Detection of a non-standard Mutation in the RET-Protoncogene, by Site Directed Mutagenesis

REAL S; PUSIOL E; PERINETTI H; MAYORGA LS; ROQUÉ M

Congreso; XXI Reunión de la Sociedad de Biología de Cuyo; 2003

Sociedad de Biología de Cuyo

Mutations in the ret protoncogene has been related to the development of type 2A multiple endocrine neoplasia (MEN2A). In 94% of the cases, mutations are detected in codon 634 of the gen ret protoncogene. Recently our group developed a PCR-based detection method for all possible mutation in this hot-spot site. The initial aim was to detect a mutation i the ret protoncogene of a MEN2A patient by this method. The result revealed that there was no mutation in the 634 codon. Therefore, the entire gene was sent abroad to be sequenced. The sequencing result corroborated that the codon 634 was normal and detected a mutation in codon 611 that changed TGC (Cys) to TTC (ala). To screen the rest of the family, we developed a PCR mutagenic strategy for this specific mutation. A forward primer introducing a mutation limiting with codon 611 was designed. In the Wild Type PCR product, the introduced mutation formed a restriction site for PvuII, distinguishable from the mutated PCR product i which the restriction site was absent. Through a posterior PAGE analysis we could differentiate the wild type allele of the mutated allele. We detected 3 unaffected sons and 1 affected seven years old son of the index patient. This young mutated patient will be preventively operated next month.