Hashimotos HypothyroiditisHashimotos hypothyroiditis was first defined in 1912 in Japan by Dr. Hakaru Hashimoto, who had been examining the thyroid results of four womens thyroidectomies, all of whom had undergone surgery due to compressive symptoms in the throat. Hashimotos is a thyroid-specific chronic autoimmune disorder. It is considered the most common autoimmune disorder, and is most prevalent in women, and affects around five percent of the total population (1-3). Hashimotos results in thyroid disintegration from apoptotic processes in the inflamed thyroid and from T-cell mediated cytotoxicity. The biochemical identifiers of the disease are included in the presence of thyroid autoantibodies (TAbs) in sera for a pair of thyroid antigens: Thyroperoxidase (TPO) and Thyroglobulin (Tg). TAbs against TPO can cause damage to the thyroid through antibody dependent T cell cytotoxicity (4). Symptoms for Hashimotos when left untreated include: weight gain, depression, lack of energy, oversensitivity to cold, dry hair and nails, increase in menstruation and inflammation of thyroid which can lead to goiter, and difficulty swallowing or breathing. CTLA-4 also known as Cytotoxic T-Lymphocyte Associated Protein 4 is both the name of the gene and the protein it regularly codes for, which is a co-stimulatory molecule meant to suppress T-cell mediated immune responses. It is a transmembrane protein and is a member of the superfamily of immunoglobulin genesCTLA-4 is induced by T-cell receptor activation whereupon it is externally expressed by the cell where the inhibitory signal can be transmitted and it is crucial in maintaining immunological self-tolerance (5). There are many HPC gens that contribute to Hashimotos but CTLA-4 is the primary one (3). Found at chromosomal position 2q33 the gene starts from kb 202 9496 at the p-terminus and spans approximately 62 kb (6). CTLA-4 has three domains: a hydrophobic transmembrane domain, an immunoglobulin V domain, a cytoplasmic domain (6). It is expressed primarily on T-cells, following activation. The vast majority of CTLA-4 is found in vesicles within the cell where it can be transported to the T-cell Receptor site at the surface of the cell (4, 7). CTLA-4 protein expression on the surface of Helper T cells inhibits the activation of the T cell through signal transmittance and negative regulation of T-cell activity by moderating apoptosis for specific antigens (4, 5, 8, 9), while the normal activation of T cells results in an increase of Cytotoxic T-cell antigen 4 expression (5). Where there is a large threshold of CTLA-4 signals, the T-cell will not undergo activation due to CD28 and T-cell Receptor site (TCR) signal activation inhibition, both of which are necessary as costimulatory molecules for T-cell activation. Figure 1 demonstrates the action of inhibition in a regulatory T-cell. The ligation of either CD80 or CD86 by themselves to CTLA-4 on the surface of the antigen presenting cell creates a signal that interferes with the TCR signaling, thereby preventing activation of the T-cell. CTLA-4 much prefers to have ligation with both CD80 and CD86 to achieve activation of the cell (7). Augmented function or a reduced expression of CTLA-4 may have an impact on thyroid autoimmunity, and many studies support the correlated link between thyroid autoimmunity and gene polymorphisms in CTLA-4, especially in the case of Hashimotos hypothyroiditis (10). It has been noted that CTLA-4 contributes to the hereditary predisposition of excessive expression of thyroid autoantibodies (11). Many monozygotic twin studies have supported the claim that the mutations and polymorphisms of CTLA-4 are hereditary and are very likely to contribute to the development of Hashimotos disease (5).CTLA-4 expression plays a large in the development of many autoimmune disorders, and is likely to develop gene polymorphisms under unique sets of environmental factors such as infection (8). Polymorphic sequences are specific variations of DNA between individuals, and are also referred to as alleles (22). For the soluble form of CTLA-4 it has been indicated that it can inhibit initial activation of T-cells by preventing the CD80 and CD86 interactions using the CD28 molecule (12). The common allelic variant of CTLA-4 has been correlated with lower mRNA levels of the alternate soluble splice variant of CTLA-4 (13). T-cell function and CTLA-4 gene polymorphism