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

• Autor(a) principal: Kiehl, Mariana Fitarelli
• Data de Publicação: 2016
• Outros Autores: Macedo, Gabriel de Souza, Schlatter, Rosane Paixão, Santos, Patrícia Koehler dos, Matte, Ursula da Silveira, Prolla, Patrícia Ashton, Giacomazzi, Juliana
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UFRGS
• Texto Completo: http://hdl.handle.net/10183/147480
• topic: Neoplasias; Predisposição genética para doença; TP53-p.R337H; RFLP; TaqMan; HRM; Sanger sequencing

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.