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 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 (RUNX1 (WT/WT) , RUNX1 (WT/L43S) , and RUNX1 (L43S/L43S) ). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1 (L43S/L43S) and RUNX1 (WT/L43S) mice had a significantly longer tail-bleeding time than RUNX1 (WT/WT) mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1 (L43S/L43S) and RUNX1 (WT/L43S) displayed impaired agonist-induced spreading and alpha -granule release, with no differences in delta -granule secretion. Levels of integrin alpha (IIb) beta (3) activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1 (L43S/L43S) and RUNX1 (WT/L43S) using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1 (L43S/L43S) and RUNX1 (WT/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 alpha (IIb) beta (3) activation, aggregation, alpha -granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

Automated summary

RUNX1-related disorder is a rare and heterogeneous disease caused by germline variants affecting the RUNX1 gene, and using murine models can help elucidate its pathogenic mechanisms.