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Thyroid Resistance to TSH Complicated by Autoimmune Thyroiditis

Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, Università di Pisa, 56124 Cisanello, 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.

Abstract

In this report we describe a 47-yr-old woman who was referred to our department for elevated serum TSH associated with normal free thyroid hormone levels, suggesting subclinical hypothyroidism. When first seen she was clinically euthyroid, and her thyroid gland was normal in size both at palpation and by ultrasound. The ultrasound of the thyroid showed a normoechogenic pattern. Serum thyroid hormone levels were confirmed to be within the normal range, whereas the serum TSH concentration was moderately elevated (13.4 µU/ml). Tests for antithyroperoxidase, antithyroglobulin, and anti-TSH receptor antibodies gave negative results. The only son of the proband, a clinically euthyroid 23-yr-old man, had a slightly elevated serum TSH concentration (5.2 µU/ml) with normal free thyroid hormone levels. The entire coding regions of the TSH receptor gene were sequenced in the proband, the son, and the father of the son. Genetic analysis in the proband showed a homozygous inactivating mutation of the TSH receptor. The mutation consisted of the substitution of an alanine in place of proline at position 162 in the extracellular portion of the receptor. The son was heterozygous for Pro¹⁶²Ala. Only the wild-type sequence was found in the father. Both the proband and her son were considered to have compensated TSH resistance and were not treated. After 2 yr of follow-up, new thyroid tests were performed in the proband and showed a marked increase in the serum TSH concentration (61 µU/ml) compared with the initially observed value; serum free T₄ and T₃ levels were in the low normal range. At that time, tests for antithyroglobulin and antithyroperoxidase antibodies gave positive results, and thyroid echography showed a gland of normal size, but with a diffuse hypoechogenic pattern. In conclusion, we describe the first case of compensated TSH resistance evolving to mild hypothyroidism due to the appearance of a chronic autoimmune thyroiditis.

THE MAJORITY OF patients with thyroid resistance to TSH have compensated hypothyroidism, defined as slightly elevated serum TSH associated with normal free thyroid hormone levels (1, 2, 3, 4). Overt hypothyroidism, with markedly reduced free thyroid hormones and markedly elevated TSH, was found in five patients with thyroid resistance reported previously (5, 6, 7, 8, 9).

The most common cause of acquired spontaneous hypothyroidism in adults is thyroid autoimmunity, characterized by the presence in the patient’s serum of antithyroperoxidase antibodies (TPOAb) and antithyroglobulin antibodies (TgAb) (10). Thus, autoimmune hypothyroidism must be distinguished from defects in the biological activity of the TSH molecule or from the rare inherited condition of thyroid resistance to TSH. This latter condition may be due to abnormalities in the TSH receptor (TSHr) (1, 2, 3, 4, 5, 6, 7, 8, 9) or defective G_s protein (in type 1 pseudohypoparathyroidism) (11, 12). Other pathogenic mechanisms, such as reduction in the expression of the TSHr or putative defects downstream of G_s, have been hypothesized, but not proven, in rare families in whom affected individuals manifest the phenotype of TSH resistance but no abnormality in the coding regions of the TSHr or G_s (13, 14).

In this report we describe a 47-yr-old woman who was initially diagnosed as having compensated hypothyroidism due to an inactivating mutation of the TSHr. During the follow-up her serum TSH showed a marked increase due to the development of autoimmune thyroiditis. This case report underscores the diagnostic difficulties that may be encountered in patients with elevated serum TSH and normal free thyroid hormone concentrations. It also supports the recommendation for long-term follow-up in patients with inherited thyroid resistance to TSH and compensated hypothyroidism.

Case Report

The propositus, a 47-yr-old woman, was referred to our department in November 1997 because a thyroid test performed for nonspecific complaints (neck constriction) had shown a moderately elevated TSH associated with normal free thyroid hormone levels. She was clinically euthyroid, with no symptoms or signs of hypothyroidism at any time before or during the first examination. At palpation her thyroid was normal in size and consistency. Laboratory investigations disclosed an elevated serum TSH level (13.4 µU/ml; normal, 0.4–4.5) with normal free T4 (FT₄) serum values (12.2 µg/dl; normal, 7–15) and FT₃ (2.8 ng/dl; normal, 2–5.5). Serum TgAb, TPOAb, and anti-TSH receptor antibodies (TRAb) were undetectable. She was 162 cm in height and 55 kg in weight. After iv TRH injection (200 µg), her serum TSH concentration increased from 13 µU/ml to a peak of 82 µU/ml at 30 min (Table 1). The TRH-induced surge of TSH was followed by an adequate production of FT₄ and FT₃ , indicating that the TSH molecule had normal bioactivity (Table 1). Serum calcium, PTH, LH, and FSH concentrations were all normal. Serum cholesterol, triglycerides, liver enzymes (alanine aminotransferase and aspartate aminotransferase), -glutamyltranspeptidase, total serum lactic dehydrogenase, alkaline phosphatase and creatine phosphokinase (CPK), were all in the normal range. Thyroid sonography showed a thyroid gland of normal size (thyroid lobes: 16 x 14 x 43 and 13 x 13 x 40 mm; volume, 8 ml) and a normal echogenic pattern. She was considered to have compensated hypothyroidism, and no substitution therapy was initiated.

