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E Chiefari

Publications and source records attributed to E Chiefari.

16 recordsLinked to original sources
PubMed2004

Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma.

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational "hot spots" in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single-strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13-16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the "hot spots" domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.

Carcinoma, Medullary↗
PubMed2002

Similarities and differences in the phenotype of members of an Italian family with hereditary non-autoimmune hyperthyroidism associated with an activating TSH receptor germline mutation.

Constitutively activating germline mutations of the TSH receptor (TSH-R) are considered the cause of hereditary non-autoimmune hyperthyroidism. In this study, 10 members (8 affected and 2 unaffected) of an Italian family with hereditary non-autoimmune hyperthyroidism were investigated for the presence of mutations in the TSH-R gene. The clinical features of the disease were also analyzed. PCR-amplified fragments of the TSH-R gene were obtained from genomic DNA extracted from peripheral blood leukocytes of each family member and analyzed by direct nucleotide sequencing and restriction analysis. An identical germline TSH-R mutation was detected in all the patients with hyperthyroidism but in none of the unaffected family members. The mutation was heterozygotic and determined the substitution of valine for methionine (codon 463; ATG-->GTG) in the second transmembrane domain of the TSH-R. When expressed in chinese hamster ovary (CHO) cells, the Val463 mutant TSH-R induced constitutive activation of the TSH receptor. Analysis of the clinical features of our family and those of other families with hereditary non-autoimmune hyperthyroidism, including one with the same Val463 mutation, revealed wide variability in the phenotypical expression of the disease. Our findings indicate that an activating germline mutation in the TSH-R gene plays a key role in hereditary non-autoimmune hyperthyroidism although the onset of clinical manifestations and the evolution of the disease seem to depend heavily on other factors, thus far unidentified. The absence of a clear correlation between mutant genotypes and phenotypic expression of the disease currently limits the prognostic value of genetic testing in families with hereditary non-autoimmune hyperthyroidism.

Adult↗
PubMed2001

RET proto-oncogene mutation in a mixed medullary-follicular thyroid carcinoma.

A case of a patient with an uncommon thyroid carcinoma, showing histological and immunohistochemical features of both follicular and parafollicular cells is described. Somatic point mutation (ATG to ACG heterozygotic mutation at codon 918) of the RET proto-oncogene was detected in tumor tissue, as confirmed by immunohistochemical expression of RET oncoprotein. Our findings suggest that constitutive RET proto-oncogene activation may be involved in the development of mixed medullary-follicular thyroid carcinoma.

Adenocarcinoma, Follicular↗
PubMed2001

A large family with hereditary MTC: role of RET genetic analysis in differential diagnosis between MEN 2A and FMTC.

Germline mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN 2A), multiple endocrine neoplasia type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). In the absence of biochemical and/or clinical evidence of pheochromocytoma and hyperparathyroidism, patients with MEN 2A disease display the same phenotype of FMTC disease, although prognosis and clinical management in both affected and unaffected familial members are quite different. We studied a family with hereditary MTC, whose proband was referred to us because of enlarged cervical nodes and increased calcitonin serum levels 28 years after the total thyroidectomy for MTC. Cervical node dissection was carried out and subsequently the presence of MTC metastasis was histologically confirmed. A RET genomic mutation at codon 634 (TGC-->TTC) was identified in the proband and in seven out of 19 familial members studied. Accordingly, a hereditary disease was suggested. However, the strong association of RET mutation at codon 634 with the presence of pheochromocytoma in MEN 2 disease suggested a more rigorous management in all gene carriers. Indeed, during the follow-up pheochromocytoma was subsequently identified in the proband. This finding suggests that all families with a pedigree suggestive of FMTC should be regarded at risk from MEN 2A disease, at least when a critical mutation in the RET cysteine domain is detected.

Adolescent↗
PubMed2001

Transcriptional regulation of human insulin receptor gene by the high-mobility group protein HMGI(Y).

