Genes Predisposing to Graves’ Disease

In the last 20 years, the impressive advancement of biomedical research has allowed a remarkable expansion in genetics, which has led to the identification of several genes involved in the predisposition to Graves’ disease.33,38 Linkage, association, and candidate genes analyses, as well as whole-genome screening, have all been used to accomplish this goal. Based on the data available, genetic susceptibility to thyroid autoimmunity in general and to Graves’ disease in particular seems to be determined by a combination of a number of genes, some which most likely still remain to be identified.^33,38,39

Infections

Over the years, both experimental and epidemiologic evidence has suggested that infections could play a role in the pathogenesis of Graves’ disease.54,55 Seasonal and geographic variations in the incidence of the disease have been reported,^56,57 although seasonal variations have not been confirmed in other studies.⁵⁸ Blood group nonsecretors, who are more prone than secretors to infections, are more frequently found among patients with Graves’ disease than in controls.⁵⁹ This observation has been interpreted as indirect evidence that infectious pathogens may be involved in the etiology of Graves’ disease, although a direct genetic effect of the secretor status could also explain these results. Evidence of a recent viral infection has been reported in a high percentage of patients with Graves’ disease.^54,55

Molecular mimicry has been invoked to explain the association between infections and Graves’ disease.⁶⁰ Molecular mimicry is based on the hypothesis that cross-reactions of some microbial antigens with a self-antigen may cause an immune response to autoantigens. In Graves’ disease, the pathogen Yersinia enterocolitica has been thoroughly studied after reports of association of this microbe with the disease. A high prevalence of circulating antibodies against Y. enterocolitica has been observed in patients with Graves’ disease, and Yersinia antibodies were found to interact with thyroid structures.^61–63 In a recent study from Denmark, it was shown that the occurrence of IgA and IgG antibodies against Yersinia not only is more frequent in Graves’ patients than in case controls but also in twins with Graves’ disease compared with their discordant twins.⁶⁴ Saturable binding sites for TSH have been found in Yersinia and were also recognized by TRAbs from patients with Graves’ disease.^65,66 In animals immunized with Yersinia proteins, antibodies developed against human thyroid epithelial cells and TSHR.^67,68 Overall, the affinity of these cross-reactive antibodies to the thyroid was low, and immune responses were transient. Low-affinity binding sites for TSH have also been found in other bacteria, including some species of Leishmania and Mycoplasma.^53,54 However, it must be noted that thyroid autoimmunity does not develop in most patients with Yersinia infections,⁶⁹ so the evidence in favor of Yersinia infections as a precipitating cause of Graves’ disease awaits confirmation.

Viruses could theoretically trigger autoimmunity through several mechanisms, including interactions with autoantigens, permanent expression of viral proteins on the surface of epithelial cells, aberrant induction of HLA antigens on epithelial cells (see later), and molecular mimicry.60,70 In 1989, the presence of retroviral (HIV-1 glycosaminoglycan protein) sequences in the thyroid and peripheral mononuclear cells of patients with Graves’ disease was reported,⁷¹ but viral sequences were not found in control thyroids. This finding, however, remained isolated and was not confirmed in subsequent studies.^72,73 Human foamy virus antigens were shown by immunofluorescence to be present in the thyroid of patients with Graves’ disease.⁷⁴ Again, further studies using more specific and sensitive techniques failed to identify foamy virus DNA and antiviral antibodies in the blood of affected subjects.^75,76 Homology between another HIV-1 protein (Nef) and human TSHR has also been reported, although sera from patients with Graves’ disease did not react with the peptide bearing the highest degree of homology.⁷⁷ Another retroviral protein, p15E, has been isolated from the thyroids of patients with Graves’ disease but not from control glands.⁷⁸ In this regard, it is worthwhile emphasizing that retroviral-like proteins, including p15E, are encoded by the normal human genome. Although their function is unclear, they may be expressed in many epithelial tissues under certain conditions such as inflammation and may modulate but not initiate the immune response.⁷⁹ The finding of retroviral sequences or proteins in the glands of patients with Graves’ disease may therefore represent a secondary rather than a causative phenomenon. Circulating antibodies against another retroviral particle, namely HIAP-1, have been found in as many as 87.5% of patients with Graves’ disease as compared with 10% to 15% of controls,⁸⁰ but HIAP-1 particles were not detected when human T cells were co-cultured with Graves’ thyrocytes.⁸¹

A highly speculative hypothesis has been raised that involves superantigens.55,60 Superantigens are endogenous or exogenous proteins, such as microbial proteins, capable of stimulating a strong immune response through molecular interactions with nonvariant parts of the T-cell receptor and the HLA class II proteins. Through this mechanism, superantigens are in theory capable of stimulating the expansion of autoreactive T cells and therefore of driving an autoimmune response.^55,60 Such a mechanism has been suggested in rheumatoid arthritis, and a similar mechanism was proposed for Graves’ disease. In vitro superantigen stimulation of glands with autoimmune thyroid disease induced expression of HLA class II molecules on thyrocytes, and this phenomenon was IFN-γ dependent.⁸² The interpretation of this observation was that superantigen-reactive T cells exist among the lymphocytes infiltrating the thyroid in autoimmune thyroid disease, and that these lymphocytes may have been activated after exposure to extrinsic superantigens.

The most recent hypothesis, the so-called “hygiene hypothesis of autoimmunity,” implies that infections may protect from, rather than precipitate, autoimmune diseases. Exposure of the immune system to infective agents may somehow allow better control of autoimmune responses. In this regard, improved living standards have been associated with decreased exposure to infections and an increased risk of autoimmune diseases. Kondrashova et al. reported a much reduced prevalence of thyroid autoantibodies in the lower-economic population, which may suggest the hygiene hypothesis may apply to thyroid autoimmune diseases.⁸³ Further studies are needed to investigate whether this is in fact the case.

