Dados do Resumo

Título

Development of a BRCA1 p.A870T Mutant Plasmid Using Overlapping Extension PCR and Functional Viability Assays in a Triple-Negative Breast Cancer Cell Line

Introdução

Triple-Negative Breast Cancer (TNBC) represents 15-20% of all breast cancers and is strongly associated with pathogenic variants (PVs) in the BRCA1 gene. BRCA1 is a tumor suppressor gene whose protein plays a key role in repairing double-strand DNA breaks (DSBs) through the homologous recombination (HR) pathway. Patients with TNBC are commonly referred for genetic testing. Approximately 10% of TNBC patients, regardless of age at diagnosis, carry pathogenic variants in BRCA1, while 2% have BRCA1 variants of uncertain significance (VUS). VUS pose clinical challenges due to their unclear impact on TNBC management. In this study, our aim is to perform functional assays using TNBC cell lines to evaluate the potential effects of these variants by using overlapping Extension PCR (OE-PCR) technique to create a BRCA1 VUS detected in patient with TNBC

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 phenotype of the protein

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 plated in P96 wells, treated with different concentrations of Cisplatin and incubated for 48 hours. MTT {bromide of [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium]} were added to the wells and read at an absorbance of Λ = 590 nm with a reference filter of Λ = 620 nm

Resultados

Primers were created for site-directed mutagenesis (SDM). Subsequently, OE-PCR was performed. Once the plasmid product containing the VUS was obtained, transformation by heat shock was carried out, followed by confirmation through amplicon sequencing. The transfection was confirmed by immunofluorescence and the MTT assay was performed in triplicates with cisplatin ranging from 100 to 600 µM and 1% DMSO. Preliminary results showed that the transfection is effective and the MTT results indicate that the VUS behaves more similarly to the pathogenic controls (p.EGFP and MDA) than to the benign control (p.P871L), suggesting that, up to this point, it has a pathogenic phenotype

Conclusões

The OE-PCR was verified by agarose gel, allowing control over the efficiency of each step. After sequencing, the presence of the G>A mutation at its expected position (2608) was confirmed. From the functional cell viability assay with MTT, it was observed that the VUS p.A870T presents a phenotype suggesting it is pathogenic. However, further tests are needed to verify its adhesion, migration, or DSB repair potential to confirm and possibly contribute to the reclassification of the VUS

Palavras Chave

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