The +869T/C polymorphism in the transforming growth factor-β1 gene is associated with the severity and intractability of autoimmune thyroid disease

Abstract

The severity of Hashimoto's disease (HD) and the intractability of Graves' disease (GD) vary among patients. To clarify whether the +869T/C polymorphism in the transforming growth factor-β1 (TGF-β1) gene, which is associated with TGF-β1 expression, is involved in the intractability of GD and severity of HD, we genotyped the TGF-β1 +869T/C polymorphism by polymerase chain reaction–restriction fragment length polymorphism method in genomic DNA samples from 33 patients with HD who developed hypothyroidism before they were 50 years old (severe HD) and 30 untreated, euthyroid patients with HD who were older than 50 years (mild HD). We also examined 48 euthyroid patients with GD who had been under treatment and were still positive for anti-thyrotropin receptor antibodies (intractable GD), 20 euthyroid patients with GD in remission and 45 healthy controls. The frequency of the T allele and the TT genotype were higher in patients with severe HD than in those with in mild HD. In contrast, the frequency of the CC genotype was higher in patients with intractable GD than in patients with GD in remission. In conclusion, the +869T/C polymorphism in the TGF-β1 gene is associated with the severity and intractability of autoimmune thyroid disease.

Introduction

Autoimmune thyroid diseases (AITD), such as Graves' disease (GD) and Hashimoto's disease (HD), are archetypes for organ-specific autoimmune diseases [1, 2]. The intractability of GD and the severity of HD vary among patients. Some patients with GD achieve remission through medical treatment, but others do not. Most patients with HD maintain a lifetime euthyroid state without any medical treatment, whereas others become hypothyroid. Although the immunological differences that underlie the differences in intractability or severity remain unclear, we have reported previously that CD8⁺ CD25⁺-activated cytotoxic T lymphocytes are involved in the severity of HD [3] and that the frequency of the T allele of the +874A/T polymorphism in the interferon (IFN)-γ gene, which is associated with high IFN-γ production, is higher in patients with severe HD than in those with mild HD [4]. It was suggested that increased IFN-γ, a major cytokine produced by T helper type 1 (Th1) cells [5], activates cytotoxic T lymphocytes and enhances cell-mediated cytotoxicity in severe HD [3, 4].

Transforming growth factor-β1 (TGF-β1) is important to induce and maintain tolerance against several self-antigens [6]. The human TGF-β1 gene, which is located on chromosome 19q13, contains seven exons that give rise to a precursor protein of 390 amino acids [7]. TGF-β1 inhibits the proliferation of T and B cells and the generation of T cell cytotoxicity [8, 9]. Furthermore, TGF-β1 also inhibits cytokine production and decreases the expression of human leucocyte class II antigens (HLA) induced by IFN-γ[10].

There is a polymorphism in the TGF-β1 gene, +869T/C in the region encoding the signal peptide, that results in a Leu to Pro substitution at amino acid position 10. The C allele (Pro) of this polymorphism shows a 2·8-fold higher secretion of TGF-β1 than the T allele (Leu) in HeLa cells [11], and the C allele is also associated with high serum concentrations of TGF-β1 [12]. This difference in serum concentration is more apparent in the CC homozygote than in the CT and TT homozygotes. Although weak associations were reported between the TGF-β1 +869T/C polymorphism and susceptibilities to rheumatoid arthritis [13] and systemic sclerosis [14], no association was observed for AITD [15]. Furthermore, the serum concentration of TGF-β1 is not associated with susceptibility to GD [16].

Because TGF-β1 modulates the immune system systematically, and its effect is not specific to the thyroid, this polymorphism may be associated with the intractability of GD and the severity of HD rather than the susceptibility to these diseases. Therefore, in the present study we investigated the association between this polymorphism and the intractability and severity of AITD.

