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Original Studies |
Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, and Dipartimento di Oncologia, Divisione di Anatomia Patologica (P.C.), Università di Pisa, 56124 Pisa, Italy
Address all correspondence and requests for reprints to: Dr. Massimo Tonacchera, Dipartimento di Endocrinologia, Università degli Studi di Pisa, Via Paradisa 2, 56124 Cisanello, Pisa, Italy. E-mail: mtonacchera{at}hot-mail.com
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Abstract |
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Genetic analysis in the propositus showed a homozygous inactivating mutation of the TSH receptor that had not been previously described. The mutation consisted of the substitution of an isoleucine in place of a highly conserved threonine at position 477 in the first extracellular loop of the receptor (T477I). The brother, one sister of the father (whose DNA was not available), the mother of the propositus, one sister, and the brother were heterozygous for T477I. All the heterozygous persons were unaffected.
After transfection in COS-7 cells, the mutant receptor displayed an extremely low expression at cell surface. At variance with cells transfected with the wild-type TSH receptor, cells transfected with the mutant T477I did not show constitutive activity for the adenylyl cyclase pathway. A dramatic reduction in the amount of cAMP accumulation after bovine TSH challenge was observed in cells transfected with the mutant T477I receptor. A structural defect in the mutant TSH receptor protein was probably responsible for the poor routing of the receptor to the cell membrane. This is the first time that a loss of function mutation of the TSH receptor is described in a patient with severe congenital hypothyroidism and absent circulating thyroglobulin due to TSH unresponsiveness and the first time that an inactivating mutation of the TSH receptor is described in the first extracellular loop.
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Introduction |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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In this report we describe a 22-yr-old female patient who was severely hypothyroid and mentally retarded. Serum T4 and T3 concentrations were below the sensitivity of the methods, with elevated serum TSH levels. Serum Tg was undetectable. A normally shaped hypoplastic gland located in the appropriate anatomical position in the neck was found at scintiscan. The gland did not respond after administration of bovine TSH either in terms of 131I uptake or thyroid hormone synthesis.
The entire coding regions of the TSHr gene were sequenced. The genetic analysis showed a homozygous inactivating mutation of the TSHr that had not been previously described. The mutation consisted in the substitution of an isoleucine in place of a highly conserved threonine at position 477 in the first extracellular loop of the receptor (T477I). The brother and one sister of the father (whose DNA was not available), the mother of the propositus, one sister, and the brother were heterozygous for T477I. All heterozygous subjects were unaffected.
In transfection experiments in COS-7 cells the mutant receptor showed a very low expression at cell surface and a poor response to bovine TSH (bTSH). We believe that the mutant TSHr is the cause of thyroid unresponsiveness to TSH and hypoplasia in this patient.
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Case Report |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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60 mg/day), but substitution treatment was given irregularly, with
several interruptions lasting from a few months to 1 yr until she was
aged 22 yr. Thyroid function was checked at the age of 9 yr by
measuring plasma protein-bound iodine, which was reported to be low,
but the precise result could not be traced. At that age the IQ was said
to have been 67. Regular menses were reported since the age of 13 yr. A
few months before the first admission to our institution, a thyroid
profile during treatment with desiccated thyroid (30–60 mg/day)
disclosed low total T4 (2.1 µg/dL; normal
values, 4.2–12) and T3 (51 ng/dL; normal values,
100–210). T3 resin uptake was 22% (normal
values, 23.5–36%), and the free T4 index was
1.6 (normal values, 4–11). The serum TSH concentration determined by
RIA was 27 µU/mL (normal range, 0.5–5). When first admitted to our
institution in 1984, the patient was 22 yr old and had received no
thyroid medication for approximately 6 weeks. She was 146 cm in height,
her span (distance between the outspread middle fingertips) was
142 cm, and she weighed 60 kg. The face was cretinoid, but the bridge
of the nose was not flattened. There was no umbilical hernia; muscles
were well developed, with pseudohypertrophy of gastrocnemic muscles.
