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Original Studies |
Klinik und Poliklinik für Kinderheilkunde, Virchow-Klinikum, Humboldt-Universität zu Berlin (H.B., H.K., A.G.); and Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin (T.S., G.S., T.G.), Berlin, Germany
Address all correspondence and requests for reprints to: Annette Grüters, M.D., Klinik und Poliklinik für Kinderheilkunde, Virchow-Klinikum, Medizinische Fakultät der Humboldt-Universität zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The hypothesis that the TSHR gene is a candidate gene for thyroid
dysgenesis was strengthened by the finding that a homozygous loss of
function mutation in a highly conserved P in the fourth transmembrane
domain of the TSHR causes hypothyroidism and thyroid hypoplasia in the
hyt/hyt mouse (6, 7). The first somatic and germline
mutations in the coding sequence of the human TSHR have been introduced
as pathogenic entities causing gain of function syndromes (8).
Recently, two different missense mutations in the large extracellular
N-terminal domain of the human TSHR were identified (9) (Fig. 1
). In contrast to patients with
congenital hypothyroidism, the three affected compound heterozygote
siblings were euthyroid and had normally sized thyroid glands, probably
due to the capability of the markedly elevated TSH level to compensate
for the receptor defect.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The patient was the first of two daughters born at term to
nonconsanguineous parents. At the time of neonatal screening the girl
had a blood TSH concentration of 89 mU/L (normal, <20 mU/L). At
reexamination, a serum TSH concentration of 82 mU/L in conjunction with
decreased serum concentrations of T4 (78 nmol/L), free
serum T4 (FT4; 8.2 pmol/L), and serum
T3 (1.4 nmol/L; Table 1) were
detected. The constellation of elevated TSH levels combined with below
normal FT4 concentrations was considered diagnostic of
congenital primary hypothyroidism. Ultrasound examinations of the
newborn revealed a small thyroid gland with a reduced volume (<0.5 mL)
compared to normal values (0.7–2.4 mL) obtained from healthy newborns
in the same moderately iodine-deficient geographic area (10). The
transplacental passage of maternal antithyroid antibodies was ruled out
by negative tests for antithyroid peroxidase, antithyroglobulin, and
TSH binding inhibitory antibodies and antibody-dependent cell-mediated
cytotoxicity in the serum of the newborn. There was no history of
excess perinatal iodine contamination, and the iodine concentration in
a randomly chosen urine sample of the newborn was normal (85 µg/L).
The clinical data collected prompted the initiation of T4
supplementation therapy with a dose of 50 µg
L-T4/day (13 µg/kg BW). Discontinuation of
T4 treatment for 4 weeks at the age of 2 yr resulted in a
marked increase in the serum TSH concentration (79 mU/L) concomitant
with decreased T4, FT4, and T3
concentrations, thus confirming the diagnosis of persistent congenital
hypothyroidism. Ultrasonography again revealed a reduced volume of the
thyroid gland compared with values obtained from an age-matched normal
population (Table 1). Consequently, L-T4
therapy was reinstalled and has not been discontinued since then.
Linear growth of the presently 5-yr-old girl was normal along the 75th
percentile, and determinations of bone age at 2 and 4 yr of life showed
no advancement with respect to chronological age. The girl’s mental
development, as studied at the age of 5 yr, applying the Hamburg
Wechsler intelligence test (11), was normal, and no neurological
abnormalities were observed.
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Serum concentrations of T4 and T3 were measured with an immunofluorometric assay (Delfia, Wallac, Freiburg, Germany). Serum free T4, antithyroid peroxidase, and antithyroglobulin antibodies were measured by RIA (Dynotest, Brahms Diagnostika, Berlin, Germany). TSH binding inhibitory antibodies were measured by TRAK assays (Brahms Diagnostika, Berlin, Germany). Antibody-dependent cell-mediated cytotoxicity was assessed using a chromium release assay in human thyroid cells (12). For ultrasonographic examinations, a 7.5-megahertz transducer (Acuson 128, Computer Sonography, Berlin, Germany) was used.
