1. Gain-of-function mutations.
On a theoretical basis, for a hormone receptor, “gain of function” may have several meanings: (i) activation in the absence of ligand (constitutivity); (ii) increased sensitivity to its normal agonist; (iii) increased, or de novo sensitivity to an allosteric modulator; (iv) broadening of its specificity. When the receptor is part of a chemostat, as is the case for the TSH receptor, the first situation is expected to cause tissue autonomy, whereas the second would simply cause adjustment of TSH to a lower value. In the third and fourth cases, inappropriate stimulation of the target will occur because the illegitimate agonists or modulators are not expected to be subject to the normal negative feedback. If a gain-of-function mutation of the first category occurs in a single cell normally expressing the receptor (somatic mutation), it will become symptomatic only if the regulatory cascade controlled by the receptor is mitogenic in this particular cell type or, during development, if the mutation affects a progenitor contributing significantly to the final organ. Autonomous activity of the receptor will cause clonal expansion of the mutated cell. If the regulatory cascade also positively controls function, the resulting tumor may progressively take over the function of the normal tissue and ultimately result in autonomous hyperfunction. If the mutation is present in all cells of an organism (germline mutation) autonomy will be displayed by the whole organ
From what we know of thyroid cell physiology it is easy to predict the phenotypes associated with gain-of-function of the cAMP-dependent regulatory cascade. Two observations provide pertinent models of this situation. Transgenic mice made to express ectopically the adenosine A2a receptor in their thyroid display severe hyperthyroidism associated with thyroid hyperplasia 1 . As the A2a adenosine receptor is coupled to Gs and displays constitutive activity due to its continuous stimulation by ambient adenosine 2 , this model mimics closely the situation expected for a gain-of-function germline mutation of the TSH receptor. Patients with the McCune-Albright syndrome are mosaïc for mutations in Gs (Gsp mutations) leading to the constitutive stimulation of adenylyl-cyclase 3 . Hyperfunctioning thyroid adenomas develop in these patients from cells harboring the mutation, making them a model for gain-of-function somatic mutations of the TSH receptor. A transgenic model in which Gsp mutations are targeted for expression in the mouse thyroid has been constructed. Though with a less dramatic phenotype this represents also a pertinent model for a gain of function of the cAMP regulatory cascade 4 .
Since the TSH receptor is capable of activating both Gs and Gq (though with lower potency) the question arises whether mutations with a different effect on the two cascades would be associated with different phenotypes. Studies in mice 5 and a recent report 6 suggests that activation of Gq may be required to observe goitrogenesis in patients with non-autoimmune familial hyperthyroidism. However, when tested in transfected non-thyroid cells, all identified gain of function mutations of the TSHR stimulate constitutively Gs, with only a minority capable of stimulating both Gs and Gq 7, 8 . Also, thyroid adenomas or multinodular goitre are frequent in McCune Albright syndrome, which is characterized by pure Gs stimulation 9 .
1.1. Familial non-autoimmune hyperthyroidism or hereditary toxic thyroid hyperplasia.
The major cause of hyperthyroidism in adults is Graves’ disease in which an autoimmune reaction is mounted against the thyroid gland and auto-antibodies are produced that recognize and stimulate the TSH receptor. This may explain why the initial description by the group of Leclère of a family showing segregation of thyrotoxicosis as an autosomal dominant trait in the absence of signs of autoimmunity was met with skepticism 10 . Re-investigation of this family together with another family from Reims identified two mutations of the TSH receptor gene, which segregated in perfect linkage with the disease 11 . A series of additional families have been studied since 6, 12-32 . [For a complete list of TSH receptor gene mutations with their functional characteristics, see "Glycoprotein-hormone receptor information system database", GRIS, at http://gris.ulb.ac.be/ 33 and 34 at http://www.ssfa-gphr.de/ ]. The functional characteristics of these mutant receptors confirm that they are constitutively stimulated (see below). This nosological entity, hereditary toxic thyroid hyperplasia (HTTH), sometimes called Leclère’s disease, is characterized by the following clinical characteristics: autosomal dominant transmission; hyperthyroidism with a variable age of onset (from infancy to adulthood, even within a given family); hyperplastic goiter of variable size, but with a steady growth; absence of clinical or biological stigmata of auto-immunity. An observation common to most cases is the need for drastic ablative therapy (surgery or radioiodine) in order to control the disease, once the patient has become hyperthyroid 11, 35 . The autonomous nature of the thyroid tissue from these patients has been elegantly demonstrated by grafting in nude mice 36 . Contrary to tissue from Graves’disease patients, HTTH cells continue to grow in the absence of stimulation by TSH or TSAb.
