Thyrotropin Receptor Polymorphisms and Thyroid Diseases

Massimo Tonacchera and Aldo Pinchera

Department of Endocrinology University of Pisa 56124 Cisanello, Pisa, Italy

Address correspondence and requests for reprints to: Aldo Pinchera, Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Cisanello, Pisa, Italy. E-mail: a.pinchera{at}endoc.med.unipi.it


    Introduction
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 Introduction
 References
 
Mutations arise as a change in the coding sequence of a gene. Point mutations occur when another replaces one base. Insertions and deletions are the addition and removal of one or more bases, respectively. Different mutations will have differing consequences for the function of the protein. A nonsense mutation, resulting from a point mutation that converts a codon to a stop codon, produces a premature termination of the polypeptide chain and usually a nonfunctional protein. Because of the redundancy of the genetic code, substitutions may not lead to the incorporation of an incorrect amino acid in the protein, and even when an incorrect amino acid is incorporated (a missense mutation) it may have little effect on the function of the protein, unless it is in a critical portion of the protein. In this case, the variant allele can be transmitted to daughter cells without causing disease. If the germ line is affected, the variant allele can be transmitted over generations with no adverse effects. This is usually referred to as a simple polymorphism. Mutations are important for two reasons. They are responsible for inherited disorders and other diseases, such as cancer, that involve alterations in genes. At the same time, mutations are the source of phenotypic variations on which natural selection is based.

Over the past few years, mutations in the TSH receptor (TSHr) gene have been identified as a cause of acquired, hereditary, or congenital thyroid diseases, as recently reviewed by Duprez et al. (1). The identification of a large series of gain-of-function mutations in the TSHr gene was one of the first examples of a new pathophysiological mechanism, in which diseases are caused by mutations that increase the basal activity of a receptor and make it constitutively active, in the absence of its normal agonist. TSH stimulates the proliferation, differentiation, and functional activity of the thyroid follicular cell. TSH exerts its effects by binding to the TSHr, a member of the family of G-protein-coupled receptors characterized by seven transmembrane domains. Activation of the TSHr stimulates Gs{alpha} and the adenylyl-cyclase cyclic adenosine 3',5'-monophospahte (cAMP) pathway, resulting in an increase in growth and hormonal secretion. The TSHr also stimulates a Gq protein and the phospholipase C-dependent pathway, which generates the intracellular signals myoinositol-1,4,5-triphosphate and dyacylglycerol, which are important in man for iodination and hormone synthesis. Somatic gain-of-function mutations of the TSHr gene have been detected in toxic thyroid adenomas. Germ line gain-of-function mutations of the TSHr gene have been found in autosomal dominant familial nonautoimmune hyperthyroidism, and de novo germ line gain-of-function mutations have been described in rare cases of children with severe congenital hyperthyroidism. Mutations must be distinguished from polymorphisms in which the amino acid substitution may have little effect on the function of the protein and do not necessarily cause disease. Three germ line polymorphisms of the TSHr have been described in the population (1). Two of them were described in the extracellular portion of the TSHr: one is a substitution of an aspartic acid to histidine in position 36 (D36H), and the other is a proline substituted for a threonine in position 52 (P52T). The third polymorphism described is a substitution of glutamic acid for aspartic acid (D727E) within the intracellular portion of the receptor.

