Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Genetics and Molecular Biology |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572016000200203 |
Resumo: | Abstract Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario. |
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Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53TP53-p.R337HRFLPTaqManHRMSanger SequencingAbstract Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario.Sociedade Brasileira de Genética2016-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572016000200203Genetics and Molecular Biology v.39 n.2 2016reponame:Genetics and Molecular Biologyinstname:Sociedade Brasileira de Genética (SBG)instacron:SBG10.1590/1678-4685-gmb-2014-0351info:eu-repo/semantics/openAccessFitarelli-Kiehl,MarianaMacedo,Gabriel S.Schlatter,Rosane PaixãoKoehler-Santos,PatriciaMatte,Ursula da SilveiraAshton-Prolla,PatriciaGiacomazzi,Julianaeng2017-03-17T00:00:00Zoai:scielo:S1415-47572016000200203Revistahttp://www.gmb.org.br/ONGhttps://old.scielo.br/oai/scielo-oai.php||editor@gmb.org.br1678-46851415-4757opendoar:2017-03-17T00:00Genetics and Molecular Biology - Sociedade Brasileira de Genética (SBG)false |
dc.title.none.fl_str_mv |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
title |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
spellingShingle |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 Fitarelli-Kiehl,Mariana TP53-p.R337H RFLP TaqMan HRM Sanger Sequencing |
title_short |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
title_full |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
title_fullStr |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
title_full_unstemmed |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
title_sort |
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53 |
author |
Fitarelli-Kiehl,Mariana |
author_facet |
Fitarelli-Kiehl,Mariana Macedo,Gabriel S. Schlatter,Rosane Paixão Koehler-Santos,Patricia Matte,Ursula da Silveira Ashton-Prolla,Patricia Giacomazzi,Juliana |
author_role |
author |
author2 |
Macedo,Gabriel S. Schlatter,Rosane Paixão Koehler-Santos,Patricia Matte,Ursula da Silveira Ashton-Prolla,Patricia Giacomazzi,Juliana |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Fitarelli-Kiehl,Mariana Macedo,Gabriel S. Schlatter,Rosane Paixão Koehler-Santos,Patricia Matte,Ursula da Silveira Ashton-Prolla,Patricia Giacomazzi,Juliana |
dc.subject.por.fl_str_mv |
TP53-p.R337H RFLP TaqMan HRM Sanger Sequencing |
topic |
TP53-p.R337H RFLP TaqMan HRM Sanger Sequencing |
description |
Abstract Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-06-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572016000200203 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572016000200203 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/1678-4685-gmb-2014-0351 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Sociedade Brasileira de Genética |
publisher.none.fl_str_mv |
Sociedade Brasileira de Genética |
dc.source.none.fl_str_mv |
Genetics and Molecular Biology v.39 n.2 2016 reponame:Genetics and Molecular Biology instname:Sociedade Brasileira de Genética (SBG) instacron:SBG |
instname_str |
Sociedade Brasileira de Genética (SBG) |
instacron_str |
SBG |
institution |
SBG |
reponame_str |
Genetics and Molecular Biology |
collection |
Genetics and Molecular Biology |
repository.name.fl_str_mv |
Genetics and Molecular Biology - Sociedade Brasileira de Genética (SBG) |
repository.mail.fl_str_mv |
||editor@gmb.org.br |
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1752122386768986112 |