Mutation detection of PKD1 identifies a novel mutation common to three families with aneurysms and/or very-early-onset disease.

It is known that several of the most severe complications of autosomal-dominant polycystic kidney disease, such as intracranial aneurysms, cluster in families. There have been no studies reported to date, however, that have attempted to correlate severely affected pedigrees with a particular genotype. Until recently, in fact, mutation detection for most of the PKD1 gene was virtually impossible because of the presence of several highly homologous loci also located on chromosome 16. In this report we describe a cluster of 4 bp in exon 15 that are unique to PKD1. Forward and reverse PKD1-specific primers were designed in this location to amplify regions of the gene from exons 11-21 by use of long-range PCR. The two templates described were used to analyze 35 pedigrees selected for study because they included individuals with either intracranial aneurysms and/or very-early-onset disease. We identified eight novel truncating mutations, two missense mutations not found in a panel of controls, and several informative polymorphisms. Many of the polymorphisms were also present in the homologous loci, supporting the idea that they may serve as a reservoir for genetic variability in the PKD1 gene. Surprisingly, we found that three independently ascertained pedigrees had an identical 2-bp deletion in exon 15. This raises the possibility that particular genotypes may be associated with more-severe disease.

Somatic mutation in individual liver cysts supports a two-hit model of cystogenesis in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), Type I is a common genetic disorder and an important cause of renal failure. The disease is characterized by progressive cyst formation in a variety of organs including the kidney, liver and pancreas. We have previously shown that in the case of PKD1, renal cyst development is likely to require somatic inactivation of the normal allele coupled to a germline PKD1 mutation. In this report, we have used unique reagents to show that intragenic, somatic mutations are common in hepatic cysts. All pathogenic mutations were shown to have altered the previously normal copy of the gene. These data extend the "two-hit" model of cystogenesis to include a second focal manifestation of the disease.

Gene conversion is a likely cause of mutation in PKD1.

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.

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 dominant polycystic kidney disease, PKD1, encodes a 14 kb mRNA that is predicted to result in an integral membrane protein of 4302 amino acids. The major challenge faced by researchers attempting to complete mutation analysis of the PKD1 gene has been the presence of several homologous loci also located on chromosome 16. Because the sequence of PKD1 and its homologs is nearly identical in the 5' region of the gene, most traditional approaches to mutation analysis cannot distinguish sequence variants occurring uniquely in PKD1. Therefore, only a small number of mutations have been identified to date and these have all been found in the 3', unique portion of the gene. In order to begin analysis of the duplicated region of PKD1, we have devised a novel strategy that depends on long-range PCR and a single gene-specific primer from the unique region of the gene to amplify a PKD1-specific template that spans exons 23-34. This 10 kb template, amplified from genomic DNA, can be employed for mutation analysis using a wide variety of sequence-based approaches. We have used our long-range PCR strategy to begin screening for sequence variants with heteroduplex analysis, and several affected individuals were discovered to have clusters of base pair substitutions in exons 23 and 25. In two patients, these changes, identified in exon 23, would be predicted to result in multiple amino acid substitutions in a short stretch of the protein. This clustering of base pair substitutions is unusual and suggests that mutation may result from unique structural features of the PKD1 gene.