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Dipartimento di Endocrinologia e Metabolismo, Centro Eccellenza AMBISEN, Università di Pisa (M.T., A.P., G.D.M., P.A., M.E.B., C.D.C., L.G., P.V., A.P.), 56124 Pisa, Italy; and Cattedra di Endocrinologia, Fondazione S. Maugeri, IRCCS (L.C.), Università di Pavia, 27100 Pavia, Italy
Address all correspondence and requests for reprints to: Dr. Massimo Tonacchera, Dipartimento di Endocrinologia, Università degli Studi di Pisa, Via Paradisa 2, Cisanello, 56124 Pisa, Italy. E-mail: mtonacchera{at}hotmail.com.
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Abstract |
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Introduction |
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We studied 42 subjects with slight to moderate elevations of circulating TSH and normal free thyroid hormone levels, in whom the diagnosis of autoimmune thyroid disease had been excluded by currently available in vitro and in vivo tests. For the purpose of this study, this condition was referred to as isolated hyperthyrotropinemia. In three families (A, B, and C), which included eight of the 42 cases, other members besides the propositus were found to have isolated hyperthyrotropinemia. The entire coding regions of the TSHr gene were sequenced, and TSHr mutations were found in five subjects from families A and B, whereas no mutations were identified in one member of family A, in members of family C, and in the 34 remaining cases of isolated hyperthyrotropinemia. The genetic analysis showed a previously described P162A mutation (5) in the proband (III.2), her mother (II.1), and the son (IV.1) in family A. A new inactivating mutation L252P in the proband (III.2) and her mother (II.2) was found in family B.
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Subjects and Methods |
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In all 42 subjects, serum thyroid hormone levels were within the normal range, and serum TSH levels were moderately elevated (5 mU/liter and, in five, between 10–20 mU/liter). Serial dilutions of the TSH immunoreactivity were performed in all cases and were always parallel to the standard curve (data not shown). Test for antithyroperoxidase (TPOAb), antithyroglobulin (TgAb), and anti-TSHr antibodies gave negative results. Ultrasound of the thyroid showed a normal gland in the proper position in the neck with a normoechogenic pattern.
After iv TRH challenge, the surge of TSH was proportional to basal TSH values and was followed by an adequate production of FT4 and FT3. These findings suggest that the TSH molecule had normal bioactivity (data not shown). Parathyroid function was normal.
In all subjects, serum cholesterol, triglycerides, liver enzymes (alanine aminotransferase and aspartate aminotransferase), 
-glutamyltranspeptidase, total serum lactic dehydrogenase, alkaline phosphatase, and creatine phosphokinase were in the normal range.
Four of eight subjects of pediatric age received L-T4 therapy, which produced a normalization of serum TSH. Most adults (four men and 18 woman) were not treated with L-T4 replacement therapy, and serum thyroid hormone levels and TSH were checked every year. After a follow-up of 3 yr, serum TSH concentrations were unchanged, with the value recorded at the first observation. None of these subjects developed in vitro or in vivo features of thyroid autoimmunity. The proband of family A and the proband of family B were treated with L-T4, which produced normalization of TSH.
The study was approved by the local ethical committee, and informed consent was obtained from all subjects.
Family A (Fig. 1A
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The propositus was born from consanguineous parents. She was a 47-yr-old woman who was referred to our department because of neck discomfort. She had isolated hyperthyrotropinemia (TSH, 13.4 mU/liter; FT4, 10.7 pmol/liter), with no symptoms or signs of hypothyroidism. Peripheral parameters of thyroid hormone action were in the normal range (Table 1). There was no history of hypothyroidism in the father of the proband (II.2; thyroid function tests were not performed because he had died some years previously); the mother (II.1) had normal free thyroid hormone levels, but her basal TSH level was above the upper limits of the normal range (TSH, 5.0 mU/liter; FT4, 16.1 pmol/liter). The only son of the proband (IV.1) was a clinically euthyroid 23-yr-old man. He had free thyroid hormone levels in the normal range, but his basal TSH level was above the upper limits of the normal range (TSH, 5.2 mU/liter; FT4, 11.7 pmol/liter). Tests for TPOAb, TgAb, and anti-TSHr antibodies gave negative results. Thyroid echography demonstrated a gland of normal size with a normoechogenic pattern. He had no signs or symptoms of hypothyroidism and some parameters of thyroid status were in the normal range (Table 1).
