Abstract
The severity of Hashimoto's disease (HD) and intractability (or inducibility to remission) of Graves' disease (GD) varies among patients. Forkhead box P3 (FoxP3) is a crucial regulatory factor for the development and function of regulatory T (Treg) cells, and deficiency of the FoxP3 gene (FOXP3) suppresses the regulatory function of Treg cells. To clarify the association of the functional polymorphisms of the FOXP3 with the prognosis of GD and HD, we genotyped −3499A/G, −3279C/A and −2383C/T polymorphisms in FOXP3 gene obtained from 38 patients with severe HD, 40 patients with mild HD, 65 patients with intractable GD, in whom remission was difficult to induce, 44 patients with GD in remission and 71 healthy volunteers. The −3279CA genotype was more frequent in patients with GD in remission than in patients with intractable GD, and the −3279AA genotype, which correlates to defective transcription of FOXP3, was absent in patients with GD in remission. The −2383CC genotype was more frequent in patients with severe HD than in those with mild HD. In conclusion, the −3279A/C polymorphism is related to the development and intractability of GD and the −2383CC genotype to the severity of HD.
Keywords: disease intractability, disease severity, FoxP3, regulatory T cell, single nucleotide polymorphism
Introduction
Autoimmune thyroid diseases (AITDs), such as Hashimoto's disease (HD) and Graves' disease (GD), are archetypes for organ-specific autoimmune diseases [1,2]. The severity of HD and intractability (that is, inducibility to remission) of GD vary among patients. Some patients develop hypothyroidism in early life, whereas others maintain a euthyroid state into old age [3,4]. Some patients with GD achieve remission through medical treatment, while others do not. However, the severity of HD and intractability of GD are difficult to predict at the first diagnosis. We have reported previously that hypothyroidism caused by severe destruction of thyroid tissue is more likely to develop in HD patients with a genetically higher production of interferon (IFN)-γ[5] or a genetically lower production of interleukin (IL)-4 [6].
Regulatory T (Treg) cells are known to play an important role in immune regulation. In a mouse model of GD, Treg cells appear to be crucial in the pathogenesis of GD [7]. Moreover, we have reported previously an apoptosis-induced decrease in the proportion of intrathyroidal Treg cells in patients with AITD [8]. Therefore, we consider that Treg may be involved in preventing the development of AITDs.
Forkhead box P3 (FoxP3) is a major regulatory factor for the development and functioning of Treg cells [9]. FoxP3 controls Treg function through co-operation with nuclear factor of activated T cells (NFAT) [10], and deficiency of the FoxP3 gene (FOXP3) impairs the suppressor function of Treg cells [11]. Some microsatellites in FOXP3 were associated with susceptibility to AITD in Caucasians, but these associations were not observed in the Japanese population [12]. There are five single nucleotide polymorphisms (SNP) in the promoter region of FOXP3. These are −924A/G (rs2232365), −1383C/T (rs2232364), −2383C/T (rs3761549), −3279C/A (rs3761548) and −3499A/G (rs3761547) polymorphisms [13], and they may affect the expression of FOXP3. In the present study, we focused upon −2383C/T, −3279C/A and −3499A/G polymorphisms, as these are common in the Japanese population according to the National Center for Biotechnology Information (NCBI) SNP (http://www.ncbi.nlm.nih.gov/snp/) and Japanese (J)-SNP databases (http://snp.ims.u-tokyo.ac.jp/). The AA genotype of the −3279C/A polymorphism causes the loss of binding with some transcription factors, such as E47 and C-Myb, leading to defective transcription of FOXP3[14]. Moreover, the A allele of this polymorphism is associated with a dramatic reduction in luciferase activity compared with the C allele [14]. Conversely, there is no report on the functional effects of −2383C/T and −3499A/G polymorphisms on gene expression.
In this study, we genotyped these three polymorphisms in Japanese patients to clarify their effects on the development and prognosis of AITDs.
