ABSTRACT
Previous studies have identified that Th17/Treg cells were involved in Graves’ disease (GD). This study aimed at clarifying the association between GD susceptibility and single nucleotide polymorphisms (SNPs) of Th17/Treg cells-related genes, including IL2RA, miR27a , miR182, and FoxO1 .A two-stage association study was performed in 650 GD patients and 1300 healthy controls.PCR-RFLP assays, real-time PCR and ELISA were performed. The result demonstrated thatsignificantly increased frequencies of IL2RA /rs3118470 TT genotype (Pc =2.212×10-3) and IL2RA /rs2104286 AA genotype (Pc =1.754×10-5) were observed in GD. Stratification analysis also revealed that rs3118470 TT genotype and rs2104286 AA genotype were associated with Graves’ ophthalmopathy (GO) susceptibility (Pc = 1.848×10-6, Pc = 1.230×10-3, respectively). Functional studies showed that carriers of the rs2104286AA genotypehad lower IL2RA mRNA expression than AG genotype carriers (P =0.021). Cytokine analyses revealed that carriers with the rs3118470 risk genotype had decreased anti-inflammatory cytokine levels (IL-10 and TGF-β1) and increased IL-17 secretion than protective genotype individuals (P < 0.05). For the rs2104286 locus, AA genotype individuals had lowerIL-10 levels (P =0.015) and increased IL-17 levels than AG genotype carriers (P =1.467×10-4). In conclusion, our findings suggested that IL2RA / (rs3118470, rs2104286) were associated with GD and GO susceptibility in Southwest Chinese Han population.
Keywords: Graves’ disease, genetic susceptibility,IL2RA , miR27a, miR182 , FoxO1
INTRODUCTION
Graves’ disease (GD), also known as toxic diffuse goiter, is one of the most common autoimmune thyroid diseases (AITDs), which accounts for more than 85% of all clinical types of hyperthyroidism (Xiaoheng et al., 2017). GD occurs in women aged 30−50 years (Veneti et al., 2019), and it affects approximately 1% of the world’s population (J. Li, Teng, Yu, Hou, & Shan, 2019). The ratio of male to female patients is approximately 1:5−1:10 (Liang et al., 2015). Approximately 50% of GD patients can develop Graves’ ophthalmopathy (GO), and severe stages can affect vision (Longo & Higgins, 2019). Immunologically, GD is characterized by an increased level of circulating thyroid stimulating antibody (TSAb) against thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg) and thyroid peroxidase (TPO), leading to diffuse thyroid enlargement and hyperthyroidism (Fang et al., 2015). The pathogenesis of GD has not yet been fully elucidated, but it is generally believed that the occurrence of GD is the interaction result of genetic susceptibility and environmental factors (Chen et al., 2018).
Studies have shown that the coincidence rate of GD in identical twins was 50%−70%, while the coincidence rate of GD in fraternal twins was 3%−25% (Hwangbo & Park, 2018). Twin studies have indicated that the genes account for 79% of the role in predisposition to GD (Lane et al., 2019). Extensive genetic association studies have identified that many genes are associated with predisposition to GD, for example, human leukocyte antigen (HLA ), cytotoxic T-lymphocyte-associated antigen-4 (CTLA4 ), protein tyrosine phosphatase 22 (PTPN22 ), Fc receptor like 3 (FCRL3 ), thyroid stimulating hormone receptor (TSHR ), and vitamin D receptor gene (VDR ) (Mehraji et al., 2017; Chu et al., 2018; Ting et al., 2016; Li et al., 2017; Khong et al., 2016; Fujii et al., 2017; M. Planck, Shahida, Malm, & Manjer, 2018 ). At present, antithyroid drugs are regarded as the preferred methods to treat GD in China, but recurrence within two years after retreat of the drug is as high as 50%−60% (J. Li, Sun, Yao, & Xia, 2018). Therefore, it is necessary to elucidate the pathogenesis of GD.
Studies have recognized that T helper 17 (Th17) cells and Regulatory T (Treg) cells are involved in the pathogenesis GD. The proportion of Th17 cells and the expression of related genes (IL17 mRNA and ROR-γt mRNA) were increased in patients with GD compared with healthy individuals, and the number of Th17 cells was directly proportional to TSAb, whereas the number of Treg cells and the relatedFoxP3 mRNA expression were lower in GD patients than in normal controls (Qin et al., 2017). The human IL2RA gene located on the chromosome 10 (10p15-p14), encoded the IL-2RA protein and constitutively expressed on the Treg cell surface, which is essential for mediating both the generation of Treg and the maintenance of immune tolerance (Wang & Chen, 2018). In the context of CD4+ T-cell differentiation, FoxO proteins promote the expression of FoxP3genes essential for the generation and function of Tregs. In addition, FoxO1 can directly inhibit the differentiation of Th17 by binding to the transcription factor RORγt (Harada et al., 2010; Laine et al., 2015). MiR27a andmiR182 can target the 3’UTR of FoxO1 , suppressingFoxO1 expression (Guttilla & White, 2009; Wang et al., 2019). A previous study showed that the frequencies of the IL2RA/ rs3118470 C allele and the CC genotype were higher in the patients with a family history of alopecia areata in a Chinese population (Miao et al., 2013). It has been reported that the IL2RA/ rs2104286 G allele and the GG genotype increased the risk of multiple sclerosis (Xia, Qin, & Zhao, 2018). An analysis showed that miR27a/ rs895819 contributed to the occurrence of gastric cancer under a co-dominant model in the Chinese population (Yun et al., 2019). Previous studies have identified that the frequency of themiR182 /rs76481776 CT/TT genotype was increased in patients with Vogt-Koyanagi-Harada(VKH) syndrome and Behcet’s disease, and increased expression of miR-182 in CT/TT cases compared with that in CC cases in anti-CD3 and anti-CD28 antibodies stimulated CD4+ T cells (Yu, Liu, Bai, Kijlstra, & Yang, 2014). TheFoxO1 /rs2297626 A allele and the AA genotype showed significantly increased frequencies in acute anterior uveitis patients with ankylosing spondylitis (Yu, Liu, Zhang, et al., 2014). The FoxO1 /rs17592236 located in 3’-UTR of the FoxO1 gene and the identified Ars17446614-Crs17592236 haplotype were associated with an increased risk of diabetic nephropathy (Ma et al., 2019). Considering these studies, we selected nine SNPs in Th17/Treg cells-related genes, including IL2RA, miR27a, miR182, and FoxO1, to explore the association between gene polymorphisms and GD susceptibility in Southwest Chinese Han population. Our results showed that IL2RA gene, but not miR27a , miR182, andFoxO1 , contributed to the predisposition of GD.