The goal of this proposal is to define the polymorphism of the human major histocompatibility complex class I HLA-A,B,C antigens and to assess the significance of the role that undetected structural variants may have as histocompatibility antigens in human allogeneic marrow transplantation. The extent of polymorphism among class I alloantigens has been recently assessed by isoelectric focusing (IEF) gel analysis demonstrating that more than 40% of seriologically-defined antigens can be further subdivided. Studies with cytotoxic T lymphocytes (CTL) have demonstrated that these structural variants behave as alloantigens. These findings have prompted us to ask whether disparity for class I subtypes might be responsible for the increased risk of graft-versus-host disease (GVHD) in patients transplanted from unrelated donors that appear to be HLA identical. The first specific aim of this project is to identify serologically undetected class I structural variants in unrelated but HLA-A,B,C phenotypically identical donors and recipients using IEF gel and CTL analysis. This will provide a measure of the frequency of these otherwise undetected structural variants that occur in clinical unrelated donor transplantation. The second specific aim is to determine the primary structure of these class I variant molecules.
This aim will be achieved by the use of polymerase chain reaction (PCR). HLA class I cDNA is specifically amplified by PCR and then sequenced. This method will facilitate the large scale sequencing experiments proposed in this project. The third specific aim is to determine the possible role of class I structural variation as a risk factor for causing GVHD.
This aim will also address the possible correlation between primary structure and immunological function of the class I molecules. The fourth specific aim is the long-range goal of establishing the reagents and methods for genotyping HLA class I alleles.
This aim will be approached using sequence specific oligonucleotide probe (SSOP) hybridization with PCR-amplified genomic DNA. A more extensive data base of DNA sequences will be necessary to develop appropriate strategies for designing SSOP. The genetic information and improved technologic approaches developed in this project will have a major impact on our understanding of the significance of HLA class I polymorphism in marrow transplantation and will enable us to increase the precision of HLA typing and improve the quality of donor selection for patients suffering from otherwise fatal congenital and malignant hematological diseases.
Showing the most recent 10 out of 32 publications