Materials and methods
Subjects
Genomic DNA was obtained from 38 patients with HD who developed moderate to severe hypothyroidism before 50 years of age and were treated daily with at least 1·5 µg thyroxine per kg body weight (severe HD), and from 40 untreated, euthyroid patients with HD who were more than 50 years of age (mild HD). All patients with HD were positive for anti-thyroid microsomal antibody (McAb) or anti-thyroglobulin antibody (TgAb) and all patients with mild HD had palpable diffuse goitre. Also examined were 65 euthyroid patients with GD who had been treated with methimazole or propylthiouracil for at least 5 years and were still positive for TRAb (intractable GD), 44 GD patients in remission who had maintained a euthyroid state and were negative for TRAb for more than 2 years without medication (GD in remission) and 71 healthy volunteers (control subjects), who were euthyroid and were negative for thyroid autoantibodies, were also examined. All patients and control subjects were Japanese and unrelated. All patients were followed-up closely for more than 5 years as out-patients at our thyroid clinic. Patients with AITD did not suffer from other autoimmune diseases. The clinical characteristics of groups of patients at the time of sampling and the ages of onset were shown in Table 1. The titres of TgAb were significantly higher in patients with severe HD than in those with mild HD, as our previous report [15].
Table 1.
Clinical characteristics of groups of patients with autoimmune thyroid disease at the time of sampling and the age of onset
| Graves' disease | Hashimoto's disease | ||||
|---|---|---|---|---|---|
| Presence of the past clinical history of thyrotoxicosis with elevated TRAb | Presence of the diffuse goitre and the positive TgAb and/or McAb | ||||
| Controls | Intractable | In remission | Severe | Mild | |
| Female/male (n) | 71 (61/10) | 65 (57/8) | 44 (40/4) | 38 (33/5) | 40 (34/6) |
| Age of onset (years) | 44·1 ± 12·2† | 35·2 ± 14·7 | 29·0 ± 12·7 | 37·3 ± 11·1 | 58·7 ± 8·9† |
| (range) | (21–67)† | (10–49) | (15–66) | (10–49) | (50–79)† |
| FreeT4 (ng/dl) | n.d. | 1·38 ± 0·68 | 1·35 ± 0·63 | 1·42 ± 0·24 | 1·43 ± 0·61 |
| FreeT3 (pg/ml) | n.d. | 4·12 ± 3·42 | 3·20 ± 1·62 | 2·82 ± 0·38 | 3·28 ± 1·42 |
| TSH (µU/ml) | n.d. | 2·59 ± 2·63 | 2·43 ± 3·47 | 1·84 ± 2·04 | 1·99 ± 1·43 |
| TRAb (IU/l) (range) | < 1·0 | 7·3 ± 12·5 (1·1–71·0) | < 1·0 | < 1·0 | < 1·0 |
| TgAb (2n × 100) | Negative | 2·9 ± 0·6 | 1·7 ± 1·5 | 7·7 ± 3·1* | 1·7 ± 2·7 |
| McAb (2n × 100) | Negative | 4·6 ± 2·6 | 5·3 ± 2·2 | 6·1 ± 2·7* | 2·4 ± 0·8 |
| Treatment | None | Methimazole or PTU | None | L-thyroxine | None |
P < 0·01 versus mild Hashimoto's disease.
Age at the time of sampling.
Data are expressed as mean ± standard deviation. n.d.: not determined; T4: thyroxine; T3: triiodothyronine; TSH: thyrotropin; PTU: propylthiouracil; TRAb: anti-thyrotropin receptor antibody; McAb: anti-thyroid microsomal antibody; TgAb: anti-thyroglobulin antibody.
Genomic DNA was isolated from ethylenediamine tetraacetic acid (EDTA)-treated peripheral blood mononuclear cells with a commercially available kit (DrGenTLE™; Takara Bio Inc., Shiga, Japan). Written informed consent was obtained from all patients and controls, and the study protocol was approved by the Ethics Committee of Osaka University.
Genotyping of −2383C/T polymorphism
The target sequence of FOXP3 gene was amplified using polymerase chain reaction (PCR). The forward primer was 5′-GCCTGGCACTCTCAGAGCTT-3′, the reverse primer was 5′-GTCTGTGGAGGCTCCGAACA-3′. The protocol for the PCR was as follows: 95°C for 3 min and 30 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min and a single final extension at 72°C for 10 min. The PCR product [942 base pairs (bp)] was digested by addition of BsrI and incubation at 65°C for 6 h.
