Platelet and erythrocyte functional defects caused by mutations of GFI1B

Abstract

GFI1B is a transcriptional repressor that is a major regulator of megakaryocytic and erythroid differentiation and development. The cause of a bleeding disorder in a four- generation Australian family, with cell changes consistent with aberrant platelet structure and function, and red blood cell morphology, showing autosomal dominant inheritance, was identified as being caused by a single nucleotide insertion into the GFI1B gene (c.880_881insC). The aims of this thesis were to study the effects of this mutant GFI1B on erythrocyte development in a cell culture model; to study how loss of Gfi1b function affects megakaryocytic development in a conditional knockout mouse model; and to examine the effects of human GFI1Bc.880_881insC on megakaryocytic / erythroid cell development in a transgenic mouse model, with correlation to the human phenotype.

Separate lines of erythroleukaemic K562 cells were transfected with GFI1BWT and GFI1Bc.880_881insC for studies into red cell formation. Inconclusive results from constant loss of transfection led to the view that a cell model derived from iPSC from affected family members would provide a more stable system for this study.

For the conditional knockout mouse model, C57BL/6J mice with floxed Gfi1b alleles were cross-bred to transgenic mice expressing Cre recombinase under the platelet factor 4 (Pf4) promoter to create a null allele. Mice with retained floxed alleles were controls. The effects of the loss of ­Gfi1b along the megakaryocytic lineage included 60-fold reduction in platelet count; aberrant megakaryocyte morphology, with high DNA ploidy and increased numbers in bone marrow and spleen; high plasma TPO level; enlarged spleens with evidence of stress erythropoiesis; and reduced red cell indices. Reported studies in the literature corroborated these findings.

C57BL/6J transgenic mice with either GFI1BWT or GFI1Bc.880_881insC with a lox-STOP-lox cassette inserted at the collagen Col1a1 locus were crossed with transgenic mice expressing Pf4-Cre for the transgenic mouse model. The premise, that the human mutant GFI1B would cause a dominant negative effect on the endogenous mouse Gfi1b, was not seen in the lack of a bleeding phenotype, and evidence of normal haematological parameters in these mice. A knock-in model with GFI1Bc.880_881insC replacing Gfi1b may be more effective.