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M Tonacchera

Publications and source records attributed to M Tonacchera.

At least 37 records · Page 2Linked to original sources
PubMed1998

Hyperfunctioning thyroid nodules in toxic multinodular goiter share activating thyrotropin receptor mutations with solitary toxic adenoma.

Toxic multinodular goiter is a cause of nonautoimmune hyperthyroidism and is believed to differ in its nature and pathogenesis from toxic adenoma. Gain-of-function mutations of the TSH receptor gene have been identified as a cause of toxic adenoma. The pathogenesis at the molecular level of hyperfunctioning nodules in toxic multinodular goiter has yet not been reported. Six patients with a single hot nodule within a multinodular goiter and 11 patients with toxic thyroid adenoma were enrolled in our study. At histology five hyperfunctioning nodules in multinodular goiters showed the features of adenomas, and one was identified as a hyperplastic nodule. The entire exon 10 of the TSH receptor gene was directly sequenced after PCR amplification from genomic DNA obtained from surgical specimens. Functional studies of mutated receptors were performed in COS-7 cells. Five out of 6 (83%) hyperfunctioning nodules within toxic multinodular goiters harbored a TSH receptor mutation. A TSH receptor mutation was also evident in the hyperfunctioning nodule that at histology had the features of noncapsulated hyperplastic nodule. Among toxic adenomas, 8 out of 11 (72%) nodules harbored a TSH receptor mutation. All the mutations were heterozygotic and somatic. Nonfunctioning nodules, whether adenomas or hyperplastic nodules present in association with hyperfunctioning nodules in the same multinodular goiters, had no TSH receptor mutation. All the mutations identified had constitutive activity as assessed by cAMP production after expression in COS-7 cells. Hyperfunctioning thyroid nodules in multinodular goiters recognize the same pathogenetic event (TSH receptor mutation) as toxic adenoma. Other mechanisms are implicated in the growth of nonfunctioning thyroid nodules coexistent in the same gland.

Adenoma↗
PubMed1996

Functional characteristics of a variant thyrotropin receptor.

A claim has been made that a variant of the human thyrotropin receptor in which Pro52 is replaced by Thr ([Thr52]thyrotropin receptor) is associated with autoimmune thyroid diseases and displays increased responsiveness to thyrotropin. We have analysed the functional characteristics of this variant receptor. Equivalent numbers of of the wild type and of the variant thyrotropin receptor, measured both by 125I-thyrotropin binding and by flow cytofluorimetry, were transiently expressed in COS-7 cells. Under these conditions, the two receptors showed the same degree of constitutive activity for the cAMP pathway, the same affinity for bovine thyrotropin, and a virtually identical responsiveness to bovine thyrotropin for activation of both the cAMP and inositol-phosphate regulatory pathways. Our results show that the [Thr52]thyrotropin receptor variant of the human thyrotropin receptor, which is present in the 12% of the population, does not affect receptor function and represents most likely a simple polymorphism.

Alleles↗
PubMed1996

Differential effects of NaCl concentration on the constitutive activity of the thyrotropin and the luteinizing hormone/chorionic gonadotropin receptors.

The TSH receptor (TSHR) and the LH/CG receptor (LHR) are members of the family of G protein-coupled receptors. Recently, point mutations conferring constitutive activity to the TSHR and LHR have been observed as a cause of toxic adenoma and familial/sporadic male pseudo-precocious puberty, respectively. When evaluated by transfection in COS-7 cells the wild-type (wt) TSHR displays definite constitutive activity towards Gs-dependent adenylylcyclase stimulation, while available evidence shows that the LHR does not. In order to compare the constitutive activity of both receptors, we performed functional studies in COS-7 cells using different assay conditions. Human TSHR and LHR cDNAs subcloned in the expression vector pSVL were transiently expressed in COS-7 cells and cAMP production was determined following incubation in a medium containing physiological concentration of NaCl [isotonic (NaCl)] or in the same medium without NaCl [hypotonic (NaCl-)] or where NaCl was replaced by an isoosmolar concentration of sucrose [isotonic (sucrose)]. Cells transfected with the TSHR showed higher basal cAMP levels over cells transfected with pSVL in all conditions tested. The effect was stronger when cells were incubated in isotonic (sucrose) buffer. Cells expressing LHR exhibited a minimal increase of cAMP levels over cells transfected with pSVL in isotonic (NaCl) buffer; however, a marked increase in basal cAMP levels was observed when cells were assayed in hypotonic (NaCl-) or isotonic (sucrose) buffers. Varying the pH or incubation temperature was without effect on the results obtained with both receptors. Our data show that despite extensive sequence similarity, the LH and TSH receptors differ markedly in their basal activity. The differential sensitivity of both receptors to low NaCl concentrations, suggests that the unliganded TSH receptor is less constrained than its LH homolog and may be more susceptible to activation by a wide spectrum of mutations.

