OC 27.4 - ZFPM2 regulates megakaryocyte proliferation, differentiation and granulogenesis and influences thrombotic risk

Background: ZFPM2 encodes zinc finger protein ZFPM2 (FOG2), a GATA transcription cofactor implicated in Tetrology of Fallot, congenital diaphragmatic hernia and sex-reversal disorders. ZFPM2 also has megakaryocyte (MK)-restricted gene expression in bone marrow. Common variants in ZFPM2 associate with platelet count (PLT) and side scatter, a proxy of platelet granularity. Although these observations suggest a role for ZFPM2 in megakaryopoiesis, there is currently little experimental evidence for this.

Aims: We explore clinical associations of common variants in ZFPM2 and describe the first in-depth functional characterisation of this gene in a MK cell model.

Methods: Variant-phenotype associations were examined through a Mendelian randomisation (MR) phenome-wide association study of genetic and health outcome data from UK Biobank. A model of reduced ZFPM2 expression was developed using RNP-based CRISPR-Cas9 editing in immortalised MK (imMKCL) cells.

Results: MR analysis revealed that PLT-associated SNP rs6993770 in ZFPM2, which is also a pQTL for multiple alpha-granule proteins, was causally associated with a reduced risk of venous thromboembolism and cardiovascular disease. CRISPR-Cas9 knockdown of ZFPM2 in progenitor imMKCL cells reduced proliferation compared to scrambled controls and increased the proportion of cells in S phase, suggesting cell cycle arrest. Following differentiation, mature ZFPM2 knockdown imMKCL cells were smaller, had lower DNA ploidy and reduced surface expression of MK-maturity marker CD42b compared with controls. The knockdown imMKCL cells also had lower side scatter, altered abundance of transcripts associated with platelet alpha granules and abnormal ultrastructure with conspicuous areas of cytoplasm devoid of organelles. Proplatelet formation was reduced compared to controls.

Conclusion(s): The effect of ZFPM2 knockdown in imMKCL cells indicates a critical role in the proliferation and differentiation of MK with manifestations including abnormal granulogenesis. These observations are consistent with prior GWAS associations between common ZFPM2 variant rs6993770 and platelet traits and provide potential mechanistic explanations for the causal associations with thrombotic disease.