Navegando por Palavras-chave "Sequencing"

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    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.
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    Analysis of KIAA1549-BRAF fusion gene expression and IDH1/IDH2 mutations in low grade pediatric astrocytomas
    (Springer, 2014-04-01) Rampazzo, Gabriela Cruz [UNIFESP]; Oliveira, Indhira Dias [UNIFESP]; Moraes, Lais [UNIFESP]; Paniago, Mario Del Giudice [UNIFESP]; Seixas Alves, Maria Teresa de [UNIFESP]; Capellano, Andrea Maria [UNIFESP]; Silva, Nasjla Saba da [UNIFESP]; Cavalheiro, Sergio [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; Toledo, Silvia Regina Caminada de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Low-grade astrocytomas comprise about 30 % of the central nervous system tumors in children. Several investigations have searched a correlation between the BRAF gene fusions alterations and mutations at IDH1 and IDH2 genes in low grade pediatric astrocytomas. This study identified the expression of KIAA1549-BRAF fusion gene and BRAF V600E mutation, mutations at exon 4 of the IDH1 and IDH2 genes in samples of pilocytic astrocytomas (PA) and grade-II astrocytomas (A-II) pediatric patients. the correlation between these alterations and the clinical profile of the patients was also evaluated. Eighty-two samples of low-grade astrocytomas (65 PA and 17 A-II) were analyzed by PCR and sequencing for each of the targets identified. We identified the KIAA1549-BRAF fusion transcript in 45 % of the samples. BRAF V600E and BRAFins598T mutations were detected in 7 and 1 % of the samples, respectively. Mutations in the R132/R172 residues of the IDH1/IDH2 genes were detected in only two samples, and the G105G polymorphism (rs11554137:C > T) was identified in ten patients. Additionally, we observed two mutations out of the usual hotspots at IDH1 and IDH2 genes. We observed a smaller frequency of mutations in IDHs genes than previously described, but since the prior studies were composed of adult or mixed (adults and children) samples, we believe that our results represent a relevant contribution to the growing knowledge in low grade childhood astrocytomas.
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    Caracterização molecular e interpretação clínica de rearranjos cromossômicos equilibrados associados com alterações fenotípicas
    (Universidade Federal de São Paulo (UNIFESP), 2018-02-02) Oliveira, Mariana Moyses [UNIFESP]; Melaragno, Maria Isabel de Souza Aranha [UNIFESP]; http://lattes.cnpq.br/0678071850781758; Universidade Federal de São Paulo (UNIFESP)
    O mapeamento preciso dos pontos de quebra de rearranjos cromossômicos equilibrados associados a alterações fenotípicas pode levar ao reconhecimento de novos loci genômicos associados a doenças e ao melhor entendimento de mecanismos de formação de alterações cromossômicas. O estudo de mulheres com alterações fenotípicas e translocações equilibradas envolvendo o cromossomo X apresenta um potencial ainda maior de contribuir para o entendimento do genoma humano, uma vez que oferece a oportunidade de se avaliar a correlação genótipo-fenótipo levando em consideração a inativação preferencial do cromossomo X normal. O presente trabalho teve como objetivo estudar os mecanismos patogênicos responsáveis pelas alterações clínicas de pacientes com translocações equilibradas, buscando genes rompidos pelos pontos de quebra ou próximos aos pontos de junção, candidatos para os seus fenótipos. Adicionalmente, objetivou-se avaliar a natureza das sequências rompidas pelas quebras cromossômicas e as cicatrizes deixadas nos pontos de junção. Primeiramente, 11 pacientes com translocações equilibradas e fenótipo alterado tiveram seus pontos de quebra mapeados por metodologias de citogenética clássica e molecular associadas com sequenciamento de nova geração. Em seguida, os pontos de junção dos rearranjos foram sequenciados, resultando numa resolução de par de base. Foi observada a ruptura de nove genes nos pontos de quebra de sete pacientes e, em dois casos, foram detectadas fusões