Sweatman, Angela Karen; (1994) Physical mapping studies of the X-linked agammaglobulinemia locus in Xq22. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease which results in a deficiency of immunoglobulins of all isotypes. This is a direct result of low numbers of circulating B cells. At the start of this work the causative gene was unknown but the disease locus was mapped to Xq22. The aim of this work was to use a positional cloning approach to identify the XLA gene. A number of DNA loci were known to map to Xq22 but the physical size of the region could only be estimated at between 10 and 20Mb. Pulsed field gel electrophoresis was used to construct two unlinked submaps spanning over 3Mb of DNA and incorporating seven polymorphic and non-polymorphic DNA markers. One of the polymorphic DNA markers, DXS101, was known to have four additional copies which map to Xq22. Single copy probes for two of these were isolated from DXS101 positive cosmids. One was found to lie within 550kb of the DXS178 locus, which is closely linked to the XLA locus. Analysis of DXS178 positive cosmids resulted in the physical mapping of the DXS265 locus to within 5kb of the DXS178 locus. In order to identify patients with deletions, screening of 46 unrelated XLA patients with DNA markers closely linked to the XLA locus was carried out. A deletion in a patient with XLA was found which encompassed the DXS442 locus, suggesting that the XLA gene may lie nearby. This work led indirectly to the identification of a new restriction fragment length polymorphism at the DXS101 locus. The gene responsible for XLA, BTK, was cloned in the last year of this work. BTK was incorporated into the physical map of the Xq22 region and the cDNA used to identify mutations in XLA patients. Southern blot analysis identified deletions in five XLA patients. In addition, the XLA patient who was deleted at the DXS442 locus was found to have a point mutation in the BTK gene.

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