TSH resistance revisited

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1 Endocrine Journal 2015, 62 (5), Review TSH resistance revisited Satoshi Narumi and Tomonobu Hasegawa Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan Abstract. Genetic defects of hormone receptors are the most common form of end-organ hormone resistance. One example of such defects is TSH resistance, which is caused by biallelic inactivating mutations in the TSH receptor gene (TSHR). TSH, a master regulator of thyroid functions, affects virtually all cellular processes involving thyroid hormone production, including thyroidal iodine uptake, thyroglobulin iodination, reuptake of iodinated thyroglobulin and thyroid cell growth. Resistance to TSH results in defective thyroid hormone production from the neonatal period, namely congenital hypothyroidism. Classically, clinical phenotypes of TSH resistance due to inactivating TSHR mutations were thought to vary depending on the residual mutant receptor activity. Nonfunctional mutations in the two alleles produce severe thyroid hypoplasia with overt hypothyroidism (uncompensated TSH resistance), while hypomorphic mutations in at least one allele produce normal-sized thyroid gland with preserved hormone-producing capacity (compensated TSH resistance). More recently, a new subgroup of TSH resistance (nonclassic TSH resistance) that is characterized by paradoxically high thyroidal iodine uptake has been reported. In this article, the pathophysiology and clinical features of TSH resistance due to inactivating TSHR mutations are reviewed, with particular attention to the nonclassic form. Key words: TSH, TSH receptor, TSH resistance, Genetics, Mutation Submitted Mar. 4, 2015; Accepted Mar.5, 2015 as EJ Released online in J-STAGE as advance publication Mar. 21, 2015 Correspondence to: Satoshi Narumi, M.D., Ph.D., Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo , Japan. sat_naru@hotmail.com The thyroid gland produces thyroid hormones (T 3 and T 4 ), which upregulate the metabolism of many types of animal cells. The production of thyroid hormones is regulated by the hypothalamus-pituitary axis. Serum thyroid hormone levels are usually kept constant in narrow ranges, but the hormone levels can be lowered in conditions when hypometabolism is beneficial (e.g., undernutrition and acute illness). The TSH receptor (TSHR) is the only receptor that can transduce the signal of TSH. TSHR is expressed in the basal plasma membrane of thyroid follicular cells. Both activating and inactivating mutations in the TSHR gene (TSHR) have been described [1, 2], and each of them drastically affects the hormone-producing capacity of the thyroid gland. Activating mutations cause ligand-independent activation of TSHR, and result in overproduction of thyroid hormones (i.e., thyrotoxicosis). This type of mutations can occur in a germline cell or a somatic cell, causing congenital hyperthyroidism [3] or functional thyroid nodule [1], respectively. Conversely, inactivating TSHR mutations cause TSH resistance, which results in congenital hypothyroidism [2]. In this review article, we focus on TSH resistance due to inactivating TSHR mutations, and discuss its pathophysiology and clinical phenotypes. Structure and functions of TSHR Structure of TSHR TSHR is a seven-transmembrane G-protein coupled receptor (GPCR). Mammals have more than 1,000 GPCRs, and most of them are odorant receptors [4]. Based on secondary and tertiary structure similarity, TSHR, along with FSH receptor and LHCG receptor, is classified as glycohormone receptor family. TSH, FSH, LH and human chorionic gonadotropin (hcg) have a common alpha subunit, and unique but structurally relevant beta subunits. Some invertebrate animals have only one glycohormone and corresponding one receptor, whereas all vertebrate animals have TSH, FSH, LH and their corresponding receptors. It is believed that gene duplication events during the invertebrate-to-vertebrate transition caused coevolution of the glycohormones and their receptors, although the The Japan Endocrine Society

