Acute graft versus host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic bone marrow transplantation. In the previous grant period we demonstrated an association between polymorphisms in a DQ- beta gene (hereafter referred to as the DQR gene) and the risk of developing GVHD following allogeneic BMT. These results suggested the existence of DNA sequences homologous to but not identical with DQ-beta, that may encode an antigen or antigens responsible for GVHD. The data also suggest that the locus or loci detected by the DNA probe and highly polymorphic and unlinked to HLA. These results provides an incentive to clone and further characterize the DQR gene. Our collaborator, Dr. Georgia Vogelsang has developed a skin-explant model for GVHD using donor lymphocytes that have been sensitized against recipient lymphocytes in vitro and co-cultured with the recipient's skin. Histologic changes compatible with GVHD are found in the positive explants. We plan to combine molecular genetic techniques and the skin explant model in order to test our hypothesis that the DQR gene encodes the minor histocompatibility antigen(s) responsible for the allo-reaction of the donor immune system against the recipient (GVHD). We are proposing a project with three main goals: (1) to clone and characterize the DQR gene; including isolating candidate clones, determining their DNA sequence, deducing the structure of a putative protein product of the gene(s) and determining whether the gene is a single or multiple gene system. (2) to evaluate polymorphisms in the gene; including characterizing polymorphisms associated with the gene (RFLPs), determining if the putative protein product is polymorphic (amino acid sequence variation), re-evaluating the relationship between genetic differences between donors and recipients (RFLPs) and the risk of developing acute GVHD and determining whether genetic non-identity between donors and recipients correlates with positive reactions in a skin explant model system. (3) to study the expression of the gene; including determining whether the gene is expressed and where the gene is expressed and where and determining whether or not the gene is expressed in cultured skin fibroblasts used in the skin explant model for GVHD and if so then clone the cDNA and determine its structure.
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