Novel TSHR germline mutation (Met463Val) masquerading as Graves' disease in a large Welsh kindred with hyperthyroidism.

Hereditary nonautoimmune hyperthyroidism is caused by activating germline mutations in the thyrotropin (TSH) receptor (TSHR) gene. We describe an extended Welsh kindred with toxic thyroid hyperplasia affecting 8 family members in three generations and a history consistent with thyrotoxicosis in a further three generations now deceased. A novel heterozygous germline mutation (ATG --> GTG; Met463Val) was identified in the second membrane spanning TSHR region in 6 relatives with thyrotoxicosis and goiter and absence of TSHR antibodies. Screening of 5 additional family members led to the identification of 2 siblings with the mutation, who were asymptomatic at the time, although subsequent thyroid function tests in these children showed suppressed serum TSH and increased serum free triiodothyronine (FT3) and free thyroxine (FT4) concentrations. Functional studies of the novel TSHR germline mutation demonstrated a constitutive activation of the cAMP pathway, which in the thyroid controls both thyroid hormone production and stimulation of thyroid growth. The molecular diagnosis in this family has clinical implications: genetic counseling is required, and primary thyroid ablation should be advocated as the preferred treatment in the patients with the constitutively activating TSHR germline mutation.     

A novel thyrotropin receptor germline mutation (Asp617Tyr) causing hereditary hyperthyroidism

Constitutively activating germline mutations of the thyrotropin receptor (TSHR) gene have been identified as a molecular cause of hereditary nonautoimmune hyperthyroidism. We describe here a Japanese kindred with two affected individuals who showed overt hyperthyroidism and mild goiter in the absence of TSHR antibodies. A novel heterozygous germline point mutation, identified in both individuals, resulted in an amino acid substitution of aspartic acid for tyrosine at codon 617 (Asp617Tyr) in the third intracellular loop of the TSHR. Screening of 7 additional family members led to the identification of the same mutation in 4 relatives: 1 had undergone thyroidectomy due to hyperthyroidism but 3 were asymptomatic with subclinical hyperthyroidism. In vitro functional studies of the Asp617Tyr TSHR demonstrated a constitutive activation of the cyclic adenosine monophosphate pathway, but not of the inositol phosphate cascade, with data similar to those of Asp619Gly, the first constitutively activating mutant TSHR identified. Treatment with inorganic iodine for 7 months successfully relieved all symptoms of hyperthyroidism in both patients.     

Interactions between the extracellular domain and the extracellular loops as well as the 6th transmembrane domain are necessary for TSH receptor activation

OBJECTIVE: The molecular mechanisms of TSH receptor (TSHR) activation and intramolecular signal transduction are largely unknown. Deletion of the extracellular domain (ECD) of the TSHR results in increased constitutive activity, which suggests a self-inhibitory interaction between the ECD and the extracellular loops (ECLs) or the transmembrane domains (TMDs). To investigate these potential interactions and to pursue the idea that mutations in the ECD affect the constitutive activity of mutants in the ECLs or TMDs we generated double mutants between position 281 in the ECD and mutants in all three ECLs as well as the 6th TMD. DESIGN: We combined mutation S281D, characterized by an impaired TSH-stimulated cAMP response, with the constitutively activating in vivo mutations I486F (1st ECL), I568T (2nd ECL), V656F (3rd ECL) and D633F (6th TMD). Further, we constructed double mutants containing the constitutively activating mutation S281N and one of the inactivating mutations D474E, T477I (1st ECL) and D633K (6th TMD). RESULTS: The cAMP level of the double mutants with S281N and the inactive mutants in the 1st ECL was decreased below the level of the inactive single mutants, demonstrating that a constitutively activating mutation in the ECD cannot bypass disruption of signal transduction in the serpentine domain. In double mutants with S281D, basal and TSH-induced cAMP and inositol phosphate production of constitutively active mutants was reduced to the level of S281D. CONCLUSION: The dominance of S281D and the dependence of constitutively activating mutations in the ECLs on the functionally intact ECD strongly suggest that interactions between these receptor domains are required for TSHR activation and intramolecular signal transduction.     

A novel activating mutation in transmembrane helix 6 of the thyrotropin receptor as cause of hereditary nonautoimmune hyperthyroidism.