In summary, although epidemiologic evidence indicates that infection may play an important role as a causative or protective factor in Graves’ disease, we are still lacking definitive identification of the etiologic organism(s) and a reasonable explanation for microbes to precipitate or protect from the disorder.

Stress

The suggestion that psychological stress may be a precipitating factor in Graves’ disease has been made as early as the first description of the disease.³ The occurrence of stressful events before the onset of Graves’ disease is a recurrent impression among clinicians, and by the end of the 19th century, Graves’ disease was considered to be a result of prolonged emotional disturbances. In cross-sectional questionnaire-based studies, some investigators have shown an increased prevalence of stressful life events in the months preceding the onset of Graves’ disease.^84–87 However, some of the recorded events (such as arguments with spouses and in the workplace) could have been influenced by the behavior of patients with a yet undetected hyperthyroidism and therefore be a consequence rather than a cause of the disease. Other events, however, were largely independent of the patient’s behavior, such as unemployment and financial difficulties. In some studies, patients were asked to rate the stressfulness of life events, and patients with Graves’ disease ranked such events more stressful than did controls.⁸⁴ Thus it is possible that the perception of life events is different in hyperthyroid patients. In cross-sectional studies, an increase in the prevalence of Graves’ disease was reported during World War II in Germany but not during the civil unrest in Ireland or during the German occupation of Belgium, which suggests that the stressful events of personal life may be more important than “social stress.”⁸⁴ On the other hand, chronic stress due to panic disorder was not associated with the occurrence of Graves’ disease.⁸⁸ Stress is associated with increased adrenocorticotropic hormone (ACTH) and cortisol secretion, which can in turn determine immune suppression, but additional non-ACTH-related immunosuppressive phenomena also occur.^89,90 Recovery from such immune suppression can be associated with rebound immune hyperactivity, which could precipitate autoimmunity. The best example of such a phenomenon is perhaps the well-documented immune suppression of pregnancy, which can be followed by new or recurrent onset of autoimmune disorders, including Graves’ disease (see later discussion).⁹¹

In summary, limited but significant evidence indicates that stress may be a contributing factor in the etiology of Graves’ disease, probably in connection with other predisposing factors. The available studies are all retrospective and therefore carry a number of possible biases. Worried patients sick with hyperthyroidism may be more prone to recall upsetting events, and subclinical undetected thyrotoxicosis present before clinical diagnosis may alter the perception of life events and even the behavior of patients. Unfortunately, prospective studies addressing the problem of stress and Graves’ disease and its interplay with genetic factors are not available to date.

Gender

Graves’ disease is typically but not exclusively a disease of women. In most series, the female-to-male ratio ranges from 5 to 10 at any age,16,17 although the difference may be smaller during childhood.⁹² The reason for the disproportionate prevalence of Graves’ disease in women is not known, but genetic and nongenetic factors must play a role. A number of studies indicate a stronger immune system in women.^93,94 Autoimmune phenomena and diseases are in general more prevalent in women.^93,94 A large body of evidence clearly indicates the existence of sexual dimorphism in normal and abnormal immune responses in spontaneous and experimental animal models, including models of autoimmune thyroiditis.^93,94 In these models, male hormones appear to down-regulate immunity and therefore protect animals from autoimmunity, whereas the effect of estrogen is not always unequivocal. Despite the evidence obtained from animal studies, little evidence in the literature supports a role for sex hormones in the high prevalence of Graves’ disease in women.^93,94 Women with normal baseline levels of estrogen, but with an increased sensitivity to the hormone as shown by the presence of melasma, had a higher prevalence of thyroid autoimmune disorders.⁹⁵ However, a clear association between exogenous estrogen administration and Graves’ disease has never been reported. Moreover, thyroid autoimmunity is often found in patients with Turner’s syndrome, who typically have low estrogen levels.⁹⁶ Conversely, male patients with primary hypogonadism such as in Klinefelter’s syndrome do not show a higher incidence of Graves’ disease or Hashimoto’s thyroiditis.⁹⁷ In these human conditions, however, the chromosomal abnormality probably plays an important role that is largely independent of sex hormone levels.

Pregnancy is an important risk factor, and it is well recognized that in any woman the risk of development of Graves’ disease increases fourfold to eightfold in the postpartum year.⁹¹ The abrupt fall in the level of pregnancy-associated immunosuppressive factors immediately after delivery (rebound immunity) is likely to be the mechanism responsible for the precipitation of Graves’ disease.⁹¹ Nevertheless, in a recent retrospective study it was found that the relative frequencies of postpartum onset of Graves’ disease were similar in relation of increasing parity, which would not support a role of the postpartum period as a major risk factor for the first appearance of Graves’ disease.⁹⁸ On the other hand, it was also shown by the same group that the postpartum period is indeed a risk factor for relapse of Graves’ thyrotoxicosis after withdrawal of antithyroid drugs.⁹⁹ The factors involved in the immune alterations of the postpartum period may include but are not limited to estrogen and progestin.^91,100

Besides sex hormones, factors on the X chromosome could explain the epidemiologic evidence of a female preponderance in Graves’ disease. A linkage analysis in families with Graves’ disease has located a putative Graves’ disease susceptibility locus on the long arm of the X chromosome.³³ Although most X-linked disorders are expressed phenotypically only in men, it is possible that a gene with a dose-dependent effect may determine more relevant clinical effects in women. This finding could help explain the higher incidence of Graves’ disease observed in women and, possibly, in patients with Turner’s syndrome. Inactivation of the X chromosome, an epigenetic phenomenon, has also been suggested to be involved in the female predisposition to thyroid autoimmunity, and recently it was shown that this phenomenon is more frequently observed in patients with Graves’ disease or autoimmune thyroiditis than in healthy controls.¹⁰¹

Smoking

A number of studies have provided evidence for an association between smoking and thyroid diseases, including Graves’ disease.102,103 Retrospective analysis shows that in smokers there is an increased risk of Graves’ disease and ophthalmopathy, as well as of relapse of hyperthyroidism following anti-thyroid drug withdrawal, which is more pronounced in the female gender. The findings may be explained both by a direct action of smoking metabolites on the immune system or by damage induced by smoking metabolites on thyrocytes, which may determine exposure of thyroid antigens to the immune system.

