In this study serum BAFF concentrations were analyzed in a group of women with GD and compared these concentrations with those of age- and BMI-matched CTRL. BAFF showed markedly elevated concentrations in GD patients and ROC curve analysis demonstrated the biomarker significance of this cytokine in disease prediction (AUC = 0.971). In addition, BAFF was positively correlated with TT3 and T4. It was also found that two promoter SNPs of the TNFSF13B gene, rs9514827 and rs9514828, were associated with susceptibility to GD, particularly at the haplotype level. Besides, the results indicated that the TT genotype of rs9514828 was associated with greater serum concentrations of BAFF. Together, these findings suggest a role for BAFF in the etio-pathogenesis of GD.

GD is an organ-specific autoimmune disorder affecting the thyroid gland, and as demonstrated by observational and experimental studies, B cells play a vital role in the pathophysiology of the disease because these cells are the source of pathogenic autoantibodies (i.e. TRAb) against the TSH receptor. Thus, binding of TRAb to the TSH receptor can activate target cells and may lead to higher levels of T3 and T4 (thyroid hormones) [29]. Studies have demonstrated that the essential survival signals for B cells are provided by BAFF, and therefore up-regulated functional activity of these cells can be associated with elevated concentrations of BAFF [30]. In fact, massive expansion of activated autoreactive B cells and autoantibody production was evident in mice in association with over-expression of BAFF [31]. Consistent with our findings, previous studies also reported elevated plasma concentrations and up-regulated gene expression of BAFF in patients with GD [11,12,13]. Furthermore, blockade of BAFF in a mouse model of GD was associated with a significant decrease in hyperthyroidism [32]. Therefore, blocking the BAFF interaction has been proposed as one of the novel therapeutic options in GD [33]. Together, these data indicate the pathological significance of elevated BAFF concentrations in the serum of patients with GD particularly in terms of prognosis (AUC = 0.971; sensitivity = 94.6%; specificity = 94.4%).

The action of BAFF on B cells is mediated through interaction with three surface receptors, namely BAFF receptor 3 (BR3), transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI), and B cell maturation antigen [30]. BR3 is essential for the development of immature B cells into transitional B cells, as this receptor is involved in mediating pro-survival signals to rescue B cells from premature death [34]. In contrast, TACI is associated with B-cell apoptosis and mediates IL-10 production by regulatory B-lymphocytes to exert an immunosuppressive function [35]. Altered expression of BR3 and TACI has recently been demonstrated in peripheral B cells of GD patients, with the former being over-expressed and the latter showing lower expression. Accordingly, the authors suggested that autoimmunity in GD could be attributed to altered expressions of BAFF and its receptors through the enhancement and inhibition of the activities of the BR3 and TACI signaling pathways, respectively [13]. Consistent with this suggestion, blocking the interaction between BAFF and BR3 was associated with decreased B cell proliferation, decreased B cell survival, and decreased autoantibody production [36]. Therefore, targeting such an interaction between BAFF and its receptors may have therapeutic potential in GD.

With respect to thyroid hormones, serum TT3 and TSH concentrations showed no significant differences between GD patients and CTRL, while T4 concentrations were significantly lower in patients. T3 and T4 are secreted by the thyroid gland after stimulation with TSH, a hormone produced by the anterior pituitary gland. Irregular production of these hormones can lead to hypothyroidism or hyperthyroidism. Hyperthyroidism is the hallmark of GD as T3 and T4 show up-regulated concentrations in patients' serum, while TSH is down-regulated [1]. This pattern of TT3, T4, and TSH concentrations in current GD patients was not followed as described above. However, the median concentration of the three hormones was within the reference range (TT3: 1.13 vs. 0.8–2.0 ng/mL; T4: 6.90 vs. 5.5–12.2 µg/dL; TSH: 2.23 vs. 0.3–4.5 µIU/mL) [37]. The normalization of TT3, T4 and TSH concentrations may be due to therapy effects as most patients (84 of 90; 93.3%) were receiving treatment with CMZ or CMZ + RAI. This is evidenced by the fact that CMZ is a well-known anti-thyroidal drug that targets the synthesis of thyroid hormones and causes a decrease in their concentrations [38].