Isolation and characterization of genomic DNA encoding the TSHR
Genomic DNA was isolated from peripheral blood leukocytes from selected family members with the help of a DNA extraction kit (QIAamp Blood Kit, Qiagen, Hilden, Germany). Exons 1–10 of the TSHR gene were amplified by PCR techniques as described previously (13). In addition, specific primer pairs were designed to amplify exon 10 of the TSHR gene as five overlapping fragments. The following pair of primers generated a 376-bp fragment of the 5'-portion of exon 10: forward primer, 5'-GAT GAG ATC ATT GGT TTT GG-3' (nucleotide positions 1080–1099); and reverse primer, 5'-TGG CAT GGT TGT AGT ACT CA-3' (nucleotide positions 1455–1436). Single strand conformational polymorphism (SSCP) analysis was carried out as described by Orita et al. (14). PCR products were further characterized by restriction endonuclease digestion with BsaAI followed by separation of resulting fragments on 2% agarose gels and by dideoxy sequencing (15).
Construction of mutant TSHR genes
To characterize functional properties of wild-type and mutant TSHRs, the complementary DNA (cDNA) coding for the human TSHR was subcloned into the polylinker of the eukaryotic expression plasmid pcD-PS (16). Novel mutant TSHRs detected in this study were created by employing standard PCR-based mutagenesis techniques (17). Thus, the PCR fragment containing the paternal mutation (TSHR-C390W) was digested with MscI/Bsu36I; the fragment containing the maternal mutant (TSH-R-419trunc) was cut with MscI/BstEII. These fragments were used to replace the corresponding segment in the wild-type TSHR. The correctness of PCR-derived products was verified by dideoxy sequencing.
Cell culture, transfection, and functional assays
COS-7 and CHO-K1 cells were cultured and transfected as described previously (18). To establish CHO-K1 cell lines permanently expressing wild-type and mutant TSHRs, receptor constructs and the plasmid pcDNAneo, (Invitrogen, Leek, The Netherlands) were cotransfected by lipofection (Life Technologies, Eggenstein, Germany). Fourteen single colonies for each receptor construct transfected that were resistant to the antibiotic G418 (750 µg/mL) were isolated and maintained in Ham’s F-12 medium in the presence of G418 (400 µg/mL). Cell clones were tested for the expression of wild-type and mutant TSHR-C390W receptor in functional assays. In the case of the wild-type TSHR, all 14 cell clones responded to bovine TSH (bTSH) with increases in cAMP levels ranging from 7- to 27-fold above basal values. In the case of TSHR-C390W, only 5 of 14 stable cell clones showed TSH-inducible cAMP accumulation, with a range of 7- to 13-fold stimulation above basal. Determination of cAMP and inositol phosphate formation was performed as outlined previously (18, 19).
Radioligand binding assay
Binding studies were performed using membrane homogenates prepared as described previously (20). Incubation buffer consisted of 50 mmol/L Tris (pH 7.4), 3 mmol/L MgCl2, 1 mmol/L ethylenediamine tetraacetate, 0.1% BSA, and 0.1 mg/mL bacitracin. Incubations were carried out for 1.5 h at 22 C in a 0.25-mL volume supplemented with 40,000 cpm [125I]bTSH (54 µCi/µg; Brahms Diagnostika). Membranes and bound ligand were separated from unbound ligand by centrifugation (10,000 x g, 10 min) through a silicon oil layer according to the method of McArdle (21). Nonspecific binding was defined as binding in the presence of 100 nmol/L bTSH. The protein content of samples was determined by the method of Bradford (22). Binding data were analyzed by a nonlinear least squares curve-fitting procedure using the computer program Ligand (23).
Immunofluorescence microscopy and enzyme-linked immunosorbent assay (ELISA)
Immunofluorescence microscopy was performed as described previously (24). A 1:4 dilution of tissue culture supernatant from the hybridoma clone 2C11 directed against the extracellular domain of the human TSHR (25) was used to detect receptor protein.
