risk. Multiple comparison tests for adjusting P-value were performed. TaqI and BsmI differences were significant (P = 0.045 and P = 0.012, respectively) after applying the Bonferroni correction. Therefore, T and b TaqI and BsmI alleles are protective against T1DM in Korean individuals.
Discussion Several studies have focused on associations between candidate genes and development of T1DM. To clarify the contribution of the VDR polymorphisms TaqI, BsmI, and ApaI to T1DM genetic susceptibility in Korean patients, 81 patients with T1DM and 113 unrelated healthy controls with no history of DM or other autoimmune diseases were tested. This is the first study on the association between VDR SNP and T1DM among Korean subjects. We demonstrate here that BsmI and TaqI polymorphisms were more commonly associated with T1DM in Korean individuals. The t allele for the TaqI polymorphism and the B allele for the BsmI polymorphism were associated with increased risk of T1DM. The VDR locus has been studied extensively for association(s) with osteoporosis,9–11 primary hyperparathyroidism,12,13 and autoimmune diseases such as Graves’ disease,14,15 Hashimoto’s thyroiditis,16 and multiple sclerosis.17 Polymorphisms in VDR are reported to be associated with insulin secretory capacity in humans.18 Interestingly, in an epidemiological study from several European countries, vitamin D supplementation in early childhood seems to be associated with a reduction in the incidence of T1DM, indicating that the immune-regulatory effects of vitamin D through VDR may modulate the disease course of T1DM.3 Four SNP in VDR (FokI, BsmI, ApaI, and TaqI) have been investigated in previous studies.5 TaqI is a silent SNP in exon 9, while both ApaI and BsmI are located in the intronic region between exons 8 and 9 and do not affect VDR protein structure.5,19,20 Some studies have reported linkage between BsmI and a variable length polymorphism in the 3′ untranslated region, known as poly (A), which may influence VDR mRNA stability.4,21 This opens up the possibility that the observed © 2015 Japan Pediatric Society
linkage points either toward another closely linked T1DM susceptibility gene or a second functional nucleotide change within the VDR gene itself.22 Associations between VDR polymorphisms and T1DM have been seen in other countries including Taiwan,19 Hungary,20 Uruguay,23 India,24 Germany,25 Croatia,23,26 Holland,27 Romania,28 Iran,29 and Japan.30 Other studies have not confirmed these findings and no associations were found in Portuguese7 or Italian populations,31 indicating that differing genetic background may be causative of these differences. A recent meta-analysis suggested that the BsmI polymorphism is associated with increased risk of T1DM, especially in Asian subjects.32 Shimada et al. demonstrated that peripheral blood mononuclear cells from T1DM patients with the BsmI BB genotype produced higher levels of interferon-γ, suggesting that this VDR polymorphism could contribute to the T-helper (Th)1 response.33 Given that the active form of vitamin D activates the expression of transforming growth factor-beta 1 and interleukin-4 cytokines, therefore inhibiting Th1-type responses and inducing regulatory T cells, it was suggested that it can also regulate differentiation and maturation of dendritic cells, which is critical in the induction of T-cell-mediated immune responses.34 Discrepancies in VDR polymorphisms associated with T1DM in different populations may be due to differences in ethnic background, diverse evolutionary lineages, and interactions with other genetic and/or environmental factors involved in the pathogenesis of T1DM.25,29 In the present study, BsmI bb homozygotes had a smaller risk of T1DM compared with B allele (BB + Bb) carriers, while TaqI TT homozygotes had a decreased risk of T1DM compared with t allele (tt+Tt) carriers. This suggests that BsmI and TaqI polymorphisms may contribute to T1DM pathogenesis, with the bb and TT genotypes protective against T1DM. This finding was recently attributed to potential interference in the VDR genotype by environmental factors, such as ultraviolet radiation (UVR).35 The association between the B allele and T1DM increased as regional winter UVR increased, and most Asian countries have a high winter UVR level,35 which may explain individual differences in susceptibility to T1DM. To the best of our knowledge, there has been no case–control study examining the association between VDR polymorphisms and T1DM in Korea. Interestingly, the ageadjusted incidence of TIDM in South Korea is low (1.1 per 100 000/year) compared with other countries.36 The lower incidence of TIDM may be linked to T and b TaqI and BsmI alleles, which are protective against T1DM in Korean individuals. Taken together, the present study is in agreement with findings from other populations, such as Indian Asian and Spanish subjects, for the BsmI polymorphism,24,37 and Bangladeshi Asian subjects for the TaqI polymorphism.18 The present study, however, has several limitations. First, the study was very small and thus it is difficult to reach a definitive conclusion on biological relevance. Susceptibility alleles are likely to individually contribute very modestly to the overall risk, and the magnitude of the effects is usually observed only in samples of hundreds of patients and controls.32 For these reason it is extraordinary that several OR in this study seem so high (or low, in the case of the alternative allele). It may be due to sample size. Second, the analysis of VDR FokI genotype was not
