New Constitutional GATA1 Variants Study Reveals the Contribution of GATA1 in MYH10 Silencing During Megakaryopoiesis
ISTH Academy. Saultier P. Jul 10, 2019; 274054; OC 72.3 Topic: Platelet Disorders, Hereditary
Dr. Paul Saultier
Dr. Paul Saultier
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OC 72.3

New Constitutional GATA1 Variants Study Reveals the Contribution of GATA1 in MYH10 Silencing During Megakaryopoiesis

P. Saultier1, S. Cabantous1, M. Puceat2, J. Van Agthoven3, M. Canault1, F. Peiretti1, J.-C. Bordet4, P.-E. Morange1, M.-C. Alessi1, M. Poggi5
1C2VN INSERM 1263, Faculté de Médecine, Marseilles, France, 2Inserm 1251, Team Physiopathology of Cardiac Development, Marseilles, France, 3Structural Biology Program, Department of Medicine, Charlestown, United States, 4HCL Hôpital Cardiologique Louis Pradel, Unité d'Hémostase Biologique, Lyon, France, 5C2VN INSERM 1263, Marseilles, France

Main Topic: Platelet Disorders
Category: Platelet Disorders, Hereditary

Background: Megakaryocyte (MK) polyploidization requires MYH10 expression silencing through the action of transcription repressors (ie RUNX1 and FLI1). We identified two new constitutional GATA1 variants c.617A>T (F1) and c.802C>A (F2) in thrombocytopenic patients.
Aims: To study the role of GATA1 in MYH10 regulation and to characterize the phenotype of the GATA1 variants´ carriers.
Methods: Platelet function, granule content and MYH10 expression were investigated (aggregation and mepacrine fluorescence assays, electron microscopy, western-blot). Patients CD34+ cells-derived MKs differentiation was evaluated (flow cytometry). Transfected cells were used to study GATA1 subcellular localizations and partner interactions. Transcriptional regulation was analyzed using luciferase reporter plasmid.
Results: Severe thrombocytopenia (15x109/l) was noticed at diagnosis in F1-patient, whereas the initially normal platelet counts observed in the two F2-affected siblings progressively dropped during childhood (>50x109/l). At the time of normal platelet count, F2 patients suffered spontaneous bleedings associated with aggregation defects. We identified platelet granule deficiencies in carriers. Bone marrow smears showed hypolobular MKs. Patients-derived MKs were smaller then controls and showed ploidy defect. MYH10 overexpression found in the GATA1 variant carriers' platelets suggests a defective silencing during megakaryopoiesis. Genetic variations did not alter GATA1 nuclear localization and disrupted GATA1-FOG1 interaction was noticed in F1. Analysis of normal megakaryocytes ChIP-seq data (Tijssen et al., 2011) showed GATA1, FLI1 and RUNX1 co-occupancy in a promoter and an intronic regions of MYH10. The two identified sequences were cloned in luciferase-reporter constructs. Each sequences associated with decreased luciferase activity in the hematopoietic-cell line HEL. In MSR-cells co-transfected with reporter construct and GATA1, only the intronic region associated with altered luciferase activity. Expression of GATA1 variants led to a reduction in regulatory activity.
Conclusions: Thrombocytopenia severity differs with the GATA1 variants. We propose MYH10 as a new GATA1 target involving both a promoter and an intronic regulatory element.

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