Mutation Analysis of the Entire PKD1 Gene: Genetic and Diagnostic Implications

Abstract

Mutation screening of the major autosomal dominant polycystic kidney disease (ADPKD) locus, PKD1, has proved difficult because of the large transcript and complex reiterated gene region. We have developed methods, employing long polymerase chain reaction (PCR) and specific reverse transcription-PCR, to amplify all of the PKD1 coding area. The gene was screened for mutations in 131 unrelated patients with ADPKD, using the protein-truncation test and direct sequencing. Mutations were identified in 57 families, and, including 24 previously characterized changes from this cohort, a detection rate of 52.3% was achieved in 155 families. Mutations were found in all areas of the gene, from exons 1 to 46, with no clear hotspot identified. There was no significant difference in mutation frequency between the single-copy and duplicated areas, but mutations were more than twice as frequent in the 3' half of the gene, compared with the 5' half. The majority of changes were predicted to truncate the protein through nonsense mutations (32%), insertions or deletions (29.6%), or splicing changes (6.2%), although the figures were biased by the methods employed, and, in sequenced areas, approximately 50% of all mutations were missense or in-frame. Studies elsewhere have suggested that gene conversion may be a significant cause of mutation at PKD1, but only 3 of 69 different mutations matched PKD1-like HG sequence. A relatively high rate of new PKD1 mutation was calculated, 1.8x10-5 mutations per generation, consistent with the many different mutations identified (69 in 81 pedigrees) and suggesting significant selection against mutant alleles. The mutation detection rate, in this study, of>50% is comparable to that achieved for other large multiexon genes and shows the feasibility of genetic diagnosis in this disorder.

Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease

“…5,6 In this study we demonstrate that WGS is a reliable, minimally labour-intensive and reproducible technique with which to overcome the challenge of pseudogene homology and thus make a molecular diagnosis of ADPKD. Using this technique we were able to make a molecular diagnosis in 86% of patients.…”

“…To date, long-range PCR (LR-PCR) amplification, followed by Sanger sequencing, has been the gold standard used by most diagnostic laboratories. 1,5,6 This technique has a diagnostic rate of approximately 90% in well-phenotyped trial cohorts and 40-60% in the commercial reference laboratory. [7][8][9] The technique is labour intensive and thus expensive.…”

Novel PKD1 deletions and missense variants in a cohort of Hellenic polycystic kidney disease families

“…29,34,38 De novo mutations reported by several groups confirm this hypothesis. 24,25,39 The great rarity of some mutations and their confinement within certain families suggests also that they must have occurred very recently. In many cases multiple polymorphisms have been detected on same alleles, being in linkage disequilibrium.…”

Mutations of the PKD1 gene among Japanese autosomal dominant polycystic kidney disease patients, including one heterozygous mutation identified in members of the same family

“…Mutation analysis of the PKD1 gene has been complicated because at least three PKD1-like homologous genes exist on chromosome 16p13.1. Several improved strategies have been developed to detect mutations in the entire PKD1 gene including the duplicated region (Peral et al 1997;Roelfsema et al 1997;Watnick et al 1997; Thomas et al 1999;Rossetti et al 2001).…”

“…These mutations resulted in various alterations of amino acids by substitution, deletion, or insertion of nucleotides (nt). Previous reports suggested that new mutations of the PKD1 gene occur at a significant rate (Peral et al 1997;Rossetti et al 2001). Approximately 80% of the mutations in the 3ЈPKD1 unique region (exons 34-46) were predicted to truncate the protein, and 37% of mutations in the 5Јpart of the gene were missense mutations (Bogdanova et al 2000).…”