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Otto Heubner Centrum für Kinderheilkunde und Jugendmedizin, Pädiatrische Endokrinologie; Charité Campus Virchow Klinikum, Humboldt Universität zu Berlin (H.B., A.G.), 13353 Berlin, Germany; Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin (T.S.), 14195 Berlin, Germany; Division of Endocrinology, Department of Pediatrics, Ohio State University (J.G.), Columbus, Ohio 43205; and Institut für Pharmakologie und Toxikologie, Fachbereich Humanmedizin, Philipps Universität Marburg (C.H., T.G.), 35033 Marburg, Germany
Address all correspondence and requests for reprints to: Dr. Annette Grüters, Otto Heubner Centrum für Kinderheilkunde und Jugendmedizin Pädiatrische Endorinologie, Charité Campus Virchow Klinikum, Humboldt Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: annette.grueters{at}charite.de
Abstract
Sporadic and familial nonautoimmune hyperthyroidism are very rarely occurring diseases. Within the last years constitutively activating TSH receptor mutations were identified as one possible pathomechanism. Except for S281N in the extracellular N-terminal domain, all other germline mutations are located in the transmembrane domains 2, 3, 5, 6, and 7 of the TSH receptor, whereas no mutation was reported in transmembrane domains 1 and 4 to date. Here we report the first family with a constitutively active TSHR mutation in transmembrane domain 1 resulting in a substitution of the conserved Gly431 for Ser. This mutation was found in the investigated patient, his father, and the paternal grandmother. As known from other familial cases of nonautoimmune hyperthyroidism, the age of onset of the disease was variable, ranging from early childhood in the patient and his father to adolescence in the grandmother. Functional characterization of this mutation showed a constitutive activation of the Gs/adenylyl cyclase system. Moreover, this germline mutation also activates the Gq/11/phospholipase C pathway. The importance of Gly431 for receptor quiescence is supported further by introduction of other mutations at this position, all leading to constitutive receptor activity. Our data show now that constitutively activating mutations can be found in the entire transmembrane domain region of the TSH receptor, indicating the important role of all parts of the transmembrane domain region for maintaining the inactive receptor conformation.
THE TSH RECEPTOR (TSHR) belongs to the subfamily of glycoprotein hormone receptors, members of the large superfamily of G protein-coupled receptors (GPCR) (1). Glycoprotein hormone receptors share the common structural feature of a large extracellular domain and a transmembrane domain (TM) consisting of seven membrane-spanning segments connected by three intracellular and three extracellular loops. Mutations leading to constitutive receptor activation were first detected in the transmembrane receptor core clustering in TM6 (2, 3). Activating germline mutations of the TSHR gene cause rarely occurring, nonautoimmune sporadic congenital hyperthyroidism (4, 5, 6, 7, 8, 9) or familial nonautoimmune hyperthyroidism (10, 11, 12, 13, 14, 15). Most gain of function mutations are located in TM2, 3, 5, 6, and 7 and at a single amino acid position (Ser281) within the extracellular domain (16). The functional consequence of these mutations is a ligand-independent activation of the Gs/adenylyl cyclase system, whereas the Gq/11/phospholipase C system appears to remain unaffected. To date only a few somatic TSHR mutations found in toxic thyroid nodules are also able to constitutively activate both the Gs/adenylyl cyclase and the Gq/11/phospholipase C pathways (17).
In this study we report the first activating TSHR mutation located in TM1 identified in a patient, his father, and grandmother, all suffering from nonautoimmune hyperthyroidism. The missense mutation leads to the substitution of the highly conserved Gly431 residue to Ser, activating both the Gs/adenylyl cyclase system and the phospholipase C pathway in a ligand-independent manner.
Case Report
The propositus is a Caucasian male. He was born at 36 wk
gestation, with a birth weight of 2500 g, without complication.
