**4.4 The cytotoxic T lymphocyte-associated factor 4 (CTLA4 Or CD152)**

It is a protein receptor that functions as an immune checkpoint and downregulates immune responses. It is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation; a phenomenon which is particularly notable in cancers [47]. It is homologous to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTL4-4 binds CD80 and CD86 with greater affinity and avidity than CD28 thus enabling it to outcompete CD28 for its ligands. CTLA4 transfers an inhibitory signal to T cells, [48] whereas CD28 transmits a stimulatory signal [48]. CTLA4 is also found in regulatory T cells and thereby contributing to their inhibitory function. CTLA4 consists of four exons encoding different functional domains such as a leader sequence and extracellular, transmembrane as well as cytoplasmic domains. The most reliable associations with GD within *CTLA4* locus were found with three polymorphisms: the

*AT-microsatellite polymorphism (ATn)* at the 3'untranslated region (3'UTR) of the gene [49]. It has been proposed that this AT-repeat allele decreased the` stability of *CTLA4* mRNA thus dampening the inhibitory function of the protein and thus diminishing the control of T-cell proliferation [50]. The *second* polymorphism implicated was *rs231775 (A49G)* in the signal peptide causing a substitution of Thr to Ala [51]. This amino acid change could influence post-translational processing leading to inefficient glycosylation of the autoimmunity predisposing variant [52]. Another widely studied genetic polymorphism in *CTLA4 gene* is *rs3087243* (*CT60)* located downstream from the 3'UTR of the *CTLA4* [53]*.* After taking into account the CT60 genotype, Examination of full-length (flCTLA-4) and sCTLA-4 expression revealed a lower expression of sCTLA-4 in persons homozygous for the G allele [54]. But, in a larger Swedish study this result was not replicated which also did not find any association between concentration of serum sCTLA4 and disease status or CT60 genotype [54]. Lately, in Japanese patients a noteworthy association with CTLA4 CT60 was found for GD with OR=2.97. The present state of knowledge does not specify evidently the mechanism behind the association of *CTLA4* with GD. However, *CTLA4* polymorphism is consistently associated with thyroid autoimmune diseases in the majority of populations.

#### **4.5 The thyrotropin receptor (TSHR)**

The thyrotropin receptor (TSHR) responding to thyrotropin (thyroidstimulating hormone, TSH) is a Gs-protein coupled receptor and stimulates the production of thyroxin (T4) and triiodothyronine (T3). It is primarily found on the surface of the thyroid epithelial cells [55], but also found on adipose tissue and fibroblasts. A G protein signal cascade is activated upon the binding of circulating ligand TSH which activates adenylyl cyclase that synthesizes cAMP from ATP and subsequently resulting in increased intracellular levels of cAMP. cAMP functions as a secondary messenger and activates all functional aspects of the thyroid cells which include thyroglobulin synthesis, iodine pumping, endocytosis, iodination, proteolysis, thyroid peroxidase activity and hormone release. *TSHR gene* is located on 14q31 [56] and consists of 13 exons [57]. The TSHR is among the susceptibility genes of GD because it encodes for a protein that is both responsible for the clinical manifestations of the disease and is the direct target of the autoimmune response in GD. The anti-TSHR antibodies in serum are the main serological manifestations of GD. Indeed, TSHR- stimulating antibodies (TSAbs) are present in nearly all cases of GD and severity of the disease correlates with TSAbs levels. One of the first non-MHC genes to be tested for association with the disease was TSHR. Three germline missence mutations (a substitution of aspartic acid (D) for histidine (H) in position 36 *(D36H)*; a substitution of a proline (P) for threonine (T) in position *52(P52T)*, and a substitution of aspartic acid (D) for glutamic acid (E) in position *727 (D727E)* were primarily described in individuals suffering from GD and proposed to be associated with the disease [3]. Of these three, *D36H* and *P52T* are the two mutations which are located in the putative ligand binding region of the extracellular domain of the TSHR, while the third one, *D727E* lies within the intracellular domain of the receptor.

