Abstract
Background
Graves' disease (GD), including Graves' ophthalmopathy or orbitopathy (GO), is an autoimmune disease with an environmental and genetic component to its etiology. The genetic contribution to the GO clinical phenotype remains unclear. Previous data from our laboratory and others have suggested that GO has no specific genetic component distinct from GD itself, while other reports have occasionally appeared suggesting that polymorphisms in genes such as CTLA4 and IL23R specifically increase the risk for GO. One of the criticisms of all these reports has been the clinical definition of the GO phenotype as distinct from hyperthyroid GD devoid of clinically significant eye involvement. The objective of this study was to take advantage of a phenotypically pure group of GD patients with GO and examine a series of genes associated with GD to determine if any were more definitively associated with GO rather than Graves' thyroid disease itself.
Methods
To further examine whether specific susceptibility genes are associated with GO, we have performed further genetic association studies using highly characterized GO patients, many of whom had undergone orbital decompression surgery for their exophthalmos. We genotyped HLA, CTLA4, IL23R, and TSHR genes in a group of 256 Caucasian patients with severe GO (n=199) and less severe GO (n=57), and 90 patients with GD but no clinically apparent GO.
Results
We found that the allele and genotype frequencies were not statistically different between GO and non-GO patients for any of the genes and gene combinations assessed.
Conclusions
These results provide further evidence that patients with GO do not have a distinct genetic susceptibility to their eye disease and again suggest that environmental and/or epigenetic influences are at play.
Introduction
Graves' disease (GD) is an autoimmune thyroid disease (AITD) that affects ∼1% of the general population and is characterized by T cell and B cell reactivity to the TSH receptor (TSHR) (1,2). TSHR antibodies act as TSH agonists and induce thyroid overactivity. It has been estimated that 25%–50% of GD patients have clinical signs of Graves' ophthalmopathy or orbitopathy (GO), with the vast majority having relatively mild disease (3,4). As with all autoimmune disorders, it is thought that GD is caused by the interaction of genetic, epigenetic, and environmental factors in a stochastic manner. As far as GO is concerned, the primary antigen is also considered to be the retro-orbital expression of the TSHR, combined with a propensity for cytokine-mediated tissue damage from retro-orbital inflammation (5,6).
A variety of gene polymorphisms have been found to be linked and/or associated with GD, and the genes involved have fallen into two distinct groupings. The first group is those genes common to many autoimmune diseases and involving immune regulatory and response genes. These include HLA-DR3 (7,8), CTLA4 (9,10), PTPN22 (11,12), CD40 (13,14), IL2RA (CD25) (15,16), FCRL3 (17,18), and the IL23R (19). The second group includes genes that are specific to the thyroid gland: the TSHR (20–24) and thyroglobulin (TG) (25,26). However, the distinct factors predisposing GD patients to the development of GO remain unclear (27). While claims for preferential genetic associations with GO (19,28) have been made, our own data have not substantiated such reports (23,27).
To further examine whether any of the known gene associations are more likely to be associated with GO rather than just GD, we have re-examined the association of some of the gene polymorphisms claimed to be associated with GO. We have used a group of well-defined patients with GD including almost 200 patients who had undergone orbital decompression surgery. In this group of GO patients, there can be no doubt about the severe clinical phenotype and no doubt about the diagnosis—a criticism made against many earlier genetic studies of patients with GD. Nevertheless, our conclusions remain unchanged. There is no evidence to date that patients with GO have a genetic signature any distinct from patients with GD who do not develop GO.
Methods
Subjects
DNA was obtained from 346 Caucasian patients with GD as described in Table 1. These studies were approved by the Mount Sinai Institutional Review Board. A total of 199 patients with severe GO had undergone orbital decompression cosmetic surgery for their disease, and 57 had less critical GO but all had Werner class IIIa or higher GO (4,29), as well as 90 patients who had no clinical signs of eye disease (non-GO, or GD without GO). All patients had over a 2-year history of AITD. GD was defined as the presence of chemical hyperthyroidism and a normal or increased diffuse thyroid radioiodine uptake and/or the presence of TSHR antibodies. GO (also known as thyroid eye disease and dysthyroid/thyroid-associated orbitopathy) was defined clinically as a chronic autoimmune inflammatory disorder impacting the orbit around the eye, presenting various eye features as described (30). A group of Caucasian controls (n=402) was available from commercial sources (Coriell Institute, Camden, NJ).
Table 1.
Characterization of Patient Groups
| Diagnosis | n | Sex | Age (mean years) | Range (years) |
|---|---|---|---|---|
| GO | 198 | Female | 52 | 22–85 |
| 58 | Male | |||
| Non-GO | 80 | Female | 44 | 21–60 |
| 10 | Male | |||
| All GD | 278 | Female | 51 | 21–85 |
| 68 | Male |
The numbers represent individual subjects.
GO, Graves' ophthalmopathy; GD, Graves' disease.
HLA-DR3 typing
Positive or negative HLA-DR3 was typed by the method of group-specific polymerase chain reaction (PCR) amplification followed by restriction enzyme digestion as described by Westman et al. (31).
Genotyping
TSHR single-nucleotide polymorphism (SNP)—TSHR-SNP-rs2268458, which we have previously found to be associated with GD (23)—is located in intron 1, and was measured using standard PCR-RFLP procedures. The protocol was the same as described in the previous study (23). Human genomic DNA (∼25 ng) was amplified by PCR and 415-bp products were generated. Primer 1 was 5′-CCAGCAGAGGGAGCA CAA-3′, and primer 2 was 5′-TAGAGAATAGAGCAGCAA GAGACT-3′.