We have previously identified two closely related nuclear binding proteins that specifically interact with two unique functional AT-rich sequences of the 5' regulatory region of the human insulin receptor gene. Expression of these nuclear binding proteins increases during myocyte and adipocyte differentiation, and in other tissues appears to correlate with insulin receptor content. We have hypothesized, therefore, that insulin receptor expression in the insulin target tissues is regulated at least in part by these nuclear proteins. Here we show data on purification and biochemical characterization of these DNA binding proteins. Using a conventional chromatographic purification procedure combined with electrophoresis mobility shift assay and immunoblot analyses, a unique approximately 15 kDa protein, either identical to or highly related to the architectural transcription factor HMGI(Y), has now been identified, suggesting an essential role for HMGI(Y) in regulating insulin receptor gene transcription. Direct evidence of HMGI(Y) insulin receptor promoter interactions is provided by functional analysis with the CAT reporter gene and by hormone binding studies in cells expressing HMGI(Y) antisense RNA. In these experiments, antisense HMGI(Y) specifically inhibits insulin receptor promoter function and insulin receptor protein expression, indicating that HMGI(Y) is required for proper transcription of insulin receptor gene. Moreover, our data consistently support the hypothesis that a putative defect in this nuclear binding protein may cause insulin receptor dysfunction with subsequent impairment of insulin signaling and action.

5' Untranslated Regions↗
PubMed2000

A Phe 486 thyrotropin receptor mutation in an autonomously functioning follicular carcinoma that was causing hyperthyroidism.

Hot nodules are rarely found to be carcinomas. We report a case of a nonmetastatic follicular carcinoma that presented as a hot nodule that was causing hyperthyroidism. A base substitution (ATC for TTC) was found in codon 486 of the TSH receptor gene and this resulted in the substitution of an isoleucine for a phenylalanine in the first extracellular loop of the receptor. This was absent in the deoxyribonucleic acid from the surrounding normal thyroid tissue indicating its somatic origin. This mutation, which was previously reported to activate both cyclic adenosine monophosphate and the inositol phosphate-diacylglycerol cascades, may have been responsible for the constitutive activation of the thyrotropin receptor and resulting hyperfunction of this follicular carcinoma.

Adenocarcinoma, Follicular↗
PubMed2000

The 3',5'-cyclic adenosine monophosphate response element binding protein (CREB) is functionally reduced in human toxic thyroid adenomas.

In human normal thyrocytes, the cAMP-responsive signaling pathway plays a central role in gene regulation, cell proliferation, and differentiation. Constitutive activation of the cAMP signal transduction system has been documented in thyroid autonomously hyperfunctioning adenomas in which activating mutations in either the TSH receptor gene or the Gsalpha protein gene (gsp oncogene) have been described. The molecular mechanism whereby cAMP induces thyrocyte proliferation is unknown, but recent evidence suggests that the transcription factor cAMP response element binding protein (CREB) may serve as an important biochemical intermediate in this proliferative response. Herein we have investigated the expression of CREB in normal and tumoral thyroid tissues from a series of ten unrelated patients with autonomously hyperfunctioning adenomas, previously screened for mutations in the TSH receptor and Gsalpha genes. In all tumors examined, the expression of the activated, phosphorylated form of CREB was markedly reduced compared with that of the corresponding paired normal thyroid tissue, and this reduction was independent of the presence of mutations in the TSH receptor gene and Gsalpha gene. Moreover, no correlation was observed in these tissues between CREB phosphorylation and either protein kinase A activity or protein phosphatase expression. Thus, these data suggest that in human hyperfunctioning thyroid adenomas, the PKA/CREB system does not play a role in cell proliferation.

Adenoma↗
PubMed2000

A novel mutation in the thyrotropin (TSH) receptor gene causing loss of TSH binding but constitutive receptor activation in a family with resistance to TSH.

Resistance to TSH is a syndrome due to reduced responsiveness of the thyroid gland to biologically active TSH. Inactivating mutations of the TSH receptor (TSH-R) have been detected in several cases of resistance to TSH, both partial and complete, sporadic and familial. In this study, we describe a family with the presence of resistance to TSH responsible for euthyroid hyperthyrotropinemia in two siblings from consanguineous parents. By direct sequencing of the TSH receptor gene, we identified a new mutation responsible for the substitution of an arginine with a cysteine at position 310, in the extracellular domain of the TSH-R. The mutation was homozygous in two brothers; heterozygous in both parents, an uncle, and an unaffected brother; and absent in the other unaffected brother. When stably transfected in Chinese hamster ovary cells, the Cys310 mutant TSH-R showed loss of response to TSH in terms of cAMP stimulation. However, a constitutive activity in terms of basal cAMP production was detected in the Cys310 mutant, compared with the wild-type TSH-R. Our data suggest that such a Cys310 TSH-R mutant may determine both the TSH resistance and the clinical euthyroidism detected in this family.

