Polymorphisms of cell cycle control genes influence the development of sporadic medullary thyroid carcinoma.

BACKGROUND: The role of key cell cycle regulation genes such as, CDKN1B, CDKN2A, CDKN2B, and CDKN2C in sporadic medullary thyroid carcinoma (s-MTC) is still largely unknown. METHODS: In order to evaluate the influence of inherited polymorphisms of these genes on the pathogenesis of s-MTC, we used TaqMan SNP genotyping to examine 45 s-MTC patients carefully matched with 98 controls. RESULTS: A multivariate logistic regression analysis demonstrated that CDKN1B and CDKN2A genes were related to s-MTC susceptibility. The rs2066827*GT+GG CDKN1B genotype was more frequent in s-MTC patients (62.22%) than in controls (40.21%), increasing the susceptibility to s-MTC (OR=2.47; 95% CI=1.048-5.833; P=0.038). By contrast, the rs11515*CG+GG of CDKN2A gene was more frequent in the controls (32.65%) than in patients (15.56%), reducing the risk for s-MTC (OR=0.174; 95% CI=0.048-0.627; P=0.0075). A stepwise regression analysis indicated that two genotypes together could explain 11% of the total s-MTC risk. In addition, a relationship was found between disease progression and the presence of alterations in the CDKN1A (rs1801270), CDKN2C (rs12885), and CDKN2B (rs1063192) genes. WT rs1801270 CDKN1A patients presented extrathyroidal tumor extension more frequently (92%) than polymorphic CDKN1A rs1801270 patients (50%; P=0.0376). Patients with the WT CDKN2C gene (rs12885) presented larger tumors (2.9±1.8 cm) than polymorphic patients (1.5±0.7 cm; P=0.0324). On the other hand, patients with the polymorphic CDKN2B gene (rs1063192) presented distant metastases (36.3%; P=0.0261). CONCLUSION: In summary, we demonstrated that CDKN1B and CDKN2A genes are associated with susceptibility, whereas the inherited genetic profile of CDKN1A, CDKN2B, and CDKN2C is associated with aggressive features of tumors. This study suggests that profiling cell cycle genes may help define the risk and characterize s-MTC aggressiveness.

Comprehensive analysis of RET gene should be performed in patients with multiple endocrine neoplasia type 2 (MEN 2) syndrome and no apparent genotype-phenotype correlation: an appraisal of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families.

BACKGROUND: We previously identified a four-generation family with medullary thyroid cancer (MTC) and a germline p.Y791F RET mutation whose cancer lacked a strong genotype-phenotype correlation. The entire gene coding region of the RET gene should be sequenced when genotype-phenotype discrepancies are observed in patients with multiple endocrine neoplasia type 2 (MEN 2), even if a RET hotspot mutation has been identified. METHODS: A new genetic test was performed in the index case of this family with the p.Y791F RET germline mutation. The entire coding region of the RET gene was investigated by direct sequencing of PCR products. Once a mutation was identified, the target exon was sequenced in all at-risk relatives. RESULTS: An additional p.C634Y germline mutation in the RET gene was identified in the reported family. The double mutation occurred in cis and segregated with the phenotype. Through the Brazilian Genetic Screening Program developed at our institution, we additionally report the combination of these two mutations (p.C634Y/p.Y791F) in the RET gene in four other unrelated families. The overall penetrance of MTC and pheochromocytoma in patients with the p.C634Y/p.Y791F mutations was 79% and 13%, respectively. CONCLUSION: Our data emphasises that a comprehensive analysis of the RET gene may reveal multiple germline mutations in MEN 2 patients who exhibit an atypical clinical course of the disease.

Genes of detoxification are important modulators of hereditary medullary thyroid carcinoma risk.

