Abstract
Vitamin D is a multi-functional immune regulator, and a low serum concentration of vitamin D promotes autoimmune inflammation. In this study, we evaluate the association between the prognosis of autoimmune thyroid disease (AITD) and the functional polymorphisms of genes that regulate vitamin D metabolism. For 139 Graves’ disease (GD) patients, 116 Hashimoto's disease (HD) patients and 76 control subjects, we genotyped the following polymorphisms using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP): vitamin D receptor (VDR): rs731236, rs7975232, rs2228570 and rs1544410; group-specific component (GC): rs7041 and rs4588; and CYP2R1: rs10741657. The frequency of the TT genotype for the rs731236 polymorphism was higher in GD patients than in HD patients (P = 0·0147). The frequency of the C allele for the rs7975232 polymorphism was higher in GD patients than in control subjects (P = 0·0349). The proportion of GD patients whose anti-thyrotrophin receptor antibody (TRAb) level was >51% was higher in those with the CC genotype than in those with the CA+AA genotypes (P = 0·0065). The frequency of the CC genotype for the rs2228570 polymorphism was higher in HD patients than in control subjects (P = 0·0174) and GD patients (P = 0·0149). The frequency of the Gc1Gc1 genotype for the GC polymorphism and the AG genotype for the CYP2R1 polymorphism were lower in intractable GD than in GD in remission (P = 0·0093 and 0·0268, respectively). In conclusion, genetic differences in the VDR gene may be involved in the development of AITD and the activity of GD, whereas the genetic differences in the GC and CYP2R1 genes may be involved with the intractability of GD.
Keywords: autoimmune thyroid disease, intractability, polymorphism, severity, vitamin D
Introduction
Autoimmune thyroid diseases (AITDs), such as Hashimoto's disease (HD) and Graves’ disease (GD), are archetypes of organ-specific autoimmune disease [1,2]. The severity of HD and intractability of GD vary among patients. Some patients develop hypothyroidism in early life, and some maintain a euthyroid state in old age despite the passage of time. Some patients with GD achieve remission through medical treatment; others do not. Although it is difficult to predict the disease severity and intractability of AITDs, we have reported previously that they are affected by the patient's genetic capability to express and produce immune regulatory factors [3–11].
Vitamin D is a multi-functional immune regulator that has been shown to inhibit the differentiation of T helper type 1 (Th1) and Th17 cells and to promote the generation of regulatory T (Treg) cells [12,13]. The serum concentrations of 25(OH)2D3 and 1·25-dihydroxy vitamin D3 [1·25 (OH)2D3], which are metabolites of vitamin D [14], have been shown to be lower in patients with autoimmune diseases, including AITDs and multiple sclerosis (MS) [15–17]. These studies suggested that reduced immune regulation of vitamin D may induce autoimmune reactions.
Vitamin D receptor (VDR) is a nuclear receptor that binds specific DNA sequences and vitamin D response elements (VDRE), and controls the transcription of regulating genes associated with calcium metabolism and immune responses [18]. The major polymorphisms of the VDR gene are rs7975232 (ApaI), rs1544410 (BsmI), rs2228570 (FokI) and rs731236 (TaqI). The functions of these polymorphisms are summarized in Table 1[19–21]. For the rs7975232 polymorphism, the frequency of the C allele is higher in Egyptians with GD than in healthy controls [15], and lower in Japanese with GD than in healthy controls [22]. For the rs1544410 polymorphism, the A allele was reported to be a risk allele for type I diabetes (T1D) and GD [20,22]. For the rs2228570 polymorphism, the frequency of the C allele was higher in Chinese with GD than in healthy controls and in Japanese with HD than in healthy controls [23,24].
Table 1.
The function of VDR polymorphism genotyped in this study
| Polymorphism | The function of polymorphism | References |
|---|---|---|
| rs7975232 C/A (ApaI) | AA genotype > CC genotype [serum 25 (OH)2D3 concentration] | [19] |
| rs1544410 A/G (BsmI) | A allele > G allele (IFN-γ production in PBMC) | [20] |
| rs2228570 C/T (FokI) | TT genotype > CC genotype [serum 25 (OH)2D3 concentration] | [19] |
| rs731236 T/C (TaqI) | T allele > C allele (VDR expression) | [21] |
IFN = interferon; PBMC = peripheral blood mononuclear cells.
Genome-wide association studies (GWAS) have shown that group-specific components (GC) and CYP2R1 are associated with circulating vitamin D levels [25,26]. The GC gene encodes vitamin D binding protein (DBP), a vitamin D transporter. There are two common polymorphisms (rs4588 and rs7041) in the GC gene that are associated with the binding affinity of GC to vitamin D [27]. There are three major isoforms of DBP, Gc1F, Gc1S and Gc2. A threonine substituted by lysine (rs4588) generates Gc2, and an aspartic acid substituted by glutamine (rs7041) generates Gc1S. The binding affinity of Gc1F to vitamin D was four times higher than that of Gc2 and two times higher than that of Gc1S [27]. In addition, the serum and plasma vitamin D concentration in Gc2 allele carriers was lower than that in Gc1 allele (Gc1F + Gc1S) carriers [28,29]. Therefore, rs4588 and rs7041 are functional polymorphisms because they allow the generation of Gc1 and Gc2 isoforms. Conversely, the functional polymorphisms in the CYP2R1 gene, rs12794714 and rs10741657, are in linkage disequilibrium [30,31]. The vitamin D serum concentration in individuals with the AA genotype of rs10741657 was higher than that in G carriers [31], while individuals with the GG genotype had a higher vitamin D serum concentration than A carriers [30]. The functions of this polymorphism have not been clarified.
