Annals of Clinical & Laboratory Science, vol. 31, no. 3, 2001 221

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- 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 Normal Thyroid Function

The goal of this review is to introduce the clinical The and anterior laboratorian to several recent advances in molecular thyrotrophs monitor free thyroid hormone levels in thyroidology. Many novel disorders involving the blood stream (Fig. 1). Unbound or free triiodo- aberrations of the hypothalamic-pituitary-thyroid thyronine (FT3) is present in the plasma and enters gland-thyroid hormone axis have been described in the parvicellular division of the paraventricular nucleus. the last 5 to 10 years. While molecular diagnosis of Within these paired hypothalamic nuclei that are such conditions is rarely indicated for clinical adjacent to the superior aspect of the third ventricle, management, knowledge of the molecular mechanisms intracellular T3 is also derived from monodeiodination of disease can greatly enhance the laboratorian’s ability of free tetraiodothyronine (free thyroxine, FT4) that to advise clinicians about appropriate thyroid testing, has entered the cell cytoplasm from the plasma. If the and to interpret the complex and sometimes confusing intracellular level of T3 declines, thyrotropin-releasing results of thyroid function tests. This review begins hormone (TRH) is released into the hypothalamic- with a brief overview of the normal hypothalamic- pituitary-portal system to be delivered to the -thyroid gland-thyroid hormone axis. pituitary gland. TRH is the tripeptide pyroGlu-His- Pro-NH2. The cyclized glutamic acid terminus and Address correspondence to William E. Winter, M.D., Department an intact amide are required for TRH bioactivity. TRH of Pathology, Immunology and Laboratory Medicine, University sensitizes the anterior pituitary thyrotrophs to release of Florida Medical School, Box 100275, Gainesville, FL 32610- 0275, USA; tel 352 392 4495; fax 352 846 2149; e-mail more thyroid stimulating hormone (TSH) if intra- [email protected]. cellular thyrotroph T3 levels are deficient.

0091-7370/01/0300/0221 $6.00; © 2001 by the Association of Clinical Scientists, Inc. 222 Annals of Clinical & Laboratory Science

TSH circulates systemically. Upon binding of TSH to the TSH receptor located on , 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 thyroxine- binding 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 Fig. 1. The hypothalamus secretes thyrotropin releasing hormone suppressed, completing the negative feedback loop for (TRH) into the hypothalamic-pituitary portal system. In turn, control of thyroid hormone synthesis and secretion. TRH regulates the responsiveness of thyrotropin (TSH) to thyroid Thyroid hormone is “trophic” for many tissues. hormone feedback. TSH circulates systemically and stimulates the Thyroid hormone is important for the growth, thyroid gland to release thyroxine (tetraiodothyronine, T4) and 3,5,3'- (T3). About 80% of circulating T3 is differentiation, and maintenance of the central nervous derived from peripheral monodeiodination of T4 to T3. In target system (very important), skeleton (very important), tissues, T4 is also converted to T3 and the T3 interacts with nuclear cardiovascular system, and gastrointestinal system. thyroid hormone receptors. Basal metabolic rate (BMR) is directly regulated by thyroid hormone. Thyroid hormone also affects and Thyrotrophs, the anterior pituitary cells that release regulates intermediary metabolism, drug metabolism, TSH in response to TRH and decreased T3, express and the activity of other hormones (eg, growth TRH receptors. When TRH binds to the TRH hormone secretion is impaired in individuals with receptor, the thyrotroph depolarizes, allowing calcium hypothyroidism). to influx into the thyrotroph cytoplasm. In turn, increased free cytosolic calcium activates the Ca2+- Overview of Molecular Thyroidology phosphatidylinositol cascade. This causes TSH release and synthesis and glycosylation of alpha and beta TSH “New explanations for old diseases” would be an subunits. Stimulation of glycosylation of TSH subunits appropriate title for much of this review. The following is relatively a greater effect of TRH than stimulation topics will be addressed: of TSH synthesis. Glycosylation is necessary for Molecular mutations causing central hypothyroidism: bioactivity of TSH. TRH also depresses T3 receptor • Isolated autosomal recessive TRH deficiency, expression. This makes the thyrotroph less sensitive to • Autosomal recessive TRH-receptor inactivating mutations, thyroid hormone negative feedback, further increasing • Pit-1 mutations, TSH release. The major site of central negative • PROP-1 mutations; feedback is the pituitary. However, injected TRH TSH beta-subunit bio-inactivating mutations: normally releases TSH and prolactin. The lactotrophs • High molecular weight bio-inactive TSH; express the TRH receptor. For all other hormones Defects in response to TSH: mutations in the TSH receptor: regulated negatively by the hypothalamus and pituitary, • TSH receptor gain-of-function mutations, the major site of negative feedback is the hypothalamus. • TSH receptor loss-of-function mutations; Review of molecular thyroidology 223