Thyroid Damage

There are reports of Graves’ disease appearing after ethanol injections performed for treatment of autonomous thyroid nodules, which has been interpreted as due to the massive release of thyroid antigens, thereby triggering an autoimmune response to TSH-R in predisposed individuals.¹⁰⁴ Furthermore, and possibly because of similar pathogenetic mechanisms (i.e., massive release of thyroid antigens), Graves’ disease or simply serum TRAb have been reported to appear following radioiodine treatment for toxic adenoma or toxic nodular goiter.¹⁰⁵ We observed a patient with Graves’ disease that appeared after a neck injury (unpublished observation), which in theory may also reflect release of thyroid antigens.

Nomenclature

The nomenclature of TRAbs is complex and largely dependent on the assay used to detect these antibodies in serum.107–111 Assays measuring displacement of radiolabeled TSH from its receptor by serum immunoglobulins detect TRAbs regardless of their functional activity. These antibodies have been termed TSH-binding inhibitory immunoglobulins (TBIIs). Assays for TSAbs use cellular systems carrying a functional TSHR and detect the release of cyclic adenosine monophosphate (cAMP) in the culture medium upon challenge with serum or purified immunoglobulins.^107–111 These antibodies are essentially the cause of hyperthyroidism in Graves’ disease. In the same bioassay system, antibodies with blocking activity on the TSHR (TBAbs) can be detected.^107–112 These antibodies characterize a subset of patients with atrophic autoimmune thyroiditis and hypothyroidism, but they can also occasionally be found in patients with Graves’ disease, in combination with TSAbs.¹¹²

Major Autoantigen in Graves’ Disease: Structure-Function Relationship of the TSHR

Definitive proof that TSHR is the target of TSAbs came from the cloning of this protein in the late 1980s.12,13,107–109 The receptor is a member of the G protein–coupled receptor superfamily. Its structure includes seven hydrophobic transmembrane domains, an extracellular N-terminal domain (ectodomain), and an intracellular C-terminal domain. Heavy glycosylation of the extracellular domain accounts for about 20% of its molecular weight of 84 kD. The primary structure of the protein consists of 744 residues. The gene is located on chromosome 14q31 and is formed by 10 exons that yield a single polypeptide. Shedding of the N-terminal extracellular 310 amino-acid residues (the so-called A subunit) may either initiate or amplify the immune response against TSHR, being immunoreactive epitopes partially sterically hindered on the holoreceptor on the plasma membrane.^108,113,114 Binding sites for TSH and TRAbs are located in the extracellular domain and are mainly conformational.^107,108 The major epitopes for TSAbs are located in the extreme N-terminal portion, whereas those for TBABs are mainly, but not exclusively, in the C-terminal portion of the extracellular domain, closer to the cell membrane.^{107,108,115–118} However, this should not be taken as a paradigm, especially since TBABs are more diverse and spread over the receptor extracellular domain.¹⁰⁸ In fact, TRAbs are to some extent heterogeneous for epitope recognition, possibly due to epitope spreading during the immune response.¹⁰⁸ Whether and to what extent glycosylation and dimerization of TSH receptor affects its recognition by antibodies is uncertain.¹⁰⁸

Assays for TSH Receptor Antibody

The pioneering era of bioassays in vivo for TRAbs11,119 has been superseded by the present period, in which a number of in vitro assays are more readily performed by many laboratories and provide more reproducible and reliable measures of TRAb levels in the serum of patients. The radioreceptor assay uses TSHR from various sources: porcine or human recombinant TSHR from transfected cell lines.^120–126 Although assay design varies in all these methods, they all rely on displacement of labeled TSH from solubilized TSHR from the serum of patients. Studies in hyperthyroid patients with Graves’ disease show positive TBII tests with these methods in 75% to 95% of patients. The recently designed second-generation radioreceptor assay attains even higher sensitivity while maintaining high specificity (99%)¹²⁶ and is commonly used by the majority of laboratories for the routine detection of TRAb. An enzyme-linked assay is also available.¹²⁷ Radioreceptor assays do not require permanent cell cultures; they are the most readily available (also commercially) and are therefore the most frequently used in clinical practice. The TBII test, however, does not give information on the functional properties of the antibody detected and can be positive in the presence of TBAb.

The functional stimulating properties of TRAbs can be studied by in vitro bioassays based on the measurement of cAMP production from cells with a functional TSHR.¹²⁰ Human thyroid follicular cells,¹²⁸ a rat thyroid cell strain (FRTL-5),^129,130 and Chinese hamster ovary cells stably transfected with human TSHR (CHO-R)^131–133 have all been used for this purpose. With these assays, TSAb can be detected in more than 90% of patients with untreated Graves’ disease (Fig. 9-3). The system using CHO-R cells has some advantages over the others: it is slightly more sensitive and relies on easier culture conditions, which also makes it more reproducible in different laboratories. As stated previously, the bioassays have the advantage of giving information on the functional properties of TRAbs and, in a modification of the assay, can also identify TBAbs.^112,133 However, they require permanent cell culture equipment and pre-purification of the immunoglobulin fraction of serum, which makes these assays not readily available to routine endocrine laboratories. The latter problem has been overcome by very sensitive assays in which activation of a transfected firefly luciferase gene produces chemiluminescence in response to TSHR stimulation by whole serum.^134,135 Even more recently, a coated-tube assay which discriminates TSAb from TBAb was developed.¹³⁶ The assay is based on the use of a chimeric receptor, where a TSAb epitope 8-165 is replaced by comparable LH receptor residues. Binding of radiolabeled TSH to this chimera can be inhibited by sera containing TBAb up to 95%.

