Abstract 1445: The functional role of promoter germline variants in breast cancer susceptibility genes

Abstract Introduction: Germline variants are associated with breast cancer risk (BC). It is estimated that inherited deleterious or pathogenic variants in high- to moderate-penetrance BC susceptibility genes such as BRCA1, BRCA2, STK11, CDH1, ATM, PALB2, and CHEK2 may explain up to 27% of the BC cases. For the remaining cases, BC develops due to the complex interaction between environmental and lifestyle risk factors and inherited familial predisposition. While low penetrance genetic variants have a limited impact on BC risk when taken individually, their combined effect on polygenic risk scores may provide a risk elevated enough to justify clinical actions. Most of the previous work in hereditary breast cancer research has focused on variants located in the coding region of the genes. and information on regulatory variants remains limited. This study aims to assess germline variants in the promoter region of BC susceptibility genes on gene expression level. We selected the single promoter genes CDH1 and STK11 and the bidirectional promoter genes PALB2/DCTN5 and ATM/NPAT. Methods: A bioinformatics approach was developed to identify and prioritize promoter variants that could have a functional impact on gene expression levels. Promoters of each gene of interest were cloned into a luciferase reporter expression system. Side-directed mutagenesis was used to introduce the variants of interest in promoter constructs. The reporter constructs were transfected in MCF7 and MCF10A cell lines and the luciferase activity induced by reference promoter was compared to that of the construct carrying the genetic variants All statistical analyses and graphical visualization were conducted in R implemented in R Studio. Results: A total of 6, 427 variants spanning the promoter regions of interest were identified. Of those, thirteen were selected based on conservation level and predicted binding of transcription regulatory proteins. We identified two variants in the promoter region of STK11 that significantly decrease the expression of Luciferase by a factor of 0.83 and -0.04-fold, almost inhibiting the expression of STK11, while the change in CDH1 has a more subtle effect with a variant significantly increase Luciferase expression by a factor of 2.41-fold. Work is on the way to finish characterizing CDH1 variants and the bidirectional promoters ATM/NPAT and PALB2/DCTN5. Conclusion: This work demonstrates the functional significance of genetic variants located in regulatory regions of BC susceptibility genes. At the clinical level, expanding genetic testing to include regulatory variants could help better define BC risk in women with negative hereditary cancer test results. Citation Format: Nelly A. Arroyo, Lenin Godoy, Doralis De León Vázquez, Sol V. Pérez Martir, Julie Dutil. The functional role of promoter germline variants in breast cancer susceptibility genes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1445.