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Genes involved in the maintenance of genomic integrity
There are two classes of proteins that act together to regulate cellular proliferation, i.e. “caretakers” and “gatekeepers” (Hanahan and Weinberg 2011). Gatekeeper proteins function as part of a system of checks that monitors the level of cell division and cell death. These proteins are in place to lessen the effects of genome/tissue damage (Thiagalingam 2015). Caretaker proteins are responsible for maintaining genome integrity (Yao and Dai 2014). Genomic instability leads to the accumulation of DNA mutations that affect either genome gatekeeper or caretaker genes and make cells more sensitive to any additional mutations (Kwei, et al. 2010). Gatekeeper genes generally code for proteins that function in cell cycle arrest and cell death (Vogelstein, et al. 2013). Typical caretaker genes include genes that function in DNA repair pathways and in the different phases of cell mitosis (Negrini, et al. 2010). Genomic instability brought on by mutated caretaker genes renders the cell selectively vulnerable to both exogenous and endogenous mutagens (Smith, et al. 2010). Cancer malignant neoplasia is aided by the mutation of two gene classes, oncogenes and tumour suppressor (TS) genes (Stephens, et al. 2012). Proto-oncogenes (normal/non-mutated oncogenes) code for proteins that drive cell growth and division and function in the gap (G) and segregation (S) phases of the cell cycle (Chow 2010). Growth factor receptors and proteins that initiate DNA replication are examples of such genes. Proto-oncogenes acquire mutations and become oncogenes that result in the activation of irregular cell proliferation and contribute to tumour growth. Oncogenes induce genome instability by activating growth signalling pathways which places the cell under replicative stress (Negrini, et al. 2010; Osborne, et al. 2004).
TS proteins restrict cell growth and promotes programmed cell death (apoptosis) (Delbridge, et al. 2012). Mutations in TS genes have a loss of function effect on cellular mechanisms that inhibit persistent cell division, ultimately resulting in malignant cell growth. The TS gene TP53 and genes that code for DNA damage recognition proteins are examples which have proved to be the most mutated genes in cancer tumours (Chow 2010; Negrini, et al. 2010). Tumour suppressors and oncogenes provide a link between cell cycle regulation and control as well as the formation of tumours and ultimately to cancer development (Chow 2010). TS genes are significantly more mutated than oncogenes (Negrini, et al. 2010) and as a result, TS genes have proved to be most useful when diagnosing BC (Osborne, et al. 2004).
The Fanconi Anemia pathway
The genetic disease, FA, is associated with mutations in genes that comprise the FA pathway. The main function of the FA pathway is to coordinate distinct pathways that resolve interstrand cross-linked DNA regions (ICL) (Filippini and Vega 2013; Kee and D’Andrea 2010). ICL’s cause lesions in the DNA double strand helix and block the progression of cellular replication and transcription (Deans and West 2011). This pathway contains elements of three classic DNA repair processes including NER, the error-prone translesion synthesis (TLS) and HR to remove cross-linked DNA (Kim and D’Andrea 2012). There are 15 genes that are associated with FA including FANCA–C, D1 (BRCA2), D2, E–G, I, J (BRIP1), L (PHF9), M (Hef), N (PALB2), RAD51C (FANCO) and SLX4 (FANCP) (Hucl and Gallmeier 2011). The FA pathway remains dormant and is only activated during the cell cycle synthesis phase (Garner and Smogorzewska 2011). During the cell cycle S-phase or upon recognition of DNA damage a kinase protein, such as ATR kinase, phosphorylates the FA core proteins. This core complex consists of eight proteins i.e. A, B, C, E, F, G, L, M and is stabilised by other weaker sub-complexes for e.g. partial interactions with additional FA-associated proteins such as FAAP24 and FAAP100 (Moldovan and D’Andrea 2009; Shuen and Foulkes 2011). Together, these proteins assemble into a multi-subunit ubiquitin ligase complex (Hucl and Gallmeier 2011). FAAP24 directly recognises cross-linked DNA and interacts with FANCM to open DNA strand replication forks and recruits the FA core complex to ubiquitinate FANCD2 and FANCI (Moldovan and D’Andrea 2009). Unique DNA repair structures (i.e. foci) are formed under the direction of the ubiquitinated FANCD2 & FANCI (Hollestelle, et al. 2010). To link the FA pathway with HR, FANCD2 co-localises with FANCD1, FANCN, FANCJ and RAD51 (Figure 1.8) (Shuen and Foulkes 2011). Specific DNA structures with FANCD2-I, FANCD1 (BRCA2), RAD51 and PCNA are vital towards promoting homologous DNA repair (Kee and D’Andrea 2010). The FANCJ-BRIP1 complex has a 5’ – 3’ helicase activity that localises to DNA repair structures associated with BRCA2 and RPA and remodels DNA structures, regulating HR and other repair processes (Moldovan and D’Andrea 2009).
Chapter 1: Literature Review
1.1. Introduction
1.2. Histopathological and molecular classification of breast cancer
1.3. Hallmarks of breast cancer development
1.4. Breast cancer risk factors
1.5. Breast cancer susceptibility
1.6. The “missing” hereditability of familial breast cancer
1.7. Next-generation sequencing methods used to discover novel breast cancer genes
1.8. Research study motivation and project aim
Chapter 2: Materials and Methods
2.1. Patient selection
2.2. Sample preparation and WE sequencing
2.3. Data processing
2.4. Variant filtration and annotation
2.5. Computational approach of candidate gene prioritisation
2.6. Sanger sequencing
2.7. In silico prediction of pathogenic variant
Chapter 3: Results of whole exome sequencing quality analysis and variant identification
3.1. Quality assessment of Illumina whole exome data
3.2. Evaluating paired-end read mapping
3.3. Variant identification and filtration
3.4. Sanger sequence confirmation
3.5. Final list of candidate genes
Chapter 4: TCHP sequence variants in South African breast and ovarian cancer families
4.1. Introduction
4.2. Materials and Methods
4.3. Results and Discussion
4.4. Conclusion
Chapter 5: EME2 gene variants in high-risk South African breast and ovarian cancer families
5.1. Introduction
5.2. Materials and Methods
5.3. Results and Discussion
5.4. Conclusion
Chapter 6: HELQ gene variants in high-risk non-BRCA1/2 South African families
6.1. Introduction
6.2. Materials and Methods
6.3. Results and Discussion
6.4. Conclusion
Chapter 7: Germline sequence variants in DNA repair genes of South African breast/ovarian cancer families with and without BRCA mutations
7.1. Introduction
7.2. Methods and Materials
7.3. Results and Discussion
7.4. Conclusion
Chapter 8: Conclusion
8.1. Whole exome sequencing of high-risk BC/OVC families
8.2. Challenges in elucidating the missing heritability of familial breast cancer
8.3. Concluding statements and future directions