Constitutively-activating germline mutations of the thyrotropin receptor (TSHR) gene are very rare and are considered the cause of hereditary nonautoimmune hyperthyroidism. We describe four affected individuals from a Caucasian family: a mother and her three children, and an unaffected father. The mother and her first two children presented in a similar manner: lifelong histories of heat intolerance, hyperactivity, fast heart rate, reduced energy, increased appetite, and scrawny build. They all developed goiter in childhood and showed a suppressed TSH and elevated thyroxine (T(4)). The last child, a 12-year-old female, presented with no clinical symptoms or palpable neck mass, but with a suppressed TSH, elevated T(4) and thyromegaly detected by ultrasound. Mutation analysis of the TSHR gene in all family members revealed a novel heterozygous germline mutation resulting in the substitution of phenylalanine (TTC) by serine (TCC) at codon 631 in transmembrane helix 6 in the mother and all three children. Functional characterization of this germline mutation showed constitutive activation of the G(s)-mediated cyclic adenosine monophosphate (cAMP) pathway, which controls thyroid hormone production and thyroid growth. Molecular characterization of F631S demonstrates that this activating mutation plays a key role in the development of hereditary hyperthyroidism in this family although the timing of onset of clinical manifestations in the subjects may depend on other, as yet unidentified, factors.     

Novel thyrotropin receptor germline mutation (Ile568Val) in a Saxonian family with hereditary nonautoimmune hyperthyroidism.

OBJECTIVE: Hereditary nonautoimmune hyperthyroidism is caused by activating thyrotropin receptor (TSHR) germline mutations. We describe a family from Saxony, Germany, with this condition. Recurrent thyrotoxicosis and goiter were prevalent in three generations, affecting in addition to the 16-year-old index patient, her father and late paternal grandmother. Hyperthyroidism in the girl was remarkable for its poor response to methimazole (40-60 mg/d) treatment. METHODS AND RESULTS: Molecular analysis of genomic DNA extracted from peripheral blood leucocytes showed a TSHR germline mutation in the girl and her father. This mutation results in a new amino acid exchange of valine for isoleucine in TSHR codon 568 (Ile568Val). Only the wild-type TSHR sequence was found in the girl's mother. On functional characterization in COS-7 cells, the novel Ile568Val TSHR mutation conferred constitutive activation of the cAMP pathway (2- to 3-fold increase of basal cyclic adenosine monophosphate [cAMP]), but not of the inositol phosphate cascade. As a consequence of the molecular findings, total thyroidectomy has been performed in the girl. She is now euthyroid on levothyroxine replacement therapy. CONCLUSION: This is the second Saxonian family with autosomal-dominant nonautoimmune hyperthyroidism, adding to a total of 13 families and 11 individuals with activating TSHR germline mutations worldwide. We suggest that the condition may indeed be more frequent than previously thought and that consequent assessment of a family history in children as well as adults with thyroid autonomy will contribute to ensure correct diagnosis and adequate treatment of patients with activating TSHR germline mutations.     

Sporadic congenital hyperthyroidism due to a germline mutation in the thyrotropin receptor gene (Leu 512 Gln) in a Japanese patient

Constitutively activating thyrotropin receptor (TSHR) germline mutations have been identified as a molecular cause of congenital hyperthyroidism. We here describe a Japanese woman who had presented with severe hyperthyroidism and advanced bone age as a neonate. She underwent neurosurgical intervention for craniosynostosis, and presented with perodactylia and mild mental retardation with hydrocephalus. Hyperthyroidism has been refractory to antithyroid drug therapy in the absence of antithyrotropin receptor antibodies during follow-up of 20 years, resulting in an enlarged goiter. Analysis of the patient's genomic DNA showed a heterozygous thymine-to-adenine point mutation in exon 10 of TSHR at position 1535 which was not present in the parents' DNA. This mutation, changing leucine to glutamine in codon 512 in the third transmembrane region, was previously identified as a somatic mutation in toxic thyroid nodules and was shown to increase basal cAMP production in vitro. To our knowledge, this is the first report of a germline mutation of TSHR causing sporadic congenital nonautoimmune hyperthyroidism in a Japanese patient.     