These primers flank the DNA fragment in TSHR gene intron 1. The PCR (25 μL) containing 1U Platinum Taq polymerase was carried out as directed by the manufacturer (Invitrogen Corporation, Carlsbad, CA). The PCR parameters were as follows: 95°C for 5 minutes, 35 cycles (95°C for 1 min, 52°C for 1 min, and 72°C for 1 min), followed by 72°C for 10 minutes. Then, 8 μL of PCR products were digested for 2 hours in 10 μL total volume with the restriction endonuclease AluI according to the manufacturer's instructions (New England BioLabs, Inc., Beverly, MA) along with a nondigested DNA control. After digestion, the digested fragments were mixed with a gel loading buffer, separated on a 3% agarose gel, visualized by ethidium bromide and UV light, documented with FluorChem®HD2 Imaging System (Alpha Innotech Corporation, San Leandro, CA), and analyzed by the genotype patterns. Since AluI digestion determines AGTT versus AGCT, this allowed the determination of each individual hetero- or homogenotype. The patterns were recorded as TT with one 333-bp DNA fragment, TC with one 333-bp and one 275-bp fragments, and CC with one 275-bp fragment.
CTLA4 SNP
The CTLA4 exon 1, A49G SNP was assayed by the same method as described above, but using a different primer pair and the BbvI restriction enzyme (New England BioLabs, Inc., Beverly, MA) (32). The forward primer was 5′-GCTCTAC TTCCTGAAGACCT-3′, and the reverse primer was 5′-AGTC TCACTCACCTTTGCAG-3′.
The A allele resulted in an undigested PCR product of 162 bp; the G allele resulted in a digested PCR product, showing 90- and 72-bp fragments viewed as one bold band on the agarose gel, and representing each individual hetero- or homogenotype.
IL23R SNP-rs10889677
The IL23R SNP-rs10889677 (located in the 3′ untranslated region 309 bases downstream the stop codon) was genotyped by the same method as described above, but using a different primer pair and the MnlI restriction enzyme (New England BioLabs, Inc., Beverly, MA) (19). The forward primer was 5′-AGGGGATTGCTGGGCCATAT-3′, and the reverse primer was 5′-TGTGCCTGTATGTGTGACCA-3′.
The A allele resulted in an undigested PCR product of 216 bp; the C allele resulted in a digested PCR product, showing 154- and 62-bp fragments on the agarose gel, and representing each individual hetero- or homogenotype.
IL23R SNP-rs2201841
The IL23R SNP-rs2201841 (located in intron 7) was genotyped by the same method as described above, but applying a fluorescent-based restriction fragment length polymorphism method and using a different primer pair and the HpyF3I restriction enzyme (19). The forward primer was 5′-GGCC TATGATTATGCTTTTTCCTG-3′, and the reverse primer was 5′-GAACATAACCCTATTGACACCCTG-3′.
The forward primer was fluorescently labeled. The A allele resulted in an undigested PCR product of 188 bp; the G allele resulted in a digested PCR product, showing 163 and 25 bp fragments; and the 163-bp fragment contained the fluorescently labeled forward primer. The PCR products were separated and visualized on an ABI 3130xl genetic analyzer, and finally analyzed by GeneMapper software (Applied Biosystems, Foster City, CA) in order to assign each allele.
Statistical analyses and power calculations
Patient groups were clearly defined for the association studies. Differences in allele frequencies and genotypes between the groups were analyzed by the Chi-square test (χ2) and Fisher's exact test (where appropriate). All tests were two tailed, and p<0.05 was considered as significant. Using the Centers for Disease Control (CDC) simulation software (Epi Info v.3.5.3.), we were able to define the limits of our analyses. This indicated that a sample patient group size of 256 GO and 90 non-GO subjects would enable detection of genetic associations, with 80% power, with odds ratios of >1.74, >1.74, >1.80, >1.87, and >1.91 for HLA-DR3, CTLA4, TSHR, IL-23R-rs10889677, and IL-23R-rs2201841, respectively, with an alpha of 0.2.
Results
Comparing GD patients with and without GO
To determine a genetic basis for the clinical phenotype known as GO, we compared patients with and without this clinical manifestation of GD. For the well-established polymorphisms associated with GD, we found no difference in the prevalence between those patients having GO and those without clinical evidence of GO. This observation applied to the presence of HLA-DR3, CTLA4, IL-23R, and the TSHR (Table 2). No significant differences were observed for any of the measured polymorphisms.
Table 2.
