Establishing functional assays for evaluation of synonymous variants associated with hereditary breast cancer

Abstract

Breast cancer is the most commonly diagnosed cancer among women worldwide and a leading cause of cancer-related deaths. While most cancer cases are sporadic, approximately 5-10% are hereditary, with early-onset cases being more frequently associated with hereditary factors. They are often linked to mutations in DNA damage repair genes such as BRCA1/2, and their interacting partners, BARD1, BRIP1, PALB2, and RAD51D, which all play a crucial role in repairing DSB (double-strand breaks) through HR (homologous recombination). Historically the focus in genetic studies has been on non-synonymous variants that alter protein sequences. In contrast, synonymous or silent variants, which do not change the encoded amino acid, have largely been overlooked due to them being presumed nonfunctional. However, recent studies have suggested that these variants can be functional and affect gene function through various mechanisms and may therefore contribute to cancer susceptibility.

This project aimed to develop and optimize functional assays to evaluate the impact of synonymous variants in BARD1, BRIP1, PALB2, and RAD51D. Two assays were used, the CFA (colony formation assay), which assesses colony formation following DNA damage, and the HDR (homology-directed repair) assay, which measures HR efficiency with the help of a fluorescent reporter gene. Endogenous gene expression was knocked down using siRNAs, and cells were transfected with plasmids containing synonymous mutant constructs.

Although assay development and validations faced several challenges, limiting the analysis to a small subset of variants for only BRIP1 and RAD51D in the CFA that yielded inconclusive results, the project provided valuable insights. Foundational work was established for future studies aimed at developing functional assays to evaluate the role of synonymous variants in hereditary breast cancer, with the long-term goal of improving genetic screening and cancer risk assessments.

Popular Abstract

When silent mutations are heard

Breast cancer is one of the most common cancers affecting women worldwide, leading to multiple deaths each year. While most cancers are caused by random mutations in your cells, some are caused by known mutations passed on to you by your parents. They are called hereditary cancers and often appear earlier in life.

People with a family history of breast cancer can undergo genetic testing where mutations known to increase the risk of developing breast cancer can be detected. If a known harmful mutation is detected they can undergo regular breast cancer screenings. This can lower the risk of developing the cancer by early detection and preventive measures. But what if you have a harmful mutation, that increases your risk of developing breast cancer, but the mutation is unknown?

This is a real concern when it comes to particular mutations called synonymous mutations, also known as silent mutations. Silent mutations change the DNA sequence but do not change the sequence of the protein. And since they do not directly change the protein, they were previously considered unimportant and often ignored. Now we know that these silent mutations are not always so silent, they can interfere with how the cells work, effecting how genes are read and how effectively the proteins are made. And that means they can play a role in diseases such as cancer.

How the silent mutations are heard
This project focused on finding ways to detect or hear these silent mutations specifically in genes that help to repair damaged DNA. Mutations in DNA repair genes are common in hereditary cancers because when DNA is not repaired, the damage builds up and can lead to cancer. To study this, two methods were used. A method called colony formation assay (CFA), where cells are treated with a drug that causes DNA damage. Healthy cells will repair the damage so they can survive and continue to divide, forming colonies in a culture dish. Cells with mutations that affect DNA repair, grow fewer and/or smaller colonies. And a method called homology-directed repair assay, where a specific DNA repair process called homologous recombination is looked at. DNA damage is created and cells that successfully repair the damage using this method become green under the microscope. If the mutation affects the repair process, fewer cells will become green.

Although the project faced some challenges, such as time limitations and incomplete assay optimization, it still provided valuable insights into assay development. Only a few mutations were tested using the CFA, and the results were inconclusive. However, the work laid critical groundwork for future projects on silent mutations using these methods. In conclusion, this research project hoped to highlight how silent mutations may not be so silent after all. It also underscored the importance of developing better tools to “hear” these overlooked silent mutations so that more women can benefit from early detection and preventive measures.

Master’s Degree Project in Molecular Biology 60 credits 2025
Department of Biology, Lund University

Advisor: Helena Persson
Clinical Sciences Lund, Oncology, Faculty of Medicine

• author: Jóhannsdóttir, Agata
• supervisor: Helena Persson
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9214509
• date added to LUP: 2025-10-24 15:29:23
• date last changed: 2025-10-24 15:29:23