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• W2011719303 abstract "SummaryAutosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous, with at least three chromosomal loci (PKD1, PKD2, and PKD3) that account for the disease. Mutations in the PKD2 gene, on the long arm of chromosome 4, are expected to be responsible for ∼15% of cases of ADPKD. Although ADPKD is a systemic disease, it shows a focal expression, because <1% of nephrons become cystic. A feasible explanation for the focal nature of events in PKD1, proposed on the basis of the two-hit theory, suggests that cystogenesis results from the inactivation of the normal copy of the PKD1 gene by a second somatic mutation. The aim of this study is to demonstrate that somatic mutations are present in renal cysts from a PKD2 kidney. We have studied 30 renal cysts from a patient with PKD2 in which the germline mutation was shown to be a deletion that encompassed most of the disease gene. Loss-of-heterozygosity (LOH) studies showed loss of the wild-type allele in 10% of cysts. Screening of six exons of the gene by SSCP detected eight different somatic mutations, all of them expected to produce truncated proteins. Overall, ⩾37% of the cysts studied presented somatic mutations. No LOH for the PKD1 gene or locus D3S1478 were observed in those cysts, which demonstrates that somatic alterations are specific. We have identified second-hit mutations in human PKD2 cysts, which suggests that this mechanism could be a crucial event in the development of cystogenesis in human ADPKD-type 2. Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous, with at least three chromosomal loci (PKD1, PKD2, and PKD3) that account for the disease. Mutations in the PKD2 gene, on the long arm of chromosome 4, are expected to be responsible for ∼15% of cases of ADPKD. Although ADPKD is a systemic disease, it shows a focal expression, because <1% of nephrons become cystic. A feasible explanation for the focal nature of events in PKD1, proposed on the basis of the two-hit theory, suggests that cystogenesis results from the inactivation of the normal copy of the PKD1 gene by a second somatic mutation. The aim of this study is to demonstrate that somatic mutations are present in renal cysts from a PKD2 kidney. We have studied 30 renal cysts from a patient with PKD2 in which the germline mutation was shown to be a deletion that encompassed most of the disease gene. Loss-of-heterozygosity (LOH) studies showed loss of the wild-type allele in 10% of cysts. Screening of six exons of the gene by SSCP detected eight different somatic mutations, all of them expected to produce truncated proteins. Overall, ⩾37% of the cysts studied presented somatic mutations. No LOH for the PKD1 gene or locus D3S1478 were observed in those cysts, which demonstrates that somatic alterations are specific. We have identified second-hit mutations in human PKD2 cysts, which suggests that this mechanism could be a crucial event in the development of cystogenesis in human ADPKD-type 2. Autosomal dominant polycystic kidney disease (ADPKD; MIM 173900) is one of the most common Mendelian disorders in humans and the most frequent genetic cause of renal failure in adults. The main feature of the disease is the bilateral progressive cystic dilation of the renal tubules, which may lead to end-stage renal disease (ESRD). Hepatic cysts, cerebral aneurysms, and cardiac valve abnormalities may also be found (Gabow Gabow, 1993Gabow PA Autosomal dominant polycystic kidney disease.N Engl J Med. 1993; 329: 332-342Crossref PubMed Scopus (810) Google Scholar). Although ADPKD is a systemic disease, it shows a focal expression, because <1% of nephrons become cystic (Baert Baert, 1978Baert L Hereditary polycystic kidney disease (adult form): a microdissection study of two cases at an early stage of the disease.Kidney Int. 1978; 13: 519-525Crossref PubMed Scopus (96) Google Scholar). ADPKD is a genetically heterogeneous condition, with at least three genes involved: (1) PKD1, located in 16p13.3, which accounts for most ADPKD cases (Reeders et al. Reeders et al., 1985Reeders ST Breuning MH Davies KE Nicholls RD Jarman AP Higgs RD Pearson PL et al.A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16.Nature. 