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ABSTRACT: Congenital hypothyroidism (CH) due to thyroglobulin (TG) deficit is an autosomal recessive disease (OMIM #274700) characterized by hypothyroidism, goiter, low serum TG, and a negative perchlorate discharge test. The aim of this study was to perform the genetic analysis of the TG gene in two sisters born from consanguineus parents and affected by CH and low serum TG levels. The index patient and her sister were identified at neonatal screening for CH and treated with L-thyroxine (L-T4). After discontinuation of L-T4 therapy, hypothyroidism was confirmed, serum TG was undetectable, and no organification defect after 123I scintigraphy and perchlorate test was shown; thyroid ultrasound showed a eutopic gland of normal size. DNA was extracted from peripheral white blood cells of the two sisters and the father. All 48 exons of TG gene were amplified by polymerase chain reaction and subjected to direct sequencing. A novel homozygous point mutation in exon 10 of TG gene was identified in the patient and her sister. The mutation determined a stop codon at position 768 (R768X) resulting in an early truncated protein or in the complete absence of the protein. The father (euthyroid) was heterozygous carrier of the mutation. Conclusion: Genetic analysis of TG gene was performed in two sisters affected by CH. A novel point mutation of the TG gene determining a stop codon at position 768 of the protein was identified. The early truncated nonfunctioning protein or the absence of the protein due to the premature degradation of abnormal mRNA may be responsible of the observed phenotype.
European Journal of Pediatrics 03/2013; · 1.88 Impact Factor
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Francesco Latrofa,
Debora Ricci, Lucia Montanelli,
Roberto Rocchi,
Paolo Piaggi,
Eleonora Sisti,
Lucia Grasso,
Fulvio Basolo,
Clara Ugolini,
Aldo Pinchera,
Paolo Vitti
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ABSTRACT: Context:Thyroglobulin autoantibodies (TgAb) have been proposed as a surrogate marker of thyroglobulin in the follow-up of differentiated thyroid carcinoma. Commercially available TgAb assays are often discordant. We investigated the causes of discrepancy.Design:TgAb were measured by three noncompetitive immunometric assays and three competitive RIA in 72 patients with papillary thyroid carcinoma and associated lymphocytic thyroiditis (PTC-T), 105 with papillary thyroid carcinoma and no lymphocytic thyroiditis (PTC), 160 with Hashimoto's thyroiditis, and in 150 normal subjects. The results of the six assays were correlated. TgAb epitope pattern, evaluated by inhibition of serum TgAb binding to thyroglobulin by TgAb-Fab regions A, B, C, and D, were compared in sera which were positive in all six assays (concordant sera) and positive in only one to five assays (discordant sera) were compared. TgAb International Reference Preparation (IRP) was measured in 2007 and 2009.Results:The correlations of the six assays ranged from -0.01 to 0.93 and were higher in PTC-T and Hashimoto's thyroiditis than in PTC and normal subjects. Two uncorrelated components, one including the three immunometric assays, the other the three RIA, explained 40 and 37% of the total variance of the results of the six assays. The levels of inhibition were higher in concordant sera than in discordant sera by TgAb-Fab region B (27.0%, 21.2-34.0 vs. 6.0%, and 2.7-12.7%) and region C (30.5%, 21.3-37.7 vs. 4.0%, and 1.0-6.5%); thus, the epitope pattern was more homogeneous in concordant sera than in discordant sera. TgAb IRP ranged from 157 to 1088 (expected 1000) IU/ml in 2009; results in 2007 were similar in all but two assays.Conclusions:TgAb assays are highly discordant. Discrepancy is lower when comparing assays with similar methodology. Results of TgAb from PTC-T are more concordant than those from PTC because their epitope pattern is more restricted. The internal standardization of TgAb is generally, but not completely, satisfactory.
The Journal of clinical endocrinology and metabolism 09/2012; · 6.50 Impact Factor
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Francesco Latrofa,
Debora Ricci, Lucia Montanelli,
Roberto Rocchi,
Paolo Piaggi,
Eleonora Sisti,
Lucia Grasso,
Fulvio Basolo,
Clara Ugolini,
Aldo Pinchera,
Paolo Vitti
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ABSTRACT: Serum thyroglobulin (Tg), the marker of residual tumor in papillary thyroid carcinoma, can be underestimated in patients with Tg autoantibodies (TgAb). TgAb are due to a coexistent lymphocytic thyroiditis (LT) or the papillary thyroid carcinoma per se. TgAb assays are highly discordant.
