Methylation levels of sodium-iodide symporter (NIS) promoter in benign and malignant thyroid tumors with reduced NIS expression.

DNA methylation regulates gene expression. Aberrant methylation plays an important role in human tumorigenesis. We have previously detected reduced NIS mRNA expression in thyroid tumors as compared to non-tumor tissues. Thus, in this study we investigated whether the methylation of the CpG-island located in the NIS gene promoter was associated with reduced mRNA expression in thyroid tumors. Methylation levels of 30 pairs of samples from 10 benign and 20 malignant thyroid tumors (T) along with matched non-tumor (NT) areas were determined by semiquantitative methylation specific-PCR. NIS methylation was detected in all samples. Methylation levels and frequencies did not differ between the groups and were not associated with BRAF mutational status. Highest methylation levels and frequencies were detected in the 5' region of the CpG-island decreasing toward the 3' end. Intraindividual analysis (T versus NT) showed high tumor methylation levels in 40 % of the samples in the benign group and 30 % in the malignant group, associated with low NIS mRNA expression. No quantitative correlation was detected between methylation levels and mRNA expression in any the groups. The results of this study showed that methylation of NIS promoter is a very frequent event in both benign and malignant tumors as well as in their surrounding tissues, and characterized a non-homogeneous methylation pattern along the CpG island. Therefore, further investigations involving other sites that may be implicated in methylation regulation of NIS expression are warranted.

Oral ingestion of a hydrolyzed gelatin meal in subjects with normal weight and in obese patients: Postprandial effect on circulating gut peptides, glucose and insulin.

Gut hormones [ghrelin, peptide YY (PYY) and glucagon-like peptide-1 (GLP-1)] are an important group of hormones that target appetite control. They are released from endocrine L cells of the small bowel in proportion to the volume, components and calories in a meal. In the current study, 20 g of gelatin (flavored and sweetened) were given to obese patients (n=12) and lean subjects (n=10). Subsequently, plasma samples were collected at-30- minute intervals up to 180 minutes and glucose, insulin, PYY, GLP-1 and ghrelin were assayed using specific and sensitive immunofluorometric and radioimmunoassays. As expected, obese patients had normal serum glucose levels, higher serum insulin, and lower plasma concentration of ghrelin at all times compared to lean subjects. GLP-1 plasma levels were significantly elevated at 60 minutes, peaking at 120 minutes in obese patients and lean subjects. As a consequence, there was a significant rise in serum insulin levels with a significantly higher peak level at 60 min (obese) and 30 min (lean). There were no significant changes in PYY plasma concentrations and no correlation was found between body mass index and concentrations of ghrelin, PYY and GLP-1 in the group of obese patients. In conclusion, a single gelatin meal induces a rise in plasma GLP-1 followed by an increase in serum levels of insulin. These findings may be applied to maximize satiety in obese patients as a means of improving adherence to calorie-controlled diets as well as provide better control of diabetic patients.

Peripheral blood levels of thyroglobulin mRNA and serum thyroglobulin concentrations after radioiodine ablation of multinodular goiter with or without pre-treatment with recombinant human thyrotropin.

We investigated the effect of therapeutic doses of radioiodine (RAI) on peripheral serum messenger thyroglobulin RNA (Tg mRNA) and serum thyroglobulin (sTg) in patients with multinodular goiter (MNG) preceded or not by treatment with recombinant human TSH (rhTSH). Fourteen patients with large MNG (91-542 ml) received RAI (550-2960 MBq). Half of the patients received 0.45 mg of rhTSH prior to the treatment (RAI+rhTSH group) and half did not (RAI group). Patients' blood samples were collected before and 24, 48, and 72 h; 7 and 30 days; and 6, 9, and 12 months after RAI treatment. Serum Tg was measured by immunoradiometric assay, serum anti-Tg by radioimmunoassay, and quantification of circulating Tg mRNA was performed by real-time PCR. The shrinkage of MNG volume was documented by serial computed tomography (CT) scans before, 6 and 12 months after RAI. Peak Tg mRNA and sTg were reached earlier in the RAI+rhTSH group (24 h and 48 h) than in the RAI group (7 days). Both declined after the peak and the lowest levels were observed at 12 months. The mean reduction of the thyroid volume was 19.8% (RAI group) and 30.3% (RAI+rhTSH group) at 6 months (ns) and 32.8% RAI and 52.5% (RAI+rhTSH group) at 12 months (p<0.05). After RAI treatment there was a significant and positive correlation between goiter volume and sTg only in the RAI group (r=0.7; p=0.032). Serum anti-Tg had a transitory and relatively small elevation in 3 and 2 patients, respectively, in the RAI and RAI+rhTSH groups. We concluded that after RAI ablation of MNG there is a rapid release of Tg into the serum possibly from the colloid, which is followed by an elevation of serum Tg mRNA that may be due to an increased release of follicular cells into the blood stream. Both phenomena are enhanced by the use of rhTSH before RAI treatment as a consequence of a more effective and prolonged radiation exposure of the thyroid follicles.

