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Title: Glycoprotein estimation and analysis of genes associated with inherited platelet disorders and complications of pregnancy
Author: Poles, A.
Awarding Body: University of the West of England, Bristol
Current Institution: University of the West of England, Bristol
Date of Award: 2013
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The archetypical inherited platelet (PLT) disorders are Glanzmann’s Thrombasthenia (GT) and Bernard Soulier Syndrome (BSS) and both are autosomal recessive disorders. GT results from mutations in the ITGB3 and/or ITGA2B genes that encode the GPIIb/IIIa complex, while BSS results from mutations in the genes (GPIba, GPIbb and GPIX) that encode the GPIb/IX/V complex. GT and BSS patients have a mild bleeding tendency which is in contrast with patients with another inherited PLT disorder known as congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is associated with mutations in the MPL gene that encodes the thrombopoietin receptor (TPOr), and develop pancytopenia which requires stem cell transplantation. The GPIIb/IIIa and GPIb/IX/V complexes are also the carrier molecules for the majority of Human Platelet Antigens (HPAs). Alloimunisation against HPA is important in foetal maternal alloimmune thrombocytopenia (FMAIT) where the mother becomes immunized against an antigen that the foetus has inherited from the father. In the GT patients, seven novel mutations were identified. One homozygous mutation in the ITGA2B gene resulted in a frameshift and premature stop codon, two heterozygous missense mutations were identified in the same patient and lastly a homozygous missense mutation was identified in a patient that also possessed a novel intronic mutation in the ITGB3 gene. A further two novel homozygous missense mutations were also detected in the ITGB3 gene. In the BSS patients five novel mutations were identified in the GPIba, GPIbb and GPIX genes. There were three single base deletions resulting in premature stop codons, one missense mutation in the leader sequence of the GPIbb gene, and a further heterozygous missense mutation also in the GPIbb gene. Five of seventeen suspected CAMT patients possessed mutations in the MPL gene. Three novel mutations were identified; one missense mutation resulting in a premature stop codon, one mutation in intron 11 and a homozygous missense mutation. Gene analysis provided confirmatory diagnosis and correlated with disease progression. In the FMAIT case, maternal antibodies reactive only with paternal platelets were localized to GPIIb/IIIa by crossmatch using the MAIPA assay. A mutation was detected in exon 23 of ITGA2B in the father and the three children. This mutation predicts a valine to leucine amino acid substitution (V740L) in the mature protein. Recombinant GPIIb glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells was specifically recognised by maternal antibodies. The mutation was not detected either in the mother or a cohort of one hundred volunteer platelet apheresis donors. Evidence from these patient cohorts indicated that misdiagnosis is not uncommon and this highlights the need for confirmatory diagnosis based on immunophenotyping and gene analysis. This was emphasised in the cohort of CAMT patients, that had variously been incorrectly diagnosed with FMAIT, Immune Thrombocytopenic Purpura and Aplastic Anaemia. Gene analysis of the ITGA2B gene permitted definition of the V740L mutation that defines a new low frequency antigen implicated in two cases of FMAIT in a single family.
Supervisor: Not available Sponsor: NHSBT
Qualification Name: Thesis (D.B.M.S.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available