We evaluated 141 patients with a clinical diagnosis of nodular thyroid disease, 32 of Hashimoto's thyroiditis, and four of Graves' disease, who underwent total thyroidectomy for an associated papillary thyroid carcinoma. Patients were classified as papillary thyroid carcinoma-lymphocytic thyroiditis (PTC-T) and papillary thyroid carcinoma (PTC) according to the presence or absence of LT on histology. Tg was measured before thyroid remnant ablation, when it is expectedly detectable, by an immunometric assay (IMA) and TgAb by three noncompetitive IMA and three competitive radioimmunoassays (RIA). The number of lymphocytes was compared with TgAb concentration.
Seventy-two of 177 patients (40.7%) were classified as PTC-T and 105 (59.3%) as PTC. Although the tumor stage was similar in the two groups, Tg was undetectable in more PTC-T (37 of 72) than PTC (12 of 105) (P < 0.01), and Tg values were lower in the former (0; 0-4.7 ng/ml) (median; 25th to 75th percentiles) than in the latter group (9.7; 2.7-24.2) (P < 0.01). Accordingly, the percent of positive TgAb by the six assays resulted in higher PTC-T (29.2-50.0%) than PTC (1.9-6.7%) (P < 0.01). Among 49 patients with undetectable Tg, TgAb were more frequently positive by IMA (57.1-63.3%) than RIA (30.6-42.9%). The number of lymphocytes correlated with TgAb concentration in all six assays (0.34 < Rho < 0.46) (all P < 0.01).
In papillary thyroid carcinoma, LT on histology must be carefully searched for because it is frequently associated with TgAb and therefore mistakenly low or undetectable Tg. TgAb can be missed by some assays. In absence of LT, TgAb are rare.
The Journal of clinical endocrinology and metabolism 04/2012; 97(7):2380-7. · 6.50 Impact Factor
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Giuseppina De Marco,
Patrizia Agretti, Lucia Montanelli,
Caterina Di Cosmo,
Brunella Bagattini,
Melissa De Servi,
Eleonora Ferrarini,
Antonio Dimida,
Andrea Claudia Freitas Ferreira,
Angelo Molinaro,
Claudia Ceccarelli,
Federica Brozzi,
Aldo Pinchera,
Paolo Vitti,
Massimo Tonacchera
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ABSTRACT: Congenital hypothyroidism (CH) associated with goiter or a gland of normal size has been linked to dual oxidase 2 (DUOX2) mutations in the presence of iodide organification defect.
Thirty unrelated children with CH or subclinical hypothyroidism (SH) identified during infancy with a eutopic thyroid gland, coming from our Screening Centre for CH or referred from other regions of Italy, were studied with the perchlorate discharge test to identify organification defects. Eleven children with iodide organification defect were considered for the genetic analysis of TPO, DUOX2, and dual oxidase maturation factor 2 (DUOXA2) genes.
Eight children with CH and three with SH and eutopic thyroid gland were included in the study. After discontinuation of therapy, a partial or complete organification defect was shown after ¹²³I scintigraphy and perchlorate test.
TPO, DUOX2, and DUOXA2 genes were analyzed, and functional activity of DUOX2 variants was studied in HeLa cells.
Sequencing of the DUOX2 gene revealed a deletion S965fsX994 in three children; two were euthyroid after 1 month of L-T₄ discontinuation but developed SH after 5 and 18 months, respectively, whereas the other child had SH. One child with SH showed H678R, R701Q, and P982A substitutions, and another child with SH showed only the P982A. One child with SH showed the Y1150C mutation, and another euthyroid child showed the A728T mutation. Functional studies confirmed that S965fsX994, Y1150C, and A728T mutations were responsible for the defect in H₂O₂ production, whereas H678R, R701Q, and P982A did not alter H₂O₂ production in vitro.
Genetic analysis of the DUOX2 gene was performed in 11 children with organification defect. Two new mutations (Y1150C and A728T) and the deletion S965FsX994 were responsible for the deficit in the organification process and the phenotypes. Three polymorphisms (H678R, P982A, and R701Q) were identified.
