Myasthenia Gravis is an autoimmune neuromuscular disorder in which antibodies are produced against several muscle antigens. Immunological evidence indicates that the muscle antigens are also expressed in thymus. In the present proposal, we plan to 1) identify not yet characterized 'non-cholinergic"""""""" antigens in MG; 2) assess their pathological importance; 3) investigate transcriptional and genetic characteristics that may lead to deregulation in the expression of muscle and thymic peptides (and proteins) that become antigens in MG and 4) develop MG familial pedigrees for future linkage analysis. The studies will be conducted employing available cDNA clones and new cDNA clones selected from expression cDNA libraries: 1) Non- cholinergic cDNA clones are selected by screening expression cDNA libraries with MG sera known to react with different muscle antigens; 2) cDNA clones that code for the four subunits of the mouse AcChR will be employed to isolate their correspondent human counterparts. Identification of non-cholinergic antigens will be carried out through cDNA sequencing of the corresponding cDNA clones and immunological identification of the native protein by antibodies raised against the corresponding recombinant peptide. To assess the pathological significance of a peptide coded for a given cDNA clone, recombinant peptides will be employed in tests devised to determine antibody titers of individual MG sera. Titers will then be correlated with disease activity. To investigate transcriptional and genetic characteristics that may account for mechanisms leading to an autoimmune attack (i.e. antigenic deregulation), the selected (cholinergic and non- cholinergic) cDNA clones will be employed to analyze muscle and thymic transcripts from normal and MG tissues (Northern blot analysis) and for the study of the genomic characteristics (Southern blot analysis) of normal and MG tissues. Fine transcriptional analysis, employing nuclease protection assays, will be conducted with full length cDNA probes. cDNA clones will also be employed for the selection of genomic DNA clones from a human genomic library. These genomic clones will be incorporated as tools in the genomic DNA analysis of MG patients and control. Establishment of MG familial pedigrees for future linkage analysis will be carried out (Core A).
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