Genetics of Graves’ Disease

of Graves’ disease including both environmental and genetic components. With respect to environmental effects, a variety of factors have been reported to contribute to the development of Graves’ disease including, for example, dietary iodine intake, infectious agents such as viruses, and stressful life events. Direct evidence of causation for any of these components awaits confirmation.

II. GENETIC SUSCEPTIBILITY

In support of a genetic component, Graves’ disease tends to cluster in families with an increased risk of thyroid disease in siblings of affected individuals (6). The concordance rate of Graves’ disease has been reported to be between 30 and 50% in monozygotic twins and less than 5% in dizygotic twins (6,7). Brix and his colleagues have recently confirmed these findings in a population-based study of Danish twins (8). They reported a proband wise concordance rate of 35% in monozygotic twins and 7% in dizygotic twins identified from the Danish twin registry. This is undoubtedly one of the most accurate estimates of concordance rates in Graves’ disease. Although starting with over 20,000 twin pairs, meticulous clinical phenotyping of twins on the register discovered only 5 twin pairs being concordant and 40 discordant for Graves’ disease. Higher concordance rates among monozygotic twins who have identical genes compared to dizygotic twins, who have approximately 50% identical genes, clearly support the involvement of the genetic factors in the development of Graves’ disease. However, since this is well below 100%, environmental factors are also playing an important role in the development of disease.

The magnitude of genetic contribution or familial clustering can be estimated by dividing the lifetime risk to a second sibling (in cases in which the first has the disease) by the population frequency of the disease to produce the lambda (λ) sib (s) statistic (9). A λs of 1 implies no genetic contribution to disease, whereas a disorder arising from rare single-gene defects with high penetrance, such as hemophilia, will have a λs of greater than 1000. We have estimated that Graves’ disease has an overall λs of 7.5–10, supporting a genetic contribution to disease (10). However, the magnitude of this value suggests that Graves’ disease develops as a result of a complex inheritance pattern to which several genetic susceptibility loci and environmental factors are likely to contribute. Analysis of familial clustering data by gender further highlights the female preponderance of Graves’ disease (6). The risk of Graves’ disease developing in a sister when her sibling already has the disease is reported to be between 5 and 10%. In contrast, the risk for a brother is 0.9–7.4%. This suggests that a sister is 5.4–12.6 times more likely and the brother 1.2– 7.4 times more likely to develop Graves’ disease than an individual in the general population when a sibling already has disease. The reasons for the female preponderance are unknown. A number of hypotheses have been advanced, including effects resulting from differences in sex hormones. Although it has also been suggested that gender differences may arise because of differences in the sex chromosomes, this needs some clarification. It seems most unlikely that gender differences could arise because of susceptibility loci on the X chromosome. If such a locus existed, a dominant effect would lead to no difference between males and females, whereas a recessive effect would lead to an increase in Graves’ disease in males (since they only have one X chromosome they only need one susceptibility allele). Gender differences could arise because of a susceptibility locus on the Y chromosome, but this would need to confer protection to males.

In an attempt to identify loci conferring susceptibility to Graves’ disease, two main approaches have been adopted: the candidate gene approach and the genome-wide search

using linkage analysis (10). Advances in the dissection of Graves’ disease susceptibility loci have been made by linkage analysis and allelic association methods. These have been greatly assisted by the emergence of detailed maps of the human genome and several collections of large population-based, case–control, family-based data sets.

Because Graves’ disease is immunologically mediated, many researchers employing the candidate gene approach have focused on genes that regulate the immune response and also the target antigen: the TSH receptor.

III. MAJOR HISTOCOMPATIBILITY COMPLEX REGION

The human major histocompatibility complex (MHC) region on chromosome 6p21 encodes proteins involved in the human leukocyte antigen (HLA) system. This chromosomal region is well researched and may influence the ultimate outcome of somatic recombination by affecting the selection of T cells and B cells. The region can be organized into three main gene clusters. The class I region genes include those encoding the α-chains of HLA-A, HLA-B, and HLA-C antigens. These are membrane-bound proteins expressed on the cell surface of the nucleated cells involved in the process of presentation of endogenous peptide to the cytotoxic (CD8 ) T lymphocytes. The class II gene region, on the other hand, encodes proteins expressed on specialized antigen-presenting cells, including the macrophages and B lymphocytes and also, under certain circumstances, other cell types. Class II molecules are likely to be involved in the autoimmune disease process as they bind products generated by the degradation of proteins in the endocytic pathway. These complexes can then stimulate T-helper (CD4 ) lymphocytes. The class III region contains genes that encode immune regulator proteins, including some of the cytokines.

Several population-based case–control studies have shown an association between Graves’ disease and polymorphisms within the MHC–HLA region. Consistent associations have been reported between class II gene polymorphisms and, specifically, HLADR3 (Table 1). The magnitude of the contribution of the HLA region to Graves’ disease is summarized in Table 1, which shows relative risks of between 1.9 and 3.8 for polymorphism in the class II region. Reports of the association between Graves’ disease and other

Table 1 Reported Associations Between MHC-HLA Region and Graves’ Disease Since 1990

class II genes are less convincing. Strong linkage disequilibrium exists across the HLA region, for example, HLA-DRB1*0304, DQB1*02, and DQA1*0501 are in tight linkage disequilibrium. This makes it difficult to ascertain which allele is exerting a primary effect (11) or whether susceptibility is conferred by a haplotype. Part of the reason for this uncertainty results from the fact that there are 14 alleles at DRB1*0304, DQB1*02 and DQA1*0501, and most case-control data sets have lacked the power to withstand the appropriate correction factor to identify an independent effect. Yanagawa et al. (12) reported for the first time an independent effect of the HLA-DQA1*0501 allele, conferring a greater risk than the HLA-DR3. Barlow and co-workers (13) subsequently reported an uncorrected significant association of HLA-DQA1*0501 in DR3-negative subjects in the United Kingdom. Using our data set that comprises more than 1000 cases and controls, we are unable to confirm the independent effect of the HLA-DQA1*0501 allele in DR3 negative subjects (Nithiyananthan, unpublished data).

The population-based case-control study, is one of the most sensitive methods for the identification of susceptibility loci exerting small effects. However, this approach lacks specificity and can lead to false-positive results as a consequence of population stratification and inadequate matching of cases and controls (10). Mismatching can be minimized by large data sets but unfortunately most published reports lack sufficient power to detect adequately a true effect and exclude a random chance event. For example, for a susceptibility locus that confers a relative risk of 2.0 for development of Graves’ disease, and a susceptibility genotype frequency of 20%, a total data set of more than 800 cases and controls would be needed to have an 80% power to achieve a result with significance of p 0.001. Most published case–control studies have used numbers well below this figure.

Population stratification can be eliminated using the intrafamilial association study approach using family-based data sets. One such approach utilizes the transmission disequilibrium test (TDT), which requires genotype data and therefore DNA from both parents and at least one affected sibling (14). This approach can be further strengthened by incorporating data from unaffected siblings to exclude segregation distortion. We have, for the first time, reported an allelic association between the HLA class II haplotypes DRB1*0304-DQB1*02-DQA1*0501 and Graves’ disease using the TDT approach (11). These data provide evidence for linkage in the presence of linkage disequilibrium between HLA and Graves’ disease.

Classic linkage analysis can also be used to identify susceptibility loci. This method has been used successfully to identify the genes that have major effects. However, it has a limited ability in detecting genes that have modest effect (15), which again, in part, relates to data sets of inadequate size. In Graves’ disease it is likely that a number of susceptibility loci, each with a modest effect, are contributing to disease development. Power calculations suggest that large numbers of Graves’ disease families are required to pick up these effects (between 600 and 1000 sib-pairs). It is not surprising, therefore, that this approach to date has met with a very limited success and largely failed to detect the HLA region as a susceptibility locus. A recent report, however, does provide weak evidence for linkage between HLA and Graves’ disease (16). As this was reported in only 77 sib-pairs, this result and certainly the magnitude of the effect at this locus should be viewed with caution.

Although the exact mechanism by which polymorphism in the HLA region confers susceptibility to Graves’ disease is unknown, it is highly likely that polymorphism within the HLA region results in differences in amino acid sequences. These differences could

affect antigen-binding sites, thereby influencing antigen-binding affinity, the nature of the stimulation of T lymphocyte, and the development of the immune response.

Although MHC-HLA association is a general feature of autoimmune diseases (17) there is evidence to suggest this region may be facilitating a tissue-specific effect. Using animal models it was possible to show that by swapping diabetogenic and autoimmune thyroid MHC haplotypes, the tissue in which the autoimmune process developed could be changed. Hence thyroiditis was initiated in an animal model of diabetes with thyroid MHC haplotype (18,19).

