Pediatric Diabetes 2014: 15: 416–421 doi: 10.1111/pedi.12105 All rights reserved

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pediatric Diabetes

Original Article

No association between type 1 diabetes and genetic variation in vitamin D metabolism genes: a Danish study Thorsen SU, Mortensen HB, Carstensen B, Fenger M, Thuesen BH, Husemoen L, Bergholdt R, Brorsson C, Pociot F, Linneberg A, Svensson J. No association between type 1 diabetes and genetic variation in vitamin D metabolism genes: a Danish study. Pediatric Diabetes 2014: 15: 416–421. Background: Vitamin D, certain single nucleotide polymorphisms (SNPs) in the vitamin D-receptor (VDR) gene and vitamin D metabolism genes have been associated with type 1 diabetes (T1D). Objective: We wanted to examine if the most widely studied SNPs in genes important for production, transport, and action of vitamin D were associated with T1D or to circulating levels of vitamin D 25-hydroxyvitamin D [25(OH)D] in a juvenile Danish population. Methods: We genotyped eight SNPs in five vitamin D metabolism genes in 1467 trios. 25(OH)D status were analyzed in 1803 children (907 patients and 896 siblings). Results: We did not demonstrate association with T1D for SNPs in the following genes: CYP27B1, VDR, GC, CYP2R1, DHCR7, and CYP24A1. Though, variants in the GC gene were significantly associated with 25(OH)D levels in the joint model. Conclusion: Some of the most examined SNPs in vitamin D metabolism genes were not confirmed to be associated with T1D, though 25(OH) levels were associated with variants in the GC gene.

Steffen U Thorsena , Henrik B Mortensena , Bendix Carstensenb , Mogens Fengerc , Betina H Thuesend , Lotte Husemoend , Regine Bergholdte , Caroline Brorssonf , Flemming Pociotf,g , Allan Linnebergd and Jannet Svenssona a Herlev

University Hospital, Herlev, Denmark; b Steno Diabetes Center, Gentofte, Denmark; c Department of Clinical Biochemistry and Molecular Biology, Hvidovre Hospital, Hvidovre, Denmark; d Research Centre for Prevention and Health, Glostrup University Hospital, Glostrup, Denmark; e Hagedorn Research Institute, Gentofte, Denmark; f Glostrup Research Institute, Glostrup University Hospital, Glostrup, Denmark; and g Department of Biomedical Science, University of Copenhagen, Copenhagen, Denmark Key words: cholecalciferol – diabetes mellitus, type 1 – polymorphism, single nucleotide – receptors – vitamin D Corresponding author: Steffen Ullitz Thorsen, MD, Herlev University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark. Tel: +45-22-28-8995; fax: +45-38-68-5012; e-mail: [email protected] Submitted 5 July 2013. Accepted for publication 1 November 2013

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T1D and SNPs in vitamin D metabolism genes Type 1 diabetes (T1D) is looked upon as a multi-factorial disease, where both polygenic and environmental factors are contributors. In search of environmental factors, a north-south gradient and seasonal variation in incidence have linked ultraviolet radiation (UVR) and hence vitamin D (25-hydroxyvitamin D (25(OH)D)) to the pathogenesis of T1D (1). So far the anti-autoimmune properties of vitamin D have mostly been studied in animal models (2, 3), which again have been supported by epidemiologic studies that have shown a decreased risk of T1D in children given vitamin D supplementation in early childhood (4–6), and lower levels of 25(OH)D in newly diagnosed patients compared to healthy controls (7–9). None of these studies included information on the genes involved in vitamin D metabolism. T1D genetic determinants of utmost importance are found within the human leukocyte antigen (HLA) class II locus though other non-HLA gene regions have been identified – among the vitamin D metabolism genes, the vitamin D-receptor (VDR) gene is the most studied (10, 11). The VDR, a ligand-activated transcription factor, is located in most tissues, including the pancreas and immune system cells. 25(OH)D is primarily hydroxylated in the kidneys by the CYP27B1 enzyme to form 1,25(OH)2 D, which exerts its systemic effects via binding to the VDR. The discovery of certain immune cells, e.g., the dendritic cell’s capability of converting 25(OH)D to its active form (1,25(OH)2 D) via CYP27B1 (1α-hydroxylase) expression promotes hypotheses on the importance of auto and paracrine stimulation for a tolerogenic immune system – these processes have been shown to rely on an optimal 25(OH)D level (12). The pleiotropic actions of vitamin D through its receptor have been proven to influence immune and other pathways (13). The role of single nucleotide polymorphisms (SNPs) in the VDR gene, e.g., Fok1, Bsm1, Taq1, and Apa1 has been unclear. Two meta-analyses have been conducted to elucidate an association between T1D risk and the aforementioned SNPs, the most comprehensive meta-analysis found Bsm1 to be associated with overall increased risk of T1D, but after subgroup analysis by ethnicity the association was only significant in the Asian population (10, 14). A few studies have found that polymorphisms in the gene that encodes the enzyme CYP27B1 increase the risk of developing T1D (15–17). Interestingly, a large genome-wide association study found that genetic variants at four loci – involved in vitamin D metabolism – were associated with 25(OH) levels: GC/4p12 (rs2282679), a gc-globulin/vitamin D-binding protein; CYP2R1/11p15 (rs10741657), an enzyme that converts cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) into 25(OH)D; Pediatric Diabetes 2014: 15: 416–421