Defects in thyroid gland formation: transcription factor degree of sialylation increases (an effect of estrogen), mutations: the half-life of TBG increases, raising TBG levels. In • TTF-2, Pax8 [1]; congenital TBG excess, an X-linked dominant Defects in peripheral thyroid hormone metabolism: condition, male hemizygotes display 3- to 5-fold • Defective intrapituitary conversion of T4 to T3 [2], elevations in TBG levels, while female heterozygotes • Hemangioma consumption of thyroid hormone; Defects in response of tissues to thyroid hormone: display 2- to 3-fold increases in TBG. Besides estrogen • Generalized thyroid hormone resistance [3], effects and congenital excess, other causes of elevated • Selective pituitary thyroid hormone resistance. TBG levels include acute liver disease and drugs (eg, phenothiazines). While many defects in thyroid hormone In familial dysalbuminemic hyperthyroxinemia, a biosynthesis have been described, their etiology is mutant dominantly-inherited form of albumin generally understood and they are not reviewed in detail (Arg218His) binds increased amounts of T4 but not in this paper. However,the clinical laboratory scientist T3, producing euthyroid hyperthyroxinemia, with should be familiar with cases of goitrous congenital normal T3 levels as well as normal FT4 levels. In hypothyroidism and non-autoimmune goitrous contrast, TBG excess raises both T4 and T3. Another hypothyroidism that result from: cause of euthyroid hyperthyroxinemia is familial • defects in transport into the thyroid gland euthyroid thyroxine excess that results from a mutant (autosomal recessive mutations in the sodium/iodide form of transthyretin (TTR, Thr119Met). An older symporter located on chromosome 19p12-13.2) [4], name for TTR is thyroxine-binding prealbumin • organification defects (autosomal recessive (TBPA). Encoded by the TTR gene on chromosome mutations of thyroperoxidase located on chromosome 18q11.2, TTR exists as a stable 55 kDa tetramer of 2p25 and associated with the Pendred syndrome 127 amino acid monomers. TTR participates in mutation located at chromosome 7q21-34), vitamin A transport by binding to the complex of • synthesis defects (autosomal vitamin A and retinol-binding protein. As a side note, recessive disorder mapping to chromosome 8q24), and more than 40 TTR mutations have been reported that • the iodine recycling disorder involving can cause familial amyloidosis affecting the heart dehalogenase. (cardiomyopathy) or nervous system (autonomic Recently the gene responsible for Pendred neuropathy or polyneuropathy). It is of interest that syndrome (hypothyroidism due to defective iodine other causes of familial amyloidoses include mutations organification of thyroglobulin associated with in apolipoprotein A-I, gelsolin, fibrinogen, and congenital or early-onset sensorineural deafness) was lysozyme. Gelsolin is a cytoplasmic and plasma cloned and named the PDS (Pendred syndrome) gene calcium-binding protein that binds to and fragments [5]. The PDS gene product is a transmembrane protein actin filaments. Many reviews of euthyroid () which transports iodide and chloride [6]. hyperthyroxinemia with normal FT4 levels have been Defects in thyroid hormone transport are also well published [9] and these disorders are not further described in the literature. These defects can cause discussed in this paper. confusion in the interpretation of elevated total T4 measurements when T-uptake or T3 resin uptake is Molecular Mutations with Central Hypothyroidism not also measured. As measurements of free T4 (FT4) replace total T4 measurements, the diagnostic problems Central hypothyroidism is diagnosed when clinical posed by thyroxine binding globulin (TBG) excess, hypothyroidism is accompanied by low FT4 (and FT3) familial dysalbuminemic hyperthyroxinemia [7], and and inappropriately normal or low TSH concentration. familial euthyroid thyroxine excess [8] should wane. Most cases of hypothyroidism are primary in etiology Located on chromosome Xq11-23, TBG is a 395 and show an elevated TSH concentration in the blood. amino acid 54-kDa acidic glycoprotein with a single Regardless of etiology (eg, either primary or iodothyronine binding site. TBG has 4 hetero- central), the clinical features of hypothyroidism include saccharide side chains with 5-9 sialic acids. As the symptoms of tiredness, constipation, cold intolerance, 224 Annals of Clinical & Laboratory Science dry hair or skin, weight gain, menstrual irregularities, diabetes insipidus could cause serious hypovolemia breast milk production, and slow mentation. Signs of when the patient’s oral intake of food and fluids is hypothyroidism can include low heart rate restricted preoperatively, or when the patient’s oral (bradycardia), decreased strength of cardiac contraction intake is restricted postoperatively and sufficient causing decline in the usual difference between systolic intravenous fluids are not administered to replace and diastolic blood pressures (low pulse pressure), excessive urinary fluid loss. If a mass lesion is discovered myxedema (nonpitting edema), hypercholesterolemia, in the hypothalamus or pituitary that requires surgery elevated creatine kinase, growth failure (including or irradiation, all aspects of anterior and posterior congenital hypothyroidism), short stature, retarded pituitary function should also be examined at the bone age, stippled growth plates, decreased reflexes, conclusion of the tumor therapy. congestive heart failure, and coma. Newborns with congenital hypothyroidism may display an enlarged Isolated autosomal recessive TRH deficiency and TRH posterior fontanelle, large tongue, prolonged jaundice receptor mutation. When anatomic hypothalamic and (delayed expression of UDP-glucuronyl transferase), pituitary pathology have been excluded, the clinician low body temperature, delayed passage of meconium, can perform a thyrotropin-releasing hormone (TRH) large body size at birth because of postmaturity (delayed test to localize the cause of the central hypothyroidism. delivery), or excessive body hair. If TRH is deficient, administration of exogenous TRH In cases of central hypothyroidism where low FT4 will raise the TSH concentrations during the TRH- is accompanied by an inappropriately low TSH level, stimulation test. This substantiates the diagnosis of the clinician’s initial obligation is to exclude by tertiary (hypothalamic) hypothyroidism. However, if radiology a tumor mass lesion or other anatomic cause TRH is unable to elicit a TSH response, the pituitary of central hypothyroidism that might require surgery is at fault. This substantiates the diagnosis of secondary or irradiation. Other types of central endocrine (pituitary) hypothyroidism. deficiencies should also be pursued and treated Assuming that all other anterior and posterior preoperatively, such as ACTH deficiency causing pituitary axes are intact, which in fact is rare, the glucocorticoid deficiency and ADH deficiency causing clinician and laboratorian should consider in their diabetes insipidus. differential diagnosis: (1) isolated autosomal recessive Failure to detect and treat glucocorticoid TRH deficiency (chromosome 3) [10] (Table 1), (2) insufficiency preoperatively could lead to fatal intra- autosomal recessive TRH-receptor inactivating operative adrenal crisis. Likewise, failure to recognize mutations (chromosome 8q23) (Table 2), and (3)

Table 1. Clinical and laboratory features of molecular mutations Table 2. Clinical and laboratory features of molecular mutations causing central hypothyroidism: Isolated autosomal recessive TRH causing central hypothyroidism: TRH receptor (TRHR) mutation deficiency (TRH gene (?); chromosome 3). (autosomal recessive; TRHR gene; chromosome 8q23).

When to consider this disorder: When to consider this disorder: • Central (TSH-deficient) hypothyroidism • Central (TSH-deficient) hypothyroidism • TSH responds to exogenous TRH administration (this • Lack of TSH response to exogenous TRH administration demonstrates that the TRH receptor and signaling to TSH (demonstrates that TRHR/pituitary is dysfunctional) secretion are intact) • Lack of prolactin response to exogenous TRH admin- • Prolactin responds to exogenous TRH administration istration (demonstrates that TRHR/pituitary is dysfunctional) (provides further evidence that the pituitary is functional; • Absence of other anterior pituitary hormone defects (rules rules against Pit-1 and Prop1 mutations) against Pit-1 and Prop1 mutations) • Absence of other anterior pituitary hormone defects (rules • Absence of anatomic hypothalamic lesions against Pit-1 and Prop1 mutations) • Absence of hypothalamic-pituitary portal system lesions • Absence of anatomic hypothalamic lesions How to diagnose this disorder definitively: • Absence of hypothalamic-pituitary portal system lesions • Clinical criteria noted above, and How to diagnose this disorder: • Sequence TRHR gene and detect mutations • Diagnosis by exclusion Differential diagnosis: Differential diagnosis: • Pituitary disease • Hypothalamic disease Review of molecular thyroidology 225 familial TSH deficiency (Table 3, discussed below). Table 3. Clinical and laboratory features of molecular mutations β Collu et al [11] have reported a child with central causing central hypothyroidism: TSH beta (TSH ) mutations: Familial autosomal recessive TSH deficiency (autosomal recessive; hypothyroidism resulting from compound hetero- TSHβ gene; chromosome 1p22). zygous mutations in the TRH receptor gene. Etiologies of central hypothyroidism are illustrated in Fig. 2. When to consider this disorder: When central anatomic lesions are absent and • Central (TSH-deficient) hypothyroidism multiple anterior pituitary hormone deficiencies are • Absent TSH response to exogenous TRH administration (demonstrates that thyrotroph is dysfunctional; alpha subunit otherwise unexplained, the clinician and laboratory rises after TRH indicating that the TRHR is functional) scientist should consider Pit-1 mutations and Prop1 • Prolactin responds to exogenous TRH administration mutations [12]. (demonstrates that TRHR is functional) • Absence of other anterior pituitary hormone defects (rules against Pit-1 and Prop1 mutations) TSH beta mutations: familial autosomal recessive TSH • Absence of anatomic hypothalamic lesions deficiency. Autosomal recessive TSH deficiency results • Absence of hypothalamic-pituitary portal system lesions from homozygosity or compound heterozygosity for How to diagnose this disorder definitively: TSH beta subunit mutations [13,14] (Table 3). Only • Clinical criteria noted above and β TSH is deficient as other anterior pituitary hormones • Sequence TSH gene and detect mutations Differential diagnoses: are intact. TSH (molecular weight 28 kD) is similar to • 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. 226 Annals of Clinical & Laboratory Science

