ROBERTO MARQUES RIBEIRO
Projetos de Pesquisa
Unidades Organizacionais
LIM/46 - Laboratório de Parasitologia Médica, Hospital das Clínicas, Faculdade de Medicina
12 resultados
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conferenceObject A NEW INSIGHT ON CFTR ALLELE FREQUENCY IN BRAZIL THROUGH NEXT GENERATION SEQUENCING(2016) NUNES, Luisa Mesquita; RIBEIRO, Roberto; SABINO, Ester; NIEWIANDONSKI, Vivian D. T.; YAMAMOTO, Guilherme Lopes; SILVA FILHO, Luiz Vicente Ribeiro F. da- HCV inter-subtype 1a/1b recombinant detected by complete-genome next-generation sequencing(2016) GASPARETO, Karine Vieira; RIBEIRO, Roberto Marques; MALTA, Fernanda de Mello; GOMES-GOUVEA, Michele Soares; MUTO, Nair Hideko; MENDES-CORREA, Maria Cassia; ROZANSKI, Andrei; CARRILHO, Flair Jose; SABINO, Ester Cerdeira; PINHO, Joao Renato RebelloNext-generation sequencing (NGS) provides a practical approach to HCV complete-genome sequencing, detecting low-frequency variants and allowing analysis of viral genetic diversity (quasispecies) in the sample, and so far, it is very useful for identifying preexisting drug-resistant mutants and emerging escape mutations, as well as detecting viral recombinants containing genomic regions from different genotypes and subtypes. The aim of this study was to analyze the complete coding region of hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) from patients with chronic infection who were direct-acting antiviral (DAA) na < ve. Next-generation sequencing (Ion Torrent (TM) PGM) was used to determine the sequence of the complete coding region of 100 HCV-monoinfected DAA-na < ve patients (51 and 49 subtypes 1a and 1b, respectively). We report the first description of nearly complete HCV genome sequences of subtype 1a and 1b isolates from a large population of Brazilian patients with chronic hepatitis C, and HCV-1a grouped in two different clades. Using this methodology, an inter-subtype 1a/1b recombinant was identified in this study.
- An alternative storage method for characterization of the intestinal microbiota through next generation sequencing(2018) RIBEIRO, Roberto Marques; SOUZA-BASQUEIRA, Marcela de; OLIVEIRA, Lea Campos de; SALLES, Flavia Cristina; PEREIRA, Natalia Bueno; SABINO, Ester CerdeiraGut microbiota has been the subject of various molecular studies mainly due to its importance and wide-ranging relationships with human hosts. However, the storage of fecal samples prior to DNA extraction is critical when characterizing the composition of intestinal microbiota. Therefore, we aimed to understand the effects of different fecal storage methods to characterize intestinal microbiota using Next Generation Sequencing (NGS) as well as to establish an alternative conservation method of bacterial genetic material in these samples using guanidine. Stool samples from 10 healthy volunteers were collected. Each sample was divided into five aliquots: one aliquot was extracted immediately after collection (fresh) and two aliquots were subjected to freezing at -20 degrees C or -80 degrees C and extracted after 48 h. The other two aliquots were stored in guanidine at room temperature or 4 degrees C and extracted after 48 h. The V4 hypervariable regions of the bacterial and archeal 16S rRNA gene were amplified by PCR and sequenced using an Ion Torrent PGM platform for NGS. The data were analyzed using QIIME software. Statistical significance was determined using a non-parametric Kruskal-Wallis test. A total of 11,494,688 reads with acceptable quality were obtained. Unweighted principal coordinate analysis (PCoA) revealed that the samples were clustered based on the host rather than by the storage group. At the phylum and genus levels, we observed statistically significant differences between two genera, Proteobacteria (p=0.013) and Suterella (p=0.004), comparing frozen samples with guanidine-stored samples. Our data suggest that the use of guanidine can preserve bacterial genetic materials as well as freezing, providing additional conveniences.