DHCR7/11q12 (rs12785878), an enzyme that converts 7-dehydrocholesterol to cholesterol and thereby removes the substrate from the synthetic pathway of 25(OH)D; and CYP24A1/20q13 (rs6013897), an enzyme that initiates degradation of 1,25(OH)2 D) to be associated with 25(OH)D levels (18). This genetic association with 25(OH)D levels has since been replicated in healthy individuals and three of the four loci were also associated with the risk of T1D (17). On the basis of a relatively large population sample of children newly diagnosed with T1D and their siblings, we have the following hypotheses: i Vitamin D metabolism genotypes are associated with the risk of developing T1D. ii Certain vitamin D metabolism genotypes influence 25(OH)D serum levels.

Methods Study population Data were obtained from a large population-based register of diabetic children called DanDiabKids – the registry has an associated biobank. Established in 1996, the register includes incidence data and clinical information from more than 4000 newly diagnosed cases for individuals with T1D aged 0–18. The biobank associated with the register contains blood samples on approximately 75% of all children and their first-degree relatives. Among the latter, there are more than 2300 healthy siblings less than 18 yr of age. About 1467 affected offspring trios of Danish origin were genotyped – all patients with measured 25(OH)D were included – independent of time of blood sampling. Additionally, 358 extra trios were available for genotyping and therefore included. For 25(OH)D analysis, a random sample of 1077 cases with blood sampling less than 3 months after onset were chosen. The date of onset was defined as the date of first insulin injection, and diabetes duration thereafter was measured in months. A random sample of 983 siblings from the biobank was matched with the cases for age, gender, month of sampling, and sample year representing the entire study period. Blood samples from the same family were taken within 1 month in 89% of the families. Of the 2060 children with available 25(OH)D, 88 (two patients and 86 siblings) were excluded because of age above 18 yr. Material was missing for two patients and one sibling. We excluded 166 patients where samples were taken within the first 2 days after onset due to a dramatic downward shift in 25(OH)D levels after the first 2 days – longitudinal sampling was not performed. The association between the eight SNPs

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Thorsen et al. and 25(OH)D levels were tested in 1803 children (907 patients and 896 siblings where 859 were females and 944 were males). The children were aged 0–18 yr, with a mean of 10.6 yr (SEM 0.13) for patients and 9.9 yr (SEM 0.12) for siblings. Serum samples have been stored at −80◦ C during the entire study period (1997–2009). The study was performed according to the criteria of the Helsinki II Declaration and was approved by the Danish National Committee on Biomedical Research Ethics (H-KA-20070009). For all the patients, their parents or guardians gave informed consent.

Genotyping Eight SNPs in or near genes associated with the metabolism of vitamin D were genotyped using predesigned TaqMan SNP genotyping assays, and run on the TaqMan 7900 HT (Applied Biosystems, Foster City, CA, USA) or the CFX384 (Bio-Rad) systems. The SNPs genotyped were Bsm1 (C>T) (rs1544410); Fok1 (C>T) (rs2228570); and Apa1 (A>C) (rs7975232) in VDR; rs4646536 (T>C) in CYP27B1; rs2282679 (A>C) in GC; rs10741657 (G>A) near CYP2R1; rs12785878 (T>G) near DHCR7; and rs6013897 (T>A) in CYP24A1. In CYP27B1, two SNPs rs4646536 (+2838 (T>C) and rs10877012 (−1260 C>A) have previously been genotyped. However, as they are in perfect linkage disequilibrium (16), we only evaluated rs4646536.

25(OH)D and parathyroid hormone Vitamin D status was measured as serum 25(OH)D by high-performance liquid chromatography (19). Detection limit for 25(OH)D was 9.5 nmol/L, with a variance of 8%. Twenty-five children had a level below detection limit and three had missing values. Parathyroid hormone (PTH) levels were measured by competitive chemiluminescent enzyme immunoassays (IMMULITE 2000 System; Siemens Healthcare

Diagnostics, Deerfield, IL, USA). Detection limit was 0.33 pmol/L, with a variance of 6.6–10%. Ten children had missing values and 589 were below detection limit.