The prevalence of hereditary toxic thyroid hyperplasia is difficult to estimate. It is likely that many cases are still mistaken for Graves’disease. This may be explained by the high frequency of thyroid auto-antibodies (anti-thyroglobulin, anti-thyroperoxidase) in the general population. It is expected that wider knowledge of the existence of the disease will lead to better diagnosis. This is not a purely academic problem, since presymptomatic diagnosis in children of affected families may prevent the developmental or psychological complications associated with infantile or juvenile hyperthyroidism (for a review, see 37 . A country-wide screening for the condition has been performed in Denmark. It was found in one out of 121 patients with juvenile thyrotoxicosis (0.8%; 95% CI: 0.02-4.6%), which corresponds to one in 17 patients with presumed non-autoimmune juvenile thyrotoxicosis (6%; 95% CI:0.15-28.69) 38 .
1.2. Sporadic toxic thyroid hyperplasia.
Cases with toxic thyroid hyperplasia have been described in children born from unaffected parents 32, 39-48 . Conspicuously, congenital hyperthyroidism was present in most of the cases and required aggressive treatment. Mutations of one TSH receptor allele were identified in the children, but were absent in the parents. As paternity was confirmed by mini- or microsatellite testing, these cases qualify as true neomutations. When comparing the aminoacid substitutions implicated in hereditary and sporadic cases, for the majority, they do not overlap (see table 1 and 37 and GRIS, at http://gris.ulb.ac.be/ 33 and 34 at http://www.ssfa-gphr.de/ ). Whereas most of the sporadic cases harbor mutations that are also found in toxic adenomas, most of the hereditary cases have “private” mutations. Although there may be exceptions, the analysis of the functional characteristics of the individual mutant receptors in COS cells, and the clinical course of individual patients, suggest an explanation for this observation: “sporadic” and somatic mutations seem to have a stronger activating effect than “hereditary” mutations 49 . From their severe phenotypes, it is likely that newborns with neomutations would not have survived, if not treated efficiently. On the contrary, from inspection of the available pedigrees, it seems that the milder phenotype of patients with ‘hereditary” mutations has only limited effect on reproductive fitness. The fact that “hereditary” mutations are rarely observed in toxic adenomas is compatible with the suggestion that they would cause extremely slow tissue growth and, accordingly, would rarely cause thyrotoxicosis if somatic. There is, however, no a priori reason for neomutations to cause stronger gain of function than hereditary mutations. Accordingly, an activating mutation of the TSH receptor gene has been found in a six month child with subclinical hyperthyroidism presenting with weight loss as the initial symptom 50 .
1.3.Somatic mutations: autonomous toxic adenomas.
Soon after mutations of G s had been found in adenomas of the pituitary somatotrophs 51 , similar mutations (also called Gsp mutations) were found in some toxic thyroid adenomas and follicular carcinomas 52-55 . 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. Toxic adenomas were found to be a fruitful source of somatic mutations activating the TSHr, probably because the phenotype is very conspicuous and easy to diagnose 56 . Whereas mutations are distributed all along the serpentine portion of the receptor and even in the extracellular amino-terminal domain 7, 56-65 , there is clearly a hotspot at the cytoplasmic end of the sixth transmembrane segment (see figure 16a-1a,b). The clustering reflects the pivotal role of this portion of the molecule in the activation mechanisms 66-70 . For a complete list of TSH receptor gene mutations with their functional characteristics, see “Glycoprotein-hormone receptor information system database”, GRIS, at http://gris.ulb.ac.be/ 33 and 34 at http://www.ssfa-gphr.de/ ].
