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Genetic and functional studies in inherited platelet disorders https://doi.org/10.17863/CAM.126386 Collins, Janine 0000000287163261 Ouwehand, Willem Platelets are anucleate blood cells containing secretory granules, which play a critical role in haemostasis, atherogenesis, thrombosis, and wound healing. Platelet number, volume, and function are genetically regulated, and genome wide association studies have identified more than 1000 loci associated with platelet traits. Common variants, with a minor allele frequency (MAF) ≥1% are associated with mild variation in platelet parameters, whereas rare variants (MAF <1%) are associated with extreme phenotypes and inherited platelet disorders (IPDs). The polygenic score (PGS) for platelet count is constructed from 739 common variants, explaining almost 20% of phenotypic variance. The accurate determination of effect sizes of rare variants causal of IPDs is essential for reliable reporting of variant pathogenicity to clinicians and patients. However, this has only recently become feasible for rare disease-causing variants with the release of the whole exome sequencing (WES) and associated clinical data for 500,000 UK Biobank (UKB) participants. The first part of this thesis reports on the interrogation of UKB data to determine, for the first time, the effect sizes on platelet parameters of reported rare pathogenic and likely pathogenic variants, deemed causal of IPDs. The contribution of PGS to incomplete penetrance of a subset of rare variants was estimated and this illustrated that potentially causal rare variants need to be considered in the context of an individual’s genetic architecture. The clinical sequelae of carrying a single pathogenic or likely pathogenic variant for a recessive IPD was explored, finding that loss-of-function variants in genes for Bernard-Soulier syndrome, Glanzmann thrombasthenia and congenital amegakaryocytic thrombocytopenia (CAMT) had a significant effect on platelet count in carriers. In the case of CAMT-causing MPL variants, there was an unexpected increase in platelet count in carriers. Despite major advances in genetic diagnosis through high-throughput sequencing diagnostic gene panels, WES and whole genome sequencing projects, approximately 50% patients with suspected IPDs remain molecularly uncharacterised; this makes patient-specific haemostasis management challenging. To date, there are 69 diagnostic-grade genes for IPDs. NBEAL2, the gene in which biallelic pathogenic variants cause gray platelet syndrome (GPS), was discovered in 2011. The second and third parts of this thesis report on studies of the largest collection of patients with GPS and a cellular model of GPS. GPS is an ultra-rare autosomal recessive disorder, characterised by macrothrombocytopenia, severe deficiency of platelet alpha (α)-granules, and a variable bleeding tendency. Patients may develop splenomegaly, and in the bone marrow, emperipolesis (the engulfment of neutrophils by megakaryocytes (MKs)) is observed, as well as an increased incidence of early onset fibrosis. Nbeal2−/− mice have defects of secretory granules in a variety of myeloid and lymphoid cells, increased susceptibility to infection, and their MKs have a proinflammatory profile. Determining the clinical relevance for patients has been challenging due to extreme rarity of cases. Through the National Institute for Health and Care Research BioResource Rare Diseases GPS study, an international collaboration was established to investigate the spectrum of pathological features in patients with GPS. The application of human phenotype ontology terms enabled systematic analysis of clinical and laboratory phenotypes, and identified novel immune abnormalities in patients, including leukopenias, autoantibody positivity, and autoimmune disease. There were widespread differences in the transcriptome and proteome of platelets in patients with GPS, but also in neutrophils, monocytes, and CD4-lymphocytes. Proteomic analysis of plasma by mass spectrometry (MS) identified increased levels of proteins associated with inflammation and the immune response in patients with GPS. Additionally, proximity extension assay analysis identified differential expression of chemokines and cytokines in the patients. This work demonstrates that in GPS, there is not only a defect of haemostasis, but also a loss of immune homeostasis. Three cellular models of GPS were generated using CRISPR-Cas9 gene-editing of NBEAL2-FTAP-tagged iPSCs, an NBEAL2 knockout and two knock-in lines, each harbouring a known disease-causing single nucleotide variant (Met2080Lys or Pro2100Leu) in homozygosity. Forward programming of iPSCs to MKs enabled comparison of the morphology and α-granule protein expression between NBEAL2 variant MKs and wild type. The cellular endophenotype of these cell models was explored through RNA-sequencing and MS proteomics analysis. The NBEAL2-FTAP-tag was used for Nbeal2 pull-downs and MS analysis, to explore the Nbeal2 interactome. In summary, this work explored the clinical consequences of rare variants for inherited platelet disorders and focused on the clinical and cellular phenotypes of an exemplar rare platelet disorder through the study of a large patient cohort and an iPSC-derived MK model. University of Cambridge 2025-07-23 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/397202 https://www.repository.cam.ac.uk/bitstreams/e822b905-1c68-4b5a-8ac6-aa7dd26b3fdd/download 1e3952e6f673f926b72016607c034ffc https://www.repository.cam.ac.uk/bitstreams/c51ad516-13ef-4abd-ab6f-6bd5f3685009/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved genetics inherited platelet