Background
Gestational diabetes mellitus (GDM) is defined as glucose intolerance diagnosed during pregnancy [
1]. The prevalence of GDM shows differences among ethnic populations and ranges from 1 to 14% [
2]. GDM is characterized by increased insulin resistance, hyperglycemia, and obesity [
1,
3‐
5]. Genetic and environmental factors play an important role in the etiology of GDM [
3]. Women with a family history of diabetes mellitus (DM) are at risk of GDM. Women with a history of GDM are at risk of type 2 DM (T2DM) in the future [
1‐
5]. Genetic variations related to ß-cell dysfunction and insulin resistance have been shown to contribute to the development of GDM [
1,
3,
5,
6]. The vitamin D receptor (
VDR) gene is actively involved in the insulin metabolic pathway. Vitamin D shows its cellular activity by binding to VDR. Vitamin D plays a role in insulin secretion [
7]. Vitamin D deficiency was associated with pre-eclampsia, insulin resistance, and GDM [
8]. Active vitamin D shows efficacy by binding to VDR and it has a wide range of genetic variations [
9]. The complex of vitamin D and its receptor is a transcription factor that plays a role in the regulation of insulin secretion from pancreatic beta cells [
10]. VDR acts as a ligand-dependent transcription factor and it is a member of the nuclear hormone receptor family. The
VDR gene is localized on chromosome 12q13.1, which consists of 11 exons [
11‐
13]. This complex affects immune system regulation [
11]. It has an effect on the proliferation, differentiation, and activation of immune cells and cytokine production, and accordingly, DM development [
10‐
12]. Vitamin D deficiency leads to defects in insulin synthesis and secretion [
10,
11,
13].
VDR polymorphisms have been associated with type 1 DM (T1DM) [
11] and T2DM [
13‐
15].
BsmI (A > G, rs1544410),
ApaI (A > C, rs7975232),
TaqI (T > C, rs731236), and
FokI (C > T, rs2228570) are human VDR single-nucleotide polymorphisms (SNPs).
VDR gene
Bsml,
ApaI, and
TaqI SNPs are found in 3 prime untranslated regions where gene expression is regulated.
FokI leads to T > C substitution at exon 2, thus the first translation initiation region is removed, and consequently transcriptional activity of VDR is changed [
13,
16]. Both insulin resistance and impaired insulin secretion play a role in the pathogenesis of GDM and T2DM [
1,
3,
6]. The association between
VDR gene SNPs and GDM has been investigated in a few studies [
12,
16‐
19].
VDR gene SNPs and GDM have not been investigated in Turkish pregnant women. The present study aimed to investigate associations between VDR gene SNPs (
Taq,
BsmI,
FokI and
ApaI) and GDM in Turkish pregnant women.
Results
Obesity (46.2 vs. 18.0%,
p = 0.001) and insulin resistance (72.4 vs. 7.2%, p = 0.001) were higher in women with GDM than in the non-GDM controls. Serum glucose, insulin, HOMA-IR, HbA1c, BMI, and BPs were higher in the GDM group than in the control group (
p < 0.05). 25(OH)D was lower in women with GDM than in the controls (
p < 0.05). The characteristics of the pregnant women are shown in Table
1. The four SNPs in the control group were within the HWE. Minor allele frequency and the HWE are shown in Table
2.
