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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4238-4242
Copyright © 2000 by The Endocrine Society

Original Studies

A Novel Mutation in the Thyrotropin (TSH) Receptor Gene Causing Loss of TSH Binding But Constitutive Receptor Activation in a Family with Resistance to TSH1

Diego Russo, Corrado Betterle, Franco Arturi, Eusebio Chiefari, Maria E. Girelli and Sebastiano Filetti

Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e Clinica (F.A., E.C., S.F.), and Dipartimento di Scienze Farmacobiologiche, Facoltà di Farmacia (D.R.), Università di Catanzaro, 88100 Catanzaro; and Cattedra di Endocrinologia, Istituto di Semeiotica Medica (C.B., M.E.G.), Università di Padova, Italy

Address correspondence and requests for reprints to: Sebastiano Filetti, M.D., Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e Clinica, Policlinico Mater Domini, Via T. Campanella 115, 88100 Catanzaro, Italy. E-mail: filetti{at}tin.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Resistance to TSH is a syndrome due to reduced responsiveness of the thyroid gland to biologically active TSH. Inactivating mutations of the TSH receptor (TSH-R) have been detected in several cases of resistance to TSH, both partial and complete, sporadic and familial. In this study, we describe a family with the presence of resistance to TSH responsible for euthyroid hyperthyrotropinemia in two siblings from consanguineous parents. By direct sequencing of the TSH receptor gene, we identified a new mutation responsible for the substitution of an arginine with a cysteine at position 310, in the extracellular domain of the TSH-R. The mutation was homozygous in two brothers; heterozygous in both parents, an uncle, and an unaffected brother; and absent in the other unaffected brother. When stably transfected in Chinese hamster ovary cells, the Cys310 mutant TSH-R showed loss of response to TSH in terms of cAMP stimulation. However, a constitutive activity in terms of basal cAMP production was detected in the Cys310 mutant, compared with the wild-type TSH-R.

Our data suggest that such a Cys310 TSH-R mutant may determine both the TSH resistance and the clinical euthyroidism detected in this family.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID RESISTANCE TO TSH syndrome is defined as reduced or absent responsiveness of the thyroid gland to biologically active TSH. The resistance may be complete or partial, sporadic and/or familial. In complete resistance to TSH, the thyroid gland is hypoplastic and the phenotype is not clinically or biochemically different from that found in the other forms of congenital hypothyroidism, with very high concentrations of biologically normal TSH and undetectable thyroid hormone serum levels. In partial resistance to TSH, also referred as euthyroid hyperthyrotropinemia, increased serum TSH levels are present with normal serum concentrations of thyroid hormones and a condition of clinically "compensated hypothyroidism" (1).

Recently, TSH receptor (TSH-R) gene mutations causing loss of the receptor function have been described in some patients with sporadic/familial hypothyroidism and unresponsiveness to TSH (2, 3, 4), as well as in a small percentage of congenital hypothyroidism (5, 6, 7).

In the present study, we describe a family with partial resistance to TSH, whose members were identified in adult age during a routine screening because they presented the phenotype of compensated hypothyroidism (clinical euthyroidism with normal serum thyroid hormone concentrations and increased serum concentrations of TSH). A new mutation (Arg->Cys310) of the TSH-R gene was detected in some individuals of this family in heterozygosis or homozygosis. When tested in vitro, the Cys310 TSH-R showed the loss of TSH-binding properties but a constitutive activity in terms of cAMP production, which may contribute to maintain adequate plasma levels of thyroid hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Family history

The propositus (DB) was a 63-yr-old man. He was married with a second-grade cousin (DL) and bore four sons (DF, DR, DS, and DD) (see Fig. 1Go). At age 50, a hypercholesterolemia was found, and for this reason he underwent thyroid function investigation. Free T4 (FT4) was 11.0 ng/L (normal range, 7.1–18.5 ng/L), free T3 (FT3) was 1.7 ng/L (normal range, 2.3–6.6 ng/L), and TSH was 6.8 mU/L (normal range, 0.2–4. mU/L). A TRH test revealed an increased response of TSH. Both ultrasonography and thyroid isotope scan revealed a gland in the normal range. Thyroid anti-thyroperoxidase, anti-thyroglobulin, and TSH-binding inhibiting immunoglobulins were negative. The physical and mental development was normal. A substitutive therapy with L-T4 was initiated, and the TSH returned in the normal range.



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  		Figure 1. Pedigree and clinical features of the family with resistance to TSH.