Thyroid-Stimulating Antibody in the Pathogenesis and Natural History of Graves’ Disease

Historically, the identification of TSAbs as the cause of hyperthyroidism and goiter in Graves’ disease came from the demonstration of a stimulating factor in the sera of hyperthyroid patients with a half-life much longer than that of TSH (LATS).¹¹ Subsequently this factor was shown to be an autoantibody.^137–139 TSAbs were shown to interact with TSHR in that they act as a potent agonist and thus cause hyperfunction of the thyroid gland.^107–110 Definitive proof that TRAbs interact with TSHR eventually came from studies with the cloned protein.^107–110 A clear-cut demonstration of the role of TSAb in the pathogenesis of hyperthyroidism is provided by the observation that the transplacental transfer of antibodies from TSAb-positive pregnant mothers to the fetus may cause a form of transient neonatal thyrotoxicosis that vanishes with the disappearance of TSAb from the serum of the newborn.¹⁴⁰

TSAbs are oligo- or pauciclonal, and this observation has suggested a primary defect at the B-cell level.107–110 TSAbs appear to be produced mainly by thyroid-infiltrating lymphocytes and lymphocytes in the draining lymph nodes.¹⁴¹ Synthesis by peripheral blood lymphocytes has been documented as well.^142–144 As mentioned earlier, TSAbs can be detected in more than 90% of patients with untreated Graves’ disease hyperthyroidism.^120–126 The observation of a small proportion of patients with undetectable TBIIs or TSAbs has been attributed to the occurrence of these autoantibodies at a serum level too low to be detected by current methods. Alternatively, restricted intrathyroidal production of TRAbs has been hypothesized.¹⁴⁵ A positive correlation between TSAb levels and serum triiodothyronine (T₃) levels, serum thyroglobulin levels, and goiter size has been observed.^107–110

TRAb levels usually fall during long-term treatment with antithyroid drugs.146,147 This phenomenon has been attributed to an immunosuppressive effect of antithyroid drugs,¹⁴⁸ but it could also result from the correction of thyrotoxicosis or even reflect the natural history of the disease.

Thyroid

The thyroid gland is usually symmetrically enlarged (Fig. 9-4), although nodular glands can be seen, especially in geographic areas of iodine deficiency, where nodular goiter often preexisted. Sometimes true nodules are difficult to distinguish from the lobulations typical of a hyperplasic gland. Goiter size is widely variable, and some patients can even have a gland of normal size. Large goiters can be associated with engorgement of the jugular veins and a positive Pemberton sign (swelling of the jugular veins upon elevation of the arms). By palpation, the consistency of the gland is generally firm, although softer than in Hashimoto’s thyroiditis. Thrills and bruits resulting from increased blood flow may be present on the gland, especially in the early phases of the disease and with a large goiter.

Skin and Appendages

The skin of a thyrotoxic patient is warm, thin, and moist; palmar erythema is common. Dermatographism and pruritus are often reported, but their significance is unclear.²¹⁰ Urticaria may also be associated. Vitiligo is frequent, being not a consequence of thyrotoxicosis but rather an associated independent skin autoimmune disease.²¹¹ The hair is friable, diffuse alopecia of mild degree is often observed, but alopecia areata is rare. Nails are soft and friable with longitudinal striations, and onycholysis (detachment of the nail from the ungual bed) is observed in long-lasting cases. Pretibial myxedema and thyroid acropachy, described elsewhere in this chapter, can also be seen.

Cardiovascular System

Heart-related symptoms are a frequent initial complaint in patients with Graves’ disease.212,213 The most common are tachycardia and palpitations. Signs and symptoms of heart failure may also develop, and edema of the lower extremities is often found in elderly patients. The heart and vascular system are major targets of thyroid hormones, which explains the high prevalence of cardiovascular symptoms in thyrotoxic patients with Graves’ disease. Cardiac complications may be a major concern in the care of these patients. Thyrotoxicosis causes an increase in both inotropism and chronotropism of the heart. Overall, vascular resistance is decreased because of peripheral vasodilatation. The net effect of these changes is increased cardiac output, which is the major pathophysiologic event. Increased cardiac workload causes increased oxygen consumption, which in turn can precipitate angina pectoris in the presence of preexisting coronary artery disease. Peripheral edema can be observed in the absence of overt heart failure. Dyspnea on exertion or at rest and chest pain may also be present and are prominent features of thyrotoxic heart disease.

On physical examination, the heart of a thyrotoxic patient is characterized by resting tachycardia. Heart sounds are accentuated. A systolic murmur may be heard on the precordium, sometimes related to associated mitral valve prolapse. Arrhythmias can range from sporadic premature beats to atrial fibrillation.