LH, FSH, and hCG: all are glycoprotein hormones mutations most commonly produce growth hormone that share a common alpha subunit (Mr 14,700, two deficiency but also commonly produce central oligosaccharide moieties; chromosome 6q21.1-q23) hypothyroidism and prolactin deficiency resulting in while each glycoprotein hormone has a unique beta a combined pituitary hormone deficiency (CPHD) subunit that is responsible for the specific bioactivity (Table 4). There is no apparent adverse consequence of the hormone. The TSH beta chain (Mr = 15,600, to being prolactin deficient. However, diagnostically, one oligosaccharide moiety) gene is located on prolactin deficiency should be sought by measuring chromosome 1p. Mutations in both TSH beta chain prolactin as part of the subject’s TRH stimulation test genes lead to TSH deficiency. if the subject is evaluated for central hypothyroidism. TSH deficiency causes congenital hypothyroidism Gonatrophs that secrete LH and FSH and cortico- with low to undetectable TSH values. Central hypo- trophs that secrete ACTH are uninvolved in cases of thyroidism will be detected in neonatal hypothyroid Pit-1 deficiency. screening programs that test for depressions in total Recessive and dominant modes of inheritance of T4. In programs that depend on elevated TSH levels Pit-related familial panhypopituitarism have been to diagnose hypothyroidism, TSH deficiency will be described. Recessive Pit-1 mutations include complete missed. deletion of the PIT-1 gene, F135C (phenylalanine — Metabolic findings in cases of TSH deficiency > cysteine), R143N (arginine —> glutamine), A158P include low basal radioactive iodine uptake (RAIU) (alanine —> proline), R172X (arginine —> stop), and that increases after administration of bovine TSH. This E250X (glutamate—> stop). For example, the A158P proves that the thyroid gland itself is normal. After mutation disturbs the formation of Pit-1 homodimers administration of exogenous TRH, intact TSH and and greatly decreases transcription activation. The the TSH beta subunit remain undetectable, while the R271W (arginine —> tryptophan) mutation and the TSH alpha subunit is increased in concentration. With P24L (proline —> leucine) mutation produce exogenous T3 replacement, the alpha subunit dominant forms of Pit-1–deficient hypopituitarism. concentration declines, demonstrating that feedback The dominant negative effect of these latter two exists centrally. Heterozygotes with one normal and mutations is not clearly understood. one abnormal TSH beta allele are clinically normal. Recurrence risk in siblings is 25%. Table 4. Clinical and laboratory features of molecular mutations The TSH beta gene has 3 exons. The following causing central hypothyroidism: Pit-1 mutations: Familial polyhormone hypopituitarism syndromes (autosomal recessive and mutations have been described: dominant forms; PIT-1 gene; chromosome 3p11).

Base Nucleotide Mutation When to consider this disorder: change position • Central (TSH-deficient) hypothyroidism G —> A 29 Missense • Absent TSH response to exogenous TRH administration G —> T 94 Transversion (demonstrates that TRHR/pituitary is dysfunctional) Base deletion 105 Frameshift • Absent prolactin response to exogenous TRH administration (demonstrates that TRHR/pituitary is dysfunctional) • Growth hormone and prolactin deficiency (other anterior Pit-1 and Prop1 mutations: Familial polyhormone pituitary hormone defects present) hypopituitarism syndromes (Combined pituitary • Normal LH and FSH response to GnRH (rules against hormone deficiency, CPHD). Transcription factors are Prop1 mutations); normal ACTH and cortisol proteins that regulate gene expression. Pit-1 and Prop1 • Absence of anatomic hypothalamic lesions • Absence of hypothalamic-pituitary portal system lesions are transcription factors that regulate the activity of • Family history may be positive for similarly affected several key genes encoding anterior pituitary hormones. individuals Encoded by the PIT-1 gene on chromosome 3p11, How to diagnose this order definitively: Pit-1 is a pituitary-specific transcription factor that • Clinical criteria noted above and binds to the DNA regulatory regions of the thyrotroph • Sequence PIT-1 gene and detect mutations Differential diagnoses: TSH beta gene, the somatotroph growth hormone • Hypothalamic or pituitary disease gene, and the lactotroph prolactin gene. Pit-1 Review of molecular thyroidology 227

Expressed at an early stage in pituitary gland transduction through the TSHR. Unfortunately, there development, the prophet of Pit-1 gene (Prop1) were no molecular analyses of the TSH beta or TSH encodes a paired-like homeodomain protein within its alpha genes. Even if both genes were normal, 3 exons. Prop1 may regulate Pit-1 [15]. Mutations in theoretically there could be aberrant Golgi processing Prop1 cause gonadotropin (LH and FSH) deficiency leading to polymerization of the TSH molecules and in addition to deficiencies of TSH beta, growth an increased molecular mass. Another possibility to hormone and prolactin [16] (Table 5). At least one consider would be a “macro-TSH” (eg, TSH bound Prop1 family has additionally been described with by a plasma immunoglobulin). This case illustrates that, ACTH deficiency [17]. This family had a 301- while most individuals with normal T4 and T3 levels 302delAG Prop1 frameshift mutation. This site is a and elevated TSH levels have subclinical hypo- hot-spot for mutation in the PROP1 gene. Another thyroidism, rare individuals may lack a TSH molecule Prop1 mutation is R120C [18]. Magnetic resonance of normal biopotency. One condition not excluded in (MR) imaging in patients with Prop1 mutations can the 1981 report was human anti-mouse monoclonal reveal congenital hypoplasia of the anterior pituitary antibodies (HAMA) that could produce a false gland [19]. elevation in measured TSH levels. The large in vivo size of the patient’s TSH argues against the possibility Thyrotropin with impaired biologic activity. In 1981, of HAMA. the case of a euthyroid adult with an elevated TSH It may seem odd to characterize this disorder as a level was reported where the TSH displayed impaired form of central hypothyroidism because the TSH level biologic activity [20] (Table 6). The TSH level was is elevated. However, because the thyroid gland can increased approximately 25-fold over the upper limit respond normally to exogenous TSH in this condition, of the reference range. Chromatographic analysis thyrotropin dysfunction appears to be a consequence demonstrated that the TSH in this individual was of of a “pituitary manufacturing problem” and thus much higher molecular weight than normal. classification of “thyrotropin with impaired biologic Furthermore, while this large form of TSH bound to activity” as a type of central hypothyroidism is the TSHR receptor normally, there was decreased signal appropriate.

Table 5. Clinical and laboratory features of molecular mutations Table 6. Clinical and laboratory features of molecular mutations causing hypothyroidism: Prop1 mutations: Familial polyhormone causing central hypothyroidism: Thyrotropin with impaired hypopituitarism syndromes (autosomal recessive, PROP1 gene; biologic activity (inheritance unknown). chromosome 5p).

