Review: Molecular Thyroidology

Transcription

1 Annals of Clinical & Laboratory Science, vol. 31, no. 3, Review: Molecular Thyroidology William E. Winter and Maria Rita Signorino Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida Abstract. Novel disorders involving aberrations of the hypothalamic-pituitary-thyroid gland-thyroid hormone axis have been described in the last 5 to 10 years. The following topics are addressed: molecular mutations causing central hypothyroidism (isolated autosomal recessive TRH deficiency; autosomal recessive TRH-receptor inactivating mutations; TSH beta-subunit bio-inactivating mutations; Pit-1 mutations; Prop1 mutations; high molecular weight bio-inactive TSH); defects in response to TSH (mutations in the TSH receptor: TSH receptor gain-of-function mutations; TSH receptor loss-of-function mutations); defects in thyroid gland formation: transcription factor mutations (TTF-2 and Pax8); defects in peripheral thyroid hormone metabolism (defective intrapituitary conversion of T4 to T3; hemangioma consumption of thyroid hormone); and defects in tissue response to thyroid hormone (generalized thyroid hormone resistance, selective pituitary thyroid hormone resistance). While molecular diagnosis of such conditions is rarely indicated for clinical management, knowledge of the molecular mechanisms of these diseases can greatly enhance the clinical laboratory scientist s ability to advise clinicians about appropriate thyroid testing and to interpret the complex and sometimes confusing results of thyroid function tests. (received 17 March 2001; accepted 20 March 2001) Key words: TRH, TRH receptor, TSH, TSH receptor, thyroid hormone receptor Introduction The goal of this review is to introduce the clinical laboratorian to several recent advances in molecular thyroidology. Many novel disorders involving aberrations of the hypothalamic-pituitary-thyroid gland-thyroid hormone axis have been described in the last 5 to 10 years. While molecular diagnosis of such conditions is rarely indicated for clinical management, knowledge of the molecular mechanisms of disease can greatly enhance the laboratorian s ability to advise clinicians about appropriate thyroid testing, and to interpret the complex and sometimes confusing results of thyroid function tests. This review begins with a brief overview of the normal hypothalamicpituitary-thyroid gland-thyroid hormone axis. Address correspondence to William E. Winter, M.D., Department of Pathology, Immunology and Laboratory Medicine, University of Florida Medical School, Box , Gainesville, FL , USA; tel ; fax ; winter.pathology@mail.health.ufl.edu. Normal Thyroid Function The hypothalamus and anterior pituitary gland thyrotrophs monitor free thyroid hormone levels in the blood stream (Fig. 1). Unbound or free triiodothyronine (FT3) is present in the plasma and enters the parvicellular division of the paraventricular nucleus. Within these paired hypothalamic nuclei that are adjacent to the superior aspect of the third ventricle, intracellular T3 is also derived from monodeiodination of free tetraiodothyronine (free thyroxine, FT4) that has entered the cell cytoplasm from the plasma. If the intracellular level of T3 declines, thyrotropin-releasing hormone (TRH) is released into the hypothalamicpituitary-portal system to be delivered to the anterior pituitary gland. TRH is the tripeptide pyroglu-his- Pro-NH 2. The cyclized glutamic acid terminus and an intact amide are required for TRH bioactivity. TRH sensitizes the anterior pituitary thyrotrophs to release more thyroid stimulating hormone (TSH) if intracellular thyrotroph T3 levels are deficient /01/0300/0221 $6.00; 2001 by the Association of Clinical Scientists, Inc.

2 222 Annals of Clinical & Laboratory Science Fig. 1. The hypothalamus secretes thyrotropin releasing hormone (TRH) into the hypothalamic-pituitary portal system. In turn, TRH regulates the responsiveness of thyrotropin (TSH) to thyroid hormone feedback. TSH circulates systemically and stimulates the thyroid gland to release thyroxine (tetraiodothyronine, T4) and 3,5,3'-triiodothyronine (T3). About 80% of circulating T3 is derived from peripheral monodeiodination of T4 to T3. In target tissues, T4 is also converted to T3 and the T3 interacts with nuclear thyroid hormone receptors. Thyrotrophs, the anterior pituitary cells that release TSH in response to TRH and decreased T3, express TRH receptors. When TRH binds to the TRH receptor, the thyrotroph depolarizes, allowing calcium to influx into the thyrotroph cytoplasm. In turn, increased free cytosolic calcium activates the Ca 2+ - phosphatidylinositol cascade. This causes TSH release and synthesis and glycosylation of alpha and beta TSH subunits. Stimulation of glycosylation of TSH subunits is relatively a greater effect of TRH than stimulation of TSH synthesis. Glycosylation is necessary for bioactivity of TSH. TRH also depresses T3 receptor expression. This makes the thyrotroph less sensitive to thyroid hormone negative feedback, further increasing TSH release. The major site of central negative feedback is the pituitary. However, injected TRH normally releases TSH and prolactin. The lactotrophs express the TRH receptor. For all other hormones regulated negatively by the hypothalamus and pituitary, the major site of negative feedback is the hypothalamus. TSH circulates systemically. Upon binding of TSH to the TSH receptor located on thyroid follicular cell, many processes are activated to increase the release of thyroid hormone into the circulation. T4 is derived only from the thyroid gland. On the other hand, only about 20% of T3 is directly generated from the thyroid gland, with about 80% of T3 being derived from peripheral monodeiodination of T4 to T3. The majority of thyroid hormone is bound to plasma proteins, including the alpha-1 globulin thyroxinebinding globulin (TBG), thyroxine-binding prealbumin (now called transthyretin), and albumin. Only 0.03% of T4 and 0.3% of T3 are unbound. The unbound or free fractions of thyroid hormone are the biologically active forms of thyroid hormone in the circulation. With a rise in plasma FT3 and intracellular T3 in the pituitary (and to a lesser degree in the hypothalamus), TSH and TRH secretion are suppressed, completing the negative feedback loop for control of thyroid hormone synthesis and secretion. Thyroid hormone is trophic for many tissues. Thyroid hormone is important for the growth, differentiation, and maintenance of the central nervous system (very important), skeleton (very important), cardiovascular system, and gastrointestinal system. Basal metabolic rate (BMR) is directly regulated by thyroid hormone. Thyroid hormone also affects and regulates intermediary metabolism, drug metabolism, and the activity of other hormones (eg, growth hormone secretion is impaired in individuals with hypothyroidism). Overview of Molecular Thyroidology New explanations for old diseases would be an appropriate title for much of this review. The following topics will be addressed: Molecular mutations causing central hypothyroidism: Isolated autosomal recessive TRH deficiency, Autosomal recessive TRH-receptor inactivating mutations, Pit-1 mutations, PROP-1 mutations; TSH beta-subunit bio-inactivating mutations: High molecular weight bio-inactive TSH; Defects in response to TSH: mutations in the TSH receptor: TSH receptor gain-of-function mutations, TSH receptor loss-of-function mutations;