Electrocardiographic findings are nonspecific and include sinus tachycardia with ST elevation, QT shortening, and PR prolongation. Atrial fibrillation or flutter can occur in up to 10% to 15% of patients, especially if elderly. Ischemic changes can be found when underlying coronary artery disease is present. The observation of reversible heart failure in younger patients with thyrotoxicosis has raised the question whether a distinct thyrotoxic cardiomyopathy exists in the absence of preexisting detectable heart disease.212,213 Technically, the high-output heart failure typically observed in these cases may not be due to “failure” of the heart pump, but instead only to the changes in the peripheral circulation induced by vasodilatation and sodium-water retention. However, long-lasting tachyarrhythmias have been shown to impair the contractility of cardiac myocytes, and this mechanism has been proposed for the thyrotoxicosis-induced heart failure observed in young patients.²¹⁴ In most cases, however, cardiac complications occur in elderly patients in whom underlying heart disease is likely to exist. In this setting, heart failure occurs mainly in the presence of atrial fibrillation or ischemic heart disease.^212,213

Gastrointestinal Tract

Increased appetite associated with weight loss is a very common complaint and is due to increased catabolism. Increased gastrointestinal motility of the bowel leads to frequent bowel movements and, less often, to diarrhea.²¹⁵ These symptoms can be associated with some degree of malabsorption and steatorrhea, which can contribute to weight loss.²¹⁶ Atrophic gastritis and/or celiac disease of autoimmune origin may be associated with Graves’ disease.²¹¹ Major toxic effects of thyroid hormone on the liver have not been reported. Nonetheless, mild elevations of liver enzymes are often detected in thyrotoxic patients.²¹⁷ Thyrotoxicosis-induced liver function abnormalities can last for months and need to be taken into account when examining patients during treatment with antithyroid drugs, because they can be mistaken for adverse reactions to thionamides.

Nervous System

Psychic and nervous symptoms are a prominent and relevant part of the clinical picture.²¹⁸ Insomnia and irritability are the most frequent complaints. Patients appear restless and agitated, and logorrhea is often present and becomes clearly evident during history taking. Concentration ability is also decreased. This picture may sometimes confound the physician, and manic disorders have initially been diagnosed in many patients with Graves’ disease. Fatigability and asthenia are often present and are important in differentiation from true manic or bipolar disorders. In some cases, nervous signs take the form of “apathetic thyrotoxicosis” with severe apathy, lethargy, and pseudodementia, a profile more commonly observed in elderly patients.²¹⁹ In rare cases, true psychoses can be precipitated by thyrotoxicosis and improve with restoration of euthyroidism.²¹⁸

The peripheral nervous system is also deeply affected.220,221 Fine distal tremor is an almost universal finding and can also be observed on protrusion of the tongue or at the eyelids. Deep tendon reflexes are brisk, with a shortened relaxation time. Clonus can be sometimes elicited. The characteristic stare of a thyrotoxic patient is due to autonomic hyperstimulation of the elevator muscle of the lid and can also be found in the absence of Graves’ ophthalmopathy. True thyrotoxic neuropathy has occasionally been reported and is characterized by areflexic flaccid quadriparesis.^220,221

Muscles

Thyrotoxic patients frequently report muscle weakness and easy exhaustion. In more severe cases, atrophy of variable degree can occur in the setting of a more general wasting syndrome. Specific diseases of the muscle can be associated with Graves’ disease. Less than 1% of patients with Graves’ disease have classic myasthenia gravis, although ocular myasthenia gravis may be more frequent in these patients.222–225 Conversely, about 3% of patients with myasthenia gravis have Graves’ disease.²²³ The pathogenic significance of this association is not known, but it is interesting that the two prototypic diseases characterized by cell surface-receptor autoimmunity can occur together. Recognition of this association is clinically important because thyrotoxic myopathy can worsen the muscular symptoms of myasthenia. Moreover, it is important to correctly distinguish the ocular manifestations of the two disorders (they both cause diplopia), because the treatment is different. Therefore, when the degree of ocular muscle dysfunction in a patient with Graves’ disease is disproportionate to the degree of proptosis and inflammatory changes, clinical and serologic (anti–acetylcholine receptor antibodies) tests for myasthenia gravis are warranted.

In some patients, Graves’ disease thyrotoxicosis can precipitate crises of periodic hypokalemic paralysis.²²⁶ The syndrome is in all regards identical to familial periodic paralysis, but thyrotoxicosis of various causes is invariably present. The reason for the more frequent association with Graves’ disease may merely be that Graves’ disease is the most frequent cause of severe and long-lasting thyrotoxicosis in susceptible populations. Periodic hypokalemic paralysis is much more frequent in Asian subjects, in whom an association with certain HLA haplotypes has been observed,²²⁷ but it has also been reported in white and Native American patients.²²⁶ Mutations of ionic (potassium) channel genes have been shown to be responsible for some cases of hypokalemic paralysis in thyrotoxic patients.²²⁸ Effective treatments include potassium replacement, β-blocking agents, and rapid correction of thyrotoxicosis. Relapse of the hypokalemic crisis has occasionally been reported in patients after definitive treatment of the thyrotoxicosis, but it is rare.²²⁶

Skeletal System

Thyrotoxicosis is known to be associated with an increased rate of bone remodeling.229–231 The disproportionate increase in bone resorption over new bone formation leads to net bone loss, and consequently hyperthyroid patients have a reduced bone mass. Bone density improves after attainment of euthyroidism, but it often remains below the normal range. The degree of osteoporosis depends on the duration of hyperthyroidism and the coexistence of other risk factors for osteoporosis. Hence postmenopausal women with a history of hyperthyroidism have an increased risk of fractures, and hyperthyroid women are found more frequently among women with fractures.^229–231 The consequences of this effect of thyrotoxicosis on public health have been demonstrated in a large epidemiologic survey showing that fracture-related mortality is significantly increased among women with a history of hyperthyroidism.²³² Mild hypercalcemia and increased levels of bone turnover markers can be observed in thyrotoxic patients. Their levels are closely correlated with those of serum thyroid hormones and return to normal after correction of thyrotoxicosis.^229–231 It has been recently found that the effects of thyrotoxicosis on bone may reflect the loss of a protective action that TSH exerts via the TNF-α pathway, rather than (or in addition to) a direct action of thyroid hormones on bone.^233–236

