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Hum Mutat ; 39(12): 2025-2039, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30204945


The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5' noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

Genes Chromosomes Cancer ; 55(12): 915-924, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27328445


Triple negative breast cancers (TNBCs) represent about 15-20% of all breast cancer cases and are characterized by a complex molecular heterogeneity. Some TNBCs exhibit clinical and pathological properties similar to BRCA-mutated tumors, without actually bearing a mutation in BRCA genes. This "BRCAness" phenotype may be explained by germline mutations in other genes involved in DNA repair. Although respond to chemotherapy with alkylating agents, they have a high risk of recurrence and progression. Some studies have shown the efficacy of neoadjuvant therapy in TNBC patients with DNA repair defects, but proper biomarkers of DNA repair deficiency are still needed. Here, we investigated if mutations in DNA repair genes may be correlated with anthracyclines/taxanes neoadjuvant therapy response. DNA from 19 TNBC patients undergoing neoadjuvant therapy were subjected to next generation sequencing of a panel of 24 genes in DNA repair and breast cancer predisposition. In this study, 5 of 19 patients (26%) carried a pathogenic mutation in BRCA1, PALB2, RAD51C and two patients carried a probable pathogenic missense variant. Moreover, VUS (Variants of Unknown Significance) in other genes, predicted to be deleterious by in silico tools, were detected in five patients. Germline mutations in DNA repair genes were found to be associated with the group of TNBC patients who responded to therapy. We conclude that a subgroup of TNBC patients have defects in DNA repair genes, other than BRCA1, and such patients respond favourably to neoadjuvant anthracyclines/taxanes therapy. © 2016 Wiley Periodicals, Inc.

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Enzimas Reparadoras do DNA/genética , Mutação em Linhagem Germinativa/genética , Terapia Neoadjuvante , Recidiva Local de Neoplasia/genética , Neoplasias de Mama Triplo Negativas/genética , Adulto , Idoso , Proteína BRCA1/genética , Estudos de Casos e Controles , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Seguimentos , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Proteínas Nucleares/genética , Prognóstico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Supressoras de Tumor/genética
Int J Mol Med ; 35(4): 950-6, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25683334


The study of BRCA1 and BRCA2 genes and their alterations has been essential to the understanding of the development of familial breast and ovarian cancers. Many of the variants identified have an unknown pathogenic significance. These include variants which determine alternative mRNA splicing, identified in the intronic regions and those are capable of destroying the splicing ability. The aim of this study was to detect BRCA1/BRCA2 aberrant transcripts resulting from alternative splicing, in women with a known family history and/or early onset of breast and/or ovarian cancer, tested wild-type for BRCA1 and BRCA2. The identification and characterization of aberrant transcripts through the analysis of mRNA levels in blood lymphocytes may help us to recognize families otherwise misclassified as wild-type BRCA1 and BRCA2. Blood samples were collected from 13 women that had a family history of breast and/or ovarian cancer and tested negative for pathogenic mutations in the BRCA1 and BRCA2 genes. Total RNA was analyzed for the presence of BRCA1 and BRCA2 naturally occurring and pathological transcripts using RT-PCR. In 2 out of the 13 samples, 2 alternative transcripts of the BRCA1 gene were identified. These were probably pathogenic as they lacked exon 17 and exon 15, respectively, giving rise to a truncated protein. In addition to these, we identified the Δ17-19 transcript in 1 patient, which gives rise to a protein with an in-frame deletion of 69 amino acids. In conclusion, this study on alternative transcripts of the BRCA1 and BRCA2 genes revealed the presence of isoforms (prevalence of 15%) in blood samples from women with breast and ovarian cancer that were probably pathogenic, that were not detected by conventional methods of mutation screening based on direct sequencing of all coding regions, intron-exons junctions and MLPA analysis.

Processamento Alternativo , Neoplasias da Mama/genética , Genes BRCA1 , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Adulto , Éxons , Feminino , Genes BRCA2 , Estudos de Associação Genética , Humanos , Pessoa de Meia-Idade , Mutação , Isoformas de RNA , RNA Mensageiro/genética , Análise de Sequência de DNA , Transcrição Genética