3 Review of molecular thyroidology 223 Defects in thyroid gland formation: transcription factor mutations: TTF-2, Pax8 [1]; Defects in peripheral thyroid hormone metabolism: Defective intrapituitary conversion of T4 to T3 [2], Hemangioma consumption of thyroid hormone; Defects in response of tissues to thyroid hormone: Generalized thyroid hormone resistance [3], Selective pituitary thyroid hormone resistance. While many defects in thyroid hormone biosynthesis have been described, their etiology is generally understood and they are not reviewed in detail in this paper. However,the clinical laboratory scientist should be familiar with cases of goitrous congenital hypothyroidism and non-autoimmune goitrous hypothyroidism that result from: defects in iodine transport into the thyroid gland (autosomal recessive mutations in the sodium/iodide symporter located on chromosome 19p ) [4], organification defects (autosomal recessive mutations of thyroperoxidase located on chromosome 2p25 and associated with the Pendred syndrome mutation located at chromosome 7q21-34), thyroglobulin synthesis defects (autosomal recessive disorder mapping to chromosome 8q24), and the iodine recycling disorder involving dehalogenase. Recently the gene responsible for Pendred syndrome (hypothyroidism due to defective iodine organification of thyroglobulin associated with congenital or early-onset sensorineural deafness) was cloned and named the PDS (Pendred syndrome) gene [5]. The PDS gene product is a transmembrane protein (pendrin) which transports iodide and chloride [6]. Defects in thyroid hormone transport are also well described in the literature. These defects can cause confusion in the interpretation of elevated total T4 measurements when T-uptake or T3 resin uptake is not also measured. As measurements of free T4 (FT4) replace total T4 measurements, the diagnostic problems posed by thyroxine binding globulin (TBG) excess, familial dysalbuminemic hyperthyroxinemia [7], and familial euthyroid thyroxine excess [8] should wane. Located on chromosome Xq11-23, TBG is a 395 amino acid 54-kDa acidic glycoprotein with a single iodothyronine binding site. TBG has 4 heterosaccharide side chains with 5-9 sialic acids. As the degree of sialylation increases (an effect of estrogen), the half-life of TBG increases, raising TBG levels. In congenital TBG excess, an X-linked dominant condition, male hemizygotes display 3- to 5-fold elevations in TBG levels, while female heterozygotes display 2- to 3-fold increases in TBG. Besides estrogen effects and congenital excess, other causes of elevated TBG levels include acute liver disease and drugs (eg, phenothiazines). In familial dysalbuminemic hyperthyroxinemia, a mutant dominantly-inherited form of albumin (Arg218His) binds increased amounts of T4 but not T3, producing euthyroid hyperthyroxinemia, with normal T3 levels as well as normal FT4 levels. In contrast, TBG excess raises both T4 and T3. Another cause of euthyroid hyperthyroxinemia is familial euthyroid thyroxine excess that results from a mutant form of transthyretin (TTR, Thr119Met). An older name for TTR is thyroxine-binding prealbumin (TBPA). Encoded by the TTR gene on chromosome 18q11.2, TTR exists as a stable 55 kda tetramer of 127 amino acid monomers. TTR participates in vitamin A transport by binding to the complex of vitamin A and retinol-binding protein. As a side note, more than 40 TTR mutations have been reported that can cause familial amyloidosis affecting the heart (cardiomyopathy) or nervous system (autonomic neuropathy or polyneuropathy). It is of interest that other causes of familial amyloidoses include mutations in apolipoprotein A-I, gelsolin, fibrinogen, and lysozyme. Gelsolin is a cytoplasmic and plasma calcium-binding protein that binds to and fragments actin filaments. Many reviews of euthyroid hyperthyroxinemia with normal FT4 levels have been published [9] and these disorders are not further discussed in this paper. Molecular Mutations with Central Hypothyroidism Central hypothyroidism is diagnosed when clinical hypothyroidism is accompanied by low FT4 (and FT3) and inappropriately normal or low TSH concentration. Most cases of hypothyroidism are primary in etiology and show an elevated TSH concentration in the blood. Regardless of etiology (eg, either primary or central), the clinical features of hypothyroidism include symptoms of tiredness, constipation, cold intolerance,