VDR polymorphisms and T1DM in Korea 873 Table 2 VDR gene polymorphisms in patients with T1DM and controls Polymorphism TaqI
Model
Genotype/Allele
Controls n (%)
T1DM n (%)
Codominant 105 8 0
(92.9) (7.1) (0.0)
66 15 0
(81.5) (18.5) (0.0)
TT+Tt tt
113 0
(100.0) (0.0)
81 0
(100.0) (0.0)
TT Tt+tt
105 8
(92.9) (7.1)
66 15
(81.5) (18.5)
2.98 (1.19–7.42) 0.33 (0.13–0.83) NA NA
Recessive
NA NA 0.015
Trend Codominant Dominant Recessive
2.98 (1.20–7.42) 0.33 (0.13–0.83) 0.015 NA 0.015 0.019
TaqI allele T t
218 8
(96.5) (3.5)
147 15
(90.7) (9.3)
BB Bb bb
1 4 108
(0.9) (3.5) (95.6)
0 13 68
(0.0) (16.0) (84.0)
BB+Bb bb
5 108
(4.4) (95.6)
13 68
(16.0) (84.0)
BB Bb+bb
1 112
(0.9) (99.1)
0 81
(0.0) (100.0)
Codominant
0.33 (0.13–0.83) NA 2.98 (1.20–7.42) 2.78 (1.15–6.72) 0.36 (0.14–0.87)
0.004
Dominant
NA 0.19 (0.06–0.61) 4.13 (1.41–12.10) 0.006
Recessive
0.24 (0.08–0.71) 4.13 (1.41–12.10) 0.040
Trend Codominant Dominant Recessive
NA NA 0.019 0.006 0.395 0.016
BsmI allele B b ApaI
OR (95%CI)
0.015 TT Tt tt
Dominant
BsmI
P-value
6 220
(2.7) (97.3)
13 149
(8.0) (92.0)
AA Aa aa
9 34 70
(8.0) (30.1) (61.9)
5 32 44
(6.2) (39.5) (54.3)
AA+Aa aa
43 70
(38.1) (61.9)
37 44
(45.7) (54.3)
AA Aa+aa
9 104
(8.0) (92.0)
5 76
(6.2) (93.8)
Codominant
0.19 (0.06–0.61) 0.24 (0.08–0.71) NA 0.31 (0.11–0.84) 3.20 (1.19–8.60)
0.385
Dominant
1.32 (0.42–4.08) 0.66 (0.36–1.20) 1.37 (0.77–2.44) 0.287 0.73 (0.41–1.30) 1.37 (0.77–2.44) 0.634
Recessive Trend Codominant Dominant Recessive
1.32 (0.42–4.08) 0.76 (0.24–2.36) 0.523 0.287 0.634 0.508
ApaI allele A a
52 174
(23.0) (77.0)
42 120
(25.9) (74.1)
0.33 (0.36–1.20) 0.73 (0.41–1.30) 1.32 (0.42–4.08) 0.85 (0.53–1.36) 1.17 (0.73–1.87)
NA, not applicable; T1DM, type 1 diabetes mellitus; VDR, vitamin D receptor.
performed in our study. Therefore, further study involving a much larger number of patients and controls including the VDR polymorphism Fok1 is needed to confirm the present results. Conclusion
The present case–control study indicates an association between two VDR SNP and T1DM among Korean subjects with a low
incidence of T1DM. We suggest that T and b TaqI and BsmI alleles are protective against T1DM in Korean individuals. Further work is required to explore genetic and/or environmental factors that potentially enhance or inhibit this role of VDR polymorphisms in T1DM, such as the contribution of related loci or UVR exposure, the immune system, and other environmental determinants of vitamin D status. © 2015 Japan Pediatric Society
874 C-K Cheon et al. Acknowledgments We thank the patients and their families for participating in this study. This study was supported by a 1 year research grant from Pusan National University. The authors declare no conflicts of interest.
20
References
21
1 Bener A, Alsaied A, Al-Ali M. High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children. Acta Diabetol. 2009; 46: 183–9. 2 Bach JF. Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr. Rev. 1994; 15: 516–42. 3 Mathieu C, Badenhoop K. Vitamin D and type 1 diabetes mellitus: State of the art. Trends Endocrinol. Metab. 2005; 16: 261–6. 4 Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene 2004; 338: 143–56. 5 Panierakis C, Goulielmos G, Mamoulakis D, Petraki E, Papavasiliou E, Galanakis E. Vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Crete, Greece. Clin. Immunol. 2009; 133: 276–81. 6 Boraska V, Skrabić V, Zeggini E et al. Family-based analysis of vitamin D receptor gene polymorphisms and type 1 diabetes in the population of South Croatia. J. Hum. Genet. 2008; 53: 210–4. 7 Lemos MC, Fagulha A, Coutinho E et al. Lack of association of vitamin D receptor gene polymorphisms with susceptibility to type 1 diabetes mellitus in the Portuguese population. Hum. Immunol. 2008; 69: 134–8. 8 Turpeinen H, Hermann R, Vaara S et al. Vitamin D receptor polymorphisms: No association with type 1 diabetes in the Finnish population. Eur. J. Endocrinol. 2003; 149: 591–6. 9 Arai H, Miyamoto K, Taketani Y et al. A vitamin D receptor gene polymorphism in the translation initiation codon: Effect on protein activity and relation to bone mineral density in Japanese women. J. Bone Miner. Res. 1997; 12: 915–21. 