Table 1
Characteristics of subjects
Age (year) | 29.13 ± 5.20 | 29.41 ± 5.02 | 0.704 |
Gestational age (weeks) | 26.62 ± 1.48 | 26.04 ± 1.67 |
0.014
|
Height (cm) | 159.01 ± 5.90 | 158.52 ± 4.95 | 0.665 |
Weight (kg) | 68.58 ± 9.65 | 75.08 ± 10.07 |
0.002
|
BMI (kg/m2) | 26.19 ± 4.01 | 29.94 ± 4.18 |
0.003
|
Glucose (mg/dl) | 76.19 ± 8.93 | 105.77 ± 7.81 |
0.005
|
İnsulin (μIU/ml) | 8.01 ± 1.69 | 12.83 ± 4.07 |
0.002
|
HOMA-IR | 1.52 ± 0.48 | 3.36 ± 1.14 |
0.003
|
HbA1c (%) | 4.99 ± 0.25 | 5.69 ± 0.38 |
0.004
|
25(OH)D | 17.60 ± 10.24 | 12.04 ± 8.51 |
0.001
|
Systolic BP (mmHg) | 101.37 ± 11.44 | 111.77 ± 14.31 |
0.008
|
Diastolic BP (mmHg) | 66.01 ± 7.46 | 71.08 ± 7.22 |
0.012
|
Table 2
Minor allele frequency and Hardy-Weinberg Equilibrium of VDR gene SNPs
Apa I rs7975232 | C | 0.54 | 0.23 |
TaqI rs731236 | C | 0.35 | 0.78 |
BsmI rs15444410 | G | 0.38 | 0.15 |
FokI rs2228570 | T | 0.29 | 0.20 |
The distributions of the
VDR gene SNPs are shown in Table
3. The frequency of
VDR gene
ApaI rs7975232,
TaqI rs731236 and
BsmI rs1544410 did not differ between women with and without GDM in a codominant model and dominant model and recessive model (
p > 0.05, each).
VDR gene
ApaI,
TaqI, and
BsmI SNPs were not associated with GDM. The frequency of
VDR gene
FokI rs2228570 differed between women with and without GDM (
p < 0.05). Compared with the controls,
FokI CT genotype (CT vs. CC, OR = 1.84, 95% CI: [1.05–3.23],
p = 0.031) and TT (TT vs. CC, OR = 3.95, 95% CI: [1.56–9.96],
p = 0.002) genotype were associated with an increased GDM risk in a codominant model, and CT/TT carriers had increased 2.2 odds of having GDM (CT/TT vs. CC, OR = 2.29, 95% CI: [1.35–3.89], p = 0.002) in a dominant model. Compared with the controls, TT genotype carriers had increased 3.02 odds of having GDM (CT/CC vs. TT, OR = 3.02, 95% CI: [1.23–7.38],
p = 0.012) in a recessive model. Gestational age was lower in
FokI-TT genotype compared with CC and CT genotype (
p < 0.05). Glucose, HbA1c, and HOMA-IR were higher in the
FokI-TT genotype compared with the CC and CT genotypes (p < 0.05) (Table
4).
FokI-T (risk allele) was positively correlated with log-HOMA-IR (r = 0.326,
p = 0.004). In the logistic regression analysis,
FokI SNPs were independently associated with GDM after adjustig for BMI and age (β = 1.63, 95% CI: [1. 2-4.2],
p = 0.012).
Table 3
Genotype analysis of VDR gene SNPs
ApaI rs7975232 (%) | | | | |
Co-dominant Wild type AA | 19.4 | 17.0 | | |
Heterozygous AC | 56.7 | 52.0 | 1.04 (0.51–2.12) | 0.985 |
Homozygous CC | 23.9 | 31.0 | 1.48 (0.67–3.25) | 0.326 |
Dominant (AA/AC + CC) | | | 1.17 (0.59–2.30) | 0.639 |
Recessive (AA+AC/CC) | | | 1.43 (0.80–2.56) | 0.225 |
TaqI rs731236 (%) |
Co-dominant Wild type TT | 40.0 | 44.0 | | |
Heterozygous CT | 49.6 | 42.0 | 0.76 (0.44–1.33) | 0.353 |
Homozygous CC | 10.4 | 14.0 | 1.22 (0.52–2.84) | 0.633 |
Dominant (TT/CT + CC) | | | 0.84 (0.50–1.43) | 0.539 |
Recessive (TT + CT/CC) | | | 1.40 (0.63–3.10) | 0.396 |
BsmI rs1544410 (%) | | | | |
Co-dominant Wild type AA | 31.9 | 42.0 | | |
Heterozygous AG | 57.0 | 44.0 | 0.58 (0.33–1.02) | 0.062 |
Homozygous GG | 11.1 | 14.0 | 0.95 (0.41–2.22) | 0.916 |
Dominant (AA/AG + GG) | | | 0.64 (0.37–1.10) | 0.109 |