The skin was cold, pale, rough, and dry. The patient had severe mental
retardation (IQ = 60), and convergent strabismus of both eyes.
Reflex relaxation was strikingly low. Eating was apparently normal. No
thyroid tissue was palpable in the neck. In vitro studies
Total serum T4 and T3 concentrations were below the sensitivity of the methods (<2 µg/dL and < 50 ng/dL, respectively). T4-binding globulin was 28 µg/mL (normal values, 8,9–30,5). Serum TSH determined by RIA was more than 80 µU/mL (normal values, 0.5–5). Serum Tg was undetectable (<2 ng/mL). Hemagglutination tests for anti-Tg and antimicrosomal or antithyroperoxidase antibodies were negative. Urinary iodine excretion was low (66 µg/L), as expected in the mountain area where the patient lived. Calcium, phosphorus, and alkaline phosphatase concentrations were within normal limits.
In vivo studies
Thyroid uptake of a tracer dose of 131I (50 µCi)
was 4% at 1 h and 4%, 3%, and 2% at 2, 3, and 24 h,
respectively. The scintiscan was performed with a K545 collimator and
revealed a normally shaped, but reduced in size, thyroid gland located
in the appropriate anatomical position in the neck (Fig. 1
). Thyroid echography confirmed the
presence of two small thyroid lobes normally located in the neck. Ten
units of bovine TSH (Ambinon) were administered im for 3 days.
On the last day of TSH administration there was no evidence of response
to bovine TSH in terms of either 131I uptake (3 h, 4%;
24 h, 3%) or serum T4, T3, and Tg
concentrations. The saliva to plasma ratio of radioiodide was measured
1 h after the oral administration of 131I. This saliva
to plasma ratio was 62 in the patient (normal, 25–140). Immunoreactive
GH and plasma cortisol showed a normal response to insulin hypoglycemia
(basal GH concentration, <1 ng/mL), which increased to 21 ng/mL 60 min
after insulin injection; the basal (0800 h) cortisol concentration was
90 ng/mL and increased to 227 ng/mL 90 min after insulin injection.
Serum gonadotropin concentrations were appropriate for the cycle phase
and showed a normal response after GnRH challenge (basal FSH, 7.0
mIU/mL; basal LH, 4.9 mIU/mL in follicular phase); after GnRH
treatment, LH increaseed to 31 mIU/mL and FSH to 11 mIU/mL. An X-ray
study of the skull showed an enlarged sella turcica.
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Family history
The father had died in a car accident years before. He was reported to have a nontoxic nodular goiter. Serum free thyroid hormones and TSH concentrations were checked in all living members of the family. The father’s siblings and the mother, two sisters, and a brother of the propositus are all euthyroid, with serum TSH concentrations within the normal limits. The mother and one of the two sisters were found to have small diffuse goiter.
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Materials and Methods |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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Serum total T4 and T3 were assayed by RIA using ARIA II kits (Becton Dickinson and Co., Milan, Italy). The serum free T4 index was calculated by multiplying total T4 by T3-resin uptake (Trilute kits, Miles S.P.A., Milan, Italy). Serum TSH was determined using the RIA-mat TSH kit (Byk-Mallinckrodt, Inc., Milan, Italy) and a sensitive immunoradiometric assay (Sucrosep TSH IRMA, Boots Celltech). Serum Tg was measured with an immunoradiometric assay kit (HTGK, Sorim Biomedia, Saluggia, Italy).
Antithyroglobulin and antimicrosomal (and thyroperoxidase) antibodies were measured by passive hemoagglutination (SERODIA-AMC and SERODIA-ATG, Fujirebio, Tokyo, Japan).
Sequence determination
Genomic DNA was extracted from peripheral lymphocytes using standard procedures (5). PCR amplification was designed to produce two overlapping fragments covering the whole length of exon 10 coding for the entire portion of the C-terminal region of the TSHr gene (all seven transmembrane segments and extracellular and intracellular loops) exactly as described previously (5). Exons 1–9 were amplified individually using couples of intronic primers as described previously (6). At least two different PCR amplifications from genomic DNA were sequenced on double stranded DNA with sense and antisense primers. To confirm the presence of a TSHr mutation, the mutation was subcloned in a plasmid, and sequences were repeated on individual clones.