1985; 317: 542-544Crossref PubMed Scopus (528) Google Scholar; Peters and Sandkuijl Peters and Sandkuijl, 1992Peters DJM Sandkuijl LA Genetic heterogeneity of polycystic kidney disease in Europe.in: Breuning MH Devoto M Romeo G Contributions to nephrology: polycystic kidney disease. Karger, Basel1992: 128-139Crossref Google Scholar; European Polycystic Kidney Disease Consortium European Polycystic Kidney Disease Consortium, The, 1994European Polycystic Kidney Disease Consortium, The The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16.Cell. 1994; 77: 881-894Abstract Full Text PDF PubMed Scopus (705) Google Scholar; Torra et al. Torra et al., 1996Torra R Badenas C Darnell A Nicolau C Volpini V Revert L Estivill X Linkage, clinical features and prognosis of ADPKD types 1 and 2.J Am Soc Nephrol. 1996; 7: 2142-2151Crossref PubMed Google Scholar); (2) PKD2, located in 4q21–q22 (Kimberling et al. Kimberling et al., 1993Kimberling WJ Kumar S Gabow PA Kenyon JB Connolly CJ Somlo S Autosomal dominant polycystic kidney disease: localization of the second gene to chromosome 4q13-q23.Genomics. 1993; 18: 467-472Crossref PubMed Scopus (279) Google Scholar; Peters et al. Peters et al., 1993Peters DJM Spruit L Saris JJ Ravine D Sandkuijl LA Fossdal R Boersma J et al.Chromosome 4 localization of a second gene for autosomal dominant polycystic kidney disease.Nat Genet. 1993; 5: 359-362Crossref PubMed Scopus (237) Google Scholar); and (3) the much rarer PKD3, not yet mapped (Daoust et al. Daoust et al., 1995Daoust MC Reynolds DM Bichet DG Somlo S Evidence for a third genetic locus for autosomal dominant polycystic kidney disease.Genomics. 1995; 25: 733-736Crossref PubMed Scopus (245) Google Scholar; de Almeida et al. De Almeida et al., 1995De Almeida S de Almeida E Peters DJM Pinto JR Tavora I Lavinha J Breuning MH et al.Autosomal dominant polycystic kidney disease: evidence for the existence of a third locus in a Portuguese family.Hum Genet. 1995; 96: 83-88Crossref PubMed Scopus (113) Google Scholar). PKD1 and PKD2 genes have been recently cloned and characterized (European Polycystic Kidney Disease Consortium European Polycystic Kidney Disease Consortium, The, 1994European Polycystic Kidney Disease Consortium, The The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16.Cell. 1994; 77: 881-894Abstract Full Text PDF PubMed Scopus (705) Google Scholar; Burn et al. Burn et al., 1995Burn TC Connors TD Dackowski WR Petry LR Van Raay TJ Millholland JM Venet M et al.Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease (PKD1) gene predicts the presence of a leucine-rich repeat.Hum Mol Genet. 1995; 4: 575-582Crossref PubMed Scopus (233) Google Scholar; Hughes et al. Hughes et al., 1995Hughes J Ward CJ Peral B Aspinwall R Clark K San Millán JL Gamble V et al.The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains.Nat Genet. 1995; 10: 151-160Crossref PubMed Scopus (725) Google Scholar; International PKD Consortium International PKD Consortium, 1995International PKD Consortium Polycystic kidney disease: the complete structure of the PKD1 gene and its protein.Cell. 1995; 81: 289-298Abstract Full Text PDF PubMed Scopus (602) Google Scholar; Mochizuki et al. Mochizuki et al., 1996Mochizuki T Wu G Hayashi T Xenophontos SL Veldhuisen B Saris JJ PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1100) Google Scholar). The PKD2 gene consists of 15 exons with an open reading frame of 2,904 bp (Mochizuki et al. Mochizuki et al., 1996Mochizuki T Wu G Hayashi T Xenophontos SL Veldhuisen B Saris JJ PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1100) Google Scholar; Hayashi et al. Hayashi et al., 1997Hayashi T Mochizuki T Reynolds DM Wu GQ Cai Y Somlo S Characterization of the exon structure of the polycystic kidney disease 2 gene (PKD2).Genomics. 