Somatic and germline mutations of the TSH receptor and thyroid diseases (2001)

The TSH receptor (TSH-R) belongs to the superfamily of G-protein-coupled receptors (GPCRs) (Nagayama & Rapoport, 1992; Vassart & Dumont, 1992), in a subfamily whose prototypic members are rhodopsin and adrenergic receptors (Helmreich & Hofmann, 1996). A common characteristic of GPCRs is the presence of seven transmembrane segments, subdividing the molecules into an extracellular domain (aminoterminal segments plus three extracellular loops), a transmembrane barrel (seven α helices) and an intracytoplasmic domain (three intracytoplasmic loops plus the carboxyl-terminal segment). Together with the other glycoprotein hormone receptors (LH/CG-R and FSH-R), the TSH-R is characterized by a particularly long aminoterminal segment (398 residues), which is responsible for high affinity binding of TSH (Parmentier et al., 1989). The intracytoplasmic domain of GPCRs contains the site(s) of interactions with G proteins, the transducers between activated receptors and the various effectors (O'Dowd et al., 1988). In human thyroid cells TSH-R interacts functionally with the Gs and Gq proteins (Fig. 1): the TSH-R is preferentially coupled to Gs leading to a stimulation of the cyclic AMP (cAMP) cascade, but at higher thyrotropin concentrations the TSH-R is also coupled to Gq, leading to the activation of the phospholipase C cascade and thus stimulating the production of diacylglycerol and inositolphosphate (IP3) (Laurent et al., 1987). As activation of phospholipase C pathway requires concentration of TSH five to 10 times higher than those needed for activation of the cAMP pathway, it is generally accepted that the main effect of TSH on the thyroid gland is to control cAMP concentrations. The cAMP pathway has been shown to positively control the function (thyroid hormones secretion) and growth of thyrocytes whereas the phospholipase C pathway is principally involved in control of iodination and thyroid hormone synthesis (Dumont et al., 1992)(Fig. 1). Regulatory cascades controlled by the TSH receptor. Because the cAMP cascade has a central role in thyroid cell function and proliferation, it is expected that interruption or inappropriate stimulation of this cascade could lead to various thyroid diseases. For a hormone receptor, ‘gain-of-function’ may have three meanings: (1) Activation in the absence of ligand (constitutive activity) (2) Increased sensitivity to its normal agonist (3) Broadening of its specificity to other agonists. In the case of the TSH-R, the first situation is expected to lead to thyroid autonomy, i.e. TSH-independent activation of thyroid cell. In the presence of normal T4 negative feedback, the second case would simply be expected to cause adjustment of TSH concentration to a lower value. In the third situation, inappropriate stimulation of the gland is expected because an ‘unnatural agonist’ is not expected to be subjected to the normal negative feed-back. If a gain of function of the first category occurs in a single thyrocyte (somatic mutation), constitutive activation of the cAMP-dependent regulatory cascade is expected to cause clonal expansion of the mutated cell and unregulated growth of autonomous hyperfunctioning tissue (Dumont et al., 1989). In case of a germline mutation, the mutation is present in all cells of the organism and autonomy will be displayed by the whole tissue expressing the gene. Such gain-of-function could theoretically occur secondary to heterozygous mutations activating in a constitutive way any of the elements of the cAMP regulatory cascade (TSH-R, Gsα, adenylylcyclase, protein kinase A…) Alternatively, it could result from homozygous mutations inactivating both alleles of a protein with a negative effect on the regulation of the cascade (cAMP phosphodiesterases, elements of the iodine metabolism.) Up until now, only activating mutations in two molecules of the cAMP regulatory cascade have been associated with thyroid hyperfunction: Gsα and the TSH-R. If a gain-of-function mutation of the TSH-R or Gsα occurs in a single cell, autonomous activity of the cAMP regulatory cascade will presumably cause the clonal expansion of a cell displaying permanent functional activity. The resulting tumour will progressively take over the function of the normal tissue, leading ultimately to its extinction. This scenario is consistent with the natural history and clinical findings of two thyroid diseases caused by tissue autonomy: solitary toxic adenomas and multinodular goitres. Solitary toxic thyroid adenomas are well defined, encapsulated benign tumours characterized by TSH-independent (autonomous) function and growth (Hamburger, 1987). As the adenoma grows, its contribution to thyroid hormone secretion increases, TSH secretion therefore decreases and finally hyperthyroidism ensues. The decrease in TSH secretion results in reduced function of the extranodular