Prognostic value of serial serum thyroglobulin determinations after total thyroidectomy for differentiated thyroid cancer.

Serial weekly serum samples (for 3 weeks) were obtained from 42 patients with differentiated thyroid cancer (DTC, papillary no.=35, follicular no.=6, Hurthle cell no.=1) for serum thyroid hormone, TSH and TG before and after total thyroidectomy. Serum specimens were also obtained one month after radioiodine (131I) therapy followed by suppressive dose of L-thyroxine (L-T4, 2.5 microg/kg). The patients were subdivided into four groups: group I: the DTC was confined to a single solid nodule (no.=1 2); group II: thyroid malignancy invaded local cervical structures but there were no lymph node metastases (no.=8); group III: DTC with lymph node metastases (no.=6); and group IV: DTC with distant metastases (no.=16). In all group I patients serum TG remained undetectable in spite of elevated serum TSH levels at the 3rd week post-surgery (PS). Only one of group II patients had a detectable serum TG value of 5.2 ng/ml (3rd week PS). By contrast, 37.5% of group III patients had detectable serum TG levels, ranging from 3.4 to 16.8 ng/ml (3rd week PS). Lymph node metastases were detected in 5 of these patients by whole body scan (WBS) and removed surgically in 3. As expected, group IV patients had elevated serum TG values ranging 33.0-958.0 ng/ml and distant metastases were confirmed in all of them by WBS. From the calculations through univariate logistic regression comparing TG concentrations at the 3rd week PS from groups I and II vs groups III and IV, we obtained a cut-off value of 2.3 ng/ml with the following efficacy features: sensitivity=74.5%; specificity=95%; positive predictive value=92.3%; negative predictive value=65.5%; and accuracy=73.8%. After 131I and L-T4 suppressive therapy, only 5 out of 36 patients of groups I, II and III had detectable serum TG levels (3.1-7.0 ng/ml) whereas serum TG was detectable in all group IV patients (ranging 2.5-8.6 ng/ml). We concluded that serum TG concentrations above 2.3 ng/ml at the 3rd week PS could be suggestive of lymph node or distant metastases in patients with DTC. Patients with serum TG above this limit could be considered at risk for metastatic disease and higher doses of diagnostic iodine-131 (131I) may be indicated for actinic ablation.

Differential reactivity for galectin-3 in Hürthle cell adenomas and carcinomas.

Hürthle cell carcinomas behave as the most aggressive variant of differentiated thyroid carcinoma of follicular origin, with frequent recurrences and higher morbidity. Its differential diagnosis with Hürthle cell adenoma remains a problem for the clinician and for the pathologist. The vertebrate lectins, galectin-1 and galectin-3 have been implicated in the regulation of cellular growth, differentiation, and malignant transformation in thyroid neoplasms. Galectin-3, a beta-galactoside binding protein, has been recently found to be highly expressed in papillary and follicular carcinomas. The current study was undertaken to investigate immunohistochemical reactivity for galectin-3 of thyroid specimen tissues with Hürthle cell adenomas (n = 14) and carcinomas (n = 17), follicular (n = 14) and papillary (n = 11) carcinomas, colloid goiter (n = 30), Hashimoto's thyroiditis (n = 11), follicular adenoma (n = 9), and normal thyroid tissues (n = 18). Follicular (78.5%) and papillary (82.0%) carcinomas were frequently reactive for galectin-3, more often when some H rthle cells were present. There was no galectin-3 immunostaining in any of the specimens from Hashimoto's thyroiditis, colloid goiters or normal thyroid samples, whereas only one case of follicular adenoma was found positive (11.1%). By contrast, galectin-3 immunostaining in Hürthle cell carcinomas was significantly higher (59%) than in H rthle cell adenomas (7.1), p < 0.05). These results suggest that galectin-3 may potentially serve as a marker in difficult differential diagnosis cases involving Hürthle cell adenomas and Hürthle cell carcinomas.