1997; 44: 131-136Crossref PubMed Scopus (80) Google Scholar). Polycystin-2, the PKD2 gene product, is predicted to be a 968-amino acid integral-membrane protein with six membrane-spanning domains and intracellular amino and carboxyl termini (Mochizuki et al. Mochizuki et al., 1996Mochizuki T Wu G Hayashi T Xenophontos SL Veldhuisen B Saris JJ PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1100) Google Scholar). At present, several germline mutations have been described in the PKD2 gene, including nonsense, splice-site, frame-shift, and two putative missense mutations (Mochizuki et al. Mochizuki et al., 1996Mochizuki T Wu G Hayashi T Xenophontos SL Veldhuisen B Saris JJ PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1100) Google Scholar; Veldhuisen et al. Veldhuisen et al., 1997Veldhuisen B Saris JJ de Haij S Hayashi T Reynolds DM Mochizuki T Elles R et al.A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).Am J Hum Genet. 1997; 61: 547-555Abstract Full Text PDF PubMed Scopus (79) Google Scholar; Viribay et al. Viribay et al., 1997Viribay M Hayashi T Tellería D Mochizuki T Reynolds DM Alonso R Lens XM et al.Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.Hum Genet. 1997; 101: 229-234Crossref PubMed Scopus (34) Google Scholar; Xenophontos et al. Xenophontos et al., 1997Xenophontos S Constantinides R Hayashi T Mochizuki T Somlo S Pierides A Constantinou-Deltas C A translation frameshift mutation induced by a cytosine insertion in the polycystic kidney disease 2 gene (PDK2).Hum Mol Genet. 1997; 6: 949-952Crossref PubMed Scopus (23) Google Scholar; Pei et al. Pei et al., 1998Pei Y He N Wang K Kasenda M Paterson AD Chan G Liang Y et al.A spectrum of mutations in the polycystic kidney disease-2 (PKD2) gene from eight Canadian kindreds.J Am Soc Nephrol. 1998; 9: 1853-1860Crossref PubMed Google Scholar; Torra et al., Torra et al., in pressTorra R, Viribay M, Tellería D, Badenas C, Watson M, Harris PC, Darnell A, et al. Seven novel mutations of the PKD2 gene in families with autosomal dominant polycystic kidney disease. Kidney Int (in press)Google Scholar). Most of the PKD2 mutations are expected to produce truncated proteins, since they also occur for mutations in the PKD1 gene. Recent studies have demonstrated that polycystin-2 interacts with polycystin-1 through its cytoplasmic carboxyl end (Qian et al. Qian et al., 1997Qian F Germino FJ Cai Y Zhang X Somlo S Germino GG PKD1 interacts with PKD2 through a probable coiled-coil domain.Nat Genet. 1997; 16: 179-183Crossref PubMed Scopus (533) Google Scholar; Tsiokas et al. Tsiokas et al., 1997Tsiokas L Kim E Arnould T Sukhatme UP Walz G Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2.Proc Natl Acad Sci USA. 1997; 94: 6965-6970Crossref PubMed Scopus (404) Google Scholar); thus, truncating mutations would interfere with this interaction. These truncating mutations, in both PKD1 and PKD2, suggest that ADPKD is caused by inadequate levels of polycystin (i.e., haploinsufficiency). The process of cystogenesis could also be explained by a model of dominant/negative function, in which a mutated form of polycystin would inactivate the normal polycystin produced by the normal allele. On the basis of loss-of-heterozygosity (LOH) studies, it has been proposed that the process of cystogenesis in PKD1 results from focal somatic second hits on the wild-type allele (Qian et al. Qian et al., 1996Qian F Watnick TJ Onuchic LF Germino GG The molecular basis of cyst formation in human autosomal dominant polycystic kidney disease type 1.Cell. 1996; 87: 979-987Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar; Brasier and Henske Brasier and Henske, 1997Brasier JL Henske EP Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.J Clin Invest. 1997; 99: 194-199Crossref PubMed Scopus (212) Google Scholar). However, the strong immunoreactivity for polycystin-1 observed in the majority of PKD1 cysts has challenged this hypothesis (Geng et al. Geng et al., 1996Geng L Segal Y Peissel B Deng N Pei Y Carone F Rennke HG et al.Identification and localization of polycystin, the PKD1 gene product.J Clin Invest. 1996; 98: 2674-2682Crossref PubMed Scopus (164) Google Scholar; Ward et al. Ward et al., 1996Ward CJ Turley H Ong AC Comley M Biddolph S Chetty R Ratcliffe PJ et al.Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.Proc Natl Acad Sci USA. 1996; 93: 1524-1528Crossref PubMed Scopus (204) Google Scholar; Ibraghimov-Beskrovnaya et al. Ibraghimov-Beskrovnaya et al., 1997Ibraghimov-Beskrovnaya O Dackowski WR Foggensteiner L Coleman N Thiru S Petry LR Polycystin: in vitro synthesis, in vivo tissue expression, and subcellular localization identifies a large membrane-associated protein.Proc Natl Acad Sci USA. 1997; 94: 6397-6402Crossref PubMed Scopus (162) Google Scholar; Ong et al., Ong et al., 1999Ong ACM Ward CJ Butler RJ Biddolph S Bowker C Torra R Pei Y et al.Coordinate expression of the ADPKD proteins, polycystin-2 and polycystin-1, in normal and cystic tissue.Am J Pathol. 1999; 154: 1721-1729Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Alternatively, it has been suggested that, as polycystin-1 interacts with polycystin-2, somatic mutations in PKD2 could cooperate with the germline mutation in PKD1 (Qian et al. Qian et al., 1996Qian F Watnick TJ Onuchic LF Germino GG The molecular basis of cyst formation in human autosomal dominant polycystic kidney disease type 1.Cell. 1996; 87: 979-987Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Wu et al. (Wu et al., 1998Wu G D'Agati V Cai Y Markowitz G Park JH Reynolds DM Maeda Y et al.Somatic inactivation of PKD2 results in polycystic kidney disease.Cell. 1998; 93: 177-188Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar) showed results that support a two-hit hypothesis in mice. Recent studies on a mouse model for PKD2 found that mice that carry a null allele and an allele that can undergo genomic rearrangements that lead to a null allele develop renal cysts at a higher frequency than those that are heterozygous for any of these alleles (Wu et al. Wu et al., 1998Wu G D'Agati V Cai Y Markowitz G Park JH Reynolds DM Maeda Y et al.Somatic inactivation of PKD2 results in polycystic kidney disease.Cell. 1998; 93: 177-188Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). More recently, Koptides et al. (Koptides et al., 1999Koptides M Hadjimichael C Koupepidou P Pierides A Constantinou Deltas C Germinal and somatic mutations in the PKD2 gene of renal cysts in autosomal dominant polycystic kidney disease.Hum Mol Genet. 1999; 8: 509-513Crossref PubMed Scopus (82) Google Scholar) showed evidence of somatic point mutations for human PKD2 cysts, but did not find evidence of LOH. The purpose of the present study was to demonstrate a loss-of-function mechanism for PKD2 in humans through the study of LOH and point somatic mutations within the PKD2 gene. The cysts studied were obtained from a kidney that belonged to a patient with ADPKD who underwent an orthotopic transplantation at age 69 years. His kidney was removed during the transplantation procedure. This patient belongs to a large family we had previously studied that had clear evidence of linkage to PKD2 (LOD score 4.0) (Torra et al. Torra et al., 1996Torra R Badenas C Darnell A Nicolau C Volpini V Revert L Estivill X Linkage, clinical features and prognosis of ADPKD types 1 and 2.J Am Soc Nephrol. 1996; 7: 2142-2151Crossref PubMed Google Scholar). This family was the only one of our families with PKD2 in which no mutation had been detected by heteroduplex or SSCP analyses (Torra et al., Torra et al., in pressTorra R, Viribay M, Tellería D, Badenas C, Watson M, Harris PC, Darnell A, et al. Seven novel mutations of the PKD2 gene in families with autosomal dominant polycystic kidney disease. Kidney Int (in press)Google Scholar). The clinical features of the persons in this family were those typical of persons with PKD2 with late-onset disease and with ESRD at a mean age of 72 years. The kidney was studied immediately after removal, which took place in the Hospital Clínic of Barcelona. The study was approved by the hospital's ethical committee. To avoid contamination from neighboring cells, we followed the procedure of Qian et al. (Qian et al., 1996Qian F Watnick TJ Onuchic LF Germino GG The molecular basis of cyst formation in human autosomal dominant polycystic kidney disease type 1.Cell. 1996; 87: 979-987Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar) and introduced some modifications. Throughout the procedure, the kidney was placed on ice in a tray (fig. 1). To obtain as few contaminating cells as possible, the surface of the cyst was first rinsed with PBS. We then drained the