tissue, so that its ability to concentrate the radionuclide decreases (Fig. 2). Proposed scheme for the evolution of radionuclide thyroid scans (upper panel) and thyroid hormone secretion (lower panel) in autonomous adenoma. The progression from stage 1 to stage 4 remains hypothetical. Stage 0: normal radionuclide thyroid scan and normal hormone production. Stage 1: an autonomous adenoma starts out as a small warm or hot nodule on the radionuclide thyroid scan depending on the contrast with the extranodular tissue. Total hormone secretion is normal with a small proportion produced by the autonomous tissue. The TSH level generally remains normal. Stage 2: the autonomous adenoma increases in size and appears now as a hot nodule on the radionuclide thyroid scan. Radionuclide uptake by the extranodular tissue is decreased. Total hormone secretion remains normal but a significant proportion comes from the autonomous tissue. The TSH level remains normal or is slightly decreased. Stage 3: the autonomous adenoma is still increasing in size. Radionuclide uptake by the extranodular tissue is suppressed. Total hormone secretion is normal or slightly increased and comes mainly from autonomous tissue. The TSH level is suppressed. Stage 4: the autonomous adenoma is still growing. Radionuclide uptake by the extranodular tissue remains suppressed. Total hormone secretion is increased (thyrotoxicosis). The TSH level is suppressed (from Corvilain, B,., Dumont, J.E. & Vassart, G. (2000) Toxic adenoma and toxic multinodular goiter. In: Braverman, L.E. & Utiger, R.D., eds. Werner and Ingbar's The Thyroid: a fundamental and clinical text, 8th edn. J.B. Lippincott Williams and Wilkins, Chapter 31, p. 565 with the permission of the editor). The pathogenesis of multinodular goitres is not completely elucidated. In the early phase of goitrogenesis, non toxic goitres are mainly diffuse goitres. With time, diffuse goitres continue to grow but also become nodular. The growth of multiple hyperactive areas causes first a non toxic multinodular goitre and finally a toxic multinodular goitre. Many of these goitres have nodules that grow autonomously but have little ability to concentrate iodine (cold nodules) explaining the heterogeneous pattern of uptake on a radionuclide thyroid scan. The pattern of evolution described above is supported by cross-sectional studies showing that prevalence of goitre, thyroid volume, thyroid nodularity and thyroid autonomy are positively correlated with age (Berghout et al., 1990; Aghini-Lombardi et al., 1999). For a review see (Hermus & Huysmans, 2000). Soon after mutations of Gsα had been found in adenomas of the pituitary somatotrophs, similar mutations (also called Gsp mutations) were found in some toxic thyroid adenomas and follicular carcinomas (Landis et al., 1989; Lyons et al., 1990) (Farfel et al., 1999). The mutated residues (Arg201, Glu227) are homologous to those found mutated in the ras proto-oncogenes:, i.e. the mutations decrease the endogenous GTPase activity of the G protein, resulting in a constitutively active molecule. The frequency of mutations in Gsα in thyroid hyperfunctioning adenomas varies from 0 to 38% in different reports (O'Sullivan et al., 1991; Russo et al., 1995b; Fuhrer et al., 1997a; Parma et al., 1997; Tonacchera et al., 1998; Tonacchera et al., 1999). However, according to studies published after 1996, they probably account for less than 10% of toxic adenomas (Tonacchera et al., 1999; Fuhrer et al., 1997a; Tonacchera et al., 1998) (Parma et al., 1997). Schematic representation of the TSH receptor with the locations of known gain-of-function mutations with increased activity after are The by that G receptors as the adrenergic receptors be constitutively activated by in the third et al., 1990; et al., 1992; et al., to a for similar natural mutations in the TSH-R (Parma et al., et al., Toxic adenomas were found to be a of mutations resulting in a activation of a known receptor, the TSH-R, probably because the is and to of the mutations are in the third or in the transmembrane segment of the receptor. The the role of this of the in the activation et al., 1997). were also found in the and in the extracellular aminoterminal domain of the receptor et al., The prevalence of TSH-R mutations in toxic adenomas has in different to be in in and in the but they have been found in the of This may be to the various of et al., 1995b; et al., or the different sensitivity of the but the is in iodine In with a of activating mutations of the TSH-R are the cause of solitary toxic for to of the (Tonacchera et al., 1998; Tonacchera et al., et al., 1997a; Parma et al., 1997). The of TSH-R mutations with Gsα mutations may have as by constitutive activation of the pathway is at the receptor level than at Gs and is therefore to a the of for activating mutations is higher in the TSH-R 10 different than in Gs lower iodine may to mutations in