Despite some dispute about the prevalence of TSH receptor mutations in toxic adenomas (which may be due to different origin of patients 71, 72 or different sensitivity of the methodology) the current consensus is that activating mutations of the TSH receptor are the major cause of solitary toxic adenomas and account for about 60 to 80% of cases 7, 61, 73-75 . Contrary to initial suggestions 71 , the same percentage of activating TSH receptor mutations is observed in Japan, an iodine-sufficient country with low prevalence of toxic adenomas 76 . In some patients with a multinodular goiter and two zones of autonomy at scintigraphy, a different mutation of the TSH receptor was identified in each nodule 77-79 . This indicates that the pathophysiological mechanism responsible for solitary toxic adenomas can be at work on a background of multinodular goiter. In agreement with this notion, activating mutations of the TSH receptor have been identified in hyperfunctioning areas of multinodular goiter 14, 24, 73, 80 . The independent occurrence of two activating mutations in a patient may seem highly improbable at first. However, the multiplicity of the possible targets for activating mutations within the TSH receptor makes this less unlikely. It is also possible that a mutagenic environment is created in glands exposed to a chronic stimulation by TSH, resulting in H2O2 generation 81 82 . Finally the involvement of TSH receptor mutations in thyroid cancers has been implicated in a limited number of follicular thyroid carcinoma 83-88 .
1.4. Structure-function relationships of the TSH receptor, as deduced from activating mutations.
Most of the activating mutations of the TSH receptor have been studied by transient expression in COS or HEK293T cells. There is no guarantee that the mutants will function in an identical way in these artificial systems as they do in thyrocytes. Arguments have been obtained for such cell-type specific effects 89 . In thyrocytes, a better relation has been observed between adenylylcyclase stimulation and differentiation than with growth 89 . However, the built-in amplification associated with transfection of constructs in COS or HEK 293T cells makes it possible to detect even slight increases in constitutive activity of the TSH receptor mutants. An important observation has been that the wild-type receptor itself displays significant constitutive activity 56, 90 . This characteristic is not unique to the TSH receptor, but interestingly, it is not shared by its close relatives, the luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) and the follitropin receptor (FSHR). The effect of activating mutations must accordingly be interpreted in terms of “increase in constitutive activity.”
Most receptor mutants found in toxic adenomas and/or toxic thyroid hyperplasia share common characteristics: (1) they increase the constitutive activity of the receptor toward stimulation of adenylylcyclase; (2) with a few notable exceptions 91 , they do not display constitutive activity toward the inositol phosphate/diacylglycerol pathway; (3) their expression at the cell surface is decreased (from slightly to severely); (4) most, but not all of them keep responding to TSH for stimulation of cAMP and inositol phosphate generation, with a tendency to do so at decreased median effective concentrations; and (5) they bind 125 I-labeled bovine TSH with an apparent affinity higher than that of the wild-type receptor.
No simple relationship exists between the position of the mutations or the nature of the amino acid substitution and their functional characteristics. Mutations found in transmembrane segments 1, 2, 3, 6, and 7 and the third cytoplasmic loop all have similar phenotypes; they involve amino acids belonging to all classes (charged, polar, hydrophobic), with substitutions not necessarily involving a shift to another class. Mutations involving Ile486 and Ile568 in the first and second extracellular loops, respectively, and Pro639 in transmembrane segment 6 are exceptional in that in addition to stimulating adenylylcyclase, they cause constitutive activation of the inositol phosphate pathway.