Aggressive metastatic follicular thyroid carcinoma with anaplastic transformation arising from a long-standing goiter in a patient with Pendred's syndrome.

In this article we describe detailed pathological and molecular genetics studies in a consanguineous kindred with Pendred's syndrome. The index patient was a 53-year-old female patient with congenital deafness and goiter. Her parents were first-degree cousins. She had a large goiter (150 g) that had been present since childhood. One of her sisters and a niece are also deaf and have goiter as well. The presence of Pendred's syndrome was confirmed by a positive perchlorate test and the demonstration of a Mondini malformation. Thyroid function tests (under levothyroxine [LT4] therapy) were in the euthyroid range with a thyrotropin [TSH] level of 2.8 microU/mL (0.2-3.2), a serum total thyroxine (T4) of 90 nmol/L (54-142), and a serum total triiodothyronine (T3) of 2.7 nmol/L (0.8-2.4). Total thyroidectomy was performed, and the mass in the right lobe was found to have invaded adjacent tissues. The histopathological findings were consistent with a follicular carcinoma with areas of anaplastic transformation and lung metastasis. The patient was treated twice with 100 mCi 131iodine (3,700 MBq) and received suppressive doses of LT4. Postoperatively, the serum thyroglobulin (Tg) levels remained markedly elevated (2,352 to 41,336 ng/mL). The patient died of a sudden severe episode of hemoptysis. Sequence analysis of the PDS gene performed with DNA from the two relatives with Pendred's syndrome revealed the presence of a deletion of thymidine 279 in exon 3, a point mutation that results in a frameshift and a premature stop codon at codon 96 in the pendrin molecule. We concluded that prolonged TSH stimulation because of iodine deficiency or dyshormonogenesis in combination with mutations of oncogenes and/or tumor suppressor genes, may result in the development of follicular thyroid carcinomas that undergo transformation into anaplastic cancers. It is likely that these pathogenetic mechanisms have been involved in the development of aggressive metastatic thyroid cancer in this unusual patient with Pendred's syndrome.

Congenital goiter with hypothyroidism caused by a 5' splice site mutation in the thyroglobulin gene.

In this work we have extended our initial molecular studies of a consanguineous family with two affected goitrous siblings (H.S.N. and Ac.S.N.) with defective thyroglobulin (Tg) synthesis and secretion because of a homozygotic deletion of a fragment of 138 nucleotides (nt) in the central region of the Tg mRNA, identified previously in H.S.N. In order to identify the intron/exon boundaries and to analyze the regions responsible for pre-mRNA processing corresponding to a 138 nt deletion, we performed a screening of a human genomic library. The intron/exon junction sequences were determined from one positive clone by sequencing both strands of the DNA template. The results showed that the deletion mapped between positions 5549 and 5686 of the Tg mRNA and corresponded to exon 30. The positions of the exon limits differed by three nucleotides from the previously reported data obtained from direct sequencing of the deleted reverse transcriptase-polymerase chain reaction fragment from H.S.N. These variations are because the intron/exon junctions in this region were not available at the time when the deletion was first described. The deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shortened by 46 residues. The same 138 nt deletion was observed in reverse transcriptase-polymerase chain reaction studies performed in the thyroid tissues from Ac.S.N. Genomic DNA analysis showed that a G to T transversion was observed at position +1 in the donor site of intron 30. Both affected patients (H.S.N. and Ac.S.N.) are homozygous for the mutation whereas the normal sister (At.S.N.) had a normal allele pattern. The functional consequences of the deletion are related to structural changes in the protein molecule that either could modify the normal routing of the translation product through the membrane system of the cell or could impair the coupling reaction. Probably the mutant Tg polypeptide might be functionally active in the production of thyroid hormone, because in the presence of a normal iodine ingestion (approximately 150 microg/day), Ac.S.N. was able to maintain normal serum levels of total triiodothyronine (T3) associated with relatively low serum total thyroxine (T4) with normal somatic development without signs of brain damage.

A novel mutation (Q40P) in PAX8 associated with congenital hypothyroidism and thyroid hypoplasia: evidence for phenotypic variability in mother and child.

