Detection of novelNF2 mutations by an RNA mismatch cleavage method

Publisher Website
Google Scholar
Abstract
Mutations in the neurofibromatosis type 2 gene (NF2) cause benign nervous system tumors. Common methods for detecting NF2 mutations (such as single stranded conformational polymorphism analysis and denaturing gradient gel electrophoresis) are laborious and time‐consuming. We adapted and improved a commercial assay, the Non‐Isotopic RNase Cleavage Assay (NIRCA™, Ambion, Austin, TX) for rapid, non‐isotopic, high‐sensitivity screening for NF2 mutations in tumors. We improved the assay by: 1) extending the typical NIRCA™ template size of < 500 bp to 1.3 kb without decreasing detection efficiency; 2) modifying the transcription step of the original protocol so that transcription of PCR products was increased by up to 50%; 3) optimizing the combination of cleavage enzymes and reaction time. With these modifications, mutations were found in 15 of 20 patients (75%) using NIRCA™. Seven of the point mutations detected (two nonsense, two missense, and three splice‐site) are novel. All mutations were confirmed by direct sequencing and no mutations were found using direct sequencing in patients that were negative by NIRCA™. The 75% NF2 mutation detection rate using this design is similar to detection rates in tumors using other mutation detection methods. Hum Mutat 15:474–478, 2000.