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1.
Identification of mutations in the repeated part of the autosomal dominant polycystic kidney disease type 1 gene, PKD1, by long-range PCR.
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R Thomas R McConnell J Whittacker P Kirkpatrick J Bradley R Sandford 《American journal of human genetics》1999,65(1):39-49
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. 相似文献
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3.
DNA structural transitions within the PKD1 gene. 总被引:7,自引:0,他引:7
Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single-stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H-y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures. 相似文献
4.
Stefan Kirsch Juanjo Pasantes Andreas Wolf Nadia Bogdanova Claudia Münch Arseni Markoff Petra Pennekamp Michael Krawczak Bernd Dworniczak Werner Schempp 《BMC evolutionary biology》2009,9(1):14-1
Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Münch C, Pennekamp P, Krawczak M, Dworniczak B, Schempp W: Chromosomal evolution of the
PKD1
gene family in primates. BMC Evolutionary Biology 2008, 8 :263 (doi:10.1186/1471-2148-8-263) 相似文献
5.
The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation. 相似文献
6.
About 400 distinct mutations have been defined in the BRCA1 gene, and these are spread fairly evenly through the 5592 bp of coding DNA. This circumstance presents a formidable challenge
for mutation screening. Apart from total direct sequencing, the preferred screening method has been single-strand conformation
polymorphism (SSCP) gels, with a smaller input from constant denaturant gradient electrophoresis (CDGE), heteroduplex (HD)
analysis, and mismatch cleavage. The protein truncation test (PTT) was used early in BRCA1 mutation screening but has not been widely adopted, perhaps because a straightforward analysis of the whole BRCA1 gene requires working with RNA and all its perceived problems. The present work was undertaken to assess the practicality
of using the PTT under routine conditions for the screening of long genes such as BRCA1 that are not highly expressed in lymphocytes. We conclude that, provided RNA preparation is carried out effectively and consistently,
the PTT approach has significant advantages over other methodologies such as SSCP gels. 相似文献
7.
K Tsuchiya M Komeda M Takahashi N Yamashita M Cigira T Suzuki K Suzuki H Nihei T Mochizuki 《Mutation research》2001,458(3-4):77-84
Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disease that causes renal failure. One of the genes that is responsible for this disease, PKD1, has been identified and characterized. Many mutations of the PKD1 gene have been identified in the Caucasian population. We investigated the occurrence of mutations in this gene in the Japanese population. We analyzed each exon in the 3' single copy region of the gene between exons 35 and 46 in genomic DNA obtained from 69 patients, using a PCR-based direct sequencing method. Four missense mutations (T3509M, G3559R, R3718Q, R3752W), one deletion mutation (11307del61bp) and one polymorphism (L3753L) were identified, and their presence confirmed by allele-specific oligonucleotide (ASO) hybridization. These were novel mutations, except for R3752W, and three of them were identified in more than two families. Mutation analysis of the PKD1 gene in the Japanese population is being reported for the first time. 相似文献
8.
Mutation detection of PKD1 identifies a novel mutation common to three families with aneurysms and/or very-early-onset disease 总被引:5,自引:0,他引:5
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Watnick T Phakdeekitcharoen B Johnson A Gandolph M Wang M Briefel G Klinger KW Kimberling W Gabow P Germino GG 《American journal of human genetics》1999,65(6):1561-1571
It is known that several of the most severe complications of autosomal-dominant polycystic kidney disease, such as intracranial aneurysms, cluster in families. There have been no studies reported to date, however, that have attempted to correlate severely affected pedigrees with a particular genotype. Until recently, in fact, mutation detection for most of the PKD1 gene was virtually impossible because of the presence of several highly homologous loci also located on chromosome 16. In this report we describe a cluster of 4 bp in exon 15 that are unique to PKD1. Forward and reverse PKD1-specific primers were designed in this location to amplify regions of the gene from exons 11-21 by use of long-range PCR. The two templates described were used to analyze 35 pedigrees selected for study because they included individuals with either intracranial aneurysms and/or very-early-onset disease. We identified eight novel truncating mutations, two missense mutations not found in a panel of controls, and several informative polymorphisms. Many of the polymorphisms were also present in the homologous loci, supporting the idea that they may serve as a reservoir for genetic variability in the PKD1 gene. Surprisingly, we found that three independently ascertained pedigrees had an identical 2-bp deletion in exon 15. This raises the possibility that particular genotypes may be associated with more-severe disease. 相似文献
9.