conferenceObject NEXT GENERATION SEQUENCING OF CFTR IN BRAZILIAN CF CHILDREN(2015) NUNES, L. M.; RIBEIRO, R.; NIEWIADONSKI, V. D.; NISHIMURA, P. Y.; SABINO, E.; SILVA FILHO, L. R. daconferenceObject Molecular Characterization of the Fecal Microbiome in Brazilian Obese NASH patients compared to lean healthy controls.(2015) OLIVEIRA, Claudia P.; STEFANO, Jose Tadeu; RIBEIRO, Roberto M.; DUARTE, Sebastiao M.; RODRIGUES, Livia; CAMPOS, Priscila B.; COSTA, Fernando G.; MAZO, Daniel F.; CARRILHO, Flair J.; SABINO, Ester C.- A new insight into CFTR allele frequency in Brazil through next generation sequencing(2017) NUNES, Luisa M.; RIBEIRO, Roberto; NIEWIADONSKI, Vivian D. T.; SABINO, Ester; YAMAMOTO, Guilherme L.; BERTOLA, Debora R.; GABURO, Nelson; SILVA FILHO, Luiz Vicente R. F. daBackgroundAs of 2013, fewer than 20% of patients in the Brazilian CF Registry had two CFTR mutations identified. The aim of this study was to sequence the coding region of the CFTR in Brazilian CF patients and determine the frequency of mutations in this cohort. MethodsPatients with CF and those with suspected atypical CF or CFTR-related disorders were invited to enroll. Total DNA was extracted from blood samples, quantified, and purified. Library preparation was performed using Ion Xpress Plus gDNA and Amplicon Library preparation kits (Life Technologies), as well as sequencing using the Ion Torrent platform (Life Technologies). ResultsA total of 141 patients were enrolled, and 45 mutations were identified. Among 126 CF patients, we identified mutations in 97.2% of alleles. The three most common mutations were F508del, G542X, and 3120+1G->A. Five novel pathogenic mutations were also identified. ConclusionsNext generation sequencing (NGS) allowed the identification of mutations in most CF alleles and confirmed allelic heterogeneity in our population.
- RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients(2017) DEZAN, Marcia R.; RIBEIRO, Ingrid Helena; OLIVEIRA, Valeria B.; VIEIRA, Juliana B.; GOMES, Francisco C.; FRANCO, Lucas A. M.; VARUZZA, Leonardo; RIBEIRO, Roberto; CHINOCA, Karen Ziza; LEVI, Jose Eduardo; KRIEGER, Jose Eduardo; PEREIRA, Alexandre Costa; GUALANDRO, Sandra F. M.; ROCHA, Vanderson G.; MENDRONE-JUNIOR, Alfredo; SABINO, Ester Cerdeira; DINARDO, Carla LuanaBackground: The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants. Aims: To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support. Methods: Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared. Results: Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion. Conclusion: The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.
- Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil(2016) MAISTRO, Simone; TEIXEIRA, Natalia; ENCINAS, Giselly; KATAYAMA, Maria Lucia Hirata; NIEWIADONSKI, Vivian Dionisio Tavares; CABRAL, Larissa Garcia; RIBEIRO, Roberto Marques; GABURO JUNIOR, Nelson; GOUVEA, Ana Carolina Ribeiro Chaves de; CARRARO, Dirce Maria; SABINO, Ester Cerdeira; DIZ, Maria del Pilar Estevez; CHAMMAS, Roger; BOCK, Geertruida Hendrika de; FOLGUEIRA, Maria Aparecida Azevedo KoikeBackground: Approximately 8-15% epithelial ovarian cancer patients are BRCA1 or BRCA2 germline mutation carriers. Brazilian inhabitants may have peculiar genetic characteristics associated with ethnic diversity, and studies focusing on the entire BRCA1/BRCA2 gene sequencing in Brazilian ovarian cancer patients are still lacking. The aim of this study was to evaluate BRCA1/2 mutations, through entire gene sequencing, in a Brazilian population of women with epithelial ovarian cancer. Methods: In a cross sectional study performed in one reference centre for cancer treatment in Sao Paulo, Brazil, 100 patients diagnosed with epithelial ovarian cancer unselected for family history of breast and/or ovarian cancer were included. The complete coding sequence of BRCA1/2 genes was evaluated through Next-Generation or capillary sequencing. Large deletions were investigated through Multiplex Ligation-dependent Probe Amplification (MLPA). Results: Nineteen pathogenic mutations (BRCA1: n = 17 and BRCA2: n = 2) featuring 14 different mutations, including two large deletions in BRCA1 (exon 1-2 deleted and exon 5-7 deleted) were identified. Three mutations were detected more than once (c.3331_3334delCAAG, c.5266dupC and c.4484G > T). Two novel frameshift mutations were identified, one in BRCA1 (c.961_962delTG) and one in BRCA2 (c.1963_1963delC). BRCA1/2 mutations were seen in 35.5% of the patients with first and/or second-degree relatives with breast and/or ovarian cancer. Nineteen variants of uncertain significance (VUS) were detected (BRCA1: n = 2 and BRCA2: n = 17), including five distinct missense variants (BRCA1: c. 5348 T > C; BRCA2: c.2350A > G, c.3515C > T, c.7534C > T, and c.8351G > A). Conclusions: Among epithelial ovarian cancer patients unselected for family history of cancer, 19% were BRCA1/2 germline mutation carriers. Almost 3/4 of the BRCA mutations, including two large deletions, were detected only once. Our work emphasizes the need of entire gene sequencing and MLPA screening in Brazil.
