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76 A Dominant Negative Mutation (p.P214L) in ETV6 is Associated with Megakaryocyte and Erythroid Transcript Misregulation

Disorders of Platelet Number or Function
Program: Oral and Poster Abstracts
Type: Oral
Session: 311. Disorders of Platelet Number or Function: Genetics of Inherited and Acquired Thrombocytopenias
Saturday, December 5, 2015: 12:45 PM
W315, Level 3 (Orange County Convention Center)

Leila Noetzli, BS1, Jesse W Rowley, PhD2*, Michael U Callaghan, MD3, Madhvi Rajpurkar, MD4, Alisa B. Lee-Sherick, MD5, Christopher C. Porter, MD6, Andrew S. Weyrich, PhD7 and Jorge A. Di Paola, MD6

1Department of Pediatrics, University of Colorado Denver Anschutz Medical Campus, Aurora, CO
2Program in Molecular Medicine University of Utah, Salt Lake City, UT
3Children's Hospital of Michigan, Detroit Medical Center, Detroit, MI
4Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, MI
5Pediatrics, University of Colorado and Children's Hospital Colorado, Aurora, CO
6Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO
7The Program in Molecular Medicine, University of Utah, Salt Lake City, UT

Background:  Our group has recently described a family with autosomal dominant thrombocytopenia, high mean corpuscular volume (MCV) of red cells, and 2 occurrences of pre B-cell acute lymphoblastic leukemia (ALL) associated with a mutation in the transcription factor ETV6. ETV6 is a transcriptional repressor which functions through dimerization with itself or with other transcription factors such as FLI1, mutations in which cause a familial form of thrombocytopenia called Paris-Trousseau syndrome. The missense mutation found in this family (p.P214L) has a dominant negative effect by dimerizing with and sequestering WT ETV6 from the nucleus. This dominant negative mutation causes a phenotype in both platelets and red cells, suggesting a disruption in megakaryocyte-erythroid differentiation. Therefore, we aimed to study the effect of p.P214L ETV6 on megakaryocyte-erythroid differentiation by studying the platelet transcriptome, protein from platelet lysates, and platelet surface markers of patients with the ETV6 mutation. To investigate whether this platelet and red cell phenotype is due to interactions with FLI1, which is essential for megakaryocyte-erythroid lineage determination, we also aimed to study the interaction of p.P214L ETV6 with FLI1.

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.

*signifies non-member of ASH