The first activating TSH receptor mutation in transmembrane domain 1 identified in a family with nonautoimmune hyperthyroidism

Sporadic and familial nonautoimmune hyperthyroidism are very rarely occurring diseases. Within the last years constitutively activating TSH receptor mutations were identified as one possible pathomechanism. Except for S281N in the extracellular N-terminal domain, all other germline mutations are located in the transmembrane domains 2, 3, 5, 6, and 7 of the TSH receptor, whereas no mutation was reported in transmembrane domains 1 and 4 to date. Here we report the first family with a constitutively active TSHR mutation in transmembrane domain 1 resulting in a substitution of the conserved Gly(431) for Ser. This mutation was found in the investigated patient, his father, and the paternal grandmother. As known from other familial cases of nonautoimmune hyperthyroidism, the age of onset of the disease was variable, ranging from early childhood in the patient and his father to adolescence in the grandmother. Functional characterization of this mutation showed a constitutive activation of the G(s)/adenylyl cyclase system. Moreover, this germline mutation also activates the G(q/11)/phospholipase C pathway. The importance of Gly(431) for receptor quiescence is supported further by introduction of other mutations at this position, all leading to constitutive receptor activity. Our data show now that constitutively activating mutations can be found in the entire transmembrane domain region of the TSH receptor, indicating the important role of all parts of the transmembrane domain region for maintaining the inactive receptor conformation.     

Sporadic nonautoimmune neonatal hyperthyroidism due to A623V germline mutation in the thyrotropin receptor gene

Neonatal hyperthyroidism is a rare disorder and occurs in two forms. An autoimmune form is associated with maternal Graves' disease, resulting from transplacental passage of maternal thyroid-stimulating antibodies and a nonautoimmune form is caused by gain of function mutations in the thyrotropin receptor (TSHR) gene. Thyrotoxicosis caused by germline mutations in the TSHR gene may lead to a variety of clinical consequences. To date, 55 activating mutations of the TSHR gene have been documented. Fourteen cases with sporadic activating TSHR germline mutations have been described. Here we report a male infant with nonautoimmune hyperthyroidism due to an activating germline TSHR mutation (A623V), whose clinical picture started in the newborn period with severe hyperthyroidism. His parents did not have the same mutation. This mutation had been previously detected as a somatic mutation in patients with toxic adenomas. This is the first report of a sporadic case of nonautoimmune congenital hyperthyroidism associated with A623V mutation.     

A new case of familial nonautoimmune hyperthyroidism caused by the M463V mutation in the TSH receptor with anticipation of the disease across generations: a possible role of iodine supplementation.

OBJECTIVE: Hereditary (familial) nonautoimmune hyperthyroidism (FNAH) is caused by activating thyroid-stimulating hormone (thyrotropin) receptor (TSHR) germline mutations. We describe a family with recurrent thyrotoxicosis and goiter across three generations, including an 8-year-old girl. MAIN OUTCOME: Sequences of the TSHR gene in the index patient, her father, her paternal grandmother, and a paternal uncle demonstrated the presence of an identical germline TSHR mutation. The mutation was heterozygous and determined the substitution of valine for methionine (codon 463; ATG-->GTG) in the second transmembrane domain of the TSHR in all the affected patients, but in none of the unaffected family members. CONCLUSIONS: We compared the clinical presentation of FNAH in the family reported by us with the other cases harboring the same mutation reported in the literature. This analysis revealed high variability in the phenotypical expression of the disease. In the family reported by us, we also observed a clear anticipation of the onset of the disease across generations, and we discussed whether such a phenomenon can be the consequence of the increased iodine supplementation in the area where the family lives.     

Premature birth and low birth weight associated with nonautoimmune hyperthyroidism due to an activating thyrotropin receptor gene mutation

OBJECTIVE: Nonautoimmune hyperthyroidism (NAH), a rare autosomal dominantly inherited condition characterized by nonremitting thyrotoxicosis and the absence of features of autoimmune thyrotoxicosis, can result from activating germline mutations in the thyrotropin receptor (TSHR) gene. We report clinical and genetic features of a new family with NAH, and highlight that premature delivery and low birth weight are important characteristics of this condition. PATIENTS AND METHODS: Thyrotoxicosis was diagnosed in two children at the ages 20 months and 4 years and in their father at the age of 9 years. Both children were born prematurely by Caesarian section at 33 and 30 weeks following early rupture of the membranes. Their birth weights were 1750 g (27th centile) and 790 g (< 3rd centile), respectively. Mutation analysis of the TSHR gene was performed in both children and their parents by direct DNA sequencing. RESULTS: A heterozygous germline mutation of the TSHR gene resulting in the substitution of serine (AGC) by asparagine (AAC) at codon 505 (S505N) was found, which co-segregated with thyrotoxicosis in the family. A review of all previously reported cases of NAH due to an activating TSHR germline mutation showed that the mean duration of gestation in these patients was significantly lower than in patients with inactivating TSHR mutations causing congenital hypothyroidism (35.8 weeks vs. 39.4 weeks, P = 0.003). In addition, the mean birth weight in patients with activating TSHR mutations was lower than in patients with inactivating TSHR mutations (2338 g vs. 3470 g, P = 0.004). CONCLUSIONS: Premature delivery and low birth weight are consistent features of NAH due to activating TSHR germline mutations. This suggests a possible role for the fetal thyroid axis in the regulation of the timing of delivery and possibly fetal growth.     