The Journal of clinical endocrinology and metabolism 05/2011; 96(8):E1335-9. · 6.50 Impact Factor
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ABSTRACT: Thyroid dysgenesis (TD) is the most common cause of congenital hypothyroidism (CH), a relatively frequent endocrine disease in newborns (1 in 3000-4000 live births). TD is a defect in the organogenesis of the gland resulting in hypoplastic, ectopic or absent-thyroid gland. TD is usually sporadic but mutations in transcription factors (PAX8, TTF1, FOXE1 and NKX2-5) involved in thyroid development have been shown to cause a minority of cases transmitted as Mendelian diseases. This review focuses on the genetics and phenomics of hypothyroidism and TD due to PAX8 and TTF1 mutations.
Molecular and Cellular Endocrinology 03/2010; 322(1-2):64-71. · 4.19 Impact Factor
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Lucia Montanelli,
Patrizia Agretti,
Giuseppina de Marco,
Brunella Bagattini,
Claudia Ceccarelli,
Federica Brozzi,
Teresa Lettiero,
Manuela Cerbone,
Paolo Vitti,
Mariacarolina Salerno,
Aldo Pinchera,
Massimo Tonacchera
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ABSTRACT: Iodide transport defects (ITDs), rare causes of congenital hypothyroidism (CH), have been shown to arise from abnormalities of the sodium/iodide symporter (NIS). We describe a 16-year-old girl with CH caused by an ITD resulting from a novel mutation of NIS.
A 16-year-old girl with CH diagnosed by a neonatal screening program received early treatment with L-thyroxine replacement therapy. A (123)I scan had failed to reveal any iodide uptake by the thyroid and salivary glands; thus, thyroid agenesis was diagnosed. Thyroglobulin (Tg) was not measured when she was a neonate or infant. Unexpectedly, at the age of 14.5 years, a nodular goiter and high serum Tg concentrations (303 ng/mL; normal, <50) were identified. Her thyroid radioactive iodine uptake was very low as was the saliva to plasma iodide ratio (0.5). Analysis of her NIS gene revealed an in-frame six-nucleotide deletion of the coding sequence (1206-1211delGTCGGC) corresponding to the deletion of amino acids 287 and 288 of the human NIS protein located at the beginning of the VIII transmembrane segment. The proband was homozygous for this deletion, whereas both unrelated parents and her brother were heterozygous. COS-7 cells transfected with the mutant NIS failed to concentrate iodide, confirming that the mutation was the direct cause of the ITD in this patient.
We describe a patient with CH caused by a previously not described mutation of the NIS gene that was inherited from her parents. We therefore recommend that thyroid ultrasonography be performed in CH patients with low radioactive iodine uptake and elevated serum Tg.
Thyroid: official journal of the American Thyroid Association 11/2009; 19(12):1419-25. · 2.60 Impact Factor
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Massimo Tonacchera,
Giuseppina De Marco,
Patrizia Agretti, Lucia Montanelli,
Caterina Di Cosmo,
Andrea Claudia Freitas Ferreira,
Antonio Dimida,
Eleonora Ferrarini,
Helton Estrela Ramos,
Claudia Ceccarelli,
Federica Brozzi,
Aldo Pinchera,
Paolo Vitti
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ABSTRACT: Some cases of congenital hypothyroidism (CH) are associated with a gland of normal size.
To explore the cause of organification defect in one child with CH and a eutopic thyroid gland, genetic analyses of TPO, DUOX2, and DUOXA2 genes were performed.
One child with CH, a eutopic thyroid gland, and a partial organification defect was shown after (123)I scintigraphy and perchlorate test.
In the child with the organification defect, TPO, DUOX2, and DUOXA2 genes were analyzed. The functional activity of the DUOX2 mutants was studied after expression in eukaryotic cells.
No TPO or DUOXA2 gene mutations were identified. Direct sequencing of the DUOX2 gene revealed a compound heterozygous genotype for S911L and C1052Y substitutions. S911L and C1052Y caused a partial defect in H(2)O(2) production after transient expression in HeLa cells.
We performed a genetic analysis in one child with CH and a eutopic thyroid gland. Two new mutations in DUOX2 gene responsible for the partial deficit in the organification process were identified.
The Journal of clinical endocrinology and metabolism 09/2009; 94(11):4309-14. · 6.50 Impact Factor
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ABSTRACT: Genetic analysis of the TSH receptor gene in seven subjects with subclinical hypothyroidism (SH), in whom the diagnosis of autoimmune thyroid disease had been excluded by laboratory and instrumental techniques currently available.