4 224 Annals of Clinical & Laboratory Science dry hair or skin, weight gain, menstrual irregularities, breast milk production, and slow mentation. Signs of hypothyroidism can include low heart rate (bradycardia), decreased strength of cardiac contraction causing decline in the usual difference between systolic and diastolic blood pressures (low pulse pressure), myxedema (nonpitting edema), hypercholesterolemia, elevated creatine kinase, growth failure (including congenital hypothyroidism), short stature, retarded bone age, stippled growth plates, decreased reflexes, congestive heart failure, and coma. Newborns with congenital hypothyroidism may display an enlarged posterior fontanelle, large tongue, prolonged jaundice (delayed expression of UDP-glucuronyl transferase), low body temperature, delayed passage of meconium, large body size at birth because of postmaturity (delayed delivery), or excessive body hair. In cases of central hypothyroidism where low FT4 is accompanied by an inappropriately low TSH level, the clinician s initial obligation is to exclude by radiology a tumor mass lesion or other anatomic cause of central hypothyroidism that might require surgery or irradiation. Other types of central endocrine deficiencies should also be pursued and treated preoperatively, such as ACTH deficiency causing glucocorticoid deficiency and ADH deficiency causing diabetes insipidus. Failure to detect and treat glucocorticoid insufficiency preoperatively could lead to fatal intraoperative adrenal crisis. Likewise, failure to recognize Table 1. Clinical and laboratory features of molecular mutations causing central hypothyroidism: Isolated autosomal recessive TRH deficiency (TRH gene (?); chromosome 3). Central (TSH-deficient) hypothyroidism TSH responds to exogenous TRH administration (this demonstrates that the TRH receptor and signaling to TSH secretion are intact) Prolactin responds to exogenous TRH administration (provides further evidence that the pituitary is functional; rules against Pit-1 and Prop1 mutations) Absence of other anterior pituitary hormone defects (rules against Pit-1 and Prop1 mutations) Absence of anatomic hypothalamic lesions Absence of hypothalamic-pituitary portal system lesions How to diagnose this disorder: Diagnosis by exclusion Differential diagnosis: Hypothalamic disease diabetes insipidus could cause serious hypovolemia when the patient s oral intake of food and fluids is restricted preoperatively, or when the patient s oral intake is restricted postoperatively and sufficient intravenous fluids are not administered to replace excessive urinary fluid loss. If a mass lesion is discovered in the hypothalamus or pituitary that requires surgery or irradiation, all aspects of anterior and posterior pituitary function should also be examined at the conclusion of the tumor therapy. Isolated autosomal recessive TRH deficiency and TRH receptor mutation. When anatomic hypothalamic and pituitary pathology have been excluded, the clinician can perform a thyrotropin-releasing hormone (TRH) test to localize the cause of the central hypothyroidism. If TRH is deficient, administration of exogenous TRH will raise the TSH concentrations during the TRHstimulation test. This substantiates the diagnosis of tertiary (hypothalamic) hypothyroidism. However, if TRH is unable to elicit a TSH response, the pituitary is at fault. This substantiates the diagnosis of secondary (pituitary) hypothyroidism. Assuming that all other anterior and posterior pituitary axes are intact, which in fact is rare, the clinician and laboratorian should consider in their differential diagnosis: (1) isolated autosomal recessive TRH deficiency (chromosome 3) [10] (Table 1), (2) autosomal recessive TRH-receptor inactivating mutations (chromosome 8q23) (Table 2), and (3) Table 2. Clinical and laboratory features of molecular mutations causing central hypothyroidism: TRH receptor (TRHR) mutation (autosomal recessive; TRHR gene; chromosome 8q23). Central (TSH-deficient) hypothyroidism Lack of TSH response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) Lack of prolactin response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) Absence of other anterior pituitary hormone defects (rules against Pit-1 and Prop1 mutations) Absence of anatomic hypothalamic lesions Absence of hypothalamic-pituitary portal system lesions Clinical criteria noted above, and Sequence TRHR gene and detect mutations Differential diagnosis: Pituitary disease

5 Review of molecular thyroidology 225 familial TSH deficiency (Table 3, discussed below). Collu et al [11] have reported a child with central hypothyroidism resulting from compound heterozygous mutations in the TRH receptor gene. Etiologies of central hypothyroidism are illustrated in Fig. 2. When central anatomic lesions are absent and multiple anterior pituitary hormone deficiencies are otherwise unexplained, the clinician and laboratory scientist should consider Pit-1 mutations and Prop1 mutations [12]. TSH beta mutations: familial autosomal recessive TSH deficiency. Autosomal recessive TSH deficiency results from homozygosity or compound heterozygosity for TSH beta subunit mutations [13,14] (Table 3). Only TSH is deficient as other anterior pituitary hormones are intact. TSH (molecular weight 28 kd) is similar to Table 3. Clinical and laboratory features of molecular mutations causing central hypothyroidism: TSH beta (TSHβ) mutations: Familial autosomal recessive TSH deficiency (autosomal recessive; TSHβ gene; chromosome 1p22). Central (TSH-deficient) hypothyroidism Absent TSH response to exogenous TRH administration (demonstrates that thyrotroph is dysfunctional; alpha subunit rises after TRH indicating that the TRHR is functional) Prolactin responds to exogenous TRH administration (demonstrates that TRHR is functional) Absence of other anterior pituitary hormone defects (rules against Pit-1 and Prop1 mutations) Absence of anatomic hypothalamic lesions Absence of hypothalamic-pituitary portal system lesions Sequence TSHβ gene and detect mutations Pituitary disease Fig. 2. The molecular mutations that cause central hypothyroidism are illustrated. See text for details. TRH = thyrotropin releasing hormone; TRHR = thyrotropin releasing hormone receptor; TSH = thyrotropin.