Genetic Association with Graves' Disease
| Chromosome Annotated gene [SNP] | Risk allele | Status | Gender | GO, n=256 (%) | Severe GO, n=199 (%) | Non-GO, n=90 (%) | GO vs. Non-GO, p-value |
|---|---|---|---|---|---|---|---|
| 6p21 HLA-DR3 |
Present/absent | Total | 115/141 (44.9) | 89/110 (44.7) | 42/48 (46.7) | 0.775 | |
| Female | 87/111 (34.0) | 66/87 (33.2) | 39/41 (43.3) | 0.466 | |||
| Male | 28/30 (10.9) | 23/23 (11.6) | 3/7 (3.3) | 0.286 | |||
| 14q31 TSHR [rs2268458] |
C | Present/absent | Total | 117/139 (45.7) | 92/107 (46.2) | 46/44 (51.1) | 0.377 |
| Female | 92/106 (35.9) | 71/82 (35.7) | 42/38 (46.7) | 0.362 | |||
| Male | 25/33 (9.8) | 21/25 (10.6) | 4/6 (4.4) | 0.855 | |||
| 2q33 CTLA4 [A49G] |
G | Present/absent | Total | 171/85 (66.8) | 132/67 (66.3) | 56/34 (62.2) | 0.432 |
| Female | 126/72 (49.2) | 97/56 (48.7) | 50/30 (55.6) | 0.859 | |||
| Male | 45/13 (17.6) | 35/11 (17.6) | 6/4 (6.7) | 0.236 | |||
| 1p31 IL23R [rs10889677] |
C | Present/absent | Total | 233/23 (91.0) | 180/19 (90.5) | 83/7 (92.2) | 0.726 |
| Female | 181/17 (70.7) | 138/15 (69.3) | 74/6 (82.2) | 0.766 | |||
| Male | 52/6 (20.3) | 42/4 (21.1) | 9/1 (10.0) | 0.974 | |||
| 1p31 IL23R [rs2201841] |
A | Present/absent | Total | 231/25 (90.2) | 178/21 (89.4) | 83/7 (92.2) | 0.576 |
| Female | 180/18 (70.3) | 137/16 (68.8) | 74/6 (82.2) | 0.669 | |||
| Male | 51/7 (19.9) | 41/5 (20.6) | 9/1 (10.0) | 0.851 |
SNP, single-nucleotide polymorphism; TSHR, TSH receptor.
HLA-DR3
About 45.4% (n=346) of patients with GD had HLA-DR3 compared with 22.9% (n=402) of controls (p<10−7). There was no significant difference in the percentage of HLA-DR3-positive patients in the GO group (44.9%) (n=256) compared to the non-GO group (46.7%) (n=90) (Fig. 1, Table 2). The non-GO group also had significantly more HLA-DR3-positive members than the control group (p=0.0000046).
FIG. 1.
HLA-DR3 in autoimmune thyroid disease. The X axis shows the genotyping results of HLA-DR3-positive (DR3+) and HLA-DR3-negative (DR3−) samples from patients with all Graves' disease (GD), Graves' ophthalmopathy (GO), GD without GO (Non-GO), and control. The Y axis shows the HLA-DR3 genotyping frequency (%) in tested samples. They were significantly different when comparing GD, GO, and Non-GO with control, respectively (p<1×10−8, p<1×10−8, and p=0.0000046). There is no significant difference between GO and non-GO (p=0.775).
CTLA4 and IL23R
The G allele of the CTLA4 A49G SNP, previously reported as more significantly associated with GO rather than non-GO patients (28), was found in 41.2% of the GO patients, which was not different from the prevalence of this polymorphism in the group of patients without GO (40.6%) (Table 3). Similarly, examining two SNPs in the IL23R gene, which have also been previously reported to be associated with GO rather than non-GO (19), were again similar with no significant differences in the prevalence of the alleles of these SNPs or their genotypes (Tables 2 and 3).
Table 3.
Allele and Genotype Frequencies in Graves' Ophthalmopathy Versus Non–Graves' Ophthalmopathy
| Chromosome Annotated gene [SNP] | Allele/genotype | GO, n=256 (%) | Non-GO, n=90 (%) | p-value |
|---|---|---|---|---|
| 2q33 CTLA4 [A49G] |
A | 301 (58.8) | 107 (59.4) | 0.878 |
| G | 211 (41.2) | 73 (40.6) | ||
| AA | 85 (33.2) | 34 (37.8) | 0.434 | |
| AG | 131 (51.2) | 39 (43.3) | ||
| GG | 40 (15.6) | 17 (18.9) | ||
| 1p31 IL23R [rs10889677] |
C | 360 (70.3) | 121 (67.2) | 0.439 |
| A | 152 (29.7) | 59 (32.8) | ||
| CC | 127 (49.6) | 38 (42.2) | 0.368 | |
| AC | 106 (41.4) | 45 (50.0) | ||
| AA | 23 (9.0) | 7 (7.8) | ||
| 1p31 IL23R [rs2201841] |
A | 359 (70.1) | 125 (69.4) | 0.866 |
| G | 153 (29.9) | 55 (30.6) | ||
| AA | 128 (50.0) | 42 (46.7) | 0.641 | |
| AG | 103 (40.2) | 41 (45.6) | ||
| GG | 25 (9.8) | 7 (7.8) | ||
| 14q31 TSHR [rs2268458] |
T | 377 (73.6) | 124 (68.9) | 0.221 |
| C | 135 (26.4) | 56 (31.1) | ||
| TT | 139 (54.3) | 44 (48.9) | 0.413 | |
| TC | 99 (38.7) | 36 (40.0) | ||
| CC | 18 (7.0) | 10 (11.1) |
TSHR
Polymorphisms in TSHR have been widely associated with GD but not Hashimoto's thyroiditis, and we have previously failed to observe a preference for patients with GO (23). The present report found the same result in this larger patient cohort dominated by patients with severe disease (Tables 2 and 3).
Comparing GD patients with severe GO and less severe GO
When severe GO was defined as only those patients undergoing decompression surgery, we also found no difference in any of the gene polymorphisms comparing such patients with those not requiring surgery and defined as less severe disease although this also most likely included patients who refused surgery (Table 4).
Table 4.