#### **4.6 FCRL3 (FC receptor-like-3)**

Fc receptor-like protein 3 is a protein that in humans is encoded by *FCRL3 gene* [58]*.* It is located on 1q23.1. This gene located on q arm of chromosome 1st is one of the several Fc receptors like glycoproteins which encode a member of the IR superfamily. The encoded protein plays a role in regulation of the immune system *Graves' Disease: Pathophysiology, Genetics and Management DOI: http://dx.doi.org/10.5772/intechopen.98238*

and in its cytoplasmic domain it contains immunoreceptor-tyrosine activation motifs and immunoreceptor-tyrosine inhibitory motifs. Rheumatoid arthritis, autoimmune thyroid disease, and systemic lupus erythematosus have been associated in the mutation of this gene [58]. FCRL3 is a novel immunoregulatory gene believed to perform similar functions as FC gamma receptors due to high structural homology between them. FCRL3 gene polymorphism is related to the susceptibility to GD with regional and ethnic variability [59]. A SNP at the position *-169A/G (rs7528684)* in promoter of *FCRL3* gene has been found to be associated with GD. Correlation of this SNP to serum levels of FT3, FT4, TSH and TRAb, gender, age, TpoAb, TgAb, severity of goiter and presence or absence of exophthalmos in GD have been widely investigated [59]. There is an association of anther SNP *rs3761959* (tagging rs7528684) with GD. Overall the available data suggest that genetic polymorphism(s) modifying susceptibility for GD do exist in the *FCRL3* region but the primarily associated variant(s) remain(s) to be found [60].

#### **4.7 Secretoglobin 3A2 (SCGB3A2) gene**

This gene is present on chromosome 5 in humans and chromosome 18 in mouse. It is a homodimeric protein thought to play a role in the modulation of inflammation and tumorigenesis. SCGB3A2 is a member of secretoglobin superfamily, a family of small, secreted proteins found in animals exclusively of mammalian lineage. *SCGB3A2* mRNA is predominantly expressed in the lung with low levels of expression in the thyroid. Variants in the promoter of the *SCGB3A2* gene encoding secretory uteroglobin-related protein 1 (UGRP1) have been found associated with GD in an extensive study of a total of ~2500 patients and controls from the Chinese population who aimed to explain signals in the chromosomal region 5q12-q33 obtained in previous studies using linkage analysis [61–63]. Song *et al.* reported the strongest association of GD with *rs1368408 (112G/A, OR=1.28, P=1.43x10-6)* and *SNP75 (−623~−622 AG/−T, OR=1.32, P=7.62x10-5)* [60, 61]. Association between GD and the A allele of *rs1368408* (OR=1.18, P=0.007) was independently confirmed in a similarly sized UK cohort [64]. Recently, further evidence for association between rs1368408 and GD was provided by a study in a Russian cohort (~1500 cases and controls, OR=1.33, P=2.910-5) [60]. It should be noted that a relatively early study in a Chinese population did not observe the effect of *rs1368408,* although this might have been caused by low power due to limited numbers of subjects (~200 cases and controls) [62]. Till present it is still not clear how variants in *SCGB3A2* predispose to GD.

There are other genes which have strong association with GD such as cytokine genes: *IL3, IL4, IL5, IL9, IL13* and the *ADRB2* gene encoding beta-2-Adrenergic receptor [65–67] were all associated with GD.

#### **5. Diagnosis of Graves' Disease**

The diagnosis to confirm the cause of Graves' hyperthyroidism is based on the clinical and biochemical manifestations of hyperthyroidism and on the clinical and laboratory features. For the presence of hyperthyroidism, measurement of serum thyrotropin is a useful screening test, because the secretion of thyrotropin is reduced by very small increase in thyroid secretion, so by the measurement of serum free thyroxine the diagnosis of hyperthyroidism must be confirmed [68]. Patients may have only increased secretion of triiodothyronine in the earliest stage of Graves' hyperthyroidism; therefore, patients with normal serum free thyroxine concentrations and low serum thyrotropin concentrations, serum free

triiodothyronine should be measured. Because of the use of certain drugs and increase in thyroid hormone–binding proteins, measurements of serum total thyroxine and triiodothyronine are less reliable as it can cause high values [69]. In patients with hyperthyroidism and a diffuse goiter, the signs of ophthalmopathy or dermopathy are sufficient to confirm the diagnosis of GD. In patients with GD, other autoimmune disorders occur more frequently (Type 1 diabetes mellitus, Addison's disease, Vitiligo, Pernicious anemia Alopecia areata, Myasthenia gravis, Celiac disease) and their presence therefore supports this diagnosis. Occasionally, in patients with pre-existing nodular goiter, GD occurs which causes confusion. The presence of a high serum concentration of thyroid peroxidase antibody which is present in about 75 percent of patients with Graves' hyperthyroidism, or a thyroid radionuclide scan demonstrating a diffuse goiter provides evidence of GD, when the diagnosis is unclear clinically. Occasionally, to distinguish between Graves' hyperthyroidism and thyrotoxicosis caused by painless, destructive (autoimmune) thyroiditis, thyroid radionuclide studies may be indicated especially in women post-partum. Patients may have a small diffuse goiter with painless thyroiditis, like those with GD. However, it is very unlikely that thyrotoxicosis due to painless thyroiditis will last longer than two months [70].