6 226 Annals of Clinical & Laboratory Science LH, FSH, and hcg: all are glycoprotein hormones that share a common alpha subunit (Mr 14,700, two oligosaccharide moieties; chromosome 6q21.1-q23) while each glycoprotein hormone has a unique beta subunit that is responsible for the specific bioactivity of the hormone. The TSH beta chain (Mr = 15,600, one oligosaccharide moiety) gene is located on chromosome 1p. Mutations in both TSH beta chain genes lead to TSH deficiency. TSH deficiency causes congenital hypothyroidism with low to undetectable TSH values. Central hypothyroidism will be detected in neonatal hypothyroid screening programs that test for depressions in total T4. In programs that depend on elevated TSH levels to diagnose hypothyroidism, TSH deficiency will be missed. Metabolic findings in cases of TSH deficiency include low basal radioactive iodine uptake (RAIU) that increases after administration of bovine TSH. This proves that the thyroid gland itself is normal. After administration of exogenous TRH, intact TSH and the TSH beta subunit remain undetectable, while the TSH alpha subunit is increased in concentration. With exogenous T3 replacement, the alpha subunit concentration declines, demonstrating that feedback exists centrally. Heterozygotes with one normal and one abnormal TSH beta allele are clinically normal. Recurrence risk in siblings is 25%. The TSH beta gene has 3 exons. The following mutations have been described: Base Nucleotide Mutation change position G > A 29 Missense G > T 94 Transversion Base deletion 105 Frameshift Pit-1 and Prop1 mutations: Familial polyhormone hypopituitarism syndromes (Combined pituitary hormone deficiency, CPHD). Transcription factors are proteins that regulate gene expression. Pit-1 and Prop1 are transcription factors that regulate the activity of several key genes encoding anterior pituitary hormones. Encoded by the PIT-1 gene on chromosome 3p11, Pit-1 is a pituitary-specific transcription factor that binds to the DNA regulatory regions of the thyrotroph TSH beta gene, the somatotroph growth hormone gene, and the lactotroph prolactin gene. Pit-1 mutations most commonly produce growth hormone deficiency but also commonly produce central hypothyroidism and prolactin deficiency resulting in a combined pituitary hormone deficiency (CPHD) (Table 4). There is no apparent adverse consequence to being prolactin deficient. However, diagnostically, prolactin deficiency should be sought by measuring prolactin as part of the subject s TRH stimulation test if the subject is evaluated for central hypothyroidism. Gonatrophs that secrete LH and FSH and corticotrophs that secrete ACTH are uninvolved in cases of Pit-1 deficiency. Recessive and dominant modes of inheritance of Pit-related familial panhypopituitarism have been described. Recessive Pit-1 mutations include complete deletion of the PIT-1 gene, F135C (phenylalanine > cysteine), R143N (arginine > glutamine), A158P (alanine > proline), R172X (arginine > stop), and E250X (glutamate > stop). For example, the A158P mutation disturbs the formation of Pit-1 homodimers and greatly decreases transcription activation. The R271W (arginine > tryptophan) mutation and the P24L (proline > leucine) mutation produce dominant forms of Pit-1 deficient hypopituitarism. The dominant negative effect of these latter two mutations is not clearly understood. Table 4. Clinical and laboratory features of molecular mutations causing central hypothyroidism: Pit-1 mutations: Familial polyhormone hypopituitarism syndromes (autosomal recessive and dominant forms; PIT-1 gene; chromosome 3p11). Central (TSH-deficient) hypothyroidism Absent TSH response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) Absent prolactin response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) Growth hormone and prolactin deficiency (other anterior pituitary hormone defects present) Normal LH and FSH response to GnRH (rules against Prop1 mutations); normal ACTH and cortisol Absence of anatomic hypothalamic lesions Absence of hypothalamic-pituitary portal system lesions Family history may be positive for similarly affected individuals How to diagnose this order definitively: Sequence PIT-1 gene and detect mutations Hypothalamic or pituitary disease

7 Review of molecular thyroidology 227 Expressed at an early stage in pituitary gland development, the prophet of Pit-1 gene (Prop1) encodes a paired-like homeodomain protein within its 3 exons. Prop1 may regulate Pit-1 [15]. Mutations in Prop1 cause gonadotropin (LH and FSH) deficiency in addition to deficiencies of TSH beta, growth hormone and prolactin [16] (Table 5). At least one Prop1 family has additionally been described with ACTH deficiency [17]. This family had a delAG Prop1 frameshift mutation. This site is a hot-spot for mutation in the PROP1 gene. Another Prop1 mutation is R120C [18]. Magnetic resonance (MR) imaging in patients with Prop1 mutations can reveal congenital hypoplasia of the anterior pituitary gland [19]. Thyrotropin with impaired biologic activity. In 1981, the case of a euthyroid adult with an elevated TSH level was reported where the TSH displayed impaired biologic activity [20] (Table 6). The TSH level was increased approximately 25-fold over the upper limit of the reference range. Chromatographic analysis demonstrated that the TSH in this individual was of much higher molecular weight than normal. Furthermore, while this large form of TSH bound to the TSHR receptor normally, there was decreased signal transduction through the TSHR. Unfortunately, there were no molecular analyses of the TSH beta or TSH alpha genes. Even if both genes were normal, theoretically there could be aberrant Golgi processing leading to polymerization of the TSH molecules and an increased molecular mass. Another possibility to consider would be a macro-tsh (eg, TSH bound by a plasma immunoglobulin). This case illustrates that, while most individuals with normal T4 and T3 levels and elevated TSH levels have subclinical hypothyroidism, rare individuals may lack a TSH molecule of normal biopotency. One condition not excluded in the 1981 report was human anti-mouse monoclonal antibodies (HAMA) that could produce a false elevation in measured TSH levels. The large in vivo size of the patient s TSH argues against the possibility of HAMA. It may seem odd to characterize this disorder as a form of central hypothyroidism because the TSH level is elevated. However, because the thyroid gland can respond normally to exogenous TSH in this condition, thyrotropin dysfunction appears to be a consequence of a pituitary manufacturing problem and thus classification of thyrotropin with impaired biologic activity as a type of central hypothyroidism is appropriate. Table 5. Clinical and laboratory features of molecular mutations causing hypothyroidism: Prop1 mutations: Familial polyhormone hypopituitarism syndromes (autosomal recessive, PROP1 gene; chromosome 5p). Central (TSH-deficient) hypothyroidism Absent TSH response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) Absent prolactin response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) Growth hormone, prolactin, LH and FSH deficiency and possible ACTH deficiency (LH/FSH deficiency rule in favor of Prop1 deficiency and against Pit-1 deficiency) Possible hypoplasia of the anterior pituitary gland on MRI Absence of hypothalamic-pituitary portal system lesions Family history may be positive for similarly affected individuals Sequence PROP1 gene and detect mutations Hypothalamic or pituitary disease Table 6. Clinical and laboratory features of molecular mutations causing central hypothyroidism: Thyrotropin with impaired biologic activity (inheritance unknown). Hyperthyrotropinemic euthyroidism (elevated TSH, normal FT4, T4, FT3, T3) TSH rises in response to exogenous TRH (pituitary is functional) Prolactin rises in response to exogenous TRH (pituitary is functional) Reduction in TSH after exogenous T3 administration (sufficient level of T3 administration can suppress TSH) Increased T4 and thyroid radioactive iodine uptake after administration of bovine TSH (TSHR is functional) Chromatographic analysis of TSH demonstrates high molecular weight TSH Subclinical primary hypothyroidism Mild TSHR loss-of-function mutation