Comparing Genetic Association with Clinical Degree of Graves' Ophthalmopathy
| Chromosome Annotated gene [SNP] | Risk allele | Status | Severe GO, n=199 (%) | Mild GO, n=57 (%) | p-value |
|---|---|---|---|---|---|
| 6p21 HLA-DR3 |
Present | 89 (44.7) | 26 (45.6) | 0.905 | |
| Absent | 110 (55.3) | 31 (54.4) | |||
| 14q31 TSHR [rs2268458] |
C | Present | 92 (46.2) | 25 (43.9) | 0.751 |
| Absent | 107 (53.8) | 32 (56.1) | |||
| 2q33 CTLA4 [A49G] |
G | Present | 132 (66.3) | 39 (68.4) | 0.768 |
| Absent | 67 (33.7) | 18 (31.6) | |||
| 1p31 IL23R [rs10889677] |
C | Present | 180 (90.5) | 53 (93.0) | 0.556 |
| Absent | 19 (9.5) | 4 (7.0) | |||
| 1p31 IL23R [rs2201841] |
A | Present | 178 (89.4) | 53 (93.0) | 0.428 |
| Absent | 21 (10.6) | 4 (7.0) |
Genetic profiling of patients with GD
Examining patients for the presence of multiple GD-associated polymorphisms showed that <1% of GD patients harbored none of the selected gene polymorphisms examined. In contrast, ∼10% of patients showed all five of the GD-associated polymorphisms (Table 5). There was no significant difference in the number of measured polymorphisms between GO and non-GO patients. The number of patients with all five polymorphisms was also similar in those groups of patients with GO and those without GO (Table 5).
Table 5.
Genetic Profiling of Graves' Disease
| Number of gene/SNP | GO, n=256 (%) | Non-GO, n=90 (%) | GO vs. non-GO, p-value; <χ2> (degree) |
|---|---|---|---|
| 0 | 1 (0.4) | 1 (1.1) | 0.819; <2.210> (5) |
| 1 | 9 (3.5) | 2 (2.2) | 0.806; <1.614> (4) |
| 2 | 29 (11.3) | 8 (8.9) | 0.740; <1.255> (3) |
| 3 | 95 (37.1) | 32 (35.6) | 0.668; <0.807> (2) |
| 4 | 95 (37.1) | 39 (43.3) | 0.463; <0.539> (1) |
| 5 | 27 (10.5) | 8 (8.9) |
Gene/SNP: HLA-DR3, CTLA4, IL23R-rs10889677, IL23R-rs2201841, and TSHR.
Gender
When the patients were divided by gender, there were no significant differences in any of the measured polymorphisms between GO and non-GO in exclusively male or female patient groups (Table 2).
Relationship of HLA-DR3 with other susceptibility genes
We further analyzed whether HLA-DR3 was directly associated with the other susceptibility genes examined. Supplementary Table S1 (Supplementary Data are available online at www.liebertonline.com/thy) shows that HLA-DR3 did not associate with any of the assessed genes in any of the subgroups, indicating that HLA-DR3 susceptibility was independent of these genes in patients with GD.
Discussion
We now know that GD is a complex genetic disease with significant genetic and environmental influences that may exert their effects via epigenetic mechanisms (26,33–35). The susceptibility genes have been identified both by the candidate gene approach and by whole genome linkage and association studies (36). The identified genes have, to date, each provided only a small contribution to genetic susceptibility, including the TSHR gene (22,23), indicating that many genes must be involved. However, the concept of major, or necessary, gene polymorphisms that have to be inherited to develop the disease does not appear to apply to GD (37,38). A further conclusion from such information is that the same clinical phenotype of GD may be obtained by the conjunction of different susceptibility genes in different individuals and in different populations.
One of the most obvious clinical phenotypes of GD is the orbitopathy. GO may be a most disabling condition with very unsatisfactory treatment approaches (30,39,40). The primary antigen appears to be the TSHR expressed on fibroblasts and adipocytes throughout the body, but they find themselves in a retroorbital environment that exacerbates the immune response to their presence (40–42). The argument over whether GO is a disorder distinct from GD and simply inherited at the same time appears less impressive now that the TSHR has been identified as an important antigen and that titers of TSHR antibodies correlate with the disease (40,41). In the present study and in our previous observations (23), TSHR gene polymorphisms were no more prevalent in the population of patients with GD and GO compared to patients without GO. Nevertheless, the thought has persisted that particular susceptibility must bias a patient toward the development of the eye phenotype rather than such patients simply having a worse form of the same disorder. While some studies have suggested that all patients with GD have a degree of eye involvement (2,27,43,44), others have suggested that GO has a genetic etiology rather than the more likely environmental cause, whether it be orbital anatomy, smoke, or trauma (19,28). Hence, CTLA4 gene polymorphisms were claimed as the immune mechanism pushing patients to a more severe retro-orbital inflammatory response (28), while most recently IL23R has been said to be responsible (19).
The CTLA4 gene is an immune regulatory molecule that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. Hence, deficient CTLA4 activity is associated with exacerbated T cell reactivity. CTLA4 gene polymorphisms have been shown to confer susceptibility to several autoimmune diseases, including AITD. However, CTLA4 was not demonstrated as a specific GO-associated gene in the present study in keeping with our previous observations (27) and those of others (45,46). The IL-23R is a type I transmembrane protein that binds with the IL-23 cytokine to activate Janus kinases. This leads to transcription of key pro-inflammatory genes, including IL-17, and interferon-γ from T regulatory cells and when defective impairs T regulatory cell function. It is already known that polymorphisms in the IL23R gene confer susceptibility to several autoimmune diseases, including Crohn's disease, psoriasis, rheumatoid arthritis, ankylosing spondylitis, and multiple sclerosis (47–50), and it was not unreasonable to expect the AITDs to show a similar risk. Although the IL23R gene was previously reported to be specifically associated with GO (19), this specific association was not seen in the present study. This may have been because this was a well-defined Caucasian group of severe GO patients. Of note, the association has also not been seen in a Japanese population (51). These results, therefore, provide no evidence that the IL23R gene confers an important specific genetic susceptibility to GO. In addition, the IL23R gene has also been shown to not confer susceptibility to several other autoimmune diseases, such as systemic sclerosis, systemic lupus erythematosus, and Sjögren's syndrome (50).