Three families where different members (2 children and 5 adults) affected by SH were studied. GENETIC ANALYSIS: Genomic DNA was extracted from peripheral lymphocytes and the entire coding sequence of the TSHr gene was sequenced. pSVL-TSHr construct harbouring a Q8fsX62 insertion was obtained by site-directed mutagenesis. COS-7 cells transfected with wild-type and mutant receptor were used for binding studies, flow cytometry, and cyclic AMP (cAMP) determination.
A four base pair (bp) duplication in position 41 (41TGCAins), leading to a premature stop of translation at codon 62 (Q8fsX62), was found to be heterozygous in the proband, the father and the sister in Family 1. In Family 2 the proband and the sister were heterozygous for the mutation D410N. In Family 3 the proband and the father were heterozygous for the mutation P162A. After transfection in COS-7 cells, the mutant receptor Q8fsX62 displayed a low expression at the cell surface, and a reduced response to bovine TSH (bTSH) in terms of cAMP production.
We identified TSH receptor mutations in seven members of three families with subclinical hypothyroidism.
Clinical Endocrinology 12/2007; 67(5):712-8. · 3.17 Impact Factor
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Massimo Tonacchera,
Maria Elena Banco, Lucia Montanelli,
Caterina Di Cosmo,
Patrizia Agretti,
Giuseppina De Marco,
Eleonora Ferrarini,
Arash Ordookhani,
Anna Perri,
Luca Chiovato,
Ferruccio Santini,
Paolo Vitti,
Aldo Pinchera
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ABSTRACT: To analyse the coding region of PAX8 in individuals with congenital (CH) or post neonatal hypothyroidism due to dysgenetic (TD) or eutopic thyroid glands.
Forty-three children with CH and TD (13 agenesis, 23 ectopia, and seven hypoplasia), one subject with post neonatal onset of hypothyroidism and thyroid ectopia, 15 children with CH and eutopic thyroid glands and six euthyroid adults with thyroid hemiagenesis were enrolled as cases, along with 120 healthy individuals as controls.
Exons 2-8 of the PAX8 were directly sequenced. HeLa and HEK293 cells were transfected with PAX8 wild-type (PAX8-WT), mutant PAX8, p300, thyroid transcription factor 1 (TTF-1) and thyroglobulin promoter pGL3 (TG prom-pGL3). Synergism of TTF-1 with PAX8-WT vs. mutant and activity of PAX8-WT vs. mutant in accompaniment with p300 on TG prom-pGL3 were also assessed. The luminescence produced by PAX8-WT and mutant PAX8 was measured.
Among patients and controls only a 15-year-old girl with thyroid ectopia showed a heterozygous transition of cytosine to thymine at position 674 in exon 6, which changed a conserved threonine at position 225 to methionine (PAX8-T225M). Her father and sister harboured PAX8-T225M without abnormal thyroid phenotypes. PAX8-T225M and PAX8-WT similarly increased luciferase activity and had a similar synergistic effect with TTF-1. At 500 ng p300, however, PAX8-T225M could not significantly increase TG promoter activity when compared to PAX8-T225M alone, while PAX8-WT +500 ng p300 induction was significantly higher than PAX8-WT alone (P < 0.001). Cotransfection of TTF-1 together with PAX8-T225M resulted in rescuing of the lack of synergism with p300.
PAX8 mutations in congenital hypothyroidism due to dysgenetic or orthotopic thyroid glands are rare. PAX8-T225M is probably a rare variant.
Clinical Endocrinology 07/2007; 67(1):34-40. · 3.17 Impact Factor
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Massimo Tonacchera,
Mariaelena Banco,
Paola Lapi,
Caterina Di Cosmo,
Anna Perri, Lucia Montanelli,
Lidia Moschini,
Gianluca Gatti,
Daniele Gandini,
Alessandro Massei,
Patrizia Agretti,
Giuseppina De Marco,
Paolo Vitti,
Luca Chiovato,
Aldo Pinchera
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ABSTRACT: Homozygous null mice for thyroid transcription factor (TTF)-2 gene exhibit cleft palate and thyroid malformation. We performed a genetic analysis of the TTF-2 gene in 2 children with congenital hypothyroidism (CH) and cleft palate, 45 children with thyroid dysgenesis, 19 children with isolated cleft palate or cleft lip, 4 patients with thyroid hemiagenesis. The entire coding-region of the TTF-2 gene was analyzed by direct sequencing. Direct sequencing of the TTF-2 gene revealed polymorphisms in the length of the polyalanine tract. The most frequent stretch length was 14 residues and it was found in 50 of 70 (71%) and in 45 of 53 (85%) normal healthy controls. A polyalanine tract of 16 residues in the heterozygous state was seen in 18 of 70 (26%) cases and in 4 of 53 (7%) normal subjects. In 1 of 4 (25%) case of hemiagenesis a polyalanine tract of 16 residues in the homozygous state was observed. In 1 of 26 agenesis the polyalanine tract consisted of 12 residues in the heterozygous state. Direct sequencing also revealed the presence of two silent polymorphisms. No mutations were identified in the TTF-2 gene. In conclusion, our results show that no genetic alteration was present in the TTF-2 gene of these patients, suggesting that defects in the TTF-2 gene are a rare event.
