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Title: The relationship between ADAMTS13 genotype and phenotype in congenital thrombotic thrombocytopenic purpura and characterisation of ADAMTS13 mutants
Author: Underwood, M. I.
ISNI:       0000 0004 5365 7081
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Congenital thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, usually involving ADAMTS13 gene defects. ADAMTS13 processes the multimeric plasma glycoprotein Von Willebrand factor making it less reactive to platelets. Patients differ in terms of disease severity and evidence suggests a relationship between ADAMTS13 genotype and disease phenotype. Over 140 mutations have been identified in patients but only ~30% of these has been expressed in vitro. The aim of this thesis was to study certain ADAMTS13 mutations identified in a homozygous form in congenital TTP patients to assess in vitro their effect on the secretion and activity of ADAMTS13 and to assess their contribution to disease phenotype. ADAMTS13 mutants (p.R102H, p.I143T and p.Y570C) and wild type (WT) were expressed in HEK293T cells. The p.R102H mutation partially affected the secretion of ADAMTS13 and reduced the catalytic efficiency of the mutant but not to the extent predicted based upon levels measured in patient plasma. Expressing this mutant with three ADAMTS13 polymorphisms (p.Q448E, p.P618A and p.A900V) which were also identified in the patient with this mutation further reduced the secretion and activity of ADAMTS13. When these three polymorphisms were expressed separately in WT ADAMTS13, the p.P618A polymorphism reduced the secretion and subsequently the activity of ADAMTS13 suggesting that this polymorphism in particular was responsible for the reduction observed. These results highlight the importance of ADAMTS13 polymorphisms. The p.I143T and p.Y570C mutations severely affected ADAMTS13 secretion. Immunofluorescence studies showed localisation of these mutants within the ER but less extensive localisation within the cis Golgi compared to WT ADAMTS13. The p.I143T mutant was characterised further and was shown to be degraded by the cell proteasome. Addition of a chemical chaperone (betaine) appeared to rescue the secretion defect caused by the p.I143T mutation. This may have future therapeutic implications for the treatment of some congenital TTP patients.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available