10 Morrison NA, Qi JC, Tokita A et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994; 367: 284–7. 11 Tokita A, Matsumoto H, Morrison NA et al. Vitamin D receptor alleles, bone mineral density and turnover in premenopausal Japanese women. J. Bone Miner. Res. 1996; 11: 1003–9. 12 Carling T, Kindmark A, Hellman P et al. Vitamin D receptor genotypes in primary hyperparathyroidism. Nat. Med. 1995; 1: 1309–11. 13 Carling T, Akerstrom G, Rastad J, Westin G. Vitamin D receptor (VDR) and parathyroid hormone mRNA levels correspond to polymorphic VDR alleles in human parathyroid tumors. J. Clin. Endocrinol. Metab. 1998; 83: 2255–9. 14 Ban Y, Ban Y, Taniyama M, Katagiri T. Vitamin D receptor initiation codon polymorphism in Japanese patients with Graves’ disease. Thyroid 2000; 10: 375–80. 15 Ban Y, Taniyama M, Ban Y. Vitamin D receptor gene polymorphism is associated with Graves’ disease in the Japanese population. J. Clin. Endocrinol. Metab. 2000; 85: 4639–43. 16 Ban Y, Taniyama M, Ban Y. Vitamin D receptor gene polymorphisms in Hashimoto’s thyroiditis. Thyroid 2001; 11: 607–8. 17 Fukazawa T, Yabe I, Kikuchi S et al. Association of vitamin D receptor gene polymorphism with multiple sclerosis in Japanese. J. Neurol. Sci. 1999; 166: 47–52. 18 Ogunkolade BW, Boucher BJ, Prahl JM et al. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D
© 2015 Japan Pediatric Society
19
22 23
24 25 26
27 28 29 30
31 32 33 34
35
36 37
status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 2002; 51: 2294–300. Chang TJ, Lei HH, Yeh JI et al. Vitamin D receptor gene polymorphisms influence susceptibility to type 1 diabetes mellitus in the Taiwanese population. Clin. Endocrinol. (Oxf) 2000; 52: 575–80. Györffy B, Vásárhelyi B, Krikovszky D et al. Gender-specific association of vitamin D receptor polymorphism combinations with type 1 diabetes mellitus. Eur. J. Endocrinol. 2002; 147: 803–8. Lowe LC, Guy M, Mansi JL et al. Plasma 25-hydroxy vitamin D concentrations, vitamin D receptor genotype and breast cancer risk in a UK Caucasian population. Eur. J. Cancer 2005; 41: 1164–9. Skrabić V, Zemunik T, Situm M, Terzić J. Vitamin D receptor polymorphism and susceptibility to type 1 diabetes in the Dalmatian population. Diabetes Res. Clin. Pract. 2003; 59: 31–5. Mimbacas A, Trujillo J, Gascue C, Javiel G, Cardoso H. Prevalence of vitamin D receptor gene polymorphism in a Uruguayan population and its relation to type 1 diabetes mellitus. Genet. Mol. Res. 2007; 6: 534–42. McDermott MF, Ramachandran A, Ogunkolade BW et al. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians. Diabetologia 1997; 40: 971–5. Pani MA, Knapp M, Donner H et al. Vitamin D receptor allele combinations influence genetic susceptibility to type 1 diabetes in Germans. Diabetes 2000; 49: 504–7. Zemunik T, Skrabic V, Boraska V et al. FokI polymorphism, vitamin D receptor, and interleukin-1 receptor haplotypes are associated with type 1 diabetes in the Dalmatian population. J. Mol. Diagn. 2005; 7: 600–4. Koeleman BP, Valdigem G, Eerligh P, Giphart MJ, Roep BO. Seasonality of birth in patients with type 1 diabetes. Lancet 2002; 359: 1246–7. Guja C, Marshall S, Welsh K et al. The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population. J. Cell. Mol. Med. 2002; 6: 75–81. Mohammadnejad Z, Ghanbari M, Ganjali R et al. Association between vitamin D receptor gene polymorphisms and type 1 diabetes mellitus in Iranian population. Mol. Biol. Rep. 2012; 39: 831–7. Ban Y, Taniyama M, Yanagawa T et al. Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population. BMC Med. Genet. 2001; 2: 7. Bianco MG, Minicucci L, Calevo MG, Lorini R. Vitamin D receptor polymorphisms: Are they really associated with type 1 diabetes? Eur. J. Endocrinol. 2004; 151: 641–2. Zhang J, Li W, Liu J et al. Polymorphisms in the vitamin D receptor gene and type 1 diabetes mellitus risk: An update by metaanalysis. Mol. Cell. Endocrinol. 2012; 355: 135–42. Shimada A, Kanazawa Y, Motohashi Y et al. Evidence for association between vitamin D receptor BsmI polymorphism and type 1 diabetes in Japanese. J. Autoimmun. 2008; 30: 207–11. Penna G, Adorini L. 1 Alpha, 25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J. Immunol. 2000; 164: 2405–11. Ponsonby AL, Pezic A, Ellis J et al. Variation in associations between allelic variants of the vitamin D receptor gene and onset of type 1 diabetes mellitus by ambient winter ultraviolet radiation levels: A meta-regression analysis. Am. J. Epidemiol. 2008; 168: 358–65. DIAMOND Project Group. Incidence and trends of childhood Type 1 diabetes worldwide 1990-1999. Diabet. Med. 2006; 23: 857–66. Audí L, Martí G, Esteban C et al. VDR gene polymorphism at exon 2 start codon (FokI) may have influenced type 1 diabetes mellitus susceptibility in two Spanish populations. Diabet. Med. 2004; 21: 39394.
