title: Detection of novel NF2 mutations by an RNA mismatch cleavage method.
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 (NIRCAtrade mark, Ambion, Austin, TX) for rapid, non-isotopic, high-sensitivity screening for NF2 mutations in tumors. We improved the assay by: 1) extending the typical NIRCAtrade mark 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 NIRCAtrade mark. 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 NIRCAtrade mark. The 75% NF2 mutation detection rate using this design is similar to detection rates in tumors using other mutation detection methods.
authors.affiliation: House Ear Institute, Los Angeles, California 90057, USA.
authors.forename: R
authors.initials: R
authors.lastname: Faudoa
authors.forename: Z
authors.initials: Z
authors.lastname: Xue
authors.forename: F
authors.initials: F
authors.lastname: Lee
authors.forename: M E
authors.initials: ME
authors.lastname: Baser
authors.forename: G
authors.initials: G
authors.lastname: Hung
mesh_terms: D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis / Q000379:methods*; D016515:Genes, Neurofibromatosis 2; D006801:Humans; D007438:Introns; D008565:Membrane Proteins / Q000235:genetics; D009154:Mutation; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics; D016518:Neurofibromatosis 2 / Q000235:genetics*; D025581:Neurofibromin 2; D018807:Polymorphism, Single-Stranded Conformational; D017384:Sequence Deletion
chemical_list: D008565:Membrane Proteins; D009363:Neoplasm Proteins; D025581:Neurofibromin 2