Introduction of a PCR-RFLP method for the detection of two mutations in VHL gene

Background: Von Hippel-Lindau disease is an inherited neoplastic disorder caused by germline mutations in the VHL gene. In Cuba, molecular diagnosis is performed by the method of single-strand conformation polymorphism of DNA of the three exons of the gene followed by sequencing. This method is expensive, complicated and time-consuming. Objective: to describe the introduction of the molecular diagnosis of mutations c.362A>G and c.481C> in the VHL gene by PCR-RFLP. Methods: computer software CLC Sequence viewer 6.5.1 was used to identify restriction enzymes with cleavage sites modified by mutation c. 362A>G in the exon 2 and c. 481C>T in exon 3 in the VHL gene. DNA samples of patients already diagnosed through SSCP-sequencing were used. Such samples were amplified by PCR method followed by the enzymatic digestion with SfaNI and BtgZI restriction enzymes. Amplified fragments were analyzed by agarose gel electrophoresis. Results obtained using both methods were compared. Results: effectiveness of the PRC-RFLP method for the diagnosis of c. 362A>G and c. 481C>T mutations in the VHL gene was standardized and proved. Conclusions: PCR-RFLP method has advantages over SSCP-sequencing strategy for establishing a fast, reproducible and reliable diagnosis of VHL disease in family cases molecularly characterized.