cyst content with a needle and a syringe. The needle was left inserted in the cyst during the washing steps. The cavity of the cyst was rinsed four to eight times with Ca2+ and Mg2+-free PBS, until the liquid that was obtained appeared to be clear. At this point, PBS/2 mM EDTA was introduced into the cysts and was energetically pulled in and out of the cyst several times to facilitate the detachment of the epithelial cells from the basement membrane. Only the cysts that remained intact were considered for the study. The cells that were obtained from the cyst cavity were suspended in PBS/2 mM EDTA and centrifuged at 14,000 rpm for 5 min, and the supernatant was discarded. The pellet was resuspended with 600 μl of urea solution (8 M urea, 0.3 M NaCl, 10 mM EDTA, 10 mM TRIS, 2% SDS) and 12 μl of proteinase K solution (10 mg/ml), incubated overnight at 37°C, and treated with a phenol/chloroform extraction. Next, 0.5 μl glycogen and 2.5 V absolute ethanol were added, and the pellet was incubated for 1 h at −80°C. After incubation, it was centrifuged for 20 min at 14,000 rpm and the supernatant was discarded. Eventually, the pellet was dried and resuspended in 45 μl 1×TE pH 7.5. To increase the amount of DNA for subsequent studies, the material obtained from cystic cells was preamplified. The reaction included 1–2 μl DNA, 6 μl 10× buffer (Perkin-Elmer), 4mM DTT, 2.5 mM MgCl2, 10 mM of each dNTPs, 5 U of Taq polymerase (Perkin-Elmer), 400 μM of each random primer (15-mer oligonucleotides in which any one of the four possible bases could be present at each position), and distilled water to 60 μl. PCR amplification was performed as follows: 5 min at 94°C; 50 cycles: 1 min at 94°C, 2 min at 37°C, and 4 min at 55°C. Each DNA sample was typed by PCR by using microsatellites D4S1542 and D4S1563 for PKD2 locus and by using D16S663, D16S291, and KG8 [PKD1-3′UTR] for PKD1. The distance of these flanking markers to the corresponding gene was <1 cM. Marker D3S1478 was used as a control. The patient was informative for all the microsatellites studied. One microliter of preamplified DNA was used for each PCR reaction. We used 5% DMSO and 1.5 mM (D16S663, KG8 [PKD1-3′UTR],D4S1563, D4S1542, and D3S1478) or 1 mM (D16S291) MgCl2. The PCR conditions were as described elsewhere (Torra et al. Torra et al., 1996Torra R Badenas C Darnell A Nicolau C Volpini V Revert L Estivill X Linkage, clinical features and prognosis of ADPKD types 1 and 2.J Am Soc Nephrol. 1996; 7: 2142-2151Crossref PubMed Google Scholar; Brasier et al. Brasier and Henske, 1997Brasier JL Henske EP Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.J Clin Invest. 1997; 99: 194-199Crossref PubMed Scopus (212) Google Scholar). The PCR products were run on an acrylamide gel and were silver stained (Bassam et al. Bassam et al., 1991Bassam BJ Caetano-Anolles G Gresshoff PM Fast and sensitive silver staining of DNA in polyacrylamide gels.Anal Biochem. 1991; 196: 80-83Crossref PubMed Scopus (2568) Google Scholar). We analyzed exons 2, 4–6, 11, and 12 of PKD2. The primers used for exon amplification and PCR conditions have been described elsewhere (Hayashi et al. Hayashi et al., 1997Hayashi T Mochizuki T Reynolds DM Wu GQ Cai Y Somlo S Characterization of the exon structure of the polycystic kidney disease 2 gene (PKD2).Genomics. 1997; 44: 131-136Crossref PubMed Scopus (80) Google Scholar; Viribay et al. Viribay et al., 1997Viribay M Hayashi T Tellería D Mochizuki T Reynolds DM Alonso R Lens XM et al.Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.Hum Genet. 1997; 101: 229-234Crossref PubMed Scopus (34) Google Scholar). PCR products were analyzed by SSCP. Three microliters of denatured PCR product were combined with loading buffer, were loaded into GeneGel Excel 12.5 acrylamide gels (Amersham Pharmacia Biotech), and were run according to the manufacturer's instructions. The different migrations were silver stained (Bassam et al. Bassam et al., 1991Bassam BJ Caetano-Anolles G Gresshoff PM Fast and sensitive silver staining of DNA in polyacrylamide gels.Anal Biochem. 