Statistical methods Association with T1D was evaluated by means of the transmission disequilibrium test (TDT) in plink version 1.07 (20). In addition the impact of genotypes on 25(OH)D and PTH levels were modeled based on a linear mixed model including a random effect and correlation within families using Proc Mixed in sas9.2. The model is further adjusted for sample year, age, and season (month of sample). The levels of vitamin D and PTH were logtransformed before analysis to meet the assumption of the statistical model (normally distributed residuals). A p value of 0.05 was considered to be statistically significant. Furthermore, the p value for the SNPs association was corrected for eight multiple tests using the Bonferroni method (8 SNPs pcorrected = 0.05/8 = 0.00625).

Results Genes The eight SNPs we selected for examination are all common SNPs with minor allele frequencies ranging from 0.21 to 0.45. We did not demonstrate association with T1D – in the 1467 trios – for any of the following SNPs: CYP27B1, Bsm1, Fok1, Apa1, GC, CYP2R1, DHCR7, and CYP24A1 [p (TDT) = 0.49, 0.22, 0.85, 0.92, 0.90, 0.88, 0.21, and 0.65, respectively) (Table 1). All SNPs were in Hardy–Weinberg equilibrium. There was no evidence of a different distribution of genes according to gender.

25(OH)D levels and genes Variants in the GC gene were significantly associated with 25(OH)D levels (p = 0.0003) even after correcting

Table 1. Results of transmission disequilibrium test (TDT) of association between single nucleotide polymorphisms (SNPs) in vitamin D-related genes and risk of type 1 diabetes (T1D) in a Danish family cohort SNP rs7975232 (A>C) rs1544410 (C>T) rs2228570 (C>T) rs4646536 (T>C) rs2282679 (A>C) rs10741657 (G>A) rs12785878 (T>G) rs6013897 (T>A)

Chromosome

Position

Transmitted

Untransmitted

Odds ratio

p value

Gene

12 12 12 12 4 11 11 20

46525104 46526102 46559162 56444255 72608383 14914878 71167449 52742479

711 666 671 623 543 734 597 483

715 712 678 648 539 728 641 469

0.99 0.94 0.99 0.96 1.01 1.01 0.93 1.03

0.9156 0.2153 0.8488 0.4832 0.9032 0.8753 0.2111 0.65

VDR (Apa1) VDR (Bsm1) VDR (Fok1) CYP27B1 GC CYP2R1 DHCR7 CYP24A1

Position: base pair position on genome build hg18; Transmitted: number of transmitted minor alleles; Untransmitted: number of untransmitted minor alleles. Odds ratio and p value from the TDT test. Gene: SNP location in or near the closest gene.

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Pediatric Diabetes 2014: 15: 416–421

T1D and SNPs in vitamin D metabolism genes Table 2. SNPs from vitamin D metabolism genes and 25(OH)D concentration (nmol/L) Median

Q1

Q3

Case

Case

Case

Patient Sibling Patient Sibling Patient Sibling Genotypes GC (A>C) AA 61.00 AC 56.39 CC 51.51 DHCR7 (T>G) TT 61.00 TG 58.00 GG 56.40 Bsm1 (C>T) CC 56.90 CT 60.06 TT 57.00 Fok1 (C>T) CC 57.07 CT 59.83 TT 62.00 Apa1 (A>C) AA 59.00 AC 59.59 CC 58.00 CYP27B1 (T>C) TT 60.00 TC 58.00 CC 60.00 CYP2R1 (G>A) GG 58.00 GA 59.00 AA 60.60 CYP24A1 (T>A) TT 58.70 TA 59.83 AA 58.00

Seasonal variation 25(OH)D levels varied significantly with months of sampling, having the highest levels from July until September (p < 0.001). There was no difference in seasonal variation between patients and siblings (p = 0.57).

Discussion 59.42 57.00 47.00

43.00 35.41 32.00

39.62 35.00 30.00

88.34 90.00 85.00

88.10 87.00 73.00

59.23 56.58 51.71

41.00 35.41 40.00

38.75 35.00 32.00

85.38 92.00 74.08

87.24 86.00 92.70

57.77 58.00 57.00

38.04 39.00 38.00

37.00 38.00 32.00

83.88 91.00 87.00

84.09 89.00 81.00

58.92 56.65 53.79

37.51 39.81 39.50

38.38 91.00 87.00 36.00 85.00 88.10 34.75 100.00 78.00

51.71 59.51 56.11

40.00 37.19 36.99

32.38 40.21 37.95

89.00 89.08 84.19

85.04 88.10 84.09

59.45 56.00 52.72

39.00 37.51 39.22

36.27 37.50 35.41

93.76 82.59 87.87

91.67 84.92 71.41

52.00 59.40 58.09

39.81 39.00 35.95

35.00 39.55 33.56

89.00 87.87 88.34

83.00 91.60 83.00

58.00 59.00 44.00

37.51 39.22 39.70

37.50 85.16 89.00 36.17 90.25 83.00 31.50 103.10 69.00

for multiple testing. 25(OH)D levels being 21.3% (CI 95% 6.8; 37.9%) higher in carriers homozygous for the major allele and 7.6% (CI 95% 5.4; 22.3%) higher in heterozygous carriers when compared to the carriers homozygous for the minor allele in the adjusted model, whereas none of the other genes were associated with 25(OH)D levels (Table 2).

