Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil

Abstract\ud \ud Background\ud 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.\ud \ud \ud Methods\ud In a cross sectional study performed in one reference centre for cancer treatment in São 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).\ud \ud \ud Results\ud 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).\ud \ud \ud Conclusions\ud Among epithelial ovarian cancer patients unselected for family history of cancer, 19% were BRCA1/2 germline mutation carriers. Almost ¾ 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.Geertruida Hendrika de Bock received a grant of Coordenação de Aperfeiçoamento\ud de Pessoal de Nível Superior (CAPES), Programa Pesquisador Visitante Especial (PVE\ud #029/2012), Simone Maistro received a postdoctoral scholarship by Coordenação\ud de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, PVE #029/2012), Giselly\ud Encinas received a PhD scholarship grant by São Paulo Research Foundation\ud (FAPESP, #2011/09572-1) and Natalia Teixeira received a scholarship grant by São\ud Paulo Research Foundation (FAPESP, #2012/05754-1).\ud This work was in part supported by a grant from Diagnósticos da América\ud -DASA and by a grant from NAP-Biobanco/USP

Gene expression study in Chagas disease patients receiving benznidazole treatment: the search for a biomarker of cure

Mais de 100 anos após a doença de Chagas (DC) ter sido descrita pela primeira vez, em alguns países, como o Brasil, o benznidazole (BZN) é o único medicamento disponível para seu tratamento e, no entanto, não existe um bom marcador de cura. A descoberta de biomarcadores associados à resposta terapêutica do BZN é essencial. O objetivo deste estudo foi determinar se os perfis de expressão gênica de células sanguíneas periféricas poderiam distinguir os indivíduos com DC que respondem positivamente ao tratamento com BZN (Respondedores) daqueles que não respondem (NRespondedores). O critério de resposta terapêutica adotado foi a negativação sérica para T. cruzi por qPCR, avaliada em três momentos, final do tratamento 60 dias (T60), seis meses (T6M) e um ano (T12M) após o início do tratamento com BZN (T0). O perfil transcriptômico foi realizado por sequenciamento massivo (RNAseq) e avaliado utilizando amostras de RNA total de oito pacientes Respondedores e 17 NRespondedores, para amostras do T0, T60 e T12M. Dos 20802 genes codificantes observados, 1882 foram expressos de forma significativa e diferencial entre os grupos, em pelo menos um desses três momentos. Destes, 13 genes foram selecionados como candidatos a biomarcadores, para validação por RT-qPCR. O gene LTF provou ser um biomarcador tardio da resposta terapêutica do BZN, apresentando diferença significativa na expressão de amostras T12M (valor de P de 0,01). Os genes AZU1 e LCN2 demonstraram perfil de biomarcadores da resposta terapêutica ao BZN logo após o término do tratamento, bem como de forma tardia, uma vez que o gene AZU1 apresentou diferença significativa de expressão para as amostras T60 e T12M (valor de P de 0,05). Assim como o gene LCN2 para as amostras T60 (valor de P de 0,05), e T12M (valor de P de 0,01). Estes três genes têm plausibilidade biológica pois estão associados a resposta imune. Desta forma estes marcadores podem ser bons candidatos a validação em grandes estudos clínicosMore than 100 years after Chagas disease (CD) was first described, in some countries, such as Brazil, benznidazole (BZN) is the only drug available for its treatment, but there is no good biomarker of cure. Therefore, the discovery of biomarkers associated with BZN therapeutic response is essential. The aim of this study was to determine whether peripheral blood cell gene expression profiles could distinguish CD subjects who respond positively to BZN (Responders) and non-responders (NR) treatment. The criterion of therapeutic response adopted was serum negative for T. cruzi by qPCR, evaluated at three moments, end of treatment - 60 days (T60), six months (T6M) and one year (T12M) after the beginning of BZN treatment. (T0). The transcriptomic profile was performed by massive sequencing (RNAseq) and evaluated using total RNA samples from eight Responding and 17 NResponding patients for T0, T60 and T12M samples. Of the 20802 coding genes observed, 1882 were expressed significantly and differently between groups at least one of these three moments. Of these, 13 genes were selected as candidate biomarkers for validation by RT-qPCR. The LTF gene proved to be a late biomarker of BZN therapeutic response, showing significant difference in T12M sample expression (P value 0.01). The AZU1 and LCN2 genes demonstrated biomarkers profile of the therapeutic response to BZN soon after treatment completion, as well as late, since the AZU1 gene showed significant difference of expression for samples T60 and T12M (P value of 0 .05). As well as the LCN2 gene for samples T60 (P value 0.05), and T12M (P value 0.01). These three genes are biological plausible because they are all related to immune response. They are candidates for validation in large scale clinical trial studie

Use of an automated pyrosequencing technique for confirmation of sickle cell disease.

BackgroundThe diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sβ0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.ObjectivesTo develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene.MethodsWe developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.ResultsWe identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified as Sβ0 thalassemia and 81 as Sβ+ thalassemia. The most frequent beta thalassemia mutations of Sβ0 and Sβ+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.DiscussionThe PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common β+ and β0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD cohort in Brazil

Additional file 1: of Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil

Supplementary Methods. Table S1. Clinical and pathological characteristics, BRCA sequencing and MLPA results. Table S2. BRCA1 gene variants. Table S3. BRCA2 gene variants. (ZIP 73.7 kb

Additional file 1: of Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil

Supplementary Methods. Table S1. Clinical and pathological characteristics, BRCA sequencing and MLPA results. Table S2. BRCA1 gene variants. Table S3. BRCA2 gene variants. (ZIP 73.7 kb