8 228 Annals of Clinical & Laboratory Science Defects in Thyroid Follicular Cell Response to TSH Mutations in the TSH receptor. TSH action on the thyroid follicular cell is mediated through a TSH receptor (TSHR)-G protein-adenyl cyclase-coupled production of intracellular cyclic adenosine monophosphate (camp). The TSHR is a member of the superfamily of G-protein-coupled receptors. Other members of this receptor superfamily include the ACTH receptor, alpha-adrenergic and beta-adrenergic catecholamine receptors, LH receptor, FSH receptor, hcg receptor, glucagon receptor, PTH receptor, and somatostatin receptor. A rise in intracellular camp in the follicular thyrocyte leads to increased iodide uptake, increased thyroperoxidase and thyroglobulin synthesis, hormone secretion, expression of type 1 deiodinase, and growth of the thyroid follicular cell. At high TSH concentrations, TSH activates the Ca 2+ -phosphatidylinositol-phosphate protein kinase C cascade, which stimulates H 2 O 2 generation and I - efflux. The 10-exon TSH receptor gene, located on chromosome 14q31, covers 60 kb. The predominant mrna is 4.3 kb,with smaller transcripts also observed. After glycosylation, the TSHR weighs ~100 kda. Prior to glycosylation, the apoprotein core weighs 84.5 kda. The N-terminal extracellular domain of 398 amino acids is encoded by the first 9 exons. There are 6 N- glycosylation sites in the extracellular domain. TSH binds to this region of the TSHR. The 346 amino acid carboxyl half of the receptor, which is encoded by a single large exon 10, contains the 7 hydrophobic transmembrane segments that are connected by 3 extraand 3 intracellular loops and the cytoplasmic portion of the TSHR. This portion of the TSHR demonstrates homology with other G protein-coupled receptors and activates the G s complex upon TSH binding to the extracellular domain of the TSHR. Upon TSH binding to the extracellular domain of the TSHR, a conformational change is believed to take place in the TSH receptor. This would allow interactions between the TSHR and the G s (G stimulatory) complex. Alternatively, the TSHR may exist in 2 forms: an on form which interacts with the G s complex and an off form that does not interact with the G s complex. In the absence of TSH, the TSHR predominantly exists in the off form and no signal transduction occurs. However with TSH binding, the equilibrium shifts to the on form and signaling continues. In the basal state, the 3 subunits of the G s complex, alpha, beta, and gamma, are associated and alpha subunit non-covalently binds guanosine diphosphate (GDP). The G protein family has more than 50 members. These proteins bind either GDP or guanosine triphosphate (GTP). The larger G proteins of 80 to 90 kda function in hormone pathways. The G s alpha subunit is located on chromosome 20q13.2. When the TSHR interacts with the basal G s complex, the alpha subunit dissociates from the beta and gamma subunits. The alpha subunit sheds GDP, which is replaced by guanosine triphosphate (GTP). This activated G s alpha subunit with GTP attached activates adenyl cyclase. Adenyl cyclase converts ATP to camp. As a mechanism of internal negative feedback regulation, the activated G s alpha subunit does acquire GTPase activity. This converts the attached GTP to GDP (plus phosphate). The G s alpha subunit with GDP then recombines with beta and gamma and G s returns to its basal inactive state (Fig. 3). Mutations in the TSHR can produce decreased or increased spontaneous activity [21,22]. The loss-offunction mutations localize to the extracellular TSHbinding domain of the TSHR (Fig. 4). Two well described loss-of-function mutations are the P162A and I167N mutations. Except for the extracellular TSH-binding domain TSHR 281 mutations (S281N and S281T), the gain-of-function mutations localize to the multiple transmembrane portions and connecting loops of the TSHR. TSHR gain-of-function mutations; (a) Thyroid neoplasms. Returning to the TSHR model of the TSH-bound- on configuration versus the TSHunbound- off configuration, gain-of-function mutations shift the equilibrium to the on configuration in the absence of the TSH ligand. Thus even without the TSH ligand, the TSHR is transducing signals leading to autonomous thyroid follicular cell hyperfunction. If the TSHR mutation occurs as a somatic mutation (eg, in a thyroid adenoma), a thyrotoxic nodule results (Table 7). About 80% of toxic thyroid adenomas (hot nodules) exhibit TSHR gainof-function mutations [23]. Multinodular goiter can also result from gain-of-function mutations [24].

9 Review of molecular thyroidology 229 Fig. 3. Thyrotropin (TSH) binds to a specific TSH receptor on thyroid follicular cells. The G s protein dissociates into the beta/gamma subunits and an alpha subunit. The alpha subunit loses GDP and gains GTP and becomes active. Through the interaction of the active G s with adenyl cyclase, adenyl cyclase acquires enzymatic activity and converts ATP to camp. Active G s also expresses an intrinsic GTPase activity. GTPase cleaves GTP to GDP plus Pi. The G s subunit with GDP is inactive and binds to the beta/gamma subunits to return to its basal state Table 7. Defects in thyroid follicular cell response to TSH: TSHR gain-of-function mutations: Thyroid neoplasias (somatic; TSHR gene; chromosome 14q31). Thyroid adenoma or multinodular goiter with or without a hot nodule or nodules (hyperthyroidism may or may not be present) Sequence TSHR transmembrane domain to detect TSHR gain-of-function mutation (if no mutations detected, study extracellular TSH-binding domain) Thyroid adenoma or multinodular goiter without TSHR mutation or with G s gain-of-function mutation Somatic gain-of-function mutations have been described at TSHR amino acid positions 281, 453, 486, 568, 619, 623, 629, 631, 632, 633, and Overproduction of thyroid hormone by toxic nodules can suppress central TSH secretion and induce a state of thyroid hypoactivity in the remainder of the thyroid gland. With sufficient autonomous hyperactivity, clinical hyperthyroidism can result from such a hot nodule. A hyperactive, autonomous nodule could present as a palpable thyroid mass or as nodular hyperthyroidism (eg, hyperthyroidism occurring in association with a palpable nodule).

