| Autor: |
Fitarelli-Kiehl M; Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.; Laboratório de Medicina Genômica, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil., Macedo GS; Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.; Laboratório de Medicina Genômica, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil., Schlatter RP; Grupo de Pesquisa e Pós-Graduação (GPPG), Hospital de Clínicas de Porto Alegre and Programa de Pós Graduação em Cardiologia, UFRGS Porto Alegre, RS, Brazil., Koehler-Santos P; Unidade de Análises Moleculares e de Proteínas (UAMP), Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil., Matte Uda S; Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.; Unidade de Análises Moleculares e de Proteínas (UAMP), Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.; Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil., Ashton-Prolla P; Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.; Laboratório de Medicina Genômica, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil.; Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil., Giacomazzi J; Laboratório de Medicina Genômica, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, RS, Brazil.; Departamento de Genética, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. |
| Abstrakt: |
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. |
