Understanding granule disorders of myeloid cells by unravelling the interactome and function of the Nbeal2 protein

We have three types of blood cells; red cells carry oxygen to our tissues, white cells fight infections and platelets prevent bleeding. Platelets are smart devices that make the blood clot in the right place, by the right amount, to stop bleeding and allow healing of the damaged vessel wall. Neutrophils, a type of white cell, fight infections by forming traps to capture bugs and producing chemicals to kill them. The flipside to their good roles is that both cells can work together to make blood clot too vigorously. This may lead to clots that prevent blood from reaching the heart or brain, causing a heart attack or stroke.

It is important to perform research to better understand how the function of these cells is regulated. As a haematology doctor I diagnose and treat patients with blood disorders and I have an idea to gain insight into how these cells work based on the very rare inherited bleeding condition Gray Platelet Syndrome (GPS). Patients with GPS bleed because their platelets do not function well and may also have autoimmune disease because their white cells do not function well either. Researchers in Cambridge have discovered that changes in the DNA code of the gene NBEAL2 cause GPS. Not a lot is known about this gene but we do know that the platelets and neutrophils lack certain types of granules. These granules are like packages containing many proteins, which the cells release to help fulfil their important functions. It is not surprising, therefore, that platelets and neutrophils from patients with GPS fail to arrest bleeding or form traps for bugs.

To better understand the function of the Nbeal2 protein researchers disabled the Nbeal2 gene in mice. As well as having problems with the blood system, these mice also have fragile bones and are protected from the spread of cancer showing that the Nbeal2 protein is involved in many important functions. It is reasonable to assume that Nbeal2 does not act alone, but works together with many other proteins to form and retain granules. Results from recent research indicates it works with 64 other proteins. The project I propose is to identify the critical proteins for granule function within this group of 64. To do this, I will use two different but complementary approaches.

Firstly, I will investigate how changes in the Nbeal2 protein identified in patients with GPS alters the interaction with its 64 partner proteins. I will use a new technology to introduce changes in the NBEAL2 gene in stem cells. I will then instruct these gene-modified stem cells to become specialised cells that make platelets. I will use these specialised cells and work with researchers in Dundee to determine whether the gene editing has caused changes in the protein interactions.

Secondly, I will work with mathematicians to investigate whether any of the 64 genes for the Nbeal2 partner proteins are changed in patients with unexplained platelet disorders. Researchers in Cambridge have worked with doctors in hospitals in the UK and overseas to engage with families diagnosed with rare platelet disorders. It is very likely that these unexplained disorders are caused by a change in their DNA code. To find these changes they have so far deciphered the entire DNA code of 1013 patients. If I succeed in identifying a presumed new disease-causing gene, then I will invite the affected families for further research studies to confirm this observation.

My belief is that the knowledge gained from my project will facilitate the discovery of new genes and proteins which are important for the formation and function of granules in blood cells. The research may bring immediate benefits to the care of patients with inherited blood cell disorders because we can readily introduce a DNA test for more rapid diagnosis. I also hope that in the long term the discoveries made by my research will bring improvements to the prevention and treatment of heart attacks and strokes.

The regulation of myeloid cell secretory granules has a key role in atherogenesis and thrombosis. Understanding granule function could lead to new treatments for haemostatic and cardiovascular diseases. I will focus on NBEAL2 variants, which cause Gray Platelet Syndrome (GPS), a rare bleeding disorder. Molecular studies on cells from patients and knockout mice show Nbeal2 is key to PLT alpha-granule and NEU specific granule regulation and function. I will use iPSC technology and CRISPR genome editing to elucidate the mechanism by which NBEAL2 variants cause granule pathology. Preliminary proteomics studies of iMKs have identified 64 proteins interacting with Nbeal2.

