Evaluation of the protein truncation test and mutation detection in the NF1 gene: mutational analysis of 15 known and 40 unknown mutations

The neurofibromatosis type 1 (NF1) gene located at 17q 11.2 contains 60 exo ns and spans 350 kb of genomic DNA. Mutation analysis has been hampered by the large size of the gene, the high rate of new mutations, a lack of mutat ional clustering and the presence of numerous homologous loci. Mutation det ection methods based on the direct analysis of a gene's RNA transcript perm it the rapid screening of large multi-exonic genes. However, the detection of frame-shift or nonsense mutations can be limited by instability of the m utant mRNA species due to nonsense-mediated decay. In order to determine th e frequency of this allelic exclusion, total lymphocyte RNA was analysed fr om 15 NF1 patients with known truncating mutations and a panel of 40 NF1 pa tients with unknown mutations. The level of expression of the mutant messag e was greatly reduced in 2 of the 15 samples (13%), and 3 of the 18 informa tive samples from the panel of 40. A coupled reverse-transcription polymera se chain reaction and protein truncation test method was subsequently appli ed to screen RNA from the panel of 40 unrelated NF1 patients. Aberrant poly peptide bands were identified and characterised in 21 samples (53%). The mu tations identified were 479del107;ins31, 495delTGTT, 1127delTGAT, R416X, R4 40X, l446del 62, 1541delAG, 2252del 74, 2537insTG, 3456delACTC, R1276X, R13 62X, 5749ins171, 6084del280, 6487insA, R2214X, 6791insA, 6858del141, 7458de lC, 7676 2A-G and 8081delC. These mutations were uniformly distributed acro ss the gene and 14 represent novel changes that contribute to the germline mutational spectrum of the NF1 gene.