10 230 Annals of Clinical & Laboratory Science Fig. 4. This schematic diagram illustrates the locations of thyrotropin receptor (TSHR) mutations that produce gain-offunction or loss-of-function effects. Table 8. Defects in thyroid follicular cell response to TSH: TSHR gain-of-function mutations: Familial hyperthyroidism and congenital hyperthyroidism (autosomal dominant; TSHR gene; chromosome 14q31). Primary hyperthyroidism (suppressed TSH, elevated FT4, T4, FT3, T3) Absence of exophthalmos Absence of pretibial myxedema in adults Absence of thyroid autoantibodies Affected first degree relatives in an autosomal dominant pattern of inheritance Sequence TSHR transmembrane domain to detect TSHR gain-of-function mutation (if no mutations detected, study extracellular TSH-binding domain) Graves disease Exogenous thyroid hormone ingestion Thyroid adenoma or multinodular goiter without TSHR mutation or with G s gain-of-function mutation (TSHR sensitivity to hcg has similar criteria but produces hyperthyroidism only during pregnancy. ) (b) Familial hyperthyroidism. If the gain-of-function TSHR mutation occurs in the germline, theoretically all thyroid cells would display a degree of autonomy in the production of thyroid hormone resulting in a nonautoimmune form of hyperthyroidism. Nonautoimmune familial hyperthyroidism has been known for at least 20 years [25] (Table 8). Because such gain-offunction mutations are inherited as autosomal dominant characteristics, familial nonautoimmune hyperthyroidism would be recognized in succeeding generations. Nevertheless, we must recall that most cases of familial hyperthyroidism do result from Graves disease where thyroperoxidase, thyroid microsomal, thyroglobulin, or TSH receptor autoantibodies are detected, as well as exophthalmus and pretibial myxedema in adults (pretibial myxedema is rare in children with Graves disease). The TSH receptor autoantibodies (TRAbs) can be assayed by determining thyroid stimulating immunoglobulins (TSIs) or thyrotropin-binding inhibitory immunoglobulins

11 Review of molecular thyroidology 231 (TBIIs). Autoimmune thyroid disease is often inherited in an autosomal dominant mode, with women more often affected than men. Among autoimmune thyroid disease cases, Hashimoto thyroiditis is more common than Graves disease, but both Graves disease and Hashimoto thyroiditis can be seen in the same family [26]. Familial gain-of-function mutations have been described at TSHR amino acid positions 505, 509, 650, 670, and 672. (c) Congenital hyperthyroidism. Cases of non-autoimmune autosomal-dominant familial hyperthyroidism of neonatal onset have been described [27]. Here TSHR gain-of-function mutation is apparently so severe that the clinical onset of disease occurs in the newborn period. Similar to adult-onset forms of nonautoimmune autosomal-dominant familial hyperthyroidism, these thyrotoxic neonates lack exophthalmus and thyroid autoantibodies. Neonatal hyperthyroidism that appears in infants born to mothers with Graves disease is usually transient and results from transplacental passage of TSHR agonist autoantibodies. (d) TSHR sensitivity to human chorionic gonadotropin (hcg). A unique form of TSHR gain-of-function mutation was described by Rodien et al [28]. A woman and her mother are described who both developed transient hyper-thyroidism that developed only during pregnancy. Analysis of the TSHR DNA sequence revealed a K183R mutation (lysine replaced by arginine at amino acid position 183). When the K183R TSHR was expressed in COS-7 cells in vitro, the COS-7 cells responded normally to the addition of bovine TSH. However, when COS-7 cells were exposed to human chorionic gonadotropin (hcg) in a concentration comparable to the second trimester of pregnancy, the cells with the mutant TSHR showed a 350% increase in camp generation (versus TSH stimulation) versus no increase in camp in cells with the wild-type TSHR. Thus, while the mutant TSHR responded normally to TSH, the mutant TSHR was able to respond to hcg, which would produce hyperthyroidism only during pregnancy when hcg levels are high. TSHR loss-of-function mutations; (a) Euthyroid hyperthyrotropinemia. Loss-of-function mutations result in decreased respond to TSH. Mild TSH-resistance can be overcome by a sufficient elevation in TSH. Such cases with compensated primary hypothyroidism (elevated TSH and normal T4) carry the eponym euthyroid hyperthyrotropinemia (Table 9). The first report was of 3 euthyroid sisters similarly affected with elevated TSH levels. They were shown to be compound heterozygotes [P162A (partially functional) and I167N (nonfunctional)] [29]. Cases of euthyroid hyperthyrotropinemia have been summarized by Gagne et al [30]. (b) Congenital TSH unresponsiveness. If the TSHresistance is more severe, a biochemical picture similar to uncompensated primary hypothyroidism appears: elevated TSH and low T4, FT4 and T3. TSHR lossof-function mutations are uncommon [31].When this mutation occurs in the germline, failure to respond to TSH in utero can lead to congenital hypothyroidism and is termed congenital TSH unresponsiveness (Table 10). Biebermann et al [32] described the first Table 9. Defects in thyroid follicular cell response to TSH: TSHR loss-of-function mutations: Euthyroid hyperthyrotropinemia (autosomal dominant; TSHR gene; chromosome 14q31). Elevated TSH with normal FT4, T4, FT3, T3 No clinical evidence of hypothyroidism (euthyroid state) Familial, autosomal dominant pattern of inheritance Sequence TSHR extracellular TSH-binding domain to detect TSHR loss-of-function mutation Subclinical primary hypothyroidism Thyrotropin with impaired biologic activity Table 10. Defects in thyroid follicular cell response to TSH: TSHR loss-of-function mutations: Congenital TSH unresponsiveness (autosomal dominant; TSHR gene; chromosome 14q31). Familial congenital primary hypothyroidism (elevated TSH, low FT4, T4) Sequence TSHR extracellular TSH-binding domain to detect TSHR loss-of-function mutation Thyroid aplasia or hypoplasia (of primary hypothyroidism) TTF-2 mutation Pax8 mutation

