Abstract It is estimated that inherited deleterious or pathogenic germline variants in high- to moderate- penetrance breast cancer (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 as a result of the complex interaction between environmental and lifestyle risk factors as well as inherited familial predisposition. While low penetrance genetic variants have 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 as a result, there is an abundance of information on the functional interpretation of such variants, while information on regulatory variants remains limited. The goal of this study was to assess the functional impact of germline variants in the promoter region of BC susceptibility genes on gene expression level. Genes evaluated include CDH1, STK11, and bidirectional promoter genes ATM/NPAT and PALB2/DCTN5. A bioinformatics approach was developed to identify and prioritize variants based on conservation level and predicted binding of transcription regulatory proteins. Selected variants will be introduced into pGL3 constructs harboring the promoter of interest using site-directed mutagenesis followed by transfection into HEK293T cells. Dual-glo luciferase expression assays are used to measure levels of promoter activities. Expression levels from promoters containing genetic variants are compared with those from constructs containing the reference sequence. The reference constructs for STK11 and CDH1 containing the genes complete promoters demonstrated functional activity. For bidirectional promoters, forwards and reverse construct were tested. Forward ATM/NPAT and PALB2/DCTN5 constructs showed luciferase expression under the influence of ATM and DCTN5 respectively. Work is on the way to assess promoter activity of the reverse complement sequence of those promoters and determine the impact of 13 candidate variants on the strength of these promoters. At the clinical level, identifying germline variants that contribute to BC risk could help better define BC risk in women with negative hereditary cancer genetic test results. Citation Format: Nelly Arlene Arroyo, Lenin J. Godoy, 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 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5238.