DISCUSSION
In this study, we showed that IL2RA /rs3118470 andIL2RA /rs2104286 were associated with GD susceptibility, while the remaining seven loci (rs7093069, rs895819, rs76481776, rs2297626, rs17592236, rs9549241, and rs12585277) were not associated with susceptibility to GD in Southwest Chinese Han population. Stratification analysis revealed that two SNPs (rs3118470 and rs2104286) also contributed to the genetic susceptibility of GO in Southwest Chinese Han population.
GD, a common organ-specific autoimmune disease, is caused by genetic susceptibility and environmental factors, resulting in immune dysfunction among individuals and unregulated thyroid hormone production(Sun et al., 2019; Kahaly, Diana, Kanitz, Frommer, & Olivo, 2020). The cytokine IL-2 is essential for the differentiation of Treg and maintenance of the suppressive function. Activated effector T cells produce IL-2 and transiently up-regulate IL-2RA, enabling them to respond to IL-2. IL2RA on the effector T cells can reduce its expression through a negative feedback when the cells come to rest (Sun et al., 2019). A protease can cleave off IL2RA on the T cell surface to produce sIL2RA that can be detected in the serum (Cerosaletti et al., 2013).
Studies have shown that the IL2RA mRNA was down-regulated in PBMCs from patients with GD (Ji et al., 2020). A study by Tang et al found that IL2RA/ rs3118470 was highly significantly associated with type 1 diabetes and was highly acetylated in T cells involved in indirect disruption of IL2RA transcription (Tang et al., 2015). Song et al conducted a GWAS study of GD in the Han population in Shanghai and Anhui province of China, and found that theIL2RA /rs3118470 polymorphism was significantly associated with susceptibility to GD in the Chinese Han population at the first phase (P = 0.019), but the significance was lost in the verification stage (Song et al., 2013).Our study found that the IL2RA /rs3118470 TT genotype can increase the GD susceptibility in Southwest Chinese Han population. It should be noted that the P-value of the association between IL2RA /rs3118470 TT genotype and GD susceptibility is relatively large in the first stage (Pc = 0.024), while lack of the association was found in the second phase.Previous study showed thatIL2RA /rs2104286 TT genotype was associated with the risk of multiple sclerosis (MS) (Xia et al., 2018). A latest study by Buhelt et al. reported that MS-associated IL2RA /rs2104286 was associated with the expression of IL2RA in CD4+ cells but not CD8+T cells, and carriers of the risk genotype had reduced surface expression of IL2RA in post-thymic expanded CD4+T cells, CD39+Treg cells, and memory cell subsets (Buhelt et al., 2019). Chistiakov et al. found that the IL2RA /rs2104286 polymorphism was not significantly associated with the GD susceptibility of the Russian population (Chistiakov, Chistiakova, Voronova, Turakulov, & Savost’anov, 2011). Our study identified the IL2RA /rs2104286 AA genotype can significantly increase the GD susceptibility in Southwest Chinese Han population, and the IL2RA mRNA expression in PBMC is relatively reduced for carrier of rs2104286 AA genotype. The reason for the inconsistent results may be due to the different genetic background of people in different regions. Brand et al found that IL2RA /rs7093069 was significantly associated with susceptibility to GD in Caucasian (Brand et al., 2007)., while the results of our study showed thatIL2RA /rs7093069 was not significantly associated with GD susceptibility in Southwest China. Recent clinical studies have indicated that low-dose recombinant IL-2 can reduce the number of Th17 cells in the peripheral blood of systemic lupus erythematosus patients and the disease activity index, while it can increase the number of Treg cells (He et al., 2016).
The miRNA, short and non-coding RNA, acts mainly via targeting and regulation messenger RNAs (Viuff, Skakkebaek, Nielsen, Chang, & Gravholt, 2019). miR27a and miR182 act on the target molecule,FoxO1 mRNA, and inhibit the expression of FoxO1 (Yang et al., 2014). A previous bioinformatic analysis showed that rs895819 is situated in the terminal loop of the pre-miR27a, suggesting that it may affect the expression of matured miR27a and affinity with target mRNAs (Strafella, 2019). However, Takuseet al. reported that there was no difference in the allele and genotype frequencies of miR27a /rs895819 between GD patients and healthy controls in a Japanese population, and rs895819 variants did not affect the expression levels of hsa-miR-27a-3p in PBMCs (Takuse et al., 2017). Our results showed that there was a lack of association betweenmiR27a /rs895819 variants and susceptibility to GD, which is consistent with the study by Takuse et al. Previous studies have identified that themiR182 /rs76481776 locus was associated with VKH syndrome and Behcet’s disease, and rs76481776 variants influenced the gene expression in CD4+ T cells (Yu, Liu, Bai, Kijlstra, & Yang, 2014). In contrast, we did not find any correlation betweenmiR182 /rs76481776 variants and GD susceptibility. This inconsistent result may be due to the fact that each disease may have specific risk genes.
FoxO1 in the nucleus promotes FoxP3 expression via binding to the FoxP3 conserved non-coding sequence 1 region and decreasing Th17 cells generation as well as transcription of Th17-related genes, including IL-17A, IL-23R and RORγt-target genes (Du et al., 2018). The study by Gong et al. and colleagues revealed that FoxO1 /rs17592236 was not associated with type 2 diabetes in a Chinese Han population (Gong et al., 2017). Previous studies showed that four SNPs (rs2297626, rs17592236, rs9549241, and rs12585277) were not associated with Behçet’s disease and VKH Syndrome in a Chinese Han population (Yu, Liu, Bai, et al., 2014). Similarly, our study showed that FoxO1/ (rs2297626, rs17592236, rs9549241, and rs12585277) were not associated with GD susceptibility in the Chinese Han population. This study is the first to explore FoxO1 gene polymorphisms and GD susceptibility, and therefore, further research is needed to verify this association in large study samples.
There are some potential limitations in this study. First, this study only investigated a limited number of SNPs, and it is possible that other as yet unknown SNPs of candidate genes might participate in the occurrence of GD. In addition, PBMCs, a large-cell population, were used to compare the difference in the mRNA expression among various genotype carriers. Further study is needed in a more purified cell subgroup, such as CD4+ T cells. Moreover, only the Chinese Han population was enrolled in our study, and thus, it is not certain whether this significant association can be generalized to the other ethnic populations.
In summary, our study identified that IL2RA/rs3118470 and IL2RA/rs2104286 were significantly associated with GD and GO susceptibility in Southwest Chinese Han population, which may be involved in the occurrence of GD and GO by affecting the mRNA expression of IL2RA gene and the cytokine production.