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Ogawa S, Kunishima S. Functional characterization of a novel GFI1B mutation causing congenital macrothrombocytopenia. J Thromb Haemost 2016; 14: 1462-9. Essentials• Two groups recently reported GFI1B as a novel causative gene for congenital macrothrombocytopenia.• We performed functional analysis of a novel GFI1B mutation and previous mutations.• An immunofluorescence analysis of the platelet CD34 expression can be useful as a screening test.• Mutant-transduced megakaryocytes produced enlarged proplatelet tips which were reduced in number.Summary. Background: GFI1B is an essential transcription factor for megakaryocyte and erythrocyte development. Two groups have recently identified GFI1B as a novel causative gene for congenital macrothrombocytopenia associated with a-granule deficiency. Methods: We performed whole exome sequencing and identified a novel GFI1B p.G272fsX274 mutation in a family with macrothrombocytopenia, and a decreased number of platelet a-granules and abnormally shaped red blood cells. p.G272fsX274 and the previous two mutations all predicted disruption of an essential DNA-binding domain in GFI1B. We therefore performed functional studies to characterize the biochemical and biological effects of these three patient-derived mutations. Results: An immunofluorescence analysis revealed decreased thrombospondin-1 and increased CD34 expression in platelets from our patient. Consistent with the previous studies, the three patient-derived mutants were unable to repress the expression of the reporter gene and had a dominantnegative effect over wild-type GFI1B. In addition, the three mutations abolished recognition of a consensusbinding site in gel shift assays. Furthermore, transduction of mouse fetal liver-derived megakaryocytes with the three GFI1B mutants resulted in the production of abnormally large proplatelet tips, which were reduced in number. Conclusions: Our study provides further proof of concept that GFI1B is an essential protein for the normal development of the megakaryocyte lineage.