In this study, we genotyped these functional polymorphisms to clarify the association of genes that regulate vitamin D for the development and prognosis of AITD.
Materials and methods
Subjects
We carefully selected 54 patients with severe HD and 42 patients with mild HD and 20 patients who could not be categorized to severe HD or mild HD groups at the time of analysis from 116 HD patients who were positive for anti-thyroid microsomal antibody (McAb) and/or anti-thyroglobulin antibody (TgAb). Patients with HD who developed moderate to severe hypothyroidism before 50 years of age and who were treated daily with thyroxine were defined as patients with severe HD, and untreated, euthyroid patients with HD who were more than 50 years of age were defined as patients with mild HD. All patients with mild HD had a palpable diffuse goitre.
We also carefully selected 61 patients with intractable GD, 40 patients with GD in remission and 38 patients who could not be categorized to intractable GD or GD in remission groups at the time of analysis from 139 GD patients who had a clinical history of thyrotoxicosis with elevated TRAb. Euthyroid patients with GD who had been treated with methimazole for at least 5 years and were still positive for TRAb were defined as patients with intractable GD, and patients with GD who had maintained a euthyroid state and were negative for TRAb for more than 2 years without medication were defined as patients with GD in remission. We genotyped 76 healthy volunteers (control subjects) who were euthyroid and negative for thyroid autoantibodies. Written informed consent was obtained from all patients and controls, and the study protocol was approved by the Ethics Committee of Osaka University. Clinical characteristics of subjects were shown in Table 2.
Table 2.
Clinical characteristics of groups of patients with autoimmne thyroid disease at the time of sampling
| Graves’ disease (GD) | Hashimoto's disease (HD) | ||||
|---|---|---|---|---|---|
| Past clinical history of thyrotoxicosis with elevated TRAb | Diffuse goitre and positive TgAb and/or McAb | ||||
| Controls | Intractable | In remission | Severe | Mild | |
| n (female/male) | 76 (49/27) | 61 (49/12) | 40 (30/10) | 54 (43/11) | 42 (34/8) |
| Age of onset (years) | 28·9 ± 11·0‡ | 33·2 ± 13·6 | 30·8 ± 11·1 | 37·2 ± 11·0 | 59·0 ± 10·2‡ |
| (range) | (21–58) | (11∼66) | (10∼58) | (10∼49) | (50∼92) |
| Free T4 (ng/dl) (normal range: 0·9–1·7 ng/dl) | n.d. | 1·25 ± 0·31 | 1·24 ± 0·16 | 1·36 ± 0·30 | 1·24 ± 0·23 |
| Free T3 (pg/ml) (normal range: 2·3–4·3 pg/ml) | n.d. | 2·76 ± 0·55 | 2·64 ± 0·34 | 2·59 ± 0·56 | 2·78 ± 0·42 |
| TSH (μU/ml) (normal range: 0·5–5·0 μU/ml) | n.d. | 1·60 ± 1·27 | 1·87 ± 1·26 | 1·83 ± 1·32 | 2·67 ± 1·78 |
| TRAb (IU/l) | <2·0 | 7·64 ± 13·1 | <2·0 | <2·0 | <2·0 |
| (range) | (2·0∼71) | ||||
| TgAb (2n × 100)* | Negative | 2·89 ± 2·93 | 2·48 ± 0·64 | 7·00 ± 3·49 | 1·56 ± 2·40 |
| McAb (2n × 100)* | Negative | 4·73 ± 2·59 | 2·71 ± 0·96 | 5·41 ± 3·18 | 3·23 ± 2·77 |
| Current treatment | None | MMI or PTU | None | L-thyroxine | None |
| Treatment time (years) | None | 12·1 ± 7·61 | 3·21 ± 1·26‡ | 11·3 ± 8·52 | None |
| Current dose of anti-thyroid drug (mg/day)† | None | 19·1 ± 34·4 | None | None | None |
| (range) | (2·5∼200) | ||||
| Current dose of L-thyroxine (μg/day) | None | None | None | 90·4 ± 37·7 | None |
| (range) | (50∼250) | ||||
When the titre of anti-thyroglobulin antibody (TgAb) or microsomal antibody (McAb) was 25600, it was expressed as 28 × 100,
Doses were expressed as the comparable dose of methimazole (MMI) [50 mg of propylthiouracil (PTU) was converted to 5 mg of MMI].
Age at the time of sampling. Data are expressed as mean ± standard deviation.
McAb = anti-thyroid microsomal antibody; n.d. = not determined; T3 = triiodothyronine; T4 = thyroxine; TRAb = anti-thyrotrophin receptor antibody; TSH = thyrotrophin.
Genotyping of polymorphisms
The polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was used for genotyping each polymorphism. The target sequences of each gene were amplified using PCR, and the resulting PCR products were digested by addition of restriction enzymes. The sequences of the forward and reverse primers, the PCR conditions and the restriction enzymes used are summarized in Table 3.
