Contributions
Abstract: S434
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 12:15 - 12:30
Location: Room N101
Background
GFI1B is a transcription factor that plays an important role in haematopoiesis. Families with a mutation of the fifth DNA-binding zinc-finger domain of GFI1B experience bleeding and have a platelet phenotype characterised by macrothrombocytopenia, increased CD34 expression and alpha-granule deficiency.
Aims
To explore the function of other zinc finger domains of GFI1B we have characterised two unrelated families with a GFI1B variant, C168F, predicted to disrupt the first Zn-finger domain and compared the phenotype with a previously described pedigree with the H294fs mutation that disrupts the fifth Zn-finger domain.
Methods
Clinical platelet phenotypes were determined by light and transmission electron microscopy and functional studies performed by light transmission and whole blood impedance aggregometry. Platelet protein expression was measured by flow cytometry and western blotting. DNA-binding of variants was determined by gel mobility shift assays (EMSA) and changes in gene transcription by luciferase assays. Cellular phenotypes were then studied in patient specific iPSC derived megakaryocytes.
Results
Individuals with GFI1B C168F are thrombocytopenic (mean platelet count =107 x109/L, n=8) but lack the collagen induced aggregation defects and bleeding symptoms observed in individuals with H294fs (ISTH BAT, P=0.015). Alpha granule content observed by microscopy and quantitated by western blotting of granule related proteins, P-selectin and fibrinogen, were similar between C168F and control platelets and this was significantly greater than that observed for the H294fs mutation (P<0.01). EMSA studies indicate that the C168F variant retains the ability to bind DNA whereas the H294fs mutation altering Zn finger 5 abrogates DNA binding. Despite retaining the ability to bind DNA, the C168F variant de-represses gene transcription at TUBB1, MEIS1 and CD34 target genes (P<0.01). This de-repression is less marked than that observed with the non-DNA-binding H294fs mutation (P<0.01). The transcriptional de-repression observed at the CD34 promoter with both Zn finger domain 1 and 5 variants was validated by an increase in platelet surface CD34 measured by flow cytometry and total CD34 protein measured by western blotting (P<0.01). This increased CD34 expression appears specific for GFI1B mutation as increased CD34 expression was not observed in platelets derived from individuals with FLI1, RUNX1 or MYH9 mutation. To validate these clinical observations, iPSCs were generated from the different pedigrees and megakaryocyte differentiation performed in vitro. Megakaryocyte CD34 expression was increased in cells derived from individuals with both C168F and H294fs variants but alpha granule deficiency was only observed in cells containing the non-DNA-binding H294fs mutation.
Conclusion
Mutations altering GFI1B Zn finger 1 cause thrombocytopenia with increased CD34 expression but these platelets retain relatively normal alpha-granule content and these individuals lack clinical bleeding symptomatology. This mutation is mechanistically distinct from the Zn finger 5 mutation that abrogates DNA binding with a subsequent phenotype characterised by alpha-granule deficiency and clinical bleeding.
Session topic: 32. Platelets disorders
Keyword(s): Thrombocytopenia, Inherited platelet disorders, transcription factor
Abstract: S434
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 12:15 - 12:30
Location: Room N101
Background
GFI1B is a transcription factor that plays an important role in haematopoiesis. Families with a mutation of the fifth DNA-binding zinc-finger domain of GFI1B experience bleeding and have a platelet phenotype characterised by macrothrombocytopenia, increased CD34 expression and alpha-granule deficiency.
Aims
To explore the function of other zinc finger domains of GFI1B we have characterised two unrelated families with a GFI1B variant, C168F, predicted to disrupt the first Zn-finger domain and compared the phenotype with a previously described pedigree with the H294fs mutation that disrupts the fifth Zn-finger domain.
Methods
Clinical platelet phenotypes were determined by light and transmission electron microscopy and functional studies performed by light transmission and whole blood impedance aggregometry. Platelet protein expression was measured by flow cytometry and western blotting. DNA-binding of variants was determined by gel mobility shift assays (EMSA) and changes in gene transcription by luciferase assays. Cellular phenotypes were then studied in patient specific iPSC derived megakaryocytes.
Results
Individuals with GFI1B C168F are thrombocytopenic (mean platelet count =107 x109/L, n=8) but lack the collagen induced aggregation defects and bleeding symptoms observed in individuals with H294fs (ISTH BAT, P=0.015). Alpha granule content observed by microscopy and quantitated by western blotting of granule related proteins, P-selectin and fibrinogen, were similar between C168F and control platelets and this was significantly greater than that observed for the H294fs mutation (P<0.01). EMSA studies indicate that the C168F variant retains the ability to bind DNA whereas the H294fs mutation altering Zn finger 5 abrogates DNA binding. Despite retaining the ability to bind DNA, the C168F variant de-represses gene transcription at TUBB1, MEIS1 and CD34 target genes (P<0.01). This de-repression is less marked than that observed with the non-DNA-binding H294fs mutation (P<0.01). The transcriptional de-repression observed at the CD34 promoter with both Zn finger domain 1 and 5 variants was validated by an increase in platelet surface CD34 measured by flow cytometry and total CD34 protein measured by western blotting (P<0.01). This increased CD34 expression appears specific for GFI1B mutation as increased CD34 expression was not observed in platelets derived from individuals with FLI1, RUNX1 or MYH9 mutation. To validate these clinical observations, iPSCs were generated from the different pedigrees and megakaryocyte differentiation performed in vitro. Megakaryocyte CD34 expression was increased in cells derived from individuals with both C168F and H294fs variants but alpha granule deficiency was only observed in cells containing the non-DNA-binding H294fs mutation.
Conclusion
Mutations altering GFI1B Zn finger 1 cause thrombocytopenia with increased CD34 expression but these platelets retain relatively normal alpha-granule content and these individuals lack clinical bleeding symptomatology. This mutation is mechanistically distinct from the Zn finger 5 mutation that abrogates DNA binding with a subsequent phenotype characterised by alpha-granule deficiency and clinical bleeding.
Session topic: 32. Platelets disorders
Keyword(s): Thrombocytopenia, Inherited platelet disorders, transcription factor