doi: 10.1111/ped.12634
Original Article
Vitamin D receptor gene polymorphisms and type 1 diabetes mellitus in a Korean population Chong-Kun Cheon,1* Hyo-Kyoung Nam,2* Kee-Hyoung Lee,2 Su Yung Kim,1 Ji Sun Song3 and Choongrak Kim4 Department of Pediatrics, Pediatric Endocrinology, Pusan National University Children’s Hospital, Pusan National University School of Medicine, Yangsan, 2Department of Pediatrics, College of Medicine, Korea University, Seoul, 3Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Gyeongnam and 4 Department of Statistics, Pusan National University, Pusan, South Korea 1
Abstract
Background: Vitamin D receptor (VDR) has been suggested to play a role in the pathogenesis of type 1 diabetes mellitus (T1DM). There has been no case–control study examining the association between VDR polymorphisms and T1DM among Korean subjects with a low incidence of T1DM. Methods: Eighty-one T1DM patients and 113 unrelated healthy controls with no history of DM or other autoimmune diseases were investigated at either Pusan National University Children’s Hospital or Korea University Anam Hospital between March 2009 and September 2013. Polymerase chain reaction–restriction fragment length polymorphism was utilized to genotype single nucleotide substitutions at TaqI, BsmI, and ApaI alleles. Results: All frequencies in T1DM and control subjects were in Hardy–Weinberg equilibrium, although ApaI in controls and TaqI in T1DM showed relatively weak equilibrium. TaqI and BsmI differences were significant (P = 0.045 and P = 0.012, respectively) after applying Bonferroni correction. The TT genotype carrier frequency among controls was higher than among the T1DM patients (P = 0.015; OR, 2.98; 95%CI: 1.19–7.42). T allele frequency was higher among controls than T1DM patients (P = 0.019; OR, 2.78; 95%CI: 1.15–6.72). The frequency of bb genotype carriers among controls was higher than among T1DM patients (P = 0.004; OR, 4.13; 95%CI: 1.4–12.10). The frequency of the b allele among controls was higher than that among T1DM patients (P = 0.016; OR, 3.20; 95%CI: 1.19–8.60). Conclusions: T and b TaqI and BsmI alleles are protective against T1DM in Korean subjects.
Key words polymorphism, type 1 diabetes mellitus, vitamin D receptor. Type 1 (insulin-dependent) diabetes mellitus (T1DM) is an autoimmune disease that results in the destruction of pancreatic beta cells due to interactions between susceptibility genes and environmental exposure.1 Most T1DM in humans is considered to be a T-cellmediated autoimmune disease.2 There is evidence for worldwide epidemics of both vitamin D deficiency and T1DM,1 and vitamin D has been identified as a contributing factor to DM.3 Vitamin D, among its many roles, acts as a modulator of the immune system by promoting monocyte differentiation and inhibiting lymphocyte proliferation and cytokine secretion.4 Four single-nucleotide polymorphisms (SNP) in the vitamin D receptor (VDR) gene have been studied: FokI F>f (rs10735810, NM_000376.2: c.2T>C), BsmI B>b (rs1544410, NM_000376.2: c.1024+283G>A), ApaI A>a (rs7975232, NM_000376.2: c.1025-49G>T), and TaqI T>t (rs731236, NM_000376.2: c.1056T>C).5 Allele F of the FokI Correspondence: Chong-Kun Cheon, MD PhD, Department of Pediatrics, Pediatric Genetics and Metabolism, Pusan National University Children’s Hospital, Pusan National University School of Medicine, Geumo-ro, Yangsan-si, Gyeongnam 602-739, South Korea. Email: [email protected] * Chong-Kun Cheon and Hyo-Kyoung Nam are co-first authors. [Correction added on 28 July 2015, after first online publication: The above statement has been added to indicate co-first authorship.] Received 26 July 2014; revised 27 January 2015; accepted 19 February 2015. © 2015 Japan Pediatric Society
SNP creates an alternative ATG initiation codon in exon 2, leading to a VDR protein that is three amino acids longer than the wild type. ApaI, BsmI and TaqI polymorphisms are located near the 3′ end of the VDR gene; BsmI and ApaI SNP are both located in intron 8, and TaqI is a silent SNP in exon 9.5 To date, a large number of studies have suggested that VDR is involved in the pathogenesis of T1DM.6 Several studies have suggested an association between one or more of the aforementioned SNP and T1DM, but others have failed to confirm these findings.7,8 This study therefore assessed the contribution of VDR polymorphisms to TIDM susceptibility in a genetically homogenous population in Korea.
