RESUMO
BRCA1, BRCA2, CHEK2 and PALB2 genes are associated with hereditary breast and ovarian cancer syndrome. Genetic testing of these genes is of increasing importance to guide therapeutic and management decisions. In this study, we evaluated the performance of a next generation sequencing (NGS) assay for the complete analysis of BRCA1, BRCA2, CHEK2 and PALB2 genes using Agilent's SureMASTR BRCA Screen that enabled the detection of single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variations (CNVs) in a single-tube PCR based library preparation. The results showed 100% sensitivity and specificity on a set of 52 known samples from de-identified patients and external quality assessment program. A concordance rate of 87.5% was achieved in the comparison of variant classification with the external laboratories. The high accuracy of the assay supports the use of SureMASTR BRCA Screen in clinical diagnostic laboratories (SureMASTR BRCA Screen is for research use only, not for use in diagnostic procedures).
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Quinase do Ponto de Checagem 2/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Variações do Número de Cópias de DNA/genética , Detecção Precoce de Câncer/métodos , Feminino , Predisposição Genética para Doença , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer's disease (AD). Although the actual risk-increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the ß-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs. RESULTS: Three patient-specific CLU mutations in the ß-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines. CONCLUSIONS: Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the α- and ß-chain of CLU.