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A cousin of the proband (IV.3) had normal thyroid function tests; her daughter (V.1) had free thyroid hormone levels in the normal range, but her TSH value was at the upper limit of the normal range (TSH, 4.9 mU/liter; FT4, 11.6 pmol/liter); in the latter subject (V.1), the tests for TPOAb and TgAb were positive, suggesting a diagnosis of chronic autoimmune thyroiditis.
Family B (Fig. 1B
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The propositus (III.2) was a 34-yr-old woman who was referred to our department because of hyperthyrotropinemia. She had free thyroid hormone levels in the normal range and a basal TSH level above the upper limit of the normal range (TSH, 8.6 mU/liter; FT4, 12.7 pmol/liter). Tests for TPOAb, TgAb, and anti-TSHr antibodies gave negative results. Thyroid echography demonstrated a gland of normal size with a normoechogenic pattern. The mother (II.2) had free thyroid hormone levels in the normal range and a basal TSH level above the upper limit of the normal range (TSH, 4.6 mU/liter; FT4, 12.9 pmol/liter). Tests for TPOAb, TgAb, and anti TSHr antibodies gave negative results. The propositus and her mother had parameters of thyroid status in the normal range (Table 1).
The brother and father (II.1 and III.1) of the propositus had normal thyroid function tests.
Family C (pedigree not shown)
The propositus was a 30-yr-old woman who was referred to our department because of hyperthyrotropinemia. She had free thyroid hormone levels in the normal range and elevated basal TSH (TSH, 18 mU/liter; FT4, 12.9 pmol/liter). Tests for TPOAb, TgAb, and anti-TSHr antibodies gave negative results. Thyroid echography demonstrated a gland of normal size with a normoechogenic pattern. Her mother had free thyroid hormone levels in the normal range and a slightly elevated serum TSH (6.8 mU/liter; FT4, 9.0 pmol/liter). Tests for TPOAb, TgAb, and anti-TSHr antibodies gave negative results. The propositus and her mother had parameters of thyroid status in the normal range (data not shown).
Laboratory evaluation of thyroid function
Serum FT4 and FT3 were measured by RIA (FT4 RIA and FT3 RIA, Liso-phase, Laboratori Bouty, Milan, Italy). TSH was assessed with a sensitive method (sensitive-TSH ICMA, Immulite 2000, Diagnostic Products Corp., Los Angeles, CA). TPOAb and TgAb were measured by immunofluorometric assay (AIA-PACK TgAb/TPOAb, Tosoh Corp., Tokyo, Japan). TSHr antibodies were measured using a commercial radioreceptor assay (TRAk human, B.R.A.H.M.S., Berlin, Germany).
Sequence determination
Genomic DNA was extracted from peripheral lymphocytes using standard procedures (9), and after PCR amplification, the TSHr gene was sequenced exactly as described previously (9). 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 the mutant gene
The pSVL-TSHr construct harboring mutation L252P was obtained by site-directed mutagenesis using the GeneTailor site-directed mutagenesis system (Invitrogen Life Technologies Carlsbad, CA). The accuracy of the recombinant construct was verified by direct sequencing.
COS-7 cells transfected with wild-type (wt) and mutant receptor were used for binding studies, flow cytometry, and cAMP determination. COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 1 mM sodium pyruvate. For the transient expression of wt and mutant TSHr, COS-7 cells were seeded at a 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 (18).
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. The cAMP assay, binding assay, and flow cytometric analysis were performed exactly as described previously (15).
