Associations between CD160 polymorphisms and autoimmune thyroid disease: a case-control study

Autoimmune thyroid diseases (AITDs) are endocrine autoimmune diseases with a prevalence of about 5% worldwide that varies with different racial populations and in areas with different amounts of iodine intake [1‐4]. AITD occurs more frequently in women than in men, and the lifetime risk of Graves’ disease (GD) is approximately 3% for women and 0.5% for men. Accounting for individuals with undiagnosed and euthyroid Hashimoto’s thyroiditis (HT), the true prevalence of HT is estimated to be about 5–10% of the general population. AITDs encompass several types, including GD, HT, atrophic autoimmune thyroiditis, postpartum thyroiditis, and Graves’ ophthalmopathy (GO) [5]. The prevalence of HT is the highest, followed by GD. Because GD and HT are the two main subtypes of AITDs and most published literature investigating the genetics of AITDs have focused on susceptibility genes of either GD or HT, we aimed to explore the susceptibility genes of GD and HT and to include only GD and HT patients in our research [6, 7]. Though its pathogenesis has not yet been clarified, it has been well accepted that genetic and environmental factors both play crucial roles in the pathogenesis of AITD, of which genetic factors contribute up to 80% of the risk factors [8‐10]. Some studies have confirmed that there is a clear familial predisposition to AITD [11]. GD is characterized by a diffuse toxic goiter caused by thyroid-stimulating hormone (TSH) receptor antibodies (TRAb) and is the most common cause of hyperthyroidism [12]. TRAb binds to TSH receptors on thyroid follicle cells and stimulates the production of thyroid hormones. However, the clinical manifestations of HT are diametrically opposite, with hypothyroidism accompanied by an increase in thyroglobulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb) [10]. HT, also known as chronic lymphocytic thyroiditis or autoimmune thyroiditis, is the most common type of AITD. It is also the most common organ-specific autoimmune disease and the most common cause of hypothyroidism. The pathological feature of HT is the local infiltration of lymphocytes, accompanied by elevated levels of TGAb and TPOAb. Environmental factors affecting the occurrence of HT mainly include high iodine intake, infection, stress, and radiation exposure [7, 13]. There is evidence that many of the susceptibility genes are shared by various autoimmune diseases, such as CTLA4 and PTPN22, which indicate that autoimmune diseases have some similar genetic and molecular pathways [14, 15]. Previous studies have reported that there are many susceptibility genes associated with AITD. Generally, most of those susceptibility genes can be classified into two types. One is thyroid-associated genes including TSHR and TG, and the other is immunity-related genes, such as FOXP3 [16], HLA-DR genes [17], and CD40 [15]. Some other novel AITD genes have also been reported, such as VDR [18, 19] and FCRL3 [20].

Herpesvirus entry mediator (HVEM) is a receptor on immune cells, which is also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14) [21]. CD160, B- and T-lymphocyte attenuating agents (BTLA), lymphotoxin-like (LIGHT), and lymphotoxin-alpha (LTa) are ligands for HVEM [22, 23]. The CD160/HVEM/LIGHT/BTLA pathway is an essential signaling pathway in regulating immunity, which is involved in the pathogeneses of infection and autoimmunity [24‐26].. Some gene polymorphisms in the CD160/HVEM/LIGHT/BTLA pathway have an obvious influence on their functions and are thus associated with host susceptibility to autoimmune diseases [27‐31].. The inhibitory co-stimulator CD160, originally named BY55, is an immunoglobulin-like activator of the NK cell receptor and belongs to the glycosylphosphatidylinositol (GPI) fixed membrane protein family. The gene is located at 1q42.3 [32, 33]. There are also several single nucleotide polymorphisms (SNPs) in CD160. A study found that CD160 rs744877 was closely related to the susceptibility of rheumatoid arthritis (RA) and may be involved in the development of RA [34]. rs3766526 of CD160 was identified with data from the 1000 Genomes Project (http://​phase3browser.​1000genomes.​org/​index.​html), and it met the selection criteria of MAF more than 0.10 and r² more than 0.8 in the linkage disequilibrium analysis. Currently, there is no study on the relationship between CD160 genetic polymorphisms and AITD, and thus the role of CD160 genetic polymorphisms in the susceptibility to AITD remains unclear. Herein, we report a case-control involving a large sample to investigate the relationship between two CD160 SNPs (rs744877 and rs3766526) and AITD in a Chinese Han population.

In this study, we explored two SNPs in CD160 and found an obvious relationship between CD160 rs744877 and GD. This correlation was also confirmed through multivariate logistic regression analysis. However, our study did not find any correlation between rs744877 and HT. To our knowledge, it is the first study on the linkage of CD160 polymorphisms to AITD.