12 232 Annals of Clinical & Laboratory Science case of congenital hypothyroidism resulting from compound heterozygosity for TSHR loss-of-function mutations: C390W (cytosine replaced by tryptophan at amino acid 390) and 419trunc (an 18 bp deletion with a novel 4 bp insertion that introduced 14 new amino acids before the appearance of a stop codon at residue 419 producing a truncated TSHR protein). Gagne et al [30] described a child with congenital hypothyroidism and measurable thyroglobulin with a G to C transversion at position +3 of the donor site of intron 6 and a 2 bp deletion of codon 655 in exon 10 (del655). Tonacchera et al [33] reported a child with unmeasurable thyroglobulin and congenital hypothyroidism from loss-of-function TSHR mutations. Previous cases of TSH resistance all displayed measurable thyroglobulin levels. To place TSH insensitivity into perspective as a cause of familial congenital hypothyroidism, Ahlom et al [34] did not find linkage of congenital hypothyroidism to the TSHR locus in 23 families with familial congenital hypothyroidism. Proposed clinical criteria for TSH insensitivity include: (1) hypothyroidism without goiter, (2) normal anatomic thyroid location (excludes ectopic thyroid gland such as a lingual thyroid), (3) low 131 I and pertechnetate 99m Tc uptake (excludes non-iodide pump forms of inborn errors in thyroid hormone biosynthesis where radioactive iodine uptake is usually high), (4) no in vivo response to exogenous TSH administration (eg, no increase in thyroid hormone release or radioactive iodide uptake after TSH injection, demonstrating that the problem is not in the TSH molecule itself), and (5) high plasma TSH (confirming primary hypothyroidism). While these criteria do not identify the etiology of the TSH resistance, they allow the clinician to consider various non-autoimmune causes of primary hypothyroidism when autoimmune thyroid disease, iodine excess/deficiency and inborn errors in thyroid hormone biosynthesis are considered to be unlikely. Generally, patients with inborn errors display goiter that is not typical of TSHR loss-offunction mutations. Mutations in the thyroperoxidase and thyroglobulin genes can cause congenital hypothyroidism [35]. Several reviews concerning TSHR mutations have recently been published [36-38]. Table 11 summarizes these disorders. Mutations in the G s alpha subunit. Gain-of-function mutations can occur in the TSH receptor by way of aberrations in the G s alpha subunit of the G s complex. If the G s alpha subunit suffers a mutation where GTPase activity is lost, the G s alpha subunit will express sustained activity leading to hyperstimulation of the thyroid gland inducing hyperthyroidism. Alternatively, the G s alpha subunit will acquire spontaneous activity by loss of GDP without receptor interaction again inducing hyperthyroidism. Somatic gain-of-function G s alpha subunit mutations have been recognized in ~4% of thyroid toxic adenomas and thyroid adenomas in McCune- Albright syndrome (Table 12). The entire thyroid gland is not hyperactive in McCune-Albright syndrome because of mosaicism. In fact, embryonic somatic Table 11: Relationship of the severity of the germline TSHR mutation to age at onset and phenotype (the disorder and the consequence). TSHR gain-of-function mutations Severity Onset Disorder Consequence Modest Child/adult Familial hyperthyroidism Hyperthyroid Modest Pregnancy TSHR sensitivity to hcg Hyperthyroid Severe In utero Congenital hyperthyroidism* Hyperthyroid TSHR loss-of-function mutations Severity Onset Disorder Consequence Mild Child/adult Euthyroid hyperthyrotropinemia** Euthyroid Severe In utero Congenital TSH unresponsiveness Hypothyroid * Persistent (non-autoimmune) neonatal hyperthyroidism. ** Euthyroid elevation of TSH with normal TSH bioactivity.