For ELISA studies, COS-7 cells were transfected with various TSHR constructs as described above. Three days later, cells were washed twice with PBS and treated with 120 µL lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediamine tetraacetate, 1% desoxycholate, 1% Nonidet P-40, 0.2 mmol/L phenylmethylsulfonylfluoride, and 10 µg/mL aprotinin]. After vigorous vortexing, followed by removal of cell debris by centrifugation, supernatants (100 µL/well) containing solubilized receptor protein were used to coat microtiter plates. After incubation for 16 h at 4 C, plates were blocked with 10% FCS in PBS. Then, 100 µL/well of a 1:4 dilution of tissue culture supernatant from the hybridoma clone 2C11 directed against the extracellular domain of the human TSHR (25) were applied and incubated for 2 h at 37 C. Plates were washed three times with PBS containing 0.05% Triton X-100 and incubated with a 1:3,000 dilution of a peroxidase-conjugated antimouse IgG antibody for 1 h at 37 C. After removal of excess unbound antibody, H2O2 and o-phenylenediamine (2.5 mmol/L each in 0.1 mol/L phosphate-citrate buffer, pH 5.0) were added to serve as substrate and chromogen, respectively. After 15 min, the enzyme reaction (carried out at room temperature) was stopped by the addition of 1 mol/L H2SO4 containing 0.05 mol/L Na2SO3, and color development was measured bichromatically at 492 and 620 nm using an ELISA reader (Titertek Multiskan MCC/340, Labsystems, Finland).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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At birth, the patient (daughter 1) presented with markedly
elevated TSH levels concomitant with low peripheral thyroid hormone
levels and a reduced thyroid volume as determined by ultrasonography.
This constellation of clinical findings warranted the diagnosis of
congenital hypothyroidism. The results of thyroid function tests and
ultrasonographic examinations of the patient at the time of initial
diagnosis and during the follow-up period are summarized in Table 1
. At
the age of 6 months, the daily T4 dose was increased to
62.5 µg to counteract elevated TSH levels and undetectable
FT4 serum concentrations measured during low dose therapy
and was maintained until the age of 2 yr. Clinical data obtained during
a 4-week discontinuation of T4 replacement therapy (Table 1) confirmed the initial diagnosis, and L-T4
therapy was reinstalled.
In the mother 2 yr before her first pregnancy, a moderately elevated
serum TSH concentration (5.5 mU/L; normal, <4 mU/L) and a borderline
FT4 level of 10.5 pmol/L (normal, 11–25 pmol/L) had been
reported by her physician. Ultrasonographic examinations at that time
indicated a rather small thyroid volume of 5 mL. Tests for antithyroid
peroxidase, antithyroglobulin, and TSH binding inhibitory antibodies
yielded negative results, and she had been treated with a daily
L-T4 dose of 75 µg since then. When
reexamined by us at the age of 29 yr, discontinuation of
L-T4 medication for 6 weeks was accompanied by
TSH levels in the normal range (Table 1). The father, who was of
Romanian origin, and the presently 1-yr-old second daughter were
euthyroid.
Genomic analysis and identification of mutations in the TSHR gene
Applying SSCP analysis, genomic DNA of the patient (daughter 1;
Fig. 2A) was screened for mutations in
exons 1–10 of the TSHR gene. PCR products representing a 376-bp
segment of exon 10 of the TSHR gene showed marked differences in
electrophoretic mobility compared with amplificates from healthy
controls (Fig. 2B). Further studies of maternal and paternal TSHR genes
identified the parents as heterozygote carriers of 2 different
abnormalities (Fig. 2B). Genomic DNA amplified from the younger
daughter (daughter 2; Fig. 2A) displayed an electrophoretic mobility
profile identical to that of the father, whereas DNA from the patient
appeared to combine properties of both parents. Subsequently, a 376-bp
fragment of exon 10 of the TSHR gene was sequenced. Different
alterations were detected in each of the 2 alleles, confirming the
initial suspicion of compound heterozygocity (Fig. 3). In 1 allele, 18 bp (nucleotide
positions 1217–1234) were deleted, and 4 novel bp were inserted
instead (Fig. 3). These alterations resulted in a frame shift and the
appearance of coding sequence for 14 novel amino acids, followed by a
premature stop codon after amino acid position 419 (Fig. 1). The
rearrangement of exon 10 within 1 allele created a novel unique
BsaAI restriction site in the 376-bp PCR fragment that could
now be used to trace this mutant to the mother (Fig. 2C). Subsequent
sequencing of the paternal allele revealed a T to G transversion at
nucleotide position 1170, resulting in the exchange of a highly
conserved C at position 390 for a W residue (Figs. 1 and 3). Sequencing
further confirmed the heterozygous state of each parent and that of
daughter 2 by the presence of 1 normal allele.