the TSH-R is not In iodine thyroid are to a stimulation by This stimulation a caused by TSH of and secondary production of et al., et al., 1996). by increasing the cell will mutations and therefore the frequency of is also that the of iodine may evolution of the mutated cell and the of a in TSH-R are also found in hyperfunctioning nodules from with a multinodular goitre et al., 1997a; et al., 1999). In some with a multinodular goitre and two autonomous a different mutation of the TSH-R in nodule et al., 1997a; Tonacchera et al., activating TSH-R mutations are also found in different autonomous areas of the multinodular goitre, in the absence of adenoma. This in hyperfunctioning areas to i.e. multiple and not by a and in areas on et Tonacchera et is therefore that TSH-R mutation is an early in the of the of hyperfunctioning areas of toxic multinodular goitre. The higher and the higher found in thyroid to iodine may therefore account for the high of mutations in the TSH-R found in with a iodine The role of these in mutations in other thyroid remains an between high in the thyroid and the of thyroid in goitre The of TSH-R mutations in thyroid has been in a proportion of follicular thyroid for high activity of et al., et al., or for high iodine uptake et al., et et 2000). the of these in less as well as the of of autonomous adenomas the benign of the cAMP pathway in thyrocytes and therefore of activating TSH-R mutations et al., et al., 1999). This in well with the role of this pathway in both and (Dumont et al., A of germline mutation activating the cAMP regulatory cascade is in with this are for mutation in Gsα in multiple leading to the constitutive activation of the cAMP regulatory cascade in various the thyroid et al., et al., thyroid adenomas in these from cells the mutation, a for gain-of-function from this only germline mutations in TSH-R have been associated with activation of the cAMP regulatory cascade in the A similar is responsible for of the resulting from activating mutations of the receptor et al., The first of a in which hyperthyroidism as an in the absence of by the of in et al., it with until of this with from to the of two germline activating mutations of the TSH-R which in with the et al., This toxic thyroid or A of have been this first they an and different mutation of the TSH-R (Tonacchera et al., Fuhrer et al., This is characterized by the clinical hyperthyroidism with a age of (from to a the goitre of but with absence of of As age at may be in by in iodine or is needed because is of of The prevalence of toxic thyroid is to as it is that have been for with negative thyroid stimulating reports have from only has been with a TSH-R mutation et al., 1999). In a with a high prevalence of hyperthyroidism mutations of the TSH-R were but only 10 et 2000). iodine in different may the in results with to mutations in the be found to they in prevalence for germline with toxic thyroid have been described in from et al., et al., et al., et al., 1997a; et al., et al., of the TSH-R were in the but were in the after of by a germline hyperthyroidism and the than in toxic thyroid hyperthyroidism is at This be in a case of hyperthyroidism with history of hyperthyroidism is caused by of thyroid stimulating the hyperthyroidism not and requires of the mutations found in have also been found in toxic In the of mutations in toxic thyroid are not in toxic This is with the mutations a less with a effect on in the absence of The of the activating mutations of the TSH-R have been by in the of the it is to increases in the constitutive activity of the TSH-R. has been that the receptor significant constitutive activity et al., 1992; et al., et al., This characteristic is not to the TSH-R & et al., et al., it is not by its the receptor. Such a could the thyroid function in TSH The effect of activating mutations be in of in constitutive different mutated residues have been found in toxic adenoma or toxic thyroid found in toxic adenomas common (1) they the constitutive activity of the receptor stimulation of (2) with a (Parma et al., they not constitutive activity the (3) at the cell is (from slightly to but not all of a to TSH by stimulation of cAMP and inositolphosphate but with a to they TSH with an affinity higher than that of the receptor. is between the level of cAMP by different in cells and level of at the cell et al., This that have different constitutive (cAMP at the cell this activity may the of receptor it is not a of the effect of the mutation in would be to the to be found between the and the level of cAMP of the level of receptor However, to its cAMP in is to the level of of constitutive TSH-R in adenomas has not been between the of the in non thyroid cells and thyrocytes in may this of a of TSH-R in cells from the natural in thyrocytes in (1) the level of of the receptor is higher in cells (2) the and concentrations of G are probably different (3) to the