gênicas. Sete desses genes tiveram a sua expressão alterada e dois deles puderam ser definidos como a causa do fenótipo dos pacientes. Quatro translocações não romperam genes, sugerindo outros mecanismos patogênicos mais complexos nesses casos. Quatro genes foram considerados como potencialmente afetados por efeito de posição e foi demonstrada a alteração da expressão de um deles. Verificou-se que a junção das extremidades não homólogas foi o mecanismo de formação inferido com maior frequência e que 65% das regiões de pontos de quebra mapeadas afetavam elementos de repetição. Dentre esses casos de rearranjos equilibrados, descrevemos uma paciente com translocação X-autossomo equilibrada, com ruptura da região promotora do gene AMMECR1 no ponto de quebra do cromossomo X, o que ocasionou a ausência de cópias funcionais desse gene. AMMECR1 é um gene de função desconhecida e a consequência clínica de sua perda de função ainda não havia sido descrita. A partir dessa paciente com translocação, e de outros quatro indivíduos do sexo masculino com variantes deletérias envolvendo o AMMECR1, foi observado que a perda de função deste gene acarreta baixa estatura, atraso do desenvolvimento neuropsicomotor, alterações ósseas e cardíacas e perda de audição. A modelagem in vivo por meio de knockdown do ortólogo do AMMECR1 em zebrafish confirmou parte dessa correlação genótipo-fenótipo. Em humanos, a superexpressão do AMMECR1L, um parálogo localizado no cromossomo 2, corresponde a um possível mecanismo de compensação da perda de função do AMMECR1. A proteína AMMECR1 é extremamente conservada ao longo da evolução e apresenta dobras (folds) que potencialmente interagem com ácido nucleico. Além disso, neste estudo foi observado que a proteína AMMECR1 é expressa no núcleo da célula e que a rede de interação desse gene é enriquecida para genes que participam do controle do ciclo celular, sugerindo que a AMMECR1 também exerça um papel nesse processo biológico. Neste estudo, também foi demonstrado que Ammecr1 e Ammecr1l são expressos em tecidos embrionários de camundongo que originam os tipos celulares afetados pelo fenótipo dos pacientes com a perda de função do gene. Para que fosse realizada a quantificação precisa da expressão gênica por RT-qPCR, foi necessário o estudo prévio da estabilidade da expressão de genes de referência comumente usados como controle interno metodológico. De maneira geral, a abordagem metodológica aplicada neste trabalho permitiu a identificação de genes candidatos para o fenótipo dos pacientes com translocações equilibradas, a exploração dos possíveis impactos dos rearranjos na organização da cromatina e a análise de seus mecanismos de formação. Em especial, a descrição das consequências fenotípicas da perda de função do gene AMMECR1 auxiliará no diagnóstico de outros pacientes com doenças genéticas, enquanto que o estudo da função do AMMECR1 levará ao melhor entendimento do controle do ciclo celular. Este trabalho reforça a relevância de se mapear os pontos de quebra de rearranjos cromossômicos equilibrados com alta resolução para a busca de novos genes candidatos para doenças humanas.
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    Frequency of human rhinovirus species in outpatient children with acute respiratory infections at primary care level in Brazil
    (Lippincott Williams & Wilkins, 2011-07-01) Moreira, Luciana Peniche [UNIFESP]; Kamikawa, Janete [UNIFESP]; Watanabe, Aripuanã Sakurada Aranha [UNIFESP]; Carraro, Emerson [UNIFESP]; Leal, Elcio; Arruda, Eurico; Granato, Celso Francisco Hernandes [UNIFESP]; Bellei, Nancy Cristina Junqueira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fed Univ Para; Universidade de São Paulo (USP)
    This study assessed the occurrence of human rhinovirus (HRV) species in outpatient children attending day-care in São Paulo, Brazil. HRV reverse transcriptase polymerase chain reaction and amplicon sequencing were done in 120 samples collected in 2008. HRV was detected in 27.5% of samples. HRV C was detected in 60.7% of wheezers, a frequency not different from that observed in nonwheezers (69.6%).
