Molecular studies on Burton's tyrosine kinase

X-Linked agammaglobulinaemia (XLA) is a humoral immunodeficiency resulting from a block in the B cell maturation pathway at the pre-B cell stage. Affected males have normal numbers of pre-B cells in the bone marrow but virtually no mature B cells in the peripheral circulation and are unable to produce immunoglobulin of all isotypes. The gene defective in XLA has been identified as a non-receptor tyrosine kinase and named Btk (Bruton's tyrosine kinase). Btk is a modular protein related to but distinct from the Src family of tyrosine kinases. However, the precise pathways in which Btk is involved and its exact role in lymphocyte maturation remain unclear This thesis describes a programme of work ranging from the identification of the genetic defects in Btk through to studies attempting to define the role of Btk in intracellular signalling by the use of gene transfer technology. Mutation analysis of Btk cDNA from individuals with XLA using single stranded conformational polymorphism (SSCP) analysis and direct sequencing resulted in the identification of 5 mutations. In three unrelated individuals the same mutation was identified suggesting the presence of a mutational hotspot in Btk. Subsequent studies investigated the expression and activity of Btk protein in primary cells from XLA patients in order to correlate the clinical phenotype with the genetic defect. However, it was shown that regardless of the mutation or clinical phenotype, there was complete lack of Btk expression and activity. These assays were also used to confirm the diagnosis of XLA in certain individuals in whom XLA was suspected but not confirmed by genetic analysis. Gene transfer technology was used to create in vitro models with which study the function of the Btk protein. Retroviral vectors encoding a copy of wild type and kinase mutant Btk were generated and used to express Btk in a fibroblast line to study the binding of Btk to candidate signalling proteins. Retroviral transfer of Btk into an EBV transformed B cell line from an XLA patient with a null phenotype was also performed in order to study reconstitution of B cell function.