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Results |
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) affecting the proline residue at position 162, which was replaced by an alanine in family A. This residue is placed in the extracellular portion of the receptor. The propositus (III.2) was homozygous for the mutation (Fig. 2A). The son (IV.1) was found to be heterozygous for the P162A substitution (Fig. 2A). The mother of the proband (II.1) was heterozygous for the mutation. The nephew (IV.2) had no TSHr mutations.
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) affecting the leucine residue at position 252, which was replaced by a proline. Leucine 252 is highly conserved among the receptors of glycoprotein hormones, lending support to a pathogenetic role of mutation L252P. The residue is placed in the extracellular portion of the receptor. The propositus (III.2) was heterozygous for the mutation (Fig. 2B). The mother (II.2) was heterozygous for this mutation. In family C, the genetic analysis of the TSHr in the affected members did not reveal any alteration. In all remaining cases of isolated hypertropinemia, the genetic analysis did not reveal any loss of function mutation of the TSHr gene. Direct sequencing of all exons of the TSHr gene revealed three polymorphic variants. In three subjects there was an allelic variant in the exon 1 (Pro52Thr; in the heterozygous state). In 26 subjects there was a polymorphism in exon 7 at nucleotide 561 (AAC/AAT; in 24 it was in the homozygous state, and in two it was in the heterozygous state). This substitution did not change the amino acid in position 187. Twenty-six subjects were homozygous for a polymorphism in exon 9 at nucleotide 855 (GCC/GCT). This substitution did not change the amino acid in position 285.
Functional characteristics of the TSHr mutation L252P
The functional characteristics of the mutant receptor L252P were studied by transient expression in COS-7 cells. Cells transfected with a cDNA construct encoding the wtTSHr or the empty pSVL vector were used as controls.
Flow cytometry.
Flow cytometric analysis using the BA8 monoclonal antibody (19) directed to the TSHr (Fig. 3A) showed a low level of expression of L252P at the cell surface. The L252P was clearly detectable within the cells. These findings suggested that the L252P receptor was synthesized and recognized by the BA8 monoclonal.
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cAMP production.
As previously reported (5, 9), COS-7 cells transfected with wtTSHr exhibited a 5-fold increased production of cAMP in the absence of the agonist (205 ± 13 pmol/dish), compared with cells transfected with vector alone (30 ± 8 pmol/dish; Fig. 4). Cells transfected with the mutant receptor L252P showed a lower cAMP production with respect to the wtTSHr, but higher than that in cells transfected with the vector alone (54 ± 13 pmol/dish; Fig. 4).
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). Cells transfected with the mutant L252P displayed an greatly reduced ability to stimulate cAMP accumulation in response to bTSH. Because TSH-stimulated cAMP production in cells transfected with the mutant L252P was very low, the EC50 could not be calculated. |
Discussion |
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protein (in type 1 pseudohypoparathyroidism) (22). We studied the prevalence of TSHr mutations in a large series of subjects with isolated hyperthyrotropinemia and no detectable signs of thyroid autoimmunity. Direct sequencing of the entire coding region of the TSHr revealed the presence of TSHr mutations in five of 42 subjects included in the study. Two inactivating mutations of the TSHr gene were identified in five subjects with isolated hyperthyrotropinemia. In family A, the previously described P162A mutation was identified in the proband (III.2) (homozygous state), his son (IV.1), and the mother of the proband (II.1) in the heterozygous state. No mutations were identified in one member of this family with isolated hyperthyrotropinemia. In family B, a new inactivating mutation of the TSHr gene was identified in the proband (III.2) and the mother (II.2), both in the heterozygous state. Genetic analysis revealed an L252P missense mutation in a highly conserved residue located in the extracellular portion of the TSHr gene. The mutation was shown to cosegregate with the disease within the family. In family C, no inactivating mutations of the TSHr gene were identified. In all remaining cases of isolated hyperthyrotropinemia, the genetic analysis did not reveal any loss of function of the TSHr gene. Most patients we investigated were adults, and they did not receive L-T4 therapy.