1991; 196: 80-83Crossref PubMed Scopus (2568) Google Scholar). DNA samples with abnormal migrations were sequenced. PCR products were purified by using the QIAquick PCR purification kit (Qiagen) and were automatically sequenced by means of the Dye Terminator Cycle Sequencing Ready Reaction (Perkin Elmer Cetus) and an automatic sequencer (ABI310). To exclude the possibility that the mutations were the result of the preamplification step, we analyzed nonpreamplified DNA from two cysts that harbor mutations that disrupted an endonuclease recognition site (see table 1) and confirmed the presence of the corresponding mutations. We also preamplified and twice sequenced the other mutations that do not abolish a restriction site, which confirmed the presence of the nucleotide changes.Table 1Somatic Mutations in the PKD2 Gene in a Polycystic Kidney of a Patient with Autosomal Dominant PKD2CystLOH for MicrosatellitesMutationExon or IntronC1IVS4+1G→AIntron 4C3934insGExon 4C5990delCaExon 4C6D4S1542, D4S1563PKD2 deletionExons 1–15C82236insAExon 11C10D4S1542, D4S1563PKD2 deletionExons 1–15C17D4S1563PKD2 deletion?C23Y429XbExon 5C252198del13Exon 11C291217delTExon 5C302207delTExon 11aMutation 990delC abolishes a BamHI restriction site.bMutation Y429X abolishes an RsaI restriction site. Open table in a new tab aMutation 990delC abolishes a BamHI restriction site. bMutation Y429X abolishes an RsaI restriction site. Five micrograms of genomic DNA from affected and normal individuals were digested with 50 U of EcoRI (Boehringer-Mannheim) and run on a 0.8% agarose gel at 40 V overnight. Southern blotting onto a nylon filter was done according to standard protocols. DNA probes were prepared by PCR amplification of genomic DNA, followed by extraction of the corresponding band from low–melting-point agarose gels. For PKD2, two probes were obtained by amplification of part of exon 1 of the gene with primers F42 and IR19d (Hayashi et al. Hayashi et al., 1997Hayashi T Mochizuki T Reynolds DM Wu GQ Cai Y Somlo S Characterization of the exon structure of the polycystic kidney disease 2 gene (PKD2).Genomics. 1997; 44: 131-136Crossref PubMed Scopus (80) Google Scholar) or by amplification of a fragment containing exons 11 and 12 with primers IF10 and IR3. A control DNA probe was obtained by amplification of exon 4 of the CFTR gene (Zielenski et al. Zielenski et al., 1991Zielenski J Rozmahel R Bozon D Kerem BS Grzelczak Z Riordan JR Rommens J et al.Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (484) Google Scholar). DNA probes were labeled with [32P]-dCTP to high specific activity by the random hexamer primer method. Hybridizations, simultaneously containing one of the PKD2 probes and the control probe, were done at 65°C. The sizes of the target EcoRI fragments were 5.2 and 5.4 kb for exon 1 and exons 11–13 of the PKD2 gene, respectively (Hayashi et al. Hayashi et al., 1997Hayashi T Mochizuki T Reynolds DM Wu GQ Cai Y Somlo S Characterization of the exon structure of the polycystic kidney disease 2 gene (PKD2).Genomics. 1997; 44: 131-136Crossref PubMed Scopus (80) Google Scholar), and 4.7 kb for exon 4 of CFTR (Zielenski et al. Zielenski et al., 1991Zielenski J Rozmahel R Bozon D Kerem BS Grzelczak Z Riordan JR Rommens J et al.Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Genomics. 1991; 10: 214-228Crossref PubMed Scopus (484) Google Scholar). The band densities of patients and normal individuals were analyzed by densitometry. The germline mutation causing the disease in this family had not been identified after repeated screenings of exonic sequences and exon-intron boundaries by SSCP or by heteroduplex analyses in previous studies (Torra et al., Torra et al., in pressTorra R, Viribay M, Tellería D, Badenas C, Watson M, Harris PC, Darnell A, et al. Seven novel mutations of the PKD2 gene in families with autosomal dominant polycystic kidney disease. Kidney Int (in press)Google Scholar). However, analysis with a frequent polymorphism recently identified within exon 1 of PKD2 (Arg28Pro, Torra et al., Torra et al., in pressTorra R, Viribay M, Tellería D, Badenas C, Watson M, Harris PC, Darnell A, et al. Seven novel mutations of the PKD2 gene in families with autosomal dominant polycystic kidney disease. Kidney Int (in press)Google Scholar) detected