IV. CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED-4 GENE

An immune response initiated by the presentation of an antigen by an MHC-HLA class II molecule to the T-cell receptor on the T lymphocyte can only progress in the presence of a second costimulatory pathway. This can either be provided by local infection (20) or by an interaction between the cluster designation-28 (CD28)/cytotoxic T-lymphocyte associated-4 (CTLA-4) molecules on the T lymphocyte and the B7 molecule of the antigen presenting cell. The interaction between CD28/CTLA-4 and B7 molecule appears to be an important step in the process of autoimmune disease (Fig. 1). Without costimulation provided by CD28/B7 binding the T lymphocyte remains in a state of anergy and may even undergo cell death.

CD28 and CTLA-4 are two T lymphocyte surface molecules that bind their ligands B7.1 and B7.2 (CD86 and CD80, respectively) on antigen-presenting cells and other activated cells (21,22). CD28 is expressed on resting and activated T lymphocytes, and its interaction with the B7.1 molecule on the antigen-presenting cell leads to expansion of the antigen-specific T-lymphocyte and cytokine production (23,24). The CTLA-4 molecule, on the other hand, is only produced after activation of T lymphocyte. Its interaction with the B7.2 on the antigen-presenting cell modulates the immune response, especially CD4 responses, by downregulation of T-cell receptor (25,26). This pathway is tightly regulated, and can strongly influence the immune response.

CD28 and CTLA-4 genes are closely located on chromosome 2q33, suggesting a common ancestral origin. Yanagawa and co-workers in 1995 reported an association between a polymorphism of the (AT)n microsatellite marker in the 3′untranslated region of the CTLA-4 gene and Graves’ disease (27). A single nucleotide polymorphism (SNP) in exon 1 of this gene (A → G polymorphism) was subsequently found to be in linkage disquilibrium with the (AT)n microsatellite, with the G allele being associated with disease in a European case–control data set. Linkage to type 1 diabetes has also been reported (28). Further reports have shown association of the A-G polymorphism of the CTLA-4 gene with Graves’ disease (29–31), Hashimoto’s thyroiditis (31,32), type 1 diabetes (33), Addison’s disease (32,34), celiac disease (35,36), and primary biliary cirrhosis (37). We have also shown intrafamilial allelic association between the G allele of the A-G polymorphism and Graves’ disease in a cohort of 179 families (30), and therefore excluded population stratification as an alternative explanation. We also demonstrated that the G allele was associated not only with the disease but also with its severity at the time of presentation by showing that the presence of the GG genotype is associated with a more severe biochemical disturbance of circulating free thyroxin levels (30).

Family linkage data are also available showing linkage of the CTLA-4 gene region to Graves’ disease in a UK data set (16) and linkage to thyroid antibody production in a

Figure 1 The thyroid-stimulating hormone receptor (TSHR) antigen is presented to the T-cell receptor (TCR) by the major histocompatibility complex (MHC) class II molecules on the antigen-presenting cell (APC), leading to the development of potentially autoreactive T cells. For the progression of an immune response, a second signal is required, which is provided by the interaction of B7 (on the APC) and CD28 (on the T lymphocyte). The interaction of B7 and cytotoxic T-lymphocyte-associated-4 (CTLA- 4) leads to a decrease of the T lymphocyte, largely by a decrease of the TCR. Disequilibrium of CD28 and CTLA-4 could lead to a more aggressive immune response and contribute to the autoimmune disease process.

US data set (38). Linkage data from affected sib-pairs in the UK, as with HLA, was in a relatively small number of families, the magnitude of the contribution reported at this locus needs verification.

Association and linkage between the CTLA-4 gene polymorphism and Graves’ disease merely suggests that there is likely to be a susceptibility locus in this chromosomal region. These data, however, do not pinpoint the locus to the CTLA-4 gene directly. Although polymorphism within the CTLA-4 gene may well confer susceptibility to Graves’ disease, it is just as likely that the polymorphisms studied are in linkage disequilibrium with a disease-causing mutation, even in a neighboring gene. Genes in this region include CD28, the inducible costimulatory molecule (ICOS) gene, Caspace 8, and Caspace 10, all of which are equally good immune response candidate genes. Only comprehensive fine mapping of all polymorphisms in the region leading to the determination of maximal point and extent of linkage disequilibrium will identify the location of the etiological mutation. Examination of one SNP in a neighboring gene such as CD28 merely excludes that polymorphism but not the gene (38). Comprehensive screening for all polymorphisms is vital.

Polymorphism of the CTLA-4 gene leading to alteration in gene function may lead to the development of the autoimmune disease process by interfering with the regulatory role of CTLA-4 in the CD28/CTLA-4 -B7 pathway. Functional studies are now emerging in a number of disease states including Graves’ disease (39), myasthenia gravis (40), and multiple sclerosis (41). Although none of these studies has demonstrated differences between subjects with and without disease, there are reports of differences in T-cell proliferation and CTLA-4 expression between individual CTLA-4 genotypes. Again, although these data are interesting and provide a potential functional link to the autoimmune disease process, they do not directly incriminate the A-G polymorphism as an etiological polymorphism.

Finally a number of candidate gene loci have been examined with respect to the development of Graves’-associated orbital disease including the CTLA-4 gene region. Although association between the CTLA-4 gene and eye disease has been reported, this was in a data set of 94 subjects with eye disease and 94 subjects without eye disease (42). These data lacked sufficient power to support the hypothesis that eye disease is related to CTLA-4 genotype. In our own data set of 323 patients with Graves’ disease without eye disease and 161 patients with eye disease (NOSPECS classification 2–6), there were no differences in CTLA-4 genotype between the groups. As might be expected, however, multiple regression analysis confirmed association between thyroid eye disease and smoking (42a).

V. GENOME-WIDE SEARCHES

There is little doubt that the MHC-HLA region and CTLA-4 gene region contribute to the genetic susceptibility to development of Graves’ disease. However, these regions do not explain all of the genetic contribution to disease. Although candidate gene studies will continue to play an important role in the identification of susceptibility loci, genome-wide searches have also been employed to identify further loci.

A number of new candidate loci have been identified by genome-wide linkage analysis. As with many other complex diseases, replication of linkage in independent data sets has proved problematic and this approach has so far failed to deliver novel candidate genes.

The GD-1 gene region is located on chromosome 14q31 (43,44). Chromosome 14 was initially screened with 14 microsatellite markers in 323 individuals from 53 families. The inclusion of additional markers within the region of linkage were used in a multipoint parametric analysis that gave a significant maximum logarithm of odds (LOD) score of 2.5 between the markers D14S81 and D14S1054 ( 3cM apart), with the data supporting a model of recessive inheritance. Further analysis has localized GD-1 to within 2cM of the multinodular goiter-1 (MNG-1) locus identified in a family pedigree of subjects with a multinodular nonautoimmune thyroid goiter (45). This raises the possibility that MNG- 1 and GD-1 are the same and that this locus confers susceptibility to both Graves’ disease and multinodular goiter. Within the chromosomal region of GD-1 are other possible thyroid autoimmune candidate genes such as the TSHR gene, immunoglobulin heavy chain gene (IgH), the T-cell receptor α gene, the insulin-dependent diabetes mellitus 11 (IDDM11) gene, and the estrogen receptor β (ESRβ) gene. However, most if not all of these candidates are outside the region of linkage identified as GD-1. Association studies have revealed conflicting results from some of the candidate genes in this region, including the

TSHR, thyroid-stimulating hormone receptor; IL-1RA, interleukin 1 receptor antagonist; LMP, large multifunctional proteasome; TNF-β, tumor necrosis factor-beta; Il-4, interleukin-4.

TSHR gene (see Table 2). It is unlikely that current markers at GD-1 or MNG-1 represent the known polymorphisms in the candidate genes listed above and both replication and fine mapping of the GD-1/MNG-1 locus are needed to identify the Graves’ disease-specific locus.

Using the same data set used to identify the GD-1, Tomer and co-workers found a second Graves’ disease locus designated GD-2 that was mapped onto 20q11.2 (46). Multipoint parametric linkage analysis of this region showed a significant maximum LOD score of 3.5 in a 6cM region between microsatellite markers D20S195 and D20S107, assuming a recessive mode of inheritance. Similar results were shown when the data were analyzed using a nonparametric method, which has the advantage of not assuming the mode of inheritance. Several candidate genes have been mapped in this region, including the interleukin-6 nuclear factor (NF-IL6). NF-IL6 gene is a good candidate gene for autoimmunity as it encodes protein that binds to several regulatory regions of other cytokines.

In 45 families collected as part of the original 53 families described above, linkage has also been reported in a region designated GD-3 on chromosome Xq21.33–22 (47). The X chromosome was initially screened with 20 microsatellite markers, and multipoint linkage analysis of 8 markers between DXS1196 to DXS1001 ( 46cM) produced a maximum LOD score of 2.5, suggestive of linkage. The same report also excluded a number of gender-related candidate genes as susceptibility loci for Graves’ disease.