CONTEXT: Different inherited profiles of genes involved in cellular mechanisms of activation and detoxification of carcinogenic products can provide specific protection or determine the risk for cancer. Low-penetrance polymorphic genes related to the biotransformation of environmental toxins have been associated with susceptibility to and the phenotype of, human tumours. OBJECTIVE: To investigate the role of germline inheritance of polymorphisms in CYP1A2*F, CYP1A1 m1, GSTP1, NAT2 and TP53 genes in hereditary medullary thyroid carcinoma (HMTC) patients. DESIGN: This study was developed in University of Campinas (Unicamp). PATIENTS: We studied 132 patients with HMTC, 88 first-degree relatives of HMTC patients and 575 control individuals. MEASUREMENTS: All patients with MTC and their relatives were sequenced for the RET gene and five genes were genotyped using TaqMan(®) system. RESULTS: We observed that the inheritance of CYP1A2*F (OR = 2·10; 95% CI = 1·11-3·97; P = 0·022), GSTP1 (OR = 4·41; 95% CI = 2·47-7·88; P < 0·001) and NAT2 (OR = 2·54; 95% CI = 1·16-5·58; P = 0·020) variants increased the risk for HMTC. In addition, multiple regression analysis showed that the inheritance of GSTP1 polymorphisms was associated with the diagnosis in older patients (B = 8·0229; 95% IC = ± 5·5735; P = 0·0054). Concerning the group of HTMC relatives, CYP1A2*F (OR = 2:40; 95% CI = 1·19-4·86; P = 0·015), CYP1A1 m1 (OR = 2·79; 95% CI = 1:04-7·51; P = 0·042), GSTP1 (OR = 2·86; 95% IC = 1·53-5·32; P < 0·001) and NAT2 (OR = 2·25; 95% IC = 1·20-4·22; P = 0·012) were associated with HMTC risk. CONCLUSIONS: We have demonstrated that the inheritance of specific genes determining the individual response to environmental toxins may contribute to the risk and phenotypic variability that exists in patients with HMTC. Moreover, we identified a group at risk in relatives of HMTC patients.

Evidence that polymorphisms in detoxification genes modulate the susceptibility for sporadic medullary thyroid carcinoma.

AIM: Polymorphic low-penetrance genes have been consistently associated with the susceptibility to a series of human tumors, including differentiated thyroid cancer. METHODS: To determine their role in medullary thyroid cancer (MTC), we used TaqMan SNP method to genotype 47 sporadic MTC (s-MTC) and a control group of 578 healthy individuals for CYP1A2*F, CYP1A1m1, GSTP1, NAT2 and 72TP53. A logistic regression analysis showed that NAT2C/C (OR=3.87; 95% CI=2.11-7.10; P=2.2×10(-5)) and TP53C/C genotypes (OR=3.87; 95% CI=1.78-6.10; P=2.8×10(-4)) inheritance increased the risk of s-MTC. A stepwise regression analysis indicated that TP53C/C genotype contributes with 8.07% of the s-MTC risk. RESULTS: We were unable to identify any relationship between NAT2 and TP53 polymorphisms suggesting they are independent factors of risk to s-MTC. In addition, there was no association between the investigated genes and clinical or pathological features of aggressiveness of the tumors or the outcome of MTC patients. CONCLUSION: In conclusion, we demonstrated that detoxification genes and apoptotic and cell cycle control genes are involved in the susceptibility of s-MTC and may modulate the susceptibility to the disease.

The Ala45Thr polymorphism of NEUROD1 is associated with type 1 diabetes in Brazilian women.

BACKGROUND: NEUROD1 encodes a transcription factor expressed in the endocrine pancreas, and involved in beta-cell development, function and mechanisms of apoptosis. In this study, we investigated the association of a frequent polymorphism in exon 2 of NEUROD1 (G > A; Ala45Thr) with Type 1 diabetes in Brazilian subjects. METHODS: A population/association study comprising 246 unrelated Type 1 diabetic and 275 nondiabetic white Brazilian subjects. The Ala45Thr variant was genotyped by a PCR-RFLP method. RESULTS: The frequency of the Thr allele was significantly higher in patients with Type 1 diabetes than in controls (42.3% vs 35.3%, P=0.02). Stratification by gender showed that homozygosity for the Thr allele was associated with Type 1 diabetes in women with odds ratio of 3.66 (95% C.I. 1.43-10.11, P=0.009) as compared to homozygosity for the Ala allele. This effect was not observed in men. CONCLUSIONS: We found a gender-specific association of the Ala45Thr variant of NEUROD1 with Type 1 diabetes in Brazilian women. Our results suggest that gender as well as ethnicity might modulate the association of NEUROD1 with Type 1 diabetes.

Gene expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors.