Congenital hypothyroidism associated with thyroid hypoplasia can be caused by several genetic defects, including mutations in the TSHbeta-subunit, the TSH receptor, the G(s)alpha-subunit, and the transcription factor PAX8. Four girls with sporadic congenital hypothyroidism and hypoplastic thyroid glands were analyzed for mutations in PAX8 and TTF2 (FKHL15). Mutations in the coding region of the TSHbeta-subunit gene, the TSH receptor gene, and exons 8 and 9 of G(s)alpha had been excluded previously. Serum TSH concentrations were 150 mU/liter or more, TG levels were within normal limits, and thyroid autoantibodies were absent. Technetium scintigraphies did not reveal the presence of thyroid tissue, but ultrasonography documented hypoplastic, normally located glands. One patient was found to harbor a heterozygous transversion 119A-->C in exon 3 of PAX8 replacing a conserved glutamine by proline in the paired box domain (Q40P). Analysis of her family members revealed that her mother, who has a thyroid gland of normal size and mild, adult-onset autoimmune hypothyroidism, is also heterozygous for this mutation. Functional analyses of the PAX8 Q40P mutation showed impaired binding to a PAX8 response element and absent trans-activation of a thyroid peroxidase promoter luciferase reporter gene. These findings confirm the important role of PAX8 in the development of the thyroid, but they indicate that PAX8 gene mutations may have a variable penetrance or expressivity. The absence of mutations in the coding sequences of the analyzed genes in the three other patients supports the concept that the pathogenesis of congenital hypothyroidism associated with thyroid hypoplasia is diverse.

Gastrointestinal side effects of orlistat may be prevented by concomitant prescription of natural fibers (psyllium mucilloid).

OBJECTIVES: This placebo-controlled open study was designed to test the hypothesis that most of the gastrointestinal (GI) side events induced by treatment of obese patients with orlistat (a gastrointestinal lipase inhibitor) could be prevented or ameliorated by concomitant use of natural fibers (psyllium mucilloid). DESIGN: Two groups of obese women (BMI>27 kg/m(2)) were treated with orlistat 120 mg three times a day. One group (A, n=30) was randomized to receive orlistat and, approximately 6.0 g of orange-flavored psyllium mucilloid dissolved in water and the other group (B, n=30) received orlistat and orange-flavored placebo. At the end of 30 days and 2 weeks of washout, group A switched to placebo and group B received psyllium while continuing orlistat three times a day. SUBJECTS: Sixty professional women, more than 21-y-old with a body mass index (BMI) between 27.3 and 48.0 kg/m(2), who were not receiving any other medication. MEASUREMENTS: Assessments included weekly visits to attending physician, filling a form in which GI events were recorded, monthly measurements of body weight, blood pressure and serum lipids. The frequency and severity of GI events were evaluated by a score system, based on information provided by the patients. RESULTS: Both groups A and B significantly lost (P<0.01) weight after 60 days of orlistat (A=96.8 to 94.9 kg and B=98.7 to 96.5 kg). Similarly, BMI values declined significantly in both groups. While in the psyllium plus orlistat group (group A) the mean +/-s.e.m. of the scores reflecting GI events was 13.0+/-1.8, the placebo plus orlistat group (B) had a value of 35.9+/-2.7 (P<0.01). When the reverse situation was instituted the placebo and orlistat group presented a mean score of 36.1+/-3.6 and the psyllium plus orlistat a mean score of 8.9+/-1.5 (P<0.01). CONCLUSIONS: Psyllium hydrophilic mucilloid concomitantly prescribed to obese patients receiving 120 mg of orlistat three times a day is an effective and safe adjunct therapy that is helpful in controlling the GI side effects of this pancreatic lipase inhibitor.

Clinical and molecular analysis of three Mexican families with Pendred's syndrome.