Animals↗
PubMed1996

Cloning of candidate autoantigen carboxypeptidase H from a human islet library: sequence identity with human brain CPH.

A number of proteins, many of them enzymes, i.e. glutamic acid decarboxylase (GAD), carboxypeptidase H, 37-40 K tyrosine phosphatase (ICA512, IA2/IA2 beta), have been proposed as islet autoantigens involved in the pathogenesis of IDDM. Until recently, progress in their characterization has been impeded by the inaccessibility of the human pancreas, resulting in many of them being cloned from animal or non-islet sources. Carboxypeptidase H, one of these enzymes, has been cloned and sequenced from human brain and from rat islets but not from human islets. In this study, we describe the production of a human islet cDNA library and the cloning of islet CPH from it. Since CPH clones were also detected in a human thyroid library, we have sequenced CPH from these two endocrine tissue libraries and compared them to the known brain sequence. The sequences from islets and thyroid were identical and differed from brain only in the absence of a second ATG in the predicted 5'non-coding region. Northern blot analysis revealed the presence of an identical 2.5 kb transcript in human islets, thyroid and brain. The confirmation of the existence of a single isoform of CPH expressed in brain and endocrine tissues simplifies future experiments to elucidate the role of CPH as autoantigen.

Animals↗
PubMed1996

Functional characteristics of three new germline mutations of the thyrotropin receptor gene causing autosomal dominant toxic thyroid hyperplasia.

We report three unrelated families in which hyperthyroidism associated with thyroid hyperplasia was transmitted in an autosomal dominant fashion, in the absence of signs of autoimmunity. Exon 10 of the TSH receptor gene was directly sequenced after PCR amplification from DNA of peripheral leukocytes. In one family, a C to A transversion resulted in an S505R substitution in the third transmembrane segment; in the second, an A to T transversion caused a N650Y substitution in the sixth transmembrane segment; and in the third family, an A to G transition resulted in an N670S substitution in the seventh transmembrane segment. When expressed by transfection in COS-7 cells, each mutated receptor displayed an increase in constitutive stimulation of cAMP production; no effect on basal accumulation of inositol phosphates (IP) could be detected. In binding studies, cells transfected with wild-type or mutated receptors showed similar levels of expression, with the mutated receptors displaying similar or slightly increased affinity for bovine TSH (bTSH) binding. Cells transfected with S505R and N650Y mutants showed a similar cAMP maximal TSH-stimulated accumulation over the cells transfected with the wild type, whereas N670S transfectants showed a blunted response with an increase in EC50. A higher IP response to 100 mU/mL bTSH over that obtained with the wild-type receptor was obtained in cells transfected with N650Y; in contrast, cells transfected with S505R showed a blunted IP production (50% less), and the N670S mutant completely lost the ability to stimulate IP accumulation in response to bTSH. The differential effects of individual mutations on stimulation by bTSH of cAMP or IP accumulation suggest that individual mutant receptors may achieve different active conformations with selective abilities to couple to Gs alpha and to Gq alpha.

Amino Acid Sequence↗
PubMed1996

Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor.

