The overall goal of this project is to identify the sequences that distinguish unique HLA-A, and C alleles and to determine the impact of disparity for HLA-A, B and C microvariants on the development of graft failure and graft-versus-host disease (GVHD) following unrelated marrow transplantation. The increased incidence of significant acute GVHD observed in recipients of unrelated marrow grafts cannot be entirely ascribed to mismatching within the HLA-D region alone. We hypothesize that disparity for unrecognized HLA class I variants may be significant cause of GVHD in HLA-DRB, DQB, DPB-matched transplants. Our rationale for this study is based upon two observations: 1) conventional serologic methods greatly underestimate the polymorphism encoded at HLA-A, B and C, necessitating a more precise molecular approach to the characterization of these alleles, and 2) our experience has shown that unrelated donor marrow transplants incompatible for a serologically- defined HLA-A or B """"""""minor"""""""" mismatch have a higher probability of developing significant GVHD than do recipients of HLA-A, B phenotypically-identical grafts. We will apply the polymerase chain reaction (PCR), single-strand conformational polymorphism (SSCP) analysis and direct DNA sequencing to study the extent of polymorphism at HLA-A, B and C loci, and to assess the degree of allelic compatibility between patients and their HLA-A, B, D phenotypically- matched unrelated donors. The objectives of this project will be organized into two components. Because HLA-C-encoded alloantigens are poorly defined by available serologic reagents and sequence information is very limited, priority will be given to sequence analysis of HLA-C alleles and to the development of an automated PCR-based direct sequencing method for typing HLA-c alleles. In contrast to HLA-C, substantially more information is known about HLA-A and B alleles, and the extent of polymorphism appears to be far greater. The focus of the HLA-A and B component will be to determine the degree to which unrelated donors and patients are mismatched for HLA-A and B variant. This will be accomplished systematically according to the extent of polymorphism already known for given HLA-A and B antigens: 1) antigens which are known to have multiple variants by isoelectric focusing (IEF) will be studied by direct sequencing methods, and 2) antigens for which no IEF variants are known to exist will be compared by a sensitive SSCP analysis. We anticipate that this strategy for identifying HLA-A, B and C microvariants will allow us to define with relative efficiency the full extent of gene polymorphism detectable within our patient population while enabling us to determine with precision the degree of molecular matching between unrelated donor-recipient pairs. Finally, we will determine whether disparity for HLA-A, B and C alleles in otherwise DRB1, DQB1-matched transplants significantly increases the risk of graft failure or GVHD. These data will be used to assess whether more stringent criteria for donor selection based on allele matching would result in a reduction of morbidity and mortality following unrelated donor marrow transplantation.
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