The CD160/HVEM/LIGHT/BTLA pathway is an essential signaling pathway in regulating immunity, which is involved in the pathogeneses of infection and autoimmunity [22, 23, 39]. CD160, BTLA, LIGHT, and LTa all are ligands for HVEM, but they exert different regulatory roles [22, 23]. The binding of LIGHT or LTa to HVEM stimulates costimulatory signals, but the binding of BTLA or CD160 to HVEM initiates co-suppression signals, and thus the CD160/HVEM/LIGHT/BTLA costimulatory/ co-suppression pathway is a bidirectional switch that adjusts T cell activation and takes an important part in regulating immune responses [24, 40, 41]. Although the binding of LIGHT or LTa to HVEM promotes T cell activation, HVEM predominantly shows negative regulatory effects mediated by BTLA and CD160 [22, 39]. The cysteine concentration 1 (CRD1) region in HVEM molecule is responsible for the combination of CD160 and BTLA, and the deletion of HVEM CRD1 region could block the combination of CD160/BTLA and HVEM and eliminate the inhibitory effect. Thus, HVEM can be transformed into a dominant co-stimulator and enhance the immune response, even causing graft rejection [42]. Some observational studies have revealed that these genes are aberrantly expressed in patients with autoimmune diseases [43‐45]. In addition, animals with knock-out of those members in the CD160/HVEM/LIGHT/BTLA signaling pathway could change the development or progression of autoimmune diseases, such as colitis and atopic dermatitis [46, 47].

Recent genetic studies have identified an association of CD160/HVEM/LIGHT/BTLA pathway-related genes with autoimmune diseases [48]. HVEM (TNFRSF14) rs6684865 was related to primary sclerosing cholangitis (PSC), RA, and multiple sclerosis (MS) [30, 48, 49]. The findings from our study prove that CD160 rs744877 is related to GD, which adds new evidence for the roles of the CD160/HVEM/LIGHT/BTLA pathway in autoimmune diseases. CD160 is an essential member of the CD160/HVEM/LIGHT/BTLA pathway, and its role in immunity has been widely recognized [50]. CD160 is highly expressed in the spleen, small intestine, and peripheral blood, but is lowly expressed in the brain, liver, heart, thymus, and other tissues [25, 32, 33]. In the immune system, CD160 is a costimulatory receptor molecule and is expressed on multiple immune cells, including intestinal intraepithelial T lymphocytes, CD56dimCD16+ NK lymphocytes, and a minor subset of CD4+ T cells and CD8+ T cells [51].. In the pathogeneses of GD, antigen-presenting cells (APC), T cells, and B cells are all involved, and the dysfunction of one type of immune cells can further lead to aberrant function of other immune cells [10]. CD160 primarily functioning as a costimulatory receptor molecule. CD160 is an essential member of the CD160/HVEM/LIGHT/BTLA pathway, and its role in immunity has been widely recognized. Therefore, CD160 plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases. More importantly, one of our recent study suggested that CD160 was differentially expressed on the surface of CD8⁺ T cells between GD patients and healthy controls (P = 0.002), which suggested that CD160 dysfunction was involved in the pathogenesis of GD [52]. CD160 SNP may lead to impairment of its immunomodulatory function by affecting its expression or function. Therefore, aberrant expression or function of CD160 can first result in the dysfunction of T cells, which may further cause the activation of antibody-producing B lymphocytes and lead to the development of GD. CD160 has a higher affinity for HVEM and can inhibit CD4⁺ T cell proliferation and cytokine secretion after cross-linking [51]. Therefore, CD160 plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases [25, 53].

Though our study indicated CD160 rs744877 locus is a susceptible factor for GD, the molecular mechanisms underlying the roles of CD160 rs744877 in autoimmune diseases are still unclear. CD160 rs744877 is an intronic SNP and is unable to act by changing the composition of amino acids or the protein structure of CD160. One possibility is that CD160 rs744877 takes part in disease development by impacting its expression or post-translational modification. CD160 polymorphisms may not be related to HT due to the different molecular mechanisms between GD and HT. Though both GD and HT are similar autoimmune diseases occurring in the same organ, they still may have some distinct molecular mechanisms [8, 54]. Previous studies also suggested several genes are differently associated with GD and HT, such as BTG1 and TSHR. Therefore, the associations of CD160 polymorphisms with GD and HT may be different, as CD160 may exert different roles in the pathogeneses of GD and HT. Another explanation was the limited number of HT patients in our study, which may cause low statistical power to detect a statistically significant relationship. Finally, subgroup analyses by gender found that rs3766526 was not associated with GD and HT in females, but it was significantly associated with HT in males for both the genotype distribution (P < 0.001) and allele frequencies (P = 0.015) (Supplementary Table 3), which suggested that the gender-specific association of rs3766526 with HT was possible. However, the finding above may be biased by the limited sample size in the subgroup analyses by gender, and studies with larger sample sizes are recommended to provide a more precise evaluation of the gender-specific association of rs3766526 with HT. Similar to other published gene-association studies, our study did not perform a replication study, which was due to the limited number of AITD DNA samples in our clinical biobank of AITD. To ensure the reliability of the findings in our study, we attempted to collect more AITD samples, and the sample size in our study is relatively large compared with other published case-control studies on the susceptibility genes of AITD. Moreover, the sample size calculation also suggested that the sample size in our study was large enough to assess associations adequately. Our study focused on the relationship between AITD and controls, and from this perspective the number of patients was sufficient to assess the relationship between CD160 SNPs and AITD. However, when subgroup analysis was done, there were fewer HT patients, which may lead to low statistical power in assessing the relationship between CD160 SNPs and HT. Nevertheless, the findings in our study still need to be validated in future studies, especially for those from other countries or populations.

In conclusion, this is the first demonstration of the association of CD160 rs744877 with GD and supports the critical role of the CD160 / HVEM / LIGHT / BTLA pathway in the pathogenesis of GD.