Hypothesis 1: GPS variants cause loss of function of one/several interactions with Nbeal2

I will introduce by CRISPR-editing the codons for GPS-causing BEACH-domain variants M2080K, P2100L and G2290W to generate mutants of the line IPSC-S4_NBEAL2_TAP for forward programming to iMKs in biological triplicates (Ghevaert lab protocol; transcription factors GATA-1, FLI-1, TAL-1; tetracycline inducible expression). I will characterise iMKs by ATAC- and RNA- seq, confocal and electron microscopy, platelet formation/function assays and perform pulldowns. Nbeal2's interactions will be defined through proteomics analysis by mass spectrometry (Lamond laboratory) and loss-of-interaction(s) will be confirmed by bi-directional immunoblots, co-localisation and proximity ligation assays.

Hypothesis 2: Proteins in the Nbeal2 interactome have increased likelihood of being candidates for inherited blood cell disorders

I will apply a new Bayesian algorithm to analyse genome sequencing data for ~750 unexplained BPD cases to determine if the 64 genes encoding Nbeal2's interactors are enriched for rare, possibly causal variants. I will verify putative candidate variants using control databases, detailed clinical phenotyping and co-segregation of pedigrees and functional studies using a CRISPR-knockout iPSC line.

1. Immediate beneficiaries

I see potential for positive impact on quality of life for a number of patient groups within the 3-year timeframe of my fellowship.

a) Study participants

Participants in the NIHR BioResource study will receive direct feedback if a new causative variant is identified. Genetic diagnosis helps guide prevention and treatment of excessive bleeding at times of haemostatic challenge (e.g. childbirth). By screening family members, I anticipate identifying some who have a bleeding phenotype who will benefit from education and perhaps pharmacoprophylaxis (e.g. for dental procedures). Genetic counselling and antenatal diagnosis can also be considered.

The impact of GPS-causing variants is of direct interest to patients with GPS. Our increased mechanistic understanding of GPS, combined with detailed clinical phenotyping of our unique collection of patients will allow us to better prognosticate the haemostatic and non-haemostatic abnormalities, enabling appropriate clinical monitoring, preventative strategies and treatment (e.g. understanding osteoporosis seen in knockout mice may translate into bone density imaging and pharmacoprophylaxis).

b) Patients with inherited BPDs

Approximately 60% patients thought to have abnormal granule function based on currently available laboratory tests do not have a molecular diagnosis. Identification of novel causative loci will increase the diagnostic sensitivity of the ThromboGenomics HTS platform. In the longer term, there is potential to improve treatment for this rare disease group.

2. Longer term beneficiaries

a) Patients with or at risk of cardiovascular disease

The regulation of myeloid cell granules has a key role in atherogenesis and thrombosis. By unpicking the function of Nbeal2 and its protein interactors, we will make progress in understanding how PLT alpha granule and NEU specific granule formation/retention is orchestrated. This may open up avenues for a new generation of therapeutics that regulate granules for prevention and/or treatment of coronary artery disease and thrombotic stroke.

b) Patients with clinical features associated with GPS

GPS is more than just a disorder of haemostasis. The vast array of proteins in alpha granules are central to processes of inflammation, angiogenesis and wound healing. We postulate that non-GPS patients with associated non-haemostatic clinical features including bone marrow fibrosis and aggressive early-onset autoimmune conditions may have variants in NBEAL2 or interacting genes, and this research could improve diagnostic pathways for these patients.

c) Pharmaceutical companies

The identification of new molecular pathways to explain granule function disorders has commercial private sector beneficiaries, through potential for development of innovative treatments to regulate granule release, with opportunity for widespread clinical benefit in common and rare disease groups as described above. Contributions to pharmaceutical developments positively impacts on the UK economy.

d) Health Policy and commissioning

I recognise it takes many years for new knowledge to translate into new treatments for patients, however, through contributing to the genomics revolution, my work will feed into established health policy infrastructure, such as the UK Strategy for Rare Diseases, which aims to provide the best possible quality of evidence based care for patients with rare diseases like inherited BPDs. Increased knowledge also supports charities to provide education and resources to patients and the public.

The identification of new genetic and molecular mechanisms for atherosclerosis will in the era of precision medicine contribute to risk stratification, relevant to clinical decision makers (e.g. GPs) and Clinical Commissioning Groups. Prioritisation of resources for preventative management strategies relies on understanding mechanisms of common diseases like atherosclerosis.