In the UK sib-pairs in which linkage was reported at HLA and CTLA-4 gene region, linkage has also been reported on chromosome 18q21 (48) and the X chromosome at Xp11, conditioned for allele sharing at the CTLA-4 gene region (49). Linkage at marker DS18S487 (48) has previously been reported in type 1 diabetes and rheumatoid arthritis, suggesting the possibility of a general autoimmunity gene at this locus. No evidence for linkage in this data set was found at GD-1 or GD-3. It should be pointed out, however, that the small size of this data set does not have the power to exclude an effect equivalent to GD-1 or GD-3. Furthermore, despite the small size of this data set, subgroup analysis

was performed to replicate GD2 (50). Such data should, therefore, be viewed with extreme caution. At the present time replication of linkage data in family data sets of sufficient size to detect loci of modest effect is needed to confirm the present findings and to quantify the contribution of linked loci to the genetic susceptibility to Graves’ disease.

Preliminary data from the UK suggest that the HLA and CTLA-4 gene regions may contribute up to 50% of the familial clustering, leaving the remaining 50% to be explained by as yet unknown loci (16).

VI. OTHER CANDIDATE GENES

Table 2 shows the results of the candidate genes examined to date. Most of these have been tested in population-based case–control studies. The inconsistent findings are almost certainly the result of inadequately sized data sets and, in some instances, poor matching of controls. At present only the HLA gene cluster and the CTLA-4 gene have been consistently reported to be associated with Graves’ disease, in both case–control and family–based data sets.

VII. CONCLUSION

Graves’ disease is a common complex autoimmune disease of unknown etiology. There is clear evidence for an immunological basis, with TSHR acting as the primary autoantigen. Clustering in families and twin data confirm an important role for genetic causes, with the same studies highlighting an equally important contribution from the environment. The HLA and CTLA-4 gene regions undoubtedly contain important susceptibility loci, although the magnitude of their effect awaits confirmation. Although genome-wide searches have identified chromosomal regions of linkage to Graves’ disease, including GD-1, GD-2, GD-3 chromosome 18, and the X chromosome, these preliminary findings need replication and novel candidate genes within these regions are eagerly awaited.

Data from HLA and the CTLA-4 gene regions suggest that susceptibility loci are common polymorphisms also present in the general ‘‘unaffected’’ population. For example, in the United Kingdom (11) the HLA susceptibility haplotype DRB1*0304- DQB1*02-DQA1*0501 is present in 47% of the patients with Graves’ disease and 32% of the controls. Similar findings have been observed for polymorphisms of the CTLA-4 gene present in 42% of the cases and 32% of the controls (30). These data support the hypothesis that common polymorphisms that protect or predispose to development of autoimmune diseases are the same common polymorphisms that confer resistance or susceptibility to development of infectious disease.

Over the last 10 years or so we have gained a greater understanding of factors leading to the development of Graves’ disease. We are beginning to unravel some of the susceptibility loci that make up the genetic contribution to the development of disease. The establishment of large case–control and family-based data sets for linkage and association analysis combined with the publication of human genome sequence data are likely to lead to further advances in our understanding of the genetic susceptibility to the development of Graves’ disease.

ACKNOWLEDGMENTS

This work is supported by the grant (No. 95/3717) from the Wellcome Trust and the Regional Research and Development NHS Executive, West Midlands, United Kingdom.

REFERENCES

1.Weetman AP. Graves’ disease. N Engl J Med 2000; 343:1236–1248.

2.McIver B, Morris JC. The pathogenesis of Graves’ disease. Endocrinol Metab Clin North Am 1998; 27:73–89.

3.Burrow GN. The management of thyrotoxicosis in pregnancy. N Engl J Med 1985; 313:562– 565.

4.Zakarija M, McKenzie JM, Munro DS. Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves’ disease. J Clin Invest 1983; 72: 1352–1356.

5.Vanderpump PJ TW. The Epidemiology of Autoimmune Thyroid Disease. In: PV, ed. Autoimmune Endocrinopathies. Totowa, NJ: Humana Press, 1999:141–162.

6.Brix TH, Kyvik KO, Hegedus L. What is the evidence of genetic factors in the etiology of Graves’ disease? A brief review. Thyroid 1998; 8:727–734.

7.Brix TH, Christensen K, Holm NV, Harvald B, Hegedus L. A population-based study of Graves’ disease in Danish twins. Clin Endocrinol (Oxf ) 1998; 48:397–400.

8.Brix TH, Kyvik KO, Christensen K, Hegedus L. Evidence for a major role of heredity in Graves’ disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001; 86:930–934.

9.Risch N. Assessing the role of HLA-linked and unlinked determinants of disease. Am J Hum Genet 1987; 40:1–14.

10.Allahabadia A, Gough SC. The different approaches to the genetic analysis of autoimmune thyroid disease. J Endocrinol 1999; 163:7–13.

11.Heward JM, Allahabadia A, Daykin J, Carr-Smith J, Daly A, Armitage M, Dodson PM, Sheppard MC, Barnett AH, Franklyn JA, Gough SC. Linkage disequilibrium between the human leukocyte antigen class II region of the major histocompatibility complex and Graves’ disease: replication using a population case control and family-based study. J Clin Endocrinol Metab 1998; 83:3394–3397.

12.Yanagawa T, Mangklabruks A, DeGroot LJ. Strong association between HLA-DQA1*0501 and Graves’ disease in a male Caucasian population. J Clin Endocrinol Metab 1994; 79:227– 229.

13.Barlow AB, Wheatcroft N, Watson P, Weetman AP. Association of HLA-DQA1*0501 with Graves’ disease in English Caucasian men and women. Clin Endocrinol (Oxf ) 1996; 44:73– 77.

14.Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 1993; 52:506–516.

15.Risch N, Merikangas K. The future of genetic studies of complex human diseases. Science 1996; 273:1516–1517.

16.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.

17.Heward J, Gough SC. Genetic susceptibility to the development of autoimmune disease. Clin Sci (Colch) 1997; 93:479–491.

18.Damotte D, Colomb E, Cailleau C, Brousse N, Charreire J, Carnaud C. Analysis of susceptibility of NOD mice to spontaneous and experimentally induced thyroiditis. Eur J Immunol 1997; 27:2854–2862.

19.Wicker LS, Todd JA, Peterson LB. Genetic control of autoimmune diabetes in the NOD mouse. Annu Rev Immunol 1995; 13:179–200.

20.Van Parijs L, Abbas AK. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 1998; 280:243–248.

21.Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol 1996; 14:233–258.

22.Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol 1993; 11:191–212.

23.Krummel MF, Allison JP. CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J Exp Med 1996; 183:2533–2540.

24.Walunas TL, Bakker CY, Bluestone JA. CTLA-4 ligation blocks CD28-dependent T cell activation. J Exp Med 1996; 183:2541–2550.

25.Lee KM, Chuang E, Griffin M, Khattri R, Hong DK, Zhang W, Straus D, Samelson LE, Thompson CB, Bluestone JA. Molecular basis of T cell inactivation by CTLA-4. Science 1998; 282:2263–2266.

26.Mangklabruks A, Cox N, DeGroot LJ. Genetic factors in autoimmune thyroid disease analyzed by restriction fragment length polymorphisms of candidate genes. J Clin Endocrinol Metab 1991; 73:236–244.

27.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.

28.Nistico L, Buzzetti R, Pritchard LE, Van der Auwera B, Giovannini C, Bosi E, Larrad MT, Rios MS, Chow CC, Cockram CS, Jacobs K, Mijovic C, Bain SC, Barnett AH, Vandewalle CL, Schuit F, Gorus FK, Tosi R, Pozzilli P, Todd JA. The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry. Hum Mol Genet 1996; 5:1075–1080.

29.Donner H, Rau H, Walfish PG, Braun J, Siegmund T, Finke R, Herwig J, Usadel KH, Badenhoop K. CTLA4 alanine-17 confers genetic susceptibility to Graves’ disease and to type 1 diabetes mellitus. J Clin Endocrinol Metab 1997; 82:143–146.

30.Heward JM, Allahabadia A, Armitage M, Hattersley A, Dodson PM, Macleod K, Carr-Smith J, Daykin J, Daly A, Sheppard MC, Holder RL, Barnett AH, Franklyn JA, Gough SC. The development of Graves’ disease and the CTLA-4 gene on chromosome 2q33. J Clin Endocrinol Metab 1999; 84:2398–2401.

31.Kotsa K, Watson PF, Weetman AP. A CTLA-4 gene polymorphism is associated with both Graves disease and autoimmune hypothyroidism. Clin Endocrinol (Oxf ) 1997; 46:551–554.

32.Donner H, Braun J, Seidl C, Rau H, Finke R, Ventz M, Walfish PG, Usadel KH, Badenhoop K. Codon 17 polymorphism of the cytotoxic T lymphocyte antigen 4 gene in Hashimoto’s thyroiditis and Addison’s disease. J Clin Endocrinol Metab 1997; 82:4130–4132.

33.Marron MP, Raffel LJ, Garchon HJ, Jacob CO, Serrano-Rios M, Martinez Larrad MT, Teng WP, Park Y, Zhang ZX, Goldstein DR, Tao YW, Beaurain G, Bach JF, Huang HS, Luo DF, Zeidler A, Rotter JI, Yang MC, Modilevsky T, Maclaren NK, She JX. Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups. Hum Mol Genet 1997; 6:1275–1282.