Michael Koptides Rolandos Constantinides George Kyriakides Michael Hadjigavriel Philippos C. Patsalis Alkis Pierides C. Constantinou Deltas 《Human genetics》1998,103(6):709-717
Loss of heterozygosity (LOH) is a molecular phenomenon that denotes the loss of one of the two alleles at a specific locus.
It is frequently associated with tumour suppressor genes in various cancers and also with hyperproliferative disorders, although
not exclusively. Interestingly, in conditions where there is an inherited germline mutation, the lost allele is always the
functional one, thereby rendering a phenotypically dominant disease of recessive character at the cellular level. A disease
more recently shown to be associated with LOH is polycystic kidney disease type 1, a systemic disorder characterized by significant
pleiotropy. The main pathology is from renal cyst formation that eventually leads to end-stage renal failure during adult
life. We describe the identification of a missense mutation in the repeated part of the PKD1 gene, exon 31, that substitutes valine for methionine. The mutation, M3375V, cosegregates with the disease phenotype in a
large Cypriot family. During transplantation of one patient, one of the polycystic kidneys was removed and DNA was isolated
from cystic epithelial cells. In 3 of 17 cysts examined with intragenic and flanking polymorphic markers on chromosome 16
we detected LOH, since the wild-type allele was lost, thereby rendering the affected kidneys of mosaic character. The degree
of LOH was extensive and varied among the three cysts, supporting the multiplicity of expression of the phenomenon on different
occasions. No LOH was detected for other selected loci examined. Our work further supports the hypothesis that the rate-limiting
step in cyst formation may be the occurrence of a second somatic hit, although other factors may be also involved. The high
frequency of mutations at this locus may, to a great extent, explain the variability in phenotype observed among patients
in the same families, and the relatively high frequency of the disease worldwide.
Received: 13 August 1998 / Accepted: 13 October 1998 相似文献
10.
Mutation detection in the PKD1 gene proved to be difficult because two-thirds of the gene is reiterated several times on chromosome 16. Long-range PCR has been used previously to overcome this limitation, but due to a 2.5-kb polypyrimidine tract in intron 21, the screening capacity of the PKD1 gene using this technique was hindered. Here we report the measures that we have used to overcome this limitation. 相似文献
11.
Polycystin-1 (PC1), the product of the polycystic kidney disease-1 (PKD1) gene, has a number of reported missense mutations whose pathogenicity is indeterminate. Previously, we utilized N-linked glycosylation reporter tags along with membrane insertion and topology assays to define the 11 membrane-spanning domains (I-XI) of PC1. In this report, we utilize glycosylation assays to determine whether two reported human polymorphisms/missense mutations within transmembrane (TM) domains VI and X affect the membrane topology of PC1. M3677T within TM VI had no effect on the topology of this TM domain as shown by the ability of two native N-linked glycosylation sites within the extracellular loop following TM VI to be glycosylated. In contrast, G4031D, within TM X, decreased the glycosylation of TM X reporter constructs, demonstrating that the substitution affected the C-terminal translocating activity of TM X. Furthermore, G4031D reduced the membrane association of TM X and XI together. These results suggest that G4031D affects the membrane insertion and topology of the C-terminal portion of polycystin-1 and represents a bona fide pathogenic mutation. 相似文献
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13.
