Germline mutations of breast cancer susceptibility genes through expanded genetic analysis in unselected Colombian patients

Abstract

Background: In Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly involve hereditary and familial cases, demonstrating a gap in the literature regarding the identification of germline mutations in unselected patients from Latin-America. Identification of pathogenic/likely pathogenic (P/LP) variants is important for shaping national genetic analysis policies, genetic counseling, and early detection strategies. The present study included 400 women with unselected breast cancer (BC), in whom we analyzed ten genes, using Whole Exome Sequencing (WES), know to confer risk for BC, with the aim of determining the genomic profile of previously unreported P/LP variants in the affected population. Additionally, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to identify Large Genomic Rearrangements (LGRs) in the BRCA1/2 genes. To ascertain the functional impact of a recurrent intronic variant (ATM c.5496 + 2_5496 + 5delTAAG), a minigene assay was conducted.

Results: We ascertained the frequency of P/LP germline variants in BRCA2 (2.5%), ATM (1.25%), BRCA1 (0.75%), PALB2 (0.50%), CHEK2 (0.50%), BARD1 (0.25%), and RAD51D (0.25%) genes in the population of study. P/LP variants account for 6% of the total population analyzed. No LGRs were detected in our study. We identified 1.75% of recurrent variants in BRCA2 and ATM genes. One of them corresponds to the ATM c.5496 + 2_5496 + 5delTAAG. Functional validation of this variant demonstrated a splicing alteration probably modifying the Pincer domain and subsequent protein structure.

Conclusion: This study described for the first time the genomic profile of ten risk genes in Colombian women with unselected BC. Our findings underscore the significance of population-based research, advocating the consideration of molecular testing in all women with cancer.

Keywords: Minigene assay; Pathogenic germline variants; Unselected breast cancer; Whole exome sequencing.

Fig. 1

Pedigrees of index cases and their relatives assessed in the segregation analysis. a Germline mutation located in BRCA2 gene: c.2808_2811delACAA, p.Ala938Profs*21; b Germline mutation located in BRCA2 gene: c.3860delA, p.Asn1287Ilefs*6; c Germline mutation located in BRCA2 gene: c.1763_1766delATAA, p.Asn588Serfs*25; d and f Germline mutation located in ATM gene: c.5496 + 2_5496 + 5delTAAG; e Germline mutation located in PALB2 gene: c.3350 + 4A > G; **individuals tested harboring germline mutation; * individuals tested not harboring germline mutation

Fig. 2

Exon skipping of exon 36 of the ATM gene due to germline mutation c.5496 + 2_5496 + 5delTAAG. a Diagram of the minigene pSpliceExpress vectors, WT which is constituted by exon 36 of ATM and exons 2 and 3 from Rat insulin (Rat Ins Ex2 and Rat Ins Ex3), and Mut which represents the presence of the germline mutation of interest. b RT-PCR, performed after transfection of the WT and Mut plasmids, in HEK-293, MCF-7, MDA-MB-231, and BT-474 cell lines, showed exon skipping in all cell lines, negative control was not transfected cells (NT). c Sanger sequencing was performed to confirm the effect in splicing observed in RT-PCR