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Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53

Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53

Authors :
Mariana Fitarelli-Kiehl
Gabriel S. Macedo
Rosane Paixão Schlatter
Patricia Koehler-Santos
Ursula da Silveira Matte
Patricia Ashton-Prolla
Juliana Giacomazzi
Source :
Genetics and Molecular Biology, Vol 39, Iss 2, Pp 203-209 (2016)
Publication Year :
2016
Publisher :
Sociedade Brasileira de Genética, 2016.

Abstract

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.

Details

Language :
English
ISSN :
16784685
Volume :
39
Issue :
2
Database :
Directory of Open Access Journals
Journal :
Genetics and Molecular Biology
Publication Type :
Academic Journal
Accession number :
edsdoj.b06f7ba767449c8dd265517066349d
Document Type :
article
Full Text :
https://doi.org/10.1590/1678-4685-gmb-2014-0351