When to consider this disorder: When to consider this disorder: • Central (TSH-deficient) hypothyroidism • Hyperthyrotropinemic euthyroidism (elevated TSH, normal • Absent TSH response to exogenous TRH administration FT4, T4, FT3, T3) (demonstrates that TRHR/pituitary is dysfunctional) • TSH rises in response to exogenous TRH (pituitary is • Absent prolactin response to exogenous TRH administration functional) (demonstrates that TRHR/pituitary is dysfunctional) • Prolactin rises in response to exogenous TRH (pituitary is • Growth hormone, prolactin, LH and FSH deficiency and functional) possible ACTH deficiency (LH/FSH deficiency rule in favor • Reduction in TSH after exogenous T3 administration of Prop1 deficiency and against Pit-1 deficiency) (sufficient level of T3 administration can suppress TSH) • Possible hypoplasia of the anterior pituitary gland on MRI • Increased T4 and thyroid radioactive iodine uptake after • Absence of hypothalamic-pituitary portal system lesions administration of bovine TSH (TSHR is functional) • Family history may be positive for similarly affected How to diagnose this disorder definitively: individuals • Clinical criteria noted above and How to diagnose this disorder definitively: • Chromatographic analysis of TSH demonstrates high • Clinical criteria noted above and molecular weight TSH • Sequence PROP1 gene and detect mutations Differential diagnoses: Differential diagnoses: • Subclinical primary hypothyroidism • Hypothalamic or pituitary disease • Mild TSHR loss-of-function mutation 228 Annals of Clinical & Laboratory Science

Defects in Thyroid Follicular Cell Response to TSH binding, the equilibrium shifts to the “on” form and signaling continues. In the basal state, the 3 subunits

Mutations in the TSH receptor. TSH action on the of the Gs complex, alpha, beta, and gamma, are thyroid follicular cell is mediated through a TSH associated and alpha subunit non-covalently binds receptor (TSHR)-G protein-adenyl cyclase-coupled guanosine diphosphate (GDP). The G protein family production of intracellular cyclic adenosine mono- has more than 50 members. These proteins bind either phosphate (cAMP). The TSHR is a member of the GDP or guanosine triphosphate (GTP). The larger G superfamily of G-protein-coupled receptors. Other proteins of 80 to 90 kDa function in hormone members of this receptor superfamily include the pathways. The Gs alpha subunit is located on ACTH receptor, alpha-adrenergic and beta-adrenergic chromosome 20q13.2. catecholamine receptors, LH receptor, FSH receptor, When the TSHR interacts with the basal Gs hCG receptor, glucagon receptor, PTH receptor, and complex, the alpha subunit dissociates from the beta somatostatin receptor. A rise in intracellular cAMP in and gamma subunits. The alpha subunit sheds GDP, the follicular thyrocyte leads to increased iodide uptake, which is replaced by guanosine triphosphate (GTP). increased thyroperoxidase and thyroglobulin synthesis, This “activated” Gs alpha subunit with GTP attached hormone secretion, expression of type 1 deiodinase, activates adenyl cyclase. Adenyl cyclase converts ATP and growth of the thyroid follicular cell. At high TSH to cAMP. As a mechanism of internal negative feedback 2+ concentrations, TSH activates the Ca -phosphatidyl- regulation, the “activated” Gs alpha subunit does inositol-phosphate protein kinase C cascade, which acquire GTPase activity. This converts the attached - stimulates H2O2 generation and I efflux. GTP to GDP (plus phosphate). The Gs alpha subunit The 10-exon TSH receptor gene, located on with GDP then recombines with beta and gamma and chromosome 14q31, covers 60 kb. The predominant Gs returns to its basal inactive state (Fig. 3). mRNA is 4.3 kb,with smaller transcripts also observed. Mutations in the TSHR can produce decreased or After glycosylation, the TSHR weighs ~100 kDa. Prior increased spontaneous activity [21,22]. The loss-of- to glycosylation, the apoprotein core weighs 84.5 kDa. function mutations localize to the extracellular TSH- The N-terminal extracellular domain of 398 amino binding domain of the TSHR (Fig. 4). Two well acids is encoded by the first 9 exons. There are 6 N- described loss-of-function mutations are the P162A glycosylation sites in the extracellular domain. TSH and I167N mutations. Except for the extracellular binds to this region of the TSHR. The 346 amino acid TSH-binding domain TSHR 281 mutations (S281N carboxyl half of the receptor, which is encoded by a and S281T), the gain-of-function mutations localize single large exon 10, contains the 7 hydrophobic to the multiple transmembrane portions and transmembrane segments that are connected by 3 extra- connecting loops of the TSHR. and 3 intracellular loops and the cytoplasmic portion of the TSHR. This portion of the TSHR demonstrates TSHR gain-of-function mutations; (a) Thyroid homology with other G protein-coupled receptors and neoplasms. Returning to the TSHR model of the activates the Gs complex upon TSH binding to the “TSH-bound-‘on’ configuration” versus the “TSH- extracellular domain of the TSHR. unbound-‘off’ configuration”, gain-of-function Upon TSH binding to the extracellular domain mutations shift the equilibrium to the “on” of the TSHR, a conformational change is believed to configuration in the absence of the TSH ligand. Thus take place in the TSH receptor. This would allow even without the TSH ligand, the TSHR is transducing interactions between the TSHR and the Gs (G signals leading to autonomous thyroid follicular cell stimulatory) complex. Alternatively, the TSHR may hyperfunction. If the TSHR mutation occurs as a exist in 2 forms: an “on” form which interacts with the somatic mutation (eg, in a ), a Gs complex and an “off” form that does not interact thyrotoxic nodule results (Table 7). About 80% of toxic with the Gs complex. In the absence of TSH, the thyroid adenomas (hot nodules) exhibit TSHR gain- TSHR predominantly exists in the “off” form and no of-function mutations [23]. Multinodular goiter can signal transduction occurs. However with TSH also result from gain-of-function mutations [24]. Review of molecular thyroidology 229

Fig. 3. Thyrotropin (TSH) binds to a specific TSH receptor on thyroid follicular cells. The Gs 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 Gs with adenyl cyclase, adenyl cyclase acquires enzymatic activity and converts ATP to cAMP. Active Gs also expresses an intrinsic GTPase activity. GTPase cleaves GTP to GDP plus Pi. The Gs 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 Somatic gain-of-function mutations have been gain-of-function mutations: Thyroid neoplasias (somatic; described at TSHR amino acid positions 281, 453, TSHR gene; chromosome 14q31). 486, 568, 619, 623, 629, 631, 632, 633, and 658- When to consider this disorder: 661. • Thyroid adenoma or multinodular goiter Overproduction of thyroid hormone by toxic • with or without a hot nodule or nodules ( nodules can suppress central TSH secretion and induce may or may not be present) a state of thyroid hypoactivity in the remainder of the How to diagnose this disorder definitively: • Clinical criteria noted above and thyroid gland. With sufficient autonomous • Sequence TSHR transmembrane domain to detect TSHR hyperactivity, clinical hyperthyroidism can result from gain-of-function mutation (if no mutations detected, study such a “hot nodule.” A hyperactive, autonomous extracellular TSH-binding domain) nodule could present as a palpable thyroid mass or as Differential diagnoses: “nodular” hyperthyroidism (eg, hyperthyroidism • Thyroid adenoma or multinodular goiter without TSHR occurring in association with a palpable nodule). mutation or with Gs gain-of-function mutation 230 Annals of Clinical & Laboratory Science

Fig. 4. This schematic diagram illustrates the locations of (TSHR) mutations that produce gain-of- function or loss-of-function effects.

