Bradley, Linda Anne Dugdale; (1995) The molecular basis of X-linked agammaglobulinemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency disease characterised by a lack of B lymphocytes in the peripheral blood. The consequent lack of immunoglobulin results in severe infection. Affected males have pre-B cells in their bone marrow, suggesting that the defect lies in the pathway of B cell maturation. At the start of this study, the gene responsible for XLA had not been isolated but the disease locus had been mapped to Xq22 by genetic linkage analysis. As part of a positional cloning approach to the isolation of the gene responsible for this disease, a YAC contig was constructed containing this region. A YAC was selected from this contig on the basis that it appeared not to be chimeric and that it contained the DXS178 locus, known to have no recombinations with XLA in over 70 informative meioses. This YAC was used in a cDNA enrichment study, with the aim of isolating candidate genes for XLA. The BTK gene was isolated in 1993 and shown to be the gene defective in this disease. Btk is a non-receptor tyrosine kinase, related to, but distinct from Src. The identification of this gene made it possible to screen the BTK gene in XLA patients for mutations, using single strand conformation polymorphism (SSCP) analysis. Mutations were found and sequenced in fifteen patients, and included amino acid substitutions, small insertions and deletions, premature stop codons, disruption of the initiation codon and splice site recognition sequence alterations. A study of the BTK gene in carrier women showed that in some families carrier detection could be improved by direct mutation analysis. The detection of an polymorphism within BTK may allow carrier detection in families where the pathological mutation has not been identified. A preliminary study of the Btk protein suggested that it will be possible to correlate the disease-causing mutation with its effect on the protein, further characterising the molecular basis of XLA in these families.

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