Dados do Resumo

Título

Construction of a Plasmid Containing the BRCA1 p.A870T Mutation via OE-PCR and Immunofluorescence Analysis in a Triple-Negative Breast Cancer Cell Line

Introdução

Triple-Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases and is closely linked to pathogenic variants (PVs) in the BRCA1 gene. BRCA1 is a tumor suppressor gene, with its protein being critical for the repair of double-strand DNA breaks (DSBs) via the homologous recombination (HR) pathway. Patients diagnosed with TNBC are frequently referred for genetic testing. Around 10% of TNBC patients, irrespective of age, carry BRCA1 pathogenic variants, while 2% have BRCA1 variants of uncertain significance (VUS). VUS present clinical challenges due to their ambiguous implications for TNBC management. In this study, we aim to conduct functional assays using TNBC cell lines to assess the effects of these variants and determine their potential pathogenic or benign roles.

Objetivo

Conduct functional assays in TNBC MDA-MB-436 (MDA) cell line, which has a mutation that produces a non-functional BRCA1 protein, to insert the VUS, aiming to observe properties such as drug resistance and DSB repair capable of associating with the pathogenic or benign.

Métodos

The OE-PCR technique was used to create the plasmid containing the VUS of interest p.A870T (c.2608G>A). The product was transformed into E. coli bacteria and subjected to amplicon library sequencing to verify the presence of the mutation. After confirmation, the product was transfected with Lipofectamine 3000 into MDA cells, along with benign (p.P871L) and pathogenic (p.EGFP-C1 plasmid) controls. The empty p.EGFP-C1 plasmid will serve as a control for the pathogenic variant, as MDA already expresses a dysfunctional BRCA1 protein. After transfection, the cells will be treated with Cisplatin to induce DSB and incubated for 24 hours. Primary and secondary antibodies will then be used to mark the BRCA1 protein and DSB, with the results observed under a fluorescence microscope.

Resultados

The OE-PCR was confirmed through agarose gel electrophoresis and its sequencing was confirmed by detecting the desired mutation, G>A at position 2608. Once the generation of the plasmid containing the VUS was completed, immunofluorescence was performed in triplicate and the preliminary results of the controls confirmed the expectations: MDA transfected with p.EGFP-C1 showed a higher percentage of DSB, reflecting the stress generated by lipofectamine and the transfection of the empty plasmid, which simulates a pathogenic variant, and MDA transfected with p.P871L showed a lower percentage of DSB, indicating a better repair capacity. In contrast, MDA transfected with p.A870T exhibited a DSB rate similar to the pathogenic control, suggesting that this variant may be pathogenic.

Conclusões

From the functional assays, it was observed that the VUS p.A870T presents a functional profile suggesting possible pathogenicity. However, for an accurate characterization of the functional impact of this variant, additional studies and experimental replications are necessary to allow a more robust evaluation and precise classification of the role of this variant in the pathological context.

Financiador do resumo

CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico

Palavras Chave

Triple-Negative Breast Cancer (TNBC); Variants of Uncertain Significance (VUS) in BRCA1; Functional Assays