Program: Oral and Poster Abstracts
Type: Oral
Session: 311. Disorders of Platelet Number or Function: Genetics of Inherited and Acquired Thrombocytopenias
Methods: RNA was isolated from leukoreduced platelets of 2 patients with the p.P214L mutation (affected), 3 unaffected family members, and 2 unrelated controls for whole transcriptome analysis. GPIX quantification from platelet lysates of 2 affected patients and 3 controls was done by Western blot. Analysis of protein dimerization was done by expression of differentially tagged ETV6 and FLI1 constructs (His or c-myc) in HEK293T cells, pull down with anti-c-myc beads, and quantification of both c-myc and His by Western blot. To determine platelet receptor levels, flow cytometry was done with antibodies against GPIIb, GPIIIa, GPIX, and GPIb on peripheral blood from one affected patient and one control.
Results: Platelet transcriptome analysis of patients with the p.P214L mutation revealed significant differential expression of more than 200 transcripts between patients and controls. Seven of the significant transcripts were key erythrocyte transcripts, which were upregulated in affected patients; and 9 transcripts were key platelet transcripts, which were downregulated. Of these, the red cell marker GYPA was upregulated 25 fold in affected patients (p < 5x10-5), and the platelet markers VWF and GPIX, a target of FLI1, were downregulated 6.4 and 5.6 fold in affected patients (p < 0.05 and p < 0.07), respectively. Platelet GPIX protein expression was also downregulated in 2 affected patients as determined by Western blot. Furthermore, flow cytometry of platelets from one patient with the p.P214L mutation indicated a marginally decreased Ib-IX:IIb-IIIa ratio (0.321) when compared to a normal control (0.500). In vitro studies confirmed that the p.P214L mutation did not disrupt dimerization with WT ETV6 or FLI1, indicating that this defect could be due to dominant negative interactions with both ETV6 and FLI1.
Conclusions: Several transcription factors, including FLI1, have been described to play a role in the fate determination between erythroid and megakaryocyte lineages. We report that a dominant negative mutation in ETV6, found in a family with both platelet and red blood cell abnormalities, is associated with differentially regulated red blood cell and platelet transcripts, decreased GPIX protein in platelet lysates, decreased Ib-IX:IIb-IIIa ratio, and dimerization with WT ETV6 and FLI1 in vitro. Our data indicate an important role for ETV6, either alone or through interaction with FLI1, in megakaryocyte-erythroid differentiation.
Disclosures: No relevant conflicts of interest to declare.
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