FRA-1 protein overexpression is a feature of hyperplastic and neoplastic breast disorders

BACKGROUND: Fos-related antigen 1 (FRA-1) is an immediate early gene encoding a member of AP-1 family of transcription factors involved in cell proliferation, differentiation, apoptosis, and other biological processes. fra-1 gene overexpression has an important role in the process of cellular transformation, and our previous studies suggest FRA-1 protein detection as a useful tool for the diagnosis of thyroid neoplasias. Here we investigate the expression of the FRA-1 protein in benign and malignant breast tissues by immunohistochemistry, Western blot, RT-PCR and qPCR analysis, to evaluate its possible help in the diagnosis and prognosis of breast neoplastic diseases. METHODS: We investigate the expression of the FRA-1 protein in 70 breast carcinomas and 30 benign breast diseases by immunohistochemistry, Western blot, RT-PCR and qPCR analysis. RESULTS: FRA-1 protein was present in all of the carcinoma samples with an intense staining in the nucleus. Positive staining was also found in most of fibroadenomas, but in this case the staining was present both in the nucleus and cytoplasm, and the number of positive cells was lower than in carcinomas. Similar results were obtained from the analysis of breast hyperplasias, with no differences in FRA-1 expression level between typical and atypical breast lesions; however the FRA-1 protein localization is mainly nuclear in the atypical hyperplasias. In situ breast carcinomas showed a pattern of FRA-1 protein expression very similar to that observed in atypical hyperplasias. Conversely, no FRA-1 protein was detectable in 6 normal breast tissue samples used as controls. RT-PCR and qPCR analysis confirmed these results. Similar results were obtained analysing FRA-1 expression in fine needle aspiration biopsy (FNAB) samples. CONCLUSION: The data shown here suggest that FRA-1 expression, including its intracellular localization, may be considered a useful marker for hyperplastic and neoplastic proliferative breast disorders