BACKGROUND: The autosomal recessive Pendred's syndrome is defined by congenital sensorineural deafness, goiter, and impaired iodide organification. It is caused by mutations in the Pendred's syndrome (PDS) gene that encodes pendrin, a chloride/iodide transporter expressed in the thyroid, the inner ear, and the kidney. OBJECTIVE: To perform a detailed clinical and molecular analysis of patients with Pendred's syndrome from four patients from three unrelated Mexican families. METHODS: Thyroid function tests, perchlorate test, thyroid scintigraphy, audiometry, computer tomography and magnetic resonance imaging were performed in all affected individuals. Haplotype analyses were performed using microsatellite markers flanking the PDS locus, and the PDS gene was submitted to direct sequence analysis. RESULTS: All patients presented with sensorineural deafness, Mondini malformations of the cochlea, an enlarged vestibular aqueduct, goiter, and a positive perchlorate test. Two patients were hypothyroid, two individuals were euthyroid. Sequence analysis revealed a complex homozygous deletion/insertion mutation at the end of exon 4 in the index patient of family 1 resulting in a premature stop codon at position 138. In family 2, the affected individuals were compound heterozygous for a splice acceptor mutation (IVS2 -1G>A) and a 1231G>C transversion substituting alanine 411 by proline (A411P). In family 3, the index patient was found to be homozygous for a transversion 412G>T in exon 4 replacing valine 138 by phenylalanine (V138F). CONCLUSIONS: All patients included in this study presented with the classic Pendred syndrome triad and molecular analysis revealed pendrin mutations as the underlying cause. The identification of three novel mutations, one of them of complex structure, expands the spectrum of mutations in the PDS gene and emphasizes that they display marked allelic heterogeneity.

Overexpression of epidermal growth factor and epidermal growth factor-receptor mRNAs in dyshormonogenetic goiters.

Thyroid malignancy has been induced by long-term endogenous thyrotropin (TSH) stimulation in experimental animals, leading to local and distant metastasis. It has been postulated that constant and prolonged endogenous TSH stimulation in dyshormonogenetic thyroid tissues could result in thyroid neoplasia. The possible role of growth factors and oncogenes in goitrogenesis and favoring neoplasia has also been mentioned. Overexpression of certain growth factors and/or their receptors, and of oncogenes implicated in growth promotion may play a significant role in the relatively frequent finding of thyroid malignancy in congenital goiters. In this study the expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGF-R), transforming growth factor-beta (TGF-beta), c-myc, and p53 mRNAs was determined in 14 thyroid tissue samples: 6 from patients with thyroid peroxidase (TPO) gene mutations, 4 with thyroglobulin (Tg) gene defects and 4 normal thyroid tissues. EGF mRNA overexpression was seen in 7 of 10 dyshormonogenetic tissues (3.5 to 12.0 arbitrary optical densitometry units [AODU]) and considered significantly higher (p < 0.01) when compared to normal thyroid tissues (0.25 to 0.32 AODU). Moreover, overexpression of EGF-R mRNA was present in 6 of 10 dyshormonogenetic tissues (2.23 to 13.03 AODU) and considered significantly higher (p < 0.01) when compared to normal thyroid tissues (0.42 to 0.65 AODU). There was no difference in c-myc, p53, and TGF-beta mRNAs expression between dyshormonogenetic and normal tissues. The overexpression of EGF and EGF-R mRNAs found in dyshormonogenetic tissues may suggest that this growth factor may play a role in cellular proliferation and contribute to goiter formation.

Prevalence and characteristics of postpartum thyroid dysfunction in São Paulo, Brazil.

OBJECTIVE: Postpartum thyroid dysfunction (PPTD) is an autoimmune disorder characterized by the development of transient hyperthyroidism and, more frequently, hypothyroidism (or both) during the first six months of the puerperal period. A variable incidence has been reported in part because of differences in the number of women studied, the frequency of thyroid assessment postpartum, diagnostic criteria and methodology. The aim of this study was to evaluate thyroid function, ultrasound images and titre of autoantibodies against thyroid antigens in a cohort of pregnant women who met the criteria of 'normal' thyroid gland structure on clinical examination and imaging and normal thyroid function tests without a significantly positive anti-thyroid peroxidase (TPO) antibody titre (i.e. < 100 U/ml) in the first trimester. DESIGN AND PATIENTS: Eight hundred nulliparous or multiparous (one to seven previous pregnancies) pregnant women (age 26.1 +/- 4.8 years, mean +/- SD), were submitted to clinical, laboratory and ultrasonographic examination in the first trimester of pregnancy. Among these forty-six patients were excluded because of thyroid dysfunction, ultrasound structural abnormalities or a positive anti-TPO antibody titre (> 100 U/ml). A total number of 754 women were available for further studies in the postpartum period. A relatively large number of these patients (386) were lost for follow-up either before or after delivery. MEASUREMENTS: A cohort of 368 puerperal women was followed up regularly at 3, 6, 12 and 24 months after delivery, with periodic thyroid function tests, random urine iodine measurements, assays for serum autoantibodies against thyroid antigens and imaging by ultrasound. RESULTS: The provisional diagnosis of PPTD was established in 78 out of 368 who had positive anti-TPO levels and ultrasonographic thyroid structural changes. Twenty-nine of these patients had a transient rise of anti-TPO autoantibodies characterizing an autoimmune reaction. These autoantibodies levels progressively declined or became negative. Moreover none of these patients had evidence for altered thyroid function during the 18-24 months of follow-up. The remaining 49 patients (13.3%) progressively developed thyroid function abnormalities (mainly hypothyroidism) indicating the presence of thyroid gland changes due to PPTD. Further follow-up studies indicated that at 18-24 months, 42 patients had serum levels of anti-TPO-Ab that were more elevated, as compared with the first year values. Predictive factors found during pregnancy for developing PPTD were: (1) relatively low levels of anti-TPO, between 60 and 100 U/ml (odds ratio 3.1 : 1), and (2) ultrasonographic thyroid structural changes in the first trimester (odds ratio 6.4 : 1). CONCLUSIONS: We conclude that the prevalence of postpartum thyroid dysfunction in our geographical area ranges from 6.7% to 13.3%, considering, respectively, all pregnant women that were examined (n = 754) or only the number of puerperal women actually followed-up (n = 368). A transient form of thyroid autoimmune reaction characterized by elevated serum levels of anti-TPO that progressively declined or disappeared was observed in 29 puerperal women. Sonographic structural and echogenicity changes in the thyroid gland and borderline positive anti-TPO levels (between 60 and 100 U/ml) during pregnancy were considered to be of predictive value for development of postpartum thyroid dysfunction.

