Bariana, Tadbir Kaur; (2018) Discovery of novel variants underlying inherited bleeding and platelet disorders by next generation sequencing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Genetic variants that affect megakaryopoiesis and platelet formation result in inherited bleeding and platelet disorders (BPD). Only 40-60% of cases will receive a diagnosis indicating the pathway at fault [1, 2]. This thesis is the result of work undertaken to discover novel variants causative of BPD, as part of the NIHR BioResource-Rare Diseases Study (NIHR BR-RD). 1,213 BPD cases of uncertain genetic aetiology were recruited from 31 international centres. 687 samples were sent for whole exome sequencing and 1118 for whole genome sequencing. Cases were systematically phenotyped and genotype-phenotype relationships were assessed to detect causal variants in known and novel candidate BPD genes, including KDSR and ABCC4. KDSR is an early enzyme in the de novo sphingolipid synthetic pathway, and several cases have recently been reported with KDSR variants, severe skin pathology and thrombocytopenia, without evaluation of the mechanism of thrombocytopenia [3, 4]. I report a pedigree in which novel compound heterozygous variants in KDSR cosegregate with a severe phenotype of neonatal-onset thrombocytopenia in two siblings and juvenile myelofibrosis in the older sibling, without significant skin pathology. Cellular studies support roles for KDSR in the regulation of growth and apoptosis, megakaryocyte yield, maturity, size and proplatelet formation. This thesis also reports on the study of a pedigree in which coinheritance of homozygous loss of function variants in novel candidate BPD gene ABCC4 and heterozygous variant in known BPD gene P2RY12 cosegregate with a lifelong mild-to-moderate bleeding disorder. ABCC4 is a membrane-bound transport glycoprotein with broad substrate specificity that includes cAMP, a powerful inhibitor of platelet activation, and intra-platelet cAMP was elevated in affected individuals. The role of ABCC4 in megakaryopoiesis is also explored using CRISPR-Cas9-mediated inhibition of ABCC4 expression in cellular models of megakaryopoiesis. This study describes collaborative approaches to the analysis of high throughput sequencing data for the discovery of potentially pathogenic variants, and emphasises the necessity of functional validation of hypothetical associations.

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