Amino Acid Substitution↗
PubMed1999

A Val 677 activating mutation of the thyrotropin receptor in a Hürthle cell thyroid carcinoma associated with thyrotoxicosis.

Thyroid nodules presenting as hot at 131I-scintigraphy are usually benign follicular adenomas. We report a 42-year-old female patient with an autonomously functioning Hürthle cell thyroid carcinoma causing thyrotoxicosis. Genetic analysis of her thyroid tumoral DNA revealed a heterozygotic activating mutation of the thyrotropin receptor (TSHR) gene that was located downstream to all of the other genetic alterations currently identified, and is due to a base substitution at codon 677 (normal cytosine replaced by guanine, CTG for GTG causing leucine substitution by valine in the seventh transmembrane domain of the receptor). This mutation was detected in the tumor, but not in the leucocytes from the same patient. The Val 677-TSHR mutant showed constitutive activity, in terms of cyclic adenosine monophosphate (cAMP) production, when permanently transfected in Chinese hamster ovary (CHO) cells. Gsp and ras oncogenes and the p53 tumor suppressor gene were not present in the Hürthle cell cancer. The TSHR mutation in this Hürthle cell carcinoma may be responsible for maintaining differentiated thyroid function and hyperthyroidism.

Adenocarcinoma↗
PubMed1999

Iodide symporter gene expression in normal and transformed rat thyroid cells.

OBJECTIVE: Decrease or loss of the Na+/I- symporter (NIS) activity profoundly affects the suitability of the use of radioiodine to detect or treat metastatic thyroid tissues. The aim of our study was to verify whether specific oncogene abnormalities were responsible for the alteration in NIS activity in thyroid cells. DESIGN AND METHODS: Expression of the NIS gene was investigated by Northern blot analysis in normal and in some oncogene-transformed cell lines with different degrees of malignancy which had lost the iodide uptake ability. RESULTS: NIS gene expression was up-regulated by TSH in a dose-dependent and time-dependent way in normal PC Cl 3 cells. The same effect was observed by activating the cAMP-dependent pathway by forskolin. Conversely, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a partial inhibitory effect on NIS gene expression. The oncogene-transformed cell lines PC v-erbA, PC HaMSV, PC v-raf, and PC E1A cells showed reduced NIS mRNA levels compared with the normal PC Cl 3 cells. Conversely, an almost complete absence of NIS gene expression was found in PC RET/PTC, PC KiMSV, PC p53(143ala), and PC PyMLV cell lines. CONCLUSIONS: Our data show that oncogene activation could play a role in affecting the iodide uptake ability in thyroid tumoral cells; different mechanisms are involved in the oncogene-dependent loss of NIS activity in transformed thyroid cells.

Animals↗
PubMed1998

Analysis of RET proto-oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) may occur either as a sporadic or familial (FMTC) disease. Multiple endocrine neoplasia (MEN) type 2, inherited as an autosomal dominant disease, is characterized by coexistence of MTC with other endocrine neoplasia. Activating mutations of the RET proto-oncogene, involving the somatic or the germinal cell lineage, are found in both inherited and acquired forms. In this study, RET mutations were screened in 47 individuals either affected by MTC or belonging to families with hereditary MTC. Exons 10, 11, 13, 14, 15 and 16 of the RET gene were amplified by polymerase chain reaction and examined by DNA sequence and/or restriction enzyme analysis to detect mutations in purified amplicons. Six MEN 2A families with a germline mutation at codon 634, one FMTC family carrying a mutation at codon 618 and two MEN 2B families with a mutation at codon 918 were identified. In affected members of a MEN 2A family no known RET mutations were observed. Besides, we identified a germline mutation in a patient with apparently sporadic MTC and in two out of three sons, indicating the presence of a sporadic misclassified familial disease. In all of the families examined we were able to distinguish the affected vs unaffected (not at risk) members. A somatic mutation of codon 918 was detected in three out of ten patients with apparently sporadic MTC.

Adult↗
PubMed1998

Thyroid hyperfunctioning adenomas with and without Gsp/TSH receptor mutations show similar clinical features.