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Functional characterization of mutant TSHRs
To characterize the functional activities of mutant TSHRs, the
wild-type TSHR cDNA was modified by site-directed mutagenesis. In
transiently transfected COS-7 cells, stimulation of the wild-type
receptor and of the paternal mutant TSHR-C390W with saturating
concentrations of TSH resulted in a robust increase in agonist-induced
cAMP accumulation (Table 2). However,
dose-response curves for TSH were shifted toward higher agonist
concentrations in the case of TSHR-C390W compared to the wild-type
receptor (Fig. 4A), whereas the maximum
number of binding sites was comparable for both receptors (Table 2).
The approximately 20-fold greater potency of TSH acting at the
wild-type compared to the mutant (TSHR-C390W) receptor was the
functional correlate of binding characteristics that were monitored in
parallel (Fig. 4B). The concentration of TSH required to displace 50%
of specifically bound [125I]bTSH was 16-fold higher for
the paternal mutant than for the wild-type receptor (Fig. 4B and Table 2), thus reflecting a significantly reduced affinity of TSH to the
mutant receptor. Expression of the truncated TSHR derived from the
altered maternal allele did not yield agonist-induced cAMP accumulation
(Fig. 4A) or detectable membranous binding sites for
[125I]bTSH (Table 2). Cotransfection of the wild-type
TSHR with each of the mutant receptors to simulate the heterozygous
state of the parents resulted in reduced efficacy, as expected because
of diminished gene dosage, yet unaltered potency of TSH to stimulate
cAMP accumulation (data not shown).
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In transient transfection experiments, only a small percentage of cells
take up expression plasmids and subsequently show unphysiologically
high receptor densities. As signal transduction characteristics vary
considerably depending on the amount of receptors expressed per cell
(27), CHO-K1 cell lines were created that permanently expressed TSHR
levels comparable to those observed in thyrocytes (28). Several
G418-resistant CHO cell lines were screened for receptor expression by
radioligand binding and functional techniques, and clonal lines were
developed. None of the CHO cell clones that had previously been shown
to express TSHR-419trunc messenger ribonucleic acid with the help of
reverse transcription PCR (data not shown) displayed specific
membranous [125I]bTSH-binding sites or TSH-stimulable
adenylyl cyclase activity (Fig. 4C). In accordance with data obtained
in COS-7 cells, the concentration of TSH required to half-maximally
increase cAMP accumulation was approximately 20-fold higher in cells
expressing the wild-type receptor compared to those expressing the
TSHR-C390W mutant (Fig. 4C). It is noteworthy, however, that to be able
to detect agonist-induced cAMP accumulation in stably transfected CHO
cells at all, TSHR-C390W clones had to be chosen that display 5- to
7-fold higher receptor densities than wild-type clones (Table 2). Even
under those conditions, the efficacy of TSH acting at the mutant is
considerably lower than that of the wild-type receptor (Fig. 4C). In
binding studies performed in parallel, the reduced potency and efficacy
of TSH interacting with the paternal mutant receptor were reflected by
a drastically reduced affinity of TSHR-C390W to
[125I]bTSH (Fig. 4D).
Cellular localization of wild-type and truncated TSHR
As altered or absent functional characteristics of mutated TSHRs
could potentially be related to aberrant cellular protein expression,
we set out to study the expression and subcellular localization of TSHR
by confocal fluorescence microscopy using a monoclonal antibody
directed against the extracellular domain of the human TSHR (25).
