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

Thromb Haemost. 2021 Sep;121(9):1193-1205. doi: 10.1055/s-0041-1723987. Epub 2021 Feb 18.


RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, 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 (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

MeSH terms

  • Animals
  • Blood Platelet Disorders / blood
  • Blood Platelet Disorders / genetics*
  • Blood Platelets / metabolism*
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Core Binding Factor Alpha 2 Subunit / blood
  • Core Binding Factor Alpha 2 Subunit / genetics*
  • Cytoplasmic Granules / genetics
  • Cytoplasmic Granules / metabolism
  • Disease Models, Animal
  • Gene Knock-In Techniques
  • Genetic Predisposition to Disease
  • Hemostasis
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Mutation*
  • Phenotype
  • Platelet Activation / genetics*
  • Platelet Glycoprotein GPIIb-IIIa Complex / metabolism
  • Secretory Pathway
  • Thrombopoiesis


  • Core Binding Factor Alpha 2 Subunit
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Runx1 protein, mouse
  • CRISPR-Associated Protein 9