Autosomal dominant polycystic kidney disease is a common disorder which causes significant morbidity and premature death for which there is no known protective therapy. The goals of this proposal are to identify the gene mutated in patients with ADPKD and to understand how its mutations interfere with the function of its normal gene product to produce the clinical pathology of this disorder. It is expected that such knowledge will ultimately guide design of effective treatment. Genetic analysis has localized the gene for the most common form of autosomal dominant polycystic kidney disease to a 500kb interval. Over 90% of this interval has been cloned in 2 sets of genomic clones. cDNA clones, representing a minimum of 60kb of coding sequence and at least 15 genes, have been isolated from this interval. Hybridization analyses have failed to identify disease-specific differences, necessitating the use of sequence-based approaches. All methods currently available are very laborious and costly. Furthermore, the genomic organization of the largest of these genes (approximately 12kb message on Northern) is extremely complicated and will be especially difficult to evaluate. This proposal seeks to identify the disease gene by a unique approach that scans for differences on a protein level. It is likely that there will be either qualitative or quantitative differences of the PKD1 gene product in affected individuals which can be detected using antisera raised against it by either 1D or 2D polyacrylamide gel electrophoresis. Polyclonal antibodies generated against the peptide products of candidate genes from the PKD1 interval will be used to screen Western blots of total cell proteins isolated from cell lines, primary cell cultures or tissue derived from affected patients. Antisera will be raised in rabbits to recombinant proteins synthesized in and extracted from bacteria. Immunocytochemical analysis will be used to compare the localization of the candidate gene products in normal and cystic kidney. The sequence of a candidate gene which encodes a protein abnormality detected by its antisera will be thoroughly analyzed in panels of affected individuals. The patients will be selected from the world's most extensive clinical database so as to represent a full spectrum of disease severity and ethnic backgrounds (including individuals with de novo mutations). Characterization of the PKD1 gene product is essential to understanding its function. Antisera raised during Phase 1 studies will be used to determine the developmental and tissue expression pattern of the PKD1 gene and to analyze putative functional motifs predicted by its sequence. DNA samples of affected individuals in the database will be screened for mutations so that genotype/phenotype correlations can be determined and important functional domains of the PKD1 gene product identified. Results of these studies will guide design of a construct that can be used to generate a transgenic mouse. The latter will help define the pathophysiology of this disorder, allow the development and testing of therapeutic compounds and allow manipulation of the environmental and genetic background to assess the relative effects of each.
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