The Clinical Spectrum of Thyrotropin Receptor Gene (TSHR) Mutations

Resistance to thyrotropin (RTSH) is a condition in which thyroid cells show reduced sensitivity to TSH. This condition is characterized by elevated serum TSH concentration, a normal or hypoplastic thyroid gland and normal to very low levels of thyroid hormones. Loss-of-function mutations in the TSH receptor gene (TSHR) lead to RTSH syndrome, presenting with either congenital hypothyroidism (CH) or subclinical hypothyroidism (Beck-Peccoz et al., 2006; Refetoff, 2003).


Introduction
Resistance to thyrotropin (RTSH) is a condition in which thyroid cells show reduced sensitivity to TSH. This condition is characterized by elevated serum TSH concentration, a normal or hypoplastic thyroid gland and normal to very low levels of thyroid hormones. Loss-of-function mutations in the TSH receptor gene (TSHR) lead to RTSH syndrome, presenting with either congenital hypothyroidism (CH) or subclinical hypothyroidism (Beck-Peccoz et al., 2006;Refetoff, 2003).

Gain-of-function mutations in TSHR
Germline gain-of-function mutations result in non-autoimmune hyperthyroidism, whereas somatic mutations that constitutively activate TSHR result in toxic thyroid nodules. Hyperthyroidism caused by germline mutations in TSHR exhibits autosomal dominant inheritance. The mutations are located mainly in exon 10, which encodes the transmembrane region and intracellular tail that constitutively activate TSHR. The phenotype of these patients is characterized by hyperthyroidism with the presence of goiter but the absence of ophtolmopathy, and a lack of thyroid autoantibodies as well as of lymphocytic infiltration in thyroid histology (Van Sande et al., 1995). The clinical spectrum of phenotypes is variable and onset can occur anywhere from birth to adulthood. The presence of either congenital or adulthood-onset hyperthyroidism, multinodular goiter (MNG) and follicular carcinoma has been reported in the same family (Karges et al., 2005). To date, more then 55 germline gainof-function mutations have been reported, about 14 of them sporadic and the others with familial occurrence (http: www.hgmd.cf.ac.uk/ac/gene.php?gene=TSHR) (Akcurin et al., 2008;Davies et al., 2005;Farid et al., 2000;Führer et al., 1997(b);Holzapfel et al., 1997;Karges et al., 2005;Khoo et al., 1999;Tonacchera et al., 1996;Van Sande et al., 1995). Hyperthyroidism in affected individuals is often resistant to the conventional treatment used in Graves' disease, and either radiotherapy or total thyroidectomy is required.

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The Clinical Spectrum of Thyrotropin Receptor Gene (TSHR) Mutations 297 Autonomous benign and malignant toxic thyroid nodules have been shown to result from a variety of somatic mutations leading to constitutive activation of TSHR and affecting cell proliferation and cell function. Somatic TSHR mutations commonly occur in the transmembrane and ECL domains, but hot spots are the sixth transmembrane domain and the third ICL where the receptor interacts with G-proteins. Toxic adenoma due to TSHRactivating mutations may occur in infancy (Kohn et al., 2009) or even in utero (Kopp et al., 1997) (OMIM#2603372) (Davies et al., 2005;; Kohn et al., 2009). To date, about 25 different somatic TSHR-activating mutations have been reported manifesting with toxic adenoma, MNG and toxic thyroid carcinoma.

Loss-of-function mutations in TSHR
About 50 different loss-of-function mutations have been described in TSHR (Table 1). Affected individuals are either homozygous, compound heterozygous or heterozygous. The degree of insensitivity to TSH depends on the type and location of the TSHR mutation; more severe loss of TSHR function manifests as CH, whereas mild mutations present with euthyroid hyperthyrotropinemia or subclinical hypothyroidism. When both alleles carry mutated receptors with complete lack of function, the result is severe hypothyroidism, commonly presenting at birth, whereas carriers of a mutation on one allele present with compensated hyperthyrotropinemia. The thyroid gland is hypoplastic or invisible in a 99m TC scan; however, in ultasonographic imaging, the gland is shown to be in a normal position and commonly of small size. TSHR mutations are distributed all along the receptor. Mutations located in the binding domain result in reduced binding capacity or decreased membrane expression of the receptor. The third ECL and the seventh intracellular domain of TSHR are hot spots for gain-of-function mutations, but some inactivating mutations have been identified in this domain as well (Alberti et al., 2002;Grasberger et al., 2007;Tiosano et al., 1999) (Fig. 1).

