A Dominant-Negative GFI1B Mutation in Gray Platelet Syndrome
Authors: Van der Reijden Bert A. , Monteferrario Davide , Bolar Nikhita , Marneth Anna , Hebeda Konnie , Bergevoet Saskia , Veenstra Hans , Gorkum Britta Laros-van , MacKenzie Marius , Khandanpour Cyrus , Botezatu Lacramioara , Fransen Erik , Van Camp Guy , Duijnhouwer Anthonie , Salemink Simone , Willemsen Brigith , Huls Gerwin , Preijers Franks , van Heerde Waander L. , Jansen Joop H. , Kempers Marlies , Loeys Bart , Van Laer Lut 
Published: October 2019

Abstract
Gray platelet syndrome (GPS) is a hereditary, usually autosomal recessive bleeding disorder caused by defective production of α-granules in platelets. GPS patients show reduced numbers of platelets that are larger and have a typical gray appearance under light microscopy, caused by the lack of α-granules. We describe a large family with an autosomal dominant type of GPS characterized by mild to severe bleeding complications. In addition to large gray platelets, other GPS-associated phenomena like myelofibrosis, thrombocytopenia, and low platelet factor 4 expression were observed in affected individuals. Histopathological examination of a BM biopsy from a patient showed a cellular marrow with increased numbers of megakaryocytes that were pleomorphic in size and shape. Megakaryocytes clustered along BM sinuses and showed dysmorphic stretched features.
To determine the disease causing mutation we performed linkage analysis and identified a candidate locus on chromosome 9q34 with a LOD score of 3.9. We considered GFI1B (Growth Factor Independence 1B), located within this region, an excellent candidate gene because of its function as a transcriptional repressor in megakaryocyte development. Sequence analysis identified a nonsense mutation in GFI1B exon 6 (c.859C>T, p.Gln287*) that completely co-segregated with the GPS disease in this family. The mutated transcript predicts a 44 amino acid C-terminally truncated protein, GFI1BTr. The truncation is located within zinc finger 5 of GFI1B, deleting all of its four amino acids that directly interact with DNA. Luciferase gene reporter assays showed that GFI1BTr was unable to repress gene expression. Importantly, GFI1BTr inhibited gene repression mediated by wild type GFI1B, indicating that the mutant interferes with wild type GFI1B in a dominant-negative manner. To validate that GFI1BTr adversely affects normal GFI1B, we expressed the mutant in mouse bone marrow cells followed by induction of megakaryocytic differentiation. Compared to control cells, GFI1BTr-positive megakaryocytes showed dysplastic features including hypolobulation of the nuclei, irregular contours and multiple separate nuclei, that were very similar to those observed in patient cells. This indicates that GFI1BTr causes megakaryocytic abnormalities and that it functions in a dominant-negative manner.
GFI1B silencing inhibits the development of human megakaryocyte colonies in vitro. We observed that megakaryocyte colony forming cells were significantly more frequent in patient bone marrow compared to controls. In addition, patient-derived megakaryocyte colonies were significantly larger compared to controls. Immunophenotypic analyses of peripheral blood showed no differences in myeloid and erythroid lineages and the platelet markers GP3B, ITGA2B and ITGB3 among affected an non-affected individuals. However, within the ITGA2B/CD41-positive platelet population, 5 of 6 affected members showed a marked decrease in the platelet surface membrane glycoprotein 1b-α (GP1BA/CD42b), compared to unaffected members. In addition, a strong expression of CD34, which is usually confined to immature hematopoietic progenitors, was detected on platelets from all studied affected individuals. Immunostaining of a BM biopsy from a patient showed the presence of ITGB3/CD61 positive megakaryocytes that intensely expressed CD34. Electron microscopy analysis showed megakaryocytes with few, small, irregularly shaped and centrally located α-granules characterized by an extensive peripheral cytoplasm with irregular proplatelets, largely devoid of cell organelles. To test whether these abnormalities were cell intrinsic, we stimulated CD34+ cells from two patients to differentiate along the megakaryocytic lineage in vitro. Megakaryocytic cells showed dysplastic features reminiscent of those observed in the bone marrow aspirates. In addition, increased CD34 and decreased GP1BA/CD42b expression were observed on megakaryocytes, indicating that GFI1BTr-induced abnormalities are intrinsic to the cell. In summary, we have identified GFI1B as a causative gene in autosomal dominant GPS. GFI1BTr acts in a dominant-negative manner over wild type GFI1B and affects the development of megakaryocytes and platelets, demonstrating a pivotal role of GFI1B in governing normal megakaryopoiesis and platelet production.

Disclosures:
No relevant conflicts of interest to declare.