OC 45.5 - Transcriptomic analysis of megakaryocytes and platelets to model inherited thrombocytopenia

Monday, June 24, 2024

15:45 – 16:00 ICT

Room: 110 A-C

Presenting Author(s)

Koenraad De Wispelaere, MS

PhD Researcher
Department of Genetics and Genomics, Icahn school of medicine at Mount Sinai
New York, New York, United States

Co-Author(s)

Ernest Turro

Associate Professor
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai
New York, New York, United States
Kathleen Freson

Professor
Department of Cardiovascular Sciences, Centre for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
CT

Chantal Thys

Technician
KU Leuven
Leuven, Vlaams-Brabant, Belgium
Fabienne Ver Donck

PHD
KU Leuven
Leuven, Vlaams-Brabant, Belgium
Kato Ramaekers, MA

PhD Candidate
KU Leuven
Leuven, Vlaams-Brabant, Belgium
KE

Koji Eto

Professor
CiRA
Kyoto, Kyoto, Japan
Veerle Labarque

Professor
Department of Pediatric Hematology and Oncology, University Hospital Leuven Gasthuisberg, Leuven
ฺฺBrussels, Brussels Hoofdstedelijk Gewest, Belgium

Background: RNA is transported from megakaryocytes (MKs) into platelets, which lack a nucleus. Platelets from patients with inherited thrombocytopenia (IT) exhibit substantial dysregulation in gene expression. We sought to investigate the extent to which such transcriptomic changes are present in MKs carrying mutations responsible for IT.

Aims: To overcome the difficulty of obtaining bone marrow-derived MKs from IT patients, we explored the use of genetically modified immortalized MK progenitor cell lines (imMKCLs). We focussed on Bernard Soulier syndrome (homozygous GP9 deletion) and IKZF5-related thrombocytopenia (heterozygous Tyr121Phe). These disorders are caused by a defect in an MK-specific glycoprotein receptor and a defect in a transcription factor, respectively.

Methods: We studied MK differentiation, polyploidisation and proplatelet formation, performing bulk RNAseq at differentiation days 0, 1, 4 and 5. We used statistical model selection to categorize genes into their patterns of expression and compared the transcriptomes of imMKCL with those of control MKs, control platelets and patient platelets.

Results: GP9-/- MK lacked GPIb/IX/V expression but differentiated to CD41+ MK with normal ploidy but severely impaired proplatelet formation (96% reduction). IKZF5Y121P/WT differentiation showed CD42+/CD41+ MK with normal ploidy and a significant though mild defect in proplatelet formation (57% reduction). RNAseq of MK during differentiation revealed broad transcriptomic effects of both mutations (Fig1). While IKZF5Y121P/WT predominantly causes changes which are constant during differentiation as expected for a transcription factor, GP9-/- mostly affects genes that are important in the late stage of differentiation. The differentiation trajectory of these MK is currently being compared to platelet transcriptomes from patients with similar variants.

Conclusion(s): These data suggest that a defect in proplatelet formation impairs transcript trafficking independent of the type of molecular defect though variant-type specific changes were noticed. This has consequences when studying platelet transcriptomes of IT patients.