X-linked agammaglobulinemia (XLA) is a recessive, congenital, antibody deficiency disease linked to Xq2. The disease results from arrest in B lymphoid development that is associated with failure of Ig H chain V(D)J recombination. There is phenotypic variability of the disease. The major phenotype has arrest at pre-B cells, with production of germ line mu chain transcripts composed of a leader sequence (LS) with C mu. The minor phenotype has arrest at immature B cells, with production of intermediate transcripts composed of DJHC(mu/delta). Previous studies have found that the failure of VH to DJH recombination in minor phenotype cells can be complemented by fusion with a mouse myeloma cell. Fusion of pre-B cells from a major phenotype patient with a D mu producing clone from a minor phenotype patient does not provide complementation, suggesting that the two phenotypes represent phenotypic variation from the single Xq2 disease locus. These results indicate that XLA gene product acts in trans on the chromosome 14 linked Ig H chain locus. Cosmid clones derived from the human X chromosome have been screened for clones that can provide complementation by transfection. Two overlapping clones have been found to cause VH to DJH recombination in D mu arrested XLA cells, indicating that they carry normal copies of the XLA gene. We propose to use the transfection/complementation system to isolate the XLA gene from these cosmid clones, and to characterize this gene. Mutations in this gene that cause XLA will be identified by comparison of patient DNA with normal. The role of germline VH gene transcription in this process of recombination will be examined. Long term plans include study of structure and function of this gene in light of the mutations in XLA and to develop strategies for simpler methods for diagnosis and carrier detection.