Because of a strong family history of hyperthyroidism, thyroid hormone
levels were analyzed annually by the primary care physician. These were
first noted to be abnormal at the age of 3 yr (Table 1
). The patient had been experiencing
sleep difficulties, hyperactive behavior, and a voracious appetite with
little weight gain, but no temperature intolerance or bowel movement
disturbances were observed. A 99mTc thyroid scan
showed a symmetrical and homogeneously enlarged gland without nodules
and mild increased trapping of isotope. The thyroid gland was mildly
and symmetrically enlarged without nodules, with an audible bruit over
the right lobe. Although his eyes were mildly prominent, magnetic
resonance imaging studies did not confirm an exophthalmus. Medical
treatment was started with propylthiouracil (PTU; 25 mg, three times
daily) at the age of 33/12 yr, and the dose was
adjusted over the ensuing months (Table 1). The following history was
complicated by increasing difficulties with hyperactive behavior,
tremor, sleeping difficulties, and enuresis. Because of persistence of
symptoms despite increasing doses of PTU (150 mg/d) and an increase in
thyroid hormone levels, he underwent total thyroidectomy at age
79/12 yr. The pathology report showed a thyroid
gland with hyperplastic features.
L-T4 replacement therapy was
initiated and resulted in euthyroidism and resolution of behavioral
abnormalities.
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The paternal grandmother developed hyperthyroidism in adolescence and underwent subtotal thyroidectomy at 15 yr of age. At the time of the present study she was receiving thyroid hormone replacement therapy and had had a unilateral goiter for the past 14 yr, surveyed by biopsies that were reported to be benign. She is clinically euthyroid. There is no evidence of thyroid disease on the maternal side of the family.
All clinical investigation and genetic analyses were conducted in accordance with the guidelines proposed in the Declaration of Helsinki, and informed consent was obtained from all family members.\.
Materials and Methods
Thyroid hormone and TSH levels were determined with Abbott Laboratories IMX kits (Ashland, OH). TSHR antibodies were assayed with the Mayo Laboratory kit (Rochester, MN).
Genomic DNA was prepared from peripheral white blood cells using a commercial kit (Blood Amp kit, QIAGEN, Hilden, Germany). Exons 1–10 of the TSHR gene were amplified with primers previously described (18). Sequencing reactions were performed with BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp., Weiterstadt, Germany) and were analyzed with an automatic sequencer (ABI 373, PE Applied Biosystems, Foster City, CA).
For restriction enzyme analysis a PCR fragment was amplified using a sense primer (5'-GAA TCC TTG AGT CCT TGA TGT G-3') and an antisense primer (5'-TGG CAT GGT TGT AGT ACT CA-3'). Restriction analyses were performed with DdeI at 37 C for 1 h, and products were separated on a 2% agarose gel.
The TSHR mutation (G431S) identified, as well as TSHR-G431A and TSHR-G431C, were generated using standard mutagenesis techniques. Mutant fragments were inserted by replacing an MscI/BstEII fragment within the pcDps-hTSHR. The correctness of the mutant was verified by restriction analysis and direct sequencing.
To functionally characterize the mutant TSHRs COS-7 cells were transiently transfected, and cAMP accumulation, inositolphosphate (IP) formation, and [125I]bovine TSH ([125I]bTSH) binding assays were performed as described previously (19).\.
Results and Discussion
Familial nonautoimmune hyperthyroidism is a very rare disease. To date nine families have been identified harboring constitutively activating TSHR mutations causing the disease. All naturally occurring activating TSHR mutations found were located in the extracellular domain (Ser281) or in the TM core, with a prevalence in TM6 and TM7 as well as in the third intracellular loop (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17), but no mutation has yet been identified in TM1 and TM4.
Herein we describe a family with nonautoimmune hyperthyroidism.
