The human antibody deficiency disease, X-linked agammaglobulinemia (XLA), results from failure of B lymphoid development. Two phenotypes are distinguished; a major phenotype with arrest at the stage of pre-B cells, and a minor phenotype with arrest at the stage of immature B cells, and a minor phenotype with arrest at the stage of immature B cells. We have identified failure of immunoglobulin variable region rearrangement in both phenotypes, suggesting that the disease results from defective production of a trans acting factor required for variable region recombination. We propose to continue our studies into the molecular basis for the failure of variable region rearrangement. We will test for complementation of the truncated mu chains composed of Leader Sequence with Cmu in XLA major phenotype pre-B cells by cell fusion with cells undergoing variable region rearrangement. We will determine whether the two phenotypes of XLA are allelic, by testing for complementation of the defect in variable region rearrangement in cell hybrids. Polymerase chain reaction technology will be applied to pre-B cells from patient bone marrow, to determine the generality of the failure of variable region rearrangement. Isolation of the gene element which encodes the Leader Sequence of the truncated mu chains will serve as a test of our hypothesis that the truncated mu chains of XLA major phenotype represent transcriptional activation of the H chain locus as a first step in the normal process of variable region rearrangement. The access model for regulation of variable region rearrangement implies that transcriptional activation of recombining gene elements is required for regulation of recombination. In XLA B cells producing DJHC because of failure of VH to DJH recombination, we will test for transcription of unrearranged VH gene elements, in comparison to normal human pre-B cells. These XLA B cells will be examined for Ig V(D)J recombinase activity to determine developmental stage. These several experiments will directly test the hypothesis that a trans acting factor which provides transcriptional access of recombining gene elements is lacking in XLA. To identify the missing factor, XLA minor phenotype B cells, shown to be susceptible to complementation by cell fusion, will be tested for complementation following transfection of genomic DNA. Complementation by transfection will then be used to isolate the genes encoding the trans acting factor which is defective in XLA.