Age and gender We found a linear decrease of 2.0% [(CI 95% 1.2; 2.8); p < 0.001] per year in 25(OH)D levels and an increase of 1.4% per year [(CI 95% 0.1; 2.7%); p = 0.04] in PTH in both patients and siblings. The increase in PTH deviated slightly from a linear increase but we have chosen to report the linear factor for simplicity. There was no difference in 25(OH)D between gender. Pediatric Diabetes 2014: 15: 416–421

The aim of our study was to actively participate in the on-going examination of whether or not common SNPs in the VDR gene or genes involved in vitamin D transport (GC), hydroxylation (CYP27B1, CYP2R1, and CYP24A1), or cholesterol synthesis (DHCR7) influence the risk of developing T1D. Furthermore, we wanted to contribute to a more thorough understanding of how – or if – such SNPs could influence 25(OH)D homeostasis. In our population of Danish children with newly diagnosed T1D, we could not confirm previous findings of an association between T1D and any of the eight tested SNPs. Though we found a significant association between genetic variation in the GC locus and 25(OH)D levels. Our findings are supported by a meta-analysis that was conducted on 42 case–control-type and familytransmission-type studies from 1997 to 2005, which focused mainly on juvenile T1D (14). Notably, we only reanalyzed data on the three most-studied SNPs in the VDR gene, and there are over 200 other SNPs in the same gene (14). Contrary to the study performed in a British population (17), we could not confirm an association between T1D and DHCR7, CYP2R1, CYP27B1 and CYP24A1. Ethnic differences in genetic polymorphisms and gene expression must also be taken into account for discrepancy in results between countries. This was shown in a recently conducted large meta-analysis, which found that the Bsm1 polymorphism was only significantly associated with T1D risk in people with Asian descent after population stratification (10). It has been proposed that the SNPs associated with T1D might not be causal variants, but rather be in linkage disequilibrium with these variants – ethnicity might cause a different distribution of these yet unknown SNPs (18). Paradoxically, the most associated locus with 25(OH)D levels, GC, in the aforementioned British study showed no significant effect on T1D risk (17). We also replicated the association between the GC locus and 25(OH) levels, but found no association with T1D. Our study consists of a homogeneous population where blood sampling was performed within 3 months after onset, which is definitely a strength in regard to 25(OH)D levels. It can be argued that the TDT analysis does not have the same power as a case–control

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Thorsen et al. association analysis to detect a difference between T1D and SNPs. We confirm a variation with season and have previously shown variation with year, thereby highlighting the importance of sampling patients and controls not only by season but also by year – the latter due to change in ‘hours of bright sunshine’ (21). Matching on age should also be considered as we found decreasing levels in older children – as have others (22). The intensive study of the vitamin D metabolism genes is driven by the hypothesis that vitamin D has an important role in the pathogenesis of T1D. Epidemiologic studies have shown that T1D patients have lower levels of 25(OH)D around or years after onset when compared to healthy controls (7–9, 17). In our previous study, we could not confirm these findings (21). Moreover, we did not find lower 25(OH)D levels in the most recent cohorts in our study period (1996–2009), which does not support the hypothesis that vitamin D is associated to the increasing incidence of T1D in Denmark – at least not around onset (21). ‘The window of opportunities’ for optimal levels of 25(OH)D could be found in utero or in the first years of life when the immune system is being ‘educated’ to become self-tolerant (23, 24). Further research in this field is anticipated with great interest.

4.

5.

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7.

8.

9.

10.

Conclusion In summary, the hypothesis that a different distribution of SNPs from vitamin D metabolism genes is associated with T1D was not confirmed by our study. Though an association between genetic variation in the GC locus and 25(OH)D levels was confirmed.

11. 12. 13.

Acknowledgement We greatly acknowledge the members of the Danish Study Group for Diabetes in children and adolescents for collecting the material.

14.

Conflict of interest The authors declare no conflicts of interest.

15.

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No association between type 1 diabetes and genetic variation in vitamin D metabolism genes: a Danish study.

Vitamin D, certain single nucleotide polymorphisms (SNPs) in the vitamin D-receptor (VDR) gene and vitamin D metabolism genes have been associated wit...
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