Table 3.
The primers, polymerase chain reaction (PCR) conditions and restricion enzymes used in this study
| Primer pairs | PCR conditions | Restriction enzymes | ||
|---|---|---|---|---|
| VDR | rs7975232 (ApaI) | 5′-CAGAGCATGGACAGGGAGCAA-3′ 5′-GCAACTCCTCATGGCTGAGGTCTC-3′ | 95°C for 5 min (95°C for 30 s, 70°C for 30 s, 72°C for 60 s) × 35cycles 72°C for 5 min | ApaI |
| rs1544410 (BsmI) | 5′-CAACCAAGACTACAAGTACCGCGTCAGTGA-3′ 5′-AACCAGCGGGAAGAGGTCAAGGG-3′ | 95°C for 3 min (95°C for 30 s, 60°C for 30 s, 72°C for 30 s) × 35cycles 72°C for 5 min | BsmI | |
| rs2228570 (FokI) | 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′ | 95°C for 5 min (95°C for 30 s, 61°C for 30 s, 72°C for 30 s) × 35cycles 72°C for 5 min | FokI | |
| rs731236 (TaqI) | 5′-CAGAGCATGGACAGGGAGCAA-3′ 5′-GCAACTCCTCATGGCTGAGGTCTC-3′ | 95°C for 5 min (95°C for 30 s, 70°C for 30 s, 72°C for 60 s) × 35cycles 72°C for 5 min | TaqI | |
| GC | rs7041 | 5′-AAATAATGAGCAAATGAAAGAAGAC-3′ 5′-CAATAACAGCAAAGAAATGAGTAGA-3′ | 95°C for 3 min (95°C for 30 s, 64°C for 30 s, 72°C for 30 s) × 30cycles 72°C for 7 min | HaeIII |
| rs4588 | 5′-AAATAATGAGCAAATGAAAGAAGAC-3′ 5′-CAATAACAGCAAAGAAATGAGTAGA-3′ | 95°C for 3 min (95°C for 30 s, 64°C for 30 s, 72°C for 30 s) × 30cycles 72°C for 7 min | StyI | |
| CYP2R1 | rs10741657 | 5′-GGGAAGAGCAATGACATGGA-3′ 5′-GCCCTGGAAGACTCATTTTG-3′ | 95°C for 5 min (95°C for 30 s, 55°C for 30 s, 72°C for 30 s) × 35cycles 72°C for 5 min | MnlI |
Thyroid function and autoantibodies
The serum concentration of free T4 (FT4), free T3 (FT3) and thyrotrophin (TSH) were measured with electro-chemiluminescence immunoassay (ECLIA) (Roche Diagnostics Ltd, Tokyo, Japan). The normal serum concentration ranges of FT4, FT3 and TSH are 0·9–1·7 ng/dl, 2·3–4·3 pg/ml and 0·5–5·0 μU/ml, respectively. TgAb and McAb were measured with a particle agglutination kit (Fujirebio Inc., Tokyo, Japan). A reciprocal titre of >1 : 100 was considered positive. Serum TRAb concentration at the time of disease onset was measured by a radioreceptor assay with a commercial kit (Cosmic Corporation, Tokyo, Japan) as part of a routine study. Serum TRAb concentration during sampling was determined with ECLIA (third-generation) (Roche Diagnostics Ltd). The normal value of TRAb was less than 10% by the radioreceptor assay and 2·0 IU/l by ECLIA.
Statistical analysis
We used the χ2 test and Fisher's exact test to evaluate the significance of the differences in the frequencies of genotypes and alleles among the different groups. Student's t-test was used to analyse differences in goitre size and serum TRAb levels. The differences in titres of TgAb and McAb were analysed using the Mann–Whitney U-test. The data were analysed with jmp10 software (SAS Institute, Inc., Tokyo, Japan). Probability values of less than 0·05 were considered significant.
Results
VDR polymorphisms
The frequency of the TT genotype for the rs731236 polymorphism was higher in GD patients than in HD patients (P = 0·0147). The C allele frequency for the rs7975232 polymorphism was higher in AITD patients, especially GD patients, than in control subjects (P = 0·0375 and 0·0349, respectively) (Table 4). The frequencies of the CC genotype and C allele for the rs2228570 polymorphism were higher in HD patients than in control subjects (P = 0·0174 and 0·0458, respectively) or GD patients (P = 0·0149 and 0·0348, respectively) (Table 4). We found no difference in the frequencies of the genotypes and alleles of the rs1544410 polymorphism between the control subjects and the patients in the HD and GD groups. The frequencies of the genotypes and alleles in these VDR polymorphisms did not differ between the patients with severe HD and those with mild HD or between the patients with intractable GD and those with GD in remission (Table 5). The frequency of the CC genotype for the rs2228570 polymorphism tends to be higher in patients with severe HD than in control subjects, but not significant (P = 0·17).
Table 4.