has been found to have a direct correspondence, in addition to the correlation between CTLA-4 protein in Helper T cells and the genotype of the CTLA-4 gene (10). There is one such variant transcript of the gene with a removal, starting at the stop codon, extending from base pair 456 to 563. The deletion results in the loss of the third exon which contains approximately 37 amino acids coming from the domain of the transmembrane, and also results in a frame shift which produces twenty-two amino acids directly prior to the stop codon at base pair 523 (6). There are three known regions of polymorphic expression in the Cytotoxic T-Lymphocyte Associated Protein 4 gene that effect the development of Hashimotos hypothyroidism, and were identified as primary constituents of the disease (5). The first is a polymorphic microsatellite in exon three in the 3' untranslated region. The second polymorphism is in exon one at position number 49 containing a guanine/adenine mutative substitution, and is located within the CTLA-4 promoter (9). The third polymorphism is newly defined and contained within a promoter at position -318 with a mutative substitution of a cytosine nucleotide to a thymine nucleotide. At this position it is noted that patients with diagnosed Hashimotos thyroiditis have an increased presence of cytosine and a lesser presence of thymine. The first exon can be seen in the gene map of CTLA-4, along with the 3 untranslating region and the start and stop codons.Furthermore the presence of the first polymorphic segment is correlated to a predisposition of Hashimotos thyroiditis only when combined with the presence of a guanine allele in exon one. Thus, a promoter variation of the CTLA-4 gene is predisposed to be a risk marker for Hashimotos hypothyroiditis, however the susceptibility is only associated with the alleles of exon one (9). It has also been indicated that a substitution of amino acids at the seventeenth codon of the signaling peptide has been able to alter the peptide to direct and interfere with CTLA-4s signal trafficking intracellularly (5). Expression in the first exon of CTLA-4 of the double guanine alleles is correlated with an increased proliferation of T-cells compared to the observed proliferation of T-cells with a double adenine alleles. The double guanine genotype of CTLA-4 was also found to have reduced the function of the CTLA-4 protein itself (10). One study was performed and identified that the polymorphisms of CTLA-4 in exon one and in the promoter had a significant increase on the amount of thyroid autoantibody production in patients with Hashimotos. In this same study it was concluded that these gene polymorphisms would be able to contribute to Hashimotos autoimmune hypothyroiditis, as the production of thyroid autoantibodies causes the inflammation and deterioration of the thyroid gland, leading to a lack of the hormone thyroxin (14). In another study in the United Kingdom it was found that when compared to controls, patients with the autoimmune form of hypothyroiditis had a significant excess of the allelic guanine in the CTLA-4 susceptibility locus. Additionally, the same patients had more frequent genotypes expressing the AG and GG alleles when compared to patients without autoimmune hypothyroiditis, indicating a correlation (15). The first microsatellite polymorphism has been detected through polymerase chain reactions used to amplify the chosen DNA and resulting DNA was gelled to correctly ascertain the polymorphisms. Through this study, among others, it was determined that the microsatellite polymorphism effected CTLA-4s role in autoimmunity (16). Allele 106, the sequence of the polymorphic satellite was found to have a significant increase in patients with Hashimotos hypothyroiditis (17). The microsatellite was found to have a polymorphism containing an extensive repeat of adenines and thymines with various lengths (18). The repeated microsatellite polymorphisms are usually found in the 3 un-translating region of the gene, and are associated with lower levels of proliferative CTLA-4 and sCTLA-4 which have been connected to autoimmune hypothyroidism (19). Autoimmune susceptibility was mapped to the location of the 3 end of the gene, and in a mouse model the susceptibility was also linked to variations of CTLA-4 splicing (13).In regards to prevention and treat, the primary causes of HT can be attributed to malfunctions of the