Caracterização molecular na deficiencia de antitrombina

Orientador: Joyce M. Anicchino-BizzacchiDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A At é o mais importante inibidor da trombina, além de inativar todos os outros fatores ativos da coagulação, com uma menor atividade sobre o fator VII ativado. O grau de inibição da trombina, do fator X ativado e do fator IX ativado é muito lenta. Esta inibição é dramaticamente aumentada na presença de heparina, tornando-se virtualmente instantânea, tanto in vivo quanto in vitro. A deficiência de AT está associada a fenômenos tromboembólicos em humanos. De fato, a deficiência de A T foi a primeira causa descrita de trombofilia hereditária. A importância da manutenção de níveis normais de A T na circulação pode ser avaliada pelo fato de que, indivíduos heterozigotos para deficiência de A T podem apresentar quadro de tromboembolismo de repetição. A freqüência de deficiência congênita de A T não é incomum, estando por volta de 1/5000 e 1/2000 na população geral. Contudo, dependendo do tipo de deficiência, esta freqüência pode ser de 1/500. A incidência de deficiência de AT em pacientes com trombofilia é de aproximadamente 3%. O modo de herança é autossômico dominante, e os heterozigotos apresentam concentração plasmática de A T ao redor de 40 - 70% do normal. A deficiência de A T foi classificada em 2 tipos: Til?o 1 é caracterizada pela redução da atividade e dos níveis de antígeno; Tipo II é caracterizada pela alteração da atividade associada a níveis normais de antígeno. O tipo II se subdivide em 3: Subtipo RS - anormalidade do sítio ativo, Subtipo HBS - anormalidade do sítio de ligação com a heparina, Subtipo PE anormalidade do sítio ativo e do sítio de ligação com a heparina. O gene da ATestá localizado no cromossomo 1, na região 1q23~1q25 e inclui 7 exons, numa extensão de 13.480 pb. De acordo com o "database" de mutações no gene da AT, publicado em 1997 por LANE et aI., foram descritas 127 mutações, sendo que 60 das substituições nucleotídicas encontradas nos propósitos eram mutações "missense", 8 eram mutações" nonsense" , e 7 eram mutações em sítio de clivagem. Dentre estas mutações 16 ocorreram em dinucleotídeos CpG. Foram descritas 12 inserções e 40 deleções. Também foram descritos um total de 13 polimorfismos. Neste trabalho foram estudados cinco pacientes com deficiência de A T que apresentaram trombose espontânea (3 pacientes) ou associada ao uso de anticoncepcional oral (2 pacientes). O estudo familiar também foi realizado, contribuindo para uma melhor identificação da deficiência hereditária de A T. O rastreamento de alterações moleculares foi realizado pelo SSCP e CSGE, que se mostraram complementares. Com o uso do método de SSCP detectou 4 padrões anormais: um padrão anormal referente a dois polimorfismos no exon 4 (7596G~A e 7626G~A) em 2 pacientes; outro padrão referente à mutação +1 IVS5 G~A em 1 paciente; outro referente à mutação 13328 G~A no exon 6, com a troca do aminoácido alanina por treonina na posição 404, em um paciente, e outro no exon 3A, provavelmente decorrente da inserção ou deleção na posição 5379. O CSGE revelou 4 padrões anormais: um padrão referente a um polimorfismo no IVS5 - 9893 G~C , em 2 pacientes; outro padrão correspondente à mesma mutação também determinada pelo SSCP, +1 IVS5 G~A em 1 paciente; outro padrão referente à mutação +5 IVS1 G~A em 1 paciente, e outro padrão anormal, também rastreado pelo SSCP, localizado no exon 3A, provavelmente decorrente da inserção ou deleção na posição 5379. Todos os polimorfismos identificados já foram descritos anteriormente, e não parecem ter relação com a deficiência de antitrombina. A mutação 13328 G~A no exon 6 já havia sido descrita em 3 outras famUias com deficiência de antitrombina, e parece interferir com o sítio de ligação com a heparina e com o sítio reativo. Em nosso paciente esta foi uma mutação de novo, confirmada por estudo de paternidade. As outras 2 mutações ainda não foram descritas e ocorreram em sítios de clivagem do RNA, com troca de um G por um A nas posições +1 e +5, nos introns 5 e 1, respectivamente. Estas regiões são altamente conservadas no gene, conhecidas com regiões consenso. Mutações que ocorrem nesse região, em outros genes são responsáveis por outras doenças, como a hemofilia A, hemofilia B, deficiência de proteína C e S, e ~-talassemia. Além disso, o estudo familiar mostrou que houve segregação da mutação com a deficiência. Apesar" da deficiência de antitrombina em heterozigose apresentar um risco relativo de trombose de 5 vezes, este estudo mostrou que muitos familiares são portadores assintomáticos, o que favorece a hipótese de que outros fatores ainda não identificados devem contribuir para a clínica de trombose. Outros fatores de risco para trombofilia como deficiência de proteína C, proteína S, e mutações no gene da protrombina, não estavam presentes em nenhum dos pacientes. As mutações no gene do fator V (fator V Leiden) e da MTHFR estavam presentes apenas no paciente com a mutação de novoAbstract: AT is the most important thrombin inhibitor, also inactivate all the other coagulation active factors, witha little activity on activated factor VII. The inhibition is dramatically increased in the presence of heparin, becoming virtually instantaneous, even in vivo as in vitro. AT deficiency is associated to thromboembolic phenomenons in humans. In fact, A T deficiency was the first described cause of hereditary thrombophilia. The importance of maintenance of normal At levels in the circulation can be evaluated by the fact that, heterozygous A T deficient individuais can present recurrent thromboembolism. The frequency of congenital AT deficiency is not uncommon, being about 1/2000 to 1/5000 in the general population. H oweve r, depending on the deficiency type, this frequency can be of 1/500. The incidence of AT deficiency in patients with thrombophilia is approximately 3%. The inheritance way is autosomic dominant, and the plasmatic A T concentration in heterozygous is about 40 to 70% of the normal. A T deficiency was classified in 2 types: Type 1 is characterized by the reduction of the activity and of the levels of antigen; Type li is characterized by the alteration of the activity associated to normal levels of antigen. The type 11 is subdivided in 3: Subtype RS - abnormality of the active site, Subtype HBS abnormality of the heparin binding site, Subtype PE - abnormality of the active sits,and of the heparin binding site. A T gene is located on chromosome 1, in 1 q23~ 1 q25 and includes 7 exons, in an extension of 13.480 pb. In agreement with the database of mutations in the A T gene, published in 1997 by LANE et aI., 127 mutations were described, and 60 of the nucleotides substitutions were missense mutations, 8 were nonsense mutations, and 7 were splice site mutations. Sixteen mutations occured at dinucleotides CpG. Twelve insertions and 40 deletions were also described. A total of 13 polymorphisms were identified. We studied five patients with A T deficiency who presented spontaneous thrombosis (3 patients) or thrombosis associated to oral contraceptive use (2 patients). The family study was also accomplished, contributing to a better identification of the hereditary A T deficiency. The molecular alterations were initially identified by SSCP and CSGE, and the results showed that they were complementary methods. Using SSCP method, it was possible to detect 4 abnormal patterns: anabnormal pattern regarding two polymorphisms in the exon 4 (7596G-?>A and 7626G-+A) in 2 patients; another related to the mutation +1 IVS5 G-?>A in 1 patient; and another regarding mutation 13328 G-?>A in the exon 6, with the change of the amino acid alanine for threonine in the position 404, in one patient. The last abnormal pattern is located in the exon 3A, and probably is related to a insertion or deletion in position 5379. The CSGE revealed 4 abnormal patterns: a pattern regarding a polymorphism in the IVS5 - 9893 G-?>C, in 2 patients; another corresponding to the same mutation also determined by SSCP, +1 IVS5 G-?>A in 1 patient; and another pattern regarding the mutation +5 IVS1 G-?>A in 1 patient. The last one, also identified by SSCP, located in the exon 3A, and probably is related to a insertion or deletion in position 5379. Ali the identified polymorphism was already described previously, and no relationship was found with AT deficiency. The mutation 13328 G-?>A in the exon 6 had already been described in 3 other families with antithrombin deficiency, and it seems to interfere with the heparin binding site and the reactive site. In our patient it was a de novo mutation confirmed by paternity study. The other two mutations were not described yet, and were located at splice ~rtes, with the change of a G to A, in the +1 and +5 positions, in the introns 5 and 1, respectively. These consensus areas are highly conserved in the gene. Mutations localized in other genes at the same areas are responsible for other diseases, like hemophilia A, hemophilia B, protein C or protein S deficiency and ~-talassemia. Besides, the family study showed that there was segregation of the mutation with the deficiency. Heterozygous AT deficiency have a five-fold increased risk for venous thrombosis. In this study a number of relatives are asymptomatic carriers, that favors the hypothesis that thrombosis is a multifactorial disease. The other alterations associated with a risk for thrombosis, like protein C and protein S deficiency, and prothrombin (20210 G-?>A) gene mutation werê not present in any patient. Mutation in the gene of factor V (factor V Leiden) and MTHFR were present only in the patient with AT de novo mutationMestradoMestre em Farmacologi