Direct sequencing of all PCR-amplified exons led to identification of a
heterozygous G to A transition in exon 10 at codon position 431. This
mutation resulted in the substitution of a highly conserved glycine
residue to serine (Gly431Ser) in TM1 (Fig. 1C). The mutation creates a new
restriction site for DdeI (Fig. 1B). Restriction of the PCR
fragment amplified from the mother’s genomic DNA showed a wild-type
(WT) pattern, whereas the father could be identified as a heterozygous
carrier of the mutation. Repeated amplification and digestion of the
grandmother’s PCR fragment revealed only a weak 168-bp band (see Fig. 1B). This indicates that the grandmother is probably not a heterozygous
carrier of the mutation, but most likely presents a mosaicism of the
altered TSHR gene. Sequencing of exon 10 confirmed the heterozygous
state for the G431S mutation in the patient and the father, whereas
sequencing of the grandmother’s fragment showed only a very small A
wave under the WT G wave (see Fig. 1C). Corresponding results were
obtained by sequencing the complementary strand. This finding
suggests a mosaicism and unveiled the grandmother as
potential carrier for this mutation. Unfortunately, the
presence of a grandmaternal mosaicism could not be supported or
verified further by investigations of other tissue (fibroblasts and
hair follicles) and material from other relatives because of the
unavailability of further material. In accordance with other reports
(10, 14), the onset of disease in this family ranged from
early childhood in the patient and his father to adolescence in the
patient’s grandmother. However, in this family the later onset of
disease in the grandmother may be caused by mosaicism rather than by
heterozygosity.
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). Hormone-stimulated cAMP formation
(Emax) found for TSHR-G431S was more pronounced
than that after WT-TSHR expression. To ensure that the increase in
basal cAMP accumulation of TSHR-G431S was not due to a higher receptor
level at the cell surface, TSHR-G431S was diluted 1:2 with the m3
muscarinic receptor. This results in a reduction of the
Emax value below the level of WT-TSHR-expressing
cells. The basal cAMP accumulation mediated by the diluted mutant
receptor remained elevated (see Fig. 2A), indicating strong basal
activity. Basal IP formation mediated by TSHR-G431S was slightly, but
significantly, higher (by t test, P <
0.001) compared with that mediated by the WT-TSHR (Fig. 2B). As
expected from the cAMP accumulation assay
[125I]TSH binding of TSHR-G431S revealed a
higher level of expression (Table 2),
whereas the affinity of TSH to the mutated receptor remained unaffected
(see Table 2).
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). Our preliminary data concerning
this specific position indicate that only a very small amino acid
residue, such as Gly, is tolerated at position 431.
The only activating mutation in TM1 within the glycoprotein
hormone receptor family was reported in a patient suffering from
male-limited precocious puberty (20). A constitutively
activating mutation of the LH receptor Ala373Val
was described corresponding to position 428 in the TSHR, which is
approximately one 
-helical turn above, as seen from the cell
interior, very close to that in the TSHR described here. Gromoll and
co-workers (20) speculated that due to the high degree of
conservation in this receptor region throughout species, this receptor
region may have an important role in maintaining receptor
quiescence.
In the present study we identify the first constitutively activating mutation in TM1 of the human TSHR, indicating that activating mutations can be distributed over the entire TM region. Functional studies that are extended to other signal transduction pathways beside the Gs/adenylyl cyclase system may also help in understanding the structure-function relationship.
Acknowledgments
We thank Rita Haubold and Katrin Huhne for excellent technical assistance. We also thank Bruce Wilson, M.D. and Denise Stevens, R.N., M.S.N. (Pediatric Endocrinology, Michigan State University, Ann Arbor, MI). We are thankful to BRAHMS Diagnostica (Henningsdorf, Germany) for the donation of [125I]bTSH.
Footnotes
This work was supported by the Sonnenfeld-Stiftung, Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.
Abbreviations: bTSH, Bovine TSH; Emax, hormone-stimulated cAMP formation; GPCR, G protein-coupled receptors; IP, inositolphosphate; PTU, propylthiouracil; TM, transmembrane domain; TSHR, TSH receptor; WT, wild-type.
Received January 26, 2001.
Accepted June 12, 2001.
References
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) and agonist-induced (100 mU/ml
bTSH; 
) are shown as the mean ± SEM of three
independent experiments performed in duplicate.