Methods Patients
The study group consisted of 81 Korean patients with T1DM (35 boys and 46 girls; mean age, 10.28 ± 3.73 years old). An additional 113 unrelated healthy control group (53 boys and 60 girls; mean age, 9.98 ± 3.56 years old) with no history of DM or other autoimmune diseases was recruited from local schools. Most samples were taken from patients diagnosed and treated at either Pusan National University Children’s Hospital or Korea University Anam Hospital between March 2009 and September 2013. T1DM was diagnosed under 15 years of age, according to the World Health
VDR polymorphisms and T1DM in Korea 871 Organization criteria. All DM patients had typical histories of ketoacidosis and diabetes-associated antibodies and required continuous insulin treatment upon diagnosis. The study was approved by the Ethics Committee of the Yangsan National University Hospital and informed consent was obtained from the participating subjects and/or parents. DNA extraction
Genomic DNA was extracted from whole blood samples using the QIAamp DNA blood kit (Qiagen) according to the manufacturer’s protocol. DNA was stored at 20°C until use. VDR genotyping
Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to genotype single-nucleotide substitutions at TaqI (rs731236), BsmI (rs1544410) and ApaI (rs7975232) polymorphisms in the VDR gene (Fig. 1). Primers were designed based on GenBank cDNA sequences (Table 1). All amplifications were performed using 40 ng DNA, 10 pmol of each primer, polymerase, and PCR master mix (Promega, Madison, WI, USA) in a total volume of 25 μL. Reactions were cycled 29 times for TaqI and 35 times for ApaI, and BsmI, with each cycle consisting of 60 s at 94°C (denaturation), 60 s at 65°C (TaqI), 68°C (BsmI), or 54°C (ApaI), and 1 min at 72°C (annealing and extension), followed by a final extension at 72°C for 5 min. PCR products were run on 2% agarose gels and visualized on ethidium bromide staining. A 100 bp ladder (Bioneer, Alameda, CA, USA) was used as a marker. The amplified products were digested using the restriction enzymes ApaI and TaqI (Fermentas; St Leon-Rot, Germany), according to the manufacturer’s protocol. Briefly, 5 μL of each associated PCR product was mixed with 0.5 μL of each restriction enzyme, 1 μL of buffer, and 3.5 μL H2O, and then
incubated for 3 h at 65°C for TaqI and 16 h at 37°C for ApaI and BsmI. After incubation, the restriction enzymes were inactivated at 65°C for 20 min (BsmI and ApaI; TaqI is not inactivated). Digested samples were separated on 2% agarose gels and visualized with ethidium bromide (0.5 mg/mL). Statistical analysis
Statistical analysis was performed using SPSS for Windows (version 20.0, SPSS Inc., Chicago, IL, USA). We estimated mode of inheritance using Fisher’s exact test for the codominant, dominant, recessive and VDR genotype, and Cochran–Armitage test for the trend model in patients and healthy controls. OR and 95%CI were calculated for each allele, genotype, and haplotype. Two-sided P < 0.05 indicated statistical significance. Hardy–Weinberg equilibrium was assessed using the chi-squared goodness-of-fit test to compare the observed and allele-based expected genotype frequencies.
Results All frequencies in T1DM and control subjects were in Hardy–Weinberg equilibrium (P > 0.05), although ApaI in controls and TaqI in T1DM showed relatively weak equilibrium. In all three polymorphisms tested, the distribution of the two VDR genotype frequencies differed significantly between T1DM patients and controls. We first compared the VDR-TaqI polymorphism between T1DM patients overall and healthy subjects. As shown in Table 2, the frequency of TT genotype carriers among controls was significantly higher than that in T1DM patients (P = 0.015; OR, 2.98; 95%CI: 1.19–7.42). A significantly higher T allele frequency was seen among controls, compared with T1DM patients (P = 0.019; OR, 2.78; 95%CI: 1.15–6.72). The frequency of bb genotype carriers among controls was significantly higher than among T1DM patients (P = 0.004; OR, 4.13; Fig. 1 Genotyping of the vitamin D receptor (VDR) gene. (a) ApaI polymorphism: lane 1, markers; lane 2, uncut product (421 bp); lane 3, genotype aa homozygous (233 and 188 bp); lane 4, genotype Aa heterozygous (421, 233 and 188 bp); lane 5, genotype AA homozygous (421 bp). (b) TaqI polymorphism: lane 1, markers; lane 2, uncut product (716 bp); lane 3, genotype TT homozygous (514 and 202 bp); lane 4, genotype Tt heterozygous (514, 237 and 169 bp). (c) BsmI polymorphism: lane 1, markers; lane 2, uncut product (801 bp); lane 3, genotype bb homozygous (500 and 301 bp); lane 4, genotype Bb heterozygous (801, 500 and 301 bp); lane 5, genotype BB homozygous (801 bp).
© 2015 Japan Pediatric Society
872 C-K Cheon et al. Table 1 PCR primers used to amplify fragments harboring VDR SNP SNP
PCR primer (5’-3’)
Fragment size (bp)
ApaI
Forward GAC GCT GAG GGA TGG Reverse GTC GGC TAG CTT CTG GAT Forward AAC TTG CAT GAG GAG GAG CAT GTC Reverse GGA GAG GAG CCT GTG TCC CAT TTG Forward GGG ACG CTG AGG GAT GGA CAG AGC Reverse GGA AAG GGG TTA GGT TGG ACA GGA
421
BsmI
TaqI
801
716
PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; VDR, vitamin D receptor.
95%CI: 1.41–12.10), and the frequency of the b allele in controls was significantly higher than among T1DM patients (P = 0.016; OR, 3.20; 95%CI: 1.19–8.60). The ApaI polymorphism did not appear to be significantly associated with overall T1DM risk. Multiple comparison tests for adjusting P-value were performed. TaqI and BsmI differences were significant (P = 0.045 and P = 0.012, respectively) after applying the Bonferroni correction. Therefore, T and b TaqI and BsmI alleles are protective against T1DM in Korean individuals.
