Mutation screening of the major autosomal dominant polycystic kidney disease gene (PKD1) has been complicated by the large transcript size (>14 kb) and by reiteration of the genomic area encoding 75% of the protein on the same chromosome (the HG loci). The sequence similarity between the PKD1 and HG regions has precluded specific analysis of the duplicated region of PKD1, and consequently all previously described mutations map to the unique 3' region of PKD1. We have now developed a novel anchored reverse-transcription- PCR (RT-PCR) approach to specifically amplify duplicated regions of PKD1, employing one primer situated within the single-copy region and one within the reiterated area. This strategy has been incorporated in a mutation screen of 100 patients for more than half of the PKD1 exons (exons 2246; 37% of the coding region), including 11 (exons 2232) within the duplicated gene region, by use of the protein-truncation test (PTT). Sixty of these patients also were screened for missense changes, by use of the nonisotopic RNase cleavage assay (NIRCA), in exons 23-36. Eleven mutations have been identified, six within the duplicated region, and these consist of three stop mutations, three frameshifting deletions of a single nucleotide, two splicing defects, and three possible missense changes. Each mutation was detected in just one family (although one has been described elsewhere); no mutation hot spot was identified. The nature and distribution of mutations, plus the lack of a dear phenotype/genotype correlation, suggest that they may inactivate the molecule. RT-PCR/PTT proved to be a rapid and efficient method to detect PKD1 mutations (differentiating pathogenic changes from polymorphisms), and we recommend this procedure as a first-pass mutation screen in this disorder.
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