Recessive (AA+AG/GG) | | | 1.30 (0.59–2.83) | 0.506 |
FokI rs2228570 (%) | | | | |
Co-dominant Wild type CC | 60.0 | 41.0 | | |
Heterozygous CT | 34.1 | 43.0 | 1.84 (1.05–3.23) |
0.031
|
Homozygous TT | 5.9 | 16.0 | 3.95 (1.56–9.96) |
0.002
|
Dominant (CC/CT + TT) | | | 2.29 (1.35–3.89) |
0.002
|
Recessive (CC + CT/TT) | | | 3.02 (1.23–7.38) |
0.012
|
Table 4
Association between the VDR gene FokI SNPs and clinical features of GDM women
Age (year) | 30.04 ± 5.24 | 29.46 ± 5.31 | 29.14 ± 3.79 | 0.515 | 0.315 | 0.588 |
Gestational age (weeks) | 26.60 ± 1.66 | 26.43 ± 1.50 | 25.42 ± 1.39 | 0.893 |
0.031
|
0.033
|
BMI (kg/m2) | 27.95 ± 4.11 | 29.16 ± 4.63 | 28.61 ± 3.56 | 0.311 | 0.264 | 0.690 |
Glucose (mg/dl) | 87.06 ± 17.04 | 91.80 ± 16.66 | 99.42 ± 14.53 | 0.152 |
0.002
|
0.020
|
İnsulin (μIU/ml) | 10.19 ± 4.01 | 9.88 ± 3.71 | 12.17 ± 3.60 | 0.380 | 0.036 |
0.009
|
HOMA-IR | 2.30 ± 1.31 | 2.33 ± 1.16 | 3.05 ± 1.20 | 0.888 |
0.009
|
0.013
|
HbA1c (%) | 5.29 ± 0.50 | 5.33 ± 0.47 | 5.47 ± 0.33 | 0.738 |
0.011
|
0.019
|
Discussion
This case-control study showed that VDR gene FokI SNPs were independently associated with having GDM in Turkish women. The frequency of the VDR gene FokI TT and CT genotype was increased in women with GDM compared with the non-GDM controls. The frequency of VDR gene ApaI, BsmI, and TaqI SNPs did not differ between women with and without GDM with no association. VDR FokI SNPs might contribute to insulin resistance in the development of GDM.
Our results showed that 25(OH)D concentrations were lower in the GDM group than in the control group. Vitamin D deficiency was associated with insulin resistance and GDM [
8]. The
VDR gene has a role in the metabolic pathway of insulin [
9].
VDR gene variations have beenshown to be correlated in the development, progression, and complications of T2DM [
13‐
15]. The present study showed that
VDR gene
FokI SNPs were independently associated with an increased risk of GDM in Turkish women (β = 1.63, 95% CI: [1. 2-4.2],
p = 0.012). Our study suggested that
VDR gene
FokI SNPs might be associated with having GDM. We found that the frequency of
VDR gene
ApaI,
TaqI, and
BsmI did not differ between women with and without GDM.
VDR gene
ApaI,
TaqI, and
BsmI SNPs were not associated with GDM. The
VDR gene
FokI SNP showed significant differences between women with and without GDM.
VDR gene
FokI (variant or heterozygotes) compared to wild-type (CC) SNP revealed a significant association.
VDR gene
FokI rs2228570 TT (TT vs. CC, OR = 3.95, 95% CI: [1.56–9.96],
p = 0.002) and CT heterozygotes (CT vs. CC, OR = 1.84, 95% CI: [1.05–3.239,
p = 0.031) were associated with having GDM, compared with the controls.
VDR gene
FokI SNPs might contribute to developing GDM in the Turkish population.
Similar to our results,
FokI homozygous SNPs were reported as prevalent in patients with DM and GDM [12, 13]. Aslani et al. reported that
VDR gene
FokI SNPs were associated with GDM in an Iranian population [
12]. Another study reported that
ApaI and
Taq SNPs were associated with GDM in an Iranian population [
16]. These results are incompatible with our study, thus we showed that
ApaI,
Taq, and
BsmI SNPs were not associated with GDM.
