An unusual pattern of mutation in the duplicated portion of PKD1 is revealed by use of a novel strategy for mutation detection

The gene for the most common and severe form of autosomal dominantpolycystic kidney disease, PKD1, encodes a 14 kb mRNA that is predicted toresult in an integral membrane protein of 4302 amino acids. The majorchallenge faced by researchers attempting to complete mutation analysis ofthe PKD1 gene has been the presence of several homologous loci also locatedon chromosome 16. Because the sequence of PKD1 and its homologs is nearlyidentical in the 5' region of the gene, most traditional approaches tomutation analysis cannot distinguish sequence variants occurring uniquelyin PKD1. Therefore, only a small number of mutations have been identifiedto date and these have all been found in the 3', unique portion of thegene. In order to begin analysis of the duplicated region of PKD1, we havedevised a novel strategy that depends on long-range PCR and a singlegene-specific primer from the unique region of the gene to amplify aPKD1-specific template that spans exons 23-34. This 10 kb template,amplified from genomic DNA, can be employed for mutation analysis using awide variety of sequence- based approaches. We have used our long-range PCRstrategy to begin screening for sequence variants with heteroduplexanalysis, and several affected individuals were discovered to have clustersof base pair substitutions in exons 23 and 25. In two patients, thesechanges, identified in exon 23, would be predicted to result in multipleamino acid substitutions in a short stretch of the protein. This clusteringof base pair substitutions is unusual and suggests that mutation may resultfrom unique structural features of the PKD1 gene.