RESUMO

Germline inactivating variants in BRCA1 lead to a significantly increased risk of breast and ovarian cancers in carriers. While the functional effect of many variants can be inferred from the DNA sequence, determining the effect of missense variants present a significant challenge. A series of biochemical and cell biological assays have been successfully used to explore the impact of these variants on the function of BRCA1, which contribute to assessing their likelihood of pathogenicity. It has been determined that variants that co-localize with structural or functional motifs are more likely to disrupt the stability and function of BRCA1. Here we assess the functional impact of 37 variants chosen to probe the functional impact of variants in phosphorylation sites and in the BRCT domains. In addition, we perform a meta-analysis of 170 unique variants tested by the transcription activation assays in the carboxy-terminal domain of BRCA1 using a recently developed computation model to provide assessment for functional impact and their likelihood of pathogenicity.

Assuntos

RESUMO

BRCA1, o primeiro gene de predisposição a câncer de mama e ovário familiar descoberto, quando alterado contribui para a maioria dos casos hereditários de câncer de mama. A inativação de ambos os alelos parece ser necessária para a progressão neoplásica em mama e ovário, sugerindo que BRCA1 atue como um gene supressor de tumor. Embora a função primária de BRCA1 ainda tenha que ser descoberta, existem várias evidências de que esta proteína está envolvida na transcrição, recombinação e no reparo de DNA. Várias mutações têm sido descritas para o gene BRCA1 e algumas predispõem aos cânceres de mama e ovário. Foi encontrada uma correlação com a predisposição a câncer e a falta da atividade transcricional in vitro, em mutações existentes no domínio BRCT, região C-terminal da proteína. Utilizamos ensaios de ativação transcricional para avaliar uma série de 117 variantes da região C-terminal de BRCA1 quanto ao risco de predisposição a câncer de mama e ovário. Mutagênese sítio-dirigida através de PCR por sobreposição e extensão foi utilizada para gerar 10 mutações missense in vitro na região C-terminal de BRCA1, que posteriormente foram usadas em ensaios de ativação transcricional em células de mamíferos para auxiliar o treinamento de um algorítmo preditivo capaz de classificar variantes missense de BRCA1. Além disso, a sublocalização celular e modificações covalentes, na presença ou ausência de dano no DNA, foram estudadas para os domínios BRCT de diversas proteínas

BRCA1, the first familial breast and ovary cancer susceptibility gene discovered, accounts when mutated for most of hereditary breast cancer cases. The inactivation of both alleles appears to be necessary for neoplastic progression in the breast and ovary, suggesting that BRCA1 acts as a tumor suppressor gene. Although the primary function of BRCA1 remains to be determined, there is good evidence thatthis protein is involved in DNA transcription, recombination and DNA repair. Several mutations have been described for the BRCA1 gene and some predispose to breast and ovarian cancer. Correlation has been found with cancer predisposition and lack of in vitro transcription activation among mutations in the BRCT domain, the Cterminalregion of the protein, We used transcription activation assays to assess a series of 117 variants of BRCA1 C-terminal region regarding the predisposition risk to breast and ovarian cancer. Site-directed mutagenesis by overlap extension PCR was used to generate 10 in vitro missense mutations in the C-terminal region of BRCA1,which were later used in transcription activation assays in mammalian cells to assist the training of a predictive algorithm capable of classifying BRCA1 missense variants. Furthermore subcellular localization and covalent modifications upon and in the absence of DNA damage were studied for the BRCT domains of several proteins

Assuntos