 
His four sons, his wife, and his brother were also studied for thyroid function. No signs or symptoms of hypothyroidism were present in any member of the family; in two sons (DD and DS), however, TSH levels were high (62 and 61 mU/L, respectively) with total T3 and FT4 in the normal range. The main data of the family are summarized in Fig. 1Go. A substitutive therapy with L-T4 was, therefore, started at a dose of 1.7 µg/kg/die. After 6 months the TSH was 2.63 mU/L, with 4.0 ng/L FT3 in DD, and 0.24 mU/L, with 13 ng/L FT4 and 5.2 ng/L FT3 in DS.

Also, the brother of the proband (DG) revealed a euthyroid hyperthyrotropinemia (FT4, 9.4 ng/L; FT3, 2.9 ng/L; TSH, 5.6 mU/L). The physical examination, the thyroid ultrasonography, and thyroid scan were in the normal range, and the autoantibodies were negative.

Genetic analysis

Genomic DNA was isolated from peripheral-blood lymphocytes using a DNA extraction kit (Amersham Pharmacia Biotech, Milano, Italy). The entirety of the TSH-R gene was sequenced using oligonucleotide primers designed on the basis of the published sequence of the human TSH-R gene (8, 9) (Table 1Go).


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  		Table 1. Primers used to amplify all the exons of the TSH-R gene and the annealing temperatures

 
PCR amplification and sequencing were performed as described by Russo et al. (10). Briefly, PCR amplification was performed with 500 ng of genomic DNA, 250 nM of each primer, 200 µM deoxy-NTPs, Taq polymerase buffer [10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, and 0.1 mg/mL gelatin], and 1 U Taq DNA polymerase (AmpliTaq; Perkin-Elmer Corp., Milano, Italy). Forty cycles of denaturation (for 1 min at 94 C), annealing (for 1 min at different temperatures), and extension (for 1 min at 72 C) were performed in a programmable heat-block (Perkin-Elmer Corp.). The primers used to amplify all the exons of the TSH-R gene and the annealing temperatures are shown in Table 1Go. Following PCR, the samples were precipitated with ethanol and resuspended in 10 mM Tris and 1 mM EDTA (pH 7.5). DNA sequencing was carried out by the dideoxynucleotide method (11), using the dsDNA cycle sequencing system kit (Promega Corp., M Medical Genenco, Florence, Italy) and the same primers as those used for the amplification. Oligonucleotides were provided by Life Technologies, Inc. (Life Technologies, Inc. Italia S.r.l., Milan, Italy). The presence of the mutation was confirmed by restriction enzyme analysis. The samples were incubated for 3 h at 37 C with HhaI restriction enzyme (Promega Corp., M Medical Genenco) and run on 2.5% agarose-Tris-borate EDTA gel containing ethidium bromide.

Mutagenesis and expression of the human TSH-R

Arginine at position 310 was substituted with cysteine using the overlap extension methods (12, 13). Briefly, a 1.04-kb mutated TSH-R complementary DNA (cDNA) fragment was generated after two rounds of PCR using as a template pSV2neoECE TSH-R cDNA, which had been previously mutated (glutamic to aspartic acid and isoleucine to leucine conservative substitutions at amino acid residues 362 and 419) without alteration in TSH-R function to create a SpeI site (14). The sequences of the nucleotides used were: 1) sense with AflII site: 5'-CGCTTTTCAGGGACTATGCAATG-3'; 2) antisense with Cys at position 310: 5'-ATTTTCTCTGGCACAAGCTCTG-3'; 3) sense with Cys at position 310: 5'-CAGAGCTTGTGCCAGAGAAAAT-3'; and 4) antisense with SpeI site: 5'-GTAGTGGCTGGTGAGGAGAATAA-3'.

The nucleotide sequences of the mutated and adjacent regions were confirmed in all clones by dideoxynucleotide sequencing. The mutagenized TSH-R PCR fragments were used as a template in the second PCR, and the 1.04-kb cDNA product, excised with AflII/SpeI, was subcloned into the same pSV2neoECE TSH-R construct. The mutant Cys310 TSH-R was then transfected by the calcium phosphate method (15) into Chinese hamster ovary (CHO) cells. Surviving colonies (about 100/dish) were selected by G418 (Geneticin; Sigma s.r.l., Milano, Italy) (400 mg/mL), pooled, grown, and used for the functional studies. Three different pools of Cys310 TSH-R-transfected CHO cells, as well as pools of CHO cells stably transfected with the vector pSV2-neo alone, the wild-type TSH-R, and the mutants Gln1 13 TSH-R (loss of function) (16) and Ser623 TSH-R (gain of function) (10), were used in the functional studies.