The strongest genetic association for the AITDs has been with the MHC region (8,52). For GD, the risk ratios associated with the HLA-DR3 haplotypes have been larger than the gene risk ratios for all the other known or suspected genes (8,52) and particularly high when analyzed on the basis of the presence of Arg 74 in the DR-binding pocket (8,35). The MHC contains an assortment of immune-regulatory genes, but the HLA-DR and DQ regions have been most consistently associated although Class I genes have also been implicated (8,52,53). Past reports have suggested that HLA-DR3 was more prevalent in patients with GO, while others were unable to confirm this (54–58). Our results simply showed that HLA-DR3 was highly associated with GD, but there was no significant predilection for the GO patients rather than the non-GO patients, indicating that HLA-DR3 is not a specific GO-associated gene. Our data also suggested that HLA-DR3 does not work in association with CTLA4, IL23R, and TSHR genes in GO.
Our attempts at genetic profiling showed that very few patients failed to show any associated genes, while 10% showed all five of the at-risk polymorphisms. Nevertheless, this was not different when comparing GO with non-GO patients. While it is possible that other minor genes or some modified genes are involved in the development of GO, we found no evidence for this opinion in this study of selected susceptibility genes.
In summary, our study demonstrated that four major reported GD-associated genes (HLA-DR3, CTLA4, IL23R, and TSHR) or gene combinations were not specifically associated with GO. The evidence continues to point to GO being a manifestation of GD exacerbated by stochastic environmental or anatomical influences (42).
Supplementary Material
Acknowledgments
This work was supported in part by DK052464 and DK69713 from NIH-NIDDK the David Owen Segal Endowment, the James J. Peters VAMC, and the VA Merit Award program. We thank our colleague Dr. Yaron Tomer for his help and advice.
Disclosure Statement
T.F.D. is a Board Member of Kronus, Inc., Star, Idaho (a distributor of thyroid antibody test kits). The other authors have no conflicts of interest to disclose.
References
- 1.Jacobson DL. Gange SJ. Rose NR. Graham NM. Epidemiology and estimated population burden of selected autoimmune diseases in the United States. Clin Immunol Immunopathol. 1997;84:223–243. doi: 10.1006/clin.1997.4412. [DOI] [PubMed] [Google Scholar]
- 2.Weetman AP. Graves' disease. N Engl J Med. 2000;343:1236–1248. doi: 10.1056/NEJM200010263431707. [DOI] [PubMed] [Google Scholar]
- 3.Khoo TK. Bahn RS. Pathogenesis of Graves' ophthalmopathy: the role of autoantibodies. Thyroid. 2007;17:1013–1018. doi: 10.1089/thy.2007.0185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.El-Kaissi S. Frauman AG. Wall JR. Thyroid-associated ophthalmopathy: a practical guide to classification, natural history and management. Intern Med J. 2004;34:482–491. doi: 10.1111/j.1445-5994.2004.00662.x. [DOI] [PubMed] [Google Scholar]
- 5.Valyasevi RW. Jyonouchi SC. Dutton CM. Munsakul N. Bahn RS. Effect of tumor necrosis factor-alpha, interferon-gamma, and transforming growth factor-beta on adipogenesis and expression of thyrotropin receptor in human orbital preadipocyte fibroblasts. J Clin Endocrinol Metab. 2001;86:903–908. doi: 10.1210/jcem.86.2.7188. [DOI] [PubMed] [Google Scholar]
- 6.Gianoukakis AG. Khadavi N. Smith TJ. Cytokines, Graves' disease, and thyroid-associated ophthalmopathy. Thyroid. 2008;18:953–958. doi: 10.1089/thy.2007.0405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Vaidya B. Imrie H. Perros P. Young ET. Kelly WF. Carr D. Large DM. Toft AD. McCarthy MI. Kendall-Taylor P. Pearce SH. The cytotoxic T lymphocyte antigen-4 is a major Graves' disease locus. Hum Mol Genet. 1999;8:1195–1199. doi: 10.1093/hmg/8.7.1195. [DOI] [PubMed] [Google Scholar]
- 8.Jacobson EM. Huber A. Tomer Y. The HLA gene complex in thyroid autoimmunity: from epidemiology to etiology. J Autoimmun. 2008;30:58–62. doi: 10.1016/j.jaut.2007.11.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Yanagawa T. Hidaka Y. Guimaraes V. Soliman M. DeGroot LJ. CTLA-4 gene polymorphism associated with Graves' disease in a Caucasian population. J Clin Endocrinol Metab. 1995;80:41–45. doi: 10.1210/jcem.80.1.7829637. [DOI] [PubMed] [Google Scholar]