In the current issue of JCEM, Muhlberg et al. (2) studied the frequency of the germ line polymorphism D727E in a large series of patients with nonautoimmune hyperfunctioning thyroid disorders in a European Caucasian population. The polymorphism consists in the heterozygous C/G transition at position 2281 within codon 727, resulting in substitution of glutamic acid for aspartic acid (D727E) within the carboxyl-terminal tail of the receptor. PCR, followed by restriction enzyme digestion, was used to genotype a total of 128 patients with toxic nonautoimmune thyroid disorders (83 with toxic adenoma, 31 with toxic multinodular goiter, and 14 with disseminated autonomy). The same genetic analysis was conducted in 99 healthy controls and 108 patients with Graves’ disease. In their report, the D727E polymorphism was found in 13.3% of patients with toxic adenoma, in 21.3% of patients with Graves’ disease, and in 16.3% of the healthy control group. Comparison of codon 727 polymorphism failed to reveal a statistically significant difference between the patients’ groups and the control group. Furthermore, patient subgroups with toxic nonautoimmune thyroid disease, including toxic adenoma (13.3%), toxic multinodular goiter (9.6%), and disseminated autonomy (21.4%) were not related to significant difference of codon 727 polymorphism frequencies when compared with the healthy control group. The results obtained by Muhlberg et al. (2) do not support an association between the codon 727 polymorphism of the TSHr gene and nonautoimmune hyperthyroidism in this European Caucasian population. A similar observation was made by Nogueira et al. (3), who detected the D727E TSHr variant in one allele of the germ line in 27% of normal healthy controls. The same authors (3) studied the functional properties of this TSHr variant after transient expression in TSA-201 cells. No significant difference in the basal and TSH-stimulated cAMP-dependent luciferase activity was noted between D727E and the wild-type TSHr. According to these data, it would seem that the D727E variant is unlikely to play a role in the genesis or evolution of toxic nonautoimmune thyroid disease.

These results are in contrast with the previous report of Gabriel et al. (4), who described a high frequency D727E variant of the TSHr in a smaller series of patients with toxic multinodular goiter. In fact, 8 of 24 (33%) patients with toxic multinodular goiter were heterozygous for the D727E polymorphism. The same heterozygous amino acid substitution was also found in 5 of 52 (9.6%) normal individuals and in 8 of 49 (16%) patients with Graves’ disease. To characterize the functional activity of the variant TSHr, Gabriel et al. (4) showed that after transient expression in vitro in COS-7 cells, the mutant TSHr revealed a significantly greater cAMP response to TSH stimulation than wild-type receptor. From these findings the authors suggested that the germ line polymorphism D727E was significantly associated with toxic multinodular goiter and that although the sequence variation was conservative in nature, the variant receptor might demonstrate altered biological behavior. In the same study, Gabriel et al. (4) failed to find somatic activating TSHr mutations in any of the seven toxic adenomas and hyperfunctioning areas of 24 toxic multinodular goiters that were examined.

Constitutively activating somatic mutations of the TSHr gene have been implicated as the major cause of hyperfunctioning thyroid adenomas (1), although controversy still exists about the frequency of such mutations in this condition. The frequency of mutations in the TSHr gene in thyroid hyperfunctioning adenomas varies from 3–80% in different reports. Differences in the methods applied may account for such discrepancies: sensitivity of the detection method used (direct sequencing more sensitive than analysis involving single-strand conformational polymorphism); the extent of the region screened for mutations; the quality of the tissue examined (mutations are more difficult to find in highly fragmented DNA extracted from paraffin-embedded tissue than frozen tissue); and the type of tissue sampling (surgical specimen vs. those obtained by fine-needle aspiration biopsy). In addition, other factors such as the genetic background and the iodine intake might influence the incidence of TSHr mutations in toxic thyroid adenomas. In addition to mutations in the TSHr, toxic adenomas may be caused by somatic activating mutations in the Gs{alpha} gene, although with lower frequency. When activating mutations of the TSHr gene were discovered in toxic thyroid adenoma, the mutation was found in each case in the heterozygous state, as expected for gain-of-function mutations, and it was confined in the adenomatous tissue. This constitutes clear demonstration that the autonomous adenoma results from the clonal expansion of a single cell affected by a somatic mutation. When TSHr mutants are transiently expressed from recombinant constructs in COS cells the result is a constitutive activation of cAMP accumulation. In addition to their effect on cAMP, some mutants also activate the phospholipase C-dependent cascade. A low prevalence of TSHr mutations in toxic adenoma have been observed in Japan and the United States where the population lives in areas with adequate iodine intake. It has also been proposed that other intricate aberrations of many complex growth-controlling mechanisms might be implicated in the pathogenesis of these hyperfunctioning nodules (5). In this regard, data of ADP ribosylation of stimulatory and inhibitory G protein {alpha} subunit, together with adenylate-cyclase activity in a group of toxic adenomas with and without mutations in the TSHr or Gs{alpha} genes, suggested that a constitutive activation of the adenylate-cyclase pathway may not be sufficient to generate these adenomas (5).