The presence of the mutant TSH-R insert in transfected CHO cells was confirmed by RT-PCR and subsequent restriction enzyme analysis. A semiquantitative RT-PCR, as described by Tanaka et al. (17), was performed to assess the amount of the TSH-R transcript expressed in the pools of transfected cells (data not shown).

Radiolabeled TSH binding and cellular cAMP measurement

Cells grown to confluence (12-well Costar plates; Costar, Cambridge, MA) in Ham’s F-12 medium supplemented with 10% FCS and antibiotics were incubated for 2 h at 37 C in 1 mL modified Hanks’ buffer without NaCl, with isotonicity maintained with 280 mM sucrose, supplemented with 0.25% BSA, 125I TSH (1 x 104 cpm), and the indicated concentrations of unlabeled bovine TSH (0.1, 1.0, 10, 100, and 1000 U/L) (Sigma s.r.l.), as described previously (16). At the end of the incubation period, the cells were rapidly rinsed three times with the same buffer (ice-cold) without TSH and solubilized with 0.5 mL 1 NaOH, and radioactivity was measured in a {gamma}-counter. Nonspecific 125I-TSH binding was determined in the presence of 10-6 M TSH.

Cellular cAMP measurement was performed as described previously (18).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the TSH-R gene

Genomic DNA extracted from the blood lymphocytes of all family members was used as template to amplify the entire sequence of the TSH-R gene by PCR. The sequence of the PCR products revealed the presence of a homozygous mutation (CGC->TGC), not previously described, at codon 310 in exon 10 of the TSH-R in two brothers (DS and DD) (Fig. 2Go); the mutation determined the substitution of a cysteine for an arginine in the extracellular domain of the receptor. Their parents (DB and DL), consanguineous, an uncle (the propositus’s brother, DG), and another brother (DF) were heterozygous for the same mutation. The mutation was absent in the other brother (DR) (Fig. 2Go). The presence of the mutation was further confirmed by restriction enzyme analysis (Fig. 3Go).



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  		Figure 2. Presence of a point mutation in codon 310 of the TSH-R gene.

 


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  		Figure 3. Pedigree of the family and confirmation of the TSH-R gene mutation by restriction enzyme analysis. Presence of a thymine at position 1028 determines loss of a HhaI restriction site. DS and DD had both alleles containing thymine at position 1028 that was resistant to digestion with HhaI, producing a 446-bp fragment of DNA (H1). DB, DL, DF, and DG had only one allele resistant to digestion with HhaI, producing an undigested 446-bp DNA fragment (H1) and two digested fragments of 384 bp (H2) and 62 bp. DR had both alleles containing the cytosine at position 1028, producing two digested fragments of 384 bp (H2) and 62 bp. The small band (62 bp) is not visible (under the resolution power of photography).

 
In vitro functional activity of the mutant Cys310 TSH-R

To verify the functional role of the cysteine for arginine substitution in the TSH receptor, a Cys310 TSH-R mutant cDNA contained in the expression vector pSV2neoECE was permanently transfected in CHO cells. Cells transfected with a cDNA construct encoding the wild-type receptor or the empty pSV2neoECE vector were used as control.

The binding of 125I TSH to Cys310 TSH-R mutant was markedly reduced. The mutant receptor showed a binding capacity that is 25% of that of the wild-type receptor. Also, the dissociation constant of the Cys310 mutant receptor was significantly lower than wild-type (250 mU/mL vs. 10 mU/mL) (Fig. 4AGo). As expected, the cells transfected with Cys310 TSH-R were also unable to increase cAMP accumulation in response to TSH stimulation (Fig. 4BGo). In the absence of the added agonist, however, the mutated receptor elicited a detectable level of cAMP that was higher than in cells transfected with the wild-type receptor (Fig. 4BGo). In both Cys310- and wild-type TSH-R-transfected CHO cells, forskolin (10-5 M) was able to enhance cAMP production (data not shown). The same results were obtained by using different pools of stably transfected CHO cells (Fig. 5Go), in which the amount of the TSH-R transcript was assessed by a semiquantitative RT-PCR method.



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  		Figure 4. A, Specific TSH binding to recombinant mutant (square), wild-type (circle) TSH receptor forms, and pSV2neoECE alone (triangle) stably expressed in CHO cells. Binding of [125I] TSH was measured in the presence of increasing concentrations of bovine TSH, as described in Materials and Methods. Each point represents the mean of two duplicate determinations; the data shown are representative of two separate experiments. B, cAMP response to TSH stimulation in pooled CHO cells transfected with wild-type TSH-R, Cys310 mutant, or pSV2neoECE alone. Incubations and cAMP assay were performed as described in Materials and Methods. Each point represents the mean of two duplicate determinations; the data shown are representative of two separate experiments.