No direct relationship is found between the level of cAMP achieved by different mutants in transfected COS cells and their level of expression at the cell membrane 92 , which means that individual mutants have widely different “specific constitutive activity” (measured as the stimulation of cAMP accumulation divided by receptor number at the cell surface). Although this specific activity may tell us something about the mechanisms of receptor activation, it is not a measure of the actual phenotypic effect of the mutation in vivo. Indeed, one of the relatively mild mutations, observed only in a family with HTTH (Cys672Tyr), is among the strongest according to this criterion. It would be logical to expect the best correlation to be found between the phenotype and the actual level of cAMP achieved, irrespective of the level of receptor expression. However, differences between the effects of the mutants in transfected COS or HEK293 cells and thyrocytes in vivo may render these correlations a difficult exercise 89 . An additional complication comes from the recent observation that internalized receptors might display continuing coupling to Gs protein and activate adenylylcyclase 93 . Although this is still debated 94 , it would make it difficult to measure “specific activity” of any given mutant.
According to a model of G protein-coupled receptor (GPCR) activation, the receptor would exist under at least two interconverting conformations: R (silent conformations) and R* (the active forms) 95, 96 . The unliganded receptor would shuttle between both forms, the equilibrium being in favor of R (see figure 16a.1a). Binding of the ligand, to the slit between the transmembrane segments (for biogenic amines) and/or residues of the N-terminal segment or extracellular loops (for neuropeptides), is believed to stabilize the R* conformations. The resulting R-to-R* transition is supposed to involve a conformational change that modifies the relative position of transmembrane helices, which in turn would translate into conformational changes in the cytoplasmic domains interacting with trimeric G proteins.
Crystallographic data are available for a handful of inactive rhodopsin-like GPCRs 69, 97-99 and for one active conformation of opsin 70 . As it belongs to the rhodopsin-like GPCR family and displays many of the cognate signatures in primary structure, the serpentine portions of the TSHR is likely to share with rhodopsin common mechanisms of activation. However, additional primary structure specificities (among them the bipartite structure, with physical separation between binding of the hormone to the ectodomain and activation of the serpentine portion) together with the particularly wide spectrum of gain of function mutations identified in TSHR (see above) suggest the existence of idiosyncrasies associated with its activation. As many somatic mutations affecting a given residue have been found repeatedly in the TSHr (and do not involve hypermutable targets), it is likely that we are close to a saturation map for spontaneous gain of function mutations (see figure 16a.1b). Attempts to translate this map into mechanisms of transition between inactive and active conformations of the receptors have been made based on modeling of the serpentine portion of the receptor using the available crystallographic templates 34, 100, 101 . Three sequence patterns affected by gain of function mutations deserve special mention and might help understanding how the TSHR is activated.
First, aspartate 6.44 (D633) belongs to the “FTD signature”, a motif specific to glycoprotein hormone receptors, at the cytoplasmic side of transmembrane helix 6 (TM6) (see figure 16a.2). When mutated to a variety of aminoacids, including alanine, the result is constitutive activation in both the TSHr and LH/CGr 7, 67, 102, 103 . This is fully compatible with the notion that the gain of function would result from the breakage of (a) bond(s), rather than the creation of novel interaction(s) by the mutated residue. The observation, in a GPHR homologue of drosophila, of a reciprocal mutation involving D6.44 of the “FTD” motif in TM-VI and N7.49 of the “NPXXY” motif in TM-VII, suggested that an interaction between D6.44 and N7.49 would exist in the inactive conformation of GPHRs 67 . Modeling suggests that constitutive activation would be the consequence of breakage of an interaction between T6.43 or D6.44 with N7.49. Interestingly, TSHR constructs bearing the N7.49A substitution can no more be activated by TSH, despite normal expression and binding of the hormone 67 . We tentatively conclude that in the inactive conformation of GPHRs, the side chain of N7.49 is normally “sequestered” by both T6.43 and D6.44, and that the active conformation(s) require(s) establishment of novel interaction(s) of N7.49. Asparagine 7.49 of the NPXXY motif is one of the most conserved residues in rhodopsin-like GPCRs. It has been suggested, on the basis of a reciprocal substitution with D2.50 in the GnRH receptor, that N7.49 would be implicated in the activation mechanism 104, 105 via creation of an interaction with D2.50. The experimental data on rhodopsin structure have comforted this suggestion 106 . Our observations suggest that, in glycoprotein hormone receptors, evolution has selected a novel motif in TM6 to control an activation switch common to all rhodopsin-like receptors.