- 10.Kavvoura FK. Akamizu T. Awata T. Ban Y. Chistiakov DA. Frydecka I. Ghaderi A. Gough SC. Hiromatsu Y. Ploski R. Wang PW. Bednarczuk T. Chistiakova EI. Chojm M. Heward JM. Hiratani H. Juo SH. Karabon L. Katayama S. Kurihara S. Liu RT. Miyake I. Omrani GH. Pawlak E. Taniyama M. Tozaki T. Ioannidis JP. Cytotoxic T-lymphocyte associated antigen 4 gene polymorphisms and autoimmune thyroid disease: a meta-analysis. J Clin Endocrinol Metab. 2007;92:3162–3170. doi: 10.1210/jc.2007-0147. [DOI] [PubMed] [Google Scholar]
- 11.Velaga MR. Wilson V. Jennings CE. Owen CJ. Herington S. Donaldson PT. Ball SG. James RA. Quinton R. Perros P. Pearce SH. The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves' disease. J Clin Endocrinol Metab. 2004;89:5862–5865. doi: 10.1210/jc.2004-1108. [DOI] [PubMed] [Google Scholar]
- 12.Zhang J. Zahir N. Jiang Q. Miliotis H. Heyraud S. Meng X. Dong B. Xie G. Qiu F. Hao Z. McCulloch CA. Keystone EC. Peterson AC. Siminovitch KA. The autoimmune disease-associated PTPN22 variant promotes calpain-mediated Lyp/Pep degradation associated with lymphocyte and dendritic cell hyperresponsiveness. Nat Genet. 2011;43:902–907. doi: 10.1038/ng.904. [DOI] [PubMed] [Google Scholar]
- 13.Tomer Y. Concepcion E. Greenberg DA. A C/T single-nucleotide polymorphism in the region of the CD40 gene is associated with Graves' disease. Thyroid. 2002;12:1129–1135. doi: 10.1089/105072502321085234. [DOI] [PubMed] [Google Scholar]
- 14.Jacobson EM. Tomer Y. The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. J Autoimmun. 2007;28:85–98. doi: 10.1016/j.jaut.2007.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Brand OJ. Lowe CE. Heward JM. Franklyn JA. Cooper JD. Todd JA. Gough SC. Association of the interleukin-2 receptor alpha (IL-2Ralpha)/CD25 gene region with Graves' disease using a multilocus test and tag SNPs. Clin Endocrinol (Oxf ) 2007;66:508–512. doi: 10.1111/j.1365-2265.2007.02762.x. [DOI] [PubMed] [Google Scholar]
- 16.Chistiakov DA. Chistiakova EI. Voronova NV. Turakulov RI. Savost'anov KV. A variant of the IL2RA/CD25 gene predisposing to Graves' disease is associated with increased levels of soluble interleukin-2 receptor. Scand J Immunol. 2011;74:496–501. doi: 10.1111/j.1365-3083.2011.02608.x. [DOI] [PubMed] [Google Scholar]
- 17.Kochi Y. Yamada R. Suzuki A. Harley JB. Shirasawa S. Sawada T. Bae SC. Tokuhiro S. Chang X. Sekine A. Takahashi A. Tsunoda T. Ohnishi Y. Kaufman KM. Kang CP. Kang C. Otsubo S. Yumura W. Mimori A. Koike T. Nakamura Y. Sasazuki T. Yamamoto K. A functional variant in FCRL3, encoding Fc receptor-like 3, is associated with rheumatoid arthritis and several autoimmunities. Nat Genet. 2005;37:478–485. doi: 10.1038/ng1540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Gu LQ. Zhu W. Zhao SX. Zhao L. Zhang MJ. Cui B. Song HD. Ning G. Zhao YJ. Clinical associations of the genetic variants of CTLA-4, Tg, TSHR, PTPN22, PTPN12 and FCRL3 in patients with Graves' disease. Clin Endocrinol (Oxf ) 2010;72:248–255. doi: 10.1111/j.1365-2265.2009.03617.x. [DOI] [PubMed] [Google Scholar]
- 19.Huber AK. Jacobson EM. Jazdzewski K. Concepcion ES. Tomer Y. Interleukin (IL)-23 receptor is a major susceptibility gene for Graves' ophthalmopathy: the IL-23/T-helper 17 axis extends to thyroid autoimmunity. J Clin Endocrinol Metab. 2008;93:1077–1081. doi: 10.1210/jc.2007-2190. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Ho SC. Goh SS. Khoo DH. Association of Graves' disease with intragenic polymorphism of the thyrotropin receptor gene in a cohort of Singapore patients of multi-ethnic origins. Thyroid. 2003;13:523–528. doi: 10.1089/105072503322238773. [DOI] [PubMed] [Google Scholar]
- 21.Hiratani H. Bowden DW. Ikegami S. Shirasawa S. Shimizu A. Iwatani Y. Akamizu T. Multiple SNPs in intron 7 of thyrotropin receptor are associated with Graves' disease. J Clin Endocrinol Metab. 2005;90:2898–2903. doi: 10.1210/jc.2004-2148. [DOI] [PubMed] [Google Scholar]
- 22.Dechairo BM. Zabaneh D. Collins J. Brand O. Dawson GJ. Green AP. Mackay I. Franklyn JA. Connell JM. Wass JA. Wiersinga WM. Hegedus L. Brix T. Robinson BG. Hunt PJ. Weetman AP. Carey AH. Gough SC. Association of the TSHR gene with Graves' disease: the first disease specific locus. Eur J Hum Genet. 2005;13:1223–1230. doi: 10.1038/sj.ejhg.5201485. [DOI] [PubMed] [Google Scholar]
