NBEAL2 Variants Cause Extensive Immune Dysregulation, Granule Defects in Gray Platelet Syndrome

Molecular and cellular characterization of gray platelet syndrome (GPS) suggests that patients with GPS have immune dysregulation accompanied by autoimmune disease, driven by abnormalities in both innate and adaptive immune cells, according to a study presented during the Virtual Edition of the 25^th European Hematology Association (EHA) Annual Congress. Results from the international collaboration were presented by Janine Collins of the University of Cambridge in the United Kingdom, on behalf of the NIHR BioResource-Rare Diseases Gray Platelet Syndrome Consortium.

“This very rare clinical condition was first described in 1971,” explained Dr Collins, “in GPS we see that platelets are large, often giants and pale or grey in appearance. The reason the platelets are gray is because…the alpha granules, which are normally fairly abundant are either severely deficient or even absent in the platelet syndrome.”

The aim of the study was to investigate the spectrum of pathological features in a large collection of patients with GPS (47 patients). In addition to genotyping genetic variants in the NBEAL2 gene, which is known to cause GPS, the investigators used mass spectrometry for RNA sequencing and proteomics to characterize platelets, neutrophils, CD4+ T cells, and monocytes from 5 patients with GPS and 5 controls. They also conducted a detailed molecular characterization of plasma in 11 patients and 13 controls using mass spectrometry.

Transcriptomic and proteomic analyses revealed that platelets, neutrophils, and monocytes, had reduced protein abundance (platelet ɑ-granules, tertiary granules, and secretory granule lumen) in GPS compared with control cells, but an upregulation of immunomodulatory proteins in CD4+ T cells. As expected, proteins related to ɑ-granules were lower in GPS platelets relative to control platelets; however, ɑ-granules were also lower in the other cell types.

Proteins typically associated with neutrophil granules were identified in GPS platelets, while other proteins known to be associated with specific and gelatinase granules of neutrophils were reduced relative to those of controls. This demonstrated the importance of NBEAL2 for granules in neutrophils as in platelets.

In NBEAL2, 72 causal variants, 33 of which were novel, were identified in the patients. Known clinical phenotypes of GPS were observed, including a wide spectrum of bleeding diatheses, macrothrombocytopenia, ɑ-granule deficiency, early-onset bone marrow fibrosis, splenomegaly, and elevated serum vitamin B12 levels. Emperipolesis was present in more than 50% of megakaryocytes. The investigators also described novel immune abnormalities among patients with GPS, including leukopenias (79%), autoantibody positivity (59%), and autoimmune disease (26%).

Proteomic analysis of the plasma from 11 patients with GPS and 13 controls revealed 51 differentially expressed proteins; 11 of these proteins were upregulated in GPS and 40 were downregulated. Elevated levels of proteins were associated with inflammation and immune response in GPS samples relative to the control samples.

“As a consequence of pathogenic NBEAL2 variants, we see changes in the count, granularity, transcriptome, and proteome, of not just platelets but innate and adaptive immune cells,” said Dr Collins. “We saw a proinflammatory signature both at the cellular and systemic plasma level, and we believe this contributes to the novel finding of immune dysregulation in this rare bleeding disorder.”

Reference

Collins J, The NIHR BioResource – GPS Consortium. Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome. Presented at: Virtual Edition of the 25^th Hematology Association (EHA) Annual Congress. June 2020. Abstract S321.