Two hypotheses proposed to explain observed relationships between HLA antigens and disease susceptibility are either that the antigens themselves play some direct role in pathogenesis or they are merely fortuitous markers for nearby specific disease susceptibility genes. This proposal seeks data to distinguish between these hypothesis by using molecular probes to directly characterize the DNA of the HLA region, particularly comparing controls and patients with autoimmune diseases. DNA from peripheral blood lymphocytes will be digested with restriction endonucleases, separated electrophoretically by size on agarose gels, and transfered to nitrocellulose filters for hybridization with 32P labeled, cloned HLA gene probes. Locus and allele specific HLA class I probes will be sought from non-homologous intragenic and flanking regions of cloned HLA class I genes, either by excision and isolation or by synthesis of the desired oligonucleotide sequences. Currently available and new probes will be tested against normal controls, HLA recombinant families, and cloned genomic HLA genes (as markers) to characterize normal HLA class I restriction fragment length polymorphism (RFLP) and its correlation with known HLA antigens. De novo fragment generation has been observed and will also be investigated, ultimately by cloning and sequencing the novel and parental fragments. RFLP of variant B27 (+) normals and B27 (+) spondyloarthropathy patients will be compared to seek disease specific markers. Identification of such markers should permit identification of the pertinent gene products and ultimately clarification of the mechanism of pathogenesis.