an apparent non-Mendelian segregation of alleles for this polymorphism in the family (fig. 2A). Thus, although most affected individuals were apparently homozygous for one allele, two affected siblings (II2 and II3) were apparently homozygous for the other allele (fig. 2B). Densitometric analyses of genomic EcoRI DNA digests with probes that could detect exon 1 or exons 11–13 of PKD2 detected half intensity of PKD2 in the affected subjects (fig. 2C). This result supports a deletion that encompasses at least exons 1–13 of the gene as the germline mutation in this family. The analysis with microsatellite markers close to the disease gene (distance <1 cM) failed to detect LOH, which indicates that the deletion is probably not very large (fig. 2A). First we performed an LOH study with two microsatellite markers (D4S1542 and D4S1563) flanking PKD2 in 30 cysts from a kidney of individual I3 (fig. 2A). We found that three cysts had lost alleles for at least one of these markers (table 1). As expected, in all cases the allele lost corresponded to the wild-type PKD2 gene, as determined by segregation analysis. LOH was not always absolute, probably because of contamination from noncystic cells (fig. 3). When the PKD2 cysts were analyzed for three microsatellite markers linked to or within the PKD1 gene or with the D3S1478 marker, we failed to detect any evidence of LOH. Because no evidence of LOH for the PKD2 region was found in 27 cysts, we concentrated on screening for subtle changes in this gene, and in the first instance we focused on six exons. After SSCP analysis, eight cases with an abnormal migration pattern were observed, which on sequencing corresponded to eight different mutations (table 1 and fig. 4). All of them should result in a truncated PKD2 protein, six being frame-shift mutations due to the insertion or deletion of one or several nucleotides—one creating a premature stop codon and one affecting the splicing of intron 4. In most cases, sequencing revealed only the somatic-mutated allele, because the germline mutation was a deletion; however, since a small amount of noncystic cells are frequently present, in some sequences we could detect traces of sequence that corresponded to the wild-type allele (fig. 4B). Mutations in the PKD2 gene are expected to be responsible for ∼15% of cases of polycystic kidney disease (Peters and Sandkuijl Peters and Sandkuijl, 1992Peters DJM Sandkuijl LA Genetic heterogeneity of polycystic kidney disease in Europe.in: Breuning MH Devoto M Romeo G Contributions to nephrology: polycystic kidney disease. Karger, Basel1992: 128-139Crossref Google Scholar; Torra et al. Torra et al., 1996Torra R Badenas C Darnell A Nicolau C Volpini V Revert L Estivill X Linkage, clinical features and prognosis of ADPKD types 1 and 2.J Am Soc Nephrol. 1996; 7: 2142-2151Crossref PubMed Google Scholar). The PKD2 disease tends to run a milder course than the more common PKD1 disease, with longer patient survival and slower progression toward end-stage renal failure (Parfrey et al. Parfrey et al., 1990Parfrey PS Bear JC Morgan J Cramer BC McManamon PJ Gault MH Churchill DN et al.The diagnosis and prognosis of autosomal dominant polycystic kidney disease.N Engl J Med. 1990; 323: 1085-1090Crossref PubMed Scopus (282) Google Scholar; Gabow et al. Gabow et al., 1992Gabow PA Johnson AM Kaehny WD Kimberling WJ Lezotte DC Duley IT Jones RH Factors affecting the progression of renal disease in autosomal dominant polycystic kidney disease.Kidney Int. 1992; 41: 1311-1319Crossref PubMed Scopus (414) Google Scholar; Ravine et al. Ravi" @default.
• W2011719303 created "2016-06-24" @default.
• W2011719303 creator A5010996262 @default.
• W2011719303 creator A5017540743 @default.
• W2011719303 creator A5030376054 @default.
• W2011719303 creator A5031153935 @default.
• W2011719303 creator A5058542952 @default.
• W2011719303 creator A5083351035 @default.
• W2011719303 date "1999-08-01" @default.
• W2011719303 modified "2023-10-13" @default.
• W2011719303 title "A Loss-of-Function Model for Cystogenesis in Human Autosomal Dominant Polycystic Kidney Disease Type 2" @default.
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