Multiple relapses of hyperthyroidism after thyroid surgeries in a patient with long term follow-up of sporadic non-autoimmune hyperthyroidism

Constitutively activating thyrotropin receptor (TSHR) germline mutations have been identified as a molecular cause of congenital hyperthyroidism. Patients with relapsing hyperthyroidism were previously treated with surgery and radioiodine. We report on a 22-year-old male patient who was treated for his multiple relapses of hyperthyroidism by repeated subtotal thyroidectomies (STE). During the 22 years of follow-up, the patient developed several relapses of hyperthyroidism, four of them after thyroid surgeries. Sequencing of the TSHR gene revealed a gain-of-function mutation with an amino acid exchange of aspartate to tyrosine in codon 633 which is located in the sixth transmembrane domain of the TSH receptor. The absence of the mutation in all other family members identifies the patient's TSHR mutation as a sporadic germline mutation. In this patient, thyroid tissue growth and hyperthyroidism could repeatedly be controlled only for limited periods by near total thyroidectomy. Therefore, this case confirms that early combined treatment with near-total thyroidectomy plus radioiodine therapy seems to be the treatment of choice for patients with sporadic non-autoimmune hyperthyroidism.     

Expanding clinical spectrum of non-autoimmune hyperthyroidism due to an activating germline mutation, p.M453T, in the thyrotropin receptor gene

Objective  To describe clinical and genetic features of a Thai family with non-autoimmune hyperthyroidism (NAH) caused by an activating germline mutation in the thyrotropin receptor ( TSHR ) gene.
Patients  Three affected individuals from the same family (a father and his two children) were studied. Clinical and imaging findings were reviewed and compared.
Genetic analysis  Genomic DNA was extracted from peripheral blood leukocytes and mutation analysis of the entire coding sequence of the TSHR gene was performed in both children and their parents by direct DNA sequencing.
Results  A heterozygous germline T to C transition in exon 10 of the TSHR gene (c.1358T→C) resulting in the substitution of methionine (ATG) by threonine (ACG) at codon 453 (p.M453T) was identified in the father and his two children. They presented with different clinical severity and variable age of onset. In addition to hyperthyroidism, ventriculomegaly and bilateral shortening of the fifth metacarpal bones and the middle phalanges of the fifth fingers were consistently found in all affected individuals.
Conclusions  Ventriculomegaly and bilateral shortening of the fifth metacarpal bones and the middle phalanges of the fifth fingers might be characteristic features of NAH because of an activating TSHR germline mutation. In addition, the shortening of the middle phalanges of the fifth fingers has never been previously described, expanding the phenotypic spectrum of the disease.     

Variable phenotype associated with Ser505Asn-activating thyrotropin-receptor germline mutation.

Constitutively activating thyrotropin-receptor (TSHR) germline mutations have been identified as a molecular cause of hereditary nonautoimmune hyperthyroidism. To date, seven cases of familial and six cases of sporadic nonautoimmune hyperthyroidism have been described associated with 13 different TSHR germline mutations, with a variable clinical course. We report the case of a 12.3-year-old girl with a history of thyrotoxicosis since the age of 11 months who developed diffuse thyroid hyperplasia at the age of 4.5 years. The patient has required continuous moderate-dose antithyroid medication, to maintain euthyroidism. There were no clinical signs of autoimmune thyroid disease and autoantibodies were negative. An activating germline mutation in the TSHR gene was suspected and was found in TSHR exon 10 (Ser505Asn) but was absent in the girl's mother. This same mutation, was first reported in a patient with severe intrauterine hyperthyroidism with early and progressive goiter development. Our patient had a significantly less severe clinical course with later onset compared to the original patient with the same TSHR germline mutation.     