34.Kemp EH, Ajjan RA, Husebye ES, Peterson P, Uibo R, Imrie H, Pearce SH, Watson PF, Weetman AP. A cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism is associated with autoimmune Addison’s disease in English patients. Clin Endocrinol (Oxf ) 1998; 49:609–613.

35.Clot F, Fulchignoni-Lataud MC, Renoux C, Percopo S, Bouguerra F, Babron MC, DjilaliSaiah I, Caillat-Zucman S, Clerget-Darpoux F, Greco L, Serre JL. Linkage and association study of the CTLA-4 region in coeliac disease for Italian and Tunisian populations. Tissue Antigens 1999; 54:527–530.

36.Djilali-Saiah I, Schmitz J, Harfouch-Hammoud E, Mougenot JF, Bach JF, Caillat-Zucman S. CTLA-4 gene polymorphism is associated with predisposition to coeliac disease. Gut 1998; 43:187–189.

37.Agarwal K, Jones DE, Daly AK, James OF, Vaidya B, Pearce S, Bassendine MF. CTLA-4

gene polymorphism confers susceptibility to primary biliary cirrhosis. J Hepatol 2000; 32: 538–541.

38.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.

39.Kouki T, Sawai Y, Gardine CA, Fisfalen ME, Alegre ML, DeGroot LJ. 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. J Immunol 2000; 165:6606–6611.

40.Huang D, Giscombe R, Zhou Y, Pirskanen R, Lefvert AK. Dinucleotide repeat expansion in the CTLA-4 gene leads to T cell hyper-reactivity via the CD28 pathway in myasthenia gravis. J Neuroimmunol 2000; 105:69–77.

41.Ligers A, Teleshova N, Masterman T, Huang W-X, Hillert J. CTLA-4 expression is influenced by promoter and exon 1 polymorphisms. Genes Immunity 2001; 2:145–152.

42.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.

42a. Allahabadia A, Heward J, Nithiyananthan R, Gibson SM, Reuser TTQ, Dodson PM, Franklyn JA, Gough SCL. MHC class II region, the CTLA-4 gene and ophthalmology in patients with Graves’. Lancet 2001; 358:984–985.

43.Tomer Y, Barbesino G, Keddache M, Greenberg DA, Davies TF. Mapping of a major susceptibility locus for Graves’ disease (GD-1) to chromosome 14q31. J Clin Endocrinol Metab 1997; 82:1645–1648.

44.Tomer Y, Barbesino G, Greenberg DA, Concepcion E, Davies TF. Linkage analysis of candidate genes in autoimmune thyroid disease. III. Detailed analysis of chromosome 14 localizes Graves’ disease-1 (GD-1) close to multinodular goiter-1 (MNG-1). International Consortium for the Genetics of Autoimmune Thyroid Disease. J Clin Endocrinol Metab 1998; 83:4321– 4327.

45.Bignell GR, Canzian F, Shayeghi M, Stark M, Shugart YY, Biggs P, Mangion J, Hamoudi R, Rosenblatt J, Buu P, Sun S, Stoffer SS, Goldgar DE, Romeo G, Houlston RS, Narod SA, Stratton MR, Foulkes WD. Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer. Am J Hum Genet 1997; 61:1123–1130.

46.Tomer Y, Barbesino G, Greenberg DA, Concepcion E, Davies TF. A new Graves diseasesusceptibility locus maps to chromosome 20q11.2. International Consortium for the Genetics of Autoimmune Thyroid Disease. Am J Hum Genet 1998; 63:1749–1756.

47.Barbesino G, Tomer Y, Concepcion E, Davies TF, Greenberg DA. Linkage analysis of candidate genes in autoimmune thyroid disease: 1. Selected immunoregulatory genes. International Consortium for the Genetics of Autoimmune Thyroid Disease. J Clin Endocrinol Metab 1998; 83:1580–1584.

48.Vaidya B, Imrie H, Perros P, Young ET, Kelly WF, Carr D, Large DM, Toft AD, KendallTaylor P, Pearce SH. Evidence for a new Graves disease susceptibility locus at chromosome 18q21. Am J Hum Genet 2000; 66:1710–1714.

49.Imrie H, Vaidya B, Perros P, Kelly WF, Toft AD, Young ET, Kendall-Taylor P, Pearce SH. Evidence for a Graves’ disease susceptibility locus at chromosome Xp11 in a United Kingdom population. J Clin Endocrinol Metab 2001; 86:626–630.

50.Pearce SH, Vaidya B, Imrie H, Perros P, Kelly WF, Toft AD, McCarthy MI, Young ET, Kendall-Taylor P. Further evidence for a susceptibility locus on chromosome 20q13.11 in families with dominant transmission of Graves’ disease. Am J Hum Genet 1999; 65:1462– 1465.

51.Badenhoop K, Schwarz G, Schleusener H, Weetman AP, Recks S, Peters H, Bottazzo GF, Usadel KH. Tumor necrosis factor beta gene polymorphisms in Graves’ disease. J Clin Endocrinol Metab 1992; 74:287–291.

52.Badenhoop K, Walfish PG, Rau H, Fischer S, Nicolay A, Bogner U, Schleusener H, Usadel KH. Susceptibility and resistance alleles of human leukocyte antigen (HLA) DQA1 and HLA DQB1 are shared in endocrine autoimmune disease. J Clin Endocrinol Metab 1995; 80:2112– 2117.

53.Yanagawa T, Mangklabruks, A, Cahng, YB, et al. Human histocompatibility leukocyte antigen DQA1*0501 allele associated with genetic susceptibility to Graves’ disease in a Caucasian population. J Clin Endocrinol Metab 1993; 76:1569.

54.Cuddihy RM, Bahn RS. Lack of an independent association between the human leukocyte antigen allele DQA1*0501 and Graves’ disease. J Clin Endocrinol Metab 1996; 81:847–849.

55.Cuddihy RM, Dutton CM, Bahn RS. A polymorphism in the extracellular domain of the thyrotropin receptor is highly associated with autoimmune thyroid disease in females. Thyroid 1995; 5:89–95.

56.Watson PF, French A, Pickerill AP, McIntosh RS, Weetman AP. Lack of association between a polymorphism in the coding region of the thyrotropin receptor gene and Graves’ disease. J Clin Endocrinol Metab 1995; 80:1032–1035.

57.Kotsa KD, Watson PF, Weetman AP. No association between a thyrotropin receptor gene polymorphism and Graves’ disease in the female population. Thyroid 1997; 7:31–33.

58.Allahabadia A, Heward JM, Mijovic C, Carr-Smith J, Daykin J, Cockram C, Barnett AH, Sheppard MC, Franklyn JA, Gough SC. Lack of association between polymorphism of the thyrotropin receptor gene and Graves’ disease in United Kingdom and Hong Kong Chinese patients: case control and family-based studies. Thyroid 1998; 8:777–780.

59.Blakemore AI, Watson PF, Weetman AP, Duff GW. Association of Graves’ disease with an allele of the interleukin-1 receptor antagonist gene. J Clin Endocrinol Metab 1995; 80:111– 115.

60.Heward J, Allahabadia A, Gordon C, Sheppard MC, Barnett AH, Franklyn JA, Gough SC. The interleukin-1 receptor antagonist gene shows no allelic association with three autoimmune diseases. Thyroid 1999; 9:627–628.

61.Allahabadia A, Heward J, Carr-Smith J, Daykin J, Barnett AH, Sheppard MC, Franklyn JA, Gough SC. Sharing of susceptibility loci between autoimmune diseases: lack of association of the insulin gene region with Graves’ disease. Thyroid 1999; 9:317–318.

62.Heward JM, Allahabadia A, Sheppard MC, Barnett AH, Franklyn JA, Gough SC. Association of the large multifunctional proteasome (LMP2) gene with Graves’ disease in a result of linkage disequilibrium with the HLA haplotype DRB1*0304-DQB1*02-DQA1*0501. Clin Endocrinol (Oxf ) 1999; 51:115–118.

63.Cavan DA, Penny MA, Jacobs KH, Kelly MA, Jenkins D, Mijovic CH, Chow CC, Cockram CS, Hawkins BR, Barnett AH. Analysis of a Chinese population suggests that the TNFB gene is not a susceptibility gene for Graves’ disease. Hum Immunol 1994; 40:135–137.

64.Hunt PJ, Marshall SE, Weetman AP, Bell JI, Wass JA, Welsh KI. Cytokine gene polymorphisms in autoimmune thyroid disease. J Clin Endocrinol Metab 2000; 85:1984–1988.

65.Heward J, Nithiyananthan R, Allahabadia A, Gibson S, Barnett AH, Franklyn J, Gough SC. No association of an interleukin-4 (IL-4) promoter polymorphism with Graves’ disease. J Clin Endocrinol Metabol 2001; 86:3861–3863.