Decreased androgen levels in massively obese men may be associated with impaired function of the gonadostat.

OBJECTIVE: In obese men, sex hormone-binding globulin (SHBG) as well as total testosterone (TT) levels are decreased. Data concerning serum free testosterone (FT) levels in obese men are discordant. FT levels are decreased in only some morbidly obese men, consistent with an impairment of the feedback regulatory mechanism. In this study we aimed to verify serum levels of TT and FT in two groups of obese men (BMI < 35.0 kg/m2 and BMI > 35.1 kg/m2) before and after weight loss. DESIGN: Two groups of obese men (group 1: BMI < or = 35 kg/m2; and group 2: BMI > or =35.1 kg/m2) were studied before and after 6 months of a low energy diet (1200 kcal/day). Every patient received a therapeutic prescription of dexfenfluramine (15 mg b.i.d.) that was maintained for 6 months. SUBJECTS: Thirty-seven obese men and 20 normal weight men. MEASUREMENTS: Serum sex hormones (TT and FT), serum luteinizing hormone (LH) and insulin were analyzed by RIA assays. Plasma insulin levels, serum TT, FT and LH concentrations were obtained before and after weight loss. RESULTS: Moderately obese men (BMI = 32.3+/- 1.9 kg/m2) presented significantly decreased TT levels (390+/-120ng/dl) as well as FT (mean+/-s.d.:16.0+/-4.8pg/ml) as compared with normal controls. FT serum levels had a significant and negative correlation with body mass index (BMI), whereas for TT concentrations this correlation was not significant. Serum LH concentrations (4.5+/-2.9mlU/ml) were normal. Insulin levels were elevated in all patients (46.3+/-30.1 microU/ml). After weight loss there was a significant (P< 0.01) increase in TT, FT and LH levels, whereas insulin concentrations significantly decreased. In massively obese men (BMI = 43.0 6.7 kg/m2), TT (320+/-110ng/dl), FT (11.0+/-2.1 pg/ml) and LH (3.1+/-1.3mlU/ml) were decreased and significantly lower as compared with the previous group and normal controls. As expected, after weight loss TT, FT and LH levels increased significantly while insulin concentrations decreased. CONCLUSIONS: We concluded that FT levels are dependent on the degree of obesity, massively obese men (BMI > or =35.1 kg/m2) being considered as candidates for consistently low FT levels. A functional decrease of LH pulse amplitude and serum LH levels as well as a possible negative action of excess of circulating leptin on the steroidogenesis may be related to the decreased androgens levels in massively obese men.

Thyrotropin receptor mutations in hyperfunctioning thyroid adenomas from Brazil.

