Deleterious variants in the BRCA1 and BRCA2 genes account for approximately 20% of cases of hereditary breast and ovarian cancer. The BRCA1 gene is found on the long arm of chromosome 17 (17q21) and plays a crucial role in DNA damage response. BRCA1 inactivating mutations lead to genetic instability, indirectly causing tumours to occur as a result of an accumulation of mutations in cell cycle regulatory genes. In Spain, families that carry the deleterious mutation in the BRCA1 gene have a 52% cumulative risk of developing breast cancer and a 22% risk of developing ovarian cancer by the age of 70 1.

The BRCA1 protein is a protein of 1863 amino acids. The two most important domains of BRCA1 are the RING domain and the BRCT domain. The RING domain is located at the amino terminus of BRCA1, between amino acids 1 and 109 (exons 2 - 7), and it is responsible for the E3·ubiquitin·ligase activity of BRCA1 2 and binds to the BARD1 protein. The BRCT domain mediates binding with phosphorylated proteins such as: Abraxas 34, BACH1/BRIP1 5, and CtlP 6, forming three mutually exclusive protein complexes named A, B and C complexes (see below). A number of other proteins also bind to the C-terminal region of BRCA1 and/or its central region 7 (Figure 1).

BRCA1 is involved in several steps of the DNA damage response process, a cell cycle control mechanisms that prevent the progression of the cell cycle while activating DNA repair routes. From a mechanistic point of view, it is known that BRCA1 forms part of 4 macro-complexes 89 BRCA1A, BRCA1B BRCA1C and BRCC. The BRCA1A complex acts as a DNA damage sensor and regulates the G2/M cell cycle checkpoint. The BRCA1B complex participates in the G1/S and intra-S-phase checkpoints, ensuring genome integrity prior to DNA synthesis. The BRCA1C complex mediates DNA repair through homologous recombination, facilitating the formation of single-stranded DNA in the step prior to homologous recombination. The BRCC complex is capable of recognising the previously formed single-strand DNA and mediating strand invasion; it is considered the effector in homologous recombination.

Interpreting variants of uncertain significance for the effect of protein function, and therefore for the risk of developing cancer, has become a challenge in clinical practice for genetic counselling services. It is necessary to develop tools that will help us reduce the high percentage of variants of uncertain significance, therefore facilitating genetic counselling process.

The variant of uncertain significance c.5434C->G (p.Pro1812Ala = Pro1812Ala) has been found in a high-risk family, where the proband is a woman diagnosed with papillary serous cystadenocarcinoma of the ovary, and her mother and maternal aunt are diagnosed with breast cancer. The c.5434C>G variant is found at position 28 of exon 23 of the BRCA1 gene. In this work we apply systems biology based mathematical modelling approaches to mechanistically determine the pathogenic effect of this variant.

In order to determine the physiological consequences of BRCA1 c.5434C->G (p.Pro1812Ala) mutation, this variant was analysed from two complementary perspectives to determine, first, the protein structural changes caused by the mutation in BRCA1 and then, the impact of these changes in the protein functionally in the context of the DNA damage response.

In order to investigate the hypothetically pathogenic role of the BRCA1 c.5434C->G (p.Pro1812Ala) mutation, a study was carried out using computational methods. A structural model was created and it was assessed from a functional and systems biology point of view.

The c.5434C->G (p.Pro1812Ala) mutation causes an important splicing defect in BRCA1, resulting in the omission of exon 23, which in turn leads to the appearance of a premature stop codon (p.Gly1803GlnfsX11) 10. However, and according to our results, it does not lead to secondary or tertiary structural changes. The structural configuration of the wild-type protein is preserved, which coincides with the findings of Drikos et al.: the c.5434C->G (p.Pro1812Ala) mutation preserves the ability to interact through the BRCT domain with proteins such as BRIP1 and RBBP8. But binding affinity and stability were substantially modified, which could alter the conformational equilibrium and therefore affect the protein's function in vivo 11.

As a result of a dysfunctional BRCT domain, the mutated protein cannot carry out its role in DNA damage response and cell cycle regulation.