Table 8. Defects in thyroid follicular cell response to TSH: TSHR (b) Familial hyperthyroidism. If the gain-of-function gain-of-function mutations: Familial hyperthyroidism and TSHR mutation occurs in the germline, theoretically congenital hyperthyroidism (autosomal dominant; TSHR gene; all thyroid cells would display a degree of autonomy chromosome 14q31). in the production of thyroid hormone resulting in a When to consider this disorder: nonautoimmune form of hyperthyroidism. Nonauto- • Primary hyperthyroidism (suppressed TSH, elevated FT4, immune familial hyperthyroidism has been known for T4, FT3, T3) at least 20 years [25] (Table 8). Because such gain-of- • Absence of exophthalmos • Absence of pretibial myxedema in adults function mutations are inherited as autosomal • Absence of thyroid autoantibodies dominant characteristics, familial nonautoimmune • Affected first degree relatives in an autosomal dominant hyperthyroidism would be recognized in succeeding pattern of inheritance generations. Nevertheless, we must recall that most How to diagnose this disorder definitively: cases of familial hyperthyroidism do result from Graves • Clinical criteria noted above and • Sequence TSHR transmembrane domain to detect TSHR disease where thyroperoxidase, thyroid microsomal, gain-of-function mutation (if no mutations detected, study thyroglobulin, or TSH receptor autoantibodies are extracellular TSH-binding domain) detected, as well as exophthalmus and pretibial Differential diagnoses: myxedema in adults (pretibial myxedema is rare in • Graves disease • Exogenous thyroid hormone ingestion children with Graves disease). The TSH receptor • Thyroid adenoma or multinodular goiter without TSHR autoantibodies (TRAbs) can be assayed by determining

mutation or with Gs gain-of-function mutation thyroid stimulating immunoglobulins (TSIs) or (TSHR sensitivity to hCG has similar criteria but produces thyrotropin-binding inhibitory immunoglobulins hyperthyroidism only during pregnancy. ) Review of molecular thyroidology 231

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

Table 9. Defects in thyroid follicular cell response to TSH: TSHR Table 10. Defects in thyroid follicular cell response to TSH: TSHR loss-of-function mutations: Euthyroid hyperthyrotropinemia loss-of-function mutations: Congenital TSH unresponsiveness (autosomal dominant; TSHR gene; chromosome 14q31). (autosomal dominant; TSHR gene; chromosome 14q31).

When to consider this disorder: When to consider this disorder: • Elevated TSH with normal FT4, T4, FT3, T3 • Familial congenital primary hypothyroidism (elevated TSH, • No clinical evidence of hypothyroidism (euthyroid state) low FT4, T4) • Familial, autosomal dominant pattern of inheritance How to diagnose this disorder definitively: How to diagnose this disorder definitively: • Clinical criteria noted above and • Clinical criteria noted above and • Sequence TSHR extracellular TSH-binding domain to • Sequence TSHR extracellular TSH-binding domain to detect TSHR loss-of-function mutation detect TSHR loss-of-function mutation Differential diagnoses: Differential diagnoses: • Thyroid aplasia or hypoplasia (of primary hypothyroidism) • Subclinical primary hypothyroidism • TTF-2 mutation • Thyrotropin with impaired biologic activity • Pax8 mutation 232 Annals of Clinical & Laboratory Science case of congenital hypothyroidism resulting from molecule itself), and (5) high plasma TSH (confirming compound heterozygosity for TSHR loss-of-function “primary” hypothyroidism). While these criteria do not mutations: C390W (cytosine replaced by tryptophan identify the etiology of the TSH resistance, they allow at amino acid 390) and 419trunc (an 18 bp deletion the clinician to consider various non-autoimmune with a novel 4 bp insertion that introduced 14 new causes of primary hypothyroidism when autoimmune amino acids before the appearance of a stop codon at thyroid disease, iodine excess/deficiency and inborn residue 419 producing a truncated TSHR protein). errors in thyroid hormone biosynthesis are considered Gagne et al [30] described a child with congenital to be unlikely. Generally, patients with inborn errors hypothyroidism and measurable thyroglobulin with a display goiter that is not typical of TSHR loss-of- G to C transversion at position +3 of the donor site of function mutations. Mutations in the thyroperoxidase intron 6 and a 2 bp deletion of codon 655 in exon 10 and thyroglobulin genes can cause congenital (del655). Tonacchera et al [33] reported a child with hypothyroidism [35]. unmeasurable thyroglobulin and congenital Several reviews concerning TSHR mutations have hypothyroidism from loss-of-function TSHR recently been published [36-38]. Table 11 summarizes mutations. Previous cases of TSH resistance all these disorders. displayed measurable thyroglobulin levels. To place TSH insensitivity into perspective as a cause of familial Mutations in the Gs alpha subunit. Gain-of-function congenital hypothyroidism, Ahlom et al [34] did not mutations can occur in the TSH receptor by way of find linkage of congenital hypothyroidism to the TSHR aberrations in the Gs alpha subunit of the Gs complex. locus in 23 families with familial congenital hypo- If the Gs alpha subunit suffers a mutation where thyroidism. GTPase activity is lost, the Gs alpha subunit will express Proposed clinical criteria for TSH insensitivity sustained activity leading to hyperstimulation of the include: (1) hypothyroidism without goiter, (2) normal thyroid gland inducing hyperthyroidism. Alternatively, anatomic thyroid location (excludes ectopic thyroid the Gs alpha subunit will acquire spontaneous activity gland such as a lingual thyroid), (3) low 131I and by loss of GDP without receptor interaction again pertechnetate 99mTc uptake (excludes non-iodide inducing hyperthyroidism. pump forms of inborn errors in thyroid hormone Somatic gain-of-function Gs alpha subunit biosynthesis where radioactive iodine uptake is usually mutations have been recognized in ~4% of thyroid high), (4) no in vivo response to exogenous TSH toxic adenomas and thyroid adenomas in McCune- administration (eg, no increase in thyroid hormone Albright syndrome (Table 12). The entire thyroid gland release or radioactive iodide uptake after TSH injection, is not hyperactive in McCune-Albright syndrome demonstrating that the problem is not in the TSH 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. Review of molecular thyroidology 233

Table 12. Defects in thyroid follicular cell response to TSH: Table 13. Defects in thyroid gland formation: Transcription factor Mutations in the Gs alpha subunit (somatic; Gs alpha subunit mutations: Thyroid Transcription Factor-2 (TTF-2) mutation gene; chromosome 20q13.2). (autosomal recessive; TTF-2 gene; chromosome 9q).

When to consider this disorder: When to consider this disorder: • Thyroid toxic adenomas and thyroid adenomas in McCune- • Familial congenital primary hypothyroidism (elevated TSH, Albright syndrome low FT4, T4) How to diagnose this disorder definitively: • Coexistent left palate and choanal atresia • Clinical criteria noted above and How to diagnose this disorder definitively: • Sequence Gs alpha subunit gene to detect Gs alpha subunit • Clinical criteria noted above and gain-of-function mutation • Sequence thyroid TTF-2 gene to detect mutation Differential diagnoses: Differential diagnoses: • Thyroid adenoma without Gs alpha subunit mutation or • Thyroid aplasia or hypoplasia (of primary hypothyroidism) with TSHR gain-of-function mutation • Congenital TSH unresponsiveness • Pax8 mutation