Discussion Several studies have focused on associations between candidate genes and development of T1DM. To clarify the contribution of the VDR polymorphisms TaqI, BsmI, and ApaI to T1DM genetic susceptibility in Korean patients, 81 patients with T1DM and 113 unrelated healthy controls with no history of DM or other autoimmune diseases were tested. This is the first study on the association between VDR SNP and T1DM among Korean subjects. We demonstrate here that BsmI and TaqI polymorphisms were more commonly associated with T1DM in Korean individuals. The t allele for the TaqI polymorphism and the B allele for the BsmI polymorphism were associated with increased risk of T1DM. The VDR locus has been studied extensively for association(s) with osteoporosis,9–11 primary hyperparathyroidism,12,13 and autoimmune diseases such as Graves’ disease,14,15 Hashimoto’s thyroiditis,16 and multiple sclerosis.17 Polymorphisms in VDR are reported to be associated with insulin secretory capacity in humans.18 Interestingly, in an epidemiological study from several European countries, vitamin D supplementation in early childhood seems to be associated with a reduction in the incidence of T1DM, indicating that the immune-regulatory effects of vitamin D through VDR may modulate the disease course of T1DM.3 Four SNP in VDR (FokI, BsmI, ApaI, and TaqI) have been investigated in previous studies.5 TaqI is a silent SNP in exon 9, while both ApaI and BsmI are located in the intronic region between exons 8 and 9 and do not affect VDR protein structure.5,19,20 Some studies have reported linkage between BsmI and a variable length polymorphism in the 3′ untranslated region, known as poly (A), which may influence VDR mRNA stability.4,21 This opens up the possibility that the observed © 2015 Japan Pediatric Society
linkage points either toward another closely linked T1DM susceptibility gene or a second functional nucleotide change within the VDR gene itself.22 Associations between VDR polymorphisms and T1DM have been seen in other countries including Taiwan,19 Hungary,20 Uruguay,23 India,24 Germany,25 Croatia,23,26 Holland,27 Romania,28 Iran,29 and Japan.30 Other studies have not confirmed these findings and no associations were found in Portuguese7 or Italian populations,31 indicating that differing genetic background may be causative of these differences. A recent meta-analysis suggested that the BsmI polymorphism is associated with increased risk of T1DM, especially in Asian subjects.32 Shimada et al. demonstrated that peripheral blood mononuclear cells from T1DM patients with the BsmI BB genotype produced higher levels of interferon-γ, suggesting that this VDR polymorphism could contribute to the T-helper (Th)1 response.33 Given that the active form of vitamin D activates the expression of transforming growth factor-beta 1 and interleukin-4 cytokines, therefore inhibiting Th1-type responses and inducing regulatory T cells, it was suggested that it can also regulate differentiation and maturation of dendritic cells, which is critical in the induction of T-cell-mediated immune responses.34 Discrepancies in VDR polymorphisms associated with T1DM in different populations may be due to differences in ethnic background, diverse evolutionary lineages, and interactions with other genetic and/or environmental factors involved in the pathogenesis of T1DM.25,29 In the present study, BsmI bb homozygotes had a smaller risk of T1DM compared with B allele (BB + Bb) carriers, while TaqI TT homozygotes had a decreased risk of T1DM compared with t allele (tt+Tt) carriers. This suggests that BsmI and TaqI polymorphisms may contribute to T1DM pathogenesis, with the bb and TT genotypes protective against T1DM. This finding was recently attributed to potential interference in the VDR genotype by environmental factors, such as ultraviolet radiation (UVR).35 The association between the B allele and T1DM increased as regional winter UVR increased, and most Asian countries have a high winter UVR level,35 which may explain individual differences in susceptibility to T1DM. To the best of our knowledge, there has been no case–control study examining the association between VDR polymorphisms and T1DM in Korea. Interestingly, the ageadjusted incidence of TIDM in South Korea is low (1.1 per 100 000/year) compared with other countries.36 The lower incidence of TIDM may be linked to T and b TaqI and BsmI alleles, which are protective against T1DM in Korean individuals. Taken together, the present study is in agreement with findings from other populations, such as Indian Asian and Spanish subjects, for the BsmI polymorphism,24,37 and Bangladeshi Asian subjects for the TaqI polymorphism.18 The present study, however, has several limitations. First, the study was very small and thus it is difficult to reach a definitive conclusion on biological relevance. Susceptibility alleles are likely to individually contribute very modestly to the overall risk, and the magnitude of the effects is usually observed only in samples of hundreds of patients and controls.32 For these reason it is extraordinary that several OR in this study seem so high (or low, in the case of the alternative allele). It may be due to sample size. Second, the analysis of VDR FokI genotype was not