Genotype and allele frequencies of the polymorphisms genotyped in patients with Graves’ disease (GD), Hashimoto's disease (HD) and in control subjects
| Control | All patients with AITD | All patients with GD | All patients with HD | |||||
|---|---|---|---|---|---|---|---|---|
| VDR | TT | 58 (77·3%) | 196 (81·3%) | 109 (87·2%) | 0·0147†§ | 87 (75·0%) | ||
| rs731236 | TC | 17 (22·7%) | 43 (17·9%) | n.s.* | 15 (12·0%) | n.s.* | 28 (24·1%) | n.s.* |
| CC | 0 (0%) | 2 (0·8%) | 1 (0·8%) | 1 (0·9%) | ||||
| T allele | 133 (88·7%) | 435 (90·2%) | n.s.* | 233 (93·2%) | n.s.* | 202 (87·1%) | n.s.* | |
| C allele | 17 (11·3%) | 47 (9·8%) | 17 (6·8%) | 30 (12·9%) | ||||
| VDR | CC | 31 (41·3%) | 114 (49·1%) | 63 (50·4%) | 51 (47·7%) | |||
| rs7975232 | CA | 32 (42·7%) | 105 (45·3%) | 0·0256* | 56 (44·8%) | 0·0280* | 49 (45·8%) | n.s.* |
| AA | 12 (16·0%) | 13 (5·6%) | 6 (4·8%) | 7 (6·5%) | ||||
| C allele | 94 (62·7%) | 333 (71·8%) | 0·037* | 182 (72·8%) | 0·0349* | 151 (70·6%) | n.s.* | |
| A allele | 56 (37·3%) | 131 (28·2%) | 68 (27·2%) | 63 (29·4%) | ||||
| VDR | CC | 25 (32·9%) | 100 (41·8%) | 46 (34·9%) | 54 (50·5%) | 0·0174*¶, 0·0149‡¶ | ||
| rs2228570 | CT | 42 (55·3%) | 113 (47·3%) | n.s.* | 70 (53·0%) | n.s.* | 43 (40·2%) | n.s.* |
| TT | 9 (11·8%) | 26 (10·9%) | 16 (12·1%) | 10 (9·3%) | ||||
| C allele | 92 (59·2%) | 313 (65·5%) | n.s.* | 162 (61·4%) | n.s.* | 151 (70·6%) | 0·0458*, 0·0348‡ | |
| T allele | 60 (40·8%) | 165 (34·5%) | 102 (38·6%) | 63 (29·4%) | ||||
| VDR | AA | 3 (4·7%) | 7 (3·4%) | 3 (2·7%) | 4 (4·1%) | |||
| rs1544410 | AG | 11 (17·2%) | 36 (17·3%) | n.s.* | 15 (13·7%) | n.s.* | 21 (21·4%) | n.s.* |
| GG | 50 (78·1%) | 165 (79·3%) | 92 (83·6%) | 73 (74·5%) | ||||
| A allele | 17 (13·3%) | 50 (12·0%) | n.s.* | 21 (9·5%) | n.s.* | 29 (14·8%) | n.s.* | |
| G allele | 111 (86·7%) | 366 (88·0%) | 199 (90·5%) | 167 (85·2%) | ||||
| GC | Gc1Gc1 | 37 (61·7%) | 131 (54·6%) | 64 (50·8%) | 67 (58·8%) | |||
| Gc1Gc2 | 19 (31·7%) | 97 (40·4%) | n.s.* | 54 (42·9%) | n.s.* | 43 (37·7%) | n.s.* | |
| Gc2Gc2 | 4 (6·6%) | 12 (5·0%) | 8 (6·3%) | 4 (3·5%) | ||||
| Gc1 allele | 93 (77·5%) | 359 (74·8%) | n.s.* | 182 (72·2%) | n.s.* | 177 (77·6%) | n.s.* | |
| Gc2 allele | 27 (22·5%) | 121 (25·2%) | 70 (27·8%) | 51 (22·4%) | ||||
| CYP2R1 | GG | 25 (40·3%) | 97 (38·2%) | 54 (38·9%) | 43 (37·4%) | |||
| rs10741657 | GA | 26 (41·9%) | 111 (43·7%) | n.s.* | 60 (43·2%) | n.s.* | 51 (44·4%) | n.s.* |
| AA | 11 (17·8%) | 46 (18·1%) | 25 (17·9%) | 21 (18·2%) | ||||
| G allele | 76 (61·3%) | 305 (60·3%) | n.s.* | 168 (60·4%) | n.s.* | 137 (59·6%) | n.s.* | |
| A allele | 48 (38·7%) | 203 (39·7%) | 110 (39·6%) | 93 (40·4%) | ||||
Analysed by χ2 tests
versus control;
versus HD;
versus GD;
TT versus TC + CC;
CC versus CT + TT;
n.s. = not significant. AITD = autoimmune thyroid disease.
Table 5.
