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Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53

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 p...

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Autores principales: Fitarelli-Kiehl, Mariana, Macedo, Gabriel S., Schlatter, Rosane Paixão, Koehler-Santos, Patricia, Matte, Ursula da Silveira, Ashton-Prolla, Patricia, Giacomazzi, Juliana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Sociedade Brasileira de Genética 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910550/
https://www.ncbi.nlm.nih.gov/pubmed/27275664
http://dx.doi.org/10.1590/1678-4685-GMB-2014-0351
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author Fitarelli-Kiehl, Mariana
Macedo, Gabriel S.
Schlatter, Rosane Paixão
Koehler-Santos, Patricia
Matte, Ursula da Silveira
Ashton-Prolla, Patricia
Giacomazzi, Juliana
author_facet Fitarelli-Kiehl, Mariana
Macedo, Gabriel S.
Schlatter, Rosane Paixão
Koehler-Santos, Patricia
Matte, Ursula da Silveira
Ashton-Prolla, Patricia
Giacomazzi, Juliana
author_sort Fitarelli-Kiehl, Mariana
collection PubMed
description 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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spelling pubmed-49105502016-07-01 Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53 Fitarelli-Kiehl, Mariana Macedo, Gabriel S. Schlatter, Rosane Paixão Koehler-Santos, Patricia Matte, Ursula da Silveira Ashton-Prolla, Patricia Giacomazzi, Juliana Genet Mol Biol Special Oncogenetics 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 Genética 2016-06-03 2016 /pmc/articles/PMC4910550/ /pubmed/27275664 http://dx.doi.org/10.1590/1678-4685-GMB-2014-0351 Text en Copyright © 2016, Sociedade Brasileira de Genética. http://creativecommons.org/licenses/by/4.0/ License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited.
spellingShingle Special Oncogenetics
Fitarelli-Kiehl, Mariana
Macedo, Gabriel S.
Schlatter, Rosane Paixão
Koehler-Santos, Patricia
Matte, Ursula da Silveira
Ashton-Prolla, Patricia
Giacomazzi, Juliana
Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53
title Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53
title_full Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53
title_fullStr Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53
title_full_unstemmed Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53
title_short Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H inTP53
title_sort comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.r337h intp53
topic Special Oncogenetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910550/
https://www.ncbi.nlm.nih.gov/pubmed/27275664
http://dx.doi.org/10.1590/1678-4685-GMB-2014-0351
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