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

<p>Abstract</p> <p>Background</p> <p>We have previously developed a long RT-PCR method for selective amplification of full-length <it>PKD1 </it>transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 an...

Full description

Bibliographic Details
Main Authors: Bunditworapoom Duangkamon, Sirinavin Chintana, Vareesangthip Kriengsak, Limwongse Chanin, Thongnoppakhun Wanna, Rungroj Nanyawan, Yenchitsomanus Pa-thai
Format: Article
Language:English
Published: BMC 2004-02-01
Series:BMC Medical Genetics
Online Access:http://www.biomedcentral.com/1471-2350/5/2
id doaj-cda685b03ba14727bbfc069a4dc562ad
record_format Article
spelling doaj-cda685b03ba14727bbfc069a4dc562ad2021-04-02T03:08:32ZengBMCBMC Medical Genetics1471-23502004-02-0151210.1186/1471-2350-5-2Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)Bunditworapoom DuangkamonSirinavin ChintanaVareesangthip KriengsakLimwongse ChaninThongnoppakhun WannaRungroj NanyawanYenchitsomanus Pa-thai<p>Abstract</p> <p>Background</p> <p>We have previously developed a long RT-PCR method for selective amplification of full-length <it>PKD1 </it>transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the <it>PKD1 </it>gene. These have provided us with an opportunity to study <it>PKD1 </it>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 <it>PKD1 </it>mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and <it>de novo PKD1 </it>mutations are identified and reported.</p> <p>Methods</p> <p>Full-length <it>PKD1 </it>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 <it>PKD1 </it>region, sequencing, direct mutation detection, and segregation analysis in the affected family.</p> <p>Results</p> <p>Five <it>PKD1 </it>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 <it>de novo</it>.</p> <p>Conclusion</p> <p>The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening <it>PKD1 </it>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 <it>de novo PKD1 </it>mutations indicates that this gene is prone to mutations.</p> http://www.biomedcentral.com/1471-2350/5/2
collection DOAJ
language English
format Article
sources DOAJ
author Bunditworapoom Duangkamon
Sirinavin Chintana
Vareesangthip Kriengsak
Limwongse Chanin
Thongnoppakhun Wanna
Rungroj Nanyawan
Yenchitsomanus Pa-thai
spellingShingle Bunditworapoom Duangkamon
Sirinavin Chintana
Vareesangthip Kriengsak
Limwongse Chanin
Thongnoppakhun Wanna
Rungroj Nanyawan
Yenchitsomanus Pa-thai
Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
BMC Medical Genetics
author_facet Bunditworapoom Duangkamon
Sirinavin Chintana
Vareesangthip Kriengsak
Limwongse Chanin
Thongnoppakhun Wanna
Rungroj Nanyawan
Yenchitsomanus Pa-thai
author_sort Bunditworapoom Duangkamon
title Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
title_short Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
title_full Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
title_fullStr Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
title_full_unstemmed Novel and <it>de novo PKD1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
title_sort novel and <it>de novo pkd1 </it>mutations identified by multiple restriction fragment-single strand conformation polymorphism (mrf-sscp)
publisher BMC
series BMC Medical Genetics
issn 1471-2350
publishDate 2004-02-01
description <p>Abstract</p> <p>Background</p> <p>We have previously developed a long RT-PCR method for selective amplification of full-length <it>PKD1 </it>transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the <it>PKD1 </it>gene. These have provided us with an opportunity to study <it>PKD1 </it>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 <it>PKD1 </it>mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and <it>de novo PKD1 </it>mutations are identified and reported.</p> <p>Methods</p> <p>Full-length <it>PKD1 </it>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 <it>PKD1 </it>region, sequencing, direct mutation detection, and segregation analysis in the affected family.</p> <p>Results</p> <p>Five <it>PKD1 </it>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 <it>de novo</it>.</p> <p>Conclusion</p> <p>The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening <it>PKD1 </it>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 <it>de novo PKD1 </it>mutations indicates that this gene is prone to mutations.</p>
url http://www.biomedcentral.com/1471-2350/5/2
work_keys_str_mv AT bunditworapoomduangkamon novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
AT sirinavinchintana novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
AT vareesangthipkriengsak novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
AT limwongsechanin novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
AT thongnoppakhunwanna novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
AT rungrojnanyawan novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
AT yenchitsomanuspathai novelanditdenovopkd1itmutationsidentifiedbymultiplerestrictionfragmentsinglestrandconformationpolymorphismmrfsscp
_version_ 1724174061566689280