immune system, giving it the distinction of an autoimmune disorder. A large chunk of these malfunctions can be attributed to the errors of CTLA-4, which come about from gene polymorphisms found in the promoter and the first two exons of the gene, which affect the gene and the corresponding T-cells through substitution of nucleotides. These polymorphisms result in the proliferation of Tabs and malfunctioning Helper T cells. Thus CTLA-4 malfunction leads to inflammation of the thyroid by creating thyroid autoantibodies which deteriorate the gland and lead to a lack of and an inability to create thyroxin. Thyroxin is one of the major hormones of the endocrine system and is used in the regulation of psychological, metabolic, and physiological development, and if hypothyroidism is present it can adversely affect this development. Treatment of HT includes the semiannual screening of thyroid hormones to determine the levels of synthetic thyroxin needed to overcome hypothyroidism, and to increase the diagnoses of subclinical hypothyroidism (20). Since Hashimotos hypothyroiditis is an autoimmune disease caused by an immune malfunction resulting in thyroid inflammation and it is officially non-preventable. While there is currently no known cause of the genetic malfunction, there can be no surefire way to prevent the autoimmunity. What is known about the cause of the disease is that multiple polymorphisms in the CTLA-4 gene exons and promoter region contribute to the malfunction of the protein, creating a surplus of thyroid autoantibodies which break the gland down and result in inadequate levels of thyroxin, which is necessary for the essential and standard functioning of the body. The polymorphisms associated with autoimmune hypothyroiditis are considered to be hereditary and are likely to develop through a persons interaction with their environment however how much of an impact the environment has on the diseases development is currently unknown. Hashimotos hypothyroiditis, however, is very treatable. This is particularly effective when the signs are recognized and it is diagnosed early (24). Early diagnosis is important as Hashimotos can interfere with development psychologically and physically in both children, adults and fetuses. Screening for hypothyroidism in neonates is the most effective way to determine if thyroxin supplementation is needed and is also useful in preventing the development of complications from central nervous system maturation when there is an absence of thyroid hormone (21). Current treatment for Hashimotos is symptom based and is focused on administrating synthetic thyroid hormone, like synthroid and levothyroxine, to supplement the lack of thyroid hormone as is needed. Treatment can also include surgical removal of goiter when it interferes with a patients breathing and swallowing (1, 22). There have been some instances however where after a period of years a patient may develop hyperthyroidism even having been diagnosed with Hashimotos hypothyroidism previously (23).

Figure 1.Method of inactivation of CTLA-4 in T Helper Cell. CTLA-4 is represented by the dark green circles embedded in the cell membrane; it has a CD80 ligand and a CD86 ligand attached to either receptor, preventing the signal transmittance and subsequent activation the cell, even with the presence of the yellow CD28 molecule activated and the T-cell Receptor. (7)

Figure 2. Goiter is represented by the enlarged butterfly shaped thyroid gland surrounding the trachea. The gland itself enlarges and compresses the throat while it grows. http://www.webmd.com/women/goiter

Figure 3. Cytotoxic T-Lymphocyte Associated Protein 4Found at chromosome 2, on the long arm, position 33, contains 4 exons and the cDNA is 6,173bp long CTLA-4Gene map of CTLA-4 variant one DNA sequence is represented. Shown in yellow, the first base is the Transcription Starts Site, the beginning of the first exon, and the beginning of the 5 untranslated region (UTR), which is a tan color. The second exon (in orange) and the Start codon (in red) both start at base pair 156. The Coding DNA sequence starts at the same location, and is represented by a salmon color. The stop codon, ATG, is represented in purple, and begins at base 825. The remaining region is the 3 UTR which begins at base 829 and ends at the Poly-A Tail, which starts at base 1976, and is represented in blue. Gene map was created by author.

