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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 4 1094-1100
Copyright © 1997 by The Endocrine Society

Pediatric Endocrinology

Two Novel Mutations in the Thyrotropin (TSH) Receptor Gene in a Child with Resistance to TSH1

R. J. Clifton-Bligh2, J. W. Gregory, M. Ludgate, R. John, L. Persani, C. Asteria, P. Beck-Peccoz and V. K. K. Chatterjee

Department of Medicine, University of Cambridge, Addenbrooke’s Hospital (R.J.C.-B., V.K.K.C.), Cambridge, United Kingdom CB2 2QQ; the Departments of Child Health (J.W.G.), Pathology (M.L.), and Medical Biochemistry (R.J.), University Hospital of Wales, Cardiff, United Kingdom CF4 4XW; and the Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore, IRCCS and Centro Auxologico Italiano IRCCS (L.P., C.A., P.B.-P.), Milan, Italy

Address all correspondence and requests for reprints to: Dr. V. K. K. Chatterjee, Department of Medicine, University of Cambridge, Level 5, Addenbrooke’s Hospital, Hills Road, Cambridge, United Kingdom CB2 2QQ.


   Abstract
Top
 Abstract
Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 Note Added In Proof
 References
 
The TSH receptor is a G protein-coupled receptor that mediates the effects of TSH in thyroid development, growth, and synthetic function. We report here that a child with features of TSH resistance, including markedly increased serum TSH concentrations and low normal thyroid hormone levels, is a compound heterozygote for two novel mutations in the TSH receptor gene. One allele has a G to A transition corresponding to an arginine to glutamine change at codon 109 (R109Q) in the extracellular domain of the receptor. The other allele has a G to A transition corresponding to a premature termination codon at tryptophan 546 (W546X) in the fourth transmembrane segment. Each parent is heterozygous for one mutation, and both parents have normal thyroid function. Cells transiently transfected with the R109Q mutant exhibited reduced membrane binding of [125I]TSH and impaired signal transduction in response to TSH. In contrast, the W546X mutant was nonfunctional, with negligible membrane radioligand binding. Our findings indicate that a single normal TSH receptor allele is sufficient for normal thyroid function, but that the compound abnormality in the proband leads to TSH resistance.


   Introduction
Top
 Abstract
 Introduction
Case Report
 Materials and Methods
 Results
 Discussion
 Note Added In Proof
 References
 
PITUITARY TSH is critical for thyroid development and function, and its actions are mediated by a transmembrane receptor, which, together with LH/CG and FSH receptors, belongs to a subfamily of G protein-coupled receptors. The principal biological effects of TSH on the thyrocyte occur by receptor-mediated activation of Gs{alpha} and subsequent generation of intracellular cAMP (1, 2). The TSH receptor gene is located on chromosome 14 (3, 4), and the extracellular domain is encoded by nine exons, with exon 10 coding transmembrane and intracellular portions of the receptor. In keeping with many other G protein-coupled receptors (5, 6), both gain and loss of function mutations have been described in the TSH receptor. Gain of function mutations are autosomal dominant and, when somatic, cause thyroid hyperfunctioning adenomas (7, 8, 9, 10, 11, 12, 13), whereas germline inheritance leads to nonautoimmune congenital hyperthyroidism (14, 15, 16, 17, 18). Autosomal recessive inheritance of TSH resistance caused by mutations in the TSH receptor was first reported in three siblings who were compound heterozygotes for mutations of two extracellular domain residues (19). All three patients were euthyroid with normal serum thyroid hormone concentrations but increased serum concentrations of TSH, indicating that the resistance in these cases was partial. Each parent was heterozygous for one of these mutations.

We describe a child with greatly increased serum TSH concentrations and low normal thyroid hormone concentrations who is a compound heterozygote for two novel mutations in the TSH receptor gene, one inherited from each parent. The mutation inherited from the mother corresponds to a TSH receptor truncated in the fourth transmembrane domain that is functionally inactive in vitro. The paternal mutation lies in the extracellular segment of the receptor and has a reduced binding affinity for TSH, resulting in impaired signal transduction.