Second, in rhodopsin, arginine 3.50 of the highly conserved “D/ERY/W” motif at the bottom of TM3 forms an ionic lock with glutamate 6.30 at the cytoplasmic end of TM6 97 , and this lock is disrupted in the active conformation of opsin 70 . This results in a dramatic movement of TM6 with the “opening” of a cavity between the cytoplasmic ends of TM3, 5 and 6 allowing interaction with- and activation of the G protein. One of the very first gain of function mutations identified in the TSHR affected aspartate 6.30 56 , indicating the importance of the TM3-TM6 ionic lock for maintenance of the TSHR in the inactive state.
Third, serine 281 belongs to a “YPSHCCAF” sequence signature, specific to glycoprotein hormone receptors, located downstream of the leucine-rich portion of the ectodomain (see figure 16a.2). After mutation of this serine residue had been shown to activate the TSH receptor constitutively 44, 62 , this segment, sometimes referred to as the “hinge” motif, was shown to play an important role in activation of all three GPHRs 107 . The functional effect of substitutions of S281 in the TSHr, or S277 in the LH/CGr, likely results of a “loss-of-structure”, locally, since the more de-structuring the substitutions, the strongest the activation 107, 108 . This observation, together with results showing that mutation of specific residues in the extracellular loops of the TSHr cause constitutive activation 59 led to the hypothesis that activation of the receptor could result from the rupture of an inhibitory interaction between the ectodomain and the serpentine domain 62 .
The hypothesis that the ectodomain would exert an inhibitory effect on an inherently noisy rhodopsin-like serpentine domain, is supported by early data showing that mild treatment of TSHr-expressing cells by trypsin causes partial activation of the receptor 109 . Definite demonstration of such an effect was made by Zhang et al 110 , who showed activation of the TSHr secondary to “beheading”, in aminoterminal truncated mutants. However, careful comparison of the activity of truncated mutants with maximally stimulated wild type TSHr, or S281 gain of function mutants, indicated that truncation of the receptor of its ectodomain resulted only in partial activation of the serpentine domain 111 . In addition, engineering activating mutations in the serpentine of an aminoterminally truncated mutant resulted in further activation of the constructs 111 . Interestingly, only mutations in the transmembrane helices were effective; substitutions in the extracellular loops of serpentine-only constructs were without effects 111 .
From these observations, the following model for activation of the TSHr was proposed 111 (see figure 16a.3). In the resting state, the ectodomain would exert an inhibitory effect on the activity of an inherently noisy rhodopsin-like serpentine, qualifying pharmacologically as an inverse agonist of the serpentine. Upon activation, by binding of the hormone, or secondary to mutation of S281 in the hinge region, the ectodomain would switch from inverse agonist to full agonist of the serpentine portion. The ability of the strongest S281 mutants to fully activate the receptor in the absence of hormone, suggests that the ultimate agonist of the serpentine domain would be the “activated” ectodomain. The ineffectiveness of mutations in the extracellular loops to activate serpentine-only constructs suggests that, in the wild type receptor, the exoloops and a portion of the ectodomain (the hinge region?) do cooperate in the generation of a structural module functioning as an agonist of the serpentine. The identification of a monoclonal antibody recognizing an epitope in the hinge region and displaying inverse agonist activity gives strong support to this model 112 .