Figure 1 The development of clinically overt Graves’ disease seems to involve a complex interplay of genetic, endogenous, and environmental factors. At present, it is, however, not clear how and to what degree, if any, the candidate genes or genetic markers interact with the endogenous or environmental risk factors. HLA, human leukocyte antigen; CTLA-4, the cytotoxic T-lymphocyte antigen 4; AITD-1, GD-1, GD-2, and GD-3, susceptibility loci for GD, located on chromosome 6, 14, 20, and X, respectively; TSHR, the thyrotropin receptor. (Modified from Ref. 7.)

II.DO ENVIRONMENTAL FACTORS PLAY A ROLE IN THE CAUSES OF GRAVES’ DISEASE?

The influence of genetic and environmental factors in the causes of ‘‘complex diseases’’ have traditionally been studied in families and twins (6). Family and twin studies have provided indisputable evidence for the contribution of both genetic and environmental factors in the development of GD (7–9). This is based on familial clustering of GD and on concordance rates for GD being significantly higher in monozygotic than in dizygotic twins. Since monozygotic twins share all genes and dizygotic twins, on average, share half of their genes, the difference in concordance rates can be explained by genetic factors. On the other hand, even with more than 25 years of follow-up, the crude probandwise concordance rates for GD are no higher than 30–60% in monozygotic twins (8,9). In other words, although they are genetically alike, in 40–70% of monozygotic twins one twin develops GD but the second twin does not, thus providing evidence that environmental factors play a causative role in GD. This is further supported by recent studies that have assessed the role of specific candidate genes or genetic markers in the causes of GD. These studies demonstrate that a large number of healthy subjects harbor one or several of the currently known genetic risk markers for GD without having the disease (3,7,10). The considerable regional variations in the prevalence of GD demonstrated in large epidemiological surveys (11–13) also point strongly toward environmental factors. Accepting, therefore, that environmental factors play a role in the causes of GD, how large is the effect?

In a recent population-based twin study comprising data from over 8,900 Danish twin pairs, it has been estimated that 79% (95% confidence interval, 64–90%) of the liability to develop clinically overt GD is attributable to additive genetic factors (heritability), whereas environmental factors explain the remaining 21% (95% confidence interval,

Table 2 Summary of the Evidence for or Against a Causal Relationship Between Specific Environmental Exposures and Graves’ Disease

abnormalities leading to thyroid disease are autonomous growth of follicular tissue and thyroid autoimmunity. Since both processes are influenced by the iodine intake level, it is not surprising that iodine has been implicated as playing an important role in the causes of most thyroid diseases.

Considerable evidence exists both in human populations (18) and in animal models (19) that iodine plays an important and probably causal role in the development of GD in genetically predisposed individuals. Epidemiological surveys have repeatedly demonstrated that differences in prevalence and/or incidence of overt GD in different parts of the world closely mimic the magnitude of the iodine intake, with GD being more prevalent in areas with the highest iodine intake (11–13,20). Furthermore, recent longitudinal data from Austria (21) demonstrate that an increase in iodine intake from low to a normal level is accompanied by a twofold increase in the incidence of hyperthyroidism secondary to GD. Although rare, iodine-induced hyperthyroidism has also been described in iodinesufficient areas (22). However, in the great majority of these patients, if not all, the induced hyperthyroidism is secondary to an underlying thyroid autonomy (22,23). On the other hand, the level of thyrotropin receptor antibodies has been shown to increase significantly in hyperthyroid patients with GD when given excess iodine (24). At present there are, however, no epidemiological data available regarding the consequences of incidental exposure to iodine excess (e.g., by amiodarone or kelp tablets, iodine-containing x-ray contrast agents, or iodine-rich foodstuffs) in subjects predisposed for GD living in iodinesufficient areas.

The above epidemiological observations suggest that iodine can affect the course of GD. It has long been recognized that antithyroid drug therapy reduces thyroidal iodine content and that patients given iodine supplementation after discontinuing drug therapy are more likely to have relapses than patients not given iodine (25). More recently it has been shown that the response to antithyroid drugs in patients with GD is more rapid and the dose required to control the disease is smaller in iodine-deficient areas than in iodinereplete areas (26,27). In line with these observations, the remission rate of GD after antithyroid drug therapy is generally lower in areas with a high iodine intake than in areas with a low to normal iodine intake (18,28). Moreover, the difference in GD remission rates between the United States and Europe has, at least in part, been attributed to the higher iodine intake in the United States (28,29). GD is more likely to recur following thyroidectomy in areas with a high iodine intake than in areas with a normal iodine intake

(30). Thus, it seems evident that iodine administered to subjects genetically predisposed to GD may result in development of a clinically overt disease.

The exact mechanisms by which iodine provokes the development of overt GD in susceptible individuals are uncertain (19). Based on the observation that iodine, in vitro, can stimulate B lymphocytes to increased production of immunoglobulin (18), it has been speculated that iodine may induce GD by enhancing the activity of lymphocytes primed by thyroid-specific antigens (31). Consonant with this view is the observation that excess iodine administered to patients with GD significantly increased the thyrotropin receptor antibody titers (24).

In summary, although the mechanisms by which iodine induces GD in predisposed individuals remain to be defined, the relation between iodine intake and overt GD is well established in terms of both epidemiology and disease course. The available data show consistency, temporality, a dose–response pattern, and the observations are all biologically plausible. In combination, these features strongly indicate a causal relationship between iodine intake and the development of overt GD in genetically susceptible subjects (Table 2).

B.Smoking

Although the global influence of cigarette smoking on the immune system is not yet fully understood, it has long been recognized that smoking has a number of immunological effects involving both the humoral and cellular components of the immune response (32). Some of these immunological effects could theoretically have implications for the genesis of autoimmunity. This is supported by several case–control studies reporting an increased prevalence of autoimmune diseases among smokers compared to nonsmokers (32). Smoking has thus emerged as an environmental factor contributing to the development of autoimmunity.

A relationship between cigarette smoking and GD with or without ophthalmopathy was first suggested in 1987 in a small series of patients by Ha¨gg and Asplund (33). Despite major differences in study designs, size of study populations, definitions of smokers and nonsmokers, iodine intake, and methods used for evaluating the degree of ophthalmopathy and thyroid function, in a large number of retrospective (34–40) and in a few prospective (41,42) studies smoking has subsequently repeatedly been associated with an increased risk of GD and especially Graves’ ophthalmopathy. It has, however, been speculated that the association between smoking and GD is an artifact and occurs indirectly through other (confounding) factors such as stress or other neurobehavioral changes related to hyperthyroidism (43,44). However, in a recent study from Japan (38), even after adjusting for stressful life events, smoking was still an independent risk factor for GD. It is also worth noting that smoking has not been found to be associated with hyperthyroidism secondary to toxic nodular goiter (Plummer’s disease) (34,37).

Although the information is not given directly, it seems that in most studies, persons with Graves’ disease, with or without ophthalmopathy, began smoking more than 1 year prior to diagnosis. This was certainly the case in our own data set (39). These observations suggest a temporal, cause and effect relationship between smoking and GD. In GD with ophthalmopathy, recent prospective data clearly establish such a temporal relationship (42).

Evidence supporting a dose–response effect between smoking and GD with ophthalmopathy (42) and without ophthalmopathy (39) has been reported. In their prospective

Figure 2 Individual intrapair pack-year difference (pack years of the proband minus pack years of the healthy co-twin) in twin pairs discordant for clinically overt Graves’ disease but concordant for smoking. Note that each bar represents a twin pair and that a positive value is obtained when pack years of the proband are higher than pack years for the corresponding co-twin; proband versus co-twin, 13 pairs, p 0.048. (Modified from Ref. 39.)

study, Pfeilschifter and Ziegler (42) found that the relative risk for eye disease increased in proportion to the current number of cigarettes smoked daily. In a recent twin study (39), it was shown that among twin pairs concordant for smoking (both twins smoke) but discordant for GD (only one of the twins has GD), the twin with GD smoked significantly more than the healthy co-twin (Fig. 2). This finding suggests a positive correlation between the cumulative cigarette consumption (counted in pack-years) and the development of GD in genetically susceptible individuals.

In sharp contrast to previous retrospective data (36,40), more recent prospective data (42) indicate that former smokers have a lower risk of developing eye disease than current smokers even with a comparable lifetime tobacco consumption. This suggests that giving up smoking may be beneficial. Unfortunately, there are no prospective data on whether cessation of smoking reduces the degree of hyperthyroidism, or a preexisting ophthalmopathy, or the risk of development or deterioration of ophthalmopathy.

As with iodine, smoking may also affect treatment outcome in GD. In a historical cohort study comprising 150 consecutive patients treated with high-dose oral prednisone and radiotherapy for severe ophthalmopathy, a response to treatment was seen in up to 93.8% of the nonsmokers but in only 68.2% of the smokers (45). Furthermore, in randomized treatment studies ophthalmopathy was three to five times more likely to improve in nonsmokers than in smokers, whereas ophthalmopathy was more likely to get worse in smokers than in nonsmokers (45,46).