2 394 Narumi et al. details of coevolution remain largely unknown. It is worth to note that physiological levels of LH or FSH cannot activate TSHR, while hcg can activate TSHR during the first trimester of pregnancy when serum hcg levels become enormous [5]. Glycohormone receptors, including TSHR, have exceptionally large extracellular ligand-binding domain in addition to seven transmembrane domains. The crystal structure of the ligand binding domain of TSHR revealed 10 leucine-rich repeats that are expected to interact with TSH [6]. Crystal structure of the seven transmembrane domain of TSHR has not been analyzed at present. Thus, detailed molecular mechanisms involving the signal transduction, which is triggered by TSH or autoantibodies, are largely unknown. Functions of TSHR TSH can stimulate several cellular responses ultimately increase thyroid hormone production. The responses include active transport of iodine into the thyroid cells, iodination of the thyroglobulin protein, endocytosis of iodinated thyroglobulin protein, and cell proliferation. It has long been known that the Gs/ camp pathway serves the principal role in transducing these cellular responses. Additionally, some observations suggested roles of Gq/Ca 2+ pathway in thyroid hormone production [7], but they had remained controversial until when genetically-engineered mice was analyzed. In 2007, Kero and colleagues generated thyroid-specific double knockout mice lacking protein expression of Gq and G11 in the thyroid [8]. These mice became hypothyroid at several months after birth. The mice have defects in TSH-dependent thyroglobulin iodination and TSH-dependent cellular growth. These data clearly showed that the Gq/Ca 2+ pathway has critical roles in thyroglobulin iodination and growth of thyrocytes. Compensated TSH resistance: the first case of inactivating TSHR mutations Cases with clinically diagnosed TSH resistance have been reported since 1960s [9]. In 1995, the first pedigree of molecularly-confirmed TSH resistance (i.e., biallelic inactivating TSHR mutation carriers) was reported by Sunthornthepvarkaui et al. [2] The proband was the second child of three sisters affected by congenital hypothyroidism. She had a high blood-spot TSH level in newborn screening for congenital hypothyroidism, and came to medical attention. She had a high serum TSH level (47 mu/l), normal thyroid hormone levels, normal thyroidal iodine uptake and normal thyroid morphology on radioiodine scintigraphy. Thyroid function of the elder sister of the proband was evaluated after the diagnosis of the proband, showing that the sister also had compensated hypothyroidism. The younger sister of the proband was diagnosed in the frame of newborn screening as the proband. These three affected siblings commonly had two heterozygous TSHR mutations (Ile167Asn transmitted from the father and Pro162Ala transmitted from the mother). In vitro expression experiments using COS7 cells revealed that loss of function of the two mutant receptors. The Ile167Asn mutant showed abrogated TSH-stimulated camp producing activity, while the Pro162Ala mutant had partial activity. The authors concluded that these in vitro data would explain the phenotype of the patients that was compensated TSH resistance. By 1997, three further papers reporting TSHR mutation carriers with the compensated TSH resistance phenotype were published [3, 10, 11], and TSH resistance due to TSHR mutations were established as a disease entity. Uncompensated TSH resistance: another phenotype In 1998, TSHR mutation carriers with a novel phenotype were reported from Belgium [12]. The probands were affected siblings born from consanguineous parents. Distinct from the other mutation-carrying cases, the patients showed overt congenital hypothyroidism. The initial diagnosis was thyroid aplasia, because no thyroidal radioiodine uptake was observed. However, thyroglobulin could be measured in the sera, indicating the presence of thyroid tissue remnant. The patients were finally diagnosed as severe thyroid hypoplasia based on ultrasonographic images showing the thyroid remnant. Genetic analysis showed a homozygous Ala553Thr mutation in the patients, and both of the parents were heterozygotes. The Ala553Thr mutation showed profound plasma membrane localization defect, when expressed in COS7 cells, and was judged as a null mutation. These data implied the molecular mechanism underlying the uncompensated TSH resistance phenotype of the probands. Since then, similar mutation-carrying cases with nonfunctional mutations were reported [13, 14].