Subjects and methods

Subjects

We screened for the +869T/C polymorphism in 48 euthyroid patients (five men and 43 women; 50·2 ± 16·1 years old) with GD who had been treated with 5–15 mg/day of methimazole for at least 5 years and were still positive for anti-thyrotropin receptor antibody (TRAb) (intractable GD) and in 20 patients (one man and 19 women; 48·9 ± 13·0 years old) with GD in remission who had maintained a euthyroid state and were negative for TRAb for more than 5 years without medication (GD in remission). We also examined 33 patients (four men and 29 women; 52·1 ± 9·9 years old) with HD who developed moderate to severe hypothyroidism before the age of 50 years and were treated daily with at least 1·5 μg thyroxine per kg body weight (severe HD) and 30 untreated, euthyroid patients (one man and 29 women; 56·2 ± 6·6 years old) with HD who were older than 50 years (mild HD). All patients with HD were positive for anti-thyroid microsomal antibody (McAb). Because the prevalence of hypothyroidism caused by autoimmune thyroid destruction increases with age [17], we classified HD patients who developed hypothyroidism before the age of 50 years, as severe HD patients and euthyroid HD patients who had not developed hypothyroidism by 50 years of age as mild HD patients. Patients with transient hypothyroidism were excluded. All patients did not have any other autoimmune diseases and autoantibodies. Forty-five healthy volunteers (five men and 40 women; 47·6 ± 12·2 years old) who were euthyroid and negative for thyroid autoantibodies were included as control subjects. All patients and control subjects were Japanese and unrelated. Written informed consent was obtained from all patients and control subjects, and the study protocol was approved by the Ethics Committee of Osaka University (Osaka, Japan).

Genotyping of the +869T/C polymorphism in the TGF-β1 gene

Genomic DNA was isolated from 1 ml of ethylenediamine tetraacetic acid (EDTA)-treated peripheral blood with a commercially available kit (Dr GenTLE™, Takara Bio Inc., Shiga, Japan). Genotyping was performed with the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Briefly, isolated genomic DNA was amplified with primers 5^′-AGGTTATTTCCGTGGGATAC-3′ (forward) and 5′-ATAGTCTTGCAGGTGGATAG-3′ (reverse). Amplification conditions consisted of one cycle of 94°C for 4 min followed by 35 cycles of 95°C for 30, 50°C for 30 and 72°C for 1 min. The PCR products [300 base pairs (bp)] were then digested with restriction enzyme MspA1 I (New England Biolabs, Beverly, MA, USA) for 4 h at 37°C. The T allele was recognized by two fragments of 244 and 64 bp, while the C allele showed three bands of 232, 66 and 12 bp. DNA fragment length polymorphisms were visualized by UV transillumination after electrophoretic separation on an 8% polyacryamide gel. Genotyping of this polymorphism was confirmed by the direct sequencing method and PCRs were repeated to confirm initial results in all samples.

Assay of thyroid function and autoantibody levels

The serum concentration of free T4 (FT4) was measured with a 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. Anti-thyrotropin receptor antibodies (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 was measured with a radioreceptor assay (Cosmic Co., Tokyo, Japan); results are expressed as percentage inhibition of binding of labelled TSH. The normal value is less than 10%.

Statistical analysis

All patients and control subjects were categorized into three groups on the basis of the +869T/C genotype: TT, CT or CC. We used the χ² test to evaluate the significance of differences between genotype frequencies in the two GD groups, and between those in the two HD groups. The number of T and C alleles was calculated in each patient group and in the control group. We used Fisher's exact test to evaluate the significance of differences between the allele frequencies in the two HD groups, between those in the two GD groups, and between those in each patient group and the control group. Data were analysed with JMP6 software (SAS Institute, Inc., Tokyo, Japan). A probability (P) values of less than 0·05 were considered significant.

Results

Statistical analysis revealed a significant association between the +869T/C polymorphism in the TGF-β1 gene and the pathological differences of HD and GD (Table 1). The frequency of the T allele was higher in patients with severe HD than in patients with mild HD (Table 1). The frequency of the TT genotype was higher in patients with severe HD than in patients with mild HD (Table 1).However, serum titres of McAb and TgAb did not differ significantly among the three groups with respect to the genotype (data not shown).

Table 1.

Genotype and allele frequencies of the +869T/C polymorphism in patients with Hashimoto's disease (HD), in patients with Graves' disease (GD), and in control subjects.

	HD				GD
	Severe (n = 33)	Mild (n = 30)	P-value	Odds ratio (95% CI)	Intractable (n = 48)	In remission (n = 20)	P-value	Odds ratio (95% CI)	Control (n = 45)
Genotype
CC	6 (18·2%)	10 (33·3%)	0·0104*		13 (27·1%)	1 (5·0%)	0·0457*		11 (24·4%)
CT	13 (39·4%)	17 (56·7%)			19 (39·6%)	13 (65·0%)			21 (46·7%)
TT	14 (42·4%)	3 (10·0%)			16 (33·3%)	6 (30·0%)			13 (28·9%)
Allele
C	25 (37·9%)	37 (61·7%)	0·0063†	2·6 (1·2–5·4)	45 (46·9%)	15 (37·5%)	n.s.†	1·5 (0·7–3·1)	43 (47·8%)
T	41 (62·1%)	23 (38·3%)			51 (53·1%)	25 (62·5%)			47 (52·2%)

^*Value calculated by the χ² test.