We describe a patient with monoclonal gammopathy who subsequently developed thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy. The pathogenesis of thyrotoxicosis in Graves' disease is due to the presence of autoantibodies that mimic the action of thyrotropin (TSH), called thyroid-stimulating antibodies (TS-ab); these antibodies may or may not inhibit the binding of TSH to the receptor (thyroid-binding inhibiting immunoglobulin, TBII). The patient's immunoglobulins were TS-ab positive and TBII negative when measured on CHO cells expressing the human TSH receptor. The pathogenetic link between the thyroid, orbit and skin is yet to be established but several candidate shared antigens have been proposed, including the TSH receptor itself. The monoclonal immunoglobulins were in evidence before the symptoms of pretibial myxedema, thyrotoxicosis and ophthalmopathy. In addition, the patient had no autoantibodies to thyroglobulin or thyroperoxidase, which are classic markers of thyroid autoimmunity. This combination led us to postulate that the monoclonal gammopathy could be the cause of all the observed pathology. One method to test this hypothesis would be to show that the monoclonal immunoglobulin is a TS-ab. Various methods were used to separate the monoclonal from the polyclonal components of the patient's serum. Preparative isoelectric focusing enabled us to obtain fractions containing only the monoclonal (as revealed by polyacrylamide gel electrophoresis), which were devoid of TS-ab activity (measured as cAMP accumulation in CHO cells expressing the human TSH receptor in a hypotonic bioassay). Subsequently, different oligoclonal fractions were shown to have varying degrees of TS-ab activity, with one fraction having faint biological activity and able to recognize a recombinant TSH receptor preparation in a Western blot. In conclusion, the monoclonal antibody does not seem to be responsible for the thyrotoxicosis, pretibial myxedema and ophthalmopathy. We confirm previous data showing that TSH receptor antibodies in patients with Graves' disease are heterogeneous in nature and we present the first demonstration of autoantibodies capable of binding the TSH receptor but devoid of TBII activity.

Adult↗
PubMed1996

Autoimmune hypothyroidism and hyperthyroidism in patients with Turner's syndrome.

A high prevalence of autoimmune thyroid disease (AITD) has been described in Turner's syndrome (TS) but the extent of this association is controversial for the prevalence of thyroid autoantibody and the clinical impact of thyroid dysfunction. In this study we searched for thyroid disease and thyroid autoantibodies in patients with TS. Seventy-five unselected TS patients (age range 3-30 years) were studied. Sera were tested for thyroid hormones, thyrotropin (TSH), thyroglobulin (TG-ab) and thyroperoxidase (TPO-ab) antibodies. The TSH-receptor antibodies with thyroid-stimulating (TS-ab) or TSH-blocking activity (TSHB-ab) were measured in the IgG fraction using a bioassay. Ten out of 75 (13.3%) TS patients had AITD: eight had autoimmune thyroiditis (AT) (six with subclinical and two with overt hypothyroidism and one with euthyroidism) and one had Graves' disease. The prevalence of AITD increased significantly (p < 0.05) from the first (15%) to the third (30%) decade of life. The prevalence of TPO-ab and/or TG-ab (20%) was higher (p < 0.05) in TS than in age-matched female controls and increased from the first (15%) to the third (30%) decade of life. Clinical AITD was diagnosed in 46% of TS patients with TPO-ab and/or TG-ab. Thyroid-stimulating antibody was detected in the hyperthyroid patient, and TSHB-ab was found in one of eight patients with hypothyroid AT. It was concluded that: TS patients are at higher than average risk of developing AITD not only in adolescence and adult age but also in childhood; hypothyroidism, mainly subclinical, is the most frequent thyroid dysfunction; elevated TPO-ab and/or TG-ab alone do not imply thyroid dysfunction; TS-ab or TSHB-ab are always associated with thyroid dysfunction although most cases of autoimmune hypothyroidism are not due to the latter antibody.

Adolescent↗
PubMed1996

Activating mutations of the TSH receptor gene cause thyroid diseases.

Spontaneous mutations have been identified in the gene encoding the thyrotropin receptor, the effect of which is to activate the receptor in the absence of hormone. When they occur within thyrocytes (somatic mutations) activating mutations cause clonal expansion of the cells into a hyperfunctional thyroid adenoma (toxic nodule). Our results demonstrate that this pathophysiologic mechanism accounts for the majority of toxic adenomas (9 mutations found out of 11 adenomas). The remaining cases are probably secondary to mutations in the G protein Gs. When similar mutations are present in the germ line, they cause a form of non-autoimmune hyperthyroidism transmitted as an autosomal dominant trait. Mutations of the tsh receptor gene have been found in five different families, including that corresponding to the original description of the syndrome by J. Leclère (Nancy). Structure/function studies of the various mutant receptors will contribute to our understanding of the mecanisms involved in the activation of G protein-coupled receptors.

