The overall goal of this project is to determine the nature and extent of allelic diversity in the HLA-DP region and to assess the relationship of this polymorphism to HLA-associated disease susceptibility. A variety of diseases, many but not all of which are autoimmune, have been associated with specific serologically defined HLA class II alleles and/or haplotypes. Recently, DNA-based typing has shown that particular DR and DQ sequence-defined alleles were associated with specific diseases; sequence comparisons of susceptible and non-susceptible alleles revealed the potential importance of individual polymorphic positions of the class II beta chains. The role of DP polymorphism in disease susceptibility has not been studied to the same extent. The distribution of DPB1 and DPA1 alleles in patients and in ethnically matched controls will be compared for a variety of diseases. Due to the strong linkage disequilibrium within the HLA region, assessing the role of DP polymorphism requires the analysis of HLA (B,DR,DQ,DP) haplotypes . We have already examined the nature and distribution of extended haplotypes in a Caucasian group. Our project will focus on families, allowing the unambiguous determination of haplotypes and we will compare the different haplotypic combinations that are found in different ethnic groups. These haplotype data are critical not only for understanding the complex patterns of HLA-disease associations but also for defining the likelihood of finding HLA-matched bone marrow and solid organ donors in different ethnic groups. Our proposal to characterize the DP sequence polymorphism, to refine our non-radioactive oligonucleotide probe DP typing system, and to apply this method (both dot blot and reverse dot blot) to disease susceptibility studies is based upon the polymerase chain reaction (PCR). Our previous work using DPB1 and DPA1 primers to PCR amplify the polymorphic second exon for sequence analysis will be extended to additional samples and populations to identify new alleles, revealed as a novel pattern of probe reactivity. New alleles are always confirmed by sequence analysis. The relationship of recombination frequency to the observed patterns of linkage disequilibrium will be examined in family studies but also by using the approach of sperm mapping, the co-amplification by PCR of linked loci from individual sperm. This method for determining recombination frequencies over short genetic distances, like those within the HLA region, can be used to compare recombination frequencies between individuals and between haplotypes. PCR amplification from sperm will also be used to try to detect the generation of new DPB1 alleles by segmental exchange, the putative gene-conversion-like mechanism inferred from the patchwork pattern of DPB1 polymorphism. A phylogenetic analysis of DPB1 polymorphism will also be carried out on a data set of non-human primate DPB1 alleles. These studies will increase our knowledge of HLA-DP polymorphism and contribute to our understanding of its role in disease susceptibility.
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