In Spearman's correlation analysis, BAFF was found to be positively correlated with TT3 and T4 in GD patients but not in CTRL. In light of this, a functional relationship can be proposed between BAFF and thyroid hormones T3 and T4 in GD patients. This topic has recently been addressed and the results showed that high systemic concentrations of TT3 and T4 are associated with over-expression of BAFF and may have effects on B-cell differentiation [39]. In addition, it has been found that elevated concentrations of TT3 can induce polarization of M1 macrophages and this may lead to increased expression of BAFF [40].

With regard to VitD, although there was no correlation with BAFF, current GD patients were predominantly characterized by VitD insufficiency/deficiency (< 30 ng/mL). Therefore, low concentrations of VitD could be promoted to become a potential risk factor associated with the etio-pathogenesis of GD as recently suggested by our group [16]. Besides being an important component of the endocrine system, studies have indicated that VitD can perform various immune functions and thus may have an essential role in the pathophysiology of autoimmune diseases [41]. However, the relationship between VitD and BAFF in GD has not been investigated, but in patients with Sjögren's syndrome (an autoimmune disorder), BAFF concentrations increased while VitD concentrations decreased, a negative relationship was also found between them but it was not significant [42]. Accordingly, simultaneous evaluation of BAFF and its receptors and VitD may represent a cornerstone in understanding the pathophysiology of GD and further studies are warranted.

Family and twin studies have revealed the role of genetic factors in conferring an individual's susceptibility to GD. In this context, several genetic loci have been described as harboring alleles and genotypes with potential for predisposition to GD [2]. It has been suggested that TNFSF13B, the gene encoding BAFF protein, is among such loci that may have a role in determining susceptibility to GD [22]. This proposal motivated us to expand our understanding of the role of BAFF in the pathogenesis of GD by analyzing two promoter SNPs of the TNFSF13B gene (rs9514827 and rs9514828) in a cohort of 90 GD patients and 70 CTRL. The two SNPs show moderate linkage disequilibrium and are located in the 5’ untranslated region within 5kb of the first exon of the gene [20]. The results were interesting and mutant alleles (rs9514827 C and rs9514828 T) and genotypes (rs9514827 CC and rs9514828 TT) were associated with an increased risk of GD. The OR for the corresponding alleles was 2.00 and 2.15, respectively (allele model), and for the corresponding genotypes it was 4.29 and 4.57, respectively (co-dominant model). Furthermore, haplotype analysis revealed that the C-T haplotype (rs9514827-rs9514828) was associated with a 2.71-fold increased risk of GD, whereas the TC haplotype was associated with a reduced risk of the disease (OR = 0.44). In addition, the mutant TT genotype of rs9514828 was associated with elevated serum BAFF concentrations compared to CC and CT genotypes. To date, one previous study has explored the association between the rs9514827 variant and GD in Tunisians, and no significant association with susceptibility to the disease was found. In addition, the rs9514827 genotypes were not associated with serum BAFF concentrations [23]. Other autoimmune diseases, such as SLE and RA, also showed no association with the rs9514827 variant [18, 20]. The present study may be the first to report a significant association between this variant and the risk of GD in Iraqi women. Ethnic difference may be responsible for this discrepancy in results and further studies are warranted. Regarding the rs9514828 SNP, only two studies have investigated the association between this variant and susceptibility to GD. In the first, no association with GD was found in a cohort from the United Kingdom [22], while in the second, the rs9514828 T allele was associated with an increased risk of GD in Tunisians [23]. The rs9514828 T allele was also found to be more prevalent in RA patients and the rs9514828 variant was considered a risk factor for the disease in a Mexican population. Moreover, the rs9514828 TT genotype was associated with elevated expression of TNFSF13B gene [20]. Taken together, these data indicate that the rs9514827 and rs9514828 SNPs may be associated with susceptibility to GD in Iraqi women, in addition to being involved in controlling BAFF production. In this context, rs9514828 polymorphism has been shown to be present in potential transcription factor-binding sites, and thus may affect the regulation of TNFSF13B gene expression [43].

The study encountered an important limitation, namely, the low sample size of GD patients (particularly ND patients of whom there were only six) and CTRL. In addition, the effects of BAFF on extra-thyroidal manifestations, such as orbitopathy and dermopathy, were not analyzed. Importantly, bioinformatic analysis of the rs9514827 and rs9514828 SNPs with respect to some of the transcription factor-binding sites in the TNFSF13B gene was not performed. Such an issue requires extensive investigation and may contribute to understanding the effects of SNP genotypes on BAFF synthesis.