Gene Expression Regulation↗
PubMed1995

The thyroperoxidase doublet is not produced by alternative splicing.

Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reducing conditions, we observed a broad signal at 105-110 kDa, which appeared to comprise two bands, with both polyclonal antibodies and the monoclonal. There was no difference in the image between the normal and the Graves' thyroid. In reducing conditions, the broad signal resolved clearly into two distinct bands, one at 105 and the other at 110 kDa. Once again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves' glands. We conclude that the TPO doublet is not the consequence of translation of TPO2.

Alternative Splicing↗
PubMed1995

Humoral thyroid autoimmunity is not involved in the pathogenesis of myxedematous endemic cretinism.

A role of thyroid autoimmunity in the pathogenesis of myxedematous endemic cretinism was suggested by reports indicating the presence of thyroid growth-blocking antibodies in the sera of these patients. To check this hypothesis, we searched for TSH receptor antibodies with thyroid growth-blocking or adenylate cyclase (AC)-inhibiting (TSH-blocking) activity in immunoglobulin G (IgG) from 18 euthyroid and 21 hypothyroid endemic cretins living in Italy and Peru. Among hypothyroid cretins, 12 had no palpable goiter. Stages I-III goiters were present in 12 of 18 euthyroid cretins. Controls included 25 euthyroid nongoitrous subjects living in the same endemic regions as cretins, and 10 normal subjects from an iodine-sufficient area. IgG from 4 selected patients with autoimmune atrophic thyroiditis and from 2 neonates with sporadic transient congenital hypothyroidism due to maternal TSH-blocking antibodies were included in the study. The blocking effect of the IgG was assessed in FRTL-5 cells by measuring TSH-stimulated [3H]thymidine incorporation, DNA accumulation, and AC activation. A radioreceptor assay was used to detect TSH-binding inhibiting antibodies (TBIAb). No IgG from hypothyroid endemic cretins without goiter contained TBIAb or inhibited TSH-stimulated cell growth or AC activation. The effect of IgG from hypothyroid nongoitrous cretins did not differ from that produced by IgG from hypothyroid cretins with goiter, euthyroid cretins with or without goiter, or normal controls. In contrast to these results, IgG from patients with autoimmune atrophic thyroiditis and from neonates with sporadic transient congenital hypothyroidism contained TBIAb that inhibited both TSH-stimulated cell growth and AC activation. In conclusion, our results indicate that, similar to other types of endemic cretinism, hypothyroid endemic cretins without goiter do not have TSH receptor antibodies able to inhibit TSH-stimulated thyroid cell growth or function. These observations argue against a role of humoral thyroid autoimmunity in the development of myxedematous endemic cretinism.

Adolescent↗
PubMed1995

Somatic mutations causing constitutive activity of the thyrotropin receptor are the major cause of hyperfunctioning thyroid adenomas: identification of additional mutations activating both the cyclic adenosine 3',5'-monophosphate and inositol phosphate-Ca2+ cascades.

A series of somatic mutations of the TSH receptor gene have been demonstrated in hyperfunctioning thyroid adenomas. The mutations studied up to now cause constitutive (i.e. TSH-independent) activation of the cAMP-regulatory cascade only. As a follow-up to our original study, we have now completely sequenced exon number 10 of the TSH receptor gene in the same series of toxic adenomas. An activating mutation was found in nine of 11 tumors. In addition to the mutations already described, two isoleucine residues belonging to the first and second extracellular loops of the receptor (Ile486 and Ile568) were found mutated. Two different adenomas were found to harbor a different amino acid substitution at residue 486 (Ile486Phe, Ile486Met). Ile568 was mutated to threonine in one. When studied by transfection in COS-7 cells, all three mutations caused very strong activation of the cAMP-regulatory cascade. In addition, the Ile486Phe and, to a lesser extent, the Ile486Met and Ile568Thr mutants stimulated constitutively the inositol phosphate-diacylglycerol cascade. Our results demonstrate that 1) the first and second extracellular loops contribute to the silencing of the unliganded TSH receptor; 2) the two regulatory cascades normally under TSH control can be constitutively activated by somatic mutations of the receptor; 3) the TSH receptor can be activated by mutation of a large number of residues distributed over the first and second extracellular loops, the third intracellular loop, and the third, sixth, and seventh transmembrane segments; 4) activating mutations of the TSH receptor constitute the major cause of toxic adenomas, accounting for about 80% of the cases.