Genotyping of −3279C/A polymorphism
The target sequence of the FOXP3 gene was amplified using PCR. The forward primer was 5′-CCTCTCCGTGCTCAGTGTAG-3′, the reverse primer was 5′-CTCACCTAGCCCAGCTCTTG-3′. The protocol for the PCR was as follows: 94°C for 5 min and 30 cycles of denaturing at 94°C for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 30 s and a single final extension at 72°C for 7 min. The PCR product (300 bp) was digested by addition of PstI and incubation at 37°C for 6 h.
Genotyping of −3499A/G polymorphism
The target sequence of the FOXP3 gene was amplified using PCR. The forward primer was 5′-CTCTGGCTCTCCATGCATGT-3′, the reverse primer was 5′-TGCAGGGCTTCAAGTTGACAG-3′. The protocol for the PCR was as follows: 94°C for 5 min and 30 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s and a single final extension at 72°C for 7 min. The PCR product (158 bp) was digested by addition of PvuII and incubation at 37°C for 6 h.
Thyroid function and autoantibodies
The serum concentration of free T4 (FT4) was measured with a commercial radioimmunoassay kit (Eiken Chemical Co. Ltd, Tokyo, Japan). The normal range of serum FT4 is 1·0–1·6 ng/dl (12·9–20·6 pmol/l). The serum concentration of free T3 (FT3) was measured with a radioimmunoassay kit (Japan Kodak diagnostic Co. Ltd, Tokyo, Japan). The normal range of serum FT3 is 2·4–4·6 pg/ml (3·8–7·2 pmol/l). The serum TSH concentration was also measured with a radioimmunoassay kit (Daiichi Radioisotope Laboratories Ltd, Tokyo, Japan). The normal range of serum TSH is 0·6–5·4 µU/ml. TgAb and McAb were measured with a particle agglutination kit (Fujirebio Inc., Tokyo, Japan). A reciprocal titre of > 1:100 was considered positive. Serum level of TRAb was determined with an enzyme-linked immunosorbent assay (ELISA) (third-generation) (Cosmic Co., Tokyo, Japan). The normal value of TRAb was less than 1·0 IU/l.
Statistical analysis
We used χ2 tests and Fisher's exact test to evaluate the significance of differences in frequencies of genotype and allele among the subject groups. Student's t-test was used to analyse difference in goitre size. A Mann–Whitney U-test was used to analyse differences in serum titres of McAb, TgAb and TRAb. Data were analysed with jmp8 software (SAS Institute, Inc., Tokyo, Japan). Probability values of less than 0·05 were considered significant.
Results
−2383C/T polymorphism
The frequency of this polymorphism did not differ between normal subjects and each group of patients with HD and GD (Table 2).
Table 2.
Genotype and allele frequencies of the forkhead box P3 polymorphisms in patients with Graves' disease, Hashimoto's disease and in control subjects
| Graves' disease | Hashimoto's disease | |||||||
|---|---|---|---|---|---|---|---|---|
| Control | Intractable | In remission | Severe | Mild | ||||
| −2383C/T | CC | 38 (61·2%) | 36 (62·1%) | 20 (69·0%) | 23 (67·6%) | 16 (41·0%) | ||
| CT | 19 (30·7%) | 16 (27·6%) | 8 (27·6%) | n.s.† | 9 (26·5%) | 20 (51·3%) | n.s.‡ | |
| TT | 5 (8·1%) | 6 (10·3%) | 1 (3·4%) | 2 (5·9%) | 3 (7·7%) | |||
| CC | 38 (61·2%) | 36 (62·1%) | 20 (69·0%) | n.s.† | 23 (67·6%) | 16 (41·0%) | P = 0·0220‡ | |
| CT + TT | 24 (38·8%) | 22 (37·9%) | 9 (31·0%) | 11 (32·4%) | 23 (59·0%) | OR = 3·01 (1·15–7·86) | ||