 


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  		Figure 5. cAMP basal levels in different pools of CHO cells stably transfected with wild-type, Cys310, Gln113, and Ser623 TSH-R. The results are corrected by the amount of TSH-R messenger RNA expression, as assessed by semiquantitative RT-PCR (see Materials and Methods). The data shown are representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A defective response to TSH stimulation determines the syndrome of resistance to TSH that may be complete or partial. In the former case, we have the feature of congenital hypothyroidism, not distinguishable by other genetically determined syndromes, with hypoplastic gland and low levels of circulating thyroid hormones; differently in the latter, elevated levels of TSH may represent the only biochemical abnormality of the affected individuals, so that this condition is usually referred as compensated hypothyroidism (1). Obviously, the two conditions seem quite different in that it concerns the clinical approach and the need for a substitutive therapy with thyroid hormones.

Recent reports suggest a pathogenic role for TSH-R gene alterations in the resistance to TSH syndromes (19, 2, 3, 4, 5, 6, 7). However, absence of TSH-R gene abnormalities, including in some cases the promoter region, has also been described in patients with congenital hypothyroidism and unresponsiveness to TSH as well as in three families with resistance to TSH (20, 21, 22), suggesting the occurrence of other postreceptorial alterations underlying these syndromes (23).

Moreover, "loss of function" TSH-R mutations are associated with a wide spectrum of clinical syndromes, ranging from severe to mild or absolutely compensated hypothyroidism. In the last situation, except when the neonatal screening reveals increased levels of serum TSH, the clinical latency of the disease is responsible for the late and the casual discovery of the condition. This was the case of the family analyzed in this study, where a subclinical hypothyroidism casually detected in a 50-yr-old individual allowed to discover a familial condition of compensated hypothyroidism. In this family, in fact, the presence of a partial resistance to TSH did not compromise at all the physical and mental development (even in two sons found homozygous for the genetic defect), as well as did not require any substitutive therapy in the development age. Moreover, when a therapy with L-T4 was started, TSH values returned in the normal range, excluding occurrence of pituitary autonomy.

In this family, a new germinal mutation in exon 10 of the TSH-R gene was found causing a cysteine for arginine substitution at codon 310 in the extracellular domain of the receptor. This Arg->Cys 310 mutation is expected to profoundly alter the receptor conformation for at least two reasons: 1) the nonconservative nature of the amino acid change; and 2) the presence of an additional cysteine in a region of the receptor in which disulfide bonds are believed to play a fundamental role in maintaining the structure (23).

Our in vitro studies demonstrated the reduced ability of the mutant receptor to bind to radiolabelled TSH, but also revealed the presence of a constitutive activity in terms of cAMP production, higher than that of the wild-type receptor and not influenced even by the highest dose of TSH tested. This finding is not fully surprising in that changes in the extracellular portion of the TSH-R have been reported also to influence the signal transduction properties of the TSH-R, both in in vitro (14) and in vivo (24, 25) studies. Furthermore, also a mutant Cys390Trp receptor, detected in a family with resistance to TSH (3) possessed a constitutive activity when transfected in COS-7 cells. It is quite intriguing that the highest dose of TSH tested in vitro does not elicit a biological response whereas, in vivo, the increase of TSH levels seems to contribute to fully compensate the hypothyroidism, although it is not possible to establish which dose of TSH may reproduce, in an in vitro system, the effects of elevated serum levels of the hormone. However, even considering all the limitations in transposing in vitro data obtained in nonthyroid cells to in vivo thyroid tissues (5), the phenotype found in our family, especially in the two sons with both alleles mutated, fits well with the suggestive hypothesis that it is the constitutive activity of the Cys310 mutant TSH-R, rather than or together with the elevated levels of TSH, that sustains a normal production of thyroid hormones in vivo.

A similar situation was described in a patient where an activating mutation in the FSH receptor was able to sustain fully developed spermatogenesis, compensating the absence of gonadotrophins owing to hypophysectomy because of a pituitary tumor (26). In our family, the same genetic alteration, on one side, determines an abnormal control of the thyroid function, but, on the other side, may provide by itself to compensate the pathological condition.

Additional studies are necessary to assess whether this situation should be considered an almost unique condition or a less rare event in the yet undiscovered spectrum of the G protein-coupled receptor-related diseases.


   Footnotes
 
1 Supported by grants from the Associazione Italiana per la Ricerca sul Cancro and MURST-Cofin 98 (to S.F.). F.A. is recipient of Dottorato di ricerca in "Basi molecolari dell’azione ormonale" at the University of Catania. Back

Received March 28, 2000.

Revised July 5, 2000.

Accepted July 13, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals