Validating the mutations identified


We have used state-of-the-art optimized gene-editing (CRISPR Cas9) methods to generate the cell models with the desired variants in the identified candidate genes.

In these, we have performed functional assays such as the COMET, cell proliferation/viability and DNA repair proficiency to inspect their ability to modify protein function and therefore modulate cancer risk.

In parallel, we have cultured the cell lines with the desired variants and performed RNA sequencing in the NovaSeq platform

Our functional assays suggest thatone of the variants, which was presumably regulatory due to its genomic position, has an impact on the DNA repair proficiency of the cells as measured by comet assay. Because genome instability is a known hallmark of cancer genes, this result adds mechanistic insights on the possible cause for cancer susceptibility driven by the affected gene. These results also validate the functional approach we have used here to study other possible predisposing variants.

Our gene expression analysis indicates that this single variant impacts on the expression of approximately ~900 genes. The signalling pathway most enriched among those genes is the AGE-RAGE, which is involved in the process of inflammation, very relevant in cancer, whereas a missense mutation in the same gene leads to deregulation of ~400 genes and the pathway enrichment analysis indicates that the most deregulated genes are involved in calcium binding and cell adhesion, a very important process for cell migration and invasion and very relevant in the metastatic process.