Identification of Mutations in the Repeated Part of the Autosomal Dominant Polycystic Kidney Disease Type 1 Gene, PKD1, by Long-Range PCR

Abstract

We have used long-range PCR to identify mutations in the duplicated part of the PKD1 gene. By means of a PKD1-specific primer in intron 1, an approximately 13.6-kb PCR product that includes exons 2-15 of the PKD1 gene has been used to search for mutations, by direct sequence analysis. This region contains the majority of the predicted extracellular domains of the PKD1-gene product, polycystin, including the 16 novel PKD domains that have similarity to immunoglobulin-like domains found in many cell-adhesion molecules and cell-surface receptors. Direct sequence analysis of exons encoding all the 16 PKD domains was performed on PCR products from a group of 24 unrelated patients with autosomal dominant polycystic kidney disease (ADPKD [MIM 173900]). Seven novel mutations were found in a screening of 42% of the PKD1-coding region in each patient, representing a 29% detection rate; these mutations included two deletions (one of 3 kb and the other of 28 bp), one single-base insertion, and four nucleotide substitutions (one splice site, one nonsense, and two missense). Five of these mutations would be predicted to cause a prematurely truncated protein. Two coding and 18 silent polymorphisms were also found. When, for the PKD1 gene, this method is coupled with existing mutation-detection methods, virtually the whole of this large, complex gene can now be screened for mutations.

Mutation detection for exons 2 to 10 of the Polycystic Kidney Disease 1 (PKD1)-gene by DGGE

“…7 In one of these regions in intron 1, close to exon 2, we selected a PCR primer for long-range PCR (pPKDin1AF). The other primer (pTr) is located in exon 11.…”

Autosomal dominant polycystic kidney disease (ADPKD, MIM 173900, PKD1 and PKD2 genes, protein products known as polycystin-1 and polycystin-2)

“…39,40 The majority of mutations are nonsense or frameshifting and predict a truncated protein product (see Table 1 for details of mutations identified). 41,42 Although large deletions of PKD1 are described the frequency of this type of mutation is not known.…”

Adult Polycystic Kidney Disease and Critical Illness Insurance

“…Many of the techniques used to analyze DNA had difficulty in distinguishing between homologous regions of DNA, and in the first few years after the discovery of APKD1, most known mutations were found in a small part of the gene that is not repeated elsewhere. Only recently has it been possible to screen the whole gene for mutations (Thomas et al 1999;Rossetti et al 2001). …”