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- ItemAcesso aberto (Open Access)Análise por sequenciamento paralelo maciço da resistência aos antirretrovirais em pacientes infectados pelo HIV-1 em terapia de resgate(Universidade Federal de São Paulo (UNIFESP), 2017-06-21) Castro, Daniela Funayama de [UNIFESP]; Komninakis, Shirley Vasconcelos [UNIFESP]; Lattes http://lattes.cnpq.br/6529080329230878; http://lattes.cnpq.br/8695544517472892; Universidade Federal de São Paulo (UNIFESP)Antiretroviral Therapy (ART) has improved the quality and life expectancy of HIV-1 infected individuals, reducing both mortality and morbidity. However, along with ART there was the emergence of resistant viral populations selected by the selective pressure of the drugs. These viral populations may lead the patient to virological failure. Although the success rates of ART are high, patients with virologic failure usually require changes in their antiretroviral regimens, which is called a "rescue therapy". Objectives: 1 - Analyze resistance mutations in samples of patients who failed ART and using rescue antiretrovirals: Darunavir, Etravirine and / or Raltegravir; 2 - Compare the mutational profile obtained by the Massive Parallel Sequencing with the one identified by conventional sequencing - Sanger's method. METHODS: Twenty-eight samples with HIV genotyping results with virological failure of antiretroviral drugs were selected between March 2014 and May 2015. The selected samples were from patients with failed ART and one or more antiretroviral drugs from this study. For the analysis of the samples the Massive Parallel Sequencing was done. Results: The most prevalent mutation among all analyzed was I84V in the protease (39.1%). Among the mutations that reduced susceptibility to Darunavir, after I84V, the most common was L33F, accounting for 26%. In mutations that confer reduced susceptibility to Etravirine, mutations K101P, Y181I and Y181V were identified, representing a frequency of 4.3% each. With regard to Raltegravir, a frequency above 21% of the N155H mutation was observed, which gives a significant reduction in the susceptibility to this drug. Mutations with minority prevalence and TAMs M41L, D67N, K70R, L210W, T215F, T215Y and K219Q were also identified. Conclusion: Minority resistance mutations are not currently identified by the method used in clinical practice, such as population sequencing (Sanger's method), whose detection limit is higher than 20-30%. Massive Parallel Sequencing makes it possible to identify mutations with frequency up to 1%. The analysis of minority mutations could, in some situations, help in the choice of a more adequate antiretroviral.The analysis of minority mutations could, in some situations, help in the choice of a more adequate antiretroviral.
- ItemSomente MetadadadosAnalysis of polymorphisms in codons 11, 72 and 248 of tp53 in brazilian women with breast cancer(Funpec-editora, 2016) Almeida, B. C. [UNIFESP]; Kleine, J. P. F. O. [UNIFESP]; Camargo-Kosugi, C. M. [UNIFESP]; Lisboa, M. R. [UNIFESP]; Franca, C. N. [UNIFESP]; Franca, J. P.; Silva, I. D. C. G. [UNIFESP]The association between TP53 gene polymorphisms and breast cancer (BC) in Brazilian women is a controversial topic. In this cross-sectional study, we evaluated the association between clinical pathological variables and three polymorphisms (TP53(star)11, TP53(star)72, and TP53(star)248) in BC patients and controls. Genomic DNA was extracted from the blood cells of 393 participants
- ItemSomente MetadadadosApoptosis- and cell cycle-related genes methylation profile in myeloproliferative neoplasms(Taylor & francis ltd, 2016) Tognon, Raquel; Nunes, Natalia S.; Ambrosio, Luciana; Souto, Elizabeth Xisto; Perobelli, Leila; Simoes, Belinda Pinto; Lima Souza, Mariana Cristina [UNIFESP]; Chauffaille, Maria de Lourdes [UNIFESP]; de Castro, Fabiola Attie