References1.P. Caturegli, A. De Remigis, N. R. Rose, Hashimoto thyroiditis: clinical and diagnostic criteria. Autoimmun Rev 13, 391-397 (2014).2.A. Syrenicz et al., [Hashimoto disease and hypothyroidism in child-bearing period--essential problem for woman and her child]. Endokrynol Pol 56, 1008-1015 (2005).3.E. M. Jacobson, Y. Tomer, THE CD40, CTLA-4, THYROGLOBULIN, TSH RECEPTOR, AND PTPN22 GENE QUINTET AND ITS CONTRIBUTION TO THYROID AUTOIMMUNITY: BACK TO THE FUTURE. Journal of autoimmunity 28, 85-98 (2007).4.K. Zaletel, S. Gaberek, Hashimoto's Thyroiditis: From Genes to the Disease. Current Genomics 12, 576-588 (2011).5.D. A. Chistiakov, Immunogenetics of Hashimoto's thyroiditis. Journal of Autoimmune Diseases 2, 1-1 (2005).6.D. Chistiakov, R. Turakulov, CTLA-4 and its role in autoimmune thyroid disease. Journal of Molecular Endocrinology 31, 21-36 (2003).7.D. M. Sansom, CD28, CTLA-4 and their ligands: who does what and to whom? Immunology 101, 169-177 (2000).8.T. F. Davies, R. Latif, X. Yin, New Genetic Insights from Autoimmune Thyroid Disease. Journal of Thyroid Research 2012, 6 (2012).9.J. Braun et al., CTLA-4 promoter variants in patients with Graves' disease and Hashimoto's thyroiditis. Tissue Antigens 51, 563-566 (1998).10.T. Kouki et al., CTLA-4 Gene Polymorphism at Position 49 in Exon 1 Reduces the Inhibitory Function of CTLA-4 and Contributes to the Pathogenesis of Graves Disease. The Journal of Immunology 165, 6606-6611 (2000).11.Y. Tomer, D. A. Greenberg, G. Barbesino, E. Concepcion, T. F. Davies, CTLA-4 and Not CD28 Is a Susceptibility Gene for Thyroid Autoantibody Production. The Journal of Clinical Endocrinology & Metabolism 86, 1687-1693 (2001).12.D. Saverino et al., Soluble CTLA-4 in autoimmune thyroid diseases: relationship with clinical status and possible role in the immune response dysregulation. Clinical immunology (Orlando, Fla.) 123, 190-198 (2007).13.H. Ueda et al., Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423, 506-511 (2003).14.K. Zaletel, B. Krhin, S. Gaberek, S. Hojker, Thyroid autoantibody production is influenced by exon 1 and promoter CTLA-4 polymorphisms in patients with Hashimoto's thyroiditis. International Journal of Immunogenetics 33, 87-91 (2006).15.R. Nithiyananthan, J. M. Heward, A. Allahabadia, J. A. Franklyn, S. C. L. Gough, Polymorphism of the CTLA-4 Gene Is Associated with Autoimmune Hypothyroidism in the United Kingdom. Thyroid 12, 3-6 (2002).16.E. H. Kemp et al., A cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism is associated with autoimmune Addison's disease in English patients. Clinical endocrinology 49, 609-613 (1998).17.K. Kotsa, P. F. Watson, A. P. Weetman, A CTLA-4 gene polymorphism is associated with both Graves disease and autoimmune hypothyroidism. Clinical endocrinology 46, 551-554 (1997).18.E. H. Kemp et al., Analysis of a microsatellite polymorphism of the cytotoxic T-lymphocyte antigen-4 gene in patients with vitiligo. The British journal of dermatology 140, 73-78 (1999).19.Z. Chen et al., CTLA4 -1661A/G and 3[prime]UTR long repeat polymorphisms are associated with ulcerative colitis and influence CTLA4 mRNA and protein expression. Genes Immun 11, 573-583 (2010).20.F. W. F. Hanna, J. H. Lazarus, M. F. Scanlon, Controversial aspects of thyroid disease. BMJ : British Medical Journal 319, 894-899 (1999).21.K. Boelaert, J. A. Franklyn, Thyroid hormone in health and disease. Journal of Endocrinology 187, 1-15 (2005).22.J. Slatosky, B. Shipton, H. Wahba, Thyroiditis: differential diagnosis and management. American family physician 61, 1047-1052, 1054 (2000).23.A. S. Alzahrani, S. Aldasouqi, S. Abdel Salam, A. Sultan, Autoimmune Thyroid Disease with Fluctuating Thyroid Function. PLoS Medicine 2, e89 (2005).24.Milas, K., Preventing Hashimotos Thyroiditis. Endocrineweb.com. Posted May 29, 2014. Retrieved March 27, 2016.