Mutation detection for exons 2 to 10 of the Polycystic Kidney Disease 1 (PKD1)-gene by DGGE

Peters, Dorien J.M.; Ariyürek, Yavuz; M, van Dijk; Mh, Breuning

doi:10.1038/sj.ejhg.5200756

Cited by 9 publications

(6 citation statements)

References 13 publications

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“…The extensive allelic heterogeneity in PKD1 and PKD2 , combined with the fact that no single mutation accounts for >2% of ADPKD‐affected families, necessitates mutation‐screening methods for accurate molecular diagnosis of ADPKD [Harris and Rossetti, 2010]. Although several indirect methods have been developed, such as denaturing gradient gel electrophoresis [Perrichot et al, 1999, 2000; Peters et al, 2001], single‐strand conformation polymorphism analysis [Veldhuisen et al, 1997], the protein‐truncation test [Roelfsema et al, 1997; Rossetti et al, 2001], DHPLC [Rossetti et al, 2002], Transgenomics SURVEYOR Nuclease and WAVE Nucleic Acid High Sensitivity Fragment Analysis [Tan et al, 2009], and high resolution melt analysis [Bataille et al, 2011], direct sequencing [Garcia‐Gonzalez et al, 2007; Rossetti et al, 2007] is undoubtedly the gold standard.…”

Section: Resultsmentioning

confidence: 99%

Autosomal dominant polycystic kidney disease: Comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients

et al. 2012

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Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disorder, is caused by mutations in PKD1 or PKD2. The molecular diagnosis of ADPKD is complicated by extensive allelic heterogeneity and particularly by the presence of six highly homologous sequences of PKD1 exons 1-33. Here, we screened PKD1 and PKD2 for both conventional mutations and gross genomic rearrangements in up to 700 unrelated ADPKD patients--the largest patient cohort to date--by means of direct sequencing, followed by quantitative fluorescent multiplex polymerase chain reaction or array-comparative genomic hybridization. This resulted in the identification of the largest number of new pathogenic mutations (n = 351) in a single publication, expanded the spectrum of known ADPKD pathogenic mutations by 41.8% for PKD1 and by 23.8% for PKD2, and provided new insights into several issues, such as the population-dependent distribution of recurrent mutations compared with founder mutations and the relative paucity of pathogenic missense mutations in the PKD2 gene. Our study, together with others, highlights the importance of developing novel approaches for both mutation detection and functional validation of nondefinite pathogenic mutations to increase the diagnostic value of molecular testing for ADPKD.

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Section: Resultsmentioning

confidence: 99%

Autosomal dominant polycystic kidney disease: Comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients

et al. 2012

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“…Filters were also hybridized with probe D16S138 (N2; Himmelbauer et al, 1991) to check equal loading. Radiation hybrid HJ145.19, which contains only the PKD1 gene and the rodent-human somatic cell hybrid N23HA, which contains only the homologous region, were used to validate the probes (The European Polycystic Kidney Disease Consortium, 1994; Peters et al, 2001). Probe intron 1 recognized the 20kb EcoRI+AflII fragment and a 30kb fragment from the homologous region.…”

Section: Resultsmentioning

confidence: 99%

Large deletions in the polycystic kidney disease 1 (PKD1) gene

et al. 2003

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Since identification of the genes mutated in patients with Autosomal Dominant Polycystic Kidney Disease, PKD1 and PKD2, a large number of different germ line mutations in both genes have been found by conventional PCR-based mutation detection methods. Nevertheless, in approximately 40% of the PKD1 families the disease-causing mutation remains to be elucidated. Complex germ line rearrangements are often not detectable by these standard diagnostic techniques. To detect large deletions in the PKD1 gene we performed Field Inversion Gel Electrophoresis (FIGE) followed by Southern blot analysis with probes selected in the unique and in the reiterated region of this gene. Our analysis revealed 4 deletions in 125 patients, indicating that large deletions in PKD1 are rare. Likely, patients with a deletion that also affects the neighbouring Tuberous Sclerosis Complex 2 (TSC2) gene will be diagnosed as patients with tuberous sclerosis. It was speculated that the exceptional polypyrimidine tract located in intron 21 and the small tract in intron 22, might play a role in the pathogenesis of ADPKD. Since this region is extremely difficult to amplify by PCR, we analysed the 5.8 kb BamHI fragment that contains the polypyrimidine tracts. We did not observe a disease-linked alteration although we detected two different rare variants either in PKD1 or in one of its homologues.

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“…The limitation of detection rate may be due to low sensitivity of SSCP for mutation screening in long cDNA fragments (>300 bp) usually present after the digestion of PCR products with restriction enzymes and also probably due to the use of only single SSCP condition. However, this detection rate is comparable to that of protein-truncation test (PTT) which is about 52% [26,27] although it is lower than that of DGGE [28-30] and DHPLC [15] which are about 60% and 70%, respectively. In the MRF-SSCP analysis, false-positive mobility shifts of the digested-cDNA fragments were found to be about 10%, which could be mainly eliminated by repeating the screening experiment or performing the analysis in more than one affected members in the same family.…”

Section: Discussionmentioning

confidence: 99%

Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)

et al. 2004

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BackgroundWe have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported.MethodsFull-length PKD1 cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family.ResultsFive PKD1 mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly de novo.ConclusionThe MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of de novo PKD1 mutations indicates that this gene is prone to mutations.

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Mutation detection for exons 2 to 10 of the Polycystic Kidney Disease 1 (PKD1)-gene by DGGE

Cited by 9 publications

References 13 publications

Autosomal dominant polycystic kidney disease: Comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients

Autosomal dominant polycystic kidney disease: Comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients

Large deletions in the polycystic kidney disease 1 (PKD1) gene

Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)

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