Alternative splicing variants in breast cancer

• Autor(a) principal: Chande, Andreia Ringler
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/156156
• topic: BRCA1; Exon 11; Breast Cancer; Splice Variants; Alternative splicing; mRNA splicing analysis; Ciências Médicas

Abstract Cancer is considered a genetic disease that incurs in uncontrolled and disproportional growth of the cells with distinct characterizing hallmarks. Breast Cancer (BC) is the most common type of cancer and a leading cause in death of women therefore remaining a target of study. Hereditary breast cancer (HBC) is tightly associated with mutations in tumor suppressor genes such as the BRCA genes. These are highly penetrant genes involved in the DNA damage response. BRCA1 codes for a multifunctional protein that, when mutated with a pathogenic variant impairs many pathways in the cell. Genetic tests have increasingly been asked for these genes, many resulting in variants of unknown significance (VUS). VUS represent a concern as their biological impact is not known thus, their study is of vital interest. Several are the factors involved in their study. These include mRNA splicing assays, seen as sometimes these VUS lead to alternative splicing of the gene, thus identified as splice variants (SV). Our work is divided in two tasks, firstly our aim was to identify potential SV in different cell lines commonly used in BC research, using one non-tumorigenic (MCF-10A) for comparative purposes and three distinct tumorigenic cell lines (MCF-7, MDA-MB-231 and SK-BR-3). We performed mRNA splicing analysis for the exon 11 (highly alternative spliced region) of the BRCA1 gene using PCR-based techniques. The results obtained for this task indicate that no SV was found in this exon for these in vitro models. In the second part of our study, we cultured an MCF-10A cell line previously cloned by our group with a VUS of the BRCA1 gene located in the exon 11, using CRISPR-Cas9, and applied the same method used for mRNA splicing analysis of the other cells. First, we had to confirm that the cells indeed carried the VUS, which was confirmed establishing a new homozygous in vitro model, and only then applied the method, which, after analysis, apparently indicated that this is not a SV. For further confirmation off the results, we performed direct RNA sequencing by nanopore. Unfortunately, this data is still under analysis and the results are not presented here.