| C allele | 95 (76·6%) | 88 (75·9%) | 48 (82·8%) | n.s.† | 55 (79·7%) | 52 (66·7%) | n.s.‡ | |
| T allele | 29 (23·4%) | 28 (24·1%) | 10 (17·2%) | 13 (20·3%) | 26 (33·3%) | |||
| −3279C/A | CC | 58 (81·7%) | 44 (71·0%) | 20 (55·6%) | 31 (81·5%) | 26 (76·5%) | ||
| CA | 8 (11·3%) | 11 (17·7%) | 16 (44·4%) | P = 0·0016† | 5 (13·2%) | 7 (20·6%) | n.s.‡ | |
| AA | 5 (7·0%) | 7 (11·3%) | 0 (0%) | 2 (5·3%) | 1 (2·9%) | |||
| AA | 5 (7·0%) | 7 (11·3%) | 0 (0%) | P = 0·0449†§ | 2 (5·3%) | 1 (2·9%) | n.s.‡§ | |
| CA + CC | 66 (93·0%) | 55 (88·7%) | 36 (100%) | 36 (94·7%) | 33 (97·1%) | |||
| CA | 8 (11·3%) | 11 (17·7%) | 16 (44·4%) | P = 0·0048† | 5 (13·2%) | 7 (20·6%) | n.s.‡ | |
| AA + CC | 63 (88·7%) | 51 (82·3%) | 20 (55·6%) | OR = 3·71 (1·47–9·36) | 33 (86·8%) | 27 (79·4%) | ||
| A allele | 18 (12·7%) | 25 (19·7%) | 16 (22·2%) | n.s.† | 9 (12·5%) | 9 (14·5%) | n.s.‡ | |
| C allele | 124 (87·3%) | 99 (80·3%) | 56 (77·8%) | 63 (87·5%) | 53 (85·5%) | |||
| −3499A/G | AA | 46 (65·7%) | 37 (56·9%) | 32 (72·7%) | 20 (62·5%) | 19 (47·5%) | ||
| AG | 20 (28·6%) | 22 (33·9%) | 11 (25·0%) | n.s.† | 11 (34·4%) | 18 (45·0%) | n.s.‡ | |
| GG | 4 (5·7%) | 6 (9·2%) | 1 (2·3%) | 1 (3·1%) | 3 (7·5%) | |||
| AA | 46 (65·7%) | 37 (56·9%) | 32 (72·7%) | n.s.† | 20 (62·5%) | 19 (47·5%) | n.s.‡ | |
| AG + GG | 24 (34·3%) | 28 (43·1%) | 12 (27·3%) | 12 (37·5%) | 21 (52·5%) | |||
| A allele | 112 (80·0%) | 96 (73·8%) | 75 (85·2%) | n.s.† | 51 (79·7%) | 56 (70·0%) | n.s.‡ | |
| G allele | 28 (20·0%) | 34 (26·2%) | 13 (14·8%) | 13 (20·3%) | 24 (30·0%) | |||
Analysed by χ2 tests or intractable Graves' disease versus Graves' disease in remission.
Severe Hashimoto's disease versus mild Hashimoto's disease.
Fisher's exact test.
n.s.: not significant; OR: odds ratio (95% confidence interval).
The CC genotype was significantly frequent in the patients with severe HD than in those with mild HD [P = 0·0220, odds ratio (OR) = 3·01] (Table 2). However, we found no difference in genotypes and allele frequencies of this polymorphism between in the patients with intractable GD and those with GD in remission (Table 2).
−3279C/A polymorphism
Genotypic distribution was significantly different between intractable GD and GD in remission groups (P = 0·0016) and the AA genotype was found in 11·3% of patients with intractable GD, whereas the proportion of AA genotype was 0% in patients with GD in remission. Moreover, the frequency of CA genotype was significantly higher in patients with GD in remission than in those with intractable GD (P = 0·0048, OR = 3·71) (Table 2).
Conversely, we found no significant difference in the genotype and allele frequencies between two HD groups (Table 2).
−3499A/G polymorphism
We found no difference in genotype and allele frequencies of −3499 A/G polymorphism among any groups (Table 2).
Association between each polymorphism and levels of autoantibody
We found no association each of TRAb levels, McAb titres and TgAb titres with examined polymorphisms (data not shown).
Discussion
Because Treg cells play a role in suppressing the development of autoimmune diseases by inhibiting autoreactive T cells [16] and FOXP3 is a master gene of Treg cells, we expected that FOXP3 genotypes related to a lower expression of FoxP3 would be frequent in AITD patients.