Constitutively activating mutations in the thyrotropin (TSH) receptor have been identified as a major molecular cause of hyperfunctioning thyroid adenomas. A smaller subset of these benign tumors is caused by constitutive activation of the adenylyl cyclase cascade by somatic mutations in the Gsalpha gene. In this study, we analyzed hyperfunctioning thyroid adenomas from seven Brazilian patients for TSH receptor and G(s)alpha gene mutations. Solitary autonomous thyroid adenomas were identified by ultrasound and scintigraphy, and DNA was extracted from adenomatous and periadenomatous tissue. Exons 9 and 10 of the TSH receptor gene, and exons 8 and 9 of the G(s)alpha gene, were amplified by polymerase chain reaction (PCR) and subjected to direct sequence analysis. Six of seven adenomas harbored heterozygous mutations known to confer constitutive activity to the TSH receptor. In one case, aspartate 619 was substituted by glycine (D619G). In four adenomas, alanine 623 was replaced by valine (A623V). Both residues are located in the third intracellular loop. In one instance, aspartate 633 located in the sixth transmembrane domain was replaced by tyrosine (D633Y). In this patient, one allele also contained a change of aspartate 727 to glutamate (D727E). This substitution is thought to be a polymorphic variant of the wild-type but it has also been associated with toxic multinodular goiters. Functional comparison of D727 with E727 did not reveal differences in basal or TSH-stimulated cyclic adenosine monophosphate (cAMP)-dependent luciferase activity in transiently transfected cells. These results demonstrate a high prevalence of activating TSH receptor mutations in toxic adenomas in this small series from Brazil (approximately 86%). These findings are in agreement with reports from other countries with a marginal iodine intake but contrast with studies from regions with a high iodine intake where these mutations appear to be less prevalent.

A novel mutation in the TPO gene in goitrous hypothyroid patients with iodide organification defect.

OBJECTIVE: To screen and subsequently sequence the TPO gene for mutations in patients with congenital goitre, hypothyroidism and evidence for an organification defect (positive perchlorate discharge test). PATIENTS: We have studied seven hypothyroid and congenitally goitrous patients from three unrelated families. DESIGN AND MEASUREMENTS: We have measured serum thyroid hormone levels, 131I uptake, serum TSH and serum Tg concentrations. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene. RESULTS: DGGE identified the presence of two frameshift mutations: a GGCC duplication in exon 8 (homozygous in one family and heterozygous in the other family) and a heterozygous insertion of a single nucleotide (C) at position 2505-2511 in exon 14. In addition, we have detected an alteration in exon 11, not yet described in the literature, derived from a single nucleotide substitution of a C to G at position 2008, altering the well-conserved amino acid domain among the peroxidases superfamily. This mutation in exon 11 was present in two families that showed heterozygous mutation for exon 8 or for exon 14. CONCLUSIONS: These results could support the hypothesis for a putative compound heterozygosity pattern in the affected patients. The altered phenotype (goitre and hypothyroidism since birth) seems justifiable in view of the possible inactivating character of this novel mutation in exon 11.

A premature stopcodon in thyroglobulin messenger RNA results in familial goiter and moderate hypothyroidism.

Impaired thyroglobulin (Tg) synthesis is one of the putative causes for dyshormonogenesis of the thyroid gland. This type of hypothyroidism is characterized by intact iodide trapping, normal organification of iodide, and usually low serum Tg levels in relation to high TSH, and when untreated the patients develop goiter. In thyroid tissue from a 13-yr-old patient suspected of a thyroglobulin synthesis defect, the Tg mRNA was studied. The complete coding region of 8307 bp was directly sequenced and revealed a homozygous point mutation: a C886T transition in exon 7. Upon translation this mutation would result in a stopcodon at amino acid position 277, replacing the arginine residue. A Tg cDNA construct containing the mutation was expressed in rabbit reticulocyte lysate resulting in a truncated protein of 30 kDa. Expression in the presence of microsomal membranes resulted in a gel shift of this Tg molecule, indicating glycosylation ability. Two other siblings had a clinical presentation like the index patient, while their parents were unaffected. Additional restriction fragment length polymorphism analysis of the pedigree verified that the homozygous nonsense mutation cosegregated with the clinical phenotype. Clinically, hypothyroidism was not severe in the affected siblings because the truncated Tg glycoprotein was still capable of thyroid hormonogenesis.

Combined ultrasonographic and cytological studies in the diagnosis of thyroid nodules.

