Abstract DICER1 is a key protein in the micro(mi)RNA biogenesis pathway responsible for cleaving precursor (pre)-miRNA stemloops with its RNase IIIa and IIIb domains into mature single stranded miRNAs. DICER1 syndrome is a pleiotropic tumor predisposition syndrome in which clinical manifestations are normally first seen during childhood. This genetic disorder is defined by the presence of inherited or de novo germline pathogenic variants (GPVs) in the DICER1 gene. While many germline loss of function variants fail to generate DICER1 protein, a subset of patients has germline missense variants that are predicted to produce full-length DICER1 protein that cluster in the DICER1 Platform domain, a region that plays a role in binding to the 5’ phosphate of pre-miRNA stemloops. Due to the uncertainty surrounding the pathogenicity of missense variants in DICER1 syndrome, clinical actionability can be limited. To determine how Platform mutations predispose to DICER1 syndrome we characterized a number of these variants using in vitro pre-miRNA binding and cleavage assays. Interestingly, four of these variants displayed impaired pre-miRNA binding and processing, with three of them localizing to a hydrophobic pocket in the Platform domain. In keeping with in vitro results, DICER1 mutants displayed impaired miRNA processing when introduced into mesenchymal stem cells. Furthermore, we identified a second somatic hit in the RNase IIIb domain of DICER1 in thyroid nodular hyperplasia DNA from a patient affected by one of these variants. Taken together, these results identify a key region in the DICER1 protein that is essential for pre-miRNA processing and help the classification of these germline variants as pathogenic to ensure proper clinical management of affected patients. Additionally, these results provide insight on the molecular basis involved in DICER1 syndrome predisposition. Citation Format: Dylan Pelletier, Anne-Laure Chong, Sophie Albert, Marc Fabian, William Foulkes. DICER1 Platform domain missense variants led to tumor susceptibility via failure of precursor miRNA binding. [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 3522.