Genotype and allele frequencies of the polymorphisms genotyped in patients with Graves’ disease (GD) and Hashimoto's disease (HD)
| GD | HD | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Control | Intractable | In remission | Severe | Mild | |||||
| VDR | TT | 58 (77·3%) | 45 (84·9%) | 32 (86·5%) | 42 (79·3%) | 30 (75·0%) | |||
| rs731236 | TC | 17 (22·7%) | 7 (13·2%) | 5 (13·5%) | n.s.* | 10 (18·9%) | 10 (25·0%) | n.s.‡ | |
| CC | 0 (0%) | 1 (1·9%) | 0 (0%) | 1 (1·8%) | 0 (0%) | ||||
| T allele | 133 (88·7%) | 97 (91·5%) | 69 (93·2%) | n.s.* | 94 (88·7%) | 70 (87·5%) | n.s.‡ | ||
| C allele | 17 (11·3%) | 9 (8·5%) | 5 (6·8%) | 12 (11·3%) | 10 (12·5%) | ||||
| VDR | CC | 31 (41·3%) | 25 (50·0%) | 16 (41·0%) | 22 (50·0%) | 18 (42·9%) | |||
| rs7975232 | CA | 32 (42·7%) | 23 (46·0%) | 19 (48·7%) | n.s.* | 18 (40·9%) | 22 (52·4%) | n.s.‡ | |
| AA | 12 (16·0%) | 2 (4·0%) | 4 (10·3%) | 4 (9·1%) | 2 (4·7%) | ||||
| C allele | 94 (62·7%) | 73 (73·0%) | 51 (65·4%) | n.s.* | 62 (70·5%) | 58 (69·0%) | n.s.‡ | ||
| A allele | 56 (37·3%) | 27 (27·0%) | 27 (34·6%) | 26 (29·5%) | 26 (31·0%) | ||||
| VDR | CC | 25 (32·9%) | 18 (36·7%) | 9 (22·5%) | 20 (45·5%) | 22 (55·0%) | |||
| rs2228570 | CT | 42 (55·3%) | 27 (55·1%) | 27 (67·5%) | n.s.* | 19 (43·2%) | 16 (40·0%) | n.s.‡ | |
| TT | 9 (11·8%) | 4 (8·2%) | 4 (10·0%) | 5 (11·3%) | 2 (5·0%) | ||||
| C allele | 92 (59·2%) | 63 (64·3%) | 45 (56·3%) | n.s.* | 59 (67·0%) | 60 (75·0%) | n.s.‡ | ||
| T allele | 60 (40·8%) | 35 (35·7%) | 35 (43·7%) | 29 (33·0%) | 20 (25·0%) | ||||
| VDR | AA | 3 (4·7%) | 0 (0%) | 1 (2·8%) | 0 (0%) | 2 (5·4%) | |||
| rs1544410 | AG | 11 (17·2%) | 4 (9·5%) | 6 (16·7%) | n.s.* | 7 (17·1%) | 9 (24·3%) | n.s.‡ | |
| GG | 50 (78·1%) | 38 (90·5%) | 29 (80·5%) | 34 (82·9%) | 26 (70·3%) | ||||
| A allele | 17 (13·3%) | 4 (4·5%) | 8 (11·1%) | n.s.*† | 7 (8·5%) | 13 (17·6%) | n.s.‡ | ||
| G allele | 111 (86·7%) | 80 (95·5%) | 64 (88·9%) | 75 (91·5%) | 61 (82·4%) | ||||
| GC | Gc1Gc1 | 37 (61·7%) | 21 (38·9%) | 24 (66·7%) | 0·0093*§ | 31 (62·0%) | 25 (62·5%) | ||
| Gc1Gc2 | 19 (31·7%) | 32 (59·3%) | 10 (27·8%) | 0·0109* | 18 (36·0%) | 15 (37·5%) | n.s.‡ | ||
| Gc2Gc2 | 4 (6·6%) | 1 (1·8%) | 2 (5·5%) | 1 (2·0%) | 0 (0%) | ||||
| Gc1 allele | 93 (77·5%) | 74 (68·5%) | 58 (80·6%) | n.s.* | 80 (80·0%) | 65 (81·3%) | n.s.‡ | ||
| Gc2 allele | 27 (22·5%) | 34 (31·5%) | 14 (19·4%) | 20 (20·0%) | 15 (18·7%) | ||||
| CYP2R1 | GG | 25 (40·3%) | 26 (42·6%) | 12 (30·0%) | 20 (37·0%) | 11 (30·5%) | |||
| rs10741657 | GA | 26 (41·9%) | 20 (32·8%) | 22 (55·0%) | n.s.* | 0·0268*¶ | 23 (42·6%) | 19 (52·8%) | n.s.‡ |
| AA | 11 (17·8%) | 15 (24·6%) | 6 (15·0%) | 11 (20·4%) | 6 (16·7%) | ||||
| G allele | 76 (61·3%) | 72 (59·0%) | 46 (57·5%) | n.s.* | 63 (58·3%) | 41 (56·9%) | n.s.‡ | ||
| A allele | 48 (38·7%) | 50 (41·0%) | 34 (42·5%) | 45 (41·7%) | 31 (43·1%) | ||||
Analysed by χ2 tests or
Fisher's exact test;
intractable GD versus GD in remission;
severe HD versus mild HD;
Gc1Gc1 versus Gc1Gc2 + Gc2Gc2;
GA versus GG + AA; n.s. = not significant.
GC polymorphism
We categorized the Gc1 (Gc1F + Gc1S) and Gc2 alleles according to the results for the rs7041 and rs4588 polymorphisms. The frequency of the Gc1Gc1 genotype was lower in patients with intractable GD than in those with GD in remission (P = 0·0093) (Table 5), although we found no difference in the genotype or allele frequencies of this polymorphism between the control subjects and the patients in the HD and GD groups. These frequencies did not differ between patients with severe HD and those with mild HD (Tables 4 and 5).