3 TSH resistance revisited 395 Genotype-phenotype correlation To date, more than 60 patients with TSH resistance due to biallelic inactivating TSHR mutations have been reported. Locations of reported inactivating TSHR mutations are shown in Fig. 1. The clinical phenotype of most of those patients can be explained in the frame of genotype-phenotype correlation originally proposed by Tiosano et al. [14]: compensated TSH resistance and uncompensated TSH resistance. In compensated TSH resistance, the thyroid hormone production level is maintained within a normal range by compensatory elevation of serum TSH levels. Thyroid size and thyroidal iodine uptake, which are positively regulated by TSH signaling, usually remain normal. Patients with compensated TSH resistance have at least one mutant allele with residual function (e.g., missense mutations). This form of TSH resistance is relatively frequent. In patients with uncompensated TSH resistance, serum thyroid hormone levels are low despite marked elevation of serum TSH levels. The thyroid gland is severely hypoplastic, resembling thyroid aplasia. Thyroidal iodine uptake is low, not only due to decreased thyroid follicular cell numbers but also due to low iodine uptake activity of each thyroid follicular cell. This form of TSH resistance is usually associated with two nonfunctional alleles, such as nonsense mutations, splice site mutations and frameshift mutations. Frequency of uncompensated TSH resistance seems lower than compensated TSH resistance. Nonclassic TSH resistance TSHR mutation carriers with a paradoxical phenotype In 2007, three consanguineous sib cases with a homozygous TSHR mutation (Leu653Val) were reported [15]. One of three probands had elevated thyroidal 123 I uptake (50% at 24 h; reference 8-40). This clinical phenotype seemed paradoxical, because high thyroidal iodine uptake is usually observed in patients with thyroid dyshoromonogenesis, such as thyroid peroxidase defect and Pendred syndrome, but never observed in patients with TSH resistance. Considering that thyroidal iodine uptake is positively regulated by the TSH signaling, thyroidal iodine uptake is expected to be low to normal in the TSH resistant condition. The patient had slightly small thyroid gland on ultrasonography, and had a normal serum thyroglobulin level, which were not compatible with typical thyroid dyshormonogenesis. Arg450His Leu653Val Fig. 1 Location of inactivating TSH receptor mutations A schematic diagram of TSH receptor (TSHR) protein, showing the location of inactivating mutations. TSHR has 10 leucine-rich repeats in the extracellular ligand-binding domain. The receptor also has a seven transmembrane domain, which is a common structure of G-protein coupled receptors, and intracellular tail in the carboxyl terminal. In this figure, inactivating mutations are classified into two groups: truncating mutations (nonsense, frameshift or splice site mutations) and missense mutations. Inactivating mutations are seen across the receptor and this is contrasting to activating mutations that are usually found in the seven transmembrane domain. An exception is the C-tail region, in which no inactivating mutations have been reported. Arrows indicate the location of the Arg450His and Leu653Val mutations that cause Gq-dominant coupling defect. Note that Arg450His and Leu653Val are the only missense mutations detected in the first intracellular loop and the third extracellular loop, respectively. To test whether TSHR mutation carriers with high iodine uptake are observed in a cohort of Japanese patients with congenital hypothyroidism, we sequenced TSHR in 24 congenitally hypothyroid patients that had high thyroidal 123 I uptake [16]. As a result, we found two unrelated TSHR mutation carrying patients ([Thr145Ile];[Arg450His] and [Arg450His];[Ile661Asnfs*10]) that had high 123 I uptake (41.8 and 43.0%). These two patients had slightly hypoplastic thyroid gland on ultrasonography as the original case described by Grasberger et al. Serum thyroglobulin levels were slightly elevated (150 and 210 ng/ml;

4 396 Narumi et al. A Physiological state B (compensated) (uncompensated) Nonclassic TSH resistance Fig. 2 Schematic diagrams of classic and nonclassic TSH resistance A, In the physiological state, TSH receptor (TSHR) activates two G-proteins (Gs and Gq) when the receptor is stimulated by TSH. The Gs/cAMP pathway mediates thyroidal iodine uptake, cell growth and thyroglobulin iodination, while the Gq/Ca 2+ pathway mediates cell growth and thyroglobulin iodination. Clinical parameters, [radioiodine uptake (RAIU), thyroid size and perchlorate discharge rate] reflect the activities of these cellular processes. Note that cell growth and thyroglobulin iodination are regulated by both Gs and Gq, whereas thyroidal iodine uptake is solely regulate by Gs. B, Left panel: In classic TSH resistance, coupling to Gs and Gq is equally affected. If the mutant receptor has significant residual function, elevated TSH stimulation can compensate the downstream signaling pathways (classic compensated TSH resistance). Center panel: If the residual function of mutant receptor is negligible, elevated TSH stimulation cannot compensate the downstream signaling pathways (classic uncompensated TSH resistance). Right panel: Nonclassic TSH resistance is characterized by Gq-dominant coupling defect. In this condition, cell growth and thyroglobulin iodination, which are regulated by Gq, are affected. However, thyroidal iodine uptake, which is solely regulated by Gs, is exaggerated. reference <30), but were lower than those seen in typical thyroid dyshormonogenesis cases (>1,000 ng/ml). These genetic screening data clearly supported the presence of TSH resistance with the paradoxical phenotype (elevated 123 I uptake in TSH resistance) in a cohort of congenital hypothyroidism, and we termed the new disease entity as nonclassic TSH resistance. Molecular basis of nonclassic TSH resistance In vitro functional analyses of the Leu653Val mutation revealed that the mutant protein had profound defect in coupling to Gq, while coupling to Gs was only mildly affected [15]. This characteristic molecular property (Gq-dominant coupling defect) was also observed in the Arg450His mutation [16], which the two Japanese patients with nonclassic TSH resistance had. A computational modeling study suggested that TSHR contacts with the alpha-subunit of Gq (Gqα) at the first intracellular loop, and the Arg450 residue contacts with Gqα directly [17]. Thus, mutations of the Arg450 residue would result in Gq-dominant coupling defect. Thyroidal iodine uptake seems regulated solely by the Gs pathway, because thyroid-specific Gq/G11 double knockout mice had normal thyroidal iodine uptake. Therefore, if a patient had a TSHR mutation with Gq-dominant coupling defect and had compensatory serum TSH elevation, cellular responses that are solely regulated by Gs (e.g., iodine uptake) can be overcompensated, while Gq-dependent cellular responses remain deteriorated (Fig. 2).

