X-linked agammaglobulinemia (XLA) is a single gene defect characterized by profound hypogammaglobulinemia and markedly reduced numbers of B cells. The abnormal gene product has not yet been identified; however, studies in the Pi's laboratory have shown that the gene defect is intrinsic to the B cell lineage and that the gene product is likely to be expressed throughout B cell differentiation. In the next budget period the PI plans to identify, isolate and characterize the gene for XLA. Linkage studies have localized the gene for XLA to a 6-10 mb segment of the X chromosome. No recombination has been seen with the probe p2l2 at Xq22, suggesting that the gene defect is likely to be within 1-2 mb of p2l2. In the first specific aim, the PI will use pulse field electrophoresis, yeast artificial chromosomes (YACs), new DNA probes and linkage analysis to further refine the DNA segment within which the XLA gene must lie. In preliminary studies, the segment of interest has been trimmed to 2-3 mb by analysis of recombinant X chromosomes. The availability of cells and DNA from two unrelated males who are likely to have deletions of the XLA gene should help further localize the gene. YACs that encompass the XLA gene locus will be used to generate new DNA probes. In the second specific aim, the PI will identify B cell specific transcripts that are encoded in the XLA gene segment. The methylation site described above will be characterized in detail. In addition, phage libraries from overlapping YACs that encompass the XLA gene segment will be used to screen B cell cDNA libraries.
In specific aim three, genomic DNA and/or B cell lineage mRNA from 21 unrelated patients with XLA will be analyzed with probes that identify XLA gene candidates. Identification and characterization of the XLA gene will undoubtedly improve diagnosis and care of affected patients. It should also increase our understanding of mechanisms involved in normal B cell differentiation.
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