To investigate the molecular events involved in the pathogenesis and/or progression of thyroid tumors, we compared the gene expression profiles of three thyroid carcinoma cell lines, which represent major tumor subtypes of thyroid cancer and normal thyroid tissue. Using cDNA array methodology, we investigated the expression of 1807 open reading frame expressed sequence tags (ORESTES), selected from head and neck tumor libraries generated through the Brazilian Human Cancer Project-LICR/FAPESP. We found that 505 transcripts were differentially expressed in the thyroid carcinoma cell lines. Using a more stringent criterion, transcripts underexpressed or overexpressed more than fivefold in 1 of 3 or 3 of 3 carcinoma cell lines, a list of 55 ESTs were detected. Five candidate genes were further validated by quantitative polymerase chain reaction (qPCR) in an independent set of 52 thyroid tumors and 22 matched normal thyroid tissues. DCN was found underexpressed in a high percentage of the follicular thyroid adenomas, follicular thyroid carcinomas, and follicular variant of papillary thyroid carcinomas. DIO1 and DIO2 were underexpressed in nearly all papillary thyroid carcinomas. These genes not only could help to better define a tumor signature for thyroid tumors, but may, in part, also become useful as potential targets for thyroid tumor treatment.

Expression of Smad4 and Smad7 in human thyroid follicular carcinoma cell lines.

Smad proteins have been shown tomediate the signal transduction pathway downstream of the transforming growth factor beta (TGFbeta). TGFbeta induces the phosphorylation of Smad2 and Smad3 which associate with Smad4 and translocate to the nucleus where they regulate gene transcription; besides these stimulatory Smads, the inhibitory Smads, Smad6 and Smad7, oppose signaling by blocking receptors and interrupting the phosphorylation of Smads2/3. The loss of TGFbeta-sensitivity, caused by inactivation of components of TGFbeta signaling, as Smad4, underlies a wide variety of human disorders, including cancer. In addition, the overexpression of the inhibitory Smad7, which prevents the phosphorylation of Smad2/3 and consequently inhibits TGFbeta signaling pathways, was observed in some diseases. In the present study we investigated the expression of Smad4 and Smad7 in thyroid cell lines (NPA papillary carcinoma, WRO follicular carcinoma and ARO anaplastic carcinoma) by RT-PCR and immunocytochemistry. Our results show that Smad4 was expressed in all thyroid cell lines and controls analyzed, differently from other classes of tumors where Smad4 expression was deleted. On the other hand, Smad7 was overexpressed in ARO anaplastic cell line, the most malignant follicular thyroid carcinoma. Our data suggest that the abrogation of the TGFbeta response by Smad7 overexpression may be a mechanism for the tumor aggressiveness observed in undifferentiated thyroid tumors.

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines.

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

Estradiol decreases iodide uptake by rat thyroid follicular FRTL-5 cells

Estradiol has well-known indirect effects on the thyroid. A direct effect of estradiol on thyroid follicular cells, increasing cell growth and reducing the expression of the sodium-iodide symporter gene, has been recently reported. The aim of the present investigation was to study the effect of estradiol on iodide uptake by thyroid follicular cells, using FRTL-5 cells as a model. Estradiol decreased basal iodide uptake by FRTL-5 cells from control levels of 2.490 0.370 to 2.085 0.364 pmol I-/æg DNA at 1 ng/ml (P<0.02), to 1.970 0.302 pmol I-/æg DNA at 10 ng/ml (P<0.003), and to 2.038 0.389 pmol I-/æg DNA at 100 ng/ml (P<0.02). In addition, 4 ng/ml estradiol decreased iodide uptake induced by 0.02 mIU/ml thyrotropin from 8.678 0.408 to 7.312 0.506 pmol I-/æg DNA (P<0.02). A decrease in iodide uptake by thyroid cells caused by estradiol has not been described previously and may have a role in goiter pathogenesis

Comparaçäo entre duas estratégias para a detecçäo precoce do hipotireoidismo congênito / Comparison between two strategies for the precocious detection of congenital hypothyroidism

