Analysis of hereditary and putative breast cancer susceptibility genes in BRCA1 and BRCA2 mutation-negative individuals

Author: Chloe Thompson-Peach

Thompson-Peach, Chloe, 2020 Analysis of hereditary and putative breast cancer susceptibility genes in BRCA1 and BRCA2 mutation-negative individuals, Flinders University, College of Medicine and Public Health

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Breast cancer is the most common cancer affecting Australian women, with many affected individuals exhibiting a strong family history of the disease. Whilst inherited mutations in BRCA1, BRCA2 and additional susceptibility genes account for approximately 30% of familial breast cancer cases, the underlying cause in the remaining 70% is unknown, suggesting that additional breast cancer susceptibility genes exist. We hypothesised that mutations within genes that play a role in the DNA damage repair and checkpoint control pathways may be involved in predisposing families to inherited breast cancer.

In order to test our hypothesis Ion Torrent Massively Parallel Sequencing and a custom targeted panel were used to sequence 51 genes of interest in a cohort of BRCA1/2 mutation-negative individuals with familial breast cancer. The gene panel consisted of 19 known breast cancer susceptibility genes (diagnostic genes) and 32 genes which play integral roles in the DNA damage repair and cell cycle control pathways and therefore are potentially involved in the development of breast cancer (discovery genes). For this study, a bespoke bioinformatics pipeline was developed for the analysis of Ion Torrent data generated from a cohort of BRCA1/2 mutation-negative individuals from South Australia. A novel three-dimensional pooling strategy (Tri-Pool-Seq) was piloted for the identification of rare variants within the patient cohort, however this failed to identify known sequence changes and therefore was not extended to the full cohort.

From the individual sequencing and analysis of patients, an average of 125 variants were identified in each sample, with rare variants analysed further. In total 166 rare variants were identified which were predicted to alter gene transcription or translation; of these 82 variants were identified as being potentially pathogenic. Moreover, a known pathogenic truncation mutation was identified in PALB2 in 2 individuals.

CRISPR/Cas9 was used to functionally validate a UIMC1 polymorphism identified in 2 patients in an attempt to establish the role of this gene in cancer development. This study indicated that cells lacking functional UIMC1 demonstrated an increased sensitivity to ionising radiation, resulting in an increase in cell death and a reduced capacity to repair DNA double stranded breaks. These results indicate that a loss of UIMC1 may play an important role in the development of hereditary breast cancer, through the loss of vital DNA damage repair capabilities and dysregulation of cell growth. Overall, this research has the potential to provide much needed diagnostic information for the identification of mutations resulting in familial breast cancer, and to identify novel breast cancer genes.

The work carried out within this thesis provides further evidence that additional genes are involved in the development of hereditary breast cancer. This South Australian population-based analysis of BRCA1 and BRCA2 mutation-negative individuals has resulted in the identification of both pathogenic disease causative mutations, and a potentially novel gene involved in cancer development.

Keywords: Breast Cancer, Genetics, BRCA1, BRCA2, Hereditary Breast Cancer, Massively Parallel Sequencing, CRISPR/Cas9

Subject: Medical Science thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Medicine and Public Health
Supervisor: Associate Professor Karen Lower