mosaicism in the gain-of-function Gs alpha subunit A loss-of-function mutation in the Gs alpha mutation explains the “patchy” pattern of skin and subunit or absence of the Gs alpha subunit have been tissue involvement in McCune-Albright syndrome. described and induce hormone resistance syndromes McCune-Albright syndrome is a sporadic disorder when the receptor ligand fails to produce the expected characterized by hyperfunction of multiple endocrine result. For example, in Albright hereditary osteo- glands, multiple cafe-au-lait spots, and polyostotic dystrophy (pseudohypoparathyroidism type Ia) there fibrous dysplasia. The endocrine disorders in McCune- is a partial congenital defect in the expression of the Albright syndrome include gonadotropin-independent Gs alpha subunit. Therefore there is defective response precocious puberty, TSH-independent hyper- to PTH with ensuing hypocalcemia and hyperphos- thyroidism, hyperparathyroidism, and pituitary phatemia. To date, no loss-of-function Gs mutations adenomas [growth hormone-secreting adenomas have been described as causes of thyroid disease. producing acromegaly (eg, somatotroph adenoma), ACTH-secreting adenomas producing Cushing Defects in Thyroid Gland Formation syndrome or ]. Gain-of-function Gs alpha subunit mutations also Mutations of thyroid transcription factor-2 (TTF-2) can cause testotoxicosis in association with pseudo- and PAX8. Defects in the intrinsic formation or hypoparathyroidism type Ia, 30-40% of non-McCune- biochemistry of the thyroid gland can produce Albright growth hormone-secreting pituitary adenomas congenital hypothyroidism [42,43]. Mutations in two (somatotrophinomas), and a small percentage of non- transcription factors have been reported as causes of secreting and ACTH-secreting pituitary adenomas congenital hypothyroidism: thyroid transcription [39]. Testotoxicosis results from a Gs alpha subunit factor-2 (TTF-2) and Pax8. The homozygous TTF-2 gain-of-function mutation associated with the LH missense mutation (Ala65Val) produced congenital receptor or from a gain-of-function mutation in the hypothyroidism in siblings and was associated with cleft LH receptor itself [40]. This disorder is characterized palate and choanal atresia [44] (Table 13). The by the development of precocious puberty in boys forkhead/winged-helix domain transcription factors are where the testes are determined to be the spontaneous often key regulators of embryogenesis. TTF-2 is a source of androgen (eg, testosterone) in the absence of member of this family. FKHL15 is the human elevated gonadotropin levels. In testotoxicosis there is homologue of mouse TTF-2 . no evidence for testicular tumor. Expression of the gain- Pax8 mutations have been detected in two sporadic of-function Gs alpha subunit mutation appears patients and in one familial case of congenital temperature-dependent: the gain-of-function mutation hypothyroidism [45] (Table 14). The authors may be expressed only in the testes, where the discovered that all three point mutations were located temperature is less than the core body temperature [41]. in the paired domain of Pax8 and resulted in a severe 234 Annals of Clinical & Laboratory Science

Table 14. Defects in thyroid gland formation: Transcription factor Defects in Extrathyroidal Thyroid Hormone mutations: Pax8 mutation (autosomal recessive or dominant; PAX8 Metabolism gene; chromosome 2q12-q14). Disordered conversion of T4 to T3 and decreased When to consider this disorder: • Familial congenital primary hypothyroidism (elevated TSH, metabolism of rT3 (reverse T3, 3,3',5'-triiodo- low FT4, T4) thyronine), leading to depressed serum T3 and elevated • Absence of coexistent left palate and choanal atresia rT3 concentrations in patients with nonthyroidal How to diagnose this disorder definitively: illness, are well described in the literature. The • Clinical criteria noted above and • Sequence PAX8 gene to detect mutation following disorders of peripheral thyroid hormone Differential diagnoses: metabolism are not so well known: (1) depressed • Thyroid aplasia or hypoplasia (of primary hypothyroidism) intracellular-pituitary thyrotroph conversion of T4 to • Congenital TSH unresponsiveness T3, producing either TSH-dependent hyperthyroidism • TTF-2 mutation or hyperthyrotropinemia after T4 replacement in primary hypothyroidism and (2) the expression of type 3 iodothyronine deiodinase in infantile hemangiomas. reduction in the DNA-binding activity of Pax8. Each of the affected individuals displayed heterozygosity for Defective intrapituitary conversion of T4 to T3. In the Pax8 mutation (R108X, R31H and L62R). In the the pituitary, conversion of T4 to T3 normally provides familial case, a mother and two of her affected children the thyrotroph with sufficient levels of intracellular T3 were heterozygous for a Pax8 mutation, implying to suppress TSH synthesis and secretion, thereby dominant inheritance. Therefore Pax8 mutations can maintaining the euthyroid state. A family has been function either as recessives or dominants. reported with TSH-dependent hyperthyroidism where In mice, knock-out of the thyroid transcription T3, but not T4, administration was able to induce a factor-1 (TTF-1) gene produces athyrosis. No coding euthyroid state [50] (Table 15). Either this family sequence mutations of TTF-1 with congenital displayed isolated pituitary T3 resistance or suffered hypothyroidism have been discovered in humans [46]. from a defect in the conversion of T4 to T3 within the However, several polymorphisms of TTF-1 exist and pituitary thyrotroph. The hypothesized cause of the some of these polymorphisms may provide an increased hyperthyroidism was that, whereas supra-normal FT3 risk for congenital hypothyroidism [47]. Several reviews levels were required to suppress TSH secretion, the of the molecular biology of congenital hypothyroidism normal extra-pituitary peripheral sensitivity of the body are available [48,49].