13 Review of molecular thyroidology 233 Table 12. Defects in thyroid follicular cell response to TSH: Mutations in the G s alpha subunit (somatic; G s alpha subunit gene; chromosome 20q13.2). Thyroid toxic adenomas and thyroid adenomas in McCune- Albright syndrome Sequence G s alpha subunit gene to detect G s alpha subunit gain-of-function mutation Thyroid adenoma without G s alpha subunit mutation or with TSHR gain-of-function mutation mosaicism in the gain-of-function G s alpha subunit mutation explains the patchy pattern of skin and tissue involvement in McCune-Albright syndrome. McCune-Albright syndrome is a sporadic disorder characterized by hyperfunction of multiple endocrine glands, multiple cafe-au-lait spots, and polyostotic fibrous dysplasia. The endocrine disorders in McCune- Albright syndrome include gonadotropin-independent precocious puberty, TSH-independent hyperthyroidism, hyperparathyroidism, and pituitary adenomas [growth hormone-secreting adenomas producing acromegaly (eg, somatotroph adenoma), ACTH-secreting adenomas producing Cushing syndrome or prolactinomas]. Gain-of-function G s alpha subunit mutations also can cause testotoxicosis in association with pseudohypoparathyroidism type Ia, 30-40% of non-mccune- Albright growth hormone-secreting pituitary adenomas (somatotrophinomas), and a small percentage of nonsecreting and ACTH-secreting pituitary adenomas [39]. Testotoxicosis results from a G s alpha subunit gain-of-function mutation associated with the LH receptor or from a gain-of-function mutation in the LH receptor itself [40]. This disorder is characterized by the development of precocious puberty in boys where the testes are determined to be the spontaneous source of androgen (eg, testosterone) in the absence of elevated gonadotropin levels. In testotoxicosis there is no evidence for testicular tumor. Expression of the gainof-function G s alpha subunit mutation appears temperature-dependent: the gain-of-function mutation may be expressed only in the testes, where the temperature is less than the core body temperature [41]. Table 13. Defects in thyroid gland formation: Transcription factor mutations: Thyroid Transcription Factor-2 (TTF-2) mutation (autosomal recessive; TTF-2 gene; chromosome 9q). Familial congenital primary hypothyroidism (elevated TSH, low FT4, T4) Coexistent left palate and choanal atresia Sequence thyroid TTF-2 gene to detect mutation Thyroid aplasia or hypoplasia (of primary hypothyroidism) Congenital TSH unresponsiveness Pax8 mutation A loss-of-function mutation in the G s alpha subunit or absence of the G s alpha subunit have been described and induce hormone resistance syndromes when the receptor ligand fails to produce the expected result. For example, in Albright hereditary osteodystrophy (pseudohypoparathyroidism type Ia) there is a partial congenital defect in the expression of the G s alpha subunit. Therefore there is defective response to PTH with ensuing hypocalcemia and hyperphosphatemia. To date, no loss-of-function G s mutations have been described as causes of thyroid disease. Defects in Thyroid Gland Formation Mutations of thyroid transcription factor-2 (TTF-2) and PAX8. Defects in the intrinsic formation or biochemistry of the thyroid gland can produce congenital hypothyroidism [42,43]. Mutations in two transcription factors have been reported as causes of congenital hypothyroidism: thyroid transcription factor-2 (TTF-2) and Pax8. The homozygous TTF-2 missense mutation (Ala65Val) produced congenital hypothyroidism in siblings and was associated with cleft palate and choanal atresia [44] (Table 13). The forkhead/winged-helix domain transcription factors are often key regulators of embryogenesis. TTF-2 is a member of this family. FKHL15 is the human homologue of mouse TTF-2. Pax8 mutations have been detected in two sporadic patients and in one familial case of congenital hypothyroidism [45] (Table 14). The authors discovered that all three point mutations were located in the paired domain of Pax8 and resulted in a severe

14 234 Annals of Clinical & Laboratory Science Table 14. Defects in thyroid gland formation: Transcription factor mutations: Pax8 mutation (autosomal recessive or dominant; PAX8 gene; chromosome 2q12-q14). Familial congenital primary hypothyroidism (elevated TSH, low FT4, T4) Absence of coexistent left palate and choanal atresia Sequence PAX8 gene to detect mutation Thyroid aplasia or hypoplasia (of primary hypothyroidism) Congenital TSH unresponsiveness TTF-2 mutation reduction in the DNA-binding activity of Pax8. Each of the affected individuals displayed heterozygosity for the Pax8 mutation (R108X, R31H and L62R). In the familial case, a mother and two of her affected children were heterozygous for a Pax8 mutation, implying dominant inheritance. Therefore Pax8 mutations can function either as recessives or dominants. In mice, knock-out of the thyroid transcription factor-1 (TTF-1) gene produces athyrosis. No coding sequence mutations of TTF-1 with congenital hypothyroidism have been discovered in humans [46]. However, several polymorphisms of TTF-1 exist and some of these polymorphisms may provide an increased risk for congenital hypothyroidism [47]. Several reviews of the molecular biology of congenital hypothyroidism are available [48,49]. Table 15. Defects in extrathyroidal thyroid hormone metabolism: Depressed intracellular-pituitary thyrotroph conversion of T4 to T3 producing TSH-dependent hyperthyroidism (mode of inheritance unknown). TSH-dependent hyperthyroidism Absence of anatomic hypothalamic or pituitary lesions Normal TRβ gene sequence Exogenous T3 administration produces a decline in TSH and induces clinical return to the euthyroid state TSH-secreting pituitary tumor Selective pituitary resistance to thyroid hormone Defects in Extrathyroidal Thyroid Hormone Metabolism Disordered conversion of T4 to T3 and decreased metabolism of rt3 (reverse T3, 3,3',5'-triiodothyronine), leading to depressed serum T3 and elevated rt3 concentrations in patients with nonthyroidal illness, are well described in the literature. The following disorders of peripheral thyroid hormone metabolism are not so well known: (1) depressed intracellular-pituitary thyrotroph conversion of T4 to T3, producing either TSH-dependent hyperthyroidism or hyperthyrotropinemia after T4 replacement in primary hypothyroidism and (2) the expression of type 3 iodothyronine deiodinase in infantile hemangiomas. Defective intrapituitary conversion of T4 to T3. In the pituitary, conversion of T4 to T3 normally provides the thyrotroph with sufficient levels of intracellular T3 to suppress TSH synthesis and secretion, thereby maintaining the euthyroid state. A family has been reported with TSH-dependent hyperthyroidism where T3, but not T4, administration was able to induce a euthyroid state [50] (Table 15). Either this family displayed isolated pituitary T3 resistance or suffered from a defect in the conversion of T4 to T3 within the pituitary thyrotroph. The hypothesized cause of the hyperthyroidism was that, whereas supra-normal FT3 levels were required to suppress TSH secretion, the normal extra-pituitary peripheral sensitivity of the body Table 16. Defects in extrathyroidal thyroid hormone metabolism: Depressed intracellular-pituitary thyrotroph conversion of T4 to T3 producing hyperthyrotropinemia on T4 replacement in primary hypothyroidism (mode of inheritance unknown). Hyperthyrotropinemia on T4 replacement in primary hypothyroidism with normal peripheral blood FT4 (and T4) concentration Normal TRβ gene sequence Exogenous T3 administration produces a decline in TSH (exogenous T4 does not suppress TSH) Inadequately treated primary hypothyroidism (insufficient dose of T4 or noncompliance) Subclinical hypothyroidism