TSHr mutations have also been searched for in toxic multinodular goiter. The term "toxic multinodular goiter" encompasses a spectrum of different clinical entities, ranging from a single hyperfunctioning nodule within an enlarged thyroid that also contain nonfunctioning nodules, to multiple hyperfunctioning areas scattered throughout the gland, barely distinguishable from nonfunctioning nodules and extranodular parenchyma. Recently, we (6) reported that similar to solitary toxic thyroid adenomas, activating TSHr mutations are present in single hyperfunctioning nodules (either adenoma or hyperplastic nodules) within a toxic multinodular goiter in which nonfunctioning nodules also coexist. The presence of activating TSHr mutations in a few cases of multiple adenomatosis has also been reported (7, 8). However, in areas of iodine deficiency, most patients with toxic or autonomous multinodular goiter show thyroid scintigraphic patterns in which hyperfunctioning areas are not superimposable onto nodules found at physical and ultrasound examination. This implies that the boundaries of scintigraphic areas with increased radioiodine uptake do not necessarily correspond to the anatomic boundaries of thyroid nodules. In a recent report (9), we have shown that activating TSHr mutations are present in the majority of nonadenomatous hyperfunctioning nodules scattered throughout the gland in patients with toxic or functionally autonomous multinodular goiter coming from an area of iodine deficiency. Most hyperfunctioning areas corresponded to aggregates of micro-macrofollicular structures not defined by a capsule (9). Thus, it would seem that TSHr mutations are also implicated in the genesis of the majority of hyperfunctioning areas of toxic multinodular goiter.

It is well recognized that the TSHr functions as a B-cell autoantigen in autoimmune thyroid diseases. 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. However, whether the TSHr is directly involved in the initiation of autoimmune thyroid diseases remains unclear. For this reason, mutations of the TSHr gene have been searched for in patients with Graves’ disease. It has been claimed that a modified antigen could have novel immunogenic properties. A first variant in the extracellular portion, D36H, was initially described as resulting from a somatic mutation and proposed as a cause of thyroid autoimmunity. This interpretation was later retracted by the same authors when it was found that the D36H was actually a germ line variant corresponding to a polymorphism that is also present in healthy people (10). The D36H variant TSHr did not show any functional impairment with regard to ligand binding and adenylate-cyclase activation when expressed in eukariotic cells (10). The second variant described in the TSHr extracellular portion, P52T, was found in about 9% of patients with Graves’ disease, but also in 12% of the normal population. As such, it qualifies as a frequent polymorphism. It has been reported that the P52T variant receptor displays an enhanced signaling efficiency toward cAMP generation when expressed in CHO cells (11). On this basis, it was speculated that the P52T could aggravate hyperthyroidism in patients with Graves’ disease. This observation was not confirmed in subsequent studies in which it has been shown that the functional properties of P52T could not be distinguished from wild-type TSHr when tested after expression in other cells (1). Moreover, this is consistent with the observation that homozygosity for P52T was found in two healthy individuals who were clinically euthyroid and had normal serum FT4 and TSH concentrations (12). This provides further evidence that in vivo the variant receptor is able to respond normally to TSH stimulation (12).

In conclusion, Muhlberg et al. (2) provide clear evidence on a large series of patients and controls of a lack of association of nonautoimmune hyperfunctioning thyroid disorders and a germ line polymorphism of codon 727 of the TSHr in a European Caucasian population. As discussed above, no firm evidence exists about a role of the two other variants identified in the extracellular portion of the TSHr in the pathogenesis of thyroid autoimmune diseases. Taking into consideration the fact that these variant TSHrs are frequently detected in the general population, it would seem that they represent simple polymorphisms and probably are not involved in the development of thyroid diseases.

Received June 12, 2000.

Accepted June 12, 2000.


    References
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 Introduction
 References
 

  1. Duprez L, Parma J, Van Sande J, et al. 1998 TSH receptor mutations and thyroid disease. Trends Endocrinol Metab. 9:133–140.[CrossRef]
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  3. Nogueira CR, Kopp P, Arseven OK, Santos CLS, Jameson JL, Medeiros-Neto G. 1999 Thyrotropin receptor mutations in hyperfunctioning thyroid adenomas from Brazil. Thyroid. 9:1063–1068.[Medline]
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