In the case of the FOXP3−3279C/A polymorphism, the A allele correlates with a reduction in FoxP3 expression [14]. Interestingly, we determined that 11·3% of patients with intractable GD had the −3279AA genotype, which shows the lowest production of FoxP3 among the three genotypes of this polymorphism [14], whereas this genotype was absent in patients with GD in remission (Table 2). Therefore, GD patients with the −3279AA genotype may have Treg cells with weak suppressor activity, and as FoxP3 controls Treg function [10] it may be difficult for such patients to achieve remission. These results are consistent with our previous report, indicating a decrease in the proportion of intrathyroidal Treg cells in patients with intractable GD [8]. Therefore, the −3279AA genotype may contribute to the severity of the immune response. Surprisingly, in addition, the −3279CA genotype (heterozygote) was more frequent in patients with GD in remission than in patients with intractable GD (P = 0·0048, OR = 3·71) (Table 2). Although the functional significance of the heterozygous −3279AC genotype of FOXP3 is still unclear, this heterozygosity may itself have unknown but considerable effects on GD pathogenesis. This possibility is supported by the previous finding that the FOXP3−3279CA genotype was also more frequent in patients with allergic rhinitis [17] which, like GD, is caused by antibodies.
For the −2383C/T polymorphism, the distribution of alleles and genotypes did not differ between normal subjects and GD patients in this study. These distributions also did not differ between normal subjects and GD patients in a UK population [18], suggesting that this polymorphism may have no effect on the pathogenesis of GD. However, in our study, the −2383CC genotype was significantly more frequent in patients with severe HD than in those with mild HD (P = 0·0220, OR = 3·01) (Table 2). Because the functional differences of each allele in this polymorphism are still unclear, we used p-Match software [19] to predict the binding sites, and attempted to estimate the effect of this polymorphism. The result of this search indicated that the sequence of the −2383T allele may be essential for the binding of a transcriptional factor Yin-Yang-1 (YY1) with FOXP3. The consensus YY-1 motif is CATNTWN (‘N’ representing any nucleotide and ‘W’ representing A or T); the −2383T allele creates the DNA sequence CAGCTAG and the −2383C allele creates CAGCCAG. As the number of mismatched bases compared with the YY-1 motif is lower for the T allele of this polymorphism than for the C allele, YY-1 may bind this sequence more strongly in the −2383T allele than in the C allele. In addition, YY-1 also binds to the promoter region and enhances promoter activity in IL4[20]. Therefore, we assumed that individuals with the CC genotype of the −2383C/T polymorphism might exhibit a lower expression of FoxP3, resulting in weak suppressor function of Treg cells. This decrease in regulatory function may increase the activity of autoreactive T cells and may cause severe autoimmune destruction of thyroid tissue in HD patients with the CC genotype. Although some interesting significances were found in this study, it may be necessary to examine a larger number of samples to perform a more detailed analysis, such as haplotype analysis, using these polymorphisms. This limitation of sample numbers was due to very strict criteria for selection of samples to exclude borderline cases and enhance the study reliability.
In conclusion, functional polymorphisms of FOXP3 are associated with the prognosis of AITDs. The −3279A/C polymorphism is related to the development and intractability of GD, and the CC genotype of the −2383C/T polymorphism is related to the severity of HD.
Acknowledgments
This study was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Science and Culture of Japan.
Disclosure
The authors have no financial conflicts of interest.
References
- 1.Volpe R. The immune system and its role in endocrine function. In: Becker KL, editor. Principles and practice of endocrinology and metabolism. Philadelphia, PA: Lippincott Williams & Wilkins; 2001. pp. 1770–81. [Google Scholar]