- ItemSomente MetadadadosApparent Mineralocorticoid Excess Syndrome in a Brazilian Boy Caused by the Homozygous Missense Mutation p.R186C in the HSD11B2 Gene(Sbem-soc Brasil Endocrinologia & Metabologia, 2008-11-01) Coeli, Fernanda Borchers; Caldas Ferraz, Lucio Fabio; Lemos-Marini, Sofia H. V. de; Pinto Rigatto, Sumara Zuanazi; Santoro Belangero, Vera Maria; Mello, Maricilda Palandi de [UNIFESP]; Universidade Estadual de Campinas (UNICAMP); Universidade Federal de São Paulo (UNIFESP)The apparent mineralocorticoid excess syndrome (AME) is a rare autosomal recessive disorder due to the deficiency of 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD2). The 11beta-HSD2 enzyme, encoded by HSD11B2 gene, metabolizes active cortisol in cortisone. Mutations on HSD11B2 gene affect the enzyme activity by leading to an excess of cortisol, which causes its inappropriate access to mineralocorticoid receptor. Therefore, cortisol will bind mineralocorticoid receptor. The human HSD11B2 gene maps to chromosome 16q22 and consists of five exons encoding a protein of 405 amino acids. We present here clinical and molecular studies on a Brazilian boy who was born pre-term after an oligodramnious pregnancy. He was diagnosed as having AME at the age of 26 months. His parents are second cousins. Molecular characterization of the HSD11B2 gene revealed the homozygous mutation p.R186C. The patient described here is the second case of HDS11B2 gene mutation reported in Brazilian patients with AME. (Arq Bras Endocrinol Metab 2008; 52/8:1277-1281)
- ItemAcesso aberto (Open Access)Efeito de mutações identificadas em pacientes com Hipotireoidismo Congênito (HC) na atividade da tireoperoxidase (TPO)(Universidade Federal de São Paulo, 2021-02-18) Frutuoso, Vitória Sousa [UNIFESP]; Rubio, Ileana Gabriela Sanchez de [UNIFESP]; http://lattes.cnpq.br/3231635049279767; http://lattes.cnpq.br/6011992632798715O hipotireoidismo congênito (HC) é caracterizado pela deficiência dos hormônios tireoidianos e é a causa mais comum de retardo mental que pode ser prevenida quando diagnosticada e tratada precocemente. O diagnóstico da doença é realizado através da triagem neonatal (popular "teste do pezinho") que possui alta sensibilidade e poucos falsos-negativos. A incidência de HC é de 1:3000 a 1:4000 nascidos vivos e pode variar de acordo com fatores étnicos e consanguinidade. Mutações em diversos genes já foram associadas ao HC, entre elas, no gene da tireoperoxidase (TPO). A TPO é uma enzima localizada na membrana apical da tireoide que desempenha papel central na síntese dos hormônios tireoidianos. Mutações que afetam sua estrutura, sua atividade enzimática ou sua localização são os defeitos genéticos mais frequentes da síntese dos hormônios tireoidianos. Em pesquisas anteriores de nosso grupo, foram identificadas diversas mutações no gene da TPO em pacientes com HC por defeito de síntese, assim este projeto teve como objetivo avaliar o efeito de duas mutações, a p.Cys296Alafs*21 (c. DelT886) e a p.Arg665Trp (c.2083C>T), identificadas em dois irmãos com bócio fetal, na atividade enzimática da TPO in vitro e verificar se poderiam ser associadas ao fenótipo dos pacientes. O plasmídeo com a TPO com a mutação p.Arg665Trp, foi obtido por mutagênese sítio dirigida utilizando o vetor pCDNA 3.1 contendo o cDNA da TPO selvagem e o plasmídeo com a mutação p.Cys296Alafs*21 foi obtido previamente no laboratório. Após padronização da técnica, as células HEK293A foram transfectadas de maneira transiente utilizando o reagente Lipofectamina 3000. Para avaliar a atividade enzimática foi utilizado o reagente Amplex UltraRed, um substrato incolor que na presença de peróxido de hidrogênio (H2O2) e por ação da TPO se converte em resorufina fluorescente e a ativida de das TPOs mutadas foi comparada com a da TPO selvagem. Nas células expressando a TPO selvagem verificamos valores de fluorescência elevados quanto que baixos valores de fluorescência foram observados em aquelas expressando os mutantes de TPO, similares aos da linhagem com o pCDNA3.1, controle negativo. Estes resultados sugerem que ambas mutações diminuem a atividade da TPO, o que explicaria o HC severo dos pacientes sugerindo uma correlação entre o genótipo e fenótipo.