Hematopoietic System

Mild leukopenia with relative lymphocytosis is a relatively common finding in patients with thyrotoxic Graves’ disease, which needs to be distinguished from antithyroid drug–induced leucopenia or agranulocytosis.²³⁷ Normocytic anemia is rare, but it can occur.²³⁸ Pernicious anemia occurs in a small minority of patients with Graves’ disease, but circulating autoantibodies to gastric parietal cells are found in a much higher percentage of cases and are a sign of associated gastric autoimmunity.^211,239 Aplastic anemia has also been reported.²⁴⁰ Graves’ disease is occasionally associated with autoimmune thrombocytopenic purpura, but nonimmunologic alterations in hemostasis have also been reported.²¹¹ Increases in factor VIII levels and fibrinogen have been reported most consistently, but the clinical relevance of these findings is unknown.²⁴¹

Reproductive System

Metabolic Changes

Significant weight loss with normal or increased caloric intake is a hallmark of thyrotoxicosis.²⁴⁷ It is explained by an increased metabolic rate, with increased heat production as a net result. Mitochondrial oxygen consumption is increased by thyroid hormones in almost every tissue. Increased mitochondrial activity and numbers were also shown in several tissues in experimental thyrotoxicosis. The use of oxygen by mitochondria is inefficient in experimental thyrotoxicosis in that fewer molecules of high-energy substrates are produced per molecule of oxygen used. A widespread increase in the use of ATP by cation transporters in tissues has been proposed to explain the increased energy use, although this point is controversial.^247,248 Whatever the mechanism, increased heat production and dispersion are manifested as a moderate rise in body temperature that is partially compensated for by increased sweating, heat intolerance, and weight loss.²⁴⁷

Peripheral utilization of carbohydrates is increased in thyrotoxicosis, in keeping with the enhanced energy consumption, and the primary mechanism seems to be an increased cellular transport of glucose.²⁴⁹ Thyrotoxicosis, however, also causes some degree of insulin resistance.²⁴⁹ Consequently, diabetes mellitus may be exacerbated by thyrotoxicosis. Type I diabetes mellitus can be associated with Graves’ disease within polyglandular autoimmune syndromes.²¹¹

Serum cholesterol and triglyceride levels are decreased in thyrotoxicosis, mainly because of a decrease in low-density lipoproteins (LDLs), in spite of an increase of hepatic lipogenesis.250,251 This reduction in lipids can result from the decrease in total body fat as a consequence of weight loss, but specific actions of thyroid hormones on lipid metabolism have also been described. Cholesterol conversion to bile acid in the liver is enhanced, and LDL receptor number on adipocytes is increased as well.²⁵⁰ These phenomena may account for the increased turnover of cholesterol and triglycerides. Thyroid function may alter adipokines, a number of biologically active substances produced by adipocyte with different physiologic functions, which include leptin, adiponectin, and resistin.^251,252 Thyrotoxicosis was reported to be associated with elevated serum levels of adiponectin, but the finding was not confirmed.^251–254 Serum leptin and resistin seem to be unaffected by thyrotoxicosis.^251–254

Protein metabolism is altered during thyrotoxicosis, with both increased protein synthesis and degradation. In most cases, however, degradation predominates and causes negative nitrogen balance. This imbalance can be partly controlled by adequate calories and protein intake.²⁴⁷

Graves’ Ophthalmopathy

Graves’ ophthalmopathy is the clinical manifestation of an inflammatory disorder of the orbit and is almost exclusively associated with Graves’ disease.255,256 The many aspects of this puzzling disorder are examined in detail elsewhere in this book. The prominence of the signs of Graves’ ophthalmopathy makes this feature the most striking physical finding in some patients (Fig. 9-5). Most of the manifestations of Graves’ ophthalmopathy are related to its central pathophysiologic event, an increase in the volume of retro-orbital tissue because of inflammation. As a consequence, the eye bulb is pushed forward and proptosis or exophthalmos results. Venous congestion causes swelling and edema of the periorbital tissue. Inflammatory changes also involve the extraocular muscles and may cause diplopia. The eyes are protruding, often asymmetrically. Lid retraction is seen and can be worsened by the concurrent thyrotoxicosis. Edema and swelling of the lids are also typical. The conjunctival mucosa is injected and edematous (chemosis). Lagophthalmos (incomplete palpebral closure) and lacrimal gland dysfunction may combine to cause drying of the mucosal and corneal surfaces and consequently irritation and (less often) corneal ulceration. Photophobia, burning sensation, retrobulbar pain, tearing, and a sandy sensation are common symptoms and may initially mislead clinicians into making a diagnosis of conjunctivitis. Optic neuropathy resulting from optic nerve compression by inflamed and swollen extraocular muscles at the orbital apex may occur in the most severe cases and cause reduction or loss of vision. When the proptosis is extremely severe, subluxation of the bulb outside the orbit may occur and pose an immediate threat to visual function.