- 23.Yin X. Latif R. Bahn R. Tomer Y. Davies TF. Influence of the TSH receptor gene on susceptibility to Graves' disease and Graves' ophthalmopathy. Thyroid. 2008;18:1201–1206. doi: 10.1089/thy.2008.0098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Colobran R. Armengol Mdel P. Faner R. Gartner M. Tykocinski LO. Lucas A. Ruiz M. Juan M. Kyewski B. Pujol-Borrell R. Association of an SNP with intrathymic transcription of TSHR and Graves' disease: a role for defective thymic tolerance. Hum Mol Genet. 2011;20:3415–3423. doi: 10.1093/hmg/ddr247. [DOI] [PubMed] [Google Scholar]
- 25.Tomer Y. Greenberg DA. Concepcion E. Ban Y. Davies TF. Thyroglobulin is a thyroid specific gene for the familial autoimmune thyroid diseases. J Clin Endocrinol Metab. 2002;87:404–407. doi: 10.1210/jcem.87.1.8291. [DOI] [PubMed] [Google Scholar]
- 26.Stefan M. Jacobson EM. Huber AK. Greenberg DA. Li CW. Skrabanek L. Conception E. Fadlalla M. Ho K. Tomer Y. Novel variant of thyroglobulin promoter triggers thyroid autoimmunity through an epigenetic interferon {alpha}-modulated mechanism. J Biol Chem. 2011;286:31168–31179. doi: 10.1074/jbc.M111.247510. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Villanueva R. Inzerillo AM. Tomer Y. Barbesino G. Meltzer M. Concepcion ES. Greenberg DA. MacLaren N. Sun ZS. Zhang DM. Tucci S. Davies TF. Limited genetic susceptibility to severe Graves' ophthalmopathy: no role for CTLA-4 but evidence for an environmental etiology. Thyroid. 2000;10:791–798. doi: 10.1089/thy.2000.10.791. [DOI] [PubMed] [Google Scholar]
- 28.Vaidya B. Imrie H. Perros P. Dickinson J. McCarthy MI. Kendall-Taylor P. Pearce SH. Cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism confers susceptibility to thyroid associated orbitopathy. Lancet. 1999;354:743–744. doi: 10.1016/S0140-6736(99)01465-8. [DOI] [PubMed] [Google Scholar]
- 29.Werner SC. Modification of the classification of the eye changes of Graves' disease: recommendations of the Ad Hoc Committee of the American Thyroid Association. J Clin Endocrinol Metab. 1977;44:203–204. doi: 10.1210/jcem-44-1-203. [DOI] [PubMed] [Google Scholar]
- 30.Kloprogge S. Kowal L. Wall J. Frauman AG. The clinicopathologic basis of Graves' ophthalmopathy: a review. Eur J Ophthalmol. 2005;15:315–323. doi: 10.1177/112067210501500301. [DOI] [PubMed] [Google Scholar]
- 31.Westman P. Kuismin T. Partanen J. Koskimies S. An HLA-DR typing protocol using group-specific PCR-amplification followed by restriction enzyme digests. Eur J Immunogenet. 1993;20:103–109. doi: 10.1111/j.1744-313x.1993.tb00099.x. [DOI] [PubMed] [Google Scholar]
- 32.Tomer Y. Greenberg DA. Barbesino G. Concepcion E. Davies TF. CTLA-4 and not CD28 is a susceptibility gene for thyroid autoantibody production. J Clin Endocrinol Metab. 2001;86:1687–1693. doi: 10.1210/jcem.86.4.7372. [DOI] [PubMed] [Google Scholar]
- 33.Yin X. Latif R. Tomer Y. Davies TF. Thyroid epigenetics: X chromosome inactivation in patients with autoimmune thyroid disease. Ann NY Acad Sci. 2007;1110:193–200. doi: 10.1196/annals.1423.021. [DOI] [PubMed] [Google Scholar]
- 34.Tomer Y. Genetic susceptibility to autoimmune thyroid disease: past, present, and future. Thyroid. 2010;20:715–725. doi: 10.1089/thy.2010.1644. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Davies TF. Yin X. Latif R. The genetics of the thyroid stimulating hormone receptor: history and relevance. Thyroid. 2010;20:727–736. doi: 10.1089/thy.2010.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Davies TF. Latif R. Yin X. Inheriting autoimmune thyroid disease. Endocr Pract. 2009;15:63–66. doi: 10.4158/EP.15.1.63. [DOI] [PubMed] [Google Scholar]
- 37.Wiebolt J. Koeleman BP. van Haeften TW. Endocrine autoimmune disease: genetics become complex. Eur J Clin Invest. 2010;40:1144–1155. doi: 10.1111/j.1365-2362.2010.02366.x. [DOI] [PubMed] [Google Scholar]
- 38.Davies TF. Really significant genes for autoimmune thyroid disease do not exist—so how can we predict disease? Thyroid. 2007;17:1027–1029. doi: 10.1089/thy.2007.1526. [DOI] [PubMed] [Google Scholar]
- 39.Bartalena L. Editorial: glucocorticoids for Graves' ophthalmopathy: how and when. J Clin Endocrinol Metab. 2005;90:5497–5499. doi: 10.1210/jc.2005-1553. [DOI] [PubMed] [Google Scholar]
- 40.Bahn RS. Graves' ophthalmopathy. N Engl J Med. 2010;362:726–738. doi: 10.1056/NEJMra0905750. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Eckstein AK. Johnson KT. Thanos M. Esser J. Ludgate M. Current insights into the pathogenesis of Graves' orbitopathy. Horm Metab Res. 2009;41:456–464. doi: 10.1055/s-0029-1220935. [DOI] [PubMed] [Google Scholar]