Adenoma↗
PubMed1995

Mapping thyroid peroxidase epitopes using recombinant protein fragments.

The identification of autoantibody epitopes is important to the understanding of autoimmune thyroid diseases. In the case of thyroid peroxidase antibodies (TPO-ab), recent reports have disagreed about the number and type of autoantibody epitopes found in human TPO. In order to clarify the nature of these epitopes, we used an approach that provides recombinant human TPO produced by bacterial cells. The cDNA of four slightly overlapping fragments of human TPO-TPO 1(Glu 17-Ser 227), TPO 2(Tyr 226-Thr 476), TPO 3(Glu 471-Ser 720) and TPO 4(Phe 709-Leu 993)--were amplified by polymerase chain reaction and subcloned into the expression vector pMAL. In addition, a TPO 3 species for an alternatively spliced form of TPO of 876 amino acids was constructed (TPO 3M). Each of these constructs encodes a fusion protein, in which the amino terminal portion is maltose-binding protein, followed by the sequence of the fragment of human TPO. The plasmid constructs were transformed in Escherichia coli and, after growth, bacterial cells were harvested, lysed and the lysate was passed over an amylose affinity column and eluted with maltose. Western blots were performed using 33 sera from patients with autoimmune thyroid disease (group 1) and 17 sera from patients with nodular goiter and focal thyroiditis (group 2), all positive for TPO-ab measured by radio-immunoassay; sera from 10 healthy people with no clinical evidence of thyroiditis and positive for TPO-ab measured by radioimmunoassay (group 3) and sera from 30 patients with antigastric parietal cell antibodies without signs or symptoms of thyroiditis, 16 negative for TPO-ab (group 4a) and 14 positive for TPO-ab (group 4b), were included in the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Induction of thyrotropin receptor (TSH-R) autoantibodies and thyroiditis in mice immunised with the recombinant TSH-R.

Two groups of 10 Balbc by Jico male mice were immunised on days 0, 15 and 35, with the extra cellular domain (ECD) of the human thyrotropin receptor (TSH-R) expressed as a fusion protein in bacteria (group 1) or with the maltose binding protein (MBP) fusion partner alone (group 2). Blood was obtained on days 0, 22, 32, 42 and 49 and samples from the individual animals pooled for each group. Serum and immunoglobulin (IgG) preparations were tested, using CHO cells expressing the human TSH-R (JP26 and JP09) for thyroid stimulating (TSAb); thyroid blocking (TBAb) and thyrotropin binding inhibiting (TBII) activities. Neither serum nor IgGs were found to contain TSAb at any time point. TBII activity was present in the serum of both groups on day 32 and in group 1 only on day 49; when the test was performed on IgGs, only the MBP-ECD day 49 preparation remained significantly positive for TBII (p < 0.005). Significant TBAb activity was present in both the serum and IgG of group 1 day 49 (p < 0.005) and to a lesser extent on 42 (p < 0.02). Following the second immunisation (day 15) both groups and had decreased circulating T4 levels (p < 0.05) when compared with day 0 in each case. Group 2 were unaffected by the third immunisation on day 35 but the MBP-ECD group again had significantly decreased T4 levels (p < 0.02) compared with MBP day 49 and (p < 0.03) when compared with MBP-ECD day 0. Histological examination of thyroids from group 1 animals revealed extensive vascularisation and an atypical lymphoblastoid infiltration which was not observed in control mice. These preliminary results indicate that care is required in interpreting data since a non-receptor antigen was shown to decrease circulating thyroxine and serum from these animals had apparent TBII like activity. However, the results obtained with the IgGs suggest that receptor autoantibodies can be induced by immunising with the human TSH-R, in addition, the immunised mice show histological evidence for the development of thyroiditis.

Animals↗