Clinical signs or symptoms of ophthalmopathy are present in about 50% of Graves’ patients, with a wide variability of its degree of severity.255,256 When present, the onset of Graves’ ophthalmopathy coincides with the onset of thyrotoxicosis in about 40% of cases, follows it in another 40%, and precedes it in 20%.^255,256 Even when the onset of the two disorders does not coincide, each occurs within 18 months from the onset of the first manifestation. As reported later in the chapter, the presence of ophthalmopathy may affect the choice of treatment of hyperthyroidism in patients with Graves’ disease.²⁵⁷

Pretibial (or Localized) Myxedema and Thyroid Acropachy

When von Basedow first described his cases of Graves’ disease,⁵ he also reported a puzzling manifestation of the skin characterized by a nonpitting swelling of the pretibial areas, brownish and reddish in color, well delimited, and containing little free fluid. This manifestation of unknown pathogenesis is relatively rare in patients with Graves’ disease and is almost invariably observed only when Graves’ ophthalmopathy is also present.²⁵⁸ Although most frequently localized to the pretibial regions, it has also been observed on the forearms and other areas.²⁵⁸ Different degrees of severity have been described (Fig. 9-6). Diffuse pretibial myxedema refers to the mildest form, with only superficial diffuse edema. Localized areas of more prominent infiltration that assume a papular aspect characterize the nodular form. In the most severe forms, elephantiasis occurs with extensive swelling and sometimes ulceration.

Histopathologic studies have shown that the swelling is caused by the inordinate accumulation of hyaluronic acid in the subcutaneous layers of the involved areas, strikingly similar to the diffuse myxedematous changes of hypothyroidism but also to some extent to the changes in orbital tissues in Graves’ ophthalmopathy. A lymphocytic infiltrate may be observed, but it is by no means a constant finding. The origin of the mucinous material (hyaluronic acid) appears to be the skin fibroblast.²⁵⁸

The cause of pretibial myxedema is unknown, as is its relationship to the pathogenetic events of Graves’ disease.258,259 Most experts in the field agree that pretibial myxedema is another autoimmune manifestation of Graves’ disease, because of their association and of the almost invariable presence of serum TRAbs in patients with myxedema.^258,259 T-cell receptor V chain restriction has been observed in the lymphocytic infiltration of patients with early pretibial myxedema and suggests the presence of an ongoing antigen-specific immune response.²⁶⁰ As for Graves’ ophthalmopathy, TSHR is the obvious candidate antigen.²⁵⁸ Using patients’ serum as a tool, putative TSH-binding sites on pretibial fibroblasts were detected.²⁶¹ In addition, expression of TSHR mRNA and TSHR immunoreactivity in human fibroblasts from the pretibial region were found, as well as immunoreactivity for TSHR in pretibial connective tissue from patients with myxedema, but not from normal subjects.^262–264 In contrast, thyroglobulin, another candidate antigen which may contribute to the pathogenesis of Graves’ ophthalmopathy by reaching orbital tissues from the thyroid,²⁶⁵ was not found in pretibial myxedema (Marinò et al., unpublished). In summary, the pathogenesis of pretibial myxedema, similar to that of Graves’ ophthalmopathy, remains obscure, although most studies support the hypothesis of an autoimmune disorder because of the ectopic expression of thyroidal antigens or the presence of cross-reacting antigens localized to restricted regions of the human skin.

Thyroid acropachy is another rare manifestation of Graves’ disease that is observed most often in long-lasting and usually severe forms of Graves’ ophthalmopathy and pretibial myxedema; it is almost invariably associated with the presence of serum TRAbs.258,266 Thyroid acropachy is characterized by clubbing and soft tissue swelling of the last phalanx of the fingers and toes, all changes that are similar to those observed in chronic respiratory insufficiency. The overlying skin is often discolored and thickened. Microscopically, increased glycosaminoglycan deposition in the skin is observed. Subperiosteal new bone formation is also present. Again, the pathogenesis and the link with the immunologic changes of Graves’ disease are unknown, being the most “popular” hypothesis similar to those formulated for Graves’ ophthalmopathy and pretibial myxedema.^258,266 The disease develops without symptoms over a period of years, and it often goes unnoticed by the patient. This manifestation of Graves’ disease is harmless and asymptomatic and generally requires no treatment.

Hormone Measurements

Although laboratory assessment of thyroid function is treated in detail elsewhere in this book, it seems appropriate to review in this section the mainstays of the diagnosis of Graves’ hyperthyroidism. Suspicion of hyperthyroidism can be confirmed by thyroid hormone measurements. TSH is the single most useful test in confirming the presence of thyrotoxicosis.²⁶⁷ By sensitive assays, TSH should be undetectable or low in all patients with thyrotoxicosis of thyroidal origin. Low TSH levels can also be observed in a number of conditions such as nonthyroidal illnesses or endogenous or exogenous corticosteroid excess. Therefore, parallel measurement of thyroid hormone levels is recommended in all patients for a correct interpretation of a low TSH level. Total thyroxine (TT₄) and triiodothyronine (TT₃) measurements are relatively inexpensive and reliable, but they can yield high values in otherwise euthyroid subjects with conditions characterized by increased levels of serum thyroxine-binding globulin (TBG), most commonly pregnancy, oral contraceptive use, and chronic liver disease.²⁶⁷ Familial excess of TBG and familial dysalbuminemic hyperthyroxinemia are rare disorders that also cause elevated TT₄ levels.²⁶⁸ Free thyroid hormone measurements, although not completely devoid of flaws, are therefore more satisfactory but more expensive.²⁶⁷ In most iodine-sufficient countries, a single free thyroxine (FT₄) measurement is sufficient to confirm or reject the suspicion of thyrotoxicosis. After the TSH level, FT₄ is the test most often used in North America for thyroid function screening and therefore the one that most clinicians are familiar with. However, in iodine-deficient countries, a significant proportion of hyperthyroid patients (up to 12%) may have normal FT₄ levels with elevated free T₃ (FT₃) levels, a condition termed T₃ toxicosis.²⁶⁷ Conversely, FT₄ can be falsely elevated in conditions causing reduced peripheral conversion of T₄ to T₃, such as amiodarone administration or high-dose propranolol treatment. In our practice, we initially measure both FT₄ and FT₃ levels together with TSH, with little additional expense, to obtain a complete baseline assessment of thyroid function status. Circulating thyroglobulin concentrations are high in hyperthyroidism and low in factitious thyrotoxicosis. Measurement of thyroglobulin in serum is therefore useful in the differential diagnosis of thyrotoxicosis in patients with no goiter or ophthalmopathy.²⁶⁹