   Case Report
Top
 Abstract
 Introduction
 Case Report
Materials and Methods
 Results
 Discussion
 Note Added In Proof
 References
 
The patient was identified after a positive result on neonatal screening for hypothyroidism. He is the only child of unrelated parents. He was delivered by caesarean section at 38 weeks gestation, weighed 2.45 kg, and had transient hypoglycemia. Thyroid function tests at 8 weeks of age showed a serum free T4 concentration of 10 pmol/L (normal range, 12–28 pmol/L) with a TSH of 92 mU/L (normal range, 0.4–4.0 mU/L). There were no clinical features of hypothyroidism. He was commenced on T4. The progress of his thyroid function tests in response to T4 administration is shown in Fig. 1Go. At 12 months of age, a T4 dose of 60 µg suppressed serum TSH into the normal range but with raised serum free T4 concentrations of 40 pmol/L. While on treatment, his mother described him as irritable; when treatment was discontinued at 2 yr of age, his behavior became more normal. After birth, he demonstrated catch-up growth to the 50th percentile, and thereafter his growth, bone maturation, and development have continued to be normal without treatment. His hearing is normal, as measured by otoacoustic emissions. A thyroid isotope scan at 2 yr of age showed a gland of normal size and location, with uniform tracer uptake. Thyroid ultrasonography was normal. A perchlorate discharge test was slightly elevated at 15%. Both patient and mother were negative for thyroid autoantibodies. Serum electrolyte, GH, PRL, LH, FSH, PTH, and calcium concentrations were all normal in the patient. He remains clinically euthyroid without therapy. His most recent thyroid function tests at 3 yr of age show a TSH of 134 mU/L and free T4 of 12 pmol/L. The results of a T3 suppression test are shown in Table 2Go. At this time the serum glycoprotein hormone {alpha}-subunit level was 2.5 ng/mL (normal range, 0.24–1.05 ng/mL), but the molar ratio of {alpha}-subunit to TSH was normal at 0.17 (normal range, <5.5). TRH tests in both parents were normal (data not shown).



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  		Figure 1. Free T4 (•) and TSH ({triangleup}) concentrations of the patient in relation to T4 dose. Normal ranges for free T4 (dotted area) and TSH (cross-hatched area) are shown. T4 therapy was discontinued at 24 months of age.

 

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  		Table 2. Thyroid function and indexes of hormone action during a T3 suppression test

 

   Materials and Methods
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
Results
 Discussion
 Note Added In Proof
 References
 
Genetic analyses

Patient lymphocytes were Epstein-Barr virus-transformed (ECACC, Porton Down, Salisbury) and grown in RPMI 1640 supplemented with 10% FCS. Ribonucleic acid and genomic DNA were extracted from the lymphocytes using TRIzol according to the manufacturer’s instructions (Life Technologies, Paisley, Scotland). First strand complementary DNA (cDNA) was synthesized using Superscript II reverse transcriptase (Life Technologies), and nested reverse transcription-PCR was subsequently performed with primers spanning the extracellular and transmembrane coding regions of the TSH receptor. Genomic DNA was isolated from peripheral blood leukocytes from each parent using standard techniques. Exons of the TSH receptor were amplified from patient and parents by PCR using recently published intronic primer sequences (20). The forward primer in each case was tagged with the universal M13 primer sequence. Direct sequencing of the PCR products was undertaken by cycle sequencing using dye-labeled universal 21M13 primers (Applied Biosystems/Perkin-Elmer, Cheshire, UK) and analyzed by electrophoresis on an ABI 373 sequencer (Applied Biosystems, Foster City, CA). Primer sequences and PCR conditions are available on request.

Functional studies

TSH bioactivity was measured in Chinese hamster ovary cells expressing recombinant human TSH receptor as previously described (21). DNA fragments bearing each mutation were replaced in full-length wild-type TSH receptor cDNA cloned into the eukaryotic expression vector pSVL (22), and constructs were verified by sequencing. JEG3 (human choriocarcinoma) cells were grown in Optimem (Life Technologies) supplemented with 2% (vol/vol) FCS and 1% (vol/vol) penicillin, streptomycin, fungizone (Life Technologies). Eighteen hours before transfection, the medium was replaced with Optimem containing 2% resin-stripped FCS. Cells were transfected by a 5-h exposure to calcium phosphate containing {alpha}LUC reporter, TSH receptor expression vector, and internal control plasmid BOS-ß-galactosidase. Twenty-four hours after transfection, the medium was replaced to include bovine TSH (Sigma, Dorset, UK) or recombinant human TSH (Genzyme, West Malling, UK) as appropriate. Sixteen hours later, cells were lysed and assayed for luciferase and galactosidase activities. Data are the mean ± SE of at least three separate experiments performed in triplicate.