^†Value calculated by Fisher's exact test. *^†The P-values were calculated in comparisons within the disease group. CI, confidence interval; n.s., not significant.

In GD patients, the frequency of the CC genotype was significantly higher in patients with intractable GD than in patients with GD in remission (Table 1), although there was no significant difference in the frequency of the C allele between patients with intractable GD and those with GD in remission (Table 1). Serum levels of TRAb did not differ significantly among the three groups with respect to genotype (data not shown).

Discussion

In the present study, we examined allele and genotypes frequencies of the +869T/C functional polymorphism in the TGF-β1 gene in Japanese patients with AITD and found that this polymorphism is associated with the occurrence of hypothyroidism in HD patients and disease intractability in GD. The frequency of the +869T allele was significantly higher in patients with severe HD than in those with mild HD (Table 1), and the frequency of the TT genotype was significantly lower in patients with mild HD than in those with severe HD (Table 1). In vitro transfection experiments that examined the change in TGF-β1 secretion by substitution of proline for leucine at 10 amino acid position in the signal peptide sequence of TGF-β1 [11] revealed that proline (+869C allele) shows 2·8-fold higer secretion of TGF-β1 than does leucine (+869T allele). Furthermore, the serum concentration of TGF-β1 is higher in individuals with the CC genotype than in those with the TT or TC genotype [12]. TGF-β1 suppresses the actions of cytotoxic T cells and expressions of IFN-γ [6], which are thought to be the main effector for thyroid destruction in HD [1–3]. Consistent with this, the proportion of activated cytotoxic T cells is lower in patients with mild HD than in those with severe HD [3]. In addition to this, previous reports show that IFN-γ levels are decreased in patients with mild HD [18] and that intracellular expression of IFN-γ in T cells is decreased in patients with mild HD [19]. Therefore, increased TGF-β1 may suppress T cell cytotoxicity in the thyroid and protect against the progression of hypothyroidism in HD patients with the +869C allele. In HD patients with the TT genotype, which is associated with low secretion of TGF-β1, T cell cytotoxicity against thyroid tissue may not be suppressed, resulting in the rapid progression of hypothyroidism. This hypothesis is supported by findings that systemic or local administration of TGF-β1 suppresses several autoimmune diseases associated with cell-mediated immune responses [20, 21].

Recently, it has been reported that TGF-β1 converts CD4⁺ CD25^- T cells to CD4⁺ CD25⁺ forkhead box P3⁺ (FoxP3⁺) regulatory T (T_reg) cells in vitro[22, 23]. TGF-β1 deficiency inhibits the suppressive function of T_reg, resulting in autoimmunity [24]. Over-expression of TGF-β1 in pancreatic islets expands the T_reg population and protects non-obese diabetic mice against type 1 diabetes [25]. In HD patients with the TGF-β1 +869C allele, the minimizing regulatory function of T_reg cells may be high autoimmune destruction of the thyroid gland.

In GD patients the frequency of the CC genotype was significantly higher in patients with intractable GD than in those with GD in remission (Table 1). Therefore, in contrast to HD, TGF-β1 may increase disease intractability or severity in GD. The reason why the effect of TGF-β1 on disease severity is different between HD and GD is unclear, but this may be due to the different aggravation mechanisms between HD and GD. Cell-mediated cytotoxicity exacerbates HD and antibody aggravates GD. Interestingly, there are many reports that TGF-β1 suppresses progression of autoimmune diseases where cell-mediated cytotoxicity plays an important role in tissue destruction, such as rheumatoid arthritis [21, 26] and autoimmune insulitis [25, 27]. In contrast, in autoimmune diseases where autoantibodies play an important role in the pathology, such as myasthenia gravis [28, 29], TGF-β1 aggravates the disease. In patients with asthma, which is caused by immunoglobulin E (IgE) antibody, TGF-β1 increases the disease severity [30]. Therefore, TGF-β1 may aggravate pathological conditions in particular immune diseases that are caused by antibodies such as autoantibodies and IgE.

In conclusion, the +869T/C polymorphism in the TGF-β1 gene is related to severity of HD and the intractability of GD patients.