Clinical and laboratory follow-up of the proband

In December 1999, during a routine laboratory control, the basal serum TSH level of the proband had increased to 61 µU/ml. The serum concentrations of FT₄ (8.2 µg/dl; normal, 7–15) and FT₃ (2.2 ng/dl; normal, 2–5.5) were in the low-normal range. Medium to high titers of TgAb (1:6400) and TPOAb (1:6400) antibodies were also detected in her serum. An increase in serum CPK (250 U/L; normal, 10–167 U/L) and total cholesterol (250 v.n. = mg/dl; normal, <200 mg/dl) were also found. Triglycerides were in the normal range (Table 2).

Materials and Methods

Laboratory evaluation of thyroid function

Serum FT₄ and FT₃ were measured by RIA (FT₄ RIA and FT₃ RIA, Lysophase, Technogenetics SpA, Milan, Italy). TSH was assessed with a sensitive method (sensitive-TSH immunoradiometric assay, Delfia, Wallac, Inc., Turku, Finland). TPOAb and TgAb were measured by passive agglutination (SERODIA-AMC and SERODIA-ATG, Fujirebio, Tokyo, Japan). TRAb were measured using a commercial RRA (TRAK assay, B.R.A.H.M.S., Berlin, Germany).

Sequence determination

Genomic DNA was extracted from peripheral lymphocytes using standard procedures (15). 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 previously described (15). Exons 1 through 9 were amplified individually using couples of intronic primers as described previously (16). At least two different PCR amplifications from genomic DNA were sequenced on double stranded DNA with sense and antisense primers. PCR products were purified (Concert Rapid PCR Purification System, Life Technologies, Inc., Grand Island, NY). Direct sequencing was performed using AmpliTaq DNA Polymerase FS, with an ABI Prism Bigdye terminator cycle sequencing kit (ABI 310 PE, PE Applied Biosystems, Foster City, CA) following the protocol of the supplier. Sequencing products were analyzed on a sequencer (model 310 A, PE Applied Biosystems). To confirm the presence of a TSHr mutation, the mutation was subcloned in a plasmid, and sequences were repeated on individual clones.

Results

Direct sequencing of exon 1 through 10 of the TSHr gene revealed the presence of a previously described anomaly (1), a C to G transversion in nucleotide 484 (CCT/GCT) affecting the proline residue at position 162, which was replaced by an alanine (Fig. 1). A homozygous, previously described, polymorphic variant in exon 1 (Pro⁵²Thr) (17) was also present. These residues are placed in the extracellular portion of the receptor. The propositus was homozygous for the mutation (Fig. 1). The son was heterozygous both for the Pro¹⁶²Ala substitution (Fig. 1) and Pro⁵²Thr. The father was heterozygous for Pro⁵²Thr.

View larger version (37K): [in this window] [in a new window]   Figure 1. Direct sequencing gel showing the genotype of the family. Direct sequencing of PCR-amplified genomic DNA from peripheral blood demonstrates an CCT/GCT mutation affecting the proline residue at position 162, which was replaced by an alanine in the first extracellular portion of the TSHr protein. The mutation was homozygous in the propositus. The son was heterozygous.

The condition of moderately elevated serum TSH associated with normal free thyroid hormone concentrations found in the proband at first examination might have been due to TSH unresponsiveness, defects in the biological activity of the TSH molecule, or chronic autoimmune thyroiditis. Chronic autoimmune thyroiditis is the most frequent cause of hypothyroidism in adults (10), and its diagnostic hallmarks are circulating TgAb, TPOAb, or TSHr-blocking antibodies. A hypoechogenic pattern of the thyroid gland at ultrasound examination is also observed in autoimmune thyroiditis (18). Serum-negative Hashimoto’s thyroiditis, although uncommon, may also occur and may be suspected on the basis of a thyroid hypoechogenic pattern at ultrasound (18) and eventually diagnosed by fine needle aspiration of the gland. In our proband the absence of TPOAb, TgAb, and TRAb in serum and the normal pattern of the thyroid ultrasound excluded autoimmune thyroiditis at first examination. A bioinactive TSH was ruled out by the normal rise of serum FT₃ and FT₄ upon TSH surge induced by TRH challenge. Thus, a diagnosis of TSH unresponsiveness was done. This condition may be due to TSHr anomalies or alterations in the proteins involved in the signaling pathway downstream from the receptor (1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12).