Functional characterization of five constitutively activating thyrotrophin receptor mutations

OBJECTIVE: Gain of function mutations of the thyrotrophin receptor (TSHR) affect several functional characteristics, such as cAMP and inositol phosphate (IP) accumulation, cell surface expression and TSH affinity. In this study we compared five constitutively activating TSHR mutations, four receptors with a point mutation (S505N, L629F, I630L, V656F) and a nine amino acid (aa) deletion mutant (aa positions 613-621) for these functional parameters in parallel transfection experiments. METHODS: The wild-type TSHR (wt) and TSHRs containing the mutations S505N, L629F, I630L, V656F and the deletion 613-621 (all cloned in the expression vector pSVL) were transiently expressed in COS-7 cells in parallel experiments. Forty-eight hours after transfection the basal and stimulated cAMP and inositol phosphate accumulation as well as the cell surface expression (by FACS and ELISA), KD-values and TSHR down regulation by different stimuli were determined. RESULTS: In contrast to the very different values for specific constitutive activity (sca) (ranging from 7.5 to 100.3-fold wt) and very different levels of receptor cell surface expression (11-94% wt level) the basal cAMP accumulation determined in transfected COS-7 cells was surprisingly uniform (6.5-8.0 over wt basal). None of the point mutated receptors constitutively activates the phospholipase C cascade. In contrast the deletion 613-621 mutant showed constitutive activity for the IP pathway with a twofold increase in basal IP accumulation compared to the wild type TSHR. All investigated TSHR-mutants showed a TSH-stimulated receptor down-regulation, which seems to be independent of the phospholipase C pathway. CONCLUSIONS: The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.     

Cloning of the cat TSH receptor and evidence against an autoimmune etiology of feline hyperthyroidism.

Cats are the only nonhuman mammalian species with a high incidence of hyperthyroidism, and a better understanding of the pathogenesis of feline hyperthyroidism is of clinical relevance for veterinary medicine. The etiology of this disease in cats remains controversial. Both an intrinsic autonomy of growth and function of follicular cells as well as an autoimmune-related mechanism have been proposed. To explore the role of the autologous TSH receptor (TSHR) in the pathogenesis of hyperthyroidism in cats, we cloned the coding sequence of the feline TSHR by RT-PCR. The open reading frame consists of 2292 nucleotides and encodes a 763-amino acid protein, one amino acid less than the human TSHR. Species comparison reveals that the cat TSHR is most closely related to the canine TSHR, with 96% identity and 97% similarity in amino acid sequence. cAMP accumulation, inositol phosphate production, and TSH binding were similar in the feline TSHR, compared with the human receptor. Analogous to the human TSHR, the cat TSHR also displays basal constitutive activity. To test the possibility that hyperthyroid cats develop antibodies that stimulate the autologous receptor, transfected cells expressing the feline TSHR were treated with sera or purified IgG obtained from 16 hyperthyroid cats. There was no increase in cAMP-dependent luciferase activity in the hyperthyroid cats, suggesting the absence of stimulatory autoantibodies. These sera were also negative for TSH-binding inhibitory Igs in an RRA. At least in the animals included in this study, there is no evidence for the presence of circulating thyroid stimulating factors as a mechanism underlying the pathogenesis of feline hyperthyroidism, and the findings support a model involving intrinsic autonomy of thyroid follicular cell growth and function.     

Follicular carcinoma presenting as autonomous functioning thyroid nodule and containing an activating mutation of the TSH receptor (T620I) and a mutation of the Ki-RAS (G12C) genes.

Most autonomous functioning thyroid nodules (AFTN) are benign thyroid follicular neoplasms. There are rare reports of malignant hot nodules, in which activating mutations of the TSH receptor (TSHR) were found. We report a case of follicular carcinoma presenting as an AFTN causing subclinical hyperthyroidism in a 64-year-old woman who had a 6-cm hot nodule in the left thyroid lobe. Genomic DNA was extracted from paraffin-embedded tissues from the tumor and extratumoral thyroid tissue. Sequence analyses revealed point mutations in two different genes: the normal ACC sequence at codon 620 of the TSHR gene was replaced by ATC, changing the threonine by isoleucine (T620I); and the wild-type GGT at codon 12 of Ki-RAS mutated to TGT, replacing glycine by cysteine (G12C). In transfection experiments the T620I mutant showed constitutive activity in terms of cyclic adenosine monophosphate (cAMP) production when permanently transfected in 3T3 cells. Here, we describe for the first time an activating mutation in 3codon 620 of the TSHR. In addition, the cancerous AFTN also contained a G12C Ki-RAS mutation. We hypothesize that the combination of these two mutations might have played an important role in both the hyperfunction of the tumor and the carcinogenetic process.     