Radiolabeled ligand binding studies were performed using membranes extracted from COS cells transiently transfected with the receptor expression vectors described above. Membranes were prepared as previously described (23), and protein was quantified by the Bradford assay. Equal amounts (30–70 µg) of protein were incubated for 2 h with 0.5 kilobecquerels [125I]TSH (RSR, Cardiff, UK) in NaCl-free assay buffer with isotonicity maintained with 280 mmol/L sucrose (23) and in the presence of increasing amounts of unlabeled bovine TSH (Sigma, Dorset, UK). Radiolabeled TSH bound after washing was determined by {gamma}-counting.


   Results
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
Discussion
 Note Added In Proof
 References
 
The results of thyroid function tests of all family members are shown in Table 1Go. The biological activity of TSH in serum from the proband was measured in Chinese hamster ovary cells expressing recombinant human TSH receptor, and the ratio of bioreactivity to immunoreactivity was normal at 0.69 (normal range, 0.6–2.1). This is in contrast to a recent report of congenital hypothyroidism caused by biologically inactive TSH due to a mutation in the TSH ß-subunit gene (24). Thus, the patient demonstrated features of TSH resistance, namely reduced circulating thyroid hormone levels together with elevated bioactive TSH. As the endocrine abnormality appeared to be confined to the thyroid, analysis of the TSH receptor gene was undertaken. Direct sequencing of the extracellular and transmembrane-coding regions of the TSH receptor showed that the patient was heterozygous for a CGG to CAG mutation in exon 4 corresponding to an arginine to glutamine change at codon 109 (R109Q) and was heterozygous for a second TGG to TAG mutation in exon 10 corresponding to a premature termination at codon 546 (W546X; data not shown). The mutations were verified by repeated sequencing of both genomic DNA and receptor cDNA isolated from the patients’ Epstein-Barr virus-transformed lymphocytes. Exons 4 and 10 were amplified and sequenced from genomic DNA isolated from each parent, showing that mother was heterozygous for the W546X mutation, and the father was heterozygous for the R109Q mutation.


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  		Table 1. Thyroid function test results at diagnosis

 
The functional consequences of each mutation were investigated using a method similar to that employed by Sunthornthepvarakul and colleagues (19). Expression vectors encoding wild-type or mutant receptor were transiently cotransfected with a reporter gene consisting of the glycoprotein hormone {alpha}-subunit promoter and luciferase gene ({alpha}LUC) into JEG3 cells. The {alpha}LUC reporter is highly responsive in this system due to tandemly repeated cAMP response elements between -146 and -111 bp of the {alpha}-subunit promoter (25). Each mutant demonstrated impaired signal transduction in response to TSH compared to that of the wild type (Fig. 2aGo). The W546X mutant did not stimulate reporter gene activity above basal levels, whereas the R109Q mutant showed a right-shifted dose-response profile, such that maximal wild-type activity was achieved at higher concentrations of TSH. Similar results, indicating impaired signal transduction, were obtained using recombinant human TSH (data not shown). To recapitulate the parental genotypes in vitro, either mutant was cotransfected in equal amounts with wild-type receptor. The resulting dose-response profiles did not differ from that seen with wild-type receptor alone, whereas cotransfection of both mutants reproduced the activity of transfection of R109Q alone (Fig. 2bGo). Thus, neither mutant was able to dominantly inhibit the activity of wild-type receptor, nor was any positive cooperativity between the mutants observed. Although constitutive activation of the cAMP cascade by unliganded TSH receptor has been described in COS cells (7, 9, 14), such basal activity of either wild-type or mutant TSH receptors could not be detected in this assay. This may be due to a limitation of the luciferase reporter system, as previous characterization of mutant TSH receptors using this assay in COS cells also failed to show constitutive receptor activity (19).



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  		Figure 2. Function of wild-type and mutant TSH receptors. a, Activation of {alpha}LUC by wild-type or mutant receptors transfected individually. Cells were transfected with 500 ng reporter, 100 ng receptor expression vector, and 100 ng BOS-ß-galactosidase. Luciferase activity was determined after incubation with 0–100 mU/mL bovine TSH and normalized for transfection efficiency using ß-galactosidase activity. The mean (±SE) responses to bovine TSH are expressed relative to the maximum response obtained with wild-type receptor, which was approximately 10-fold. b, Coexpression of wild type with each mutant receptor expression vector. Cells were transfected with reporter and reference plasmids, as described in a, and 50 ng receptor expression vectors in the indicated combinations. For comparison 50 ng R109Q vector were transfected individually with 50 ng pSVL to correct for DNA concentration. Hormone-dependent activation after incubation with 0–100 mU/mL bovine TSH was determined as described above. Where values are less than 10% of the mean, error bars have been omitted for clarity. The combinations of wild-type and each mutant receptor reflect the heterozygous nature of each parent (mother and father), whereas the combination of both mutant receptors represents the compound state of the proband.