Prevalence of loss-of-function mutations
The exact prevalence of inactivating TSHR mutations is not known. A prevalence of 4.3% biallelic TSHR mutations was found among 134 Japanese infants with CH (Narumi et al., 2009). Among 38 children with non-autoimmune subclinical hypothyroidism, 11 (29%) were carriers of TSHR mutations (Nicoletti et al., 2009). A prevalence of 12% TSHR mutations was shown in 42 subjects with non-autoimmune isolated hyperthyrotropinemia in Italy; all were with familial occurrence (Tonacchera et al., 2004). Camilot et al. (2005) identified 13 patients with heterozygous mutations (11%) out of 116 pediatric patients with asymptomatic euthyroid hyperthyrotropinemia. A rate of 0.6% for carriers of W546X-mutated TSHR was identified in Welsh euthyroid individuals (Jordan et al., 2003). We found up to 2.4% carriers of two known mutations in a highly consanguineous population in the northern region of Israel (Tenebaum-Rakover et al., 2009). Moreover, the coexistence of two different novel mutations of TSHR in each of two separate clans has been shown (Sriphrapradang et al., 2011). In view of these data, it may be speculated that the occurrence of inactivating TSHR mutations in certain populations is not so rare, and therefore screening for TSHR mutations is indicated in cases with non-autoimmune subclinical hypothyroidism in those populations.

Clinical characteristics
Loss-of-function mutations manifest with a variable clinical spectrum of phenotypes. Severe uncompensated RTSH presents with CH, partially compensated RTSH manifests with subclinical hypothyroidism, and fully compensated RTSH presents with euthyroid hyperthyrotropinemia or even normal thyroid function. The diagnosis of TSHR defect is based on the absence of thyroid antibodies, a lack of goiter, measurable serum thyroglobulin, and familial occurrence of hyperthyrotropinemia or hypothyroidism. CH is commonly detected by TSH-based neonatal screening but may missed by total T 4 (TT 4)based screening since, in many cases, TT 4 levels are within the normal range at birth ( Table  1). The degree of CH is variable and depends on the genotype. Severe forms manifest as overt CH (Bretones et al., 2001;Gagne et al., 1998;Jeziorowska et al., 2006;Park et al., 2004;Tonacchera et al., 2000), moderate forms as hypothyroidism identified by neonatal screening without clinical symptoms of hypothyroidism (Abramowicz et al., 1997;Jordan et al., 2003), and mild forms present with hyperthyrotropinemia and normal thyroid hormones (de Roux et al., 1996;Nagashima et al., 2001;Narumi et al., 2009;Tenenbaum-Rakover et al., 2009). Gagne et al. (1998) described a case of CH with persistent neonatal jaundice, myxedematous facies, large fontanelle and absence of ossification centers of the knee on x-rays, indicating severe prenatal deficiency of thyroid hormone. Most of the described cases of CH are detected by neonatal screening with elevated TSH and normal TT 4 levels, but without any www.intechopen.com The Clinical Spectrum of Thyrotropin Receptor Gene (TSHR) Mutations 299 clinical symptoms or signs of hypothyroidism (de Roux et al., 1996;Tenenbaum-Rakover et al., 2009). Nevertheless, L-T 4 therapy is initiated in most cases to prevent future consequences of untreated CH. At the age of 2 to 3 years, when L-T 4 is withdrawn, thyroid hormones remain low in the severe mutations (Abramowicz et al., 1997;Biebermann et al., 1997;Tonacchera et al., 2000); however in milder mutations, despite extremely elevated TSH, thyroid hormone levels are normal, indicating compensated hypothyroidism (Clifton-Bligh et al., 1997;Tenenbaum-Rakover et al., 2009). 99m TC scan commonly reveals a normal or hypoplastic gland but in some cases, an absence of thyroid gland has been demonstrated, suggesting thyroid agenesis ( Table 1). The presence of detectable thyroglobulin as well as the demonstration of a thyroid gland in the normal position in ultrasonographic imaging exclude thyroid agenesis and indicate a diagnosis of RTSH. In a few reports, an enlarged thyroid gland has been described (de Roux et al., 1996;Grasberger et al., 2007). Inactivating TSHR mutations at older ages present with either subclinical hypothyroidism or euthyroid hyperthyrotropinemia without thyroid autoantibodies. The affected patients are commonly identified by routine laboratory tests and are asymptomatic. Most of the described cases are heterozygous for TSHR mutations, but biallelic mutations have been reported as well (Kanda et al., 2006;Russo et al., 2000;Sriphrapradang et al., 2011;Tenenbaum-Rakover et al., 2009;Tonacchera et al., 2001Tonacchera et al., , 2007.