CYP2R1 polymorphism
The frequency of the AG genotype of the rs10741657 polymorphism was lower in patients with intractable GD than in those with GD in remission (P = 0·0268) (Table 5). We found no differences in the frequencies of the genotypes or alleles of this polymorphism between the control subjects and the patients in the HD and GD groups. These frequencies did not differ between patients with severe HD and those with mild HD (Tables 4 and 5).
Clinical characteristics and genotype frequencies
For the rs7975232 polymorphism, the proportion of GD patients whose anti-TRAb level at the onset of disease was >51% was significantly higher for patients with the CC genotype (58·3%) than in those with the AA + CA genotypes (16·7%) (P = 0·0065) (Fig. 1). However, we found no association between TRAb level, anti-thyroid McAb titre, anti-TgAb titre or goitre size and any other polymorphisms.
Fig. 1.
Serum levels of thyrotrophin receptor antibody (TRAb) at the onset of disease in Graves’ disease (GD) patients with different VDR rs7975232 genotypes. (a) We set the cut-off value for TRAb level at the onset of disease at 51%.
Discussion
In this study, the frequencies of the CC genotype and C allele for the VDR rs2228570 polymorphism, which were correlated with a lower concentration of serum vitamin D, were higher in the HD group than in control subjects (Table 4). These data are consistent with that of a previous report [23]. Because the C allele was also associated with a higher production of interleukin (IL)-12, which induces Th1 differentiation [32] and thyroid destruction in HD patients, possibly through cytotoxic T lymphocytes and Th1 cells [33,34], the CC genotype may be associated with the induction of autoimmune thyroid destruction. Conversely, the frequency of the C allele for the VDR rs7975232 polymorphism was higher in AITD patients, especially GD patients (Table 3). In a previous study, however, the frequency of this allele was lower in GD patients [22]. We randomly selected a sample of subjects and genotyped this polymorphism again using the direct sequencing method, and we confirmed all our results. Therefore, we concluded that the frequency of the C allele may be higher in GD patients, and our results may have been inconsistent with those of the previous study because of differences in the GD patient populations of the two studies. In our study, interestingly, the TRAb level at the onset of disease was significantly higher in GD patients with the CC genotype than in those with the AA + CA genotypes (Fig. 1). These results suggest that the C allele is associated with the development and activity of GD through the regulation of TRAb production. The frequency of the TT genotype for the rs731236 polymorphism, which was correlated with higher VDR expression, was lower and the frequency of the CC genotype for the rs2228570 polymorphism was higher in HD patients than in GD patients (Table 4). These results suggest that immune regulation by VDR may be suppressed in HD patients in comparison to GD patients. This was supported by the fact that the prevalence of vitamin D deficiency was greater in HD patients than in GD patients [35]. Therefore, genetic differences in the VDR gene may be a factor in the development of GD and HD.
For the GC polymorphism, the frequency of the Gc1Gc1 genotype, which was correlated with a higher vitamin D concentration, was lower in patients with intractable GD than in those with GD in remission (Table 5). It has been shown previously that vitamin D inhibits the differentiation of Th17 cells and promotes the generation of Treg cells [13]. The possible mechanisms of Th17 suppression may be reduction of IL-6, IL-17A and IL-23 production, and retinoic acid-related orphan nuclear receptor γT ((RORγt) expression [36]. IL-6, IL-17A, transforming growth factor (TGF)-β and RORγt were critical for Th17 differentiation and IL-23 maintains the activity of Th17 cells [37]. Therefore, the suppression of these factors may profoundly affect Th17 cells. In addition, we reported previously that the proportion of Th17 cells in AITD patients, especially in those with intractable GD, was higher than in controls [34]. Therefore, we propose that the suppression of Th17 by vitamin D may inhibit the intractability of GD. Interestingly, for the CYP2R1 rs10741657 polymorphism, the frequency of the AG genotype (heterozygote) was lower in patients with intractable GD than in those with GD in remission (Table 5). The functional difference between the heterozygosity and homozygosity of this polymorphism is still unclear, which indicates that heterozygosity has some unknown but considerable effects on the pathogenesis of GD. Supporting this possibility, we reported previously that the CA genotype (heterozygote) of the −3279C/A polymorphism in the forkhead box protein 3 (FoxP3) gene was associated with the intractability of GD [7].
A limitation of this study may be the small sample numbers. This is because we categorized patients very strictly, and excluded many obscure cases. In this study, however, we could find statistically significant differences between patients’ groups despite the moderate numbers of samples, and so we think that such differences would be major.
In conclusion, genetic differences in the VDR gene may be involved in the development of AITD and the activity of GD, whereas the genetic differences in the GC and CYP2R1 genes may be involved in the intractability of GD.
Disclosure
The authors have no conflicts of interest to declare.