 
Binding studies with radiolabeled ligand were undertaken using the same receptor expression vectors transiently transfected in COS-7 cells (Fig. 3Go). The R109Q mutant showed a binding capacity of 1140 cpm, which is 60% that of wild-type receptor (1900 cpm), and raised EC50 (20 vs. 7 mU of the bovine TSH preparation we used; data not shown), consistent with reduced binding affinity for TSH. When experiments were performed on whole cells, the binding to R109Q, although higher than that on W546X or vector alone, was too low to obtain a curve, indicating very poor surface expression of the mutant (data not shown). The W546X mutant receptor showed negligible specific binding for TSH on either whole cells or membranes, which is probably indicative of very little surface expression of this receptor, although the possibility that the truncated receptor still inserts into the membrane but fails to bind TSH through lacking C-terminal residues cannot be excluded. Nevertheless, poor cellular expression of prematurely truncated receptors is well described (26, 27, 28).



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  		Figure 3. A representative experiment showing binding of [125I]TSH to membrane-associated TSH receptors. COS-7 cells were transfected with the same receptor expression plasmids as in Fig. 2Go, and membranes were incubated with [125I]TSH and 0–100 mU/mL unlabeled bovine TSH. Mean (±SE) binding values of triplicate determinations, expressed as counts per min, are shown.

 
Persistent elevation of serum TSH concentrations in this child (Fig. 1Go) has prompted concern as to whether there may be a risk of developing pituitary autonomy. Magnetic resonance imaging (MRI) performed at 2 yr of age showed two small areas of hypoattenuation after the administration of gadolinium (Fig. 4Go). A T3 suppression test was subsequently performed, which showed brisk reduction of TSH into the normal range after the administration of T3 (Table 2Go). Creatine kinase, cholesterol, and triglyceride levels showed a normal response to exogenous T3, although there was little change in serum sex hormone-binding globulin or alkaline phosphatase levels. Overall, these results suggest normal pituitary and peripheral tissue responsiveness to T3 in the patient. Sequencing of the thyroid hormone receptor ß gene in the proband was also normal (data not shown).



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  		Figure 4. Pituitary MRI scan in the proband at 2 yr of age. Two areas of hypoattenuation after the administration of gadolinium are indicated (arrows). The contours of the gland are normal.

 

   Discussion
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
Note Added In Proof
 References
 
We have described a child with persistent hyperthyrotropinemia together with normal serum thyroid hormone concentrations associated with two novel mutations in the TSH receptor gene. When tested individually, each mutant receptor showed impaired function in transfection studies. Although the R109Q mutant receptor showed only mild impairment, clinical data from the proband suggest that this mutation results in markedly abnormal thyroid function. It is possible that the limited dynamic range (10-fold) of the transfection assay may underestimate the in vivo consequences of the R109Q mutation. Neither mutant receptor affected wild-type receptor function when coexpressed, which is concordant with the observation that both parents have normal thyroid function. Coexpression of both mutant receptors results in some residual functional activity, which correlates with the partial TSH resistance seen in the patient. As expected from the autosomal recessive nature of this disorder, neither mutant was able to dominantly inhibit the function of wild-type receptor.

Our case is the second recorded example of loss of function mutations in the TSH receptor gene (19). All cases described hitherto have been euthyroid, indicating that TSH resistance in each case is only partial, such that the elevated TSH levels are able to stimulate adequate thyroid hormone secretion. In contrast, the phenotype of more severe TSH resistance is represented by the recessively inherited hyt/hyt hypothyroid mouse (29). Here, fetal onset of profound hypothyroidism is associated with greatly elevated TSH levels. The homozygous mutant thyroid gland is hypoplastic and demonstrates diminished follicular size and reduced colloid. Recently, a point mutation in the TSH receptor gene was identified in this mouse, corresponding to a Pro to Leu change at codon 556 in the transmembrane region, which abolished TSH binding and response to TSH in vitro (30).

The locations of loss of function mutations in the TSH receptor identified to date are shown in Fig. 5Go. Study of these mutations has defined some important residues for TSH binding and receptor function. The previous report indicated that mutation of proline at codon 162 to alanine retained some biological activity, whereas mutation of isoleucine at codon 167 to asparagine virtually abolished signal transduction (19). A structural model for the hormone-binding site has been proposed on the basis of similarity between the extracellular domain of the TSH receptor and the crystal structure of the ribonuclease inhibitor (31, 32). From this model, the arginine at codon 109, which is mutated in our case, may be expected to project into solution, contributing to the TSH binding cavity. This hypothesis is confirmed by studies indicating that mutation of this residue to glutamine interferes with TSH binding. Both arginine at codon 109 and tryptophan at codon 546 are conserved in the TSH receptor from a number of species. Furthermore, tryptophan at codon 546 in the TSH receptor is conserved at homologous positions in both the LH/CG and FSH receptors (33, 34). Curiously, in the LH/CG receptor, the residue homologous to arginine 109 is glutamine. However, the transfected R109Q mutant TSH receptor did not mediate signal transduction in response to human LH (data not shown).