Activating mutations of Gs alpha protein (gsp) and TSH receptor (TSH-R) identified in autonomously hyperfunctioning thyroid adenomas have been proposed as the primary event responsible for this disease. Since mutations have not been detected in 100% (ranging from less than 10% to 90%) of the patients, we evaluated whether the presence of gsp and TSH-R mutations cause differences in the clinical and biochemical parameters of the affected patients. Fifteen consecutive patients (11 women and 4 men) with autonomously hyperfunctioning thyroid adenomas who underwent thyroidectomy, previously examined for the presence of gsp or TSH-R mutations, were investigated. In all of the patients we examined plasma free T3, free T4, TSH levels and ultrasound volume of the nodules. The patients with mutations in gsp or TSH-R were similar to the patients without mutations for clinical presentation, sex distribution and mean age. Furthermore, basal serum FT3, TSH and tumor volume in the patients with mutations were not significantly different from the group without mutations. Our preliminary data demonstrate that no significant differences are present in the two groups of patients examined, suggesting that factors other than gsp or TSH-R mutations play a role in the clinical presentation of the disease.

Adenoma↗
PubMed1998

Iodide symporter gene expression in human thyroid tumors.

Expression of the Na+/I- symporter (NIS) gene was investigated by RT-PCR in a selected series of 26 primary thyroid carcinomas (19 papillary, 5 follicular, and 2 anaplastic). Fifteen follicular adenomas (11 "cold" and 4 "hot" adenomas) were also studied. Five of 19 papillary thyroid cancer did not express NIS messenger ribonucleic acid (mRNA). In all but 1 follicular cancer, NIS transcript was fully detected. In anaplastic tissue, NIS mRNA was only barely detected in 1 case. All of the follicular thyroid adenomas except 1 expressed the NIS gene. In contrast, all tumors studied excluding the anaplastic histotype fully expressed thyroglobulin and thyroid peroxidase mRNA transcripts. In 2 patients, a lower expression (3- to 5-fold) of NIS mRNA was found in metastasis by dot blot analysis compared with those in both normal and primary neoplastic thyroid tissue. Four of 8 differentiated thyroid cancer patients selected for the presence of metastases with negative posttherapy 131I total body scan showed the lack of NIS gene expression in their primary cancer. This defect, at least in these cases, is a somatic and intrinsic lesion of the primary cancer cells and is not due to a dedifferentiation process in the metastatic tissue. The early detection of the loss of NIS gene expression in the primary cancer, therefore, may provide useful information for the management of differentiated thyroid cancer patients.

Adenocarcinoma, Follicular↗
PubMed1997

A case of metastatic medullary thyroid carcinoma: early identification before surgery of an RET proto-oncogene somatic mutation in fine-needle aspirate specimens.

Medullary thyroid carcinoma (MTC) management requires determination of the sporadic or familial nature of the disease. RET proto-oncogene mutation analysis in the tumor tissue obtained at surgery and in the peripheral blood identifies somatic vs. germinal mutations. We now report a case of MTC in which a RET somatic mutation at codon 918 was detected in fine-needle aspiration specimens obtained from both the thyroid nodule and two enlarged neck lymph nodes but not in peripheral blood. Therefore, a diagnosis of sporadic MTC was made before surgery. Thus, this approach, by excluding preoperatively multiple endocrine neoplasia disease, permitted immediate thyroidectomy without search for pheochromocytoma. PCR-based genetic analysis in fine-needle aspiration biopsy specimens, therefore, preoperatively identifies genetic abnormalities at an early and easily manageable stage and may well contribute to the management strategy of MTC.

Base Sequence↗
PubMedDate not supplied

Thyrotropin receptor: a role for thyroid tumourigenesis?

Human thyroid tumours represent an example of the interplay of genetic and non genetic carcinogenesis. Recently, genetic abnormalities in the elements of the Thyrotropin receptor (TSH-R) dependent cAMP regulatory cascade have been found to be involved both in benign and malignant thyroid tumours. The presence of activating mutations has been demonstrated in the TSH-R gene as well as in the Gs alpha protein gene in thyroid toxic adenoma resulting in the constitutive activation of the cAMP pathway and it has been hypothesised that these genetic alterations may play a causative role in the disease. However, recent observations suggest more caution in accepting such a hypothesis. The presence of activating TSH-R mutations has also been demonstrated in differentiated thyroid carcinomas. At present, the percentage of such a modification is low, unless referred to selected series of tumours. Activating mutations of the TSH-R gene have been detected in a group of differentiated carcinomas with high basal adenylyl cyclase activity, and in a few cases of hyperfunctioning thyroid carcinoma. However, the role of the TSH-R-related cAMP pathway alterations in thyroid transformation remains to be elucidated. In this review, the role of TSH-R gene alterations in benign and malignant thyroid neoplasia is examined.

Adenoma↗