Construction and expression of mutant genes
The pSVL-TSHr construct harboring mutation T477I was obtained by replacing, within the original wild-type construct (wtTSHr) an SpeI-BstEII segment directly amplified from the genomic DNA of the patient. To facilitate the insertion of T477I, a unique SpeI restriction site within the sequence encoding the end of transmembrane segment I of wtTSHr was created as described previously (7). Constructs containing the mutant TSHr sequences were obtained by replacing a SpeI-CvnI segment (positions 1322–1603) from this new construct with the corresponding segments of the mutant, obtained by direct PCR amplification of genomic DNA. The sequence of the resulting mutant construct was verified by double stranded sequencing.
COS-7 cells transfected with wild-type and mutant receptors were used for binding studies, flow cytometry, and cAMP determination. COS-7 cells were grown in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL fungizone, and 1 mmol/L sodium pyruvate. For the transient expression of wild-type and mutant TSHr, COS-7 cells were seeded at the concentration of 150,000 cells/3-cm dish. One day after seeding, cells were transfected using the diethylaminoethyl-dextran method followed by a 2-min 10% dimethylsulfoxide shock (8).
Functional assays
Forty-eight hours after transfection, cells were used for cAMP production assay, [125I]TSH binding studies, and flow cytometric analysis. All experiments were performed in triplicate, and each experiment was repeated at least three times. Results were expressed as the mean ± SE.
cAMP assay. Cells were washed with Krebs-Ringer-HEPES buffer and preincubated for 30 min at 37 C in Krebs-Ringer-HEPES buffer. This was followed by a 1-h incubation at 37 C in the same medium supplemented with 0.5 mmol/L isobutylmethylxanthine, as a cAMP phosphodiesterase inhibitor, in the absence of bTSH (basal values) or in the presence of various concentrations of bTSH (Sigma, St. Louis, MO). At the end of the incubation period the medium was removed, and replaced by 0.1 mol/L HCl. The cell extracts were dried in a vacuum concentrator (Savant Instruments, Farmingdale, NY), and cAMP was determined using a commercial RIA kit (code 432, TRK Amersham Pharmacia Biotech, Arlington Heights, IL). Results were expressed as picomoles per dish.
Binding assays. Forty-eight hours after transfection, cells
were washed once with Hanks’ solution in which NaCl was replaced by
280 mmol/L sucrose containing 0.2% BSA and 2.5% low fat milk. Binding
studies were performed by incubating cells in that same medium at room
temperature for 4 h in the presence of 80,000 cpm
[125I]TSH (TRAK Assays, BRAHMS Diagnostica, Berlin,
Germany; 35 µCi/µg, 40 U/mg) and the appropriate concentrations of
unlabeled TSH. At the end of the incubation period, cells were rinsed
twice with ice-cold Hanks’ medium and solubilized with 1 N
NaOH, and bound radioactivity was determined in a 
-scintillation
counter.
The addition of 2.5% low fat milk decreased nonspecific binding, i.e. [125I]TSH binding to cells not expressing TSHr or the binding to plastic dishes (7). Under these conditions (and contrary to other protocols we have explored) nonspecific binding (defined as the radioactivity bound to the dishes in the presence of 100 mU/mL cold TSH) was identical to that bound to mock-transfected cells (1% of the total counts introduced). In the absence of a consensus about the bioactivity of pure bovine TSH (9), we expressed all TSH or TSHr concentrations as milliunits per mL, assuming a 1:1 stoichiometry for TSH binding to its receptor. The binding competition curves have been fitted by nonlinear regression, assuming a single receptor-binding site (10).