Nonpermeabilized COS-7 cells transfected with a cDNA coding for the
wild-type TSHR and for TSHR-C390W showed an intense staining of the
plasma membrane (Fig. 5, A and C),
whereas cells transfected with TSHR-419trunc cDNA completely lacked any
cell surface staining (Fig. 5E). Studies with permeabilized cells,
however, revealed that considerable amounts of wild-type and mutant
TSHRs were retained inside the cell (Fig. 5, B, D, and F). It should
not go unnoticed, however, that a high percentage of cells expressing
the truncated receptor protein contained large immunoreactive
conglomerates, preferably located in the vicinity of the nucleus (Fig. 5F).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit gene of glycoprotein hormones (29). The demonstration of
the first expression of the TSHR gene on day 14 of mouse development, a
time when the thyroid anlage has just arrived in the position of the
anterior neck and differentiation of thyroid follicles starts (5),
suggests that TSH function might not be important for the early events
of thyroid development, but may regulate the subsequent growth and
differentiation of the gland. Thus, the clinical consequence of loss of
function mutations may cover a range from hyperthyrotropinemia to
hypoplasia with hypothyroidism, but not to athyrosis or ectopy.
Assuming that TSH plays a critical role in promoting growth as well as
differentiation of the human thyroid gland, the TSHR can be considered
a candidate gene, possibly altered in congenital hypothyroidism due to
hypoplasia of the gland (30). Therefore, we screened for mutations in
the TSHR gene in patients with congenital hypothyroidism and thyroid
hypoplasia. Two novel TSHR mutants were identified in a girl suffering
from congenital hypothyroidism. She is a compound heterozygote, having
inherited distinct defective alleles from either parent.
Functional characterization of the mutant paternal TSHR harboring a
missense mutation at nucleotide position 1170 revealed a loss of
affinity and a subsequent loss of potency of TSH acting at the mutant
TSHR-C390W. The human TSHR contains 11 cysteine residues in the
N-terminal extracellular domain that are assumed to be involved in
disulfide bonding to preserve the overall three-dimensional structure
of the ligand-binding domain and to attach the extracellular
-subunit to the membrane-spanning ß-subunit after cleavage of the
receptor protein in the cell membrane (31). C-390 is strictly conserved
within the family of glycoprotein hormone receptors (3). It was
speculated that C-301 and C-390 are linked by a disulfide bond to
preserve a protein conformation required for high affinity hormone
interaction and for binding of blocking anti-TSHR antibodies (32).
Thus, it was not surprising that the disruption of a putative disulfide
bridge and the introduction of a bulky hydrophobic indol ring into a
hydrophilic environment, as realized in the mutant TSHR-C390W,
interfered with hormone binding and signal transduction of the mutant
receptor. In consonance with our findings, mutation of C-390 to ser
decreased the affinity of the receptor for TSH and the potency of the
hormone in terms of cAMP formation approximately 25- to 30-fold
(32).
It is noteworthy, however, that the phenotype of the paternal mutation at the cellular level was more prominent in CHO cells permanently expressing TSHRs than in transiently transfected COS-7 cells. In the case of mutant V2 vasopressin receptors, it has been shown that functional data obtained in COS-7 cells may differ dramatically from those derived from stable cell lines (33). Although only a small fraction of COS cells incorporate TSHR cDNA during transient transfection procedures, the few cells that become transfected will substantially overexpress receptor protein. Under such circumstances it has frequently been found that an increase in receptor density is associated with a decrease in the EC50 value for the agonist, and artifactual G protein coupling patterns may occur (summarized in 27 . Thus, after transient transfection of COS cells, caution has to be exercised when interpreting signal transduction processes in physiological terms. CHO cell lines chosen for our studies represent homogeneous cell populations expressing wild-type TSHRs at physiological densities (28). In contrast to studies with the wild-type receptor, TSH-induced cAMP accumulation via the mutant TSHR-C390W was hardly detectable if expressed at comparably low densities (10–50 fmol/mg protein). Therefore, we had to resort to a stable cell line homogeneously expressing higher densities of mutant receptors to functionally characterize TSHR-C390W. In summary, one may surmise that in thyroid follicular cells, signal transduction involving TSHR-C390W is severely impaired regardless of elevated serum TSH concentrations.
