TY - JOUR
T1 - Mutation analysis of the entire PKD1 gene
T2 - Genetic and diagnostic
AU - Rossetti, Sandro
AU - Strmecki, Lana
AU - Gamble, Vicki
AU - Burton, Sarah
AU - Sneddon, Vicky
AU - Peral, Belén
AU - Roy, Sushmita
AU - Bakkaloglu, Aysin
AU - Komel, Radovan
AU - Winearls, Christopher G.
AU - Harris, Peter C.
N1 - Funding Information:
We wish to thank S. Butler, J. Sloane-Stanley, and K. Clark for technical assistance; K. Zerres and S. Ozen for supplying samples; the patients and their families for taking part in the study; and V. E. Torres and D. J. Weatherall for support and encouragement. The work was supported by the Medical Research Council (United Kingdom), the Mayo Foundation, Telethon (Italy), NATO/Royal Society (United Kingdom), EMBO, Oxford Kidney Unit Trust Fund, and the Wellcome Trust.
PY - 2001
Y1 - 2001
N2 - 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, ∼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, 1.8 × 10-5 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.
AB - 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, ∼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, 1.8 × 10-5 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.
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U2 - 10.1086/316939
DO - 10.1086/316939
M3 - Article
C2 - 11115377
AN - SCOPUS:0035171856
SN - 0002-9297
VL - 68
SP - 46
EP - 63
JO - American journal of human genetics
JF - American journal of human genetics
IS - 1
ER -