Characterization of the Platelet Phenotype Caused By a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting RUNX1 gene. This rare and heterogeneous disorder has no specific clinical and laboratory phenotype, making genetic diagnosis necessary. Despite international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Therefore, murine models may be useful not only to settle definitively the controversy about the pathogenicity of certain RUNX1 variants, but also to elucidate the mechanisms of molecular pathogenesis. Thus, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1^WT/WT, RUNX1^WT/L43S, RUNX1^L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1^L43S/L43S and RUNX1^WT/L43S mice had significantly prolonged bleeding-tail time than RUNX1^WT/WT, indicating a role of the variant in hemostasis. Nevertheless, only homozygous mice displayed mild-thrombocytopenia. RUNX1^L43S/L43S and RUNX1^WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of activation of integrin α_IIbβ₃, fibrinogen-binding, and aggregation were significantly lower in platelets from RUNX1^L43S/L43S and RUNX1^WT/L43S using PMA, ADP, and high-doses of thrombin. Lower levels of PKC phosphorylation in RUNX1^L43S/L43S and RUNX1^WT/L43S suggested that PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin α_IIbβ₃ activation, aggregation, α-granule secretion, and platelet spreading; mimicking the phenotype associated with RUNX1 variants in the clinical setting.