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  		Figure 5. Location of loss of function mutations in the TSH receptor structure. Mutations of proline at codon 162, isoleucine at codon 167, and proline at codon 556 have been identified previously (19, 30). The R109Q and W546X mutations are arrowed.

 
Our case contributes two more important findings. First, it describes the only occurrence hitherto of a TSH receptor with a premature termination codon within its serpentine portion, resulting in a biologically inactive product. The mother of the proband is thus effectively hemizygous for functional TSH receptors, suggesting that only a single normal TSH receptor allele is required to sustain normal thyroid function. Second, TSH levels in the proband are the highest of those reported to date. Elevated serum TSH levels are characteristic of TSH resistance and probably represent resetting of the pituitary threshold for TSH suppression by thyroid hormones (19), although the mechanism remains obscure. In keeping with this, there is no evidence for resistance to thyroid hormones in our patient. The occurrence of possible pituitary abnormalities on a MRI scan in our case in the context of persistently high TSH levels was disturbing, although the significance of these findings remains uncertain. The evidence of brisk inhibition of serum TSH levels during a T3 suppression test mitigates against pituitary autonomy. It is also encouraging that the secretion of other anterior pituitary hormones, the {alpha}-subunit/TSH molar ratio, and the growth pattern are all normal. Nevertheless, enlargement of the sella has been shown to occur in long term juvenile and untreated congenital hypothyroidism (35, 36), which may be proportional to the degree of TSH elevation (36). Moreover, thyrotrope hyperplasia is evident at autopsy in long term primary hypothyroidism (37). It is known from animal studies that prolonged hypothyroidism may ultimately lead to thyrotrope neoplasia (38). We intend to follow the pituitary changes closely, with further imaging at regular intervals.

It is clear that activating mutations of the TSH receptor increase both thyrocyte growth and function (39). In contrast, impaired TSH receptor function has not yet been associated with disordered thyroid development in humans. A role for TSH in thyroid ontogeny is suggested by evidence that thyroid development is arrested in late gestation in mice homozygous for a knockout of the glycoprotein hormone {alpha}-subunit common to TSH, LH, and FSH (40), and that similarly in humans, thyroid dysgenesis has been reported in some cases of TSH deficiency caused by homozygous mutations in TSH ß-subunit gene (41, 42). By analogy with the hyt/hyt hypothyroid mouse, we speculate that a combination of severe loss of function TSH receptor mutations will be found in some cases of thyroid dysgenesis. However, it is also relevant to note that cases of congenital hypothyroidism and TSH hyporesponsiveness have been described in which the TSH receptor gene is reportedly normal (43, 44), suggesting that the molecular basis of athyreosis is likely to be heterogeneous. Partial TSH resistance with normal gland development, low or normal thyroid hormone concentrations, and elevated bioactive TSH levels may be a more readily identifiable entity. The study of such cases will continue to provide valuable insights into the function of the TSH receptor.


   Note Added In Proof
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 Note Added In Proof
References
 
Since the submission of this manuscript, de Roux et al. have reported compound heterozygosity for the W546X and another mutation in the TSH receptor in a case of TSH resistance. N. de Roux, M. Misrahi, R. Brauner, M. Houang, J. C. Carel, M. Granier, Y. Le Bouc, N. Ghinea, A. Boumedienne, J. E. Toublanc, E. Milgrom. 1996 Four families with loss of function mutations of the thyrotropin receptor. J Clin Endocrinol Metab. 81:4229–4235.


   Acknowledgments
 
The authors are indebted to Prof. G. Vassart for providing the wild-type TSH receptor cDNA cloned in pSVL.


   Footnotes
 
1 This work was supported by the Wellcome Trust (to V.K.K.C.), the Medical Research Council (to M.L.), and Murst and CNR, Rome (to P.B.P.). Back

2 Commonwealth Foundation Research Scholar. Back

Received November 7, 1996.

Revised December 9, 1996.

Accepted December 30, 1996.


   References
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 Note Added In Proof
 References
 

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