- ItemAcesso aberto (Open Access)Genotipagem da gp41 do Vírus da Imunodeficiência Humana tipo 1 (HIV-1) em indivíduos respondedores e não respondedores ao inibidor de fusão T20(Universidade Federal de São Paulo (UNIFESP), 2009-10-28) Azevedo, Rafael Gonçalves de [UNIFESP]; Komninakis, Shirley Vasconcelos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: the failure to HAART becomes important to development of new drugs that target different steps of the life cycle of HIV-1. The Enfuvirtide (T20) is a synthetic peptide that mimics the HR2 region of gp41 of HIV-1, preventing its fusion and entry into the host cell. The presence of primary resistance mutations to T20 can lead to lack of sustained virologic response (SVR) in people in the HAART failure. Objectives: to genotype the gp41 of HIV-1 subjects considered responders and nonresponders to T20; verify the presence of primary resistance mutations could influence the SVR, SVR status correlate with aspects of viral, immunological and tropism. Methodology: genomic DNA from baseline (before treatment), 6 and 12 months after treatment with T20, was purified using QIAamp DNA Mini kit (Qiagen ®, Valencia, California, USA). All patients received optimal therapy more T20 at the beginning. 506 bp referring to the HR1 and HR2 regions of gp41 were amplified by PCR nested. PCR nested the V3 region to study viral tropism amplified 654 bp. The PCR was purified using Montage ® PCR Centrifugal Filter Devises (Millipore ®). The PCRs of purified V3 and gp41 regions were sequenced in ABI Prism 3130 Genetic Analyzer (Applied Biosystems, CA, USA) with commercial kit BigDye ® Terminator Cycle Sequencing version 3.1 (Applied Biossystems, Foster City. California, USA). Initiators of the second stage of PCR nested were used for sequencing. Results: seven patients had a higher response to T20 for 12 months, 4 for at least 6 months and two did not respond between 6 and 12 months. The average age was 44.92 ± 5.39, and 46.16% female and 53.84% male. Of the 13 patients analyzed, 12 belong to subtype B and 1 to F1. Eight patients had the coreceptor R5 and five the X4. There were no mutations at positions 36 to 45 of HR1 in 12 months. We acquired the N42S, which is responsible for decreased susceptibility to T20. From a total of 59 amino acids analyzed in HR1, we observed 18.64% of change and in HR2 region, 38.88%. In HR1 within 8 months or more compared to the baseline, we found no changes in positions 36 to 45. From a total of 59 amino acids analyzed, we observed 15.25% of change and in HR2, 36.11%. We checked the E137K without the presence of N43D, which caused resistance. We checked the S138T mutation as the primary persistence of 12 months without the N43D and the lack of response to treatment. On the count of CD4 + T cells, we found no statistical difference between the means of different groups (ANOVA - p = 0.1). The viral loads after 6 months were undetectable (<50 copies / mL) in 69.23% of patients. There was a decrease in viral load baseline to 6 months (p = 0.034) and for 6 months and 8 months or more. The use of the drug had significant interference when compared to baseline and 6 months (p = 0.004) and between baseline and 12 months (p = 0.022), but between 6 and 12 months the interference was not significant, showing that after 6 months it remains sustainable. Among the variables, responders and R5 genotype, there were high value of similarity (76.98). There was a grouping between the two samples of responders with X4 genotype with mutations 306, 311 and 320 which characterize it. Discussion: regarding tropism, 38.47% of the patients studied had correceptor X4 after a few years of infection and multiple HAART regimens. This result shows agreement with the literature, which describes an average rate of 50%. The N42S, observed in our study is associated with decreased drug susceptibility. However, only one patient did not respond to treatment, without increase of his immune. Conclusion: the tropism of HIV-1 in the majority of people who responded to the T20 is strongly associated with the R5, which causes rapid progression. The presence of S138T was sufficient for the lack of response to T20, but no association with N43D. It was clearly shown SVR and immune recovery with the T20, but that was not enough to remove these people from the risk of contracting opportunistic diseases. Our study showed the importance of starting the rescue when the count of CD4 + T cells are above 200 cells/mm3.
- ItemAcesso aberto (Open Access)Investigação de rearranjos cromossômicos no gene BRAF em carcinomas pediátricos da tiroide(Universidade Federal de São Paulo (UNIFESP), 2017-06-20) Sisdelli, Luiza de Mello Oliveira [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; Carvalheira, Gianna Maria Griz [UNIFESP]; http://lattes.cnpq.br/2792461820772788; http://lattes.cnpq.br/1384038091754225; http://lattes.cnpq.br/8310429990740701; Universidade Federal de São Paulo (UNIFESP)The incidence of thyroid carcinoma, mainly of the papillary histological subtype (PTC), has increased in most populations, including pediatric patients. Genetic alterations leading to the MAPK pathway activation are highly prevalent in PTC. It has been demonstrated that BRAF V600E mutation is the most prevalent alteration found in sporadic PTC in adult (~40%). However, this event is rarely identified in pediatric PTC. In contrast, chromosomal rearrangements involving BRAF have been identified, mainly in radiation-exposed pediatric PTC. Our group described, for the first time, the presence of AGK-BRAF fusion in sporadic pediatric PTC. This suggests that it might be an alternative mechanismof aberrant BRAF activation in pediatric cases. Thus, the objective of this work was to investigate rearrangements involving BRAF. In the first part of the study, we performed the investigation on 22 pediatric PTC, by FISH break-apart (FISH BA), 5'RACE and sequencing. By FISH BA, it was revealed four positive cases for the BRAF gene breakage, with 10%, 11%, 17% and 36% of the primary tumor cells carrying the rearrangement. Using 5'RACE and sequencing techniques, it was possible to identify the AGK/BRAF rearrangement in one of them. In the second part of the study, we expanded our analysis to 52 pediatric PTC. AGK/BRAF fusion was investigated by RT-PCR and BRAF V600E mutation by PCR and sequencing methodologies. The AGK/BRAF fusion was identified in 27% of the cases and associated with younger ages while BRAF V600E mutation was found in 8% and associated with older ages. PTC with BRAF alterations tends to have a more aggressive biological behavior.