How smoking contributes to the development of GD with or without ophthalmopathy is at present unclear, although several mechanisms have been suggested (40,43,47).

Regardless of the exact mechanisms, the association between smoking and GD, and especially GD with ophthalmopathy, is strong and well established (Table 2).

C.Stress

The triggering role of stressful life events in disease development has been suggested frequently since the very first descriptions of GD in the early 19th century (48,49). Thanks to the development of standardized methods for identification, measurement, and scoring of negative/adverse (stressful) as well as positive life events, this hypothesis is now sup-

ported by substantial data. The first study to use a standardized methodology in the evaluation of the impact of stressful life events on the development of GD was published in 1991 by Winsa and colleagues (50). The impact of stressful life events was evaluated by comparing the answers to a standardized questionnaire about life changes from 208 subjects with newly diagnosed GD to those from 372 matched control subjects. Compared with controls, subjects with GD reported more negative life events in the 12 months preceding the diagnosis, and negative life event scores were also significantly higher (odds ratio 6.3, 95% confidence interval 2.7–14.7 for the group with the highest negative score). In spite of methodological differences, similar results have been reported from England (51), Italy (52), Hong Kong (53), and Yugoslavia (54). These studies have, however, been criticized for inappropriate control of a number of pertinent modulating factors such as social support, mood disturbances, coping skills, and smoking, which all may influence the immune system (32,55). In one recent study (38), stressful life events were still strongly associated with GD (odds ratio 7.7, 95% confidence interval, 2.2–27) even after adjustment for daily hassles, social support, coping skills, smoking and drinking habits.

Only two studies (38,50) have evaluated whether the association between stressful life events and GD show a dose–response pattern. In both studies, the relative risk of GD increased as the life event score increased, suggesting a dose–response relationship. Although a dose–response effect strongly supports a causal relationship, especially when coupled with a large relative risk, its presence does not exclude bias. Bias may, however, very well be present, since all published studies have been retrospective. Such a study design is highly vulnerable to bias, especially recall bias (48). Subjects with GD may be more prone to remember negative life events than controls, especially when such life changes are thought to be the cause of their disease. Thus, if present, recall bias is a serious problem because it will overestimate the strength of the association (48). In our opinion, recall bias is, to some degree, present in all of these studies. Another serious concern is the combination of the insidious onset of GD and information on stressful life events gathered in a retrospective manner, where by definition both the purported cause (stressful life events) and the effect (GD) are measured at the same time. This combination makes it almost impossible to determine which came first. Since it is absolutely necessary for a cause (stressful life events) to precede an effect (GD), the lack of such a sequence is a strong argument against a causal relationship. This is not to say that the right temporal relationship between stressful life events and GD is lacking, but rather that no film conclusions can be made due to the lack of prospective data.

The mechanisms by which stress might precipitate GD in predisposed individuals remains to be clarified. According to current thinking, the role of stress is explained by tight relations between the hypothalamic–pituitary–adrenocortical axis, the central nervous system, and the immune system (48,49). The suggested pathways are that stress stimulates the hypothalamic–pituitary–adrenocortical axis with a consequent increase in serum glucocorticoids and activation of the autonomic nervous system, followed by release of catecholamines. This altered neuroendocrine equilibrium has a profound effect on the immune system through direct interaction of hormones and neuropeptides with specific receptors, leading to a change in the profile of cytokine secretion.

In summary, the observed association between stress and GD is consistent, strong, probably dose-dependent, and biologically plausible. However, due to the lack of prospective data, no firm conclusions can be made with respect to the temporal sequence of stress (cause) and GD (effect).

D.Infections

The idea that an infection might trigger the development of overt GD in genetically predisposed individuals has long been a popular theory (31,56,57). Infectious agents may induce thyroid autoimmunity by various well-described mechanisms (56), such as inducing alterations or modifications of self antigens, mimicking self antigens, activating superantigeninduced T cells or inducing expression of human leukocyte antigen (HLA) molecules on thyroid cells.

One of the best-studied infectious agents in relation to GD is Yersinia enterocolitica (56,57). A possible relationship between Yersinia enterocolitica and GD was first suggested in the mid 1970’s by Bech and colleagues (58). In this study, antibodies against Yersinia enterocolitica (serotype 3) were found in 110 of 185 patients (59.5%) with newly diagnosed GD, compared to 27.7% in the control population. Results from subsequent retrospective case–control studies from different parts of the world have, however, been contradictory (57). Moreover, in studies showing an association, the antibody titers against Yersinia enterocolitica are generally low and there are no obvious correlations with age, gender, thyroid antibodies, or disease severity (57,58). In line with these observations is the finding that most patients with Yersinia infection do not develop GD including those who produce antithyrotropin receptor antibodies (56).

In summary, although the proposed association between Yersinia enterocolitica infection and GD is biologically plausible, it does not show consistency, specificity, or temporality, and there is at present no evidence for a causal relationship.

Numerous other infectious agents, such as influenza B virus, various retroviruses, human foamy virus, coxsackie B virus, and Mycoplasma species have also been investigated, but no convincing evidence has been produced. Thus, at present the role of infection in precipitating GD in humans remains purely hypothetical (1,56,57).

E. Other Environmental Factors

Additional proposed environmental risk factors for GD include certain drugs, in particular lithium (59) and amiodarone (23), as well as an adverse intrauterine environment reflected by low birth weight (60,61). For the time being, the evidence of a causal relationship between these environmental factors and GD is rather weak. Lithium-associated hyperthyroidism has only been reported in sporadic case reports or in small retrospective crosssectional studies with inappropriate control populations (59). Amiodarone-induced hyperthyroidism is a well-known condition (17,22), but prospective data show that long-term treatment with amiodarone (in an iodine-sufficient area) does not increase the prevalence of thyroid autoantibodies. This suggests that thyroid autoimmunity plays little, if any, role in the development of hyperthyroidism in amiodarone-treated subjects without underlying thyroid disorders (23). In a cross-sectional study of 305 women aged 60–71 years, the proportion of women with thyroglobulin and thyroid peroxidase antibodies decreased with increasing birth weight (60). In contrast, a recent population-based twin control study did not find any effect of birth weight, or several other birth characteristics, on the risk of developing clinically overt autoimmune thyroid disease (62).

IV. CONCLUSIONS

From this brief review of the role of environmental factors in the genesis of GD one can draw several conclusions. First, the influences of environmental exposures have seldom

been studied in a prospective manner. Nevertheless, at present there are convincing epidemiological and clinical observations strongly indicating that the association between GD and iodine intake and smoking is causal.

Second, with very few exceptions (38,50) the impact of various environmental factors on disease development has only been studied in isolation. That is, no study has taken the presence of a possible interaction between two or more environmental risk factors into consideration. It is clear that variation in iodine intake modulates the effect of smoking on the thyroid, with the predominant effect of smoking being goitrogenic and/or antithyroid when the iodine intake is low, and immunogenic when it is adequate (47). The relationship between smoking habits and stress is another example, demonstrating that environmental risk factors for GD can interact and thereby influence disease risk. Future studies aimed at clarifying the role of environmental factors in GD should, therefore, analyze as many factors as possible simultaneously with a multivariate statistical method in order to determine both their independent and combined influences on disease development.

Third, besides two recent twin case–control studies (39,62) the impact of environmental factors on the development of GD has never been studied in conjunction with the genetic background. Neither is it clear whether the environmental risk factors interact with the presently known susceptibility genes or genetic markers for GD, such as HLA-DR3 and CTLA-4 (3,10). In the near future a large number of new susceptibility genes and their numerous allelic variants probably will be identified. Thus, it will be important to assess how modifiable environmental risk factors, such as iodine intake or smoking or both in combination, interact with these susceptibility genes to influence disease risk. At present, the known environmental risk factors in GD, as in most chronic diseases, have a very poor predictive value for disease occurrence. Therefore, stratifying according to genetic susceptibility at one or more loci will greatly improve the predictive value for disease occurrence among biologically susceptible individuals and may thus help us to target preventive and therapeutic interventions.

ACKNOWLEDGMENTS

The present work has been supported by grants from the Agnes & Knut Mørks Foundation and the Clinical Research Institute, University of Southern Denmark.

REFERENCES

1.Weetman AP, McGregor AM. Autoimmune thyroid disease: further developments in our understanding. Endocr Rev 1994; 15:788–830.

2.Smith B, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev 1988; 9:106–121.

3.Brix TH, Kyvik KO, Hegedu¨s L. What is the evidence of genetic factors in the etiology of Graves’ disease?—a brief review. Thyroid 1998; 8:627–634.

4.Philippou G, McGregor AM. The aetiology of Graves’ disease: what is the genetic contribution? Clin Endocrinol 1998; 48:393–395.

5.Davies TF. Autoimmune thyroid disease genes come in many styles and colors. J Clin Endocrinol Metab 1998; 83:3391–3393.

