Detection of novel NF2 mutations by an RNA mismatch cleavage method

Mutations in the neurofibromatosis type 2 gene (NF2) cause benign nervous s ystem tumors. Common methods for detecting NF2 mutations (such as single st randed conformational polymorphism analysis and denaturing gradient gel ele ctrophoresis) are laborious and time-consuming. We adapted and improved a c ommercial assay, the Non-Isotopic RNase Cleavage Assay (NIRCA(TM), Ambion, Austin, TX) for rapid, non-isotopic, high-sensitivity screening for NF2 mut ations in tumors. We improved the assay by: 1) extending the typical NIRCA( TM) template size of < 500 bp to 1.3 kb without decreasing detection effici ency; 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 modification s, mutations were found in 15 of 20 patients (75%) using NIRCA(TM). Seven o f the point mutations detected (two nonsense, two missense, and three splic e-site) are novel. All mutations were confirmed by direct sequencing and no mutations were found using direct sequencing in patients that were negativ e by NIRCA(TM). The 75% NF2 mutation detection rate using this design is si milar to detection rates in tumors using other mutation detection methods. Hum Mutat 15:474-478, 2000. (C) 2000 Wiley-Liss, Inc.