VDR polymorphisms and T1DM in Korea 873 Table 2 VDR gene polymorphisms in patients with T1DM and controls Polymorphism TaqI
Model
Genotype/Allele
Controls n (%)
T1DM n (%)
Codominant 105 8 0
(92.9) (7.1) (0.0)
66 15 0
(81.5) (18.5) (0.0)
TT+Tt tt
113 0
(100.0) (0.0)
81 0
(100.0) (0.0)
TT Tt+tt
105 8
(92.9) (7.1)
66 15
(81.5) (18.5)
2.98 (1.19–7.42) 0.33 (0.13–0.83) NA NA
Recessive
NA NA 0.015
Trend Codominant Dominant Recessive
2.98 (1.20–7.42) 0.33 (0.13–0.83) 0.015 NA 0.015 0.019
TaqI allele T t
218 8
(96.5) (3.5)
147 15
(90.7) (9.3)
BB Bb bb
1 4 108
(0.9) (3.5) (95.6)
0 13 68
(0.0) (16.0) (84.0)
BB+Bb bb
5 108
(4.4) (95.6)
13 68
(16.0) (84.0)
BB Bb+bb
1 112
(0.9) (99.1)
0 81
(0.0) (100.0)
Codominant
0.33 (0.13–0.83) NA 2.98 (1.20–7.42) 2.78 (1.15–6.72) 0.36 (0.14–0.87)
0.004
Dominant
NA 0.19 (0.06–0.61) 4.13 (1.41–12.10) 0.006
Recessive
0.24 (0.08–0.71) 4.13 (1.41–12.10) 0.040
Trend Codominant Dominant Recessive
NA NA 0.019 0.006 0.395 0.016
BsmI allele B b ApaI
OR (95%CI)
0.015 TT Tt tt
Dominant
BsmI
P-value
6 220
(2.7) (97.3)
13 149
(8.0) (92.0)
AA Aa aa
9 34 70
(8.0) (30.1) (61.9)
5 32 44
(6.2) (39.5) (54.3)
AA+Aa aa
43 70
(38.1) (61.9)
37 44
(45.7) (54.3)
AA Aa+aa
9 104
(8.0) (92.0)
5 76
(6.2) (93.8)
Codominant
0.19 (0.06–0.61) 0.24 (0.08–0.71) NA 0.31 (0.11–0.84) 3.20 (1.19–8.60)
0.385
Dominant
1.32 (0.42–4.08) 0.66 (0.36–1.20) 1.37 (0.77–2.44) 0.287 0.73 (0.41–1.30) 1.37 (0.77–2.44) 0.634
Recessive Trend Codominant Dominant Recessive
1.32 (0.42–4.08) 0.76 (0.24–2.36) 0.523 0.287 0.634 0.508
ApaI allele A a
52 174
(23.0) (77.0)
42 120
(25.9) (74.1)
0.33 (0.36–1.20) 0.73 (0.41–1.30) 1.32 (0.42–4.08) 0.85 (0.53–1.36) 1.17 (0.73–1.87)
NA, not applicable; T1DM, type 1 diabetes mellitus; VDR, vitamin D receptor.
performed in our study. Therefore, further study involving a much larger number of patients and controls including the VDR polymorphism Fok1 is needed to confirm the present results. Conclusion
The present case–control study indicates an association between two VDR SNP and T1DM among Korean subjects with a low
incidence of T1DM. We suggest that T and b TaqI and BsmI alleles are protective against T1DM in Korean individuals. Further work is required to explore genetic and/or environmental factors that potentially enhance or inhibit this role of VDR polymorphisms in T1DM, such as the contribution of related loci or UVR exposure, the immune system, and other environmental determinants of vitamin D status. © 2015 Japan Pediatric Society
874 C-K Cheon et al. Acknowledgments We thank the patients and their families for participating in this study. This study was supported by a 1 year research grant from Pusan National University. The authors declare no conflicts of interest.
20
References
21
1 Bener A, Alsaied A, Al-Ali M. High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children. Acta Diabetol. 2009; 46: 183–9. 2 Bach JF. Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr. Rev. 1994; 15: 516–42. 3 Mathieu C, Badenhoop K. Vitamin D and type 1 diabetes mellitus: State of the art. Trends Endocrinol. Metab. 2005; 16: 261–6. 4 Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene 2004; 338: 143–56. 5 Panierakis C, Goulielmos G, Mamoulakis D, Petraki E, Papavasiliou E, Galanakis E. Vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Crete, Greece. Clin. Immunol. 2009; 133: 276–81. 6 Boraska V, Skrabić V, Zeggini E et al. Family-based analysis of vitamin D receptor gene polymorphisms and type 1 diabetes in the population of South Croatia. J. Hum. Genet. 2008; 53: 210–4. 7 Lemos MC, Fagulha A, Coutinho E et al. Lack of association of vitamin D receptor gene polymorphisms with susceptibility to type 1 diabetes mellitus in the Portuguese population. Hum. Immunol. 2008; 69: 134–8. 8 Turpeinen H, Hermann R, Vaara S et al. Vitamin D receptor polymorphisms: No association with type 1 diabetes in the Finnish population. Eur. J. Endocrinol. 2003; 149: 591–6. 9 Arai H, Miyamoto K, Taketani Y et al. A vitamin D receptor gene polymorphism in the translation initiation codon: Effect on protein activity and relation to bone mineral density in Japanese women. J. Bone Miner. Res. 1997; 12: 915–21. 10 Morrison NA, Qi JC, Tokita A et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994; 367: 284–7. 