Table 16. Defects in extrathyroidal thyroid hormone metabolism: Depressed intracellular-pituitary thyrotroph conversion of T4 to Table 15. Defects in extrathyroidal thyroid hormone metabolism: T3 producing hyperthyrotropinemia on T4 replacement in primary Depressed intracellular-pituitary thyrotroph conversion of T4 to hypothyroidism (mode of inheritance unknown). T3 producing TSH-dependent hyperthyroidism (mode of inheritance unknown). When to consider this disorder: • Hyperthyrotropinemia on T4 replacement in primary When to consider this disorder: hypothyroidism with normal peripheral blood FT4 (and T4) concentration • TSH-dependent hyperthyroidism β • Absence of anatomic hypothalamic or pituitary lesions • Normal TR gene sequence • Normal TRβ gene sequence How to diagnose this disorder definitively: How to diagnose this disorder definitively: • Clinical criteria noted above and • Clinical criteria noted above and • Exogenous T3 administration produces a decline in TSH • Exogenous T3 administration produces a decline in TSH (exogenous T4 does not suppress TSH) and induces clinical return to the euthyroid state Differential diagnoses: Differential diagnoses: • Inadequately treated primary hypothyroidism (insufficient • TSH-secreting pituitary tumor dose of T4 or noncompliance) • Selective pituitary resistance to thyroid hormone • Subclinical hypothyroidism Review of molecular thyroidology 235 to elevated FT3 levels produced clinical hyper- TBG, albumin, or TTR binding of thyroid hormone. thyroidism. A more perplexing condition is euthyroid hyperthy- Another abnormality of peripheral conversion also roxinemia with elevated FT4 levels. However, if we involves the intrapituitary conversion of T4 to T3. A consider the concept of hormone resistance syndromes, 63-yr old woman with primary hypothyroidism was the situation is straightforward. If there is generalized reported where decreased intrapituitary conversion of resistance to the effects of thyroid hormone, TSH levels T4 to T3 presumably lead to a persistent elevation of rise to stimulate the thyroid gland to produce the higher TSH while she was receiving T4 replacement, whereas total and free thyroid hormone levels that are necessary T3 replacement returned the TSH to normal [51] to maintain the euthyroid state. Thus the phenotype (Table 16). If the pituitary gland were able to convert of thyroid hormone resistance includes mild, diffuse T4 to T3 normally, exogenous T4 should have thyromegaly, clinically euthyroid to mildly hypothyroid suppressed TSH. However, only exogenous T3 was able phenotype, elevated T4, FT4, T3, FT3, and high to suppress TSH. The type II 5'-deiodinase converts normal to mildly elevated TSH (Table 18). Resistance T4 to T3 in the pituitary gland. to thyroid hormone results from mutations in the thyroid hormone receptor-beta gene (TRβ) and its Hemangioma expression of type 3 iodothyronine transcribed protein. deiodinase. Hypothyroidism has been reported in an infant with a hemangioma that hypermetabolized T4 Actions of thyroid hormone at the target tissue. In order and T3 [52]. The increased thyroid hormone turnover understand how TRβ mutations cause thyroid (eg, consumption via deiodination) led to thyroid hormone resistance, we must examine the effects of hormone insufficiency and primary hypothyroidism thyroid hormone on the tissues [53,54]. Normally T4 (Table 17). The deficiency of thyroid hormone in such and T3 enter the cytoplasm of the target cell. T4 cases might be analogous to decreased levels of clotting undergoes deiodination to T3 intracellularly. T3 next factors that occur as part of a consumptive enters the nucleus to bind to the thyroid hormone coagulopathy. The patient’s hemangioma and 3 of 5 receptor (TR) [55]. TR can enter the nucleus from other hemangiomas were shown in vitro to express the cytoplasm without binding T3. TR functions as a increased levels of the type 3 iodothyronine deiodinase. nuclear transcription factor (TF). TFs are proteins that regulate gene expression: therefore upon T3 binding, Defects in Tissue Response to Thyroid Hormone TR regulates gene transcription within the cell nuclei. Biologically, TR is a member of the steroid/thyroid Euthyroid hyperthyroxinemia with normal FT4 levels was briefly discussed as the result of abnormalities of Table 18. Defects in tissue response to thyroid hormone: Generalized thyroid hormone resistance [autosomal dominant Table 17. Defects in extrathyroidal thyroid hormone metabolism: (predominantly) and autosomal recessive; thyroid hormone Expression of type 3 iodothyronine deiodinase in infantile receptor β (TRβ) gene; chromosome 3]. hemangiomas (consequence of a somatic tumor). When to consider this disorder: When to consider this disorder: • Euthyroid hyperthyroxinemia with elevated FT4, T4, FT3, • Development of primary hypothyroidism in an infant with T3 a massive hemangioma • TSH high normal or mildly elevated How to diagnose this disorder definitively: • Clinically euthyroid or mildly hypothyroid • Clinical criteria noted above and • Mild goiter common • Expression of type 3 iodothyronine deiodinase in the How to diagnose this disorder definitively: hemangiomas • Clinical criteria noted above and Differential diagnoses: • Sequence TRβ domains I and II to detect TRβ mutation (if • Acquired primary hypothyroidism (eg, lingual thyroid gland, no mutation found, sequence outside of domains I and II) post-thyroidectomy, post-irradiation, antithyroid medical Differential diagnoses: therapy for primary hyperthyroidism, inborn errors in thyroid • Apathetic hyperthyroidism (eg, biochemical hyper- hormone biosynthesis, autoimmune thyroid disease, etc.) thyroidism without the complete clinical picture of hyperthyroidism; can be observed in the elderly) 236 Annals of Clinical & Laboratory Science

Fig. 5. The genes for the thyroid hormone receptor alpha (TR-alpha) and beta (TR-beta) are in boxes. Below each gene are the various mRNAs that can be transcribed: TR-alpha 1 and TR-alpha 2; and TR-beta 1 and TR-beta 2. hormone superfamily. Other members of the steroid/ heterodimers do so with TRs called thyroid receptor thyroid hormone superfamily include the receptors for accessory (auxiliary) proteins (TRAPs). An example of retinoic acid, vitamin D, sex steroids, glucocorticoids, a TRAP is the retinoid X receptor (RXR). and mineralocorticoids. In the normal state when the heterodimer TR- There are two TR genes: TRα and TRβ, TRAP is bound to the TRE in the absence of T3, gene respectively, encoded on chromosomes 17 and 3 (Fig. transcription is inactivated as a co-repressor is bound 5). Alternative splicing of the TRα mRNA produces to the TR-TRAP complex and the basal transcriptional two forms of TRα proteins: TRα1 and TRα2. machinery [57-59] (Fig. 7). Upon T3 binding, the co- Similarly, alternative splicing of the TRβ mRNA repressor leaves the TR-TRAP complex and a co- produces two forms of TRβ proteins: TRβ1 and TRβ2. activator associates with the complex. The co-activator Paradoxically, TRα2 does not bind T3. TRα1, TRα2, can subsequently interact with the basal transcriptional TRβ1, and TRβ2 are expressed to different degrees in machinery to activate gene transcription. various tissues. The sizes of the TRs are given in Table 19 [56]. Table 19. Thyroid hormone receptor (TR) sizes. Typical of TFs, TR proteins display a central DNA binding domain; the carboxy-terminal portion of the Receptor Amino acids kDa TF-TR contains the T3-ligand binding region, the TRα1 410 47 transactivation, and the dimerization domains (Fig. 6). TRα2 490* 55 TF-TR can form homo or heterodimers. TRs bind to TRβ1 461 53 thyroid response elements in the target genes. Thyroid TRβ2 514 ** 58 response elements are part of the regulatory DNA * extended C-terminal region (does not bind T3). region that controls gene transcription. TRs that form ** extended N-terminal region. Review of molecular thyroidology 237

Fig. 6. The intranuclear thyroid hormone receptor functions as a transcription factor with characteristic transactivation, DNA-binding/ dimerization (DBD), hinge, and ligand-binding domains. Homo or heterodimers of the thyroid hormone receptors may form that can bind co-repressors or co-activators.

Fig. 7. Upper left: Thyroid hormone receptors (TRs) that form heterodimers do so with TRs called thyroid receptor accessory (auxiliary) proteins (TRAPs). An example of a TRAP is the retinoid X receptor (RXR). In the normal state when the heterodimer TR-TRAP is bound to the thyroid hormone response element (TRE) in the absence of T3, gene transcription is inactivated as a co-repressor is bound to the TR-TRAP complex and the basal transcriptional machinery (BTM). The BTM is bound to the TATA box. Lower left and upper right: Upon T3 binding, the co-repressor leaves the TR-TRAP complex and a co-activator associates with the TR-TRAP complex. Lower right: The co-activator can subsequently interact with the basal transcriptional machinery to activate gene transcription. 238 Annals of Clinical & Laboratory Science

Fig. 8. Upper and lower left: If a thyroid hormone receptor (TR) homodimer (TR-TR) is bound to the thyroid hormone response element (TRE), T3 binding to TR-TR liberates TR-TR from the TRE. Upper right : Liberation of the TR homodimer allows TR and a thyroid receptor accessory (auxiliary) protein (TRAP) to bind to the TRE. Lower right: With TR-TRAP binding to the TRE, gene transcription can begin once T3 and a co-activator bind.

