[Development and establishment of a yeast-based stop codon assay for detection of NF2 gene premature-terminating...

Neurofibromatosis type 2 (NF2) is an autosomally inherited disorder, caused by a mutation in NF2 tumor suppressor gene on chromosome 22q12, being characterized by multiple intracranial tumors including schwannomas, meningiomas and ependymomas. The protein encoded by the NF2 gene has a similarity to ezrin, radixin and moesin (ERM) proteins that link membrane proteins to the cytoskeleton. It has been reported that the majority of NF2 gene mutations are nonsense mutations that result in a premature termination of translation. We have developed and established a yeast-based stop codon assay for detection of NF2 gene premature terminating mutations. This assay utilizes an autonomously replicating yeast vector that expresses an NF2::ADE2 chimera protein, which gives a normal white colony when a sample NF2 cDNA is homologously recombinated, while it gives a red colony when the sample cDNA contains a nonsense mutation. The assay gave 8.0 +/- 3.5 (mean +/- SD) background red colonies when tested on clinical samples which did not contain an NF2 gene mutation. A total of 16 schwannomas (including three NF2 cases) were tested by the assay. NF2 gene mutations were detected as red colonies more than 10% in 13 of the 16 cases. Sequence analyses of plasmids recovered from the red colonies showed single base substitutions giving stop codons in five cases, and base deletions leading to frameshift and premature termination in four. Additionally, in-frame exon skippings were found in three cases. One case that gave 14% red colonies did not show a clonal mutation. This study demonstrates that the newly established assay is capable of an efficient detection of nonsense mutations of NF2 gene in clinical samples.