Flow cytometric analysis
Cells were detached from culture dishes with 5 mmol/L each of ethylenediamine tetraacetate and ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid in phosphate-buffered saline (PBS) and transferred to Falcon tubes (2052, Falcon Labware, Cockeysville, MD). Cells were washed with PBS plus 0.1% BSA, centrifuged at 500 x g at 4 C for 3 min, and appropriately treated for the nonpermeabilized or permeabilized cell assay as described previously (11). Nonpermeabilized cells were incubated at room temperature for 30 min with 200 µL of a monoclonal antibody directed at the TSHr (BA8) (12) diluted in PBS plus 0.1% BSA. BA8 was a gift from Dr. Costagliola, Brussels, Belgium. A blank sample was prepared by incubating cells with 200 µL PBS plus 0.1% BSA. For the permeabilized cell assay, cells were fixed with 2% PBS-paraformaldehyde (UCB, Brussels, Belgium) and then treated for 30 min with PBS plus 0.1% BSA and 0.2% saponin (Sigma). To detect the TSHr monoclonal antibody, cells were washed in PBS and 0.1% BSA and then incubated for 30 min at room temperature in the dark with a goat antimouse IgG fluorescein-conjugated (Becton Dickinson and Co., San Jose, CA) diluted 1:20 in PBS with 0.1% BSA. After washing, cells were resuspended in 1 mL propidium iodide (PI) (Sigma) solution containing 10 µg/mL PI in PBS and 12 µL ribonuclease (Calbiochem, San Diego, CA) and incubated for 30 min at 4 C in the dark. Flow cytometric analysis was performed using a FACSort flow cytometer (Becton Dickinson and Co.) equipped with a laser for an excitation at 488 nm to detect monoclonal antibodies conjugated with fluorescein 5-isothiocyanate and PI. Fluorescence emissions of fluorescein 5-isothiocyanate and PI from single cells were separated and measured using the standard optics of the FACSort. Cell doublets and debris were excluded by gating on FL2 width and FL2 area dot plots. The CellQuest software program (Becton Dickinson and Co.) was used to acquire and analyze data. A minimum of at least 20,000 cells were analyzed.
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Results |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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, A and B). Thr477 is highly
conserved among the receptors of glycoprotein hormones, lending support
to a pathogenetic role of mutation T477I. The residue is placed at the
beginning of the first extracellular loop (Fig. 2B), immediately after
the second transmembrane segment of the TSHr. The propositus was
homozygous for the mutation (Fig. 2A and 3). The brother and one sister of the
father (whose DNA was not available because he died years before), the
mother of the propositus, one sister, and the brother were found to be
heterozygous for T477I (Figs. 2A and 3). All of the heterozygous
members of this family were unaffected. This pattern is consistent with
an autosomal recessive inheritance (Fig. 3).
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The functional characteristics of the mutant receptor were studied by transient expression in COS-7 cells. Cells transfected with a complementary DNA construct encoding the wtTSHr or the empty pSVL vector were used as controls.
To measure the total number of receptors (TSH binding capacity)
expressed at the surface of cells transfected with the different
constructs and their relative dissociation constants
(Kd), binding studies were performed with a
bovine [125I]TSH tracer as described in Materials
and Methods. The mutant TSHr construct showed an extremely low
level of expression at the cell surface (Fig. 4), with a total receptor
concentration value of 0.043 ± 0.003 mU/mL compared to
0.84 ± 0.07 mU/mL for the wtTSHr in a representative experiment.
The observed dissociation constant of the mutant receptor was
significantly smaller than that of the wild-type receptor (0.38 ±
0.03 vs. 2.8 ± 0.20 mU/mL, respectively; Fig. 4).
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). The T477I
was clearly detectable within the cells. These findings suggested that
the T477I receptor was synthesized and recognized by the BA8
monoclonal. The very low expression of T477I at the cell surface might
be attributed to a defect in folding of the mutant protein resulting in
abnormal routing to the plasma membrane.