Similarities and differences in the phenotype of members of an Italian family with hereditary non-autoimmune hyperthyroidism associated with an activating TSH receptor germline mutation

Constitutively activating germline mutations of the TSH receptor (TSH-R) are considered the cause of hereditary non-autoimmune hyperthyroidism. In this study, 10 members (8 affected and 2 unaffected) of an Italian family with hereditary non-autoimmune hyperthyroidism were investigated for the presence of mutations in the TSH-R gene. The clinical features of the disease were also analyzed. PCR-amplified fragments of the TSH-R gene were obtained from genomic DNA extracted from peripheral blood leukocytes of each family member and analyzed by direct nucleotide sequencing and restriction analysis. An identical germline TSH-R mutation was detected in all the patients with hyperthyroidism but in none of the unaffected family members. The mutation was heterozygotic and determined the substitution of valine for methionine (codon 463; ATG-->GTG) in the second transmembrane domain of the TSH-R. When expressed in chinese hamster ovary (CHO) cells, the Val463 mutant TSH-R induced constitutive activation of the TSH receptor. Analysis of the clinical features of our family and those of other families with hereditary non-autoimmune hyperthyroidism, including one with the same Val463 mutation, revealed wide variability in the phenotypical expression of the disease. Our findings indicate that an activating germline mutation in the TSH-R gene plays a key role in hereditary non-autoimmune hyperthyroidism although the onset of clinical manifestations and the evolution of the disease seem to depend heavily on other factors, thus far unidentified. The absence of a clear correlation between mutant genotypes and phenotypic expression of the disease currently limits the prognostic value of genetic testing in families with hereditary non-autoimmune hyperthyroidism.     

A novel activating mutation in the thyrotropin receptor gene in an autonomously functioning thyroid nodule developed by a Japanese patient

OBJECTIVE: A number of activating mutations of the thyrotropin receptor (TSHR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese patients because no TSHR activating mutation has been found by previous incomplete studies. DESIGN: A typical AFTN developed in a 69-year-old Japanese woman was studied. METHODS: The entire exon 10 of the TSHR cDNA was sequenced. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. RESULTS: We identified a novel heterozygous TSHR gene mutation, Leu512-->Arg (L512R; CTG-->CCG), from the AFTN. The mutation was not detected in the adjacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TSHR expression vector exhibited a 3.3-fold increase in basal cAMP level compared with that of cells transfected with wild-type TSHR DNA, confirming that the mutation was the direct cause of the AFTN. TSHR activating mutations involving the third transmembrane helix reported to date are S505R/N and V509A as well as L512R. An in vitro site-directed mutagenesis study encompassing residues 505-513 revealed that mutations involving residues other than these three did not show constitutive activation. CONCLUSION: This is the first TSHR activating mutation found in a Japanese patient, although true prevalence of TSHR activating mutations in AFTNs developed in Japanese patients remains to be elucidated. In addition, functional studies suggested that amino acid residues in the third transmembrane helix maintaining inactive conformation of the TSHR seem to be located on the same surface of the alpha-helix, possibly making interhelical bonds with another helix.     

Deletion of thyrotropin receptor residue Asp403 in a hyperfunctioning thyroid nodule provides insight into the role of the ectodomain in ligand-induced receptor activation

Somatic mutations of the TSH receptor (TSHR) gene are the main cause of autonomously functioning thyroid nodules. Except for mutations in ectodomain residue S281, all of the numerous reported activating mutations are in the TSHR membrane-spanning region. Here, we describe a patient with a toxic adenoma with a novel heterozygous somatic mutation caused by deletion of ectodomain residue Asp403 (Del-D403). Subsequent in vitro functional studies of the Del-D403 TSHR mutation demonstrated greatly increased ligand-independent constitutive activity, 8-fold above that of the wild-type TSHR. TSH stimulation had little further effect, indicating that the mutation produced near maximal activation of the receptor. In summary, we report only the second TSHR ectodomain activating mutation (and the first ectodomain deletion mutation) responsible for development of a thyroid toxic adenoma. Because Del-D403 causes near maximal activation, our finding provides novel insight into TSHR structure and function; residue D403 is more likely to be involved in the ligand-mediated activating pathway than in the ectodomain inverse agonist property.