BRCA1 activity that depends on proteins that interact with other domains, such as interaction of the RING domain with the BARD1 protein, is not expected to be affected by this mutation 31.

In the c.5434C->G (p.Pro1812Ala) variant, p53 is not activated by the altered BRCT domain. The initiation of the apoptotic response may therefore be compromised 24. Although this process will be much more fragile and sensitive to future mutations, it is expected that BRCA1 will retain a certain ability to stop the cell cycle. The activation of p53 and other key mediators of the cell cycle and apoptosis does not depend on BRCA1 alone.

The results of structural and mechanical analyses mean that the c.5434C->G (p.Pro1812Ala) variant can be reclassified from a variant of unknown significance to a pathogenic variant. The following evidence supports this statement:

Segregation: the c.5434C->G (p.Pro1812Ala) variant was suspected of being harmful in at least one family 32.

Population Frequency: The c.5434C->G (p.Pro1812Ala) mutation has been identified in patients with breast cancer or ovarian cancer, in both Spanish and non-Ashkenazi Jewish women 32.

Evolutionary Preservation: At position 1812 of the BRCA1 protein the amino acid proline is highly conserved between species, suggesting that the normal function of the protein plays an essential role. The c.5434C->G (p.Pro1812Ala) variant causes a change in the splicing pattern of BRCA1 10. Transcriptions expressed from the mutant c.5434C->G (p.Pro1812Ala) exhibit an omission of exon 23 in 75% of cases.

Functional assay: Our study did not indicate any significant change in structure. However, Drikos et al. observed a lower thermal stability that could influence conformational equilibrium. Assays of c.5434C->G (p.Pro1812Ala) mutation activity showed slightly reduced gene activity 11. An in vivo reporter gene assay showed that in c.5434C->G (p.Pro1812Ala) mutation activity clearly affects function 32. This assay assessed the BRCT domain's ability to induce transcriptional activation 10.

BRCA1 mutations with a similar splicing effect, including the neighbouring variants c.4987-3C>G 33 and c.5075-1G>C 34, were classified as pathogenic. Another study that examined a missense variant of unknown significance in the C-terminus region, close to variant c.5434C->G (p.Pro1812Ala), revealed significant alterations in BRCT structure and function 35.

Systems biology tools were used to evaluate the impact of the BRCA1 c.5434C->G (p.Pro1812Ala) mutation on DNA damage response from a mechanistic perspective. The results of this analysis support the findings of structural and functional methods explaining how the c.5434C->G (p.Pro1812Ala) mutation leads to impaired DNA repair and cell cycle control. This conclusion can be extrapolated to all mutations that cause loss of BRCT domain activity.

As regards DNA repair pathways, Mismatch Repair (MMR) and homologous recombination can be strongly altered in cells that harbour the c.5434C->G (p.Pro1812Ala) variant, whereas the effect on non-homologous end joining (NHEJ) and nucleotide excision repair (NER) seem to be less significant. Each of these mechanisms plays an independent and irreplaceable role in DNA repair. Failure of any one of them makes it impossible to repair a certain type of damage, which will eventually lead to an accumulation of mutations.

Together with structural and functional analysis, mechanistic insight provides a sound basis for defining the c.5434C->G (p.Pro1812Ala) mutation as a pathogenic variant. The BRCA1 c.5434C->G (Pro1812Ala) mutation causes deletion of the second BRCT domain, which plays a key role in critical processes, such as DNA repair and cell cycle control. It is therefore to be expected that this variant will not be able to exert its tumour suppressor action.

In conclusion, the c.5434C->G (p.Pro1812Ala) mutation creates an environment with increased risk of binding and tolerance to damage, which promotes the accumulation of mutations. Despite some compensation mechanisms, DNA damage whose repair is not induced, cell cycle arrest or stimulation of the apoptotic machinery may go unnoticed. This phenomenon can lead to mutations in other tumour suppressor genes or oncogenes, which causes cancer to develop. This hypothesis is consistent with Knudson's two-hit hypothesis 36 and with increased cancer susceptibility associated with BRCA1 37.