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As previously reported (5, 7), COS-7 cells transfected with wtTSHr
exhibited a 3-fold increased production of cAMP in the absence of the
agonist (4.8 ± 0.6 pmol/dish), compared with that in cells
transfected with vector alone (1.60 ± 0.22 pmol/dish; Fig. 6). Cells transfected with the mutant
receptor showed a low cAMP production that did not differ from that
found in cells transfected with the vector alone (1.68 ± 0.25
pmol/dish; Fig. 6).
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). Cells transfected with the mutant T477I displayed a greatly reduced
ability to stimulate cAMP accumulation in response to bTSH. Because
TSH-stimulated cAMP production in cells transfected with the mutant
T477I was very low, the EC50 could not be
calculated. |
Discussion |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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After cloning of the human TSHr gene (13, 14, 15), loss of function mutations of the TSHr were identified in hypothyroid mice and in humans with either euthyroid hyperthyrotropinemia or hypothyroidism due to thyroid unresponsiveness to TSH. A loss of function mutation (P556L) in the TSHr was first identified in the hyt/hyt mouse (16). This mouse strain exhibits autosomal recessive congenital hypothyroidism with a normally located hypoplastic thyroid gland (16). The mouse thyroid gland shows diminished follicular size, reduced colloid, and reduced number of microvilli and mitochondria at histological examination (16). TSH unresponsiveness in these mice resulted from defective TSH binding to the mutant receptor, which was apparently expressed at the cell surface membrane (17). In humans, two missense mutations in the extracellular domain of the TSHr were found to produce a condition of partial TSH unresponsiveness with euthyroid hyperthyrotropinemia in affected compound heterozygous siblings (18). After this initial report, other families with this condition due to compound heterozygous or homozygous inactivating mutations of the TSHr gene were described (19, 20). All of these patients had thyroid hormone levels in the normal range, in the majority of cases in the low normal range. Subsequently, loss of function TSHr mutations were described in four neonates identified by systematic screening who had permanently low serum T4 concentrations and markedly hypoplastic thyroid gland located in the normal position in the neck (11, 21, 22). Two siblings were overtly hypothyroid with barely detectable serum T4 due to a homozygous missense mutation (A553T) in the fourth transmembrane domain of the TSHr gene (11). Another child had milder hypothyroidism (free T4, 8 pmol/L) and was compound heterozygote for a missense mutation and a deletion-insertion mutation producing a stop codon, both in the extracellular domain of the TSHr gene (21). The fourth male baby described by Gagné et al. (22) had severe hypothyroidism and was compound heterozygote for two inactivating TSHr mutations. The maternal allele carried a splicing mutation, and the other allele had a deletion of two nucleotides, causing premature termination of translation at codon 656. In these four neonates with congenital hypothyroidism due to thyroid unresponsiveness to TSH, serum Tg concentrations were in the high normal range (11, 21, 22) for newborns (66–84 ng/mL). The finding of high serum levels of Tg in these neonates at the time of diagnosis was quite surprising and was not easily explained in the context of a very hypoplastic gland and the inability of TSH to stimulate thyroid function. Incomplete polarization of follicular cells contained in these hypoplastic glands, abnormal routing of Tg due to accumulation of misfolded TSHr mutant protein in the rough endoplasmic reticulum, or intercellular leakage of Tg from dysplastic follicles were hypothesized to explain the disproportionately high serum Tg levels (11, 22). The observation of circulating Tg in neonates with inactivating mutations of the TSHr was also in sharp contrast with the reported absence of serum Tg in children and adults originally described as having congenital hypothyroidism with thyroid unresponsiveness to TSH (1, 2, 3, 4). In two of these patients, who were siblings, Takeshita et al. did not find mutations in the TSHr gene or in its promoter (23).
The phenotype of our patient, who was thoroughly investigated for the
first time at the age of 22 yr, is very much in keeping with the
originally described syndrome of congenital hypothyroidism due to
thyroid unresponsiveness to TSH (1). She was severely hypothyroid, with
a constantly elevated TSH level and had a small thyroid gland normally
located in the neck that failed to respond to bTSH in terms of
131I uptake, thyroid hormone secretion, and Tg release.