5 TSH resistance revisited 397 Table 1 Classification of TSH resistance Uncompensated Compensated Serum TSH level (mu/l) >50 mu/l mu/l >40 mu/l Serum thyroid hormone levels Very low Normal Low Thyroid size Very small Normal Slightly small Thyroidal iodine uptake Low Normal High TSHR mutation Null mutations a in two Hypomorphic mutation b alleles in at least one allele a Null mutations include nonsense mutations, frame shift mutations and splice site mutations. b Most missense mutations have residual receptor functions in vitro, and thus are hypomorphic. c At present, two mutations (Arg450His and Leu653Val) were shown to have the defect. Nonclassic TSH resistance Mutation with Gq-dominant coupling defect c in at least one allele Nonetheless, one question remained to be answered. The Arg450His mutation is common in Japan, accounting for about 70% of total inactivating TSHR mutations in the population [18]. However, except for our two cases [16], no Arg450His mutation carriers with high thyroidal iodine uptake have been reported, implying that nonclassic TSH resistance phenotype is not solely caused by the presence of Arg450His mutation. We pointed out that the emergence of the nonclassic phenotype requires an additional factor: high serum TSH levels [16]. TSHRs with Gq-dominant coupling defect have partial resistance to Gs, thus overcompensation of the Gs pathway requires high TSH levels. Relationship between thyroidal 123 I uptake and serum TSH levels among TSHR mutation carriers suggests a threshold level of about 40 mu/l for the emergence of nonclassic TSH resistance [16]. Because patients with homozygous for Arg450His typically have mild elevation of serum TSH levels (10-20 mu/l), their 123 I uptake levels would remain within the normal range. Conclusions Most of TSH resistance cases are classic type, and the thyroid phenotypes (hormone producing capacity, gland size, iodine uptake) are determined by the residual mutant receptor activity (Table 1). However, TSHR mutations with Gq-dominant coupling defect can cause TSH resistance with paradoxically high thyroidal iodine uptake (Table 1). Physicians should remember that high thyroidal iodine uptake does not necessarily exclude the possibility of TSHR mutations. Key features to suspect nonclassic TSH resistanece among patients with high thyroidal iodine uptake include thyroid morphology (slightly hypoplastic) and serum thyroglobulin levels (only mildly elevated). There are a plenty of G-protein coupled receptors associated with congenital endocrine disorders, including receptors for FSH, LH, GHRH and AVP etc. Theoretically, mutations that affect specific subtype of G protein can occur in those other receptors than TSHR. Such G-protein subtype-specific coupling defects are likely rare, but would be important for clarifying signaling pathways of endocrine cells. Acknowledgments The authors thank Prof. Takao Takahashi for fruitful discussion. This work was supported by a Grantin-Aid for Scientific Research (C) ( ) from the Japan Society for the Promotion of Science. Disclosure The authors have no financial relationships relevant to this article to disclose. References 1. Parma J, Duprez L, Van Sande J, Cochaux P, Gervy C, et al. (1993) Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365: Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S (1995) Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 332: de Roux N, Misrahi M, Brauner R, Houang M, Carel JC, et al. (1996) Four families with loss of function mutations of the thyrotropin receptor. J Clin Endocrinol Metab 81:

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