- ItemSomente MetadadadosProcessing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity(Elsevier Science Bv, 2017) Gilio, Joyce M. [UNIFESP]; Marcondes, Marcelo F. [UNIFESP]; Ferrari, Debora; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Oliveira, Vitor [UNIFESP]; Machado, Mauricio F. M. [UNIFESP]Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active
- ItemSomente MetadadadosStudy of hTERT and Histone 3 Mutations in Medulloblastoma(Karger, 2017) Viana-Pereira, Marta; Almeida, Gisele Caravina; Stávale, João Norberto [UNIFESP]; Malheiro, Susana [UNIFESP]; Clara, Carlos; Lobo, Patricia; Pimentel, Jose; Reis, Rui ManuelHotspot activating mutations of the telomerase reverse transcriptase (hTERT) promoter region were recently described in several tumor types. These mutations lead to enhanced expression of telomerase, being responsible for telomere maintenance and allowing continuous cell division. Additionally, there are alternative telomere maintenance mechanisms, associated with histone H3 mutations, responsible for disrupting the histone code and affecting the regulation of transcription. Here, we investigated the clinical relevance of these mechanistically related molecules in nnedulloblastoma. Sixty-nine medulloblastomas, formalin fixed and paraffin embedded, from a cohort of patients aged 1.5-70 years, were used to investigate the hotspot mutations of the hTERT promoter region, i.e. H3F3A and HIST1H3B, using Sanger sequencing. We successfully sequenced hTERT in all 69 medulloblastoma samples and identified a total of 19 mutated cases (27.5%). c.-124:G>A and c.-146:G>A mutations were detected, respectively, in 16 and 3 samples. Similar to previous reports, hTERT mutations were more frequent in older patients (p < 0.0001), being found only in 5 patients <20 years of age. In addition, hTERT-mutated tumors were more frequently recurrent (p = 0.026) and hTERT mutations were significantly enriched in tumors located in the right cerebellar hemisphere (p = 0.039). No mutations were found on the H3F3A or HIST1H3B genes. hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in nnedulloblastoma. (C) 2016 S. Karger AG, Basel
- ItemSomente MetadadadosValor diagnóstico de mutações BRAF, Nraseptertem punções aspirativas indeterminadas de nódulos tiroidianos(Universidade Federal de São Paulo (UNIFESP), 2021) Alves, Thalita Goulart [UNIFESP]; Martins, Joao Roberto Maciel [UNIFESP]; Universidade Federal de São PauloIntroduction: Thyroid nodules are common events and present in 37-68% of the population when imaging tests are used for diagnosis, such as US, computed tomography (CT), nuclear magnetic resonance (MRI) and PET-CT scan. However, only 5 to 15% correspond to malignant neoplasms, and this proportion varies according to gender, age, family history, among others. For this reason, clinical investigation aims to distinguish between benign and malignant nodules in order to avoid unnecessary surgeries. The best diagnostic test is fine needle aspiration biopsy (FNAB), which provides results based on the Bethesda cytopathological classification. However, in 20-30% of cases the results are undetermined or suspicious, and of these 6-75% are malignant, representing an important clinical challenge. Approximately 90% of all thyroid cancers (TC) present a well-known genetic event in their etiopathogenesis. Objective: Thus, the main objective was to evaluate the role of molecular testing of a panel of frequently mutated CT genes (BRAF, NRAS and pTERT), in a series of indeterminate FNABs, to investigate their possible diagnostic and prognostic role. Patients and methods: We obtained 240 cases of patients with undetermined cytological results. DNA was extracted from FNAB slides, needle wash and ThinPrep, and PCR was performed to amplify the genes of interest. After verification of the reaction products by agarose gel electrophoresis, Sanger sequencing was performed for mutational analysis of BRAF, NRAS and pTERT genes. Results: BRAF and pTERT mutations were found in 15% of the FNABs, whose histopathology confirmed malignancy. As expected, NRAS mutations (5% of FNABs) were found in both benign and malignant nodules. The mutational analysis showed high specificity (86%) and good accuracy (71%), but low sensitivity (28%). The positive predictive value (PPV) was 42% and the negative predictive value (NPV) was 77%, indicating that, in general, genetic profiling has great power to exclude malignancy when no mutations are found. Conclusion: Our results suggest that the gene panel may be a useful additional diagnostic/prognostic tool in the investigation of indeterminate FNABsand contribute to avoid unnecessary surgery due to a high negative predictive value.