1.5. Familial gestational hyperthyroidism.
Some degree of stimulation of the thyroid gland by human chorionic gonadotrophin (hCG) is commonly observed during early pregnancy. It is usually responsible for decrease in serum thyrotropin with an increase in free thyroxine concentrations that remains within the normal range (for references see 113 ). When the concentrations of hCG are abnormally high, like in molar pregnancy, true hyperthyroidism may ensue. The pathophysiological mechanism is believed to be the promiscuous stimulation of the TSH receptor by excess hCG, as suggested by the rough direct or inverse relation between serum hCG and free T4 or TSH concentrations, respectively 114 . A convincing rationale is provided by the close structural relationships of the glycoprotein hormones and their receptors, respectively 101 . A new syndrome has been described in 1998 in a family whith dominant transmission of hyperthyroidism limited to pregnancy (see fig.16a-4) 115 . The proposita and her mother had severe thyrotoxicosis together with hyperemesis gravidarum during the course of each of their pregnancies. When non pregnant they were clinically and biologically euthyroid. Both patients were heterozygous for a K183R mutation in the extracellular aminoterminal domain of the TSH receptor gene. When tested by transient transfection in COS cells, the mutant receptor displayed normal characteristics towards TSH. However, providing a convincing explanation to the phenotype, it showed higher sensitivity to stimulation by hCG, when compared with wild type TSH receptor 115 . The aminoacid substitution responsible for the promiscuous stimulation of the TSHr by hCG is surprisingly conservative. Also surprising is the observation that residue 183 is a lysine in both the TSH and LH/CG receptors. When placed on the available three dimensional model of the hormone-binding domain of the TSH receptor 116 , residue 183 belongs to one of the beta sheets which constitute the putative surface of interaction with the hormones 117 . In search for a structural explanation of the phenotype, Lys183 was mutated to a variety of aminoacids with different physicochemical properties 118 . Unexpectedly, these experiments indicated that the gain of function of the Lys183Arg mutant toward hCG was not due to the presence of the Arg but to the loss of the Lys. Indeed, most mutants displayed a phenotype similar to that of Lys183Arg 118 . A detailed functional analysis of site-directed mutagenesis constructs designed after molecular dynamic simulations led to the conclusion that the widening of specificity toward hCG was due to the release of the side chain of Glu157 (a neighbor of Lys183 in the tridimensional structure of the ectodomain) from an ionic bond with Lys183, making it available for interaction with hCG. This view of the molecular mechanism responsible for the broadening of specificity of the mutant has been fully confirmed when the three-dimensional structure of the ectodomain of the FSH receptor complexed with FSH became available 119 . It is likely that an arginine in position 183 would confer a slight increase in stability to the illegitimate hCG/TSHr complex 120 . This would be enough to cause signal transduction by the hCG concentrations achieved in pregnancy, but not by the LH concentrations observed after menopause. Indeed the mother of the proposita remained euthyroid after menopause. This is compatible with a relatively modest gain-of-function of the K183R mutant for stimulation by hCG. Contrary to other mammals, human and primates rely on chorionic gonadotropin for maintenance of corpus luteum in early pregnancy 121 . The frequent partial suppression of TSH observed at peak hCG levels during normal pregnancy indicates that evolution has selected physiological mechanisms operating very close to the border of thyrotoxicosis. This may provide a rationale to the observation that, in comparison to other species, the glycoprotein hormones of primates display a lower biological activity due to positive selection by evolution of specific aminoacid substitutions in their alpha subunits 122 . Up to now no spontaneous mutation has been identified which would increase de bioactivity of hCG. An interesting parallel may be drawn between familial gestational hyperthyroidism and cases of spontaneous ovarian hyperstimulation syndrome (sOHSS) 123, 124 . In sOHSS, mutations of the FSH receptor gene render the receptor abnormally sensitive to hCG. The result is recurrent hyperstimulation of the ovary, on the occasion of each pregnancy
