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Developmental mutation identified in gray platelet syndrome

Issue: January 10, 2014

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NEW ORLEANS — Researchers have identified growth factor independent 1B as a causative gene in autosomal dominant gray platelet syndrome, altering the development of megakaryocytes and platelets, according to study findings presented at the ASH Annual Meeting and Exposition.

Patients with gray platelet syndrome — a hereditary bleeding disorder — exhibit reduced numbers of platelets, which are enlarged with a gray appearance caused by the shortage of alpha-granules.

 

Bert A. Van der Reijden

Bert A. Van der Reijden, PhD, from Radboud University Medical Center in Nijmegen, the Netherlands, and colleagues examined a large family with an autosomal dominant type of gray platelet syndrome characterized by mild to severe bleeding complications. Gray platelet syndrome-associated phenomena like myelofibrosis, thrombocytopenia and low platelet factor 4 expression were observed in affected individuals.

Histopathological examination of a bone marrow biopsy from a patient showed a cellular marrow with increased numbers of megakaryocytes that were pleomorphic in size and shape. Megakaryocytes were clustered along the bone marrow sinuses and displayed dysmorphic stretched features.

To establish the disease-causing mutation, researchers performed linkage analysis and identified a candidate locus on chromosome 9q34 with a LOD score of 3.9. Growth factor independent 1B (GFI1B), located within this region, was regarded as an excellent candidate gene due to its function as a transcriptional repressor in megakaryocyte development.

Through sequence analysis, Van der Reijden and colleagues identified a nonsense mutation in GFI1B exon 6 that completely cosegregated with the gray platelet syndrome disease in this family. The mutated transcript predicts a 44 amino acid C-terminally truncated protein, GFI1B^Tr.

Although luciferase gene reporter assays demonstrated that GFI1B^Tr was unable to repress gene expression, GFI1B^Tr was observed to inhibit gene repression mediated by wild-type GFI1B, indicating that the mutant interferes with wild-type GFI1B in a dominant-negative manner.

To confirm that GFI1B^Tr adversely affected normal GFI1B, the researchers expressed the mutant in mouse bone marrow cells followed by induction of megakaryocytic differentiation. Compared to control cells, GFI1B^Tr-positive megakaryocytes showed dysplastic features, including hypolobulation of the nuclei, irregular contours and multiple separate nuclei.

Van der Reijden and colleagues observed that megakaryocyte colony-forming cells were significantly more frequent in patient bone marrow vs. controls. In addition, patient-derived megakaryocyte colonies were significantly larger compared with controls.

Immunophenotypic analyses of peripheral blood showed no differences in myeloid and erythroid lineages and the platelet markers GP3B, ITGA2B and ITGB3 among affected and non-affected individuals. However, within the ITGA2B/CD41-positive platelet population, five of six affected members showed a marked decrease in the platelet surface membrane glycoprotein 1b-alpha (GP1BA/CD42b) vs. unaffected members. In addition, researchers detected a strong expression of CD34 — typically confined to immature hematopoietic progenitors — on platelets from all affected individuals.

Immunostaining of a bone marrow biopsy from a patient showed the presence of ITGB3/CD61 positive megakaryocytes that strongly expressed CD34. Electron microscopy analysis showed megakaryocytes with few, small, irregularly shaped and centrally located alpha-granules characterized by an extensive peripheral cytoplasm with irregular proplatelets, largely devoid of cell organelles.

To test whether these abnormalities were cell intrinsic, researchers 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 GFI1B^Tr-induced abnormalities are intrinsic to the cell.

For more information:

Van der Reijden B. Abstract LBA-3. Presented at: 55th ASH Annual Meeting and Exposition; Dec. 7-10, 2013; New Orleans.

Disclosure: The researchers report no relevant financial disclosures.