Thyroid 09/2004; 14(8):584-8. · 4.79 Impact Factor
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Ferruccio Santini,
Paolo Vitti,
Luca Chiovato,
Giovanni Ceccarini,
Marco Macchia, Lucia Montanelli,
Gianluca Gatti,
Veronica Rosellini,
Claudia Mammoli,
Enio Martino,
Inder J Chopra,
Joshua D Safer,
Lewis E Braverman,
Aldo Pinchera
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ABSTRACT: Creams containing thyroid hormone are commonly employed for cosmetic purposes. To verify whether T(4) applied to the skin surface can enter the bloodstream either directly or as a metabolite, a cream containing L-T(4) [3,5,3',5'-tetraiodothyronine (T(4))] was self-applied by volunteers for 2 wk. No significant variations in urinary iodide, TSH, and serum (total and free) T(4) and T(3) concentrations were observed at any time relative to pretreatment values, whereas rT(3) concentrations increased significantly 6 and 12 h after cream application. The increased rT(3) concentration led us to investigate the presence of inner ring type III deiodinase (D3) activity in human skin. Using human surgical discard skin, we found that T(4) can be carried across human epidermis in a liposome cream. Substantial inner ring deiodination was suggested by the fact that only 10% of transferred thyroid hormone remained as T(4), and T(3) was not detected. We then measured D3 activity in a surgical skin specimen. The K(m) for T(3) was 1.74 nmol/liter, and the maximum velocity was 23.5 fmol/microg microsomal protein/h. In conclusion, our study indicates that normal human skin serves as a substantial, but incomplete, barrier to T(4) passage. D3 plays an important role in augmenting T(4) blockade by inactivating T(4) to rT(3).
Journal of Clinical Endocrinology & Metabolism 07/2003; 88(6):2825-30. · 6.50 Impact Factor
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ABSTRACT: Chemotherapy represents the only therapeutic option in most poorly differentiated thyroid carcinomas, although its effect is limited and short lasting. The aim of this study was to evaluate whether increasing the metabolic rate of thyroid cancer cells by TSH stimulation might result in higher response rate to chemotherapy. Fourteen patients with poorly differentiated thyroid carcinoma and nonfunctioning diffuse lung metastases detected at computed tomography scan, entered this study. A combination of carboplatinum and epirubicin was administered at 4- to 6-wk intervals for six courses. TSH stimulation was achieved by reduction of the daily dose of L-thyroxine resulting in mild hypothyroidism (eight patients) or by administration of recombinant human TSH (six patients). Two additional patients did not complete the therapeutic protocol due to severe hematological side effects. Results were evaluated by comparison of lung computed tomography scans before and after therapy. One patient had a complete remission. Five patients had partial remission, and seven patients had disease stabilization. One patient progressed to death. The overall rate of positive responses was 37% that rose to 81% when including patients with stable disease. Serum thyroglobulin after chemotherapy declined more than 50% in six patients, with respect to basal levels. Apparently, no difference in the response rate was observed between exogenous or endogenous TSH stimulation. At the time of this analysis, among the patients who completed the treatment courses, 9 of 14 patients (64.3%) are still alive (median survival from start of chemotherapy = 21 months, range: 15-34). Six of these patients did not show progression of lung disease, whereas regrowth of lung metastases was observed in three patients after 19, 20, and 21 months from chemotherapy, respectively. Five patients died of their disease, including the one who had progression of lung disease during chemotherapy, three who died for brain or bone metastases, and one who died for refractory local tumor invasion. No progression of lung metastases was observed until death in these four patients. In conclusion, the response rate of poorly differentiated thyroid cancer to chemotherapy observed in this study was favorable and promising. TSH stimulation may have contributed to these results.
Journal of Clinical Endocrinology & Metabolism 10/2002; 87(9):4160-5. · 6.50 Impact Factor
