Molecular diagnosis of autosomal dominant polycystic kidney disease (ADPKD) has proven difficult because of genetic and allelic heterogeneity and the complex structure of the major gene, PKD1. Consequently, the degree to which the marked phenotypic variability in the severity of renal disease and expression of extrarenal manifestations correlates with genotype is largely unknown. Using recent improvements to specifically amplify the PKDI gene and temperature modulated heteroduplex analysis (TMHA), we propose to develop rapid and accurate genetic characterization of the ADPKD genes. Mutation detection rates of >80 percent for PKD1 and >90 percent for PKD2 will be achieved during the project. The fate and stability of mutant ADPKD proteins, polycystin-1 and -2, will be investigated in patient-derived lymphoblastoid cell lines, epithelial cell lines derived from a single cyst lining, and knockout mouse models of PkdI. The mutational mechanism will be further explored by the isolation of individual cyst lining, characterized by their immuno-reactivity to the polycystin proteins, using laser capture microdissection. Genetic analysis of these cells will reveal the importance of somatic events at the ADPKD genes and elsewhere for cyst initiation and expansion. Phenotype/genotype correlations will be explored in defined patient groups: renal insufficient; geographically defined without referral or recognition bias; very early onset disease; late onset disease; severe liver disease and vascular abnormalities. The ADPKD gene is known to be a strong indicator of renal disease severity (PKD1 more severe than PKD2) but the relative contribution of the two genes to extra-renal disease is unknown. The prevalence of PKD2 in the general population is also largely unknown. Correlations between the type and position of mutation in PKD1 and PKD2 will be made to the severity of renal disease and to the different phenotypic groups. These results will show if there are clear phenotype/genotype correlations, that may have prognostic implications, reveal more about the mutational mechanism and highlight important regions of the polycystin proteins. Specific mutational mechanisms, such as an early embryonic somatic mutation or the modifying effect of variants at the ADPKD allele inherited from the normal parent, will be analyzed in early onset cases. This study will help resolve questions about the mutational mechanism in ADPKD, determine the role of somatic events, show the extent to which the ADPKD genotype dictates clinical outcomes and generate phenotypically and genotypically well characterized ADPKD populations that will be suitable for testing the role of other genetic modifying factors.
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