H Himmelbauer M Pohlschmidt A Snarey G G Germino D Weinstat-Saslow S Somlo S T Reeders A M Frischauf 《Genomics》1992,13(1):35-38
Autosomal dominant polycystic kidney disease (PKD1) is linked to the alpha-globin locus near the telomere of chromosome 16p. We established the existence of a conserved linkage group in mouse by mapping conserved sequences and cDNAs from the region surrounding the PKD1 gene in the mouse genome. Results obtained with the BXD recombinant strain system and somatic cell hybrids show the homologous region to be located on mouse chromosome 17 near the globin pseudogene Hba-ps4, an unprocessed alpha-like globin gene. The markers we mapped are widely distributed over the region known to contain the PKD1 gene, and it is therefore likely that the mouse homologue of PKD1 is also located on mouse chromosome 17. 相似文献
14.
Chisato Fujimoto Yoshiro Ishimaru Yuka Katano Takumi Misaka Tatsuya Yamasoba Tomiko Asakura Keiko Abe 《Biochemical and biophysical research communications》2011,404(4):497
The polycystic kidney disease 1-like 3 (PKD1L3)–polycystic kidney disease 2-like 1 (PKD2L1) complex functions as a Ca2+-permeable, non-selective cation channel that is activated by acid and its subsequent removal; this is called an off-response. In this study, we identified a single aspartic residue in PKD2L1 that is responsible for the Ca2+ permeation of the PKD1L3/PKD2L1 complex. Calcium imaging analysis using point mutants of negatively charged amino acids present in the putative pore regions of PKD1L3 and PKD2L1 revealed that neutralization of the aspartic residue in PKD2L1 (D523N), which is conserved among PKD2 family members, abolished Ca2+ permeation, despite robust cell surface expression. In contrast, neutralization of the other negatively charged residues of PKD1L3 (D2049N and E2072Q) and PKD2L1 (D525N and D530N) as well as substitution of Asp523 with a glutamate residue (D523E) had little effect on Ca2+ permeation properties. These results demonstrate that Asp523 in PKD2L1 is a key determinant of Ca2+ permeation into the PKD1L3/PKD2L1 complex and that PKD2L1 contributes to forming the pore of the PKD1L3/PKD2L1 channel. 相似文献
15.
C. Daniells Magitha Maheshwar Lazarus Lazarou Felicity Davies Gerry Coles David Ravine 《Human genetics》1998,102(2):216-220
A search has been conducted for disease-causing mutations in the PKD1 gene in 147 unrelated ADPKD index cases. Using the polymerase chain reaction with primer pairs located in the 3′ single copy
region of the gene and single-strand conformation polymorphism analysis, we detected novel aberrant bands in five individuals
that were absent in 100 control samples. Sequencing revealed three nonsense mutations (Q4010X, E4024X, Q4041X), a frameshift
mutation (12262 del 2 bp), and a missense mutation (G4031D). In addition, three polymorphisms were detected [12346 + 19delG,
heterozygosity (0.13), I4044V (0.23), 12212-34C > A (0.07)]. The mutational mechanism for the recurrent mutation (Q4041X)
is likely to be slipped mispairing of an adjacent direct imperfect repeat sequence.
Received: 5 April 1997 / Accepted: 26 August 1997 相似文献
16.
Afzal AR Hand M Ternes-Pereira E Saggar-Malik A Taylor R Jeffery S 《Human genetics》1999,105(6):648-653
Mutation screening in 90 unrelated ADPKD1 patients was carried out on some of the exons in the single copy area (37, 38, 39, 44, 45) using genomic PCR and SSCP. Four novel mutations were found: a 15 bp in-frame deletion in exon 39 [nt11449 (del 15)], a 2 bp deletion in exon 44 [nt12252 (del 2)], a G insertion in exon 44 [nt12290 (Ins G)], and a GTT in-frame deletion in exon 45 [nt12601 (del 3)]. 相似文献
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18.
