OC 64.2 - Platelet RNA sequencing and generation of an imMKCL-based cell model for SLFN14 K219N deficiency show evidence of increased cellular stress

Tuesday, July 12, 2022

3:00 PM – 3:15 PM

Location: ExCel Center, ICC Capital Suite Room 14-16

Presentation Format: In-Person

Presenting Author(s)

Fabienne Ver Donck, MS

PhD student
Center for Molecular and Vascular Biology, University of Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium

Co-Author(s)

KR

Kato Ramaekers

PhD student
Center for Molecular and Vascular Biology, University of Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
CT

Chantal Thys

Technician
KU Leuven
Leuven, Vlaams-Brabant, Belgium
KP

Kathelijne Peerlinck

MD, PhD
University Hospitals Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
CV

Chris Van Geet

MD
Paediatric Hemato-Oncology, University Hospitals Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
KE

Koji Eto

professor
Center for iPS Cell Research and Application, Kyoto University
Kyoto, Kyoto, Japan
VL

Veerle Labarque

MD
Paediatric Hemato-Oncology, University Hospitals Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
Kathleen Freson, PhD

Professor
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium.
Leuven, Vlaams-Brabant, Belgium

Background: Single nucleotide variants in SLFN14, which encodes an RNA endoribonuclease protein, are known to cause inherited thrombocytopenia. Patients with a SLNF14 variant also present with reduced platelet aggregation and ATP secretion. Despite mild laboratory defects, these patients display an obvious bleeding phenotype. The function of SLFN14 in megakaryocyte and platelet biology is not studied.

Aims: This study aims to characterize the platelet transcriptome in patients with a SLFN14 K219N variant using total RNA sequencing and to model the disease using the immortalized MegaKaryocyte Cell Line imMKCL (Nakamura et al. Cell Stem Cell 2014).

Methods: Patients’ platelet RNA was extracted and used for total RNA sequencing. Bioinformatics analysis was performed in R using DESeq2 and WGCNA packages. The K219N variant was introduced in imMKCL using CRISPR/Cas9.

Results: The SLFN14 K219N variant was detected by WES in thrombocytopenia patients from a large pedigree. Platelet RNA was sequenced for two patients and 12 healthy controls. Differential gene expression analysis yielded 2831 and 2860 significantly (|log2FC|>1, FDR < 0.05) upregulated and downregulated genes, respectively (Figure 1). Top 500 up- and downregulated genes were used for Reactome pathway analysis. Upregulated genes were enriched for mRNA splicing, rRNA processing and regulation of HSF1-mediated heat shock response (all indicative of increased cellular stress). Downregulated genes were enriched for platelet function pathways (mainly integrin αIIbβ3). Gene co-expression network analysis confirmed these pathways. Heterozygous SLFN14 K219N imMKCL showed a pronounced defect in megakaryocyte differentiation and proplatelet-formation compared to the wild type condition. Heterozygous SLFN14 defective megakaryocytes contain numerous multilamellar bodies that occur after increased cellular stress. An almost complete block in megakaryopoiesis was detected for the homozygous SLFN14 K219N imMKCL.

Conclusion(s): Our results indicate dysregulation of gene transcription and translation as potential disease mechanism underlying SLFN14-related thrombocytopenia. We are currently characterizing the endoribonuclease properties of mutant SLFN14.