Mechanism of loss-of-function mutations
The mechanism leading to loss-of-function of TSHR includes abnormal binding affinity, abnormal receptor synthesis, accelerated degradation, defective receptor targeting to the cell membrane and abnormal signal transduction (Tao, 2006). Mutations may exert their activity by causing protein misfolding, misassembly or aberrant oligomerization. Loss-of-function mutations are located all along the TSHR (Biebermann et al., 2010) (Fig 1). The function of TSHR is assessed in vitro by cAMP response, IP accumulation, TSH binding and cell-surface expression of the mutated receptor. The analysis is performed with COS-7 cells transfected with the mutant receptor. Each mutation has a different effect on binding capacity, membrane expression and cAMP and IP accumulation, depending on its type and location along the TSHR. In in-vitro studies, it has been shown that TSHR mutations differ in their effect on the Gs and Gq pathways, which may lead to more severe loss of one pathway compared to the other (Claus et al., 2005). The third ECL represents an important domain for intermolecular TSHR signal transduction and single amino acids play different roles in receptor folding and cAMP and IP signaling (Claus et al., 2005). We identified a biallelic L653V mutation located in the third ECL in three sisters presenting with marked hyperthyrotropinemia and increased thyroid radioiodine uptake (Grasberger et al., 2007). Normal ligand binding, slightly reduced cell expression and mildly reduced basal and stimulated cAMP accumulation with markedly reduced IP formation were found in in-vitro studies using transfected COS-7 cells. These in-vitro findings explained the phenotype of the affected subjects manifesting compensated hyperthyrotropinemia concomitant with increased iodide uptake, and this was the first report to provide in-vivo evidence of the important role of the IP/Ca +2 pathway in the regulation of thyroid hormone synthesis. Narumi et al. (2011) recently reported two patients with CH and high iodide uptake harboring biallelic TSHR mutations (R450H+T145I in one and R450H+I166fs in the other), supporting our previous findings. They termed this apparently discrepant phenotype nonclassic TSH resistance.

Heterozygosity for loss-of-function mutations
Heterozygous subjects carrying loss-of-function mutations in TSHR are commonly mildly affected, presenting with euthyroid hyperthyrotropinemia but with variable expressivity (Camilot et al., 2005;Sriphrapradang et al., 2011;Tenenbaum-Rakover et al., 2009). Heterozygotes are typically diagnosed with slightly increased TSH but normal free T 4 (FT 4 ) levels. At least one case of neonatal hypothyroidism has been reported as well (Camilot et al., 2005). In in-vitro models expressing the combination of wild-type and mutated TSHR, it has been shown that basal and TSH-stimulated cAMP production are reduced compared to cells transfected with wild-type receptor, albeit less severely than in biallelic mutations.