the cell is non This is for by the that the TSH-R only cAMP production in thyroid but both the cAMP cascade and phospholipase C cascade it is in The that in a of residues over the of the TSH-R causes in its constitutive activity is with the for activation of G receptors et al., This that activation is by the of a in the of the et al., 1992; et al., et al., et al., et al., 1996). The that mutations in residues over of the of the receptor are in activating it not to be a characteristic in all that the TSH-R be less than The constitutive activity of the receptor is with this the TSH-R would be to by a of The of toxic adenomas is by the prevalence of the mutation in all its has been by other et al., et al., that the adenoma from the of cell in which the mutation has other or to the of the remains an a of the of to a adenoma. cell cell by i.e. a of 1 a of 1 or 1 would a of The of in the adenomas is with this et al., that a normal thyroid cell in an times this a in the in of the of the in toxic adenomas of cells in the is increased by a of as with normal tissue, in the absence of cell would to a of et 2000). This is with a at the of the and varies from adenoma to is therefore a between the of cell to a tumour over a and the of cell at the of two The is is the are A similar on clinical findings In a in the in which were for the size of the adenoma increased by 1 or in only in and the adenoma in size in the (Hamburger, In the of of hyperthyroidism et al., et al., The evolution after therefore with the of growth to an (Hamburger, 1987). this stage the adenoma cells are therefore at the of (Hamburger, 1987). This may theoretically result from an between cell and but in in the adenoma as by the et 2000). The therefore that the of autonomous adenomas and other are not the of the Many of the of iodine and T4 and synthesis and secretion are by cAMP and are increased in toxic adenomas because of an in the cAMP pathway caused by mutations of the TSH-R or For cAMP of the in T4 and secretion and this is increased in toxic the of the is by cAMP and the high uptake of in autonomous adenomas is by an in and presumably and thyroid hormone synthesis are by the of et al., an of In human thyroid these are controlled positively by the phospholipase cascade and by the cAMP This well with the that is in autonomous adenomas et al., 2000). The of iodination and T4 and synthesis are higher in adenomas than in the tissue only because is increased et al., 1988). The TSH-R mutations which lead to a stimulation of the phospholipase C cascade would be expected to cause increases in thyroid hormone synthesis and thus to lead to The level of thyroid hormone secretion by autonomous adenomas be from the of of The first be by the of the into account that cell may by a of be at by The of the of to the of the nodule and the of by iodine in this The activity cell is to only be from the (thyroid hormone iodine is in with adenomas of less than in or less than in volume, i.e. the normal thyroid therefore be that the activity of the cells be increased by than a of et al., 1996). activity cell is from nodule to remains an If so it would for a hyperthyroidism would the nodule a size. A is in the between the size of the nodule and the of in a some are for a nodule of less than in whereas some are not for a above This may the of from nodule to to various decreases in or the of the of the active nodule particularly in the presence of As not it may also different However, it that activating mutations of the TSH-R a cause of toxic it to that the of the activation of that varies from mutation to and may lead to different activity cell but also different uptake and of growth and therefore different As the functional studies of the various TSH-R were in a in which high concentrations of receptors are this of the functional of the a the results may not with the activity of the adenoma the receptor in to the of only a of the to constitutively both the cAMP and the regulatory cascades (Parma et al., In the human thyroid cAMP and thyroid hormone the regulatory pathway is in the control of hormone synthesis TSH-R with on the cAMP cascade only or on both the cAMP and cascades would be expected to different the would be less in stimulating thyroid hormone production and lead to hot nodules which would less The would be expected to cause of normal tissue and and to lead to nodules which different activity cell, also that different may lead to different growth However, this is to the adenoma is at the of its and the between the mutation and the of a adenoma is not Because of the of mutations and the size of has been to a between the of the mutations and the of the However, were found the clinical of different particularly between those mutation activating both regulatory cascades and those with only stimulation of cAMP is therefore that other than the of the mutations may the of the The that in toxic thyroid the age