PB0355 - Knock-in of homozygous NBEAL2 variants to model gray platelet syndrome in megakaryocytes derived from CRISPR-Cas9-edited pluripotent stem cells

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Janine Collins, MB BChir MRCP (she/her/hers)

Clinical Research Training Fellow
University of Cambridge
Loughton, England, United Kingdom

Co-Author(s)

HF

Holly R. Foster

Postdoctoral researcher
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre
Cambridge, England, United Kingdom
LM

Louisa Mayer

Research Associate
University of Cambridge
New York, New York, United States
AC

Alexi Crosby, PhD

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom
AE

Amanda L. Evans

Research Associate
University of Cambridge
Cambridge, England, United Kingdom
WL

Winnie Lau

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom
SM

Samantha Mason

PhD Fellow
University of Cambridge
Cambridge, England, United Kingdom
RM

Rebecca McDonald

PhD Fellow
University of Cambridge
Cambridge, England, United Kingdom
TV

Thomas Vallance

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom
Amie K. Waller, PhD

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom
WO

Willem H. Ouwehand

Group Coordinator and Principal Investigator
University of Cambridge
Cambridge, England, United Kingdom
Ernest Turro, PhD

Associate Professor
Icahn School of Medicine at Mount Sinai
New York, New York, United States
Cedric Ghevaert, MD PhD

Professor of Transfusion Medicine
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre
Cambridge, England, United Kingdom
JG

Jose A. Guerrero

Postdoctoral researcher
University of Cambridge
Cambridge, England, United Kingdom

Background: Gray Platelet Syndrome (GPS) is an ultra-rare autosomal recessive disorder caused by rare variants in NBEAL2. GPS is characterised by macrothrombocytopenia and platelet alpha-granule deficiency, with heterogeneous clinical features including bleeding, bone marrow fibrosis, and immune dysfunction.

Aims: To characterise the cellular consequences of function-altering amino acid changes in NBEAL2 using transcriptomic and proteomic analysis of iPSC-derived megakaryocytes (MKs) carrying homozygous GPS-causing variants.

Methods: Two single nucleotide variants in the NBEAL2 BEACH-domain, causative of GPS and predicted to lead to loss-of-function by crystallography modelling, were introduced into wild-type (WT) iPSCs using CRISPR-Cas9 gene-editing. The WT, homozygous knock-in (M2080K, P2100L), and NBEAL2 knockout (KO) iPSCs were differentiated into megakaryocytes (MKs). Following flow cytometry assessment of CD41/CD42a expression to determine MK maturity, RNA was extracted and sequenced, and MK lysates were analysed by protein tandem mass tag mass spectrometry (MS).

Results: MK protein abundance and gene expression were positively correlated (r = 0.32, p < 2.2 x10-16). RNA-sequencing revealed 90 differentially expressed genes in M2080K, 339 in P2100L, and 343 in KO MKs compared with WT (posterior probability of differential expression >0.5). Protein MS identified 37, 95, and 73 differentially abundant proteins in M2080K, P2100L, and KO MKs compared with WT, respectively (absolute log2-fold-change >1; Benjamini-Hochberg-adjusted p< 0.05) (Figure 1). The knock-ins shared 25 differentially abundant proteins, of which three, including a transcription factor at 1q21.3 were up-regulated, and 22 were down-regulated, including NBEAL2, thrombospondin, and 10 additional alpha-granule proteins (Figure 2). NBEAL2 abundance was lower in KO than knock-in MKs (Figures 1&2). PPBP was the only shared protein with a significantly differentially expressed transcript in knock-in MKs.

Conclusion(s): Homozygous GPS-causing variants result in an altered cellular endophenotype, in keeping with our observations in platelets from GPS patients. Interestingly, this new model of GPS has revealed novel differences in protein abundance, including increased transcription factor levels.