Many aspects of thyroid nodule evaluation and management remain controversial. Widespread application of ultrasonography has resulted in frequent discovery of incidental nodules in the general population which has created a management dilemma for physicians. In this paper we have introduced a novel approach for evaluation of solid nodules, using an index derived from ultrasonographic and cytologic studies. Briefly thyroid nodules were classified ultrasonographically into four grades, with increasing score numbers (1-4) as progression to malignantly suspicious lesions was present. Similarly, four grades of a cytologic classification of fine needle biopsy aspirates were introduced with scores of 1-6 (benign to malignant diagnosis). The sum of the ultrasonographic and cytologic scores were the basis of a diagnostic index: benign (2-4), doubtful (5), suspicious (6) and malignant (7-10). Sixty patients with an index equal or higher than 6 were submitted to thyroidectomy and the prevalence of thyroid cancer (n = 46) in the excised nodules was 76.6%. Most series report a 10% to 30% incidence of malignancy in excised nodules with suspicious diagnosis. We concluded that using an index derived from combined ultrasonographic and cytologic studies will result in a better patient selection for surgery.

Structural analysis of the thyrotropin receptor in four patients with congenital hypothyroidism due to thyroid hypoplasia.

Sporadic congenital hypothyroidism is most commonly caused by developmental abnormalities of the thyroid gland. More rarely, it is due to defects in gene products involved in the regulation of the hypothalamic-pituitary-thyroid axis or thyroid hormone synthesis. Loss of function mutations in the thyrotropin (TSH) receptor have been shown to result in resistance to biologically active TSH. In complete resistance to TSH, the thyroid gland is hypoplastic and unable to synthesize and secrete sufficient amounts of thyroid hormones. In partial resistance, referred to as euthyroid hyperthyrotropinemia, the size of the gland and the thyroid hormone levels are normal at the expense of an elevated TSH. Four patients with sporadic congenital hypothyroidism and properly located hypoplastic thyroid glands were included in this study. Serum TSH concentrations were 150 mU/L or higher, serum thyroglobulin levels were within normal limits (6.1 to 8.2 ng/mL; normal range: 2.1 to 32 ng/mL), and thyroid autoantibodies were absent. The coding region of the TSHbeta subunit gene, the TSH receptor gene, and exons 8 and 9 of Gsalpha were analyzed by direct sequencing and found to be normal in all patients. One patient was heterozygous for a G to A transition in the TSHbeta gene resulting in a substitution of alanine by threonine at position -7 of the signal peptide. This substitution was also found in her euthyroid father. In addition, Southern analysis of the TSH receptor gene excluded major structural alterations. These findings support previous reports that indicate that TSH resistance is genetically heterogeneous. In addition to mutations in the TSH receptor or the Gsalpha genes, other genetic defects can lead to an identical phenotype. These observations also suggest that TSH receptor mutations might be a relatively rare cause of congenital thyroid hypoplasia.

Elevated anti-galactosyl antibody titers in endemic goiter.

Anti-Gal is a human polyclonal antibody that constitutes approximately 1% of the circulating immunoglobulin G (IgG), interacts specifically with the mammalian carbohydrate alpha-galactosyl epitope. Furthermore, it was found to mimic in vitro thyrotropin (TSH) effects regarding stimulation for cyclic adenosine monophosphate (cAMP) synthesis, 125I uptake, and cellular proliferation on cultured porcine thyrocytes and on Graves' disease thyrocytes, but not on normal human thyrocytes. As immune activation in sporadic and endemic goiters might play a secondary role in regulating thyrocyte proliferation and function, we evaluated anti-Gal titers in endemic goiter. Serum was obtained from 109 Chagas'-negative patients living in an endemic goiter area of Brazil (Grao Mogol, MG) and 160 controls. The patients were divided into 3 groups, according to their goiter size (World Health Organization [WHO] classification): grade 0 (group 1, n = 24), grade I-II (group 2, n = 41), and grade III-IV (group 3, n = 44). Anti-Gal was assessed by a radioimmunological procedure (results expressed as the percentage of bound radioactivity/total activity [%B/T]). The antibody titer was significantly more elevated in group 1 (mean +/- SEM: 9.27%+/-0.80%), in group 2 (mean +/- SEM: 16.17%+/-0.97%), and in group 3 (20.97%+/-1.30%) than in normal controls (6.46%+/-0.33%). Analysis of the male and female data separately for anti-Gal titer did not substantially alter these results. We concluded that the anti-Gal titer is higher in patients with endemic goiter and presented a possible relationship with the size of goiter. Whether these antibodies contribute to the pathogenesis of the disease needs further clarification.