Structural polymorphanisms in class I and II HLA molecules are the greatest immunological barrier to clinical transplantation in humans. For transplantation of bone marrow and solid organs, survival of graft and patient survival are improved with good HLA matching. During the last 15 years evidence has accumulated that traditional serological typing does not, and probably cannot, distinguish all the different HLA molecules. Moreover, the serologically undetected differences are often those stimulating strong alloreactive T cell responses which are likely to contribute to transplant rejection. Other disadvantages of serological HLA typing are the complexity, low titer, and limited supply of the typing alloantisera. These and other factors have resulted in relatively poor definition of the HLA molecules of American Blacks, Native Americans, and other minority ethnic populations in the United states. This decreases the probability of finding a good match between transplant donors and recipients in those populations. For these reasons, new HLA typing methods based on the nucleotide sequences encoding the polymorphic polypeptides of the class I and II molecules. The proposed research aims first to provide the essential nucleotide sequence information from class I HLA alleles that is a prerequisite for design of DNA-based typing, and second to develop strategies for typing based on polymerase chain on polymerase chain amplification of DNA and oligonucleotide hybridization. This investigation will build on extensive experience in the study of Class I HLA molecules. Alleles of the HLA-A and B loci will be isolated and sequenced with emphasis on those found at high frequency in American Blacks and Amerindians. Application of automated DNA sequencing to speed the acquisition of this data will be made. A complicating factor in the development of DNA-based typing for class I HLA molecules is the presence in the human genome of 15 genes, pseudogenes and gene fragments that cross- hybridize with HLA-A, B, and C. The structure and polymorphism of those additional genes will be characterized and this information will reveal potential problems and permit their avoidance. The accumulated sequence information will be used to develop """"""""typing"""""""" strategies that discriminate HLA-A, B, C alleles on the basis of hybridization of sequence specific oligonucleotides to segments of genomic DNA amplified by the polymerase chain reaction. The alleles of each locus will be divided into groups and in the initial stages typing within a group will be developed. Initial groups for study are the HLA-A2/A28 family and the HLA-B17/B5/B35 family. Once accurate, discriminating typing for these groups has been established then further groups will be selected for study. In addition to laying a foundation for accurate, routine, and comprehensive class I HLA typing for transplantation this project will develop information and reagents invaluable to future research on T-cell responses in human diseases.
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