References
- 1.Weetman AP. Chronic autoimmune thyroiditis. In: Braverman L, Utiger R, editors. The thyroid: a fundamental and clinical text. Philadelphia: Lippincott Williams & Wilkins; 2000. pp. 721–732. [Google Scholar]
- 2.Menconi F, Oppenheim YL, Tomer Y. Graves’ disease. In: Shoenfeld Y, Cervera R, Gershwin ME, editors. Diagnostic criteria in autoimmune diseases. Totowa, NJ: Humana Press; 2008. pp. 231–235. [Google Scholar]
- 3.Nanba T, Watanabe M, Akamizu T, Iwatani Y. The –590CC genotype in the IL4 gene as a strong predictive factor for the development of hypothyroidism in Hashimoto disease. Clin Chem. 2008;54:621–623. doi: 10.1373/clinchem.2007.099739. [DOI] [PubMed] [Google Scholar]
- 4.Ito C, Watanabe M, Okuda N, Watanabe C, Iwatani Y. Association between the severity of Hashimoto's disease and the functional +874A/T polymorphism in the interferon-gamma gene. Endocr J. 2006;53:473–478. doi: 10.1507/endocrj.k06-015. [DOI] [PubMed] [Google Scholar]
- 5.Hayashi F, Watanabe M, Nanba T, Inoue N, Akamizu T, Iwatani Y. Association of the –31C/T functional polymorphism in the interleukin-1beta gene with the intractability of Graves’ disease and the proportion of T helper type 17 cells. Clin Exp Immunol. 2009;158:281–286. doi: 10.1111/j.1365-2249.2009.04034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Inoue N, Watanabe M, Morita M, et al. Association of functional polymorphisms in promoter regions of IL5, IL6 and IL13 genes with development and prognosis of autoimmune thyroid diseases. Clin Exp Immunol. 2011;163:318–323. doi: 10.1111/j.1365-2249.2010.04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Inoue N, Watanabe M, Morita M, et al. Association of functional polymorphisms related to the transcriptional level of FOXP3 with prognosis of autoimmune thyroid diseases. Clin Exp Immunol. 2010;162:402–406. doi: 10.1111/j.1365-2249.2010.04229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Inoue N, Watanabe M, Nanba T, Wada M, Akamizu T, Iwatani Y. Involvement of functional polymorphisms in the TNFA gene in the pathogenesis of autoimmune thyroid diseases and production of anti-thyrotrophin receptor antibody. Clin Exp Immunol. 2009;156:199–204. doi: 10.1111/j.1365-2249.2009.03884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Tomizawa R, Watanabe M, Inoue N, et al. Association of functional GITR gene polymorphisms related to expression of glucocorticoid-induced tumour necrosis factor-receptor (GITR) molecules with prognosis of autoimmune thyroid disease. Clin Exp Immunol. 2011;165:141–147. doi: 10.1111/j.1365-2249.2011.04414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Inoue N, Watanabe M, Yamada H, et al. Associations between autoimmune thyroid disease prognosis and functional polymorphisms of susceptibility genes, CTLA4, PTPN22, CD40, FCRL3 and ZFAT, previously revealed in genome-wide association studies. J Clin Immunol. 2012;32:1243–1252. doi: 10.1007/s10875-012-9721-0. [DOI] [PubMed] [Google Scholar]
- 11.Morita M, Watanabe M, Inoue N, et al. Functional polymorphisms in TBX21 and HLX are associated with development and prognosis of Graves’ disease. Autoimmunity. 2011;45:129–136. doi: 10.3109/08916934.2011.622013. [DOI] [PubMed] [Google Scholar]
- 12.Almerighi C, Sinistro A, Cavazza A, Ciaprini C, Rocchi G, Bergamini A. 1Alpha,25-dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in human monocytes. Cytokine. 2009;45:190–197. doi: 10.1016/j.cyto.2008.12.009. [DOI] [PubMed] [Google Scholar]
- 13.Peelen E, Knippenberg S, Muris AH, et al. Effects of vitamin D on the peripheral adaptive immune system: a review. Autoimmun Rev. 2011;10:733–743. doi: 10.1016/j.autrev.2011.05.002. [DOI] [PubMed] [Google Scholar]
- 14.Smolders J, Peelen E, Thewissen M, et al. The relevance of vitamin D receptor gene polymorphisms for vitamin D research in multiple sclerosis. Autoimmun Rev. 2009;8:621–626. doi: 10.1016/j.autrev.2009.02.009. [DOI] [PubMed] [Google Scholar]
- 15.El Gawad SS, Samee ER, Metwali AA, El Gawad MS. Vitamin D receptor gene polymorphism and its association with 1,25-dihydroxyvitamin D3 in patients with graves disease in Egyptian population: a pilot study. Endocr Pract. 2011;18:132–139. doi: 10.4158/EP11131.OR. [DOI] [PubMed] [Google Scholar]
- 16.Correale J, Ysrraelit MC, Gaitan MI. Immunomodulatory effects of vitamin D in multiple sclerosis. Brain. 2009;132:1146–1160. doi: 10.1093/brain/awp033. [DOI] [PubMed] [Google Scholar]
- 17.Tamer G, Arik S, Tamer I, Coksert D. Relative vitamin D insufficiency in Hashimoto's thyroiditis. Thyroid. 2011;21:891–896. doi: 10.1089/thy.2009.0200. [DOI] [PubMed] [Google Scholar]