The mutant maternal allele described in the present study codes for a truncated receptor protein that comprises most of the N-terminal hormone-binding domain (TSHR-419trunc). In mammalian cells it has been difficult to produce conformationally intact TSHR ectodomain that still had the ability to bind hormone and were secreted from the cell (34, 35). Truncated LH/CG receptors, however, displayed high affinity hormone binding, but were also reported by some researchers to be trapped intracellularly (36, 37), although other investigators described the secretion of truncated LH/CG receptors (38, 39). Immunofluorescence studies revealed that TSHR-419trunc cDNA was transcribed and translated. Most noticeably, the truncated protein was retained intracellularly and was not inserted into the plasma membrane. As TSHR-419trunc cDNA encodes the extracellular TSHR domain nearly in its entirety, including the signal sequence for translocation through endoplasmic reticulum (ER) membranes, it appears reasonable to assume that the reticular fluorescence pattern observed resulted from synthesis of the truncated protein at the rough ER. Failure of an aberrant protein to fold correctly may lead to retention in the ER (40), resulting in the formation of large immunopositive intracellular aggregates, as observed by us in transfected COS-7 cells.
Hypothyroidism of the patient, as assessed by thyroid function tests, was not as severe as that in patients suffering from athyrosis or profound defects of thyroid hormone biosynthesis. Serum T4 concentrations at the time of clinical diagnosis and after discontinuation of replacement therapy were within the range determined in patients with thyroid ectopy (32–130 nmol/L) or thyroid dyshormogenesis (20–154 nmol/L) (41). During hormone replacement therapy, close monitoring and a steady increase in the dose of L-T4 was necessary, because a decrease in the T4 dose per kg BW was accompanied by a pronounced increase in serum TSH levels and a decrease in T4 and free T4 levels, probably reflecting the limited biosynthetic capacity of the hypoplastic gland. This observation is at variance with other cases of mild hypothyroidism, for instance due to thyroid ectopy, which can be treated with lower daily L-T4 doses for the first 3 yr of life without evidence of decreasing T4 levels (42). In summary, the diagnosis of persistent congenital hypothyroidism is substantiated by clinical and biochemical findings. These clinical data are in good agreement with results obtained in in vitro expression experiments that emphasize a severely impaired signal transduction via mutant TSHRs. The low residual T4 secretion observed in our patient may result from the residual signal transduction capability of the paternal TSHR-C390W and may reflect basal T4 secretion as well.
The father and one younger sister who bear the TSHR-C390W mutation were euthyroid. The mother carrying the TSHR-419trunc allele, however, was reported to have undergone a period of mild hypothyroidism in the past. Upon reexamination by us, the latter diagnosis could not be confirmed. However, we cannot rule out that in certain situations, such as puberty, pregnancy, or iodine deficiency, wild-type/TSHR-419trunc heterozygocity may develop the clinical correlate of hyperthyrotropinemia or even mild hypothyroidism. These observations notwithstanding, genetic and clinical studies suggest an autosomal recessive mode of inheritance of congenital hypothyroidism in the affected family.
