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.

Figure 1

Diagram showing the fragments employed for mutation screening of the PKD1 gene and the mutations detected. The PKD1 gene (top) and transcript (bottom), showing the intron/exon structure, duplicated region (blue), single-copy coding exons (solid boxes), and 5′ and 3′ UTRs (open boxes). The positions of fragments to amplify the transcript specifically (Spec; blue), plus the PTT fragments (PTT; red) and cDNA sequencing products (cSeq; green), are shown at the bottom. The locations of the PKD1-specific and anchored genomic fragments (Gen; blue), plus the genomic sequencing products (GenSeq; green), are illustrated at the top. The sites of the mutations detected in the 81 different PKD1 pedigrees (P) are shown in the center. Different mutation types are color coded and grouped: splicing mutations, turquoise; frameshifting deletions or insertions, purple; in-frame deletions or insertions, green; nonsense mutations, red; and missense mutations, dark blue. Newly described mutations are shown in boldface type.

Figure 2

Examples of PTT gels. A, Fragment PTT3, transcripted and translated from cDNA of mouse; human (Normal); somatic-cell hybrids, 77-2/1 and P-MWH2A (HG only; lane 2 and 6, respectively); P-SHH1H; whole chromosome 16 (16 hybrid); and the radiation hybrid, Hy145.19 (PKD1 only). The product is only generated in PKD1-containing samples showing the specificity of this PTT assay. Analysis of PKD1 patient and normal cDNA by PTT assays, PTT1 (B) and PTT4 (C). The pedigree number and detected mutation are shown for each sample. Mutant polypeptides are seen as smaller products on the gel.

Figure 3

Examples of direct sequence analysis of patient DNA. A, The missense substitution E2771K, caused by G→A transition at position 8522 nt. B, Two nonsense mutations S225X and Q227X, caused by C→A and C→T substitutions at positions 885 and 890 nt, respectively. C, Normal and patient DNA with the mutation F1992L, 1993delT, caused by a single codon deletion, 6187del3. Note the double peak at the site of a substitution (A and B) and continued double peaks after the deletion (C).

Figure 4

Segregation of PKD1 mutations in pedigrees. A, IVS7+1G→A segregates in pedigree 1 (P1). Restriction digest of PKD1 specifically amplified DNA (GenSeq 7) digested with MnI1. A 234-bp fragment (arrow) is found in the affected individuals because of loss of a restriction site. Samples numbered in the pedigree are in the corresponding gel lane. B, Genomic heteroduplex analysis of the mutation 224del13 in pedigree 103 (P103). Heteroduplexes (arrow) and two homoduplex fragments, normal and deleted, can be seen in the affected individuals.