- 18.Guillot X, Semerano L, Saidenberg-Kermanac'h N, Falgarone G, Boissier MC. Vitamin D and inflammation. Joint Bone Spine. 2010;77:552–557. doi: 10.1016/j.jbspin.2010.09.018. [DOI] [PubMed] [Google Scholar]
- 19.Li K, Shi Q, Yang L, et al. The association of vitamin D receptor gene polymorphisms and serum 25-hydroxyvitamin D levels with generalized vitiligo. Br J Dermatol. 2012;167:815–821. doi: 10.1111/j.1365-2133.2012.11132.x. [DOI] [PubMed] [Google Scholar]
- 20.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;30:207–211. doi: 10.1016/j.jaut.2007.09.002. [DOI] [PubMed] [Google Scholar]
- 21.Agliardi C, Guerini FR, Saresella M, et al. Vitamin D receptor (VDR) gene SNPs influence VDR expression and modulate protection from multiple sclerosis in HLA-DRB1*15-positive individuals. Brain Behav Immun. 2011;25:1460–1467. doi: 10.1016/j.bbi.2011.05.015. [DOI] [PubMed] [Google Scholar]
- 22.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;85:4639–4643. doi: 10.1210/jcem.85.12.7038. [DOI] [PubMed] [Google Scholar]
- 23.Ban Y, Taniyama M, Ban Y. Vitamin D receptor gene polymorphisms in Hashimoto's thyroiditis. Thyroid. 2001;11:607–608. doi: 10.1089/105072501750302967. [DOI] [PubMed] [Google Scholar]
- 24.Chen RH, Chang CT, Chen HY, Chen WC, Tsai CH, Tsai FJ. Association between vitamin-D receptor gene FokI polymorphism and Graves’ disease among Taiwanese Chinese. J Clin Lab Anal. 2007;21:173–177. doi: 10.1002/jcla.20163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Ahn J, Yu K, Stolzenberg-Solomon R, et al. Genome-wide association study of circulating vitamin D levels. Hum Mol Genet. 2010;19:2739–2745. doi: 10.1093/hmg/ddq155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Wang TJ, Zhang F, Richards JB, et al. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet. 2010;376:180–188. doi: 10.1016/S0140-6736(10)60588-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Arnaud J, Constans J. Affinity differences for vitamin D metabolites associated with the genetic isoforms of the human serum carrier protein (DBP) Hum Genet. 1993;92:183–188. doi: 10.1007/BF00219689. [DOI] [PubMed] [Google Scholar]
- 28.Abbas S, Linseisen J, Slanger T, et al. The Gc2 allele of the vitamin D binding protein is associated with a decreased postmenopausal breast cancer risk, independent of the vitamin D status. Cancer Epidemiol Biomarkers Prev. 2008;17:1339–1343. doi: 10.1158/1055-9965.EPI-08-0162. [DOI] [PubMed] [Google Scholar]
- 29.Lauridsen AL, Vestergaard P, Hermann AP, et al. Plasma concentrations of 25-hydroxy-vitamin D and 1,25-dihydroxy-vitamin D are related to the phenotype of Gc (vitamin d-binding protein): a cross-sectional study on 595 early postmenopausal women. Calcif Tissue Int. 2005;77:15–22. doi: 10.1007/s00223-004-0227-5. [DOI] [PubMed] [Google Scholar]
- 30.Bu FX, Armas L, Lappe J, et al. Comprehensive association analysis of nine candidate genes with serum 25-hydroxy vitamin D levels among healthy Caucasian subjects. Hum Genet. 2010;128:549–556. doi: 10.1007/s00439-010-0881-9. [DOI] [PubMed] [Google Scholar]
- 31.Ramos-Lopez E, Bruck P, Jansen T, Herwig J, Badenhoop K. CYP2R1 (vitamin D 25-hydroxylase) gene is associated with susceptibility to type 1 diabetes and vitamin D levels in Germans. Diabetes Metab Res Rev. 2007;23:631–636. doi: 10.1002/dmrr.719. [DOI] [PubMed] [Google Scholar]
- 32.van Etten E, Verlinden L, Giulietti A, et al. The vitamin D receptor gene FokI polymorphism: functional impact on the immune system. Eur J Immunol. 2007;37:395–405. doi: 10.1002/eji.200636043. [DOI] [PubMed] [Google Scholar]
- 33.Watanabe M, Yamamoto N, Maruoka H, et al. Independent involvement of CD8+ CD25+ cells and thyroid autoantibodies in disease severity of Hashimoto's disease. Thyroid. 2002;12:801–808. doi: 10.1089/105072502760339370. [DOI] [PubMed] [Google Scholar]
- 34.Nanba T, Watanabe M, Inoue N, Iwatani Y. Increases of the Th1/Th2 cell ratio in severe Hashimoto's disease and in the proportion of Th17 cells in intractable Graves’ disease. Thyroid. 2009;19:495–501. doi: 10.1089/thy.2008.0423. [DOI] [PubMed] [Google Scholar]
- 35.Kivity S, Agmon-Levin N, Zisappl M, et al. Vitamin D and autoimmune thyroid diseases. Cell Mol Immunol. 2011;8:243–247. doi: 10.1038/cmi.2010.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Zhang H, Shih DQ, Zhang X. Mechanisms underlying effects of 1,25-dihydroxyvitamin D3 on the Th17 cells. Eur J Microbiol Immunol (Bp) 2013;3:237–240. doi: 10.1556/EuJMI.3.2013.4.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol. 2009;27:485–517. doi: 10.1146/annurev.immunol.021908.132710. [DOI] [PubMed] [Google Scholar]