- 2.Davies TF. Pathogenesis of Graves' disease. In: Braverman LE, Utiger RD, editors. The thyroid: a fundamental and clinical text. Philadelphia, PA: Lippincott Williams & Wilkins; 2000. pp. 518–31. [Google Scholar]
- 3.Amino N, Hagen SR, Yamada N, Refetoff S. Measurement of circulating thyroid microsomal antibodies by the tanned red cell haemagglutination technique: its usefulness in the diagnosis of autoimmune thyroid diseases. Clin Endocrinol (Oxf) 1976;5:115–25. doi: 10.1111/j.1365-2265.1976.tb02822.x. [DOI] [PubMed] [Google Scholar]
- 4.Yoshida H, Amino N, Yagawa K, et al. Association of serum antithyroid antibodies with lymphocytic infiltration of the thyroid gland: studies of seventy autopsied cases. J Clin Endocrinol Metab. 1978;46:859–62. doi: 10.1210/jcem-46-6-859. [DOI] [PubMed] [Google Scholar]
- 5.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-γ gene. Endocr J. 2006;53:473–8. doi: 10.1507/endocrj.k06-015. [DOI] [PubMed] [Google Scholar]
- 6.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;54:621–3. doi: 10.1373/clinchem.2007.099739. [DOI] [PubMed] [Google Scholar]
- 7.Saitoh O, Nagayama Y. Regulation of Graves' hyperthyroidism with naturally occurring CD4+CD25+ regulatory T cells in a mouse model. Endocrinology. 2006;147:2417–22. doi: 10.1210/en.2005-1024. [DOI] [PubMed] [Google Scholar]
- 8.Nakano A, Watanabe M, Iida T, et al. Apoptosis-induced decrease of intrathyroidal CD4+CD25+ regulatory T cells in autoimmune thyroid diseases. Thyroid. 2007;17:25–31. doi: 10.1089/thy.2006.0231. [DOI] [PubMed] [Google Scholar]
- 9.Yagi H, Nomura T, Nakamura K, et al. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol. 2004;16:1643–56. doi: 10.1093/intimm/dxh165. [DOI] [PubMed] [Google Scholar]
- 10.Wu Y, Borde M, Heissmeyer V, et al. FOXP3 controls regulatory T cell function through cooperation with NFAT. Cell. 2006;126:375–87. doi: 10.1016/j.cell.2006.05.042. [DOI] [PubMed] [Google Scholar]
- 11.Okumura A, Ishikawa T, Sato S, et al. Deficiency of forkhead box P3 and cytotoxic T-lymphocyte-associated antigen-4 gene expressions and impaired suppressor function of CD4CD25 T cells in patients with autoimmune hepatitis. Hepatol Res. 2008;38:896–903. doi: 10.1111/j.1872-034X.2008.00349.x. [DOI] [PubMed] [Google Scholar]
- 12.Ban Y, Tozaki T, Tobe T, et al. The regulatory T cell gene FOXP3 and genetic susceptibility to thyroid autoimmunity: an association analysis in Caucasian and Japanese cohorts. J Autoimmun. 2007;28:201–7. doi: 10.1016/j.jaut.2007.02.016. [DOI] [PubMed] [Google Scholar]
- 13.Bassuny WM, Ihara K, Sasaki Y, et al. A functional polymorphism in the promoter/enhancer region of the FOXP3/Scurfin gene associated with type 1 diabetes. Immunogenetics. 2003;55:149–56. doi: 10.1007/s00251-003-0559-8. [DOI] [PubMed] [Google Scholar]
- 14.Shen Z, Chen L, Hao F, et al. Intron-1 rs3761548 is related to the defective transcription of Foxp3 in psoriasis through abrogating E47/c-Myb binding. J Cell Mol Med. 2008;14:226–41. doi: 10.1111/j.1582-4934.2008.00370.x. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 15.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;12:801–8. doi: 10.1089/105072502760339370. [DOI] [PubMed] [Google Scholar]
- 16.Aricha R, Feferman T, Fuchs S, Souroujon MC. Ex vivo generated regulatory T cells modulate experimental autoimmune myasthenia gravis. J Immunol. 2008;180:2132–9. doi: 10.4049/jimmunol.180.4.2132. [DOI] [PubMed] [Google Scholar]
- 17.Zhang L, Zhang Y, Desrosiers M, et al. Genetic association study of FOXP3 polymorphisms in allergic rhinitis in a Chinese population. Hum Immunol. 2009;70:930–4. doi: 10.1016/j.humimm.2009.08.001. [DOI] [PubMed] [Google Scholar]
- 18.Owen CJ, Eden JA, Jennings CE, et al. Genetic association studies of the FOXP3 gene in Graves' disease and autoimmune Addison's disease in the United Kingdom population. J Mol Endocrinol. 2006;37:97–104. doi: 10.1677/jme.1.02072. [DOI] [PubMed] [Google Scholar]
- 19.Chekmenev D, Haid C, Kel A. P-Match version 1.0. Available at: http://www.gene-regulation.com/cgi-bin/pub/programs/pmatch/bin/p-match.cgi (accessed 14 April 2010) [DOI] [PMC free article] [PubMed]
- 20.Guo J, Casolaro V, Seto E, et al. Yin-Yang 1 activates interleukin-4 gene expression in T cells. J Biol Chem. 2001;276:48871–8. doi: 10.1074/jbc.M101592200. [DOI] [PubMed] [Google Scholar]