The mutant L252P TSHr showed a very low expression at the cell surface, a low constitutive activity for the adenylyl cyclase pathway, and extremely impaired response to bTSH after transient expression in COS cells. The loss of function observed in mutant TSHr described in the literature can be due to a reduced amount (decreased synthesis or increased degradation) of the mutant receptor, failure of targeting to the membrane, or a reduced binding affinity for TSH. Like other loss of function mutations, a structural defect of the L252P due to faulty folding of the molecule is probably responsible for the poor routing of the receptor to the cell membrane.
Germline homozygous TSHr mutations have been described as a cause of thyroid resistance to TSH. 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 (5). After this initial report, other families with this condition due to compound heterozygous or homozygous inactivating mutations of the TSHr gene were described (5, 6, 7, 8, 9, 10). Recently, Alberti et al. (11) reported a higher prevalence of TSHr mutations in a series of 10 patients with the phenotype of thyroid resistance to TSH. Several factors contributed to the high prevalence of TSHr mutations in this series, because subjects were selected at neonatal screening, and familiarity for hyperthyrotropinemia was documented in eight of 10 subjects. All of these patients had thyroid hormones in the low normal range and increased serum TSH levels. Mild, borderline elevations of TSH were observed in the heterozygous parents of the original family and the other families reported with loss of function TSHr mutations (5, 6, 7, 8, 9, 10, 11). The molecular mechanisms responsible for these observations are not known and might involve haploinsufficiency or dominant negative influence of mutant receptors on wt receptor function. Similarly, in the mouse, thyroid transcription factor-1 haploinsufficiency produces hypothyroidism, mainly through a reduction in TSHr gene expression, which is partially compensated by an increase in serum TSH (23).
We were able to identify TSHr mutations in two of three families with isolated hyperthyrotropinemia. In the third family, no TSHr mutations were identified. Xie et al. (24) targeted the TSHr gene for analysis in three families with isolated resistance to TSH and were unable to detect mutations in the TSHr gene in any of them. Moreover, using intragenic polymorphic markers to perform linkage analysis, they were able to exclude the TSHr gene as the gene responsible for the phenotype in two of the families. Patients with sporadic or familial hyperthyrotropinemia may indeed represent forms of autoimmune thyroiditis without in vitro evidence of thyroid autoimmunity. A longer clinical follow-up can help to answer this question. Our study indicates that TSHr mutations are a very rare event in the pathogenesis of sporadic cases of SH, and the genetic analysis of this gene should be restricted to familial cases of SH without evidence of autoimmunity. The data presented in this paper also suggest that TSHr mutations may not account for all forms of hormone resistance, and other defects in other components of the signaling cascade might impair hormonal action. Similarly, patients with pseudohypoparathyroidism type 1b manifest target resistance only to PTH, but nucleotide sequence analysis of the gene and the cDNA encoding the type 1 PTH receptor as well as linkage analysis using intragenic polymorphisms have excluded this candidate gene as the basis for pseudohypoparathyroidism type 1b (25). Defects in other proteins mediating TSH signaling or controlling TSHr expression have been suggested as a cause of TSH resistance. Defects in G protein-coupled receptor kinase-5, different isoforms of adenylate cyclase or phosphodiesterase, and modifications of thyroid transcription factor-1, PAX-8, or cAMP response element-binding protein are also possibilities, but given their widespread expression, it seems unlikely that the phenotype would be restricted to the thyroid.
In conclusion, in five of eight familial cases, but in none of 34 sporadic cases, of isolated hyperthyrotropinemia, inactivating mutations of the TSHr were identified. The question of whether the latter cases represent subtle forms of autoimmune thyroiditis or might bear as yet unidentified genetic defects remains a subject of future studies.
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Footnotes |
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Received June 29, 2004.
Accepted August 12, 2004.
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References |
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gene in a Japanese patient with pseudohypoparathyroidism. J Endocrinol Invest 19:236–241[Medline]
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