Circulating Autoantibodies

Although by no means necessary, tests for circulating thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) antibodies may be useful in confirming the presence of thyroid autoimmunity. TPOAbs can be detected by commercial radioimmunoassays in up to 90% of patients with untreated Graves’ disease,270,271 whereas TgAbs are less frequently positive, in about 50% to 60% of cases, depending on the sensitivity of the method used.^272,273 Both antibodies can be detected in a relatively high percentage (up to 25%) of normal subjects, especially elderly women or in patients with other nonautoimmune thyroid disorders such as nodular goiter or thyroid carcinoma.^273,274 Therefore, tests for TPOAb and TgAb have limited diagnostic value and must be considered complementary to the diagnosis.

TRAb assay is very specific and sensitive for hyperthyroid Graves’ disease. Up to 98% of untreated patients are positive with second-generation radioreceptor assays, with very few false-positive results.¹²⁶ The radioreceptor test for TRAb is widely available in commercial laboratories but quite expensive. It is needed in selected situations in which confirmation of the nature of thyrotoxicosis is required or when the clinical picture or thyroid function tests are not clear. These situations include the differential diagnosis of thyrotoxicosis in pregnancy, the nodular variants of Graves’ disease, which must be differentiated from toxic nodular goiter,²⁷⁵ and patients with exophthalmos without thyrotoxicosis (euthyroid Graves’ disease).²²⁴

Thyroid Radioiodine Uptake and Scanning

Before the introduction of accurate thyroid hormone and TSH measurements, a radioactive iodine uptake (RAIU) test was always required for the evaluation of thyrotoxicosis. Normal ranges for RAIU vary according to the status of iodine supply in the population.276,277 In iodine-replete countries, the upper limit of normal RAIU 24 hours after the administration of a tracer dose of radioiodine is around 25%, whereas it may reach 40% in areas with mild to moderate iodine deficiency. In patients with Graves’ disease, a high value is always found after 24 hours, and in some cases the value after 3 or 6 hours can be even higher as a consequence of the rapid iodine turnover typical of Graves’ disease glands. The RAIU test is not readily available as an office-based approach and is not always required. It can be very useful, however, to rule out silent or subacute thyroiditis, factitious thyrotoxicosis, and type II amiodarone-induced thyrotoxicosis²⁷⁸ (Table 9-4). RAIU results can also be used before radioiodine treatment of hyperthyroidism to calculate the dose needed (see relevant heading). RAIU, like any other radioisotopic in vivo procedure, is absolutely contraindicated during pregnancy.

Thyroid imaging with radioisotopes can be performed with radioiodine at the time that RAIU is performed or by using pertechnetate 99m (Fig. 9-7A). Thyroid scanning in Graves’ disease is useful when coexisting nodules are detected by palpation and their functional status needs to be evaluated.

Thyroid Ultrasound

The thyroid gland is an excellent candidate for ultrasound studies, given its superficial anatomic location and the presence of a high number of liquid-solid interfaces (virtually one at every follicle/colloid-lining surface), which in normal conditions produce high reflectivity. Although the equipment is still relatively quite costly, it is now affordable at the office level and requires little maintenance. Thyroid ultrasound is an invaluable tool in the diagnosis of thyroid nodular disorders and has become more and more an extension of the physical examination for many endocrinologists. Its application to non-nodular disorders of the thyroid has come from early observations of typical changes of the gland’s echoic structure in thyroid autoimmune diseases.²⁷⁹ In hyperthyroid Graves’ disease, the echoic pattern undergoes diffuse changes. The tissue, possibly because of the reduction in colloid content, the increase in thyroid vascularity, and the lymphocytic infiltrate, becomes typically hypoechoic (Fig. 9-7B). This pattern is similar to the one observed in chronic thyroiditis and, when diffuse, is almost pathognomonic of thyroid autoimmunity.²⁸⁰ Therefore, thyroid ultrasound can be useful during the evaluation of thyrotoxicosis to confirm the suspicion of thyroid autoimmunity. Thyroid ultrasound also allows accurate measurement of thyroid size,²⁸¹ information that can help in the decision-making process when definitive treatment is being planned (see later). Moreover, careful definition of coexisting nodules can be obtained when needed. Therefore, thyroid ultrasonography provides very useful information in the initial evaluation of patients with Graves’ disease, although it is seldom strictly required for purely diagnostic purposes.

Color flow Doppler (CFD) techniques have been applied to the study of glands with Graves’ disease. CFD allows semiquantitative measurement of blood flow to the thyroid gland.²⁸² In untreated Graves’ disease, a distinct CFD pattern characterized by markedly increased signals with a patchy distribution is usually observed (Fig. 9-7C). This pattern, in conjunction with a hypoechoic pattern, allows distinction from Hashimoto’s thyroiditis. In this setting, CFD studies of the thyroid gland can be useful in distinguishing hyperthyroidism of Graves’ disease from thyrotoxicosis of other causes, such as amiodarone-induced thyrotoxicosis, subacute thyroiditis, painless thyroiditis and factitious thyrotoxicosis. For this reason, CFD evaluation may become a valid substitute for RAIU when these conditions are included in the differential diagnosis.