6.Martin N, Boomsma D, Machin G. A twin-pronged attack on complex traits. Nat Genet 1997; 17:387–392.

7.Brix TH, Hegedu¨s L. Genetic predisposition versus environmental factors in autoimmune thy-

roid disease. In: Pe´ter F, Wiersinga W, Hostalek U, eds. The Thyroid and Environment. Stuttgart: Schattauer 2000:105–119.

8.Brix TH, Christensen K, Holm NV, Harvald B, Hegedu¨s L. A population based study of Graves’ disease in Danish twins. Clin Endocrinol 1998; 48:397–400.

9.Brix TH, Kyvik KO, Christensen K, Hegedu¨s L. Evidence for a major role of heredity in Graves’ disease—a population based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001; 86:930–934.

10.Gough SC. The genetics of Graves’ disease. Endocrinol Metab Clin North Am 2000; 29:255– 266.

11.Reinwein D, Benker G, Ko¨nig MP, Pinchera A, Schatz H, Schleusener H. Hyperthyroidism in Europe: clinical and laboratory data of a prospective multicentre survey. J Endocrinol Invest 1986; 9(Suppl 2):1–35.

12.Laurberg P, Pedersen KM, Vestergaard H, Sigurdsson G. High incidence of multinodular toxic goitre in the elderly population in a low iodine intake area vs. high incidence of Graves’ disease in the young in a high iodine intake area: Comparative surveys of thyrotoxicosis epidemiology in East Jutland Denmark and Iceland. J Int Med 1991; 229:415–420.

13.Vanderpump MPJ, Tunbridge WMG. The epidemiology of thyroid diseases. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text. Philadelphia: Lippincott–Raven, 2000:467–474.

14.Rothman KJ, Greenland S. Causation and causal inference. In: Rothman KJ, Greenland S, eds. Modern Epidemiology. Philadelphia: Lippincott–Raven, 1998:7–28.

15.Hill AB. The environment and disease: association or causation? Proc R Soc Med 1965; 58: 295–300.

16.Phillips DIW, Lazarus JH, Hall R. Iodine metabolism and the thyroid. J Endocr 1988; 119: 361–363.

17.Roti E, Vagenakis AG. Effect of excess iodide: Clinical aspects. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text. Philadelphia: Lippincott–Raven Publishers, 2000:316–327.

18.McGregor AM, Weetman AP, Ratanachaiyavong S, Owen GM, Ibbertson HK, Hall R. Iodine: an influence on the development of autoimmune thyroid disease? In: Hall R, Ko¨bberling J, eds. Thyroid Disorders Associated with Iodine Deficiency and Excess. New York: Raven Press, 1985:209–216.

19.Drexhage HA, de Wit HJ, Mooij P. Iodine and thyroid autoimmune disease. In: Pe´ter F, Wiersinga W, Hostalek U, eds. The Thyroid and Environment. Stuttgart: Schattauer, 2000:163– 177.

20.Wang C, Crapo LM. The epidemiology of thyroid disease and implications for screening. Endocrinol Metab Clin North Am 1997; 26:189–218.

21.Mostbeck A, Galvan G, Bauer P, Eber O, Atefie K, Dam K, Feichtinger H, Fritzsche H, Haydl H, Kohn H, Korig B, Koriska K, Kroiss A, Lind P, Markt B, Maschek W, Pesl H, RamschakSchwarzer S, Riccabona G, Stockhammer M, Zechmann W. The incidence of hyperthyroidism in Austria from 1987 to 1995 before and after an increase in salt iodization in 1990. Eur J Nucl Med 1998; 25:367–374.

22.Stanbury JB, Ermans AE, Bourdoux P, Todd C, Oken E, Tonglet R, et al. Iodine-induced hyperthyroidism: occurrence and epidemiology. Thyroid 1998; 8:83–100.

23.Trip MD, Wiersinga W, Plomp TA. Incidence, predictability, and pathogenesis of amiodaroneinduced thyrotoxicosis and hypothyroidism. Am J Med 1991; 91:507–511.

24.Wilson R, Mc Killop JH, Thomson JA. The effect of pre-operative potassium iodide therapy on antibody production. Acta Endocrinol 1990; 123:531–534.

25.Alexander WD, Harden RM, Koutras DA, Wayne E. Influence of iodine intake after treatment with antithyroid drugs. Lancet 1965; 11:866–868.

26.Azizi F. Environmental iodine intake affects the response to methimazole in patients with diffuse toxic goitre. J Clin Endocrinol Metab 1985; 61:374–377.

27.Benker G, Vitti P, Kahaly G, Raue F, Tegler L, Hirche H, Reinwein D. Response to methimazole in Graves’ disease. The European Multicenter Study Group. Clin Endocrinol 1995; 43: 257–263.

28.Wartofsky L. Low remission after therapy for Graves’ disease: possible relation of dietary iodine with antithyroid therapy results. JAMA 1973; 226:1083–1088.

29.Solomon BL, Evaul JE, Burman KD, Wartofsky L. Remission rates with antithyroid drug therapy: continuing influence of iodine intake? Ann Intern Med 1987; 107:510–512.

30.Thjodleifsson B, Hedley AJ, Donald D, Chesters MI, Kjeld M, Beck JS, Crooks J, Michie W, Hall R. Outcome of sub-total thyroidectomy for thyrotoxicosis in Iceland and Northeast Scotland. Clin Endocrinol 1977; 7:367–376.

31.Safran M, Paul TL, Roti E, Braverman LE. Environmental factors affecting autoimmune thyroid disease. Endocrinol Metab Clin North Am 1987; 16:327–342.

32.George J, Levy Y, Shoenfeld Y. Smoking and immunity: an additional player in the mosaic of autoimmunity. Scand J Immunol 1997; 45:1–6.

33.Ha¨gg E, Asplund K. Is endocrine ophthalmopathy related to smoking? Br Med J 1987; 295: 634–635.

34.Bartalena L, Martino E, Marcocci C, Bogazzi F, Panicucci M, Velluzzi F, Loviselli A, Pinchera A. More on smoking habits and Graves’ ophthalmopathy. J Endocrinol Invest 1989; 12:733–737.

35.Shine B, Fells P, Edwards OM, Weetman AP. Association between Graves’ ophthalmopathy and smoking. Lancet 1990; 335:1261–1264.

36.Tellez M, Cooper J, Edmonds C. Graves’ ophthalmopathy in relation to cigarette smoking and ethnic origin. Clin Endocrinol 1992; 36:291–294.

37.Prummel MF, Wiersinga WM. Smoking and risk of Graves’ disease. JAMA 1993; 269:479– 482.

38.Yoshiuchi K, Kumano H, Nomura S, Yoshimura H, Ito K, Kanaji Y, Ohashi Y, Kuboki T, Suematsu H. Stressful life events and smoking were associated with Graves’ disease in women, but not in men. Psychosom Med 1998; 60:182–185.

39.Brix TH, Hansen PS, Kyvik KO, Hegedu¨s L. Cigarette smoking and risk of clinically overt thyroid disease: a population based twin case–control study. Arch Intern Med 2000; 160:661– 666.

40.Bertelsen JB, Hegedu¨s L. Cigarette smoking and the thyroid. Thyroid 1994; 4:327–331.

41.Winsa B, Mandahl A, Karlsson FA. Graves’ disease, endocrine ophthalmopathy and smoking. Acta Endocrinol 1993; 128:156–160.

42.Pfeilschifter J, Ziegler R. Smoking and endocrine ophthalmopathy: impact of smoking severity and current vs lifetime cigarette consumption. Clin Endocrinol 1996; 45:477–481.

43.Bartalena L, Bogazzi F, Tanda ML, Manetti L, Dell’Unto E, Martino E. Cigarette smoking and the thyroid. Eur J Endocrinol 1995; 133:507–512.

44.Perkins KA, Grobe JE. Increased desire to smoke during acute stress. Br J Addict 1992; 87: 1037–1040.

45.Bartalena L, Marcocci C, Tanda ML, Manetti L, Dell’Unto E, Bartolomei MP, Nardi M, Martino E, Pinchera A. Cigarette smoking and treatment outcomes in Graves’ ophthalmopathy. Ann Intern Med 1998; 129:632–635.

46.Tallstedt L, Lundell G, Taube A. Graves’ ophthalmopathy and tobacco smoking. Acta Endocrinol 1993; 129:147–150.

47.Utiger RD. Cigarette smoking and the thyroid. N Engl J Med 1995; 333:1001–1002.

48.Chiovato L, Pinchera A. Stressful life events and Graves’ disease. Eur J Endocrinol 1996; 134:680–682.

49.Lecle`re JF. Stress and autoimmune thyroid disease. In: Pe´ter F, Wiersinga W, Hostalek U, eds. The Thyroid and Environment. Stuttgart: Schattauer, 2000:155–162.

50.Winsa B, Adami H-O, Bergstro¨m R, Gamstedt A, Dahlberg PA, Adamson U, Jansson R, Karlsson A. Stressful life events and Graves’ disease. Lancet 1991; 338:1475–1479.