The phenotype of this patient with congenital hypothyroidism and thyroid hypoplasia differs from the clinical picture of three siblings identified to be compound heterozygote for two different TSHR mutations in the extracellular domain (P162A/I167N), who were characterized to have normal thyroid function with elevated TSH levels and normal thyroid volume (9). At the time of submission of our manuscript, de Roux et al. (43) reported on four new cases of hyperthyrotropinemia due to mutant TSHRs. Because of these different phenotypes we would like to put forward the hypothesis that loss of function mutations in the TSHR may result in a spectrum of clinical manifestations ranging from mild, subclinical hyporesponsiveness to TSH as first described by Sunthornthepvarakul et al. (9) to severe hypothyroidism and thyroid hypoplasia as exemplified by the hyt/hyt mouse (6, 7) and by the patient described in this study. The resulting phenotype may reflect the critical amount of residual TSHR activity necessary for normal human thyroid development. The same differences in phenotype depending on the degree of loss of function mutations have been reported for the LH receptor, another member of the glycoprotein hormone receptor subfamily including the FSH and TSH receptors. Although homozygosity of a truncated LH receptor resulted in the severe phenotype of male pseudohermophroditism, a single amino acid substitution in the transmembrane domain with only reduced transduction activity was characterized by the less severe phenotype of micropenis (44). However, it should be mentioned that differences in phenotype of even the same mutation exist depending on the individual genetic background and environmental factors, as shown for activating germ-line mutations of the TSHR (45).
In the present study we demonstrate for the first time that germ-line loss of function mutations in the TSHR gene can result in persistent congenital hypothyroidism due to hypoplasia of the thyroid gland. However, loss of function mutations in the TSHR are unlikely to provide a molec-ular explanation for the majority of patients suffering from congenital hypothyroidism because most of them are characterized by thyroid ectopy or athyrosis. Moreover, the autosomal recessive inheritance demonstrated for TSHR loss of function mutations is incompatible with the hallmark of sporadic occurrence in almost all cases with congenital hypothyroidism, implying other genetic mechanisms or environmental factors as the cause of thyroid dysgenesis.
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Acknowledgments |
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Footnotes |
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Received December 31, 1996.
Revised June 10, 1997.
Accepted June 20, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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T3–1 cells. Mol Cell
Endocrinol. 87:95–103.[CrossRef][Medline]
-subunit produces hypogonadal and hypothyroid mice. Genes Dev. 9:2007–2019.This article has been cited by other articles:
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M. Tonacchera, A. Perri, G. De Marco, P. Agretti, M. E. Banco, C. Di Cosmo, L. Grasso, P. Vitti, L. Chiovato, and A. Pinchera Low Prevalence of Thyrotropin Receptor Mutations in a Large Series of Subjects with Sporadic and Familial Nonautoimmune Subclinical Hypothyroidism J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5787 - 5793. [Abstract] [Full Text] [PDF]
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N. Jordan, N. Williams, J. W. Gregory, C. Evans, M. Owen, and M. Ludgate The W546X Mutation of the Thyrotropin Receptor Gene: Potential Major Contributor to Thyroid Dysfunction in a Caucasian Population J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1002 - 1005. [Abstract] [Full Text] [PDF]
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R. S. Brown and L. A. Demmer The Etiology of Thyroid Dysgenesis--Still an Enigma after All These Years J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4069 - 4071. [Full Text] [PDF]
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L. Alberti, M. C. Proverbio, S. Costagliola, R. Romoli, B. Boldrighini, M. C. Vigone, G. Weber, G. Chiumello, P. Beck-Peccoz, and L. Persani Germline Mutations of TSH Receptor Gene as Cause of Nonautoimmune Subclinical Hypothyroidism J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2549 - 2555. [Abstract] [Full Text] [PDF]
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),
TSHR-C390W (
), and TSHR-419trunc (
) cDNAs. Cells were incubated
with various concentrations of bTSH, and increases in intracellular
cAMP levels were determined as described in Subjects and
Methods. Responses are expressed as the fold increase in cAMP
content over basal levels (147 ± 28 cpm for COS-7; 137 ± 26
cpm for CHO-K1 cells) determined in the absence of bTSH. To
characterize binding properties of wild-type and mutant receptors,
displacement studies were performed, incubating membranes from
transfected cell lines with approximately 40,000 cpm
[125I]bTSH (per tube) and increasing concentrations of
displacing bTSH. Data are given as the percentage of specifically bound
[125I]bTSH (<10% of total counts per min) in the
absence of displacing ligand. Data represent the mean ±
SEM of three independent experiments.