INTRODUCTION AND OBJECTIVES: Germline mutations in the tumor suppressor gene BRCA1 are associated with increased lifetime risk of hereditary breast and ovarian cancers. Genetic testing is widely used to identify pathogenic variants and to guide clinical management of individuals at increased cancer risk. However, many mutations do not have information regarding their pathogenicity and risk of cancer, being classified as variants of uncertain clinical significance (VUS). BRCA1 is an essential protein in the DNA damage response by homologous recombination, interacting with several partners, like PALB2. The interaction between BRCA1 and PALB2 is mediated by the direct binding of residues “a” and “d” of their coiled-coil domains, located in the protein C and N-terminal regions, respectively. BRCA1 C-terminal region also encloses the tandem BRCT domain, which is crucial to BRCA1 function and has an intrinsically transcriptional activation capacity. Mutations that compromise the BRCA1-PALB2 interaction or BRCT transcriptional activity are associated with cancer predisposition. Our goal is to functionally evaluate natural missense variants classified as VUS located in residues “a” of BRCA1 coiled-coil domain through PALB2 interaction ability and BRCT transcription activation. MATERIAL AND METHODS: Natural missense variants positioned in the “a” residues R1397 and L1404 of BRCA1 coiled-coil domain were identified by all possible point mutations in these residues. All variants (a total of 10) were considered for the study and generated through site-directed mutagenesis strategy. The interaction between BRCA1 (wt or variant) with PALB2 were evaluated by a mammalian two-hybrid assay and the BRCA1 transcriptional activity by a luciferase-reporter approach, both in HEK293FT cells. RESULTS AND CONCLUSION: Our results showed only one variant (R1397K) that exhibited both PALB2 interaction and transcription activation capacity similar to BRCA wt protein, which suggests a non-pathogenic behavior. On the other hand, two variants (L1404R and L1404V) present an important reduction in transcriptional activation and also in the PALB2 interaction ability, which indicates a functional impairment and putative pathogenic behavior. Interestingly, one variant (R1397W) showed increased levels of transcriptional activity and in the interaction with PALB2, suggesting an altered behavior. Similarly, variant R1397M displayed increased levels of PALB2 interaction but a transcriptional activity similar to positive control. All remaining variants (R1397G, R1397S, R1397T, L1404M and L1404Q) presented transcription activity similar to BRCA1 wt protein but exhibited reduced levels of interaction with PALB2. Collectively, these data reveal the functional behavior of BRCA1 missense variants located in a critical position of PALB2 binding site, allowing for more accurate genetic counseling and clinical management.