51.Harris T, Creed F, Brugha TS. Stressful life events and Graves’ disease. Br J Psychiatry 1992; 161:535–541.

52.Sonino N, Girelli ME, Boscaro M, Fallo F, Busnardo B, Fava GA. Life events in the pathogenesis of Graves’ disease. A controlled study. Acta Endocrinol 1993; 128:293–296.

53.Kung AWC. Life events, daily stresses and coping in patients with Graves’ disease. Clin Endocrinol 1995; 42:303–308.

54.Radosavljevic VR, Jankovic SM, Marinkovic JM. Stressful life events in the pathogenesis of Graves’ disease. Eur J Endocrinol 1996; 134:699–701.

55.Ursin H. Stress, distress, and immunity. Ann NY Acad Sci 1994; 741:204–211.

56.Tomer Y, Davies T. Infection, thyroid disease, and autoimmunity. Endocr Rev 1993; 14:107– 121.

57.Wenzel BF, Peters A, Zubaschev I. Bacterial virulence antigens and the pathogenesis of autoimmune thyroid diseases (AITD). Exp Clin Endocrinol Diabetes 1996; 104(Suppl. 4):75–78.

58.Bech K, Nerup J, Larsen JH. Yersinia enterocolitica infection and thyroid diseases. Acta Endocrinol 1977; 84:87–92.

59.Barclay ML, Brownlie BEW, Turner JG, Wells JE. Lithium associated thyrotoxicosis: a report of 14 cases, with statistical analysis of incidence. Clin Endocrinol 1994; 40:759–764.

60.Phillips DIW, Cooper C, Fall C, Prentice L, Osmond C, Barker DJP, Rees Smith B. Fetal growth and autoimmune thyroid disease. Q J Med 1993; 86:247–253.

61.Wiersinga WM. Environmental factors in autoimmune thyroid disease. Exp Clin Endocrinol Diabetes 1999; 107(Suppl 3):67–70.

62.Brix TH, Kyvik KO, Hegedu¨s L. Low birth weight is not associated with clinically overt thyroid disease: a population based twin case-control study. Clin Endocrinol 2000; 53:171– 176.

values of control populations (3–6%) (17,20,24). This relative increase, however, does not reach the level of significance in some studies (20). The occurrence of thyroid autoantibodies, moreover, does not necessarily identify patients who develop thyroid dysfunction because of autoimmune thyroid disease. Therefore, their routine measurement in myasthenia gravis patients without clinical signs of thyroid disease seems also of little prognostic help.

Marino and colleagues (25) have found that myasthenia gravis associated with autoimmune thyroid disease (Graves’ disease and Hashimoto’s thyroiditis) has a mild clinical expression with preferential ocular involvement and lower frequency of thymic disease and of acteylcholine receptor antibodies. They suggest that concurrent ocular myasthenia gravis and endocrine ophthalmopathy may be due to immunological cross-reactivity against common autoimmune targets in the eye muscle as well as to a common genetic background.

The findings of Spurkland et al. (7) substantiate the latter part of this hypothesis to a certain extent: they have pointed out that myasthenia gravis patients with concurrent thymoma tend to be less frequently positive for human leukocyte antigen (HLA)-B8 and DR 3 markers than their controls. Only patients with concomitant thymic hyperplasia had a relatively higher frequency of HLA B8 and DR3, a constellation typical for Graves’ disease. The percentage of patients with thymoma in Marino et al.’s (25) group with concurrent Graves’ disease and myasthenia gravis had indeed the lowest occurrence rate of thymoma (8.9% vs. 19.4% in control).

REFERENCES

1.Ko¨hler W, Sieb P. Myasthenia gravis. Bremen: UNI-MED Verlag AG, 2000:58–70.

2.Barton JJS, Fouladvand M. Ocular aspects of myasthenia gravis. Semin Neurol 2000; 20:7– 20.

3.Rennie GE. Exophthalmic goitre combined with myasthenia gravis. Rev Neurol Psychiatry 1908; 6:229–233.

4.Boyages SC. The neuromuscular system and brain in thyrotoxicosis. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text. 8th ed. Philadelphia: Lippincott Williams & Wilkins, 2000:631–633.

5.Farid NR, Stone E, Johnson G. Graves’ disease and HLA: clinical and epidemiologic associations. Clin Endocrinol 1980; 15:535–539.

6.Ragheb S, Lisak RP. The immunopathogenesis of acquired (autoimmune) myasthenia gravis. In: Lisak RP, ed. Handbook of Myasthenia Gravis and Myasthenic Syndromes. New York: Marcel Dekker, 1994:239–276.

7.Spurkland A, Gilhus NE, Ronnigen KS, Aarli JA, Vartdal F. Myasthenia gravis patients with thymus hyperplasia and myasthenia gravis patients with thymoma display different HLA associations. Tissue Antigens 1991; 37:90–93.

8.Osserman KE, Silver S. The differential diagnosis of myopathy as seen in hyperthyroidism and myasthenia gravis. In: Pitt-Rivers R, ed. Advances in Thyroid Research. Oxford: Pergamon Press, 1961:100–117.

9.Ohno M, Hamada N, Yamakawa J, Noh J, Morii H, Ito K. Myasthenia gravis associated with Graves’ disease in Japan. Jpn J Med 1987; 26:2–6.

10.Sahay BM, Blendis LM, Greene R. Relation between myasthenia gravis and thyroid disease. Br Med J 1965; 1:762–766.

11.Kuroiwa Y. Epidemiological aspects of myasthenia gravis in Japan. In: Japan Medical Research Foundation, eds. Myasthenia Gravis. Tokyo: University of Tokyo Press, 1981:9–20.

12.Somnier FE, Keiding N, Paulson OB. Epidemiology of myasthenia gravis in Denmark. A longitudinal and comprehensive population survey. Arch Neurol 1991; 48:733–739.

13.Phillips LH. The epidemiology of myasthenia gravis. Neurol Clin 1994; 12:263–271.

14.Lavrnic D, Jarebinski M, Rakocevic-Stojanovic V, Stevic Z, Lavrnic S, Pavlovic S, Trikic R, Tripkovic I, Neskovic V, Apostolski S. Epidemiological and clinical characteristics of myasthenia gravis in Belgrade, Yugoslavia (1983–1992). Acta Neurol Scand 1999; 100:168–174.

15.Jacobson DM. Acetylcholine receptor antibodies in patients with Graves’ ophthalmopathy. J Neuroophthalmol 1995; 15:166–170.

16.Marino M, Barbesino G, Manetti L, Chiovato L, Ricciardi R, Rossi B, Muratorio A, Mariotti S, Braverman LE, Pinchera A. Mild clinical expression of myasthenia gravis associated with autoimmune thyroid disease—author’s response. Letter to the Editor. J Clin Endocrinol Metab 1997; 82:3905–3906.

17.Kiessling WR, Pflughaupt KW, Ricker K, Haubitz I, Mertens H-G. Thyroid function and circulating antithyroid antibodies in myasthenia gravis. Neurology 1981; 31:771–774.

18.Christensen PB, Jensen TS, Tsiropoulos I, Sorensen T, Kjaer M, Hojer-Pedersen E, Rasmussen MJ, Lehfeldt E. Associated autoimmune disease in myasthenia gravis. A population-based study. Acta Neurol Scand 1995; 91:192–195.

19.Thorlacius S, Aarli JA, Riise T, Matre R, Johnsen HJ. Associated disorders in myasthenia gravis: autoimmune diseases and their relation to thymectomy. Acta Neurol Scand 1989; 80: 290–295.

20.Weissel M, Mayr N, Zeitlhofer J. Clinical significance of autoimmune thyroid disease in myasthenia gravis. Exp Clin Endocrinol Diabetes 2000; 108:63–65.

21.Dos Remedios LV, Weber PM, Feldman R, Schurr DA, Tsoi TG. Detecting unsuspected thyroid dysfunction by the free thyroxine index. Arch Intern Med 1980; 140:1045–1049.

22.Eggertson R, Petersen K, Lundberg P-A, Nystrom E, Lindstedt G. Screening for thyroid disease in a primary care unit with a thyroid stimulating hormone assay with a low detection limit. Br Med J 1988; 297:1586–1592.

23.Vanderpump MPJ, Tunbridge WMG, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F, et al. The incidence of thyroid disorders in the community: a twenty year follow-up of the Whickham survey. Clin Endocrinol 1995; 43:55– 68.

24.Tunbridge WMJ, Evered DC, Hall R, Appleton D, Brewis M, Clark F, Evans JG, Young E, Bird T, Smith PA. The spectrum of thyroid disease in the community: The Whickham survey. Clin Endocrinol 1977; 7:481–493.

25.Marino M, Ricciardi R, Pinchera A, Barbesino G, Manetti L, Chiovato L, Braverman LE, Rossi B, Muratorio A, Mariotti S. Mild clinical expression of myasthenia gravis associated with autoimmune thyroid disease. J Clin Endocrinol Metab 1997; 82:438–443.