If a TR homodimer (TR-TR) is bound to the TRE, Thyroid hormone receptor beta mutations and thyroid T3 binding to TR-TR liberates TR-TR from the TRE, hormone resistance. While TRβ mutations cause THR, allowing TR and a TRAP to bind to the TRE (Fig. 8). no TRα mutations have been reported and 10% of With TR-TRAP binding to the TRE, gene trans- families with clinical THR do not exhibit either TRα cription can begin once T3 and a co-activator bind. or TRβ mutations [60]. More than 30 TRβ mutations The various configurations of TRE are given in Table 20. There are a large number of nuclear factors that Table 21. Nuclear Factors Interacting with TRs can interact with the TRs as shown in Table 21. General TFs: TAFII110, TFIIB, TBP Table 20. The thyroid hormone response elements (TRE): Corepressors: N-CoR, SMRT Specific TRE sequences that TRs bind to: Coactivators: GRIP-1 (TIF2), SRC-1, p/CIP, coactivator- binding coactivators (CBP, p300) -—————> —————> TFs: Pit-1 (specific), AP-1 (general)

Direct repeats 5'-AGGTCA N4 AGGTCA-3' TRAPs: RXR, RAR, VDR, PPAR

—————> <—————- TAF: TATA box binding protein associated factor Palindrome 5'-AGGTCA TGACCT-3' TBP: TATA binding protein TF: transcription factor <————— —————> N-CoR: nuclear receptor corepressor

Inverted 5'-TGACCT N4 AGGTCA-3' Pit-1: pituitary TF

palindrome N6 ppar: peroxisome proliferator-activator receptor RAR: retinoic acid receptor

——————> RXR: retinoid X receptor Monomer site 5'-TAAGGTCA-3' SRC-1: protooncogene SRC, Rous sarcoma (chromosome 20q12-q13) N = nucleotide not specified VDR: vitamin D receptor Review of molecular thyroidology 239

Fig. 9. Mutations in the thyroid hormone receptor beta protein are located predominantly in 2 domains: mutant domain 1 and mutant domain 2, although some mutations are located outside of these domains. One mutation in the dimerization domain outside of the mutant domains has been strongly associated with Fig. 11. If the thyroid hormone receptor (TR) is mutated and isolated pituitary resistance. doesn’t bind T3 effectively, there will be decreased release of TR- TR homodimers and decreased transcription of genes normally have been reported including missense (amino acid activated by thyroid hormone. substitution), codon deletion, nonsense (stop), and If TRβ is mutated and either does not bind T3 frameshift mutations. Most TRβ mutations localize efficiently or does not transactivate the TR-TRAP basal to the carboxy terminus in exons 7 through 10 (Fig. transcriptional machinery interaction, the rate of gene 9). Two mutational domains have therefore been transcription will be impeded (Figs. 10, 11). This low described: domain I includes amino acids 310 through rate of gene transcription is overcome only when TSH 347 and domain II encompasses amino acids 438 concentrations rise and stimulate increased thyroid through 461. These regions of the TR-TF are involved hormone secretion to raise intracellular T3 levels in hormone binding and dimerization. The novel sufficiently to increase gene transcription and complete Arg383His mutation that is outside of these domains the negative feedback loop. Readers may recall that appears to be uniquely associated with selective negative feedback of thyroid hormone centrally occurs pituitary resistance as noted above. The autosomal primarily at the level of the pituitary and secondarily dominant nature of THR is explained by the dominant at the level of the hypothalamus. negative effect of TF mutations. Generalized versus selective pituitary resistance to thyroid hormone. In generalized thyroid hormone resistance, TSH rises in response to exogenous TRH administration and TSH falls in response to exogenous T3 administration. Clinical features of generalized thyroid hormone resistance include hyperactivity, learning disabilities, and occasional hearing deficits or deafness. In patients whose free thyroid hormone levels are able to compensate for the resistant state, cholesterol, triglycerides, carotene, and creatine kinase will not be depressed in spite of elevated FT4 and FT3 levels. If the elevations in free thyroid hormone levels are not sufficient to compensate for the resistant state, short stature, delayed bone age, and delayed dentition can be observed as features of mild hypothyroidism. Fig. 10. If the thyroid hormone receptor (TR) is mutated and doesn’t bind T3 effectively, there will be decreased transcription of However, only a minority of thyroid-hormone-resistant genes normally activated by thyroid hormone. patients are hypothyroid. Thyroid hormone resistance 240 Annals of Clinical & Laboratory Science

is inherited predominantly as an autosomal dominant Table 23. Findings suggestive of TSH-producing trait, although autosomal recessive forms have been described. Males and females are equally affected. (1) Failure of TSH to increase appropriately in response to TRH Thyroid hormone receptor beta (TRβ) mutations cause (2) Failure of TSH to decrease in response to supraphysiological doses of thyroid hormone the thyroid hormone resistance syndrome. (3) Increase of the alpha subunit-to-whole-TSH ratio If thyroid hormone resistance occurs only, or predominantly, in the pituitary gland, TSH hyper- secretion produces peripheral hyperthyroidism (Table strengthened by Safer et al [61], when they described 22). In this latter rare case, a TSH-secreting pituitary a case clinically compatible with isolated pituitary adenoma must be excluded as a cause of TSH- resistance and a novel TRβ mutation not observed in dependent hyperthyroidism. In contrast, in generalized generalized thyroid hormone resistance. This R383H resistance to thyroid hormone, both the pituitary and mutation was also located outside domain I and periphery are resistant to the effects of thyroid domain II of TRβ where previously almost all TRβ hormone. The clinical features of TSH-dependent mutations had been identified. hyperthyroidism include typical clinical hyper- thyroidism, diffuse thyromegaly, lack of exophthalmus, Therapy elevated T4, FT4, T3, FT3, high normal to mildly elevated TSH, and absence of thyroid and TSHR Therapy for THR disorders depends upon the autoantibodies. To exclude a pituitary adenoma as the clinical status of the patient. Euthyroid subjects require source of excess TSH, the glycoprotein alpha subunit no treatment and should certainly not be treated with must be measured and a computed tomograph (CT) antithyroid medications. The misdiagnosis of THR or magnetic resonance image (MRI) of the pituitary euthyroid hyperthyroxinemia as hyperthyroidism with must be obtained. In cases of TSH-secreting tumors, subsequent treatment with antithyroid medication has the alpha subunit can be elevated and a mass is induced hypothyroidism. This therapeutic “mis- radiologically observed. If the alpha subunit is not adventure” has produced serious mental and learning elevated and no mass is observed on CT/MRI, selective disabilities in children so treated. If hypothyroid, the pituitary resistance is likely. Furthermore, TSH- affected individual should be administered sodium-l- secreting tumors are unresponsive to exogenous thyroxine until the TSH is suppressed to the normal administration of TRH. The features of TSH-secreting or near normal range and clinical euthyroidism is tumors are summarized in Table 23. There has been achieved. In rare patients with selective pituitary debate whether or not isolated pituitary resistance resistance, typical antithyroid medications such as actually exists. The argument for this disorder was propylthiouracil or methimazole can be prescribed. Overzealous dosing with antithyroid medications should be avoided in patients with selective pituitary Table 22. Defects in tissue response to thyroid hormone: Selective resistance, as the therapy can induce hypothyroidism. pituitary thyroid hormone resistance (sporadic, mode of inheritance unknown; thyroid hormone receptor β (TRβ) gene; chromosome 3). Table 24. Molecular causes of congenital hypothyroidism. When to consider this disorder: • TSH-dependent hyperthyroidism Inborn errors in thyroid hormonogenesis • Absence of anatomic hypothalamic or pituitary lesions • Sodium/iodide symporter mutations • No increase in alpha subunit concentrations • Thyroperoxidase mutations • TSH suppressed by T3 administration • Thyroglobulin mutations How to diagnose this disorder definitively: • Dehalogenase defects • Clinical criteria noted above and • PDS gene mutations β • Sequence TR gene to detect mutation TSH beta subunit mutations (TSH deficiency) Differential diagnoses: TSH Receptor loss-of-function mutations • TSH-secreting pituitary tumor Transcription factor mutations • Depressed intracellular-pituitary thyrotroph conversion of • Thyroid transcription factor-2 mutations T4 to T3 producing TSH-dependent hyperthyroidism • Pax8 mutations Review of molecular thyroidology 241

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