3120 - Dominant-Negative GFI1B Mutations are Causal in Rare Inherited Platelet Disorders and Cause Defects in Stress Thrombopoiesis

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Inherited platelet disorders represent a heterogeneous group of rare diseases often characterized by lifelong bleeding diatheses. Specific therapies are lacking because the affected molecular processes are poorly understood. Previously, heterozygous nonsense mutations in Growth Factor Independence 1b (GFI1B) in families with autosomal-dominant bleeding disorders were identified. This disease is characterized by a plethora of megakaryocyte (MK) and platelet abnormalities, including macro thrombocytopenia, dysmorphic megakaryocytes, paucity of platelet /-granules, CD34 expression on MKs and platelets, increased MK numbers and their dislocation in the marrow. GFI1B is a transcription factor that contains an evolutionary conserved N-terminal SNAG domain and six C-terminal zinc fingers of which zinc finger three to five are crucial for DNA binding. Through the SNAG domain GFI1B recruits chromatin-modifying enzymes to promoters of target genes to regulate gene expression. Many mutations in the GFI1B gene found in inherited platelet disorders are located in zinc finger five, resulting in a DNA binding defective protein. To study the consequences of these mutations in vivo, we used Crispr/Cas technology and established three independent mouse lines that carry one Gfi1b allele in which the 5th and 6th zinc fingers are truncated or eliminated. All three lines of GFI1B DN mice had a mild but significant reduction of platelet counts resembling the situation found in patients. Moreover, GFI1B DN mice showed an expansion MKs and Mk precursors as well as hematopoietic stem cells. To test whether the DN mutations affect platelet recovery, we injected GFI1B DN mice and controls with an anti GP-IB antibody, which leads to an almost complete depletion of platelets. Clearly, GFI1B DN mice showed a delayed recovery of platelet numbers compared to controls. In addition, in many cases the platelets of DN mice were larger and appeared less granular after recovery than their counterparts in wt mice. Our data provide evidence for a causal relationship between inherited mutations in GFI1B in patients with bleeding disorders and reveal a new role for GFI1B in stress thrombopoiesis.