BsmI and
FokI SNPs were not associated with GDM in a Saudi Arabian population [
17]. Vural and Maltas et al. showed that
TaqI SNPs were not associated with T2DM in a Turkish population [
15]. Dilmec et al. reported that
TaqI SNPs were associated with T2DM, but
ApaI and
FokI SNPs were not associated with T2DM in a Turkish population [
14]. Previous studies investigating the
VDR gene in Turkish patients with T2DM were compatible with our study. Hence, we supposed that
Taq and
ApaI were not associated with having T2DM in the Turkish population.
VDR gene
Taq,
BsmI or
ApaI SNPs were not associated with diabetic microvascular complications but only
FokI SNPs were associated with diabetic neuropathy in a Caucasian population [
13]. Meta-analysis reported that only
FokI SNPs were found as a risk factor for T2DM.
Taq,
BsmI or
ApaI SNPs were not associated with DM [
21]. These reports were similar to the present study; we showed that only
FokI SNPs were associated with having GDM. A meta-analysis showed that only BsmI SNPs were associated with autoimmune T1DM in an Asian population [
11]. We supposed that autoimmunity might contribute to the association between
BsmI SNPS and having T1DM. The inconsistency between studies might result from ethnic diversity and environmental factors on
VDR variations in different populations [
12].
The present study showed that the FokI-T (risk allele) was positively correlated with log-HOMA-IR. Assessment of allele frequency distribution showed a significant association of the FokI variant allele (T) on susceptibility toward to GDM. We supposed that the FokI variant might contribute to impaired insulin resistance and metabolic disorder in developing GDM. Hence, FokI SNPs might have a role in the pathogenesis of GDM.
BsmI,
ApaI, and
TaqI polymorphisms of the
VDR gene are found in the three-primer untranslated region (3′-UTR) and have been shown to be in strong linkage disequilibrium (LD) [
21]. The
FokI polymorphism was reported as an independent marker of the
VDR gene because it has not been shown to be in linkage disequilibrium with any of other
VDR polymorphisms [
12]. Our study reported that
VDR gene
FokI, ApaI, BsmI and
TaqI haplotypes were not associated with GDM, and
ApaI, BsmI and
TaqI polymorphisms were not shown in LD.
ApaI and
BsmI polymorphisms of the
VDR gene, both in intron 8, are considered as silent SNPs. These polymorphisms do not change the amino acid sequence of the encoded protein, but they might affect gene expression by modulating stability of mRNA [
21]. The
TaqI polymorphism is located at codon 352 in exon 9 of the
VDR gene. The
TaqI TT genotype (absence of restriction site) is related to lower active vitamin D3 [
21]. The only locus with impact on the structure of VDR protein is the
FokI polymorphism, which is located on the 5′ end region of the
VDR gene. The
VDR gene
FokI polymorphism is functional because it is found in a coding sequence. The
FokI polymorphism is located in the first ATG starting code of VDR protein.
FokI is involved in thymine to cytosine (T/C) substitution at exon 2, the first translation initiation region is removed, and transcriptional activity of VDR is changed [
12,
13,
16,
22]. It alters the ACG codon, which is found ten base pairs upstream from the translation starting codon and leads to the generation of an additional starting codon. Two different VDR isoforms occur with transition of allele T to C in ATG. When initiating translation starts from this alternative site in the thymine variant, it generates a longer VDR protein comprised of 427 amino acids. The gene is transcribed in normal length if there is a restriction site. Thus, the C/C allele codes a 424-amino acid protein and the T/T allele codes a 42 7-amino acid protein. The longer VDR protein has low activity in transcription, accordingly activation is decreased in target cells [
12,
13]. The
FokI T/T genotype,
FokI C/C, showed 1.7-fold greater function in vitamin D-dependent transcriptional activation of a reporter through the regulation of a vitamin D response element [
22]. The
FokI rs2228570 polymorphism is the only
VDR gene polymorphism involved in the generation of altered protein expression [
12]. Apart from obesity and insulin resistance, complex genetic (ethnicity) and non-genetic (epigenetic) mechanisms may have a role in the etiology of GDM [
9].
The cross-sectional design, small sample size, and absence of postpartum follow-up are the limitations of this study.