Genotype-phenotype association
The phenotype of the affected subjects correlates with the severity of the mutation, which is dependent on its location and type, and whether it is mono-or biallelic. Most of the described cases reveal a direct association between the severity of the mutation and the phenotype, which is reflected by the extent of increase in TSH and decrease in FT 4 levels. The more severe mutations manifest in infancy with persistent CH, while the mild monoallelic mutations manifest as asymptomatic mild hyperthyrotropinemia. We identified 33 subjects carrying two novel TSHR gene mutations (P68S and L653V) in a large consanguineous kindred occurring as homozygous L653V (5 subjects), heterozygous P68S (4 subjects), heterozygous L653V (20 subjects), and compound heterozygous L653V/P68S (4 subjects). Our finding in a large cohort of affected members enabled us to assess the genotype-phenotype association. All homozygotes and compound heterozygotes presented with compensated RTSH, 9 out of 24 heterozygotes showed mild hyperthyrotropinemia and the others had normal TSH values. The clinical results were supported by in-vitro studies in which the L653V-mutated TSHR resulted in more severely impaired signal transduction than the other genotype combinations. However, large variability was found to exist between affected members. Among those with the homozygous L653V mutation, one child had CH and the other four, aged 3 to 20 years, had markedly elevated TSH, but FT 4 levels were within the normal range; among the heterozygous members for the two different mutations, variable hyperthyrotropinemia was observed, with a few of the affected subjects showing normal thyroid function (Tenenbaum-Rakover et al., 2009).

Outcome
Despite several reports of patients affected with TSHR mutations, there are limited data on the long-term outcome of this condition. In subjects with TSHR mutations, it has been shown that TSH levels remain stable and they do not develop hypothyroidism; in contrast, in autoimmune thyroid disease (AITD), overt hypothyroidism commonly develops over the years. In our abovementioned large cohort of affected family members, cross-sectional analysis showed neither a decrease nor an increase in TSH levels with age, suggesting stable compensated RTSH with an appropriately adjusted set point of pituitary-thyroid feedback (Tenenbaum-Rakover et al., 2009). In contrast to subclinical hypothyroidism in the context of AITD, the thyroidal compensation in mild to moderate RTSH is expected to be clinically stable with no progression toward true hypothyroidism or spontaneous regression toward normal TSH levels. Patients with homozygous or compound heterozygous mutations who are detected in infancy by neonatal screening to have CH may have normal FT 4 levels despite elevated TSH levels after L-T 4 withdrawal and in these patients, L-T 4 replacement may not be needed. In contrast, development of overt hypothyroidism at the age of 15 years was shown in a patient homozygous for the R540H mutation who presented with compensated hypothyroidism in infancy (Mizuno et al., 2009), but not in an additional four subjects with the same genotype after long-term follow-up. Asymptomatic heterozygotes for TSHR mutations have normal or slightly elevated TSH levels with negative thyroid antibodies (Camilot et al., 2005). However, coexistence of thyroid autoantibodies has been reported in some cases of compensated RTSH, leading to overt hypothyroidism (Tonacchera et al., 2001). It is possible that carriers of TSHR mutations are at increased risk for AITD. TSHR is involved in AITD, TSH-stimulating autoantibodies in Graves' disease and TSH-blocking antibodies in Hashimoto thyroiditis. Therefore, it has been speculated that modification of TSH structure by the mutated receptor may lead to AITD (Tonacchera et al., 2001). Fluctuation of TSH levels from slightly above normal to normal values have been observed in some cases by us and others (Tenenbaum-Rakover et al., 2009;Tonacchera et al., 2001). In view of the variability in outcome among affected individuals, careful long-term follow-up is recommended.