Patients with the syndrome of TSH unresponsiveness described previously have compensated (1, 2, 3, 4) or overt hypothyroidism (5, 6, 7, 8, 9). Their thyroid gland is located in the normal position in the neck and may be of either normal or reduced size. Serum TSH levels are invariably increased, and the bioactivity of the TSH molecule is intact (1, 2, 3, 4, 5, 6, 7, 8, 9). Loss of function TSHr mutations have been described as the major cause of TSH unresponsiveness (1, 2, 3, 4, 5, 6, 7, 8, 9). Other pathogenic mechanisms, such as reduction in the expression of the TSHr or putative defects downstream of G_s, have been hypothesized, but not proven, in families in whom affected individuals manifest the phenotype of resistance to TSH without abnormalities in the coding regions of the TSHr or G_s (13, 14).

The proband described in this paper was clinically euthyroid with elevated TSH only discovered during laboratory investigations performed for nonspecific complaints. The genetic analysis in this patient showed a homozygous mutation yielding a Pro¹⁶²Ala substitution in the extracellular domain of the receptor molecule. The Pro¹⁶²Ala mutation was previously detected in the patients studied by Refetoff et al. (1) (as a compound heterozygosis together with Ile¹⁶⁷Asn) and as a homozygous mutation in two probands described by de Roux et al. (2). Serum TSH concentrations in our proband were moderately elevated, whereas her son, who was heterozygous for the Pro¹⁶²Ala, had serum TSH levels just above the upper limit of the normal range. The Pro¹⁶²Ala mutation has been demonstrated to be responsible for the phenotype described previously (1, 19). Functional studies performed after transfection of the mutated TSHr in eukaryotic cells clearly showed that the mutant Pro¹⁶²Ala receptor has 1/10th the normal activity of the wild-type TSHr (19). The Pro¹⁶² residue belongs to the putative amino-terminal hormone- binding region of the receptor and is located within the portion of the molecule that has been shown to be made of leucine-rich motifs (19). It has been proposed (19) that substitutions of Pro¹⁶² might affect the binding characteristics of the receptor without dramatically interfering with its overall structure.

In the proband of family 4 studied by de Roux et al. (2) who harbored a germline homozygous Pro¹⁶²Ala TSHr mutation, the serum TSH level was 99 µU/ml. Within the same family, the proband’s sibling, who also harbored the Pro¹⁶²Ala mutation, had a serum TSH level of 14 µU/ml. Thus, similar to what was shown for activating germline mutations of the TSHr (17), the same inactivating mutation may determine a phenotype of different severity, possibly due to a different genetic background and/or environmental factors. It is therefore not surprising that in our proband the serum TSH concentration was 13 µU/ml.

The peculiarity of the phenotype in our patient was that during a follow-up laboratory investigation 2 yr after the initial evaluation her serum TSH level had increased to 62 µU/ml in concomitance with the appearance of circulating TgAb and TPOAb. Serum CPK and cholesterol also were increased above normal limits. At the same time a typical hypoechogenic pattern of the thyroid was observed at ultrasonography. Thus, a diagnosis of chronic autoimmune thyroiditis was formulated. Although we believe that the association of a TSHr mutation and the development of autoimmune thyroiditis occurred by chance, it has been mentioned that the TSHr may function as an autoantigen in autoimmune thyroid diseases (17). Evidence for the involvement of this receptor in autoimmune thyroid diseases includes the presence of thyroid-stimulating antibodies in Graves’ disease and TSH binding-inhibiting antibodies in Hashimoto’s thyroiditis (17). It has been claimed that a modified TSHr could have novel immunogenic properties, possibly leading to the development of autoimmune thyroid disease. Thus, modifications in the primary structure of the amino-terminal portion of the TSHr have been searched for in patients with Graves’ disease. A first variant in the extracellular portion, Asp³⁶His, was initially described as resulting from a somatic mutation and was proposed as a cause of thyroid autoimmunity. This interpretation was later retracted when it was found that the Asp³⁶His was actually a germline variant corresponding to a polymorphism that is also present in healthy people (17). The second variant described in the TSHr extracellular portion, Pro⁵²Thr, was found in about 9% of patients with Graves’ disease as well as in 12% of the normal population (7). As such it qualifies as a frequent polymorphism. In conclusion, we describe the first case of compensated TSH resistance evolving to mild hypothyroidism due to the appearance of chronic autoimmune thyroiditis.

Acknowledgments

Footnotes

This work was supported by the following grants: Ministero dell’Università e della Ricerca Scientifica (MURST), Programma di Ricerca: Le Malattie della Tiroide: Dalle Basi Molecolari alla Clinica; Ministero dell’Università e della Ricerca Scientifica (MURST), Programma di Ricerca: Strategie per la Valutazione degli Effetti Disruptivi dei Contaminanti Ambientali sul Sistema Endocrino degli Animali e dell’Uomo; CNR Progetto Biotecnologie CTB 99.00.224; and PF 31: Basi Molecolari delle Neoplasie Benigne e Maligne della Tiroide.

Abbreviations: CPK, Creatine phosphokinase; FT₃, free T₃; FT₄, free T₄; TgAb, anti-Tg antibodies; TPOAb, antithyroperoxidase antibodies; TRAb, anti-TSH receptor antibodies; TSHr, TSH receptor.

Received February 27, 2001.

Accepted May 15, 2001.

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