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    Hereditary hemochromatosis: Mutations in genes involved in iron homeostasis in Brazilian patients
    (Elsevier B.V., 2011-04-15) Santos, Paulo C. J. L.; Cancado, Rodolfo D.; Pereira, Alexandre C.; Schettert, Isolmar T.; Soares, Renata A. G.; Pagliusi, Regina A.; Hirata, Rosario D. C.; Hirata, Mario H.; Teixeira, Ana C.; Figueiredo, Maria Stella [UNIFESP]; Chiattone, Carlos S.; Krieger, Jose E.; Guerra-Shinohara, Elvira M.; Universidade de São Paulo (USP); Santa Casa Med Sch; Novo Atibaia Hosp; Adolfo Lutz Inst; Universidade Federal de São Paulo (UNIFESP)
    Background: p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1). the low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non-HFE genes may be linked to HH phenotype.Aim: To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population.Materials and methods: Fifty-one patients with primary iron overload (transferrin saturation >= 50% in females and >= 60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed.Results: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. the most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). in addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found.Conclusions: the HE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. the HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G2045 mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated. (C) 2011 Elsevier Inc. All rights reserved.
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    HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?
    (Elsevier B.V., 2010-12-15) Santos, Paulo C. J. L.; Pereira, Alexandre C.; Cancado, Rodolfo D.; Schettert, Isolmar T.; Sobreira, Tiago J. P.; Oliveira, Paulo S. L.; Hirata, Rosario D. C.; Hirata, Mario H.; Figueiredo, Maria Stella [UNIFESP]; Chiattone, Carlos S.; Krieger, Jose E.; Guerra-Shinohara, Elvira M.; Universidade de São Paulo (USP); Santa Casa Med Sch; Novo Atibaia Hosp; Universidade Federal de São Paulo (UNIFESP)
    Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. the low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. the main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation >50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. the effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. the most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. in silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and beta 2-microglobulin ((beta 2M) in the same way the p.C282Y mutation does compared with the native HFE protein. in conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. the novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. (C) 2010 Elsevier Inc. All rights reserved.
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    Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies
    (Elsevier B.V., 2007-11-01) Ferreira, A. H. P.; Cristofoletti, P. T.; Lorenzini, D. M.; Guerra, L. O.; Paiva, P. B.; Briones, M. R. S.; Terra, W. R.; Ferreira, C.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. for this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. the predicted midgut microvillar proteins from T molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, a-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase.. serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypepticlase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. the novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins. (c) 2007 Elsevier B.V. All rights reserved.
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    Phylogenetic classification and clinical aspects of a new putative Deltapapillomavirus associated with skin lesions in cattle
    (Funpec-editora, 2014-01-01) Melo, Tatiana C. [UNIFESP]; Carvalho, Rodrigo Franco de; Mazzucchelli-de-Souza, Jacqueline; Diniz, Nara Soares Pessoa; Vasconcelos, S.; Assaf, Suely Lucia Muro Rais; Araldi, Rodrigo Pinheiro; Ruiz, Renato de Mello; Kerkis, Irina [UNIFESP]; Beçak, Willy; Stocco, Rita de Cassia; Inst Butantan; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Universidade Federal de Pernambuco (UFPE); Univ Fed Integracao Latino Amer
    Bovine papillomaviruses (BPVs) are recognized as causal agents of benign and malignant tumors in cattle. Thirteen types of BPVs have already been described and classified into 3 distinct genera. Divergences in the nucleotide sequence of the L1 gene are used to identify new viral types through the employment of PCR assays with degenerated primers. in the present study, a method for identifying BPVs based on PCR-RFLP and DNA sequencing allowed the identification of a new putative Deltapapillomavirus, designated JN/3SP (JQ280500.1). the analysis of the L1 gene showed that this strain was most closely related to the BPVs -1, -2, -13, and OaPV1 (71-73% genetic similarity). in this study, we describe the detection of this new putative Deltapapillomavirus type and verify its phylogenetic position within the genus.