2. Loss of function mutations.
Loss-of-function mutations in the TSH receptor gene are expected to cause a syndrome of “resistance to TSH.” The expected phenotype is likely to resemble that of patients with mutations in TSH itself. These mutations have been described early because of the prior availability of information on TSH alpha and beta genes 122 . Mouse models of resistance to TSH are available as natural (hyt/hyt mouse) 125 or experimental TSH receptor mutant lines 126, 127 . Interestingly, and contrary to the situation in human (see below), the thyroid of newborn TSH receptor knockout mice is of normal size. As expected, the homozygote animals displayed profound hypothyroidism. Their thyroid do not express the sodium-iodide symporter, but showed significant (non-iodinated) thyroglobulin production. From this information one would expect patients with two TSH receptor mutated alleles to exhibit a degree of hypothyroidism in accordance with the extent of the loss of function, going from mild, compensated, hypothyroidism, to profound neonatal hypothyroidism with absent iodide trapping. Heterozygous carriers are expected to be normal or display minimal increase in plasma TSH.
Clinical cases with the mutations identified (figure 5).
A few patients with convincing resistance to TSH had been described before molecular genetics permitted identification of the mutations 128, 129 . The first cases described in molecular terms were euthyroid siblings with elevated TSH 130 . Sequencing of the TSH receptor gene identified a different mutation in each allele of the affected individuals, which made them compound heterozygotes. The substitutions were in the extracellular amino-terminal portion of the receptor (maternal allele, Pro162Ala; paternal allele, Ile167Asn). The functional characteristics of the mutant receptors showed that the paternal allele was virtually completely nonfunctional, whereas the maternal allele displayed an increase in the median effective TSH concentration for stimulation of cAMP production. Current knowledge of the tridimensional structure of part of the ectodomain of the receptor 131, 132 allows to establish structure-function relationships for mutations affecting this portion of the receptor 133, 134 .
A large number of familial cases with loss-of-function mutations of the TSH receptor have been identified in the course of screening programs for congenital hypothyroidism 135-148 ([For a complete list of TSH receptor gene mutations with their functional characteristics, see 33 at http://gris.ulb.ac.be/ and 34 at http://www.ssfa-gphr.de/]. Some of the patients displayed the usual criteria for congenital hypothyroidism, including high TSH, low free T4, and undetectable trapping of 99Tc . In some cases, plasma thyroglobulin levels were normal or high. The patients can be compound heterozygotes for complete loss of function mutations 136 , or homozygotes, born to consanguineous 135 or apparently unrelated parents 142 . In agreement with the phenotype of knock-out mice with homozygous invalidation of the TSH receptor, patients with complete loss-of-function of the receptor display an in-place, thyroid with completely absent iodide or 99Tc trapping. However, in contrast with the situation in mice, the gland is hypoplastic. Activation of the cAMP pathway, while dispensable for the anatomical development of the gland and thyroglobulin production, is thus absolutely required for expression of the NIS gene and, at least in human, for normal growth of the tissue during fetal life. This explains that in the absence of thyroglobulin measurements or expert echography, loss-of-function mutations of the TSH receptor may easily be misdiagnosed as thyroid agenesis.
In the heterozygous state, complete loss of function mutations of the TSH receptor is a cause of moderate hyperthyrotropinemia (subclinical hypothyroidism), segregating as an autosomal dominant trait 149 .
Resistance to thyrotropin not linked to the TSH receptor gene
Finally, it must be stressed that an autosomal dominant form of partial resistance to TSH has been demonstrated in families in which linkage to the TSH receptor gene has been excluded 150 . A locus has been identified on chromosme 15q25.3-26.1 but the gene responsible for the phenotype has not been identified yet 151 .
Research in the laboratory of the author was supported by the Belgian Programme on University Poles of Attraction initiated by the Belgian State, Prime Minister’s office, Service for Sciences, Technology and Culture. Also supported by grants from the fonds de la Recherche Scientifque M”dicale, the FNRS, Télévie, the European Union (Biomed), Association Belge contre le Cancer and Association de Recherche Biomédicale et de Diagnostic.
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