Actually, her circulating Tg was undetectable despite serum TSH
concentrations that had remained elevated for years due to inadequate
substitution treatment. Genetic analysis revealed a T477I missense
mutation in a highly conserved residue of the TSHr. The mutation was
shown to cosegregate with the disease within the family. It was located
in the first extracellular loop of the receptor close to the second
transmembrane segment. This is the first time that an inactivating
mutation of the TSHr has been described in the first extracellular
loop, where up to now only gain of function somatic TSHr mutations were
described in toxic thyroid adenomas (7). The functional properties of
the T477I mutant receptor were studied after transient expression in
COS-7 cells. In this expression system the wtTSHr displays a
constitutive activity toward the adenylyl cyclase pathway (5, 7).
Binding-displacement curves revealed an extremely low expression of the
mutant T477I receptor at the cell surface compared with that of the
wtTSRr. The very low expression was confirmed using a monoclonal
antibody by flow cytofluorometry. At variance with the wtTSHr, cells
transfected with the T477I mutant did not show a constitutive activity
for the adenylyl cyclase pathway; the basal production of cAMP was
identical to that of cells transfected with the vector alone. This
finding suggests an impaired interaction of the mutant TSHr with the
Gs
protein. Compared to COS-7 cells
transfected with the wtTSHr, cells transfected with the mutant T477I
showed a dramatic reduction in the amount of cAMP accumulated after
bTSH challenge. The apparent decrease in the Kd
observed with the mutant TSHr is likely to result from the small number
of receptors inserted in the cell membrane, as this parameter
normalizes when the wtTSHr and mutant receptor are expressed at similar
levels (data not shown). The mechanism responsible for this effect is
poorly understood, but it was previously reported for the wtTSHr either
transiently expressed in COS-7 cells or stably expressed in CHO cells
(11, 24). Thus, the mutant T477I TSHr showed absent constitutive
activity for the adenylyl cyclase pathway, extremely impaired response
to bTSH, and very low expression at the cell surface. A structural
defect of the mutant TSHr was probably responsible for the poor routing
of the receptor to the cell membrane.
This is the first time that a loss of function TSHr mutation is described in a patient with severe congenital hypothyroidism and absent circulating Tg. Similar to the originally described cases of congenital hypothyroidism due to TSH unresponsiveness (1, 2, 3, 4), our patient was not investigated during the neonatal period. Thus, it might be argued that serum Tg was detectable in the neonatal period and then disappeared from the circulation due to the reduced TSH drive consequent to replacement therapy. However, clinical and laboratory evidence indicates that our patient, due to inadequate substitution treatment, had remained hypothyroid with an elevated TSH level up to 22 yr of age, when she was first thoroughly investigated. The reason why other patients with congenital hypothyroidism due to inactivating TSHr mutations had circulating Tg (11, 22) remains unclear. A possible explanation might be that Tg release from the neonatal thyroid gland is partially TSH independent.
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Acknowledgments |
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Footnotes |
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Received May 27, 1999.
Revised September 27, 1999.
Revised October 26, 1999.
Accepted November 30, 1999.
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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E. Doherty, P. Pakarinen, A. Tiitinen, A. Kiilavuori, I. Huhtaniemi, S. Forrest, and K. Aittomaki A Novel Mutation in the FSH Receptor Inhibiting Signal Transduction and Causing Primary Ovarian Failure J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1151 - 1155. [Abstract] [Full Text] [PDF]
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M. Tonacchera, P. Agretti, G. De Marco, A. Perri, A. Pinchera, P. Vitti, and L. Chiovato Thyroid Resistance to TSH Complicated by Autoimmune Thyroiditis J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4543 - 4546. [Abstract] [Full Text] [PDF]
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 W. E. Winter and M. R. Signorino Molecular Thyroidology Ann. Clin. Lab. Sci., July 1, 2001; 31(3): 221 - 244. [Abstract] [Full Text] [PDF]
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