Objetivo. Comparar em recém-nascidos (RN) duas estratégias diferentes para o rastreamento do hipotiroidismo congênito (HC), a dosagem primária de TSH no sangue colhido do cordao umbilical (método 1) e a dosagem primária de T4 no sangue colhido por punçao de calcanhar no 2 dia de internaçao (método 2). Métodos. Os autores compararam as duas estratégias em 10.000 RN. Dosaram o TSH por método imunofluorimétrico sensível em papel de filtro e o T4 por radioimunoensaio em papel de filtro. A coleta de sangue do calcanhar foi realizada no 2 dia de vida. Resultados. Os dois programas diagnosticaram todos os casos de HC nos RN (4 casos, 1/2.500 RN). O índice de rechamada por coleta inadequada foi nulo no método 1 e de 8,5 por cento (850RN) no método 2. O índice de reconvocaçao para confirmaçao de resultados foi de 0,06 por cento (6RN) no método 1 e 2,25 por cento (225 RN) no método 2; quando este método incluía também a dosagem suplementar de TSH, o índice baixou para 1,63 por cento (163 RN). Conclusao. Os dados dos autores evidenciam a superioridade técnica da coleta de sangue a partir do cordao umbilical em relaçao à punçao de calcanhar, assim como da dosagem primária de TSH em relaçao à de T4, uma vez que apresentam índices muito menores de reconvocaçao.

Valor preditivo da dosagem das iodotironinas na avaliaçäo prognóstica de doentes graves / Predictiove value of the measurement of iodotironines on the prognosis of patients with severe nonthyroidal illness

Os valores das dosagens de T3 e de T4 diferenciaram os pacientes com boa e ma evoluçao durante a internaçao em unidade de terapia intensiva. Objetivo. Procurar indicadores para o prognóstico de doentes graves por meio do estudo seqüencial dos níveis séricos dos hormonios tiroidianos. Métodos. Os autores mediram as iodotironinas (T3, T4 e rT3) por ocasiao da entrada e da alta de 42 pacientes internados em unidade de terapia intensiva. Verificaram, também, os dados referentes à última coleta de outros 17 doentes, transferidos para a UTI após o início do quadro clínico. Resultados. Comparando pacientes que evoluíram bem com aqueles que foram a óbito, observaram, nos primeiros, níveis iniciais normais de T4 em 76 por cento dos casos, valores que se mantiveram estaveis ou se elevaram em 65 por cento dos pacientes durante a internaçao, de tal forma que níveis normais de T4 estavam presentes em 70 por cento dos casos por ocasiao de sua alta. Ao contrario, 56 por cento dos pacientes que evoluíram mal ja apresentavam T4 inicial baixo, que diminuiu ainda mais de 95 por cento dos pacientes durante a internaçao, notando-se valores baixos em 81 por cento dos casos por ocasiao da ultima amostra. Os valores de T3 e T4 em conjunto também diferenciaram os pacientes com boa e m evoluçao. Conclusao. Os autores sugerem que a observaçao dos níveis séricos das iodotironinas pode oferecer importante subsídio na avaliaçao prognóstica de doentes em estado grave.

Enzyme-linked thyroglobulin immunoassay

A one-step enzyme linked sandwich immunoassay using Silicone rods coated with rabbit anti-human thyroglobulin anti-Tg) immunoglobulin G (Fab') conjugated with beta-D-galactosidase was established for the measurement of thyroglobulin in human sera. The volume of serum needed for the assay was 2 mul The sensitivity of the assay was 1.52 amol/tube, corresponding to O.5 ng/ml. The precision was proven by coefficients of variation: intra-assay, 7.0 to 9.1 per cent: inter-assay, 5.3 to 7.4 per cent. The correlation between this EIA and RIA was O.91, p < O.O1.

An enzymeimmunoassay for the determination of thyrotropin (TSH) in drieb blood on filter paper: application to a screening program for detection of congenital hypothyroidism

An enzymeimmunoassay (EIA) for H-TSH (human thyrotropin) in dried blood on filter paper using an anti-H-TSH conjugate with ß-D-galactosidase and tubes coated with an anti-H-TSH was performed fo the screening program for detection of congenital hypothyroidism. The blood volume needed in this assay was 8.7 µl. The precision was evaluated by coefficients of variance within and between assays: 11.86 percent and 14.36 percent for H-TSH levels of 18.5 µU/ml and 35 µU/ml. A good correlation was observed between H-TSH concentration measured by EIA and RIA (r=0.91)