I McIntosh S D Dreyer M V Clough J A Dunston W Eyaid C M Roig T Montgomery S Ala-Mello I Kaitila A Winterpacht B Zabel M Frydman W G Cole C A Francomano B Lee 《American journal of human genetics》1998,63(6):1651-1658
Nail-patella syndrome (NPS), a pleiotropic disorder exhibiting autosomal dominant inheritance, has been studied for >100 years. Recent evidence shows that NPS is the result of mutations in the LIM-homeodomain gene LMX1B. To determine whether specific LMX1B mutations are associated with different aspects of the NPS phenotype, we screened a cohort of 41 NPS families for LMX1B mutations. A total of 25 mutations were identified in 37 families. The nature of the mutations supports the hypothesis that NPS is the result of haploinsufficiency for LMX1B. There was no evidence of correlation between aspects of the NPS phenotype and specific mutations. 相似文献
19.
Fujimoto C Ishimaru Y Katano Y Misaka T Yamasoba T Asakura T Abe K 《Biochemical and biophysical research communications》2011,404(4):946-951
The polycystic kidney disease 1-like 3 (PKD1L3)-polycystic kidney disease 2-like 1 (PKD2L1) complex functions as a Ca(2+)-permeable, non-selective cation channel that is activated by acid and its subsequent removal; this is called an off-response. In this study, we identified a single aspartic residue in PKD2L1 that is responsible for the Ca(2+) permeation of the PKD1L3/PKD2L1 complex. Calcium imaging analysis using point mutants of negatively charged amino acids present in the putative pore regions of PKD1L3 and PKD2L1 revealed that neutralization of the aspartic residue in PKD2L1 (D523N), which is conserved among PKD2 family members, abolished Ca(2+) permeation, despite robust cell surface expression. In contrast, neutralization of the other negatively charged residues of PKD1L3 (D2049N and E2072Q) and PKD2L1 (D525N and D530N) as well as substitution of Asp(523) with a glutamate residue (D523E) had little effect on Ca(2+) permeation properties. These results demonstrate that Asp(523) in PKD2L1 is a key determinant of Ca(2+) permeation into the PKD1L3/PKD2L1 complex and that PKD2L1 contributes to forming the pore of the PKD1L3/PKD2L1 channel. 相似文献
20.
Mutation detection in Machado-Joseph disease using repeat expansion detection. 总被引:3,自引:0,他引:3
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K. Lindblad A. Lunkes P. Maciel G. Stevanin C. Zander T. Klockgether T. Ratzlaff A. Brice G. A. Rouleau T. Hudson G. Auburger M. Schalling 《Molecular medicine (Cambridge, Mass.)》1996,2(1):77-85
BACKGROUND: Several neurological disorders have recently been explained through the discovery of expanded DNA repeat sequences. Among these is Machado-Joseph disease, one of the most common spinocerebellar ataxias (MJD/SCA3), caused by a CAG repeat expansion on chromosome 14. A useful way of detecting repeat sequence mutations is offered by the repeat expansion detection method (RED), in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA. We have used RED to detect CAG expansions in families with either MJD/SCA3 or with previously uncharacterized spinocerebellar ataxia (SCA). MATERIALS AND METHODS: Five MJD/SCA3 families and one SCA family where linkage to SCA1-5 had been excluded were analyzed by RED and polymerase chain reaction (PCR). RESULTS: An expansion represented by RED products of 180-270 bp segregated with MJD/SCA3 (p < 0.00001) in five families (n = 60) and PCR products corresponding to 66-80 repeat copies were observed in all affected individuals. We also detected a 210-bp RED product segregating with disease (p < 0.01) in a non-SCA1-5 family (n = 16), suggesting involvement of a CAG expansion in the pathophysiology. PCR analysis subsequently revealed an elongated MJD/SCA3 allele in all affected family members. CONCLUSIONS: RED products detected in Machado-Joseph disease families correlated with elongated PCR products at the MJD/SCA3 locus. We demonstrate the added usefulness of RED in detecting repeat expansions in disorders where linkage is complicated by phenotyping problems in gradually developing adult-onset disorders, as in the non-SCA1-5 family examined. The RED method is informative without any knowledge of flanking sequences. This is particularly useful when studying diseases where the mutated gene is unknown. We conclude that RED is a reliable method for analyzing expanded repeat sequences in the genome. 相似文献