of may from to the members of the the mutation the of other et al., in the of mutations may in the of the as well as in as the of iodine or in the However, the of mutations found in toxic adenomas with germline mutations found in to the that they only The a mutation in common with toxic adenomas have a with an hyperthyroidism than the A is that this a as a single cell, to a tumour the adenoma mutation be to be by the clinical In mutations a stimulation are expected to cause hyperthyroidism only in all thyrocytes as in is therefore between the activating mutation of the receptor and the This may be to (1) The in a The is generally by the of hyperthyroidism and the size of the adenoma. of may the of A of only the in of iodine between the with a of iodine and the is to cause hyperthyroidism in with autonomous nodule et al., As of iodine is generally not different iodine may the of the of The size of adenoma may be in some a of the of active is that a of the activity cell will the with the of the mutation in the TSH-R. is also that other generally not as the of with the (2) The of functional of in cells with the activity of the adenoma the receptors in may a role in and the of the For a secondary in cAMP activity described in thyroid cells from autonomous adenoma et 2000). (3) The that stimulation of the cAMP pathway in thyrocytes by the mutated receptor lead to secondary or responsible for different in the This is by the of clinical in of by may also be responsible for of thyroid in with germline mutation et al., The of the TSH-R cell in adenoma in of stimulation of the thyroid gland by human is early is responsible for decrease in TSH with increases in concentrations that the normal 1997). the concentrations of are as in hyperthyroidism may The is to be the stimulation of the TSH-R by as by the between and T4 concentrations et al., 1989; et al., A is by the of the glycoprotein hormones and et al., 1997). In in human thyroid in TSH from its receptor and and secretion et al., A has been described in in a with of hyperthyroidism to et al., The and had with the of of they were and were heterozygous for a mutation in the extracellular aminoterminal domain of the TSH-R gene. by in the receptor displayed normal However, a for the it higher sensitivity to stimulation by with TSH-R et al., This would be to cause by the concentrations in but not by the concentrations after the the of the after the This is with a gain-of-function of the for stimulation by This gain-of-function mutation is an of of a specificity to other agonists. Schematic representation of the TSH receptor with the locations of known mutations in TSH-R or in the G protein that the receptor to are expected to cause a of to The first mutation responsible for of the TSH-R found in the are to a of thyroid which et al., this would with two mutated alleles to a of in with the of the are expected to be normal or to in this is it is to cause or in the cell and thus A with to TSH were described of the mutations et al., et al., mutations were first described in a with or TSH i.e. with a normal thyroid normal level of and but TSH et al., The and high TSH concentrations to the and to a normal level of thyroid goitre that the of TSH the for thyroid hormone synthesis and thyroid The to be in an as it found in three from normal the TSH-R a different mutation in the extracellular domain of the receptor in of the The functional of the receptor were by in the displayed the displayed some activity with in for stimulation of cAMP production by were normal in both heterozygous for a of of the TSH to be a in cells of the and mutated receptors not for negative of the A of other have been published this first et al., et al., 1997; Russo et al., Tonacchera et al., with of mutations of the TSH-R have been in the of for et al., 1997; et al., 1997; et al., The displayed the for high T4 and of The were small and at were and were heterozygous However, TSH-R are responsible for only a proportion of with thyroid is as normal for than of the alleles in a A of germline of the TSH-R have been A role for these has been in thyroid diseases with As the TSH-R is also an it that in the of the aminoterminal domain of the TSH-R will it TSH-R mutations have been in some with However, it is that these a to the TSH-R has been in a of et al., of of human TSH-R associated with multinodular goitre, that its presence may be an in the pathogenesis of multinodular goitre et al., 1999). This in and not in a et 2000). As for other TSH-R mutations and for studies in this the of studies in the absence of TSH-R may also account for different TSH-R sensitivity a this may of TSH for a is to and probably of little clinical in the TSH-R account for a significant of of hyperthyroidism but are an cause of The of the caused by mutations of the TSH-R a of between and Because of the of mutation and the of other the between the and the is to are to some cause than