Gene expression profiles in thyroid reveal thal DCN, DI01 and DI02 are under expressed in thyroid carcinomas

BV UNIFESP: Teses e dissertaçõe

A mutation in the KCNE3 potassium channel gene is associated with susceptibility to thyrotoxic hypokalemic periodic paralysis

Hypokalemic Periodic Paralyses comprise diverse diseases characterized by acute and reversible attacks of severe muscle weakness, associated with low serum potassium. the most common causes are Familial Hypokalemic Periodic Paralysis (FHypoKPP), an autosomal dominant disease, and Thyrotoxic Hypokalemic Periodic Paralysis (THypoKPP), secondary to thyrotoxicosis. Symptoms of paralysis are similar in both diseases, distinguished by thyrotoxicosis present in THypoKPP. FHypoKPP is caused by mutations in ionic channel genes calcium (CACNIAS), sodium (SCN4A) and potassium (KCNE3). Since both diseases are similar, we tested the hypothesis that THypoKPP could carry the same mutations described in FHypoKPP, being the paralysis a genetically conditioned complication of thyrotoxicosis. in 15 patients with THypoKPP, using target-exon PCR, CSGE screening, and direct sequencing, we excluded known mutations in CACNIAS and SCN4A genes. On the other hand, we were able to identify the R83H mutation in the KCNE3 gene in one sporadic case of THypoKPP, a man who had been asymptomatic until developing thyrotoxicosis caused by Graves' disease; we confirmed the disease-causing mutation in 2 of 3 descendants. R83H was recently found in two FHypoKPP unrelated families, in which the mutant decreased outward potassium flux, resulting in a more positive resting membrane potential. We, therefore, identified the first genetic defect in THypoKPP, a mutation in the KCNE3 gene

Familial combined pituitary hormone deficiency due to a novel mutation R99Q in the hot spot region of prophet of Pit-1 presenting as constitutional growth delay

Combined pituitary hormone deficiency (CPHD) is characterized by impaired production of GH and one or more of the other anterior pituitary hormones. Prophet of Pit-1 (PROP-1), one of the pituitary specific homeodomain transcription factors, is involved in the differentiation of the anterior pituitary cells (somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs), and PROP-1 gene mutations may interfere with the development of these cells, resulting in CPHD.We performed molecular analyses of the PROP-1 gene in two siblings, born to consanguineous parents, who presented with short stature. the index patient, a boy, was initially diagnosed with constitutional growth delay based on familial short stature, low parental target height, normal GH secretion, and imaging of the pituitary gland. On follow-up, auxological data and pubertal delay prompted a thorough reevaluation, which documented GH, TSH, and gonadotropin deficiencies. Direct sequencing of the PROP-1 gene revealed a novel homozygous transition 296G-->A in exon 2 in the two affected siblings. the mutation substitutes a highly conserved arginine by a glutamine at codon 99 (R99Q) in the second helix of the DNA-binding domain of the PROP-1 protein. Compared with wild-type PROP-1, R99Q displays a significant decrease in DNA binding on a paired box response element (PRDQ9) and trans-activation of a luciferase reporter gene.The findings emphasize the importance of repeated evaluations and illustrate that patients with CPHD associated with PROP-1 mutations present with a phenotypic spectrum, suggesting that the consequences of distinct PROP-1 mutations may be diverse and/or that additional factors, such as modifier genes, may have an impact on their expressivity.Universidade Federal de São Paulo, Escola Paulista Med, Dept Med, Div Endocrinol, BR-04039002 São Paulo, SP, BrazilNorthwestern Univ, Feinberg Sch Med, Div Endocrinol Metab & Mol Med, Chicago, IL 60611 USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Med, Div Endocrinol, BR-04039002 São Paulo, SP, BrazilWeb of Scienc