- Resistance-associated variants in HCV subtypes 1a and 1b detected by Ion Torrent sequencing platform(2016) GASPARETO, Karine V.; RIBEIRO, Roberto M.; MALTA, Fernanda de Mello; GOMES-GOUVEA, Michele S.; MUTO, Nair H.; ROMANO, Camila M.; MENDES-CORREA, Maria C.; CARRILHO, Flair J.; SABINO, Ester C.; PINHO, Joao R. RebelloBackground: As a result of increased understanding of the HCV life cycle, a new generation of drugs known as direct-acting antivirals (DAAs) was developed and is constantly being improved. At baseline, HCV variants resistant to DAA therapy may pre-exist, increasing the likelihood of treatment failure. The aim of this study was to investigate the presence of resistance-associated variants (RAVs) in treatment-naive patients infected with HCV subtypes 1a and 1b. Methods: Next-generation sequencing was used to assess the frequencies of NS3-4A, NS5A and NS5B RAVs in 100 HCV monoinfected DAA-naive patients (HCV-1a: n= 51; HCV-1b: n= 49). Results: Complete HCV sequence information was obtained for most samples. RAVs were detected in the NS3-4A (T54S, V55A, Q80K and R155K), NS5A (Q30H/R, H58P and Y93C/H/N) and NS5B (A421V) regions in 10%, 22% and 8%, respectively, of patients infected with HCV subtype-1a. Among the patients infected with HCV subtype-1b, mutations in the NS3-4A (F43I, T54S, Q80H, D168E and M175L), NS5A (L28M, R30Q, L31M, Q54H, A92T and Y93H) and NS5B (L159F, C316N, A421V and S556G) regions were observed in 12%, 53% and 31% of patients, respectively. Conclusions: High-throughput DNA sequencing allows an easier and more complete analysis of DAA RAVs, including mutations that represent only a minor variant of the whole viral population. RAVs to the three different classes of DAAs were found in our population. The characterization of their profile in the circulating virus is relevant to determine the better treatment option for infected individuals or to guide the implementation of treatment policies.
- Genomic and epidemiological characterisation of a dengue virus outbreak among blood donors in Brazil(2017) FARIA, Nuno R.; COSTA, Antonio Charlys da; LOURENCO, Jose; LOUREIRO, Paula; LOPES, Maria Esther; RIBEIRO, Roberto; ALENCAR, Cecilia Salete; KRAEMER, Moritz U. G.; VILLABONA-ARENAS, Christian J.; WU, Chieh-Hsi; THEZE, Julien; KHAN, Kamran; BRENT, Shannon E.; ROMANO, Camila; DELWART, Eric; CUSTER, Brian; BUSCH, Michael P.; PYBUS, Oliver G.; SABINO, Ester C.Outbreaks caused by Dengue, Zika and Chikungunya viruses can spread rapidly in immunologically naive populations. By analysing 92 newly generated viral genome sequences from blood donors and recipients, we assess the dynamics of dengue virus serotype 4 during the 2012 outbreak in Rio de Janeiro. Phylogenetic analysis indicates that the outbreak was caused by genotype II, although two isolates of genotype I were also detected for the first time in Rio de Janeiro. Evolutionary analysis and modelling estimates are congruent, indicating a reproduction number above 1 between January and June, and at least two thirds of infections being unnoticed. Modelling analysis suggests that viral transmission started in early January, which is consistent with multiple introductions, most likely from the northern states of Brazil, and with an increase in within-country air travel to Rio de Janeiro. The combination of genetic and epidemiological data from blood donor banks may be useful to anticipate epidemic spread of arboviruses.