Treatment
The question of whether to treat patients with TSHR mutations with L-T 4 is a matter of debate (Utiger, 1995). In cases with loss-of-function mutations in TSHR presenting with CH, early initiation of L-T 4 therapy is recommended to prevent late-effect consequences of hypothyroidism as in other etiologies of CH. However, withdrawal of L-T 4 at the age of 2 to 3 years revealed transient hypothyroidism in some cases, putting the need for lifelong replacement therapy into question (Alberti et al., 2002;Tenenbaum-Rakover et al., 2009). Euthyroid hyperthyrotropinemia caused by TSHR mutations with mild to moderate loss of function maintains stable compensated RTSH and may not necessitate thyroid hormone replacement. Moreover, most patients with RTSH do not present with symptoms of hypothyroidism or with biochemical parameters of uncompensated hypothyroidism, such as elevated CPK and liver enzymes and hyperlipidemia (Tenenbaum-Rakover et al., 2009). The presence of normal FT 4 levels argues against the need for replacement treatment, especially when inadvertent overtreatment, producing subclinical hyperthyroidism, can have undesirable effects (Samuels et al., 2008). In our long experience, no clinical benefit has been observed with L-T 4 therapy. Contrasting with this approach, it has been shown that some subjects with RTSH have a slight decrease in FT 4 levels compared to controls, although remaining within the normal range, which may point to subclinical hypothyroidism in these affected patients. In addition, the possibility of secondary pituitary enlargement in patients with extreme hyperthyrotropinemia may support L-T 4 replacement therapy. In view of the variability of phenotypes in different types of mutations, as well as between individuals with the same genotypes, it is recommended that careful follow-up and cautious administration of L-T 4 be considered based on individual thyroid hormone levels in the clinical context.

Differential diagnosis
The diagnostic work-up of RTSH should exclude PAX8 mutations, which are characterized by thyroid dysgenesis associated with kidney abnormalities (Grüters et al., 2003;Park & Chatterjee, 2005), and mutations in guanine nucleotide binding subunit 1 (GNAS1), which encodes Gsα subunit and causes pseudohypoparathyroidism (PHP) type Ia. The latter inactivating mutations in Gsα lead to a syndrome of resistance to multiple hormones, including TSH (Mantovani et al., 2002). Another form of RTSH is an autosomal dominantly inherited disease characterized by euthyroid hyperthyrotropinemia, for which the specific gene has not yet been identified. This condition has been linked to a locus on chromosome 15q25.3-26.1 (Grasberger et al., 2005(b)). In many of the cases with clinical characteristics of RTSH, no mutations have been found in TSHR, suggesting that additional genes are involved in RTSH syndrome (Xie et al., 1997). Bigenic defects in thyroid-synthesis pathways have been recently described. Coexistence of mutations in TPO (Sriphrapradang et al., 2011) and GNAS (Lado-Abeal et al., 2011), in addition to mutations in TSHR, has been reported in the same individuals. In those reports, the coexistence of mutated TPO and TSHR in the same individuals belonging to the same kindred did not aggravate the severity of the RTSH phenotype (Sriphrapradang et al., 2011); similar observations were made for the presence of a monoallelic TSHR mutation coexisting with a GNAS mutation (Lado-Abeal et al., 2011). It is therefore suggested that in cases where TSHR mutations do not explain the phenotype, additional genes that are involved in thyroid hormone synthesis be screened. RTSH must be differentiated from AITD (Ross, 2000), the most common cause of subclinical hypothyroidism in the adult population. The presence of autoantibodies as well as a typical hypoechogenic pattern of the thyroid in ultasonographic imaging support the diagnosis of AITD. This is important from a clinical standpoint since in RTSH, hyperthyrotropinemia is almost always stable while in AITD, hypothyroidism develops with time in about 30% of the cases.

Conclusion
To date, about 50 different TSHR mutations have been reported presenting with a spectrum of phenotypes ranging from overt CH to mild euthyroid hyperthyrotropinemia. Subjects with euthyroid hyperthyrotropinemia commonly have stable TSH levels and do not develop overt hypothyroidism with time. The phenotype correlates with the genotype as the latter is reflected by the severity of hyperthyrotropinemia and the decrease in FT 4 levels. Screening for TSHR mutations should be considered in individuals with apparent non-autoimmune subclinical hypothyroidism. In view of the variability in phenotypes and in outcome among individuals in this condition, careful long-term follow-up is recommended and replacement www.intechopen.com

Acknowledgments
Thanks to Camille Vainstein for professional language editing, to Dr. Zaki Kraiem for fruitful discussion and to Dr. Morad Khayat for useful assistance.