11 Tokita A, Matsumoto H, Morrison NA et al. Vitamin D receptor alleles, bone mineral density and turnover in premenopausal Japanese women. J. Bone Miner. Res. 1996; 11: 1003–9. 12 Carling T, Kindmark A, Hellman P et al. Vitamin D receptor genotypes in primary hyperparathyroidism. Nat. Med. 1995; 1: 1309–11. 13 Carling T, Akerstrom G, Rastad J, Westin G. Vitamin D receptor (VDR) and parathyroid hormone mRNA levels correspond to polymorphic VDR alleles in human parathyroid tumors. J. Clin. Endocrinol. Metab. 1998; 83: 2255–9. 14 Ban Y, Ban Y, Taniyama M, Katagiri T. Vitamin D receptor initiation codon polymorphism in Japanese patients with Graves’ disease. Thyroid 2000; 10: 375–80. 15 Ban Y, Taniyama M, Ban Y. Vitamin D receptor gene polymorphism is associated with Graves’ disease in the Japanese population. J. Clin. Endocrinol. Metab. 2000; 85: 4639–43. 16 Ban Y, Taniyama M, Ban Y. Vitamin D receptor gene polymorphisms in Hashimoto’s thyroiditis. Thyroid 2001; 11: 607–8. 17 Fukazawa T, Yabe I, Kikuchi S et al. Association of vitamin D receptor gene polymorphism with multiple sclerosis in Japanese. J. Neurol. Sci. 1999; 166: 47–52. 18 Ogunkolade BW, Boucher BJ, Prahl JM et al. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D
© 2015 Japan Pediatric Society
19
22 23
24 25 26
27 28 29 30
31 32 33 34
35
36 37
status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 2002; 51: 2294–300. Chang TJ, Lei HH, Yeh JI et al. Vitamin D receptor gene polymorphisms influence susceptibility to type 1 diabetes mellitus in the Taiwanese population. Clin. Endocrinol. (Oxf) 2000; 52: 575–80. Györffy B, Vásárhelyi B, Krikovszky D et al. Gender-specific association of vitamin D receptor polymorphism combinations with type 1 diabetes mellitus. Eur. J. Endocrinol. 2002; 147: 803–8. Lowe LC, Guy M, Mansi JL et al. Plasma 25-hydroxy vitamin D concentrations, vitamin D receptor genotype and breast cancer risk in a UK Caucasian population. Eur. J. Cancer 2005; 41: 1164–9. Skrabić V, Zemunik T, Situm M, Terzić J. Vitamin D receptor polymorphism and susceptibility to type 1 diabetes in the Dalmatian population. Diabetes Res. Clin. Pract. 2003; 59: 31–5. Mimbacas A, Trujillo J, Gascue C, Javiel G, Cardoso H. Prevalence of vitamin D receptor gene polymorphism in a Uruguayan population and its relation to type 1 diabetes mellitus. Genet. Mol. Res. 2007; 6: 534–42. McDermott MF, Ramachandran A, Ogunkolade BW et al. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians. Diabetologia 1997; 40: 971–5. Pani MA, Knapp M, Donner H et al. Vitamin D receptor allele combinations influence genetic susceptibility to type 1 diabetes in Germans. Diabetes 2000; 49: 504–7. Zemunik T, Skrabic V, Boraska V et al. FokI polymorphism, vitamin D receptor, and interleukin-1 receptor haplotypes are associated with type 1 diabetes in the Dalmatian population. J. Mol. Diagn. 2005; 7: 600–4. Koeleman BP, Valdigem G, Eerligh P, Giphart MJ, Roep BO. Seasonality of birth in patients with type 1 diabetes. Lancet 2002; 359: 1246–7. Guja C, Marshall S, Welsh K et al. The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population. J. Cell. Mol. Med. 2002; 6: 75–81. Mohammadnejad Z, Ghanbari M, Ganjali R et al. Association between vitamin D receptor gene polymorphisms and type 1 diabetes mellitus in Iranian population. Mol. Biol. Rep. 2012; 39: 831–7. Ban Y, Taniyama M, Yanagawa T et al. Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population. BMC Med. Genet. 2001; 2: 7. Bianco MG, Minicucci L, Calevo MG, Lorini R. Vitamin D receptor polymorphisms: Are they really associated with type 1 diabetes? Eur. J. Endocrinol. 2004; 151: 641–2. Zhang J, Li W, Liu J et al. Polymorphisms in the vitamin D receptor gene and type 1 diabetes mellitus risk: An update by metaanalysis. Mol. Cell. Endocrinol. 2012; 355: 135–42. Shimada A, Kanazawa Y, Motohashi Y et al. Evidence for association between vitamin D receptor BsmI polymorphism and type 1 diabetes in Japanese. J. Autoimmun. 2008; 30: 207–11. Penna G, Adorini L. 1 Alpha, 25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J. Immunol. 2000; 164: 2405–11. Ponsonby AL, Pezic A, Ellis J et al. Variation in associations between allelic variants of the vitamin D receptor gene and onset of type 1 diabetes mellitus by ambient winter ultraviolet radiation levels: A meta-regression analysis. Am. J. Epidemiol. 2008; 168: 358–65. DIAMOND Project Group. Incidence and trends of childhood Type 1 diabetes worldwide 1990-1999. Diabet. Med. 2006; 23: 857–66. Audí L, Martí G, Esteban C et al. VDR gene polymorphism at exon 2 start codon (FokI) may have influenced type 1 diabetes mellitus susceptibility in two Spanish populations. Diabet. Med. 2004; 21: 39394.