- 42.Rapoport B. Alsabeh R. Aftergood D. McLachlan SM. Elephantiasic pretibial myxedema: insight into and a hypothesis regarding the pathogenesis of the extrathyroidal manifestations of Graves' disease. Thyroid. 2000;10:685–692. doi: 10.1089/10507250050137761. [DOI] [PubMed] [Google Scholar]
- 43.Jacobson DH. Gorman CA. Endocrine ophthalmopathy: current ideas concerning etiology, pathogenesis, and treatment. Endocr Rev. 1984;5:200–220. doi: 10.1210/edrv-5-2-200. [DOI] [PubMed] [Google Scholar]
- 44.Bartalena L. Pinchera A. Marcocci C. Management of Graves' ophthalmopathy: reality and perspectives. Endocr Rev. 2000;21:168–199. doi: 10.1210/edrv.21.2.0393. [DOI] [PubMed] [Google Scholar]
- 45.Allahabadia A. Heward JM. Nithiyananthan R. Gibson SM. Reuser TT. Dodson PM. Franklyn JA. Gough SC. MHC class II region, CTLA4 gene, and ophthalmopathy in patients with Graves' disease. Lancet. 2001;358:984–985. doi: 10.1016/s0140-6736(01)06125-6. [DOI] [PubMed] [Google Scholar]
- 46.Petrone A. Giorgi G. Galgani A. Alemanno I. Corsello SM. Signore A. Di Mario U. Nistico L. Cascino I. Buzzetti R. CT60 single nucleotide polymorphisms of the cytotoxic T-lymphocyte-associated antigen-4 gene region is associated with Graves' disease in an Italian population. Thyroid. 2005;15:232–238. doi: 10.1089/thy.2005.15.232. [DOI] [PubMed] [Google Scholar]
- 47.Duerr RH. Taylor KD. Brant SR. Rioux JD. Silverberg MS. Daly MJ. Steinhart AH. Abraham C. Regueiro M. Griffiths A. Dassopoulos T. Bitton A. Yang H. Targan S. Datta LW. Kistner EO. Schumm LP. Lee AT. Gregersen PK. Barmada MM. Rotter JI. Nicolae DL. Cho JH. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006;314:1461–1463. doi: 10.1126/science.1135245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Farago B. Magyari L. Safrany E. Csongei V. Jaromi L. Horvatovich K. Sipeky C. Maasz A. Radics J. Gyetvai A. Szekanecz Z. Czirjak L. Melegh B. Functional variants of interleukin-23 receptor gene confer risk for rheumatoid arthritis but not for systemic sclerosis. Ann Rheum Dis. 2008;67:248–250. doi: 10.1136/ard.2007.072819. [DOI] [PubMed] [Google Scholar]
- 49.Cargill M. Schrodi SJ. Chang M. Garcia VE. Brandon R. Callis KP. Matsunami N. Ardlie KG. Civello D. Catanese JJ. Leong DU. Panko JM. McAllister LB. Hansen CB. Papenfuss J. Prescott SM. White TJ. Leppert MF. Krueger GG. Begovich AB. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet. 2007;80:273–290. doi: 10.1086/511051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Safrany E. Melegh B. Functional variants of the interleukin-23 receptor gene in non-gastrointestinal autoimmune diseases. Curr Med Chem. 2009;16:3766–3774. doi: 10.2174/092986709789104975. [DOI] [PubMed] [Google Scholar]
- 51.Ban Y. Tozaki T. Taniyama M. Nakano Y. Yoneyama K. Hirano T. Association studies of the IL-23R gene in autoimmune thyroid disease in the Japanese population. Autoimmunity. 2009;42:126–130. doi: 10.1080/08916930802422265. [DOI] [PubMed] [Google Scholar]
- 52.Tomer Y. Davies TF. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev. 2003;24:694–717. doi: 10.1210/er.2002-0030. [DOI] [PubMed] [Google Scholar]
- 53.Simmonds MJ. Howson JM. Heward JM. Carr-Smith J. Franklyn JA. Todd JA. Gough SC. A novel and major association of HLA-C in Graves' disease that eclipses the classical HLA-DRB1 effect. Hum Mol Genet. 2007;16:2149–2153. doi: 10.1093/hmg/ddm165. [DOI] [PubMed] [Google Scholar]
- 54.Frecker M. Mercer G. Skanes VM. Farid NR. Major histocompatibility complex (MHC) factors predisposing to and protecting against Graves' eye disease. Autoimmunity. 1988;1:307–315. doi: 10.3109/08916938809010684. [DOI] [PubMed] [Google Scholar]
- 55.Stenszky V. Kozma L. Balazs C. Farid NR. HLA-DR associations with Graves' disease in eastern Hungary. Clin Invest Med. 1983;6:181–184. [PubMed] [Google Scholar]
- 56.Weetman AP. So AK. Warner CA. Foroni L. Fells P. Shine B. Immunogenetics of Graves' ophthalmopathy. Clin Endocrinol (Oxf ) 1988;28:619–628. doi: 10.1111/j.1365-2265.1988.tb03853.x. [DOI] [PubMed] [Google Scholar]
- 57.Kendall-Taylor P. Stephenson A. Stratton A. Papiha SS. Perros P. Roberts DF. Differentiation of autoimmune ophthalmopathy from Graves' hyperthyroidism by analysis of genetic markers. Clin Endocrinol (Oxf ) 1988;28:601–610. doi: 10.1111/j.1365-2265.1988.tb03851.x. [DOI] [PubMed] [Google Scholar]
- 58.Farid NR. Balazs C. The genetics of thyroid associated ophthalmopathy. Thyroid. 1998;8:407–409. doi: 10.1089/thy.1998.8.407. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.