NOVEL MANIFESTATIONS OF IMMUNE DYSREGULATION AND GRANULE DEFECTS IN GRAY PLATELET SYNDROME
Author(s): ,
Janine Collins
Affiliations:
Department of Haematology,University of Cambridge,Cambridge,United Kingdom;Department of Haematology,Barts Health NHS Trust,London,United Kingdom;NHS Blood and Transplant,Cambridge Biomedical Campus,Cambridge,United Kingdom
The NIHR BioResource - GPS Consortium
Affiliations:
Cambridge University Hospitals,Cambridge Biomedical Campus,Cambridge,United Kingdom
(Abstract release date: 05/14/20) EHA Library. Collins J. 06/12/20; 295140; S321
Janine Collins
Janine Collins
Contributions
Abstract

Abstract: S321

Type: Oral Presentation

Session title: New insights into mechanisms and management of bleeding disorders

Background
Gray platelet syndrome (GPS) is an autosomal recessive disorder caused by pathogenic variants in NBEAL2 and characterized by bleeding symptoms, macrothrombocytopenia and severe deficiency of platelet alpha (ɑ)-granules. Nbeal2-/- mice also have an increased susceptibility to infection, defects of secretory granules of neutrophils, monocytes, natural killer and mast cells and their megakaryocytes (MKs) have a pro-inflammatory profile. Therefore, NBEAL2 is important for normal granule function in a variety of myeloid and lymphoid cells in mice. Determining the clinical relevance of this for patients has been challenging because GPS is an ultra-rare inherited disease.

Aims

Our aim was to investigate the spectrum of pathological features in the largest collection of GPS patients to date. We focused on features related to immunity and followed this up with a detailed molecular characterization of plasma, platelets, and three different leukocyte populations in GPS patients.

Methods
We established an international collaboration, recruiting 47 patients from 21 hospitals. Application of human phenotype ontology codes enabled systematic analysis of clinical and laboratory phenotypes. RNA sequencing and proteomics by mass spectrometry (MS) were performed on platelets, neutrophils, monocytes and CD4+ T cells from 5 patients and controls. The plasma of 11 patients and 13 controls was analysed by protein MS.

Results

We identified 72 causal variants (33 novel) in NBEAL2. We catalogued known clinical phenotypes, including a wide spectrum of bleeding diatheses, macrothrombocytopenia, ɑ-granule deficiency, early-onset bone marrow (BM) fibrosis, splenomegaly and elevated serum vitamin B12 levels. Emperipolesis, the engulfment of intact neutrophils, was present in >50% MKs. We also identified novel immune abnormalities in patients, leucopenias (79%), autoantibody positivity (59%) and autoimmune disease (26%).

There were widespread differences in the transcriptome and proteome of not just GPS platelets, but also neutrophils, monocytes and CD4+ T cells. Proteomics analysis showed that in platelets, neutrophils and monocytes, the majority of differentially abundant proteins were down in GPS with an enrichment in gene ontology terms relevant to cell granules. As expected, GPS platelets were markedly diminished in proteins known to localise to ɑ-granules. We also observed the ectopic presence of proteins normally resident in neutrophil granules, in GPS platelets, suggesting that emperipolesis is not just a transient BM phenomenon but impacts circulating platelets. We found a significant over-representation of down proteins in specific and gelatinase granules of neutrophils, suggesting NBEAL2 is critical to retention of granules in neutrophils as well as platelets. In contrast to the other cells, the majority of differentially abundant proteins were up in GPS CD4+ T cells, with an enrichment of immunomodulatory proteins.

Proteomic analysis of GPS plasma identified 51 differentially expressed proteins, with increased levels of proteins associated with inflammation and immune response. One quarter of plasma proteins increased in GPS are synthesized outside of haematopoietic cells, predominantly in the liver. 

Conclusion
We demonstrate that, alongside the haemostasis defect, there is loss of immune homeostasis in GPS, accompanied by autoimmune disease in a quarter of patients. This immune dysregulation appears causally related to abnormalities of the transcriptome and proteome of both innate and adaptive immune cells, resulting in a chronic inflammatory state, reflected in the plasma proteome. 

Session topic: 32. Platelets disorders

Keyword(s): Genetic, Platelet, Proteomics

Abstract: S321

Type: Oral Presentation

Session title: New insights into mechanisms and management of bleeding disorders

Background
Gray platelet syndrome (GPS) is an autosomal recessive disorder caused by pathogenic variants in NBEAL2 and characterized by bleeding symptoms, macrothrombocytopenia and severe deficiency of platelet alpha (ɑ)-granules. Nbeal2-/- mice also have an increased susceptibility to infection, defects of secretory granules of neutrophils, monocytes, natural killer and mast cells and their megakaryocytes (MKs) have a pro-inflammatory profile. Therefore, NBEAL2 is important for normal granule function in a variety of myeloid and lymphoid cells in mice. Determining the clinical relevance of this for patients has been challenging because GPS is an ultra-rare inherited disease.

Aims

Our aim was to investigate the spectrum of pathological features in the largest collection of GPS patients to date. We focused on features related to immunity and followed this up with a detailed molecular characterization of plasma, platelets, and three different leukocyte populations in GPS patients.

Methods
We established an international collaboration, recruiting 47 patients from 21 hospitals. Application of human phenotype ontology codes enabled systematic analysis of clinical and laboratory phenotypes. RNA sequencing and proteomics by mass spectrometry (MS) were performed on platelets, neutrophils, monocytes and CD4+ T cells from 5 patients and controls. The plasma of 11 patients and 13 controls was analysed by protein MS.

Results

We identified 72 causal variants (33 novel) in NBEAL2. We catalogued known clinical phenotypes, including a wide spectrum of bleeding diatheses, macrothrombocytopenia, ɑ-granule deficiency, early-onset bone marrow (BM) fibrosis, splenomegaly and elevated serum vitamin B12 levels. Emperipolesis, the engulfment of intact neutrophils, was present in >50% MKs. We also identified novel immune abnormalities in patients, leucopenias (79%), autoantibody positivity (59%) and autoimmune disease (26%).

There were widespread differences in the transcriptome and proteome of not just GPS platelets, but also neutrophils, monocytes and CD4+ T cells. Proteomics analysis showed that in platelets, neutrophils and monocytes, the majority of differentially abundant proteins were down in GPS with an enrichment in gene ontology terms relevant to cell granules. As expected, GPS platelets were markedly diminished in proteins known to localise to ɑ-granules. We also observed the ectopic presence of proteins normally resident in neutrophil granules, in GPS platelets, suggesting that emperipolesis is not just a transient BM phenomenon but impacts circulating platelets. We found a significant over-representation of down proteins in specific and gelatinase granules of neutrophils, suggesting NBEAL2 is critical to retention of granules in neutrophils as well as platelets. In contrast to the other cells, the majority of differentially abundant proteins were up in GPS CD4+ T cells, with an enrichment of immunomodulatory proteins.

Proteomic analysis of GPS plasma identified 51 differentially expressed proteins, with increased levels of proteins associated with inflammation and immune response. One quarter of plasma proteins increased in GPS are synthesized outside of haematopoietic cells, predominantly in the liver. 

Conclusion
We demonstrate that, alongside the haemostasis defect, there is loss of immune homeostasis in GPS, accompanied by autoimmune disease in a quarter of patients. This immune dysregulation appears causally related to abnormalities of the transcriptome and proteome of both innate and adaptive immune cells, resulting in a chronic inflammatory state, reflected in the plasma proteome. 

Session topic: 32. Platelets disorders

Keyword(s): Genetic, Platelet, Proteomics

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