SummaryTo clarify the association between factors regulating DNA methylation and the prognosis of autoimmune thyroid diseases (AITDs), we genotyped single nucleotide polymorphisms in genes encoding DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B, methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), which are enzymes essential for DNA methylation. Subjects for this study included 125 patients with Hashimoto's disease (HD), including 48 patients with severe HD and 49 patients with mild HD; 176 patients with Graves' disease (GD), including 79 patients with intractable GD and 47 patients with GD in remission; and 83 healthy volunteers (control subjects). The DNMT1 +32204GG genotype was more frequent in patients with intractable GD than in patients with GD in remission. Genomic DNA showed significantly lower levels of global methylation in individuals with the DNMT1 +32204GG genotype than in those with the AA genotype. The MTRR +66AA genotype was observed to be more frequent in patients with severe HD than in those with mild HD. The DNMT1 +14395A/G, DNMT3B -579G/T, MTHFR +677C/T and +1298A/C polymorphisms were not correlated with the development or prognosis of AITD. Our study indicates that the DNMT1 +32204GG genotype correlates with DNA hypomethylation and with the intractability of GD, and that the MTRR +66AA genotype may correlate with the severity of HD.

Chemokines induce leukocyte chemotaxis and contribute to chronic inflammation. To clarify the association between functional polymorphisms in genes encoding some chemokines and the pathogenesis of Autoimmune thyroid disease (AITD), we genotyped IL8 -251T/A, Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) - 403G/A, -28C/G, MIG rs2276886G/A, IP10 -1596C/T, Monocyte Chemoattractant Protein1 (MCP1) - 2518G/A and IL16 -295T/C polymorphisms. We genotyped these polymorphisms using the PCR-RFLP method in 149 Graves' disease (GD) patients, including 59 patients with intractable GD and 53 patients with GD in remission, as well as 131 Hashimoto's disease (HD) patients, including 54 patients with severe HD, 46 patients with mild HD and 99 healthy controls. The IL8 -251TT genotype and MIG rs2276886 A allele were more frequent in patients with AITD (p = 0.0139 and p = 0.0005, respectively). The RANTES - 403AA and -28GG genotypes were less frequent in patients with AITD (p = 0.0164 and p = 0.0221, respectively). The MCP1 -2518GG genotype was more frequent in HD patients (p = 0.0323). The MIG rs2276886 AG genotype was less frequent in patients with intractable GD (p = 0.0051). Interestingly, the age of onset in GD patients with the RANTES - 28CC genotype was younger than in those with -28CG and GG genotypes (p = 0.0028). In this study, we first reported that the polymorphisms in IL8, RANTES and MIG genes are associated with the development of AITD, and that the MIG rs2276886 AG genotype is associated with the intractability of GD. The RANTES - 28CC genotype is associated with young onset of GD.

Sirtuin1 (SIRT1) is a Class 3 nicotinamide adenine dinucleotide-dependent histone deacetylase (HDAC) that is thought to be implicated in the protection against autoimmune diseases. However, an association between SIRT1 and autoimmune thyroid disease (AITD) has not been reported. In this study, we selected four single nucleotide polymorphisms (SNPs) in the SIRT1 gene, rs12049646 T/C (termed SNP1), rs12778366 T/C (termed SNP2), rs3758391 T/C (termed SNP3), and rs4746720 T/C (termed SNP4). We genotyped each of these polymorphic sites in 185 patients with Graves' disease (GD), including 76 patients with intractable GD and 57 patients with GD in remission; 151 patients with Hashimoto's disease (HD), including 68 patients with severe HD and 54 patients with mild HD; and 96 healthy volunteers. SNP1 and SNP3 were genotyped by the PCR-RFLP method; SNP2 and SNP4 were genotyped using TaqMan® SNP genotyping assays. We also measured the levels of SIRT1 mRNA in CD4(+) T cells from 18 control subjects, 16 patients with GD in remission and 14 patients with mild HD using a real-time PCR method. In patients with GD and HD, the C carriers (TC + CC genotypes) of SNP3 showed significantly higher titers of McAb than the TT genotype (p = 0.0261 and p = 0.0309, respectively). Additionally, the T carriers (TT + TC genotypes) of SNP4 showed significantly higher titers of McAb than the CC genotype in patients with GD (p = 0.0079). In conclusion, the polymorphisms in the SIRT1 gene were associated with a greater production of thyroid autoantibodies.

Dicer and Drosha are RNase III enzymes that are necessary for the biogenesis of most miRNAs. However, there are no reports on the association of Dicer and Drosha with the pathogenesis of autoimmune thyroid disease (AITD). We genotyped DICER rs3742330A/G and rs1057035T/C as well as DROSHA rs644236C/T and rs10719C/T polymorphisms in 255 Hashimoto's disease (HD) patients, in 255 Graves' disease (GD) patients and in 128 healthy controls by the polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) method. We also examined the expression of DICER and DROSHA gene in peripheral blood mononuclear cells (PBMCs) by quantitative RT-PCR (qRT-PCR) methods. The TT genotype of the DICER rs1057035 polymorphism was less frequent in GD patients (p = 0.0098) than in healthy subjects. The CC genotype of DROSHA rs644236 polymorphism were more frequent in GD patients than in HD patients (p = 0.0171). The gene expression of DICER was lower in patients with AITD compared with that in control subjects (p = 0.0064) and was lower in patients with GD in remission than in patients with intractable GD (p = 0.0213). In addition, the expression of DROSHA was lower in patients with AITD than that in control subjects (p < 0.0001) and was lower in patients with severe HD than in patients with mild HD (p = 0.0440). In conclusion, the DICER rs1057035 TT genotype and DROSHA rs644236 CC genotype were associated with the development of GD and the differentiation between GD and HD, respectively. The expression levels of DICER and DROSHA genes were low in AITD and differed depending on the intractability of GD and the severity of HD, respectively.

Graves' disease (GD) and Hashimoto's disease (HD) are autoimmune thyroid diseases (AITDs), and the prognosis of AITDs is different for each patient. We examined the association of polymorphisms in the Thyroglobulin (TG) gene with the pathogenesis of AITD. We genotyped TG rs180195G/A, rs853326G/A, rs2076740C/T, rs2703013G/T, rs2958692C/T and rs733735A/G polymorphisms in 137 HD patients, 131 GD patients and 89 healthy controls and also examined the levels of TG mRNA expression and serum TG. The TG rs180195 GG genotype was more frequent in HD patients (p = .0277), and the proportion of CD4 cells with high levels of TG mRNA was greater in individuals with the GG genotype than in A carriers (p = .0107). The TG rs2703013 TT genotype was less frequent in AITD (p = .0186), and serum TG levels were lower in individuals with the TT genotype than in G carriers (p = .0170). In the TG rs2958692 polymorphism, the T allele was more frequent in intractable GD than in GD in remission (p = .0055), and serum titres of anti-thyroglobulin antibody (TgAb) were lower in GD patients with the TT genotype than in C carriers (p = .0151). In the TG rs2076740 polymorphism, serum titres of TgAb were higher in HD patients who were T carriers than in those with the CC genotype (p = .0359). SNPs in the TG gene were associated with the development of HD and GD, the intractability of GD, and the levels of TG mRNA expression, serum TG, and serum TgAb.